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Dendritic cells (DCs) are recognized as a key orchestrator of immune response and homeostasis, deregulation of which may lead to autoimmunity such as experimental autoimmune encephalomyelitis (EAE). Herein we show that the phosphatase PP2Cδ played a pivotal role in regulating DC activation and function, as PP2Cδ ablation caused aberrant maturation, activation, and Th1/Th17-priming of DCs, and hence induced onset of exacerbated EAE. Mechanistically, PP2Cδ restrained the expression of the essential subunit of mTORC2, Rictor, primarily through de-phosphorylating and proteasomal degradation of the methyltransferase NSD2 via CRL4DCAF2 E3 ligase. Loss of PP2Cδ in DCs accordingly sustained activation of the Rictor/mTORC2 pathway and boosted glycolytic and mitochondrial metabolism. Consequently, ATP-citrate lyse (ACLY) was increasingly activated and catalyzed acetyl-CoA for expression of the genes compatible with hyperactivated DCs under PP2Cδ deletion. Collectively, our findings demonstrate that PP2Cδ has an essential role in controlling DCs activation and function, which is critical for prevention of autoimmunity.

Comprehensive immune responses are essential for eliminating pathogens but must be tightly controlled to avoid sustained immune activation and potential tissue damage. The engagement of TLR4, a canonical pattern recognition receptor, has been proposed to trigger inflammatory responses with different magnitudes and durations depending on TLR4 cellular compartmentalization. In the present study, we identify an unexpected role of Lamtor5, a newly identified component of the amino acid-sensing machinery, in modulating TLR4 signaling and controlling inflammation. Specifically, Lamtor5 associated with TLR4 via their LZ/TIR domains and facilitated their colocalization at autolysosomes, preventing lysosomal tethering and the activation of mTORC1 upon LPS stimulation and thereby derepressing TFEB to promote autophagic degradation of TLR4. The loss of Lamtor5 was unable to trigger the TFEB-driven autolysosomal pathway and delay degradation of TLR4, leading to sustained inflammation and hence increased mortality among Lamtor5 haploinsufficient mice during endotoxic shock. Intriguingly, nutrient deprivation, particularly leucine deprivation, blunted inflammatory signaling and conferred protection to endotoxic mice. This effect, however, was largely abrogated upon Lamtor5 deletion. We thus propose a homeostatic function of Lamtor5 that couples pathogenic insults and nutrient availability to optimize the inflammatory response; this function may have implications for TLR4-associated inflammatory and metabolic disorders.

Summary The increasing rising of multiple drug-resistant Staphylococcus aureus has become a major public health concern, underscoring a pressing need for developing therapies essentially based on the understanding of host defensive mechanism. In the present study, we showed that microRNA (miR)-127 played a key role in controlling bacterial infection and conferred a profound protection against staphylococcal pneumonia. The protective effect of miR-127 was largely dependent on its regulation of macrophage bactericidal activity and the generation of IL-22, IL-17, and anti-microbial peptides (AMPs), the pathway primarily driven by STAT3. Importantly, we revealed that the ubiquitin-editing enzyme A20, a genuine target of miR-127, specifically interacted with and repressed K63-ubiquitination of STAT3, thereby compromising its phosphorylation upon bacterial infection. Thus, our data not only identify miR-127 as a non-coding molecule with anti-bacterial activity but also delineate an unappreciated mechanism whereby A20 regulates STAT3-driven anti-microbial signaling via modulating its ubiquitination., Graphical Abstract, Highlights • miR-127 confers the protection against staphylococcal pneumonia • miR-127 augments macrophage anti-microbial responses by regulating STAT3 activity • A20 directly interacts and represses STAT3 K63-ubiquitination • The A20/STAT3 axis mediates the anti-microbial role of miR-127, Molecular Mechanism of Behavior; Immunology; Microbiology; Bacteriology

Uncontrolled inflammatory responses cause tissue injury and severe immunopathology. Pharmacological interference of intracellular pro-inflammatory signaling may confer a therapeutic benefit under these conditions. Daphnetin, a natural coumarin derivative, has been used to treat inflammatory diseases including bronchitis. However, the protective effect of daphnetin in inflammatory airway disorders has yet to be determined, and the molecular basis for its anti-inflammatory properties is unknown. This paper shows that daphnetin treatment conferred substantial protection from endotoxin-induced acute lung injury (ALI), in parallel with reductions in the production of inflammatory mediators, symptoms of airway response, and infiltration of inflammatory cells. Further studies indicate that activation of macrophage and human alveolar epithelial cells in response to lipopolysaccharide (LPS) was remarkably suppressed by daphnetin, which was related to the down-regulation of NF-κB-dependent signaling events. Importantly, this study demonstrates that TNF-α-induced protein 3 (TNFAIP3), also known as A20, was significantly induced by daphnetin, which appeared to be largely responsible for the down-regulation of NF-κB activity through modulation of nondegradative TRAF6 ubiquitination. Accordingly, the deletion of TNFAIP3 in primary macrophages reversed daphnetin-elicited inhibition of immune response, and the beneficial effect of daphnetin in the pathogenesis of ALI was, partially at least, abrogated by TNFAIP3 knockdown. These findings demonstrate the anti-inflammatory and protective functions of daphnetin in endotoxin-induced lung inflammation and injury and also reveal the key mechanism underlying its action in vitro as well as in vivo.

Introduction Curcumin has remarkable anti-inflammatory and antioxidant properties. However, its effects on bacterium-induced acute lung injury (ALI) are not fully understood. Objective To investigate the protective effects of curcumin on a mouse model of S. aureus-induced ALI. Methods Mice were pretreated with intraperitoneal injection of curcumin or vehicle 2 h before Staphylococcus aureus instillation. The survival rate and bacterial burden after infection were recorded. Mice were sacrificed for the analyses of severity of pneumonia, integrity of lung barrier, disorder of coagulation cascades and extent of inflammation 12 h postinfection. The production of proinflammatory cytokines and chemokines in the lung and bronchoalveolar lavage fluid was detected. Results Pretreatment with curcumin markedly attenuated S. aureus-induced pneumonia, barrier disruption, lung edema and vascular leakage. Activation of plasminogen activator inhibitor-1 and infiltration of neutrophils were reduced by curcumin, together with lower levels of proinflammatory cytokines and chemokines. Conclusion Curcumin can alleviate S. aureus-induced ALI through multiple pathways.

Chlamydia pneumonia (C. pneumonia) remains one of the leading causes of bacterial pneumonia and has been implicated in the pathogenesis of some inflammation-related diseases, such as asthma, chronic obstructive pulmonary disease, and vascular diseases. Heat shock protein 60 is one of the pathogenic components of C. pneumonia that is closely associated with the inflammatory disorders. However, the molecular basis for the immunopathologic property of chlamydial heat shock protein (cHSP60) has not been elucidated. In this article, we report that MAPK kinase 3 (MKK3) is essential for cHSP60-induced lung inflammation, because MKK3-knockout mice displayed significantly reduced lung neutrophil accumulation and decreased production of proinflammatory mediators, correlating with the alleviated inflammatory response in lung tissues. Mechanistically, p38 kinase was selectively activated by MKK3 in response to cHSP60 and activated NF-κB by stimulating the nuclear kinase, mitogen- and stress-activated protein kinase 1. The specific knockdown of mitogen- and stress-activated protein kinase 1 in macrophages resulted in a defective phosphorylation of NF-κB/RelA at Ser276 but had no apparent effect on RelA translocation. Furthermore, TGF-β–activated kinase 1 was found to relay the signal to MKK3 from TLR4, the major receptor that sensed cHSP60 in the initiation of the inflammatory response. Thus, we establish a critical role for MKK3 signaling in cHSP60 pathology and suggest a novel mechanism underlying C. pneumonia–associated inflammatory disorders.

MicroRNAs play an important role in regulating the inflammatory response, and are critically involved in the development of inflammatory disorders, including those affecting the lungs. While the microRNA miR-221 is involved in embryonic lung branching morphogenesis and epithelial cell development, its importance in lung inflammation has not been previously explored. In our current study, expression of miR-221 was selectively decreased by exposure to lipopolysaccharides (LPS) both in vitro and in vivo. Enforced expression of miR-221 significantly increased the production of proinflammatory cytokines TNF-α and IL-6, and enhanced the activation of NF-κB and MAPKs upon LPS stimulation. Accordingly, intratracheal stimulation of miR-221 was shown to aggravate endotoxin-induced acute lung injuries and inflammation in mice. Mechanistic studies showed that miR-221 directly targets A20, a master regulator of NF-κB and MAPK signaling, and thus represses inflammatory signaling. Restoration of A20 in macrophages abolished the stimulatory effect of miR-221 on production of proinflammatory cytokines. Together, these results indicate the presence of a novel miRNA-mediated feed-back mechanism that controls inflammation, and suggest involvement of aberrant miR-221 expression in the development of inflammatory lung disorders.

A polarized macrophage response is presumed to have a pivotal role in a variety of immunological pathophysiology. However, the molecular mechanism underlying macrophage functional shaping remains largely unknown. In this study, we reveal a pivotal role of miR-127 in macrophage development and thereby the pathogenesis of inflammation and lung injury. In particular, miR-127 was demonstrated to be prominently induced upon TLR engagement and repressed by the M2-prone cytokines. Enforced expression of miR-127 in macrophages resulted in significantly increased production of proinflammatory cytokines, whereas deletion of miR-127 impaired M1 gene expression and led to a M2-biased response. Accordingly, intratracheal administration of miR-127 resulted in an exaggerated pulmonary inflammation and injury. Conversely, antagonizing of miR-127 suppressed production of the proinflammatory cytokines and rendered the mice more refractory to the inflammation-associated pathology. Mechanistically, miR-127 demonstrated to target B cell lymphoma 6 (Bcl6) and remarkably downregulated its expression and subsequently dual specificity phosphatase 1 (Dusp1), which in turn enhanced the activation of JNK kinase and hence the development of proinflammatory macrophages. Thereby, reconstitution with the expression of Bcl6 or Dusp1 or inhibition of JNK activity impaired miR-127–mediated skewing of M1 proinflammatory macrophages, whereas interference of Bcl6 or Dusp1 expression abrogated the anti-inflammatory property of anti–miR-127. Together, these data establish miR-127 as a molecular switch during macrophage development and as a potential target for treatment of inflammatory diseases.

Bcl6, a POZ transcription factor, plays an essential role in the tumorgenensis, inflammation and development, but its role in the antiviral response has never been established. Here, we demonstrate that Bcl6 unexpectedly functions to inhibit RIG-I-mediated antiviral signaling and impedes the clearance of viral infection. More importantly, we identify that miR-127 as a upstream regulator of Bcl6, and the subsequent co-repressive complex in the initiation of RIG-I-driven response. This finding extends our knowledge about mitochondrial antiviral mechanisms and proposes a novel corepressor network required for the coordination of the innate immune response.

Hepatitis B virus (HBV) infection is a crucial cause of hepatocyte death, cirrhosis, and hepatocellular carcinoma. However, the molecular mechanism causing the death of hepatocytes during HBV infection was not completed understood. A20, also named tumor necrosis factor alpha-induced protein 3 (TNFAIP3), is a key regulator in inflammation and immunity, and was identified as a target of miR-125a. Here, we report that miR-125a is induced by hepatitis B virus X protein (HBx) and leading to repression of A20. We also found HBx or miR-125a transfection of hepatoma cell line L-O2 significantly increased its sensitivity to TRAIL-induced apoptosis. The increasing of TRAIL sensitivity was associated with repression of A20 protein expression, and HBx activates caspase-8, due to the direct cleavage of RIP1 by A20-inhibition. We also found caspase-8 K63-linked polyubiquitination was repressed by HBx. Thus, our study indicates that HBx sensitizes hepatocytes to TRAIL-induced apoptosis through upregulating miR-125a and targeting A20 through caspase-8 activation and the regulation of RIP1 ubiquitination. So these results suggest that miR-125a and A20 represent key diagnostic markers and potential therapeutic targets for the prevention and treatment of HBV infection.

Heat shock protein (HSP) 60 is the major component of Chlapidia pneumonia (C. pneumonia), and has been recognized to be related with inflammatory disorders clinically. Nevertheless, much remains to be learned about the immunopathologic property of C. pneumonia HSP60 (cHSP60). Here we report that the MAPK kinase 3 (Mkk3) is required for cHSP60-induced lung inflammation in mice, as mice with deletion of Mkk3 displayed profoundly reduced infiltration of neutrphils, production of proinflammaoty mediators, and inflammatory symptoms in lung tissue. P38 is selectively activated by Mkk3, and plays a critical role in activating production of inflammatory cytokines through transcriptional and post-transcriptional mechanisms. We also identified MSK1 to act downstream of p38 and contribute to the inflammatory response upon cHSP60 stimulation. Specific knockdown of MSK1 led to a defective phosphorylation of NF-κB/RelA at Ser276, causing the alleviated inflammatory reaction. We thus reveal the biological function of cHSP60 in vivo as well as in in vitro, and establish that MKK3/p38/MSK1 axis is essential for its pathological role, and maybe C. pneumonia-associated inflammatory diseases.