Contribution Of PD-1to Liver Kupffer Cell Dysfunction In Mice With Sepsis

ObjectiveSepsis is defined as a systemic inflammatory response syndrome caused by infectionand remains the leading cause of deaths in critically ill patients. At the late stage of sepsis,the immune system is paralyzed. Thereinto, macrophage dysfunction contributes tosepsis-associated immunosuppression. Therefore the mechanism responsible formacrophage dysfunction deserves investigation.Programmed cell death1(PD-1) is a co-inhibitory receptor and plays an important rolein peripheral tolerance and chronic viral infection. In2009, Huang et al reported that PD-1gene deficiency significantly protected mice from cecal ligation and puncture (CLP)induced lethality, suggesting a critical role of PD-1in sepsis-associatedimmunosuppression. Based on this, we investigated the expression of PD-1on kupffercells and the role of PD-1in sepsis-induced kupffer cell dysfunction as well as thepotential signal mechanism in CLP model in mice.Methods1. Experimental sepsis was induced by CLP. C57BL/6mice were randomly dividedinto Sham group (n=8) and CLP group (n=8). Liver was harvested24h after surgery. Livernonparenchyma cells (NPCs) were separated and stained with F4/80and PD-1. PD-1expression on kupffer cells was detected by flow cytometry.2. Wild type C57BL/6mice and PD-1gene knockout (PD-1-/-) mice were randomlydivided into WT Sham group (n=4), PD-1-/-Sham group (n=4), WT CLP group (n=6) andPD-1-/-CLP group (n=6). Liver was harvested24h after surgery. Liver nonparenchymacells (NPCs) were separated and stained with F4/80, MHCΠ, CD69, CD80and CD86.Expression of MHCΠ, CD69, CD80and CD86on kupffer cells were detected by flowcytometry.3. Wild type C57BL/6mice and PD-1gene knockout (PD-1-/-) mice were randomlydivided into WT Sham group (n=4), PD-1-/-Sham group (n=4), WT CLP group (n=4) andPD-1-/-CLP group (n=4). Liver was harvested24h after surgery. Liver nonparenchymacells (NPCs) were separated and kupffer cells were isolated by2h adherence to the plate.PHrodoTME. coli BioParticles Conjugate was added to the medium for1h andphagocytosis of kupffer cells was detected by flow cytometry. 4. Wild type C57BL/6mice and PD-1gene knockout (PD-1-/-) mice were randomlydivided into WT Sham group (n=4), PD-1-/-Sham group (n=4), WT CLP group (n=6) andPD-1-/-CLP group (n=6). Liver was harvested24h after surgery. Liver nonparenchymacells (NPCs) were separated and kupffer cells were isolated by2h adherence to the plate.LPS (1μg/ml) or PBS was added to the medium for24h. The levels of TNF-α, IL-1β, IL-6,IL-10, IL-12p40and MCP-1in the medium were examined by enzyme-linked immunesorbent assay.5. Wild type C57BL/6mice and PD-1gene knockout (PD-1-/-) mice were randomlydivided into WT Sham group (n=4), PD-1-/-Sham group (n=4), WT CLP group (n=6) andPD-1-/-CLP group (n=6). Liver was harvested24h after surgery. Liver nonparenchymacells (NPCs) were separated and kupffer cells were isolated by2h adherence to the plate.LPS (1μg/ml) was added to the medium for24h and kupffer cells were lysated fordetection of cleaved caspase-3by Western blot.6. Wild type C57BL/6mice and PD-1gene knockout (PD-1-/-) mice were randomlydivided into WT Sham group (n=4), PD-1-/-Sham group (n=4), WT CLP group (n=6) andPD-1-/-CLP group (n=6). Liver was harvested24h after surgery. Liver nonparenchymacells (NPCs) were separated and kupffer cells were isolated by2h adherence to the plate.LPS (1μg/ml) was added to the medium for24h. Kupffer cells were stained by TUNELand examined by fluorescence microscope.7. Wild type C57BL/6mice and PD-1gene knockout (PD-1-/-) mice were randomlydivided into WT Sham group (n=4), PD-1-/-Sham group (n=4), WT CLP group (n=6) andPD-1-/-CLP group (n=6). Liver was harvested24h after surgery. Liver nonparenchymacells (NPCs) were separated and kupffer cells were isolated by2h adherence to the plate.LPS (1μg/ml) was added to the medium for10min and kupffer cells were lysated fordetection of Akt phosphorylation and total Akt by Western blot.8. Wild type C57BL/6mice and PD-1gene knockout (PD-1-/-) mice were randomlydivided into WT Sham group (n=4), PD-1-/-Sham group (n=4), WT CLP group (n=6) andPD-1-/-CLP group (n=6). Liver was harvested24h after surgery. Liver nonparenchymacells (NPCs) were separated and kupffer cells were isolated by2h adherence to the plate.LPS (1μg/ml) was added to the medium for24h and kupffer cells were lysated fordetection of p38phosphorylation and total p38by Western blot.9. Wild type C57BL/6mice and PD-1gene knockout (PD-1-/-) mice were randomlydivided into WT Sham group (n=4), PD-1-/-Sham group (n=4), WT CLP group (n=6) and PD-1-/-CLP group (n=6). Heart, liver and kidney were harvested24h after surgery andwere homogenized. Akt phosphorylation, total Akt, STAT3phosphorylation and totalSTAT3in heart, liver and kidney were detected by Western blot.Results1. Twenty-four hours after surgery, PD-1expression on kupffer cells of CLP mice wassignificantly higher than that of Sham mice (p <0.001).2. Twenty-four hours after surgery, expression of MHCΠ and CD86on kupffer cellsof WT CLP mice significantly decreased than those of WT Sham mice (p <0.001) andCD80expression on kupffer cells of WT CLP mice significantly increased than that of WTSham mice (p <0.001). PD-1gene deficiency partly and significantly restored theexpression of MHCΠ, CD80and CD86on kupffer cells (p <0.001). No significantdifference of CD69expression existed between groups.3. Twenty-four hours after surgery, phagocytosis of kupffer cells of WT CLP micewas significantly lower than that of WT Sham mice (p <0.01). PD-1gene deficiencysignificantly enhanced phagocytosis of kupffer cells (p <0.001).4. Twenty-four hours after surgery, with respect to their LPS-inducible cytokineproductive capacity, we noted that septic WT mouse kupffer cells produced markedlylower levels of TNF-α, IL-6, IL-12, MCP-1, IL-1β and IL-10than kupffer cells fromsham-treated mice, whereas kupffer cells derived from CLP-treated PD-1-/-mice exhibiteda marked, albeit partial, restoration of the release of these inflammatory cytokines.5. Twenty-four hours after surgery, LPS-stimulated cleaved caspase-3in kupffer cellsof WT CLP mice was significantly higher than that of WT Sham mice (p <0.001). PD-1gene deficiency significantly decreased the level of cleaved caspase-3in kupffer cells (p <0.001).6. Twenty-four hours after surgery, LPS-stimulated TUNEL positive kupffer cells ofWT CLP mice were significantly more than that of WT Sham mice. PD-1gene deficiencysignificantly decreased TUNEL positive kupffer cells.7. Twenty-four hours after surgery, the ratio of phosphorylated Akt/total Akt inkupffer cells of WT CLP mice was significantly lower than that of WT Sham mice (p <0.001). PD-1gene deficiency significantly increased the ratio of phosphorylated Akt/totalAkt in kupffer cells (p <0.001).8. Twenty-four hours after surgery, the ratio of phosphorylated p38/total p38in kupffer cells of WT CLP mice was significantly higher than that of WT Sham mice (p <0.001). PD-1gene deficiency significantly decreased the ratio of phosphorylated p38/totalp38in kupffer cells (p <0.001).9. Twenty-four hours after surgery, the ratio of phosphorylated Akt/total Akt in heart,liver and kidney of WT CLP mice was significantly lower than those of WT Sham mice.PD-1gene deficiency significantly increased the ratio of phosphorylated Akt/total Akt inliver and kidney, not heart. The ratio of phosphorylated STAT3/total STAT3in heart, liverand kidney of WT CLP mice was significantly higher than those of WT Sham mice. PD-1gene deficiency significantly decreased the ratio of phosphorylated STAT3/total STAT3inheart and kidney, not liver.ConclusionsPD-1contributed to liver kupffer cell dysfunction in mice with sepsis, possiblythrough its influence on Akt phosphorylation and p38phosphorylation in kupffer cells.Meanwhile, PD-1was involved with sepsis-induced multiple organ dysfunction, whichwas associated with Akt phosphorylation and STAT3phosphorylation.