B75-28 Reprogramming the Suppressed Pulmonary Immune Response After Sepsis via Targeting Irf3-socs3 Signaling With Piceatannol and Interferon-β Treatment

Rationale Sepsis remains one of the leading causes of mortality worldwide while effective therapies are still lacking. In our previous study using a murine cecal ligation and puncture (CLP) model, we found that on postoperative day 4 (POD4), pulmonary immune mediators including tnf, il6, ccl2, and il10 were markedly suppressed, while only socs3 remained elevated, suggesting an overall transcriptional failure during the late septic state. Given that IRF3 acts as a context-dependent enhancer of sepsis and that piceatannol (PIC) suppresses IRF3 activation, we hypothesized that modulation of the IRF3-SOCS3 axis may restore cytokine responsiveness after sepsis. Methods Mild polymicrobial sepsis was induced in male C57BL/6 mice using CLP with a 23-gauge needle. On POD4, lungs were harvested and enzymatically digested into single cell suspension using the gentleMACS® system, followed by running EasySep® magnetic column separation kit to isolate mononuclear cells. Isolated mononuclear cells were stimulated with LPS (10ng/ml) in the presence or absence of PIC (30µM) and/or Interferon(IFN)-ß (15000 u/ml). Macrophage cell-line RAW264.7 cells were similarly treated. Expression of inflammatory (tnf,cxcl1,ifnb1) and inhibitory mediators (socs3) was quantified by RT-qPCR. Results In Raw264.7 cells, LPS stimulation significantly increased mRNA levels of tnf, cxcl1, ifnb1, and socs3. PIC significantly suppressed their expression, particularly strongly inhibiting socs3. In lung monocytes 4 days after sepsis, cxcl1 expression was significantly suppressed, whereas tnf and socs3 expression showed no significant suppression. PIC significantly suppressed all mRNAs. IFN-ß treatment failed to restore cxcl1 expression and only partially restored socs3 expression, but significantly and substantially restored the tnf response. Discussion In vitro analysis using cell lines revealed that PIC strongly suppresses TLR4 signaling other than tnf signaling, indicating the IRF3 pathway is strongly involved in interferon stimulating gene expression.In the ex vivo model on day 4 post-sepsis, PIC further enhanced the suppression of sepsis-induced immune unresponsiveness, confirming IRF3 pathway involvement. Co-culture results with IFN-ß suggested that, beyond IRF3, reactivation of another pathway by IFN-ß may also contribute to tnf induction following sepsis.These results suggest that precise rebalancing of immune signaling, rather than uniform suppression, is required to restore immune responsiveness following infection. Piceatannol may thus serve as a useful compound to understand the mechanisms of post-septic immune paralysis and identify potential targets for immunoreactivation therapy. This abstract is funded by: The Japan Society for the Promotion of Science