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Tissue-specific reduction in DC-SIGN expression correlates with progression of pathogenic simian immunodeficiency virus infection
ARTICLE IN PRESS Developmental and Comparative Immunology (2008) 32, 1510–1521
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Tissue-specific reduction in DC-SIGN expression correlates with progression of pathogenic simian immunodeficiency virus infection$ Jennifer H. Yearleya, Sarah Kanagya, Daniel C. Andersonb, Karen Daleckia, Douglas R. Pauleya, Carolyn Suwynb, Robert M. Donahoec, Harold M. McClureb, Shawn P. O’Neila, a
Division of Comparative Pathology, New England Primate Research Center, Harvard Medical School, One Pine Hill Dr. P.O. Box 9102, Southborough, MA 01772, USA b Yerkes National Primate Research Center, Emory University School of Medicine, 954 Gatewood Rd NE, Atlanta, GA 30329, USA c Department of Pathology, University of Utah, 15 North Medical Dr. East, Salt Lake City, UT 84112, USA Received 22 February 2008 Received in revised form 21 May 2008; accepted 6 June 2008 Available online 9 July 2008
KEYWORDS DC-SIGN; HIV; SIV; Sooty mangabey; Pig-tailed macaque; Spleen; Bone marrow
Summary Studies were undertaken to determine whether previously described reductions in splenic DC-SIGN expression in simian acquired immune deficiency syndrome (AIDS) are limited to pathogenic simian immunodeficiency virus (SIV) infection. DC-SIGN expression was evaluated by immunohistochemistry in lymphoid tissues from AIDS-susceptible Asian macaque monkeys as compared with AIDS-resistant sooty mangabey monkeys in the presence and absence of SIV infection. The phenotype of DC-SIGN+ cells in susceptible and resistant species was identical and most consistent with macrophage identity. Significantly lower levels of DC-SIGN expression were identified in spleen, mesenteric lymph node, and bone marrow of macaques with AIDS (Po0.05). Reduced levels of splenic DC-SIGN correlated significantly with CD4 T cell depletion in long-term pathogenic infection of macaques (Po0.01), whereas SIV-infected mangabeys retained high levels of DC-SIGN expression in spleen despite persistent infection. Reduced expression of DC-SIGN in spleen specifically characterizes pathogenic forms of SIV infection, correlates with disease progression, and may contribute to SIV pathogenesis. & 2008 Elsevier Ltd. All rights reserved.
$ Research support provided by NIH Public Health Service grants RR00165, RR00168, DE012936, DA010440, T32 RR007000, and K01RR24120. Corresponding author. Tel.: +1 508 624 8195; fax: +1 508 624 8181. E-mail address: [email protected] (S.P. O’Neil).
0145-305X/$ - see front matter & 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2008.06.006
Introduction Simian immunodeficiency virus (SIV) infection of Asian macaque species has long provided an important animal model of HIV infection, with similar properties of disease
ARTICLE IN PRESS Lymphoid tissue DC-SIGN in SIV infection progression and characteristic end-stage development of acquired immune deficiency syndrome (AIDS) [1–4]. In contrast to the highly pathogenic disease course observed in species such as the rhesus monkey (Macaca mulatta) and pig-tailed macaque (Macaca nemestrina), SIV infection of the sooty mangabey monkey (Cercocebus atys), a natural reservoir species, does not lead to clinical disease despite persistent high viral replication, levels of plasma viremia comparable to those seen in pathogenic infection, and profound, early, and persistent mucosal CD4T cell loss matching that seen in AIDS-susceptible species [5,6]. Despite these similar features, to date only a single case of AIDS in an SIV-infected sooty mangabey has been well documented, though rare instances of possibly SIV-associated disease in mangabeys and other SIV natural reservoir species have been reported [7–12]. It has been shown that sooty mangabeys do not develop the chronic immune activation with SIV infection that typifies pathogenic infections, instead maintaining normal T cell turnover without persistent increases in T cell proliferation, ongoing increased levels of T cell apoptosis, and ultimate loss of T cell regenerative potential seen in infections that ultimately progress to AIDS [5,6,13,14]. Nevertheless, the means by which sooty mangabeys and other natural reservoir species manage to consistently avoid the clinical deterioration characteristic of pathogenic SIV infection in the face of persistently high viral replication and viral loads is still poorly understood. Because of this, thorough characterization of features that distinguish pathogenic SIV infection from apathogenic infection may provide important insights into the pathogenesis of progressive SIV-mediated disease, and thereby into the pathogenesis of HIV-mediated disease. It has previously been observed that expression of the C-type lectin dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) markedly decreases in spleens of SIV-infected rhesus monkeys that have progressed to AIDS, a change not found in lymph nodes or other examined tissues [15,16]. DC-SIGN, expressed on both dendritic cells and a subset of tissue macrophages [17–20], has been the focus of significant attention as a facilitator of dendritic cellmediated HIV and SIV infection of T cells [18,21–23], as an adhesion molecule involved in dendritic cell trafficking [24], as a broad-spectrum pathogen recognition receptor [25–32], and as a surface molecule able to engage in high efficiency uptake of antigens for processing and presentation to CD4T cells [33]. Tissue distribution of DC-SIGN-expressing cells in the presence and absence of SIV infection has been previously evaluated in rhesus macaques [15,16,34–37], and the presence of large numbers of DC-SIGN-expressing cells has been documented in peripheral lymph nodes of African green monkeys (Chlorocebus sabaeus), which like sooty mangabeys serve as a natural reservoir species for SIV. However, broader lymphoid tissue distribution of DC-SIGN in the presence and absence of SIV infection has not been previously examined in any natural reservoir species. Given the importance of DC-SIGN as a facilitator of HIV and SIV infection, its role in trafficking of antigen-presenting cells, and its increasingly recognized function as an innate immune receptor for pathogen-associated molecular patterns, significant changes in levels of expression could have
1511 important consequences for innate immune function and progression of lentivirus-mediated disease. Furthermore, while the immunophenotype of DC-SIGN-expressing cells in tissue has been at least partially defined in rhesus monkeys [15,20,37], the uniformity of this phenotype across AIDSsusceptible and -resistant species has not previously been investigated and could have bearing on the roles played by cells expressing this antigen. In the current report, distribution and levels of DC-SIGN expression are evaluated in a spectrum of primary and secondary lymphoid tissues collected from SIV-infected and uninfected sooty mangabey monkeys and pig-tailed macaques at necropsy. Splenic tissue from a cohort of chronically SIV-infected rhesus monkeys serves as a comparison group, and provides information on the relationship between splenic DC-SIGN levels and peripheral blood CD4T cell counts. Immunophenotypes of DC-SIGN+ cells in spleen and bone marrow are examined for differences as a function of species, and the presence or absence of SIV infection.
Materials and methods Animals and viral inocula Formalin-fixed, paraffin-embedded lymphoid tissues from 12 sooty mangabey monkeys (C. atys), 9 pig-tailed macaques (M. nemestrina), and 14 rhesus macaques (M. mulatta) were evaluated. Prior to death, all animals were housed at the Yerkes National Primate Research Center in accordance with standards of the American Association for Accreditation of Laboratory Animal Care and the institutional Animal Care and Use Committee. Eight of the 12 sooty mangabeys became SIV infected through natural exposure in the mangabey colony at the Yerkes Center, one was experimentally infected with SIVmac239 as part of an unrelated study [38], and the remaining three were SIV-negative at time of death. Six of the 9 SIV-infected pig-tailed macaques were inoculated intravenously with the PGm/mln isolate of SIVsmmFGb [39], and the remaining three were SIVnegative. The 14 rhesus macaques were all inoculated intravenously with SIVsmm9, the characteristics of which have been previously described [40]. All SIV-positive pig-tailed macaques were euthanized due to development of acquired immune deficiency syndrome or other severe SIV-related diseases, which were confirmed at necropsy. All sooty mangabeys were humanely euthanized as a result of non-SIV-related, naturally occurring injury or disease. SIV-positive rhesus macaques were all infected for a period of greater than 2 years and were euthanized due to eventual development of severe SIV-related disease or were sacrificed at conclusion of the study in which they were enrolled. SIV-negative pig-tailed macaques were clinically normal animals euthanized to serve as controls. All animals received complete necropsies. Bone marrow was collected at necropsy from the femoral diaphysis of each animal. Sections of bone were collected and the marrow cavity accessed through application of a Stryker saw and bone rongeurs prior to formalin fixation. Fixed marrow was extracted from the opened bone for placement into tissue cassettes for routine processing and paraffin embedding into
ARTICLE IN PRESS 1512 blocks. All tissues were collected into 10% neutral-buffered formalin.
Immunohistochemistry Formalin-fixed, paraffin-embedded tissues were sectioned at 5 mm for routine, single-label immunohistochemistry followed by an ABC immunostaining technique as previously described [34]. Cell populations were characterized using antibodies specific for DC-SIGN (polyclonal, gift of R. Doms, University of Pennsylvania, Philadelphia, PA; clone DCN46, 551249, BD Pharmingen, Franklin Lakes, NJ) and CD68 (clone KP1, M 0814, DakoCytomation, Carpinteria, CA). Sections were deparaffinized in xylene and rehydrated through graded ethanols, followed by blocking of endogenous peroxidase by incubation in 3% H2O2 in phosphate-buffered saline. Antigen retrieval consisted of microwaving in citrate buffer (Vector Laboratories, Burlingame, CA). Sections were incubated with primary antibody followed by an avidin-biotin block (Vector) to block endogenous biotin, and sequential incubation with biotinylated secondary antibody and horseradish peroxidase-conjugated avidin (ABC Standard or ABC Elite, Vector). Antigen–antibody complex formation was detected by use of 3,30 diaminobenzidine (DAB) chromogen (DakoCytomation) and tissues were counterstained with Mayer’s hematoxylin. Irrelevant primary antibodies were used in place of the test antibody as negative controls in all immunohistochemical studies.
In situ hybridization In situ hybridization for SIV RNA in sections of spleen was performed as previously described [39]. Briefly, tissue sections were deparaffinized and rehydrated in xylene and graded ethanols. Endogenous alkaline phosphatase activity was blocked in 5 mM levamisole (Sigma Chemical Co., St. Louis, MO). Sections were hydrolyzed in 0.2 N HCl (Sigma), digested with proteinase K (Roche Diagnostics Corp., Indianapolis, IN), acetylated in acetic anhydride (Sigma), and hybridized overnight at 50 1C with digoxigeninlabeled antisense riboprobe that spans the entire genome of the SIVsmmPGm5.3 molecular clone of SIVsmmFGb (Lofstrand Laboratories, Gaithersburg, MD). Bound probe was detected by immunohistochemistry using alkaline phosphatase-conjugated sheep anti-digoxigenin F(ab) fragments (Roche) and the chromogen nitro blue tetrazolium/ 5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP, Roche). Sections were counterstained with nuclear fast red (Vector). Splenic tissue from an SIV-positive macaque monkey hybridized with antisense riboprobe served as positive control. Splenic tissue from an SIV-negative macaque hybridized with antisense riboprobe and from an SIV-positive macaque hybridized with sense riboprobe served as negative controls.
Scoring of antigen immunolabeling Positive immunohistochemical signal was evaluated and scored according to the following scale: 0 ¼ no signal, 1 ¼ low frequency signal, 2 ¼ moderate frequency signal,
J.H. Yearley et al. 3 ¼ high frequency signal, with increasing signal frequency representing increasing numbers of positively immunolabeled cells within the section. All sections were evaluated on a minimum of 3 separate occasions to ensure consistency of scoring criteria.
Lymphocyte subset analysis Specimens of whole blood were collected into ethylenediaminetetraacetic acid (EDTA) prior to necropsy for complete blood count and flow cytometric determination of percentages of CD3+/CD4+ T lymphocytes. Binding of fluorochrome-conjugated anti-CD3 (clone SP34, BD Pharmingen) and anti-CD4 (clone SK3, BD Pharmingen) were evaluated through gating on the lymphocyte population.
Confocal microscopy Double-label immunofluorescence confocal microscopy was performed on paraffin sections using antibodies specific for DC-SIGN (polyclonal, gift of R. Doms; clone DCN46), CD68 (clone KP1), Iba-1 (polyclonal, Wako, Richmond, VA), CD83 (clone 1H4b, NCL-CD83, Novocastra, Newcastle upon Tyne, UK), HLA-DP, DQ, DR (hereafter referred to as ‘‘MHC class II’’; clone CR3/43, DakoCytomation), and fascin (clone 55K-2, M3567, DakoCytomation). Briefly, sections were routinely deparaffinized, rehydrated, subjected to antigen retrieval as described for single-label immunohistochemistry, washed in 1 phosphate-buffered saline in ultrafiltered water with 0.2% fish skin gelatin (Sigma Aldrich, St Louis, MO) and 0.1% Triton X-100 (Sigma) (PBS-FSG-Triton), and blocked with 10% normal goat serum diluted in PBS-FSG-Triton. Monoclonal primary antibodies were incubated on sections overnight. Polyclonal primary antibodies were incubated on sections for 30 min. Fluorochrome-conjugated secondary antibodies, (anti-rabbit IgG Alexa 488, anti-mouse IgG1 Alexa 568, anti-mouse IgG2b Alexa 488, anti-rabbit Alexa 568, as appropriate, (Molecular Probes Inc., Eugene, OR)), were incubated on sections for 30 min. To-Pro3 (Molecular Probes) was incubated on sections for 5 min to stain nuclei. Confocal microscopy was performed using a Leica TCS SP laser scanning microscope equipped with 3 lasers (Leica Microsystems, Exton, PA). Fluorescence of individual fluorochromes was captured separately in a sequential mode, after optimization to reduce bleed-through between channels using Leica software. Colocalization of antigens was demonstrated by the addition of colors.
Statistical analysis Linear regression analysis and statistical comparisons between groups were performed with commercially available software (SigmaStat 3.1, Systat Software Inc., Richmond, CA). Data pairs were evaluated using the t test or Mann–Whitney rank sum test, as appropriate. Probability values of Po0.05 were interpreted as significant.
ARTICLE IN PRESS Lymphoid tissue DC-SIGN in SIV infection
Results DC-SIGN expression in mesenteric lymph nodes is significantly downregulated in SIV-infected pigtailed macaques Inguinal lymph nodes from both pig-tailed macaques and sooty mangabeys contained large numbers of DC-SIGN positive cells, irrespective of SIV infection status (Figure 1A–C). Positive cells were expressed most prominently within the medullary sinuses (Figure 1A), with lower numbers of positive cells also commonly observed in the subcapsular sinuses, and in the interfollicular zones of the cortex extending through the paracortex (Figure 1B). The distribution and morphology of these populations were most consistent with macrophage identity. Uninfected pig-tailed macaques and sooty mangabeys showed DC-SIGN expression levels and distribution in mesenteric lymph nodes similar to those identified in inguinal lymph nodes (Figure 1D). Many SIV-infected animals also retained abundant DC-SIGN expression in mesenteric lymph nodes; however, a subgroup of animals of both species demonstrated markedly lower levels of DC-SIGN expression (Figure 1D–F). This decrease resulted in significantly lower levels of DC-SIGN expression among SIV-infected pig-tailed macaques as a group relative to uninfected pig-tailed macaques (Po0.05). The SIV-infected mangabey group showed substantially lower levels of DCSIGN in one inguinal and one mesenteric lymph node as compared with uninfected mangabeys, but these findings occurred in two separate animals, both of which had been naturally SIV-infected within the mangabey colony.
Reduction of DC-SIGN expression in spleen during pathogenic SIV infection correlates with peripheral CD4T cell depletion Uninfected sooty mangabeys and pig-tailed macaques all showed moderate to profuse DC-SIGN expression in spleen, with large numbers of positive cells throughout the red pulp, within periarterial lymphatic sheaths, and in perifollicular marginal zones where they displayed especially intense signal (Figure 2A–C). In contrast, SIV-infected pig-tailed macaques as a group showed moderate to severe loss of
1513 splenic DC-SIGN signal (Figure 2D) a phenomenon not observed among SIV-infected sooty mangabeys (Figure 2E). DC-SIGN expression in spleens of SIV-infected pig-tailed macaques was significantly lower than that of SIV-infected sooty mangabeys (Po0.05; Figure 2A). Furthermore, in a cohort of 14 chronically SIV-infected rhesus monkeys, levels of DC-SIGN expression in spleen correlated significantly with peripheral CD4T cell count (Po0.01), such that more severe CD4T cell depletion was associated with greater loss of splenic DC-SIGN expression (Figure 2F). In addition, terminal viral loads also showed a strong inverse correlation with levels of splenic DC-SIGN expression in this cohort (Po0.01, R ¼ 0.731). In contrast, no significant correlations were identified between levels of splenic DC-SIGN expression and frequency of splenic SIV-infected cells as determined by in situ hybridization, levels of HLA-DR expression by CD8+ cells in peripheral blood, or expression of CD25 by CD4+ cells in peripheral blood (data not shown). Plasma viral loads among SIV-infected pig-tailed macaques were significantly higher than among SIV-infected mangabeys (6.9370.67 versus 4.6770.33 log10 SIV RNA copies/ml, P ¼ 0.006) consistent with the increased levels of viral replication seen with loss of immune competence in AIDS; however, no correlations were identified between plasma viral load and levels of splenic DC-SIGN for either of these groups. In addition, no correlation was present between levels of DC-SIGN expression and age among sooty mangabeys, irrespective of SIV infection status.
DC-SIGN expression in bone marrow is significantly reduced in pathogenic SIV infection Numbers of DC-SIGN+ cells in bone marrow of SIV-negative pig-tailed macaques and SIV-negative sooty mangabeys were moderate to high and widespread throughout the tissue (Figure 3A–C). Numbers of DC-SIGN+ cells in bone marrow of SIV-positive animals of both species were substantially decreased, with marrow from SIV-positive pig-tailed macaques containing significantly less DC-SIGN signal than marrow from SIV-negative pig-tailed macaques (Po0.05; Figure 3A, D, and E). The morphology of DC-SIGN positive cells in bone marrow differed between the two species. Marrow from SIV-negative
Figure 1 DC-SIGN expression in lymph nodes. A: Inguinal lymph node from SIV-negative pig-tailed macaque demonstrating high DC-SIGN expression. Bar ¼ 200 mm. (DC-SIGN immunohistochemistry with DAB chromogen (brown product) and Mayer’s hematoxylin counterstain. 200 magnification). B: Inguinal lymph node from SIV negative pig-tailed macaque demonstrating prominent plasma membrane DC-SIGN expression by cells within the subcapsular sinus and interfollicular zones. Bar ¼ 50 mm. (DC-SIGN immunohistochemistry with DAB chromogen and Mayer’s hematoxylin counterstain. Main image at 200 magnification, inset at 1000 magnification). C: DC-SIGN scores in inguinal lymph nodes across groups. SIV-PT ¼ SIV-negative pig-tailed macaques, SIVMgby ¼ SIV-negative sooty mangabeys, SIV+PT ¼ SIV-positive pig-tailed macaques, SIV+Mgby ¼ SIV-positive sooty mangabeys. D: DC-SIGN scores in mesenteric lymph nodes across groups. Scores among SIV-infected pig-tailed macaques were significantly lower than those among uninfected pig-tailed macaques (Po0.05). SIV-PT ¼ SIV-negative pig-tailed macaques, SIV-Mgby ¼ SIV-negative sooty mangabeys, SIV+PT ¼ SIV-positive pig-tailed macaques, SIV+Mgby ¼ SIV-positive sooty mangabeys. E: Mesenteric lymph node from SIV-positive pig-tailed macaque demonstrating low DC-SIGN expression. Arrows highlight small clusters of DC-SIGN+ cells, shown at greater magnification in inset. Bar ¼ 100 mm. (DC-SIGN immunohistochemistry with DAB chromogen and Mayer’s hematoxylin counterstain. Main image at 100 magnification, inset at 1000 magnification). F: Mesenteric lymph node from SIV-positive sooty mangabey demonstrating low DC-SIGN expression. Arrows highlight scattered, often pale-staining, DC-SIGN+ cells, shown at greater magnification in inset. Bar ¼ 100 mm. (DC-SIGN immunohistochemistry with DAB chromogen and Mayer’s hematoxylin counterstain. Main image at 100 magnification, inset at 1000 magnification).
ARTICLE IN PRESS 1514 pig-tailed macaques contained fairly uniform populations of ovoid to polygonal cells with abundant cytoplasm and intense, peripheral, membranous DC-SIGN signal (Figure 3B), closely resembling the DC-SIGN+ cells observed in lymph nodes (Figure 1B). Marrow from SIV-negative sooty mangabeys contained populations of DC-SIGN positive cells with more variable morphology, ranging from markedly dendritiform to small, round, and densely stained (Figure 3C). These distinctions in cytomorphology between species were in most cases preserved in SIV infection (Figure 3D and E).
J.H. Yearley et al.
DC-SIGN+ cells in spleen and bone marrow from pig-tailed macaques and sooty mangabeys share the same immunophenotype The great majority of DC-SIGN+ cells in both species coexpressed CD68 (Figure 4A and B). Fine resolution of signal demonstrated co-expression in spleen to consist almost entirely of discrete cytoplasmic CD68 expression with thin, encircling, membranous DC-SIGN signal, and minimal colocalizing overlap of the two antigens (Figure 4A). In contrast,
ARTICLE IN PRESS Lymphoid tissue DC-SIGN in SIV infection
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Figure 2 DC-SIGN expression in spleen. A: DC-SIGN scores in spleen across groups. DC-SIGN expression in spleens of SIV-infected pigtailed macaques was significantly lower than in spleens of SIV-infected sooty mangabeys (Po0.05). SIV-PT ¼ SIV-negative pig-tailed macaques, SIV-Mgby ¼ SIV-negative sooty mangabeys, SIV+PT ¼ SIV-positive pig-tailed macaques, SIV+Mgby ¼ SIV-positive sooty mangabeys. B: Spleen from SIV-negative pig-tailed macaque demonstrating high DC-SIGN expression. Bar ¼ 100 mm (DC-SIGN immunohistochemistry with DAB chromogen and Mayer’s hematoxylin counterstain. 100 magnification). C: Spleen from SIVnegative sooty mangabey demonstrating high DC-SIGN expression. Bar ¼ 100 mm (DC-SIGN immunohistochemistry with DAB chromogen and Mayer’s hematoxylin counterstain. 100 magnification). D: Spleen from SIV-positive pig-tailed macaque demonstrating low DC-SIGN expression. Bar ¼ 100 mm (DC-SIGN immunohistochemistry with DAB chromogen and Mayer’s hematoxylin counterstain. 100 magnification). E: Spleen from SIV-positive sooty mangabey demonstrating high DC-SIGN expression. Bar ¼ 100 mm (DC-SIGN immunohistochemistry with DAB chromogen and Mayer’s hematoxylin counterstain. 100 magnification). F: Splenic DC-SIGN scores correlated significantly with peripheral CD4 T cell counts at the time of euthanasia in a cohort of chronically SIV-infected rhesus monkeys (Po0.01).
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Figure 3 DC-SIGN expression in bone marrow. A: DC-SIGN scores in bone marrow across groups. DC-SIGN expression in bone marrow for both pig-tailed macaques and sooty mangabeys decreased in SIV-infected animals. The decrease among pig-tailed macaques was statistically significant (Po0.05). SIV-PT ¼ SIV-negative pig-tailed macaques, SIV-Mgby ¼ SIV-negative sooty mangabeys, SIV+PT ¼ SIV-positive pig-tailed macaques, SIV+Mgby ¼ SIV-positive sooty mangabeys. B: Bone marrow from SIV-negative pig-tailed macaque demonstrating high DC-SIGN expression. Bar ¼ 50 mm (DC-SIGN immunohistochemistry with DAB chromogen and Mayer’s hematoxylin counterstain. Main image at 200 magnification, inset at 1000 magnification). C: Bone marrow from SIV-negative sooty mangabey demonstrating high DC-SIGN expression. Bar ¼ 50 mm (DC-SIGN immunohistochemistry with DAB chromogen and Mayer’s hematoxylin counterstain. Main image at 200 magnification, inset at 600 magnification). D: Bone marrow from SIV-infected pig-tailed macaque demonstrating low DC-SIGN expression. Bar ¼ 50 mm (DC-SIGN immunohistochemistry with DAB chromogen and Mayer’s hematoxylin counterstain. Main image at 200 magnification, inset at 1000 magnification). E: Bone marrow from SIV-infected sooty mangabey demonstrating low DC-SIGN expression. Asterisks (*) indicate the presence of hemosiderin. Bar ¼ 50 mm (DC-SIGN immunohistochemistry with DAB chromogen and Mayer’s hematoxylin counterstain. Main image at 200 magnification, inset at 1000 magnification).
ARTICLE IN PRESS Lymphoid tissue DC-SIGN in SIV infection
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Figure 4 Co-expression of DC-SIGN and CD68. A: Spleen. SIV-negative sooty mangabey. High magnification image of delicate membranous DC-SIGN signal co-expressed with central, cytoplasmic CD68. Cross-section of a full DC-SIGN+ cell is illustrated centrally, with several isolated dendritiform cellular processes also containing central CD68 and peripheral DC-SIGN signal present to the cell’s right. (DC-SIGN and CD68 immunofluorescence. 1600 magnification). B: Bone marrow. SIV-negative pig-tailed macaque. Membranous DC-SIGN signal co-expressed with cytoplasmic CD68, demonstrating abundant interface colocalization of the two antigens. (DC-SIGN and CD68 immunofluorescence. 400 magnification).
interface colocalization of CD68 and DC-SIGN signal within cells in bone marrow, indicated by the occurrence of yellow signal in the combined image and demonstrating overlap of DC-SIGN and CD68 immunolabels in the cytoplasmic periphery, was often prominent (Figure 4B). DC-SIGN+ cells in both species also showed frequent and often extensive membrane-level colocalizing overlap of signal with the macrophage/microglial calcium-binding protein [41] Iba-1 (Figure 5A and B). Co-expression of DC-SIGN and MHC class II antigen or DC-SIGN and the mature dendritic cell marker CD83 [42,43] was rare, and co-expression between DC-SIGN
and the mature dendritic cell marker fascin [44–46] was uniformly absent (data not shown). While DC-SIGN expression decreased significantly in spleen and bone marrow from SIV-infected pig-tailed macaques and showed a decreasing trend in bone marrow from SIV-infected mangabeys, CD68 expression in these tissues remained high in both infected and uninfected animals (Table 1; Figure 5C and D). While DC-SIGN+ cells almost uniformly expressed CD68, the converse did not apply and large numbers of CD68+ cells were negative for DC-SIGN. In keeping with this fact, DC-SIGN scores and CD68
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Figure 5 Co-expression of DC-SIGN and Iba-1 in spleen. Retention of CD68 expression in pathogenic and apathogenic SIV infection. A: Spleen from SIV-negative pig-tailed macaque demonstrating frequent co-expression of DC-SIGN and Iba-1. (DC-SIGN and Iba-1 immunofluorescence. 400 magnification). B: Spleen from SIV-infected sooty mangabey showing abundant colocalization of DC-SIGN and Iba-1. (DC-SIGN and Iba-1 immunofluorescence. 400 magnification). C: Spleen from SIV-infected pig-tailed macaque showing preserved high levels of CD68 expression. Bar ¼ 100 mm (CD68 immunohistochemistry with DAB chromogen and Mayer’s hematoxylin counterstain. Main image at 100 magnification, inset at 600 magnification). D: Bone marrow from SIV-infected sooty mangabey showing preserved high levels of CD68 expression. Bar ¼ 50 mm (CD68 immunohistochemistry with DAB chromogen and Mayer’s hematoxylin counterstain. Main image at 200 magnification, inset at 1000 magnification).
a population still present, though selective depletion of the DC-SIGN+ cell populations cannot be ruled out.
Table 1 SIV status
Species
+ +
Pig-tailed macaque Sooty mangabey Pig-tailed macaque Sooty mangabey
CD68 bone CD68 marrow spleen score score 2.870.2 2.770.3 2.870.2 2.170.3
3.070 3.070 3.070 3.070
scores did not significantly correlate (P40.3), an expected outcome given the lack of bilateral interdependence of these two antigens. The presence of extensive CD68 expression in spleen and marrow from SIV-infected animals despite DC-SIGN loss suggests downregulation of DC-SIGN by
Discussion In all tissues examined, uninfected sooty mangabeys demonstrated patterns and levels of DC-SIGN expression comparable to those of uninfected pig-tailed macaques. Expression of splenic DC-SIGN decreased significantly in SIVinfected pig-tailed macaques, as has previously been described in rhesus monkeys with AIDS [15,16]. Furthermore, in a cohort of long-term SIV-infected rhesus monkeys, significant correlations were demonstrated between peripheral CD4T cell numbers, terminal viral loads, and levels of splenic DC-SIGN expression, indicating a strong connection between progression of pathogenic SIV infection and loss of DC-SIGN in spleen. While such correlations were not
ARTICLE IN PRESS Lymphoid tissue DC-SIGN in SIV infection identified among SIV-infected pig-tailed macaques, the infected pig-tailed macaque cohort consisted entirely of animals that had progressed to end-stage disease, with splenic DC-SIGN scores clustered at the lower end of the score range. The SIV-infected rhesus cohort, in contrast, consisted of animals representing a spectrum of disease progression extending from long-term non-progressors to AIDS. Chronically SIV-infected sooty mangabeys, all nonprogressors, maintained levels of splenic DC-SIGN expression comparable to those of uninfected animals, showing that apathogenic SIV infection is not associated with splenic DC-SIGN loss. Unexpectedly, SIV-infected pig-tailed macaques showed a significant drop in DC-SIGN expression in mesenteric lymph nodes and bone marrow, as well as in spleen. Previous reports documenting levels of DC-SIGN expression in lymph nodes have not reported changes with SIV infection [15,16,22], however, lymph nodes evaluated in these reports were either peripheral [22] or undefined as to site of origin [15,16]. As the gastrointestinal tract is a site of catastrophic virus-induced damage in both SIV and HIV infection [47–49] as well as serving as a major site for opportunistic infections in AIDS, the specificity of the noted change to mesenteric lymph nodes may be an indirect consequence of virusinduced gut pathology. SIV-infected mangabeys and pigtailed macaques showed similar patterns of mesenteric lymph node DC-SIGN expression, but lymph node DC-SIGN expression in mangabeys was more variable overall than it was for pig-tailed macaques even among SIV-negative animals, suggesting that factors other than SIV infection may be responsible for the differences in lymph node DC-SIGN expression in the examined mangabeys. Patterns and levels of DC-SIGN expression in bone marrow of non-human primates has not been previously reported, and both pig-tailed macaques and sooty mangabeys demonstrated robust populations of DC-SIGN+ cells in marrow in the absence of SIV infection. Numbers of DC-SIGN+ cells dropped by a similar magnitude in both species in the context of SIV infection, identifying this as a change that is not limited to pathogenic infection, and which SIV-infected mangabeys appear to be able to tolerate despite its sometimes striking character. It should be noted that the decrease in DC-SIGN expression reached statistical significance only for pig-tailed macaques, though this may be an artifact of small sample sizes. DC-SIGN+ cells in marrow prominently co-expressed CD68, and while morphology of the cells varied between the two species, they were predominantly consistent with forms commonly taken by macrophages in tissues. Pig-tailed macaque marrow DC-SIGN+ cells strongly resembled those seen in the subcapsular and medullary sinuses of lymph nodes, while the majority of sooty mangabey marrow DC-SIGN+ cells resembled the dendritiform macrophages which are observed at high frequency in spleen. Distribution, frequency, and CD68 co-expression were similar in marrow DC-SIGN+ cells of both species. DC-SIGN+ cells in spleen and bone marrow shared the same patterns of antigenic expression in both species, with the majority of cells appearing to be of probable macrophage phenotype based on morphology, distribution, expression of CD68 and lack of expression of mature or activated dendritic cell markers such as CD83 and fascin.
1519 While the possibility of a strongly CD68-expressing lineage of non-human primate dendritic cells cannot be ruled out, it appears that differences in phenotype of DC-SIGN+ cells between sooty mangabeys and AIDS-susceptible species are unlikely to account for differences in patterns of DC-SIGN expression loss, or progression of SIV-mediated disease. The majority of current literature on properties of DC-SIGN is centered on expression by dendritic cells. However, constitutive and induced DC-SIGN expression has been identified in several subpopulations of human macrophages [19,50–53], and co-expression of DC-SIGN with macrophage markers in tissues has also been described in rhesus monkeys [15,20]. Nevertheless, the tissue distribution of macrophages expressing DC-SIGN appears to be considerably more limited in humans than in examined non-human primates, highlighting a potentially important species difference in this property [19,51]. As reduced DC-SIGN expression in bone marrow appears to occur in both pathogenic and apathogenic SIV infection, this change either does not contribute meaningfully to progression of pathogenic SIV infection, or represents a pathologic change to which mangabeys have found some means of adaptation. In contrast, the depletion of DC-SIGN in spleen in SIV-infected pig-tailed macaques and rhesus monkeys represents a clear point of difference from the pattern observed in apathogenic SIV infection of sooty mangabeys. DC-SIGN expression has been shown to be upregulated in the context of Th2-type cytokines such as IL-4 and IL-13 [52,54,55], and ligand binding of DC-SIGN itself has been associated with IL-10 production in the context of concurrent TLR2, TLR4, or TNFa stimulation in vitro [56]. There is evidence suggesting that SIV-infected mangabeys maintain a Th2-type cytokine bias that differentiates them from SIVinfected rhesus monkeys and it is possible that at a tissue level, such differences in cytokine profile may play a role in determining the phenotype of cells present, representing an avenue for further exploration of this phenomenon [13,38]. Accounting for the tissue specificity of DC-SIGN loss in SIV infection will require further investigation, but clearly loss of DC-SIGN expression within the spleen is a feature which strictly characterizes pathogenic SIV infection, which may be contributory to or an indirect consequence of progressive infection, and which correlates significantly with loss of immune competence as infection progresses.
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Tissue-specific reduction in DC-SIGN expression correlates with progression of pathogenic simian immunodeficiency virus infection$ Jennifer H. Yearleya, Sarah Kanagya, Daniel C. Andersonb, Karen Daleckia, Douglas R. Pauleya, Carolyn Suwynb, Robert M. Donahoec, Harold M. McClureb, Shawn P. O’Neila, a
Division of Comparative Pathology, New England Primate Research Center, Harvard Medical School, One Pine Hill Dr. P.O. Box 9102, Southborough, MA 01772, USA b Yerkes National Primate Research Center, Emory University School of Medicine, 954 Gatewood Rd NE, Atlanta, GA 30329, USA c Department of Pathology, University of Utah, 15 North Medical Dr. East, Salt Lake City, UT 84112, USA Received 22 February 2008 Received in revised form 21 May 2008; accepted 6 June 2008 Available online 9 July 2008
KEYWORDS DC-SIGN; HIV; SIV; Sooty mangabey; Pig-tailed macaque; Spleen; Bone marrow
Summary Studies were undertaken to determine whether previously described reductions in splenic DC-SIGN expression in simian acquired immune deficiency syndrome (AIDS) are limited to pathogenic simian immunodeficiency virus (SIV) infection. DC-SIGN expression was evaluated by immunohistochemistry in lymphoid tissues from AIDS-susceptible Asian macaque monkeys as compared with AIDS-resistant sooty mangabey monkeys in the presence and absence of SIV infection. The phenotype of DC-SIGN+ cells in susceptible and resistant species was identical and most consistent with macrophage identity. Significantly lower levels of DC-SIGN expression were identified in spleen, mesenteric lymph node, and bone marrow of macaques with AIDS (Po0.05). Reduced levels of splenic DC-SIGN correlated significantly with CD4 T cell depletion in long-term pathogenic infection of macaques (Po0.01), whereas SIV-infected mangabeys retained high levels of DC-SIGN expression in spleen despite persistent infection. Reduced expression of DC-SIGN in spleen specifically characterizes pathogenic forms of SIV infection, correlates with disease progression, and may contribute to SIV pathogenesis. & 2008 Elsevier Ltd. All rights reserved.
$ Research support provided by NIH Public Health Service grants RR00165, RR00168, DE012936, DA010440, T32 RR007000, and K01RR24120. Corresponding author. Tel.: +1 508 624 8195; fax: +1 508 624 8181. E-mail address: [email protected] (S.P. O’Neil).
0145-305X/$ - see front matter & 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2008.06.006
Introduction Simian immunodeficiency virus (SIV) infection of Asian macaque species has long provided an important animal model of HIV infection, with similar properties of disease
ARTICLE IN PRESS Lymphoid tissue DC-SIGN in SIV infection progression and characteristic end-stage development of acquired immune deficiency syndrome (AIDS) [1–4]. In contrast to the highly pathogenic disease course observed in species such as the rhesus monkey (Macaca mulatta) and pig-tailed macaque (Macaca nemestrina), SIV infection of the sooty mangabey monkey (Cercocebus atys), a natural reservoir species, does not lead to clinical disease despite persistent high viral replication, levels of plasma viremia comparable to those seen in pathogenic infection, and profound, early, and persistent mucosal CD4T cell loss matching that seen in AIDS-susceptible species [5,6]. Despite these similar features, to date only a single case of AIDS in an SIV-infected sooty mangabey has been well documented, though rare instances of possibly SIV-associated disease in mangabeys and other SIV natural reservoir species have been reported [7–12]. It has been shown that sooty mangabeys do not develop the chronic immune activation with SIV infection that typifies pathogenic infections, instead maintaining normal T cell turnover without persistent increases in T cell proliferation, ongoing increased levels of T cell apoptosis, and ultimate loss of T cell regenerative potential seen in infections that ultimately progress to AIDS [5,6,13,14]. Nevertheless, the means by which sooty mangabeys and other natural reservoir species manage to consistently avoid the clinical deterioration characteristic of pathogenic SIV infection in the face of persistently high viral replication and viral loads is still poorly understood. Because of this, thorough characterization of features that distinguish pathogenic SIV infection from apathogenic infection may provide important insights into the pathogenesis of progressive SIV-mediated disease, and thereby into the pathogenesis of HIV-mediated disease. It has previously been observed that expression of the C-type lectin dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) markedly decreases in spleens of SIV-infected rhesus monkeys that have progressed to AIDS, a change not found in lymph nodes or other examined tissues [15,16]. DC-SIGN, expressed on both dendritic cells and a subset of tissue macrophages [17–20], has been the focus of significant attention as a facilitator of dendritic cellmediated HIV and SIV infection of T cells [18,21–23], as an adhesion molecule involved in dendritic cell trafficking [24], as a broad-spectrum pathogen recognition receptor [25–32], and as a surface molecule able to engage in high efficiency uptake of antigens for processing and presentation to CD4T cells [33]. Tissue distribution of DC-SIGN-expressing cells in the presence and absence of SIV infection has been previously evaluated in rhesus macaques [15,16,34–37], and the presence of large numbers of DC-SIGN-expressing cells has been documented in peripheral lymph nodes of African green monkeys (Chlorocebus sabaeus), which like sooty mangabeys serve as a natural reservoir species for SIV. However, broader lymphoid tissue distribution of DC-SIGN in the presence and absence of SIV infection has not been previously examined in any natural reservoir species. Given the importance of DC-SIGN as a facilitator of HIV and SIV infection, its role in trafficking of antigen-presenting cells, and its increasingly recognized function as an innate immune receptor for pathogen-associated molecular patterns, significant changes in levels of expression could have
1511 important consequences for innate immune function and progression of lentivirus-mediated disease. Furthermore, while the immunophenotype of DC-SIGN-expressing cells in tissue has been at least partially defined in rhesus monkeys [15,20,37], the uniformity of this phenotype across AIDSsusceptible and -resistant species has not previously been investigated and could have bearing on the roles played by cells expressing this antigen. In the current report, distribution and levels of DC-SIGN expression are evaluated in a spectrum of primary and secondary lymphoid tissues collected from SIV-infected and uninfected sooty mangabey monkeys and pig-tailed macaques at necropsy. Splenic tissue from a cohort of chronically SIV-infected rhesus monkeys serves as a comparison group, and provides information on the relationship between splenic DC-SIGN levels and peripheral blood CD4T cell counts. Immunophenotypes of DC-SIGN+ cells in spleen and bone marrow are examined for differences as a function of species, and the presence or absence of SIV infection.
Materials and methods Animals and viral inocula Formalin-fixed, paraffin-embedded lymphoid tissues from 12 sooty mangabey monkeys (C. atys), 9 pig-tailed macaques (M. nemestrina), and 14 rhesus macaques (M. mulatta) were evaluated. Prior to death, all animals were housed at the Yerkes National Primate Research Center in accordance with standards of the American Association for Accreditation of Laboratory Animal Care and the institutional Animal Care and Use Committee. Eight of the 12 sooty mangabeys became SIV infected through natural exposure in the mangabey colony at the Yerkes Center, one was experimentally infected with SIVmac239 as part of an unrelated study [38], and the remaining three were SIV-negative at time of death. Six of the 9 SIV-infected pig-tailed macaques were inoculated intravenously with the PGm/mln isolate of SIVsmmFGb [39], and the remaining three were SIVnegative. The 14 rhesus macaques were all inoculated intravenously with SIVsmm9, the characteristics of which have been previously described [40]. All SIV-positive pig-tailed macaques were euthanized due to development of acquired immune deficiency syndrome or other severe SIV-related diseases, which were confirmed at necropsy. All sooty mangabeys were humanely euthanized as a result of non-SIV-related, naturally occurring injury or disease. SIV-positive rhesus macaques were all infected for a period of greater than 2 years and were euthanized due to eventual development of severe SIV-related disease or were sacrificed at conclusion of the study in which they were enrolled. SIV-negative pig-tailed macaques were clinically normal animals euthanized to serve as controls. All animals received complete necropsies. Bone marrow was collected at necropsy from the femoral diaphysis of each animal. Sections of bone were collected and the marrow cavity accessed through application of a Stryker saw and bone rongeurs prior to formalin fixation. Fixed marrow was extracted from the opened bone for placement into tissue cassettes for routine processing and paraffin embedding into
ARTICLE IN PRESS 1512 blocks. All tissues were collected into 10% neutral-buffered formalin.
Immunohistochemistry Formalin-fixed, paraffin-embedded tissues were sectioned at 5 mm for routine, single-label immunohistochemistry followed by an ABC immunostaining technique as previously described [34]. Cell populations were characterized using antibodies specific for DC-SIGN (polyclonal, gift of R. Doms, University of Pennsylvania, Philadelphia, PA; clone DCN46, 551249, BD Pharmingen, Franklin Lakes, NJ) and CD68 (clone KP1, M 0814, DakoCytomation, Carpinteria, CA). Sections were deparaffinized in xylene and rehydrated through graded ethanols, followed by blocking of endogenous peroxidase by incubation in 3% H2O2 in phosphate-buffered saline. Antigen retrieval consisted of microwaving in citrate buffer (Vector Laboratories, Burlingame, CA). Sections were incubated with primary antibody followed by an avidin-biotin block (Vector) to block endogenous biotin, and sequential incubation with biotinylated secondary antibody and horseradish peroxidase-conjugated avidin (ABC Standard or ABC Elite, Vector). Antigen–antibody complex formation was detected by use of 3,30 diaminobenzidine (DAB) chromogen (DakoCytomation) and tissues were counterstained with Mayer’s hematoxylin. Irrelevant primary antibodies were used in place of the test antibody as negative controls in all immunohistochemical studies.
In situ hybridization In situ hybridization for SIV RNA in sections of spleen was performed as previously described [39]. Briefly, tissue sections were deparaffinized and rehydrated in xylene and graded ethanols. Endogenous alkaline phosphatase activity was blocked in 5 mM levamisole (Sigma Chemical Co., St. Louis, MO). Sections were hydrolyzed in 0.2 N HCl (Sigma), digested with proteinase K (Roche Diagnostics Corp., Indianapolis, IN), acetylated in acetic anhydride (Sigma), and hybridized overnight at 50 1C with digoxigeninlabeled antisense riboprobe that spans the entire genome of the SIVsmmPGm5.3 molecular clone of SIVsmmFGb (Lofstrand Laboratories, Gaithersburg, MD). Bound probe was detected by immunohistochemistry using alkaline phosphatase-conjugated sheep anti-digoxigenin F(ab) fragments (Roche) and the chromogen nitro blue tetrazolium/ 5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP, Roche). Sections were counterstained with nuclear fast red (Vector). Splenic tissue from an SIV-positive macaque monkey hybridized with antisense riboprobe served as positive control. Splenic tissue from an SIV-negative macaque hybridized with antisense riboprobe and from an SIV-positive macaque hybridized with sense riboprobe served as negative controls.
Scoring of antigen immunolabeling Positive immunohistochemical signal was evaluated and scored according to the following scale: 0 ¼ no signal, 1 ¼ low frequency signal, 2 ¼ moderate frequency signal,
J.H. Yearley et al. 3 ¼ high frequency signal, with increasing signal frequency representing increasing numbers of positively immunolabeled cells within the section. All sections were evaluated on a minimum of 3 separate occasions to ensure consistency of scoring criteria.
Lymphocyte subset analysis Specimens of whole blood were collected into ethylenediaminetetraacetic acid (EDTA) prior to necropsy for complete blood count and flow cytometric determination of percentages of CD3+/CD4+ T lymphocytes. Binding of fluorochrome-conjugated anti-CD3 (clone SP34, BD Pharmingen) and anti-CD4 (clone SK3, BD Pharmingen) were evaluated through gating on the lymphocyte population.
Confocal microscopy Double-label immunofluorescence confocal microscopy was performed on paraffin sections using antibodies specific for DC-SIGN (polyclonal, gift of R. Doms; clone DCN46), CD68 (clone KP1), Iba-1 (polyclonal, Wako, Richmond, VA), CD83 (clone 1H4b, NCL-CD83, Novocastra, Newcastle upon Tyne, UK), HLA-DP, DQ, DR (hereafter referred to as ‘‘MHC class II’’; clone CR3/43, DakoCytomation), and fascin (clone 55K-2, M3567, DakoCytomation). Briefly, sections were routinely deparaffinized, rehydrated, subjected to antigen retrieval as described for single-label immunohistochemistry, washed in 1 phosphate-buffered saline in ultrafiltered water with 0.2% fish skin gelatin (Sigma Aldrich, St Louis, MO) and 0.1% Triton X-100 (Sigma) (PBS-FSG-Triton), and blocked with 10% normal goat serum diluted in PBS-FSG-Triton. Monoclonal primary antibodies were incubated on sections overnight. Polyclonal primary antibodies were incubated on sections for 30 min. Fluorochrome-conjugated secondary antibodies, (anti-rabbit IgG Alexa 488, anti-mouse IgG1 Alexa 568, anti-mouse IgG2b Alexa 488, anti-rabbit Alexa 568, as appropriate, (Molecular Probes Inc., Eugene, OR)), were incubated on sections for 30 min. To-Pro3 (Molecular Probes) was incubated on sections for 5 min to stain nuclei. Confocal microscopy was performed using a Leica TCS SP laser scanning microscope equipped with 3 lasers (Leica Microsystems, Exton, PA). Fluorescence of individual fluorochromes was captured separately in a sequential mode, after optimization to reduce bleed-through between channels using Leica software. Colocalization of antigens was demonstrated by the addition of colors.
Statistical analysis Linear regression analysis and statistical comparisons between groups were performed with commercially available software (SigmaStat 3.1, Systat Software Inc., Richmond, CA). Data pairs were evaluated using the t test or Mann–Whitney rank sum test, as appropriate. Probability values of Po0.05 were interpreted as significant.
ARTICLE IN PRESS Lymphoid tissue DC-SIGN in SIV infection
Results DC-SIGN expression in mesenteric lymph nodes is significantly downregulated in SIV-infected pigtailed macaques Inguinal lymph nodes from both pig-tailed macaques and sooty mangabeys contained large numbers of DC-SIGN positive cells, irrespective of SIV infection status (Figure 1A–C). Positive cells were expressed most prominently within the medullary sinuses (Figure 1A), with lower numbers of positive cells also commonly observed in the subcapsular sinuses, and in the interfollicular zones of the cortex extending through the paracortex (Figure 1B). The distribution and morphology of these populations were most consistent with macrophage identity. Uninfected pig-tailed macaques and sooty mangabeys showed DC-SIGN expression levels and distribution in mesenteric lymph nodes similar to those identified in inguinal lymph nodes (Figure 1D). Many SIV-infected animals also retained abundant DC-SIGN expression in mesenteric lymph nodes; however, a subgroup of animals of both species demonstrated markedly lower levels of DC-SIGN expression (Figure 1D–F). This decrease resulted in significantly lower levels of DC-SIGN expression among SIV-infected pig-tailed macaques as a group relative to uninfected pig-tailed macaques (Po0.05). The SIV-infected mangabey group showed substantially lower levels of DCSIGN in one inguinal and one mesenteric lymph node as compared with uninfected mangabeys, but these findings occurred in two separate animals, both of which had been naturally SIV-infected within the mangabey colony.
Reduction of DC-SIGN expression in spleen during pathogenic SIV infection correlates with peripheral CD4T cell depletion Uninfected sooty mangabeys and pig-tailed macaques all showed moderate to profuse DC-SIGN expression in spleen, with large numbers of positive cells throughout the red pulp, within periarterial lymphatic sheaths, and in perifollicular marginal zones where they displayed especially intense signal (Figure 2A–C). In contrast, SIV-infected pig-tailed macaques as a group showed moderate to severe loss of
1513 splenic DC-SIGN signal (Figure 2D) a phenomenon not observed among SIV-infected sooty mangabeys (Figure 2E). DC-SIGN expression in spleens of SIV-infected pig-tailed macaques was significantly lower than that of SIV-infected sooty mangabeys (Po0.05; Figure 2A). Furthermore, in a cohort of 14 chronically SIV-infected rhesus monkeys, levels of DC-SIGN expression in spleen correlated significantly with peripheral CD4T cell count (Po0.01), such that more severe CD4T cell depletion was associated with greater loss of splenic DC-SIGN expression (Figure 2F). In addition, terminal viral loads also showed a strong inverse correlation with levels of splenic DC-SIGN expression in this cohort (Po0.01, R ¼ 0.731). In contrast, no significant correlations were identified between levels of splenic DC-SIGN expression and frequency of splenic SIV-infected cells as determined by in situ hybridization, levels of HLA-DR expression by CD8+ cells in peripheral blood, or expression of CD25 by CD4+ cells in peripheral blood (data not shown). Plasma viral loads among SIV-infected pig-tailed macaques were significantly higher than among SIV-infected mangabeys (6.9370.67 versus 4.6770.33 log10 SIV RNA copies/ml, P ¼ 0.006) consistent with the increased levels of viral replication seen with loss of immune competence in AIDS; however, no correlations were identified between plasma viral load and levels of splenic DC-SIGN for either of these groups. In addition, no correlation was present between levels of DC-SIGN expression and age among sooty mangabeys, irrespective of SIV infection status.
DC-SIGN expression in bone marrow is significantly reduced in pathogenic SIV infection Numbers of DC-SIGN+ cells in bone marrow of SIV-negative pig-tailed macaques and SIV-negative sooty mangabeys were moderate to high and widespread throughout the tissue (Figure 3A–C). Numbers of DC-SIGN+ cells in bone marrow of SIV-positive animals of both species were substantially decreased, with marrow from SIV-positive pig-tailed macaques containing significantly less DC-SIGN signal than marrow from SIV-negative pig-tailed macaques (Po0.05; Figure 3A, D, and E). The morphology of DC-SIGN positive cells in bone marrow differed between the two species. Marrow from SIV-negative
Figure 1 DC-SIGN expression in lymph nodes. A: Inguinal lymph node from SIV-negative pig-tailed macaque demonstrating high DC-SIGN expression. Bar ¼ 200 mm. (DC-SIGN immunohistochemistry with DAB chromogen (brown product) and Mayer’s hematoxylin counterstain. 200 magnification). B: Inguinal lymph node from SIV negative pig-tailed macaque demonstrating prominent plasma membrane DC-SIGN expression by cells within the subcapsular sinus and interfollicular zones. Bar ¼ 50 mm. (DC-SIGN immunohistochemistry with DAB chromogen and Mayer’s hematoxylin counterstain. Main image at 200 magnification, inset at 1000 magnification). C: DC-SIGN scores in inguinal lymph nodes across groups. SIV-PT ¼ SIV-negative pig-tailed macaques, SIVMgby ¼ SIV-negative sooty mangabeys, SIV+PT ¼ SIV-positive pig-tailed macaques, SIV+Mgby ¼ SIV-positive sooty mangabeys. D: DC-SIGN scores in mesenteric lymph nodes across groups. Scores among SIV-infected pig-tailed macaques were significantly lower than those among uninfected pig-tailed macaques (Po0.05). SIV-PT ¼ SIV-negative pig-tailed macaques, SIV-Mgby ¼ SIV-negative sooty mangabeys, SIV+PT ¼ SIV-positive pig-tailed macaques, SIV+Mgby ¼ SIV-positive sooty mangabeys. E: Mesenteric lymph node from SIV-positive pig-tailed macaque demonstrating low DC-SIGN expression. Arrows highlight small clusters of DC-SIGN+ cells, shown at greater magnification in inset. Bar ¼ 100 mm. (DC-SIGN immunohistochemistry with DAB chromogen and Mayer’s hematoxylin counterstain. Main image at 100 magnification, inset at 1000 magnification). F: Mesenteric lymph node from SIV-positive sooty mangabey demonstrating low DC-SIGN expression. Arrows highlight scattered, often pale-staining, DC-SIGN+ cells, shown at greater magnification in inset. Bar ¼ 100 mm. (DC-SIGN immunohistochemistry with DAB chromogen and Mayer’s hematoxylin counterstain. Main image at 100 magnification, inset at 1000 magnification).
ARTICLE IN PRESS 1514 pig-tailed macaques contained fairly uniform populations of ovoid to polygonal cells with abundant cytoplasm and intense, peripheral, membranous DC-SIGN signal (Figure 3B), closely resembling the DC-SIGN+ cells observed in lymph nodes (Figure 1B). Marrow from SIV-negative sooty mangabeys contained populations of DC-SIGN positive cells with more variable morphology, ranging from markedly dendritiform to small, round, and densely stained (Figure 3C). These distinctions in cytomorphology between species were in most cases preserved in SIV infection (Figure 3D and E).
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DC-SIGN+ cells in spleen and bone marrow from pig-tailed macaques and sooty mangabeys share the same immunophenotype The great majority of DC-SIGN+ cells in both species coexpressed CD68 (Figure 4A and B). Fine resolution of signal demonstrated co-expression in spleen to consist almost entirely of discrete cytoplasmic CD68 expression with thin, encircling, membranous DC-SIGN signal, and minimal colocalizing overlap of the two antigens (Figure 4A). In contrast,
ARTICLE IN PRESS Lymphoid tissue DC-SIGN in SIV infection
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Figure 2 DC-SIGN expression in spleen. A: DC-SIGN scores in spleen across groups. DC-SIGN expression in spleens of SIV-infected pigtailed macaques was significantly lower than in spleens of SIV-infected sooty mangabeys (Po0.05). SIV-PT ¼ SIV-negative pig-tailed macaques, SIV-Mgby ¼ SIV-negative sooty mangabeys, SIV+PT ¼ SIV-positive pig-tailed macaques, SIV+Mgby ¼ SIV-positive sooty mangabeys. B: Spleen from SIV-negative pig-tailed macaque demonstrating high DC-SIGN expression. Bar ¼ 100 mm (DC-SIGN immunohistochemistry with DAB chromogen and Mayer’s hematoxylin counterstain. 100 magnification). C: Spleen from SIVnegative sooty mangabey demonstrating high DC-SIGN expression. Bar ¼ 100 mm (DC-SIGN immunohistochemistry with DAB chromogen and Mayer’s hematoxylin counterstain. 100 magnification). D: Spleen from SIV-positive pig-tailed macaque demonstrating low DC-SIGN expression. Bar ¼ 100 mm (DC-SIGN immunohistochemistry with DAB chromogen and Mayer’s hematoxylin counterstain. 100 magnification). E: Spleen from SIV-positive sooty mangabey demonstrating high DC-SIGN expression. Bar ¼ 100 mm (DC-SIGN immunohistochemistry with DAB chromogen and Mayer’s hematoxylin counterstain. 100 magnification). F: Splenic DC-SIGN scores correlated significantly with peripheral CD4 T cell counts at the time of euthanasia in a cohort of chronically SIV-infected rhesus monkeys (Po0.01).
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Figure 3 DC-SIGN expression in bone marrow. A: DC-SIGN scores in bone marrow across groups. DC-SIGN expression in bone marrow for both pig-tailed macaques and sooty mangabeys decreased in SIV-infected animals. The decrease among pig-tailed macaques was statistically significant (Po0.05). SIV-PT ¼ SIV-negative pig-tailed macaques, SIV-Mgby ¼ SIV-negative sooty mangabeys, SIV+PT ¼ SIV-positive pig-tailed macaques, SIV+Mgby ¼ SIV-positive sooty mangabeys. B: Bone marrow from SIV-negative pig-tailed macaque demonstrating high DC-SIGN expression. Bar ¼ 50 mm (DC-SIGN immunohistochemistry with DAB chromogen and Mayer’s hematoxylin counterstain. Main image at 200 magnification, inset at 1000 magnification). C: Bone marrow from SIV-negative sooty mangabey demonstrating high DC-SIGN expression. Bar ¼ 50 mm (DC-SIGN immunohistochemistry with DAB chromogen and Mayer’s hematoxylin counterstain. Main image at 200 magnification, inset at 600 magnification). D: Bone marrow from SIV-infected pig-tailed macaque demonstrating low DC-SIGN expression. Bar ¼ 50 mm (DC-SIGN immunohistochemistry with DAB chromogen and Mayer’s hematoxylin counterstain. Main image at 200 magnification, inset at 1000 magnification). E: Bone marrow from SIV-infected sooty mangabey demonstrating low DC-SIGN expression. Asterisks (*) indicate the presence of hemosiderin. Bar ¼ 50 mm (DC-SIGN immunohistochemistry with DAB chromogen and Mayer’s hematoxylin counterstain. Main image at 200 magnification, inset at 1000 magnification).
ARTICLE IN PRESS Lymphoid tissue DC-SIGN in SIV infection
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Figure 4 Co-expression of DC-SIGN and CD68. A: Spleen. SIV-negative sooty mangabey. High magnification image of delicate membranous DC-SIGN signal co-expressed with central, cytoplasmic CD68. Cross-section of a full DC-SIGN+ cell is illustrated centrally, with several isolated dendritiform cellular processes also containing central CD68 and peripheral DC-SIGN signal present to the cell’s right. (DC-SIGN and CD68 immunofluorescence. 1600 magnification). B: Bone marrow. SIV-negative pig-tailed macaque. Membranous DC-SIGN signal co-expressed with cytoplasmic CD68, demonstrating abundant interface colocalization of the two antigens. (DC-SIGN and CD68 immunofluorescence. 400 magnification).
interface colocalization of CD68 and DC-SIGN signal within cells in bone marrow, indicated by the occurrence of yellow signal in the combined image and demonstrating overlap of DC-SIGN and CD68 immunolabels in the cytoplasmic periphery, was often prominent (Figure 4B). DC-SIGN+ cells in both species also showed frequent and often extensive membrane-level colocalizing overlap of signal with the macrophage/microglial calcium-binding protein [41] Iba-1 (Figure 5A and B). Co-expression of DC-SIGN and MHC class II antigen or DC-SIGN and the mature dendritic cell marker CD83 [42,43] was rare, and co-expression between DC-SIGN
and the mature dendritic cell marker fascin [44–46] was uniformly absent (data not shown). While DC-SIGN expression decreased significantly in spleen and bone marrow from SIV-infected pig-tailed macaques and showed a decreasing trend in bone marrow from SIV-infected mangabeys, CD68 expression in these tissues remained high in both infected and uninfected animals (Table 1; Figure 5C and D). While DC-SIGN+ cells almost uniformly expressed CD68, the converse did not apply and large numbers of CD68+ cells were negative for DC-SIGN. In keeping with this fact, DC-SIGN scores and CD68
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Figure 5 Co-expression of DC-SIGN and Iba-1 in spleen. Retention of CD68 expression in pathogenic and apathogenic SIV infection. A: Spleen from SIV-negative pig-tailed macaque demonstrating frequent co-expression of DC-SIGN and Iba-1. (DC-SIGN and Iba-1 immunofluorescence. 400 magnification). B: Spleen from SIV-infected sooty mangabey showing abundant colocalization of DC-SIGN and Iba-1. (DC-SIGN and Iba-1 immunofluorescence. 400 magnification). C: Spleen from SIV-infected pig-tailed macaque showing preserved high levels of CD68 expression. Bar ¼ 100 mm (CD68 immunohistochemistry with DAB chromogen and Mayer’s hematoxylin counterstain. Main image at 100 magnification, inset at 600 magnification). D: Bone marrow from SIV-infected sooty mangabey showing preserved high levels of CD68 expression. Bar ¼ 50 mm (CD68 immunohistochemistry with DAB chromogen and Mayer’s hematoxylin counterstain. Main image at 200 magnification, inset at 1000 magnification).
a population still present, though selective depletion of the DC-SIGN+ cell populations cannot be ruled out.
Table 1 SIV status
Species
+ +
Pig-tailed macaque Sooty mangabey Pig-tailed macaque Sooty mangabey
CD68 bone CD68 marrow spleen score score 2.870.2 2.770.3 2.870.2 2.170.3
3.070 3.070 3.070 3.070
scores did not significantly correlate (P40.3), an expected outcome given the lack of bilateral interdependence of these two antigens. The presence of extensive CD68 expression in spleen and marrow from SIV-infected animals despite DC-SIGN loss suggests downregulation of DC-SIGN by
Discussion In all tissues examined, uninfected sooty mangabeys demonstrated patterns and levels of DC-SIGN expression comparable to those of uninfected pig-tailed macaques. Expression of splenic DC-SIGN decreased significantly in SIVinfected pig-tailed macaques, as has previously been described in rhesus monkeys with AIDS [15,16]. Furthermore, in a cohort of long-term SIV-infected rhesus monkeys, significant correlations were demonstrated between peripheral CD4T cell numbers, terminal viral loads, and levels of splenic DC-SIGN expression, indicating a strong connection between progression of pathogenic SIV infection and loss of DC-SIGN in spleen. While such correlations were not
ARTICLE IN PRESS Lymphoid tissue DC-SIGN in SIV infection identified among SIV-infected pig-tailed macaques, the infected pig-tailed macaque cohort consisted entirely of animals that had progressed to end-stage disease, with splenic DC-SIGN scores clustered at the lower end of the score range. The SIV-infected rhesus cohort, in contrast, consisted of animals representing a spectrum of disease progression extending from long-term non-progressors to AIDS. Chronically SIV-infected sooty mangabeys, all nonprogressors, maintained levels of splenic DC-SIGN expression comparable to those of uninfected animals, showing that apathogenic SIV infection is not associated with splenic DC-SIGN loss. Unexpectedly, SIV-infected pig-tailed macaques showed a significant drop in DC-SIGN expression in mesenteric lymph nodes and bone marrow, as well as in spleen. Previous reports documenting levels of DC-SIGN expression in lymph nodes have not reported changes with SIV infection [15,16,22], however, lymph nodes evaluated in these reports were either peripheral [22] or undefined as to site of origin [15,16]. As the gastrointestinal tract is a site of catastrophic virus-induced damage in both SIV and HIV infection [47–49] as well as serving as a major site for opportunistic infections in AIDS, the specificity of the noted change to mesenteric lymph nodes may be an indirect consequence of virusinduced gut pathology. SIV-infected mangabeys and pigtailed macaques showed similar patterns of mesenteric lymph node DC-SIGN expression, but lymph node DC-SIGN expression in mangabeys was more variable overall than it was for pig-tailed macaques even among SIV-negative animals, suggesting that factors other than SIV infection may be responsible for the differences in lymph node DC-SIGN expression in the examined mangabeys. Patterns and levels of DC-SIGN expression in bone marrow of non-human primates has not been previously reported, and both pig-tailed macaques and sooty mangabeys demonstrated robust populations of DC-SIGN+ cells in marrow in the absence of SIV infection. Numbers of DC-SIGN+ cells dropped by a similar magnitude in both species in the context of SIV infection, identifying this as a change that is not limited to pathogenic infection, and which SIV-infected mangabeys appear to be able to tolerate despite its sometimes striking character. It should be noted that the decrease in DC-SIGN expression reached statistical significance only for pig-tailed macaques, though this may be an artifact of small sample sizes. DC-SIGN+ cells in marrow prominently co-expressed CD68, and while morphology of the cells varied between the two species, they were predominantly consistent with forms commonly taken by macrophages in tissues. Pig-tailed macaque marrow DC-SIGN+ cells strongly resembled those seen in the subcapsular and medullary sinuses of lymph nodes, while the majority of sooty mangabey marrow DC-SIGN+ cells resembled the dendritiform macrophages which are observed at high frequency in spleen. Distribution, frequency, and CD68 co-expression were similar in marrow DC-SIGN+ cells of both species. DC-SIGN+ cells in spleen and bone marrow shared the same patterns of antigenic expression in both species, with the majority of cells appearing to be of probable macrophage phenotype based on morphology, distribution, expression of CD68 and lack of expression of mature or activated dendritic cell markers such as CD83 and fascin.
1519 While the possibility of a strongly CD68-expressing lineage of non-human primate dendritic cells cannot be ruled out, it appears that differences in phenotype of DC-SIGN+ cells between sooty mangabeys and AIDS-susceptible species are unlikely to account for differences in patterns of DC-SIGN expression loss, or progression of SIV-mediated disease. The majority of current literature on properties of DC-SIGN is centered on expression by dendritic cells. However, constitutive and induced DC-SIGN expression has been identified in several subpopulations of human macrophages [19,50–53], and co-expression of DC-SIGN with macrophage markers in tissues has also been described in rhesus monkeys [15,20]. Nevertheless, the tissue distribution of macrophages expressing DC-SIGN appears to be considerably more limited in humans than in examined non-human primates, highlighting a potentially important species difference in this property [19,51]. As reduced DC-SIGN expression in bone marrow appears to occur in both pathogenic and apathogenic SIV infection, this change either does not contribute meaningfully to progression of pathogenic SIV infection, or represents a pathologic change to which mangabeys have found some means of adaptation. In contrast, the depletion of DC-SIGN in spleen in SIV-infected pig-tailed macaques and rhesus monkeys represents a clear point of difference from the pattern observed in apathogenic SIV infection of sooty mangabeys. DC-SIGN expression has been shown to be upregulated in the context of Th2-type cytokines such as IL-4 and IL-13 [52,54,55], and ligand binding of DC-SIGN itself has been associated with IL-10 production in the context of concurrent TLR2, TLR4, or TNFa stimulation in vitro [56]. There is evidence suggesting that SIV-infected mangabeys maintain a Th2-type cytokine bias that differentiates them from SIVinfected rhesus monkeys and it is possible that at a tissue level, such differences in cytokine profile may play a role in determining the phenotype of cells present, representing an avenue for further exploration of this phenomenon [13,38]. Accounting for the tissue specificity of DC-SIGN loss in SIV infection will require further investigation, but clearly loss of DC-SIGN expression within the spleen is a feature which strictly characterizes pathogenic SIV infection, which may be contributory to or an indirect consequence of progressive infection, and which correlates significantly with loss of immune competence as infection progresses.
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