Disordered Cutaneous And Intestinal Microbiota And Fecal Microbiota Transplantation In The Treatment Of Systemic Lupus Erythematosus

Background:Systemic lupus erythematosus(SLE)is a classic autoimmune disease characterized by facial butterfly erythematosus.In addition to the skin,kidney,blood,joints and other organ systems could also be affected.The etiology of SLE has not been fully elucidated,it is currently believed that the occurrence of SLE in genetically susceptible individuals is the result of the interaction of multiple factors such as environment and hormones.Excessive activation of immune cells and abnormal production of a large number of autoantibodies and inflammatory cytokines will cause inflammatory damage to multiple tissues and organs,which seriously affect human health.Clinically,the diagnosis of SLE is complicated and tedious.There are sevreal toxic and side effects of standard treatment drugs such as glucocorticoids and immunosuppressant,and some patients do not respond well to conventional drugs.Therefore,more specific and sensitive biomarkers,as well as safer and more effective therapeutic options are urgently needed.In recent years,the role of microbiome in the pathogenesis of SLE has also attracted researchers’ attention.At present,studies have found that imbalanced skin microbiome is involved in the occurrence of skin diseases.Skin lesions are one of the most common manifestation in SLE patients and are closely related to SLE activity.However,the disordered cutaneous microbiome in SLE lesions has not been reported.Alterations in intestinal microbiota have been observed in Rheumatoid arthritis(RA),Primary Sjogren’s syndrome(pSS)and other autoimmune diseases.The previous study of our group also found that fecal microbes from lupus mice could aggravate pristane-induced autoimmune response in lupus mice,indicating that intestinal microbiota is closely related to the occurrence and development of SLE and other autoimmune diseases.Fecal Microbiota transplantation(FMT)is considered as an effective treatment for recurrent Clostridium difficile(CD)infection.At the same time,a growing number of studies are finding more potential clinical indications for FMT beyond gastrointestinal diseases.However,there are few reports on autoimmune diseases.Previous studies of our group found that the intestinal microbiota of healthy mice could reduce the pathological phenotypes of lupus mice.However,whether FMT could restore intestinal microbiome in SLE patients and the safety and efficacy of FMT in SLE patients has not been reported.Based on the above background,comprehensively mapping skin and intestinal microbiota in SLE patients,and carring out the first clinical trial of FMT in SLE patients to explore its safety,efficacy and immune effect,will help to further clarify the role of microbiota in the pathogenesis of SLE,and to provide a solid theoretical and experimental basis for microbiota targeting therapy for SLE.Objective:To explore the alteration of cutaneous microbiota in patients with SLE,and to screen the specific skin microbiota related to SLE.To explore the alteration of intestinal microbiota in patients with SLE,and to screen the specific gut microbiota related to SLE.To explore the safety,efficacy and mechanisms of fecal microbiota transplantation in patients with active SLE.Methods:(1)69 SLE patients and 20 dermatomyositis patients with obvious erythema lesions on the face,and 49 healthy controls were included.The samples from SLE patients were divided into two groups:skin lesions and adjacent unaffected skin.The skin microbiota of each group was detected by 16SrRNA gene sequencing technology,and the diversity and species composition of microorganisms in each group were analyzed by bioinformatics method.The differential metabolic function of SLE skin microbiome were predicted by PICRUST software combined with KEGG database.(2)18 patients with active SLE and 20 healthy controls were included.The gut microbiota were detected by 16S rRNA gene sequencing.The microbial diversity,composition and enterotype in each group were analyzed by bioinformatics.Random forest algorithm was used to screen important species to build a SLE diagnosis prediction model based on gut microbiota.(3)20 SLE patients with SLEDAI>6 were recruited.FMT was administered once a week for three consecutive weeks along with standard treatment and the patients were followed for 12 weeks.At weeks 0,1,2,4,8,and 12,clinical manifestations,laboratory tests and adverse events were recorded,along with stool,serum,and peripheral blood mononuclear cell(PBMC)samples were collected.Safety was evaluated throughout the trial.The primary endpoint was the SLE Responder Index-4(SRI-4)at week 12.Microbial 16S rRNA gene sequencing and metagenomic sequencing were used to detect the alterations in gut microbiota abundance and microbial function of patients following FMT treatment,as well as to explore the gut microbiome characteristics of SRI-4 responders.The levels of short chain fatty acids(SCFAs)in the gut were detected by targeted metabolomics.Flow cytometry and flow protein quantitative detection were used to explore the dynamic changes of peripheral blood immune cell subsets and cytokines.Correlation analysis was used to explore the potential association between immunological changes,clinical laboratory tests,gut microbiota and metabolic profile.Results:(1)Compared with healthy controls,the a diversity in lesions and unaffected skin of SLE patients were significantly decreased.,and were significantly higher than those in DM lesions.The microbial P diversity in SLE lesions and unaffected skin was significantly higher than that in healthy controls.Compared with healthy controls,the relative abundance of Cutibacterium,Klebsiella and Prussiella 7 decreased significantly,while staphylococci and Corynebacterium 1 increased significantly in lesions.At species level,Staphylococcus aureus,Staphylococcus epidermidis and Staphylococcus hominis were significantly enriched in SLE lesions,while Staphylococcus wallis was significantly enriched in unaffected skin.Combining Staphylococcus aureus,Staphylococcus epidermidis and Staphylococcus hominis,the AUC value for distinguishing SLE lesions from healthy skin was 0.9832(95%CI:0.9650-1.001),and that for distinguishing healthy skin from SLE unaffected skin was 0.9346(95%CI:0.8880-0.9812).There were 11 differential microbiota related functional pathways between SLE lesions and healthy controls.Staphylococci was positively correlated with the up-regulated Staphylococcus aureus infection pathway in skin lesions(r=0.95,P<0.0001).Cutibacterium was significantly positively correlated with the down-regulated pentose phosphate pathway in skin lesions(r=0.736,P<0.0001).(2)Compared with healthy controls,the α diversity of gut microbiota in SLE patients were decreased,and the β diversity were increased,the gut microbiota structure was significantly biased compared with healthy controls.66.67%SLE patients belonged to Blautia type,27.78%belonged to Bacteroides type,and 5.56%belonged to Prevotella type.The bacterial taxa that significantly decreased in SLE patients were Bacteroides,Faecalibacterium,Prevotella,Roseburia Agathobacter,and Lachnoclostridium,while significantly increased bacterial taxa including Blautia,Bifidobacterium,Anaerostipes and Streptococcus.The AUC value of the combination of Blautia,Faecalibacterium and Bacteroides for distinguishing SLE and healthy controls was 0.95(95%CI:0.88-1),the sensitivity of the best critical point was 89%,and the specificity was 90%.(3)No serious adverse events were recorded during the study period,and the capsulized FMT therapy was well tolerated in SLE patients.The most frequent AEs were gastrointestinal complaints,which were mild,transient,and self-limited.The SLEDAI-2K score began to decrease significantly at week 2 after FMT treatment and persisted to week 12.The proportion of patients achieving SRI-4 response was 42.12%(95%CI:63.72%-23.14%)at the primary endpoint of week 12.The ratios of urine protein to creatinine(P/C ratio)were also significantly decreased at week 2 and week 8 compared with baseline.A significant decrease in the level of serum anti-dsDNA antibody were observed at week 12.FMT treatment significantly increased the gut microbial a diversity in SLE patients and SCFAs-producing taxa,while taxa which were associated with systemic inflammation were significantly decreased.After FMT treatment,the abundance of gut microbiota were mainly increased,including Gemmiger formicilis、Dorea longicatena、Blautia obeum、Dorea formicigenerans,while the abundance of Bacteroides fragilis decreased significantly.SRI-4 responders presented different gut microbiota from non-responders both before and after FMT treatment.Before FMT treatment,the combination of species with the top 14 contributions for distinguishing SRI-4 responders from non-responders with an AUC of 0.89(95%CI:0.74-1).After FMT treatment,there was a significant increase in the abundance of Bifidobacterium longum,Bifidobacterium bifidum,Bifidobacterium breve and unclassified Bifidobacterium in SRI-4 responders.At week 4,the gut microbiota related lipid metabolism pathway,fatty acid biosynthesis pathway,and glycerophospholipid metabolism pathway was significantly up-regulated,and the aging pathway and neurodegenerative disease pathway were significantly down-regulated.The abundance of glycoside hydrolase increased significantly after FMT treatment.The levels of most SCFAs in the gut increased significantly after FMT treatment,including acetic acid,butyric acid,valeric acid,isovaleric acid,caproic acid and heptanoic acid.After FMT treatment,the proportion of CD4~~+CD45RA~~+naive T cell subsets was significantly increased.The proportion of CD4~~+CD45RO~~+memory T cell subsets decreased significantly.In SRI-4 responders,the level of IL-6 in serum was significantly reduced at week 12 after FMT.There was a significant negative correlation between the abundance of bifidobacterium longum and serum IL-6 concentration;Bacteroides fragilis was negatively correlated with serum IL-10 and anti-dsDNA antibody.The urine protein to creatinine ratio(P/C ratio)was negatively correlated with the concentration of acetic acid in the gut.Conclusion:(1)Our research has verified the dysbiosis of cutaneous micobiome in SLE patients.Several bacterial taxa such as Staphylococcus and Corynebacterium were increased in SLE lesions,while Cutibacterium were decreased.Meanwhile staphylococci was closely related to the up-regulated Staphylococcus aureus infection pathway in SLE lesions.Staphylococcus aureus,Staphylococcus epidermidis and Staphylococcus hominis,which are significantly enriched in skin lesions could be potential microbial biomarkers for SLE diagnosis.(2)Our research has verified the dysbiosis of gut microbiome in SLE patients.SLE patients presented a new enterotype driven by Blautia,which is different from that of healthy people.Bacteroides and Faecalibacterium were significantly reduced in SLE patients,while Blautia was significantly enriched.The AUC value of the combination of this three bacterial taxa could be potential microbial biomarkers for SLE diagnosis with good sensitivity and specificity.(3)This is the first study demonstrated that FMT is a feasible,safe,and potentially effective therapy for SLE patients,which significantly reduced SLEDAI-2K score,urinary protein/creatinine ratio,and serum anti-dsDNA antibody in patients.FMT alters effectively the gut microbial community and metabolic function from a pro-inflammatory style to an anti-inflammatory style,thereby reducing levels of serum IL-6 and peripheral CD4~~+ memory/naive ratio to improve lupus activity.