MCP-Induced Protein 1 Participates in Macrophage-Dependent Endotoxin Tolerance.

Endotoxin tolerance is a state of hyporesponsiveness to LPS, triggered by previous exposure to endotoxin. Such an immunosuppressive state enhances the risks of secondary infection and has been associated with the pathophysiology of sepsis. Although this phenomenon has been extensively studied, its molecular mechanism is not fully explained. Among candidates that play a crucial role in this process are negative regulators of TLR signaling, but the contribution of MCP-induced protein 1 (MCPIP1; Regnase-1) has not been studied yet. To examine whether macrophage expression of MCPIP1 participates in endotoxin tolerance, we used both murine and human primary macrophages devoid of MCPIP1 expression. In our study, we demonstrated that MCPIP1 contributes to LPS hyporesponsiveness induced by subsequent LPS stimulation and macrophage reprogramming. We proved that this mechanism revolves around the deubiquitinase activity of MCPIP1, which inhibits the phosphorylation of MAPK and NF-κB activation. Moreover, we showed that MCPIP1 controlled the level of proinflammatory transcripts in LPS-tolerized cells independently of its RNase activity. Finally, we confirmed these findings applying an in vivo endotoxin tolerance model in wild-type and myeloid MCPIP1-deficient mice. Taken together, this study describes for the first time, to our knowledge, that myeloid MCPIP1 participates in endotoxin tolerance and broadens the scope of known negative regulators of the TLR4 pathway crucial in this phenomenon.

Uremic Toxin Indoxyl Sulfate Promotes Macrophage-Associated Low-Grade Inflammation and Epithelial Cell Senescence.

In this study, we investigated the impact of the uremic toxin indoxyl sulfate on macrophages and tubular epithelial cells and its role in modulating the response to lipopolysaccharide (LPS). Indoxyl sulfate accumulates in the blood of patients with chronic kidney disease (CKD) and is a predictor of overall and cardiovascular morbidity/mortality. To simulate the uremic condition, primary macrophages and tubular epithelial cells were incubated with indoxyl sulfate at low concentrations as well as concentrations found in uremic patients, both alone and upon LPS challenge. The results showed that indoxyl sulfate alone induced the release of reactive oxygen species and low-grade inflammation in macrophages. Moreover, combined with LPS (proinflammatory conditions), indoxyl sulfate significantly increased TNF-α, CCL2, and IL-10 release but did not significantly affect the polarization of macrophages. Pre-treatment with indoxyl sulfate following LPS challenge induced the expression of aryl hydrocarbon receptor (Ahr) and NADPH oxidase 4 (Nox4) which generate reactive oxygen species (ROS). Further, experiments with tubular epithelial cells revealed that indoxyl sulfate might induce senescence in parenchymal cells and therefore participate in the progression of inflammaging. In conclusion, this study provides evidence that indoxyl sulfate provokes low-grade inflammation, modulates macrophage function, and enhances the inflammatory response associated with LPS. Finally, indoxyl sulfate signaling contributes to the senescence of tubular epithelial cells during injury.

Antiviral activity of temporin-1CEb analogues against gingival infection with herpes simplex virus type 1.

Introduction: Oral herpes infections caused by herpes simplex virus type 1 (HSV-1) are one of the most common in the human population. Recently, they have been classified as an increasing problem in immunocompromised patients and those suffering from chronic inflammation of the oral mucosa and gums. Treatment mainly involves nucleoside analogues, such as acyclovir and its derivatives, which reduce virus replication and shedding. As drug-resistant strains of herpes emerge rapidly, there is a need for the development of novel anti-herpes agents. The aim of the study was to design an antiviral peptide, based on natural compounds, non-toxic to the host, and efficient against drug-resistant HSV-1. Here, we designed a lysine-rich derivative of amphibian temporin-1CEb conjugated to peptides penetrating the host cell membrane and examined their activity against HSV-1 infection of oral mucosa. Methods: We assessed the antiviral efficiency of the tested compound in simple 2D cell models (VeroE6 and TIGKs cells) and a 3D organotypic model of human gingiva (OTG) using titration assay, qPCR, and confocal imaging. To identify the molecular mechanism of antiviral activity, we applied the Azure A metachromatic test, and attachment assays techniques. Toxicity of the conjugates was examined using XTT and LDH assays. Results: Our results showed that temporin-1CEb analogues significantly reduce viral replication in oral mucosa. The mechanism of peptide analogues is based on the interaction with heparan sulfate, leading to the reduce attachment of HSV-1 to the cell membrane. Moreover, temporin-1CEb conjugates effectively penetrate the gingival tissue being effective against acyclovir-resistant strains. Collectively, we showed that temporin-1CEb can be regarded as a novel, naturally derived antiviral compound for HSV-1 treatment.

Macrophage-Specific MCPIP1/Regnase-1 Attenuates Kidney Ischemia-Reperfusion Injury by Shaping the Local Inflammatory Response and Tissue Regeneration.

Sterile inflammation either resolves the initial insult or leads to tissue damage. Kidney ischemia/reperfusion injury (IRI) is associated with neutrophilic infiltration, enhanced production of inflammatory mediators, accumulation of necrotic cells and tissue remodeling. Macrophage-dependent microenvironmental changes orchestrate many features of the immune response and tissue regeneration. The activation status of macrophages is influenced by extracellular signals, the duration and intensity of the stimulation, as well as various regulatory molecules. The role of macrophage-derived monocyte chemoattractant protein-induced protein 1 (MCPIP1), also known as Regnase-1, in kidney ischemia-reperfusion injury (IRI) and recovery from sterile inflammation remains unresolved. In this study, we showed that macrophage-specific Mcpip1 deletion significantly affects the kidney phenotype. Macrophage-specific Mcpip1 transgenic mice displayed enhanced inflammation and loss of the tubular compartment upon IRI. We showed that MCPIP1 modulates sterile inflammation by negative regulation of Irf4 expression and accumulation of IRF4+ cells in the tissue and, consequently, suppresses the post-ischemic kidney immune response. Thus, we identified MCPIP1 as an important molecular sentinel of immune homeostasis in experimental acute kidney injury (AKI) and renal fibrosis.

MCPIP-1 Restricts Inflammation via Promoting Apoptosis of Neutrophils.

Monocyte chemoattractant protein-induced protein-1 (MCPIP-1) is a potent inhibitor of inflammatory response to pathogens. Acting as endonuclease against transcripts of inflammatory cytokines or transcription factors MCPIP-1 can significantly reduce the cytokine storm, thus limiting the tissue damage. As the adequate resolution of inflammation depends also on the efficient clearance of accumulated neutrophils, we focused on the role of MCPIP-1 in apoptosis and retention of neutrophils. We used peritoneal neutrophils from cell-specific MCPIP-1 knockout mice and showed prolonged survival of these cells. Moreover, we confirmed that MCPIP-1-dependent degradation of transcripts of antiapoptotic genes, including BCL3, BCL2A1, BCL2L1, and for the first time MCL-1, serves as an early event in spontaneous apoptosis of primary neutrophils. Additionally, we identified previously unknown miRNAs as potential binding partners to the MCPIP-1 transcript and their regulation suggest a role in MCPIP-1 half-life and translation. These phenomena may play a role as a molecular switch that balances the MCPIP-1-dependent apoptosis. Besides that, we determined these particular miRNAs as integral components of the GM-CSF-MCPIP-1 axis. Taken together, we identified the novel anti-inflammatory role of MCPIP-1 as a regulator of accumulation and survival of neutrophils that simultaneously promotes an adequate resolution of inflammation.

Murine myeloid cell MCPIP1 suppresses autoimmunity by regulating B-cell expansion and differentiation.

Myeloid-derived cells, in particular macrophages, are increasingly recognized as critical regulators of the balance of immunity and tolerance. However, whether they initiate autoimmune disease or perpetuate disease progression in terms of epiphenomena remains undefined.Here, we show that depletion of MCPIP1 in macrophages and granulocytes (Mcpip1fl/fl-LysMcre+ C57BL/6 mice) is sufficient to trigger severe autoimmune disease. This was evidenced by the expansion of B cells and plasma cells and spontaneous production of autoantibodies, including anti-dsDNA, anti-Smith and anti-histone antibodies. Consequently, we document evidence of severe skin inflammation, pneumonitis and histopathologic evidence of glomerular IgG deposits alongside mesangioproliferative nephritis in 6-month-old mice. These phenomena are related to systemic autoinflammation, which secondarily induces a set of cytokines such as Baff, Il5, Il9 and Cd40L, affecting adaptive immune responses. Therefore, abnormal macrophage activation is a key factor involved in the loss of immune tolerance.Overall, we demonstrate that deficiency of MCPIP1 solely in myeloid cells triggers systemic lupus-like autoimmunity and that the control of myeloid cell activation is a crucial checkpoint in the development of systemic autoimmunity.

Deletion of Mcpip1 in Mcpip1fl/flAlbCre mice recapitulates the phenotype of human primary biliary cholangitis.

Primary biliary cholangitis (PBC) is an autoimmune disease characterized by progressive destruction of the intrahepatic bile ducts. The immunopathology of PBC involves excessive inflammation; therefore, negative regulators of inflammatory response, such as Monocyte Chemoattractant Protein-1-Induced Protein-1 (MCPIP1) may play important roles in the development of PBC. The aim of this work was to verify whether Mcpip1 expression protects against development of PBC. Genetic deletion of Zc3h12a was used to characterize the role of Mcpip1 in the pathogenesis of PBC in 6-52-week-old mice. We found that Mcpip1 deficiency in the liver (Mcpip1fl/flAlbCre) recapitulates most of the features of human PBC, in contrast to mice with Mcpip1 deficiency in myeloid cells (Mcpip1fl/flLysMCre mice), which present with robust myeloid cell-driven systemic inflammation. In Mcpip1fl/flAlbCre livers, intrahepatic bile ducts displayed proliferative changes with inflammatory infiltration, bile duct destruction, and fibrosis leading to cholestasis. In plasma, increased concentrations of IgG, IgM, and AMA autoantibodies (anti-PDC-E2) were detected. Interestingly, the phenotype of Mcpip1fl/flAlbCre mice was robust in 6-week-old, but milder in 12-24-week-old mice. Hepatic transcriptome analysis of 6-week-old and 24-week-old Mcpip1fl/flAlbCre mice showed 812 and 8 differentially expressed genes, respectively, compared with age-matched control mice, and revealed a distinct set of genes compared to those previously associated with development of PBC. In conclusion, Mcpip1fl/flAlbCre mice display early postnatal phenotype that recapitulates most of the features of human PBC.