Hanzhi Liu

Blocking IL-17A promotes the resolution of pulmonary inflammation and fibrosis via TGF-beta1-dependent and -independent mechanisms.

Pulmonary fibrosis is the pathologic basis for a variety of incurable human chronic lung diseases. IL-17A, a glycoprotein secreted from IL-17–producing cells, has recently been shown to be a proinflammatory cytokine involved in chronic inflammation and autoimmune disease. In this study, we report that IL-17A increased the synthesis and secretion of collagen and promoted the epithelial–mesenchymal transition in alveolar epithelial cells in a TGF-β1–dependent manner. Using in vivo fibrotic models, we found IL-17A expression to be elevated and IL-17A–associated signaling pathways to be activated in fibrotic lung tissues. Neutralization of IL-17A in vivo promoted the resolution of bleomycin-induced acute inflammation, attenuated pulmonary fibrosis, and increased survival. Additionally, IL-17A antagonism inhibited silica-induced chronic inflammation and pulmonary fibrosis. Targeting IL-17A resulted in a shift of the suppressive immune response in fibrotic lung tissue toward a Th1-type immune response, and it effectively induced autophagy, which promoted the autophagic degradation of collagen and autophagy-associated cell death. Moreover, IL-17A was found to attenuate the starvation-induced autophagy, and autophagy modulators regulated collagen degradation in the alveolar epithelial cells in a TGF-β1–independent manner. Administration of 3-methylamphetamine, an autophagy inhibitor, reversed the therapeutic efficacy of IL-17A antagonism in pulmonary fibrosis. Our studies indicate that IL-17A participates in the development and progression of pulmonary fibrosis in both TGF-β1–dependent and –independent manners and that the components of the IL-17A signaling pathway are potential therapeutic targets for the treatment of fibroproliferative lung diseases.

Targeting TLR2 Attenuates Pulmonary Inflammation and Fibrosis by Reversion of Suppressive Immune Microenvironment

Pulmonary fibrosis is a consequence of chronic lung injury and is associated with a high mortality. Despite the pathogenesis of pulmonary fibrosis remaining as an enigma, immune responses play a critical role in the deregulation of wound healing process after lung injury, which leads to fibrosis. Accumulating evidence argues the rationales for current treatments of pulmonary fibrosis using immunosuppressive agents such as corticosteroids. In this study, we report that bleomycin (BLM), a well-known fibrogenic agent functioning as a TLR2 agonist, induced the maturation of dendritic cells and release of cytokines. The BLM activation of TLR2 mediated a time-dependent alteration of immune responses in the lung. These responses resulted in an increase in the tissue-infiltrating proinflammatory cells and cytokines in the early period initially following BLM exposure and an increase in the tissue-infiltrating suppressive immune cells and factors during the later period following BLM exposure. TLR2 deficiency, however, reduced pulmonary inflammation, injury, and subsequently attenuated pulmonary fibrosis. Targeting TLR2 by a TLR2-neutralizing Ab not only markedly decreased animal death but also protected animals from the development of pulmonary fibrosis and reversed the established pulmonary fibrosis through regulating BLM-induced immunosuppressive microenvironments. Our studies suggest that TLR2 is a promising target for the development of therapeutic agents against pulmonary fibrosis and that eliminating immunosuppressive cells and factors via immunostimulants is a novel strategy for fibro-proliferative diseases. Moreover, combining BLM with an anti-TLR2 Ab or TLR2 antagonist for cancer therapy will improve the BLM therapeutic profile by enhancing anti-cancer efficacy and reducing systemic inflammation and pulmonary fibrosis.

Toll‐like receptor 4 activity protects against hepatocellular tumorigenesis and progression by regulating expression of DNA repair protein Ku70 in mice

Hepatocellular carcinoma (HCC) is a devastating consequence of chronic inflammatory liver diseases. The goal of this study was to investigate whether Toll‐like receptor 4 (TLR4) activity contributes to HCC initiation and progression in mice. A mouse model of diethylnitrosamine (DEN)‐induced HCC was generated with wild‐type and TLR4 mutant mice, and the development and progression of HCC and senescent responses were assessed using morphologic, immunological, and biochemical criteria. We found that genetic or pharmacologic blocking of TLR4 increased susceptibility to DEN‐induced HCC carcinogenesis and progression, which was indicated by increases in number of tumor nodules, tumor volume, and animal death. The enhanced HCC was associated with a broad‐spectrum reduction of immune response to DEN liver injury, as indicated by decreases in the liver‐infiltrating F4/80+ macrophages, the apoptosis signal‐regulating kinase 1/p38 mitogen‐activated protein kinase/NF‐κB and IRF3 signaling activities, and the expression of inflammatory cytokines. Suppressed immune networks resulted in a halt of cellular senescence induction in TLR4 mutant liver tissue, which promoted proliferation and suppressed programmed cell death. Moreover, TLR4 mutation resulted in a suppressed capacity of DNA repair due to a decrease in TLR4‐medicated expression of DNA repair proteins Ku70/80 in liver tissue and cells. Isotopic expression of Ku70 in TLR4 mutant mice restored senescence and interrupted the positive feedback loop of DNA damage and oxidative stress, which reversed TLR4 mutation–deteriorated HCC carcinogenesis and progression. Conclusion: TLR4 plays an integrated defense role against HCC carcinogenesis by enhancing the expression and function of DNA repair protein Ku70. Our studies provide novel insight into TLR4 activity in the regulation of HCC tumorigenesis, which may be useful for the prevention of HCC development. (HEPATOLOGY 2013)

Blocking TLR2 activity diminishes and stabilizes advanced atherosclerotic lesions in apolipoprotein E-deficient mice.

Aim:Toll-like receptor 2 (TLR2) signaling plays a critical role in the initiation of atherosclerosis. The aim of this study was to investigate whether blocking TLR2 activity could produce therapeutic effects on advanced atherosclerosis.Methods:Forty-week old apolipoprotein E-deficient (ApoE−/−) mice fed on a normal diet were intravenously injected with a TLR2-neutralizing antibody or with an isotype-matched IgG for 18 weeks. Double-knockout ApoE−/−Tlr2−/− mice were taken as a positive control. At the end of the treatments, the plasma lipid levels were measured, and the plaque morphology, pro-inflammatory cytokines expression and apoptosis in arteries were analyzed. In the second part of this study, 6-week old ApoE−/− and ApoE−/−Tlr2−/− mice fed on a high-cholesterol diet for 12 to 24 weeks, the expression levels of TLR2 and apoptotic markers in arteries were examined.Results:Blockade of TLR2 activity with TLR2-neutralizing antibody or knockout of Tlr2 gene did not alter the plasma lipid levels in ApoE−/− mice. However, the pharmacologic and genetic manipulations significantly reduced the plaque size and vessel stenosis, and increased plaque stability in the brachiocephalic arteries. The protective effects of TLR2 antagonism were associated with the suppressed expression of pro-inflammatory cytokines IL-6 and TNF-α and the inactivation of transcription factors NF-κB and Stat3. In addition, blocking TLR2 activity attenuated ER stress-induced macrophage apoptosis in the brachiocephalic arteries, which could promote the resolution of necrotic cores in advanced atherosclerosis. Moreover, high-cholesterol diet more prominently accelerated atherosclerotic formation and increased the expression of pro-apoptotic protein CHOP and apoptosis in ApoE−/− mice than in ApoE−/−Tlr2−/− mice.Conclusion:The pharmacologic or genetic blockade of TLR2 activity diminishes and stabilizes advanced atherosclerotic lesions in ApoE−/− mice. Thus, targeting TLR2 signaling may be a promising therapeutic strategy against advanced atherosclerosis.

Interleukin-17A is involved in development of spontaneous pulmonary emphysema caused by Toll-like receptor 4 mutation

Aim:To explore the pathogenic role of Th17 cells and interleukin-17A (IL-17A)-associated signaling pathways in spontaneous pulmonary emphysema induced by a Toll-like receptor 4 mutant (TLR4mut).Methods:Lungs were obtained from wild-type (WT) or TLR4mut mice that were treated with or without recombinant mouse IL-17A (1 μg·kg−1·d−1, ip) from the age of 3 weeks to 3 months. Pulmonary emphysema was determined using histology, immunochemistry, and biochemical analysis. T cell polarization was determined with flow cytometry, the levels of cytokines were measured using ELISA, and the levels of IL-17A-associated signaling molecules were detected using Western blot.Results:Compared to WT mice, 3 month-old TLR4mut mice were characterized by significantly reduced infiltration of Th17 cells into lungs (2.49%±1.13 % νs 5.26%±1.39%), and significantly reduced expression levels of IL-17A (3.66±0.99 pg/μg νs 10.67±1.65 pg/μg), IL-23 (12.43±1.28 pg/μg νs 28.71±2.57 pg/μg) and IL-6 (51.82±5.45 pg/μg νs 92.73±10.91 pg/μg) in bronchoalveolar lavage fluid. In addition, p38 MAPK phosphorylation and AP-1 expression were decreased to 27%±9% and 51%±8%, respectively, of that in WT mice. Treatment of TLR4mut mice with IL-17A increased the infiltration of Th17 cells into lungs and expression levels of IL-17A, IL-6, and IL-23 in bronchoalveolar lavage fluid, attenuated MDA and apoptosis, and improved emphysema accompanied with increased phosphorylation of p38 MAPK and expression of AP-1.Conclusion:Th17 cells, in particular the cytokine IL-17A, play a crucial role in the pathogenesis of TLR4mut-induced spontaneous pulmonary emphysema. Both of them are potential targets for therapeutic strategies for pulmonary emphysema.