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Interleukin-27 (IL-27) is known to control primary CD4 + T cell responses during a variety of different infections, but its role in regulating memory CD4 + T responses has not been investigated in any model. In this study, we have examined the functional importance of IL-27 receptor (IL-27R) signaling in regulating the formation and maintenance of memory CD4 + T cells following malaria infection and in controlling their subsequent reactivation during secondary parasite challenge. We demonstrate that although the primary effector/memory CD4 + T cell response was greater in IL-27R-deficient (WSX-1 −/− ) mice following Plasmodium berghei NK65 infection than in wild-type (WT) mice, there were no significant differences in the size of the maintained memory CD4 + T population(s) at 20 weeks postinfection in the spleen, liver, or bone marrow of WSX-1 −/− mice compared with WT mice. However, the composition of the memory CD4 + T cell pool was slightly altered in WSX-1 −/− mice following clearance of primary malaria infection, with elevated numbers of late effector memory CD4 + T cells in the spleen and liver and increased production of IL-2 in the spleen. Crucially, WSX-1 −/− mice displayed significantly enhanced parasite control compared with WT mice following rechallenge with homologous malaria parasites. Improved parasite control in WSX-1 −/− mice during secondary infection was associated with elevated systemic production of multiple inflammatory innate and adaptive cytokines and extremely rapid proliferation of antigen-experienced T cells in the liver. These data are the first to demonstrate that IL-27R signaling plays a role in regulating the magnitude and quality of secondary immune responses during rechallenge infections.

Chronic inflammation in arterial wall that is driven by immune cells and cytokines plays pivotal roles in the development of atherosclerosis. Interleukin 27 (IL-27) is a member of the IL-12 family of cytokines that consists of IL-27p28 and Epstein-Barr virus induced gene 3 (EBI3) and has anti-inflammatory properties that regulate T cell polarization and cytokine production. IL-27-deficient ( Ldlr−/− Ebi3−/−) and IL-27 receptor-deficient ( Ldlr−/− WSX-1−/−) Ldlr−/− mice were generated and fed with a high-cholesterol diet to induce atherosclerosis. Roles of bone marrow-derived cells in vivo and macrophages in vitro were studied using bone marrow reconstitution by transplantation and cultured peritoneal macrophages, respectively. We demonstrate that mice lacking IL-27 or IL-27 receptor are more susceptible to atherosclerosis compared with wild type due to enhanced accumulation and activation of macrophages in arterial walls. The number of circulating proinflammatory Ly6Chimonocytes showed no significant difference between wild-type mice and mice lacking IL-27 or IL-27 receptor. Administration of IL-27 suppressed the development of atherosclerosis in vivo and macrophage activation in vitro that was indicated by increased uptake of modified low-density lipoprotein and augmented production of proinflammatory cytokines. These findings define a novel inhibitory role for IL-27 in atherosclerosis that regulates macrophage activation in mice.

IFN-β, IL-27, and IL-10 have been shown to exert a range of similar immunoregulatory effects in murine and human experimental systems, particularly in Th1- and Th17-mediated models of autoimmune inflammatory disease. In this study we sought to translate some of our previous findings in murine systems to human in vitro models and delineate the interdependence of these different cytokines in their immunoregulatory effects. We demonstrate that human IL-27 upregulates IL-10 in T cell–activated PBMC cultures and that IFN-β drives IL-27 production in activated monocytes. IFN-β–driven IL-27 is responsible for the upregulation of IL-10, but not IL-17 suppression, by IFN-β in human PBMCs. Surprisingly, IL-10 is not required for the suppression of IL-17 by either IL-27 or IFN-β in this model or in de novo differentiating Th17 cells, nor is IL-27 signaling required for the suppression of experimental autoimmune encephalomyelitis (EAE) by IFN-β in vivo. Furthermore, and even more surprisingly, IL-10 is not required for the suppression of Th17-biased EAE by IL-27, in sharp contrast to Th1-biased EAE. In conclusion, IFN-β and IL-27 both induce human IL-10, both suppress human Th17 responses, and both suppress murine EAE. However, IL-27 signaling is not required for the therapeutic effect of IFN-β in EAE. Suppression of Th17-biased EAE by IL-27 is IL-10–independent, in contrast to its mechanism of action in Th1-biased EAE. Taken together, these findings delineate a complex set of interdependent and independent immunoregulatory mechanisms of IFN-β, IL-27, and IL-10 in human experimental models and in murine Th1- and Th17-driven autoimmunity.

Rationale: Atherosclerosis is a chronic inflammatory disease of the arterial wall. Several proinflammatory cytokines are known to promote atherosclerosis, but less is known about the physiological role of anti-inflammatory cytokines. Interleukin (IL)-27 is a recently discovered member of the IL-6/IL-12 family. The IL-27 receptor is composed of IL-27 receptor A (WSX-1) and gp130 and is required for all established IL-27 signaling pathways. The expression of the IL-27 subunit Ebi3 is elevated in human atheromas, yet its function in atherosclerosis remains unknown. Objective: The aim of this study was to test the role of IL-27 receptor signaling in immune cells in atherosclerosis development. Methods and Results: Atherosclerosis-prone Ldlr −/− mice transplanted with Il27ra −/− bone marrow and fed Western diet for 16 weeks developed significantly larger atherosclerotic lesions in aortic roots, aortic arches, and abdominal aortas. Augmented disease correlated with increased accumulation of CD45 + leukocytes and CD4 + T cells in the aorta, which produced increased amounts of IL-17A and tumor necrosis factor. Several chemokines, including CCL2, were upregulated in the aortas of Ldlr −/− mice receiving Il27ra −/− bone marrow, resulting in accumulation of CD11b + and CD11c + macrophages and dendritic cells in atherosclerotic aortas. Conclusions: The absence of anti-inflammatory IL-27 signaling skews immune responses toward T-helper 17, resulting in increased production of IL-17A and tumor necrosis factor, which in turn enhances chemokine expression and drives the accumulation of proatherogenic myeloid cells in atherosclerotic aortas. These findings establish a novel antiatherogenic role for IL-27 receptor signaling, which acts to suppress the production of proinflammatory cytokines and chemokines and to curb the recruitment of inflammatory myeloid cells into atherosclerotic aortas.

Structure-activity relationship (SAR) investigations of a novel class of triazolopyridazinone p38α mitogen activated protein kinase (MAPK) inhibitors are disclosed. From these studies, increased in vitro potency was observed for 2,6-disubstituted phenyl moieties and N-ethyl triazolopyridazinone cores due to key contacts with Leu108, Ala157 and Val38. Further investigation led to the identification of three compounds, 3g, 3j and 3m that are highly potent inhibitors of LPS-induced MAPKAP kinase 2 (MK2) phosphorylation in 50% human whole blood (hWB), and possess desirable in vivo pharmacokinetic and kinase selectivity profiles.

Inflammation driven by immune cells and pro-inflammatory cytokines is implicated in pancreatic β-cell injury, leading to the development of diabetes mellitus. IL-27, a cytokine consisting of IL-27p28 and Epstein-Barr virus-induced gene 3 (EBI3), binds a membrane-bound heterodimeric receptor consisting of the IL-27 receptor α chain (WSX-1) and gp130. IL-27 has anti-inflammatory properties that regulate T-cell polarization and cytokine production. We evaluated blood glucose and islet proinsulin concentrations, inflammatory cell infiltration in islets, and expression of IL-1β mRNA in pancreas in wild-type (WT), EBI3(-/-), and WSX-1(-/-) mice treated with streptozotocin (STZ). Hyperglycemia was augmented in EBI3(-/-) and WSX-1(-/-) mice compared with WT mice. Islet proinsulin levels after STZ treatment were lower in EBI3(-/-) and WSX-1(-/-) mice than in WT mice. The infiltration of islets by F4/80(+)CD11c(-)7/4(-) macrophages, CD4(+) T cells, and CD8(+) T cells was increased in EBI3(-/-) and WSX-1(-/-) mice compared with WT mice. The administration of recombinant IL-27, compared with control, decreased the blood glucose level, immune cell infiltration into islets, and IL-1β mRNA expression in the pancreas and increased islet proinsulin levels in WT and EBI3(-/-) mice. Thus, IL-27 inhibits STZ-induced hyperglycemia and pancreatic islet inflammation in mice and represents a potential novel therapeutic approach for β-cell protection in diabetes.

Mechanisms regulating intestinal T-cell accumulation during inflammation have considerable therapeutic value. In this study, LPS increased Staphylococcus aureus enterotoxin A-specific T cells in the gut through induction of IL-12 family members. Mice deficient in IL-12 (p35(-/-)) favored T(h)17 differentiation in lamina propria, whereas mice lacking both IL-12 and IL-23 (p40(-/-)) produced significantly fewer T(h)17 cells. However, serum analysis revealed that IL-27p28 was much higher and sustained following LPS injection than other IL-12 family cytokines. Strikingly, WSX-1 (IL-27Rα) deficiency resulted in log-fold increases in lamina propria T(h)17 cells without affecting T(h)1 numbers. These results may be explained by increased expression of α4β7 on WSX-1-deficient T cells after immunization. WSX-1-deficient regulatory T cells (Tregs) were also perturbed, producing more IL-17 and less IL-10 than wild-type Tregs. Thus, IL-27 blockade may provide a new pathway to improve mucosal vaccination.

The heterodimeric cytokine interleukin 27 (IL-27) signals through the IL-27Rα subunit of its receptor, combined with gp130, a common receptor chain used by several cytokines, including IL-6. Notably, the IL-27 subunits p28 (IL-27p28) and EBI3 are not always expressed together, which suggests that they may have unique functions. Here we show that IL-27p28, independently of EBI3, antagonized cytokine signaling through gp130 and IL-6-mediated production of IL-17 and IL-10. Similarly, the ability to generate antibody responses was dependent on the activity of gp130-signaling cytokines. Mice transgenic for expression of IL-27p28 showed a substantial defect in the formation of germinal centers and antibody production. Thus, IL-27p28, as a natural antagonist of gp130-mediated signaling, may be useful as a therapeutic for managing inflammation mediated by cytokines that signal through gp130.

Successful resolution of malaria infection requires induction of proinflammatory immune responses that facilitate parasite clearance; however, failure to regulate this inflammation leads to immune-mediated pathology. The pathways that maintain this immunological balance during malaria infection remain poorly defined. In this study, we demonstrate that IL-27R–deficient (WSX-1−/−) mice are highly susceptible to Plasmodium berghei NK65 infection, developing exacerbated Th1-mediated immune responses, which, despite highly efficient parasite clearance, lead directly to severe liver pathology. Depletion of CD4+ T cells—but not CD8+ T cells—prevented liver pathology in infected WSX-1−/− mice. Although WSX-1 signaling was required for optimal IL-10 production by CD4+ T cells, administration of rIL-10 failed to ameliorate liver damage in WSX-1−/− mice, indicating that additional, IL-10–independent, protective pathways are modulated by IL-27R signaling during malaria infection. These data are the first to demonstrate the essential role of IL-27R signaling in regulating effector T cell function during malaria infection and reveal a novel pathway that might be amenable to manipulation by drugs or vaccines.

The p38α mitogen-activated protein (MAP) kinase is a central signaling molecule in many proinflammatory pathways, regulating the cellular response to a multitude of external stimuli including heat, ultraviolet radiation, osmotic shock, and a variety of cytokines especially interleukin-1β and tumor necrosis factor α. Thus, inhibitors of this enzyme are postulated to have significant therapeutic potential for the treatment of rheumatoid arthritis, inflammatory bowel disease, and Crohn’s disease, as well as other diseases where aberrant cytokine signaling is the driver of disease. In this communication, we describe a novel class of 7-alkyl-1,5-bis-aryl-pyrazolopyridinone-based p38α inhibitors. In particular, compound 3f is highly potent in the enzyme and cell-based assays, selective in an Ambit kinase screen, and efficacious (ED50 ≤ 0.01 mg/kg) in the rat collagen induced arthritis (CIA) model.

We report that the asthma drugs cromolyn disodium and nedocromil sodium are potent G-protein-coupled receptor 35 (GPR35) agonists. We utilized calcium flux and inositol phosphate accumulation assays to examine the pharmacology of these asthma drugs on the human, mouse and rat GPR35. The compounds were more potent on the human GPR35 than on mouse and rat receptors. In contrast, zaprinast, a known GPR35 agonist, was more potent on mouse and rat GPR35 than the human ortholog. We show by quantitative PCR that GPR35 is expressed in human mast cells, human basophils and human eosinophils. We also demonstrate that GPR35 mRNA is upregulated upon challenge with IgE antibodies. We show that, unlike zaprinast, a potent phosphodiesterase 5 (PDE5) inhibitor, cromolyn disodium and nedocromil sodium lack inhibitory activity towards PDE5. These findings suggest that GPR35 may play an important role in mast cell biology and be a potential target for the treatment of asthma.

The p38 mitogen-activated protein kinase (MAPK) is a central signaling molecule in many proinflammatory pathways, regulating the cellular response to a multitude of external stimuli including heat, ultraviolet radiation, osmotic shock, and a variety of cytokines especially interleukin-1beta and tumor necrosis factor alpha. Thus, inhibitors of this enzyme are postulated to have significant therapeutic potential for the treatment of rheumatoid arthritis, inflammatory bowel disease, osteoporosis, and many other diseases where aberrant cytokine signaling is the driver of disease. Herein, we describe a novel class of 3-amino-7-phthalazinylbenzoisoxazole-based inhibitors. With relatively low molecular weight, these compounds are highly potent in enzyme and cell-based assays, with minimal protein shift in 50% human whole blood. Compound 3c was efficacious (ED 50 = 0.05 mg/kg) in the rat collagen induced arthritis (CIA) model.

Excessive inflammation occurs during infection and autoimmunity in mice lacking the α-subunit of the interleukin 27 (IL-27) receptor. The molecular mechanisms underlying this increased inflammation are incompletely understood. Here we report that IL-27 upregulated IL-10 in effector T cells that produced interferon-γ and expressed the transcription factor T-bet but did not express the transcription factor Foxp3. These IFN-γ+T-bet+Foxp3− cells resembled effector T cells that have been identified as the main source of host-protective IL-10 during inflammation. IL-27-induced production of IL-10 was associated with less secretion of IL-17, and exogenous IL-27 reduced the severity of adoptively transferred experimental autoimmune encephalomyelitis by a mechanism dependent on IL-10. Our data show that IL-27-induced production of IL-10 by effector T cells contributes to the immunomodulatory function of IL-27.

Interleukin 10 (IL-10) has a prominent function in regulating the balance between protective and pathological T cell responses. Consistent with that activity, many sources of this cytokine are found in vivo, including from myeloid cells and a variety of T cell subsets. However, although there are many pathways that regulate innate production of IL-10, the factors that govern its synthesis by the adaptive response are poorly understood. Here we report that IL-27 and IL-6 induced T helper type 1 and type 2 cells, as well as T helper cells that produce IL-17, to secrete IL-10. This effect was dependent on the transcription factors STAT1 and STAT3 for IL-27 and on STAT3 for IL-6. Our studies identify a previously unknown pathway that allows the immune system to temper inflammatory responses.

Suppressor of cytokine signaling (SOCS) proteins regulate the intensity and duration of cytokine responses. SOCS3 is expressed in peripheral T cells, and recent reports have suggested that overexpression of SOCS3 modulates antigen- and/or costimulation-induced T-cell activation. To study the role of SOCS3 in the regulation of T-cell activation, we used a conditional gene-targeting strategy to generate mice that lack SOCS3 in T/natural killer T cells (Socs3(DeltaLck/DeltaLck) mice). SOCS3-deficient CD8 T cells showed greater proliferation than wild-type cells in response to T-cell receptor (TCR) ligation despite normal activation of signaling pathways downstream from TCR or CD28 receptors. Signaling in response to the gp130 cytokines interleukin (IL)-6 and IL-27 was prolonged in Socs3(DeltaLck/DeltaLck) T cells, and T cells from gp130(Y757F/Y757F) mice, in which the SOCS3-binding site on gp130 is ablated, showed a striking similarity to SOCS3-deficient CD8 T cells. Although the proliferative defect of Socs3(DeltaLck/DeltaLck) T cells was not rescued in the absence of IL-6, suppression of IL-27 signaling was found to substantially reduce anti-CD3-induced proliferation. We conclude that enhanced responses to TCR ligation by SOCS3-deficient CD8 T cells are not caused by aberrant TCR-signaling pathways but, rather, that increased IL-27 signaling drives unregulated proliferation in the absence of SOCS3.

G-CSF and GM-CSF play important roles in regulating neutrophil production, survival, differentiation, and function. However, we have shown previously that G-CSF/GM-CSF double-deficient [knockout (KO)] mice still develop a profound neutrophilia in bone marrow and blood after infection with Candida albicans. This finding suggests the existence of other systems, which can regulate emergency neutrophil production. We have now developed an “in vitro” technique to detect and characterize a neutrophil-promoting activity (NPA) in the media conditioned by mouse embryonic fibroblasts (MEFs) derived from G-CSF−/−/GM-CSF−/− mice. NPA is produced in vitro by the MEFs after stimulation with LPS or heat-inactivated C. albicans. Although M-CSF added directly to bone marrow cultures does not sustain granulocyte production, our studies indicate that production of NPA requires activation of the M-CSF receptor (c-fms). First, G-CSF−/−/GM-CSF−/− MEFs produce high levels of NPA after stimulation with LPS or C. albicans, and G-CSF/GM-CSF/M-CSF triple-KO MEFs do not. Second, the production of NPA by the G-CSF−/−/GM-CSF−/− MEFs is reduced significantly upon incubation with neutralizing antibodies to M-CSF or c-fms. Third, NPA production by G-CSF−/−/GM-CSF−/−/M-CSF−/− fibroblasts is enhanced by supplementing culture medium with M-CSF. Thus, stimulation of c-fms by M-CSF is a prerequisite for the production of NPA.

Although the ability of IL-27 to promote T cell responses is well documented, the anti-inflammatory properties of this cytokine remain poorly understood. The current work demonstrates that during infection with Toxoplasma gondii, IL-27R-deficient mice generate aberrant IL-2 responses that are associated with the development of a lethal inflammatory disease. Because in vivo depletion of IL-2 prolongs the survival of infected IL-27R−/− mice, these data suggest that IL-27 curbs the development of immunopathology by limiting parasite-induced IL-2 production. Consistent with this hypothesis, IL-27R−/− CD4+ T cells produce more IL-2 than wild-type counterparts during in vitro differentiation, and when rIL-27 is introduced, it can suppress the expression of IL-2 mRNA and protein by the latter group. Additionally, these studies reveal that, like IL-27, IL-12 can inhibit IL-2 production, and although each employs distinct mechanisms, they can synergize to enhance the effect. In contrast, this property is not shared by closely related cytokines IL-6 and IL-23. Thus, while traditionally viewed as proinflammatory agents, the present findings establish that IL-27 and IL-12 cooperate to limit the availability of IL-2, a potent T cell growth and survival factor. Moreover, because the current studies demonstrate that both can induce expression of suppressor of cytokine signaling 3, a protein that tempers cytokine receptor signaling, they also suggest that IL-27 and IL-12 share additionally inhibitory properties.

Although recent studies have described IL-27 and its receptor, WSX-1, as promoters of Th1 differentiation in naive CD4+ T cells, the data presented here indicate that signaling through this receptor is involved in limiting the intensity and duration of T cell activity. When WSX-1-deficient mice are infected with the intracellular pathogen Toxoplasma gondii, they establish protective T cell responses, characterized by production of inflammatory cytokines and control of parasite replication. However, infected WSX-1-/- mice are unable to downregulate these protective responses, and develop a lethal, T cell-mediated inflammatory disease. This pathology was characterized by the excessive production of IFN-gamma, persistence of highly activated T cells, and enhanced T cell proliferation in vivo. Together, these findings demonstrate that WSX-1 is not required for the generation of IFN-gamma-mediated immunity to this parasitic infection and identify a novel function for this receptor as a potent antagonist of T cell-mediated, immune hyperactivity.

The p38 mitogen-activated protein kinase (MAPK) plays an important role in the production of proinflammatory cytokines, making it an attractive target for the treatment of various inflammatory diseases. A series of pyridazinopyridinone compounds were designed as novel p38 kinase inhibitors. A structure-activity investigation identified several compounds possessing excellent potency in both enzyme and human whole blood assays. Among them, compound 31 exhibited good pharmacokinetic properties and showed excellent selectivity against other related kinases. In addition, 31 demonstrated efficacy in a collagen-induced arthritis disease model in rats.

The p38alpha mitogen-activated protein (MAP) kinase is a central signaling molecule in many proinflammatory pathways, regulating the cellular response to a multitude of external stimuli including heat, ultraviolet radiation, osmotic shock, and a variety of cytokines especially interleukin-1beta and tumor necrosis factor alpha. Thus, inhibitors of this enzyme are postulated to have significant therapeutic potential for the treatment of rheumatoid arthritis, inflammatory bowel disease, and Crohn's disease, as well as other diseases where aberrant cytokine signaling is the driver of disease. In this communication, we describe a novel class of 7-alkyl-1,5-bis-aryl-pyrazolopyridinone-based p38alpha inhibitors. In particular, compound 3f is highly potent in the enzyme and cell-based assays, selective in an Ambit kinase screen, and efficacious (ED(50)or = 0.01 mg/kg) in the rat collagen induced arthritis (CIA) model.

IL-27 limits CD4+ TH17 cell development in vitro and during inflammatory responses in the CNS. However, whether IL-27-IL-27R interactions regulate the homeostasis or function of CD4+ T cell populations in the intestine is unknown. To test this, we examined CD4+ T cell populations in the intestine of wild-type and IL-27R−/− mice. Naive IL-27R−/− mice exhibited a selective decrease in the frequency of IFN-γ producing CD4+ TH1 cells and an increase in the frequency of TH17 cells in gut-associated lymphoid tissues. Associated with elevated expression of IL-17A, IL-27R−/− mice exhibited earlier onset and significantly increased severity of clinical disease compared with wild-type controls in a murine model of intestinal inflammation. Rag−/−/IL-27R−/− mice were also more susceptible than Rag−/− mice to development of dextran sodium sulfate-induced intestinal inflammation, indicating an additional role for IL-27-IL-27R in the regulation of innate immune cell function. Consistent with this, IL-27 inhibited proinflammatory cytokine production by activated neutrophils. Collectively, these data identify a role for IL-27-IL-27R interaction in controlling the homeostasis of the intestinal T cell pool and in limiting intestinal inflammation through regulation of innate and adaptive immune cell function.

A novel class of pyrazolopyridazine p38α mitogen-activated protein kinase (MAPK) inhibitors is disclosed. A structure activity relationship (SAR) investigation was conducted driven by the ability of these compounds to inhibit the p38α enzyme, the secretion of TNFα in a LPS-challenged THP1 cell line and TNFα-induced production of IL-8 in the presence of 50% human whole blood (hWB). This study resulted in the discovery of several inhibitors with IC50 values in the single-digit nanomolar range in hWB. Further investigation of the pharmacokinetic profiles of these lead compounds led to the identification of three potent and orally bioavailable p38α inhibitors 2h, 2m, and 13h. Inhibitor 2m was found to be highly selective for p38α/β over a panel of 402 other kinases in Ambit screening, and was highly efficacious in vivo in the inhibition of TNFα production in LPS-stimulated Lewis rats with an ED50 of ca. 0.08 mg/kg.

Studies have focused on the events that influence the development of interleukin 17 (IL-17)-producing T helper cells (T(H)-17 cells) associated with autoimmunity, such as experimental autoimmune encephalitis, but relatively little is known about the cytokines that antagonize T(H)-17 cell effector responses. Here we show that IL-27 receptor-deficient mice chronically infected with Toxoplasma gondii developed severe neuroinflammation that was CD4+ T cell dependent and was associated with a prominent IL-17 response. In vitro, treatment of naive primary T cells with IL-27 suppressed the development T(H)-17 cells induced by IL-6 and transforming growth factor-beta, which was dependent on the intracellular signaling molecule STAT1 but was independent of inhibition of IL-6 signaling mediated by the suppressor protein SOCS3. Thus IL-27, a potent inhibitor of T(H)-17 cell development, may be a useful target for treating inflammatory diseases mediated by these cells.

Previous reports have focused on the ability of IL-27 to promote naive T cell responses but the present study reveals that surface expression of WSX-1, the ligand-specific component of the IL-27R, is low on these cells and that highest levels are found on effector and memory CD4+ and CD8+ T cells. Accordingly, during infection with Toxoplasma gondii, in vivo T cell activation is associated with enhanced expression of WSX-1, and, in vitro, TCR ligation can induce expression of WSX-1 regardless of the polarizing (Th1/Th2) environment present at the time of priming. However, while these data establish that mitogenic stimulation promotes expression of WSX-1 by T cells, activation of NK cells and NKT cells prompts a reduction in WSX-1 levels during acute toxoplasmosis. Together, with the finding that IL-2 can suppress expression of WSX-1 by activated CD4+ T cells, these studies indicate that surface levels of the IL-27R can be regulated by positive and negative signals associated with lymphoid cell activation. Additionally, since high levels of WSX-1 are evident on resting NK cells, resting NKT cells, effector T cells, regulatory T cells, and memory T cells, the current work demonstrates that IL-27 can influence multiple effector cells of innate and adaptive immunity.

Although previous studies have investigated the role of IL-27/WSX-1 interactions in the regulation of Th1 responses, little is known about their role in regulating Th2-type responses. Studies presented in this work identify a direct role for IL-27/WSX-1 interactions in the negative regulation of type 2 responses independent of effects on type 1 cytokines. WSX-1−/− mice infected with the gastrointestinal helminth Trichuris muris displayed accelerated expulsion of parasites and the development of exaggerated goblet cell hyperplasia and mastocytosis in the gut due to increased production of Th2 cytokines. Enhanced mast cell activity in the absence of WSX-1 was consistent with the ability of wild-type mast cells to express this receptor. In addition, IL-27 directly suppressed CD4+ T cell proliferation and Th2 cytokine production. Together, these studies identify a novel role for IL-27/WSX-1 in limiting innate and adaptive components of type 2 immunity at mucosal sites.

There are conflicting reports on the requirements for the IL-27-WSX-1 pathway in the development of Th type 1 responses and resistance to intracellular pathogens; although early IFN-γ production and resistance to Leishmania major are impaired in the absence of WSX-1 signaling, WSX-1−/− mice generate robust IFN-γ responses and control infection with other intracellular protozoan pathogens. In this report, we resolve these conflicting observations and demonstrate that, in the absence of IL-4, WSX-1 is not required for early IFN-γ production and control of L. major. Thus, the requirement for WSX-1 signaling in Th type 1 cell differentiation is restricted to conditions in which IL-4 is produced.

Infection with influenza virus can result in massive pulmonary infiltration and potentially fatal immunopathology. Understanding the endogenous mechanisms that control immunopathology could provide a key to novel adjunct therapies for this disease. Here we show that the cytokine IL-27 plays a crucial role in protection from exaggerated inflammation during influenza virus infection. Using Il-27ra −/− mice, IL-27 was found to limit immunopathology, neutrophil accumulation, and dampened TH1 or TH17 responses via IL-10–dependent and -independent pathways. Accordingly, the absence of IL-27 signals resulted in a more severe disease course and in diminished survival without impacting viral loads. Consistent with the delayed expression of endogenous Il-27p28 during influenza, systemic treatment with recombinant IL-27 starting at the peak of virus load resulted in a major amelioration of lung pathology, strongly reduced leukocyte infiltration and improved survival without affecting viral clearance. In contrast, early application of IL-27 impaired virus clearance and worsened disease. These findings demonstrate the importance of IL-27 for the physiological control of immunopathology and the potential value of well-timed IL-27 application to treat life-threatening inflammation during lung infection., Author Summary Annual epidemics of influenza result in 3 to 5 million cases of severe illness and approximately 300,000 deaths around the world. Although most patients infected with normal circulating influenza A viruses recover from the illness, complications arise during infections with highly pathogenic strains of the virus, resulting in increased mortality associated with severe immunopathology and acute respiratory distress. Previous studies suggested a major contribution of the vigorous immune response to lung damage. How the immune system constrains the negative impact of inflammation might therefore be of significant importance for future therapies. Our study in a mouse model of influenza shows that the cytokine IL-27 plays a crucial role in survival by protecting against lung damage. Its actions include regulation of innate (neutrophil influx) and adaptive (inflammatory cytokine production of T cells) arms of immunity during the acute respiratory infection. The data also suggest a therapeutic potential of IL-27, as mice treated with recombinant cytokine at later stages of infection exhibited decreased immunopathology and showed improved survival. The findings uncover an important role of IL-27 in limiting the collateral damages of anti-viral immunity and provide initial evidence that these mechanisms might be exploited for the management of severe immunopathology after infection.

IL-27 is an important and non-redundant regulator of effector T cell accumulation in non-lymphoid tissues during infection. Using malaria as a model systemic pro-inflammatory infection, we demonstrate that the aberrant accumulation of CD4⁺ T cells in the liver of infected IL27R(-/-) (WSX-1(-/-)) mice is a result of differences in cellular recruitment, rather than changes in T cell proliferation or cell death. We show that IL-27 both inhibits the migratory capacity of infection-derived CD4⁺ T cells towards infection-derived liver cells, but also suppresses the production of soluble liver-derived mediator(s) that direct CD4⁺ T cell movement towards the inflamed tissue. Although CCL4 and CCL5 expression was higher in livers of infected WSX-1(-/-) mice than infected WT mice, and hepatic CD4⁺ T cells from WSX-1(-/-) mice expressed higher levels of CCR5 than cells from WT mice, migration of CD4⁺ T cells to the liver of WSX-1(-/-) mice during infection was not controlled by chemokine (R) signalling. However, anti-IL-12p40 treatment reduced migration of CD4⁺ T cells towards infection-derived liver cells, primarily by abrogating the hepatotropic migratory capacity of T cells, rather than diminishing soluble tissue-derived migratory signals. These results indicate that IL-27R signalling restricts CD4⁺ T cell accumulation within the liver during infection primarily by suppressing T cell chemotaxis, which may be linked to its capacity to repress Th1 differentiation, as well as by inhibiting the production of soluble, tissue-derived chemotaxins.

IL-27 is a member of the IL-12 cytokine family initially implicated in promoting Th1 cell differentiation. IL-27 is elevated in the intestines of inflammatory bowel disease patients and in a murine model of this disease, although the influence of IL-27 on intestinal inflammation remains unclear. To address this, we employed IL-27R−/− mice and the dextran sodium sulfate (DSS) model of acute intestinal inflammation. Following exposure to 5% DSS, IL-27R−/− mice exhibited early and severe disease, as defined by increased weight loss, increased severity of edema and inflammatory lesions in the colon, and increased diarrhea and intestinal bleeding. This pathology lead to high mortality in IL-27R−/− mice during the first five days of DSS treatment. Cytokines IL-6 and IL-17 were elevated in IL-27R−/− mice, while other pro-inflammatory cytokines such as IFNγ remained unchanged. IL-17 was produced both by CD4+ T cells and by non-T cells. Finally, the lamina propria of DSS-treated IL-27R−/− mice contained increased frequencies of innate immune cells following treatment. These results indicate that endogenous IL-27-IL-27R signaling can regulate both innate and adaptive immune cells and contribute to prevention of severe intestinal inflammation.

IL-12 is a potent inducer of IFN-γ production and promotes a protective cell-mediated immune response after Mycobacterium tuberculosis infection. Recently, the IL-12-related cytokine IL-27 was discovered, and WSX-1 was identified as one component of the IL-27R complex. To determine the functional significance of IL-27/WSX-1 during tuberculosis, we analyzed the course of infection and the immune response in WSX-1-KO mice after aerosol infection with M. tuberculosis. In the absence of WSX-1, an increased production of the proinflammatory cytokines TNF and IL-12p40 resulted in elevated CD4+ T cell activation and IFN-γ production, which enhanced macrophage effector functions and reduced bacterial loads. This is the first occasion of a selectively gene-deficient mouse strain showing higher levels of protective immunity against M. tuberculosis infection than wild-type mice. However, a concomitantly increased chronic inflammatory response also accelerated death of infected WSX-1-KO mice. In vitro, IL-27 induced STAT3 phosphorylation and inhibited TNF and IL-12 production in activated peritoneal macrophages, indicating a novel feedback mechanism by which IL-27 can modulate excessive inflammation. In conclusion, IL-27 both prevents optimal antimycobacterial protection and limits the pathological sequelae of chronic inflammation.