Activated γδ T cells inhibit osteoclast differentiation and resorptive activity in vitro

Extensive evidence suggests that the immune system exerts powerful effects on bone cells, particularly in chronic disease pathologies such as rheumatoid arthritis (RA). The chronic inflammatory state in RA, particularly the excessive production of T cell‐derived proinflammatory cytokines such as tumour necrosis factor (TNF)‐α and interleukin (IL)‐17, triggers bone erosions through the increased stimulation of osteoclast formation and activity. While evidence supports a role for IL‐17 and TNF‐α secreted by conventional CD4⁺ T cells in RA, recent evidence in animal models of RA have implicated γδ T cells as a major producer of pathogenic IL‐17. However, the capacity of γδ T cells to influence osteoclast formation and activity in humans has not yet been investigated widely. To address this issue we investigated the effects of γδ T cells on osteoclast differentiation and resorptive activity. We have demonstrated that anti‐CD3/CD28‐stimulated γδ T cells or CD4⁺ T cells inhibit human osteoclast formation and resorptive activity in vitro. Furthermore, we assessed cytokine production by CD3/CD28‐stimulated γδ T cells and observed a lack of IL‐17 production, with activated γδ T cells producing abundant interferon (IFN)‐γ. The neutralization of IFN‐γ markedly restored the formation of osteoclasts from precursor cells and the resorptive activity of mature osteoclasts, suggesting that IFN‐γ is the major factor responsible for the inhibitory role of activated γδ T cells on osteoclastogenesis and resorptive activity of mature osteoclasts. Our work therefore provides new insights on the interactions between γδ T cells and osteoclasts in humans.     

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.     

Pulmonary inflammation in mice with collagen‐induced arthritis is conditioned by complete Freund's adjuvant and regulated by endogenous IFN‐γ

Following immunization with collagen II (CII) in complete Freund's adjuvant (CFA), DBA/1 mice develop arthritis of major joints. This collagen‐induced arthritis (CIA) is used as a model for rheumatoid arthritis (RA) in man. Inflammatory changes in lung tissue commonly occur in RA. However, evidence for pulmonary inflammation in CIA is scarce and ambiguous. Here, we demonstrate pulmonary inflammation accompanying CIA in wild‐type DBA/1 mice. In IFN‐γ receptor‐deficient (IFN‐γR KO) mice, inflammation was more frequent and more severe. Injection of CFA only (without CII) proved to be as efficient in eliciting pulmonary inflammation as immunization with CFA + CII, though being less effective in causing arthritis. Significant correlation in severity between joint and pulmonary involvement could not be demonstrated. Macroscopic, microscopic, and functional characteristics of pulmonary inflammation in the mice resembled those seen in human RA. Increased inflammation in IFN‐γR KO mice was accompanied by augmented expression of various cytokines and chemokines, as measured by RT‐PCR on affected tissue. Treatment with a TNF‐α inhibitor ameliorated lung pathology. We conclude that CIA in DBA/1 mice is accompanied by pulmonary inflammation. Although both disease processes are kept in check by endogenous IFN‐γ, lack of strict parallelism indicates that overlap in their pathogeneses is partial.     

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.     

Expression and Identification of Immunological Activities of the HIV‐gp120N‐Human Interferon Gamma Fusion Protein

The human immunodeficiency virus type 1 (HIV‐1) envelope glycoprotein gp120 is a vaccine immunogen that has been studied extensively. To enhance the immune response of cells against HIV‐1 gp120, we tested the coexpression of gp120N with interferon‐γ (IFN‐γ) as an immune adjuvant. Two recombinant prokaryotic plasmids were constructed: the pET44b‐HIV‐1‐gp120N plasmid construct carried the HIV‐1 gp120N gene (pET44‐gp120N), whereas the pET44b‐HIV‐1‐gp120N‐IFN‐γ plasmid construct carried a fusion gp120N‐IFN‐γ gene (pET44b‐gp120N‐IFN‐γ). Target protein expression was achieved in E. coli BL21 (DE3) cells by chemical induction. To test the immunological activity of the proteins, mice were injected with a control, gp120N, or the fusion gp120N‐IFN‐γ protein. The serum and spleen cells of the mice were collected for immunological detection. Results showed that specific T lymphocyte proliferation and the expression of the Th1‐type cytokines (IL‐2 and IFN‐γ) were higher in the gp120N‐IFN‐γ group than the other two groups (P < 0.05). No difference was observed in the expression levels of the Th2‐type cytokines (IL‐4 and IL‐10; P > 0.05). These results suggest that IFN‐γ plays a prominent role as an immune adjuvant when coexpressed with HIV‐1 gp120N. IFN‐γ enhances the specific cell immune response of mice against HIV‐1 gp120. Anat Rec, 292:381–386, 2009. © 2009 Wiley‐Liss, Inc.     

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.     

Paeoniflorin ameliorates collagen‐induced arthritis via suppressing nuclear factor‐κB signalling pathway in osteoclast differentiation

Paeoniflorin (PF), extracted from the root of Paeonia lactiflora Pall, exhibits anti‐inflammatory properties in several autoimmune diseases. Osteoclast, the only somatic cell with bone resorbing capacity, was the direct cause of bone destruction in rheumatoid arthritis (RA) and its mouse model, collagen‐induced arthritis (CIA). The objective of this study was to estimate the effect of PF on CIA mice, and explore the mechanism of PF in bone destruction. We demonstrated that PF treatment significantly ameliorated CIA through inflammatory response inhibition and bone destruction suppression. Furthermore, PF treatment markedly decreased osteoclast number through the altered RANKL/RANK/OPG ratio and inflammatory cytokines profile. Consistently, we found that osteoclast differentiation was significantly inhibited by PF through down‐regulation of nuclear factor‐κB activation in vitro. Moreover, we found that PF suppressed nuclear factor‐κB activation by decreasing its translocation to the nucleus in osteoclast precursor cells. Taken together, our new findings provide insights into a novel function of PF in osteoclastogenesis and demonstrate that PF would be a new therapeutic modality as a natural agent for RA treatment and other autoimmune conditions with bone erosion.     

B cells expressing IFN‐γ suppress Treg‐cell differentiation and promote autoimmune experimental arthritis

Clinical efficacy in the treatment of rheumatoid arthritis with anti‐CD20 (Rituximab)‐mediated B‐cell depletion has garnered interest in the mechanisms by which B cells contribute to autoimmunity. We have reported that B‐cell depletion in a murine model of proteoglycan‐induced arthritis (PGIA) leads to an increase in Treg cells that correlate with decreased autoreactivity. Here, we demonstrate that the increase in Treg cells after B‐cell depletion is due to an increase in the differentiation of naïve CD4⁺ T cells into Treg cells. Since the development of PGIA is dependent on IFN‐γ and B cells are reported to produce IFN‐γ, we hypothesized that B‐cell‐specific IFN‐γ plays a role in the development of PGIA. Accordingly, mice with B‐cell‐specific IFN‐γ deficiency were as resistant to the induction of PGIA as mice that were completely IFN‐γ deficient. Importantly, despite a normal frequency of IFN‐γ‐producing CD4⁺ T cells, B‐cell‐specific IFN‐γ‐deficient mice exhibited a higher percentage of Treg cells compared with that in WT mice. These data indicate that B‐cell IFN‐γ production inhibits Treg‐cell differentiation and exacerbates arthritis. Thus, we have established that IFN‐γ, specifically derived from B cells, uniquely contributes to the pathogenesis of autoimmunity through prevention of immunoregulatory mechanisms.     

Neonatal macrophages express elevated levels of interleukin‐27 that oppose immune responses

Microbial infections are a major cause of infant mortality worldwide because of impaired immune defences in this population. The nature of this work was to further understand the mechanistic limitations of the neonatal and infant immune response. Interleukin‐27 (IL‐27) is a heterodimeric cytokine of the IL‐12 family that is produced primarily by antigen‐presenting cells and is immunosuppressive toward a variety of immune cell types. We show that IL‐27 gene expression is elevated in cord blood‐derived macrophages relative to macrophages originating from healthy adults. We also evaluated the duration over which elevated IL‐27 gene expression may impact immune responses in mice. Age‐dependent analysis of IL‐27 gene expression indicated that levels of IL‐27 remained significantly elevated throughout infancy and then declined in adult mice. Flow cytometric analysis of intracellular cytokine‐stained splenocytes further confirmed these results. Interleukin‐27 may be induced during pregnancy to contribute to the immunosuppressive environment at the fetal–maternal interface because we demonstrate dose‐responsive gene expression to progesterone in macrophages. Neutralization of IL‐27 in neonatal macrophages improved the ability of these cells to limit bacterial replication. Moreover, neutralization of IL‐27 during incubation with the Mycobacterium bovis bacillus Calmette–Guérin vaccine augmented the level of interferon‐γ elicited from allogeneic CD4⁺ T lymphocytes. This suggests that blocking IL‐27 during vaccination and infection may improve immune responses in newborn and infant populations. Furthermore, mice will be a suitable model system to further address these possibilities.     

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.     

Innate IFN‐γ Production by Subsets of Natural Killer Cells,Natural Killer T Cells and γδ T Cells in Response to Dying Bacterial‐Infected Macrophages

Interferon‐γ (IFN‐γ) activation of macrophages is a crucial step in the early innate defence against bacterial infection. This innate IFN‐γ is thought to be produced mainly by natural killer (NK) cells through activation with interleukin (IL)‐12p70 secreted by macrophages and dendritic cells (DCs) that have sensed bacterial products. However, a number of reports have shown that bacterial stimuli are unable to induce macrophages and/or DCs to produce sufficient amounts of IL‐12p70 unless these cells are primed by IFN‐γ. It remains, therefore, unsettled how initial IFN‐γ is produced. In a previous study, we reported a novel IFN‐γ production pathway that was associated with cell death in macrophages caused by intracellular bacteria like Listeria monocytogenes (LM) and Shigella flexneri. In this study, we showed that cell death of bone‐marrow‐derived macrophage (BMM) cells following in vitro infection with Staphylococcus aureus (SA), an extracellular bacterium, can also stimulate this IFN‐γ production pathway. We also unequivocally demonstrated by using BMM cells from IL‐12‐deficient mice that the bacterial‐infected macrophage cell death‐mediated IFN‐γ production can occur without IL‐12 although the magnitude of the response is much smaller than that in the presence of IL‐12. The enhancing effect of IL‐12 on this response proved to be attributable to the negligible amounts (0.5～1.5 pg/ml) of IL‐12p70 but not to the large amounts of IL‐12p40 that were both secreted by SA‐ and LM‐infected macrophages. Taken all together, we propose that macrophage cell death caused by bacteria may trigger the initial IFN‐γ production at an early stage of bacterial infection.     

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.     

Regulation of IFN‐γ by IL‐13 dictates susceptibility to secondary postinfluenza MRSA pneumonia

Superinfection in mice at day 7 postinfluenza infection exacerbates bacterial pneumonia at least in part via downstream effects of increased IFN‐γ signaling. Here we show that up to 3 days postinfluenza infection, mice have reduced susceptibility to superinfection with methicillin‐resistant Staphylococcus aureus (MRSA), but that superinfection during that time exacerbated influenza disease. This was due to IL‐13 signaling that was advantageous for resolving MRSA infection via inhibition of IFN‐γ, but was detrimental to the clearance of influenza virus. However, if superinfection did not occur until the near resolution of influenza infection (day 7), IL‐13 signaling was inhibited, at least in part by upregulation of IL‐13 decoy receptor (IL‐13Rα2), which in turn caused increases in IFN‐γ signaling and exacerbation of bacterial infection. Understanding these cytokine sequelae is critical to development of immunotherapies for influenza‐MRSA coinfection since perturbations of these sequelae at the wrong time could increase susceptibility to MRSA and/or influenza.     

RORγt antagonist suppresses M3 muscarinic acetylcholine receptor‐induced Sjögren's syndrome‐like sialadenitis

We showed recently that M3 muscarinic acetylcholine receptor (M3R)‐reactive CD3⁺ T cells play a pathogenic role in the development of murine autoimmune sialadenitis (MIS), which mimics Sjögren's syndrome (SS). The aim of this study was to determine the effectiveness and mechanism of action of retinoic acid‐related orphan receptor‐gamma t (RORγt) antagonist (A213) in MIS. Splenocytes from M3R knockout (M3R^–/–) mice immunized with murine M3R peptide mixture were inoculated into recombination‐activating gene 1 knockout (Rag‐1^–/–) mice (M3R^–/–→Rag‐1^–/–) with MIS. Immunized M3R^–/– mice (pretransfer treatment) and M3R^–/–→Rag‐1^–/– mice (post‐transfer treatment) were treated with A213 every 3 days. Salivary volume, severity of sialadenitis and cytokine production from M3R peptide‐stimulated splenocytes and lymph node cells were examined. Effects of A213 on cytokine production were analysed by enzyme‐linked immunosorbent assay (ELISA) and on T helper type 1 (Th1), Th17 and Th2 differentiation from CD4⁺ T cells by flow cytometry. Pretransfer A213 treatment maintained salivary volume, improved MIS and reduced interferon (IFN)‐γ and interleukin (IL)‐17 production significantly compared with phosphate‐buffered saline (PBS) (P < 0·05). These suppressive effects involved CD4⁺ T cells rather than CD11c⁺ cells. Post‐transfer treatment with A213 increased salivary volume (P < 0·05), suppressed MIS (P < 0·005) and reduced IFN‐γ and IL‐17 production (P < 0·05). In vitro, A213 suppressed IFN‐γ and IL‐17 production from M3R‐stimulated splenocytes and CD4⁺ T cells of immunized M3R^–/– mice (P < 0·05). In contrast with M3R specific responses, A213 suppressed only IL‐17 production from Th17 differentiated CD4⁺ T cells without any effect on Th1 and Th2 differentiation in vitro. Our findings suggested that RORγt antagonism is potentially suitable treatment strategy for SS‐like sialadenitis through suppression of IL‐17 and IFN‐γ production by M3R‐specific T cells.     

Molecular analysis for patients with IL‐12 receptor β1 deficiency

Autosomal recessive interleukin‐12 receptor β1 (IL‐12Rβ1) deficiency has been described as the most common cause of Mendelian susceptibility to mycobacterial disease (MSMD), characterized by clinical disease due to weakly virulent mycobacteria such as Bacille Calmette–Guérin (BCG) vaccines and environmental mycobacteria (EM) in children who are normally resistant to most infectious agents. Here, we report the cases of five patients with mycobacterial infection, including one with systemic lupus erythematosus (SLE). Blood samples from patients and healthy controls were activated in vitro with BCG, BCG+IL‐12, and BCG+IFN‐γ. The results showed reduced or no production of IFN‐γ after IL‐12 stimulation in all samples. IL‐12Rβ1 expression on the cell surface was negligible or absent. Genetic analysis showed five novel mutations.     

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.