The pathogenesis of COVID-19 is associated with a hyperinflammatory response; however, the precise mechanism of SARS-CoV-2-induced inflammation is poorly understood. Here we investigated direct inflammatory functions of major structural proteins of SARS-CoV-2. We observed that spike (S) protein potently induced inflammatory cytokines and chemokines including IL-6, IL-1b, TNFa, CXCL1, CXCL2, and CCL2, but not IFNs in human and mouse macrophages. No such inflammatory response was observed in response to membrane (M), envelope (E), and nucleocapsid (N) proteins. When stimulated with extracellular S protein, human and mouse lung epithelial cells also produced inflammatory cytokines and chemokines. Interestingly, epithelial cells expressing S protein intracellularly were non-inflammatory, but elicited an inflammatory response in macrophages when co-cultured. Biochemical studies revealed that S protein triggers inflammation via activation of the NF-kB pathway in a MyD88-dependent manner. Further, such an activation of the NF-kB pathway was abrogated in Tlr2-deficient macrophages. Consistently, administration of S protein induced IL-6, TNF-a, and IL-1b in wild-type, but not Tlr2-deficient mice. Notably, upon recognition of S protein, TLR2 dimerizes with TLR1 or TLR6 to activate the NF-kB pathway. Together these data reveal a mechanism for the cytokine storm during SARS-CoV-2 infection and suggest that TLR2 could be a potential therapeutic target for COVID-19.

In this study, we elucidated the mechanism by which adiponectin modulates hepatic stellate cell activation and fibrogenesis. Adiponectin-overexpressing transgenic mice receiving thioacetamide were resistant to fibrosis, compared with controls. In contrast, adiponectin-null animals developed severe fibrosis. Expression of collagen ␣1(I) and ␣-smooth muscle actin (␣-SMA) mRNAs were significantly lower in adiponectin-overexpressing mice, compared with controls. In wild-type stellate cells exposed to a lentivirus encoding adiponectin, expression of peroxisome proliferator-activated receptor-␥ (PPAR␥), SREBP1c, and CEBP␣ mRNAs was significantly increased (3.2-, 4.1-, and 2.2-fold, respectively; n ‫؍‬ 3; P < 0.05, adiponectin virus versus control), consistent with possible activation of an adipogenic transcriptional program. Troglitazone, a PPAR␥ agonist, strongly suppressed upregulation of collagen ␣1(I) and ␣-SMA mRNA in stellate cells isolated from wild-type mice; however, stellate cells from adiponectin-null animals failed to respond to troglitazone. Furthermore, in isolated stellate cells in which PPAR␥ was depleted using an adenovirus-Cre-recombinase system and in which adiponectin was also overexpressed, collagen ␣1(I) and ␣-SMA were significantly inhibited. We conclude that the PPAR␥ effect on stellate cell activation and the fibrogenic cascade appears to be adiponectin-dependent; however, the inhibitory effect of adiponectin on stellate cell activation was not dependent on PPAR␥, suggesting the presence of PPAR␥-dependent as well as independent pathways in stellate cells.

Liver wound healing is an integrated process in which hepatic stellate cells play a major role. We hypothesized that the cellextracellular signaling protein integrin-linked kinase (ILK) is important in transducing signals from the extracellular matrix to stellate cells and thus plays a critical role in stellate cell activation and fibrogenesis during liver injury. Liver injury and subsequent stellate cell activation led to a 3-fold increase in ILK expression and increased kinase activity. Overexpression of ILK in isolated stellate cells led to enhanced motility and adhesion as well as increases in smooth muscle ␣-actin and type I collagen mRNA expression. The effects of ILK on stellate cell phenotypes were phosphatidylinositol 3-kinase-dependent. Forced expression of ILK in vivo led to increases in type I collagen, smooth muscle ␣-actin, transforming growth factor-␤, and extra domain A (EDA) fibronectin mRNAs (by 3.2-, 3.5-, 2.5-, and 2.2-fold, respectively; n ‫؍‬ 8, p < 0.05 for each versus the control), whereas inhibition of ILK in vivo led to significant reductions in these mRNAs. Morphometric analysis revealed that ILK overexpression led to a 31.4% increase in liver collagen content (n ‫؍‬ 8, p < 0.05 versus the control); in contrast ILK knockdown in vivo led to a significant reduction in fibrogenesis. We conclude that ILK plays an important pathophysiological role in vivo in liver wound healing.The liver responds to injury by wound healing, leading to fibrosis. The multiple integrated systems leading to fibrosis are complex and are similar mechanistically to that in skin and other parenchymal organs. A remarkable increase in extracellular matrix production after wounding is characteristic and is typified by a 4 -6-fold increase in collagens (i.e. types I, III, and IV, etc.) as well as an increase in many other extracellular matrix constituents (1). Investigation over the past two decades has helped to establish a cellular mechanism for the woundhealing response to liver injury (2). A critical feature of this wounding response is the transformation of resident stellate cells (also known as lipocytes, Ito cells, or perisinusoidal cells) from the "quiescent" state in the normal liver to an "activated" state in the injured liver. This transition is characterized by both morphologic and functional changes, including loss of vitamin A, acquisition of stress bundles, development of prominent rough endoplasmic reticulum, and a striking increase in the secretion of extracellular matrix proteins, as well as enhanced stellate cell contractility, motility, proliferation, adhesion, and responsiveness to various cytokines and peptides found in the wounding milieu (3).Integrin-linked kinase (ILK) 2 is a newly described signaling molecule important in transducing signals from the extracellular matrix to cellular machinery through the cytoplasmic portion of integrins and adaptor proteins to a variety of signaling cascades (4 -8). For example, ILK interaction with integrins is important in cell growth, differentiation, cell ...

Integrin-linked kinase (ILK) is a multidomain focal adhesion protein implicated in signal transduction between integrins and growth factor/extracellular receptors. We have previously shown that ILK expression is increased in liver fibrosis and that ILK appears to be a key regulator of fibrogenesis in rat hepatic stellate cells, effectors of the fibrogenic response. Here we hypothesized that the mechanism by which ILK mediates the fibrogenic phenotype is by engaging the small GTPase, Rho in a signal transduction pathway linked to fibrogenesis.MethodsILK function in quiescent (non fibrogenic) and activated (fibrogenic) stellate cells was examined in cells isolated from rat livers. ILK, Rho, and Gα12/13 signaling were manipulated using established chemical agents or specific adenoviral constructs.ResultsILK activity was minimal in quiescent stellate cells, but prominent in activated stellate cells; inhibition of ILK activity had no effect in quiescent cells, but had prominent effects in activated cells. Overexpression of ILK in activated stellate cells increased Rho activity, but had no effect in quiescent cells. Further, endothelin-1 (ET-1) stimulated Rho activity in activated stellate cells, but not in quiescent cells. Rho, RhoGEF and Gα12/13 expression were increased after stellate cell activation. Inhibition of Gα12/13 signaling, by expression of the RGS domain of the p115-Rho- specific guanine nucleotide exchange factor (p115-RGS) in activated stellate cells, significantly inhibited type I collagen and smooth muscle α actin expression, both classically upregulated after stellate cell activation. The data suggest that ILK mediates Rho dependent functional effects in activated stellate cells, and raise the possibility that ILK is important in cross talk with the GPCR system.

Background Portal hypertension results from endothelial dysfunction after liver injury caused in part by abnormal production of endothelial cell derived nitric oxide synthase (eNOS). Here, we have postulated that endothelial mechanosensing pathways involving integrin linked kinase (ILK) may play a critical role in portal hypertension, eNOS expression and function. Aims In this study, we investigated the role of ILK and the small GTP-binding protein, Rho, in sinusoidal endothelial cell eNOS regulation and function. Methods Primary liver sinusoidal endothelial cells (SECs) were isolated using standard techniques. Liver injury was induced by performing bile duct ligation (BDL). To examine the expression of Rho and ILK in vivo during wound healing, SECs were infected with constitutively active Rho (V14), a dominant negative Rho (N19) and constructs encoding ILK and a short hairpin-inhibiting ILK. Results ILK expression was increased in SECs after liver injury; endothelin-1, vascular endothelial growth factor, and transforming growth factor beta-1 stimulated ILK expression in SECs. ILK expression in turn led to eNOS upregulation and to enhanced eNOS phosphorylation and NO production. ILK knockdown or ILK (kinase) inhibition reduced eNOS mRNA expression, promoter activity, eNOS expression, and ultimately NO production. In contrast, ILK over-expression had the opposite effect. Inhibition of ILK activity also disrupted the actin cytoskeleton in isolated SECs. Rho overexpression suppressed phosphorylation of the serinethreonine kinase, Akt, and inhibited eNOS phosphorylation. Finally, inhibition of Rho function with the RGS domain of the p115-Rho-specific GEF (p115-RGS) significantly increased eNOS phosphorylation. Conclusions Our data suggest a potential role for ILK, the cytoskeleton, and ILK signaling partners including Rho in regulating intrahepatic SEC eNOS expression and function.

People living with HIV (PLWH) have to take an antiretroviral therapy (ART) for life and show noncommunicable illnesses such as chronic inflammation, immune activation, and multiorgan dysregulation. Recent studies suggest that long-term use of ART induces comorbid conditions and is one of the leading causes of heart failure in PLWH. However, the molecular mechanism of antiretroviral drugs (ARVs) induced heart failure is unclear. To determine the mechanism of ARVs induced cardiac dysfunction, we performed global transcriptomic profiling of ARVs treated neonatal rat ventricular cardiomyocytes in culture. Differentially expressed genes were identified by RNA-sequencing. Our data show that ARVs treatment causes upregulation of several biological functions associated with cardiotoxicity, hypertrophy, and heart failure. Global gene expression data were validated in cardiac tissue isolated from HIV patients having a history of ART. Interestingly, we found that homeodomain-only protein homeobox (HOPX) expression was significantly increased in cardiomyocytes treated with ARVs and in the heart tissue of HIV patients. Furthermore, we found that HOPX plays a crucial role in ARVs mediated cellular hypertrophy. Mechanistically, we found that HOPX plays a critical role in epigenetic regulation, through deacetylation of histone, while the HDAC inhibitor, Trichostatin A, can restore the acetylation level of histone 3 in the presence of ARVs.

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