Pig HMG1/HMGB1 ELISA Kit

Background: High-mobility group box 1 (HMGB1) is one of the delayed pro-inflammatory cytokines produced in the later stages of pathogenesis and plays an important role in the progression of various inflammatory and autoimmune diseases. High-mobility group box 1 is able to stimulate interaction between integrins and cell adhesion molecules to facilitate cell-cell aggregation in "tissue-specific" endothelium; however, whether and how HMGB1 affects the adhesive capability of early acting immune cells in bloodstream remains largely unknown. Methods: Human peripheral blood samples were collected from healthy adult donors. The CD4 T cells were isolated from blood using CD4 T cell isolation kit and identified using flow cytometry and immunofluorescence staining. The effect of HMGB1 on adhesive ability of CD4 T cells was accessed by cell self-aggregation assay and endothelial adhesion assay. The migratory ability of CD4 T cells was evaluated by cell migration assay. Secretion of pro-inflammatory cytokines or chemokine C-X-C motif chemokine 12 (CXCL12) were detected by ELISA. Expression of integrins beta 1, beta 7, and alpha 4 beta 7 were determined by flow cytometric analysis. Inhibition of integrins was achieved with anti-integrin antibodies or cyclic peptide inhibitors. Activation of signal transducers and activators of transcription 3 (STAT3) was measured by flow cytometry and fluorescent staining. Results: High-mobility group box 1 facilitated CD4 T cell self-aggregation with simultaneous reduction of CD4 T single-cell counts in the bloodstream. The CD4 T cell self-aggregation induced by HMGB1 resulted in upregulation of integrins beta 1, beta 7, and alpha 4 beta 7; release of other pro-inflammatory cytokines or chemokine CXCL12; and activation of STAT3 signaling. Intriguingly, pro-inflammatory cytokines induced by HMGB1 could further amplify CD4 T cell self-aggregation. HMGB1 induced CD4 T cell apoptosis via activation of caspase-3/7. Furthermore, HMGB1 promoted migration and adhesion of CD4 T cells to endothelial cells. Conclusions: These results provide proof of concept that HMGB1 promotes CD4 T cell self-aggregation before homing to inflammatory sites and highlight the potential of blocking immune cell self-aggregation in blood as a novel therapeutic approach against the development and progression of HMGB1-related inflammatory diseases.

Inflammasomes are an important innate immune host defense against intracellular microbial infection. Activation of inflammasomes by microbial or host ligands results in cleavage of caspase-1 (canonical pathway) or caspase-11 (noncanonical pathway), release of interleukin (IL)-1 beta, IL-18, high mobility group box 1 (HMGB1), and inflammatory cell death known as pyroptosis.Ehrlichiaare obligate, intracellular, gram-negative bacteria that lack lipopolysaccharide but cause potentially life-threatening monocytic ehrlichiosis in humans and mice that is characterized by liver injury followed by sepsis and multiorgan failure. Employing murine models of mild and fatal ehrlichiosis caused by infection with mildly and highly virulentEhrlichia muris(EM) andIxodes ovatus Ehrlichia(IOE), respectively, we have previously shown that IOE infection triggers type I interferon (IFN-I) response and deleterious caspase-11 activation in liver tissues, which promotes liver injury and sepsis. In this study, we examined the contribution of IFN-I signaling in hepatocytes (HCs) toEhrlichia-induced liver injury. Compared to EM infection, we found that IOE enter and replicatein vitrocultured primary murine HCs and induce secretion of IFN beta and several chemokines, including regulated upon activation, normal T-cell expressed, and secreted (RANTES), monocyte chemoattractant protein 1 (MCP1), monokine induced by gamma (MIG)/chemokine (C-X-C motif) ligand 9 (CXCL9), macrophage inflammatory protein 1 alpha (MIP1 alpha), keratinocyte-derived chemokine (KC), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Notably,in vitrostimulation of uninfected andEhrlichia-infected HCs with recombinant IFN beta triggered activation of caspase-1/11, cytosolic translocation of HMGB1, and enhanced autophagy and intracellular bacterial replication. Secretion of HMGB1 by IOE-infected HCs was dependent on caspase-11. Primary HCs from IOE- but not EM-infected mice also expressed active caspase-1/11.Conclusion:HC-specific IFN-I signaling may exacerbate liver pathology during infection with obligate intracellularEhrlichiaby promoting bacterial replication and detrimental caspase-11-mediated inflammasome activation.