The Role Of TLR5-RA In Lamina Propria Dendritic Cells Induced Defense Against Intestinal Bacterial Translocation After Traumatic Hemorrhagic Shock

The First PartThe study on mechanisms of indigenous bacterial translocation from gastrointestinal tract after traumatic hemorrhagic shockBackgroundTraumatic hemorrhagic shock(THS)induced indigenous bacterial and their toxins'translocation from gastrointestinal tract are important factors for endotoxemia,systemic infection,sepsis,followed by multiple organ failure(MOF)and even death.But the true mechanisms are undefined.Intestinal bacterial overgrowth,damage to the intestinal mucosal barrier and deficiencies in host immune defenses are regarded as the primary reasons for intestinal bacterial translocation.However,whether the three mechanisms mentioned above are also involved in THS induced intestinal bacterial translocation haven't reached a unified understanding.AimThis study is to investigate whether THS could induce intestinal bacterial translocation and the underlying mechanisms,using the three primary mechanisms that promote the translocation of indigenous bacteria from the GI tract as entry points,on the basis of mouse THS model.Materials and MethodsC57 Mice were randomly divided into Sham group and THS group.Sham group underwent the same anesthetic and surgical procedures,as well as fluid resuscitation,but was not subject to femoral fracture or bleeding,and in THS group,after an open mid-diaphyseal transverse fracture,mice were bled to achieve an MAP of 30 ± 5 mmHg,and 90 min later,mice were resuscitated with lactated Ringer's solution.Besides,in this course,the dynamic changes of MAP and intestinal microvascular perfusion were monitored.What's more,the results of indigenous bacterial translocation from GI tract were detected by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry(MALDI-TOF MS),and intestinal microbiota was analyzed by 16s rRNA sequencing.Clustering analysis,PCA analysis and rarefaction curve analysis were also performed to observe the effects of THS on intestinal bacteria composition,bacterial diversity and so on.Furthermore,HE stainings was used to observe intestinal mucosal change,FITC-Dextran was used to test intestinal permeability,ussing chamber was used to measure the intestinal barrier resistance and laser Doppler imaging was used to evaluate microvascular tissue perfusion in the intestine lamina muscularis again.Finally,systemic immune status was assessed by flow cytometry and ProcartaPlex multiplex cytokine kit.ResultsIn this study,we successfully established a THS model with C57 mice,and observed indigenous bacterial translocation to MLN,liver,spleen and even blood,with most of the translocating bacteria being Hearst Escherichia Coli.The rate of bacterial translocation in THS group was obviously higher than Sham group.To further analyze the results,we observed intestinal flora disturbance after THS,manifesting with Escherichia genus overgrowth,bacterial diversity decrease and obvious bacteria composition alteration.Besides,intestinal mucosal barrier was damaged dramatically,presented with subepithelial edema and disruption of epithelial cell tight junctions and so on.Moreover,THS group showed significantly higher intestinal permeability and lower resistance than Sham group.Furthermore,the blood flow in the lamina muscularis,which is an indicator of microvascular tissue perfusion,decreased dramatically in THS group and sustained persistently.Low microvascular perfusion induced intestinal hypoxia and PH value change were probably the main reasons for damage to intestinal mucosal barrier and intestinal flora disturbance.In addition,marked suppression in cell-mediated immunity after THS seemed to be another important factor.Wherever in blood or spleen,Th1/Th2 balance shifted to Th2,regulatory T cell increased,Th17 decreased,and CD11c+ DC decreased.The three reasons probably contributed to indigenous bacterial translocation cooperatively after THS.ConclusionIn a word,THS induced more indigenous bacterial translocation to MLN,liver,spleen and even blood,with most of the translocating bacteria being Hearst Escherichia Coli,and this is associated with intestinal flora disturbance,Escherichia genus overgrowth,bacterial diversity decrease,intestinal mucosal barrier damage and marked suppression in cell-mediated immunity.Moreover,low microvascular perfusion induced intestinal hypoxia,PH value change and ischemia-reperfusion induced xanthine-oxidase-generated hydroxyl radicals were probably the main reasons for intestinal flora disturbance and intestinal mucosal barrier disruption.And the underlying mechanisms of immunosuppression deserve more deep investigations.The Second Part The role of TLR5-RA in lamina propria dendritic cells induced defense against intestinal bacterial translocation after traumatic hemorrhagic shockBackgroundSevere systemic infection secondary to trauma and hemorrhage is considered as one of the major causes for the death induced by traumatic hemorrhagic shock(THS).Intestinal lamina propria dendritic cells(LPDCs)which specifically expresse toll like receptor 5(TLR5)are regarded as the antigen presenting cells extensively localized beneath the intestinal epithelium and they are much easier to get an access to enteric bacteria antigens such as TLR5's specific ligand flagellin.After stimulation of flagellin,TLR5+ LPDCs can induce the polarization of Th1 by producing retinoic acid.However,whether LPDCs and Thl polarized by LPDCs take part in post-THS intestinal bacterial translocation are still unknown.AimThis study was to investigate the role of TLR5+ LPDCs in intestinal bacterial translocation after THS and the underlying mechanisms.Materials and MethodsWild Type(WT)or tlr5 knockout(TLR5-/-)mice were divided into Sham or THS group.After an open mid-diaphyseal transverse fracture,mice were bled to achieve an MAP of 30 ± 5 mmHg,and 90 min later,mice were resuscitated with lactated Ringer's solution.Besides,dynamic changes of MAP and intestinal microvascular perfusion were monitored.Moreover,homogenates of mesenteric lymph nodes(MLNs),liver,spleen and blood were plated on LB agar and cultured overnight 5 hours after bioluminescent citrobacter rodentium(C.R.)gavage,and bacterial colony forming units(CFUs)were determined and calculated under in vivo imaging system(IVIS).Meanwhile,trans-epithelial electrical resistance(TEER)and intestinal permeability were determined at different time points post-THS as well.Furthermore,LPDCs subgroup were sorted on the basis of CD11c and MHC ? expression by FACS sorting.RA expressed by LPDCs and T cell polarization induced by LPDCs was analyzed by flow cytometer.Finally,RA was orally gavaged to test its therapy effect on inhibiting bacterial translocation and prolonging mice's survival.ResultsWe found in WT mice,THS induced more C.R.translocation to spleen,liver and blood,but it was not noticed in TLR5-/-mice,whether it was subjected to THS or not.No statistical differences were noticed in the changes of intestinal mechanical barrier reflected by intestinal TEER and permeability between WT mice and TLR5-/-mice,and both types of mice had the same low microvascular perfusion,which might result in mucosa disruption.What's more,the proportion of Th1 in situ in lamina propria of WT moce decreased after THS,however,in TLR5-/-mice,Thl quantity there was always in low number.Furthermore,we found that mice lamina propria sourced TLR5+ LPDCs'ability of producing RA and inducing Thl differentiation were in the similar tend.In WT mice,RA production and Thl differentiation decreased after THS,whereas they were not influenced by THS attack in TLR5-/-mice.And Thl polarization induced by LPDCs of WT mice could be abrogated by RA receptor antagonist.Finally,RA treatment could alleviate bacterial translocation of WT mice post-THS,but not in TLR5-/-mice.ConclusionTLR5+ LPDCs work against bacterial translocation by inducing "local" Th1 cells polarization through TLR5 stimulation and RA production,but they become fragile after suffering THS.On the other hand,RA can alleviate bacterial translocation probably by imitating TLR5-RA dependent immune function of LPDCs.