The anti-inflammatory effects of Akkermansia muciniphila and its derivates in HFD/CCL4-induced murine model of liver injury.

Inflammation plays a critical role in the promotion of hepatocyte damage and liver fibrosis. In recent years the protective role of Akkermansia muciniphila, a next-generation beneficial microbe, has been suggested for metabolic and inflammatory disorders. In this study, we aimed to evaluate the effects of live and pasteurized A. muciniphila and its extra cellular vesicles (EVs) on inflammatory markers involved in liver fibrosis in a mouse model of a high-fat diet (HFD)/carbon tetrachloride (CCl4)-induced liver injury. Firstly, the responses of hepatic stellate cells (HSCs) to live and pasteurized A. muciniphila and its EVs were examined in the quiescent and LPS-activated LX-2 cells. Next, the anti-inflammatory effects of different forms of A. muciniphila were examined in the mouse model of HFD/CCl4-induced liver injury. The gene expression of various inflammatory markers was evaluated in liver, colon, and white adipose tissues. The cytokine secretion in the liver and white adipose tissues was also measured by ELISA. The results showed that administration of live and pasteurized A. muciniphila and its EVs leads to amelioration in HSCs activation. Based on data obtained from the histopathological analysis, an improvement in gut health was observed through enhancing the epithelium and mucosal layer thickness and strengthening the intestinal integrity in all treatments. Moreover, live A. muciniphila and its EVs had inhibitory effects on liver inflammation and hepatocytes damage. In addition, the tissue cytokine production and inflammatory gene expression levels revealed that live A. muciniphila and its EVs had more pronounced anti-inflammatory effects on liver and adipose tissues. Furthermore, EVs had better effects on the modulation of gene expression related to TLRs, PPARs, and immune response in the liver. In conclusion, the present results showed that oral administration of A. muciniphila and its derivatives for four weeks could enhance the intestinal integrity and anti-inflammatory responses of the colon, adipose, and liver tissues and subsequently prevent liver injury in HFD/CCL4 mice.

Albumin Might Attenuate Bacteria-Induced Damage on Kupffer Cells for Patients with Chronic Liver Disease.

Chronic liver diseases (CLDs) are complex diseases that cause long-term inflammation and infection, which in turn accelerate their development. The usage of albumin in patients with CLDs has been debated for years. Human serum albumin (HSA) plays a key role in immunomodulation during the process of CLDs. The correlation between albumin and C-reactive protein (CRP) in CLD patients was analyzed by linear regression with the Pearson statistic. The damage of THP-1 and primary cells was evaluated by measuring the lactate dehydrogenase (LDH) in the supernatant. Immunofluorescence staining was performed to determine underlying pathways in Kupffer cells (KCs). Albumin negatively correlated with infection in patients with CLDs. In vitro experiments with THP-1 cells and KCs showed that albumin reduced LDH release after stimulation with bacterial products, while no differences in hepatic stellate cells (HSCs) and sinusoidal endothelial cells (SECs) were detected. Moreover, immunofluorescence staining revealed an increase of p-ERK and p-NF-kB p65 density after albumin treatment of KCs stimulated by bacterial products. In conclusion, albumin could assist CLD patients in alleviating inflammation caused by bacterial products and might be beneficial to patients with CLDs by securing KCs from bacteria-induced damage, providing a compelling rationale for albumin therapy in patients with CLDs.

Odontogenic infection by Porphyromonas gingivalis exacerbates fibrosis in NASH via hepatic stellate cell activation.

Odontogenic infection of Porphyromonas gingivalis (P.g.), a major periodontal pathogen, exacerbates pathological progression of non-alcoholic steatohepatitis (NASH). In this study, we aimed to clarify the detailed mechanism in which P.g. induced hepatic stellate cells (HSCs; key effector cells in liver fibrosis) activation. In the liver of high fat diet-induced NASH mouse model with P.g. odontogenic infection, immunolocalization of P.g. was detected. The number of hepatic crown-like structure, which was macrophage aggregation and related to liver fibrosis, was drastically increased and fibrosis area was also increased through upregulating immunoexpression of Phosphorylated Smad2 (key signaling molecule of TGF-ß1) and Galectin-3. P.g.-secreted trypsin-like enzyme [gingipain; an activator of protease-activated receptor 2 (PAR2)] stimulated HSC proliferation and differentiation through Smad and ERK signaling induced by TGF-ß1 produced from HSCs with P.g.-infection. Further, Galectin-3 produced from HSCs with P.g. infection and P.g.-derived LPS/lipoprotein stimulation stabilized TGFß-receptor II resulting in increasing sensitivity for TGF-ß1, finally leading to HSC differentiation via activating Smad and ERK signaling. In addition to them, hepatocytes (main component cells of liver) contributed to HSC activation through TGF-ß1 and Galectin-3 production in paracrine manner. Collectively, P.g.-odontogenic infection exacerbates fibrosis of NASH by HSC activation through TGF-ß1 and Gal-3 production from HSCs and hepatocytes.

Brucella abortus Infection Elicited Hepatic Stellate Cell-Mediated Fibrosis Through Inflammasome-Dependent IL-1ß Production.

In human brucellosis, the liver is frequently affected. Brucella abortus triggers a profibrotic response on hepatic stellate cells (HSCs) characterized by inhibition of MMP-9 with concomitant collagen deposition and TGF-ß1 secretion through type 4 secretion system (T4SS). Taking into account that it has been reported that the inflammasome is necessary to induce a fibrotic phenotype in HSC, we hypothesized that Brucella infection might create a microenvironment that would promote inflammasome activation with concomitant profibrogenic phenotype in HSCs. B. abortus infection induces IL-1ß secretion in HSCs in a T4SS-dependent manner. The expression of caspase-1 (Casp-1), absent in melanoma 2 (AIM2), Nod-like receptor (NLR) containing a pyrin domain 3 (NLRP3), and apoptosis-associated speck-like protein containing a CARD (ASC) was increased in B. abortus-infected HSC. When infection experiments were performed in the presence of glyburide, a compound that inhibits NLRP3 inflammasome, or A151, a specific AIM2 inhibitor, the secretion of IL-1ß was significantly inhibited with respect to uninfected controls. The role of inflammasome activation in the induction of a fibrogenic phenotype in HSCs was determined by performing B. abortus infection experiments in the presence of the inhibitors Ac-YVAD-cmk and glyburide. Both inhibitors were able to reverse the effect of B. abortus infection on the fibrotic phenotype in HSCs. Finally, the role of inflammasome in fibrosis was corroborated in vivo by the reduction of fibrotic patches in liver from B. abortus-infected ASC, NLRP, AIM2, and cCasp-1/11 knock-out (KO) mice with respect to infected wild-type mice.

Brucella abortus Promotes a Fibrotic Phenotype in Hepatic Stellate Cells, with Concomitant Activation of the Autophagy Pathway.

The liver is frequently affected in patients with active brucellosis. The present study demonstrates that Brucella abortus infection induces the activation of the autophagic pathway in hepatic stellate cells to create a microenvironment that promotes a profibrogenic phenotype through the induction of transforming growth factor-ß1 (TGF-ß1), collagen deposition, and inhibition of matrix metalloproteinase-9 (MMP-9) secretion. Autophagy was revealed by upregulation of the LC3II/LC3I ratio and Beclin-1 expression as well as inhibition of p62 expression in infected cells. The above-described findings were dependent on the type IV secretion system (VirB) and the secreted BPE005 protein, which were partially corroborated using the pharmacological inhibitors wortmannin, a phosphatidyl inositol 3-kinase inhibitor, and leupeptin plus E64 (inhibitors of lysosomal proteases). Activation of the autophagic pathway in hepatic stellate cells during Brucella infection could have an important contribution to attenuating inflammatory hepatic injury by inducing fibrosis. However, with time, B. abortus infection induced Beclin-1 cleavage with concomitant cleavage of caspase-3, indicating the onset of apoptosis of LX-2 cells, as was confirmed by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay and Hoechst staining. These results demonstrate that the cross talk of LX-2 cells and B. abortus induces autophagy and fibrosis with concomitant apoptosis of LX-2 cells, which may explain some potential mechanisms of liver damage observed in human brucellosis.

The Effector Protein BPE005 from Brucella abortus Induces Collagen Deposition and Matrix Metalloproteinase 9 Downmodulation via Transforming Growth Factor ß1 in Hepatic Stellate Cells.

The liver is frequently affected in patients with active brucellosis. In the present study, we identified a virulence factor involved in the modulation of hepatic stellate cell function and consequent fibrosis during Brucella abortus infection. This study assessed the role of BPE005 protein from B. abortus in the fibrotic phenotype induced on hepatic stellate cells during B. abortus infection in vitro and in vivo. We demonstrated that the fibrotic phenotype induced by B. abortus on hepatic stellate (LX-2) cells was dependent on BPE005, a protein associated with the type IV secretion system (T4SS) VirB from B. abortus. Our results indicated that B. abortus inhibits matrix metalloproteinase 9 (MMP-9) secretion through the activity of the BPE005-secreted protein and induces concomitant collagen deposition by LX-2 cells. BPE005 is a small protein containing a cyclic nucleotide monophosphate binding domain (cNMP) that modulates the LX-2 cell phenotype through a mechanism that is dependent on the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway. Altogether, these results indicate that B. abortus tilts LX-2 cells to a profibrogenic phenotype employing a functional T4SS and the secreted BPE005 protein through a mechanism that involves the cAMP and PKA signaling pathway.

Infection as a Trigger for Portal Hypertension.

BACKGROUND: Microbial infections are a relevant problem for patients with liver cirrhosis. Different types of bacteria are responsible for different kinds of infections: Escherichia coli and Klebsiella pneumoniae are frequently observed in spontaneous bacterial peritonitis or urinary tract infections, and Streptococcus pneumoniae and Mycoplasma pneumoniae in pulmonary infections. Mortality is up to 4-fold higher in infected patients with liver cirrhosis than in patients without infections. Key Messages: Infections in patients with liver cirrhosis are due to three major reasons: bacterial translocation, immune deficiency and an increased incidence of systemic infections. Nonparenchymal liver cells like Kupffer cells, sinusoidal endothelial cells and hepatic stellate cells are the first liver cells to come into contact with microbial products when systemic infection or bacterial translocation occurs. Kupffer cell (KC) activation by Toll-like receptor (TLR) agonists and endothelial sinusoidal dysfunction have been shown to be important mechanisms increasing portal pressure following intraperitoneal lipopolysaccharide pretreatment in cirrhotic rat livers. Reduced intrahepatic vasodilation and increased intrahepatic vasoconstriction are the relevant pathophysiological pathways. Thromboxane A2 and leukotriene (LT) C4/D4 have been identified as important vasoconstrictors. Accordingly, treatment with montelukast to inhibit the cysteinyl-LT1 receptor reduced portal pressure in cirrhotic rat livers. Clinical studies have demonstrated that activation of KCs, estimated by the amount of soluble CD163 in the blood, correlates with the risk for variceal bleeding. Additionally, intestinal decontamination with rifaximin in patients with alcohol-associated liver cirrhosis reduced the portal pressure and the risk for variceal bleeding. CONCLUSIONS: TLR activation of nonparenchymal liver cells by pathogens results in portal hypertension. This might explain the pathophysiologic correlation between microbial infections and portal hypertension in patients with liver cirrhosis. These findings are the basis for both better risk stratifying and new treatment options, such as specific inhibition of TLR for patients with liver cirrhosis and portal hypertension.

Hypoxia-inducible factor-1alpha regulates autophagy to activate hepatic stellate cells.

The role of autophagy in Hif-1α modulated activation of hepatic stellate cells was illustrated in current work. Autophagy markers were determined in livers of Schistosoma japonicum infected mice and hypoxia or LPS treated human hepatic stellate cell, LX-2 cells. The action of Hif-1 to autophagy was defined as increase of autophagy markers was significantly suppressed in Hif-1α siRNA transfected cells upon hypoxia or LPS stimulation. The function of autophagy in activation of LX-2 cells was assessed as increase of activation markers was blocked using autophagy inhibitors under hypoxia and LPS stimulation. Conclusively, Hif-1α regulates activation of hepatic stellate cell by modulating autophagy.