Autophagic induction modulates splenic plasmacytoid dendritic cell mediated immune response in cerebral malarial infection model

Microbes and Infection xxx (xxxx) xxx

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Original article

Autophagic induction modulates splenic plasmacytoid dendritic cell mediated immune response in cerebral malarial infection model Anirban Sengupta a, Tarun Keswani b, Samrat Sarkar a, Soubhik Ghosh a, Saikat Mukherjee a, Arindam Bhattacharyya a, * a

Immunology Laboratory, Department of Zoology, University of Calcutta, 35, Ballygunge Circular Road, Kolkata, 700019, India Basic and Clinical Immunology of Parasitic Diseases, Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 e UMR 8204 - CIIL - Centre of Infection and Immunity Lille, F-59000 Lille, France, 1 Rue du Professeur Calmette, 59019, Lille, France b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 December 2018 Accepted 28 May 2019 Available online xxx

Splenic plasmacytoid dendritic cells (pDC) possess the capability to harbor live replicative Plasmodium parasite. Isolated splenic pDC from infected mice causes malaria when transferred to naïve mice. Incomplete autophagic degradation might cause poor antigen processing and poor immune response. Induction of autophagic flux by rapamycin treatment led to better prognosis by boosting pDC centered immune response against the pathogen. Splenic pDC from rapamycin-treated infected mice, caused less parasitemia in naïve mice. The downregulation of adhesion with unaltered phagocytic potential of the cells post autophagic induction restricted excessive parasite burden within them. Rapamycin-treated pDC played a better role in antigen presentation. They showed higher expression of co-stimulatory molecules CD80, CD86, DEC205, MHCI. Rapamycin-treated pDC induced CD28 expression on CD8þ T cells and suppressed FasL level. This cells also influenced differentiation of effector, memory T cell population. The increase in IL10: TNFa ratio, Treg: Th17 ratio and lowering of myeloid DC: plasmacytoid DC ratio was observed. It shifted the overaggressive inflammation mediated Th1 pathway that is reported to incur host damage, to a better well-balanced cytokine profile exhibiting Th2 pathway. Autophagic flux induction within pDC proved to be beneficial in combating malarial pathogenicity. © 2019 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Keywords: Cerebral malaria Plasmodium berghei Autophagy pDC-antigen presentation mDC-pDC ratio Th1-Th2 immune response

Plasmodium falciparum (Pf) causes cerebral malaria, which is the leading cause of malarial death [1]. In the latest malaria report of World Health Organization (WHO), an estimate of 219 million cases of malaria across the globe with 435,000 deaths was reported in 2017 [1]. Mice model of cerebral malaria is well established with the Pf homologs strain Plasmodium berghei ANKA (PbA) in C57BL/6 [2] and Swiss albino mice [3]. Experimental Cerebral Malaria (ECM) exhibits most of its characteristics with human counterpart [4,5]. With almost 100% lethality within two weeks post infection, anaemia, paralysis, neurological damage, and coma are a few common symptoms of this disease [4,5]. Enlargement of the spleen is another hallmark symptom of the disease [4e6]. Spleen being an important lymphoid organ plays a

* Corresponding author. E-mail addresses: [email protected] (A. Sengupta), [email protected] (T. Keswani), [email protected] (S. Sarkar), [email protected] (S. Ghosh), [email protected] (S. Mukherjee), arindam19@gmail. com (A. Bhattacharyya).

critical role in Fighting against the blood stage of the disease [6]. Previous studies highlighted the role of various cell types within the spleen against the malaria pathogens [6,7]. However, still there lies certain ambiguity in the role of splenic plasmacytoid dendritic cell (pDC) subsets in eliciting the immune response against the Plasmodium. pDC have long been thought to be quite tolerogenic dendritic cell subset [7]. However, in multiple scenarios, they show the difference in their natural characteristics by acting as immunemodulator, capable of directing the immune responses [8,9]. Studies revealed that rodent blood-stage PbA infection has a tropism for splenic pDC [10,11]. A recent report regarding pDC harboring live Plasmodium parasite in rodent spleen, retaining its infective and replicative property in vivo; having ability to cause malaria when transferred to naïve mice, is one of the major concerns in malaria [10]. pDC also persists for a more extended period compared to other DC subsets which makes it best fit for harboring the live parasite [12]. These cells have also been reported to harbor Toxoplasma gondii and act as the trojan horse for the pathogen [13]. Macroautophagy (hereafter autophagy) plays a vital role in pathogen degradation and processing in antigen presenting cells

https://doi.org/10.1016/j.micinf.2019.05.004 1286-4579/© 2019 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Please cite this article as: A. Sengupta et al., Autophagic induction modulates splenic plasmacytoid dendritic cell mediated immune response in cerebral malarial infection model, Microbes and Infection, https://doi.org/10.1016/j.micinf.2019.05.004

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(APC) [14]. Upregulation of autophagic pathway gene expression, the formation of autophagolysosome, accumulation of LC3B in the autophagic vacuoles are few crucial steps of the autophagic flux [15]. Impairment in autophagic flux in APC results in poor disease outcome in different infections [16,17]. Alteration in adhesion potential of the cells and phagocytosis of the pathogen can also influence the downstream autophagic response [18]. Although pDCs are not as efficient as that of myeloid dendritic cells (mDC) for presenting the antigens, yet they express MHC molecules as well as the costimulatory molecules, CD80 and CD86, and can present antigens to T cells [9,19]. pDCs have recycling endosomes in which peptides can be continuously loaded on MHC class I molecules, which enables efficient presentation [20]. Another unique characteristic in pDCs is to retain the capacity to present antigens via DEC-205 following TLR activation [21,22]. Plasmodium infection causes increased systemic release of proinflammatory cytokines that contribute to the pathogenesis of malaria [23]. Rapid secretion of large amounts of IFNa and release of IL12 by activated pDC was reported previously [24,25]. The previous report also shows, matured pDC secretes anti-inflammatory cytokine IL10 leading to CD4þT cell stimulation, suggesting their possible role in inhibiting excessive T-cell responses [26]. Plasma concentration of IL10 and IL10/TNFa ratios suggests pro-inflammatory and antiinflammatory cytokines imbalance and is related to disease development [27]. Ratio between forkhead box P3 (FOXP3) expressing regulatory T cell (Treg) and RAR-related orphan receptor gamma t (RORgt) expressing Th17 cell also shapes Th1/Th2 immune response [28]. Drop in IL10/TNFa expression ratio, regulatory T cells/Th17 (Treg/Th17) ratio and increase in mDC/pDC ratio are all associated with the activation of the Th1 pathway, whereas activation of the Th2 pathway is associated with increased ratio [27e29]. Treatment of the ECM with rapamycin was previously found to inhibit parasite growth and increase the mice survival [30]. Rapamycin possesses microbicidal property against Plasmodium parasites [31]. Artemisinin-derived antimalarial compounds possess an active metabolite called dihydroartemisinin that inhibits expression of mammalian target of rapamycin (mTOR) [32]. Another inhibitor of the same pathway, torin2, also reported clearing Plasmodium infection [33]. We chose rapamycin, an FDA approved drug, as a compound of interest to suppress mTOR pathway which results in autophagic induction. Lysosomotropic agent ammonium chloride is used as an autophagic inhibitor [34]. It accumulates within the lysosomes increasing its pH and thus blocks the autophagic flux [34]. Our previous report showed the impact of autophagy within splenic red pulp macrophages in ECM [35]. Similar to our previous study [35], here also we have treated the PbA infected mice with either autophagic inducer (rapamycin) or with autophagic inhibitor (ammonium chloride) and studied the effect of autophagic pathway modulation in splenic pDCs. The cells were sorted from different experimental groups and have been checked for their infective property, parasite load along with the status of the autophagic flux and its impact on host immune response. We studied the expression of antigen processing and presentation molecules on them and also the cytokine receptors on their surface. The co-culture with both CD4þ and CD8þ T cells has demonstrated the nature of activation and downstream immune responses along with the T cell maturation, differentiation, and alteration in cytokine profile. 1. Material-methods 1.1. Materials The materials used for this study including the antibodies, primers, kits, dyes and other reagents are provided in the supplementary material under ‘Materials’ section.

1.2. Experimental cerebral malaria model Swiss Albino mice (Male, ~25 g each; 6e8 weeks age) were housed five mice per cage. They were provided with rodent chow (National Institute of Nutrition, India) and filtered water ad libitum. Experiments and handling of the animals were carried out by following strictly the guidelines of ‘Committee for the purpose of Control and Supervision of Experimental Animals (CPCSEA)’, Government of India (Registration No: 885/ac/05/CPCSEA). It is approved by the Institutional Animal Ethics Committee (IAEC), University of Calcutta, and confirms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. P. berghei ANKA parasite strain was obtained from National Institute of Malaria Research Center, New Delhi. Parasitized mouse red blood cells (pRBC) stored in Liquid N2 were injected (1  106 pRBC, in 100 ml PBS) in the reservoir mice. Post-amplification, the experimental mice were inoculated with PbA (1  106 pRBC from reservoir mice, in 100 ml PBS) by intraperitoneal (IP) injection. Uninfected erythrocytes were injected to the control mice in equal number. Thin blood smears prepared from the tail snips, were monitored daily by Giemsa staining for the parasite load. The percentage of parasitemia was calculated as follows: parasitemia (%) ¼ [(number of infected erythrocytes)/(total number of erythrocytes counted) X 100]. Mice survival were also observed daily. 1.3. In vivo rapamycin and ammonium chloride treatment Mice dose was provided as according to our previous published work [35]. IP injection for rapamycin treatment was provided at 15 mg/kg body weight and ammonium chloride (Amm. Chl.) at 50 mg/kg orally once every day. The dose was given starting from one day before infection to the day of sacrifice (8th-day post infection). Three hours before sacrifice last dose was provided. The experiments were performed on the pDC, isolated from the control and three groups i.e. only PbA infected, PbA þ rapamycin-treated, PbA þ ammonium chloride-treated. Each of these four groups consisted of a set of five mice (n ¼ 5) for each experiment replicate. Total of four rounds of replicates for every experiment were performed. Hence five mice/set X 4 treatment groups X 4 replicates ¼ 80 mice were used for the study. 106 splenic pDCs, isolated from either the control or different treatment groups, were injected to naive mouse sets for assessing their role in causing malaria, prior and post autophagic induction. 1.4. Flow cytometry and cell sorting The detailed methodology for this section is provided in the Supplementary ‘Methods 2.1’ section. In brief, the single cell splenocyte suspension was prepared by mechanical tapping of the harvested spleen and passing through the cell strainer. They were then treated with RBC Lysis Buffer, washed and incubated for the antibodies specific for the target. For intracellular proteins, permeabilization buffer was used and washed carefully before antibody incubation. The cells were blocked by blocking antibody before target antibody incubation. Matching isotype control was used, and compensation for fluorescence spillover from other filters was done. For detection of serine phosphorylation of beclin-s15 and p62s403 permeabilized cells were incubated with the specific phosphorylation antibodies (1:1000 dilution) followed by conjugation with tetramethylrhodamine (TRITC)-conjugated anti-rabbit secondary antibody (1:10000 dilution). Excess antibodies were washed thrice with PBS, followed by centrifugation at 1000 rpm for

Please cite this article as: A. Sengupta et al., Autophagic induction modulates splenic plasmacytoid dendritic cell mediated immune response in cerebral malarial infection model, Microbes and Infection, https://doi.org/10.1016/j.micinf.2019.05.004

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5 min at 4  C. The cells were resuspended in cell staining buffer (CSB) and were processed for the detection of the mean fluorescence. The cell sorting was done on BD FACS Verse III, and flow cytometry detection was done on BD C6 Accuri. CD11clowCD317high pDC and CD4T and CD8T cells were sorted. Some of the key proteins and markers studied via flow cytometry were merozoite surface protein (MSP1) for detection of Plasmodium load, antigen processing marker DEC205 (CD205), Toll-like-receptor 9 (TLR9), adhesion molecule SiglecH (CD33), Th17 cell marker (RORgt), Treg cell marker (FOXP3), chemokine receptor CCR7, Fas ligand (FasL) on T cells. The antibody details were provided in Supplementary ‘Materials’ section. The gating strategy for the flow cytometry plots generated in the figures, is provided in the Supp Fig. 4. 1.5. Real-time gene expression study Whole RNA extraction from isolated pDC or brain tissues were performed using Trizol reagent (1 ml for 106 cells or 100 mg tissue). They were then reverse transcribed. Sybr Green reagent (Thermo Fisher: 4309155) was used for performing the real-time gene expression study with the primers (Supplementary Table 1) and 100 ng of cDNA as template for 35 cycles with GAPDH as the housekeeping control. Data acquisition and analysis was done by Applied Biosystem StepOne RealTime Machine and its software. The methodology details are provided in Supplementary ‘Methods 2.2’ section. 1.6. Autophagolysosome detection 105 isolated pDCs were subjected to the treatment as in manufacture's protocol (ENZ: 51031) and also as published previously from our group [35] and other groups [36,37]. The detailed methodology is provided in the Supplementary ‘Methods 2.3’ section. Briefly, the pDCs were treated with the buffer and cyto-id dye. Excess dye was washed and cells resuspended for detection by BD C6 Accuri Flow cytometry machine.

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by our group [35] (explained in details in Supplementary ‘Methods 2.6’ section). In brief, 106 calcein-labelled-iRBC were co-incubated with 105 isolated pDC from different treatment groups in proper cell culture medium in the CO2 incubator for two hours. They were washed by PBS. Fluorescence emitted from labelled-iRBC adhered on the pDC surface were detected by BD C6 Accuri machine. 1.10. DC-T co-culture The sorted pDC from respective control and treatment groups were co-cultured with either CD8 or CD4T cells isolated from the control mice. They are put in DC: T coculture at 1:2 ratio (DC: 105 cells and T cells: 2  105) with proper culture medium (DMEM, 10% FBS) in a 96 well plate and incubated at 37  C, 5% CO2 cell culture incubator. The cells were harvested after 24 h of incubation for detection of cytokines in the supernatant or the target protein detection on their surface. 1.11. ELISA Enzyme-Linked Immunosorbent Assay (ELISA) for detection of secretory cytokines were performed on the cell culture supernatant of DC-CD8T co-culture. The 100 mL supernatant media was collected after 24 h post incubation. The samples were checked for individual cytokines by following manufacturer's protocol of respective Sandwich ELISA kit. The details of the ELISA kit and methodology details are provided in the Supp. ‘Materials’ section and Supplementary ‘Methods 2.7’ section respectively. The reading was taken at 450 nm in Multiskan Go (Thermo Fisher). 1.12. Derivation of mDC:pDC ratio Total mDC and pDC were sorted from the mice splenocytes of individual experimental groups (Fig. 1A, 5th panel). Sorting was done with fixed gating for all samples and the cell count for both subtypes were taken into consideration for deriving the ratio. The derivation is explained in details in Supplementary ‘Methods 2.8’ Section.

1.7. Vacuole-bound LC3B level detection 1.13. Statistical analysis The same method followed as according to our published work [35] and as performed by other group [37] according to the manufacture's protocol (Millipore: FCCH100171) and explained in details in Supplementary ‘Methods 2.4’ section. In brief, 105 isolated pDCs were serially treated with the kit provided buffer to wash off free cytoplasmic LC3B and then incubated with LC3B-FITC antibody according to the datasheet. The vacuole bound LC3B was detected by the green filter of BD C6 Accuri flow cytometry machine.

Data from different treatment groups were analyzed by ANOVA and Tukey Test as and where applied. Data are expressed as mean ± SEM. Statistical analysis was performed by GraphPad Prism Software. The significance of the differences between mean values was determined using student's t-test. Significant differences were assessed by the Log-rank test. p < 0.05 were considered significant (*p < 0.05, **p < 0.01 and ***p < 0.001).

1.8. Phagocytosis assay

2. Results

Phagocytosis assay was performed as previously reported by our group [35] and explained in Supplementary ‘Methods 2.5’ Briefly, 105 pDCs were co-incubated with fluorochrome tagged zymosan-A granules in proper cell culture medium for 2 h in CO2 incubator. Post masking of non-ingested granules by trypan blue the cells were finally resuspended in CSB after washing with PBS. Fluorochrome signal from the ingested granules within pDC was detected by the green filter of BD C6 accuri flow cytometry machine.

2.1. Rapamycin treatment enhanced survival with parasitemia reduction in blood, brain, and pDC

1.9. Adhesion assay Infected RBC (iRBC) was extracted by mice heart puncture on the day of sacrifice. They were labeled with calcein AM according to the manufacturer's protocol (Sigma: 17783) or as previously reported

The sorted out splenic CD11clow CD317high pDC (Fig. 1A) from control and three infected groups were checked for the parasite load. The Plasmodium load in pDC subset under different dose conditions were measured by amplifying the Plasmodium 18s rRNA from the cDNA prepared for real-time analysis (Fig. 1B). pDCs isolated from the rapamycin-treated infected mice have shown a low parasite load as compared to pDCs from the no-treatment group (Fig. 1B). The same was also validated by staining the sorted cells with merozoite surface protein 1 (msp1) abundance (Fig. 1C, Supp.Fig. 1A) with a low clinical score (Supp.Fig. 1B). The parasitemia reduction in whole blood samples post rapamycin

Please cite this article as: A. Sengupta et al., Autophagic induction modulates splenic plasmacytoid dendritic cell mediated immune response in cerebral malarial infection model, Microbes and Infection, https://doi.org/10.1016/j.micinf.2019.05.004

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Fig. 1. Rapamcyin treatment enhanced parasite clearance and PbA infected mice survival: A) The cell sorting strategy for pDC. The single cells were gated out from the live cell population. CD8T cells were sorted from it and CD11clow CD317high pDCs were isolated from the remaining population. Extreme right panel shows our post sort analysis of enriched pDC subset. B) The isolated pDCs were subjected to realtime PCR for ANKA18s rRNA showed a downregulation of the parasite load as compared to non-treated infected group. C) The same trend of parasite reduction within pDC was also observed by determining merozoite surface protein (msp1) load flow cytometry. D) Drop in parasitemia post rapamycin treatment, in mice blood infected with iRBC and also when i-pDC from rapamycin treated mice were inoculated to second batch of naïve mice. E) iRBC infected mice survival enhanced post rapamycin treatment as compared to no-treatment and ammonium chloride treated groups. F) Whole RNA extracted from the homogenized brain tissue were subjected to realtime PCR for ANKA 18S rRNA. Results shows the protective role of rapamycin in preventing a bulk of the parasite load to get into the brain as observed within notreatment or ammonium chloride treated groups. Data in graphs are the representative images derived from at least four independent experiments. (*p < 0.05, **p < 0.01 and ***p < 0.001).

treatment was observed by Giemsa staining (Fig. 1D). An equal number of infected pDCs were isolated from no-treatment and rapamycin-treated mice and injected to naïve mice. Parasitemia in mice blood getting pDCs from the no-treatment group was markedly higher than the rapamycin-treated group (Fig. 1D). Consequentially, the rapamycin-treated mice set have shown a better survival as in comparison to any other groups (Fig. 1E). A reduction in parasite load in the brain as measured by ANKA 18s rRNA expression in the rapamycin-treated mice group was observed as compared to both no-treatment infected and ammonium chloridetreated group (Fig. 1F). The quantitative real-time PCR revealed that the expression of autophagy pathway genes increased post PbA infection followed by a small increment in beclin1, atg5; after rapamycin treatment (Fig. 2A). Enhanced phosphorylation of serine residues of beclin-s15p and p62-s403p confirmed the positive induction of autophagic flux after rapamycin treatment (Fig. 2B). 2.2. pDCs from rapamycin-treated mice show an enhanced autophagosome formation, vacuolar LC3B deposition, and DC-LAMP level with a drop in the siglecH expression The catiophillic amphitracer dye (Enzo) detected the increase in autophagolysosome formation within rapamycin-treated pDC in comparison to other groups (Fig. 2C). The same trend was observed in vacuolar LC3B deposition detected after washing off the free

cytoplasmic LC3B (Fig. 2D). Autophagic induction also lead to upregulation of dendritic cell lysosome-associated membrane protein (DC-LAMP), i.e., CD208, within pDC (Fig. 2E). This protein is almost exclusively expressed by mature dendritic cells (DC) and is found in the MHC-II compartment immediately before the translocation of MHC-II molecules to the cell surface. We have found a significant reduction in the expression level of adhesion molecule SiglecH (CD33) post rapamycin treatment as compared with the no-treatment infected group (Fig. 2F). 2.3. Reversal of Treg: Th17 and mDC:pDC ratio in rapamycintreated mice as compared to the no-treatment infected group In the co-culture experiment with pDC (isolated from respective groups) and CD4T cell (isolated from control mice), we have checked the influence of pDC in modulating the fate and differentiation of CD4T cells. We found an increased population of regulatory T cell (Treg) expressing FOXP3 along with a significant reduction of RORgt expressing Th17 cells post rapamycin treatment as compared to no-treatment PbA group (Fig. 3A, B). The significant influence of pDC from no-treatment infected mice was observed in the differentiation of CD4T cells to Th17 cell population. The situation reverses for the rapamycin treated pDC; where pronounced differentiation of CD4T cell towards the generation of more Treg subtypes was observed (Fig. 3A, B).

Please cite this article as: A. Sengupta et al., Autophagic induction modulates splenic plasmacytoid dendritic cell mediated immune response in cerebral malarial infection model, Microbes and Infection, https://doi.org/10.1016/j.micinf.2019.05.004

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Fig. 2. Rapamycin treatment enhanced autophagic flux within pDC by upregulating protein phosphorylation, autophagolysosome formation, vacuolar LC3B deposition, DC-LAMP expression and suppressing SiglecH expression: A) The real-time analysis for expression of the genes of autophagic pathway shows an upregulation in only PbA infected mice with subtle increase post rapamycin treatment. Ammonium chloride treatment suppress the expression. B) The phosphorylation level of serine-403 of sqstm1 and serine-15 of beclin1 increased, reconfirming the autophagic induction post rapamycin treatment. C) Autophagolysosome formation and D) Vacuolar LC3B deposition increased post autophagic induction by rapamycin treatment. E) Dendritic Cell Lysosomal Associated Protein (DC-LAMP) expression enhanced in the PbA infected mice getting rapamycin treatment, indicating a better lysosomal activity F) SiglecH (CD33) expression level dropped after rapamycin treatment as compared to the infected mice having no treatment. Data in graphs are the representative images derived from at least four independent experiments. (*p < 0.05, **p < 0.01 and ***p < 0.001).

Fig. 3. Rapamycin treatment shifted overaggressive Th1 immune response pathway in PbA infection to tolerogenic Th2 pathway; with reduced adhesion property and unaltered phagocytic potential by pDC: A) Increase in the expression of FOXP3, the marker of Treg cells, was observed on the CD4T cell surface post rapamycin treatment (third plot, upper-left quadrant) as compared to the other groups. Th17 cells was marked by RORgt expression. Its level increased on the CD4T cells during PbA infection (second plot, lower right quadrant), which was downregulated post rapamycin treatment (third plot, lower right quadrant). Enhancement of Treg cell population and lowering of Th17 results in a change in Treg-Th17 balance which critically influences the disease pathogenesis and outcome. pDC isolated from respective treatment groups incubated with CD4T cells isolated from control mice were assessed with respective parasite load and treatment condition. B) The bar graph representation of the cell populations from the flow cytometry plots of Fig. 3A showed a significant increase in Treg cell population and a decrease in the Th17 cell population post rapamycin treatment. C) The increased ratio of mDC over pDC in PbA infection, dropped post rapamycin treatment indicating a Th2 type immune response post rapamycin treatment. The ratio was derived by taking into consideration of the number of mDC isolated in parallel with the pDC isolation (as shown in Fig. 1A, fifth panel). The derivation process was explained in methods 1.12 section. D) Drop in the adhesion property of the isolated pDC in the autophagy induced samples was observed post rapamycin treatment. An insignificant change in phagocytic property as in comparison to the infected samples was observed. Flurochrome tagged zymosan A granule ingestion was used for assessing the phagocytosis property of the isolated pDC. The ability of pDC to bind calcein AM labeled iRBC was taken as the measure for the adhesion property of the cells. Data in graphs are the representative images derived from at least four independent experiments. (*p < 0.05, **p < 0.01 and ***p < 0.001).

Please cite this article as: A. Sengupta et al., Autophagic induction modulates splenic plasmacytoid dendritic cell mediated immune response in cerebral malarial infection model, Microbes and Infection, https://doi.org/10.1016/j.micinf.2019.05.004

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We found a drop in the mDC:pDC ratio post rapamycin treatment as compared with the no-treatment group (Fig. 3C). Hence increase in Treg: Th17 ratio and drop in mDC:pDC ratio shifted the balance of the immune response from Th1 to less aggressive Th2 pathway post induction of autophagy by rapamycin treatment. 2.4. Drop in adhesion property of pDCs with an unchanged phagocytic potential between rapamycin-treated and no-treatment infected groups The adhesion properties of the isolated pDCs from different treatment groups were analyzed by their capacity to adhere to calceinAM labeled control RBC (cRBC) or infected RBC (iRBC). The percentage of pDCs having calcein AM signal indicated a reduction in adhesion property of the rapamycin treated pDCs as compared to that of the no-treatment infected group (Fig. 3D). An unaltered trend in the phagocytosis rate of fluorochrome tagged zymosan granules by the pDCs was found between notreatment or rapamycin-treated group (Fig. 3D). Hence, we can conclude that the rapamycin-induced autophagic change in pDC is associated with a downregulation in adhesive property of the cells keeping the phagocytic property unchanged in ECM. 2.5. Co-stimulatory markers, MHC molecule expression, antigen presentation marker of pDCs enhanced post rapamycin treatment There was a small increase in the CD80-CD86 double-positive cell population, post rapamycin treatment as compared to notreatment infected group (Fig. 4A, B). Rapamycin treatment enhanced MHC-I expression with insignificant change in MHC-II expression on the pDC surface as compared with the no-treatment group (Fig. 4C). MHC-I level increased even after ammonium chloride treatment to a certain extent (Fig. 4C). Limited autophagic inhibition usually results in better pathogenic peptide fragment generation to be presented by MHCs. Rapamycin-treated pDC population expressed high TLR9 (Fig. 4D) and CD205 (Fig. 4E). TLR9 expression, an essential receptor of Plasmodium CpG DNA, correlated with the enhanced CD205 expression (Fig. 4F). From this, we can infer that a minor enhancement in costimulatory activity (CD80, CD86) is taking place along with higher antigen processing (CD205) and antigen presentation molecule (MHCI) expression post rapamycin treatment as compared with no-treatment PbA infected group. 2.6. Alteration in Effector and Memory CD8T cells population observed with an enhancement of effector cell population post rapamycin treatment Sorted CD8T cells from control mice were co-incubated with pDC under different treatment conditions along with cRBC or iRBC as accordingly. The differentiation of CD8T cell population was checked for their effector and memory cell populations. Although there was an increase in terminally differentiated effector cells (CCR7- CD45RAþ) during infection (Fig. 5A: lower left quadrant), post rapamycin treatment. The effector cell population dropped post ammonium chloride treatment. The effector memory T cell population (CCR7- CD45RA) reduced post rapamycin treatment in comparison to the no-treatment infected group (Fig. 5A: upper left quadrant). The central memory T cell population (CCR7þ CD45RA) was higher post autophagic induction by rapamycin as compared to no-treatment infected group (Fig. 5A: lower right quadrant). Less naïve T cells (CCR7þ CD45RAþ) was observed in a rapamycintreated group than any other groups (Fig. 5A: upper right

quadrant). A representative bar graph analysis of the different cell types is provided for easy comparison (Fig. 5B). 2.7. Rapamycin-treated pDC influences a drop in FasL expression on CD8T cell with an unchanged CD28 level as compared to the notreatment infected group pDC subsets isolated from respective sets were co-cultured with CD8T cells from control mice along with the respective cRBC or iRBC. We found enhanced expression of costimulatory marker CD28 on CD8T cells during PbA infection with a subtle increase during rapamycin treatment. The expression level dropped post ammonium chloride treatment (Fig. 5C). Fas ligand (FasL) is expressed on cytotoxic T lymphocytes. The marked reduction in its expression (Fig. 5C) post rapamycin treatment correlated well with an increase in live CD8þT cell population (Sup Fig. 3) isolated during cell sorting as shown in Fig. 1A, fourth panel. 2.8. Altered cytokine profile leads to downregulation of proinflammatory response in rapamycin-treated pDC: CD8T co-culture Co-culture supernatant media of pDC (isolated from four groups) and CD8T cells (isolated from the control mice) were collected and measured for pro and anti-inflammatory cytokines expression. Relative downregulation of pro-inflammatory cytokines like TNFa and IL6 in rapamycin-treated group than other groups was observed (Fig. 5D). Positive induction of antiinflammatory cytokines like IL10 was observed from rapamycintreated mice pDC (Fig. 5D). Rapamycin-treated pDC secreted a high IFNa (Fig. 5D). This cytokine profile again indicates a Th2 response is overruling the Th1 pathway post rapamycin treatment (as previously stated in section 2.3). The pDCs were checked for the respective cytokine receptors by flow cytometry. TNF receptor (TNFR2R) expression enhanced and a drop in IL10 receptor (IL10R) in rapamycin-treated pDC as compared with a no-treatment infected group (Fig. 5E). This was in contrast to the respective cytokine profile found via ELISA (Fig. 5D). IL2 receptor (IL2R), critical for controlling a whole set of immune regulation was significantly upregulated as well (Fig. 5E). 3. Discussion The beneficial role of rapamycin in the treatment of ECM was reported previously [30e32]. Different dose regimen in terms of differential concentration, duration, and number of doses have been investigated [30,31]. Here we tested a high dose for a longer duration than the previously reported studies and modulated the autophagic flux to investigate its effect on pDC. It has been reported that CD317high pDC subset harbors live Plasmodium parasite and can cause malaria in naïve mice [10,11]. The rapamycin treatment offered a better prognosis by eliciting an immune response and reduce Plasmodium load within splenic CD317high pDC (Fig. 1B, C, Supp.Fig. 1A). The systemic parasite counts in whole blood and brain decreased (Fig. 1D, F) resulting in low clinical score (Supp.Fig. 1B) and survival enhancement was observed (Fig. 1E). PbA infection itself caused an enhanced expression of autophagic marker genes, with a small increment post rapamycin treatment (Fig. 2A). However, the treatment was effective in the upregulation of autophagic flux within pDC which no-treatment PbA group was not exhibiting. It was confirmed by the increase in serine phosphorylation of beclin1 and p62 (Fig. 2B), autophagolysosome formation (Fig. 2C), vacuolar LC3B accumulation (Fig. 2D), as compared to no-treatment PbA group. Increase in serine

Please cite this article as: A. Sengupta et al., Autophagic induction modulates splenic plasmacytoid dendritic cell mediated immune response in cerebral malarial infection model, Microbes and Infection, https://doi.org/10.1016/j.micinf.2019.05.004

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Fig. 4. pDC exhibited an enhanced co-stimulation, antigen processing, and presentation markers post rapamycin treatment: A) Isolated pDC exhibits enhancement of CD80-CD86 double positive population post rapamycin treatment, indicating a better co-stimulation activity. B) Bar graph representation of the plots provided in Fig. 4A, is showing a small increase in pDC population possessing both CD80 and CD86 markers in rapamycin treatment group. The population dropped after ammonium chloride treatment. C) The MHC-I expression on isolated pDC surface enhanced post rapamycin treatment as compared to other groups. MHC-II expression levels remain unaltered in PbA infected group having either no-treatment or rapamycin-treatment. However, its level dropped post ammonium chloride-treatment. D) TLR9 expression and E) DEC-205 (CD205) expression in pDC, enhanced within rapamycin-treated group as compared to the non-treated PbA group. F) The observed expression of TLR9 increased in parallel with CD205 expression. Data in graphs are the representative images derived from at least four independent experiments. (*p < 0.05, **p < 0.01 and ***p < 0.001).

phosphorylation of beclin-s15p and p62-s403p (Fig. 2B) marks a positive induction of the autophagic pathway [38,39]. Wykes MN et al., 2011 [10,11]; found that pDC, isolated from infected mice, can cause PbA infection in naïve mice. This was what we have also observed (Fig. 1D). An equal number of infected pDCs (ipDC) from either the infected or infected þ rapamycin-treated mice were injected to naïve mice. The i-pDCs from rapamycin-treated mice caused less parasitemia and showed better survival response in new mice batch (Fig. 1D, Supp.Fig. 2). It needs to be noted over here, only a small proportion of parasite is viable within pDC, and this is not the viable way to spread parasite in the wild. The immune system of the first mice batches (from where the pDCs were isolated) and the naïve mice (to which infected pDCs were inoculated) also plays a role in controlling pathogenesis. Further investigations on measuring the viable parasites load and effect of immune system effect from both the mice batches will provide a clearer picture. Rapamycin treatment was accompanied by a downregulation of siglec-H (Fig. 2F), which is specifically expressed on mouse pDC [40]. Its downregulation marks pDC activation [40,41]. The loss in siglecH adhesion molecules was associated with a decrease in adhesion property of the cells to calceinAM labeled iRBC (Fig. 3D). However, it did not alter or reduce the phagocytic potential of the pDC as measured by zymosan granule ingestion (Fig. 3D). We hypothesized that probably too much pathogenic load adhered to the pDC surface is detrimental for its proper activation and impose a negative impact on downstream immune response. Hence partial decrement in adhesion property may not only be beneficial for its activation but also leads into the negligible chance of having unprocessed pathogen to harbor.

Although pDC is not the most potent antigen-presenting DC subset as compared to mDC, literature report suggests that in many cases pDC acts at par with any other APCs [7e9]. The pDC express costimulatory molecule CD80, CD86 in different conditions [7]. We have found a small increase of the double positive pDC population in rapamycin-treated group in comparison to other infected groups (Fig. 4A, B). The same trend was observed in the expression of CD205, a marker for antigen processing and presentation [21]. CD205 increased with rapamycin treatment, in positive correlation with the TLR9 expression (Fig. 4DeF). The treatment also induced MHC-I expression (Fig. 4C). Suppression of basal autophagic flux post ammonium chloride treatment was associated with MHC-II downregulation (Fig. 4C). Induction of co-stimulatory, antigen processing and presentation in rapamycin-treated PbA group directed us to study their influence in stimulating CD4T and CD8T cells. We co-cultured either CD8T or CD4T cells isolated from the control mice with that of isolated pDCs from four experimental groups. Both of these control T cells had not previously been influenced by either of the rapamycin or ammonium chloride treatment or even by the parasite. Hence any alteration in their profile was strictly restricted to the pDC influence alone. CD8T cells showed significant upregulation of costimulatory marker CD28 in both non-treated infected and rapamycin-treated groups, depicting the maintenance of immune elicitation even after rapamycin treatment (Fig. 5C). Moreover, the same rapamycin-treated cells showed downregulation of FasL (Fig. 5C) which is reported to inhibit T cell proliferation [42]. Hence, its downregulation in rapamycin-treated pDC: CD8T goes in context with an increment of the CD8þ T cell profile (Sup Fig. 3). A relatively low population of

Please cite this article as: A. Sengupta et al., Autophagic induction modulates splenic plasmacytoid dendritic cell mediated immune response in cerebral malarial infection model, Microbes and Infection, https://doi.org/10.1016/j.micinf.2019.05.004

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Fig. 5. Rapamycin-treated pDC influence CD8T differentiation to effector and memory cell population, CD28 expression, FasL level and cytokine profile: A) Flow cytometry plots and B) bar diagram representation of CD8T cells stained with CCR7 and CD45RA. Rapamycin treatment resulted into a lowering of effector memory cells (CCR7 CD45RA) and increase in central memory (CCR7þ CD45RA) cell population compared with no-treatment group. Naïve CD8T cell population (CCR7þ CD45RAþ) decreased as enhancement in differentiated effector cells (CCR7 CD45RAþ) was observed post rapamycin treatment. C) CD28 costimulatory molecule expression on CD8T cells enhanced while FAS-L level dropped in rapamycin-treated group as compared with no-treatment group. CD28 expression reduced after ammonium chloride treatment. D) Pro-inflammatory cytokine IL6 and TNFa level decreased and the upregulation of IFNa, IL2 and IL10 level was observed by ELISA post rapamycin treatment. ELISA were performed on the cell culture supernatant of pDC (isolated from respective experimental groups) incubated with CD8T cells (isolated from control mice) with respective parasite load and treatment condition. E) The cytokine receptors of TNFa, IL2, IL12 increased and IL10R decreased in pDC, post rapamycin treatment. The cytokine receptors were checked on pDC post isolation from the respective experimental groups. Data in graphs are the representative images derived from at least four independent experiments. (*p < 0.05, **p < 0.01 and ***p < 0.001).

effector memory cells (CCR7 CD45RA) but higher central memory cells (CCR7þ CD45RA) were observed in the rapamycintreated group in comparison with the only infected sample (Fig. 5A, B). Reduction in naïve T cells (CCR7þ CD45RAþ) and expansion of terminally differentiated effector CD8þT cells (CCR7CD45RAþ) by rapamycin-treated pDC indicates a better immune response. While studying the pDC: CD4T co-culture, we have found rapamycin-treated pDC influenced CD4þFOXP3þ Treg population to increase; downregulating CD4þRORgt þ Th17 cell population (Fig. 3A, B). This reversed the imbalance created in Treg: Th17 ratio during Plasmodium infection [28]; a potent cause for poor outcome of the host. Thus, we infer that rapamycin-treated pDC shapes a better CD4T and CD8T cell response. The tilt towards high mDC:pDC ratio leads to induced Th1 pathway mediated overaggressive immune-regulation leading to poor prognosis for the host [29]. A critical level of autophagic induction might be essential for the high ratio. Induction by rapamycin or even inhibition by ammonium chloride leads to lowering of the ratio (Fig. 3B). Shifting the Treg: Th17 balance towards the increased Treg population (Fig. 3A, B) along with the increase in regulatory pDC, post autophagic induction by rapamycin (Fig. 3C) dampens the inflammatory response and promotes Th2 pathway. To confirm it further we have checked cytokine profile (in pDC: CD8T coculture supernatant) and expression of the cytokine receptors on pDC. The upregulation of pro-inflammatory cytokine production during malarial infection is one of the alarming causes for poor prognosis of malaria [23]. Autophagy induction suppresses proinflammatory response [43]. Activation of the pDC is well marked by its capability of secreting IFNa [24]. Its secretion boosted up post rapamycin treatment (Fig. 5D), confirming functional activation of pDC again. Previously mentioned downregulation of siglecH

(Fig. 2F) on rapamycin treated pDC surface is also a crucial factor for IFNa upregulation [44]. The strict control of pro-inflammatory cytokine expression like TNFa is beneficial for the disease pathogenesis [23]. pDCs have been found to induce the production of the anti-inflammatory cytokine IL10 after maturation [45]. IL10 helps in the expression of costimulatory molecules CD80/CD86 [46]. Rapamycin treatment induced IL10 secretion, suppressed TNFa and thus reversed the IL10/TNFa ratios as observed in no-treatment infected group (Fig. 5D). Similarly, the expression level of pro-inflammatory cytokine IL6 and its receptor expression decreased in rapamycintreated group resulting in an overall downregulation of proinflammatory cytokines (Fig. 5D). IL2 secretion tightly regulates mounting and dampening of immune responses primarily via its direct effects on T cells [47]. Rapamycin treatment caused Plasmodium-infected pDC to secrete high levels of IL2 and its receptor (IL2R) (Fig. 5E). The report indicates that the expression of the IL2R is relevant to pDC activation [48,49]. IL2R level increases primarily due to TLR9-CpG DNA binding stimulation [48]. We have found a significant increase of IL2R in our PbA infection model (Fig. 5E). As autophagy in a cell is induced by proinflammatory cytokine [50], hence the upregulation of TNFa receptor might be a strategy offered by pDC to make the best use of whatever proinflammatory cytokine available to maintain the autophagic induction within it (Fig. 5E). Our study is limited to a certain extent due to the lack of any known potential autophagic inducer that can specifically affect the splenic pDC and not any other cells. As rapamycin can influence multiple cell types, the extent of sole contribution by pDC for better host response is yet to be deciphered. Nevertheless, in both in-vivo and in-vitro studies, pDC itself exhibits a better immune response in rapamycin-treated autophagy induced set. The study thus concludes that the pathogen clearance can be achieved by inducing the autophagic flux by rapamycin treatment.

Please cite this article as: A. Sengupta et al., Autophagic induction modulates splenic plasmacytoid dendritic cell mediated immune response in cerebral malarial infection model, Microbes and Infection, https://doi.org/10.1016/j.micinf.2019.05.004

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The role of pDC which acts as the Trojan horse and carries the live Plasmodium within it, can be reversed. pDC shows better antigen processing, co-stimulation, antigen presentation, and influence both CD4T and CD8T cells. pDC regulates the shifting of aggressive Th1 immune pathway to a more tolerogenic Th2 pathway as elucidated by the change in the ratio of Treg: Th17, mDC: pDC and IL10: TNFa. Deciphering these mechanisms in more intricate details can infer potent ways to design a therapeutic approach targeting autophagic pathways of pDC, which might lead to a successful treatment procedure in near future. Conflict of interest All the authors of this manuscript declared that there is no conflict of interest in publishing the article. Acknowledgment The study is funded by the following funding agencies: Department of Science and Technology (DST), India (SB/SO/HS-106/ 2013, dated 21/11/2014), Department of Atomic Energy- Board of Research in Nuclear Sciences (DAE -BRNS), India: (37(1)/14/54/ 2014-BRNS/1740 dated 28.10.2014), West Bengal Department of Biotechnology (WB-DBT), India (22(Sanc)/BT (Estt)/RD-20/2013 dated 07/01/2015). Calcutta University- BD Centre of Excellence Flow Cytometry Facility [CU-BD CoE (CRNN)] for cell sorting and flow cytometry. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.micinf.2019.05.004. References [1] World Health Organization. World Malaria report 2018. 2018. https://www. who.int/malaria/media/world-malaria-report-2018/en/. [2] Torre S, Langlais D, Gros P. Genetic analysis of cerebral malaria in the mouse model infected with Plasmodium berghei. Mamm Genome 2018;29(7e8): 488e506. https://doi.org/10.1007/s00335-018-9752-9. Epub 2018 Jun 19. [3] Aghahowa S. Okolocha K Comparative effects of parenteral antimalarials in Swiss albino mice after chronic exposure to Plasmodium berghei. Anim Model Exp Med 2018 Sep 25;1(3):235e41. https://doi.org/10.1002/ame2.12029. eCollection 2018 Sep. [4] Miller LH, Baruch DI, Marsh K, Doumbo OK. The pathogenic basis of malaria. Nature 2002;415:673e9. [5] Brian de Souza J, Hafalla JC, Riley EM, Couper KN. Cerebral malaria: why experimental murine models are required to understand the pathogenesis of disease. Parasitology 2009;137(5):755e72. ISSN 0031-1820, https://doi.org/ 10.1017/S0031182009991715. [6] Del Portillo HA, Ferrer M, Brugat T, Martin-Jaular L, Langhorne J, Lacerda MV. The role of the spleen in malaria. Cell Microbiol 2012 Mar;14(3):343e55. https://doi.org/10.1111/j.1462-5822.2011.01741.x. Epub 2012 Feb 2. [7] Swiecki M, Colonna M. The multifaceted biology of plasmacytoid dendritic cells. Nat Rev Immunol 2015 Aug;15(8):471e85. https://doi.org/10.1038/ nri3865. Epub 2015 Jul 10. Review. PMID:26160613. [8] Schlecht G, Garcia S, Escriou N, Freitas AA, Leclerc C, Dadaglio G. Murine plasmacytoid dendritic cells induce effector/memory CD8þ T-cell responses in vivo after viral stimulation. Blood 2004;104(6):1808e15. View at Publisher View at Google Scholar View at Scopus. [9] Villadangos JA, Young L. Antigen-presentation properties of plasmacytoid dendritic cells. Immunity 2008;29(3):352e61. [10] Wykes MN, Kay JG, Manderson A, Liu XQ, Brown DL, Richard DJ, et al. Rodent blood-stage Plasmodium survive in dendritic cells that infect naive mice. Proc Natl Acad Sci U S A 2011;108:11205e10. [11] Wykes MN, Liu XQ, Beattie L, Stanisic DI, Stacey KJ, Smyth MJ, et al. Plasmodium strain determines dendritic cell function essential for survival from malaria. PLoS Pathog 2007;3:e96. [12] O'Keeffe M, Hochrein H, Vremec D, Caminschi I, Miller JL, Anders EM, et al. Mouse plasmacytoid cells: long-lived cells, heterogeneous in surface phenotype and function, that differentiate into CD8(þ) dendritic cells only after microbial stimulus. J Exp Med 2002;196(10):1307e19.

9

[13] Bierly AL, Shufesky WJ, Sukhumavasi W, Morelli AE, Denkers EY. Dendritic cells expressing plasmacytoid marker PDCA-1 are trojan horses during Toxoplasma gondii infection. J Immunol 2008;181:8485e91. [14] Deretic V, Saitoh T, Akira S. Autophagy in infection, inflammation and immunity. Nat Rev Immunol 2013 Oct;13(10):722e37. https://doi.org/10.1038/ nri3532. Review. [15] Yoshii SR, Mizushima N. Monitoring and measuring autophagy. Int J Mol Sci 2017 Aug 28;18(9):E1865. https://doi.org/10.3390/ijms18091865. Review. [16] Bah A, Vergne I. Macrophage autophagy and bacterial infections. Front Immunol 2017 Nov 6;8:1483. https://doi.org/10.3389/fimmu.2017.01483. eCollection 2017. Review. [17] Puleston DJ, Simon AK. Autophagy in the immune system. Immunology 2014 Jan;141(1):1e8. https://doi.org/10.1111/imm.12165. Review. [18] Kenific CM, Wittmann T, Debnath J. Autophagy in adhesion and migration. J Cell Sci 2016 Oct 15;129(20):3685e93. Epub 2016 Sep 26. Review. [19] Das M, Kaveri SV, Bayry J. Cross-presentation of antigens by dendritic cells: role of autophagy. Oncotarget 2015 Oct 6;6(30):28527e8. https://doi.org/ 10.18632/oncotarget.5268. No abstract available.
rrez-Martínez E, Plane s R, Anselmi G, Reynolds M, Menezes S, Adiko AC, [20] Gutie et al. Cross-presentation of cell-associated antigens by MHC class I in dendritic cell subsets. Front Immunol 2015 Jul 17;6:363. https://doi.org/10.3389/ fimmu.2015.00363. eCollection 2015. Review. [21] Tel J, Benitez-Ribas D, Hoosemans S, Cambi A, Adema GJ, Figdor CG, et al. DEC-205 mediates antigen uptake and presentation by both resting and activated human plasmacytoid dendritic cells. Eur J Immunol 2011;41: 1014e23. [22] Pichyangkul S, Yongvanitchit K, Kum-Arb U, Hemmi H, Akira S, Krieg AM, et al. Malaria blood stage parasites activate human plasmacytoid dendritic cells and murine dendritic cells through a toll-like receptor 9-dependent pathway. J Immunol 2004;172:4926e33. [23] Hunt NH, Grau GE. Cytokines: accelerators and brakes in the pathogenesis of cerebral malaria. Trends Immunol 2003 Sep;24(9):491e9. Review. € nnblom L, Eloranta ML, Alm GV. Role of natural interferon-alpha producing [24] Ro cells (plasmacytoid dendritic cells) in autoimmunity. Autoimmunity 2003 Dec;36(8):463e72. Review. [25] Pepper M, Dzierszinski F, Wilson E, Tait E, Fang Q, Yarovinsky F, et al. Plasmacytoid dendritic cells are activated by Toxoplasma gondii to present antigen and produce cytokines. J Immunol 2008 May 1;180(9):6229e36. [26] Ito T, Yang M, Wang YH, Lande R, Gregorio J, Perng OA, et al. Plasmacytoid dendritic cells prime IL-10eproducing T regulatory cells by inducible costimulator ligand. J Exp Med 2007;204:105e15. [27] Boeuf PS, Loizon S, Awandare GA, Tetteh JK, Addae MM, Adjei GO, et al. Insights into deregulated TNF and IL-10 production in malaria: implications for understanding severe malarial anaemia. Malar J 2012 Aug 1;11:253. https:// doi.org/10.1186/1475-2875-11-253. [28] Keswani T, Bhattacharyya A. Differential role of T regulatory and Th17 in Swiss mice infected with Plasmodium berghei ANKA and Plasmodium yoelii. Exp Parasitol 2014 Jun;141:82e92. https://doi.org/10.1016/j.exppara.2014.03.003. Epub 2014 Mar 24. [29] Jangpatarapongsa K, Chootong P, Sattabongkot J, Chotivanich K, Sirichaisinthop J, Tungpradabkul S, et al. Plasmodium vivax parasites alter the balance of myeloid and plasmacytoid dendritic cells and the induction of regulatory T cells. Eur J Immunol 2008 Oct;38(10):2697e705. https://doi.org/ 10.1002/eji.200838186. [30] Gordon EB, Hart GT, Tran TM, Waisberg M, Akkaya M, Skinner J, et al. Inhibiting the mammalian target of rapamycin blocks the development of experimental cerebral malaria. mBio 2015;6. e00725-15. ~ o-Villarreal JH, Reynolds JS, De Niz M, Thompson A, Marti M, [31] Mejia P, Trevin et al. A single rapamycin dose protects against late-stage experimental cerebral malaria via modulation of host immunity, endothelial activation and parasite sequestration. Malar J 2017 Nov 9;16(1):455. https://doi.org/10.1186/ s12936-017-2092-5. [32] Odaka Y, Xu B, Luo Y, Shen T, Shang C, Wu Y, et al. Dihydroartemisinin inhibits the mammalian target of rapamycin-mediated signaling path- ways in tumor cells. Carcinogenesis 2014;35:192e200. ~o AS, Buchholz K, Prude ^ncio M, HermanOrnelas JD, [33] Hanson KK, Ressurreiça Rebelo M, et al. Torins are potent antimalarials that block replenishment of Plasmodium liver stage parasitophorous vacuole membrane proteins. Proc Natl Acad Sci U S A 2013;110:E2838e47. https://doi.org/10.1073/ pnas.1306097110. [34] Sun R, Luo Y, Li J, Wang Q, Li J, Chen X, et al. Ammonium chloride inhibits autophagy of hepatocellular carcinoma cells through SMAD2 signaling. Tumour Biol 2015 Feb;36(2):1173e7. https://doi.org/10.1007/s13277-0142699-x. Epub 2014 Oct 24. [35] Sengupta A, Sarkar S, Keswani T, Mukherjee S, Ghosh S, Bhattacharyya A. Impact of autophagic regulation on splenic red pulp macrophages during cerebral malarial infection. Parasitol Int 2019 Mar 11;71:18e26. https:// doi.org/10.1016/j.parint.2019.03.008. [36] Chan LL, Shen D, Wilkinson AR, Patton W, Lai N, Chan E, et al. Qiu J A novel image-based cytometry method for autophagy detection in living cells. Autophagy 2012 Sep;8(9):1371e82. https://doi.org/10.4161/auto.21028. Epub 2012 Aug 16. [37] Puleston DJ, Zhang H, Powell TJ, Lipina E, Sims S, Panse I, et al. Autophagy is a critical regulator of memory CD8(þ) T cell formation. Elife 2014 Nov 11:3. https://doi.org/10.7554/eLife.03706.

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A. Sengupta et al. / Microbes and Infection xxx (xxxx) xxx

[38] Wei Y, An Z, Zou Z, Sumpter R, Su M, Zang X, et al. The stress-responsive kinases MAPKAPK2/MAPKAPK3 activate starvation-induced autophagy through Beclin 1 phosphorylation. Elife 2015 Feb 18:4. https://doi.org/ 10.7554/eLife.05289. [39] Matsumoto G, Wada K, Okuno M, Kurosawa M, Nukina N. Serine 403 phosphorylation of p62/SQSTM1 regulates selective autophagic clearance of ubiquitinated proteins. Mol Cell 2011 Oct 21;44(2):279e89. https://doi.org/ 10.1016/j.molcel.2011.07.039. [40] Zhang J, Raper A, Sugita N, Hingorani R, Salio M, Palmowski MJ, et al. Characterization of Siglec-H as a novel endocytic receptor expressed on murine plasmacytoid dendritic cell precursors. Blood 2006;107:3600e8. [41] Singh A, Sen E. Reciprocal role of SIRT6 and Hexokinase 2 in the regulation of autophagy driven monocyte differentiation. Exp Cell Res 2017;360:365e74. [42] Guo CL, Yang XH, Cheng W, Xu Y, Li JB, Sun YX, et al. Expression of Fas/FasL in CD8þ T and CD3þ Foxp3þ Treg cells–relationship with apoptosis of circulating CD8þ T cells in hepatocellular carcinoma patients. Asian Pac J Cancer Prev APJCP 2014;15:2613e8. [43] Deretic V, Levine B. Autophagy balances inflammation in innate immunity. Autophagy 2018;14(2):243e51. https://doi.org/10.1080/15548627.2017.140 2992. Epub 2018 Jan 17. Review.

[44] Fitzgerald-Bocarsly P, Dai J, Singh S. Plasmacytoid dendritic cells and type I IFN: 50 years of convergent history. Cytokine Growth Factor Rev 2008;19:3e19. [45] Dasgupta S, Erturk-Hasdemir D, Ochoa-Reparaz J, Reinecker HC, Kasper. Plasmacytoid dendritic cells mediate anti-inflammatory responses to a gut commensal molecule via both innate and adaptive mechanisms. Cell Host Microbe 2014 Apr 9;15(4):413e23. https://doi.org/10.1016/j.chom.2014.03.006. [46] Zhou F, Ciric B, Li H, Yan Y, Li K, Cullimore M, et al. IL-10 deficiency blocks the ability of LPS to regulate expression of tolerance-related molecules on dendritic cells. Eur J Immunol 2012 Jun;42(6):1449e58. https://doi.org/10.1002/ eji.201141733. Epub 2012 May 23. [47] Bachmann MF, Oxenius A. Interleukin 2: from immunostimulation toimmunoregulation and back again. EMBO Rep 2007;8:1142e8. €tz A, Tang MS, Ty MC, Arama C, Ongoiba A, Doumtabe D, et al. Atypical [48] Go activation of dendritic cells by Plasmodiumfalciparum. Proc Natl Acad Sci U S A 2017;114:E10568e77.
mez M, Oliva H, Climent N, Ferna
ndez MA, Ruiz-Riol M, Bofill M, [49] Naranjo-Go et al. Expression and function of the IL-2 receptor in activated human plasmacytoid dendritic cells. Eur J Immunol 2007;37:1764e72. [50] Ma Y, Galluzzi L, Zitvogel L, Kroemer G. Autophagy and cellular immune responses. Immunity 2013;39:211e27.

Please cite this article as: A. Sengupta et al., Autophagic induction modulates splenic plasmacytoid dendritic cell mediated immune response in cerebral malarial infection model, Microbes and Infection, https://doi.org/10.1016/j.micinf.2019.05.004