Suppression of inflammatory immune responses in celiac disease by experimental hookworm infection

We present immunological data from two clinical trials where the effect of experimental human hookworm (Necator americanus) infection on the pathology of celiac disease was evaluated. We found that basal production of Interferon- (IFN-)γ and Interleukin- (IL-)17A from duodenal biopsy culture was suppressed in hookworm-infected participants compared to uninfected controls. Increased levels of CD4+CD25+Foxp3+ cells in the circulation and mucosa are associated with active celiac disease. We show that this accumulation also occurs during a short-term (1 week) oral gluten challenge, and that hookworm infection suppressed the increase of circulating CD4+CD25+Foxp3+ cells during this challenge period. When duodenal biopsies from hookworm-infected participants were restimulated with the immunodominant gliadin peptide QE65, robust production of IL-2, IFN-γ and IL-17A was detected, even prior to gluten challenge while participants were strictly adhering to a gluten-free diet. Intriguingly, IL-5 was produced only after hookworm infection in response to QE65. Thus we hypothesise that hookworm-induced TH2 and IL-10 cross-regulation of the TH1/TH17 inflammatory response may be responsible for the suppression of these responses during experimental hookworm infection.     

Differential requirements for Th1 and Th17 responses to a systemic self-antigen

T cell-APC interactions are essential for the initiation of effector responses against foreign and self-antigens, but the role of these interactions in generating different populations of effector T cells in vivo remains unclear. Using a model of CD4(+) T cell responses to a systemic self-antigen without adjuvants or infection, we demonstrate that activation of APCs augments Th17 responses much more than Th1 responses. Recognition of systemic Ag induces tolerance in self-reactive CD4(+) T cells, but induction of CD40 signaling, even under tolerogenic conditions, results in a strong, Ag-specific IL-17 response without large numbers of IFN-γ-producing cells. Transfer of the same CD4(+) T cells into lymphopenic recipients expressing the self-antigen results in uncontrolled production of IL-17, IFN-γ, and systemic inflammation. If the Ag-specific T cells lack CD40L, production of IL-17 but not IFN-γ is decreased, and the survival time of recipient mice is significantly increased. In addition, transient blockade of the initial MHC class II-dependent T cell-APC interaction results in a greater reduction of IL-17 than of IFN-γ production. These data suggest that Th17 differentiation is more sensitive to T cell interactions with APCs than is the Th1 response, and interrupting this interaction, specifically the CD40 pathway, may be key to controlling Th17-mediated autoimmunity.     

Robust antigen specific th17 T cell response to group A Streptococcus is dependent on IL-6 and intranasal route of infection

Group A streptococcus (GAS, Streptococcus pyogenes) is the cause of a variety of clinical conditions, ranging from pharyngitis to autoimmune disease. Peptide-major histocompatibility complex class II (pMHCII) tetramers have recently emerged as a highly sensitive means to quantify pMHCII-specific CD4+ helper T cells and evaluate their contribution to both protective immunity and autoimmune complications induced by specific bacterial pathogens. In lieu of identifying an immunodominant peptide expressed by GAS, a surrogate peptide (2W) was fused to the highly expressed M1 protein on the surface of GAS to allow in-depth analysis of the CD4+ helper T cell response in C57BL/6 mice that express the I-A(b) MHCII molecule. Following intranasal inoculation with GAS-2W, antigen-experienced 2W:I-A(b)-specific CD4+ T cells were identified in the nasal-associated lymphoid tissue (NALT) that produced IL-17A or IL-17A and IFN-γ if infection was recurrent. The dominant Th17 response was also dependent on the intranasal route of inoculation; intravenous or subcutaneous inoculations produced primarily IFN-γ+ 2W:I-A(b+) CD4+ T cells. The acquisition of IL-17A production by 2W:I-A(b)-specific T cells and the capacity of mice to survive infection depended on the innate cytokine IL-6. IL-6-deficient mice that survived infection became long-term carriers despite the presence of abundant IFN-γ-producing 2W:I-A(b)-specific CD4+ T cells. Our results suggest that an imbalance between IL-17- and IFN-γ-producing CD4+ T cells could contribute to GAS carriage in humans.     

4-1BB triggering ameliorates experimental autoimmune encephalomyelitis by modulating the balance between Th17 and regulatory T cells

Agonistic anti-4-1BB Ab is known to ameliorate experimental autoimmune encephalomyelitis. 4-1BB triggering typically leads to the expansion of CD8(+) T cells, which produce abundant IFN-γ, and this in turn results in IDO-dependent suppression of autoimmune responses. However, because neutralization of IFN-γ or depletion of CD8(+) T cell only partially abrogates the effect of 4-1BB triggering, we sought to identify an additional mechanism of 4-1BB-triggered suppression of autoimmune responses using IFN-γ- or IFN-γR-deficient mice. 4-1BB triggering inhibited the generation of Th17 cells that is responsible for experimental autoimmune encephalomyelitis induction and progression, and increased Foxp3(+)CD4(+) regulatory T (Treg) cells, particularly among CD4(+) T cells. This was not due to a direct effect of 4-1BB signaling on CD4(+) T cell differentiation: 4-1BB signaling not only reduced Th17 cells and increased Treg cells in wild-type mice, which could be due to IFN-γ production by the CD8(+) T cells, but also did so in IFN-γ-deficient mice, in that case by downregulating IL-6 production. These results show that although secondary suppressive mechanisms evoked by 4-1BB triggering are usually masked by the strong effects of IFN-γ, 4-1BB signaling seems to modulate autoimmune responses by a number of mechanisms, and modulation of the Th17 versus Treg cell balance is one of those mechanisms.     

A crucial role of IL-17 and IFN-γ during acute rejection of peripheral nerve xenotransplantation in mice

Nerve injuries causing segmental loss require nerve grafting. However, autografts and allografts have limitations for clinical use. Peripheral nerve xenotransplantation has become an area of great interest in clinical surgery research as an alternative graft strategy. However, xenotransplant rejection is severe with cellular immunity, and Th1 cells play an important role in the process. To better understand the process of rejection, we used peripheral nerve xenografts from rats to mice and found that mononuclear cells expressing IFN-γ and IL-17 infiltrated around the grafts, and IFN-γ and IL-17 producing CD4+ and CD8+ T cells increased during the process of acute rejection. The changes of IL-4 level had no significant difference between xenotransplanted group and sham control group. The rejection of xenograft was significantly prevented after the treatment of IL-17 and IFN-γ neutralizing antibodies. These data suggest that Th17 cells contribute to the acute rejection process of peripheral nerve xenotransplant in addition to Th1 cells.     

IL-17-dependent, IFN-gamma-independent tumor rejection is mediated by cytotoxic T lymphocytes and occurs at extraocular sites, but is excluded from the eye

Although intraocular tumors reside in an immune-privileged site where immune responses are suppressed, some tumors are rejected. An example of this is the rejection of intraocular adenovirus-induced (adenovirus type 5 early region 1 [Ad5E1]) tumors in C57BL/6 mice. We previously identified an Ad5E1 tumor clone in which the rejection is IFN-γ dependent and culminates in the destruction of both the tumor and the eye. Although Ad5E1 tumors are not rejected when transplanted into the eyes of IFN-γ KO mice, they are rejected after s.c. transplantation. Thus, outside of the eye Ad5E1 tumors elicit a form of tumor immunity that is IFN-γ independent. In this article, we demonstrate that IFN-γ-independent s.c. rejection requires both CD4(+) and CD8(+) T cells. Furthermore, s.c. tumor rejection requires IL-17, which is produced by IFN-γ-deficient CD4(+) T cells in response to tumor Ags (TAs). Splenocytes from CD4-depleted IFN-γ KO mice produce significantly less IL-17 compared with splenocytes from isotype-treated IFN-γ KO animals in response to TAs. Furthermore, depletion of IL-17 decreases CTL activity against Ad5E1 tumor cells. In this model we propose that, in the absence of IFN-γ, CD4(+) T cells produce IL-17 in response to TAs, which increases CTL activity that mediates tumor rejection; however, this does not occur in the eye. IL-6 production within the eye is severely reduced, which is consistent with the failure to induce Th17 cells within the intraocular tumors. In contrast, the s.c. environment is replete with IL-6 and supports the induction of Th17 cells. Therefore, IFN-γ-independent tumor rejection is excluded from the eye and may represent a newly recognized form of ocular immune privilege.     

Macrophage migration inhibitory factor: a downregulator of early T cell-dependent IFN-gamma responses in Plasmodium chabaudi adami (556 KA)-infected mice

Neutralization of macrophage migration inhibitory factor (MIF) increases anti-tumor cytotoxic T cell responses in vivo and IFN-γ responses in vitro, suggesting a plausible regulatory role for MIF in T cell activation. Considering that IFN-γ production by CD4(+) T cells is pivotal to resolve murine malaria and that secretion of MIF is induced by Plasmodium chabaudi adami parasites, we investigated the effect of MIF deficiency on the infection with this pathogen. Infections with P. c. adami 556 KA parasites were more efficiently controlled in MIF-neutralized and MIF-deficient (knockout [KO]) BALB/c mice. The reduction in parasitemia was associated with reduced production of IL-4 by non-T/non-B cells throughout patent infection. At day 4 postinfection, higher numbers of activated CD4(+) cells were measured in MIF KO mice, which secreted more IFN-γ, less IL-4, and less IL-10 than did CD4(+) T cells from wild-type mice. Enhanced IFN-γ and decreased IL-4 responses also were measured in MIF KO CD4(+) T cells stimulated with or without IL-12 and anti-IL-4 blocking Ab to induce Th1 polarization. However, MIF KO CD4(+) T cells efficiently acquired a Th2 phenotype when stimulated in the presence of IL-4 and anti-IL-12 Ab, indicating normal responsiveness to IL-4/STAT6 signaling. These results suggest that by promoting IL-4 responses in cells other than T/B cells during early P. c. adami infection, MIF decreases IFN-γ secretion in CD4(+) T cells and, additionally, has the intrinsic ability to render CD4(+) T cells less capable of acquiring a robust Th1 phenotype when stimulated in the presence of IL-12.     

BALB/c小鼠感染不同疟原虫CD4＋ Th应答和凋亡特点的比较研究

探讨不同疟原虫感染BALB/c小鼠的免疫应答特点。BALB/c小鼠经腹腔注射致死型约氏疟原虫（P.y17XL）和夏氏疟原虫（P.cAS）感染的红细胞,计数红细胞感染率;ELISA动态检测感染小鼠脾细胞培养上清中IFN-γ和IL-4水平;流式细胞术检测脾中CD4＋T细胞凋亡数量。约氏疟原虫（P.y17XL）感染早期,小鼠原虫血症持续上升;IFN-γ和IL-4分泌水平仅在感染后第3天出现一过性有意义的升高,而且峰值较低;脾中CD4＋T细胞大量凋亡,小鼠全部死亡;而夏氏疟原虫（P.cAS）感染小鼠,原虫血症上升缓慢;IFN-γ分泌水平在感染后第5天达峰值;IL-4分泌水平在感染后第10天达峰值,且峰值较高维持时间较长;脾中CD4＋T细胞凋亡细胞于感染后8 d出现有意义升高,小鼠全部存活。抗疟保护性免疫有赖于Th1和Th2型免疫应答的有效建立和协调过渡,感染期间CD4＋T细胞凋亡的时相和数量可能是影响免疫应答的强度或引起宿主免疫抑制的原因,从而影响宿主疟原虫感染的结局。     

Functional modulation of the pathway between dendritic cells (DCs) and CD4+T cells by the neuropeptide: methionine enkephalin (MENK)

MENK, the endogenous neuropeptide, is suggested to be involved in the regulatory loop between the immune and neuroendocrine systems, with modulation of various functions of cells related to both the innate and adaptive immune systems. Our present research findings show that MENK serves as an immune modulator to the pathway between DCs and CD4+T cells. We studied changes of DCs in key surface molecules, the activity of acid phosphatases (ACPs), the production of IL-12, and the effects on murine CD4+T cell expansion and their cytokine production by MENK alone, and in combination with interkeukin-2 (IL-2) or interferon-γ (IFN-γ). In fact, we found that MENK could markedly induce the maturation of DCs through the addition of surface molecules such as MHC class II, CD86, and CD40 on murine DCs, the production of IL-12, and the down-regulation of ACP inside DCs, (which occurs when phagocytosis of DCs is decreased, and antigen presentation increased with maturation). We also found that MENK alone or in combination with IL-2 or IFN-γ, could markedly up-regulate both CD4+T cell expansion and the CD4 molecule expression in vivo and in vitro and that MENK alone, or MENK + IL-2, could enhance the production of interferon-γ from CD4+T cells. Moreover, MENK alone, or MENK + IFN-γ, could enhance the production of IL-2 from CD4+T cells. It is therefore concluded that MENK can exert positive modulation to the pathway between dendtritic cells and CD4+T cells.     

T and B cells are not required for clearing Staphylococcus aureus in systemic infection despite a strong TLR2-MyD88-dependent T cell activation

Staphylococcus aureus infection elicits through its mature lipoproteins an innate immune response by TLR2-MyD88 signaling, which improves bacterial clearing and disease outcome. The role of dendritic cells (DCs) and T cells in this immune activation and the function of T and B cells in defense against S. aureus infection remain unclear. Therefore, we first evaluated DC and T cell activation after infection with S. aureus wild type (WT) and its isogenic mutant, which is deficient in lipoprotein maturation, in vitro. Lipoproteins in viable S. aureus contributed via TLR2-MyD88 to activation of DCs, which promoted the release of IFN-γ and IL-17 in CD4(+) T cells. This strong effect was independent of superantigens and MHC class II. We next evaluated the function of T cells and their cytokines IFN-γ and IL-17 in infection in vivo. Six days after systemic murine infection IFN-γ, IL-17, and IL-10 production in total spleen cells were MyD88-dependent and their levels increased until day 21. The comparison of CD3(-/-), Rag2(-/-), and C57BL/6 mice after infection revealed that IFN-γ and IL-17 originated from T cells and IL-10 originated from innate immune cells. Furthermore, vaccination of mice to activate T and B cells did not improve eradication of S. aureus from organs. In conclusion, S. aureus enhances DC activation via TLR2-MyD88 and thereby promotes T(H)1 and T(H)17 cell differentiation. However, neither T cells and their MyD88-regulated products, IFN-γ and IL-17, nor B cells affected bacterial clearing from organs and disease outcome.     

Hapten application to the skin induces an inflammatory program directing hapten-primed effector CD8 T cell interaction with hapten-presenting endothelial cells

Contact hypersensitivity is a CD8 T cell-mediated response to hapten sensitization and challenge of the skin. Effector CD8 T cell recruitment into the skin parenchyma to elicit the response to hapten challenge requires prior CXCL1/KC-directed neutrophil infiltration within 3-6 h after challenge and is dependent on IFN-γ and IL-17 produced by the hapten-primed CD8 T cells. Mechanisms directing hapten-primed CD8 T cell localization and activation in the Ag challenge site to induce this early CXCL1 production in response to 2,4-dinitrofluorobenzene were investigated. Both TNF-α and IL-17, but not IFN-γ, mRNA was detectable within 1 h of hapten challenge of sensitized mice and increased thereafter. Expression of ICAM-1 was observed by 1 h after challenge of sensitized and nonsensitized mice and was dependent on TNF-α. The induction of IL-17, IFN-γ, and CXCL1 in the challenge site was not observed when ICAM-1 was absent or neutralized by specific Ab. During the elicitation of the contact hypersensitivity response, endothelial cells expressed ICAM-1 and produced CXCL1 suggesting this as the site of CD8 T cell localization and activation. Endothelial cells isolated from challenged skin of naive and sensitized mice had acquired the hapten and the ability to activate hapten-primed CD8 T cell cytokine production. These results indicate that hapten application to the skin of sensitized animals initiates an inflammatory response promoting hapten-primed CD8 T cell localization to the challenge site through TNF-α-induced ICAM-1 expression and CD8 T cell activation to produce IFN-γ and IL-17 through endothelial cell presentation of hapten.     

IL-21 promotes skin recruitment of CD4(+) cells and drives IFN-γ-dependent epidermal hyperplasia

Psoriasis is a chronic inflammatory disorder of the skin characterized by epidermal hyperplasia and infiltration of leukocytes into the dermis and epidermis. T cell-derived cytokines, such as IFN-γ and IL-17A, play a major role in the psoriasis-associated epidermal hyperplasia, even though factors/mechanisms that regulate the production of these cytokines are not fully understood. We have recently shown that IL-21 is synthesized in excess in psoriatic skin lesions and causes epidermal hyperplasia when injected intradermally in mice. Moreover, in the human psoriasis SCID mouse model, neutralization of IL-21 reduces both skin thickening and expression of inflammatory molecules, thus supporting the pathogenic role of IL-21 in psoriasis. However, the basic mechanism by which IL-21 promotes skin pathology remains unknown. In this study, we show that CD4(+) cells accumulate early in the dermis of IL-21-treated mice and mediate the development of epidermal hyperplasia. Indeed, IL-21 fails to induce skin damage in RAG1-deficient mice and CD4(+) cell-depleted wild-type mice. The majority of CD4(+) cells infiltrating the dermis of IL-21-treated mice express IFN-γ and, to a lesser extent, IL-17A. Studies in cytokine knockout mice show that IFN-γ, but not IL-17A, is necessary for IL-21-induced epidermal hyperplasia. Finally, we demonstrate that IFN-γ-producing CD4(+) cells infiltrating the human psoriatic plaque express IL-21R, and abrogation of IL-21 signals reduces IFN-γ expression in cultures of psoriatic CD4(+) cells. Data indicate that IL-21 induces an IFN-γ-dependent pathogenic response in vivo, thus contributing to elucidate a mechanism by which IL-21 sustains skin-damaging inflammation.     

Regulation of autologous immunity to the mouse 5T4 oncofoetal antigen: implications for immunotherapy

Effective vaccination against tumour-associated antigens (TAA) such as the 5T4 oncofoetal glycoprotein may be limited by the nature of the T cell repertoire and the influence of immunomodulatory factors in particular T regulatory cells (Treg). Here, we identified mouse 5T4-specific T cell epitopes using a 5T4 knock out (5T4KO) mouse and evaluated corresponding wild-type (WT) responses as a model to refine and improve immunogenicity. We have shown that 5T4KO mice vaccinated by replication defective adenovirus encoding mouse 5T4 (Adm5T4) generate potent 5T4-specific IFN-γ CD8 and CD4 T cell responses which mediate significant protection against 5T4 positive tumour challenge. 5T4KO CD8 but not CD4 primed T cells also produced IL-17. By contrast, Adm5T4-immunized WT mice showed no tumour protection consistent with only low avidity CD8 IFN-γ, no IL-17 T cell responses and no detectable CD4 T cell effectors producing IFN-γ or IL-17. Treatment with anti-folate receptor 4 (FR4) antibody significantly reduced the frequency of Tregs in WT mice and enhanced 5T4-specific IFN-γ but reduced IL-10 T cell responses but did not reveal IL-17-producing effectors. This altered balance of effectors by treatment with FR4 antibody after Adm5T4 vaccination provided modest protection against autologous B16m5T4 melanoma challenge. The efficacy of 5T4 and some other TAA vaccines may be limited by the combination of TAA-specific T regs, the deletion and/or alternative differentiation of CD4 T cells as well as the absence of distinct subsets of CD8 T cells.     

MEKK3 regulates IFN-gamma production in T cells through the Rac1/2-dependent MAPK cascades

MEKK3 is a conserved Ser/Thr protein kinase belonging to the MAPK kinase kinase (MAP3K) family. MEKK3 is constitutively expressed in T cells, but its function in T cell immunity has not been fully elucidated. Using Mekk3 T cell conditional knockout (T-cKO) mice, we show that MEKK3 is required for T cell immunity in vivo. Mekk3 T-cKO mice had reduced T cell response to bacterial infection and were defective in clearing bacterial infections. The Ag-induced cytokine production, especially IFN-γ production, was impaired in Mekk3-deficient CD4 T cells. The TCR-induced ERK1/2, JNK, and p38 MAPKs activation was also defective in Mekk3-deficient CD4 T cells. In vitro, MEKK3 is not required for Th1 and Th2 cell differentiation. Notably, under a nonpolarizing condition (Th0), Mekk3 deficiency led to a significant reduction of IFN-γ production in CD4 T cells. Furthermore, the IL-12/IL-18-driven IFN-γ production and MAPK activation in Mekk3-deficient T cells was not affected suggesting that MEKK3 may selectively mediate the TCR-induced MAPK signals for IFN-γ production. Finally, we found that MEKK3 activation by TCR stimulation requires Rac1/2. Taken together, our study reveals a specific role of MEKK3 in mediating the TCR signals for IFN-γ production.     

Retinal self-antigen induces a predominantly Th1 effector response in Axl and Mertk double-knockout mice

The TAM family of receptors (Tyro3, Axl, and Mertk) plays an important role in the negative regulation of response of dendritic cells (DCs) and macrophages to pathogenic stimuli, and mice lacking this receptor family develop spontaneous lupus-like systemic autoimmunity against a variety of tissues, including retina. To study the molecular mechanism underlying the TAM regulation of APC functions and subsequent effects on the induction of an autoimmune response against the eye, we examined CD4 T cell differentiation following retinal self-antigen immunization. CD4 T cells prepared from naive or interphotoreceptor retinoid-binding protein (IRBP)1-20-immunized Axl and Mertk double-knockout (dko) mice reacted to activation using anti-CD3 and anti-CD28 Abs or to bolster by self-antigen in vitro with a predominantly Th1 effector response, as characterized by increased IFN-γ production and higher frequency of IFN-γ-positive CD4 T cells. The Th17 effector response to IRBP immunization was similar in dko mice to that in wild-type controls, as shown by ELISA measurement of IL-17A in the culture medium and flow cytometric analysis of IL-17A-secreting CD4 T cells. Interestingly, APCs or DCs isolated from IRBP-immunized dko mice exhibited a greater ability to drive the Th1 response. The production of two driving cytokines for Th1 differentiation, IL-12 and IL-18, was dramatically increased in dko DCs and macrophages, and LPS stimulation bolstered their production. The preferential development into the Th1 subset in dko mice suggests that the cytokine milieu produced by the mutant mice in vivo or by mutant APCs in vitro selectively creates a differentiation environment favoring the Th1 effector response.     

Outer membrane protein A (OmpA) of Shigella flexneri 2a links innate and adaptive immunity in a TLR2-dependent manner and involvement of IL-12 and nitric oxide

We determine that OmpA of Shigella flexneri 2a is recognized by TLR2 and consequently mediates the release of proinflammatory cytokines and activates NF-κB in HEK 293 cells transfected with TLR2. We also observe that in RAW macrophages TLR2 is essential to instigate the early immune response to OmpA via NF-κB activation and secretion of cytokines and NO. Consistent with these results, TLR2 knockdown using siRNA abolishes the initiation of immune responses. Processing and presentation of OmpA depend on TLR2; MHCII presentation of the processed antigen and expression of CD80 significantly attenuated in TLR2 knockdown macrophages. The optimum production of IFN-γ by the macrophages:CD4(+) T cells co-culture depends on both TLR2 activation and antigen presentation. So, TLR2 is clearly recognized as a decisive factor in initiating host innate immune response to OmpA for the development of CD4(+) T cell adaptive response. Furthermore, we demonstrate in vivo that intranasal immunization of mice with OmpA selectively enhances the release of IFN-γ and IL-2 by CD4(+) T cells. Importantly, OmpA increases the level of IFN-γ production in Ag-primed splenocytes. The addition of neutralizing anti-IL-12p70 mAb to cell cultures results in the decreased release of OmpA-enhanced IFN-γ by Ag-primed splenocytes. Moreover, coincubation with OmpA-pretreated macrophages enhances the production of IFN-γ by OmpA-primed CD4(+) T cells, representing that OmpA may enhance IFN-γ expression in CD4(+) T cells through the induction of IL-12 production in macrophages. These results demonstrate that S. flexneri 2a OmpA may play a critical role in the development of Th1 skewed adaptive immune response.     

IL-27 affects helper T cell responses via regulation of PGE2 production by macrophages

IL-27 is a heterodimeric cytokine that regulates both innate and adaptive immunity. The immunosuppressive effect of IL-27 largely depends on induction of IL-10-producing Tr1 cells. To date, however, effects of IL-27 on regulation of immune responses via mediators other than cytokines remain poorly understood. To address this issue, we examined immunoregulatory effects of conditional medium of bone marrow-derived macrophages (BMDMs) from WSX-1 (IL-27Rα)-deficient mice and found enhanced IFN-γ and IL-17A secretion by CD4⁺ T cells as compared with that of control BMDMs. We then found that PGE₂ production and COX-2 expression by BMDMs from WSX-1-deficient mice was increased compared to control macrophages in response to LPS. The enhanced production of IFN-γ and IL-17A was abolished by EP2 and EP4 antagonists, demonstrating PGE₂ was responsible for enhanced cytokine production. Murine WSX-1-expressing Raw264.7 cells (mWSX-1-Raw264.7) showed phosphorylation of both STAT1 and STAT3 in response to IL-27 and produced less amounts of PGE₂ and COX-2 compared to parental RAW264.7 cells. STAT1 knockdown in parental RAW264.7 cells and STAT1-deficiency in BMDMs showed higher COX-2 expression than their respective control cells. Collectively, our result indicated that IL-27/WSX-1 regulated PGE₂ secretion via STAT1–COX-2 pathway in macrophages and affected helper T cell response in a PGE₂-mediated fashion.