PD-L1Expression On Intestinal Epithelial Cell And Its Role In Sepsis-induced Intestinal Barrier Dysfunction

Background and ObjectiveSepsis remains the majorreason of deaths in critically ill patients. Studies suggestedthatintestinal barrier dysfunction and increased intestinal permeability are morbid statesthat often accompany developing of sepsis. Increased intestinal permeability maycontribute to bacterial translocation and the development of multiple organ failure.Programmed cell death ligand1(PD-L1, also called B7-H1), a member of the B7family ofimmune-regulatory cell-surface proteins, plays an important role in the negative regulationof cell-mediated immune responses through its interaction with its receptor, programmedcell death-1(PD-1).Recent studies demonstrated that these molecules are involved ofintestinal mucosal inflammation and the regulation of immune tolerance. Previous studiesin our laboratory also have found that PD-1or PD-L1deficiency could protect mice fromsepsis induced intestine injury. The lining of intestinal epithelial cells (IECs) is the keycomponent of intestinal physical barrier. IECs arealso responsible for the immunologicalpart of the intestinal barrier as IECs can function as antigen-presenting cells (APC)andpromote T lymphocyte reaction. However, little is known about this immune co-inhibitory molecule expression pattern on the IECs and its role in the intestinal barrierdysfunction during sepsis. The objective of this study was to assess the expression of PD-L1in mouse IECs under normal and sepsis condition and its role in the intestinal barrierdysfunction during polymicrobial sepsis.Measurements1. Primary IECs were isolated from naive male C57BL/6mice, PD-L1mRNA andprotein expression in IECs was detected by qPCR and Western blot, respectively.2. C57BL/6mice underwent cecal ligation and puncture (CLP)(n=18) or shamsurgery (n=24). IECs were isolated at6h,24h, or48h post surgery, PD-L1mRNA andprotein expression in IECs was detected by qPCR and Western blot,respectively.3. Wild type (WT) C57BL/6mice and PD-L1gene deficiency (PD-L1-/-) mice wererandomly distributedto WT Sham group,WT CLP group,PD-L-/-Sham group,and PD-L1-/-CLP group,Sham group had4mice and CLP group had6mice.In vivo intestinalpermeability was measured using an invivo ligated loop model24h after surgery. 4. WT C57BL/6mice and PD-L1-/-mice were randomly distributedto WT Shamgroup,WT CLP group,PD-L-/-Sham group,and PD-L1-/-CLP group,Sham group had6mice and CLP group had8mice. The TNF-α, IL-6, IL-10and MCP-1level in the ileumwas measured by ELISA24h following sepsis. The expression of tight junction proteinsZO-1, Occludin, and Claudin-1in the ileum were measured by Western blot24h aftersepsis.5. Human colonic epithelial cell line Caco-2cells were cultured, PD-L1mRNA andprotein expression were assessed with or without recombianant human TNF-α and/or IFN-γ stimulation by qPCR, Western blot, or Flow cytometry.6. Caco-2cells seeded in transwell to form monolayer, then monolayers were treatedin four groups: Non-stimulation control group, TNF-α and IFN-γ stimulation group, anti-human PD-L1antibody pretreatment+TNF-α and IFN-γ stimulation group, ISO controlantibody+TNF-α and IFN-γ stimulation group. Paracellular permeability was measured72h after treatment to evaluate barrier function. The expression and distribulation of tightjunction proteins ZO-1and Occludin were analyzed by immunofluorescence.Results1. PD-L1mRNA and protein were detected constiutively in isolated IECs from na vemice.2. PD-L1mRNA expression in IECs of septic mice was obviously higner than that ofsham mice (p<0.05) at all6h,24h, and48h post surgery. PD-L1protein expression in IECswas not changed at6h while significantly increased at24h and48h following CLP(p<0.05).3. Sepsis significantly increased intestinal epithelial permeability of the ileum in WTmice (p<0.05). Absence of PD-L1significantly decreased in vivo intestinal epithelialpermeability of the ileum24h following sepsis (p<0.05).4. Absence of PD-L1decreased TNF-α, IL-6, and MCP-1level (p<0.05) andprevented the decline expression of tight junction protein ZO-1, Occludin, and Claudin-1in the ileum24h following CLP compared with WT mice (p<0.05). 5.PD-L1mRNA and protein expression were detected in cultured Caco-2cells, andwere strongly enhanced by stimulation with recombinant human TNF-α and/or IFN-γ(p<0.05).6. Anti-human PD-L1antibody significantly attenuated paracellular permeabilityincreasing in Caco-2monolayer treated with recombinant human TNF-α and IFN-γ(p<0.05). Anti-human PD-L1antibody also dramatically alleviated recombinant humanTNF-α and IFN-γ induced morphological alteration of tight junction protein ZO-1andOccludin.ConclusionsMouse primary intestinal epithelial cells and human colonic epithelial cell lineconstitutively express PD-L1. Sepsis increased PD-L1expression in IECs and TNF-αand/or IFN-γ stimulation up-regulated PD-L1expression in Caco-2cells. Absence of PD-L1protected mice from intestinal barrier dysfunction by decreasing cytokine levels andrestoring the tight junction protein expression during sepsis, which was also confirmed invitro recombinant human TNF-α and IFN-γ induced Caco-2monolayer barrier dysfunction.Taken together, theresults suggestedthat PD-L1expressed on IECs contributes to sepsisinduced intestinal barrier dysfunction.