Role of IL-1β in Experimental Cystic Fibrosis upon P. aeruginosa Infection

Cystic fibrosis is associated with increased inflammatory responses to pathogen challenge. Here we revisited the role of IL-1β in lung pathology using the experimental F508del-CFTR murine model on C57BL/6 genetic background (Cftr ^tm1eur or d/d), on double deficient for d/d and type 1 interleukin-1 receptor (d/d X IL-1R1^−/−), and antibody neutralization. At steady state, young adult d/d mice did not show any signs of spontaneous lung inflammation. However, IL-1R1 deficiency conferred partial protection to repeated P. aeruginosa endotoxins/LPS lung instillation in d/d mice, as 50% of d/d mice succumbed to inflammation, whereas all d/d x IL-1R1^−/− double mutants survived with lower initial weight loss and less pulmonary collagen and mucus production, suggesting that the absence of IL-1R1 signaling is protective in d/d mice in LPS-induced lung damage. Using P. aeruginosa acute lung infection we found heightened neutrophil recruitment in d/d mice with higher epithelial damage, increased bacterial load in BALF, and augmented IL-1β and TNF-α in parenchyma as compared to WT mice. Thus, F508del-CFTR mice show enhanced IL-1β signaling in response to P. aeruginosa. IL-1β antibody neutralization had no effect on lung homeostasis in either d/d or WT mice, however P. aeruginosa induced lung inflammation and bacterial load were diminished by IL-1β antibody neutralization. In conclusion, enhanced susceptibility to P. aeruginosa in d/d mice correlates with an excessive inflammation and with increased IL-1β production and reduced bacterial clearance. Further, we show that neutralization of IL-1β in d/d mice through the double mutation d/d x IL-1R1^−/− and in WT via antibody neutralization attenuates inflammation. This supports the notion that intervention in the IL-1R1/IL-1β pathway may be detrimental in CF patients.     

Efficient, Repeated Adenovirus-Mediated Gene Transfer in Mice Lacking both Tumor Necrosis Factor Alpha and Lymphotoxin α

The efficiency of adenovirus-mediated gene transfer is now well established. However, the cellular and the humoral immune responses triggered by vector injection lead to the rapid elimination of the transduced cells and preclude any efficient readministration. The present investigation focuses on the role of tumor necrosis factor alpha (TNF-α), a proinflammatory cytokine, and the related cytokine lymphotoxin α (LTα), in mounting an immune reaction against recombinant adenovirus vectors. After gene transfer in the liver, mice genetically deficient for both cytokines (TNF-α/LTα^−/−), in comparison with normal mice, presented a weak acute-phase inflammatory reaction, a reduction in cellular infiltrates in the liver, and a severely impaired T-cell proliferative response to both Adenoviral and transgene product antigens. Moreover, we observed a strong reduction in the humoral response to the vector and the transgene product, with a drastic reduction of anti-adenovirus immunoglobulin A and G antibody isotypes. In addition, the reduction in antibody response observed in TNF-α/LTα^−/− and TNF-α/LTα^+/− mice versus TNF-α/LTα^+/+ mice links antibody levels to TNF-α/LTα gene dosage. Due to the absence of neutralizing antibodies, the TNF-α/LTα knockout mice successfully express a second gene transduced by a second vector injection. The discovery of the pivotal role played by TNF-α in controlling the antibody response against adenovirus will allow more efficient adenovirus-based strategies for gene therapy to be proposed.     

Optimal Attenuation of Experimental Autoimmune Encephalomyelitis by Intravenous Immunoglobulin Requires an Intact Interleukin-11 Receptor

Background

Intravenous immunoglobulin (IVIg) has been used to treat a variety of autoimmune disorders including multiple sclerosis (MS); however its mechanism of action remains elusive. Recent work has shown that interleukin-11 (IL-11) mRNAs are upregulated by IVIg in MS patient T cells. Both IVIg and IL-11 have been shown to ameliorate experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The objective of this study was to determine whether the protective effects of IVIg in EAE occur through an IL-11 and IL-11 receptor (IL-11R)-dependent mechanism.

Methods

We measured IL-11 in the circulation of mice and IL-11 mRNA expression in various organs after IVIg treatment. We then followed with EAE studies to test the efficacy of IVIg in wild-type (WT) mice and in mice deficient for the IL-11 receptor (IL-11Rα^−/−). Furthermore, we evaluated myelin-specific Th1 and Th17 responses and assessed spinal cord inflammation and demyelination in WT and IL-11Rα^−/− mice, with and without IVIg treatment. We also examined the direct effects of mouse recombinant IL-11 on the production of IL-17 by lymph node mononuclear cells.

Results

IVIg treatment induced a dramatic surge (>1000-fold increase) in the levels of IL-11 in the circulation and a prominent increase of IL-11 mRNA expression in the liver. Furthermore, we found that IL-11Rα^−/− mice, unlike WT mice, although initially protected, were resistant to full protection by IVIg during EAE and developed disease with a similar incidence and severity as control-treated IL-11Rα^−/− mice, despite initially showing protection. We observed that Th17 cytokine production by myelin-reactive T cells in the draining lymph nodes was unaffected by IVIg in IL-11Rα^−/− mice, yet was downregulated in WT mice. Finally, IL-11 was shown to directly inhibit IL-17 production of lymph node cells in culture.

Conclusion

These results implicate IL-11 as an important immune effector of IVIg in the prevention of Th17-mediated autoimmune inflammation during EAE.     

Differential Modulation by IL-17A of Cholangitis versus Colitis in IL-2Rα Deleted Mice

IFN-γ is a signature Th1 cell associated cytokine critical for the inflammatory response in autoimmunity with both pro-inflammatory and potentially protective functions. IL-17A is the hallmark of T helper 17 (Th17) cell subsets, produced by γδT, CD8+ T, NK and NKT cells. We have taken advantage of our colony of IL-2Rα^−/− mice that spontaneously develop both autoimmune cholangitis and inflammatory bowel disease. In this model CD8+ T cells mediate biliary ductular damage, whereas CD4+ T cells mediate induction of colon-specific autoimmunity. Importantly, IL-2Rα^−/− mice have high levels of interferon γ (IFN-γ), and interleukin-17A (IL-17A). We produced unique double deletions of mice that were either IL-17A^−/−IL-2Rα^−/− or IFN-γ^−/−IL-2Rα^−/− to specifically address the precise role of these two cytokines in the natural history of autoimmune cholangitis and colitis. Of note, deletion of IL-17A in IL-2Rα^−/− mice led to more severe liver inflammation, but ameliorated colitis. In contrast, there were no significant changes in the immunopathology of double knock-out IFN-γ^−/− IL-2Rα^−/− mice, compared to single knock-out IL-2Rα^−/− mice with respect to cholangitis or colitis. Furthermore, there was a significant increase in pathogenetic CD8+ T cells in the liver of IL-17A^−/−IL-2Rα^−/− mice. Our data suggest that while IL-17A plays a protective role in autoimmune cholangitis, it has a pro-inflammatory role in inflammatory bowel disease. These data take on particular significance in the potential use of anti-IL-17A therapy in humans with primary biliary cirrhosis.     

Blockade of Retinol Metabolism Protects T Cell-Induced Hepatitis by Increasing Migration of Regulatory T Cells

Retinols are metabolized into retinoic acids by alcohol dehydrogenase (ADH) and retinaldehyde dehydrogenase (Raldh). However, their roles have yet to be clarified in hepatitis despite enriched retinols in hepatic stellate cells (HSCs). Therefore, we investigated the effects of retinols on Concanavalin A (Con A)-mediated hepatitis.Con A was injected into wild type (WT), Raldh1 knock-out (Raldh1^−/−), CCL2^−/− and CCR2^−/− mice. For migration study of regulatory T cells (Tregs), we used in vivo and ex vivo adoptive transfer systems. Blockade of retinol metabolism in mice given 4-methylpyrazole, an inhibitor of ADH, and ablated Raldh1 gene manifested increased migration of Tregs, eventually protected against Con A-mediated hepatitis by decreasing interferon-γ in T cells. Moreover, interferon-γ treatment increased the expression of ADH3 and Raldh1, but it suppressed that of CCL2 and IL-6 in HSCs. However, the expression of CCL2 and IL-6 was inversely increased upon the pharmacologic or genetic ablation of ADH3 and Raldh1 in HSCs. Indeed, IL-6 treatment increased CCR2 expression of Tregs. In migration assay, ablated CCR2 in Tregs showed reduced migration to HSCs. In adoptive transfer of Tregs in vivo and ex vivo, Raldh1-deficient mice showed more increased migration of Tregs than WT mice. Furthermore, inhibited retinol metabolism increased survival rate (75%) compared with that of the controls (25%) in Con A-induced hepatitis.These results suggest that blockade of retinol metabolism protects against acute liver injury by increased Treg migration, and it may represent a novel therapeutic strategy to control T cell-mediated acute hepatitis.     

Oroxylin A Accelerates Liver Regeneration in CCI4-Induced Acute Liver Injury Mice

Introduction

Based on the previous research that oroxylin A can suppress inflammation, we investigated the hepatoprotective role of oroxylin A against CCl₄-induced liver damage in mice and then studied the possible alteration of the activities of cytokine signaling participating in liver regeneration. Wild type (WT) mice were orally administrated with oroxylin A (60 mg/kg) for 4 days after CCl₄ injection, the anti-inflammatory effects of oroxylin A were assessed directly by hepatic histology and indirectly by measuring serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and Albumin. Proliferating cell nuclear antigen (PCNA) staining was performed to evaluate the role of oroxylin A in promoting hepatocyte proliferation. Serum IL-1β, TNF-α, IL-6 and IL-1Ra levels were measured by enzyme-linked immunosorbent assay (ELISA) and liver HGF, EGF, TNF-α, IL-6, IL-1Ra and IL-1β gene expression was determined by quantitative real-time PCR. The data indicated that the IL-6 and TNF-α mRNA of oroxylin A administered group significantly increased higher than the control within 12 hours after CCl₄ treatment. Meanwhile, oroxylin A significantly enhanced the expression of IL-1Ra at the early phase, which indicated that oroxylin A could facilitate the initiating events in liver regeneration by increasing IL-1Ra which acts as an Acute-Phase Protein (APP). In addition, a lethal CCl₄-induced acute liver failure model offers a survival benefit in oroxylin A treated WT mice. However, oroxylin A could not significantly improve the percent survival of IL-1RI^−/− mice with a lethal CCl₄-induced acute liver failure.

Conclusions

Our study confirmed that oroxylin A could strongly promote liver structural remodeling and functional recovery through IL-1Ra/IL-1RI signaling pathway. All these results support the possibility of oroxylin A being a therapeutic candidate for acute liver injury.     

Cooperative Interactions between TLR4 and TLR9 Regulate Interleukin 23 and 17 Production in a Murine Model of Gram Negative Bacterial Pneumonia

Toll like receptors play an important role in lung host defense against bacterial pathogens. In this study, we investigated independent and cooperative functions of TLR4 and TLR9 in microbial clearance and systemic dissemination during Gram-negative bacterial pneumonia. To access these responses, wildtype Balb/c mice, mice with defective TLR4 signaling (TLR4^lps-d), mice deficient in TLR9 (TLR9^−/−) and TLR4/9 double mutant mice (TLR4^lps-d/TLR9^−/−) were challenged with K. pneumoniae, then time-dependent lung bacterial clearance and systemic dissemination determined. We found impaired lung bacterial clearance in TLR4 and TLR9 single mutant mice, whereas the greatest impairment in clearance was observed in TLR4^lps-d/TLR9^−/− double mutant mice. Early lung expression of TNF-α, IL-12, and chemokines was TLR4 dependent, while IFN-γ production and the later expression of TNF-α and IL-12 was dependent on TLR9. Classical activation of lung macrophages and maximal induction of IL-23 and IL-17 required both TLR4 and TLR9. Finally, the i.t. instillation of IL-17 partially restored anti-bacterial immunity in TLR4^lps-d/TLR9^−/− double mutant mice. In conclusion, our studies indicate that TLR4 and TLR9 have both non-redundant and cooperative roles in lung innate responses during Gram-negative bacterial pneumonia and are both critical for IL-17 driven antibacterial host response.     

Cold-Inducible RNA-Binding Protein Is an Important Mediator of Alcohol-Induced Brain Inflammation

Binge drinking has been associated with cerebral dysfunction. Ethanol induced microglial activation initiates an inflammatory process that causes upregulation of proinflammatory cytokines which in turn creates neuronal inflammation and damage. However, the molecular mechanism is not fully understood. We postulate that cold-inducible RNA-binding protein (CIRP), a novel proinflammatory molecule, can contribute to alcohol-induced neuroinflammation. To test this theory male wild-type (WT) mice were exposed to alcohol at concentrations consistent to binge drinking and blood and brain tissues were collected. At 5 h after alcohol, a significant increase of 53% in the brain of CIRP mRNA was observed and its expression remained elevated at 10 h and 15 h. Brain CIRP protein levels were increased by 184% at 10 h and remained high at 15 h. We then exposed male WT and CIRP knockout (CIRP^−/−) mice to alcohol, and blood and brain tissues were collected at 15 h post-alcohol infusion. Serum levels of tissue injury markers (AST, ALT and LDH) were significantly elevated in alcohol-exposed WT mice while they were less increased in the CIRP^−/− mice. Brain TNF-α mRNA and protein expressions along with IL-1β protein levels were significantly increased in WT mice, which was not seen in the CIRP^−/− mice. In cultured BV2 cells (mouse microglia), ethanol at 100 mM showed an increase of CIRP mRNA by 274% and 408% at 24 h and 48 h respectively. Corresponding increases in TNF-α and IL-1β were also observed. CIRP protein levels were markedly increased in the medium, suggesting that CIRP was secreted by the BV2 cells. From this we conclude that alcohol exposure activates microglia to produce and secrete CIRP and possibly induce pro-inflammatory response and thereby causing neuroinflammation. CIRP could be a novel mediator of alcohol-induced brain inflammation.     

Deletion of Nardilysin Prevents the Development of Steatohepatitis and Liver Fibrotic Changes

Nonalcoholic steatohepatitis (NASH) is an inflammatory form of nonalcoholic fatty liver disease that progresses to liver cirrhosis. It is still unknown how only limited patients with fatty liver develop NASH. Tumor necrosis factor (TNF)-α is one of the key molecules in initiating the vicious circle of inflammations. Nardilysin (N-arginine dibasic convertase; Nrd1), a zinc metalloendopeptidase of the M16 family, enhances ectodomain shedding of TNF-α, resulting in the activation of inflammatory responses. In this study, we aimed to examine the role of Nrd1 in the development of NASH. Nrd1^+/+ and Nrd1^−/− mice were fed a control choline-supplemented amino acid-defined (CSAA) diet or a choline-deficient amino acid-defined (CDAA) diet. Fatty deposits were accumulated in the livers of both Nrd1^+/+ and Nrd1^−/− mice by the administration of the CSAA or CDAA diets, although the amount of liver triglyceride in Nrd1^−/− mice was lower than that in Nrd1^+/+ mice. Serum alanine aminotransferase levels were increased in Nrd1^+/+ mice but not in Nrd1^−/− mice fed the CDAA diet. mRNA expression of inflammatory cytokines were decreased in Nrd1^−/− mice than in Nrd1^+/+ mice fed the CDAA diet. While TNF-α protein was detected in both Nrd1^+/+ and Nrd1^−/− mouse livers fed the CDAA diet, secretion of TNF-α in Nrd1^−/− mice was significantly less than that in Nrd1^+/+ mice, indicating the decreased TNF-α shedding in Nrd1^−/− mouse liver. Notably, fibrotic changes of the liver, accompanied by the increase of fibrogenic markers, were observed in Nrd1^+/+ mice but not in Nrd1^−/− mice fed the CDAA diet. Similar to the CDAA diet, fibrotic changes were not observed in Nrd1^−/− mice fed a high-fat diet. Thus, deletion of nardilysin prevents the development of diet-induced steatohepatitis and liver fibrogenesis. Nardilysin could be an attractive target for anti-inflammatory therapy against NASH.     

Endothelial Dysfunction in Tristetraprolin-deficient Mice Is Not Caused by Enhanced Tumor Necrosis Factor-α Expression

Cardiovascular events are important co-morbidities in patients with chronic inflammatory diseases like rheumatoid arthritis. Tristetraprolin (TTP) regulates pro-inflammatory processes through mRNA destabilization and therefore TTP-deficient mice (TTP^−/− mice) develop a chronic inflammation resembling human rheumatoid arthritis. We used this mouse model to evaluate molecular signaling pathways contributing to the enhanced atherosclerotic risk in chronic inflammatory diseases. In the aorta of TTP^−/− mice we observed elevated mRNA expression of known TTP targets like tumor necrosis factor-α (TNF-α) and macrophage inflammatory protein-1α, as well as of other pro-atherosclerotic mediators, like Calgranulin A, Cathepsin S, and Osteopontin. Independent of cholesterol levels TTP^−/− mice showed a significant reduction of acetylcholine-induced, nitric oxide-mediated vasorelaxation. The endothelial dysfunction in TTP^−/− mice was associated with increased levels of reactive oxygen and nitrogen species (RONS), indicating an enhanced nitric oxide inactivation by RONS in the TTP^−/− animals. The altered RONS generation correlates with increased expression of NADPH oxidase 2 (Nox2) resulting from enhanced Nox2 mRNA stability. Although TNF-α is believed to be a central mediator of inflammation-driven atherosclerosis, genetic inactivation of TNF-α neither improved endothelial function nor normalized Nox2 expression or RONS production in TTP^−/− animals. Systemic inflammation caused by TTP deficiency leads to endothelial dysfunction. This process is independent of cholesterol and not mediated by TNF-α solely. Thus, other mediators, which need to be identified, contribute to enhanced cardiovascular risk in chronic inflammatory diseases.     

Absence of the Common Gamma Chain (γc), a Critical Component of the Type I IL-4 Receptor,Increases the Severity of Allergic Lung Inflammation

The T_H2 cytokines, IL-4 and IL-13, play critical roles in inducing allergic lung inflammation and drive the alternative activation of macrophages (AAM). Although both cytokines share receptor subunits, IL-4 and IL-13 have differential roles in asthma pathogenesis: IL-4 regulates T_H2 cell differentiation, while IL-13 regulates airway hyperreactivity and mucus production. Aside from controlling T_H2 differentiation, the unique contribution of IL-4 signaling via the Type I receptor in airway inflammation remains unclear. Therefore, we analyzed responses in mice deficient in gamma c (γ_c) to elucidate the role of the Type I IL-4 receptor. OVA primed CD4⁺ OT-II T cells were adoptively transferred into RAG2^−/− and γ_c ^−/− mice and allergic lung disease was induced. Both γ_c ^−/− and γ_cxRAG2^−/− mice developed increased pulmonary inflammation and eosinophilia upon OVA challenge, compared to RAG2^−/− mice. Characteristic AAM proteins FIZZ1 and YM1 were expressed in lung epithelial cells in both mouse strains, but greater numbers of FIZZ1+ or YM1+ airways were present in γ_c ^−/− mice. Absence of γ_c in macrophages, however, resulted in reduced YM1 expression. We observed higher T_H2 cytokine levels in the BAL and an altered DC phenotype in the γ_c ^−/− recipient mice suggesting the potential for dysregulated T cell and dendritic cell (DC) activation in the γ_c-deficient environment. These results demonstrate that in absence of the Type I IL-4R, the Type II R can mediate allergic responses in the presence of T_H2 effectors. However, the Type I R regulates AAM protein expression in macrophages.     

Impaired Interleukin-1β and c-Fos Expression in the Hippocampus Is Associated with a Spatial Memory Deficit in P2X7 Receptor-Deficient Mice

Recent evidence suggests that interleukin-1β (IL-1β), which was originally identified as a proinflammatory cytokine, is also required in the brain for memory processes. We have previously shown that IL-1β synthesis in the hippocampus is dependent on P2X₇ receptor (P2X₇R), which is an ionotropic receptor of ATP. To substantiate the role of P2X₇R in both brain IL-1β expression and memory processes, we examined the induction of IL-1β mRNA expression in the hippocampus of wild-type (WT) and homozygous P2X₇ receptor knockout mice (P2X₇R^−/−) following a spatial memory task. The spatial recognition task induced both IL-1β mRNA expression and c-Fos protein activation in the hippocampus of WT but not of P2X₇R^−/− mice. Remarkably, P2X₇R^−/− mice displayed spatial memory impairment in a hippocampal-dependant task, while their performances in an object recognition task were unaltered. Taken together, our results show that P2X₇R plays a critical role in spatial memory processes and the associated hippocampal IL-1β mRNA synthesis and c-Fos activation.     

ApoE Production in Human Monocytes and Its Regulation by Inflammatory Cytokines

The apoE production by tissue macrophages is crucial for the prevention of atherosclerosis and the aim of this study was to further elucidate how this apolipoprotein is regulated by cytokines present during inflammation. Here we studied apoE production in peripheral blood mononuclear cells (PBMC) and analysis was made with a newly developed apoE ELISpot assay. In PBMC, apoE secretion was restricted to monocytes with classical (CD14⁺⁺CD16⁻) and intermediate (CD14⁺CD16⁺) monocytes being the main producers. As earlier described for macrophages, production was strongly upregulated by TGF-β and downregulated by bacterial lipopolysaccharide (LPS) and the inflammatory cytokines IFN-γ, TNF-α and IL-1β. We could here show that a similar down-regulatory effect was also observed with the type I interferon, IFN-α, while IL-6, often regarded as one of the more prominent inflammatory cytokines, did not affect TGF-β-induced apoE production. The TNF-α inhibitor Enbrel could partly block the down-regulatory effect of IFN-γ, IFN-α and IL-1β, indicating that inhibition of apoE by these cytokines may be dependent on or synergize with TNF-α. Other cytokines tested, IL-2, IL-4, IL-12, IL-13, IL-17A and IL-23, had no inhibitory effect on apoE production. In contrast to the effect on monocytes, apoE production by primary hepatocytes and the hepatoma cell line HepG2 was more or less unaffected by treatment with cytokines or LPS.     

IL-22-Producing RORγt-Dependent Innate Lymphoid Cells Play a Novel Protective Role in Murine Acute Hepatitis

Retinoid-related orphan receptor (ROR) γt is known to be related to the development and function of various immunological compartments in the liver, such as Th17 cells, natural killer T (NKT) cells, and innate lymphoid cells (ILCs). We evaluated the roles of RORγt-expressing cells in mouse acute hepatitis model using RORγt deficient (RORγt^−/−) mice and RAG-2 and RORγt double deficient (RAG-2^−/− × RORγt^−/−) mice. Acute hepatitis was induced in mice by injection with carbon tetrachloride (CCl₄), to investigate the regulation of liver inflammation by RORγt-expressing cells. We detected RORC expression in three compartments, CD4⁺ T cells, NKT cells, and lineage marker-negative SCA-1⁺Thy1^high ILCs, of the liver of wild type (WT) mice. CCl₄-treated RORγt^−/− mice developed liver damage in spite of lack of RORγt-dependent cells, but with reduced infiltration of macrophages compared with WT mice. In this regard, ILCs were significantly decreased in RAG-2^−/− × RORγt^−/− mice that lacked T and NKT cells. Surprisingly, RAG-2^−/− × RORγt^−/− mice developed significantly severer CCl₄-induced hepatitis compared with RAG-2^−/− mice, in accordance with the fact that hepatic ILCs failed to produce IL-22. Lastly, anti-Thy1 monoclonal antibody (mAb), but not anti-NK1.1 mAb or anti-asialo GM1 Ab administration exacerbated liver damage in RAG-2^−/− mice with the depletion of liver ILCs. Collectively, hepatic RORγt-dependent ILCs play a part of protective roles in hepatic immune response in mice.     

Herpes simplex virus type 2-induced mortality following genital infection is blocked by anti-tumor necrosis factor alpha antibody in CXCL10-deficient mice

The role of tumor necrosis factor alpha (TNF-α) was evaluated for CXCL10-deficient (CXCL10^−/−) mice which succumbed to genital herpes simplex virus type 2 (HSV-2) infection and possessed elevated levels of virus and TNF-α but not other cytokines in the central nervous system (CNS) and vaginal tissue within the first 7 days following virus exposure. Anti-TNF-α but not control antibody treatment offsets the elevated mortality rate of CXCL10^−/− mice, despite increased CNS viral titers. In addition, TNF-α neutralization suppressed recruitment of leukocyte subpopulations into the CNS, which is associated with reduced CCL2 and CXCL9 expression. Collectively, the results implicate TNF-α as the principal mediator of mortality in response to genital HSV-2 infection.     

Epithelial PBLD attenuates intestinal inflammatory response and improves intestinal barrier function by inhibiting NF-κB signaling

Intestinal barrier function defects and dysregulation of intestinal immune responses are two key contributory factors in the pathogenesis of ulcerative colitis (UC). Phenazine biosynthesis-like domain-containing protein (PBLD) was recently identified as a tumor suppressor in gastric cancer, hepatocellular carcinoma, and breast cancer; however, its role in UC remains unclear. Therefore, we analyzed colonic tissue samples from patients with UC and constructed specific intestinal epithelial PBLD-deficient (PBLD^IEC−/−) mice to investigate the role of this protein in UC pathogenesis. We found that epithelial PBLD was decreased in patients with UC and was correlated with levels of tight junction (TJ) and inflammatory proteins. PBLD^IEC−/− mice were more susceptible to dextran sulfate sodium (DSS)- and 2,4,6-trinitrobenzene sulfonic acid-induced colitis compared with wild-type (WT) mice. In DSS-induced colitis, PBLD^IEC−/− mice had impaired intestinal barrier function and greater immune cell infiltration in colonic tissue than WT mice. Furthermore, TJ proteins were markedly reduced in PBLD^IEC−/− mice compared with WT mice with colitis. Nuclear factor (NF)-κB activation was markedly elevated and resulted in higher expression levels of downstream effectors (C–C motif chemokine ligand 20, interleukin [IL]-1β, IL-6, and tumor necrosis factor [TNF]-α) in colonic epithelial cells isolated from PBLD^IEC−/− mice than WT mice with colitis. PBLD overexpression in intestinal epithelial cells (IECs) consistently inhibited TNF-α/interferon-γ-induced intestinal barrier disruption and TNF-α-induced inflammatory responses via the suppression of NF-κB. In addition, IKK inhibition (IKK-16) rescued excessive inflammatory responses induced by TNF-α in PBLD knockdown FHC cells. Co-immunoprecipitation assays showed that PBLD may interact with IKKα and IKKβ, thus inhibiting NF-κB signaling, decreasing inflammatory mediator production, attenuating colonic inflammation, and improving intestinal barrier function. Modulating PBLD expression may provide a novel approach for treatment in patients with UC.Subject terms: Ulcerative colitis, Chronic inflammation     

γδ T Cells Are Required for Pulmonary IL-17A Expression after Ozone Exposure in Mice: Role of TNFα

Ozone is an air pollutant that causes pulmonary symptoms. In mice, ozone exposure causes pulmonary injury and increases bronchoalveolar lavage macrophages and neutrophils. We have shown that IL-17A is important in the recruitment of neutrophils after subacute ozone exposure (0.3 ppm for 24–72 h). We hypothesized that γδ T cells are the main producers of IL-17A after subacute ozone. To explore this hypothesis we exposed wildtype mice and mice deficient in γδ T cells (TCRδ^−/−) to ozone or room air. Ozone-induced increases in BAL macrophages and neutrophils were attenuated in TCRδ^−/− mice. Ozone increased the number of γδ T cells in the lungs and increased pulmonary Il17a mRNA expression and the number of IL-17A⁺ CD45⁺ cells in the lungs and these effects were abolished in TCRδ^−/− mice. Ozone-induced increases in factors downstream of IL-17A signaling, including G-CSF, IL-6, IP-10 and KC were also decreased in TCRδ^−/− versus wildtype mice. Neutralization of IL-17A during ozone exposure in wildtype mice mimicked the effects of γδ T cell deficiency. TNFR2 deficiency and etanercept, a TNFα antagonist, also reduced ozone-induced increases in Il17a mRNA, IL-17A⁺ CD45⁺ cells and BAL G-CSF as well as BAL neutrophils. TNFR2 deficient mice also had decreased ozone-induced increases in Ccl20, a chemoattractant for IL-17A⁺ γδ T cells. Il17a mRNA and IL-17A⁺ γδ T cells were also lower in obese Cpe^fat versus lean WT mice exposed to subacute ozone, consistent with the reduced neutrophil recruitment observed in the obese mice. Taken together, our data indicate that pulmonary inflammation induced by subacute ozone requires γδ T cells and TNFα-dependent recruitment of IL-17A⁺ γδ T cells to the lung.     

Accelerated Calvarial Healing in Mice Lacking Toll-Like Receptor 4

The bone and immune systems are closely interconnected. The immediate inflammatory response after fracture is known to trigger a healing cascade which plays an important role in bone repair. Toll-like receptor 4 (TLR4) is a member of a highly conserved receptor family and is a critical activator of the innate immune response after tissue injury. TLR4 signaling has been shown to regulate the systemic inflammatory response induced by exposed bone components during long-bone fracture. Here we tested the hypothesis that TLR4 activation affects the healing of calvarial defects. A 1.8 mm diameter calvarial defect was created in wild-type (WT) and TLR4 knockout (TLR4^−/−) mice. Bone healing was tested using radiographic, histologic and gene expression analyses. Radiographic and histomorphometric analyses revealed that calvarial healing was accelerated in TLR4^−/− mice. More bone was observed in TLR4^−/− mice compared to WT mice at postoperative days 7 and 14, although comparable healing was achieved in both groups by day 21. Bone remodeling was detected in both groups on postoperative day 28. In TLR4^−/− mice compared to WT mice, gene expression analysis revealed that higher expression levels of IL-1β, IL-6, TNF-α,TGF-β1, TGF-β3, PDGF and RANKL and lower expression level of RANK were detected at earlier time points (≤ postoperative 4 days); while higher expression levels of IL-1β and lower expression levels of VEGF, RANK, RANKL and OPG were detected at late time points (> postoperative 4 days). This study provides evidence of accelerated bone healing in TLR4^−/− mice with earlier and higher expression of inflammatory cytokines and with increased osteoclastic activity. Further work is required to determine if this is due to inflammation driven by TLR4 activation.     

Acetylbritannilactone Modulates MicroRNA-155-Mediated Inflammatory Response in Ischemic Cerebral Tissues

Inflammatory responses play a critical role in ischemic brain injury. MicroRNA-155 (miR-155) induces the expression of inflammatory cytokines, and acetylbritannilactone (ABL) exerts potent antiinflammatory actions by inhibiting expression of inflammation-related genes. However, the functions of miR-155 and the actual relationship between ABL and miR-155 in ischemia-induced cerebral inflammation remain unclear. In this study, cerebral ischemia of wild-type (WT) and miR-155^−/− mice was induced by permanent middle cerebral artery occlusion (MCAO). pAd-miR-155 was injected into the lateral cerebral ventricle 24 h before MCAO to induce miR-155 overexpression. MCAO mice and oxygen-glucose deprivation (OGD)-treated BV2 cells were used to examine the effects of ABL and miR-155 overexpression or deletion on the expression of proinflammatory cytokines. We demonstrated that ABL treatment significantly reduced neurological deficits and cerebral infarct volume by inhibiting tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) expression in ischemic cerebral tissue and OGD-treated BV2 cells. Mechanistic studies suggested that the observed decrease in TNF-α and IL-1β expression was attributable to the ABL-induced suppression of the expression of nuclear factor-kappa B (NF-κB) and Toll-like receptor 4 (TLR4). We further found that miR-155 promoted TNF-α and IL-1β expression by upregulating TLR4 and downregulating the expression of suppressor of cytokine signaling 1 (SOCS1) and myeloid differentiation primary response gene 88 (MyD88), while ABL exerted an inhibitory effect on miR-155-mediated gene expression. In conclusion, miR-155 mediates inflammatory responses in ischemic cerebral tissue by modulating TLR4/MyD88 and SOCS1 expression, and ABL exerts its antiinflammatory action by suppressing miR-155 expression, suggesting a novel miR-155-based therapy for ischemic stroke.