Inhibition of Triggering Receptor Expressed on Myeloid Cells 1 Ameliorates Inflammation and Macrophage and Neutrophil Activation in Alcoholic Liver Disease in Mice

Alcoholic liver disease (ALD) is characterized by macrophage and neutrophil leukocyte recruitment and activation in the liver. Damage- and pathogen-associated molecular patterns contribute to a self-perpetuating proinflammatory state in ALD. Triggering receptor expressed on myeloid cells 1 (TREM-1) is a surface receptor that amplifies inflammation induced by toll-like receptors (TLRs) and is expressed on neutrophils and monocytes/macrophages. We hypothesized that TREM-1 signaling contributes to proinflammatory pathway activation in ALD. Using an in vivo ALD model in mice, we tested the effects of ligand-independent TREM-1 inhibitory peptides that were formulated into human high-density lipoprotein (HDL)-mimicking complexes GF9-HDL and GA/E31-HDL. As revealed in vitro, macrophages endocytosed these rationally designed complexes through scavenger receptors. A 5-week alcohol feeding with the Lieber-DeCarli diet in mice resulted in increased serum alanine aminotransferase (ALT), liver steatosis, and increased proinflammatory cytokines in the liver. TREM-1 messenger RNA (mRNA) expression was significantly increased in alcohol-fed mice, and TREM-1 inhibitors significantly reduced this increase. TREM-1 inhibition significantly attenuated alcohol-induced spleen tyrosine kinase (SYK) activation, an early event in both TLR4 and TREM-1 signaling. The TREM-1 inhibitors significantly inhibited macrophage (epidermal growth factor-like module-containing mucin-like hormone receptor-like 1 [F4/80], clusters of differentiation [CD]68) and neutrophil (lymphocyte antigen 6 complex, locus G [Ly6G] and myeloperoxidase [MPO]) markers and proinflammatory cytokines (monocyte chemoattractant protein 1 [MCP-1], tumor necrosis factor alpha [TNF-alpha], interleukin-1beta [IL-1beta], macrophage inflammatory protein 1alpha [MIP-1alpha]) at the mRNA level compared to the HDL vehicle. Administration of TREM-1 inhibitors ameliorated liver steatosis and early fibrosis markers (alpha-smooth muscle actin [alphaSMA] and procollagen1alpha [Pro-Col1alpha]) at the mRNA level in alcohol-fed mice. However, the HDL vehicle also reduced serum ALT and some cytokine protein levels in alcohol-fed mice, indicating HDL-related effects. Conclusion: HDL-delivered novel TREM-1 peptide inhibitors ameliorate early phases of inflammation and neutrophil and macrophage recruitment and activation in the liver and attenuate hepatocyte damage and liver steatosis. TREM-1 inhibition represents a promising therapeutic approach for further investigations in ALD

Chronic alcohol-induced neuroinflammation involves CCR2/5-dependent peripheral macrophage infiltration and microglia alterations

BACKGROUND: Chronic alcohol consumption is associated with neuroinflammation, neuronal damage, and behavioral alterations including addiction. Alcohol-induced neuroinflammation is characterized by increased expression of proinflammatory cytokines (including TNFalpha, IL-1beta, and CCL2) and microglial activation. We hypothesized chronic alcohol consumption results in peripheral immune cell infiltration to the CNS. Since chemotaxis through the CCL2-CCR2 signaling axis is critical for macrophage recruitment peripherally and centrally, we further hypothesized that blockade of CCL2 signaling using the dual CCR2/5 inhibitor cenicriviroc (CVC) would prevent alcohol-induced CNS infiltration of peripheral macrophages and alter the neuroinflammatory state in the brain after chronic alcohol consumption. METHODS: C57BL/6J female mice were fed an isocaloric or 5% (v/v) ethanol Lieber DeCarli diet for 6 weeks. Some mice received daily injections of CVC. Microglia and infiltrating macrophages were characterized and quantified by flow cytometry and visualized using CX3CR1(eGFP/+) CCR2(RFP/+) reporter mice. The effect of ethanol and CVC treatment on the expression of inflammatory genes was evaluated in various regions of the brain, using a Nanostring nCounter inflammation panel. Microglia activation was analyzed by immunofluorescence. CVC-treated and untreated mice were presented with the two-bottle choice test. RESULTS: Chronic alcohol consumption induced microglia activation and peripheral macrophage infiltration in the CNS, particularly in the hippocampus. Treatment with CVC abrogated ethanol-induced recruitment of peripheral macrophages and partially reversed microglia activation. Furthermore, the expression of proinflammatory markers was upregulated by chronic alcohol consumption in various regions of the brain, including the cortex, hippocampus, and cerebellum. Inhibition of CCR2/5 decreased alcohol-mediated expression of inflammatory markers. Finally, microglia function was impaired by chronic alcohol consumption and restored by CVC treatment. CVC treatment did not change the ethanol consumption or preference of mice in the two-bottle choice test. CONCLUSIONS: Together, our data establish that chronic alcohol consumption promotes the recruitment of peripheral macrophages into the CNS and microglia alterations through the CCR2/5 axis. Therefore, further exploration of the CCR2/5 axis as a modulator of neuroinflammation may offer a potential therapeutic approach for the treatment of alcohol-associated neuroinflammation

Extracellular vesicle isolation: present and future

Comment on: A novel affinity-based method for the isolation of highly purified extracellular vesicles. [Sci Rep. 2016

Exploring Differential Connexin Expression across Melanocytic Tumor Progression Involving the Tumor Microenvironment

The incidence of malignant melanoma, one of the deadliest cancers, continues to increase. Here we tested connexin (Cx) expression in primary melanocytes, melanoma cell lines and in a common nevus, dysplastic nevus, and thin, thick, and metastatic melanoma tumor progression series involving the tumor microenvironment by utilizing in silico analysis, qRT-PCR, immunocyto-/histochemistry and dye transfer tests. Primary melanocytes expressed GJA1/Cx43, GJA3/Cx46 and low levels of GJB2/Cx26 and GJC3/Cx30.2 transcripts. In silico data revealed downregulation of GJA1/Cx43 and GJB2/Cx26 mRNA, in addition to upregulated GJB1/Cx32, during melanoma progression. In three melanoma cell lines, we also showed the loss of GJA1/Cx43 and the differential expression of GJB1/Cx32, GJB2/Cx26, GJA3/Cx46 and GJC3/Cx30.2. The dominantly paranuclear localization of connexin proteins explained the ~10⁻90 times less melanoma cell coupling compared to melanocytes. In melanocytic tumor tissues, we confirmed the loss of Cx43 protein, fall of cell membrane and elevated paranuclear Cx32 with moderately increased cytoplasmic Cx26 and paranuclear Cx30.2 positivity during tumor progression. Furthermore, we found Cx43, Cx26 and Cx30 proteins upregulated in the melanoma adjacent epidermis, and Cx43 in the tumor flanking vessels. Therefore, differential connexin expression is involved in melanocytic tumor progression where varying connexin isotypes and levels reflect tumor heterogeneity-related bidirectional adaptive interactions with the microenvironment

Alcohol-induced miR-155 and HDAC11 inhibit negative regulators of the TLR4 pathway and lead to increased LPS responsiveness of Kupffer cells in alcoholic liver disease

Inflammation promotes the progression of alcoholic liver disease. Alcohol sensitizes KCs to gut-derived endotoxin (LPS); however, signaling pathways that perpetuate inflammation in alcoholic liver disease are only partially understood. We found that chronic alcohol feeding in mice induced miR-155, an inflammatory miRNA in isolated KCs. We hypothesized that miR-155 might increase the responsiveness of KCs to LPS via targeting the negative regulators of LPS signaling. Our results revealed that KCs that were isolated from alcohol-fed mice showed a decrease in IRAK-M, SHIP1, and PU.1, and an increase in TNF-alpha levels. This was specific to KCs, as no significant differences were observed in these genes in hepatocytes. We found a causal effect of miR-155 deficiency on LPS responsiveness, as KCs that were isolated from miR-155 KO mice showed a greater induction of IRAK-M, SHIP1, and suppressor of cytokine signaling 1 after LPS treatment. C/EBPbeta, a validated miR-155 target, stimulates IL-10 transcription. We found a higher induction of C/EBPbeta and IL-10 in KCs that were isolated from miR-155 KO mice after LPS treatment. Gain- and loss-of-function studies affirmed that alcohol-induced miR-155 directly regulates IRAK-M, SHIP1, suppressor of cytokine signaling 1, and C/EBPbeta, as miR-155 inhibition increased and miR-155 overexpression decreased these genes in LPS or alcohol-pretreated wild-type KCs. HDAC11, a regulator of IL-10, was significantly increased and IL-10 was decreased in KCs that were isolated from alcohol-fed mice. Functionally, knockdown of HDAC11 with small interfering RNA resulted in an IL-10 increase in LPS or alcohol-pretreated Mvarphi. We found that acetaldehyde and NF-kappaB pathways regulate HDAC11 levels. Collectively, our results indicate that the alcohol-induced responsiveness of KCs to LPS, in part, is governed by miR-155 and HDAC11

Abnormal neutrophil traps and impaired efferocytosis contribute to liver injury and sepsis severity after binge alcohol use

International audienceBACKGROUND & AIMS: Neutrophil extracellular traps (NETs) are an important strategy utilized by neutrophils to immobilize and kill invading microorganisms. Here we studied NET formation and its clearance by macrophages (MØ) (efferocytosis) in acute sepsis following binge drinking.METHODS: Healthy volunteers consumed 2 mL of vodka/kg body weight and blood endotoxin and 16s rDNA was measured. Peripheral neutrophils were isolated and exposed to alcohol followed by phorbol 12-myristate 13-acetate (PMA) stimulation. Mice were treated with three alcohol binges and i.p. LPS to assess the dynamics of NET formation and efferocytosis. In vivo, anti-Ly6G antibody (IA8) was used for neutrophil depletion.RESULTS: Inducers of NETs (endotoxin and bacterial DNA) significantly increased in the circulation after binge alcohol drinking in humans. Ex vivo, alcohol alone increased NET formation but attenuated NET formation upon PMA stimulation. Binge alcohol in mice in vivo resulted in a biphasic response to LPS. Initially, binge alcohol reduced LPS-induced NET formation and resulted in a diffuse distribution of neutrophils in the liver compared to alcohol-naïve mice. Moreover, indicators of NET formation including citrullinated histone H3, neutrophil elastase, and neutrophil myeloperoxidase were decreased at an early time point after LPS challenge in mice with alcohol binge suggesting decreased NET formation. However, in the efferocytosis phase (15 h after LPS) citrullinated histone-H3 was increased in the liver in alcohol binge mice, suggesting decreased clearance of NETs. In vitro alcohol treatment reduced efferocytosis and phagocytosis of NETosing neutrophils and promoted expression of CD206 on MØ. Finally, depletion of neutrophils prior to binge alcohol ameliorated LPS-induced systemic inflammation and liver injury in mice.CONCLUSIONS: Dysfunctional neutrophil NETosis and efferocytosis after binge drinking exacerbates liver injury associated with sepsis.LAY ABSTRACT: Disease severity in alcoholic liver disease (ALD) is associated with significant liver neutrophil presence. It remains unknown how alcohol affects the capacity of neutrophils to control infection, a major hallmark of ALD. We found that binge alcohol drinking impaired important strategies used by neutrophils to contain and resolve infection resulting in increased liver injury during ALD

Extracellular vesicles from mice with alcoholic liver disease carry a distinct protein cargo and induce macrophage activation through heat shock protein 90

A salient feature of alcoholic liver disease (ALD) is Kupffer cell (KC) activation and recruitment of inflammatory monocytes and macrophages (MOs). These key cellular events of ALD pathogenesis may be mediated by extracellular vesicles (EVs). EVs transfer biomaterials, including proteins and microRNAs, and have recently emerged as important effectors of intercellular communication. We hypothesized that circulating EVs from mice with ALD have a protein cargo characteristic of the disease and mediate biological effects by activating immune cells. The total number of circulating EVs was increased in mice with ALD compared to pair-fed controls. Mass spectrometric analysis of circulating EVs revealed a distinct signature for proteins involved in inflammatory responses, cellular development, and cellular movement between ALD EVs and control EVs. We also identified uniquely important proteins in ALD EVs that were not present in control EVs. When ALD EVs were injected intravenously into alcohol-naive mice, we found evidence of uptake of ALD EVs in recipient livers in hepatocytes and MOs. Hepatocytes isolated from mice after transfer of ALD EVs, but not control EVs, showed increased monocyte chemoattractant protein 1 mRNA and protein expression, suggesting a biological effect of ALD EVs. Compared to control EV recipient mice, ALD EV recipient mice had increased numbers of F4/80(hi) cluster of differentiation 11b (CD11b)(lo) KCs and increased percentages of tumor necrosis factor alpha-positive/interleukin 12/23-positive (inflammatory/M1) KCs and infiltrating monocytes (F4/80(int) CD11b(hi) ), while the percentage of CD206(+) CD163(+) (anti-inflammatory/M2) KCs was decreased. In vitro, ALD EVs increased tumor necrosis factor alpha and interleukin-1beta production in MOs and reduced CD163 and CD206 expression. We identified heat shock protein 90 in ALD EVs as the mediator of ALD-EV-induced MO activation. CONCLUSION: Our study indicates a specific protein signature of ALD EVs and demonstrates a functional role of circulating EVs containing heat shock protein 90 in mediating KC/MO activation in the liver

The relevance of dawsonite precipitation in CO2 sequestration in the Mihalyi-Repcelak area, NW Hungary

Raman spectra of dawsonite, tabl

The relevance of dawsonite precipitation in CO2 sequestration in the Mihalyi-Repcelak area, NW Hungary

Raman spectra of dawsonit