Alterations of Gut Microbiome Accelerate Multiple Myeloma Progression By Increasing the Relative Abundances of Nitrogen Recycling Bacteria
Authors: Jian Xingxing , Zhu Yinghong , Ouyang Jian , Lei Qian , Xia Jiliang , Zhang Jingyu , Guo Jiaojiao , Wang Jinuo , Lin Jingchao , Su Mingming , Guan Yongjun , Li Guancheng , Wu Minghua , Li Jian , Xiang Juanjuan , Xie Lu , Qiu Lugui , Jia Wei , Zhou Wen 
Published: February 2020

Background: Gut microbiome alterations are closely related to human health and linked to a variety of diseases. Although great efforts have been made to understand the risk factors for multiple myeloma (MM), little is known about the role of the gut microbiome and alterations of its metabolic functions in the development of MM patients.
Materials and Methods: From a cohort of 19 newly diagnosed patients with MM and 18 gender- and age-matched healthy controls (HCs), fresh fecal samples and fasting serum samples were collected, respectively, for metagenomic sequencing and metabolomic detection. Subsequently, the high-quality reads were used for taxonomic classification using Kraken and for functional annotation using MetaCV, and metabolic profiling was statistically analyzed using SIMCA-P. On the R platform, bacterial diversity was analyzed using vegan package and differential taxa were identified using DESeq2 package. In addition, we performed fecal microbiota transplantation (FMT) experiments in C57BL/KaLwRij mice which can be artificially induced to develop MM by injecting 5TGM1 MM cells.
Results: Here, significant differences in bacterial composition between MM and HC were first discovered (PERMANOVA, P=0.001), and greater bacterial richness in MM was evaluated by Shannon index (P=0.045). Several species with significant difference between the two groups were further confirmed by using qPCR of 16S rDNA V1-V2 regions in an expend cohort. Specifically, short-chain-fatty-acid producing bacteria such as Clostridium butyrate were substantially shrunk, while nitrogen recycling bacteria such as Klebsiella sp. were significantly enriched in MM. And, the MM-enriched bacteria showed higher abundance in MM patients with ISS-Ⅲ than those of MM patients with ISS-Ⅱ.
We measured much more urea in the serum of MM than those in HC (P&lt;0.001). Meanwhile, the MM-enriched bacteria were significantly correlated to the differential metabolites in host serum, suggesting strong metabolic interactions between microbes and the host. The MM-enriched bacteria were also considered to cause the enhanced urease (URE, P=0.016) and glutamine synthase activities (GS, P=0.015).
To investigate the function of the microbe-host interactions in MM progression, we performed FMT experiments in C57BL/KaLwRij mice with MM and HC feces suspension (three groups: FMT_MM, FMT_HC, PBS). As a result, we observed faster MM progression in FMT_MM mice and slower MM progression in FMT_HC mice than that of PBS mice. Notably, much more L-glutamine (Gln) in the bone marrow of FMT_MM mice was detected (FMT_MM=2.07±0.04 mM, FMT_HC=1.23±0.10 mM, PBS=1.33±0.09 mM, P&lt;0.05). With one accord, more Gln was also measured in the serum and cecum of FMT_MM mice. Therefore, we speculated that MM-enriched bacteria efficiently hydrolyze urea to de novo synthesize Gln, which may accelerate MM development. Indeed, in the cecum contents of FMT_MM mice, more urea and higher URE and GS activities were all detected (P&lt;0.05). Moreover, the accumulating urea was probably due to the declining renal function, as the experimental mice all exhibit more protein deposition of IgG2b kappa on the renal tubules than that of normal mice. Particularly, FMT_MM mice had the highest amount of IgG2b kappa, followed by FMT_HC mice and PBS mice (OD: FMT_MM=0.15±0.02, FMT_HC=0.08±0.02, PBS=0.09±0.02, Normal=0.04±0.00, P&lt;0.05). Further, we performed additional experiments by gavage, and the results validated that the increase of Klebsiella pneumoniae abundance accelerated MM progression in vivo, while Clostridium butyrate had an opposite effect.
Conclusions: Taken together, we showed that the gut microbiome in MM patients played an active role in malignant progression and that the microbe-host interactions were predominantly involved in nitrogen recycling and utilization in MM, which open new avenues for MM treatment via monitoring and manipulation of intestinal flora. Furthermore, these findings lead us to propose a broad mechanism, in which the increasing urea or NH4+ alters the gut bacterial composition, leading to preferential accumulation of nitrogen-recycling bacteria and suppression of the bacteria producing short-chain fatty acids. The altered gut microbiome should be conducive to nitrogen recycling and utilization by the host-microbe superorganism.

Disclosures
No relevant conflicts of interest to declare.