Fecal microbiota from MRL/lpr mice exacerbates pristane-induced lupus

Fecal microbiota from MRL/lpr mice aggravated lupus autoimmunity

To investigate the effects of FMT on the development of lupus disease, we transferred the fecal microbiota of MRL/lpr mice, that of MRL/Mpj mice or PBS gavage to the pristane-induced lupus-like mouse model (respectively termed as FMT-Lpr, FMT-Mpj, and FMT-PBS) (Fig. 1A). In the final observation stage, the urine protein levels of the FMT-Lpr mice were significantly elevated compared to those of the FMT-Mpj mice and FMT-PBS mice (p FMT-Lpr VS FMT-Mpj = 0.0142; p FMT-Lpr VS FMT-PBS = 0.0349) (Fig. 1B). Hematoxylin and eosin (H&E) staining and immunofluorescence of the renal sections revealed more severe glomerular abnormalities (Fig. 1C) as well as more IgG and C3 depositions in the glomeruli of the FMT-Lpr mice (Fig. 1D,E). We further detected the mRNA expression levels of proinflammatory cytokines in the kidney and found significantly increased Il12a and Il18 mRNA expression in FMT-Lpr mice compared to FMT-Mpj mice and PBS-gavaged mice (Il12a: p FMT-Lpr VS FMT-Mpj = 0.0112; p FMT-Lpr VS FMT-PBS = 0.0427. l118: p FMT-Lpr VS FMT-Mpj = 0.0111; p FMT-Lpr VS FMT-PBS = 0.0484) (Fig. 1F, Figure S2A). The percentages of T and B lymphocytes in the renal tissues were not significantly different among the three groups (Figure S2B).

Fig. 1figure 1

FMT-Lpr mice accelerated the lupus pathogenesis. A The schematic timeline of the experiment design. B Dynamic changes of urine protein levels in FMT-Lpr mice, FMT-Mpj mice, and FMT-PBS mice with the disease progression (upper) and urine protein levels measured at the final week of the experiment period (down). C Representative images of HE staining of kidney sections. Representative images of IgG (D) and C3 (E) depositions in the kidney detected by immunofluorescence. Scale bar, 100 μm. magnification × 400. F Il12a, Il18 mRNA expression levels in the kidney. G Flow cytometric analysis of the plasma cell percentages gated on the CD45 + cells in the spleens and lymph nodes (SP: spleen; DLN: draining lymph node; MLN: mesentery lymph nodes; PP: peyer’s patch). *p < 0.05. FMT-Lpr, n = 10 ~ 11; FMT-Mpj, n = 9 ~ 10; FMT-PBS, n = 6 ~ 7

Fecal microbiota transplantation had no obvious effect on the production of the anti-dsDNA antibodies (Figure S2C). The percentages of Th1, Th2, and Th17 cells in the spleen and lymph nodes were not significantly different among the three groups (Figure S2D). The increased percentages of plasma cells could be identified in the lymph nodes rather than the spleens of the FMT-Lpr mice, including draining lymph nodes, mesentery lymph nodes, and Peyer’s patches. (DLN: p FMT-Lpr VS FMT-Mpj = 0.0033; p FMT-Lpr VS FMT-PBS = 0.0274. MLN: p FMT-Lpr VS FMT-Mpj = 0.0193. PP: p FMT-Lpr VS FMT-Mpj = 0.0077; p FMT-Lpr VS FMT-PBS = 0.0108) (Fig. 1G). Therefore, the transplantation of fecal microbiota from MRL/lpr mice exacerbates the renal damage of the pristane-induced lupus mouse model.

FMT changed the intestinal immunological states in pristane-induced lupus mice

Pristane-induced lupus mice gavaged with fecal microbiota, regardless of the donor source being MRL/lpr mice or MRL/Mpj mice, maintained intestinal structural integrity without obvious inflammatory infiltration in the small or large intestine, which was identified by H&E staining (Fig. 2A,B). To test whether the intestinal permeability was affected during the fecal microbiota transplantation, we measured the transcription level of ZO-1, a tight junction protein and found the decreased mRNA expression of ZO-1 in the small and large intestines of the FMT-Lpr mice (small intestine: p FMT-Lpr VS FMT-Mpj = 0.0190; p FMT-Mpj VS FMT-PBS = 0.0463. large intestine: p FMT-Lpr VS FMT-Mpj = 0.0243), which was further validated by the immunofluorescence (Fig. 2C,D). Therefore, FMT from MRL/lpr mice may affect intestinal barrier function. We also detected the transcript levels of proinflammatory cytokines and revealed that the mRNA expression of Tnf, Il18, Ifng, and Il6 was significantly elevated in the small or large intestines of FMT-Lpr mice compared with FMT-Mpj mice or FMT-PBS mice (Fig. 2E,F, Figure S3A-B).

Fig. 2figure 2

FMT-Lpr mice have altered intestinal permeabilities and immunological states. Representative images of HE staining of the small intestine (A) and the large intestine (B). Scale bar, 50 μm. magnification × 400. ZO-1 expressions detected by immunofluorescence and the mRNA levels measured by RT-PCR in the small intestine (C) and the large intestine (D). Tnf, Il18, and Ifng mRNA expression levels in the small intestine (E) and Il6, Tnf, Il18, and Ifng mRNA expression levels in the large intestine (F). Flow cytometric analysis of the percentages of CD11b + cells gated on the CD45 + CD3 − CD19 − cells in the small intestine (G) and the percentages of B220 − IgD − CD138 + cell characterized as the plasma cells gated on the CD45 + cells in the large intestine (H). *p < 0.05. **p < 0.001. FMT-Lpr, n = 8 ~ 11; FMT-Mpj, n = 5 ~ 10; FMT-PBS, n = 5 ~ 7

Furthermore, to investigate the composition of immune cells in the intestinal mucosa, we performed flow cytometry to analyze the percentages of T cells, B cells, innate lymphoid cells (ILCs), and CD11b + cell subsets (Figure S1C). The proportions of ILC1s and ILC3s were not significantly different among the three groups. However, the ILC2 proportion was increased in the FMT-Lpr mice and FMT-Mpj mice compared with FMT-PBS mice in the large intestine (Figures S3C-D). Furthermore, in both FMT-Lpr mice and FMT-Mpj mice, the CD8 + T-cell percentages were decreased in the small intestine, and the CD4 + T-cell percentages were reduced in the large intestine compared with those of FMT-PBS mice (Figure S3E-F), indicating that the altered tendencies of T lymphocytes are FMT-related instead of lupus-related. CD4 + and CD8 + T cells in both sites were dominated by the effector subtype characterized by increased proportions of the CD44 + CD62L − subset (Figure S3G-H). The CD11b + cell percentage in the large intestine was also associated with FMT (Figure S3I). We further subclassified CD11b + cells based on the Ly6C and Ly6G markers, of which the Ly6C-Ly6G + subset was considered Ly6G + neutrophils and the Ly6C + Ly6G − subset was representative of Ly6C + monocytes/macrophages. The percentage of Ly6G + neutrophils was significantly increased in the large intestine of the FMT-Lpr mice and FMT-Mpj mice compared with the FMT-PBS mice, while the percentage of Ly6C + monocytes/macrophages showed no significant differences among the three groups (Figure S2J). Remarkably, FMT from MRL/lpr mice but not MRL/Mpj mice increased the CD11b + cell percentage in the small intestine (p FMT-Lpr VS FMT-Mpj = 0.0318) (Fig. 2G) as well as the plasma cell percentage in the large intestine (p FMT-Lpr VS FMT-Mpj = 0.0311; p FMT-Mpj VS FMT-PBS = 0.0098) (Fig. 2H). Therefore, FMT can affect the intestinal immunological status of pristane-induced lupus mice. Feces from MRL/lpr mice could alter specific immune cell types, such as plasma cells in the intestine. These findings also indicated that fecal microbiota from different origins has differentiated pathological and immunological effects.

FMT altered the gut microbial structure and composition in the pristane-induced lupus mice

We next investigated whether the gut microbial structure and composition were associated with FMT and disease progression. Fecal samples from the three groups were collected at the beginning (the first week after the intraperitoneal injection of pristane, termed T1), middle (the 5th month, termed T2), and end of the experiment (the 9th month, at the end of the experiment, termed T3), and 16S rRNA sequencing was performed to clarify the most significant discrepancy in the gut microbial structure at different stages of the lupus process. According to the 16S rRNA sequencing results, we found no distinct differences in alpha diversities among the three groups in terms of richness, evenness, and overall diversity at each time point of sample collection, as indicators such as the Shannon, Simpson, and Chao indices were compared among the three groups (Table S2). However, during the progression of lupus, the gut bacterial community of FMT-Lpr mice became gradually distinct from that of FMT-Mpj mice and FMT-PBS mice, especially at the end of the experiment, consistent with the phenotypic changing trends (Fig. 3A). Moreover, the gut microbiome could be optimally partitioned into two distinct clusters based on the structure of advantageous flora at the phylum level, as indicated by the Calinski‒Harabasz index (Figure S4A), and could be classified as one of two types of enterotypes. Enterotype 1 was mainly composed of Firmicutes, and enterotype 2 was dominated by Bacteroidetes. The gut microflora of the FMT-Lpr mice was dominated by enterotype 1, while the FMT-Mpj mice and FMT-PBS mice had similar microbial enterotype compositions, with enterotype 2 occupying an advantage (Figure S4B), which further validated that the gut microbial structure of the FMT-Lpr mice was distinct from that of the FMT-Mpj mice and FMT-PBS mice.

Fig. 3figure 3

FMT changed the microbial community structures and compositions. A The sample distribution of the gut microbiome in the principal coordinate analysis (PCoA) plot at different time points. Different color represents different group (red: FMT-Lpr, blue: FMT-Mpj, green: FMT-PBS). T1, T2, and T3 respectively refer to the beginning, middle and end of the experiment. B Profiles of the major phylum with the relative abundance > 0.01 per group. C The Firmicute/Bacteroide ratio among the three groups. D Profiles of the relative abundances of the top 20 predominant taxa per group at the genus level. E Characteristic bacterial taxa for each group shown by LDA score > 2.0 at the genus level. F The relative abundances of the prevotella, alloprevotella, streptococcus, and bilophila among the three groups. *p < 0.05. **p < 0.001. FMT-Lpr, n = 11; FMT-Mpj, n = 10; FMT-PBS, n = 7

Next, we performed metagenomic sequencing to provide deep insight into the taxonomic and structural differences among the three groups at T3, the timepoint at which the groups displayed significantly discrepant phenotypes and microbial structural distinctions as indicated by the 16S rRNA sequencing results. In fact, the results were further verified by PCoA on the basis of the metagenome data, revealing the separation of the sample distributions among the three groups (Figure S4C). Focusing on the microbiota taxonomic composition, we found that Bacteroides, Firmicutes, Actinobacteria, and Proteobacteria were still the four major phyla at the phylum level (Fig. 3B). The ratio of Firmicutes/Bacteroidetes increased gradually from FMT-Lpr mice to FMT-Mpj mice to FMT-PBS mice (Fig. 3C), which is consistent with previous results that a lower Firmicutes/Bacteroidetes ratio could be detected in SLE-related dysbiosis. At the genus level, Bacteroides and Prevotella were the dominant taxa with varied relative abundances among the three groups (Fig. 3D). According to the Kruskal‒Wallis test, the number of microbial taxa at the genus level distinguishing one group from the other two groups was 149 for FMT-Lpr mice, 35 for FMT-Mpj mice, and 118 for FMT-PBS mice. Incorporated with linear discriminant analysis (LDA), we screened out the representative genera for pristane-induced lupus mice with different fecal microbiota interventions (Fig. 3E). Prevotella, Alloprevotella, and Bacteroides were enriched in FMT-Lpr mice; Candidatus_arthromitus were enriched in FMT-Mpj mice, and Adlercreutzia, Olsenella, Atopobium, Collinsella, Holdemania, Turicibacter, Streptococcus, and Bilophila, and Unclassified_f__Coriobacteriaceae were enriched in FMT-PBS mice. Notably, among these representative taxa, the relative abundances of Prevotella, Alloprevotella, Bilophila, and Streptococcus exhibited gradually altering tendencies from FMT-Lpr mice to FMT-Mpj mice to FMT-PBS mice (Fig. 3F, Figure S5). By means of metagenome sequencing’s advantages in species-level bacterial classification, we found that several species belonging to the above characteristic genera were also meaningful as discriminative signatures for the three groups (Figure S6). In particular, several strains of Prevotella were predominantly increased in FMT-Lpr mice and may serve as microbial biomarkers of aggravated states of lupus.

FMT led to a disordered metabolic profile in the pristane-induced lupus mice

The metabolomes of fecal samples from the three groups were characterized and compared based on the LC‒MS platform. Distinct clusters related to pristane-induced lupus mice with different fecal microbiota transplantations could be visualized in the PLS-DA diagram, and the separations of the metabolic distributions among the three groups were significant (Fig. 4A). Metabolites that had distinctive levels among the three groups with p < 0.05 were mostly annotated in the metabolism pathway especially amino acid metabolism (Fig. 4B). The top 30 differentiated metabolites were presented through a heatmap, and we found that the levels of the majority of significant metabolites were reduced in the FMT-Lpr mice and elevated in the FMT-PBS mice while the FMT-Mpj mice showed transitional changing trends (Fig. 4C). Next, the significant metabolites with VIP scores > 1.0 in multivariate statistical analysis were compared in pairs and visualized in bubble diagrams respectively. Many metabolites could be used to distinguish pristane-induced lupus mice receiving different fecal microbiota interventions. For example, sorbitan stearate, alpha-D-galacturonic acid, and L-glutamate could differentiate FMT-Lpr mice from FMT-Mpj mice (Figure S7), sorbitan stearate, and L-isoleucine could differentiate FMT-Lpr mice from FMT-PBS mice (Figure S8), and linoleoyl ethanolamide, dihydrocoumarin, LPC, and L-valine could differentiate FMT-Mpj mice from FMT-PBS mice (Figure S9). The discriminative metabolites annotated in the KEGG pathway database are listed in Table S3, and some metabolites, such as L-valine, L-isoleucine, choline, and pantothenic acid, had differentiated relative abundances among the three groups, which represented different pathological states of the pristane-induced lupus mice. Therefore, these findings suggested that the metabolites of the gut microbiota may play important roles in the disease progression and pathophysiology of lupus.

Fig. 4figure 4

FMT altered the gut metabolite profiles. A Partial least squares discriminant analysis (PLS-DA) of fecal metabolomics data from three groups. Each dot represents one fecal sample. Different color represents different group (purple = FMT-Lpr, red = FMT-Mpj, blue = FMT-PBS). B The KEGG functional pathways enriched by differentiated metabolites. C The top 30 differentiated metabolites in the FMT-Lpr, FMT-Mpj, and FMT-PBS mice visualized on a heat map. FMT-Lpr, n = 11; FMT-Mpj, n = 10; FMT-PBS, n = 7

Characteristic functional pathways in the pristane-induced lupus mice with FMT

We found that the gut microbial and metabolite components were significantly different in pristane-induced lupus mice under FMT. It is necessary to investigate how these microbiota and metabolites are interrelated and affect the functional variations in different microbial ecosystems. The significantly differentiated KEGG pathways with LDA scores > 2.0 based on the metagenome results were identified and compared among the three groups. The number of functional KEGG pathways enriched by differential microbiota in the pairwise comparison was 27 for differentiating the FMT-Lpr mice and FMT-Mpj mice (Fig. 5A), 42 for differentiating the FMT-Lpr mice and FMT-PBS mice (Fig. 5B), and 3 for differentiating the FMT-Mpj mice and FMT-PBS mice (Fig. 5C). The KEGG pathways enriched by differentiated fecal metabolites based on the metabolomics results were also analyzed in pairs, presented in the form of bar graphs ordered by the enriched p value (Fig. 5D–F). The overlapping pathways, which involved the roles of characteristic microbial taxa and fecal metabolites, were considered highly important in the role of lupus progression. Functional pathways such as cyanoamino acid metabolism, ABC transporters, and glycerophospholipid metabolism made sense in discriminating FMT-Lpr mice and FMT-Mpj mice. Cyanoamino acid metabolism; valine, leucine, and isoleucine degradation; and protein digestion and absorption were associated with disparities in FMT-Lpr mice and FMT-PBS mice. There were no significant KEGG pathways enriched by both differentiated microbes and metabolites between FMT-Mpj and FMT-PBS.

Fig. 5figure 5

Differential enriched functional KEGG pathway in the pristane-induced lupus mice with different FMT. The characteristic KEGG functional pathways for each group based on the metagenome results were analyzed in the pairwise comparison, shown by LDA score > 2.0 between the FMT-Lpr and FMT-Mpj (A), the FMT-Lpr and FMT-PBS (B), and the FMT-Mpj and FMT-PBS (C). The KEGG functional pathway enriched by the differential metabolites analyzed in pairs between the FMT-Lpr and FMT-Mpj (D), the FMT-Lpr and FMT-PBS (E), the FMT-Mpj and FMT-PBS (F). G The relative abundances of the overlapped KEGG pathways including the cyanoamino acid metabolism (upper), valine, leucine, and isoleucine degradation (down) among the three groups. H The relative abundances of the L-isoleucine, L-valine, L-glutamate, L-tyrosine, and L-threonine among the three groups. *p < 0.05. **p < 0.001. FMT-Lpr, n = 11; FMT-Mpj, n = 10; FMT-PBS, n = 7

Remarkably, among these representative KEGG pathways, we observed a gradual decreasing tendency of the relative abundances of cyanoamino acid metabolism (p FMT-Lpr VS FMT-Mpj = 0.0294; p FMT-Lpr VS FMT-PBS = 0.0326), and valine, leucine, and isoleucine degradation (p FMT-Lpr VS FMT-PBS = 0.0214) from the FMT-Lpr mice to FMT-Mpj mice to FMT-PBS mice (Fig. 5G, Figure S10). Significant metabolites such as L-isoleucine, L-valine, L-glutamate, L-tyrosine, and L-threonine participated in cyanoamino acid metabolism as well as valine, leucine, and isoleucine degradation. The abundances of these characteristic metabolites showed corresponding variation trends with the abundances of the two above pathways (L-isoleucine: p FMT-Lpr VS FMT-PBS = 0.0022; p FMT-Mpj VS FMT-PBS = 0.0221, L-valine: p FMT-Lpr VS FMT-PBS = 0.0122; p FMT-Mpj VS FMT-PBS = 0.0337. L-tyrosine: p FMT-Lpr VS FMT-PBS = 0.0229; p FMT-Lpr VS FMT-Mpj = 0.0527. L-threonine: p FMT-Lpr VS FMT-PBS = 0.0399) (Fig. 5H). Therefore, amino acid metabolism, such as cyanoamino acid metabolism and valine, leucine, and isoleucine degradation, may be of great importance in promoting the development of SLE.

Strong associations between Prevotella and characteristic functional pathways

Although we determined the representative taxa and the changed functional pathways accounting for the distinct phenotypes of the pristane-induced lupus mice due to the fecal microbiota transplantation from different strains of mice, we still needed to gain insight into how specific taxa acted in the characteristic metabolic pathways. Therefore, we investigated the correlations between the microbial taxa and the characteristic KEGG pathway. At the genus level, Prevotella and Alloprevotella were highly correlated with both cyanoamino acid metabolism (Fig. 6A) and valine, leucine, and isoleucine degradation (Fig. 6B). In particular, the correlation coefficients of Prevotella and the above two functional pathways were > 0.7 or almost reached 0.7, indicating strong correlations between Prevotella and cyanoamino acid metabolism and valine, leucine, and isoleucine degradation (Fig. 6A,B). Moreover, several strains of Prevotella were also positively related to the two characteristic pathways. The degree of the correlation was significant and strong, such as Prevotella_sp._CAG:873 and Prevotella_corporis with cyanoamino acid metabolism (Fig. 6C) as well as Prevotella_sp._CAG:873, Prevotella_sp._CAG:485, Prevotella_denticola, Prevotella_bivia, Prevotella_amni, Prevotella_corporis, and Prevotella_sp._P6B1 with valine, leucine, and isoleucine degradation (Fig. 6D,E), which means that the levels of the discriminating functional pathways and the significant taxa, such as Prevotella_sp._CAG:873 and Prevotella_corporis, were synchronously changed. Therefore, the abundance alterations in specific taxa may affect these microbiota and metabolite-associated functional pathways, thus contributing to the progression of lupus.

Fig. 6figure 6

The associations between the abundances of characteristic taxa and disordered functional pathways. A The association of the abundances of prevotella and alloprevotella with the cyanoamino acid metabolism. B The association of the abundances of prevotella and alloprevotella with the valine, leucine, and isoleucine degradation. C The association of the abundances of prevotella species with the cyanoamino acid metabolism (correlation coefficient values > 0.7 are shown). D, E The association of the abundances of prevotella species with the valine, leucine, and isoleucine degradation (correlation coefficient values > 0.7 are shown). Spearman’s rank correlation coefficient, r-values, and p-values are shown. FMT-Lpr, n = 11; FMT-Mpj, n = 10; FMT-PBS, n = 7

The connections between the specific Prevotella taxa and the phenotypic changes in the pristane-induced lupus mice

The pristane-induced lupus mice had different degrees of renal injuries, manifested as increased urine protein levels and more IgG and C3 deposition in the glomeruli of the mice receiving fecal microbiota from MRL/lpr mice compared with the FMT-Mpj mice and FMT-PBS mice. How the gut microbiota influences renal functions is still unknown. However, we found that the abundances of specific microbiota, such as Prevotella, Alloprevotella, Bilophila, and Osenella, were correlated with urine protein levels. In particular, the abundance of Alloprevotella showed a strong association with the levels of proteinuria (Fig. 7A). At the species level, the abundances of Prevotella_sp._CAG:891, Prevotella_sp._CAG:755, Prevotella_sp._CAG:617, Prevotella_sp._oral_taxon_473, Prevotella_sp._DNF00663 (Fig. 7B), Alloprevotella tannerae, and Alloprevotella_rava (Fig. 7C) were also strongly positively related to the proteinuria levels. Therefore, these characteristic microbes may play roles in the progression of renal dysfunction.

Fig. 7figure 7

The associations between the urine protein levels and the abundances of characteristic taxa. A The association of the abundances of prevotella, alloprevotella, bilophila, and oslenella with the urine protein levels. B The association of the abundances of the specific prevotella taxa with the urine protein levels (correlation coefficient values > 0.7 are shown). C The association of the abundances of the specific alloprevotella taxa with the urine protein levels (correlation coefficient values > 0.7 are shown). Spearman’s rank correlation coefficient, r-values, and p-values are shown. FMT-Lpr, n = 11; FMT-Mpj, n = 10; FMT-PBS, n = 7

Increased plasma cell percentages may be considered one of the predominant immunological features in the intestinal tissues and lymph nodes of the pristane-induced lupus mice. The Prevotella species exhibited connections with alterations in plasma cells to some degree. The abundances of Prevotella_sp._CAG:891, Prevotella_sp._CAG:755, Prevotella_sp._CAG:5226, Prevotella_sp._oral_taxon_473, and Prevotella_sp._CAG:617 were associated with the plasma cell percentages in the large intestine (Figure S11A). The abundances of Prevotella_bivia, Prevotella_sp._MA2016, Prevotella_corporis, Prevotella_stercorea, Prevotella_melaninogenica, and Prevotella_sp._P6B1 were associated with plasma cell levels in Peyer’s patches (Figure S11B). Prevotella_bivia, Prevotella_stercorea, and Prevotella_buccae were associated with the plasma cell alteration tendency in the mesenteric lymph nodes (Figure S11C). Taken together, the characteristic taxa, especially the strains of Prevotella, were possibly involved in the changed immunological states of lupus mice. Although it is still unclear whether the increased abundances of the specified taxa promoted lupus renal dysfunction or whether systemic phenotypic changes contributed to intestinal disorders, targeting specific taxa could be useful for disease therapies.

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