PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Immunol. Author manuscript; available in PMC 2010 October 1.
Published in final edited form as:
PMCID: PMC2749572
NIHMSID: NIHMS130734

IL-27 regulates IL-10 and IL-17 from CD4+ cells in non-healing Leishmania major infection1

Abstract

Control of infection caused by Leishmania major requires the development of IFN-γ+CD4+ lymphocytes for the induction of microbicidal activity in host macrophages. We recently reported on the inability of conventionally resistant C57BL/6 mice to successfully resolve infection by an isolate of L. major, despite a strong IFN-γ response by the host. Susceptibility was caused by antigen-specific IL-10 from CD4+ cells that were also producing IFN-γ. In the present studies, we have explored the role for IL-27 in the regulation of IL-10 from Th1 cells in Leishmaniasis. Cytokine analysis of CD4+ cells in the lesions and draining lymph nodes of infected IL-27R deficient (WSX-1-/-) mice revealed diminished IL-10 from IFN-γ+ CD4+ cells, which was accompanied by a reduction in total IFN-γ+ CD4+ cells and an increase in IL-4. Despite the inhibition of IL-10 from CD4+ cells, no significant change in parasite numbers was observed, due both to the shift in the Th1/Th2 balance and to residual levels of IL-10. Strikingly, infected WSX-1-/- mice developed more severe lesions that were associated with the appearance of IL-17+ CD4+ cells, demonstrating a function for IL-27 in blocking the development of inappropriate Th17 cells during L. major infection. The results demonstrate the pleiotropic effects that IL-27 has on L. major-driven Th1, Th2, and Th17 development, and reinforce its function as a key regulatory cytokine that controls the balance between immunity and pathology.

Keywords: Parasitic-protozoan, T cells, cytokines, skin

Introduction

The intracellular protozoan Leishmania can produce a spectrum of clinical diseases, ranging from a single cutaneous ulcer that spontaneously heals, to chronic cutaneous or mucocutaneous lesions that are non-healing or slow to resolve, to visceral disease that is generally fatal in the absence of treatment. It is well established that successful control of infection caused by Leishmania major requires the development of IFN-γ+CD4+ lymphocytes for the induction of microbicidal activity by parasitized macrophages. Experimental models for cutaneous leishmaniasis have historically relied upon the disparate disease phenotypes displayed by C57BL/6 and BALB/c mouse strains to identify immunological mechanisms underlying, respectively, host resistance and susceptibility (1). However, the key conclusion that Th2 dominance controls susceptibility has failed to adequately explain non-healing or reactivated forms of cutaneous or visceral leishmaniasis (VL) in humans.

IL-10 has pleiotropic, primarily anti-inflammatory properties that include suppression of dendritic cell functions, and rendering macrophages unresponsive to activation signals (reviewed in (2). While its upregulation is considered a homeostatic mechanism to limit the tissue damage caused by excessive inflammation, effective clearance of Leishmania can also be compromised. Analysis of chronic or re-activating lesions in humans has revealed increased expression of pro-inflammatory cytokines, high levels of IL-10, but low or undetectable amounts of Th2 associated cytokines (3-6). In human VL, elevated levels of IFN-γ mRNA have been found in target organs, such as the spleen and bone marrow, accompanied by increased levels of IL-10 (7-9), the predominant source of which is Foxp3-CD25-CD3+ cells (10). Accumulating evidence from mouse models of non-healing or disseminating forms of leishmaniasis have reinforced pathogenetic mechanisms that take into account the presence of parasite-driven Th1 responses that are suppressed either in magnitude or function by IL-10 (reviewed (11)).

We have introduced a model of non-healing L. major in conventionally resistant C57BL/6 mice, in which IL-10 functions in a Th1 polarized setting to prevent clinical cure, and have argued that this model better reflects the conditions underlying non-healing forms of clinical disease (12). A notable feature of this infection is the presence of IFN-γ+CD4+ cells that also produce IL-10. IL-10 from this cellular source was necessary and sufficient to mediate susceptibility, as specific ablation of IL-10 from this subset resulted in enhanced clearance of infection (13). The factors that regulate IL-10 production by Th1 cells in this setting are unknown.

The IL-12-related cytokine, IL-27, is a heterodimer composed of EBI3 and p28, and is produced by innate cells, such as macrophages and dendritic cells (14). The receptor for this cytokine is composed of gp130, a sub-unit utilized by other growth factors including IL-6 and IL-11, that is paired with the unique IL-27R (WSSX, TCCR). Initial studies into the biological function of IL-27 implicated its role in promoting Th1 development from naïve cells, as WSX-1 deficient mice exhibited defective early Th1 responses to Leishmania major, though these mice eventually healed (15). The requirement for IL-27 in early Th1 development in L. major infection was later described to be restricted to its suppression of the early burst of IL-4 that normally occurs in C57BL/6 mice, as neutralization of IL-4 in WSX-1-/- mice restored Th1 development and resistance to wild type levels (16).

IL-27 has more recently been recognized for its anti-inflammatory properties in various models of infectious diseases and autoimmunity (for a review see (14)). Several recent studies have shown IL-27 to mediate anti-inflammatory activity through its ability to suppress Th17 cells (17, 18), and through the induction of IL-10 from activated CD4+ cells. Under neutral in vitro culture conditions, exogenous IL-27 exclusively induced IL-10 from naïve CD4+ cells, and enhanced IL-10 production when cells were activated in the presence of Th1 or Th2 polarizing cytokines (19-21). In vivo, CD4+ cells from WSX-1-/- mice infected with T. gondii or Listeria monocytogenes produced less IL-10 than wild type counterparts (19, 21). In the mouse model for multiple sclerosis, exogenous IL-27 suppressed EAE induced by adoptive transfer of pathogenic CD4+ cells in an IL-10 dependent manner (20). The present studies were designed to assess the function of IL-27 in the regulation of IL-10 by CD4+ effector cells responding in an IL-10 dependent, non-curing model of L. major infection, and how this regulation affects parasite control and pathology.

Materials and Methods

Mice and reagents

C57BL/6 mice were purchased from Taconic Farms (Germantown, NY), maintained in the National Institute of Allergy and Infectious Diseases animal care facility under specific pathogen-free conditions, and used under a study protocol approved by the NIAID Animal Care and Use Committee. IL27Ra-/- (WSX-1-/-) mice, backcrossed more than nine generations onto C57BL/6 mice (15), were provided by C. Saris (Amgen, Thousand Oaks, CA) and were housed and bred in specific pathogen-free facilities in the Department of Pathobiology at the University of Pennsylvania in accordance with institutional guidelines. IL27Ra-/- and IL27Ra+/+ mice, each backcrossed at least 10 generations onto C57BL/6 mice (22) were also supplied by Genentech, Inc (San Francisco, CA), and were housed and bred in specific-pathogen free conditions in the NIAID animal care facility.

Infections and in vivo treatments

L. major strain NIH/Sd (MHOM/SN/74/SD) was cultured in Medium 199 with 20% heat-inactivated FCS (Gemini Bio-Products, Woodland, CA), 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, 40 mM HEPES, 0.1 mM adenine (in 50 mM Hepes), 5 mg/ml hemin (in 50% triethanolamine), and 1 mg/ml 6-biotin (M199/S). Infective-stage metacyclic promastigotes of L. major were isolated from stationary cultures by density gradient centrifugation as described previously (23). One thousand metacyclic promastigotes were inoculated into the ear dermis using a 30 gauge needle in a volume of ~5-10 μl. Lesion development was monitored by measuring the diameter of the ear nodule with a direct-reading vernier caliper (Thomas Scientific, Swedesboro, NJ). Neutralizing anti-IL-4 mAb (11B11) (National Cancer Institute Biological Resource Branch, Frederick, MD) or isotype control antibody (GL113) (Harlan Bioproducts for Science, Inc., Madison, WI) was administered intraperitoneally beginning one day prior to infection and then weekly for the first five weeks with 3mg mAb/injection. Anti-IL-10αR mAb (1B1.3a) (Harlan Bioproducts for Science, Inc., Madison, WI), or an isotype control antibody (GL113) was administered intraperitoneally beginning six weeks after inoculation, with bi-weekly injections of 0.25mg mAb/injection for three weeks. Anti-IL-10αR mAb (0.25 mg mAb/injection) plus anti-IL-4 mAb (1.5 mg mAb/injection) were co-administered beginning six weeks after infection, with bi-weekly injections for 2.5 weeks.

Processing of ear tissue and estimation of parasite load

Parasite titrations were performed as previously described (24). The two sheets of infected ear dermis were separated, deposited in DMEM containing 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.2 mg/ml Liberase CI purified enzyme blend (Roche Diagnostics Corp., Indianapolis, IN) and incubated for 2 h at 37°C. Digested tissue was placed in a grinder and processed in a Medimachine tissue homogenizer (Becton Dickenson). Tissue homogenates were filtered through a 70-μm cell strainer (Falcon Products, St. Louis, MO). Parasite titrations were determined by serially diluting samples in 96-well flat-bottom microtiter plates containing biphasic medium prepared using 50 μl of NNN medium containing 30% of defibrinated rabbit blood overlaid with 50 μl of M199/S. The number of viable parasites in each sample was determined from the highest dilution at which promastigotes could be detected after 7 to14 days of incubation at 25°C.

Lymphocyte isolation and in vitro re-stimulation

To characterize leukocytes in the inoculation site, the ears were collected, and the ventral and dorsal dermal sheets were prepared as described above. Following preparation, cells were analyzed for surface phenotype by flow cytometry. For in vitro re-stimulation of dermal lymphocytes, cells stimulated with 10 ng/ml PMA and 500 ng/ml ionomycin in the presence of monensin (Golgistop, BD Biosciences) for four hours. The cells were then analyzed for surface markers and intracytoplasmic staining for cytokines. For in vitro re-stimulation of draining lymph node cells, CD4+ cells from cervical draining lymph node were purified by negative selection (Miltenyi Biotec, Auburn, CA) and co-cultured with CD11c+ splenic APCs (Miltenyi Biotec) isolated from naïve C57BL/6 mice at a T cell:APC ratio of 4:1 in 200 μl RPMI containing 10% FCS, 10 mM Hepes, glutamine, penn/strep in round bottom 96-well plates with or without 50 μg/ml of freeze-thaw Leishmania antigen prepared from NIH/Sd stationary phase promastigotes. Where indicated, recombinant IL-27 (R&D Systems, Inc., Minneapolis, MN) was added at a final concentration of 20 ng/ml. After 72 hours, culture supernatants were collected for ELISA measurements, or cells were stimulated with PMA and ionomycin in the presence of Monensin for four hours prior to intracellular cytokine staining.

Immunolabeling and flow cytometry analysis and cytokine measurements

The following antibodies used for immunophenotyping were purchased from BD Biosciences (San Diego, CA). FITC anti-mouse TCRβ chain (H57-597), PE-Cy7 anti-mouse CD4(L3T4) (RM4-5), APC anti-mouse F4/80 (BM8), PE anti-mouse Ly6G and Ly-6C (GR-1) (RB6-8C5), APC anti-mouse IFN-γ (XMG-1.2), PE or APC anti-mouse IL-10 (JES5-16E3), and PE anti-mouse IL-17A (TC11-18H10). FITC anti-Foxp3 staining was performed using eBioscience (San Diego, CA) reagents according to manufacture’s protocol. The isotype controls used (all from BD PharMingen) were rat IgG2b (A95-1), rat IgG2a (R35-95), and hamster IgG, group 2 (Ha4/8). Before staining, LN or dermal cells were incubated with an anti-Fc III/II receptor mAb (2-4G.1) in PBS containing 0.1% BSA and 0.01% NaN3. The staining of surface and intracytoplasmic markers was performed sequentially: the cells were stained first for their surface markers, followed by fixation/permeabilization and staining for INF-γ, IL-10, and IL-17A. For each sample, 400,000 cells were acquired for analysis. The data were acquired using a BD FACSCanto II flow cytometer (BD Biosciences), and analyzed with FlowJo software (Tree Star, Inc.). The lymphocytes from ear cells were identified by characteristic size (forward light scatter (FSC)) and granularity (side light scatter (SSC)), and by lymphocyte surface phenotype. ELISA measurements for IFN-γ, IL-10, IL-4, and IL-17A were performed using eBioscience (San Diego, CA) kits according to manufacture’s instructions.

Real-time PCR

For analysis of gene expression, ears were removed and immediately placed in RNAlater (Qiagen, Valencia, CA). Ear tissue was then disrupted mechanically using liquid nitrogen and a mortar and pestle. Homogenates were then passed through Qiashredder columns and RNA was purified using RNAeasy mini kit (Qiagen) according to the manufacture’s protocol. Reverse transcription was performed using Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen Life Technologies, Carlsbad, CA). Real-time PCR was performed on an ABI Prism 7900 sequence detection system (Applied Biosystems, Foster City, CA). PCR primer probe sets were pre-developed gene expression assays designed by Applied Biosystems and the quantities of products were determined by the comparative threshold cycle method using the equation 2-ΔΔcT to determine the fold increase in product. Each gene of interest was normalized to the 18s rRNA endogenous control and the fold change in expression are displayed as relative to naïve controls.

Statistical analyses

P values were determined using unpaired, two-tail t tests with Welch’s correction.

Results

IL-27 enhances IL-10 production from Foxp3-IFN-γ+CD4+ cells

We previously demonstrated the presence of Foxp3-CD4+ cells that produced antigen specific IL-10 in a Th1 polarized setting during a non-healing L. major infection in C57BL/6 mice. Following on the recent findings regarding IL-27 regulation of IL-10 production by naïve and activated T cells, the expression of IL-27 during L. major NIH/Sd infection, and its influence on the evolution of the non-curing phenotype, was explored. A kinetic analysis of IL-27 mRNA in the infection site revealed a progressive increase over the first 8 weeks in transcripts for both IL-27 subunits, with p28 showing upregulated expression by week 2, while elevated EBI3 was not detectable until week 5 (Fig. 1). The absolute Ct values for p28 and EBI3 in naïve ears were 17.26 +/- 0.55 and 14.39 +/- 0.95, respectively. The increase of IL-27 correlated with the upregulation of IL-10. To determine if IL-27 had the potential to regulate IL-10 in this infection, purified CD4+ T cells from draining lymph nodes of chronically infected mice were co-cultured with splenic CD11c+ antigen presenting cells (APCs) from naïve mice and re-stimulated with parasite antigen alone, or in combination with exogenous IL-27. Intracellular cytokine staining of CD4+ T cells demonstrated that following antigen stimulation a dominant source of IL-10 was from cells that co-produced IFN-γ (Fig. 2A). Although there was a substantial population that was IFN-γ-IL-10+, nuclear staining demonstrated that these were not Foxp3+ regulatory T cells. While rIL-27 in the absence of antigen did not induce an increase in the frequency of cytokine producing cells (data not shown), the addition of rIL-27 to cultures containing antigen resulted in an enhanced population of both IL-10+IFN-γ+CD4+ cells (3.7% vs. 4.6%) and IL-10+IFN-γ-CD4+ cells (3.5% vs. 5.2%). The latter were again Foxp3- cells. In agreement with flow cytometry analysis, rIL-27 increased IL-10 secretion as measured by ELISA, and concomitantly decreased IFN-γ secretion (Fig. 2B). The latter effect was due to increased IL-10, as IFN-γ levels were restored by antibody blockade of the IL-10 receptor. Therefore, IL-27 signaling in antigen-primed, Foxp3-CD4+ cells enhanced IL-10 production. Since re-stimulation with antigen alone efficiently induced the CD4+ T cells to produce IL-10, the requirement for endogenous IL-27 in these cultures was investigated using anti-IL-27 antibodies. While no effect of IL-27 neutralization was observed (not shown), the possibility remained that IL-27 plays an instructional role during the priming stage for development of Th1 cells capable of IL-10 secretion upon subsequent encounter with antigen.

Figure 1
Coordinate upregulation of IL-27 and IL-10 mRNA expression in L. major lesions. C57BL/6 mice were infected in the ear dermis with 1000 metacyclic L. major NIH/Sd parasites. At the indicated times after inoculation, mice were sacrificed and ear tissue ...
Figure 2
CD4+ effector cells increase Ag-dependent IL-10 production in response to IL-27. CD4+ T cells were purified from the draining lymph nodes of mice 8 weeks post-infection in the ear dermis with L. major NIH/Sd. CD4+ cells were pooled and cultured with splenic ...

Mice lacking the IL-27R have decreased IL-10 and increased IL-17

To determine if an absence of IL-27 signaling during Th1 development results in reduced IL-10 production by IFN-γ+CD4+ cells during infection, thereby increasing host resistance, IL-27R deficient (WSX-1-/-) mice were infected intradermally with a physiological dose of L. major NIH/Sd. Following inoculation, WSX-1-/- mice developed more severe lesions that were evident at six weeks after infection, and necessitated euthanasia by eleven weeks due to extensive necrosis and dermal erosion (Fig. 3A). Despite the increased pathology, there were no differences in the lesional parasite burdens comparing C57BL/6 and WSX-1-/- mice at either time-point (Fig. 3B), suggesting a role for IL-27 in restraining immunopathology. CD4+ cells isolated from the lesions of C57BL/6 and WSX-1-/- mice were compared for intracellular IFN-γ and IL-10 production, following ex-vivo re-stimulation with PMA and ionomycin. WSX-1-/- mice had a reduced percentage of CD4+ cells simultaneously producing IFN-γ and IL-10 (6.3% vs. 1.6%), which was accompanied by a reduction in cells producing IFN-γ alone (34.0% vs. 18.3%) (Fig. 4A). Because WSX-1-/- mice had increased pathology compared to C57BL/6 mice, and because IL-27 has been demonstrated to inhibit the development of pathological IL-17+CD4+ cells, intracellular cytokine analysis for IL-17A was performed. This revealed a large percentage of CD4+ cells producing IL-17A in WSX-1-/- mice that was nearly absent in C57BL/6 mice (17.5% vs. 2.3%). This population did not co-produce IFN-γ (Fig. 4A) or IL-10 (not shown). There were no differences between the two strains in the proportion of CD4+ cells that expressed Foxp3, and IL-10 production from Foxp3+CD4+ cells from the lesions was comparable (Fig. 4B). The severe pathology and IL-17+CD4+ cells were also associated with an increase in cellular infiltrate expressing macrophage and neutrophil markers (Fig. 4C). In agreement with the data from lesional cells, antigen re-stimulated dLN CD4+ cells from WSX-1-/- mice had a reduced frequency of IL-10+IFN-γ+ producers (1.4% vs. 0.3%) and reduced IL-10-IFN-γ+ producers (4.0% vs. 2.0%) when compared to wild type mice (Fig. 5A). IL-17A was also detected in WSX-1-/- mice, but not from wild type mice. Further analysis of lymph node cells revealed that IL-17A was not detected from non-CD4+ cells (data not shown). To confirm that the cytokines analyzed at the single cell level corresponded to total amounts of cytokines produced, the supernatants of antigen re-stimulated dLN CD4+ cells were analyzed by ELISA. IFN-γ was secreted in high concentration by cells from C57BL/6 mice, and modestly though significantly reduced by cells from WSX-1-/- mice (Fig. 5B). IL-17 was secreted in concentrations comparable with IFN-γ in WSX-1-/- mice, while nearly absent in C57BL/6 mice. IL-10 was significantly reduced in WSX-1-/- mice, but not absent, revealing the presence of an IL-27 independent pathway for the induction of IL-10. Finally, IL-4 was produced at higher amounts in WSX-1-/- mice, consistent with the known ability of IL-27 to suppress Th2 cells. IL-4 was secreted in concentrations ~1000 lower than IFN-γ or IL-17A, and was not detectable by flow cytometry. Taken together, the results demonstrate multiple roles for IL-27 that influence immunopathology and cytokine regulation of Th1, Th2, and Th17 cells.

Figure 3
Infection of WSX-1-/- mice with L. major NIH/Sd results in increased pathology but equivalent parasite burdens compared to C57BL/6 mice. C57BL/6 and WSX-1-/- mice were infected intradermally with 1000 metacyclic L. major NIH/Sd parasites. Lesion scores ...
Figure 4
Lesions of L. major NIH/Sd infected WSX-1-/- mice contain CD4+ cells producing less IL-10 and IFN-γ and more IL-17. (A) Pooled CD4+ cells from the ear lesions of mice, 11 weeks post-infection, re-stimulated with PMA and ionomycin in vitro and ...
Figure 5
CD4+ cells from draining lymph nodes of L. major NIH/Sd infected WSX-1-/- mice have altered cytokine production. Eleven weeks after intradermal inoculation, mice were sacrificed and CD4+ cells from the draining lymph nodes were purified, and pooled cells ...

Neutralization of IL-4 partially restores Th1 development in WSX-1-/- mice

Although IL-10 production in WSX-1-/- mice was reduced, it was accompanied by reciprocal changes in IFN-γ and IL-4, suggesting the lack of increased effector function may have been the result of suppressed Th1 development mediated by IL-4. Previous studies involving a healing L. major infection model in C57BL/6 mice, for which Th1 responses were not subject to such powerful IL-10 mediated control, demonstrated that an early and transient burst of IL-4 was subject to suppression by IL-27, thereby dictating the requirement for IL-27 in early Th1 development. Neutralization of IL-4 early in the infection, or the absence of IL-4 at later stages in the infection, rendered IL-27 dispensable (16). To determine if the increased IL-4 in WSX-1-/- mice infected with L. major NIH/Sd was responsible for preventing the increased parasite killing that was expected to occur as a consequence of reduced IL-10, anti-IL-4 neutralizing antibody was administered during the first five weeks of infection. When analyzed at 11 weeks post-infection, the anti-IL-4 treated WSX-1-/- mice had a slight increase in the proportion of CD4+ cells producing IFN-γ at the infection site compared to control treated mice (Fig. 6A), although still reduced compared with C57BL/6 mice. Importantly, the increase in IFN-γ was not associated with a concomitant increase in IL-10. The treatment also resulted in an increase in IL-17+CD4+ cells in WSX-1-/- mice, indicating that IL-4 was also suppressing development of Th17 cells. The proportion of IFN-γ+CD4+ cells in dLN of the anti-IL-4 treated WSX-1-/- mice was not increased over that in control treated mice, and was again lower than that in wild type mice (Fig. 6B). Most importantly, the treatment resulted in a modest, though statistically significant, reduction in parasite burdens in WSX-1-/- mice (Fig. 6C). By contrast, the wild type mice showed no change in IFN-γ and IL-10 following IL-4 neutralization, and no reduction in parasite burdens, consistent with previous data demonstrating that IL-4 was inconsequential to the non-cure phenotype of C57BL/6 mice (12). Taken together, the results show that, in the absence of IL-4, the increased parasite control resulting from decreased IL-10 in WSX-1-/- mice can be revealed. However, the increase in IL-4 in WSX-1-/- mice is only partly responsible for suppressing Th1 responses, and the role for IL-27 in promoting Th1 development during a chronic infection is not restricted to its regulation of IL-4 in the initial weeks of infection.

Figure 6
Neutralization of IL-4 in WSX-1-/- mice early in infection enhances resistance. Mice infected intradermally with L. major NIH/Sd were given weekly injections of neutralizing antibody to IL-4, beginning one day prior to infection and then weekly during ...

IL-27-independent IL-10 in WSX-1-/- is sufficient to inhibit the effector response

While IL-10 from T cells was significantly reduced in WSX-1-/- mice, its production may still have been sufficient to inhibit the effector response and prevent parasite clearance. To determine this, infected mice were treated with blocking antibody against the IL-10 receptor for a period of three weeks, beginning at six weeks post infection, and examined at 10 weeks. As previously described (12), anti-IL-10R treated C57BL/6 mice exhibited a significantly lower parasite burden (~100 fold) compared to control treated mice (Fig. 7A). WSX-1-/- mice receiving the same regimen also had a significantly reduced parasite burden, though not as great as C57BL/6 mice (~10 fold). Additionally, anti-IL-10R treated WSX-1-/- mice had an increase in lesion pathology compared to the control group (Fig. 7B), revealing that the IL-10 in WSX-1-/- mice can function to suppress pathology independently of IL-27. Treated C57BL/6 mice did not display increased pathology compared to controls. Cytokine analysis of WSX-1-/- lesions revealed an increase in the proportion of CD4+ cells producing IFN-γ (22.5% vs. 39.9%) (Fig. 7C), demonstrating that the IL-10 produced in the WSX-1-/- mice, while significantly reduced compared to C57BL/6 mice, was still sufficient to inhibit the effector response, particularly in the context of the shift in the Th1/Th2 balance that results from the absence of IL-27 signaling. Importantly, the frequency of IL-17+CD4+ cells in WSX-1-/- mice was unchanged following treatment, suggesting that, in the context of a mixed Th1/Th17 response, IL-10 functions preferentially to suppress Th1 cells.

Figure 7
IL-10 produced independently of IL-27 is sufficient to inhibit microbicidal activity. Chronically infected mice were treated with anti-IL-10R antibody once a week for a period of three weeks beginning at 6 weeks post-infection, and then sacrificed one ...

To determine if the inability of the anti-IL-10R treatment to more efficiently clear parasites from the chronic lesions in the WSX-1-/- mice was due to the continued influence of upregulated IL-4, IL27Ra-/- mice were co-injected with anti-IL10R and anti-IL-4 antibodies beginning at six weeks post-infection. Of note, the IL27R sufficient mice used in this experiment were generated from IL27Ra+/+ littermates backcrossed to C57BL/6 mice, rather than the C57BL/6 wild type mice used in the previous experiments. The comparison of lesion scores again revealed significant exacerbation of the dermal pathology in the IL27Ra-/- mice compared to IL27Ra+/+ mice beginning at four weeks post-infection (Fig. 8A). Following five bi-weekly injections, the parasite burdens in the lesions failed to reveal an additive effect of the combined anti-IL-4/IL-10R treatment, with each treatment group showing a comparable 10-20 fold reduction in the number of parasites in the site relative to the control treated mice (Fig 8B). Of note, the reduced parasite loads in the control treated IL27Ra+/+ compared to the control treated IL27Ra-/- mice, while not significant, may reflect the comparison in this experiment with the more appropriate backcrossed mice.

Figure 8
Combined treatment with anti-IL-10R and anti-IL-4 antibodies does not promote greater resistance compared to treatment with each antibody alone. (A) Leison scores in IL27Ra-/- (closed triangles) and IL27Ra+/+ mice (closed circles) following intradermal ...

Discussion

IL-10 has broad suppressive activity, functioning to limit collateral tissue damage during inflammation. The downside of this regulation can be an immune compromise leading to chronic infection. While IL-10 can be produced by a variety of cell types, we recently reported that CD4+ effectors simultaneously secreting IFN-γ and IL-10, cytokines with opposing functions, was sufficient to confer susceptibility in a non-healing model of L. major infection (13). The current studies were designed to address the role for IL-27 in regulating IL-10 from CD4+ cells in this model. In the absence of IL-27 signaling, antigen-specific IL-10 from CD4+ effectors in the lesion and draining lymph node was substantially reduced, accompanied by a decrease in the frequency of CD4+ cells secreting IFN-γ alone, and an increase in IL-4. This shift in the Th1/Th2 balance, reported previously in WSX-1-/- mice infected with a healing strain of L. major (15, 16), was sufficient, along with residual levels of IL-10, to offset any enhanced resistance that ablation of IL-27 dependent IL-10 production might confer. Importantly, there was a striking upregulation of IL-17+CD4+ cells, which along with compromised IL-10 secretion by CD4+ T cells, was associated with severe pathology at the infection site. Thus, by its ability to promote T-bet induction (25), and its inhibitory effects on parasite driven Th2 and Th17 development, the findings illustrate the multifaceted role for IL-27 as a key instructional cytokine for Th1 polarization, and its subsequent modulation of this response via a mechanism of IL-10 mediated feedback control.

One possible explanation for the reduction in IL-10+IFN-γ+ cells in WSX-1-/- mice is that the population of CD4+ cells producing IL-10 is directly proportional to IFN-γ, possibly due to intrinsic programs of cytokine gene expression during Th1 development, and/or direct effects of IFN-γ on IL-10 induction. The latter possibility is consistent with the observations that IL-12, or IL-12-induced IFN-γ, can activate Th1 cells to produce IL-10 (26-28). In the current studies, the increase in IFN-γ following anti-IL-4 or anti-IL-10R treatment in the WSX-1-/- mice did not result in a concomitant increase in IL-10, arguing for an IL-27 mediated mechanism of IL-10 induction that is independent of its effects on Th1 development.

The most striking observation of the current study is the development of severe pathology that correlates with both the reduced levels of IL-10, and the appearance of IL-17+CD4+ cells in the absence of IL-27 signaling. Th17 cells appear to be involved in host defense against certain extracellular bacterial and fungal pathogens, but they also mediate severe immunopathologies (29). There is limited information on the role of Th17 cells in experimental leishmaniasis. Local injection of IL-1β following L. amazonensis infection accelerated disease progression that was associated with, among other changes, increased activation of Th17 cells (30). IL-17 deficient BALB/c mice developed dramatically smaller lesions despite only a modest reduction in parasite loads (31), consistent with our observations that the elevated IL-17 conferred no beneficial effect in controlling parasite replication, but contributed to lesion pathology. The abundance of neutrophils in the lesion is likely due to the ability of IL-17 to regulate granulopoiesis, through the induction of G-CSF, and neutrophil recruitment, through regulation of CXC chemokines (32).

While IL-27 is known to inhibit Th17 development (17, 18), there are diseases where they do not develop in WSX-1-/- mice (33, 34). It is therefore likely that L. major infection induces factors, such as IL-6 and TGF-β that in the absence of IL-27 signaling, promote the development of Th17 cells. And while Th17 development can be inhibited by IFN-γ and IL-4 (35, 36), the mixed Th1/Th2/Th17 response seen in the L. major infected WSX-1-/- mice suggests that Th17 cells are able to develop in the presence of IFN-γ and IL-4. The current data also do not support a role for IL-10 in the inhibition of IL-17 from CD4+ cells, as has been reported (20, 37), since following IL-10R blockade initiated during chronic infection, the amount of IL-17 that was measured in the dLN was decreased relative to controls (not shown), and the proportion of IL-17+CD4+ cells in the lesions was unchanged. It is possible that the increased IFN-γ that resulted from the treatment was itself inhibitory to the Th17 response, or that the effect of IL-10 on Th17 development is confined to the initial priming of naïve cells.

The predominant function of IL-27 in promoting early Th1 development versus suppressing an exuberant inflammatory response is likely to be contextual. In Toxoplasma gondii infection, normal Th1 development and parasite clearance occurred in the absence of IL-27 signaling. The infection, however, resulted in a lethal immunopathology (38). Trypanosoma cruzi infection in WSX-1-/- mice resulted in heightened Th2 and Th1 responses, the former responsible for high parasitemia, the latter producing more severe pathology (39). Experimental tuberculosis in WSX-1-/- mice resulted in normal Th1 development, decreased bacterial burdens and increased pathology (40, 41). Similarly, L. donovani appeared not to require IL-27 for normal Th1 development, as TCCR-/- mice controlled parasite growth better than the wild type mice. The mice, however, developed more severe liver pathology (42). In the current studies, as in the prior studies involving L. major, IL-27 signaling was required for normal early Th1 development. The difference, particularly in reference to L. donovani, might be the early burst of IL-4 following L. major but not L. donovani infection in mice, requiring IL-27 mediated suppression for normal Th1 development in the former but not the latter. In the present studies a requirement for IL-27 in instructing early Th1 development appeared not to be limited to its suppression of IL-4, since neutralization of IL-4 did not restore full Th1 development. One explanation for this may be the comparatively low levels of IL-12 induced by L. major, independent of IL-4 (43), which in contrast to other infectious agents, e.g. T. gondii and M. tuberculosis, dictates the early requirement for IL-27 as a co-factor to increase responsiveness to IL-12. This requirement may explain why even the combined treatment with anti-IL-10R and anti-IL-4 failed to promote more efficient resistance in the WSX-1-/- mice, since strong Th1 inducing signals were still absent in these mice. While the influence of IL-27 signaling on Th1 development may represent a point of departure for these various pathogens, they all converge with respect to the role of IL-27 in limiting inflammation. The present data reinforce the critical role of IL-27 in inducing IFN-γ and IL-10 from CD4+ T cells, and in suppressing inappropriate Th17 subset development, in order to achieve a balance between protective immunity and pathology in response to an intracellular parasitic infection.

Acknowledgments

We thank Nico Ghilardi and Dragana Jankovic for provision of the IL27Ra-/- and IL27Ra+/+ mice, and Kim Beacht for expert technical assistance.

Footnotes

1This research was supported in part by the Intramural Research program of the NIH, NIAID, and in part by NIH grants AI 142334 and D43 TW007127.

References

1. Sacks D, Noben-Trauth N. The immunology of susceptibility and resistance to Leishmania major in mice. Nature Reviews. Immunology. 2002;2:845–858. [PubMed]
2. Moore KW, de Waal Malefyt R, Coffman RL, O’Garra A. Interleukin-10 and the interleukin-10 receptor. Annual Review of Immunology. 2001;19:683–765. [PubMed]
3. Melby PC, Andrade-Narvaez FJ, Darnell BJ, Valencia-Pacheco G, Tryon VV, Palomo-Cetina A. Increased expression of proinflammatory cytokines in chronic lesions of human cutaneous leishmaniasis. Infection & Immunity. 1994;62:837–842. [PMC free article] [PubMed]
4. Louzir H, Melby PC, Ben Salah A, Marrakchi H, Aoun K, Ben Ismail R, Dellagi K. Immunologic determinants of disease evolution in localized cutaneous leishmaniasis due to Leishmania major. J Infect Dis. 1998;177:1687–1695. [PubMed]
5. Gasim S, Elhassan AM, Khalil EA, Ismail A, Kadaru AM, Kharazmi A, Theander TG. High levels of plasma IL-10 and expression of IL-10 by keratinocytes during visceral leishmaniasis predict subsequent development of post-kala-azar dermal leishmaniasis. Clinical & Experimental Immunology. 1998;111:64–69. erratum appears in Clin Exp Immunol 1998 Jun;112(3):547. [PubMed]
6. Ismail A, El Hassan AM, Kemp K, Gasim S, Kadaru AE, Moller T, Kharazmi A, Theander TG. Immunopathology of post kala-azar dermal leishmaniasis (PKDL): T-cell phenotypes and cytokine profile. J Pathol. 1999;189:615–622. [PubMed]
7. Ghalib HW, Piuvezam MR, Skeiky YA, Siddig M, Hashim FA, el-Hassan AM, Russo DM, Reed SG. Interleukin 10 production correlates with pathology in human Leishmania donovani infections. J Clin Invest. 1993;92:324–329. [PMC free article] [PubMed]
8. Karp CL, el-Safi SH, Wynn TA, Satti MM, Kordofani AM, Hashim FA, Hag-Ali M, Neva FA, Nutman TB, Sacks DL. In vivo cytokine profiles in patients with kala-azar. Marked elevation of both interleukin-10 and interferon-gamma. Journal of Clinical Investigation. 1993;91:1644–1648. [PMC free article] [PubMed]
9. Kenney RT, Sacks DL, Gam AA, Murray HW, Sundar S. Splenic cytokine responses in Indian kala-azar before and after treatment. Journal of Infectious Diseases. 1998;177:815–818. [PubMed]
10. Nylen S, Maurya R, Eidsmo L, Manandhar KD, Sundar S, Sacks D. Splenic accumulation of IL-10 mRNA in T cells distinct from CD4+CD25+ (Foxp3) regulatory T cells in human visceral leishmaniasis. Journal of Experimental Medicine. 2007;204:805–817. [PMC free article] [PubMed]
11. Sacks D, Anderson C. Re-examination of the immunosuppressive mechanisms mediating non-cure of Leishmania infection in mice. Immunol Rev. 2004;201:225–238. [PubMed]
12. Anderson CF, Mendez S, Sacks DL. Nonhealing infection despite Th1 polarization produced by a strain of Leishmania major in C57BL/6 mice. Journal of Immunology. 2005;174:2934–2941. [PubMed]
13. Anderson CF, Oukka M, Kuchroo VJ, Sacks D. CD4(+)CD25(-)Foxp3(-) Th1 cells are the source of IL-10-mediated immune suppression in chronic cutaneous leishmaniasis. Journal of Experimental Medicine. 2007;204:285–297. [PMC free article] [PubMed]
14. Kastelein RA, Hunter CA, Cua DJ. Discovery and biology of IL-23 and IL-27: related but functionally distinct regulators of inflammation. Annual Review of Immunology. 2007;25:221–242. [PubMed]
15. Yoshida H, Hamano S, Senaldi G, Covey T, Faggioni R, Mu S, Xia M, Wakeham AC, Nishina H, Potter J, Saris CJ, Mak TW. WSX-1 is required for the initiation of Th1 responses and resistance to L. major infection. Immunity. 2001;15:569–578. [PubMed]
16. Artis D, Johnson LM, Joyce K, Saris C, Villarino A, Hunter CA, Scott P. Cutting edge: early IL-4 production governs the requirement for IL-27-WSX-1 signaling in the development of protective Th1 cytokine responses following Leishmania major infection. Journal of Immunology. 2004;172:4672–4675. [PubMed]
17. Stumhofer JS, Laurence A, Wilson EH, Huang E, Tato CM, Johnson LM, Villarino AV, Huang Q, Yoshimura A, Sehy D, Saris CJ, O’Shea JJ, Hennighausen L, Ernst M, Hunter CA. Interleukin 27 negatively regulates the development of interleukin 17-producing T helper cells during chronic inflammation of the central nervous system. Nature Immunology. 2006;7:937–945. [PubMed]
18. Batten M, Li J, Yi S, Kljavin NM, Danilenko DM, Lucas S, Lee J, de Sauvage FJ, Ghilardi N. Interleukin 27 limits autoimmune encephalomyelitis by suppressing the development of interleukin 17-producing T cells. Nature Immunology. 2006;7:929–936. see comment. [PubMed]
19. Stumhofer JS, Silver JS, Laurence A, Porrett PM, Harris TH, Turka LA, Ernst M, Saris CJM, O’Shea JJ, Hunter CA. Interleukins 27 and 6 induce STAT3-mediated T cell production of interleukin 10. Nat Immunol. 2007;8:1363–1371. [PubMed]
20. Fitzgerald DC, Zhang G-X, El-Behi M, Fonseca-Kelly Z, Li H, Yu S, Saris CJ, Gran B, Ciric B, Rostami A. Suppression of autoimmune inflammation of the central nervous system by interleukin 10 secreted by interleukin 27-stimulated T cells. Nat Immunol. 2007;8:1372–1379. [PubMed]
21. Batten M, Kljavin NM, Li J, Walter MJ, de Sauvage FJ, Ghilardi N. Cutting edge: IL-27 is a potent inducer of IL-10 but not FoxP3 in murine T cells. J Immunol. 2008;180:2752–2756. [PubMed]
22. Chen Q, Ghilardi N, Wang H, Baker T, Xie MH, Gurney A, Grewal IS, de Sauvage FJ. Development of Th1-type immune responses requires the type I cytokine receptor TCCR. Nature. 2000;407:916–920. [PubMed]
23. Spath GF, Beverley SM. A lipophosphoglycan-independent method for isolation of infective Leishmania metacyclic promastigotes by density gradient centrifugation. Experimental Parasitology. 2001;99:97–103. [PubMed]
24. Belkaid Y, Mendez S, Lira R, Kadambi N, Milon G, Sacks D. A natural model of Leishmania major infection reveals a prolonged “silent” phase of parasite amplification in the skin before the onset of lesion formation and immunity. Journal of Immunology. 2000;165:969–977. [PubMed]
25. Lucas S, Ghilardi N, Li J, de Sauvage FJ. IL-27 regulates IL-12 responsiveness of naive CD4+ T cells through Stat1-dependent and -independent mechanisms. Proceedings of the National Academy of Sciences of the United States of America. 2003;100:15047–15052. [PubMed]
26. Gerosa F, Paganin C, Peritt D, Paiola F, Scupoli MT, Aste-Amezaga M, Frank I, Trinchieri G. Interleukin-12 primes human CD4 and CD8 T cell clones for high production of both interferon-gamma and interleukin-10. J Exp Med. 1996;183:2559–2569. [PMC free article] [PubMed]
27. Windhagen A, Anderson DE, Carrizosa A, Williams RE, Hafler DA. IL-12 induces human T cells secreting IL-10 with IFN-gamma. J Immunol. 1996;157:1127–1131. [PubMed]
28. Shaw MH, Freeman GJ, Scott MF, Fox BA, Bzik DJ, Belkaid Y, Yap GS. Tyk2 negatively regulates adaptive Th1 immunity by mediating IL-10 signaling and promoting IFN-gamma-dependent IL-10 reactivation. J Immunol. 2006;176:7263–7271. [PubMed]
29. Weaver CT, Hatton RD, Mangan PR, Harrington LE. IL-17 family cytokines and the expanding diversity of effector T cell lineages. Annual Review of Immunology. 2007;25:821–852. [PubMed]
30. Vargas-Inchaustegui DA, Xin L, Soong L. Leishmania braziliensis infection induces dendritic cell activation, ISG15 transcription, and the generation of protective immune responses. J Immunol. 2008;180:7537–7545. [PMC free article] [PubMed]
31. Lopez Kostka S, Dinges S, Griewank K, Iwakura Y, Udey MC, von Stebut E. IL-17 promotes progression of cutaneous leishmaniasis in susceptible mice. J Immunol. 2009;182:3039–3046. [PMC free article] [PubMed]
32. Ouyang W, Kolls JK, Zheng Y. The biological functions of T helper 17 cell effector cytokines in inflammation. Immunity. 2008;28:454–467. [PMC free article] [PubMed]
33. Shainheit MG, Saraceno R, Bazzone LE, Rutitzky LI, Stadecker MJ. Disruption of interleukin-27 signaling results in impaired gamma interferon production but does not significantly affect immunopathology in murine schistosome infection. Infection & Immunity. 2007;75:3169–3177. [PMC free article] [PubMed]
34. Cao Y, Doodes PD, Glant TT, Finnegan A. IL-27 induces a Th1 immune response and susceptibility to experimental arthritis. Journal of Immunology. 2008;180:922–930. [PubMed]
35. Harrington LE, Hatton RD, Mangan PR, Turner H, Murphy TL, Murphy KM, Weaver CT. Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nature Immunology. 2005;6:1123–1132. [PubMed]
36. Park H, Li Z, Yang XO, Chang SH, Nurieva R, Wang YH, Wang Y, Hood L, Zhu Z, Tian Q, Dong C. A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nature Immunology. 2005;6:1133–1141. [PMC free article] [PubMed]
37. Yongpeng Gu JYXOWLHLJYJBS-HCLMJCUHX. Interleukin 10 suppresses Th17 cytokines secreted by macrophages and T cells. European Journal of Immunology. 2008;38:1807–1813. [PMC free article] [PubMed]
38. Villarino A, Hibbert L, Lieberman L, Wilson E, Mak T, Yoshida H, Kastelein RA, Saris C, Hunter CA. The IL-27R (WSX-1) is required to suppress T cell hyperactivity during infection. Immunity. 2003;19:645–655. [PubMed]
39. Hamano S, Himeno K, Miyazaki Y, Ishii K, Yamanaka A, Takeda A, Zhang M, Hisaeda H, Mak TW, Yoshimura A, Yoshida H. WSX-1 is required for resistance to Trypanosoma cruzi infection by regulation of proinflammatory cytokine production. Immunity. 2003;19:657–667. [PubMed]
40. Pearl JE, Khader SA, Solache A, Gilmartin L, Ghilardi N, deSauvage F, Cooper AM. IL-27 signaling compromises control of bacterial growth in mycobacteria-infected mice. Journal of Immunology. 2004;173:7490–7496. [PubMed]
41. Holscher C, Holscher A, Ruckerl D, Yoshimoto T, Yoshida H, Mak T, Saris C, Ehlers S. The IL-27 receptor chain WSX-1 differentially regulates antibacterial immunity and survival during experimental tuberculosis. Journal of Immunology. 2005;174:3534–3544. [PubMed]
42. Rosas LE, Satoskar AA, Roth KM, Keiser TL, Barbi J, Hunter C, de Sauvage FJ, Satoskar AR. Interleukin-27R (WSX-1/T-cell cytokine receptor) gene-deficient mice display enhanced resistance to leishmania donovani infection but develop severe liver immunopathology. American Journal of Pathology. 2006;168:158–169. [PubMed]
43. Carrera L, Gazzinelli RT, Badolato R, Hieny S, Muller W, Kuhn R, Sacks DL. Leishmania promastigotes selectively inhibit interleukin 12 induction in bone marrow-derived macrophages from susceptible and resistant mice. J Exp Med. 1996;183:515–526. [PMC free article] [PubMed]