Long‐Term Therapy With Omega‐3 Ameliorates Myonecrosis and Benefits Skeletal Muscle Regeneration in Mdx Mice

In Duchenne muscle dystrophy (DMD) and in the mdx mouse model of DMD, a lack of dystrophin leads to myonecrosis and cardiorespiratory failure. Several lines of evidence suggest a detrimental role of the inflammatory process in the dystrophic process. Previously, we demonstrated that short‐term therapy with eicosapentaenoic acid (EPA), at early stages of disease, ameliorated dystrophy progression in the mdx mouse. In the present study, we evaluated the effects of a long‐term therapy with omega‐3 later in dystrophy progression. Three‐month‐old mdx mice received omega‐3 (300 mg/kg) or vehicle by gavage for 5 months. The quadriceps and diaphragm muscles were removed and processed for histopathology and Western blot. Long‐term therapy with omega‐3 increased the regulatory protein MyoD and muscle regeneration and reduced markers of inflammation (TNF‐α and NF‐kB) in both muscles studied. The present study supports the long‐term use of omega‐3 at later stages of dystrophy as a promising option to be investigated in DMD clinical trials. Anat Rec, 298:1589–1596, 2015. © 2015 Wiley Periodicals, Inc.     

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.     

Gal‐3 regulates the capacity of dendritic cells to promote NKT‐cell‐induced liver injury

Galectin‐3 (Gal‐3), an endogenous lectin, exhibits pro‐ and anti‐inflammatory effects in various disease conditions. In order to explore the role of Gal‐3 in NKT‐cell‐dependent pathology, we induced hepatitis in C57BL/6 WT and Gal‐3‐deficient mice by using specific ligand for NKT cells: α‐galactosylceramide, glycolipid Ag presented by CD1d. The injection of α‐galactosylceramide significantly enhanced expression of Gal‐3 in liver NKT and dendritic cells (DCs). Genetic deletion or selective inhibition of Gal‐3 (induced by Gal‐3‐inhibitor TD139) abrogated the susceptibility to NKT‐cell‐dependent hepatitis. Blood levels of pro‐inflammatory cytokines (TNF‐α, IFN‐γ, IL‐12) and their production by liver DCs and NKT cells were also downregulated. Genetic deletion or selective inhibition of Gal‐3 alleviated influx of inflammatory CD11c⁺CD11b⁺ DCs in the liver and favored tolerogenic phenotype and IL‐10 production of liver NKT and DCs. Deletion of Gal‐3 attenuated the capacity of DCs to support liver damage in the passive transfer experiments and to produce pro‐inflammatory cytokines in vitro. Gal‐3‐deficient DCs failed to optimally stimulate production of pro‐inflammatory cytokines in NKT cells, in vitro and in vivo. In conclusion, Gal‐3 regulates the capacity of DCs to support NKT‐cell‐mediated liver injury, playing an important pro‐inflammatory role in acute liver injury.     

Caspase activation during spontaneous and radiation‐induced apoptosis in the murine intestine

The aim of this study was to characterize the activation of caspase‐3 along the crypt/villus axis in the normal and irradiated intestine and to compare active caspase‐3 expression with existing apoptosis detection techniques. Small and large intestine were removed from mice at various time points after exposure to 8 Gy γ‐radiation. Positive apoptotic cells stained with an antibody against active caspase‐3, haematoxylin and eosin (H&E) or TUNEL were scored in histological sections of small and large intestinal crypts and villi. In the control intestine, active caspase‐3 expression was rarely observed; however, expression was markedly increased following exposure to radiation and was predominantly confined to apoptotic bodies. Measurement of apoptosis in intestinal crypts using active caspase‐3 expression gave similar results to apoptosis detected from H&E‐stained sections. In the normal villus, active caspase‐3 expression was observed infrequently and did not significantly increase following radiation, consistent with a lack of apoptotic body formation from H&E sections. This study indicates that caspase‐3 is activated in intestinal crypts but not in villi following γ‐radiation. Active caspase‐3 detection compared favourably with existing immunological techniques, suggesting that it is a suitable alternative method for apoptosis quantification. Copyright © 2001 John Wiley & Sons, Ltd.     

Role of Cytotoxic Protease Granzyme‐b in Neuronal Degeneration During Human Stroke

Infiltration of leukocytes into post‐ischemic cerebrum is a well‐described phenomenon in stroke injury. Because CD‐8⁺ T‐lymphocytes secrete cytotoxic proteases, including granzyme‐b (Gra‐b) that exacerbates post‐ischemic brain damage, we investigated roles of Gra‐b in human stroke. To study the role of Gra‐b in stroke, ischemic and non‐ischemic tissues (from post‐mortem stroke patients) were analyzed using immunoblotting, co‐immunoprecipitation, terminal deoxy uridine nick end labeling (TUNEL) and Annexin–V immunostaining, and in vitro neuron survival assays. Activated CG‐SH cells and supernatants were used to model leukocyte‐dependent injury. Non‐ischemic brain tissues were used as non‐pathological controls. Non‐activated CG‐SH cells and supernatants were used as controls for in vitro experiments. Human stroke (ischemic) samples contained significantly higher levels of Gra‐b and interferon‐gamma inducible protein‐10 (IP‐10/CXCL10) than non‐ischemic controls. In stroke, poly (ADP‐ribose) polymerase‐1 and heat shock protein‐70 were cleaved to canonical proteolytic “signature” fragments by Gra‐b. Gra‐b was also found to bind to Bid and caspase‐3. Gra‐b also co‐localized with Annexin‐V⁺/TUNEL⁺ in degenerating neurons. Importantly, Gra‐b inhibition protected both normal and ischemia‐reperfused neurons against in vitro neurotoxicity mediated by activated CG‐SH cells and supernatants. These results suggest that increased leukocyte infiltration and elevated Gra‐b levels in the post‐stroke brain can induce contact‐dependent and independent post‐ischemic neuronal death to aggravate stroke injury.     

Characteristics of Schistosoma japonicum infection induced IFN‐γ and IL‐4 co‐expressing plasticity Th cells

Schistosoma japonicum infection can induce granulomatous inflammation and cause tissue damage in the mouse liver. The cytokine secretion profile of T helper (Th) cells depends on both the nature of the activating stimulus and the local microenvironment (e.g. cytokines and other soluble factors). In the present study, we found an accumulation of large numbers of IFN‐γ⁺ IL‐4⁺ CD4⁺ T cells in mouse livers. This IFN‐γ⁺ IL‐4⁺ cell population increased from 0·68 ± 0·57% in uninfected mice to 7·05 ± 3·0% by week 4 following infection and to 9·6 ± 5·28% by week 6, before decreasing to 6·3 ± 5·9% by week 8 in CD4 T cells. Moreover, IFN‐γ⁺ IL‐4⁺ Th cells were also found in mouse spleen and mesenteric lymph nodes 6 weeks after infection. The majority of the IFN‐γ⁺ IL‐4⁺ Th cells were thought to be related to a state of immune activation, and some were memory T cells. Moreover, we found that these S. japonicum infection‐induced IFN‐γ⁺ IL‐4⁺ cells could express interleukin‐2 (IL‐2), IL‐9, IL‐17 and high IL‐10 levels at 6 weeks after S. japonicum infection. Taken together, our data suggest the existence of a population of IFN‐γ⁺ IL‐4⁺ plasticity effector/memory Th cells following S. japonicum infection in C57BL/6 mice.     

The atypical IκB protein IκBNS is important for Toll‐like receptor‐induced interleukin‐10 production in B cells

Although a major function of B cells is to mediate humoral immunity by producing antigen‐specific antibodies, a specific subset of B cells is important for immune suppression, which is mainly mediated by the secretion of the anti‐inflammatory cytokine interleukin‐10 (IL‐10). However, the mechanism by which IL‐10 is induced in B cells has not been fully elucidated. Here, we report that IκB_NS, an inducible nuclear IκB protein, is important for Toll‐like receptor (TLR)‐mediated IL‐10 production in B cells. Studies using IκB_NS knockout mice revealed that the number of IL‐10‐producing B cells is reduced in IκB_NS^?/? spleens and that the TLR‐mediated induction of cytoplasmic IL‐10‐positive cells and IL‐10 secretion in B cells are impaired in the absence of IκB_NS. The impairment of IL‐10 production by a lack of IκB_NS was not observed in TLR‐triggered macrophages or T‐cell‐receptor‐stimulated CD4⁺ CD25⁺ T cells. In addition, IκB_NS‐deficient B cells showed reduced expression of Prdm1 and Irf4 and failed to generate IL‐10⁺ CD138⁺ plasmablasts. These results suggest that IκB_NS is selectively required for IL‐10 production in B cells responding to TLR signals, so defining an additional role for IκB_NS in the control of the B‐cell‐mediated immune responses.     

NF‐κB‐inducing kinase is a key regulator of inflammation‐induced and tumour‐associated angiogenesis

Angiogenesis is essential during development and in pathological conditions such as chronic inflammation and cancer progression. Inhibition of angiogenesis by targeting vascular endothelial growth factor (VEGF) blocks disease progression, but most patients eventually develop resistance which may result from compensatory signalling pathways. In endothelial cells (ECs), expression of the pro‐angiogenic chemokine CXCL12 is regulated by non‐canonical nuclear factor (NF)‐κB signalling. Here, we report that NF‐κB‐inducing kinase (NIK) and subsequent non‐canonical NF‐κB signalling regulate both inflammation‐induced and tumour‐associated angiogenesis. NIK is highly expressed in endothelial cells (ECs) in tumour tissues and inflamed rheumatoid arthritis synovial tissue. Furthermore, non‐canonical NF‐κB signalling in human microvascular ECs significantly enhanced vascular tube formation, which was completely blocked by siRNA targeting NIK. Interestingly, Nik^?/? mice exhibited normal angiogenesis during development and unaltered TNFα‐ or VEGF‐induced angiogenic responses, whereas angiogenesis induced by non‐canonical NF‐κB stimuli was significantly reduced. In addition, angiogenesis in experimental arthritis and a murine tumour model was severely impaired in these mice. These studies provide evidence for a role of non‐canonical NF‐κB signalling in pathological angiogenesis, and identify NIK as a potential therapeutic target in chronic inflammatory diseases and tumour neoangiogenesis. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

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.     

Exogenous interleukin‐33 targets myeloid‐derived suppressor cells and generates periphery‐induced Foxp3+ regulatory T cells in skin‐transplanted mice

Interleukin-33 (IL-33) has been a focus of study because of its variety of functions shaping CD4⁺ T-cell biology. In the present work, we evaluated the modulatory effect of IL-33 on suppressor cells in an in vivo transplantation model. C57BL/6 wild-type mice were grafted with syngeneic or allogeneic skin transplants and treated with exogenous IL-33 daily. After 10 days of treatment, we analysed draining lymph node cellularity and found in allogeneic animals an increment in myeloid-derived suppressor cells, which co-express MHC-II, and become enriched upon IL-33 treatment. In line with this observation, inducible nitric oxide synthase and arginase 1 expression were also increased in allogeneic animals upon IL-33 administration. In addition, IL-33 treatment up-regulated the number of Foxp3⁺ regulatory T (Treg) cells in the allogeneic group, complementing the healthier integrity of the allografts and the increased allograft survival. Moreover, we demonstrate that IL-33 promotes CD4⁺ T-cell expansion and conversion of CD4⁺ Foxp3⁻ T cells into CD4⁺ Foxp3⁺ Treg cells in the periphery. Lastly, the cytokine pattern of ex vivo-stimulated draining lymph nodes indicates that IL-33 dampens interferon-γ and IL-17 production, stimulating IL-10 secretion. Altogether, our work complements previous studies on the immune-modulatory activity of IL-33, showing that this cytokine affects myeloid-derived suppressor cells at the cell number and gene expression levels. More importantly, our research demonstrates for the first time that IL-33 allows for in vivo Foxp3⁺ Treg cell conversion and favours an anti-inflammatory or tolerogenic state by skewing cytokine production. Therefore, our data suggest a potential use of IL-33 to prevent allograft rejection, bringing new therapeutics to the transplantation field.     

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.     

PIAS1 and STAT‐3 impair the tumoricidal potential of IFN‐γ‐stimulated mouse dendritic cells generated with IL‐15

Primarily defined by their antigen‐presenting property, dendritic cells (DCs) are being implemented as cancer vaccines in immunotherapeutic interventions. DCs can also function as direct tumor cell killers. How DC cytotoxic activity can be efficiently harnessed and the mechanisms controlling this nonconventional property are not fully understood. We report here that the tumoricidal potential of mouse DCs generated from myeloid precursors with GM‐CSF and IL‐15 (IL‐15 DCs) can be triggered with the Toll‐like receptor (TLR) 4 ligand lipopolysaccharide to a similar extent compared with that of their counterparts, conventionally generated with IL‐4 (IL‐4 DCs). The mechanism of tumor cell killing depends on the induction of iNOS expression by DCs. In contrast, interferon (IFN)‐γ induces the cytotoxic activity of IL‐4 but not IL‐15 DCs. Although the IFN‐γ‐STAT‐1 signaling pathway is overall functional in IL‐15 DCs, IFN‐γ fails to induce iNOS expression in these cells. iNOS expression is negatively controlled in IFN‐γ‐stimulated IL‐15 DCs by the cooperation between the E3 SUMO ligase PIAS1 and STAT‐3, and can be partially restored with PIAS1 siRNA and STAT‐3 inhibitors.     

CD8+ T cells producing IL‐3 and IL‐5 in non‐IgE‐mediated eosinophilic diseases

The cytokines IL‐5, IL‐3, and GM‐CSF are crucial for eosinophil development, survival, and function. To better understand their role in non‐IgE‐mediated eosinophilic diseases, we investigated plasma levels of these cytokines as well as cytokine expression in peripheral blood T cells. While we did not find any evidence for an involvement of T‐cell‐derived GM‐CSF, some of these patients did show an increased proportion of IL‐5‐ or IL‐3‐producing CD4⁺ T cells. However, in a significant proportion of patients, IL‐5‐producing CD8⁺ T cells, so‐called Tc2 cells, which in healthy donors can only be detected at very low levels, were prominent. Furthermore, increased IL‐3 production by CD8⁺ T cells was also observed, strongly supporting the notion that CD8⁺ T cells, not just CD4⁺ T cells, must also be considered as a potential source of the cytokines promoting eosinophilia.     

Protective effect of γ‐tocopherol‐enriched diet on N‐methyl‐N‐nitrosourea‐induced epithelial dysplasia in rat ventral prostate

Despite recent advances in understanding the biological basis of prostate cancer (PCa), the management of this disease remains a challenge. Chemoprotective agents have been used to protect against or eradicate prostate malignancies. Here, we investigated the protective effect of γ‐tocopherol on N‐methyl‐N‐nitrosourea (MNU)‐induced epithelial dysplasia in the rat ventral prostate (VP). Thirty‐two male Wistar rats were divided into four groups (n = 8): control (CT): healthy control animals fed a standard diet; control+γ‐tocopherol (CT+γT): healthy control animals without intervention fed a γ‐tocopherol‐enriched diet (20 mg/kg); N‐methyl‐N‐nitrosourea (MNU): rats that received a single dose of MNU (30 mg/kg) plus testosterone propionate (100 mg/kg) and were fed a standard diet; and MNU+γ‐tocopherol (MNU+γT): rats that received the same treatment of MNU plus testosterone and were fed with a γ‐tocopherol‐enriched diet (20 mg/kg). After 4 months, the VPs were excised to evaluate morphology, cell proliferation and apoptosis, as well as cyclooxygenase‐2 (Cox‐2), glutathione‐S‐transferase‐pi (GST‐pi) and androgen receptor (AR) protein expression, and matrix metalloproteinase‐9 (MMP‐9) activity. An increase in the incidence of epithelial dysplasias, such as stratified epithelial hyperplasia and squamous metaplasia, in the MNU group was accompanied by augmented cell proliferation, GST‐pi and Cox‐2 immunoexpression and pro‐MMP‐9 activity. Stromal thickening and inflammatory foci were also observed. The administration of a γ‐tocopherol‐enriched diet significantly attenuated the adverse effects of MNU in the VP. The incidence of epithelial dysplasia decreased, along with the cell proliferation index, GST‐pi and Cox‐2 immunoexpression. The gelatinolytic activity of pro‐MMP‐9 returned to the levels observed for the CT group. These results suggest that γ‐tocopherol acts as a protective agent against MNU‐induced prostatic disorders in the rat ventral prostate.     

γδ T cells are indispensable for interleukin‐23‐mediated protection against Concanavalin A‐induced hepatitis in hepatitis B virus transgenic mice

Hepatitis B virus surface antigen (HBsAg) carriers are highly susceptible to liver injury triggered by environmental biochemical stimulation. Previously, we have reported an inverse correlation between γδ T cells and liver damage in patients with hepatitis B virus (HBV). However, whether γδ T cells play a role in regulating the hypersensitivity of HBsAg carriers to biochemical stimulation‐induced hepatitis is unknown. In this study, using HBV transgenic (HBs‐Tg) and HBs‐Tg T‐cell receptor‐δ‐deficient (TCR‐δ^−/−) mice, we found that mice genetically deficient in γδ T cells exhibited more severe liver damage upon Concanavalin A (Con A) treatment, as indicated by substantially higher serum alanine aminotransferase levels, further elevated interferon‐γ (IFN‐γ) levels and more extensive necrosis. γδ T‐cell deficiency resulted in elevated IFN‐γ in CD4⁺ T cells but not in natural killer or natural killer T cells. The depletion of CD4⁺ T cells and neutralization of IFN‐γ reduced liver damage in HBs‐Tg and HBs‐Tg‐TCR‐δ^−/− mice to a similar extent. Further investigation revealed that HBs‐Tg mice showed an enhanced interleukin‐17 (IL‐17) signature. The administration of exogenous IL‐23 enhanced IL‐17A production from Vγ4 γδ T cells and ameliorated liver damage in HBs‐Tg mice, but not in HBs‐Tg‐TCR‐δ^−/− mice. In summary, our results demonstrated that γδ T cells played a protective role in restraining Con A‐induced hepatitis by inhibiting IFN‐γ production from CD4⁺ T cells and are indispensable for IL‐23‐mediated protection against Con A‐induced hepatitis in HBs‐Tg mice. These results provided a potential therapeutic approach for treating the hypersensitivity of HBV carriers to biochemical stimulation‐induced liver damage.