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.

Proteolytic Activity-Independent Activation of the Immune Response by Gingipains from Porphyromonas gingivalis.

Porphyromonas gingivalis, a keystone pathogen in periodontitis (PD), produces cysteine proteases named gingipains (RgpA, RgpB, and Kgp), which strongly affect the host immune system. The range of action of gingipains is extended by their release as components of outer membrane vesicles, which efficiently diffuse into surrounding gingival tissues. However, away from the anaerobic environment of periodontal pockets, increased oxygen levels lead to oxidation of the catalytic cysteine residues of gingipains, inactivating their proteolytic activity. In this context, the influence of catalytically inactive gingipains on periodontal tissues is of significant interest. Here, we show that proteolytically inactive RgpA induced a proinflammatory response in both gingival keratinocytes and dendritic cells. Inactive RgpA is bound to the cell surface of gingival keratinocytes in the region of lipid rafts, and using affinity chromatography, we identified RgpA-interacting proteins, including epidermal growth factor receptor (EGFR). Next, we showed that EGFR interaction with inactive RgpA stimulated the expression of inflammatory cytokines. The response was mediated via the EGFR-phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway, which when activated in the gingival tissue rich in dendritic cells in the proximity of the alveolar bone, may significantly contribute to bone resorption and the progress of PD. Taken together, these findings broaden our understanding of the biological role of gingipains, which in acting as proinflammatory factors in the gingival tissue, create a favorable milieu for the growth of inflammophilic pathobionts. IMPORTANCE Gingipain cysteine proteases are essential virulence factors of Porphyromonas gingivalis, an oral bacterium implicated in development of periodontitis. Gingipains diffusing from anaerobic periodontal pockets lose proteolytic activity in the oxygenated environment of gingival tissues. We found that despite the loss of activity, gingipains still elicit a strong inflammatory response, which may contribute to the progression of periodontitis and bone resorption. Moreover, we identified the host molecules utilized by the pathogen as receptors for proteolytically inactivated gingipains. The broad distribution of those receptors in human tissue suggests their involvement in systemic diseases associated with periodontal pathogens.

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.

Subversion of Lipopolysaccharide Signaling in Gingival Keratinocytes via MCPIP-1 Degradation as a Novel Pathogenic Strategy of Inflammophilic Pathobionts.

Periodontal disease (PD) is an inflammatory disease of the supporting tissues of the teeth that develops in response to formation of a dysbiotic biofilm on the subgingival tooth surface. Although exacerbated inflammation leads to alveolar bone destruction and may cause tooth loss, the molecular basis of PD initiation and progression remains elusive. Control over the inflammatory reaction and return to homeostasis can be efficiently restored by negative regulators of Toll-like receptor (TLR) signaling pathways such as monocyte chemoattractant protein-induced protein 1 (MCPIP-1), which is constitutively expressed in gingival keratinocytes and prevents hyperresponsiveness in the gingiva. Here, we found that inflammophilic periodontal species influence the stability of MCPIP-1, leading to an aggravated response of the epithelium to proinflammatory stimulation. Among enzymes secreted by periodontal species, gingipains-cysteine proteases from Porphyromonas gingivalis-are considered major contributors to the pathogenic potential of bacteria, strongly influencing the components of the innate and adaptive immune system. Gingipain proteolytic activity leads to a rapid degradation of MCPIP-1, exacerbating the inflammatory response induced by endotoxin. Collectively, these results establish a novel mechanism of corruption of inflammatory signaling by periodontal pathogens, indicating new possibilities for treatment of this chronic disease. IMPORTANCE Periodontitis is a highly prevalent disease caused by accumulation of a bacterial biofilm. Periodontal pathogens use a number of virulence strategies that are under intensive study to find optimal therapeutic approaches against bone loss. In our work, we present a novel mechanism utilized by the key periodontal pathogen Porphyromonas gingivalis, based on the selective degradation of the negative regulator of inflammation, MCPIP-1. We found that the diminished levels of MCPIP-1 in gingival keratinocytes-cells at the forefront of the fight against bacteria-cause sensitization to endotoxins produced by other oral species. This results in an enhanced inflammatory response, which promotes the growth of inflammophilic pathobionts and damage of tooth-supporting tissues. Our observation is relevant to understanding the molecular basis of periodontitis and the development of new methods for treatment.

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.

Triggering NETosis via protease-activated receptor (PAR)-2 signaling as a mechanism of hijacking neutrophils function for pathogen benefits.

Neutrophil-derived networks of DNA-composed extracellular fibers covered with antimicrobial molecules, referred to as neutrophil extracellular traps (NETs), are recognized as a physiological microbicidal mechanism of innate immunity. The formation of NETs is also classified as a model of a cell death called NETosis. Despite intensive research on the NETs formation in response to pathogens, the role of specific bacteria-derived virulence factors in this process, although postulated, is still poorly understood. The aim of our study was to determine the role of gingipains, cysteine proteases responsible for the virulence of P. gingivalis, on the NETosis process induced by this major periodontopathogen. We showed that NETosis triggered by P. gingivalis is gingipain dependent since in the stark contrast to the wild-type strain (W83) the gingipain-null mutant strain only slightly induced the NETs formation. Furthermore, the direct effect of proteases on NETosis was documented using purified gingipains. Notably, the induction of NETosis was dependent on the catalytic activity of gingipains, since proteolytically inactive forms of enzymes showed reduced ability to trigger the NETs formation. Mechanistically, gingipain-induced NETosis was dependent on proteolytic activation of protease-activated receptor-2 (PAR-2). Intriguingly, both P. gingivalis and purified Arg-specific gingipains (Rgp) induced NETs that not only lacked bactericidal activity but instead stimulated the growth of bacteria species otherwise susceptible to killing in NETs. This protection was executed by proteolysis of bactericidal components of NETs. Taken together, gingipains play a dual role in NETosis: they are the potent direct inducers of NETs formation but in the same time, their activity prevents P. gingivalis entrapment and subsequent killing. This may explain a paradox that despite the massive accumulation of neutrophils and NETs formation in periodontal pockets periodontal pathogens and associated pathobionts thrive in this environment.

MCPIP-1, Alias Regnase-1, Controls Epithelial Inflammation by Posttranscriptional Regulation of IL-8 Production.

Pattern recognition receptors are critical for the detection of invading microorganisms. They activate multiple pathways that lead to the induction of proinflammatory responses and pathogen clearance. The intensity and duration of this immune reaction must be tightly controlled spatially and temporally in every tissue by different negative regulators. We hypothesized that monocyte chemoattractant protein-1-induced protein-1 (MCPIP-1) might play a role in maintaining immune homeostasis in the epithelium both under physiological conditions and upon bacterial infection. To this end, we examined the distribution of the MCPIP-1 transcript and protein in various tissues. The MCPIP-1 protein level was higher in epithelial cells than in myeloid cells. MCPIP-1 exerted RNase activity towards the interleukin (IL)-8 transcript and the lifespan of IL-8 was determined by the presence of the stem-loops/hairpin structures at the 3'UTR region of IL-8 mRNA. Moreover, using fully active, purified recombinant MCPIP-1 protein, we elucidated the mechanism by which MCPIP-1 controls the IL-8 mRNA level. In conclusion, we uncovered a novel IL-8-dependent mechanism via which MCPIP-1 maintains epithelial homeostasis. This study reveals for the first time that MCPIP-1 plays a crucial anti-inflammatory role not only in myeloid cells but also in epithelial cells.