Direct effect of dsRNA mimetics on cancer cells induces endogenous IFN‐β production capable of improving dendritic cell function

Viral double‐stranded RNA (dsRNA) mimetics have been explored in cancer immunotherapy to promote antitumoral immune response. Polyinosine–polycytidylic acid (poly I:C) and polyadenylic–polyuridylic acid (poly A:U) are synthetic analogs of viral dsRNA and strong inducers of type I interferon (IFN). We describe here a novel effect of dsRNA analogs on cancer cells: besides their potential to induce cancer cell apoptosis through an IFN‐β autocrine loop, dsRNA‐elicited IFN‐β production improves dendritic cell (DC) functionality. Human A549 lung and DU145 prostate carcinoma cells significantly responded to poly I:C stimulation, producing IFN‐β at levels that were capable of activating STAT1 and enhancing CXCL10, CD40, and CD86 expression on human monocyte‐derived DCs. IFN‐β produced by poly I:C‐activated human cancer cells increased the capacity of monocyte‐derived DCs to stimulate IFN‐γ production in an allogeneic stimulatory culture in vitro. When melanoma murine B16 cells were stimulated in vitro with poly A:U and then inoculated into TLR3^?/? mice, smaller tumors were elicited. This tumor growth inhibition was abrogated in IFNAR1^?/? mice. Thus, dsRNA compounds are effective adjuvants not only because they activate DCs and promote strong adaptive immunity, but also because they can directly act on cancer cells to induce endogenous IFN‐β production and contribute to the antitumoral response.     

IFN‐λ‐mediated IL‐12 production in macrophages induces IFN‐γ production in human NK cells

With increasing interest in alternative options to interferon‐alpha‐based treatments, IFN‐λ has shown therapeutic promise in a variety of diseases. Although the antiviral activity of IFN‐λ has been extensively studied, there is limited knowledge regarding the immunological functions of IFN‐λ and how these differ from those of other classes of IFNs. In this study, we investigated the effects of IFN‐λ on primary human NK cells, both in a direct and indirect capacity. We demonstrate that in contrast to interferon‐alpha, IFN‐λ is unable to directly stimulate NK cells, due to the absence of IFN‐λ receptor chain 1 (IFN‐λR1) on NK cells. However, IFN‐λ, in combination with TLR4 challenge, is able to induce the production of select members of the IL‐12 family of cytokines in monocyte‐derived macrophages. We further show that through macrophage‐mediated IL‐12 production, IFN‐λ is able to indirectly affect NK cells and ultimately induce IFN‐γ production.     

Regulatory effects of IFN‐β on production of osteopontin and IL‐17 by CD4+ T Cells in MS

IFN‐β currently serves as one of the major treatments for MS. Its anti‐inflammatory mechanism has been reported as involving a shift in cytokine balance from Th1 to Th2 in the T‐cell response against elements of the myelin sheath. In addition to the Th1 and Th2 groups, two other important pro‐inflammatory cytokines, IL‐17 and osteopontin (OPN), are believed to play important roles in CNS inflammation in the pathogenesis of MS. In this study, we examined the potential effects of IFN‐β on the regulation of OPN and IL‐17 in MS patients. We found that IFN‐β used in vitro at 0.5–3 ng/mL significantly inhibited the production of OPN in primary T cells derived from PBMC. The inhibition of OPN was determined to occur at the CD4⁺ T‐cell level. In addition, IFN‐β inhibited the production of IL‐17 and IL‐21 in CD4⁺ T cells. It has been described that IFN‐β suppresses IL‐17 production through the inhibition of a monocytic cytokine, the intracellular translational isoform of OPN. Our further investigation demonstrated that IFN‐β also acted directly on the CD4⁺ T cells to regulate OPN and IL‐17 expression through the type I IFN receptor‐mediated activation of STAT1 and suppression of STAT3 activity. Administration of IFN‐β to EAE mice ameliorated the disease severity. Furthermore, spinal cord infiltration of OPN⁺ and IL‐17⁺ cells decreased in IFN‐β‐treated EAE mice along with decreases in serum levels of OPN and IL‐21. Importantly, decreased OPN production by IFN‐β treatment contributes to the reduced migratory activity of T cells. Taken together, the results from both in vitro and in vivo experiments indicate that IFN‐β treatment can down‐regulate the OPN and IL‐17 production in MS. This study provides new insights into the mechanism of action of IFN‐β in the treatment of MS.     

IL‐18‐induced expression of high‐affinity IL‐2R on murine NK cells is essential for NK‐cell IFN‐γ production during murine Plasmodium yoelii infection

Early production of pro‐inflammatory cytokines, including IFN‐γ, is essential for control of blood‐stage malaria infections. We have shown that IFN‐γ production can be induced among human natural killer (NK) cells by coculture with Plasmodium falciparum infected erythrocytes, but the importance of this response is unclear. To further explore the role of NK cells during malaria infection, we have characterized the NK‐cell response of C57BL/6 mice during lethal (PyYM) or nonlethal (Py17XNL) P. yoelii infection. Ex vivo flow cytometry revealed that NK cells are activated within 24 h of Py17XNL blood‐stage infection, expressing CD25 and producing IFN‐γ; this response was blunted and delayed during PyYM infection. CD25 expression and IFN‐γ production were highly correlated, suggesting a causal relationship between the two responses. Subsequent in vitro experiments revealed that IL‐18 signaling is essential for induction of CD25 and synergizes with IL‐12 to enhance CD25 expression on splenic NK cells. In accordance with this, Py17XNL‐infected erythrocytes induced NK‐cell CD25 expression and IFN‐γ production in a manner that is completely IL‐18‐ and partially IL‐12‐dependent, and IFN‐γ production is enhanced by IL‐2. These data suggest that IL‐2 signaling via CD25 amplifies IL‐18‐ and IL‐12‐mediated NK‐cell activation during malaria infection.     

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.     

IFN‐γ stimulates CpG‐induced IL‐10 production in B cells via p38 and JNK signalling pathways

The production of IL‐10, a potent immunosuppressive cytokine, must be strictly regulated to ensure a balanced immune response. IFN‐γ, a key cytokine in multiple immune processes and pathologies, is known as an inhibitor of IL‐10 production by monocytes and macrophages, but also has some regulatory functions. In the present study, we explored the role of IFN‐γ on Toll‐like receptor (TLR)‐induced IL‐10 production in murine peritoneal and spleen cells and in human peripheral blood mononuclear cells. IFN‐γ inhibited IL‐10 production induced by TLR2, TLR3, TLR4 and TLR7/8 agonists, but stimulated IL‐10 production when cells were triggered with CpG oligodeoxynucleotides, a specific TLR9 agonist. The stimulatory effect of IFN‐γ on TLR9‐induced IL‐10 was restricted to B cells. In line with the increased IL‐10, B cells stimulated with CpG and IFN‐γ profoundly inhibited CD4 T cell proliferation. Further research into the mechanisms involved, revealed that the mitogen‐activated protein kinases p38 and JNK are essential players in this stimulatory effect, and that the phosphatase MKP1 – an inhibitor of p38 and JNK activity – is downregulated after combined stimulation with IFN‐γ and CpG. Our data may represent a novel immunoregulatory role of IFN‐γ in B cells after triggering of TLR9, by stimulating IL‐10 production.     

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.     

TLR‐mediated STAT3 and ERK activation controls IL‐10 secretion by human B cells

IL‐10‐producing B cells have a regulatory effect in various mouse models for immune‐mediated disorders via secretion of IL‐10, a potent immunoregulatory cytokine. However, currently, the signaling pathways that regulate IL‐10 production in B cells are not well understood. Here, we show that TLR signaling, but not BCR activation or CD40 ligation, induces potent production of IL‐10 in human B cells. We demonstrate that the activation of STAT3 and ERK is required for TLR‐induced IL‐10 production by B cells, since inhibition of STAT3 or ERK activation abrogates TLR‐induced IL‐10 production. We also uncover a novel function of the TLR‐MyD88‐STAT3 pathway in B cells, namely controlling IL‐10 production, in addition to the known role for this pathway in antibody production. Furthermore, IFN‐α, a member of the type I IFN family, differentially modulates TLR7/8‐ and TLR9‐activated STAT3 and ERK in B cells, which provides an explanation for our findings that IFN‐α enhances TLR7/8‐induced, but not TLR9‐induced IL‐10 production. These results yield insights into the mechanisms by which TLR signaling regulates IL‐10 production in B cells and how type I IFN modulates TLR‐mediated IL‐10 production by B cells, therefore providing potential targets to modulate the function of IL‐10‐producing B cells.     

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.     

IFN‐β therapy modulates B‐cell and monocyte crosstalk via TLR7 in multiple sclerosis patients

The implication of B lymphocytes in the immunopathology of multiple sclerosis (MS) is increasingly recognized. Here we investigated the response of B cells to IFN‐β, a first‐line therapy for relapsing‐remitting MS patients, upon stimulation with TLR. IFN‐β restored the frequency of TLR7‐induced IgM and IgG‐secreting cells in MS patients to the levels found in healthy donors, showing a specific deficiency in the TLR7 pathway. However, no difference was observed in the TLR9 response. Furthermore, in MS‐derived PBMCs, TLR7‐mediated production of IL‐6 and the ex vivo expression of B‐cell‐activating factor of the TNF family, two crucial cytokines for B‐cell differentiation and survival, were induced by IFN‐β. Depletion of monocytes, which are key producers of both IL‐6 and B‐cell‐activating factor of the TNF family, showed that TLR7‐mediated B‐cell differentiation into Ig‐secreting cells is strongly dependent on the cross‐talk between B cells and monocytes. Accordingly, impaired expression of TLR7 mRNA was observed in PBMCs and monocytes isolated from MS‐affected individuals as compared with those from healthy donors, which was rescued by IFN‐β therapy. Collectively, our data unveil a novel TLR7‐regulated mechanism in in vivo IFN‐β‐stimulated whole leukocytes that could be exploited to define new TLR7‐based strategies for the treatment of MS.     

IL‐18, but not IL‐15, contributes to the IL‐12‐dependent induction of NK‐cell effector functions by Leishmania infantum in vivo

Activation of NK cells is a hallmark of infections with intracellular pathogens. We previously showed that the protozoan parasite Leishmania infantum triggered a rapid NK‐cell response in mice that required TLR9‐positive myeloid DC and IL‐12, but no IFN‐α/β. Here, we investigated whether IL‐15 or IL‐18 mediate the activity of IL‐12 or function as independent activators of NK cells. In contrast to earlier studies that described IL‐15 as crucial for NK‐cell priming in response to TLR ligands, the expression of IFN‐γ, FasL, perforin and granzyme B by NK cells in L. infantum‐infected mice was completely preserved in the absence of IL‐15, whereas the proliferative capacity of NK cells was lower than in WT mice. IFN‐γ secretion, cytotoxicity and FasL expression of NK cells from infected IL‐18^?/? mice were significantly reduced compared with controls, but, unlike IL‐12, IL‐18 was not essential for NK‐cell effector functions. Part of the NK‐cell‐stimulatory effect of IL‐12 was dependent on IL‐18. We conclude that IL‐15 is not functioning as a universal NK‐cell priming signal and that IL‐18 contributes to the NK‐cell response in visceral leishmaniasis. The cytokine requirements for NK‐cell activation appear to differ contingent upon the infectious pathogen.     

Enhanced protection from fibrosis and inflammation in the combined absence of IL‐13 and IFN‐γ

Persistent or dysregulated IL‐13 responses are key drivers of fibrosis in multiple organ systems, and this identifies this cytokine as an important therapeutic target. Nevertheless, the mechanisms by which IL‐13 blockade leads to the amelioration of fibrosis remain unclear. Because IFN‐γ exhibits potent anti‐fibrotic activity, and IL‐4Rα signalling antagonizes IFN‐γ effector function, compensatory increases in IFN‐γ activity following IL‐13/IL‐4Rα blockade might contribute to the reduction in fibrosis. To investigate the role of IFN‐γ, we developed novel IL‐13^?/?/IFN‐γ^?/? double cytokine‐deficient mice and examined disease progression in models of type 2‐driven fibrosis. As predicted, we showed that fibrosis in the lung and liver are both highly dependent on IL‐13. We also observed increased IFN‐γ production and inflammatory activity in the tissues of IL‐13‐deficient mice. Surprisingly, however, an even greater reduction in fibrosis was observed in IL‐13/IFN‐γ double deficient mice, most notably in the livers of mice chronically infected with Schistosoma mansoni. The increased protection was associated with marked decreases in Tgfb1, Mmp12, and Timp1 mRNA expression in the tissues; reduced inflammation; and decreased expression of important pro‐inflammatory mediators such as TNF‐α. Experiments conducted with neutralizing monoclonal antibodies to IL‐13 and IFN‐γ validated the findings with the genetically deficient mice. Together, these studies demonstrate that the reduction in fibrosis observed when IL‐13 signalling is suppressed is not dependent on increased IFN‐γ activity. Instead, by reducing compensatory increases in type 1‐associated inflammation, therapeutic strategies that block IFN‐γ and IL‐13 activity simultaneously can confer greater protection from progressive fibrosis than IL‐13 blockade alone. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.     

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.     

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.     

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.     

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.