Splenic Damage during SIV Infection

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The American Journal of Pathology, Vol. -, No. -, - 2016

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Splenic Damage during SIV Infection Role of T-Cell Depletion and Macrophage Polarization and Infection Q40

Dionna W. Williams,* Elizabeth L. Engle,* Erin N. Shirk,* Suzanne E. Queen,* Lucio Gama,* Joseph L. Mankowski,*yz M. Christine Zink,*y and Janice E. Clements*yz From the Departments of Molecular and Comparative Pathobiology,* Pathology,y and Neurology,z Johns Hopkins University School of Medicine, Baltimore, Maryland Accepted for publication March 25, 2016. Address correspondence to Janice E. Clements, Ph.D., Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, 733 N Broadway, MRB 831, Baltimore, MD 21205. E-mail: [email protected].

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The effects of HIV infection on spleen and its cellular subsets have not been fully characterized, particularly for macrophages in which diverse populations exist. We used an accelerated SIV-infected macaque model to examine longitudinal effects on T-cell and macrophage populations and their susceptibilities to infection. Substantial lymphoid depletion occurred, characterized by follicular burn out and a loss of CD3 T lymphocytes, which was associated with cellular activation and transient dysregulations in CD4/CD8 ratios and memory effector populations. In contrast, the loss of CD68 and CD163þCD68þ macrophages and increase in CD163 cells was irreversible, which began during acute infection and persisted until terminal disease. Mac387 macrophages and monocytes were transiently recruited into spleen, but were not sufﬁcient to mitigate the changes in macrophage subsets. Type I interferon, M2 polarizing genes, and chemokine-chemokine receptor signaling were up-regulated in spleen and drove macrophage alterations. SIV-infected T cells were numerous within the white pulp during acute infection, but were rarely observed thereafter. CD68, CD163, and Mac387 macrophages were highly infected, which primarily occurred in the red pulp independent of T cells. Few macrophages underwent apoptosis, indicating that they are a long-lasting target for HIV/SIV. Our results identify macrophages as an important contributor to HIV/SIV infection in spleen and in promoting morphologic changes through the loss of speciﬁc macrophage subsets that mediate splenic organization. (Am J Pathol 2016, -: 1e20; http://dx.doi.org/10.1016/j.ajpath.2016.03.019)

The functions, organization, and cellular constituents of the spleen drastically change during HIV infection, as it is infected early during primary disease.1 Substantial morphologic changes occur as a result of the persistent infection, including white pulp depletion, perivascular hyalinization, necrosis, extramedullary hematopoiesis, and inﬂammatory cell inﬁltrates.2 Although not as extensively characterized as other lymphoid organs, the spleen represents an anatomical viral reservoir3e5 that occurs, in part, because of the limited penetration of some antiretroviral drugs to the organ.6 The spleen also contains potential T-cell and macrophage cellular reservoirs.7 HIV-induced splenic T lymphocyte dysfunction is well characterized and consists of a signiﬁcant decline in the numbers of CD4 cells, the

recruitment of virus-speciﬁc cytotoxic T lymphocytes, and the presence of latently infected memory cells.8e17 Macrophages are also important during infection and promote HIV persistence and damage to the spleen.5,18 In addition to producing infectious virus, macrophages may also enter into a latently infected state and contribute to viral reservoirs.7,19e29 Furthermore, macrophages are immune to the cytopathic effects of HIV infection.5,30e33 This suggests that once infected, even if present in a latent state or infected at low levels, these long-lived cells have the potential to Supported by NIH grants P40 OD013117, P01MH070306, NS077869, Q1 NS076357, U19-0AI076113, and R56-AI118753. Q2 Disclosures: None declared.

Copyright ª 2016 American Society for Investigative Pathology. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0). http://dx.doi.org/10.1016/j.ajpath.2016.03.019

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Williams et al reactivate virus, produce low-level replication, and present infectious virus to T cells over the course of many years.34e39 Peripheral blood monocytes may also contribute to splenic macrophage populations during HIV infection and promote virus-induced damage, as they can accumulate in the spleens of infected individuals.23 Diverse macrophage subsets reside within the red and white pulp compartments of the spleen, and fulﬁll different functions as a result of their localization.40 The four major splenic macrophage populations include tingible body, metallophilic, marginal zone, and red pulp macrophages. Marginal zone and metallophilic macrophages reside in the marginal zone and are critical for eliciting antimicrobial functions by clearance of blood-borne bacteria and viruses.41 Follicular tingible body macrophages remove apoptotic B cells during germinal center reactions and are essential for the generation of antigen-speciﬁc, high-afﬁnity plasma and memory cells.42 Follicular macrophages are also important for presenting antigen to T cells and initiating the adaptive immune response to pathogens.43 Finally, red pulp macrophages are laid down embryonically and mediate erythrocyte phagocytosis, hemoglobin/haptoglobin scavenging, iron recycling, and storage. Splenic macrophages are characterized by their unique functions, distinct localizations, and expression of differential cell markers.41 In addition to their unique markers, macrophages also express CD163 and CD68, less speciﬁc, general markers that can be used to collectively identify both resident and newly entered splenic cells.41 CD163, a glycoprotein member of the scavenger receptor cysteine-rich group B family, and CD68, a low-density lipoprotein binding glycoprotein, are restricted to cells of the monocyte/ macrophage lineage.44e46 CD163, a hemoglobin/haptoglobin scavenger receptor, is associated with the resolution of inﬂammation and is highly expressed on alternatively activated M2-polarized macrophages.47,48 In contrast, CD68 is a widely used immunohistochemical marker present on many resident tissue macrophages, and is considered a pan-macrophage marker.49 Mac387, an antibody that has speciﬁcity for the L1 antigen, may also be used to identify splenic macrophages, and is indicative of a blood monocyteederived subset.50 These newly inﬁltrated macrophages have a distinct morphology from resident macrophages and also have different functional capacities.51,52 The resident and recently inﬁltrated macrophage subsets may have differing levels of HIV infectivity, and may also promote distinct antiviral and/or inﬂammatory responses within the spleen. However, the effects of HIV on these different splenic macrophage subsets are not completely understood. It is important to characterize more extensively the diverse macrophage subsets as these cells are long-lived, are highly susceptible to infection, and play critical roles in maintaining splenic function. Herein, we used an accelerated, consistent SIV-infected pigtail macaque model to examine the longitudinal effects of SIV infection on the spleen. We examined splenic CD163,

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CD68, and Mac387 macrophage phenotypes and differential SIV infection of these cells from acute to late stage disease. We found that in addition to morphologic and T cell alterations, there were profound and long-lasting changes in macrophage populations within the spleen. SIV infection promoted a global increase in CD163 macrophages, and a simultaneous decrease in CD68 cells only partially because of apoptosis of these cells. In addition, there was a substantial depletion of fetal-derived red pulp CD163þCD68þ macrophages, which may compromise splenic iron scavenging. Gene expression proﬁling demonstrated that the pronounced increase of interferon mediators, M2 polarizing factors, and chemokine-chemokine receptor signaling promoted the regulation of macrophage subsets. All macrophage populations characterized contained SIV RNA, as determined by in situ hybridization analyses, although the subsets had differential patterns and levels of infectivity.

Materials and Methods Animals and Ethics Statement All animal studies were approved by the Johns Hopkins University Institutional Animal Care and Use Committee and performed according to the guidelines of the US Department of Agriculture Animal Welfare Act and the NIH Guide for the Care and Use of Laboratory Animals.53 Sixty Q4 pigtailed macaques were involved in the study, 53 of which were i.v. dual inoculated with SIV/DeltaB670 (50, 50% animal infectious doses) and SIV/17E-Fr (10,000 50% animal infectious doses) as previously described.54,55 Macaques were euthanized at 4 (eight animals), 7 (six animals), 10 (six animals), 14 (ﬁve animals), 21 (ﬁve animals), 56 (nine animals), and 84 (14 animals) days post infection (dpi), in accordance with federal guidelines and institutional policies. Seven animals were mock infected and served as uninfected controls. Macaques were perfused with sterile saline at euthanasia to remove blood from the vasculature and tissues.

Sample Preparation Blood samples were collected at multiple time points before inoculation, and at 4, 7, 10, 14, 21, 56, and 84 dpi. Blood was collected into acid citrate dextrose (Sigma-Aldrich, St. Louis, MO) and a portion used for whole blood immunostaining and ﬂow cytometric analyses. Plasma was separated from the remainder of the blood by centrifugation for the determination of plasma viral load.56e60 At euthanasia and concurrent with blood sampling, spleens were harvested, weighed, and sectioned to provide intact tissue for viral load determination, NanoString analyses, immunohistochemistry, and immunohistochemistry with in situ hybridization. Repeated sampling was not performed for these analyses, as spleen samples were isolated from independent animals euthanized at each time point after inoculation. Therefore,

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HIV/SIV Spleen Dysfunction all splenic observations constitute a cross-sectional study. For example, data points at days 4 and 7 are not directly related, but instead represent samples obtained from different populations of animals infected with SIV for 4 and 7 days, respectively. Repeated sampling was performed for blood analyses, however, and these data were therefore examined longitudinally.

Quantitation of Viral Load in Plasma and Viral RNA in Tissue

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Plasma viral load was determined throughout infection, as well as at the terminal time point. RNA was isolated from 140 mL of plasma using the QIAamp Viral RNA Mini kit (Qiagen, Valencia, CA) in accordance with the manufacturer’s protocol with the modiﬁcation of on column DNase digestion (RNase free DNase kit; Qiagen) using RQ1 RNase free DNase (Promega, Madison, WI) in the enzyme mix. Spleen viral RNA was measured at the terminal time point using 25 mg of spleen homogenized using RNA Stat-60 (AMSBio, Cambridge, MA) and RNA isolated using the RNeasy PLUS Mini Kit according to the manufacturer’s protocol with the modiﬁcation of on column DNase digestion (RNase free DNase kit; Qiagen) using TURBO DNase (Life Technologies, Grand Island, NY) in the enzyme mix. Quantiﬁcation of SIV RNA (for plasma viral load and spleen RNA levels) was performed by RT-PCR using the following primers and probes: forward primer (SGAG21) 50 -GTCTGCGTCATCTGGTGCATTC-30 ; reverse primer (SGAG22) 50 -CACTAGGTGTCTCTGCACTATCTGTTTTG-30 , probe (pSGAG23) 50 -(FAM)CTTCCTCAGTGTGTTTCACTTTCTCTTCTG-(BHQ_1)-30 , as previously described.54 Viral loads were log-transformed for statistical analyses.

Immunostaining and Flow Cytometry of PBMC and Spleen Single-Cell Suspensions Whole blood samples were stained with ﬂuorochromecoupled CD3, CD4, CD8, HLA-DR, and CD14 antibodies for 20 minutes at room temperature and ﬁxed for 10 minutes with BD FACS Lysing Solution (Becton Dickinson, Franklin Lakes, NJ). Panels with information for all anti½T1 bodies are listed in Table 1. Cells were immunostained and assayed for ﬂow cytometry immediately following initial isolation, or were cryopreserved in 90% fetal bovine serum with 10% dimethyl sulfoxide and stored at 140 C until further analysis. This cryopreservation method is well established and does not result in loss of cell surface markers.61 Spleen cells were isolated for ﬂow cytometric analyses by mechanical agitation and teasing from 10 g of Q6 tissue in RPMI (Gibco, Gaithersburg, MD) with 10% fetal bovine serum (Atlanta Biologicals, Norcross, GA) using 18 gauge needles with the thumb-end of a 5 mL syringe and passing through a 100 mm cell strainer. On obtaining singlecell suspensions, red blood cells were lysed with mild

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Table 1 List Immunostaining

of

Antibodies

Used

for

Flow

Cytometry Q39

Antibody

Company

Clone

CD3 CD4 CD8 CD11b CD14 CD16 CD20 CD28 CD45 CD69 CD95 HLA-DR

BD Invitrogen eBioScience Biolegend Invitrogen eBioScience eBioScience BD BD Biolegend BD Invitrogen

SP34-2 S3.5 RPA-T8 ICRF44 Tuk4 3G8 2H7 CD28.2 2D1 FN50 DX2 Tu36

treatment of ammonium chloride. Single-cell suspensions derived from spleen were stained with ﬂuorochromecoupled CD45, CD3, CD4, CD69, CD8, CD28, CD95, CD11b, CD14, CD16, HLA-DR, and CD20 antibodies (Table 1). Cells were stained and ﬁxed as described above and as previously described.62 Samples were analyzed using a BD LSRFortessa or FACSCalibur cytometer and Diva software version 6.1.3. Q7 FACS data were analyzed using FlowJo version 9.5.1. All Q8 antibodies were tested for speciﬁcity by FMO. To enumerate plasma CD4 T lymphocytes (deﬁned as CD3þCD4þ cells), the frequency of CD4 T cells relative to the total peripheral blood CD3 population was multiplied by the whole blood complete blood cell count. To enumerate plasma monocytes (deﬁned as CD14þHLA-DRþ cells), the frequency of CD14 monocytes in the peripheral blood was multiplied by the whole blood complete blood cell count. Spleen B lymphocytes were deﬁned as CD45þCD20þ HLA-DRþ. T lymphocytes were deﬁned as CD45þCD3þ, followed by the further division of CD3þ cells into CD8þ and CD4þ populations. CD4 T-cell subsets were characterized according to CD3 positivity, CD8 negativity, CD69 as a marker of activation,63e65 and on the basis of CD28 and CD95 expression into naïve (CD28þCD95), central memory (CD28þCD95þ), and effector memory (CD28CD95þ) subsets.66 Spleen monocytes were deﬁned according to light scatter characteristics, CD11b positivity, CD14high, CD45high, and HLA-DR expression. Monocytes were characterized further to determine the frequency of cells with surface CD16. Gating strategies for identiﬁcation of splenic T lymphocytes and monocytes are illustrated in Supplemental Figure S1.

Histopathology of Spleen Spleen sections were ﬁxed in Streck tissue ﬁxative (Streck, Omaha, NE) for 7 days and parafﬁn embedded, and hematoxylin and eosin staining was performed. To determine histological differences in the spleen during longitudinal

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Williams et al SIV infection, three random ﬁelds of each slide were examined by microscope in a blinded manner by a pathologist (M.C.Z.). Lymphoid depletion is characterized by a loss of follicles, a decreased size of the periarteriolar lymphoid sheath, reduced size of the white pulp, the presence of few to no apparent germinal centers, a loss of overall architecture of the marginal and mantle zones, and irregularly shaped follicles. Lymphoid hyperplasia is deﬁned as polymorphous and enlarged follicles, reactive mantle zones, large germinal centers, and the presence of increased numbers of white pulp follicles.

Immunohistochemistry in Spleen CD163, CD68, and Mac387 were used to evaluate splenic macrophages. Collectively, these three markers identify the majority of spleen macrophage populations.41,43 Immunohistochemistry was performed on Streck-ﬁxed, parafﬁnembedded spleen sections. Slides were deparafﬁnized by heating at 60 C for 60 minutes and rehydrated in a graded series of ethanol and water. Slides were pretreated with 10 mmol/L sodium citrate, pH 6.0 (JT Baker, Center Valley, PA), before blocking with Fc receptor Block (Innovex Biosciences, Richmond, CA) and Power Block (Biogenex, San Ramon, CA). Primary antibodies to anti-CD163 (1:5000, clone EDHu-1; AbD Serotec, Raleigh, NC), antiCD68 (1:2500, clone KP1; Dako, Carpinteria, CA), and anti-Mac387 (1:5000, clone Mac 387; Dako) were applied for 1 hour, followed by DAB HRP Peroxidase Substrate (Vector Laboratories, Burlingame, CA) for 10 minutes. For double and triple immunohistochemistry staining, the appropriate antibodies were added sequentially for 1 hour, followed by DAB HRP Peroxidase Substrate. In addition, after immunostaining with each antibody, slides were blocked with Mouse on Mouse Blocking Reagent (Vector Laboratories), a peroxidase block (3% H2O2 in methanol), and diluted horse serum to minimize nonspeciﬁc signal. Appropriate isotype-matched tissue controls were used. CD163, CD68, and Mac387 macrophages were examined by microscope in a blinded manner by three independent individuals (M.C.Z., J.E.C., and D.W.W.). To quantitate the presence of the macrophage populations in the germinal center, mantle zone, marginal zone, and red pulp, numerical scores of 0 (none), 1 (minimal), 2 (moderate), or 3 (many) were assigned. The scores for all sections were totaled and divided by the total number of ﬁelds to give a mean score for the frequency of macrophages. For our semiquantitative analyses, the mean score for each splenic area was set to one and a fold-change relative to uninfected macaques was determined.

NanoString Analysis Spleens were harvested from macaques at 4 (six animals), 7 (six animals), 14 (ﬁve animals), 21 (ﬁve animals), and 56 (nine animals) dpi. Four mock-infected animals served as

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controls. RNA was isolated as described above, 250 ng sent to the Johns Hopkins Deep Sequencing and Microarray Core, and NanoString analyses performed as previously described.67 Brieﬂy, in the NanoString technology, molecular barcodes attached to target-speciﬁc probes enable multiplexed measurement of gene expression without the need of reverse transcription or DNA ampliﬁcation. In this highly sensitive and reproducible system, barcodes for genes of interest are combined with those for negative (genes whose sequences lack speciﬁcity and cross-reactivity with mammalian genes) and positive (serially diluted probes that enable quality assurance) controls. In our study, barcodes for 76 genes of interest (including actn, GAPDH, HPRT, and PBGD as housekeeping genes) were combined with those for six positive and eight negative controls to generate CodeSets according to the company’s guidelines, based on rhesus macaque annotated sequences (NanoString, Seattle, WA). Raw data, which are proportional to gene copy numbers, were normalized by the geometric mean of the four housekeeping genes. The four housekeeping genes were not signiﬁcantly altered as a result of SIV infection and had comparable copy numbers in both infected and uninfected macaques (data not shown). After normalization, the copy numbers for each target gene were compared to those for the negative controls. Target genes with normalized copy numbers below that of the negative controls were assigned values of 1. Genes that consistently received values of 1 throughout longitudinal infection were excluded because of low hybridization or probe efﬁciency. One gene (of 76) met criteria for exclusion. A portion of the animals in this NanoString analyses were included in a recently published study.67 Our NanoString analyses were performed on tissue homogenates representative of the whole organ, and it is not possible to ascribe changes in gene expression to any speciﬁc cell population.

Combined Immunohistochemistry and in Situ Hybridization To examine SIV RNA-positive CD163, CD68, and Mac387 macrophages in the spleen, combined immunohistochemistry and in situ hybridization were performed as previously described.58 Brieﬂy, ﬁxed, parafﬁn-embedded spleen sections were deparafﬁnized, rehydrated in a graded ethanol series, and pretreated with 25 mg/mL Proteinase K (Roche, Indianapolis, IN). After post-ﬁxing with 4% paraformaldehyde (SigmaAldrich), in situ hybridization was performed using a SIVmac239 digoxigenin-UTP labeled riboprobe. Immunohistochemistry was then performed for CD163, CD68, or Mac387. Appropriate isotype-matched tissue controls were used. Uninfected and SIV-infected tissues were used as negative and positive controls, respectively. The number of SIV-RNA CD163, CD68, and Mac387 macrophages was determined by manually counting doublelabeled cells in ﬁve random ﬁelds of spleen. In addition, the number of small, punctate SIV-RNA positive cells in the

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HIV/SIV Spleen Dysfunction white pulp that lacked positivity for the macrophage immunohistochemical markers (indicative of T lymphocytes) was determined by manually counting in ﬁve follicles.

Cleaved Caspase-3 Immunohistochemistry Cleaved caspase-3 analysis was analyzed using the Leica Bond RX automated immunostainer (Leica Microsystems, Wetzlar, Germany). Cleaved caspase-3 antibody (0.252 mg/ mL, Cell Signaling) was used and immunohistochemistry performed using the Leica Bond Polymer Reﬁne Detection Kit according to manufacturer’s protocol. The Leica Bond Polymer Reﬁne Red Detection Kit was used for double staining with cleaved caspase-3 (0.126 mg/mL, Cell Signaling) and CD68 and CD163 antibodies according to manufacturer’s protocol.

Statistical Analysis Q9

Statistical analyses were performed using Prism 6.0 software (GraphPad Software, Inc., San Diego, CA). The Kruskal-Wallis analysis of variance test was performed to determine signiﬁcant changes in gene expression during SIV infection as compared to uninfected control animals (a 0.05), followed by Dunn’s multiple comparisons test (P 0.05). Linear regression was used to determine correlations between interferon (IFN)-b and interferon stimulated genes. The Mann-Whitney test was used to determine statistical signiﬁcance for all other data (P 0.05).

Results Cellular and Architectural Changes Occur in the Spleen during SIV Infection The spleen has abundant lymphocytes and macrophages, the two major cellular targets of HIV/SIV infection, which represent potential viral reservoirs and pose major obstacles to HIV eradication. Of these cell types, splenic macrophages have been largely understudied. To provide a more complete understanding of splenic alterations during SIV infection, we used a well-established, consistent, accelerated pigtail macaque model. In this model, the long progression to disease, typical of lentiviral infection, occurs within 84 dpi, and is characterized by acute (4, 7, and 10 dpi), asymptomatic (14 and 21 dpi), and chronic (56 dpi) stages, after which time terminal disease is achieved at 84 dpi 54,55 ½F1 (Figure 1A). During the progression from acute to terminal disease, plasma viremia increased and remained high, and CD4 T lymphocytes declined, which is characteristic of HIV/SIV infection (Figure 1A). We characterized the T lymphocyte populations during the course of SIV infection. There was a signiﬁcant decrease in the percentage of splenic CD4 T lymphocytes during acute infection (7 dpi), with a concomitant increase in the percentage of CD8þ cells at the same time point, as determined

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by ﬂow cytometry (Figure 1B). However, the relative frequencies of these T lymphocyte subsets were restored by 21 dpi to levels similar to that of uninfected macaques, and were maintained until terminal disease. Despite the restoration in the relative frequencies of spleen T lymphocytes, the CD4 cells that remained had alterations in cellular activation and dysregulated memory subsets, as determined by CD69 and CD28/CD95 ﬂow cytometric analyses, respectively. In uninfected macaques, 23.4% 1.8% of CD4 T cells expressed CD69, an early marker of T cell activation (Figure 1C). During acute infection (7 dpi), expression of CD69 on CD4 T lymphocytes signiﬁcantly increased to 41.4% 3.2%, decreased during asymptomatic infection (26.0% 6.6%), but later increased again during chronic infection at every time point examined, relative to uninfected animals (Figure 1C). In addition to cellular activation, there were also changes in the memory populations of CD4 T cells. In uninfected animals, central memory cells comprised the majority of spleen CD4 T lymphocytes, and naïve cells represented the second most populous subset (Figure 1D). There was also a minor population of effector memory cells in uninfected animals. During acute infection (7 dpi), there was a signiﬁcant increase in the effector memory subset, a decrease in central memory cells, and an almost complete loss of naïve CD4 T cells (Figure 1D). These naïve cells were restored during the asymptomatic phase at 21 dpi, suggesting that additional CD4 T cells were recruited into the spleen from the peripheral blood. During chronic and terminal disease, the memory subsets more closely resembled the frequencies of lymphocytes found in uninfected animals, where central memory cells comprised the major CD4 cell subpopulation (Figure 1D). Pathologic changes in the spleens of SIV-infected and uninfected animals were examined by histochemical staining. A pathologist examined the hematoxylin and eosine stained slides for lymphoid depletion, as characterized by a loss of follicles and decreased size of the periarteriolar lymphoid sheath, and lymphoid hyperplasia, indicated by enlarged and numerous follicles, in a blinded manner. There Q10 was substantial lymphoid depletion in SIV-infected macaques in both acute and chronic/terminal disease (Table 2). ½T2 There was some recovery of lymphocytes during asymptomatic infection, when most of the spleens had a normal appearance with no abnormalities. Representative images of hematoxylin and eosin staining illustrate lymphoid hyperplasia at 4 dpi, and lymphoid depletion at 56 and 84 dpi (Figure 1E). The splenic lymphoid depletion that occurred in our animal model is consistent with studies from individuals with HIV/AIDS, particularly to those from the Q11 pre-cART era.2,9,10

Spleen Macrophages Undergo Dynamic Changes in Cellular Markers Macrophages represent one of the ﬁrst cell types infected during primary HIV/SIV disease and can continue to harbor

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A

B 1200

6

900

4

600

2

300

Figure 1

100

(%)

C

E

8

*

80

*

L

60

40

*

20

* 0

0

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10

14

21

56

84

0

0

Uninfected

7

21

56

84

Uninfected

7

21

56

84

D 60

110

(%)

(%)

C *

100 90 80

40

70 60 50 40

20

30 20 10

0

Uninfected

7

21

56

0

84

E

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Williams et al

dpi

dpi

dpi

dpi

dpi

infectious virus, even after effective cART and plasma viral suppression.68e70 To evaluate spleen macrophage populations during longitudinal SIV infection, we used three speciﬁc macrophage markers: CD163 (a monocyte/ macrophage speciﬁc haptoglobin/hemoglobin scavenger receptor71), CD68 (a general macrophage marker and glycoprotein scavenger receptor), and Mac387 (an antibody that detects the L1 antigen, an intracellular calcium binding protein highly expressed in myelomonocytic cells72). Collectively, these markers account for the majority of splenic macrophage subsets, including tingible body, marginal zone, metallophilic, and red pulp macrophages.41 Each macrophage marker was assessed by immunohistochemistry and analyzed in the germinal center, mantle zone, marginal Table 2

Analysis of spleen T cells and pathology during longitudinal SIV infection. Pigtailed macaques were inoculated with an immunosuppressive swarm and a neurovirulent clone of SIV. A: Peripheral blood was obtained from SIV-infected macaques and RNA isolated at 4, 7, 10, 14, 21, 56, and 84 dpi to determine plasma viral load (open squares) by quantitative RT-PCR. CD4 T lymphocytes (closed circles) were enumerated in whole blood by ﬂow cytometry. The acute (light shading), asymptomatic, and chronic (dark shading) phases of SIV infection are depicted. BeD: Single cell suspensions were obtained from spleen at 7, 21, 56, and 84 dpi and cells analyzed by ﬂow cytometry. B: The frequency of CD8 (open squares) and CD4 T lymphocytes (closed circles) relative to the total spleen CD3 T lymphocyte population was determined by ﬂuorescence-activated cell sorter analyses. C: The percentage of splenic CD4 T lymphocytes that expressed cell surface CD69 was examined, as an indicator of cellular activation. D: The frequency of CD28þ naïve (gray bars), CD95þ effector memory (white bars), and CD28þCD95þ central memory (black bars) CD4 T lymphocytes in the spleen was determined by ﬂow cytometry. E: Representative hematoxylin and eosinestained spleen sections from uninfected and SIV-infected macaques euthanized at 4, 10, 21, 56, and 84 dpi. Solid lines connecting data points denote cumulative and longitudinal blood sampling of the same animals at every time point after inoculation. Dashed lines connecting data points indicate cross-sectional study of independent groups of animals after euthanasia at each time point. Statistical signiﬁcance was determined by Mann-Whitney test. Data are expressed as means SD (). *P 0.05 versus the preceding day after inoculation. Original magniﬁcation, 2 (E).

Q21

Q22

Q23 Q24

zone, and red pulp in three random ﬁelds, in a blinded Q12 manner. We identiﬁed four distinct macrophage populations in the spleen of uninfected animals. Macrophages were phenotypically identiﬁed by expression of CD68, CD163, or Mac387. CD68 single positive cells primarily resided in the germinal center, and represented tingible body macrophages43 (Figure 2, A and C). Although not as prevalent as in the ½F2 germinal center, CD68 macrophages were also present in the other areas of the spleen. In contrast, CD163 and Mac387 cells were not present in the germinal center and represented only a small population of the macrophages in the red pulp. CD163 and Mac387 macrophages primarily resided in the mantle and marginal zones (Figure 2, A and C). To further

Prevalence of Spleen Pathology during SIV Infection

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Acute infection

Asymptomatic infection

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No pathology Lymphoid hyperplasia Lymphoid depletion

72 14 14

22 15 63

48 19 33

22 21 57

Data are given as percentage.

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Figure 2 Analysis of spleen macrophage populations. Immunohistochemistry was performed on spleen sections for CD68 (gray), CD163 (red), CD163/CD68 (gray and red), and Mac387 (brown) macrophages in the white (A) and red pulp (B) of uninfected and SIV-infected macaques. Representative images are shown. Boxed insets show higher magniﬁcation of macrophages in the white (A) and red pulp (B) of uninfected and SIV-infected animals. C: A representative image of the white pulp in an uninfected macaque demonstrates the germinal center, mantle zone, marginal zone, and red pulp, as indicated by dashed line demarcations. DeG: To quantitate the presence of macrophages in spleen, numerical scores of 0 to 3 were assigned for each marker in the splenic regions and averaged. The fold-change of the mean scores for infected macaques was determined relative to uninfected controls, which was set to 1. The fold-change for CD68 (gray line with squares), CD163 (red line with circles), CD163/CD68 (purple line with diamonds), and Mac387 (yellow line with triangles) macrophages was determined for the germinal center (D), mantle zone (E), marginal zone (F), and red pulp (G) during the acute, asymptomatic, and chronic phases of infection. The acute (light shading), asymptomatic, and chronic (dark shading) phases of SIV infection are depicted. Cross-sectional study of independent groups of animals after euthanasia at each time point was performed and the data depicted using solid lines connecting data points for better visualization. Statistical signiﬁcance was determined by Mann-Whitney test. P 0.05 versus the preceding day after inoculation for all comparisons. Data are expressed as means SD (). *CD163 macrophages; yCD163/CD68 macrophages; zMac387 macrophages; and xCD68. Original magniﬁcations: 10 (A); and 20 (B).

identify the phenotypes, triple immunostaining was performed for the macrophage markers. Macrophages that expressed both CD163 and CD68 were predominantly localized to the red pulp and comprised the majority of macrophages in this area of the spleen (Figure 2B).

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CD163þCD68þ macrophages were also present at the proximal region of the marginal zone, directly adjacent to the red pulp. Mac387 cells appeared to be a unique population, as they did not express either of the other two macrophage markers, which is consistent with the work of others.51 Data

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Williams et al 869 summarizing the macrophage populations present in the 870 ½T3 uninfected spleen are listed in Table 3. 871 There were profound and signiﬁcant changes in all 872 splenic macrophage populations during SIV infection, the 873 majority of which began during acute infection and per874 sisted during terminal disease. First, there was a global 875 decrease in CD68 macrophages, relative to control animals 876 (Figure 2, A and B). The signiﬁcant loss of CD68 cells 877 (0.56 0.06-fold change, relative to uninfected controls) 878 879 consistently occurred at every phase of disease, and was 880 most prominent in the germinal center (Figure 2D). A 881 similar decline in CD68 macrophages also occurred in the 882 marginal zone and red pulp (Figure 2, F and G). Concom883 itant with the down-regulation of CD68 was a signiﬁcant 884 increase in CD163 macrophages, as compared to uninfected 885 macaques (Figure 2, DeG). This up-regulation in CD163 886 occurred at every stage of SIV infection, but was particu887 larly striking during the acute phase, where the greatest 888 increase occurred in the germinal center (2.33 0.25-fold 889 change, relative to uninfected controls) and the red pulp 890 891 (2.50 0.26-fold change, relative to uninfected controls) 892 (Figure 2, D and G). There was a signiﬁcant decrease in 893 red pulp CD163þCD68þ macrophages during acute 894 (0.34 0.06-fold change), asymptomatic (0.53 0.06-fold 895 change), and chronic/terminal disease (0.40 0.05-fold 896 change), as compared to controls (Figure 2G). The decline 897 of these cells has major implications for splenic function, as 898 they are a fetal-derived macrophage subset that represented 899 the major macrophage red pulp cellular constituent in un900 infected macaques73 (Figure 2B). 901 In contrast to the long-lasting decreases of CD68 and 902 903 CD163þCD68þ macrophages, and the increase in CD163 904 cells, there was also an increase in Mac387 macrophages 905 that occurred transiently, at distinct phases of disease. This 906 signiﬁcant increase in Mac387 cells occurred in the 907 germinal center (1.56 0.29-fold change) and marginal 908 zone (1.41 0.10-fold change) during asymptomatic dis909 ease, as compared to uninfected animals (Figure 2, A, D, 910 and F). This was preceded by a signiﬁcant decrease in 911 912 913 Table 3 Macrophage Populations Present in the Uninfected 914 Spleen 915 Germinal Mantle Marginal 916 Variable center zone zone Red pulp 917 918 CD68 2.3 ± 0.2 1.1 ± 0.1 1.8 0.1 1.3 0.3 919 CD163 00 0.1 0.1 1.3 0.2 1.0 0.3 920 CD68/CD163 0.3 0.2 0.1 0.1 2.1 ± 0.3 3.0 ± 0 921 Mac387 00 1.0 ± 0.2 2.2 ± 0.2 1.4 0.2 922 To quantitate the presence of each macrophage subset in seven unin923 fected macaques, three random ﬁelds were analyzed and numerical scores 924 of 0, 1, 2, or 3 were assigned. In this scoring system, 0 represented an 925 absence of the macrophage population, 1 indicated a minimal presence 926 (1 20 cells), 2 indicated a moderate presence of the macrophages (20 to 927 100 cells), and 3 indicated the presence of many macrophages in spleen ( 928 100 cells). Data are expressed as means SD. Bold text indicates the 929 predominant macrophage subset(s) within each area of spleen. 930

8

Mac387 cells in the mantle zone (0.60 0.09-fold change) during acute infection (Figure 2E), suggesting that these macrophages were recruited from the mantle zone into the other splenic locations. The loss of mantle zone Mac387 macrophages was short-lived, as they returned to baseline levels in the asymptomatic phase of infection (Figure 2E). Similarly, the presence of Mac387 cells in the germinal center and marginal zone also resolved during chronic/ terminal disease. Finally, there was increased cellularity and a loss of higher-order red pulp structure at end-state disease, which began to occur during the asymptomatic stage (Figure 2B). The only other notable change during late stage disease was the presence of CD163þCD68þ germinal center macrophages, which were not observed at any other time during infection (Figure 2A).

Type I Interferon and M2 Polarizing Gene Products Are Produced during SIV Infection To examine the molecular changes in spleen that accompanied the alterations in splenic lymphocytes and macrophages, we examined global changes in spleen gene expression using NanoString barcoding technology. In this technology, molecular barcodes were attached to targetspeciﬁc probes to enable multiplexed measurement of gene expression for 76 genes of interest, four of which were housekeeping genes that were used for normalization. The normalized copy numbers for the uninfected macaques were then set to one, and a fold-change relative to uninfected animals was determined. Forty-three genes were signiﬁcantly changed during SIV infection, as compared to uninfected macaques. Type I interferon responses were the most abundant class of genes up-regulated in the spleen. IFN-b (Figure 3A), MxA (Figure 3B), IRF7 (Figure 3C), STAT2 ½F3 (Figure 3G), and SOCS3 (Figure 3I) were all signiﬁcantly increased compared to control animals. IFN-a and IL-12 were also increased during SIV infection, but to a lesser extent than IFN-b and the other interferon-stimulated genes (Figure 3, J and K). This may be because of the increase in the anti-inﬂammatory cytokine IL-10 (Figure 3F), which can inhibit IFN-a production.74 In contrast to IFN-b, type II interferon responses were minimally elicited in spleen, as there was only a small, but signiﬁcant, increase in IFN-g (Supplemental Figure S2A). In addition to changes in expression of genes related to interferon signaling, genes involved in macrophage polarization pathways were also altered during SIV infection. CXCL10 (alias IP-10) (Figure 3D), IL-27 (Figure 3E), and IDO-1 (Figure 3H) were signiﬁcantly increased in infected macaques. These factors, as well as IL-10, promote M2 macrophage polarization74e76 and may have contributed to the increase in CD163 macrophages in the spleen (Figure 2). In agreement with a skewing toward M2 polarization, there was a signiﬁcant decrease in the M1 polarizing cytokine IL12b in SIV-infected animals77,78 (Figure 3L). In addition, cytokines produced by M1-polarized macrophages were not

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HIV/SIV Spleen Dysfunction

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Figure 3 Quantitation of interferon-stimulated genes. RNA was isolated from the spleen and gene expression analyses performed by NanoString barcoding technology. The relative counts of each gene product were determined on normalizing to four housekeeping genes. The fold-change of each gene was determined relative to uninfected controls, which was set to 1. The longitudinal fold-changes in interferon (IFN)-b (A), MxA (B), IRF7 (C), CXCL10 (D), IL-27 (E), IL-10 (F), STAT2 (G), IDO-1 (H), SOCS3 (I), IFN-a (J), IL-12 (K), and IL-12b (L) in the spleen. The acute (light shading), asymptomatic, and chronic (dark shading) phases of SIV infection are depicted. Statistical signiﬁcance was determined by Kruskal-Wallis analysis of variance test followed by Dunn’s multiple comparison analysis. Vertical lines indicate a signiﬁcant difference between the indicated dpi and uninfected animals, as determined by the Dunn’s multiple comparison analysis. Data are expressed as medians SD (AeL). *P 0.05 for infected animals at all time points relative to uninfected macaques.

elicited in spleen, including IL-1b, TNF-a, and IL-6, further indicating a shifting toward an M2 macrophage phenotype (Supplemental Figure S2, BeD). IFN-b may directly contribute to a skewing toward M2 macrophage polarization, as it was signiﬁcantly positively correlated with CXCL10 (R2 Z 0.6985, P < 0.0001), IL-27 (R2 Z 0.5832, P < 0.0001), IL-10 (R2 Z 0.4670, P < 0.0001), and IDO-1 (R2 Z 0.5989, P < 0.0001), and negatively correlated with IL-12b (R2 Z 0.3716, P Z 0.002). In contrast, there was no correlation among the macrophage polarizing factors and IFN-a expression (data

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not shown). These data indicate that an anti-inﬂammatory immune response is induced in the spleen during SIV infection as a result of an increase in IFN-b, which promotes M2 macrophage polarization.

Peripheral Blood Monocytes Are Recruited into the Spleen by Monocyte Chemokines and Chemokine Receptors To understand the changes in macrophage subsets and polarization, ﬂow cytometry was used to characterize the

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cellular populations in spleen, particularly monocytes because they contribute to splenic macrophages in the marginal and mantle zones. The frequency of monocytes, as well as T and B lymphocytes, relative to the total number of splenic leukocytes, was determined. There was a signiﬁcant increase in monocytes in the spleen of SIV-infected macaques, as compared to uninfected animals during acute infection (Figure 4A). There was a further increase during asymptomatic infection, which was maintained throughout chronic/terminal disease. In contrast to the changes in monocytes, the T lymphocyte population declined during both acute and asymptomatic disease (Figure 4A). There were no signiﬁcant changes in the frequency of B lymphocytes in the spleen during SIV infection (uninfected macaques, 38.2% 3.6%; acute infection, 41.8% 3.9%; asymptomatic infection, 43.2% 7.2%; chronic infection, 42.3% 3.0%), suggesting that the alterations in monocyte and T lymphocyte subsets were speciﬁc and did not occur as a result of changes in all splenic leukocytes. In addition to increasing in number, splenic monocytes underwent changes in cell surface CD16 expression, an

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activation/maturation marker that is up-regulated on differentiation of monocytes into tissue macrophages.79,80 In uninfected macaques, only 14.1% 0.4% of splenic monocytes expressed CD16 (Figure 4B). This was signiﬁcantly increased during SIV infection as early as 7 dpi, and peaked at 21 dpi where 88.4% 2.0% of cells expressed CD16. To determine whether the increase in spleen monocytes occurred as a result of extravasation from the peripheral blood, we next enumerated blood monocytes in infected and uninfected macaques. Although not signiﬁcantly changed, there was a trend toward a decline in peripheral blood monocytes during acute infection at 7 dpi (P Z 0.06). This was followed by an increase in monocytes within the blood during asymptomatic disease at 14 dpi (Figure 4D). This number restored to basal levels at day 10, before increasing again during chronic disease at 56 dpi (Figure 4D). The increase in spleen monocytes that we observed using ﬂow cytometry was conﬁrmed with NanoString analyses, as there was a twofold increase in CD14 in the spleen of infected macaques as compared to uninfected animals

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1117 1118 1119 1120 1121 1122 1123 1124 1125 ½F4 1126 1127 1128 1129 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169 1170 1171 1172 1173 1174 1175 1176 1177 1178

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Figure 4 Changes in spleen monocyte cells during SIV infection. A: Single-cell suspensions were generated from spleens isolated from uninfected and SIVinfected macaques during the acute (4, 7, and 10 dpi), asymptomatic (14 and 21 dpi), and chronic (56 and 84 dpi) phases of infection. Cell suspensions were analyzed by ﬂow cytometry to determine the frequency of monocytes (gray bars) and T lymphocytes (white bars) in the spleen leukocyte population. B: Spleen cell suspensions were analyzed by ﬂow cytometry to determine the frequency of monocytes that expressed cell surface CD16. C: RNA was isolated from the spleen and CD14 gene expression analyses performed by NanoString barcoding technology. The relative count of CD14 was determined on normalizing to four housekeeping genes, and the fold-change in the SIV-infected animals relative to the uninfected controls, which was set to 1, determined. D: Whole blood was collected and immunostained with ﬂuorochrome-coupled anti-CD14 to determine the frequency of peripheral blood CD14 monocytes by ﬂow cytometry. The acute (light shading), asymptomatic, and chronic (dark shading) phases of SIV infection are depicted. Solid lines connecting data points denote cumulative and longitudinal blood sampling of the same animals at every time point after inoculation. Dashed lines connecting data points indicate cross-sectional study of independent groups of animals after euthanasia at each time point. A, B, and D: Statistical signiﬁcance was determined by Mann-Whitney test. Statistical signiﬁcance was determined by Kruskal-Wallis analysis of variance test followed by Dunn’s multiple comparison analysis. C: Vertical lines indicate a signiﬁcant difference between the indicated dpi and uninfected animals, as determined by the Dunn’s multiple comparison analysis. Data are expressed as means SD (). *P 0.05, T cells versus the preceding day after inoculation; yP 0.05 for monocytes versus the preceding day after inoculation; zP 0.05 relative to the preceding day after inoculation; xP 0.05 for infected animals at all time points relative to uninfected macaques.

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HIV/SIV Spleen Dysfunction 1303 1241 infection in response to the up-regulation of monocyte (Figure 4C). To characterize the mechanisms that might 1304 1242 chemoattractants and chemokine receptors that promote the contribute to this increase in spleen monocytes, we analyzed 1305 1243 recruitment of peripheral blood derived cells. our NanoString data focusing on monocyte chemokines and 1306 1244 chemokine receptors. There was a pronounced production of 1307 1245 chemokine ligand (CCL) 8 during SIV infection, relative to SIV-Infected CD163, CD68, and Mac387 Macrophages 1308 1246 Are Primarily Present in the Red Pulp 1309 1247 ½F5 controls (Figure 5A). With an almost 200-fold increase, CCL8 was the predominant monocyte chemokine produced 1310 1248 in the spleen. Even when approaching terminal disease, To determine the extent to which macrophages contributed 1311 1249 there was still a 15-fold increase in CCL8 as compared to to SIV replication in the spleen, we performed single color 1312 1250 1313 1251 uninfected macaques (Figure 5A). Although produced to a immunohistochemistry for CD163, CD68, or Mac387 with 1314 1252 much lower extent than CCL8, additional monocyte chetandem in situ hybridization. We calculated the number of 1315 1253 mokines were also signiﬁcantly increased in the spleen as a SIV RNA positive macrophages in ﬁve random ﬁelds of 1316 1254 result of SIV infection, including CCL7 (Figure 5B), CCL2 spleen at 4, 7, 10, 14, 21, 56, and 84 dpi. We ﬁrst analyzed 1317 1255 (Figure 5C), CXCL2 (Figure 5D), CXCL3 (Figure 5E), and the white pulp, which is composed of both T lymphocytes 1318 1256 CCL5 (Figure 5F). There was also up-regulation of the and macrophages. Analysis of the white pulp indicated that 1319 1257 chemokine receptors CCR1 (receptor to CCL8, CCL7, and none of the in situ positive cells colocalized with the much 1320 1258 CCL5) (Figure 5G), CCR2 (receptor to CCL2, CCL7, and larger and morphologically complex CD163 macrophages 1321 1259 CCL8) (Figure 5H), and to a lesser extent, CCR5 (receptor (Figure 6A). The in situ positive cells in the white pulp also ½F6 1322 1260 to CCL5 and CCL8) (Figure 5I). These data suggest that failed to colocalize with the immunochemistry stains for 1323 1261 monocytes accumulate within the spleen during SIV CD68 or Mac387 macrophages (data not shown). In contrast 1324 1262 1325 1263 1326 1264 1327 1265 A B C 1328 1266 * * * 1329 1267 1330 1268 1331 1269 1332 1270 1333 1271 1334 1272 1335 1273 1336 1274 1337 1275 D E F 1338 1276 * * * 1339 1277 1340 1278 1341 1279 1342 1280 1343 1281 1344 1282 1345 1283 1346 1284 1347 1285 1348 1286 G H I * 1349 1287 * * 1350 1288 1351 1289 1352 1290 1353 1291 1354 1292 1355 1293 1356 1294 1357 1295 Figure 5 Expression of monocyte chemoattractants and chemokine receptors. RNA was isolated from the spleen and gene expression analyses performed Q32 1358 1296 by NanoString barcoding technology. The relative counts of each gene product were determined on normalizing to four housekeeping genes. The fold-change 1359 1297 of each gene was determined relative to uninfected controls, which was set to 1. The longitudinal fold-changes in chemokine ligand (CCL) 8 (A), CCL7 (B), CCL2 1360 1298 (C), CXCL2 (D), CXCL3 (E), CCL5 (F), CCR1 (G), CCR2 (H), and CCR5 (I) in the spleen are shown. The acute (light shading), asymptomatic, and chronic (dark 1361 1299 shading) phases of SIV infection are depicted. Statistical signiﬁcance was determined by Kruskal-Wallis analysis of variance test followed by Dunn’s multiple 1362 1300 comparison analysis. Vertical lines indicate a signiﬁcant difference between the indicated dpi and uninfected animals, as determined by the Dunn’s multiple 1363 1301 Q33 comparison analysis. Data are expressed as medians SD (AeI). *P 0.05 for infected animals at all time points relative to uninfected macaques. 1364 1302

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Figure 6 Determination of macrophage infection in red and white pulp. Immunohistochemistry with in situ hybridization were performed on spleen sections at 4, 7, 10, 14, 21, 56, and 84 dpi to identify SIV RNA positive (blue staining) macrophages. Representative images demonstrate CD163 macrophages in the white (A) and red pulp (B), CD68 red pulp macrophages (C), and Mac387 red pulp macrophages (D). Red staining indicates the respective macrophage populations. Blue staining indicates SIV RNA. Boxed insets show higher magniﬁcation of macrophages in the white (A) and red (BeD) pulp. SIV RNA positive macrophages are indicated by arrows. The absolute number of SIV RNA positive T lymphocytes in the white pulp (E) and red pulp CD163 (F), CD68 (G), and Mac387 (H) macrophages in ﬁve random ﬁelds was determined. I: RNA was isolated from spleen tissue and viral load determined by quantitative RT-PCR. The acute (light shading), asymptomatic, and chronic (dark shading) phases of SIV infection are depicted. Dashed lines connecting data points indicate crosssectional study of independent groups of animals after euthanasia at each time point. Statistical signiﬁcance was determined by Mann-Whitney test. Data are expressed as means SD (). *P 0.05 versus the preceding day after inoculation. Original magniﬁcation, 20 (AeD).

to the cells that were positive for any of the macrophage immunohistochemistry markers, the white pulp in situ positive cells were small and punctate (Figure 6A), and were likely T lymphocytes. These cells were quantitated, and

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during early infection (4 dpi) when considerable SIV RNA was produced (5.03 0.21 log SIV RNA copy eq./mL) (Figure 6I), there were also a large number of SIV RNA positive T lymphocytes in the white pulp (Figure 6E), as

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HIV/SIV Spleen Dysfunction evidenced by intense blue staining (Figure 6A). However, the number of in situ positive T lymphocytes dramatically decreased at 7 dpi (22.76 8.68), as compared to those present at 4 dpi (64.00 15.21) (Figure 6E). This substantial reduction in in situ positive T lymphocytes at 7 dpi was easily discernable on visual examination (Figure 6A). SIV RNA positive T lymphocytes continued to occur at relatively low levels in the white pulp throughout the remainder of acute infection and into the asymptomatic phase (Figure 6, A and E). Although there was a moderate increase in the number of in situ positive T cells at 56 dpi, this was short-lived, as SIV RNA positive T lymphocytes declined by 84 dpi (Figure 6E). Despite being in close proximity to the in situ positive T lymphocytes, SIV RNA was rarely observed in CD163 (Figure 6A), CD68, or Mac387 (data not shown) white pulp macrophages. In contrast to the follicle, macrophages comprised the predominant SIV RNA positive cells in the red pulp. CD163 macrophages were the major cells that were in situ positive in this area of the spleen and were readily observed at every examined time point during infection (Figure 6B). SIV RNA positive CD163 macrophages were widely distributed in the red pulp, and most frequently occurred in dense clusters of SIV infected cells in close proximity to each other. In contrast to CD163 cells, CD68 macrophages were infected in a biphasic manner. Although present early (7 and 10 dpi) (Figure 6C), SIV RNA positive CD68 macrophages decreased signiﬁcantly at 14 dpi, and increased again later at 21 and 56 dpi (Figure 6, C and G). At these later time points, the SIV RNA positive CD68 macrophages were more numerous and also had more intense in situ staining. In contrast, the few SIV RNA positive CD68 macrophages at 7 and 14 dpi were faintly stained, indicating a lower level of SIV RNA in the cells (Figure 6C). Although present throughout the entire red pulp, SIV RNA positive CD68 macrophages were widely scattered with only three or four positive cells present in close proximity to each other. This is in contrast to CD163 macrophages, in which SIV RNA positive cells were highly clustered. Mac387 macrophages were also infected in a biphasic manner. Similar to the CD68 SIV positive cells, a high frequency of SIV RNA positive Mac387 macrophages were identiﬁed during acute infection at 7 and 10 dpi, which decreased in number at 14 dpi, and ﬁnally increased during asymptomatic infection at 21 dpi (Figure 6D). When the SIV RNA positive Mac387 cells were at their highest levels, these macrophages were densely packed and occurred in close proximity to other positive cells (Figure 6D). These cells were also large and had a complex morphology. In contrast, when the Mac387 SIV RNA positive cells were less frequent, they had a smaller, rounder morphology and were not clustered (Figure 6D). The number of SIV RNA positive CD163, CD68, and Mac387 macrophages was enumerated in ﬁve random ﬁelds of the red pulp. In addition, in situ positive T lymphocytes

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were quantiﬁed within the white pulp. At 4 dpi when there was robust T lymphocyte infection in the white pulp, macrophages comprised only a minor portion of the SIV RNA positive cells in the spleen. Of the macrophage populations, red pulp CD163 cells represented the primary subset that was in situ positive at 4 dpi, as CD68 and Mac387 macrophages were minimally infected at this time point (Figure 6, FeH). However, as white pulp infection became less prominent, likely because of the death of infected T cells, macrophages represented the majority of the SIV RNA positive cells in the spleen. This occurred as a result of a signiﬁcant increase in the number of SIV RNA positive CD68 and Mac387 cells, which was most notable at 7, 10, and 21 dpi (Figure 6, G and H). In contrast to the other macrophage populations, SIV RNA positive CD163 cells remained consistently numerous throughout longitudinal SIV infection. In fact, CD163 macrophages represented the primary SIV RNA positive cell type in the entire spleen at 14 dpi. At this time point during the asymptomatic phase of disease, there were low numbers of in situ positive CD68 and Mac387 macrophages (Figure 6, G and H), as well as T lymphocytes (Figure 6E). Despite a signiﬁcant decrease in the number of other cell types that were SIV RNA positive during this time, CD163 macrophages alone were sufﬁcient to maintain SIV production, as spleen viral load was not signiﬁcantly altered at 14 dpi as compared to 10 dpi (Figure 6I). Together, these ﬁndings indicate that macrophages constitute a major cellular target of SIV in the spleen, and at certain phases of infection comprise the predominant population of infected cells in spleen.

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Figure 7

Quantitation of apoptotic cells in red pulp during SIV infection. A: Immunohistochemistry for cleaved caspase-3 was performed on spleen sections obtained from uninfected animals and during the acute, asymptomatic, and chronic phase of infection to identify apoptotic cells. Images from one animal, that are representative of nine examined, are shown. Brown staining indicates cleaved caspase-3, denoted by arrows. B: Quantiﬁcation was performed using Nikon Elements software and the percentage of cells with cleaved caspase-3 immunoreactivity per mm2 within the red pulp was obtained. Spleen sections were double labeled for cleaved caspase-3 and CD68 (C) or CD163 (D). Images from one animal, representative of six animals, are shown. Cleaved caspase-3 positive CD68 (C) and CD163 (D) macrophages are indicated by arrows. Statistical signiﬁcance was determined by Kruskal-Wallis analysis of variance test. Data are expressed as means SD (). *P 0.05. Original magniﬁcation, 20 (A, C, and D).

processes typically present on CD68 red pulp macrophages. In contrast, only a minor population of CD163 macrophages was positive for cleaved caspase-3 (Figure 7D). These data indicate that apoptosis contributes to a partial loss of CD68 red pulp macrophages during SIV infection.

Discussion Lymphoid organs are one of the main sites of HIV replication in the human body and contribute greatly to viral production. The effects of HIV infection in the spleen have received considerably less characterization than that of other lymphoid organs, including lymph nodes and thymus. The spleen undergoes substantial morphologic alterations during HIV infection, which is characterized by hyposplenism, white pulp depletion, and germinal center burn out. This is primarily associated with decreased T cell and humoral immune responses. The spleen is composed not only of lymphocytes, but also contains specialized macrophages, which are highly susceptible to HIV infection and fulﬁll many major functions of the spleen. Despite their roles in mediating HIV persistence, few studies have focused on HIV infection of splenic macrophages.

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Using our SIV macaque model, we studied longitudinal changes in macrophage subpopulations from acute to terminal stages of disease. We identiﬁed a striking global up-regulation of CD163 in macrophages in both the red and white pulp, which occurred with a concomitant decrease in CD68 positive macrophages. In addition, there was a rapid depletion of fetal-derived CD163þCD68þ red pulp macrophages, which occurred, in part, because of apoptosis of these cells that preceded a loss of higher-order structure and splenic integrity. The loss of CD68 and CD163þCD68þ macrophages and increase in CD163 cells occurred early during the acute phase of infection, and persisted until terminal disease. In addition to these long-lasting changes, there was also a transient, signiﬁcant inﬂux of Mac387 macrophages and peripheral bloodederived monocytes into the spleen during the asymptomatic phase of infection. The newly recruited monocytes may have contributed to splenic macrophage subsets on differentiation, as they expressed high levels of the activation/maturation marker CD16. However, monocyte recruitment was not sufﬁcient to restore splenic macrophage subsets to those present before infection. A schematic representation of SIV-induced changes in macrophage populations in spleen is depicted in Figure 8.

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HIV/SIV Spleen Dysfunction

Figure 8 Schematic representation of changes in macrophage populations in spleen during longitudinal SIV infection. During homeostasis in the uninfected spleen, CD68 and CD163þCD68þ cells represent the predominant macrophage subsets in the germinal center and red pulp, respectively. Although relatively devoid of macrophages, the mantle zone (M) primarily consists of CD68 and Mac387 cells. There are numerous CD68, CD163þCD68þ, and Mac387 cells in the macrophage-rich marginal zone (MZ). Within this area of the spleen, the CD68 cells reside in closer proximity to the mantle zone, whereas the CD163þCD68þ macrophages are proximal to the red pulp. Macrophages undergo changes in polarization and substantially alter their expression of cell markers after SIV infection. During the acute phase of infection, there is a global up-regulation of CD163 throughout the entire spleen. In contrast, there is a simultaneous and substantial decrease in CD68 macrophages. In addition, CD163 and Mac387 cells replace the fetal-derived CD163þCD68þ macrophages in the red pulp in the acute phase. During asymptomatic infection, peripheral blood monocytes are recruited into the spleen and rapidly disseminate to the germinal center. A limited recovery of CD68 macrophages in the mantle and marginal zones happens during this time. The dysregulation of mantle zone, marginal zone, and red pulp macrophages are maintained during chronic and late-stage infection. Notably during this late time, CD163þCD68þ macrophages become present in the germinal center, perhaps as a ﬁnal compensatory mechanism in an effort to recover the CD68 cells that were lost acutely. Therefore, macrophages are altered in every area of the spleen during SIV infection, and never fully recover to the previous state found in uninfected animals.

Whole-organ, gene expression analyses demonstrated that the profound changes in splenic macrophages occurred, at least in part, because of an up-regulation of interferon and interferon-stimulated genes, M2 macrophage polarization genes, and monocyte/macrophage chemokines and chemokine receptor signaling. With an approximately 200-fold increase relative to uninfected macaques, the

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monocyte/macrophage chemoattractant CCL8 was the most differentially regulated transcript during SIV infection. Because the NanoString analyses were performed using homogenates of whole spleen, rather than RNA isolated from puriﬁed cell populations, we cannot determine which cells produced our observed changes in interferon, M2 polarizing, and chemokine and chemokine receptor genes. In situ hybridization analyses for SIV RNA demonstrated that CD163, CD68, and Mac387 macrophages were highly infected. Macrophage subsets were in situ positive even when T lymphocytes no longer expressed SIV RNA. This direct comparison of infection of lymphocytes and macrophages demonstrates that macrophages had a signiﬁcant impact on splenic SIV replication in spleen and were sufﬁcient to maintain viral production in the absence of T cells. Our ﬁndings provide strong evidence that SIV promotes irreparable damage to the splenic mononuclear phagocyte system, and that splenic pathogenesis is largely driven by alterations and infection of macrophage subsets, which begins early after infection and is sustained until end-stage disease. Our comprehensive analyses provided the examination of splenic T cell populations and morphology concurrent with our macrophage analyses. We conﬁrmed that substantial splenic morphologic changes occurred during SIV infection, which has been observed previously during HIV/SIV infection.2 The most prominent morphologic ﬁnding was lymphoid depletion, characterized by a reduced, disrupted follicular architecture. Flow cytometry conﬁrmed the histological ﬁnding of lymphoid depletion, as there was a decrease in the frequency of CD3þ T cells. Longitudinal analysis enabled further characterization of splenic T cell subsets during SIV infection. During acute infection there were dysregulated CD4/CD8 ratios, markers of cellular activation, and memory/effector subsets, as compared to that present in uninfected counterparts. With the exception of cellular activation, T cell subset proﬁles resolved and were restored to levels similar to that of uninfected animals by terminal disease. This provides a more complete understanding of HIV/SIV-induced changes in splenic T cell populations. In addition, our ﬁndings underline the importance of longitudinal study. Although T cell activation was previously implicated in spleen pathology during acute SIV infection,81 many studies have been limited to crosssectional analysis, which does not completely capture the dynamic nature of T cell responses. The transient changes in T cell populations in the spleen provide further support to the substantial contribution of macrophages in promoting splenic destruction, as their SIV-induced ﬂuctuations remained present until end-stage disease. Differential regulation of macrophage expression of CD68 and CD163 has great implications for splenic function and integrity in the context of HIV infection. Decreased macrophage functional markers were shown to impair the activation of these cells, which promoted an abrogation of their ﬁlter functions and was associated with splenic

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Williams et al pathology, poor clinical outcomes, and AIDS-deﬁning opportunistic infections in HIV seropositive individuals.18 It is unclear whether CD68 and CD163 dysregulations resulted from changes in the relative frequencies of these macrophage populations, transit of cells from spleen, or whether differential expression of these macrophage markers occurred. Rapid changes in macrophage expression of CD68 and CD163 can occur on polarization with appropriate stimuli. In support of this, our NanoString data indicated a skewing toward M2 polarization, suggesting that our observed change in immunochemistry signal was because of the regulation in expression of these markers. However, morphologic analyses indicated that the number of red pulp macrophages also decreased, as evidenced by irregularly shaped cells, increased cellularity, and notable gaps in CD163þCD68þ macrophages within this area of the spleen. This loss of red pulp macrophages was partially mediated by apoptosis of CD68 cells. However, <5% of cells in the red pulp were apoptotic. We therefore hypothesize that both M2 polarization within the white pulp, and cell death of a subpopulation of red pulp macrophages, contributed to the overall loss of CD68 and increase in CD163 immunochemistry signal. Regardless of the mechanisms by which the alterations occurred, a shifting of the CD68 and CD163þCD68þ macrophages to a CD163 phenotype has functional implications in the ability of these cells to control opportunistic infections, mount adaptive immune responses, clear damaged erythrocytes, and scavenge iron, deﬁcits which are associated with HIV infection. In addition, apoptosis of red pulp CD68 macrophages has implications for splenic function as these cells are of embryonic origin and once lost, have limited potential for restoration during adulthood. In addition to the changes in CD68 and CD163, there was a signiﬁcant increase in Mac387 macrophages in the spleen during SIV infection, which was primarily restricted to the asymptomatic phase of disease. In agreement with our ﬁnding of a temporal restriction of Mac387 cells, newly disseminated Mac387 macrophages were shown to occur transiently in the lymph nodes of SIV-infected macaques.82 This suggests that Mac387 macrophages enter into tissues after high levels of viremia, and may be an indicator of early SIV/HIV-mediated damage. Newly inﬁltrated splenic Mac387 macrophages may be deleterious, as they promote lymphoid destruction, viremia, and pathology.82,83 Alternatively, the recruitment of these cells may represent a protective response, in an attempt to mitigate the loss of and changes to CD68 and CD163 macrophage populations during SIV infection. Peripheral blood monocytes were also recruited to the spleen after infection. Our gene expression data indicate that a temporal production of monocyte chemokines and chemokine receptors regulated the inﬂux of these cells into the spleen. CCL8 was the most prominently increased chemokine relative to uninfected controls, although other chemoattractants were also signiﬁcantly produced. Flow cytometric analysis demonstrated that on entering into the

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spleen, the newly inﬁltrated monocytes expressed high levels of the activation/maturation marker CD16, suggesting that they were primed for macrophage differentiation. In support of this, CD16 monocytes were shown to be precursors for CD163 macrophages and were associated with HIV disease progression.84 This suggests that newly inﬁltrated monocytes may directly contribute to the increase in CD163 cells. CD68, CD163, and Mac387 macrophages were all susceptible to SIV infection. However, their patterns of infectivity differed. Mac387 cells had the lowest rates of infection, which may have occurred because they are monocytederived cells, and are the most immature of all of the splenic macrophage subsets. Monocyte susceptibility to HIV increases with maturation and on macrophage differentiation.85 CD68 macrophages were more permissive to SIV than Mac387 cells, but their infection was temporally regulated. CD163 cells were distinct from the other macrophage subsets as they were highly susceptible to SIV and incredibly resilient in their level of infection. Remarkably, CD163 cells maintained high levels of infection even when there was a signiﬁcant decrease in splenic viral load, and when the other macrophages were infected at low levels. T cells were minimally infected, as compared to CD163 macrophages, and were highly infected only during acute infection. The numbers of SIV RNA positive T cells signiﬁcantly decreased after acute infection and continued to remain low for the remainder of the disease. When present, T cell infection occurred primarily within the follicles of the white pulp. This is in contrast to macrophages that were predominantly infected in the red pulp. The localized restriction of SIV RNA expression for the two cell types indicates that SIV infection of myeloid cells in spleen occurred independently of T cells. The red pulp comprises almost 80% of the spleen,86,87 and macrophage infection within this compartment contributes greatly to the burden of virus within the organ and potentially viral load in plasma. In addition, red pulp macrophages constitute a source of prolonged viral production, as infected macrophages are not effectively killed by cytotoxic T lymphocytes. Even though SIV-speciﬁc cytotoxic T lymphocytes are numerous within the red pulp,88 they do not efﬁciently promote macrophage death.89 In addition to contributing to SIV infection in the spleen, macrophages are immune to the cytopathic effects of HIV/SIV and may represent a source of latent virus. Our data represent a minimum estimate of the contribution of macrophages to SIV infection in the spleen because of the method used to quantitate infected cells, further underlining the importance of macrophages in HIV/SIV infection. As a result of virus infection, expression of type I interferons was increased in the spleen during SIV infection. IFN-b was signiﬁcantly increased early after infection, and although still elevated at terminal disease relative to uninfected animals, was present to a much lower extent than it was during acute disease. This same expression pattern occurred for many of the interferon stimulated genes, including MxA, IRF7, STAT2, and SOCS1. This ﬁnding of

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HIV/SIV Spleen Dysfunction robust interferon responses early during infection is consistent with our previous work that focused on the brain.59 Although IFN-a is typically produced in concert with and to a similar magnitude as IFN-b, we found that it was only minimally produced during SIV infection. IL-10 may have directly resulted in a silencing of IFN-a and its interferon-stimulated gene IL-12,74,90 as we found that the immunosuppressive cytokine was highly produced in the spleen. We previously demonstrated that the expression of IFN-b and IL-10 occurred without a concomitant upregulation of IFN-a in the central nervous system.59,60,91 Our current work supports this, and we now postulate that limited IFN-a production occurs because of a macrophagedriven, rather than T cell, pathogenesis. Furthermore, we hypothesize that a hindrance of IFN-a responses occurs in tissues in which HIV/SIV infection primarily occurs in myeloid cells, including brain and spleen. Similar to IFN-a, the interferon stimulated gene SOCS3 did not have the same expression pattern of IFN-b. Instead, SOCS3 maintained a consistent increase over uninfected controls at every examined time point after inoculation. This persistent up-regulation of SOCS3 may directly contribute to the increase in CD163 macrophages, as its expression in myeloid cells promotes M2 polarization and an antiinﬂammatory phenotype.92 Our Nanostring data indicated that the increased production of M2 polarizing cytokines IL10, IDO-1, CXCL10, and IL-27 may also have contributed to a shift in macrophage phenotype. There was also a persistent down-regulation of the M1 gene Il12b and a failure to induce M1-associated cytokines, which provides further support of a SIV-induced M2 macrophage polarization phenotype. Spleen-derived macrophages transiently polarize when exposed to M1 or M2-inducing substances, and revert back to a resting phenotype on removal of the stimuli.93 This plasticity is of paramount importance in restoring homeostasis after cellular activation and on pathogen clearance. As M2 cells are of an anti-inﬂammatory nature, a perpetual M2 macrophage phenotype may compromise the ability of the spleen to elicit an appropriate inﬂammatory response on exposure to pathogensda function that is integral to the blood ﬁltering functions of the organ. The balance of M1/M2 polarization may also directly affect viral production in the spleen. Macrophage CD163 expression, an indicator of M2 polarization, was shown to promote the HIV infectivity of these cells.94 The implications of this were demonstrated in an in vivo setting, where CD163 macrophages directly contributed to viral burden in both heart95 and brain.96 In addition, polarization is extremely important as it shifts macrophages from a state of active to latent viral infection.97 A model was proposed in which macrophages changed from M1 to M2 polarization during the transition from early to late stage disease, which promoted their latent infection and the formation of a steady-state viral reservoir.98 The loss of a polarized phenotype promoted active macrophage viral replication and the production of infectious virus,97 suggesting that

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polarization-reversing agents must be considered when developing shock and kill therapeutic strategies. HIV/SIV infection of the spleen is not only important for this organ, but also has consequences for other macrophagerich organs. We previously demonstrated that viral sequences that originated in the spleen later presented in the brain, suggesting that the spleen may facilitate new or reinfection of other areas of the body that were previously uninfected or virologically quiescent.58 This might occur through the transit of splenic monocytes into peripheral circulation.99,100 Monocytes that resided in the spleen were exposed to microbial infection while present in this organ, and on efﬂux into distal sites promoted widespread pathogen dissemination. As the Trojan Horse nature of these cells is implicated in viral seeding, controlling SIV/HIV infection of the spleen must be considered when developing HIV cure strategies. Our ﬁndings indicate that macrophages are a long-lasting and major cellular target for HIV/SIV in the spleen, and that these cells drive splenic pathology. Therapeutic strategies must also focus on restoring macrophage populations and functions, and limiting their infection to maintain splenic homeostasis during HIV infection.

Acknowledgments We thank all of the members of the retrovirus laboratory for their valuable insights and helpful discussions.

Supplemental Data Supplemental material for this article can be found at http://dx.doi.org/10.1016/j.ajpath.2016.03.019.

References 1. Nyberg M, Suni J, Haltia M: Isolation of human immunodeﬁciency virus (HIV) at autopsy one to six days postmortem. Am J Clin Pathol 1990, 94:422e425 2. Diaz LK, Murphy RL, Phair JP, Variakojis D: The AIDS autopsy spleen: a comparison of the pre-anti-retroviral and highly active antiretroviral therapy eras. Mod Pathol 2002, 15:406e412 3. Jung A, Maier R, Vartanian J-P, Bocharov G, Jung V, Fischer U, Meese E, Wain-Hobson S, Meyerhans A: Recombination: multiply infected spleen cells in HIV patients. Nature 2002, 418:144 4. Deeks SG, Autran B, Berkhout B, Benkirane M, Cairns S, Chomont N, Chun T-W, Churchill M, Di Mascio M, Katlama C: Towards an HIV cure: a global scientiﬁc strategy. Nat Rev Immunol 2012, 12:607e614 5. Costiniuk CT, Jenabian M-A: HIV reservoir dynamics in the face of highly active antiretroviral therapy. AIDS Patient Care STDs 2015, 29:55e68 6. Di Mascio M, Srinivasula S, Bhattacharjee A, Cheng L, Martiniova L, Herscovitch P, Lertora J, Kiesewetter D: Antiretroviral tissue kinetics: in vivo imaging using positron emission tomography. Antimicrob Agents Chemother 2009, 53:4086e4095 7. McElrath MJ, Steinman RM, Cohn ZA: Latent HIV-1 infection in enriched populations of blood monocytes and T cells from seropositive patients. J Clin Invest 1991, 87:27

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Williams et al 8. Doitsh G, Galloway NLK, Geng X, Yang Z, Monroe KM, Zepeda O, Hunt PW, Hatano H, Sowinski S, Munoz-Arias I, Greene WC: Cell death by pyroptosis drives CD4 T-cell depletion in HIV-1 infection. Nature 2014, 505:509e514 9. Okoye AA, Picker LJ: CD4þ T-cell depletion in HIV infection: mechanisms of immunological failure. Immunol Rev 2013, 254: 54e64 10. Doitsh G, Cavrois M, Lassen KG, Zepeda O, Yang Z, Santiago ML, Hebbeler AM, Greene WC: Abortive HIV infection mediates CD4 T cell depletion and inﬂammation in human lymphoid tissue. Cell 2010, 143:789e801 11. Williams KC, Burdo TH: HIV and SIV infection: the role of cellular restriction and immune responses in viral replication and pathogenesis. APMIS 2009, 117:400e412 12. Nie C, Sato K, Misawa N, Kitayama H, Fujino H, Hiramatsu H, Heike T, Nakahata T, Tanaka Y, Ito M, Koyanagi Y: Selective infection of CD4þ effector memory T lymphocytes leads to preferential depletion of memory T lymphocytes in R5 HIV-1-infected humanized NOD/SCID/IL-2Rgnull mice. Virology 2009, 394:64e72 13. Sun Z, Denton PW, Estes JD, Othieno FA, Wei BL, Wege AK, Melkus MW, Padgett-Thomas A, Zupancic M, Haase AT: Intrarectal transmission, systemic infection, and CD4þ T cell depletion in humanized mice infected with HIV-1. J Exp Med 2007, 204:705e714 14. Boasso A, Vaccari M, Hryniewicz A, Fuchs D, Nacsa J, Cecchinato V, Andersson J, Franchini G, Shearer GM, Chougnet C: Regulatory T-cell markers, indoleamine 2,3-dioxygenase, and virus levels in spleen and gut during progressive simian immunodeﬁciency virus infection. J Virol 2007, 81:11593e11603 15. Mattapallil JJ, Douek DC, Hill B, Nishimura Y, Martin M, Roederer M: Massive infection and loss of memory CD4þ T cells in multiple tissues during acute SIV infection. Nature 2005, 434: 1093e1097 16. Haase A: Population biology of HIV-1 infection: viral and CD4þ T cell demographics and dynamics in lymphatic tissues. Annu Rev Immunol 1999, 17:625e656 17. Cheynier R, Henrichwark S, Hadida F, Pelletier E, Oksenhendler E, Autran B, Wain-Hobson S: HIV and T cell expansion in splenic white pulps is accompanied by inﬁltration of HIV-speciﬁc cytotoxic T lymphocytes. Cell 1994, 78:373e387 18. Falk S, Stutte HJ: The spleen in HIV infection: morphological evidence of HIV-associated macrophage dysfunction. Res Virol 1990, 141:161e169 19. Chun T-W, Carruth L, Finzi D, Shen X, DiGiuseppe JA, Taylor H, Hermankova M, Chadwick K, Margolick J, Quinn TC, Kuo YH, Brookmeyer R, Zeiger MA, Barditch-Crovo P, Siliciano RF: Quantiﬁcation of latent tissue reservoirs and total body viral load in HIV-1 infection. Nature 1997, 387:183e188 20. Rappaport J, Volsky D: Role of the macrophage in HIV-associated neurocognitive disorders and other comorbidities in patients on effective antiretroviral treatment. J Neurovirol 2015, 21:235e241 21. Matusali G, Dereuddre Bosquet N, Le Tortorec A, Moreau M, Satie A-P, Mahé D, Roumaud P, Bourry O, Sylla N, BernardStoecklin S, Pruvost A, Le Grand R, Dejucq-Rainsford N: Detection of SIV in semen, urethra and male reproductive organs during efﬁcient HAART. J Virol 2015, 89:5772e5787 22. Abbas W, Tariq M, Iqbal M, Kumar A, Herbein G: Eradication of HIV-1 from the macrophage reservoir: an uncertain goal? Viruses 2015, 7:1578e1598 23. Fischer T, Wyatt CM, D’Agati VD, Croul S, McCourt L, Morgello S, Rappaport J: Mononuclear phagocyte accumulation in visceral tissue in HIV encephalitis: evidence for increased monocyte/macrophage trafﬁcking and altered differentiation. Curr HIV Res 2014, 12:201 24. Hernandez-Vargas EA, Middleton RH: Modeling the three stages in HIV infection. J Theor Biol 2013, 320:33e40 25. Pasternak AO, Lukashov VV, Berkhout B: Cell-associated HIV RNA: a dynamic biomarker of viral persistence. Retrovirology 2013, 10:41

18

26. Wang T, Xu Y, Zhu H, Andrus T, Ivanov SB, Pan C, Dolores J, Dann GC, Zhou M, Forte D, Yang Z, Holte S, Corey L, Zhu T: Successful isolation of infectious and high titer human monocytederived HIV-1 from two subjects with discontinued therapy. PLoS One 2013, 8:e65071 27. Kallianpur KJ, Shikuma C, Kirk GR, Shiramizu B, Valcour V, Chow D, Souza S, Nakamoto B, Sailasuta N: Peripheral blood HIV DNA is associated with atrophy of cerebellar and subcortical gray matter. Neurology 2013, 80:1792e1799 28. Cummins N, Badley A: Anti-apoptotic mechanisms of HIV: lessons and novel approaches to curing HIV. Cell Mol Life Sci 2012, 70: 3355e3363 29. Zhu T: HIV-1 in peripheral blood monocytes: an underrated viral source. J Antimicrob Chemother 2002, 50:309e311 30. Meltzer MS, Nakamura M, Hansen BD, Turpin JA, Kalter DC, Gendelman HE: Macrophages as susceptible targets for HIV infection, persistent viral reservoirs in tissue, and key immunoregulatory cells that control levels of virus replication and extent of disease. AIDS Res Hum Retroviruses 1990, 6:967e971 31. Koenig S, Gendelman HE, Orenstein JM, Dal Canto M, Pezeshkpour GH, Yungbluth M, Janotta F, Aksamit A, Martin MA, Fauci AS: Detection of AIDS virus in macrophages in brain tissue from AIDS patients with encephalopathy. Science 1986, 233: 1089e1093 32. Ho DD, Rota TR, Hirsch MS: Infection of monocyte/macrophages by human T lymphotropic virus type III. J Clin Invest 1986, 77:1712 33. Gartner S, Markovits P, Markovitz DM, Kaplan MH, Gallo RC, Popovic M: The role of mononuclear phagocytes in HTLV-III/LAV infection. Science 1986, 233:215e219 34. Duncan CJ, Williams JP, Schiffner T, Gärtner K, Ochsenbauer C, Kappes J, Russell RA, Frater J, Sattentau QJ: High-multiplicity HIV1 infection and neutralizing antibody evasion mediated by the macrophage-T cell virological synapse. J Virol 2014, 88:2025e2034 35. Costiniuk CT, Jenabian M-A: Cell-to-cell transfer of HIV infection: implications for HIV viral persistence. J Gen Virol 2014, 95: 2346e2355 36. Alexaki A, Liu Y, Wigdahl B: Cellular reservoirs of HIV-1 and their role in viral persistence. Curr HIV Res 2008, 6:388 37. Carr J, Hocking H, Li P, Burrell C: Rapid and efﬁcient cell-to-cell transmission of human immunodeﬁciency virus infection from monocyte-derived macrophages to peripheral blood lymphocytes. Virology 1999, 265:319e329 38. Crowe SM, Mills J, Elbeik T, Lifson JD, Kosek J, Marshall JA, Engleman EG, McGrath MS: Human immunodeﬁciency virusinfected monocyte-derived macrophages express surface gp120 and fuse with CD4 lymphoid cells in vitro: a possible mechanism of T lymphocyte depletion in vivo. Clin Immunol Immunopathol 1992, 65:143e151 39. Crowe S, Mills J, Kirihara J, Boothman J, Marshall J, McGrath M: Full-length recombinant CD4 and recombinant gp120 inhibit fusion between HIV infected macrophages and uninfected CD4-expressing T-lymphoblastoid cells. AIDS Res Hum Retroviruses 1990, 6: 1031e1037 40. Bronte V, Pittet Mikael J: The spleen in local and systemic regulation of immunity. Immunity 2013, 39:806e818 41. Davies LC, Jenkins SJ, Allen JE, Taylor PR: Tissue-resident macrophages. Nat Immunol 2013, 14:986e995 42. Cesta MF: Normal structure, function, and histology of the spleen. Toxicol Pathol 2006, 34:455e465 43. den Haan JM, Martinez-Pomares L: Macrophage heterogeneity in lymphoid tissues. Semin Immunopathol 2013, 35:541e552 44. Högger P, Dreier J, Droste A, Buck F, Sorg C: Identiﬁcation of the integral membrane protein RM3/1 on human monocytes as a glucocorticoid-inducible member of the scavenger receptor cysteinerich family (CD163). J Immunol 1998, 161:1883e1890 45. Gordon S: Homeostasis: a scavenger receptor for haemoglobin. Curr Biol 2001, 11:R399eR401

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HIV/SIV Spleen Dysfunction 46. Holness C, Simmons D: Molecular cloning of CD68, a human macrophage marker related to lysosomal glycoproteins. Blood 1993, 81:1607e1613 47. Zwadlo G, Voegeíi R, Osthoff KS, Sorg C: A monoclonal antibody to a novel differentiation antigen on human macrophages associated with the down-regulatory phase of the inﬂammatory process. Pathobiology 1987, 55:295e304 48. Porcheray F, Viaud S, Rimaniol AC, Leone C, Samah B, DereuddreBosquet N, Dormont D, Gras G: Macrophage activation switching: an asset for the resolution of inﬂammation. Clin Exp Immunol 2005, 142:481e489 49. Gordon S, Plüddemann A, Martinez Estrada F: Macrophage heterogeneity in tissues: phenotypic diversity and functions. Immunol Rev 2014, 262:36e55 50. Brandtzaeg P, Flavell DJ, Fagerhol MK: Mac 387 antibody and detection of formalin resistant myelomonocytic L1 antigen. J Clin Pathol 1988, 41:963e970 51. Soulas C, Conerly C, Kim W-K, Burdo TH, Alvarez X, Lackner AA, Williams KC: Recently inﬁltrating MAC387þ monocytes/macrophages. Am J Pathol 2011, 178:2121e2135 52. Lichtnekert J, Kawakami T, Parks WC, Dufﬁeld JS: Changes in macrophage phenotype as the immune response evolves. Curr Opin Pharmacol 2013, 13:555e564 53. Committee for the Update of the Guide for the Care and Use of Laboratory Animals; National Research Council: Guide for the Care and Use of Laboratory Animals: Eighth Edition. Washington, DC, National Academies Press, 2011 54. Zink MC, Suryanarayana K, Mankowski JL, Shen A, Piatak M, Spelman JP, Carter DL, Adams RJ, Lifson JD, Clements JE: High viral load in the cerebrospinal ﬂuid and brain correlates with severity of simian immunodeﬁciency virus encephalitis. J Virol 1999, 73: 10480e10488 55. Zink MC, Clements JE: A novel simian immunodeﬁciency virus model that provides insight into mechanisms of human immunodeﬁciency virus central nervous system disease. J Neurovirol 2002, 8: 42e48 56. Zink MC, Brice AK, Kelly KM, Queen SE, Gama L, Li M, Adams RJ, Bartizal C, Varrone J, Rabi SA, Graham DR, Tarwater PM, Mankowski JL, Clements JE: Simian immunodeﬁciency virus-infected macaques treated with highly active antiretroviral therapy have reduced central nervous system viral replication and inﬂammation but persistence of viral DNA. J Infect Dis 2010, 202:161e170 57. Dinoso JB, Rabi SA, Blankson JN, Gama L, Mankowski JL, Siliciano RF, Zink MC, Clements JE: A simian immunodeﬁciency virus-infected macaque model to study viral reservoirs that persist during highly active antiretroviral therapy. J Virol 2009, 83: 9247e9257 58. Ravimohan S, Gama L, Engle EL, Zink MC, Clements JE: Early emergence and selection of a SIV-LTR C/EBP site variant in SIVinfected macaques that increases virus infectivity. PLoS One 2012, 7:e42801 59. Witwer KW, Gama L, Li M, Bartizal CM, Queen SE, Varrone JJ, Brice AK, Graham DR, Tarwater PM, Mankowski JL, Zink MC, Clements JE: Coordinated regulation of SIV replication and immune responses in the CNS. PLoS One 2009, 4:e8129 60. Zaritsky LA, Gama L, Clements JE: Canonical type I IFN signaling in simian immunodeﬁciency virus-infected macrophages is disrupted by astrocyte-secreted CCL2. J Immunol 2012, 188:3876e3885 61. Carruth LM, Christine Zink M, Tarwater PM, Miller MD, Li M, Queen LA, Mankowski JL, Shen A, Siliciano RF, Clements JE: SIV-speciﬁc T lymphocyte responses in PBMC and lymphoid tissues of SIV-infected pigtailed macaques during suppressive combination antiretroviral therapy. J Med Primatol 2005, 34:109e121 62. Gama L, Shirk EN, Russell JN, Carvalho KI, Li M, Queen SE, Kalil J, Zink MC, Clements JE, Kallas EG: Expansion of a subset of CD14highCD16negCCR2low/neg monocytes functionally similar to

The American Journal of Pathology

-

63.

64.

65.

66.

67.

68.

69. 70.

71.

72.

73.

74.

75.

76. 77.

78.

79.

80.

myeloid-derived suppressor cells during SIV and HIV infection. J Leukoc Biol 2012, 91:803e816 Shinoda K, Tokoyoda K, Hanazawa A, Hayashizaki K, Zehentmeier S, Hosokawa H, Iwamura C, Koseki H, Tumes DJ, Radbruch A: Type II membrane protein CD69 regulates the formation of resting T-helper memory. Proc Natl Acad Sci U S A 2012, 109:7409e7414 Simms PE, Ellis TM: Utility of ﬂow cytometric detection of CD69 expression as a rapid method for determining poly- and oligoclonal lymphocyte activation. Clin Diagn Lab Immunol 1996, 3:301e304 Lindsey W, Lowdell M, Marti G, Abbasi F, Zenger V, King K, Lamb L Jr: CD69 expression as an index of T-cell function: assay standardization, validation and use in monitoring immune recovery. Cytotherapy 2007, 9:123e132 Pitcher CJ, Hagen SI, Walker JM, Lum R, Mitchell BL, Maino VC, Axthelm MK, Picker LJ: Development and homeostasis of T cell memory in rhesus macaque. J Immunol 2002, 168:29e43 Hosseini I, Gama L, Mac Gabhann F: Multiplexed component analysis to identify genes contributing to the immune response during acute SIV infection. PLoS One 2015, 10:e0126843 Chakrabarti L, Isola P, Cumont M-C, Claessens-Maire M-A, Hurtrel M, Montagnier L, Hurtrel B: Early stages of simian immunodeﬁciency virus infection in lymph nodes: evidence for high viral load and successive populations of target cells. Am J Pathol 1994, 144:1226 Kumar A, Abbas W, Herbein G: HIV-1 latency in monocytes/macrophages. Viruses 2014, 6:1837e1860 Le Douce V, Herbein G, Rohr O, Schwartz C: Molecular mechanisms of HIV-1 persistence in the monocyte-macrophage lineage. Retrovirology 2010, 7:32 Buechler C, Ritter M, Orso E, Langmann T, Klucken J, Schmitz G: Regulation of scavenger receptor CD163 expression in human monocytes and macrophages by pro-and antiinﬂammatory stimuli. J Leukoc Biol 2000, 67:97 Goebeler M, Roth J, Teigelkamp S, Sorg C: The monoclonal antibody MAC387 detects an epitope on the calcium-binding protein MRP14. J Leukoc Biol 1994, 55:259e261 Yona S, Kim K-W, Wolf Y, Mildner A, Varol D, Breker M, StraussAyali D, Viukov S, Guilliams M, Misharin A: Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis. Immunity 2013, 38:79e91 Ito S, Ansari P, Sakatsume M, Dickensheets H, Vazquez N, Donnelly RP, Larner AC, Finbloom DS: Interleukin-10inhibits expression of both interferon ae and interferon ge induced genes by suppressing tyrosine phosphorylation of STAT1. Blood 1999, 93: 1456e1463 Wang X-F, Wang H-S, Wang H, Zhang F, Wang K-F, Guo Q, Zhang G, Cai S-H, Du J: The role of indoleamine 2,3-dioxygenase (IDO) in immune tolerance: focus on macrophage polarization of THP-1 cells. Cell Immunol 2014, 289:42e48 Martinez FO, Gordon S: The M1 and M2 paradigm of macrophage activation: time for reassessment. F1000Prime Rep 2014, 6:13 van der Lans AA, Boon MR, Haks MC, Quinten E, Schaart G, Ottenhoff TH, van Marken Lichtenbelt WD: Cold acclimation affects immune composition in skeletal muscle of healthy lean subjects. Physiol Rep 2015, 3:e12394 Brown BN, Ratner BD, Goodman SB, Amar S, Badylak SF: Macrophage polarization: an opportunity for improved outcomes in biomaterials and regenerative medicine. Biomaterials 2012, 33: 3792e3802 Buckner CM, Calderon TM, Eugenin EA, Carvallo L, Williams D, Lopez L, Belbin T, Berman JW: Characterization of stages of monocyte maturation/differentiation that facilitate their transmigration across the blood brain barrier and infection by HIV: implications for NeuroAIDS. Cell Immunol 2011, 267:109e123 Williams DW, Calderon TM, Lopez L, Carvallo-Torres L, Gaskill PJ, Eugenin EA, Morgello S, Berman JW: Mechanisms of HIV entry into the CNS: increased sensitivity of HIV infected CD14þCD16þ

ajp.amjpathol.org

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Q18

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2357 2358 2359 2360 2361 2362 2363 2364 2365 2366 2367 2368 2369 2370 2371 2372 2373 2374 2375 2376 2377 2378 2379 2380 2381 2382 2383 2384 2385 2386 2387 2388 2389 2390 2391 2392 2393 2394 2395 2396 2397 2398 2399 2400 2401 2402 2403 2404 2405 2406 2407 2408 2409 2410 2411 2412 2413 2414 2415 2416 2417 2418

Williams et al

81.

82.

83.

84.

85.

86. 87. Q19

88.

89.

90.

20

monocytes to CCL2 and key roles of CCR2, JAM-A, and ALCAM in diapedesis. PLoS One 2013, 8:e69270 Wang X, Xu H, Alvarez X, Pahar B, Moroney-Rasmussen T, Lackner AA, Veazey RS: Distinct expression patterns of CD69 in mucosal and systemic lymphoid tissues in primary SIV infection of rhesus macaques. PLoS One 2011, 6:e27207 Otani I, Mori K, Sata T, Terao K, Doi K, Akari H, Yoshikawa Y: Accumulation of MAC387þ macrophages in paracortical areas of lymph nodes in rhesus monkeys acutely infected with simian immunodeﬁciency virus. Microbes Infect 1999, 1:977e985 Lakritz JR, Bodair A, Shah N, O’Donnell R, Polydefkis MJ, Miller AD, Burdo TH: Monocyte trafﬁc, dorsal root ganglion histopathology, and loss of intraepidermal nerve ﬁber density in SIV peripheral neuropathy. Am J Pathol 2015, 185:1912e1923 Fischer-Smith T, Tedaldi EM, Rappaport J: CD163/CD16 coexpression by circulating monocytes/macrophages in HIV: potential biomarkers for HIV infection and AIDS progression. AIDS Res Hum Retroviruses 2008, 24:417e421 Williams DW, Eugenin EA, Calderon TM, Berman JW: Monocyte maturation, HIV susceptibility, and transmigration across the blood brain barrier are critical in HIV neuropathogenesis. J Leukoc Biol 2012, 91:401e415 Pretlow T, Pretlow T: Analysis and separation of stromal cells inﬁltrating tumors. Cell Separat Methods Selected Appl 2014, 2:63 Pochedly CE: Disorders of the Spleen: Pathophysiology and Management. Taylor & Francis, 1989 Connick E, Folkvord JM, Lind KT, Rakasz EG, Miles B, Wilson NA, Santiago ML, Schmitt K, Stephens EB, Kim HO: Compartmentalization of simian immunodeﬁciency virus replication within secondary lymphoid tissues of rhesus macaques is linked to disease stage and inversely related to localization of virus-speciﬁc CTL. J Immunol 2014, 193:5613e5625 Rainho JN, Martins MA, Cunyat F, Watkins IT, Watkins DI, Stevenson M: Nef is dispensable for resistance of SIVeinfected macrophages to CD8þ T cell killing. J Virol 2015, 89:10625e10636 D’Andrea A, Aste-Amezaga M, Valiante NM, Ma X, Kubin M, Trinchieri G: Interleukin 10 (IL-10) inhibits human lymphocyte interferon gamma-production by suppressing natural killer cell

91.

92.

93.

94.

95.

96.

97.

98. 99.

100.

stimulatory factor/IL-12 synthesis in accessory cells. J Exp Med 1993, 178:1041e1048 Zaritsky LA, Dery A, Leong WY, Gama L, Clements JE: Tissuespeciﬁc interferon alpha subtype response to SIV infection in brain, spleen, and lung. J Interferon Cytokine Res 2013, 33:24e33 Qin H, Holdbrooks AT, Liu Y, Reynolds SL, Yanagisawa LL, Benveniste EN: SOCS3 deﬁciency promotes M1 macrophage polarization and inﬂammation. J Immunol 2012, 189:3439e3448 Mulder R, Banete A, Basta S: Spleen-derived macrophages are readily polarized into classically activated (M1) or alternatively activated (M2) states. Immunobiology 2014, 219:737e745 Tuluc F, Meshki J, Spitsin S, Douglas SD: HIV infection of macrophages is enhanced in the presence of increased expression of CD163 induced by substance P. J Leukoc Biol 2014, 96:143e150 Yearley JH, Pearson C, Shannon RP, Mansﬁeld KG: Phenotypic variation in myocardial macrophage populations suggests a role for macrophage activation in SIV-associated cardiac disease. AIDS Res Hum Retroviruses 2007, 23:515e524 Nowlin BT, Burdo TH, Midkiff CC, Salemi M, Alvarez X, Williams KC: SIV encephalitis lesions are composed of CD163þ macrophages present in the central nervous system during early SIV infection and SIV-positive macrophages recruited terminally with AIDS. Am J Pathol 2015, 185:1649e1665 Cassol E, Cassetta L, Rizzi C, Alfano M, Poli G: M1 and M2a polarization of human monocyte-derived macrophages inhibits HIV-1 replication by distinct mechanisms. J Immunol 2009, 182:6237e6246 Herbein G, Varin A: The macrophage in HIV-1 infection: from activation to deactivation. Retrovirology 2010, 7:1e15 Swirski FK, Nahrendorf M, Etzrodt M, Wildgruber M, CortezRetamozo V, Panizzi P, Figueiredo J-L, Kohler RH, Chudnovskiy A, Waterman P, Aikawa E, Mempel TR, Libby P, Weissleder R, Pittet MJ: Identiﬁcation of splenic reservoir monocytes and their deployment to inﬂammatory sites. Science 2009, 325:612e616 van der Laan AM, ter Horst EN, Delewi R, Begieneman MPV, Krijnen PAJ, Hirsch A, Lavaei M, Nahrendorf M, Horrevoets AJ, Niessen HWM, Piek JJ: Monocyte subset accumulation in the human heart following acute myocardial infarction and the role of the spleen as monocyte reservoir. Eur Heart J 2014, 35:376e385

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Supplemental Figure S1 Flow cytometric gating strategy. Peripheral blood mononuclear cells and single-cell suspensions derived from spleen were analyzed using a BD LSRFortessa or FACSCalibur cytometer and Diva software version 6.1.3. FACS data were analyzed using FlowJo version 9.5.1. A: Forward scatter height (FSC-H) versus area parameters were used to analyze single cell events by doublet discrimination on samples acquired with the LSRFortessa. B: The pan-leukocyte marker CD45 was used as a primary gating strategy, followed by discrimination of cells according to CD3 positivity (C). D: CD3 positive cells were then gated according to CD4 expression. Finally, CD3þCD4þ cells were analyzed according to CD69 (E) or CD28 and CD95 positivity (F). Cells that lacked CD3 surface expression were further examined for CD14 (G) and CD11b and HLA-DR positivity (H). I: The remaining CD3 cells were identiﬁed as CD20þHLADRþ.

Supplemental Figure S2 Expression of M1 polarizing genes. RNA was isolated from the spleen and gene expression analyses performed by NanoString barcoding technology. The relative counts of each gene product were determined on normalizing to four housekeeping genes. The fold-change of each gene was determined relative to uninfected controls, which was set to 1. The longitudinal fold-changes in interferon (IFN)-g (A), IL-1b (B), tumor necrosis factor (TNF)-a (C), and IL-6 (D) in the spleen are shown. The acute (light shading), asymptomatic, and chronic (dark shading) phases of SIV infection are depicted. Statistical signiﬁcance was determined by Kruskal-Wallis analysis of variance test followed by Dunn’s multiple comparison analysis. Data are expressed as Q16 Q17 medians SD (). *P 0.05 for infected animals at all time points relative to uninfected macaques.

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Splenic Damage during SIV Infection

Splenic Damage during SIV Infection

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