IL‐27 stimulates human NK‐cell effector functions and primes NK cells for IL‐18 responsiveness

IL‐27, a member of the IL‐12 family of cytokines, is produced by APCs, and displays pro‐ and anti‐inflammatory effects. How IL‐27 affects human NK cells still remains unknown. In this study, we observed that mature DCs secreted IL‐27 and that blockade of IL‐27R (CD130) reduced the amount of IFN‐γ produced by NK cells during their coculture, showing the importance of IL‐27 during DC–NK‐cell crosstalk. Accordingly, human rIL‐27 stimulated IFN‐γ secretion by NK cells in a STAT1‐dependent manner, induced upregulation of CD25 and CD69 on NK cells, and displayed a synergistic effect with IL‐18. Preincubation experiments demonstrated that IL‐27 primed NK cells for IL‐18‐induced IFN‐γ secretion, which was associated with an IL‐27‐driven upregulation of T‐bet expression. Also, IL‐27 triggered NKp46‐dependent NK‐cell‐mediated cytotoxicity against Raji, T‐47D, and HCT116 cells, and IL‐18 enhanced this cytotoxic response. Such NK‐cell‐mediated cytotoxicity involved upregulation of perforin, granule exocytosis, and TRAIL‐mediated cytotoxicity but not Fas‐FasL interaction. Moreover, IL‐27 also potentiated Ab‐dependent cell‐mediated cytotoxicity against mAb‐coated target cells. Taken together, IL‐27 stimulates NK‐cell effector functions, which might be relevant in different physiological and pathological situations.     

IL‐10 interferes directly with TCR‐induced IFN‐γ but not IL‐17 production in memory T cells

IL‐10 is a potent immunoregulatory and anti‐inflammatory cytokine. However, therapeutic trials in chronic inflammation have been largely disappointing. It is well established that IL‐10 can inhibit Th1 and Th2 cytokine production via indirect effects on APC. Less data are available about the influence of IL‐10 on IL‐17 production, a cytokine which has been recently linked to chronic inflammation. Furthermore, there are only few reports about a direct effect of IL‐10 on T cells. We demonstrate here that IL‐10 can directly interfere with TCR‐induced IFN‐γ production in freshly isolated memory T cells in the absence of APC. This effect was independent of the previously described effects of IL‐10 on T cells, namely inhibition of IL‐2 production and inhibition of CD28 signaling. In contrast, IL‐10 did not affect anti‐CD3/anti‐CD28‐induced IL‐17 production from memory T cells even in the presence of APC. This might have implications for the interpretation of therapeutic trials in patients with chronic inflammation where Th17 cells contribute to pathogenesis.     

Inhibition of murine γδ lymphocyte expansion and effector function by regulatory αβ T cells is cell‐contact‐dependent and sensitive to GITR modulation

γδ T cells are highly cytolytic lymphocytes that produce large amounts of pro‐inflammatory cytokines during immune responses to multiple pathogens. Furthermore, their ability to kill tumor cells has fueled the development of γδ‐T‐cell‐based cancer therapies. Thus, the regulation of γδ‐T‐cell activity is of great biological and clinical relevance. Here, we show that murine CD4⁺CD25⁺ αβ T cells, the vast majority of which express the Treg marker, Foxp3, abolish key effector functions of γδ T cells, namely the production of the pro‐inflammatory cytokines, IFN‐γ and IL‐17, cytotoxicity, and lymphocyte proliferation in vitro and in vivo. We further show that suppression is dependent on cellular contact between Treg and γδ T cells, results in the induction of an anergic state in γδ lymphocytes, and can be partially reversed by manipulating glucocorticoid‐induced TNF receptor‐related protein (GITR) signals. Our data collectively dissect a novel mechanism by which the expansion and pro‐inflammatory functions of γδ T cells are regulated.     

IL‐18Rα‐deficient CD4+ T cells induce intestinal inflammation in the CD45RBhi transfer model of colitis despite impaired innate responsiveness

IL‐18 has been implicated in inflammatory bowel disease (IBD), however its role in the regulation of intestinal CD4⁺ T‐cell function remains unclear. Here we show that murine intestinal CD4⁺ T cells express high levels of IL‐18Rα and provide evidence that IL‐18Rα expression is induced on these cells subsequent to their entry into the intestinal mucosa. Using the CD45RB^hi T‐cell transfer colitis model, we show that IL‐18Rα is expressed on IFN‐γ⁺, IL‐17⁺, and IL‐17⁺IFN‐γ⁺ effector CD4⁺ T cells in the inflamed colonic lamina propria (cLP) and mesenteric lymph node (MLN) and is required for the optimal generation and/or maintenance of IFN‐γ‐producing cells in the cLP. In the steady state and during colitis, TCR‐independent cytokine‐induced IFN‐γ and IL‐17 production by intestinal CD4⁺ T cells was largely IL‐18Rα?dependent. Despite these findings however, IL‐18Rα?deficient CD4⁺ T cells induced comparable intestinal pathology to WT CD4⁺ T cells. These findings suggest that IL‐18‐dependent cytokine induced activation of CD4⁺ T cells is not critical for the development of T‐cell‐mediated colitis.     

A role for IL‐18 in protective immunity against Mycobacterium tuberculosis

Tuberculosis remains the most hazardous bacterial infection worldwide. The causative agent, Mycobacterium tuberculosis, is a facultative intracellular pathogen of resting MΦ. IFN‐γ secreted by natural killer, CD4 Th 1 and CD8 T cells upon instruction by IL‐12 and ‐18 activates MΦ to restrict mycobacterial growth. Production of both cytokines is induced by TLR signalling in DC and MΦ. Mice deficient for the TLR adaptor, MyD88, are highly susceptible to M. tuberculosis infection. Shared usage of MyD88 by signalling cascades for TLR and receptors for IL‐1 and IL‐18 prompted us to revisit the role of IL‐18 during experimental infection with M. tuberculosis. We show that mice deficient for IL‐18 and MyD88 but not for IL‐18 receptor promptly succumbed to M. tuberculosis infection in contrast to WT or TLR‐2/‐4 double KO mice indicating that lack of IL‐18 contributes to the high susceptibility of MyD88 KO mice to M. tuberculosis. Without IL‐18, the protective Th1 response was decreased and hence, mycobacterial propagation was favoured. Neutrophil‐driven lung immunopathology concomitant with unrestrained growth of tubercle bacilli are most likely responsible for the premature death of IL‐18 KO mice. Thus, IL‐18 plays a decisive role in protective immunity against tuberculosis.     

Both IFN‐γ and IL‐17 are required for the development of severe autoimmune gastritis

IL‐17, produced by a distinct lineage of CD4⁺ helper T (Th) cells termed Th17 cells, induces the production of pro‐inflammatory cytokines from resident cells and it has been demonstrated that over‐expression of IL‐17 plays a crucial role in the onset of several auto‐immune diseases. Here we examined the role of IL‐17 in the pathogenesis of autoimmune gastritis, a disease that was previously believed to be mediated by IFN‐γ. Significantly higher levels of IL‐17 and IFN‐γ were found in the stomachs and stomach‐draining lymph nodes of mice with severe autoimmune gastritis. Unlike IL‐17, which was produced solely by CD4⁺ T cells in gastritic mice, the majority of IFN‐γ‐producing cells were CD8⁺ T cells. However, CD8⁺ T cells alone were not able to induce autoimmune gastritis. T cells that were deficient in IL‐17 or IFN‐γ production were able to induce autoimmune gastritis but to a much lower extent compared with the disease induced by wild‐type T cells. These data demonstrate that production of neither IL‐17 nor IFN‐γ by effector T cells is essential for the initiation of autoimmune gastritis, but suggest that both are required for the disease to progress to the late pathogenic stage that includes significant tissue disruption.     

EAE mediated by a non‐IFN‐γ/non‐IL‐17 pathway

Previous studies have shown that EAE can be elicited by the adoptive transfer of either IFN‐γ‐producing (Th1) or IL‐17‐producing (Th17) myelin‐specific CD4⁺ T‐cell lines. Paradoxically, mice deficient in either IFN‐γ or IL‐17 remain susceptible to EAE following immunization with myelin antigens in CFA. These observations raise questions about the redundancy of IFN‐γ and IL‐17 in autoimmune demyelinating disease mediated by a diverse, polyclonal population of autoreactive T cells. In this study, we show that an atypical form of EAE, induced in C57BL/6 mice by the adoptive transfer of IFN‐γ‐deficient effector T cells, required IL‐17 signaling for the development of brainstem infiltrates. In contrast, classical EAE, characterized by predominant spinal cord inflammation, occurred in the combined absence of IFN‐γ and IL‐17 signaling, but was dependent on GM‐CSF and CXCR2. Our findings contribute to a growing body of data, indicating that individual cytokines vary in their importance across different models of CNS autoimmunity.     

IL‐33 promotes innate IFN‐γ production and modulates dendritic cell response in LCMV‐induced hepatitis in mice

Recent studies have revealed IL‐33 as a key factor in promoting antiviral T‐cell responses. However, it is less clear as to how IL‐33 regulates innate immunity. In this study, we infected wild‐type (WT) and IL‐33^?/? mice with lymphocytic choriomeningitis virus and demonstrated an essential role of infection‐induced IL‐33 expression for robust innate IFN‐γ production in the liver. We first show that IL‐33 deficiency resulted in a marked reduction in the number of IFN‐γ⁺ γδ T and NK cells, but an increase in that of IL‐17⁺ γδ T cells at 16 h postinfection. Recombinant IL‐33 (rIL‐33) treatment could reverse such deficiency via increasing IFN‐γ‐producing γδ T and NK cells, and inhibiting IL‐17⁺ γδ T cells. We also found that rIL‐33‐induced type 2 innate lymphoid cells were not involved in T‐cell responses and liver injury, since the adoptive transfer of type 2 innate lymphoid cells neither affected the IFN‐γ and TNF‐α production in T cells, nor liver transferase levels in lymphocytic choriomeningitis virus infected mice. Interestingly, we found that while IL‐33 was not required for costimulatory molecule expression, it was critical for DC proliferation and cytokine production. Together, this study highlights an essential role of IL‐33 in regulating innate IFN‐γ‐production and DC function during viral hepatitis.     

Donor and host B cell‐derived IL‐10 contributes to suppression of graft‐versus‐host disease

Graft‐versus‐host disease (GvHD) is a frequent life‐threatening complication following allogeneic HSC transplantation (HSCT). IL‐10 is a regulatory cytokine with important roles during GvHD, yet its relevant sources, and mode of action, remain incompletely defined in this disease. Using IL‐10‐deficient donor or host mice (BALB/c or C57BL/6, respectively) in a MHC‐mismatched model for acute GvHD, we found a strongly aggravated course of the disease with increased mortality when either donor or host cells could not produce this cytokine. A lack of IL‐10 resulted in increased allogeneic T‐cell responses and enhanced activation of host DCs in spleen and MLNs. Remarkably, IL‐10 was prominently produced by host‐ and donor‐derived CD5^intCD1d^intTIM‐1^int B cells in this disease, and consistent with this, allogeneic HSCT resulted in exacerbated GvHD when mice lacking IL‐10 expression in B cells were used as donor or host, compared with controls. Taken together, this study demonstrates that host and donor B cell‐derived IL‐10 provides a unique mechanism of suppression of acute GvHD, and suggests that DCs are the targets of this B cell‐mediated suppressive effect. These findings open novel therapeutic possibilities based on the use of B cells to increase the feasibility of allogeneic HSCT.     

Lipopolysaccharide (LPS)‐induced Interferon (IFN)‐gamma Production by Decidual Mononuclear Cells (DMNC) is Interleukin (IL)‐2 and IL‐12 Dependent

Citation Negishi M, Izumi Y, Aleemuzzaman S, Inaba N, Hayakawa S. Lipopolysaccharide (LPS)‐induced interferon (IFN)‐gamma production by decidual mononuclear cells (DMNC) is interleukin (IL)‐2 and IL‐12 dependent. Am J Reprod Immunol 2011; 65: 20–27 Problem Th1‐shifted immune response is believed to be harmful for successful pregnancy because of activation of maternal cytotoxic T lymphocytes and natural killer cells. However, its effects on Toll‐like receptor (TLR)‐mediated innate immune response are so far unknown and this study has been undertaken to address the issue. Method of study Decidual tissues were obtained from 16 pregnant women undergoing elective termination during the first trimester pregnancy for socioeconomic reasons. Decidual Mononuclear Cells (DMNC) were stimulated with suboptimal doses of IL‐2 and IL‐12 with/without LPS, considered to be a TLR4 ligand, for 48 hr. Productions of IFN‐γ and tumor necrosis factor (TNF)‐α in culture supernatant were measured with ELISA. Results (i) IFN‐γ production was induced with LPS alone which was strongly up‐regulated in the presence of IL‐2 and IL‐12. (ii) TNF‐α was also induced by LPS but was not affected by the presence of IL‐2 and IL‐12. Conclusion IL‐2 and IL‐12 up‐regulated the production of IFN‐γ in DMNC through increasing their susceptibility to LPS. TNF‐α production is independent of such a mechanism.     

GM‐CSF therapy inhibits chronic graft‐versus‐host disease via expansion of regulatory T cells

Regulatory T cells (Tregs) attenuate excessive immune responses, making their expansion beneficial in immune‐mediated diseases, including allogeneic bone marrow transplantation associated with graft‐versus‐host disease (GVHD). In addition to interleukin‐2, Tregs require T‐cell receptor and costimulatory signals from antigen‐presenting cells, such as DCs, for their optimal proliferation. Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) increases DC number and may promote DC‐dependent Treg proliferation. Here, we demonstrate that GM‐CSF treatment increases CD4⁺CD8^– DCs, which are associated with Treg expansion. In a mouse model of chronic GVHD (cGVHD), GM‐CSF therapy expanded Tregs, protected against the development of skin GVHD, and regulated both Th1 and Th17 responses in the peripheral lymph nodes, resulting in an attenuation of skin cGVHD. Notably, the expanded Tregs were instrumental to GM‐CSF‐mediated cGVHD inhibition, which was dependent upon an increased ratio of Tregs to conventional T cells rather than augmentation of suppressive function. These data suggest that GM‐CSF induces Treg proliferation by expanding CD4⁺CD8^? DCs, which in turn regulate alloimmune responses in a cGVHD mouse model. Thus, GM‐CSF could be used as a therapeutic DC modulator to induce Treg expansion and to inhibit excessive alloimmune responses in immune‐related diseases.     

Type I interferon potentiates T‐cell receptor mediated induction of IL‐10‐producing CD4+ T cells

Type I interferons (IFNs) have the dual ability to promote the development of the immune response and exert an anti‐inflammatory activity. We analyzed the integrated effect of IFN‐α, TCR signal strength, and CD28 costimulation on human CD4⁺ T‐cell differentiation into cell subsets producing the anti‐ and proinflammatory cytokines IL‐10 and IFN‐γ. We show that IFN‐α boosted TCR‐induced IL‐10 expression in activated peripheral CD45RA⁺CD4⁺ T cells and in whole blood cultures. The functional cooperation between TCR and IFN‐α efficiently occurred at low engagement of receptors. Moreover, IFN‐α rapidly cooperated with anti‐CD3 stimulation alone. IFN‐α, but not IL‐10, drove the early development of type I regulatory T cells that were mostly IL‐10⁺ Foxp3^? IFN‐γ^? and favored IL‐10 expression in a fraction of Foxp3⁺ T cells. Our data support a model in which IFN‐α costimulates TCR toward the production of IL‐10 whose level can be amplified via an autocrine feedback loop.     

Efficacy of azithromycin in preventing lethal graft‐versus‐host disease

Acute graft‐versus‐host disease (GVHD) following allogeneic bone marrow transplantation (BMT) is initiated by donor T lymphocytes that recognize histocompatibility antigens presented by recipient dendritic cells (DCs). Current approaches to reduce GVHD are focused on suppressing donor T lymphocyte responses to alloantigens. However, these strategies may be inadequate in the setting of allogeneic transplants (particularly histoincompatible transplants), may increase the risk of tumour relapse and are associated with high rates of opportunistic infections. We hypothesized that inhibition of recipient DCs might suppress GVHD. We recently demonstrated in vitro that azithromycin, a macrolide antibiotic, also acts as a nuclear factor (NF)‐κB inhibitor of murine DCs and inhibits their maturation and functions, including allogeneic responses. We investigated whether azithromycin could prevent alloreactions in a murine histoincompatibility model. Oral administration of azithromycin to recipient mice for 5 days during major‐histoincompatible BMT suppressed lethal GVHD significantly, whereas ex‐vivo lymphocyte function was not affected by the drug. These data suggest that azithromycin has potential as a novel prophylactic drug for lethal GVHD.     

Secreted IL‐1α promotes T‐cell activation and expansion of CD11b+Gr1+ cells in carbon tetrachloride‐induced liver injury in mice

Interleukin‐1α is mainly expressed on the cell membrane, but can also be secreted during inflammation. The roles of secreted and membrane IL‐1α in acute liver inflammation are still not known. Here, we examined the functions of secreted and membrane IL‐1α in a mouse model of carbon tetrachloride‐induced acute liver injury. We show that secreted IL‐1α aggravates liver damage and membrane IL‐1α slightly protects mice from liver injury. Further studies showed that secreted IL‐1α promotes T‐cell activation. It also increased the expansion of CD11b⁺Gr1⁺ myeloid cells, which may serve as a negative regulator of acute liver inflammation. Moreover, secreted IL‐1α induced IL‐6 production from hepatocytes. IL‐6 neutralization reduced the proliferation of CD11b⁺Gr1⁺ myeloid cells in vivo. CCL2 and CXCL5 expression was increased by secreted IL‐1α in vitro and in vivo. Antagonists of the chemokine receptors for CCL2 and CXCL5 significantly reduced the migration of CD11b⁺Gr1⁺ myeloid cells. These results demonstrate that secreted and membrane IL‐1α play different roles in acute liver injury. Secreted IL‐1α could promote T‐cell activation and the recruitment and expansion of CD11b⁺Gr1⁺ myeloid cells through induction of CCL2, CXCL5, and IL‐6. The controlled release of IL‐1α could be a critical regulator during acute liver inflammation.