The Protective Effect Of HSF1on Multiple Organ Injury In Mice With Endotoxemia And Its Mechanism

Sepsis is a systemic inflammatory response syndrome (SIRS) triggered by infection. Although the mechanisms of sepsis have not yet been fully elucidated, it is well established that lipopolysaccharide (LPS) or endotoxin plays an important role in the pathogenesis of sepsis by initiating the immune system in vivo. LPS can activate several signal transduction pathways and initiate the host defense response, resulting in overexpression of a large number of inflammatory mediators and activation of the systemic inflammatory cells. The key mechanism in the pathogenesis of sepsis is uncontrolled inflammatory response to infection and its main characteristics and mechanisms is excessive neutrophil (polymorphonuclear neutrophil, PMN) infiltration which ultimately results in multiple organ injury.Heat shock factor1(HSF1) is the major transcriptional factor that regulates the heat shock response. Our previous study found that HSF1knockout mice (HSF1-/-) were more sensitive to LPS; and when treated with LPS, these mice showed lower survival rate. This indicates that HSF1is essential in protection against the systemic inflammation induced by bacterial endotoxin. However, the protective mechanism remains obscure. Although LPS-induced endotoxemia is characterized with infiltration of PMNs in the lungs, liver, and kidneys, whether does HSF1have an effect on PMN infiltration is still unclear.To explore the protective effect of HSF1on the multiple organ injury in LPS-induced endotoxemia mice and underlying mechanisms, we prepared a mouse model of endotoxemia and measured a series of parameters of tissue injury and performed histology analysis at the overall animal level. The results showed that:1)4h after the injection of LPS, higher level of serum inflammatory mediators such as IL-6and G-CSF, and enzymatic indicators such as LDH, ALT and BUN were observed in HSF1-/-mice than WT mice.2)4h after the injection of LPS and comparing with WT mice, microvascular permeability and wet/dry ratio of lung and renal tissues increased significantly in HSF1-/-mice.3) Consistent with lower survival rate, PMN infiltration and histopathology in multiple susceptible organs (lungs, liver and kidneys) were significantly exacerbated in HSF1-/-mice when treated with LPS, suggesting that HSF1has a protective effect on LPS-induced multiple organ injury, which is associated with the suppression of excessive PMN infiltration.PMN infiltration is a highly regulated multistep process that involves adhesion and chemotaxis. Recently, we prepared an endotoxemia model to screen HSF1-regulated inflammation-related genes from the lung tissue by microarray with384inflammatory cytokine genes. We found that HSF1may inhibit the expression of P-selectin glycoprotein ligand1(PSGL-1, a cell adhesion molecule) and XCR1(a chemokine receptor), suggesting that HSF1plays a potential inhibitory role in PMN adhesion and chemotaxis in LPS-induced endotoxemia.Next we decided to explore the effect of HSF1on PMN adhesion in LPS-induced endotoxemia. By anti-granulocyte staining, we demonstrated that PMNs in the HSF1-/-mice after LPS challenge were more adhesive to endothelium than that in the WT mice. Then, the mRNA level of PSGL-1was measured by quantitative real-time PCR, and the protein level of PSGL-1on PMN surface was measured by flow cytometry analysis. The expression levels of CD11b on PMN surface and intercellular adhesion molecule-1(ICAM-1) on endothelium were detected by flow cytometry and immunohistochemical analysis, respectively. We found that the protein levels of PSGL-1and CD11b on HSF1-/-PMNs at baseline were higher than that of the WT PMNs. LPS induced the surface expression of PSGL-1and CD11b on the PMNs of WT and HSF1-/-mice. However, greater surface expression of PSGL-1and CD11b was shown on the PMN surface of HSF1-/-mice after LPS treatment. These results indicate that HSF1is capable of down-regulating both constitutive and inducible expressions of PSGL-1and CDllb on PMNs. In addition, LPS induced higher level of ICAM-1expression on the endothelium of HSF1-/-mice. Nevertheless, the effect of LPS treatment on Hsp70protein levels in PMNs of WT and HSF1-/-mice is minimal. These results suggest that HSF1inhibits PMN adhension to endothelium by suppressing the surface expression of PSGL-1and CD11b on PMNs as well as ICAM-1on endothelium during endotoxemia.Chemotaxis is the movement of phagocytes toward an increasing concentration of attractants such as bacterial factors (LPS etc.) and chemokines. To explore the effect of HSF1on PMN chemotaxis and its mechanisms, chemotactic chamber assay was conducted to evaluate the migratory capacity of WT and HSF1-/-BM PMNs to multiple migration-inducing stimuli, including LPS (0.6μg/ml) and lung homogenates from endotoxemia mice. We found that the chemotactic activities of PMNs from the HSF1-/-mice were significantly higher than that from the WT mice. The levels of serum MIP2and XCL1were measured by ELISA. CXCR2expression on PMN surface was detected by flow cytometry analysis. The mRNA level of XCR1in BM PMNs was measured by quantitative real-time PCR. The protein expression of XCR1on circulating PMNs or BM PMNs was evaluated by immunocytochemical analysis. It was found that LPS reduced CXCR2expression on PMNs from WT and HSF11-/-mice. However, the differences in CXCR2expression on circulating PMN of the WT and HSF1-/-mice after LPS injection were negligible. There was no significant difference between serum MIP2production of the WT and the HSF1-/-mice at basal levels or after LPS exposure, although the levels of serum MIP2significantly increased in WT and HSF1-/-mice after LPS treatment. However, LPS induced higher level of serum XCL1in HSF1-/-mice and greater surface expression of XCR1on circulating PMN of the HSF1-/-mice. LPS induced a marked increase in both mRNA and protein level of XCR1in PMNs of HSF1-/-, but not WT mice. XCR1expression on HSF1-/-BM PMNs increased progressively during the in vitro LPS treatment, and this effect was not observed in the WT BM PMNs. Notably, the basal expression of XCR1on HSF1-/-PMNs was lower than that on WT PMNs. This suggests an inhibitory role of HSF1in LPS-induced XCR1expression on PMNs. Changes in cytoskeleton and podosome formation are hallmarks of migrating cells. To determine whether HSF1-/-alters cytoskeleton rearrangement, F-actin reorganization was examined in circulating and BM PMNs from WT and HSF1-/-mice. Formation of lamellipodia in response to LPS was seen in the WT BM PMNs, whereas HSF1-/-cells displayed a significant increase in F-actin-rich extensions. This is in consistent with enhanced chemotaxis and migration in HSF1-/-BM PMNs. Moreover, F-actin polymerization in HSF1-/-BM PMNs increased progressively during the in vitro LPS treatment. Once F-actin was disrupted by cytochalasin B, an inhibitor of actin polymerization, the chemotactic response of WT and HSF1-/-BM PMNs to LPS was shown to be completely abolished, indicating that HSF1inhibits LPS-induced F-actin polymerization in PMNs. These results suggest that HSF1inhibits PMN chemotaxis and migration by suppressing the surface expression of XCR1on PMN and serum XCL1production, as well as F-actin polymerization in PMN during endotoxemia.Previously, our bioinformatic analysis found a heat shock element located at the upstream of the transcription starting sites of the psgl-1and xcr1genes. Therefore, as a transcription factor, HSF1may repress psgl-1and xcr1directly at the transcriptional level.In summary, this study demonstrated for the first time that, the protective role of HSF1in LPS-induced multiple organ injury is related to the suppression of excessive PMN infiltration. Mechanistically, during endotoxemia,1) HSF1inhibits PMN adhesion to vascular endothelial cells by suppressing the expression of LPS-induced PSGL-1and CD11b on PMN surface and ICAM-1on vascular endothelial cell surface;2) HSF1inhibits PMN chemotaxis and migration by suppressing XCL1production, the XCR1receptor expression and F-actin polymerization in PMN. This study has provided not only a novel model for the pathogenesis of sepsis, but also new ideas and clues for the prevention and treatment of endotoxemia and sepsis.