| Objective:Meibomian gland dysfunction（MGD）is the main cause of many types of ocular surface diseases such as dry eye disease（DED）,keratitis and conjunctivitis associated with the eyelid margin.MGD and has become an important disease that seriously affects ocular surface health[1].Studies have found that the human ocular surface is rich in microbial diversity,and microbial infection and dysbacteriosis are closely related to the occurrence and development of MGD.But their interaction relationship is still unclear.NOD-like receptors and commensal bacteria constitute the body’s"immune sentinel",helping the host immune system in defending against infectious microorganisms and maintaining environmental homeostasis[2].However,there are few study the mechanism of NOD-like receptors in the microbial infection of MGD.In this study the differences between the meibum microorganisms in healthy people and people of DED causing by MGD was researched through 16SrRNA high-throughput sequencing as well as microbial markers and preliminarily screen potential probiotics.At the same time,the pathogenic bacteria in the meibum samples of MGD patients were isolated by traditional culture methods,and the interaction between the two bacteria in vitro was preliminarily explored through bacterial co-culture confrontation experiments in vitro.Furthermore,pathogenic bacteria and potential probiotics were used to intervene in C57 mice in vitro and in vivo to observe the structure of meibomian gland tissue,changes in MGD disease biomarkers and NLRP3/Caspase-1-mediated changes in cell pyrosis pathway.This research will explore the mechanism of bacteria participating in MGD and the role of potential probiotics,which may find some new way for the research and treatment of MGD.Methods:1.Through cluster sampling,cross-sectional investigation and collection of palpebral samples were carried out on individuals aged≥40 in six regions of Yunnan Province,and the differences between the microorganisms of meibum in healthy people and those with DED combine with MGD were compared by 16SrRNA high-throughput sequencing in order to search for microbial markers of disease and normal populations and potential probiotic.2.In order to study the pathogenic mechanism of bacteria,the meibum of clinically confirmed MGD patients were collected,the pathogenic bacteria with the highest positive rate（Staphylococcus epidermidis）were isolated,and the strains were grouped according to the severity of MGD disease to detect the activity of virulence factors:biofilm,neutral protease,and phospholipase.3.In order to clarify the interaction between pathogenic bacteria and potential probiotics,the standard strain of potential probiotics was used to co-culture with clinically isolated Staphylococcus epidermidis to observe the results of the confrontation between the two bacteria in vitro,and the biofilm adhesion,neutral protease and phospholipase activities of Staphylococcus epidermidis were detected.4.Through Transwell co-culture method,Staphylococcus epidermidis SE,SE and probiotic mixture were used to intervene in the in vitro transplantation model of mouse meibomian gland,observe the changes of the histological structure of mouse meibomian gland,and detect MGD disease biomarkers（KRT-1\KRT14\PPAR-γ）and NLRP3\Caspase-1\GSDMD\IL-1β\by the methods of immunofluorescence staining,immunohistochemistry,WB,QPCR.5.The chronic infection model of Staphylococcus epidermidis was continuously stimulated by dropwise stimulation of the ocular surface of C57 mice,and the clinical indicators of ocular surface status were observed by corneal fluorescence staining,meibomian infrared photography,tear secretion experiment.6.Pathogenic bacteria and probiotic co-culture solution,probiotic exosomes and probiotic lysate were dropped in the ocular surface of model mice.The changes of meibomian gland tissue structure was observed and the expression changes of MGD disease biomarkers（KRT-1\KRT14\PPAR-γ）and NLRP3\Caspase-1\GSDMD\IL-1β\IL-18 were showed by immunofluorescence staining,immunohistochemistry,Western blot,q PCR methods.7.All experimental data are statistically analyzed and plotted using Graphpad Prim and R software;Quantitative analysis of immunohistochemistry results using Image J software;For continuous variables,independent sample t-test was used in only two groups,one-way ANOVA was used when there were three or more groups,and LSD-t test or Dunnett-t test was used for pair-by-two comparison after there was statistical significance;Categorical variables were analyzed using chi-square test or Fisher exact probability method;AUC value measures diagnostic value,and time-dependent ROC calculates prognostic value;The correlation of continuous variables is measured by the correlation coefficient r.Results:1.In the meibum samples,the microbiota species diversity was greater in the DED group,and there was a significant difference in community structure between the DED group and the Normal group（P<0.001）.2.At the Phylum level,the microbial F/B ratio in the DED group was significantly lower than in the normal group（P<0.001）,suggesting that the alteration of the tarsal lipid microbial composition was similar to the alteration of the intestinal microbial imbalance which showed that even the alteration of individual components may affect ocular surface immune homeostasis,trigger DED and promote its vicious cycle.3.29 discriminative biomarkers in the DED group and 20 in the normal group were identified.The top five biomarkers in the DED group were Gammaproteobacteria（D2）,Burkholderiaceae（D4）,Betaproteobacteria（D3）,Ralstonia（D5）,and Pelomonas（D5）.The top five biomarkers in the normal group were Rhizobiales（D3）,Bradyrhizobium（D5）,Xanthobacteraceae（D4）,Alphaproteobacteria（D2）,and Aeromonadaceae（D4）.4.Six microbial genera showed an ability to jointly distinguish DED samples from Normal samples:Bradyrhizobium,Ralstonia,Burkholderia-Caballeronia-Paraburkholderia,Archaeon,Aeromonas,and Variovorax.The ability to predict DED with a sensitivity of 84.8%and a specificity of 67.3%.5.Bradyrhizobium is the most abundant microbial genera in normal samples,and has the largest number of positive and negative correlations with the largest number of other characteristic microorganisms（both DED group and Normal group）,which may play an important role in the regulation of the host microbial environment.6.Bradyrhizobium is negatively correlated with the degree of meibomian gland deletion,and positively correlates with tear film rupture time and OSDI scale score.Zygomycetes were positively correlated with meibomian gland loss.Sphingomonas are negatively correlated with wind speed and radiation,and positively correlated with temperature.Methyl bacteria are positively correlated with drinking habits.Meso-rhizobia is inversely correlated with smoking status.Archaea were positively correlated with ethnicity but negatively correlated with annual sunshine time.7.The phosphatidylinositol-3 kinase（PI3K）-protein kinase B（AKT）signaling pathway,phosphotransferase system（PTS）,phospholipase D signaling pathway,mammalian rapamycin signaling pathway target,Apelin signaling pathway,glycosylphosphatidylinositol（GPI）-anchored protein,biofilm formation,and bacterial invasion epithelial cells were enriched.Rhizoa bradyphyllis and Burkholderia-Caballronia-Suberkholderia were positively correlated with bacterial invasion of epithelial cells,glycosylphosphatidylinositol（GPI）anchor proteins,and phosphotransferase system（PTS）.8.Among the 231 blepharposis samples collected clinically,138 samples detected bacteria（detection rate of 59.7%）,and 76 strains of Staphylococcus epidermidis（SE）were detected among positive bacteria,with a positive rate of 56%.Among them,the number of SE strains isolated from patients with mild MGD was 7 strains（41.1%）,the number of SE isolated from patients with moderate MGD was 48 strains（60%）,and the number of SE isolated from patients with severe MGD was 21（60%）.SE remains the first place in clinically isolated pathogen of MGD.9.The main virulence factors of SE:neutral protease and phospholipase activity and biofilm formation capacity,varied with SE strains isolates from different grade of disease severity.10.After co-culture with Bradyrhizobium live bacteria,Bradyrhizobium exosomes and Bradyrhizobium lysate,the number of SE colonies,the neutral protease and phospholipase activities of SE and the adhesion ability of biofilms were reduce.11.SE intervention and stimulation of in vitro implants of mouse tarsal plates and ocular surfaces of living mice can increase the expression of MGD disease marker KRT1\KRT14\PPAR-γand activate NLRP3/Caspase-1 cell pyrosis pathway which will promote the release of IL-1β\IL-18,and increase the inflammatory damage of meibomian gland tissue.The intervention of Bradyrhizobium,its exosomes and lysate can inhibited the expression of KRT1\KRT14\PPAR-γ,and reduced the damage of meibomian tissue caused by the intervention of the activation of NLRP3/Caspase-1pathway and the release of downstream inflammatory factors.Conclusions:1.Meibum has a rich and stable microbial taxa which will be less contaminated by conjunctival sac bacteria,and may become a more stable ocular surface microbial detection specimen in the future.Changes in the abundance and species of microbial in meibum can prompt the occurrence and development of DED with MGD.2.Staphylococcus epidermidis is the pathogenic bacterium with the highest clinical isolation rate of MGD,which can cause abnormal expression of meibomian keratinizing factor and lipid differentiation factor,promote keratinization of the glands and changes in lipid composition,thereby causing or aggravating MGD.3.Staphylococcus epidermidis can activate NLRP3/Caspase-1-mediated cell pyrosis pathway,causing inflammatory damage of meibomian gland histiocyte cells,and the degree of activation of NLRP3/Caspase-1 varies between SE strains isolated from different disease severity groups.4.Bradyrhizobium is a potential probiotic mined in the 16SrRNA sequencing information.Bradyrhizobium and its bacterial products（exosomes,lysate）can inhibit the colony growth of SE in vitro,reduce its virulence factor activity and bacterial biofilm adhesion.At the same time,the intervention of Bradyrhizobium could inhibit the overexpression of keratinizing factor and lipid differentiation factor KRT1\KRT14\PPAR-γin mouse meibomian gland tissue caused by SE stimulation.Bradyrhizobium can regulate the activation degree of NLRP3/Caspase-1,reduce the release of inflammatory factors,thereby reducing meibomian tissue damage caused by SE.Bradyrhizobium and the bacterial products are expected to become a new treatments way for MGD. |
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