Wolbachia heat shock protein 60 induces pro-inflammatory cytokines and apoptosis in monocytes in vitro

Recombinant Wolbachia heat shock protein 60 (rWmhsp60) induces gene expression of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α in human monocytic cell line THP-1. In addition, it inhibits the phagocytic activity and does not alter the nitric oxide production by differentiated THP-1 macrophages, which corroborates with no significant change in inducible nitric oxide synthase gene expression in rWmhsp60 treated THP-1 monocytes. Further, 24 h stimulation of peripheral blood mononuclear cells from normal individuals by rWmhsp60 reveals that monocytes enter the late apoptotic stage, while lymphocytes do not show apoptosis. Thus these findings suggest that rWmhsp60 may contribute to inflammation mediated monocyte dysfunction in filarial pathogenesis.     

gp130 on macrophages/granulocytes modulates inflammation during experimental tuberculosis

gp130 is a common receptor chain for cytokines such as interleukin (IL)-27 and IL-6. During experimental tuberculosis (TB), IL-27 prevents optimal antimycobacterial protection and limits the pathological sequelae of chronic inflammation. The anti-inflammatory properties of IL-27 have been attributed mainly to its suppressive effect on T helper (TH) cells. However, because gp130 cytokines also suppress the inflammatory immune response of macrophages, IL-27 may also regulate inflammation by limiting the secretion of pro-inflammatory cytokines. To specifically address the role of gp130 cytokines on macrophages, the outcome of experimental TB was analysed in macrophage/neutrophil-specific gp130-deficient (LysM(cre) gp130(loxP/loxP)) mice. In these mice, the enhanced induction of inflammatory cytokines and increased expression of the inducible nitric oxide synthase (NOS2) and LRG47 was linked to a greatly augmented TH17 immune response and matrix metalloproteinase (MMP)-9 expression. However, this amplified inflammatory immune response in Mtb-infected LysM(cre) gp130(loxP/loxP) mice was not associated with reduced bacterial loads and/or accelerated pathology. Our study revealed an immunoregulatory function of gp130 cytokines on macrophages/granulocytes, which is, however, not critical for modulating the outcome of TB.     

Phospholipase C-γ2 promotes intracellular survival of mycobacteria

Mycobacterium tuberculosis (Mtb) infects millions of people each year. These bacilli can survive inside macrophages. To favor their survival, pathogen alters various signal transduction pathways in host cells. Phospholipase C (PLC) signaling regulates various processes in mammalian cells but has never been investigated for their roles in regulating phagocytosis and killing of mycobacteria by macrophages. Here, we report that infection with Mtb but not Mycobacterium smegmatis (MS) induces phosphorylation of PLC-γ2 at tyrosine 1217 in J774A.1 cells. Small interfering RNA–mediated knockdown of PLC-γ2 expression leads to the enhanced killing of both MS and Mtb by these cells suggesting that Mtb activates PLC-γ2 to promote its intracellular survival within macrophages. Knockdown of PLC-γ2 also lead to increased uptake of Mtb but not MS by J774.A.1 cells. Further, we have observed that PLC-γ2 was required for Mtb-induced inhibition of expression of proinflammatory cytokine tumor necrosis factor-α, inducible nitric oxide synthase, and chemokine (C-C motif) ligand 5 (RANTES). Altogether, our results for the first time demonstrate that Mtb induces activation of macrophages PLC-γ2 to inhibit their mycobactericidal response.     

Long noncoding RNA THRIL promotes foam cell formation and inflammation in macrophages

Tumor necrosis factor-α (TNF-α) and heterogenous nuclear ribonucleoprotein L (hnRNPL)-related immunoregulatory lincRNA (THRIL) is a long noncoding RNA (lncRNA) involved in various inflammatory diseases. However, its role in atherosclerosis is not known. In this study, we aimed to investigate the function of THRIL in mediating macrophage inflammation and foam cell formation. The expression of THRIL was quantified in THP-1 macrophages after treatment with oxidized low-density lipoprotein (oxLDL). The effect of THRIL overexpression and knockdown on oxLDL-induced inflammatory responses and lipid accumulation was determined. THRIL-associated protein partners were identified by RNA pull-down and RNA immunoprecipitation assays. We show that THRIL is upregulated in macrophages after oxLDL treatment. Knockdown of THRIL blocks oxLDL-induced expression of interleukin-1β (IL-1β), IL-6, and TNF-α and lipid accumulation. Conversely, ectopic expression of THRIL enhances inflammatory gene expression and lipid deposition in oxLDL-treated macrophages. Moreover, THRIL depletion increases cholesterol efflux from macrophages and the expression of ATP-binding cassette transporter (ABC) A1 and ABCG1. FOXO1 is identified as a protein partner of THRIL and promotes macrophage inflammation and lipid accumulation. Furthermore, overexpression of FOXO1 restores lipid accumulation and inflammatory cytokine production in THRIL-depleted macrophages. In conclusion, our data suggest a model where THRIL interacts with FOXO1 to promote macrophage inflammation and foam cell formation. THRIL may represent a therapeutic target for atherosclerosis.     

MiR‐140 modulates the inflammatory responses of Mycobacterium tuberculosis‐infected macrophages by targeting TRAF6

This study aimed to examine miR‐140 expression in clinical samples from tuberculosis (TB) patients and to explore the molecular mechanisms of miR‐140 in host‐bacterial interactions during Mycobacterium tuberculosis (M tb) infections. The miR‐140 expression and relevant mRNA expression were detected by quantitative real‐time PCR (qRT‐PCR); the protein expression levels were analysed by ELISA and western blot; M tb survival was measured by colony formation unit assay; potential interactions between miR‐140 and the 3′ untranslated region (UTR) of tumour necrosis factor receptor‐associated factor 6 (TRAF6) was confirmed by luciferase reporter assay. MiR‐140 was up‐regulated in the human peripheral blood mononuclear cells (PBMCs) from TB patients and in THP‐1 and U937 cells with M tb infection. Overexpression of miR‐140 promoted M tb survival; on the other hand, miR‐140 knockdown attenuated M tb survival. The pro‐inflammatory cytokines including interleukin 6, tumour necrosis‐α, interleukin‐1β and interferon‐γ were enhanced by M tb infection in THP‐1 and U937 cells. MiR‐140 overexpression reduced these pro‐inflammatory cytokines levels in THP‐1 and U937 cells with M tb infection; while knockdown of miR‐140 exerted the opposite actions. TRAF6 was identified to be a downstream target of miR‐140 and was negatively modulated by miR‐140. TRAF6 overexpression increased the pro‐inflammatory cytokines levels and partially restored the suppressive effects of miR‐140 overexpression on pro‐inflammatory cytokines levels in THP‐1 and U937 cells with M tb infection. In conclusion, our results implied that miR‐140 promoted M tb survival and reduced the pro‐inflammatory cytokines levels in macrophages with M tb infection partially via modulating TRAF6 expression.     

Caffeic acid phenethyl ester protects mice from lethal endotoxin shock and inhibits lipopolysaccharide-induced cyclooxygenase-2 and inducible nitric oxide synthase expression in RAW 264.7 macrophages via the p38/ERK and NF-kappaB pathways

Caffeic acid phenethyl ester has been shown to have anti-inflammatory and anti-cancer effects. We examined the effects of caffeic acid phenethyl ester on lipopolysaccharide-induced production of nitric oxide and prostaglandin E(2), and expression of inducible nitric oxide synthase and cyclooxygenase-2 in RAW 264.7 macrophages. We also investigated the effects of caffeic acid phenethyl ester on lipopolysaccharide-induced septic shock in mice. Our results indicate that caffeic acid phenethyl ester inhibits lipopolysaccharide-induced nitric oxide and prostaglandin E(2) production in a concentration-dependent manner and inhibits inducible nitric oxide synthase and cyclooxygenase-2 in RAW 264.7 cells, without significant cytotoxicity. To further examine the mechanism responsible for the inhibition of inducible nitric oxide synthase and cyclooxygenase-2 expression by caffeic acid phenethyl ester, we examined the effect of caffeic acid phenethyl ester on lipopolysaccharide-induced nuclear factor-kappaB activation and the phosphorylation of mitogen-activated protein kinases. Caffeic acid phenethyl ester treatment significantly reduced nuclear factor-kappaB translocation and DNA-binding in lipopolysaccharide-stimulated RAW 264.7 cells. This effect was mediated through the inhibition of the degradation of inhibitor kappaB and by inhibition of both p38 mitogen-activated protein kinase and extracellular signal-regulated kinase phosphorylation, at least in part by inhibiting the generation of reactive oxygen species. Furthermore, caffeic acid phenethyl ester rescued C57BL/6 mice from lethal lipopolysaccharide-induced septic shock, while decreasing serum levels of tumor necrosis factor-alpha and interleukin-1beta. Collectively, these results suggest that caffeic acid phenethyl ester suppresses the induction of cytokines by lipopolysaccharide, as well as inducible nitric oxide synthase and cyclooxygenase-2 expression, by blocking nuclear factor-kappaB and p38/ERK activation. These findings provide mechanistic insights into the anti-inflammatory and chemopreventive actions of caffeic acid phenethyl ester in macrophages.     

Fatty acylCoA synthetase FadD13 regulates proinflammatory cytokine secretion dependent on the NF‐κB signalling pathway by binding to eEF1A1

Mycobacterium tuberculosis (Mtb) manipulates multiple host defence pathways to survive and persist in host cells. Understanding Mtb–host cell interaction is crucial to develop an efficient means to control the disease. Here, we applied the Mtb proteome chip, through separately interacting with H37Ra and H37Rv stimulated macrophage lysates, screened 283 Mtb differential proteins. Through primary screening, we focused on fatty acylCoA synthetase FadD13. Mtb FadD13 is a potential drug target, but its role in infection remains unclear. Deletion of FadD13 in Mtb reduced the production of proinflammatory cytokines IL‐1β, IL‐18, and IL‐6. Bimolecular fluorescence complementation and colocalization showed that the binding partner of FadD13 in macrophage was eEF1A1 (a translation elongation factor). Knockdown eEF1A1 expression in macrophage abrogated the promotion of proinflammatory cytokines induced by FadD13. In addition, ΔfadD13 mutant decreased the expression of the NF‐κB signalling pathway related proteins p50 and p65, so did the eEF1A1 knockdown macrophage infected with H37Rv. Meanwhile, we found that deletion of FadD13 reduced Mtb survival in macrophages during Mtb infection, and purified FadD13 proteins induced broken of macrophage membrane. Taken together, FadD13 is crucial for Mtb proliferation in macrophages, and it plays a key role in the production of proinflammatory cytokines during Mtb infection.     

Increased peroxynitrite activity in AIDS dementia complex: implications for the neuropathogenesis of HIV-1 infection

Oxidative stress is suggested to be involved in several neurodegenerative diseases. One mechanism of oxidative damage is mediated by peroxynitrite, a neurotoxic reaction product of superoxide anion and nitric oxide. Expression of two cytokines and two key enzymes that are indicative of the presence of reactive oxygen intermediates and peroxynitrite was investigated in brain tissue of AIDS patients with and without AIDS dementia complex and HIV-seronegative controls. RNA expression of IL-1beta, IL-10, inducible nitric oxide synthase, and superoxide dismutase (SOD) was found to be significantly higher in demented compared with nondemented patients. Immunohistochemical analysis showed that SOD was expressed in CD68-positive microglial cells while inducible nitric oxide synthase was detected in glial fibrillary acidic protein (GFAP)-positive astrocytes and in equal amounts in microglial cells. Approximately 70% of the HIV p24-Ag-positive macrophages did express SOD, suggesting a direct HIV-induced intracellular event. HIV-1 infection of macrophages resulted in both increased superoxide anion production and elevated SOD mRNA levels, compared with uninfected macrophages. Finally, we show that nitrotyrosine, the footprint of peroxynitrite, was found more intense and frequent in brain sections of demented patients compared with nondemented patients. These results indicate that, as a result of simultaneous production of superoxide anion and nitric oxide, peroxynitrite may contribute to the neuropathogenesis of HIV-1 infection.     

Nitric oxide augments oridonin-induced efferocytosis by human histocytic lymphoma U937 cells via autophagy and the NF-κB-COX-2-IL-1β pathway

Abstract We previously demonstrated that oridonin-induced autophagy enhanced efferocytosis (phagocytosis of apoptotic cells) by macrophage-like U937 cells through activation of the inflammatory pathways. In this study, exposure of U937 cells to 2.5 μM oridonin caused up-regulation of inducible nitric oxide synthase (iNOS) expression and continuous endogenous generation of nitric oxide (NO), which was reversed by pre-treatment with the inhibitors of nitric oxide synthase 1400 W (dihydrochloride) or L-NAME (hydrochloride). NO donor sodium nitroprusside (SNP) and efferocytosis irritant lipopolysaccharide (LPS) could also exert NO generation and iNOS expression. Moreover, oridonin-induced stimulation of efferocytosis was significantly suppressed by 1400 W or L-NAME. In addition, 1400 W or L-NAME impaired oridonin-induced autophagy. Inhibition of autophagy with 3-methyladenine (3MA) or Beclin-1 siRNA attenuated the uptake of apoptotic cells with a slight increase in the production of NO. The pro-inflammatory cytokine interleukin-1β (IL-1β) has been reported to be involved in oridonin-induced efferocytosis in U937 cells and interact with NO to contribute to inflammatory responses. 1400 W or L-NAME blocked the secretion of IL-1β and the activation of NF-κB and COX-2. Provision of SNP or LPS in place of oridonin resulted in the similar enhancement of efferocytosis, autophagy, the release of IL-1β and the expression of signal protein. NO augmented the oridonin-induced efferocytosis by mediating autophagy and activating the NF-κB-COX-2-IL-1β pathway. Inhibition of NF-κB or COX-2 in turn decreased the production of NO and the expression of iNOS. There exists a positive feedback loop between NO generation and NF-κB-COX-2-IL-1β pathway.     

Activation of thromboxane receptor modulates interleukin-1β-induced monocyte adhesion--a novel role of Nox1

Activation of thromboxane receptors (TPr) may promote atherosclerosis by enhancing oxidative stress and inflammation. This study examined the role of Nox1, an NADPH-oxidase subunit, in the enhancement of interleukin (IL)-1β-induced monocyte adhesion by TPr. In cultured rat aortic vascular smooth muscle cells (VSMCs), U46619, a stable thromboxane A(2) mimetic, together with interleukin-1β significantly enhanced Nox1 mRNA expression, as well as adhesion of THP-1 monocytes. Activation of TPr also enhanced IL-1β-induced vascular cell adhesion molecule (VCAM)-1 expression, but inhibited inducible nitric oxide synthase (iNOS) expression. Silencing Nox1 expression by siRNA prevented the U46619 enhancement of IL-1β-induced monocyte adhesion, but had no significant effect on VCAM-1 or iNOS expression. Furthermore, monocyte adhesion was inhibited by superoxide dismutase, enhanced by a specific iNOS inhibitor, l-N(6)-(1-iminoethyl)-lysine, but not influenced by catalase. U46619 inhibited IL-1β-induced cyclic GMP production, and the inhibition was partially prevented by superoxide dismutase. In conclusion, activation of TPr enhances IL-1β-induced Nox1 expression in VSMCs, which is responsible for the up-regulation of monocyte adhesion. The effect of Nox1 is independent of the changes in VCAM-1 and iNOS expression, but depends on the inactivation of nitric oxide via generation of superoxide anion.     

Down-regulation of miR-20a-5p triggers cell apoptosis to facilitate mycobacterial clearance through targeting JNK2 in human macrophages

Induction of cell apoptosis is one of the major host defense mechanisms through which macrophages control Mycobacterium tuberculosis (Mtb) infection. However, the mechanisms underlying macrophage apoptosis triggered by Mtb infection are still largely unknown. In this study, a microarray profiling survey revealed 14 miRNAs were down-regulated in CD14+ monocytes from active pulmonary tuberculosis patients, and only the reduction of miR-20a-5p could be reversed after successful anti-tuberculosis treatment. Validation of miR-20a-5p expression was confirmed using real time qPCR. Moreover, miR-20a-5p expression also decreased in differentiated THP-1 macrophages after mycobacterial infection in vitro. Functional assays through forced or inhibited expression of miR-20a-5p in THP-1 macrophages demonstrated that miR-20a-5p functioned as a negative regulator of mycobacterial-triggered apoptosis. Importantly, inhibition of miR-20a-5p expression resulted in more efficient mycobacterial clearance from infected THP-1 macrophages while miR-20a-5p overexpression promoted mycobacterial survival. Mechanistically, miR-20a-5p was demonstrated to regulate Bim expression in a JNK2-dependent manner, unlike Bcl2, and luciferase assay showed JNK2 was a novel direct target of miR-20a-5p. Together, our findings indicate that downregulation of miR-20a-5p triggers macrophage apoptosis as a novel mechanism for host defense against mycobacterial infection.     

Overexpression of Dimethylarginine Dimethylaminohydrolase 1 Attenuates Airway Inflammation in a Mouse Model of Asthma

Levels of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, are increased in lung, sputum, exhaled breath condensate and plasma samples from asthma patients. ADMA is metabolized primarily by dimethylarginine dimethylaminohydrolase 1 (DDAH1) and DDAH2. We determined the effect of DDAH1 overexpression on development of allergic inflammation in a mouse model of asthma. The expression of DDAH1 and DDAH2 in mouse lungs was determined by RT-quantitative PCR (qPCR). ADMA levels in bronchoalveolar lavage fluid (BALF) and serum samples were determined by mass spectrometry. Wild type and DDAH1-transgenic mice were intratracheally challenged with PBS or house dust mite (HDM). Airway inflammation was assessed by bronchoalveolar lavage (BAL) total and differential cell counts. The levels of IgE and IgG1 in BALF and serum samples were determined by ELISA. Gene expression in lungs was determined by RNA-Seq and RT-qPCR. Our data showed that the expression of DDAH1 and DDAH2 was decreased in the lungs of mice following HDM exposure, which correlated with increased ADMA levels in BALF and serum. Transgenic overexpression of DDAH1 resulted in decreased BAL total cell and eosinophil numbers following HDM exposure. Total IgE levels in BALF and serum were decreased in HDM-exposed DDAH1-transgenic mice compared to HDM-exposed wild type mice. RNA-Seq results showed downregulation of genes in the inducible nitric oxide synthase (iNOS) signaling pathway in PBS-treated DDAH1-transgenic mice versus PBS-treated wild type mice and downregulation of genes in IL-13/FOXA2 signaling pathway in HDM-treated DDAH1-transgenic mice versus HDM-treated wild type mice. Our findings suggest that decreased expression of DDAH1 and DDAH2 in the lungs may contribute to allergic asthma and overexpression of DDAH1 attenuates allergen-induced airway inflammation through modulation of Th2 responses.