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.     

Innate IFN‐γ promotes development of experimental autoimmune encephalomyelitis: A role for NK cells and M1 macrophages

The role of IFN‐γ in the pathogenesis of autoimmune diseases is controversial. Although Th1 cells can induce experimental autoimmune encephalomyelitis (EAE), IFN‐γ can suppress Th17 cells that are pathogenic in EAE. Here we show that NK cells provide an early source of IFN‐γ during development of EAE. Depletion of NK cells or neutralization of IFN‐γ delayed the onset of EAE and was associated with reduced infiltration of IL‐17⁺ and GM‐CSF⁺ T cells into the CNS. In the passive transfer model, immune cells from myelin oligodendrocyte glycoprotein (MOG)‐immunized IFN‐γ^?/? mice failed to induce EAE, despite producing IL‐17 and GM‐CSF. The macrophages expressed markers of M2 activation and the T cells had low very late antigen‐4 (VLA‐4) expression and failed to infiltrate the CNS. Addition of recombinant IFN‐γ to immune cells from the IFN‐γ^?/? mice activated M1 macrophages and restored VLA‐4 expression, migratory, and encephalitogenic activity of T cells. Furthermore, treatment of recipient mice with anti‐VLA‐4 neutralizing antibody abrogated EAE induced by transfer of T cells from WT mice. Our findings demonstrate IFN‐γ‐producing T cells are not required for development of EAE, but NK cell‐derived IFN‐γ has a key role in promoting M1 macrophage expansion and VLA‐4‐mediated migration of encephalitogenic T cells into the CNS.     

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.     

Commensal microflora and interferon‐γ promote steady‐state interleukin‐7 production in vivo

IL‐7 is a major regulator of lymphocyte homeostasis; however, little is known about the mechanisms that regulate IL‐7 production. To study Il7 gene regulation in vivo, we generated a novel IL‐7‐reporter mouse, which allows the non‐invasive quantification of Il7 gene activity in live mice and, additionally, the simultaneous activation/inactivation of target genes in IL‐7‐producing cells. With these IL‐7‐reporter mice, we identify thymus, skin and intestine as major sources of IL‐7 in vivo. Importantly, we show that IFN‐γ and the commensal microflora promote steady‐state IL‐7 production in the intestine. Furthermore, we demonstrate that the blockade of IFN‐γ signaling in intestinal epithelial cells strongly reduces their IFN‐γ‐driven IL‐7 production. In summary, our data suggest a feedback loop in which commensal bacteria drive IFN‐γ production by lymphocytes, which in turn promotes epithelial cell IL‐7 production and the survival of IL‐7‐dependent lymphocytes.     

Interleukin‐27 suppresses osteoclastogenesis via induction of interferon‐γ

Interleukin (IL)‐27 is a heterodimeric cytokine that is known to have both stimulatory and inhibitory functions during immune responses. We investigated the effects of IL‐27 on arthritis and bone erosion in the murine collagen‐induced arthritis (CIA) model. We demonstrate that the inhibitory effect of IL‐27 on osteoclastogenesis is associated with interferon‐γ (IFN‐γ) production by using an IFN‐γ knockout mouse model. The IL‐27‐Fc was injected into both CIA and IFN‐γ‐deficient mice. The effects of IL‐27‐Fc on osteoclast differentiation were evaluated both in vitro and in vivo. The IL‐27‐Fc‐injected mice showed significantly lower arthritis indices and fewer tartrate‐resistant acid‐phosphatase‐positive osteoclasts in their joint tissues than untreated mice. Interleukin‐27 inhibited osteoclastogenesis from bone marrow‐derived mononuclear cells in vitro, which was counteracted by the addition of anti‐IFN‐γ antibody. The IL‐27‐Fc did not affect arthritis in IFN‐γ knockout mice. Interleukin‐27 also suppressed osteoclast differentiation in human and intriguingly, it could promote the expression of IFN‐γ on priming osteoclasts. These results imply that IL‐27 suppressed the generation of CIA and osteoclastogenesis, which were mediated by the induction of IFN‐γ.     

A unique DNA methylation signature defines a population of IFN‐γ/IL‐4 double‐positive T cells during helminth infection

Th1 and Th2 cell fates are traditionally viewed as mutually exclusive, but recent work suggests that these lineages may be more plastic than previously thought. When isolating splenic CD4⁺ T cells from mice infected with the parasitic helminth Schistosoma mansoni, we observed a defined population of IFN‐γ/IL‐4 double‐positive cells. These IFN‐γ⁺IL‐4⁺ cells showed differences in DNA methylation at the Ifng and Il4 loci when compared with IFN‐γ⁺IL‐4^? (Th1) and IFN‐γ^?IL‐4⁺ (Th2) cells, demonstrating that they represent a distinct effector cell population. IFN‐γ⁺IL‐4⁺ cells also displayed a discrete DNA methylation pattern at a CpG island within the body of the Gata3 gene, which encodes the master regulator of Th2 identity. DNA methylation at this region correlated with decreased Gata3 levels, suggesting a possible role in controlling Gata3 expression. These data provide important insight into the molecular mechanisms behind the co‐existence of Th1 and Th2 characteristics.     

Reverse plasticity: TGF‐β and IL‐6 induce Th1‐to‐Th17‐cell transdifferentiation in the gut

Th17 cells are a heterogeneous population of pro‐inflammatory T cells that have been shown to mediate immune responses against intestinal bacteria. Th17 cells are highly plastic and can transdifferentiate to Th1/17 cells or unconventional Th1 cells, which are highly pathogenic in animal models of immune‐mediated diseases such as inflammatory bowel diseases. A recent European Journal of Immunology article by Liu et al. (Eur. J. Immunol. 2015. 45:1010–1018) showed, surprisingly, that Th1 cells have a similar plasticity, and could transdifferentiate to Th17 cells. Thus, IFN‐γ‐producing Th1 effector cells specific for an intestinal microbial antigen were shown to acquire IL‐17‐producing capacities in the gut in a mouse model of colitis, and in response to TGF‐β and IL‐6 in vitro. TGF‐β induced Runx1, and together with IL‐6 was shown to render the ROR‐γt and IL‐17 promoters in Th1 cells accessible for Runx1 binding. In this commentary, we discuss how this unexpected plasticity of Th1 cells challenges our view on the generation of Th1/17 cells with the capacity to co‐produce IL‐17 and IFN‐γ, and consider possible implications of this Th1‐to‐Th17‐cell conversion for therapies of inflammatory bowel diseases and protective immune responses against intracellular pathogens.     

CCR6 and NK1.1 distinguish between IL‐17A and IFN‐γ‐producing γδ effector T cells

γδ T cells are a potent source of innate IL‐17A and IFN‐γ, and they acquire the capacity to produce these cytokines within the thymus. However, the precise stages and required signals that guide this differentiation are unclear. Here we show that the CD24^low CD44^high effector γδ T cells of the adult thymus are segregated into two lineages by the mutually exclusive expression of CCR6 and NK1.1. Only CCR6⁺ γδ T cells produced IL‐17A, while NK1.1⁺ γδ T cells were efficient producers of IFN‐γ but not of IL‐17A. Their effector phenotype correlated with loss of CCR9 expression, particularly among the NK1.1⁺ γδ T cells. Accordingly, both γδ T‐cell subsets were rare in gut‐associated lymphoid tissues, but abundant in peripheral lymphoid tissues. There, they provided IL‐17A and IFN‐γ in response to TCR‐specific and TCR‐independent stimuli. IL‐12 and IL‐18 induced IFN‐γ and IL‐23 induced IL‐17A production by NK1.1⁺ or CCR6⁺ γδ T cells, respectively. Importantly, we show that CCR6⁺ γδ T cells are more responsive to TCR stimulation than their NK1.1⁺ counterparts. In conclusion, our findings support the hypothesis that CCR6⁺ IL‐17A‐producing γδ T cells derive from less TCR‐dependent selection events than IFN‐γ‐producing NK1.1⁺ γδ T cells.     

Mycobacterium tuberculosis PstS1 amplifies IFN‐γ and induces IL‐17/IL‐22 responses by unrelated memory CD4+ T cells via dendritic cell activation

The immunological mechanisms that modulate protection during Mycobacterium tuberculosis (Mtb) infection or vaccination are not fully understood. Secretion of IFN‐γ and, to a lesser extent, of IL‐17 by CD4⁺ T cells plays a major role both in protection and immunopathology. Few Mtb Ags interacting with DCs affect priming, activation, and regulation of Ag‐unrelated CD4⁺ T‐cell responses. Here we demonstrate that PstS1, a 38 kDa‐lipoprotein of Mtb, promotes Ag‐independent activation of memory T lymphocytes specific for Ag85B or Ag85A, two immunodominant protective Ags of Mtb. PstS1 expands CD4⁺ and CD8⁺ memory T cells, amplifies secretion of IFN‐γ and IL‐22 and induces IL‐17 production by effector memory cells in an Ag‐unrelated manner in vitro and in vivo. These effects were mediated through the stimulation of DCs, particularly of the CD8α^? subtype, which respond to PstS1 by undergoing phenotypic maturation and by secreting IL‐6, IL‐1β and, to a lower extent, IL‐23. IL‐6 secretion by PstS1‐stimulated DCs was required for IFN‐γ, and to a lesser extent for IL‐22 responses by Ag85B‐specific memory T cells. These results may open new perspectives for immunotherapeutic strategies to control Th1/Th17 immune responses in Mtb infections and in vaccinations against tuberculosis.     

Predominance of heterosubtypic IFN‐γ‐only‐secreting effector memory T cells in pandemic H1N1 naive adults

The 2009/10 pandemic (pH1N1) highlighted the need for vaccines conferring heterosubtypic immunity against antigenically shifted influenza strains. Although cross‐reactive T cells are strong candidates for mediating heterosubtypic immunity, little is known about the population‐level prevalence, frequency, and cytokine‐secretion profile of heterosubtypic T cells to pH1N1. To assess this, pH1N1 sero‐negative adults were recruited. Single‐cell IFN‐γ and IL‐2 cytokine‐secretion profiles to internal proteins of pH1N1 or live virus were enumerated and characterised. Heterosubtypic T cells recognising pH1N1 core proteins were widely prevalent, being detected in 90% (30 of 33) of pH1N1‐naïve individuals. Although the last exposure to influenza was greater than 6 months ago, the frequency and proportion of the IFN‐γ‐only‐secreting T‐cell subset was significantly higher than the IL‐2‐only‐secreting subset. CD8⁺ IFN‐γ‐only‐secreting heterosubtypic T cells were predominantly CCR7^?CD45RA^? effector‐memory phenotype, expressing the tissue‐homing receptor CXCR3 and degranulation marker CD107. Receipt of the 2008–09 influenza vaccine did not alter the frequency of these heterosubtypic T cells, highlighting the inability of current vaccines to maintain this heterosubtypic T‐cell pool. The surprisingly high prevalence of pre‐existing circulating pH1N1‐specific CD8⁺ IFN‐γ‐only‐secreting effector memory T cells with cytotoxic and lung‐homing potential in pH1N1‐seronegative adults may partly explain the low case fatality rate despite high rates of infection of the pandemic in young adults.     

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.     

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.     

T‐bet is essential for Th1‐mediated,but not Th17‐mediated,CNS autoimmune disease

T cells that produce both IL‐17 and IFN‐γ, and co‐express ROR‐γt and T‐bet, are often found at sites of autoimmune inflammation. However, it is unknown whether this co‐expression of T‐bet with ROR‐γt is a prerequisite for immunopathology. We show here that T‐bet is not required for the development of Th17‐driven experimental autoimmune encephalomyelitis (EAE). The disease was not impaired in T‐bet^?/? mice and was associated with low IFN‐γ production and elevated IL‐17 production among central nervous system (CNS) infiltrating CD4⁺ T cells. T‐bet^?/? Th17 cells generated in the presence of IL‐6/TGF‐β/IL‐1 and IL‐23 produced GM‐CSF and high levels of IL‐17 and induced disease upon transfer to naïve mice. Unlike their WT counterparts, these T‐bet^?/– Th17 cells did not exhibit an IL‐17→IFN‐γ switch upon reencounter with antigen in the CNS, indicating that this functional change is not critical to disease development. In contrast, T‐bet was absolutely required for the pathogenicity of myelin‐responsive Th1 cells. T‐bet‐deficient Th1 cells failed to accumulate in the CNS upon transfer, despite being able to produce GM‐CSF. Therefore, T‐bet is essential for establishing Th1‐mediated inflammation but is not required to drive IL‐23‐induced GM‐CSF production, or Th17‐mediated autoimmune inflammation.     

MD‐2 is involved in the stimulation of matrix metalloproteinase‐1 expression by interferon‐γ and high glucose in mononuclear cells – a potential role of MD‐2 in Toll‐like receptor 4‐independent signalling

We reported recently that treatment of diabetic apolipoprotein E‐deficient mice with the Toll‐like receptor 4 (TLR4) antagonist Rs‐LPS, a lipopolysaccharide isolated from Rhodobacter sphaeroides, inhibited atherosclerosis. Since it is known that Rs‐LPS antagonizes TLR4 by targeting TLR4 co‐receptor MD‐2, this finding indicates that MD‐2 is a potential target for the treatment of atherosclerosis. In this study, we determined if MD‐2 is involved in the gene expression regulated by signalling pathways independent of TLR4. Given that interferon‐γ (IFNγ) and hyperglycaemia play key roles in atherosclerosis, we determined if MD‐2 is involved in IFN‐γ and high‐glucose‐regulated gene expression in mononuclear cells. Results showed that IFN‐γ and high glucose synergistically stimulated matrix metalloproteinase 1 (MMP‐1), a proteinase essential for vascular tissue remodelling and atherosclerosis, in U937 mononuclear cells, but Rs‐LPS inhibited the MMP‐1 stimulation. To provide more evidence for a role of MD‐2 in IFN‐γ‐stimulated MMP‐1, studies using antibodies and small interfering RNA demonstrated that MD‐2 blockade or knockdown attenuated the effect of IFN‐γ on MMP‐1. Furthermore, studies using PCR arrays showed that MD‐2 blockade had a similar effect as IFN‐γ receptor blockade on the inhibition of IFN‐γ‐stimulated pro‐inflammatory molecules. Although these findings indicate the involvement of MD‐2 in IFN‐γ signalling, we also observed that MD‐2 was up‐regulated by IFN‐γ and high glucose. We found that MD‐2 up‐regulation by IFN‐γ played an essential role in the synergistic effect of IFN‐γ and LPS on MMP‐1 expression. Taken together, these findings indicate that MD‐2 is involved in IFN‐γ signalling and IFN‐γ‐augmented MMP‐1 up‐regulation by LPS.     

Accelerated tumour metastasis due to interferon‐γ receptor‐mediated dissociation of perivascular cells from blood vessels

Angiostasis mediated by interferon (IFN)‐γ is a key mechanism of anti‐tumour immunity; however, the effect of IFN‐γ on host vascular endothelial growth factor A (VEGFA)‐expressing cells during tumour progression is still elusive. Here, we developed transgenic mice with IFN‐γ receptor (IFNγR) expression under control of the Vegfa promoter (V‐γR). In these mice, the IFN‐γ responsiveness of VEGFA‐expressing cells led to dramatic growth suppression of transplanted lung carcinoma cells. Surprisingly, increased mortality and tumour metastasis were observed in the tumour‐bearing V‐γR mice, in comparison with the control wild‐type and IFNγR‐deficient mice. Further study showed that perivascular cells were VEGFA‐expressing cells and potential IFN‐γ targets. In vivo, tumour vascular perfusion and pericyte association with blood vessels were massively disrupted in V‐γR mice. In vitro, IFN‐γ inhibited transforming growth factor‐β signalling by upregulating SMAD7, and therefore downregulated N‐cadherin expression in pericytes. Importantly, IFN‐γ neutralization in vivo with a monoclonal antibody reduced tumour metastasis. Together, the results suggest that IFNγR‐mediated dissociation of perivascular cells from blood vessels contributes to the acceleration of tumour metastasis. Thus, the inhibition of tumour growth via IFN‐γ‐induced angiostasis might also accelerate tumour metastasis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

The regulatory role of interferon‐γ producing gamma delta T cells via the suppression of T helper 17 cell activity in bleomycin‐induced pulmonary fibrosis

Interstitial pneumonia (IP) is a chronic progressive interstitial lung disease associated with poor prognosis and high mortality. However, the pathogenesis of IP remains to be elucidated. The aim of this study was to clarify the role of pulmonary γδT cells in IP. In wild‐type (WT) mice exposed to bleomycin, pulmonary γδT cells were expanded and produced large amounts of interferon (IFN)‐γ and interleukin (IL)‐17A. Histological and biochemical analyses showed that bleomycin‐induced IP was more severe in T cell receptor (TCR‐δ‐deficient (TCRδ^–/–) mice than WT mice. In TCRδ^–/– mice, pulmonary IL‐17A⁺CD4⁺ Τ cells expanded at days 7 and 14 after bleomycin exposure. In TCRδ^–/– mice infused with γδT cells from WT mice, the number of pulmonary IL‐17A⁺ CD4⁺ T cells was lower than in TCRδ^–/– mice. The examination of IL‐17A^–/– TCRδ^–/– mice indicated that γδT cells suppressed pulmonary fibrosis through the suppression of IL‐17A⁺CD4⁺ T cells. The differentiation of T helper (Th)17 cells was determined in vitro, and CD4⁺ cells isolated from TCRδ^–/– mice showed normal differentiation of Th17 cells compared with WT mice. Th17 cell differentiation was suppressed in the presence of IFN‐γ producing γδT cells in vitro. Pulmonary fibrosis was attenuated by IFN‐γ‐producing γδT cells through the suppression of pulmonary IL‐17A⁺CD4⁺ T cells. These results suggested that pulmonary γδT cells seem to play a regulatory role in the development of bleomycin‐induced IP mouse model via the suppression of IL‐17A production.     

IFN‐γ directly inhibits murine B‐cell precursor leukemia‐initiating cell proliferation early in life

The early‐life immune environment has been implicated as a modulator of acute lymphoblastic leukemia (ALL) development in children, with infection being associated with significant changes in ALL risk. Furthermore, polymorphisms in several cytokine genes, including IL‐10 and IFN‐γ, are associated with leukemia development. However, the mechanisms and timing of these influences remain unknown. Here, we use the Eμ‐ret transgenic mouse model of B‐cell precursor ALL to assess the influence of IFN‐γ on the early‐life burden of leukemia‐initiating cells. The absence of IFN‐γ activity resulted in greater numbers of leukemia‐initiating cells early in life and was associated with accelerated leukemia onset. The leukemia‐initiating cells from IFN‐γ‐knockout mice had reduced suppressor of cytokine signaling (SOCS‐1) expression, were significantly more sensitive to IFN‐γ, and exhibited more rapid expansion in vivo than their wild‐type counterparts. However, sensitivity to this inhibitory pathway was lost in fully transformed IFN‐γ‐knockout leukemia cells. These results demonstrate that the influence of IFN‐γ on ALL progression may not be mediated by selection of nascent transformed cells but rather through a general SOCS‐mediated reduction in B‐cell precursor proliferation. Thus, while cytokine levels may influence leukemia at multiple points during disease progression, our study indicates a significant early influence of basal, infection‐independent cytokine production on leukemogenesis.