High-mobility Group Box 1 Facilitates CD4 T Cell Self-aggregation Via Integrin and STAT3 Activation Before Homing.

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 β1, β7, and α4β7 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 β1, β7, and α4β7; 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.HMGB1 induces CD4 T cell self-aggregation in blood resulting in upregulation of integrins expression and release of pro-inflammatory cytokines/chemokines via activation of STAT3 signaling. This study highlights the potential of preventive and therapeutic intervention on immune cell self-aggregation in the bloodstream.