A Novel Lipopolysaccharide-binding Protein CRISPLD2: Expression And Significance In Sepsis

Sepsis is major cause of death for critical ill patients not only in China, but also inEuropean countries and USA. Although broad-spectrum antibiotics were used and newtreaments emerged, mortiality of sepsis paitents remains high in ICU. Recombinantactivated protein C (of drotrecogin alfa, a recombinant activated protein C) and eritorantetrasodium (a novel anti-Toll-like receptor (TLR)-4compound) in1and2clinical trialsproved to be effective, but most recently further clinical trial found no clear effect in thetreatment of severe sepsis. The important reason is the lack of a suitable marker ofstratification. To find a suitable biomarker for the diagnosis and treatment of sepsis isvery important.Our previous work had shown that Cysteine-rich secretory protein containingLCCL domain2(CRISPLD2/Crispld2) is also a LPS-binding protein that exhibitssignificant LPS-binding affinity. We found that the affinity of CRISPLD2for LPS issimilar to that for CD14and MD2, but higher than that for TLR4, lower than that forLBP. The stimulation of PBMCs with LPS can promote secretion of CRISPLD2in vitroor in vivo; treatment with recombinant CRISPLD2has been shown to inhibit the bindingof LPS with target cells, reduce LPS-induced TNF-α and IL-6production in vitro, andprotect mice from endotoxin shock. In previous work, we determined CRISPLD2concentration in serum (or plasma) using immunoblotting assay and found that its levelbe significantly higher than of LBP, soluble CD14and etc. Of note, CRISPLD2level ininfant umbilical cord blood plasma was significantly lower than that in adult.We used LPS-induced endotoxin-shock model in our previous study, and it did notreflect the real clinical status of polymicrobial infection; however, recombinantCRISPLD2significantly reduced the mortality in this experimental model. Up to nowlittle is known about the regulatory effect of CRISPLD2on host immune responses inpolymicrobial sepsis. Currently, cecal ligation and puncture (CLP) in rodents has beenwidely used for experimental sepsis, and is considered as a reliable model mimickinghuman polymicrobial sepsis. The distinct locations of cecal ligation, puncturing needlesize, and number of puncture determine the different mortality correlating to degrees ofsepsis. In the present study, three degrees of CLP-induced sepsis were used to determinewhether serum Crispld2is correlated with the sepsis severity in polymicrobial sepsis.Moreover, serum Crispld2was upregulated and then downregulated to assess itsregulatory potential in polymicrobial infected mice. The aim of this study is to further assess the value of CRISPLD2in prognosis and treatment of polymicrobial sepsis as aninnate-immune regulator.The existing experiments had show that CpG ODN (ssCpG DNA) protects animalsagainst bacterial infection. Recently studies had showed CRISPLD the R3H domain canbinding with oligonucleotide. We found that CRISPLD2contains a RAAIH (R3H)structure, but there is no knowledge whether CRISPLD2combinaed with single-strandedDNA. Glucocorticoids receptor can bind with specific DNA sequences (GRE)CRISPLD2gene promoter region.In the first part of this study, we confirmed CRISPLD2is not only anendotoxin-binding protein, but also a single-stranded DNA binding protein. A variety ofmicrobial antigens such as ssCPG-DNA, PGN-ssGPC-DNA and double-stranded RNA(PolyI: C) can stimulate peripheral mononuclear cells to secrete CRISPLD2. CRISPLD2binding with endotoxin inhibited LPS combined with the target cells; but a combinationof CRISPLD2with single-stranded DNA does not block the combination of LPS withtarget cells.In the second part of this study, we use the model of cecal ligation and perforationto explore expression patterns of CRISPLD2in sepsis and the feasibility of treatment ofsepsis targeted to CRISPLD2. The high mortality of the acute phase of septic rats withexcessive inflammatory response and the CRISPLD2the failure to increase related.Upregulation of CRISPLD2level through Pre-injected with endotoxin can reduce themortality of septic mice. We found that injection with the recombinant CRISPLD2protein raise the level of serum CRISPLD2and reduce sepsis mortality. Glucocorticoidraised the CRISPLD2level in healthy rat, small-dose glucocorticoids increaseCRISPLD2level in septic rats to improve the survival; high-dose glucocorticoids failedto increase serum CRISPLD2level in sepsis, and at the same time did not preventlealthelity.At the last, we assay serum level CRISPLD2in healthy populations and patients.Serum CRISPLD2level in healthy volunteers fluctuate with considerabe amplitude.Patients with mild and morderate sepsis had a higher level of CRISPLD2than normal.CRISPLD2concentration fail to up-regulate in severe forms of sepsis and associatedwith PCT, APACHEII and SOFA score.Summary: CRISPLD2is not only an endotoxin-binding protein, also is assCPG-DNA binding protein; high mortality in CLP sepsis model is assiodtced with low level of CRISPLD2combined with high PCT; up-regulation serum level of CRISPLD2in mice through preconditioned with LPS (by intraperitoneal injection) or injection ofrecombinant CRISPLD2, reduce the mortality of septic mice; glucocorticoid canincrease serum level of CRISPLD2in a pose-depended manner; but only low-dosesteroids,not middle or high dose can promote CRISPLD2secretion and reduce themortality of sepsis in rats; CRISPLD2shows normal distribution in healthy people andfluctuate with considerable amplitude; CRISPLD2in mild and morderate sepsis had ahigher level of CRISPLD2than normal, but it failed to up-regulate in severe forms ofsepsis.