SIRPα enhances macrophage alternative activation by preventing a phosphatase from acting on cytokine receptors.
Group A Streptococcus (GAS) is a major pathogen that causes simple and invasive infections. GAS requires iron for metabolic processes and pathogenesis, and heme is its preferred iron source. We previously described the iron-regulated hupZ in GAS, showing that a recombinant HupZ-His6 protein binds and degrades heme. The His6 tag was later implicated in heme iron coordination by HupZ-His6. Hence, we tested several recombinant HupZ proteins, including a tag-free protein, for heme binding and degradation in vitro. We established that HupZ binds heme but without coordinating the heme iron. Heme-HupZ readily accepted exogenous imidazole as its axial heme ligand, prompting degradation. Furthermore, HupZ bound a fragment of heme c (whose iron is coordinated by the cytochrome histidine residue) and exhibited limited degradation. GAS, however, did not grow on a heme c fragment as an iron source. Heterologous HupZ expression in Lactococcus lactis increased heme b iron use. A GAS hupZ mutant showed reduced growth when using hemoglobin as an iron source, increased sensitivity to heme toxicity, and decreased fitness in a murine model for vaginal colonization. Together, the data demonstrate that HupZ contributes to heme metabolism and host survival, likely as a heme chaperone. HupZ is structurally similar to the recently described heme c-degrading enzyme, Pden_1323, suggesting that the GAS HupZ might be divergent to play a new role in heme metabolism.
Intrauterine inflammation during pregnancy can cause prenatal brain injury, and is associated with preterm birth. Gardnerella vaginalis (GV) is a gram variable rod associated with bacterial vaginosis, pelvic inflammatory disease, bacteremia, and preterm birth. Bacterial vaginosis samples demonstrate up-regulation of Toll-like Receptor 2 and 4 mRNA and secretion of IL-1β in cultured trophoblasts. It has not been clearly demonstrated that isolated GV specimens are capable of the characteristic inflammatory downstream effectors in monocytes. We set out to determine if GV causes inflammatory effector recruitment and expression of proinflammatory cytokines in a human monocyte cell line, THP-1. An ASC-YFP overexpression system was used for immunofluorescent detection of inflammasome components, the Annexin-V apoptosis assay was used for viability, and cytokine levels were quantified by ELISA. Immunofluorescent detection showed co-localization of both NLRP3 and NLRC4 with ASC-YFP in GV-treated and LPS/ATP-treated cells. GV-treated cells exhibited a statistically significant IL-1β, IL-18, and TNFα response over untreated cells, however, IL-18 was significantly increased compared to LPS/ATP treatment. GV-treated cells remained viable through 24 h which demonstrates that GV causes significant cytokine elevation in THP-1 cells, without significant cell death. These results suggest that GV infection may lead to significant sequelae by production of proinflammatory cytokines.
The Th2 cytokines IL-4 and IL-13 through activation of their shared receptor IL-4Rα direct macrophage alternative activation to promote immunosuppression and wound healing. However, the mechanisms that control macrophage responses to IL-4/13 are not fully understood. Apart from driving JAK-STAT and PI3K-Akt pathways to polarize macrophages toward the alternative phenotype, the activated IL-4/13 receptors recruit negative regulators SHP-1 and SHP-2, which dephosphorylate IL-4Rα and decrease its signaling. Here we report that SIRPα spatially restricts SHP-2 and, by such, promotes IL-4/13 signaling and macrophage alternative activation. SIRPα executes this regulation via its cytoplasmic ITIMs/ITSMs that undergo phosphorylation by IL-4/13-induced, Src kinase-activated Brutons tyrosine kinase (Btk), resulting in recruitment of SHP-2 and preclusion of SHP-2 from binding to and inhibiting IL-4/13 receptors. Despite that this regulation occurs independent of CD47, extracellular CD47 ligation of SIRPα facilitates its cytoplasmic phosphorylation and SHP-2 sequestration, leading to stronger IL-4/13 signaling and enhanced macrophage expression of IL-10, TGFβ, CD206, arginase-1, etc. Conversely, deficiency of SIRPα allows SHP-2 to freely bind to γC or IL-13Rα1 and through which dephosphorylate IL-4Rα, dampening its signaling. Consistent with these findings, impaired wound healing in Sirpα-/- mice under experimental colitis correlated with a deficit of immunosuppressive macrophages in the colon, a condition that was corrected by transfusion of ex vivo-produced SIRPαhigh alternatively activated macrophages.
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