Activation Of Liver Macrophages Research Articles

Myeloid AMPK signaling restricts fibrosis but is not required for metformin improvements during CDAHFD-induced NASH in mice

Metabolic programming underpins inflammation and liver macrophage activation in the setting of chronic liver disease. Here, we sought to identify the role of an important metabolic regulator, AMP-activated protein kinase (AMPK), specifically within myeloid cells during the progression of non-alcoholic steatohepatitis (NASH) and whether treatment with metformin, a firstline therapy for diabetes and activator of AMPK could stem disease progression. Male and female Prkaa1fl/fl/Prkaa2fl/fl (Flox) control and Flox-LysM-Cre+ (MacKO) mice were fed a low-fat control or a choline-deficient, amino acid defined 45% Kcal high-fat diet (CDAHFD) for 8 weeks, where metformin was introduced in the drinking water (50 or 250 mg/kg/day) for the last 4 weeks. Hepatic steatosis and fibrosis were dramatically increased in response to CDAHFD-feeding compared to low-fat control. While myeloid AMPK signaling had no effect on markers of hepatic steatosis or circulating markers, fibrosis as measured by total liver collagen was significantly elevated in livers from MacKO mice, independent of sex. Although treatment with 50 mg/kg/day metformin had no effect on any parameter, intervention with 250 mg/kg/day metformin completely ameliorated hepatic steatosis and fibrosis in both male and female mice. While the protective effect of metformin was associated with lower final body weight, and decreased expression of lipogenic and Col1a1 transcripts, it was independent of myeloid AMPK signaling. These results suggest that endogenous AMPK signaling in myeloid cells, both liver-resident and infiltrating, acts to restrict fibrogenesis during CDAHFD-induced NASH progression but is not the mechanism by which metformin improves markers of NASH.

Isoliquiritigenin limits inflammasome activation of macrophage via docking into Syk to alleviate murine non-alcoholic fatty liver disease.

Isoliquiritigenin (ISL) is a chalcone-type flavonoid derived from the root of licorice with antioxidant, anti-inflammatory, anti-tumour and neuroprotective properties. ISL has been proven to downregulate the productions of IL-1β, TNF-α and IL-6 by macrophages. However, detailed molecular mechanisms of this modulation remain elusive. Here, ISL suppressed Syk phosphorylation and CD80, CD86, IL-1β, TNF-α and IL-6 expressions in lipopolysaccharide-stimulated macrophages exvivo. ApoC3-transgenic (ApoC3TG) mice had more activated macrophages. ISL was also able to downregulate the inflammatory activities of macrophages from ApoC3TG mice. Administration of ISL inhibited Syk activation and inflammatory activities of macrophages in ApoC3TG mice invivo. The treatment of ISL further alleviated MCD-induced non-alcoholic fatty liver disease (NAFLD) in wild-type and ApoC3TG mice, accompanied by less recruitment and activation of liver macrophages. Due to the inhibition of Syk phosphorylation, ISL-treated macrophages displayed less production of cytoplasmic ROS, NLRP3, cleaved-GSDMD and cleaved-IL-1β, suggesting less inflammasome activation. Finally, the molecular docking study demonstrated that ISL bound to Syk directly with the Kd of 1.273 × 10-8 M. When the Syk expression was knocked down by its shRNA, the inhibitory effects of ISL on activated macrophages disappeared, indicating that Syk was at least one of key docking-molecules of ISL. Collectively, ISL could alleviate MCD-induced NAFLD in mice involved with the inhibition of macrophage inflammatory activity by the blockade of Syk-induced inflammasome activation.

Improvement of hepatic innate immunity in chemically-injured livers to develop hepatocarcinoma by a serine type-protease inhibitors enriched extract from Chenopodium quinoa.

Food ingredients have critical effects on the maturation and development of the immune system, which innate - lymphoid (ILCs) and myeloid - cells play key roles as important regulators of energy storage and hepatic fat accumulation. Therefore, the objective of this study is to define potential links between a dietary immunonutritional induction of the selective functional differentiation of monocytes-derived macrophages, ILCs and lipid homeostasis in hepatocarcinoma (HCC)-developing mice. Hepatic chemically injured (diethylnitrosamine/thiacetamide) Rag2-/- and Rag2-/-Il2-/- mice were administered with serine-type protease inhibitors (SETIs) obtained from Chenopodium quinoa. Early HCC-driven immunometabolic imbalances (infiltrated macrophages, glucose homeostasis, hepatic lipid profile, ILCs expansion, inflammatory conditions, microbiota) in animals put under a high-fat diet for 2 weeks were assessed. It was also approached the potential of SETIs to cause functional adaptations of the bioenergetics of human macrophage-like cells (hMLCs) in vitro conditioning their capacity to accumulate fat. It is showed that Rag2-/-Il2-/- mice, lacking ILCs, are resistant to the SETIs-induced hepatic macrophages (CD68+F4/80+) activation. Feeding SETIs to Rag2-/- mice, carrying ILCs, promoted the expansion towards ILC3s (CD117+Nkp46+CD56+) and reduced that of ILC2s (CD117+KLRG1+) into livers. In vitro studies demonstrate that hMLCs, challenged to SETIs, develop a similar phenotype of that found in mice and bioenergetic adaptations leading to increased lipolysis. It is concluded that SETIs promote liver macrophage activation and ILCs adaptations to ameliorate HCC-driven immunometabolic imbalances.

Bcl-6 expression by CD4+ T cells determines concomitant immunity and host resistance across distinct parasitic infections

Cluster of differentiation (CD4+) T cells consist of multiple subtypes, defined by expression of lineage-specific transcription factors, that contribute to the control of infectious diseases by providing help to immune and nonimmune target cells. In the current study, we examined the role of B cell lymphoma (Bcl)-6, a transcriptional repressor and master regulator of T follicular helper cell differentiation, in T cell-mediated host defense against intestinal and systemic parasitic infections. We demonstrate that while Bcl-6 expression by CD4+ T cells is critical for antibody-mediated protective immunity against secondary infection with the nematode Heligmosoides polygyrus bakeri, it paradoxically compromises worm expulsion during primary infection by limiting the generation of interleukin-10 (IL-10)-producing Gata3+ T helper 2 cells. Enhanced worm expulsion in the absence of Bcl-6 expressing T cells was associated with amplified intestinal goblet cell differentiation and increased generation of alternatively activated macrophages, effects that were reversed by neutralization of IL-10 signals. An increase in IL-10 production by Bcl-6-deficient CD4+ T cells was also evident in the context of systemic Leishmania donovani infection, but in contrast to Heligmosoides polygyrus bakeri infection, compromised T helper 1-mediated liver macrophage activation and increased susceptibility to this distinct parasitic challenge. Collectively, our studies suggest that host defense pathways that protect against parasite superinfection and lethal systemic protozoal infections can be engaged at the cost of compromised primary resistance to well-tolerated helminths.

Toll-like receptor 2 activation in monocytes contributes to systemic inflammation and alcohol-associated liver disease in humans.

In the context of gut leakiness and translocation of microbial products in alcohol-associated liver disease (ALD), it is possible that systemic and liver inflammation involve the activation of circulating monocyte through gut-derived factors. We explored the association between monocytes, microbial translocation, systemic inflammation, and ALD. Patients with alcohol use disorder following a rehabilitation program were compared with healthy controls. We determined the circulating number and proportion of monocyte subsets by FACS. The activation of signaling pathways by gut-derived microbes was analyzed by quantitative PCR in isolated monocytes. Cytokines secretion by monocytes and phagocytosis were assessed in vitro. Serum microbial translocation markers and cytokines were measured by ELISA and multiplex assay, respectively. ALD severity and liver inflammatory responses were analyzed in liver biopsies by various methods. In patients with alcohol use disorder, the number of blood monocytes increased compared with controls. Monocytes from patients with alcohol use disorder upregulated IL-1β and IL-8 together with toll-like receptor 2 and downstream AP-1, while fungal sensor CARD9 was downregulated. IL-1β and IL-8 were actively secreted upon stimulation in vitro with the toll-like receptor 2 ligand peptidoglycan. Exposure with Escherichia coli confirmed preserved bacterial phagocytic activity. In contrast, Candida albicans stimulation leads to downregulation of IL-1β and TNFα compared with controls. Systemic cytokines and monocyte changes correlated with microbial translocation. Hepatic IL-1β and IL-8 increased with ALD severity together with liver macrophage activation and upregulation of chemokines involved in monocyte attraction. Our results point to the contribution of activated monocytes to systemic inflammation and ALD. Monocytes likely infiltrate the liver, transform into monocyte-derived macrophages and release IL-1β and IL-8 in response to peptidoglycan and toll-like receptor 2 activation.

CB2R agonist GW405833 alleviates acute liver failure in mice via inhibiting HIF-1α-mediated reprogramming of glycometabolism and macrophage proliferation.

The inflammatory responses involving infiltration and activation of liver macrophages play a vital role in acute liver failure (ALF). In the liver of ALF mice, cannabinoid receptor 2 (CB2R) is significantly upregulated on macrophages, while CB2R agonist GW405833 (GW) could protect against cell death in acute liver damage. In this study, we investigated the molecular mechanisms underlying the protective effects of GW against ALF in vivo and in vitro from a perspective of macrophage glycometabolism. Mice were pretreated with GW (10 mg/kg, i.p.), then were injected with D-GalN (750 mg/kg, i.p.) and LPS (10 mg/kg, i.p.) to induce ALF. We verified the protective effects of GW pretreatment in ALF mice. Furthermore, GW pretreatment significantly reduced liver macrophage infiltration and M1 polarization, and inhibited the release of inflammatory factors TNF-α and IL-1β in ALF mice. These protective effects were eliminated by CB2R antagonist SR144528 or in CB2R-/- ALF mice. We used LPS-stimulated RAW264.7 cells as an in vitro M1 macrophage-centered model of inflammatory response, and demonstrated that pretreatment with GW (10 μM) significantly reduced glucose metabolism by inhibiting glycolysis, which inhibited LPS-induced macrophage proliferation and inflammatory cytokines release. We verified these results in a stable CB2R-/- RAW264.7 cell line. Moreover, we found that GW significantly inhibited the expression of hypoxia inducible factor 1α (HIF-1α). Using a stable HIF-1α-/- RAW264.7 cell line, we confirmed that GW reduced the release of inflammatory cytokines from macrophages and inhibited glycolysis by downregulating HIF-1α expression. In conclusion, activation of CB2Rs inhibits the proliferation of hepatic macrophages and release of inflammatory factors in ALF mice through downregulating HIF-1α to inhibit glycolysis.

Targeting Notch1-YAP Circuit Reprograms Macrophage Polarization and Alleviates Acute Liver Injury in Mice

Hepatic immune system disorder plays a critical role in the pathogenesis of acute liver injury. The intrinsic signaling mechanisms responsible for dampening excessive activation of liver macrophages are not completely understood. The Notch and Hippo-YAP signaling pathways have been implicated in immune homeostasis. In this study, we investigated the interactive cell signaling networks of Notch1/YAP pathway during acute liver injury. Myeloid-specific Notch1 knockout (Notch1M-KO) mice and the floxed Notch1 (Notch1FL/FL) mice were subjected to lipopolysaccharide/D-galactosamine toxicity. Some mice were injected via the tail vein with bone marrow-derived macrophages transfected with lentivirus-expressing YAP. Some mice were injected with YAP siRNA using an invivo mannose-mediated delivery system. We found that the activated Notch1 and YAP signaling in liver macrophages were closely related to lipopolysaccharide/D-galactosamine-induced acute liver injury. Macrophage/neutrophil infiltration, proinflammatory mediators, and hepatocellular apoptosis were markedly ameliorated in Notch1M-KO mice. Importantly, myeloid Notch1 deficiency depressed YAP signaling and facilitated M2 macrophage polarization in the injured liver. Furthermore, YAP overexpression in Notch1M-KO livers exacerbated liver damage and shifted macrophage polarization toward the M1 phenotype. Mechanistically, macrophage Notch1 signaling could transcriptionally activate YAP gene expression. Reciprocally, YAP transcriptionally upregulated the Notch ligand Jagged1 gene expression andwas essential for Notch1-mediated macrophage polarization. Finally, dual inhibition of Notch1 and YAP in macrophages further promoted M2 polarization and alleviated liver damage. Our findings underscore a novel molecular insight into the Notch1-YAP circuit for controlling macrophage polarization in acute liver injury, raising the possibility of targeting macrophage Notch1-YAP circuit as an effective strategy for liver inflammation-related diseases.

Liver Fetuin-A at Initiation of Insulin Resistance

Hepatokines (liver secreted proteins with possible distant action) are emerging potential players in insulin resistance in type 2 diabetic patients. Here, we explored the effect of a high-fat diet on the expression of fetuin-A, one of those candidate liver proteins, and its relationship with liver macrophage activation. Mice were fed a normal diet or a high-fat diet for 3 days, known to initiate steatosis and liver insulin resistance. A preventive liver macrophage depletion was obtained by intravenous injection of clodronate-loaded liposomes. The mRNA and protein expression of fetuin-A was evaluated by qPCR, Western blot and immunofluorescence on different insulin-sensitive tissues (liver, adipose tissue, and muscle). Short-term high-fat diet-induced steatosis, liver macrophage activation, and hepatic insulin resistance together with a significantly increased expression of liver AHSG (α2-HS glycoprotein/fetuin-A) mRNA and serum fetuin-A concentration. On immunofluorescence, fetuin-A was mostly expressed in centrilobular hepatocytes. This increase in fetuin-A under high-fat diet was not evidenced in other peripheral insulin-sensitive tissues (skeletal muscle and adipose tissue). The mRNA expression of α2-HS glycoprotein was 800 times higher within the liver compared with the adipose tissue or the muscle. Liver macrophage depletion that significantly ameliorated insulin sensitivity was associated with a significant decrease in α2-HS glycoprotein mRNA expression. In conclusion, this study demonstrated liver fetuin-A overexpression at the initiation of high-fat diet feeding, concurrent with hepatic steatosis and insulin resistance. Targeting liver macrophages in this setting reduced liver α2-HS glycoprotein expression suggesting that fetuin-A acts as an hepatokine with proinsulin resistance effects.

HAO1 negatively regulates liver macrophage activation via the NF-κB pathway in alcohol-associated liver disease

Inflammation is a key factor contributing to the progression of alcohol-associated liver disease (ALD). Accumulating data have shown that ethyl alcohol (EtOH) induced liver macrophages activation along with an inflammatory response that contributes to the development of ALD. The liver-specific peroxisomal enzyme hydroxyacid oxidase 1 (HAO1) has been found to be associated with chronic liver disease. But the role of HAO1 remains unknown in ALD. In our study, HAO1 was found to be decreased in ALD patients and EtOH-fed mice. Interestingly, HAO1 expression was reduced in primary hepatocytes, whereas HAO1 was elevated in peripheral blood monocytes from ALD patients and EtOH-fed mice liver macrophages as well as LPS-treated RAW264.7 cells. Moreover, HAO1 knockdown exacerbated the inflammatory response, while HAO1 overexpression inhibited inflammation in LPS-stimulated RAW264.7 cells. Additionally, overexpression or silencing of HAO1 in vitro significantly affected NF-κB signaling pathway. Collectively, the results revealed a key role of HAO1-mediated macrophage activation and may provide a potential target for treating ALD.

SerpinB3 as a Pro-Inflammatory Mediator in the Progression of Experimental Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is becoming the most common chronic liver disease worldwide. In 20-30% of patients, NAFLD can progress into non-alcoholic steatohepatitis (NASH), eventually leading to fibrosis, cirrhosis and hepatocellular carcinoma development. SerpinB3 (SB3), a hypoxia-inducible factor-2α dependent cysteine protease inhibitor, is up-regulated in hepatocytes during progressive NAFLD and proposed to contribute to disease progression. In this study we investigated the pro-inflammatory role of SB3 by employing phorbol-myristate acetate-differentiated human THP-1 macrophages exposed in vitro to human recombinant SB3 (hrSB3) along with mice overexpressing SB3 in hepatocytes (TG/SB3) or knockout for SB3 (KO/SB3) in which NASH was induced by feeding methionine/choline deficient (MCD) or a choline-deficient, L-amino acid defined (CDAA) diets. In vivo experiments showed that the induction of NASH in TG/SB3 mice was characterized by an impressive increase of liver infiltrating macrophages that formed crown-like aggregates and by an up-regulation of hepatic transcript levels of pro-inflammatory cytokines. All these parameters and the extent of liver damage were significantly blunted in KO/SB3 mice. In vitro experiments confirmed that hrSB3 stimulated macrophage production of M1-cytokines such as TNFα and IL-1β and reactive oxygen species along with that of TGFβ and VEGF through the activation of the NF-kB transcription factor. The opposite changes in liver macrophage activation observed in TG/SB3 or KO/SB3 mice with NASH were associated with a parallel modulation in the expression of triggering receptor expressed on myeloid cells-2 (TREM2), CD9 and galectin-3 markers, recently detected in NASH-associated macrophages. From these results we propose that SB3, produced by activated/injured hepatocytes, may operate as a pro-inflammatory mediator in NASH contributing to the disease progression.

Fetuin-A in Activated Liver Macrophages Is a Key Feature of Non-Alcoholic Steatohepatitis.

Fetuin-A, a plasma multifunctional protein known to play a role in insulin resistance, is usually presented as a liver secreted protein. However, fetuin-A adipose tissue production has been also described. Here, we evaluated fetuin-A production by the liver and the adipose tissue during metabolic dysfunction-associated fatty liver disease (MAFLD)-non-alcoholic steatohepatitis (NASH) development. Fetuin-A was evaluated by enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), Western blot, and immunofluorescence in male foz−/− mice fed a normal diet (ND) or a high fat diet (HFD) at various timepoints and in MAFLD-NASH patients. Foz−/− mice fed a short-term HFD developed liver steatosis, insulin resistance, and increased circulating levels of fetuin-A compared to ND-fed mice. In mice and patients with NASH, fetuin-A was located not only in healthy or steatotic hepatocytes but also in some macrophages forming lipogranulomas. In both mice and humans, a significant amount of fetuin-A was present in the adipose tissue compared to the liver. However, messenger ribonucleic acid levels and cell culture experiments indicate that fetuin-A is produced by the liver but not by the adipose tissue. In conclusion, fetuin-A is produced by steatotic hepatocytes at early timepoints in MAFLD and correlates with insulin resistance both in mice and humans. In NASH, fetuin-A also co-localizes with activated liver macrophages and could be interpreted as a signal released by damaged hepatocytes.

ARRB1 suppresses the activation of hepatic macrophages via modulating endoplasmic reticulum stress in lipopolysaccharide-induced acute liver injury

Acute liver injury (ALI) caused by multiple inflammatory responses is a monocyte-/macrophage-mediated liver injury that is associated with high morbidity and mortality. Liver macrophage activation is a vital event that triggers ALI. However, the mechanism of liver macrophage activation has not been fully elucidated. This study examined the role of β-arrestin1 (ARRB1) in wild-type (WT) and ARRB1-knockout (ARRB1-KO) mouse models of ALI induced by lipopolysaccharide (LPS), and ARRB1-KO mice exhibited more severe inflammatory injury and liver macrophage activation compared to WT mice. We found that LPS treatment reduced the expression level of ARRB1 in Raw264.7 and THP-1 cell lines, and mouse primary hepatic macrophages. Overexpression of ARRB1 in Raw264.7 and THP-1 cell lines significantly attenuated LPS-induced liver macrophage activation, such as transformation in cell morphology and enhanced expression of proinflammatory cytokines (tumor necrosis factor-α, interleukin-1β, and interleukin-6), while downregulation of ARRB1 by small interfering RNA and ARRB1 deficiency in primary hepatic macrophages both aggravated macrophage activation. Moreover, overexpression of ARRB1 suppressed LPS-induced endoplasmic reticulum (ER) stress in liver macrophages, and inhibition of ER stress impeded excessive hepatic macrophage activation induced by downregulation of ARRB1. Our data demonstrate that ARRB1 relieves LPS-induced ALI through the ER stress pathway to regulate hepatic macrophage activation and that ARRB1 may be a potential therapeutic target for ALI.

Programmed Death Ligand 1 Is Overexpressed in Liver Macrophages in Chronic Liver Diseases, and Its Blockade Improves the Antibacterial Activity Against Infections

Background and AimsBacterial infections are common and severe in cirrhosis, but their pathogenesis is poorly understood. Dysfunction of liver macrophages may play a role, but information about their function in cirrhosis is limited. Our aims were to investigate the specific profile and function of liver macrophages in cirrhosis and their contribution to infections. Macrophages from human cirrhotic livers were characterized phenotypically by transcriptome analysis and flow cytometry; function was assessed in vivo by single photon emission computerized tomography in patients with cirrhosis. Serum levels of specific proteins and expression in peripheral monocytes were determined by ELISA and flow cytometry. In vivo phagocytic activity of liver macrophages was measured by spinning disk intravital microscopy in a mouse model of chronic liver injury.Approach and ResultsLiver macrophages from patients with cirrhosis overexpressed proteins related to immune exhaustion, such as programmed death ligand 1 (PD‐L1), macrophage receptor with collagenous structure (MARCO), and CD163. In vivo phagocytic activity of liver macrophages in patients with cirrhosis was markedly impaired. Monocytes from patients with cirrhosis showed overexpression of PD‐L1 that paralleled disease severity, correlated with its serum levels, and was associated with increased risk of infections. Blockade of PD‐L1 with anti‐PD‐L1 antibody caused a shift in macrophage phenotype toward a less immunosuppressive profile, restored liver macrophage in vivo phagocytic activity, and reduced bacterial dissemination.ConclusionLiver cirrhosis is characterized by a remarkable impairment of phagocytic function of macrophages associated with an immunosuppressive transcriptome profile. The programmed cell death receptor 1/PD‐L1 axis plays a major role in the impaired activity of liver macrophages. PD‐L1 blockade reverses the immune suppressive profile and increases antimicrobial activity of liver macrophages in cirrhosis.

Myeloid Endoplasmic Reticulum Resident Chaperone GP96 Facilitates Inflammation and Steatosis in Alcohol-Associated Liver Disease.

Cellular stress–mediated chaperones are linked to liver macrophage activation and inflammation in alcohol‐associated liver disease (ALD). In this study, we investigate the role of endoplasmic reticulum (ER) resident stress chaperone GP96/HSP90B1/GRP94, paralog of the HSP90 family, in ALD pathogenesis. We hypothesize that ER resident chaperone, heat shock protein GP96, plays a crucial role in alcohol‐associated liver inflammation and contributes to liver injury. We show high expression of GP96/HSP90B1 and GRP78/HSPA5 in human alcohol‐associated hepatitis livers as well as in mouse ALD livers with induction of GP96 prominent in alcohol‐exposed macrophages. Myeloid‐specific GP96 deficient (M‐GP96KO) mice failed to induce alcohol‐associated liver injury. Alcohol‐fed M‐GP96KO mice exhibit significant reduction in steatosis, serum endotoxin, and pro‐inflammatory cytokines compared with wild‐type mice. Anti‐inflammatory cytokines interleukin‐10 and transforming growth factor β, as well as activating transcription factor 3 and triggering receptor expressed on myeloid cells 2, markers of restorative macrophages, were higher in alcohol‐fed M‐GP96KO livers. M‐GP96KO mice exhibit protection in a model of endotoxin‐mediated liver injury in vivo, which is in agreement with reduced inflammatory responses during ex vivo lipopolysaccharide/endotoxin– stimulated bone marrow–derived macrophages from M‐GP96KO mice. Furthermore, we show that liver macrophages from alcohol‐fed M‐GP96KO mice show compensatory induction of GRP78 messenger RNA, likely due to increased splicing of X‐box binding protein‐1. Finally, we show that inhibition of GP96 using a specific pharmacological agent, PU‐WS13 or small interfering RNA, alleviates inflammatory responses in primary macrophages. Conclusion: Myeloid ER resident GP96 promotes alcohol‐induced liver damage through activation of liver macrophage inflammatory responses, alteration in lipid homeostasis, and ER stress. These findings highlight a critical role for liver macrophage ER resident chaperone GP96/HSP90B1 in ALD, and its targeted inhibition represents a promising therapeutic approach in ALD.

Leaky Gut Driven by Dysbiosis Augments Activation and Accumulation of Liver Macrophages via RIP3 Signaling Pathway in Autoimmune Hepatitis.

The gut–liver axis has been increasingly recognized as a major autoimmunity modulator. However, the implications of intestinal barrier in the pathogenesis of autoimmune hepatitis (AIH) remain elusive. Here, we investigated the functional role of gut barrier and intestinal microbiota for hepatic innate immune response in AIH patients and murine models. In this study, we found that AIH patients displayed increased intestinal permeability and pronounced RIP3 activation of liver macrophages. In mice models, intestinal barrier dysfunction increased intestinal bacterial translocation, thus amplifying the hepatic RIP3-mediated innate immune response. Furthermore, GSK872 dampened RIP3 activation and ameliorated the activation and accumulation of liver macrophages in vitro and in vivo experiments. Strikingly, broad-spectrum antibiotic ablation significantly alleviated RIP3 activation and liver injury, highlighting the causal role of intestinal microbiota for disease progression. Our results provided a potentially novel mechanism of immune tolerance breakage in the liver via the gut-liver axis. In addition, we also explored the therapeutic and research potentials of regulating the intestinal microbiota for the therapy of AIH.

Alpha-1 antitrypsin suppresses macrophage activation and promotes islet graft survival after intrahepatic islet transplantation.

Alpha-1 antitrypsin (AAT) has protective functions in animal islet transplantation models. While the therapeutic effect of AAT therapy is currently being tested in clinical trials, we investigated the mechanism of AAT protection in a clinically relevant marginal intrahepatic human islet transplantation model. In recipients receiving islets and AAT, 68.9% (20/29) reached normoglycemia, compared to 35.7% (10/28) in those receiving islets only, at 60days posttransplant (PT). AAT-treated mice had lower serum levels of inflammatory cytokines immediately PT. Reduced M1 macrophages were observed in livers of AAT-treated recipients compared to controls as evidenced by flow cytometry and RNA-seq transcriptional profiling analysis. In vitro AAT suppressed IFN-γ-induced M1 macrophage activation/polarization via suppression of STAT1 phosphorylation and iNOS production. AAT inhibits macrophage activation induced by cytokines or dying islets, and consequently leads to islet cell survival. In a macrophage depletion mouse model, the presence of M1 macrophages in the liver contributed to graft death. AAT, through suppressing macrophage activation, protected transplanted islets from death and dysfunction in the human islet and NOD-SCID mouse model. The protective effect of AAT was confirmed in a major mismatch allogeneic islet transplantation model. Taken together, AAT suppresses liver macrophage activation that contributes to graft survival after transplantation.

NLRP12 negatively regulates EtOH-induced liver macrophage activation via NF-κB pathway and mediates hepatocyte apoptosis in alcoholic liver injury

Alcohol-induced liver injury is characterized by abnormal liver dysfunction and excessive inflammation response. Recent years a wealth of data have been yielded indicating that EtOH (ethyl alcohol)-induced macrophage activation along with liver inflammation plays a dominating role in the progression of alcohol-induced liver injury. Here we found high expression of NLRP12 (Nucleotide-binding oligomerization domain protein 12, which is generally considered to be a negative regulator of inflammatory response) in EtOH-fed mouse liver tissue, primary Kupffer cells and EtOH-induced RAW264.7 cells. Additionally, overexpression of NLRP12 following Ad (adenovirus)-NLRP12-EGFP contributed to the attenuation of steatosis and inflammation in EtOH-fed mice model and EtOH-primed RAW264.7 cells. In parallel, Knockdown of NLRP12 aggravated the inflammatory response in RAW264.7 cells triggered by EtOH. Meanwhile, after administration of overexpression or inhibition of NLRP12 expression in vitro, the expression of phosphorylated protein of NF-kB signaling pathway was significantly affected. After increasing or decreasing the expression of NLRP12 in RAW264.7 cells, AML-12 cells were cultured with the supernatant of RAW264.7 cells stimulated by EtOH, and the percent of apoptosis ratio of AML-12 cells was remarkably altered. The study suggested that reduced inflammatory response induced by NLRP12-mediated inhibition of NF-kB pathway participated in the decrease of hepatocyte apoptosis in alcohol-induced liver injury. Collectively, these findings suggested the significance of NLRP12-mediated macrophage activation in alcohol-induced liver injury.

Negative feedback loop of ERK/CREB/miR-212-3p inhibits HBeAg-induced macrophage activation.

The activation of liver macrophages is closely related to liver injury after HBV infection. Our previous results demonstrated that HBeAg played a key role in inducing macrophage activation. As we all know, miRNAs are involved in the regulation of multiple immune cell functions. Meanwhile, we have shown that miR‐155 positively regulates HBeAg‐induced macrophage activation and accelerates liver injury. Subsequently, based on our previous miRNA sequencing results, we further evaluated the role of miR‐212‐3p called ‘neurimmiR’ in HBeAg‐induced macrophages in this study. First, miR‐212‐3p expression was significantly elevated in HBeAg‐treated macrophages. Meanwhile, we found up‐regulation of miR‐212‐3p significantly decreased the production of cytokines, whereas knockdown of miR‐212‐3p held the opposite effect by gains and losses of function. Mechanically, although MAPK signal pathway, including ERK, JNK and p38, was activated in HBeAg‐induced macrophages, only ERK promoted the expression of miR‐212‐3p via transcription factor CREB, which was able to bind to the promoter of miR‐212‐3p verified by ChIP assay. Moreover, we further indicated that up‐regulated miR‐212‐3p inhibited HBeAg‐induced inflammatory cytokine production through targeting MAPK1. In conclusion, miR‐212‐3p was augmented in HBeAg‐stimulated macrophages via ERK/CREB signal pathway and the elevated miR‐212‐3p suppressed inflammatory cytokine production induced by HBeAg through targeting MAPK1.

The effect of acute intragastric vs. intravenous alcohol administration on inflammation markers, blood lipids and gallbladder motility in healthy men.

Ethanol intake increases plasma concentrations of triglycerides and chronic ethanol use impairs lipid metabolism and causes chronic inflammation. The gut plays an important role in metabolic handling of nutrients, including lipids, and a leaky gut associated with alcohol intake, allowing inflammatory signals to the portal vein, has been proposed to constitute a mechanism by which ethanol induces hepatic inflammation. We compared the effects of enteral and parenteral administration of ethanol on a range of circulating inflammation markers (including soluble CD163, a marker of liver macrophage activation), lipids, cholecystokinin (CCK) and fibroblast growth factor 19 (FGF19) as well as gallbladder volume. On two separate and randomized study days, we subjected healthy men (n=12) to double-blinded intragastric ethanol infusion (IGEI) and isoethanolemic intravenous ethanol infusion (IVEI). Blood was sampled and ultrasonographic evaluation of gallbladder volume was performed at frequent intervals for 4h after initiation of ethanol administration on both days. Little or no effects were observed on plasma levels of inflammation markers during IGEI and IVEI, respectively. Circulating levels of total, low-density lipoprotein and high-density lipoprotein cholesterol decreased after ethanol administration independently of the administration form. Triglyceride and very low-density lipoprotein (VLDL) cholesterol concentrations increased more after IGEI compared to IVEI. IVEI had no effect on plasma CCK and caused an increased gallbladder volume whereas IGEI elicited a CCK response (P<0.0001) without affecting gallbladder volume. Circulating FGF19 concentrations decreased equally in response to both ethanol administration forms. In conclusion, by evaluating a range of circulating inflammation markers during IGEI and IVEI we were not able to detect signs of systemic low-grade inflammation originating from the presence of ethanol in the gut. IVEI increased gallbladder volume whereas IGEI increased plasma CCK (with neutral effect on gallbladder volume), increased plasma VLDL cholesterol and triglyceride concentrations; indicating that the enteral route of administration may influence ethanol's effects on lipid metabolism.

Serum soluble sialic acid-binding immunoglobulin-like lectin-7 concentration as an indicator of liver macrophage activation and advanced fibrosis in patients with non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is a leading cause of liver disease worldwide. Because liver fibrosis is associated with the long-term prognosis of patients with NAFLD, there is an urgent need for non-invasive markers of liver fibrosis. Sialic acid-binding immunoglobulin-like lectin-7 (Siglec-7) is an immunomodulatory molecule expressed on various immune cells, including macrophages, which plays a key role in liver inflammation and fibrosis in NAFLD. We aimed to determine whether serum levels of soluble Siglec-7 (sSiglec-7) could have utility at a marker of fibrosis in this patient population. We examined serum samples from 93 NAFLD patients and 19 healthy donors for macrophage-associated protein, including sSiglec-7, soluble CD163, and YKL-40, and examined their correlation with liver fibrosis scores, tissue elastography, and histological findings. Independent factors associated with advanced fibrosis were analyzed using a logistic regression model and a decision tree. To clarify the source of sSiglec-7, we examined its expression in liver tissue-derived macrophages and cultured monocyte-derived macrophages. Serum sSiglec-7 levels were significantly higher in NAFLD patients compared with healthy donors, and correlated positively with sCD163 and YKL-40 levels. Serum sSiglec-7 was an independent diagnostic marker with high specificity (96.3%) for advanced fibrosis (F3 and F4) in NAFLD patients. Siglec-7 was mainly expressed on CCR2+ macrophages in the liver, and sSiglec-7 production by monocyte-derived macrophages invitro was increased after stimulation by pro-inflammatory factors. Elevated serum sSiglec-7 could serve as an independent marker with high specificity for advanced liver fibrosis in patients with NAFLD.