p38 MAPK Activity Is Required to Prevent Hyperactivation of NLRP3 Inflammasome.

Inflammation contributes to the pathogenesis and morbidity of wide spectrum of human diseases. The inflammatory response must be actively controlled to prevent bystander damage to tissues. Yet, the mechanisms controlling excessive inflammatory responses are poorly understood. NLRP3 inflammasome plays an important role in innate immune response to cellular infection or stress. Its activation must be tightly regulated because uncontrolled inflammasome activation is associated with a number of human diseases. p38 MAPK signaling plays an essential role in the regulation of inflammation. The role of p38 MAPK in inflammatory response associated with the expression of proinflammatory molecules is known. However, the anti-inflammatory functions of p38 MAPK are largely unknown. In this study, we show that pharmacologic inhibition or genetic deficiency of p38 MAPK leads to hyperactivation of NLRP3 inflammasome, resulting in enhanced Caspase 1 activation and IL-1ß and IL-18 production. The deficiency of p38 MAPK activity induced an increase of cytosolic Ca2+ and excessive mitochondrial Ca2+ uptake, leading to exacerbation of mitochondrial damage, which was associated with hyperactivation of NLRP3 inflammasome. In addition, mice with deficiency of p38 MAPK in granulocytes had evidence of in vivo hyperactivation of NLRP3 inflammasome and were more susceptible to LPS-induced sepsis compared with wild-type mice. Our results suggest that p38 MAPK negatively regulates NLRP3 inflammasome through control of Ca2+ mobilization. Hyperactivity of inflammasome in p38-deficient mice causes lung inflammation and increased susceptibility to septic shock.

Autophagy Is Required for Neutrophil-Mediated Inflammation.

Autophagy, an intracellular degradation and energy recycling mechanism, is emerging as an important regulator of immune responses. However, the role of autophagy in regulating neutrophil functions is not known. We investigated neutrophil biology using myeloid-specific autophagy-deficient mice and found that autophagy deficiency reduced neutrophil degranulation in vitro and in vivo. Mice with autophagy deficiency showed reduced severity of several neutrophil-mediated inflammatory and autoimmune disease models, including PMA-induced ear inflammation, LPS-induced breakdown of blood-brain barrier, and experimental autoimmune encephalomyelitis. NADPH oxidase-mediated reactive oxygen species generation was also reduced in autophagy-deficient neutrophils, and inhibition of NADPH oxidase reduced neutrophil degranulation, suggesting NADPH oxidase to be a player at the intersection of autophagy and degranulation. Overall, this study establishes autophagy as an important regulator of neutrophil functions and neutrophil-mediated inflammation in vivo.

Critical role for IL-18 in spontaneous lung inflammation caused by autophagy deficiency.

Autophagy is an important component of the immune response. However, the functions of autophagy in human diseases are much less understood. We studied biological consequences of autophagy deficiency in mice lacking the essential autophagy gene Atg7 or Atg5 in myeloid cells. Surprisingly, these mice presented with spontaneous sterile lung inflammation, characterized by marked recruitment of inflammatory cells, submucosal thickening, goblet cell metaplasia, and increased collagen content. Lung inflammation was associated with increase in several proinflammatory cytokines in the bronchoalveolar lavage and in serum. This inflammation was largely driven by IL-18 as a result of constitutive inflammasome activation. Following i.p. LPS injection, autophagy-deficient mice had higher levels of proinflammatory cytokines in lungs and in serum, as well as increased mortality, than control mice. Intranasal bleomycin challenge exacerbated lung inflammation in autophagy-deficient mice and produced more severe fibrotic changes than in control mice. These results uncover a new and important role for autophagy as negative regulator of lung inflammation.

Regulation of IL-4 receptor signaling by STUB1 in lung inflammation.

RATIONALE: IL-4Rα, the common receptor component for IL-4 and IL-13, plays a critical role in IL-4- and IL-13-mediated signaling pathways that regulate airway inflammation and remodeling. However, the regulatory mechanisms underlying IL-4Rα turnover and its signal termination remain elusive. OBJECTIVES: To evaluate the role of STUB1 (STIP1 homology and U-Box containing protein 1) in regulating IL-4R signaling in airway inflammation. METHODS: The roles of STUB1 in IL-4Rα degradation and its signaling were investigated by immunoblot, immunoprecipitation, and flow cytometry. The involvement of STUB1 in airway inflammation was determined in vivo by measuring lung inflammatory cells infiltration, mucus production, serum lgE levels, and alveolar macrophage M2 activation in STUB1(-/-) mice. STUB1 expression was evaluated in airway epithelium of patients with asthma and lung tissues of subjects with chronic obstructive pulmonary disease. MEASUREMENTS AND MAIN RESULTS: STUB1 interacted with IL-4Rα and targeted it for ubiquitination-mediated proteasomal degradation, terminating IL-4 or IL-13 signaling. STUB1 knockout cells showed increased levels of IL-4Rα and sustained STAT6 activation, whereas STUB1 overexpression reduced IL-4Rα levels. Mice deficient in STUB1 had spontaneous airway inflammation, alternative M2 activation of alveolar macrophage, and increased serum IgE. STUB1 levels were increased in airways of subjects with asthma or chronic obstructive pulmonary disease, suggesting that up-regulation of STUB1 might be an important feedback mechanism to dampen IL-4R signaling in airway inflammation. CONCLUSIONS: Our study identified a previously uncharacterized role for STUB1 in regulating IL-4R signaling, which might provide a new strategy for attenuating airway inflammation.

Toll-like receptor 4 mediates lipopolysaccharide-induced muscle catabolism via coordinate activation of ubiquitin-proteasome and autophagy-lysosome pathways.

Cachectic muscle wasting is a frequent complication of many inflammatory conditions, due primarily to excessive muscle catabolism. However, the pathogenesis and intervention strategies against it remain to be established. Here, we tested the hypothesis that Toll-like receptor 4 (TLR4) is a master regulator of inflammatory muscle catabolism. We demonstrate that TLR4 activation by lipopolysaccharide (LPS) induces C2C12 myotube atrophy via up-regulating autophagosome formation and the expression of ubiquitin ligase atrogin-1/MAFbx and MuRF1. TLR4-mediated activation of p38 MAPK is necessary and sufficient for the up-regulation of atrogin1/MAFbx and autophagosomes, resulting in myotube atrophy. Similarly, LPS up-regulates muscle autophagosome formation and ubiquitin ligase expression in mice. Importantly, autophagy inhibitor 3-methyladenine completely abolishes LPS-induced muscle proteolysis, while proteasome inhibitor lactacystin partially blocks it. Furthermore, TLR4 knockout or p38 MAPK inhibition abolishes LPS-induced muscle proteolysis. Thus, TLR4 mediates LPS-induced muscle catabolism via coordinate activation of the ubiquitin-proteasome and the autophagy-lysosomal pathways.

Src kinase-mediated phosphorylation stabilizes inducible nitric-oxide synthase in normal cells and cancer cells.

Src kinases are key regulators of cellular proliferation, survival, motility, and invasiveness. They play important roles in the regulation of inflammation and cancer. Overexpression or hyperactivity of c-Src has been implicated in the development of various types of cancer, including lung cancer. Src inhibition is currently being investigated as a potential therapy for non-small cell lung cancer in Phase I and II clinical trials. The mechanisms of Src implication in cancer and inflammation are linked to the ability of activated Src to phosphorylate multiple downstream targets that mediate its cellular effector functions. In this study, we reveal that inducible nitric-oxide synthase (iNOS), an enzyme also implicated in cancer and inflammation, is a downstream mediator of activated Src. We elucidate the molecular mechanisms of the association between Src and iNOS in models of inflammation induced by lipopolysaccharide and/or cytokines and in cancer cells and tissues. We identify human iNOS residue Tyr(1055) as a target for Src-mediated phosphorylation. These results are shown in normal cells and cancer cells as well as in vivo in mice. Importantly, such posttranslational modification serves to stabilize iNOS half-life. The data also demonstrate interactions and co-localization of iNOS and activated Src under inflammatory conditions and in cancer cells. This study demonstrates that phosphorylation of iNOS by Src plays an important role in the regulation of iNOS and nitric oxide production and hence could account for some Src-related roles in inflammation and cancer.

Glioma pathogenesis-related protein 1 exerts tumor suppressor activities through proapoptotic reactive oxygen species-c-Jun-NH2 kinase signaling.

Glioma pathogenesis-related protein 1 (GLIPR1), a novel p53 target gene, is down-regulated by methylation in prostate cancer and has p53-dependent and -independent proapoptotic activities in tumor cells. These properties suggest an important tumor suppressor role for GLIPR1, yet direct genetic evidence of a tumor suppressor function for GLIPR1 is lacking and the molecular mechanism(s), through which GLIPR1 exerts its tumor suppressor functions, has not been shown. Here, we report that the expression of GLIPR1 is significantly reduced in human prostate tumor tissues compared with adjacent normal prostate tissues and in multiple human cancer cell lines. Overexpression of GLIPR1 in cancer cells leads to suppression of colony growth and induction of apoptosis. Mice with an inactivated Glipr1 gene had significantly shorter tumor-free survival times than either Glipr1(+/+) or Glipr1(+/-) mice in both p53(+/+) and p53(+/-) genetic backgrounds, owing to their development of a unique array of malignant tumors. Mechanistic analysis indicated that GLIPR1 up-regulation increases the production of reactive oxygen species (ROS) leading to apoptosis through activation of the c-Jun-NH(2) kinase (JNK) signaling cascade. Thus, our results identify GLIPR1 as a proapoptotic tumor suppressor acting through the ROS-JNK pathway and support the therapeutic potential for this protein.