Mechanistic Study On Intestinal E. Cloacae In Promoting Osteolytic Bone Disease Of Multiple Myeloma

Background:Multiple myeloma（MM）is characterized by the manifestations of destructive bone lesions,kidney injury,anemia,and hypercalcemia.Among them,bone disease is a cardinal complication of MM,which often lead to skeletal morbidity,usually referred to as skeletal-related events（SREs）.Defining as severe bone pain,pathological fractures,and spinal cord compression,SREs affect not only the quality of life but also their overall survival,requiring us to explore new angles for the prediction of severe SREs,elucidate the pathogenesis and then intervene with them to reduce the incidence of severe SREs.The gut microbiota is becoming increasingly recognized for its influence on diseases and therapeutic responses.Based on the evidence of the dominance of pathogenic bacteria in MM patients,it is significant to elucidate the characteristic gut microbiota of MM patients with osteolytic lesions,reveal the potential predictors,and identify new pathogenesis and potential strategies of MM patients with osteolytic lesions.Methods:（1）Shotgun metagenomic sequencing of fecal samples was applied to analyze the differential bacteria in MM patients with osteolytic lesions;q PCR was used to validate the sequenced results.Integrated with clinical data,the differential bacteria of MM patients with osteolytic lesions were screened.（2）In vivo experiments of fecal microbiota/single bacteria transplantation were utilized to confirm whether the differential bacteria promoted osteolysis by detecting bone destruction of ELISA and Micro-CT and tumor burden of ELISA and in vivo imaging in MM mice.（3）With the chemical chromogenic method,the concentrations of NH₄⁺in MM patients/mice were detected and correlated with the abundance of differential bacteria.A gavage experiment with NH₄⁺was administered to detect the osteolysis in vivo,together with osteoclastogenesis/osteoblastogenesis was performed to testify the promotive function of NH₄⁺in vitro.The protein expression of CCL3 was detected by WB,and the concentrations of CCL3 in MM patients/mice were examined by ELISA,together with correlated analysis with NH₄⁺concentration.Further,IP was performed to confirm the acetylation of CCL3 in MM cells.（4）The differentially expressed deaminase genes of E.cloacae were verified by q PCR in the MM mice/patients.Then,the strains of E.cloacae-dcd ^Delwere constructed,followed by the verification of the reduction of NH₄⁺in vitro and in vivo.Subsequently,in vivo experiment was performed to explore the alleviation of E.cloacae-dcd ^Delcompared with the wild-type strain.As well,whether probiotics combined with E.cloacae would remit the osteolysis in MM was conducted.Results:（1）The analysis of shotgun metagenomic sequencing demonstrated the characteristic microbiome in MM patients with osteolytic lesions,which defined the domination of pathological bacteria and the reduced probiotics.Especially,the abundance of intestinal E.cloacae was significantly increased in MM patients with osteolytic lesions,as well as the significant correlation of the severity of osteolysis was defined.（2）Fecal microbiota transplantation from MM patients with severe osteolysis showed more severe osteolysis in MM mice.Longitudinal analysis of the colonization demonstrated the gradual domination of E.cloacae,suggesting the potential function of osteolysis.Furthermore,single bacteria transplantation with E.cloacae further confirmed the favorable osteolysis in MM mice.（3）The concentration of NH₄⁺was significantly increased in MM patients with osteolysis and was positively correlated with the abundance of E.cloacae.Analysis of clinical characteristics demonstrated a positive correlation between the concentration of NH₄⁺and osteolysis in MM patients.Furthermore,NH₄⁺was confirmed to promote osteolysis in MM mice,as well as accelerate osteoclastogenesis in vitro.And an increased expression of CCL3 was detected by WB in MM cells and ELISA in MM mice,which was acetylated by NH₄⁺in vitro experiment.（4）The differential expression of E.cloacae deaminase gene dcd was demonstrated in MM mice.Furthermore,the strain of E.cloacae-dcd ^Delwas testified to alleviate osteolysis in MM mice,and so did the combination of probiotics and E.cloacae.Conclusion:（1）MM patients with osteolysis are characterized by the dominance of intestinal E.cloacae,which is significantly related to osteolysis in MM.（2）E.cloacae promote osteolysis by accumulating NH₄⁺in MM.（3）Accumulated NH₄⁺accelerate osteoclastogenesis by acetylate CCL3,thereby promoting osteolysis in MM.（4）Modulation of gut microbiota（modified engineering bacteria and supplements of probiotics）may alleviate osteolysis in MM.Figures:48,tables:48,references:132...