Study On Preparation Of Recombinant Lactococcus Lactis Expressing IL-35 Ameliorated Experimental Colitis In Mice

Ulcerative colitis(UC)is a kind of non-specific inflammatory disease that occurs in the colon and rectum,which is characterized by abdominal pain,diarrhea and bloody stool.Although the etiology of UC is unclear,it has been shown that excessive pro-inflammatory response induced by immune cells and their cytokines contributes to the development of UC.Moreover,inhition of over-activated pro-inflammatory cells or enhancement of regulatory T cell(Treg),one type of immunosuppressive cells,could significantly ameoliate UC.Previous work showed that interleukin-35(IL-35),a relatively newly discovered anti-inflammatory cytokine secreted by Treg,promoted Treg generation and thereby compromising inflammation.Systemic administration of IL-35 via intravenous injection showed that UC-associated inflammation was delayed and the progression of colitis was slowed down in a UC-like mouse model.However,an increased risk of adverse drug reactions,as well as the high cost and inconvenience for patients are provoked by this approach of systemic administration.Searching for an economic and convenient strategy for IL-35 administration for UC attunation is considered to be more necessary.Lactic acid bacteria,serving as probiotics in gut,is one type of well-documented engineering strains.Accumulating evidence has demonstrated that recombinant lactic acid bacteria,expressing specific function protein,could be used to diease prevention or migitation,suggesting a role for recombinant lactic acid bacteria in delivery of IL-35 and thereby ameliorating UC.In this work,we first designed and synthesized murine IL-35 gene to contruct expressive plasmid p NZ8148/IL-35 and thereby preparing recombinant lactic acid bacteria,NZ9000/IL-35.Subsequently,we demonstrated that the isolated recombinant IL-35 could promote Treg differentiation in vitro,which exhibit 44.9%,65.8% and 65.1% under the 0.5,1 and 2 ng/ml recombinant IL-35 conditions,respectively.Moreover,we observed that intestinal IL-35 was accumulated to 79.06±38.64,115.52±41.74 and 119.08±54.58pg/mg on 7,14 and 21 day respectively induced by oral administration of NZ9000/IL-35 in mice.Furthermore,we established a UC-like mouse model through dextran sodium sulfate(DSS)drinking for evaluation of the impact of NZ9000/IL-35 on UC ameloration.When administrated preventatively,NZ9000/IL-35-gavaged mice exhibited decreased weight loss,disease activity index(DAI)score,colon shortening as well as colitis-associated histopathological changes in colon,indicating that the oral administration of NZ9000/IL-35 contributed to the suppression of DSS-induced colitis progression.Moreover,much less pro-inflammantory Th17 cells,and higher level of Treg cells in lamina propria,as well as increased colon and serum levels of IL-10 with a concomitant reduced pro-inflammatory cytokines,IL-6,IL-17 A,IFN-γ,and TNF-α were apparently regulated by NZ9000/IL-35 in colitis mice.Taken together,we put forward direct evidence pinpointing the effectiveness of NZ9000/IL-35 in preventing UC-like mouse colitis,implying a potential candidate of this recombinant NZ9000/IL-35 that prevent UC progression.