Hongzhen Yang

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.

TLR4 activity is required in the resolution of pulmonary inflammation and fibrosis after acute and chronic lung injury

Pulmonary fibrosis is an inflammation-driven lung disease with a poor prognosis and no cure. Here we report that basal toll-like receptor 4 (TLR4) activity is critical for the resolution of acute and chronic inflammation and pulmonary fibrosis in mouse models of lung injury. We found that genetic or pharmacologic inhibition of TLR4 exacerbates bleomycin-induced pulmonary inflammation, fibrosis, dysfunction, and animal death through promoting formation of an immunosuppressive tissue microenvironment and attenuating autophagy-associated degradation of collagen and cell death in the fibrotic lung tissues. In contrast, pharmacologic activation of TLR4 resulted in a quick resolution of acute inflammation, reversed the established pulmonary fibrosis, improved lung function, and rescued mice from death. Similarly, blocking TLR4 impaired the resolution of silica-induced chronic inflammation and fibrosis. Importantly, altering autophagic activity could reverse the TLR4-regulated lung inflammation, fibrosis, dysfunction, and animal death. Rapamycin, an autophagy activator, reversed the effects of TLR4 antagonism. In contrast, inhibition of autophagy by 3-methyladenine reversed the proresolving and antifibrotic roles of TLR4 agonists and increased animal death. These results not only highlight a pivotal role for TLR4-mediated basal immunity, particularly autophagic activity, in the proresolution of inflammation and fibrosis after chemical-induced lung injury but also provide proof for the concept for activating TLR4 signaling, particularly TLR4-mediated autophagy, as a novel therapeutic strategy against chronic fibroproliferative diseases that are unresponsive to current therapy.

Blocking TLR2 Activity Attenuates Pulmonary Metastases of Tumor

Background Metastasis is the most pivotal cause of mortality in cancer patients. Immune tolerance plays a crucial role in tumor progression and metastasis. Methods and Findings In this study, we investigated the potential roles and mechanisms of TLR2 signaling on tumor metastasis in a mouse model of intravenously injected B16 melanoma cells. Multiple subtypes of TLRs were expressed on B16 cells and several human cancer cell lines; TLR2 mediated the invasive activity of these cells. High metastatic B16 cells released more heat shock protein 60 than poor metastatic B16-F1 cells. Importantly, heat shock protein 60 released by tumor cells caused a persistent activation of TLR2 and was critical in the constitutive activation of transcription factor Stat3, leading to the release of immunosuppressive cytokines and chemokines. Moreover, targeting TLR2 markedly reduced pulmonary metastases and increased the survival of B16-bearing mice by reversing B16 cells induced immunosuppressive microenvironment and restoring tumor-killing cells such as CD8+ T cells and M1 macrophages. Combining an anti-TLR2 antibody and a cytotoxic agent, gemcitabine, provided a further improvement in the survival of tumor-bearing mice. Conclusions and Significance Our results demonstrate that TLR2 is an attractive target against metastasis and that targeting immunosuppressive microenvironment using anti-TLR2 antibody is a novel therapeutic strategy for combating a life-threatening metastasis.

TRB3 interacts with SMAD3 promoting tumor cell migration and invasion

Tribbles homolog 3 (TRB3, also known as TRIB3, NIPK and SKIP3), a human homolog of Drosophila Tribbles, has been found to interact with a variety of signaling molecules to regulate diverse cellular functions. Here, we report that TRB3 is a novel SMAD3-interacting protein. Expression of exogenous TRB3 enhanced the transcriptional activity of SMAD3, whereas knocking down endogenous TRB3 reduced the transcriptional activity of SMAD3. The kinase-like domain (KD) of TRB3 was responsible for the interaction with SMAD3 and the regulation of SMAD3-mediated transcriptional activity. In addition, TGF-β1 stimulation or overexpression of SMAD3 enhanced the TRB3 promoter activity and expression, suggesting that there is a positive feedback loop between TRB3 and TGF-β–SMAD3 signaling. Mechanistically, TRB3 was found to trigger the degradation of SMAD ubiquitin regulatory factor 2 (Smurf2), which resulted in a decrease in the degradation of SMAD2 and phosphorylated SMAD3. Moreover, TRB3–SMAD3 interaction promoted the nuclear localization of SMAD3 because of the interaction of TRB3 with the MH2 domain of SMAD3. These effects of TRB3 were responsible for potentiating the SMAD3-mediated activity. Furthermore, knockdown of endogenous TRB3 expression inhibited the migration and invasion of tumor cells in vitro, which were associated with an increase in the expression of E-cadherin and a decrease in the expression of Twist-1 and Snail, two master regulators of epithelial-to-mesenchymal transition, suggesting a crucial role for TRB3 in maintaining the mesenchymal status of tumor cells. These results demonstrate that TRB3 acts as a novel SMAD3-interacting protein to participate in the positive regulation of TGF-β–SMAD-mediated cellular biological functions.

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 2 mediates invasion via activating NF-κB in MDA-MB-231 breast cancer cells

MDA-MB-231 breast cancer cells have a high invasive potential, yet the mechanisms involved are not known. This study showed that Toll-like receptor 2 (TLR2) was highly expressed in MDA-MB-231 cells and played a critical role in cell invasion. Compared with the poorly invasive MCF-7 cells, MDA-MB-231 cells expressed 10.5-fold more TLR2. Using TLR2 agonist pg-LPS and TLR2 neutralizing antibody, we found that TLR2 activation significantly promoted MDA-MB-231 invasion, whereas TLR2 blockade diminished this capacity. TLR2 activation enhanced the activity of NF-kappaB and induced phosphorylation of TAK1 and IkappaBalpha in the TLR2/NF-kappaB signaling pathway in MDA-MB-231, but not in MCF-7 cells. TLR2 activation increased IL-6, TGF-beta, VEGF and MMP9 secretion, which are associated with TLR2-NF-kappaB signaling. We demonstrated that TLR2 is a critical receptor responsible for NF-kappaB signaling activity and highly invasive capacity of MDA-MB-231 cells.

Loss of immunity-supported senescence enhances susceptibility to hepatocellular carcinogenesis and progression in Toll-like receptor 2-deficient mice

Hepatocellular carcinoma (HCC) is a complication at the endstage of chronic inflammatory liver diseases with dismal prognosis. Targeting of Toll‐like receptor (TLR) 2 attenuates tumor metastases; we hypothesized that blocking TLR2 might also play a crucial role in reducing hepatocarcinogenesis. Surprisingly, we found that the genetic deletion of TLR2 increased susceptibility to diethylnitrosamine (DEN), a genotoxic carcinogen that can induce HCC. Indeed, TLR2‐deficient mice showed a significant increase in carcinogenesis and progression of HCC as indicated by increases in tumor nodule size, tumor volume, and animal death. The enhanced susceptibility to DEN‐induced HCC was associated with a broad‐spectrum reduction in the immune response to DEN‐induced liver injury. We found that TLR2 deficiency caused a decrease in the infiltration of macrophages and an attenuation of apoptosis signal regulating kinase 1 (ASK1) / p38 mitogen‐activated protein kinase (p38 MAPK) / nuclear factor kappa B (NF‐κB) signaling, which led to a decrease in the expression of interferon‐gamma (IFN‐γ), tumor necrosis factor alpha (TNF‐α), interleukin (IL)‐1α/β, IL‐6, and Cxcl‐2 as well as suppression of autophagy flux and increases in oxidative stress and p62 aggregation in liver tissue. The defects in immune networks resulted in suppressed p21‐ and p16/pRb‐dependent senescence, which caused an increase in proliferation and a decrease in apoptotic and autophagy‐associated cell death in mouse livers. Restoring cellular senescence and autophagy flux by treating TLR2‐deficient mice with IFN‐γ, a T helper 1 (Th1) cytokine and positive modulator of senescence and autophagy, could attenuate the carcinogenesis and progression of HCC associated with TLR2‐deficient animals. Conclusion: The loss of immune networks supporting cellular senescence and autophagy flux is attributed to enhanced susceptibility to DEN‐induced hepatocellular carcinogenesis and progression in TLR2‐deficient mice. These findings may be used to prevent the development of liver cancer. (HEPATOLOGY 2013)

Intracellular or extracellular heat shock protein 70 differentially regulates cardiac remodelling in pressure overload mice

AIMS Innate and adaptive immune responses are associated with the development of hypertension-induced myocardial hypertrophy and fibrosis. As a result, we investigated whether heat shock protein (HSP) 70, which is a molecule of damage-associated molecular patterns, could induce inflammation in the myocardium and promote the development of hypertension-induced cardiac hypertrophy and fibrosis. METHODS AND RESULTS We found that HSP70 serum levels, as well as the amount of HSP70 translocation to the cardiomyocyte membranes and the interstitial space, were elevated in the hypertensive mice caused by abdominal aortic constriction (AAC). Transcriptional inhibition of HSP70 expression by a specific heat shock transcript factor inhibitor, KNK437, reduced the serum level, and the re-distribution of HSP70. It promoted myocardial hypertrophy and cardiac dysfunctions although it protected animals from AAC-induced cardiac fibrosis. On the other hand, the functional antagonism of HSP70 by an anti-HSP70 antibody attenuated AAC-induced cardiac hypertrophy and fibrosis without adverse haemodynamic effects. The cardioprotective effect of the anti-HSP70 antibody was largely attributed to its ability to block AAC-activated immune response in the heart, as was indicated by suppressing the hypertension-enhanced conjugation of HSP70 with toll-like receptor 4, reducing heart-infiltrating macrophages, decreasing the expression of pro-inflammatory factor monocyte chemoattractant protein-1 and profibrotic factor transforming growth factor beta 1, and attenuating pro-hypertrophy signal MAPK P38 and ERK. CONCLUSION These results indicate that intracellular and extracellular HSP70 have different roles in the regulation of cardiac remodelling and function in response to hypertension. Extracellular HSP70 is a potential therapeutic target against cardiac hypertrophy and fibrosis.

Bacillus Calmette-Guérin and TLR4 Agonist Prevent Cardiovascular Hypertrophy and Fibrosis by Regulating Immune Microenvironment

Hypertension-induced cardiovascular hypertrophy and fibrosis are critical in the development of heart failure. The activity of TLRs has been found to be involved in the development of pressure overload-induced myocardial hypertrophy and cardiac fibrosis. We wondered whether vaccine bacillus Calmette-Guérin (BCG), which activated TLR4 to elicit immune responses, modulated the pressure overload-stimulated cardiovascular hypertrophy and cardiac fibrosis in the murine models of abdominal aortic constriction (AAC)-induced hypertension. Before or after AAC, animals received BCG, TLR4 agonist, IFN-γ, or TLR4 antagonist i.p. BCG and TLR4 agonist significantly prevented AAC-induced cardiovascular hypertrophy and reactive cardiac fibrosis with no changes in hemodynamics. Moreover, TLR4 antagonist reversed the BCG- and TLR4 agonist-induced actions of anti-cardiovascular hypertrophy and cardiac fibrosis. BCG decreased the expression of TLR2 or TLR4 on the heart tissue but TLR4 agonist increased the expression of TLR2 or TLR4 on the immune cells that infiltrate into the heart tissue. This led to an increased expression ratio of IFN-γ/TGF-β in the heart. The cardiac protective effects of BCG and TLR4 agonist are related to their regulation of ERK-Akt and p38-NF-κB signal pathways in the heart. In conclusion, the activity of TLR4 plays a critical role in the mediation of pressure overload-induced myocardial hypertrophy and fibrosis. The regulation of immune responses by BCG and TLR4 agonist has a great potential for the prevention and treatment of hypertension-induced myocardial hypertrophy and cardiac fibrosis.

Timing Is Critical for an Effective Anti-Metastatic Immunotherapy: The Decisive Role of IFNγ/STAT1-Mediated Activation of Autophagy

Background Immunotherapy is often recommended as an adjuvant treatment to reduce the chance of cancer recurrence or metastasis. Interestingly, timing is very important for a successful immunotherapy against metastasis, although the precise mechanism is still unknown. Methods and Findings Using a mouse model of melanoma metastasis induced by intravenous injection of B16-F10 cells, we investigated the mechanism responsible for the diverse efficacy of the prophylactic or therapeutic TLR4 and TLR9 agonist complex against metastasis. We found that the activation of TLR4 and TLR9 prevented, but did not reverse, metastasis because the potency of this combination was neither sufficient to overcome the tumor cell-educated immune tolerance nor to induce efficacious autophagy in tumor cells. The prophylactic application of the complex promoted antimetastatic immunity, leading to the autophagy-associated death of melanoma cells via IFNγ/STAT1 activation and attenuated tumor metastasis. IFNγ neutralization reversed the prophylactic benefit induced by the complex by suppressing STAT1 activation and attenuating autophagy in mice. However, the therapeutic application of the complex did not suppress metastasis because the complex could not reverse tumor cell-induced STAT3 activation and neither activate IFNγ/STAT1 signaling and autophagy. Suppressing STAT3 activation with the JAK/STAT antagonist AG490 restored the antimetastatic effect of the TLR4/9 agonist complex. Activation of autophagy after tumor inoculation by using rapamycin, with or without the TLR4/9 agonist complex, could suppress metastasis. Conclusion and Significance Our studies suggest that activation of IFNγ/STAT1 signaling and induction of autophagy are critical for an efficacious anti-metastatic immunotherapy and that autophagy activators may overcome the timing barrier for immunotherapy against metastasis.