Differential Induction of SOCS Isoforms by Leishmania Donovani Impairs Macrophage−T Cell Cross-Talk and Host Defense

Differential Induction of SOCS Isoforms by Leishmania donovani Impairs Macrophage−T Cell Cross-Talk and Host Defense

This information is current as Pragya Chandrakar, Naveen Parmar, Albert Descoteaux and of September 30, 2021. Susanta Kar J Immunol published online 27 December 2019 http://www.jimmunol.org/content/early/2019/12/26/jimmun ol.1900412 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published December 27, 2019, doi:10.4049/jimmunol.1900412 The Journal of Immunology

Differential Induction of SOCS Isoforms by Leishmania donovani Impairs Macrophage–T Cell Cross-Talk and Host Defense

Pragya Chandrakar,*,† Naveen Parmar,*,† Albert Descoteaux,‡ and Susanta Kar*,†

Immune evasion strategies adopted by Leishmania donovani involve the exploitation of suppressor of cytokine signaling (SOCS) proteins that are well-known negative regulators of the JAK/STAT pathway. However, the cellular mechanism underpinning the induction of SOCS isoforms and their role in breaching the multilevel regulatory circuit connecting the innate and adaptive arms of immunity are still ambiguous during experimental visceral leishmaniasis. Using bone marrow–derived macrophages (BMMvs) and CD4+ T cells, we observed that L. donovani preferentially upregulates SOCS1 and SOCS3 expression in macrophages and T cells, respectively, whereas the SOCS1 level remains consistently high in BMMvs and SOCS3 expression is pronounced and Downloaded from long lasting in T cells. Consequently, this inhibits STAT1-mediated IL-12 induction in macrophages & STAT4-mediated IFN-g synthesis in T cells. Mechanistically, PI3K/Akt–mediated SRF activation promotes nuclear translocation and binding of Egr2 to SOCS1 promoter for its early induction in infected BMMvs. Additionally, L. donovani activates IDO/kynurenine/AHR signaling in BMMvs to maintain prolonged SOCS1 expression. Later, PGE2, secreted from infected BMMvs induces cAMP–PKA pathway by binding to the EP2/EP4 receptor of CD4+ T cells, leading to SP1, CREB, and GATA1 activation and SOCS3 expression. Small interfering RNA–mediated silencing of SOCS1 and SOCS3 in macrophage and T cells, respectively, restored IL-12 and IFN-g http://www.jimmunol.org/ cytokine levels and BMMv–T cell interaction. Vivo morpholino–mediated silencing of SOCS1 and SOCS3 resulted in protective cytokine responses, thereby reducing organ parasite burden signiﬁcantly in L. donovani–infected BALB/c mice. Collectively, our results imply that L. donovani orchestrates different SOCS isoforms to impair macrophage–T cell cross-talk and preserve its own niche. The Journal of Immunology, 2020, 204: 000–000.

he severity of visceral leishmaniasis (VL) is primarily due or Th2 mode (3). Early studies using mouse models of VL have to parasite migration to the vital organs, such as the liver, revealed a clear dichotomy between Th1-associated cytokines me- spleen and bone marrow of the mammalian host, that diating protection and Th2-associated cytokines mediating suscep-

T by guest on September 30, 2021 eventually causes immunological dysfunctions of T cells, NK cells, tibility (2, 4). In human and experimental models of VL, control and macrophages (1, 2). In different forms of leishmaniasis, it of infection is critically dependent upon the early induction of an has been well documented that disease progression or resolution is IL-12–driven Th1-type immune response and the production of ultimately dictated by CD4+ T cell differentiation to either the Th1 IFN-g by CD4+ T cells (4, 5). IFN-g plays a critical role in the activation of macrophages by inducing production of NO, which is critical for the elimination of parasites (2). *Division of Molecular Parasitology and Immunology, Council of Scientific and Industrial Research-Central Drug Research Institute, Lucknow 226031, India; Most cytokines use the so-called JAK/STAT pathways for †Academy of Scientific and Innovative Research, Ghaziabad, Uttar Pradesh 201002, immune-functioning, and suppressor of cytokine signaling (SOCS) India; and ‡Institut National de la Recherche Scientifique-Centre Armand-Frappier Sante´ Biotechnologie, Laval, Quebec H7V 1B7, Canada proteins are one of the crucial negative regulators of these pathways and cytokine-mediated immune homeostasis (6). Studies using ORCIDs: 0000-0002-0633-5309 (A.D.); 0000-0002-9907-539X (S.K.). conditional knockout (KO) mice have shown that SOCS proteins Received for publication April 10, 2019. Accepted for publication November 24, 2019. are key physiological and pathological regulators of immune ho- This work was supported by the Indian National Science Academy (SP/YSP/115/ meostasis, as they bind to JAK or cytokine receptors, thereby sup- 2015), the Department of Science and Technology (Grant SB/FT/LS-310/2012), and pressing subsequent signaling events associated with host immune the Council of Scientific and Industrial Research (CSIR) (NWP BSC0114). P.C. response (7). SOCS, consisting of eight members (SOCS1 to received a fellowship from CSIR (New Delhi) and N.P. received a fellowship from the University Grants Commission (New Delhi). A.D. is the holder of the Canada SOCS7 and CIS) all share a central SH2 domain and a C-terminal Research Chair on the Biology of Intracellular Parasitism. The funders had no role in SOCS box (8). The expression of SOCS is induced by multiple study design, data collection and analysis, decision to publish, or preparation of the cytokines, growth factors (9), LPS (10), and Gram-positive bacteria manuscript. This manuscript has Central Drug Research Institute Communication number 10010. (11). The SOCS group of proteins positively and negatively regulate Address correspondence and reprint requests to Dr. Susanta Kar, Council of macrophage and dendritic cell activation and are essential for Scientific and Industrial Research-Central Drug Research Institute, Sector-10, T cell development and differentiation (12). It has been postulated Jankipuram Extension, Sitapur Road, Lucknow 226031, Uttar Pradesh, India. that the JAK/STAT signaling is interrupted by lack of appropriate E-mail address: [email protected] phosphorylation of STAT transcription factors, which inhibits The online version of this article contains supplemental material. macrophage activation. For example, SOCS1 and 3 inhibit STAT1 Abbreviations used in this article: BMMv, bone marrow–derived macrophage; CDRI, Central Drug Research Institute; ChIP, chromatin immunoprecipitation; CSIR, phosphorylation, SOCS3 and 4 inhibit STAT3 phosphorylation, Council of Scientific and Industrial Research; KO, knockout; LPG, lipophosphogly- SOCS3 and 5 modulate STAT4 and STAT6 activation, respectively can; PKA, protein kinase A; siRNA, small interfering RNA; SOCS, suppressor of (13). These, in turn, regulate synthesis of different cytokines, such cytokine signaling; VL, visceral leishmaniasis; WT, wild-type. as STAT1, which is essential for biological effects of IFN-g; Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 STAT4 is important for the cellular effect of IL-12; STAT3 plays

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900412 2 SOCS1 AND SOCS3 BREACH MACROPHAGE–T CELL CROSS-TALK IN VL a conserved role in IL-10 signaling; and STAT6 is important determined on cultures run in parallel. Infection was allowed to proceed for 4 h; cells were washed at least four times to remove uningested par- for IL-4– and IL-13–mediated response (14). Moreover, the role + of SOCS isoforms in modulating cellular immune response in asites and left for 20 h. CD4 T cells were activated (cultured with plate- bound anti-CD3, 2 mg/ml) and were incubated with L. donovani–infected various infectious diseases is well documented. For instances, BMMv-derived supernatant for different time periods, and later, T cells Toxoplasma gondii induces SOCS1 expression, leading to inhi- were isolated and analyzed for different immunological assays. bition of cytokine signaling and suppression of immune responses Ethics statement (15). Mycobacterium bovis also increases SOCS1 and SOCS3 to inhibit IFN-g–induced STAT1 activation (16, 17). Similarly, mice All animal experiments for this study were officially approved by the lacking SOCS1 were protected from Plasmodium berghei, Animal Ethics Committee of the Council of Scientific and Industrial Research (CSIR)–Central Drug Research Institute (CDRI), Lucknow, which cause cerebral malaria, and expression of SOCS1 ham- India (Institutional Animal Ethics Committee approval number: IAEC/ pered Chlamydia pneumonia clearance (18, 19). In the context 2017/191). All animals were housed in a pathogen-free environment and of leishmaniasis, to date, few facts have been highlighted, such as were maintained under experimental conditions, such as room temperature in SOCS1-deficient mice, uncontrolled levels of IFN-g increased (24 6 2˚C) and 40–60% relative humidity. Experimental methods for this resistance to Leishmania major infection, and it was also seen that study were as per the strict guidelines by the Committee for the Purpose of Control and Supervision of Experiments on Animals (New Delhi, India). SOCS3 is induced by L. donovani and L. major with variable All the efforts were carried out to minimize the suffering of animals, which expression levels (20), but comprehensive studies are still lacking. were euthanized by CO2 inhalation. A recent report also suggested that SOCS1 and -3 inductions Real-time PCR by L. donovani subvert macrophage apoptotic machinery by pre- venting oxidative burst (15). However, these reports only dem- In brief, total RNA from L. donovani–infected macrophage cells or from Downloaded from + onstrated the induction of specific SOCS isoforms (SOCS1 and 3) cocultured CD4 T cells was isolated using the RNeasy Mini Kit (QIAGEN). RNA integrity was further confirmed, and purity was assessed by the following Leishmania infection, but the cellular mechanism be- NanoDrop spectrophotometer (Thermo Fisher Scientific). An equal amount hind this induction is completely unknown. Comprehensive un- of RNA (1 mg) from each experimental group was subjected to cDNA derstanding is still ambiguous, such as in-depth knowledge about synthesis by using RevertAid First Strand cDNA Synthesis Kit (Fermentas). how L. donovani establish a cross-talk between the innate and Quantitative real-time PCRs were then carried out using SYBR Green–based detection primers. The PCR amplification conditions were as follows: adaptive immune system by inducing a SOCS isoform for its own http://www.jimmunol.org/ 40 cycles of 95˚C for 15 s and optimized annealing temperature for 30 s, survival and hamper the general cross-talk between immune cells. extension at 72˚C for 30 s. GAPDH, a housekeeping gene, was chosen as an Further, the upstream pathways responsible for transcriptional and internal standard to control for variability in amplification. Relative quanti- translational regulation of SOCS during experimental VL remain tation was determined by using the comparative ΔΔCt method, and data were an unmet goal and are yet to be elucidated. normalized to GAPDH mRNA levels and expressed as fold change compared with uninfected controls. All samples were run in triplicate. The primer sequences were as follows: Egr2 forward, 59-TGCTAGCCCTTT- Materials and Methods CCGTTGA-39 and reverse, 59-TCTTTTCCGCTGTCCTCGAT-39;AHR Isolation and culture of bone marrow–derived macrophages forward, 59-CGGAGCGCTGCTTCCTCC AC-39 and reverse, 59-GCTGC- CCTTTGGCATCACAACC-39; IDO forward, 59-AGG ATCCTTGAAGA-

Isolation of bone marrow–derived macrophages (BMMvs) was performed CCACCA-39 and reverse, 59-CCAATAGAGAGACGAGGAAG-39. by guest on September 30, 2021 as described earlier, with some modification. Briefly, femoral and tibia bones were aseptically removed from the BALB/c mice of the 6–8 wk age Protein extraction and immunoblotting group. Each bone end was cut off, and bone marrow was flushed out by BMMvs and T cells were harvested and washed with cold 13 PBS. 3 using sterile PBS and then was centrifuged at 150 g for 10 min. These Whole cell lysates from the samples were prepared as described previously cells were resuspended in complete RPMI 1640 with 10% heat-inactivated (22). Cell lysates (30 mg) were further resolved in 10% SDS-PAGE and 3 6 FCS and were layered in six-well plates (0.8–1.5 10 cells per well), then were transferred to a PVDF membrane (Millipore). This was pro- with 10 ng/ml M-CSF and 100 U/ml penicillin and 100 mg/ml strepto- ceeded with blocking of the membrane with 5% BSA (1 h) and then was mycin (Invitrogen) in 5% CO2 atmosphere at 37˚C. The medium was subsequently incubated with primary Ab (overnight) and then secondary changed every 2 d, and cells were cultured for 7 d. Ab (1 h). For detection, bands on the membrane were visualized using Purification of CD4+ T cells SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Sci- entific), and the image was acquired by using a Bio-Rad Laboratories Mice were sacrificed, and their spleens were dissected aseptically and ChemiDoc imager. further processed for the single-cell suspensions. Briefly, spleens were choppedintopieces,minced,andpassedovera40-mm cell strainer Cytokine estimation by ELISA to remove clumps. The cell suspension was further centrifuged at Cytokine levels from harvested supernatants of infected BMMvsand 1200 rpm for 10 min at 4˚C, and the residing erythrocytes were lysed from the T cell cultures with infected BMMvs supernatant were measured using splenocyte using RBC lysis buffer. Thereafter, the cells were centrifuged + selective kits, as per manufacturer’s instructions (BD Biosciences). again and resuspended in ice-cold MACS buffer, and CD4 T cells were Briefly, ELISA plates (Nunc MaxiSorp; Thermo Fisher Scientific) were purified using a CD4+ MACS kit from Miltenyi Biotec (Bergisch Glad- + coated with specific capture Abs at 4˚C, followed by blocking with assay bach, Germany). The splenocyte fraction was incubated with CD4 T cell diluents (PBS containing FBS) and were then incubated with superna- 3 6 beads (1 ml beads per 1 10 cells) at 4˚C for 20 min. These suspended tants for 2 h at room temperature, washed, and incubated with appro- cells were passed from a MACS MS Column and were flushed out by using + + priate biotinylated detection Ab for 2 h, followed by incubation with MACS buffer to obtain CD4 T cells. Isolated CD4 T cells were also streptavidin–HRP. After appropriate washes, the signal was developed by further checked for their purity by flow cytometer (BD FACSAria; BD addition of 3,39,5,59-tetramethylbenzidine (TMB), and OD was mea- Biosciences) and were constantly found to have ∼90% CD4+ T cells. sured at 450 nm after addition of 1 M H3PO4 as stop solution. Parasites, macrophage infection, and culture of T cells with PGE2 was assayed using the Prostaglandin E2 Metabolite EIA Kit, v according to the manufacturer’s instructions (Cayman Chemicals). The infected BMM supernatant cAMP was measured by using a cAMP Assay Kit from Cayman Leishmania donovani strain MHOM/IN/80/Dd8, L. donovani 1S Sudan Chemicals, according to the manufacturer’s protocol. strain, and the isogenic lpg1-KO mutant (21) were maintained as pro- Preparation of nuclear and cytoplasmic extracts mastigotes in M199 medium (Sigma-Aldrich) supplemented with 10% heat-inactivated FBS (Invitrogen) at 24 6 2˚C. L. donovani amastigotes Infected or uninfected BMMvs and cocultured CD4+ T cells were MHOM/IN/80/Dd8 were freshly isolated from the splenocytes of infected taken, washed with PBS (pH 7.4), and homogenized in ice-cold lysis BALB/c mice. BMMvs were infected with L. donovani promastigotes or buffer (10 mM HEPES [pH 7.9], 1.5 mM MgCl2, 10 mM KCl, 0.5 mM freshly isolated amastigotes at a parasite-to-macrophage ratio of 8:1 for phenylmethylsulphonyl fluoride, 0.5 mM DTT, and 1 mg/ml of aprotinin, different time intervals. Before infection, macrophage numbers were Leupeptin, and Pepstatin A), using a Dounce homogenizer. Homogenates The Journal of Immunology 3 were incubated on ice for 45 min, NP-40 was added to a final concentration groups were analyzed for statistical significance by Student t test or one-way of 0.5%, and the mixture was centrifuged at 5000 3 g for 10 min at 4˚C. ANOVA followed by a Tukey posttest using GraphPad Prism software. The supernatant was saved for analysis of cytosolic proteins. The pellets were extracted in nuclear extraction buffer (20 mM HEPES [pH 7.8], Results 400 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 0.5 mM Effect of L. donovani infection on cytokine synthesis and SOCS phenylmethylsulphonyl fluoride, 0.5 mM DTT, and 1 mg/ml of aprotinin, Leupeptin, and Pepstatin A for 30 min on ice and were centrifuged at expression in isolated macrophages and T cells 3 15,000 g for 15 min. Protein concentrations were determined, and The mounting of an effective immune response during pathogenic extracts were stored at 280˚C. challenge requires the coordinated function of both the innate and Small interfering RNA mediated knockdown in macrophages the adaptive arms of the immune system, and cytokines play an and CD4+ T cells essential role in this regard. In VL, it is well documented that disease resolution or progression is ultimately dictated by shifting Small interfering RNA (siRNA) targeted against SOCS1, SOCS3, SRF, Egr2, AHR, SP1, GATA1, CREB, or control siRNA were obtained from Santa Cruz of cytokine synthesis to either Th1 or Th2 mode. Macrophage Biotechnology and were transfected into BMMvsorCD4+ T cells according mediated early induction of IL-12–driven Th1-type immune re- to siRNA transfection protocol. Briefly, 50 nM siRNA were added to mac- sponse by increasing IFN-g synthesis by T cells, and the scenario rophages in siRNA transfection medium along with siRNA transfection re- is vice versa. Thus, inhibition of these cytokines may be one of agent for 5 h. After transfection, macrophages or T cells were incubated for an additional 24 h in antibiotic-free medium. BMMvs were then infected the crucial adaptive strategies that help in the successful prop- with L. donovani for different time periods, and siRNA-treated CD4+ Tcells agation of the Leishmania parasite within the hostile environ- were incubated with infected BMMv-derived supernatants. ment of macrophages. To check these, we first isolated BMMvs

and stimulated them with rIFN-g in the presence or absence of Downloaded from Phagocytosis assay L. donovani promastigotes for 24 and 48 h, respectively, and BMMvs were transfected (24 h) with either control, SOCS1, SOCS3, or checked for IL-12 synthesis. We observed that L. donovani dras- both siRNA, followed by incubation with GFP–L. donovani promastigotes tically downregulate IL-12 production in BMMvs, even in pres- at a ratio of 1:10 (1 macrophage to 10 parasites) at 37˚C, 5% CO2 for 4 h. Cells were then washed with ice-cold PBS for at least five times and ence of IFN-g stimulus at both time points (Fig. 1A). Further, to immediately resuspended in ice-cold MACS buffer and analyzed by flow check the effect of L. donovani on IFN-g secretion by T cells, we + cytometry (FACSCalibur; BD Biosciences). isolated and stimulated splenic CD4 T cells by rIL-12 in the http://www.jimmunol.org/ Chromatin immunoprecipitation assay presence or absence of supernatants derived from L. donovani– infected BMMvs for 24 and 48 h, respectively. This is because Chromatin immunoprecipitation (ChIP) assay was performed using the L. donovani could not infect T cells but secreted a wide array of SimpleChIP Enzymatic Chromatin IP Kit from Cell Signaling Technology as virulence factors and anti-inflammatory mediators from infected per the manufacturer’s guidelines. Briefly, uninfected and infected BMMvs (4 3 106 cells) or CD4+ T cells (treated with infected BMMv-derived su- macrophages that inhibit T cell–mediated immunity. We observed pernatants) were cross-linked with 1% formaldehyde for 10 min at 37˚C, that supernatants derived from infected BMMvs downregulate followed by addition of glycine solution for 5 min at room temperature. The IFN-g production in CD4+ T cells in presence of IL-12 cytokine cells were further processed as described previously (22). PCR was carried stimulus (Fig. 1B). These clearly suggested that L. donovani

out with denaturation at 94˚C for 30 s, optimized annealing temperature by guest on September 30, 2021 (Egr2, 50˚C; AHR, 55.5˚C; SRF 52˚C; SP1, 60˚C; GATA1. 51˚C; CREB, impaired IL-12– and IFN-g–driven macrophage–T cell cross- 53.3˚C) and extension at 72˚C for 30 s for 40 cycles followed by 10 min at talk, which is important for host defensive Th1 kind of immu- 72˚C using the following primers designed to amplify the specific regions nity. Most cytokines use the so-called JAK/STAT pathways for involving the putative transcription factor binding sites: SP1 forward, 59- immune-functioning, and SOCS proteins are one of the crucial GTTCCAGGAATCGGGGGGCGGG-39 and reverse, 59-CCCCCTCTGGC- negative regulators of these pathways and cytokine-mediated TTCGCTGCTCC-39;CREBforward,59-GCACAGCCTTTCAGTGCA-39 and reverse, 59-GAGACAGCGGTCGTAAGAGCA-39; GATA1 forward, 59- immune homeostasis (23). Thus, we hypothesized that SOCS GCTTTGTCTCCCTCTCGGTGAGT-39 and reverse, 59-AGTGTAGAGTC- proteins might be exploited by L. donovani for its survival in the AGAGTTAGAGCC-39; SRF forward, 59-GTCCACCGGCTGCAAATC-39 host. To confirm this, we first carried out expression profiling of all and reverse, 59-GCAGGTCTTCAATACCCGTGA-39;Egr2forward,59- SOCS isoforms at different time interval in BMMvs following CTTCAAAGGAAGCCTAA GGCG-39 and reverse, 59-CCACGTAGTAA- + GAGTGCAGAG-39; AHR forward, 59-AGCAGAGAGAACTGCGGCC-39 L. donovani infection and in splenic CD4 T cells incubated with and reverse, 59-GTGGCATCCTCGACCCTGC-39. the supernatant derived from L. donovani–infected or noninfected macrophage. Real-time PCR analysis suggested that L. donovani Vivo morpholino–mediated knockdown of SOCS1 and SOCS3 infection-induced SOCS1 and SOCS3 expression in BMMvs, in BALB/c mice whereas specific induction of SOC3 was observed in CD4+ T cells Vivo morpholino oligonucleotides for SOCS1 59-CCAACAGACCCCA- incubated with the supernatant derived from L. donovani–infected AGGAGCG AGAGC-39,SOCS359-AAACTTGCTGTGGGTGACCATG-39 macrophages (Table I). In infected BMMvs, maximum induc- and control morpholino 59-CCTCTTACCTCAGTTACAATTTATA-39 were tion of SOCS1 was observed at 24 h (8.3-fold as compared with purchased from Gene Tools. Vivo morpholinos were resuspended in sterile PBS at a concentration of 0.5 mM and, thereafter, injected into the BALB/c control cells), whereas maximum induction of SOCS3 was ob- mice (n = 5) via the tail vein route. Respective morpholinos were injected served at 12 h (5.8-fold as compared with control cells, Fig. 1C). twice a week at a dose of 10 nmol (5 mg/kg). Dosing of morpholinos was Similar induction of SOCS1 and SOCS3 expression was also started 1 wk prior to the infection and was continued up to the sixth week observed in protein levels (Fig. 1D) as studied by immunoblot postinfection (as mentioned in the diagram, Fig. 7A). Knockdown efficiency was determined at the second, fourth, and sixth weeks postinfection in the analysis. Interestingly, although the induction of SOCS1 was whole cell lysate of the splenocytes of control, SOCS1 or SOCS3, and found to be stable up to 36 h in infected macrophages, SOCS3 SOCS1 and SOCS3 morpholino-treated animals via immunoblotting. expression was significantly reduced in both mRNA and protein Splenic and liver parasite loads were measured each week and were repre- after 12 h of infection in infected BMMvs. sented as Leishman Donovan Units. Cytokines were measured at the second, In contrast, expression of SOCS3 was more pronounced as fourth, and sixth weeks postinfection in splenocytes obtained from various experimental groups of BALB/c mice (as mentioned in the Fig. 7A). compared with other SOCS isoforms (10.2-fold at 24 h as compared with uninfected control) in splenic CD4+ T cells incubated Statistical analysis with the supernatant derived from infected macrophages. In fact, + Data are represented as the mean 6 SD of individual experiments (n =3), the expression level of SOCS3 was much higher in CD4 T cells which were performed in replicate, and differences between the compared than what we observed in infected macrophages and was stable up 4 SOCS1 AND SOCS3 BREACH MACROPHAGE–T CELL CROSS-TALK IN VL Downloaded from http://www.jimmunol.org/

FIGURE 1. Effect of L. donovani infection on the modulation of proinflammatory cytokine and SOCS1/SOCS3 expression. (A) BMMvs were prepared from the femurs and tibias of BALB/c mice and stimulated with rIFN-g (2 ng/ml) either in presence or absence of L. donovani promastigote for 24 and 48 h. IL-12 level in macrophage supernatants was measured using a sandwich ELISA kit. (B) CD4+ T cells were isolated from splenocytes and stimulated by (4 ng/ml) IL-12, respectively, either in presence or absence of L. donovani–infected BMMv–derived supernatants for 24 and 48 h. IFN-g level in T cells mixture was measured using a sandwich ELISA kit. (C) Real-time PCR was performed to check SOCS1 and SOCS3 expression in L. donovani–infected BMMvs. The expression changes were calculated relative to uninfected control macrophages after normalizing with GAPDH as endogenous control. (D) BMMvs were infected with stationary-phase L. donovani promastigotes for indicated time intervals, and expression of SOCS1 and SOCS3 was analyzed by Western blotting. (E) Real-time PCR was performed to check SOCS3 expression in T cells incubated with L. donovani–infected macrophage by guest on September 30, 2021 supernatant, and the expression changes were calculated relative to control after normalizing with GAPDH as endogenous control. (F) T cells were incubated with L. donovani–infected macrophage supernatant, and expression of SOCS3 was analyzed by Western blotting. Further equal protein loading was ensured by b-actin. (G) BMMvs were transfected with scrambled control siRNA or SOCS1 or SOCS3 or SOCS1 and SOCS3 both siRNA for 24 h, followed by treatment as mentioned in (A). Concentrations of IL-12 were measured by ELISA in the cell-free culture supernatants. (H) T cells were transfected with SOCS3 or scrambled control siRNA for 24 h, followed by treatment as mentioned in (B). Concentrations of IFN-g were measured by ELISA. (I) BMMvs were transfected with SOCS1 or SOCS3 or SOCS1 and SOCS3 both or scrambled control siRNA for 24 h, followed by infection with L. donovani promastigotes for 48 h. Numbers of surviving intramacrophagic amastigotes were counted by Giemsa staining. (J) BMMvs were transfected with SOCS1 or scrambled siRNA followed by stimulation with (2 ng/ml) rIFN-g, either in presence or absence of L. donovani promastigote for 8 h. Levels of total and phosphorylated STAT1 were measured by immunoblotting. (K) Splenic T cells were transfected with SOCS3 or scrambled control siRNA followed by stimulation with rIL-12 (4 ng/ml), either in presence or absence of L. donovani-infected macrophage- derived mixture for 8 h. Levels of total and phosphorylated STAT4 were measured by immunoblotting. (L) BMMvs were transfected with SOCS1 or scrambled siRNA followed by stimulation with (2 ng/ml) rIFN-g, either in presence or absence of L. donovani promastigote for 8 h. Nuclear extracts were prepared and translocation of STAT1 was analyzed by immunoblotting. Histone H3 was used as an endogenous control for nuclear protein. (M) Splenic T cells were transfected with SOCS3 or scrambled control siRNA, followed by stimulation with rIL-12 (4 ng/ml), either in presence or absence of supernatant derived from L. donovani-infected macrophage for 8 h. Nuclear extracts were prepared, and translocation of STAT4 was analyzed by immunoblotting. Significance was determined for infected versus uninfected groups. *p , 0.05, **p , 0.005, ***p , 0.0003. to 36 h as determined by both real-time (Fig. 1E) PCR and im- and efficacy of SOCS1 and SOCS3 siRNA were determined by munoblot (Fig. 1F) analysis. Because the amastigote stage of the immunoblotting at 24 h posttransfection. Expressions of both Leishmania parasites is the disease-causing intracellular form, SOCS1 and SOCS3 in macrophages (Supplemental Fig. 1B), as which can reside and multiply for prolonged duration in phago- well as SOCS3 in T cells (Supplemental Fig. 1C), were consider- cytic cells, we also determined the expression of SOCS1 in ably reduced by treatment with respective siRNAs as compared BMMvs infected with L. donovani amastigotes. We observed with control siRNA treatment. Further, to check whether siRNA that infection of BMMvs with freshly isolated amastigotes also treatment (SOCS1, SOCS3, or both) could affect the phagocyto- resulted in an upregulation of SOCS1 at the protein level in a sis of the Leishmania parasite, we determined the uptake of time-dependent fashion. However, unlike promastigotes, the in- GFP-transfected promastigotes in siRNA-treated BMMvsbyflow duction of SOCS1 by amastigotes was less pronounced and was cytometry. We did not observe any remarkable difference in intra- observed at later time points of infection (Supplemental Fig. 1A). cellular parasite uptake by control or siRNA-treated macrophages We next studied the effect of SOCS1/SOCS3 silencing on the (Supplemental Fig. 1D), which suggested that siRNA treatment modulation of IL-12 synthesis in BMMvs stimulated with rIFN-g in could not affect the phagocytosis of BMMvs. Knockdown of the presence or absence of L. donovani infection. The specificity SOCS1 in infected BMMvs led to a marked induction in the level The Journal of Immunology 5

of IL-12 (1588 pg/ml in L. donovani plus IFN-g plus SOCS1 siRNA-treated cells as compared with 710 pg/ml in L. donovani 0.4 0.5 0.5 0.4 0.4 0.7 0.3 0.5 plus IFN-g plus control siRNA-treated cells) (Fig. 1G), whereas 6 6 6 6 6 6 6 6 36h

Materials and SOCS3 knockdown could not signiﬁcantly change the IL-12 level in infected BMMvs. Because the SOCS3 level was markedly induced in T cells incubated with the supernatants derived from infected macrophages, we thought it would be worthwhile to check its role in inhibiting IL-12–mediated IFN-g + 0.4 9.2 0.4 1.5 0.6 1.6 0.6 1.4 0.4 1.4 0.7 1.8 0.5 1.2 0.4 1.5 synthesis in CD4 T cells. We observed marked induction in the 6 6 6 6 6 6 6 6 +

24h level of IFN-g by silencing SOCS3 in splenic CD4 T cells T cell 1.6 1.5 1.6 1.3 which underwent incubation with supernatants derived from infected macrophages plus IL-12 (2153 pg/ml in L. donovani plus IL-12 plus SOCS1 siRNA-treated cells as compared with 954 pg/ml in L. donovani plus IL-12 plus control siRNA-treated cells) (Fig. 1H). This was further reﬂected in in vitro para- 0.4 10.8 0.7 0.4 1.7 0.4 0.4 1.9 0.4 0.4 1.6 0.5 sitemia, as amastigote multiplication was signiﬁcantly attenu- 6 6 6 6 6 6 6 6 12h ated in infected BMMvs that underwent SOCS1 and SOCS3 knockdown and is more pronounced in infected macrophages

that underwent knockdown of both SOCS1 and SOCS3 iso- Downloaded from forms (Fig. 1I). We next, therefore, studied the outcome of silencing of SOCS1 and SOCS3 in the modulation of upstream 0.3 7.9 0.5 1.8 0.3 1.5 0.5 1.4 0.5 1.6 0.5 1.5 0.5 1.3 0.4 1.2 JAK/STAT signaling (i.e., STAT1 and STAT4 phosphorylation 6 6 6 6 6 6 6 6

36h that is crucial for IL-12 and IFN-g synthesis). For this, we –infected macrophage supernatant, which was carried out as described in checked the phosphorylation of STAT1 and STAT4 in SOCS1/

SOCS3 siRNA-treated infected macrophages as well as in http://www.jimmunol.org/ T cells incubated with L. donovani–infected macrophage- L. donovani derived supernatant. siRNA-mediated knockdown of SOCS1

SD. in L. donovani–infected BMMvs restored STAT1 phosphory- 0.7 1.8 0.3 1.6 0.3 1.2 0.7 1.6 0.8 7.1 0.3 1.5 0.3 1.6 0.4 1.3 6 6 6 6 6 6 6 6 6 lation, whereas knockdown of SOCS3 in T cells restored STAT4 24h phosphorylation (Fig. 1J, 1K). This was also reﬂected in the Macrophage nuclear translocation of both STAT1 and STAT4. We observed reduced nuclear translocation of STAT1 in L. donovani plus IFN-g–treated macrophages and also reduced nuclear translocation of STAT4 in the nucleus of T cells incubated with parasite- by guest on September 30, 2021 s and T cells incubated with 0.4 2.6 0.6 1.9 0.4 1.1 0.4 1.8 0.7 8.5 0.4 1.6 0.6 1.9 0.6 1.5 v infected macrophage-derived supernatant, respectively, which 6 6 6 6 6 6 6 6 12h was restored after siRNA-mediated knockdown of SOCS1 and 6.1 1.7 1.3 1.5 6.5 1.4 1.7 1.4 SOCS3 in BMMvs and T cells, respectively, under similar conditions (Fig. 1L, 1M). Collectively, these results suggested

–infected BMM that L. donovani preferentially upregulated SOCS1 and SOCS3 in BMMvs and T cells, respectively, to inhibit STAT1/STAT4 phosphorylation and impair IL-12 and IFN-g synthesis. L. donovani and and and and and and and and 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 Transcriptional regulation of SOCS1 in 9 L. donovani–infected macrophages EnhancedSOCS1expressioninL. donovani–infected BMMvs could be the result of altered SOCS1 transcriptional regulation in these cells. To check this, we took the help of existing reports that suggested the possible involvement of Egr2, AHR, and IRF1 transcription factors in SOCS1 induction (24–26). We ﬁrst checked their binding to the endogenous SOCS1 promoter by Primer Sequence ChIP analysis. L. donovani infection in BMMvs increases the association of Egr2 and AHR with the SOCS1 promoter in a GATAGGACGAGTTCCCCACA-3 -CAAGACCTTCAGCTCCAAAA-3 -GGAAGGGCTCCTTCAAAATC-3 -TGTGCAAGGATAAACGGACA-3 -GGCACTGCGTCTACAGATGA-3 -AGACCTTCGACTGCCTTTTC-3 -CCTTACAGCTGGGACTGAGC-3 -CCCAGAGGAAGTGACAGAGG-3 -CCCCAGTGCCTGTATGTTCT-3 -TTTCATAGGCGAAGGAATGG-3 -GACGCTCAACGTGAAGAAGT-3 -TTGTCAACCTGGGTTTCCTC-3 -TCACTCGCATCTCCATCTTG-3 -ACGGAGTACCGGGTTAAGAG-3 -GGGTCTTTGTGGAACAGGAA-3 -GGGTGCTGTCTCGAACTAGG-3 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 time-dependent fashion (Fig. 2A, 2B), in which optimal re- 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 cruitment was observed between 8 and 16 h postinfection. On the

erse, contrary, we did not observe any detectable binding of tran- reverse, reverse, reverse, reverse, reverse, reverse, reverse, rev scription factor IRF1 on SOCS1 promoter after L. donovani in- Forward, Forward, Forward, Forward, fection (data not shown). Chromatin fractions from uninfected macrophages exhibited no binding of Egr2 and AHR to the SOCS1 promoter, suggesting that the basal expression level of SOCS1 is not modulated by these transcription factors. The result was additionally validated by PCR analysis of the positive con-

. Assays were performed at least three times in replicate, andtrol values are the mean fold changes relative(input), to uninfected controls which indicated that the soluble chromatin samples SOCS4 Forward, SOCS3 Forward, SOCS7 SOCS2 Forward, SOCS6 SOCS1 Forward, SOCS5 Gene Name CIS

Real-time PCR expression proﬁling of SOCS isoforms at various time points in obtained from each time point had equal amounts of chroma- Methods Table I. Expression proﬁling of different SOCS proteins in macrophage and T cell at different time points tin fragments containing the SOCS1 promoter. To further gain 6 SOCS1 AND SOCS3 BREACH MACROPHAGE–T CELL CROSS-TALK IN VL

FIGURE 2. Egr2 and AHR-mediated induction of SOCS1 in L. donovani–infected BMMvs. (A and B) ChIP assays were performed to observe the binding of Egr2 and AHR to the SOCS1 promoter in L. donovani–infected and control macrophages. BMMvs were left uninfected or infected with stationary-phase L. donovani promastigotes for respective time periods. Chromatin was prepared, and ChIP analysis was performed as described in the Materials and Methods section. PCR was carried out using primers speciﬁc to SOCS1 promoter region after immunoprecipitation with anti-Egr2, anti-AHR, or control IgG Ab. (C and D) BMMvs were infected with L. donovani promas-

tigotes for respective time periods, and expression Downloaded from of Egr2 and AHR was analyzed (C) by real-time PCR and (D) by immunoblotting. (E and F) Mac- rophages were infected with L. donovani promastigotes for respective time periods, nuclear and cytosolic extract were prepared, and translocation of Egr2 and AHR was analyzed by immunoblot-

ting. Histone H3 and GAPDH were used as an http://www.jimmunol.org/ endogenous control for nuclear and cytosolic proteins. (G) Macrophages were transfected (24 h) with either control, Egr2, AHR, or both siRNA followed by infection with L. donovani promastigotes for 24 h. The expression of SOCS1 was evaluated by immunoblot analysis. Signiﬁcance was determined for infected versus uninfected group. **p , 0.005, ***p = 0.0009. by guest on September 30, 2021

insight into the transcriptional induction of SOCS1 by Egr2 and in infected cells treated with Egr2 plus AHR siRNA (Fig. 2G). AHR, we next checked the relative expression of Egr2 and AHR Collectively, these data suggested that Egr2 and AHR is important in infected BMMvs by quantitative real-time PCR and immuno- for SOCS1 induction in L. donovani–infected macrophages. blot analysis. Egr2 mRNA expression was increased in infected macrophages in a time-dependent fashion (3.8-, 6.9-, and 4.4-fold Signaling events behind Egr2 and AHR induction in increase at 4, 8, and 16 h postinfection as compared with control L. donovani–infected macrophages macrophages), whereas the mRNA level of AHR remained the Because Egr2 and AHR are important for upregulating SOCS1 in same in infected macrophages as compared with control macro- L. donovani–infected macrophages, we first sought to identify the phages (Fig. 2C). A similar trend of increased expression for Egr2 transcriptional events behind the upregulation of Egr2 in infected was also observed at protein levels in infected BMMvs, whereas macrophages. To this end, we first checked whether L. donovani AHR expression remains unchanged (Fig. 2D). Activation of these exploits SRF to increase Egr2 expression in macrophages, as SRF transcription factors was also assessed by studying their cytosol- is a well-known transcriptional regulator of Egr2 (27, 28). For this, ic and nuclear distribution in BMMvs at 4–16 h postinfec- phosphorylation-mediated activation of SRF was observed in in- tion (Fig. 2E, 2F). Immunoblot analysis showed that leishmanial fected-BMMvs, and we found that L. donovani increases phos- infection-induced nuclear translocation of both Egr2 and AHR in phorylation of SRF in infected macrophages in a time-dependent BMMvs, which is evident from the gradual increase of both these fashion (Fig. 3A). Activation of SRF was also assessed by mon- proteins in the nuclear fraction along with a subsequent decrease itoring its subcellular distribution in infected macrophages, and in the cytosolic fraction at 12 and 16 h, respectively (Fig. 2E, 2F). we observed increased translocation of SRF to the nucleus To further validate the functional role of Egr2 and AHR in reg- throughout the time course of infection (being maximum at 12 and ulating SOCS1 expression in L. donovani–infected macrophages, 16 h postinfection) with a concomitant decrease of expression in we used a siRNA-mediated knockdown approach. Efficacy of both cytosolic extract (Fig. 3B, 3C). Transcriptional activation of Egr2 Egr2 and AHR siRNA knockdown was also determined by im- by SRF was also assessed in L. donovani–infected BMMvsby munoblot analysis (Supplemental Fig. 1B). Knockdown of Egr2 ChIP analysis. Chromatin fractions from infected macrophages and AHR markedly reduced SOCS1 expression in L. donovani– exhibited increased association of SRF to the Egr2 promoter, infected BMMvs, in which the maximum decrease was observed suggesting that L. donovani infection induces the expression of The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 3. Upstream pathways involved in Egr2 and AHR induction in L. donovani–infected BMMvs. (A) BMMvs were infected with L. donovani promastigotes for respective time periods, and phosphorylation of SRF was analyzed by immunoblotting. (B and C) BMMvs were infected with L. donovani promastigotes for the respective time periods, nuclear and cytosolic extract were prepared, and translocation of SRF was analyzed by immunoblotting. Histone H3 and GAPDH were used as endogenous control for nuclear and cytosolic fraction, respectively. (D) ChIP assays were performed to observe the binding of SRF to the Egr2 promoter in L. donovani–infected and uninfected macrophages. Macrophages were left untreated or infected with L. donovani for the respective time periods. Chromatin was prepared, and ChIP analysis was performed as described in Materials and Methods section. PCR was carried out using primers speciﬁc to Egr2 promoter region after immunoprecipitation with anti-SRF or control IgG Ab. (E) BMMvs were

transfected (24 h) with either control or SRF siRNA followed by infection with L. donovani promastigotes for 12 h. Expression of Egr2 was evaluated by by guest on September 30, 2021 immunoblot analysis. (F) BMMvs were infected with L. donovani for respective time periods, and phosphorylation of Akt was analyzed by immunoblotting. (G–I) Macrophages were treated with PI3K/Akt pathway inhibitor wortmannin (1 mM) or LY294002 (10 mM) for 1 h followed by infection with L. donovani promastigotes for 16 h. Cytosolic and nuclear extracts were prepared, and nuclear translocation of SRF, Egr2, AHR (G and H) and expression of SOCS1 (I) was evaluated by immunoblot analysis. (J) BMMvs were infected with L. donovani promastigotes for 4–24 h. The level of kynurenine in the supernatant of infected macrophages was analyzed at respective time periods. (K and L) Expression of IDO at mRNA and protein level in infected macrophages at different time points were analyzed by real-time PCR and immunoblot analysis. (M–O) Macrophages were transfected (24 h) with either control or IDO siRNA followed by infection with L. donovani promastigotes. (M and N) Cytosolic and nuclear distribution of AHR at 16 h postinfection and (O) expression of SOCS1 at 24 h postinfection were evaluated by immunoblot analysis. Signiﬁcance was determined for infected versus uninfected groups. **p , 0.005, ***p , 0.0001. ns, not signiﬁcant.

Egr2 by SRF, as we did not observe any association in chro- Time-dependent phosphorylation and activation of Akt were ob- matin fraction of uninfected macrophages (Fig. 3D). PCR served in L. donovani–infected BMMvs (Fig. 3F), and to check it analysis of the positive control (input) indicated that the soluble after effect, BMMvs were treated with a selective PI3K/Akt in- chromatin samples obtained from each time point had equal hibitor (i.e., wortmannin and LY294002) prior to L. donovani amounts of chromatin fragments containing the Egr2 promoter infection, and the nuclear translocation of SRF, Egr2, and AHR (Fig. 3D). Moreover, siRNA-mediated knockdown of SRF mark- were assessed. In a similar experimental system, we observed edly reduced Egr2 expression in L. donovani–infected BMMvs SOCS1 induction at 24 h postinfection. We observed that blockade (Fig. 3E). Efficacy of siRNA knockdown of SRF was evaluated by of PI3K/Akt pathway by a pharmacologic inhibitor markedly at- immunoblot analysis (Supplemental Fig. 1B). This suggests that tenuated L. donovani–induced nuclear translocation of SRF and SOCS1 expression in L. donovani–infected macrophages is me- Egr2 as well as SOCS1 expression, whereas nuclear translocation diated through SRF/Egr2–mediated axis. of AHR was not affected (Fig. 3G–I). These results suggest that Earlier reports suggested that G-CSF induced Egr1 expression in L. donovani induces SOCS1 expression in BMMvs through the myeloid leukemia cells through the interaction of SRF and Fli-1 PI3K/Akt/SRF/Egr2 axis, but this pathway does not directly reg- with the SRE-1 sequence through upstream activation of PI3K- ulate AHR-mediated SOCS1 induction. dependent pathway (29). Moreover, the tumor-promoting agent, AHR, which is activated by a range of ligands of environmental TPA, also activates SRF through PI3K-dependent and T cell– or dietary origin, has emerged recently as an important regulator of specific factor–independent pathway [58]. Because Leishmania innate immunity (25). The tryptophan metabolite kynurenine ac- also activates PI3K/Akt signaling in infected macrophages tivates the AHR and thus stimulates its translocation to nucleus. (30, 31) to promote anti-inflammatory responses, we next checked Thus, to check the possible involvement of this pathway in AHR whether PI3K/Akt activation regulates SRF-, Egr2-, and AHR- induction, we first performed a time course experiment to mea- mediated upregulation of SOCS1 in L. donovani–infected BMMvs. sure the level of kynurenine synthesis in L. donovani–infected 8 SOCS1 AND SOCS3 BREACH MACROPHAGE–T CELL CROSS-TALK IN VL

BMMvs. L. donovani infection in BMMvs increased the in- involvement in SOCS1 expression in BMMvs. Earlier it has been tracellular concentration of kynurenine, which started at 8 h reported that L. major LPG could induce both SOCS1 and SOCS3 postinfection, peaking at 12 h (17.2 nM) and elevated up to 24 h, in murine macrophages as a negative regulator of cytokine sig- as observed (Fig. 3J). Kynurenine is the first tryptophan me- naling (34). To check the possible involvement LPG, we first in- tabolite of the IDO pathway, so we checked expression of the fected the BMMvs with either L. donovani Dd8, L. donovani 1S, IDO gene in L. donovani–infected macrophages by real-time or isogenic LPG-defective L. donovani 1S lpg1-KO promastigotes PCR and immunoblot analysis and observed time-dependent for 24 h and determined the expression of SOCS1 at the protein induction of IDO at both the mRNA and protein level in level. The induction of SOCS1 was found to be much higher in BMMvs, being maximum at 8 h (Fig. 3K, 3L). siRNA-mediated BMMvs infected with wild-type (WT) parasites but less pro- knockdown of IDO in L. donovani–infected macrophages, mark- nounced in the BMMvs infected with the lpg1-KO mutant. edly reduced AHR translocation to the nucleus, as AHR expres- However, expression of SOCS1 in lpg1-KO–infected macrophages sion was retained in the cytosol but undetectable in the nucleus of was not completely abrogated and remained higher than that of IDO siRNA-treated infected macrophages (Fig. 3M, 3N). The the basal expression level observed in control macrophages, sug- efficacy of siRNA knockdown of IDO was determined by im- gesting that the virulence factor LPG plays a partial role in the munoblot analysis (Supplemental Fig. 1B). Similarly, silencing of induction of SOCS1 (Fig. 4A). To gain mechanistic insight, we IDO markedly abolished the expression of SOCS1 in L. donovani– next checked whether transcriptional activation of SOCS1 by Egr2 infected macrophages (Fig. 3O). Collectively these data suggested or AHR is under the control of LPG and checked the nuclear that L. donovani also exploits the IDO/kynurenine pathway for translocation of both these transcription factors in BMMvs in- transcriptional activation of AHR that, in turn, induces the ex- fected with either WT or lpg1-KO L. donovani 1S parasites. In the Downloaded from pression of SOCS1 in infected macrophages. absence of LPG, L. donovani promastigotes failed to induce the nuclear translocation of Egr2, as observed by reduced nuclear Role of lipophosphoglycan and gp63 from L. donovani in the accumulation and increased cytosolic abundance of Egr2 (Fig. 4B, SOCS1 induction 4C). On the contrary, cytosolic-to-nuclear translocation of tran- Subversion of immune cell signaling by using pathogen-derived scription factor AHR was unperturbed in BMMvs infected with

virulence factor is a major adoptive strategy exploited by intra- either WT parasites (LD1S) or lpg1-KO L. donovani parasite, http://www.jimmunol.org/ cellular pathogens, and Leishmania can use surface-expressed thereby suggesting that transcriptional control of SOCS1 is partially lipophosphoglycan (LPG) and secreted metalloprotease gp63 to modulated by LPG (Fig. 4B, 4C). achieve this. In fact, phagocytic dysfunction is induced by both We next evaluated the functional signiﬁcance of L. donovani LPG and gp63, as they have been shown to inactivate macrophage gp63 (a Zn2+-dependent metalloprotease) for SOCS1 induction in microbicidal function and thwart host defense, thereby allowing BMMv, and for this, we took dual approaches. First, L. donovani parasite survival and disease progression (32, 33). To this end, we promastigotes were incubated with 1,10-Phenanthroline (a Zn2+ checked for the possible involvement of Leishmania LPG or gp63, chelator and an inhibitor of Zn2+ metalloproteases gp63) (35, 36) the two predominant and abundant virulence factors, for their or vehicle (dH2O) for 12 h, and then BMMv were infected with by guest on September 30, 2021

FIGURE 4. Effect of L. donovani LPG and gp63 on the induction of SOCS1. (A) BMMvs were infected with either WT or lpg1-KO L. donovani (1S strain) or L. donovani (Dd8) promastigotes for 24 h, and expression of SOCS1 was analyzed by immunoblotting. (B and C) Macrophages were infected with either WT or lpg1-KO L. donovani (1S strain) or L. donovani (Dd8) promastigotes for 12 h. (B and C) Nuclear and cytosolic extract were prepared, and translocation of Egr2 and AHR was analyzed by immunoblotting. Histone H3 and GAPDH were used as an endogenous control for nuclear and cytosolic proteins. (D) L. donovani promastigotes were incubated with 1,10-Phenanthroline (a Zn2+ chelator and an inhibitor of Zn2+ metalloproteases gp63) (25 mM) or vehicle (dH2O) for 12 h, then BMMvs were infected with these parasites for 24 h to check SOCS1 induction by Western blotting. (E) L. donovani promastigotes were preincubated with a rabbit polyclonal Ab raised against L. donovani gp63 (5 mg/ml) or normal rabbit IgG for 1 h, then BMMv were infected with these parasites for 24 h to check the expression of SOCS1. (F) BMMvs were infected with either stationary-phase L. donovani promastigotes (1:8), or with heat-killed L. donovani (1:8), latex beads (0.005%) for 24 h, and expression of SOCS1 was analyzed by Western blotting. The Journal of Immunology 9

L. donovani (Dd8) promastigotes to check SOCS1 induction. However, no detectable binding of AP1 to the SOCS3 promoter Second, promastigotes were pretreated with a rabbit polyclonal was observed in CD4+ T cells cultured with the supernatant de- Ab raised against L. donovani gp63 (37) or control rabbit IgG rived from L. donovani–infected BMMvs (data not shown). Re- for1h,andthenBMMv were infected with these parasites to placement of SP1, GATA1, and CREB Ab with control IgG in the check SOCS1 induction. Our results showed that preblocking ChIP assay failed to yield any amplicon, suggesting the specific- of leishmanial gp63 had no role in SOCS1 induction, as 1,10- ity of our assay. Transcriptional activation of SP1, GATA1, and Phenanthroline or anti-gp63 Ab failed to inhibit L. donovani– CREB was also assessed by checking their subcellular distribution induced SOCS1 upregulation in infected macrophages (Fig. 4D, (cytosolic and nuclear) at the protein level in CD4+ T cells in- 4E). To further check whether SOCS1 induction is dependent on cubated with L. donovani–infected macrophage-derived superna- the phagocytosis of L. donovani or if it requires the viable and tant (Fig. 5D, 5E). Immunoblot analysis revealed that coculture of active parasites, we checked the expression level of SOCS1 in T cells with infected macrophage-derived supernatant markedly BMMvs incubated with heat-killed parasites or an inert particle induced nuclear translocation of SP1, GATA1, and CREB, which (i.e., latex bead). Phagocytosis of latex beads did not induce ex- is evident from the gradual increase of these transcription factors pression of SOCS1, whereas less pronounced but significant in- at nuclear fraction along with subsequent decrease in cytosolic duction of SOCS1 was observed in heat-killed parasite-treated fraction (being prominent at 12 and 18 h). The functional role BMMvs as compared with control macrophages (Fig. 4F). This of SP1, GATA1, and CREB in regulating SOCS3 expression was may be due to the fact that LPG is intact in the heat-killed parasite also validated by siRNA-mediated knockdown of all these and can partially induce SOCS1 expression in BMMvs. Collec- transcription factors in splenic CD4+ T cells prior to incubation tively, our results suggested that LPG from L. donovani partially with L. donovani–infected macrophage-derived supernatant. Downloaded from regulates SOCS1 induction in infected BMMvs by transcriptional The efficacy and specificity of siRNA-mediated knockdown were activation of EGR2. also assessed by immunoblotting (Supplemental Fig. 1C). Expres- sions of SP1, GATA1, and CREB were significantly reduced by Signaling events involved behind SOCS3 induction in splenic respective siRNAs as compared with control siRNA treatment. T cells during infection Knockdown of SP1, GATA1, and CREB markedly reduced SOCS3

The enhanced SOCS3 expression in T cells incubated with expression in these T cells (Fig. 5F) during coculture experiments. http://www.jimmunol.org/ L. donovani–infected BMMv-derived supernatant could be the These data suggested that SP1, GATA1, and CREB are impor- result of an altered SOCS3 transcriptional regulation in these cells. tant for inducing SOCS3 expression in splenic T cells during Earlier studies suggested that various transcription factors, such as L. donovani infection. SP1, GATA1, AP1, and CREB could induce the transcription of SOCS3 in immune cells (38–41). We therefore checked for the PGE2/cAMP/PKA pathway is involved behind the activation of binding of these transcription factors to the SOCS3 promoter by SP1-, GATA1-, and CREB-mediated SOCS3 induction in ChIP analysis in CD4+ T cells cultured in the presence of super- T cells during infection natant derived from L. donovani–infected BMMvs. We observed Previous studies have reported that L. donovani–infected mac- significant enrichment of SP1, GATA1, and CREB to the SOCS3 rophages produce cyclooxygenase PGE2, which influences im- by guest on September 30, 2021 promoter region (Fig. 5A–C), which is increased in a time- mune cell function (42, 43), and PGE2 inhibits the synthesis dependent manner and maximum at 12 and 18 h, respectively. of inflammatory cytokines/chemokines and IL-17 in VL (44).

FIGURE 5. CREB, GATA1, and SP1 mediated induction of SOCS3 in T cells during infection. (A–C) ChIP assays were performed to observe the binding of CREB, GATA1, and SP1 to SOCS3 promoter. CD4+ T cells were left untreated or incubated with L. donovani–infected macrophage-derived supernatant for 3–18 h. Chromatin was prepared, and ChIP analysis was performed as described in the Materials and Methods section. PCR was carried out using primers speciﬁc to SOCS3 promoter region after immunoprecipitation with anti-CREB, anti-SP1, and anti-GATA1 or control IgG Ab. (D and E) T cells were left untreated or incubated with L. donovani–infected macrophage-derived supernatant for respective time periods. The nuclear and cytosolic extracts were prepared, and translocation of CREB, GATA1, and SP1 were analyzed by immunoblotting. (F) T cells were transfected with control, CREB, GATA1, or SP1 siRNA for 24 h followed by incubation with L. donovani– infected macrophage-derived supernatant for 24 h. The expression of SOCS3 was evaluated by immunoblot analysis. 10 SOCS1 AND SOCS3 BREACH MACROPHAGE–T CELL CROSS-TALK IN VL

However, the role of secreted PGE2 on modulating T cell sig- (AH6809) and 100 nM EP4 antagonist (GW627368) and cocul- naling during VL is unknown, despite T cells having functional tured with L. donovani–infected macrophage-derived supernatant receptors for PGE2 (i.e., EP1, EP2, EP3, and EP4). We observed to check the expression of SOCS3. A significant decrease in that L. donovani promastigote triggers a significant and time- SOCS3 expression was observed in AH6809- and GW627368- dependent increase in PGE2 synthesis in BMMvs, with a treated T cells and in NS-398–treated infected BMMv-derived maximum level (.2300 pg/ml) observed at 24 h postinfection supernatant-incubated T cells as indicated by immunoblot analysis (Fig. 6A). PGE2 is recognized to transduce cellular effects on (Fig. 6B). These observations further confirmed that PGE2 is T cells specifically by interacting with PGE2-specific divergent G secreted in the extracellular mixture of L. donovani–infected protein–coupled receptors (i.e., EP2 and EP4) (45, 46). Thus, we BMMvs and binds to EP2/EP4 receptors in T cells for SOCS3 next ascertained whether PGE2 secreted from infected BMMvs expression. EP2 and EP4 receptors are well known to activate and the EP2/EP4 receptor present in CD4+ T cells regulate SOCS3 adenylate cyclase and stimulate the intracellular cAMP level. A induction during infection. To this end, BMMvs were pre- time-dependent increase in intracellular cAMP level was observed incubated with 1 mM COX2 inhibitor NS-398 (COX2 inhibition in T cells incubated with L. donovani–infected BMMvs super- prevents PGE2 synthesis in macrophages) followed by infection natant, peaking at 8 h (10.2 pmol/ml), and thereafter maintained with L. donovani for 24 h. The supernatant was collected from up to 12 h postincubation (Fig. 6C). To further ascertain the role of these BMMvs, CD4+ T cells were treated with this mixture for PGE2 derived from infected BMMvs in inducing cAMP level in 24 h, and SOCS3 expression at the protein level was assessed. T cells, supernatant was derived from NS-398 (COX-2 inhibitor)– Similarly, T cells were preincubated with 10 mM EP2 antagonist pretreated L. donovani–infected macrophages and incubated with Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 6. PGE2/cAMP/PKA–mediated transcriptional activation of SOCS3 in T cells during infection. (A) BMMvs were isolated and infected with L. donovani promastigotes for respective time periods, and PGE2 level in culture supernatants were measured using a PGE2 sandwich ELISA kit. (B) T cells were pretreated with 10 nM PGE2, AH6809 (EP2 antagonist, 10 mM), or GW627368 (EP4 antagonist, 100 nM) followed by incubation with infected macrophage-derived supernatant or NS-398 (COX2 inhibitor, 1 mM)–treated infected macrophage-derived supernatant for 24 h and checked for expression of SOCS3. (C) T cells were incubated with L. donovani–infected macrophage supernatant for respective time periods, and level of cAMP was measured using a sandwich ELISA kit. (D) T cells were pretreated with 10 nM PGE2, AH6809 (10 mM), GW627368 (100 nM), or AH6809 plus GW627368, followed by incubation with L. donovani–infected macrophage supernatant or NS-398–treated infected macrophage-derived supernatant for 8 h and mixture was checked for the production of cAMP. (E) CD4+ T cells were incubated with L. donovani–infected (24 h) macrophage-derived mixture for different time points, and PKA activation was analyzed through immunoblot analysis. (F and G) T cells were pretreated with PKA inhibitor (10 mM H89 or KT5720) for 1 h followed by incubation with L. donovani–infected macrophage supernatant. Nuclear and cytosolic extracts were prepared, and subcellular distribution of SP1, GATA1, and CREB was checked through immunoblot analysis. (H and I) T cells were preincubated with 10 nM PGE2, 10 mM H89, or 10 mM AH6809 or GW627368 (100 nM), followed by incubation with L. donovani–infected macrophage-derived supernatant. Nuclear and cytosolic distribution of SP1, GATA1, and CREB was analyzed by immunoblotting. Significance was determined for infected versus uninfected groups. *p , 0.05, **p , 0.005, ***p , 0.0004. ns, not significant. The Journal of Immunology 11 isolated splenic CD4+ T cells. In another set, T cells were treated burdens were found to be reduced in infected BALB/c mice treated with AH6809 or GW627368 (EP2 or EP4 antagonist) followed by with SOCS1 (48.1 and 51.02% reduction, respectively) and SOCS3 incubation with L. donovani–infected macrophage-derived super- morpholino (53.04 and 55.1% reduction, respectively) com- natants. We observed that L. donovani–infected BMMv-derived pared with control morpholino-treated mice at 6 wk postinfection supernatant and 10 nM PGE2 (positive control) increases the (Fig. 7E, 7F). However, reduction in organ parasite burden was production of cAMP to a similar extent, whereas supernatant maximum in infected BALB/c mice treated with SOCS1 plus derived from NS-398–pretreated infected BMMv-derived super- SOCS3 morpholino (77.1 and 74.8% reduction of liver and spleen natants failed to induce cAMP synthesis in T cells (Fig. 6D). parasite burden, respectively), further establishing the role of Similarly, pretreatment of T cells with AH6809 or GW627368 SOCS1 and SOCS3 in parasite survival (Fig. 7E, 7F). We then (EP2 or EP4 antagonist) followed by incubation with L. donovani– sought to determine the consequences of SOCS1 and SOCS3 in- infected BMMv-derived supernatant failed to induce expression hibition on the in vivo pro/anti-inflammatory cytokine balance. of cAMP in T cells (Fig. 6D). Because cAMP activates protein In vivo silencing of SOCS1 and SOCS3 in infected BALB/c mice kinase A (PKA), we next checked for its activation/phosphorylation resulted in increased levels of IL-12 (2.96- and 2.2-fold, respec- via immunoblot analysis by incubating T cells with the tively, compared with control morpholino-treated infected mice at L. donovani–infected macrophage mixture. A time-dependent in- the 4 wk postinfection) (Fig. 7G) and IFN-g (2.41- and 3.11-fold, crease in PKA phosphorylation was observed, which (Fig. 6E) respectively, compared with control morpholino-treated infected implies that PGE2 secreted by L. donovani–infected macrophages mice at 4 wk postinfection) (Fig. 7H). This was followed by a activates the cAMP/PKA pathway in T cells during macrophage– concomitant decrease in IL-10 (31.2 and 40.7% reduction in

T cell cross-talk. Next, we checked whether activation of SP1, SOCS1 and SOCS3 morpholino-treated mice, respectively, com- Downloaded from GATA1, and CREB in T cells was under the control of the cAMP/ pared with control morpholino-treated infected mice) (Fig. 7I) PKA pathway. For this, T cells were treated with PKA inhibitor and TGF-b synthesis (36.5 and 40.4% reduction in SOCS1 and (H89 or KT5720) prior incubation with L. donovani–infected SOCS3 morpholino-treated mice, respectively, compared with (24 h) macrophage-derived mixture, and nuclear translocation of control morpholino-treated infected mice) (Fig. 7J) at 4 wk SP1, GATA1, and CREB were checked by immunoblot analysis. postinfection. However, switching of anti-inﬂammatory to proin-

We observed marked reduction in translocation of SP1, GATA1, flammatory response was markedly enhanced in SOCS1 plus http://www.jimmunol.org/ and CREB to the nucleus in H89- or KT5720-treated cells, as their SOCS3 morpholino-treated mice, in which we observed 4.5- and expression is retained in the cytosolic extract (Fig. 6F, 6G). Fur- 4.7-fold induction of IL-12 and IFN-g, respectively, compared ther blocking PGE2 secretion in L. donovani–infected macro- with control morpholino-treated infected mice (Fig. 7G, 7H) with phage supernatant by NS-398 and pretreatment of T cell with a significant decrease in IL-10 and TGF-b synthesis (55.7 and AH6809 or GW627368 (EP2, EP4 antagonist) markedly reduced 65.8% compared with control morpholino-treated infected nuclear translocation of SP1, GATA1, and CREB (Fig. 6H, 6I) mice, Fig. 7I, 7J) at 4 wk postinfection. These results sug- in T cells during coculture experiments. Because elevated PGE2 gested that a strong upregulation of SOCS1 and SOCS3 in vivo secretion was observed in infected macrophages for a long du- might be associated with an anti-inflammatory response that, in ration, we also checked the effect of L. donovani amastigotes on turn, facilitated parasite survival in the BALB/c mice model of by guest on September 30, 2021 PGE2 secretion by BMMvs. Like promastigotes, L. donovani experimental VL. amastigotes could also induce PGE2 secretion from BMMvs at late hours of infection, although the level was less than that Discussion of promastigote-infected macrophages with a maximum level Cytokine cross-talk between macrophage and T cell eventually (.1450 pg/ml) observed at 48 h postinfection (Supplemental determines the Th1 or Th2 type of immune response in combating Fig. 1E). Collectively, these results suggested that PGE2/EP2/ various immunological disorders and defines the fate of host im- EP4 axis–mediated activation of cAMP/PKA signaling induces munity during infection. A strong circuitry between these cells nuclear translocation of the SP1, GATA1, and CREB transcrip- is established by IL-12 and IFN-g, in which IL-12 is synthesized tion factor in T cells for SOCS3 induction. by macrophages after stimulation by Th1 cell–derived IFN-g. Thereafter, the secreted IL-12, in turn, acts as a stimulant for Role of SOCS1 and SOCS3 in in vivo parasite survival and IFN-g synthesis by Th1 cells, thereby connecting both arms of pro/anti-inflammatory cytokine balance during experimental VL immunity. This positive cross-talk between macrophages–T cells We next validated our observation in vivo by analyzing the ex- is crucial for combating harmful pathogens and mounting effec- pression of SOCS1 and SOCS3 in splenocytes of L. donovani– tive innate and adaptive immune responses. For instance, IL-12 infected BALB/c mice that were evaluated by real-time PCR and is crucial for host-protective immunity against Mycobacterium immunoblot analysis (Fig. 7B). Similar to the data obtained ex avium infection, and endogenous IL-12 mediates protection against vivo, the splenocytes of infected mice showed induction in the Yersinia enterocolitica in C57BL/6 mice by triggering IFN-g pro- levels of SOCS1 and SOCS3 at both the mRNA level and protein duction in NK and CD4+ T cells. Similarly, mice challenged with level, where maximum induction was observed at 4 wk postin- Plasmodium yoelii sporozoites can be completely protected against fection (Fig. 7B, 7C). Further, to evaluate the role of SOCS1 and malaria sporozoite challenge when pretreated with CpG oligo- SOCS3 in in vivo disease progression, both isoforms were deoxynucleotides, and this depends on IL-12 and IFN-g generation. knocked down by SOCS1 and SOCS3 morpholino (antisense In case of VL, too, Th1 associated cytokines are essential oligos that can block translation; reviewed in the administration for controlling disease progression (47). L. donovani has evolved scheme in Fig. 7A). The efficacy and specificity of morpholino on several well-equipped machineries to impair the proinflammatory SOCS1 and SOCS3 expression were further evaluated by immu- cytokine response in infected macrophages (48). However, the noblot analysis in splenocytes of infected mice at various time mechanistic details underlying the strategies employed by periods of postinfection (Fig. 7D). SOCS1 and SOCS3 morpho- L. donovani to breach the multilevel barriers maintained by the lino led to a substantial reduction in the SOCS1 and SOCS3 cross-talk between innate and adaptive immunity are sorely protein levels as compared with infected mice administered with lacking. In the current study, we unveil the mechanistic pathway control morpholino (Fig. 7D). Both liver and spleen parasite by which parasite L. donovani interrupts this cross-talk between 12 SOCS1 AND SOCS3 BREACH MACROPHAGE–T CELL CROSS-TALK IN VL Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 7. Effect of SOCS1 and SOCS3 knockdown on parasite survival and cytokine balance in vivo. (A) Schematic representation of the experimental protocol for vivo morpholino–mediated translation blocking of SOCS1 and SOCS3 in BALB/c mice to determine parasite burden and cellular immune response. BALB/c mice were infected with 1 3 107 L. donovani promastigotes via an i.v. route. Splenocytes were isolated from infected mice of different groups, and expression of SOCS1 and SOCS3 was evaluated at second, fourth, and sixth week by (B) by real-time PCR as well as (C) by immunoblotting. (D) Western blot analysis of SOCS1 and SOCS3 protein in splenocytes isolated from L. donovani–infected mice treated either with control vivo morpholino or SOCS1- and SOCS3-specific vivo morpholino at different time intervals as indicated. (E and F) Hepatic and splenic parasitemia was determined at fourth and sixth week postinfection from different groups of infected mice and expressed as the Leishman Donovan (Figure legend continues) The Journal of Immunology 13 macrophages and T cells and escapes immune surveillance by Similarly, T. gondii induces Egr2-mediated SOCS1 induction in inhibiting IL-12 and IFN-g cytokine production, which is achieved infected RAW 264.7 cells (54), and Egr2 mediated SOCS1 ex- by biphasic induction of SOCS proteins. We observed that during pression is also reported during splenic DC development (55). early hours of infection L. donovani preferentially induced SOCS1 ChIP analysis also showed direct interaction of Egr2 and/or Egr3 expression in BMMvs to breach the first-line of host immune de- in the SOCS1 promoter that is important to control the activation fense. At a later phase, evasion of host-adaptive immunity is per- of STAT1-mediated cytokine signaling in B and T cells (24, 56). petrated by significant upregulation of SOCS3 in T cells incubated Collectively, these results suggested that the Egr group of tran- with L. donovani–infected BMMvs. scription factors (Egr1, Egr2, and Egr3) might play a similar role Our results clearly demonstrated that SOCS1 expression was in the induction of SOCS1 in different cellular contexts. In our robust and stable in infected macrophages, although surprisingly, case, the induction of SOCS1 is regulated by the Egr2 isoform we also observed expression of SOCS3 in infected macrophages, along with transcription factor AHR in infected BMMvs. The which induced early, albeit for a short period of time. Whereas induction of AHR is in accordance with the previous studies, previous studies have reported an early induction of SOCS3 in which showed that AHR modulates expression of various SOCS macrophages following Leishmania infection, there is a notable family members in a different cellular context (25, 57, 58). temporal variation; for instance, Bertholet et al. (49) has reported Parasite surface molecules not only play an important role during the induction of SOCS3 at 45 minutes after the infection in human initial interaction and phagocytosis but also induce numerous macrophages, whereas Srivastav et al. (50) have reported induc- macrophage dysfunctions to evade host immunity. LPG and gp63 tion of SOCS3 at 6 hours in infected macrophages, which is are the two predominant and abundant virulence factors of the similar to our observation, in which we found that the induction of Leishmania species, which are well known for subverting mac- Downloaded from SOCS3 started as early as 6 hours postinfection in BMMv, being rophage signaling and microbicidal function. An earlier report maximum at 12 hours, and then gradually declined. This temporal suggested that L. major LPG could induce the expression of both variation in SOCS3 induction in different reports may be attrib- SOCS1 and SOCS3 in infected murine macrophages (34). This is uted to both the difference in the leishmanial strain concerned as also true in the case of L. donovani, as we observed partial but well as the origin of the cell used for the in vitro study. Never- significant reduction of SOCS1 expression in lpg1-KO–infected

theless, we further validated that inhibition of SOCS1 upregulated BMMv. This may be due to the fact that we observed tran- http://www.jimmunol.org/ the IL-12 cytokine level in infected macrophages, whereas SOCS3 scriptional activation of EGR2 but not AHR is inhibited in lpg1- inhibition failed to restore its level, suggesting that the brief and KO–infected BMMvs. Moreover, we observed that the PI3K/Akt early induction of SOCS3 in infected macrophages may contribute pathway regulates Egr2 activation, which led us to speculate that to the reactive oxygen species–mediated apoptotic signaling cas- LPG either directly or indirectly activates this pathway to induce cade, as demonstrated earlier (50). Conversely, we found that the Egr2 nuclear translocation. This is because a recent finding expression of SOCS3 was robust and stable in T cells incu- reported that L. donovani LPG induced the autophagy of neutro- bated with L. donovani–infected macrophage-derived supernatant. phils by activating the PI3K/Akt signaling pathway (59). How- A correlation between the upregulation of SOCS1 and SOCS3 ever, unlike LPG, we did not observe any role of gp63 in isoforms in macrophages and T cells was observed with subse- regulating SOCS1 induction, as inhibition of this metalloprotease by guest on September 30, 2021 quent downregulation of IL-12 and IFN-g, respectively, and is did not alter the SOCS protein level in infected BMMvs. achieved by inhibiting phosphorylation of STAT1 and STAT4. In the quest of looking for mediators that modulate macrophage– Our results support the observation that SOCS1 could effectively T cell cross-talk during infection, we observed L. donovani in- inhibit phosphorylation of STAT1 and that SOCS3 could inhibit duces PGE2 secretion from infected macrophages, which is a phosphorylation of STAT4 (51, 52). major negative regulator of T cell–derived IFN-g synthesis. PGE2 Apart from Leishmania, several parasites induce SOCS ex- is an arachidonic acid metabolite that inhibits T cell proliferation, pression, for instance, Toxoplasma gondii suppresses the IFN-g– differentiation, expression of membrane receptors, secretion of di- mediated activation of murine macrophages, through the induction verse cytokines, cytotoxicity, and other specific effector functions in of SOCS1 (15). M. bovis also increases SOCS1 and SOCS3 to in- cellular immune reactions (60). Moreover, PGE2 is known to be hibit IFN-g induced STAT1 (16). Similarly, mice lacking SOCS1 induced by a variety of pathogens. For instance, Mycobacterium are protected from P. berghei, which causes cerebral malaria, and intracellulare induces PGE2 synthesis, inhibiting the production of expression of SOCS1 has hampered C. pneumonia clearance lymphokines in infected macrophages and suppressing an effective (18, 19). In addition to these reports, our finding elaborates that immune response (61). PGE2 promotes Burkholderia pseudomallei L. donovani induces SOCS1 expression in macrophages by acti- intracellular survival through the activation of arginase 2, thereby vating two independent signaling pathways (i.e., activation of limiting NO production. Recent studies have confirmed that EP2 transcription factor Egr2 via PI3K/Akt–mediated SRF axis and and EP4 are the major receptor subtypes (G protein–coupled activation of transcription factor AHR via IDO/kynurenine sig- receptors) to mediate the actions of PGE2 in human and murine naling). The Egr group of transcription factors is known to reg- CD4+ T cells (62), and cAMP/PKA is well-known secondary ulate the transcription of SOCS family members, and various Egr messenger that is activated by this G protein–coupled receptor. isoforms, such as Egr1, Egr2, and Egr3, are known to regulate the Different pathogens activate cAMP/PKA pathway by increas- transcription of SOCS1. For instance, Egr1 was reported to in- ing PGE2 secretion; for instance, Salmonella-induced PGE2 teract with the SOCS promoter in RAW 264.7 cells in response activates the PKA pathway and upregulates IL-10 production to to LPS as well as during infection with L. donovani (50, 53), promote an immunosuppressive phenotype. In our study, PGE2

Units 6 SD for five mice per group.(G–J) Level of pro- and anti-inflammatory cytokines, such as (G) IL-12, (H) IFN-g,(I) IL-10, and (J) TGF-b in culture supernatants isolated from splenocytes of control, L. donovani–, L. donovani plus control morpholino–, L. donovani plus SOCS1 and SOCS3 morpholino (either alone or both)–treated mice at fourth and sixth week postinfection were determined by ELISA. Significance is indicated for the control morpholino- treated versus SOCS1- or SOCS3- or SOCS1 plus SOCS3 morpholino-treated groups. *p , 0.05, **p , 0.005, ***p , 0.0001. 14 SOCS1 AND SOCS3 BREACH MACROPHAGE–T CELL CROSS-TALK IN VL derived from L. donovani–infected macrophages was capable of cytokine signaling 3 and suppresses signal transduction in response to the activating factor IFN-gamma. J. Immunol. 163: 2640–2647. inducing EP2/EP4 receptor-mediated cAMP/PKA-driven activa- 11. Stoiber, D., S. Stockinger, P. Steinlein, J. Kovarik, and T. Decker. 2001. Listeria tion of SP1, GATA1, and CREB transcription factors. This, in monocytogenes modulates macrophage cytokine responses through STAT serine turn, induces SOCS3 expression in T cells, which further inhibits phosphorylation and the induction of suppressor of cytokine signaling 3. J. Immunol. 166: 466–472. STAT4-mediated IFN-g synthesis, and helps to establish an al- 12. Yoshimura, A., T. Naka, and M. Kubo. 2007. SOCS proteins, cytokine signalling ternate circuit between macrophages and T cells. and immune regulation. Nat. Rev. Immunol. 7: 454–465. Our study reveals that L. donovani modulates host-protective, 13. Kasprzycka, M., M. Marzec, X. Liu, Q. Zhang, and M. A. Wasik. 2006. Nucleophosmin/anaplastic lymphoma kinase (NPM/ALK) oncoprotein induces cytokine-mediated macrophage–T cell cross-talk and estab- the T regulatory cell phenotype by activating STAT3. Proc. Natl. Acad. Sci. USA lishes an alternate cross-talk by inducing SOCS1 and SOCS3 in 103: 9964–9969. 14. Trengove, M. C., and A. C. Ward. 2013. SOCS proteins in development and macrophage and T cell, respectively, to protect its own niche disease. Am. J. Clin. Exp. Immunol. 2: 1–29. during infection. Because strategies for therapeutic manipulation 15. Zimmermann, S., P. J. Murray, K. Heeg, and A. H. Dalpke. 2006. Induction of of signaling intermediates exist, an increased understanding of the suppressor of cytokine signaling-1 by Toxoplasma gondii contributes to immune evasion in macrophages by blocking IFN-gamma signaling. J. Immunol. 176: regulation of cytokine and immune cell signaling strategies 1840–1847. employed by Leishmania will soon have significance not only for 16. Imai, K., T. Kurita-Ochiai, and K. Ochiai. 2003. Mycobacterium bovis bacillus nonhealing leishmaniasis but also for other similar types of dis- Calmette-Gue´rin infection promotes SOCS induction and inhibits IFN-gamma- stimulated JAK/STAT signaling in J774 macrophages. FEMS Immunol. Med. eases. This is because manipulation of host–defense machinery by Microbiol. 39: 173–180. exploiting SOCS isoforms is not unique for Leishmania. Many 17. The´ry, C., M. Ostrowski, and E. Segura. 2009. Membrane vesicles as conveyors of immune responses. Nat. Rev. Immunol. 9: 581–593. other infectious diseases are propagated by exploiting these pro- 18. Bullen, D. V., D. S. Hansen, M. A. Siomos, L. Schofield, W. S. Alexander, and teins, as a crucial negative regulator of major host cell signal- E. Handman. 2003. The lack of suppressor of cytokine signalling-1 (SOCS1) Downloaded from ing pathways (such as JAK/STAT, TLR/NF-kBpathway,etc.), protects mice from the development of cerebral malaria caused by Plasmodium berghei ANKA. Parasite Immunol. 25: 113–118. for instance, disease propagated by T. gondi, Mycobacterium, 19. Yang, T., P. Stark, K. Janik, H. Wigzell, and M. E. Rottenberg. 2008. SOCS-1 C. pneumonia, and Plasmodium. Altogether, comprehensive in- protects against Chlamydia pneumoniae-induced lethal inflammation but ham- sights into host cell inactivation and immune evasion gained from pers effective bacterial clearance. J. Immunol. 180: 4040–4049. 20. Ryo, A., F. Suizu, Y. Yoshida, K. Perrem, Y. C. Liou, G. Wulf, R. Rottapel, our study also have relevance for other chronic infectious diseases S. Yamaoka, and K. P. Lu. 2003. Regulation of NF-kappaB signaling by Pin1- and autoimmune situations. dependent prolyl isomerization and ubiquitin-mediated proteolysis of p65/RelA. http://www.jimmunol.org/ Mol. Cell 12: 1413–1426. 21. Prive´, C., and A. Descoteaux. 2000. Leishmania donovani promastigotes evade the activation of mitogen-activated protein kinases p38, c-Jun N-terminal kinase, Acknowledgments and extracellular signal-regulated kinase-1/2 during infection of naive macro- We thank Director, CSIR-CDRI for providing the research facilities and phages. Eur. J. Immunol. 30: 2235–2244. encouragement. We are thankful to Dr. Chandrima Shaha, National Institute 22. Parmar, N., P. Chandrakar, P. Vishwakarma, K. Singh, K. Mitra, and S. Kar. of Immunology, New Delhi, for help in procuring the WT and lpg1-KO 2018. Leishmania donovani exploits tollip, a multitasking protein, to impair TLR/IL-1R signaling for its survival in the host. J. Immunol. 201: 957–970. (LD 1S) parasites. We are thankful to Dr. Amogh A. Sahasrabuddhe, 23. Tamiya, T., I. Kashiwagi, R. Takahashi, H. Yasukawa, and A. Yoshimura. 2011. CSIR-CDRI, for providing the GFP-tagged L. donovani strain. We also Suppressors of cytokine signaling (SOCS) proteins and JAK/STAT pathways: thank Dr. Suvendra N. Bhattacharya and Dr. Nahid Ali, CSIR-Indian regulation of T-cell inflammation by SOCS1 and SOCS3. Arterioscler. Thromb.

Institute of Chemical Biology, Kolkata, for providing the gp63 inhib- Vasc. Biol. 31: 980–985. by guest on September 30, 2021 24. Li, S., T. Miao, M. Sebastian, P. Bhullar, E. Ghaffari, M. Liu, A. L. Symonds, itor and Ab. We are thankful for the sophisticated analytical instrumen- and P. Wang. 2012. The transcription factors Egr2 and Egr3 are essential for the tation facility of CSIR-CDRI in providing support with the flow cytometry control of inflammation and antigen-induced proliferation of B and T cells. experiments. Immunity 37: 685–696. 25. Barroso, A., M. Gualdroń-Lo´pez, L. Esper, F. Brant, R. R. Arau´jo,M.B.Carneiro, T. V. A´ vila, D. G. Souza, L. Q. Vieira, M. A. Rachid, et al. 2016. The aryl hy- Disclosures drocarbon receptor modulates production of cytokines and reactive oxygen species and development of myocarditis during Trypanosoma cruzi infection. Infect. The authors have no financial conflicts of interest. Immun. 84: 3071–3082. 26. Madonna, S., C. Scarponi, R. 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Supplementary Figure 1. (A) BMMфs were infected with L. donovani amastigote for indicated time intervals and expression of SOCS1 was analyzed by western blotting. (B) The siRNA efficacy determination in BMMфs. BMMфs were transfected (24 h) with either control or SOCS1, SOCS3, Egr2, AHR, SRF or IDO siRNA followed by infection with L. donovani promastigotes for respective hour (SOCS1: 24 h, SOCS3:12 h, Egr2: 12 h, AHR:12 h, SRF: 8 h, IDO: 12 h). Expression of individual protein was evaluated by immunoblot analysis. (C) CD 4+ T cells were transfected with control, SOCS3, CREB, GATA1 or SP1 siRNA for 24 h followed by incubation with L. donovani-infected macrophage derived supernatant for respective hour (SOCS3:24 h, CREB: 12h, GATA1: 12 h, and SP1: 12 h). Expression of individual protein was evaluated by immunoblot analysis. (D) BMMфs were transfected (24 h) with either control, SOCS1, SOCS3 or both siRNA followed by infection with GFP transfected L. donovani (Dd8) promastigotes for 4 h. Cells were washed with PBS and were analyzed by flow cytometry. FACS histograms showing the percentages of GFP- expressing infected macrophage. (E) BMMфs were infected with L. donovani amastigotes for respective time periods. PGE2 level in culture supernatants were measured using a PGE2 sandwich ELISA Kit.