Antibiotics given post disease onset attenuated lupus

The onset of autoimmune responses in female MRL/lpr mice is as early as 6 weeks of age26. To determine the effects of antibiotics on active disease in lupus-prone MRL/lpr mice, we treated female mice with a combination of antibiotics (ampicillin, neomycin, metronidazole and vancomycin5) started at 9 weeks of age and post disease onset. The treatment led to enlarged ceca as expected for antibiotic treatments, whereas the overall body weight did not change (data not shown). Spleen and mesenteric lymph node (MLN) weights were significantly decreased with antibiotic treatment compared to controls (Fig. 1A). The serum level of IgG autoantibodies against double-stranded (ds) DNA was also significantly reduced by the mixed antibiotic treatment (Fig. 1B). As kidney inflammation (or lupus nephritis) affects up to 60% of lupus patients27,28, we determined the renal function by measuring proteinuria and kidney histopathology. Both glomerular and tubulointerstitial scores were significantly decreased with antibiotic treatment compared to controls (Fig. 1C). The antibiotics also significantly reduced proteinuria (Fig. 1D), suggesting improvement of renal function. Together, these results indicate amelioration of lupus-like disease in female MRL/lpr mice with post-disease-onset antibiotic treatment.

Figure 1 Antibiotic treatment started after lupus onset led to disease attenuation. (A) Spleen and MLN tissue weight to body weight ratio (%) at 16 weeks of age (n ≥ 8 per group). Control: no antibiotics. Abx-9w: oral antibiotics were given starting from 9 weeks of age and post disease onset. (B) Level of anti-dsDNA IgG in the mouse serum, total IgG, and the ratio of anti-dsDNA IgG to total IgG at 16 weeks of age (n ≥ 8 per group). (C) Renal histopathology at 16 weeks of age (n ≥ 8 per group). Left: representative PAS-stained kidney sections; bar equals 200 µm. Middle: glomerular score. Right: tubulointerstitial (TI) score. (D) Level of proteinuria over time (n ≥ 8 per group). *p < 0.05, **p < 0.01, ***p < 0.001. Full size image

We next sought to understand the underlying mechanism of how antibiotic treatment ameliorated lupus in female MRL/lpr mice by examining immune cell differentiation and inflammatory mediator production (Fig. 2). IL-6, known to promote lupus disease in both human and mouse due to its ability to increase T-helper (Th)17 differentiation and IL-17 production29,30, was significantly decreased in the serum with antibiotic treatment (Fig. 2A). We then quantified IL-17-producing cells in the spleen and kidney by using flow cytometry. As anticipated, antibiotic treatment significantly reduced the percentages of IL-17+ cells (Fig. 2B), CD3+CD4+RORγ+ Th17 cells and Lin−CD4−RORγ+ group 3 innate lymphoid cells (ILC3s) (Fig. 2C). Both Th17 cells and ILC3s are known producers of IL-17 in the spleen31, and their decrease suggests systemic downregulation of IL-17 with antibiotic treatment. In the kidney, the double negative (DN) T cells and Th17 cells are major sources of IL-1732 and they both were significantly reduced with post-disease-onset antibiotic treatment (Fig. 2D and Fig. 2E, respectively). As IL-17 was too low to be detected in the circulation, we measured IL-17 protein in the splenic extract that reflected the systemic level of IL-17. Post-disease-onset antibiotic treatment significantly reduced the level of splenic IL-17 (Fig. 2F). These results suggest that antibiotics may attenuate lupus-like disease in female MRL/lpr mice by suppressing the production of IL-17 from Th17 cells and ILC3s in the spleen and from DN-T and Th17 cells in the kidney. Interestingly, we also found a significant increase in serum IL-10 with antibiotic treatment (Fig. 2G) that correlated with an increase in the number of IL-10-producing cells in the MLN (Fig. S1A). IL-10 is known as a protective cytokine in MRL/lpr mice33.

Figure 2 Antibiotic treatment decreased IL-17-producing cells in the spleen and kidney and increased IL-10 in the circulation. (A) Serum level of IL-6 at 16 weeks of age (n ≥ 7 per group). (B) Intracellular staining of IL-17 and the percentage of IL-17-producing cells in the spleen at 16 weeks of age (n = 4 per group). (C) FACS analysis of CD4+RORγ+ Th17 cells and CD4−RORγ+ ILC3 cells in the spleen at 16 weeks of age (n = 4 per group). (D,E) FACS analysis of DN-T cells (D) and Th17 cells (E) in the kidney at 16 weeks of age (n = 4 per group). (F) Protein level of IL-17 in the spleen at 16 weeks of age (n ≥ 7 per group). (G) Serum level of IL-10 in the mouse serum at 16 weeks of age (n ≥ 7 per group). *p < 0.05, **p < 0.01. Full size image

Antibiotics given post disease onset reshaped gut microbiota

It has been recently reported that Th17 cells can migrate from the gut to the kidney to facilitate the development of lupus nephritis34, whereas the generation of Th17 cells in the gut is dependent on the gut microbiota35. We thus characterized the gut microbiota in antibiotics-treated mice. While antibiotic treatment initiated post disease onset did not decrease the bacterial diversity (Fisher index, Fig. S1B), it did reduce the bacterial load of fecal microbiota by 2 magnitudes (Fig. S1C). Based on 16 S rRNA sequencing analysis, the overall structure of the remaining gut bacteria was distinct from untreated animals (Fig. 3A; p < 0.01, PERMANOVA; red vs. blue symbols). In addition, the overall structure of the gut microbiota was different between the time points before (3 and 8 weeks of age) and after (11 and 15 weeks of age) antibiotic treatment initiation at 9 weeks of age (also in Fig. 3A; p < 0.01, PERMANOVA; comparison within the red symbols). Moreover, the antibiotic-treated group before given the treatment (3 and 8 weeks of age) shared similar gut microbiota composition with the Control group, but the antibiotic treatment initiated at 9 weeks of age appears to have altered the bacterial composition at 11 and 15 weeks of age (Fig. 3B). Further analysis on specific bacteria groups revealed that antibiotic treatment significantly increased the abundance of Lactobacillus spp. and significantly decreased the abundance of Lachnospiraceae (Fig. 3C), two groups of bacteria previously shown to be associated with improved or deteriorated symptoms in MRL/lpr mice, respectively3. L. agilis, L. brevis, L. mucosae and L. reuteri, in particular, were below detection limit before antibiotic treatment, whereas their abundance increased to about 5% after antibiotics-mediated enrichment. In addition to these changes, treatment with antibiotics removed significant amounts of Bacteroidales and Clostridiales while increasing the relative abundance of Bacillales (Fig. 3D). These results suggest that antibiotics given post disease onset reshaped the gut microbiota, removing potentially harmful bacteria (e.g., Lachnospiraceae) and enriching those that are associated with better disease outcomes (e.g., Lactobacilli).

Figure 3 Composition of gut microbiota changed with antibiotic treatment. (A) Principal component analysis of fecal microbiota. w: weeks of age. p < 0.01, PERMANOVA. (B) Time-dependent changes of fecal microbiota. Bacterial taxa at the order level are shown. (C) Relative abundance of detectable Lactobacillus spp. (Lacto., left panel) and that of Lachnospiraceae (Lachno., right panel) (n = 6 per group). The detectable Lactobacillus spp. were L. agilis, L. brevis, L. mucosae and L. reuteri, and the sum of their relative abundance is shown. Before: 3 and 8 weeks of age. After: 11 and 15 weeks of age. (D) Relative abundance of Bacteroidales, Clostridiales, and Bacillales before and after antibiotic treatment initiated at 9 weeks of age (n = 6 per group). *p < 0.05, ***p < 0.001, ****p < 0.0001. Full size image

Vancomycin recapitulated the disease-attenuating effects of mixed antibiotics

A cocktail of 4 different antibiotics is impractical to implement as a treatment for lupus and more likely to induce resistance. Vancomycin alone, however, can remove Gram-positive bacteria such as Clostridial species (Lachnospiraceae) but spares Lactobacilli17,18,19,20, making it a favorable choice as a potential intervention against lupus progression in MRL/lpr mice. Importantly, vancomycin is not absorbed in the intestine36,37 and its effects are limited to targeting commensal bacteria in the gut lumen. We thus examined whether oral treatment of vancomycin initiated at 9 weeks of age could attenuate lupus. As anticipated, the relative abundance of Lactobacillus spp. was significantly elevated with vancomycin treatment (Fig. 4A). While the overall body weight did not change (Fig. S2A), vancomycin treatment significantly decreased the weight of spleen, MLN and major lymph nodes (Fig. 4B). Furthermore, vancomycin significantly reduced the level of circulating anti-dsDNA IgG (Fig. 4C), proteinuria (Fig. 4D), and renal histopathological scores (Fig. 4E). In contrast, neomycin, an antibiotic with a broad spectrum of activity against both Gram-positive and Gram-negative bacteria38, did not affect the severity of lupus disease when given starting from 9 weeks of age. This indicates that the decrease in bacterial load, achieved by both vancomycin and neomycin treatments, was not the reason for disease attenuation. Together, these results suggest that vancomycin given post disease onset recapitulated the attenuated disease phenotype seen with mixed antibiotic treatment.

Figure 4 Vancomycin but not neomycin treatment started post disease onset ameliorated lupus-like disease. (A) Relative abundance of Lactobacillus spp. in the fecal microbiota at 15 weeks of age (n = 4 per group). Control: no antibiotics. Van-9w: vancomycin was given starting from 9 weeks of age. (B) Tissue to body weight ratio (%) for the spleen, MLN and major lymph nodes (main LN) including mesenteric, renal, inguinal, lumbar, superficial, axillary/brachial, mediastinal lymph nodes at 15 weeks of age. Neo-9w: neomycin was given starting from 9 weeks of age. (C) Level of anti-dsDNA IgG in the mouse serum and its ratio to total IgG at 15 weeks of age. (D) Level of proteinuria over time. (E) Renal histopathology at 15 weeks of age. Left: representative PAS-stained kidney sections; bar equals 200 µm. Middle: glomerular score. Right: tubulointerstitial score. In B-E, n = 12 in Control and Van-9w groups, n = 4 in the Neo-9w group. *p < 0.05, **p < 0.01, n.s.: not statistically significant. Letters a and b represent statistically significant difference among the groups. Full size image

Vancomycin reshaped the gut microbiota and differentially affected KEGG pathways

We collected weekly fecal samples from vancomycin-treated mice and determined longitudinal changes of gut microbiota composition by using 16 S rRNA sequencing. The diversity of gut microbiota was largely reduced upon vancomycin administration (Fisher index, Fig. S2B). Similar to the mixed antibiotic treatment, vancomycin removed Clostridiales right after treatment initiation and Bacteroidales at most of the observed time points (Fig. 5A). Many other groups of bacteria were also removed by vancomycin, including Desulfovibrionales and Turicibacterales, whereas Enterobacteriales were enriched. Anaeroplasmatales, on the other hand, was increased by vancomycin treatment at the later time points. These results suggest that vancomycin given during active disease reshaped the gut microbiota in MRL/lpr mice.

Figure 5 Vancomycin treatment reshaped the gut microbiota. (A) Time-dependent changes of the relative abundance of gut bacteria at the order level. The first label “o__” means an order with uncultured bacteria. (B) Mathematical networks generated based on the longitudinal changes of gut microbiota at the phylum level. 1) Actinobacteria, 2) Bacteroidetes, 3) Cyanobacteria, 4) Firmicutes, 5) Proteobacteria, 6) Tenericutes, and 7) Verrucomicrobia. Blue, positive influence. Red, negative influence. The thicker the line, the stronger the relationship. Full size image

We next established mathematical networks to model the gut microbiota changes at the phylum level (Fig. 5B). While some relationships remain the same with and without vancomycin treatment, such as the positive influence of Bacteriodetes on Verrucomicrobia (nodes 2 and 7), overall, treatment with vancomycin significantly affected the interaction among different bacterial groups. Firmicutes (node 4), for example, were shown to positively influence Verrucomicrobia (node 7) only in the vancomycin-treated group. Both Lactobacillaceae (“good” bacteria) and Lachnospiraceae (possibly “harmful” bacteria in this model) belong to the phylum Firmicutes. Another example is Proteobacteria (node 5), which are commonly used to represent Gram-negative bacteria. In untreated mice, Proteobacteria were involved in a complex interaction network with Bacteroidetes (node 2), Actinobacteria (node 1) and Verrucomicrobia (node 7), suggesting that Gram-negative bacteria may contribute to lupus pathogenesis in MRL/lpr mice. However, these interactions were absent in vancomycin-treated mice, suggesting that Gram-negative bacteria no longer play an important role to promote lupus in the presence of vancomycin.

To get insights into functional categories and pathways affected by antibiotics treatment, we performed PICRUSt analyses, and analyzed KEGG level 3 pathways with DESeq. 2. The analysis showed that the presentation of functional pathways was relatively stable over time for the control group, whereas treatment with vancomycin produced the most significant changes of the functional pathways at 9 weeks of age and 4 days after the initiation of antibiotic treatment (Fig. 6A). Some of the changes sustained beyond 9 weeks of age in the vancomycin group, while others returned to the baseline levels. Among the pathways with significant changes (Table S1), many exhibited similar trends as in our previous publication3. These include the vancomycin-mediated upregulation of Peptidoglycan biosynthesis and Transcriptional factors pathways that were associated with improved lupus-like symptoms in MRL/lpr mice, as well as vancomycin-mediated downregulation of Glyoxylate and dicarboxylate metabolism, Histidine metabolism, and Phenylalanine, tyrosine and tryptophan biosynthesis pathways that were associated with deteriorated lupus-like symptoms in MRL/lpr mice.

Figure 6 Vancomycin treatment differentially affected KEGG pathways in a time-dependent manner. (A) Changes of level 3 functional pathways over time. The result of PICRUSt analysis was plotted with DESeq. 2. Raw data can be found in Table S1. Note that the 9-week microbiota samples were collected 4 days after the initiation of vancomycin treatment for the Van group. (B) Changes of level 3 functional pathways when data from 9–15 weeks were averaged within each treatment group. 9w-Van, vancomycin was given starting from 9 weeks of age. (C) Changes of the average level of 12 LPS-related functional genes over time. PICRUSt analysis was performed at the ortholog level. Raw data with the names of the LPS-related functional genes can be found in Table S2. Full size image

In addition to analysis of the time course, we also determined differential presentation of functional pathways between control and vancomycin groups when data from 9–15 weeks were averaged (Fig. 6B). This analysis showed 75 functional pathways that were significantly altered, including the Phenylalanine, tyrosine and tryptophan biosynthesis pathway that was associated with more severe lupus disease3 and significantly downregulated by vancomycin treatment. Another important pathway, Lipopolysaccharide biosynthesis, was also significantly downregulated by vancomycin regardless of sampling time. Detailed analysis of PICRUSt data on the ortholog level (Fig. S3 and Table S2) revealed vancomycin-mediated downregulation of 12 functional genes within the Lipopolysaccharide biosynthesis pathway. When the relative levels of these genes were plotted over time, they exhibited the same pattern of a sharp decrease at 9 weeks of age, followed by gradual recovery from 10–11 weeks of age (Fig. 6C). Importantly, a majority of these genes were Lpx genes involved in lipid A biosynthesis39. Lipid A is the endotoxic component of LPS. These analyses suggest that vancomycin may attenuate lupus-like disease in MRL/lpr mice by downregulating the relative abundance of Gram-negative bacteria and the LPS endotoxin.

Vancomycin decreased intestinal permeability and the plasma level of LPS

LPS accelerates lupus progression in several lupus-prone mouse models21,22,23,24,25. A leaky gut may allow for the translocation of Gram-negative bacteria across the intestinal epithelium, leading to an increase of LPS—a cell wall component of Gram-negative bacteria—in the circulation. We thus determined the intestinal permeability of vancomycin-treated mice by measuring the diffusion of orally gavaged (for small intestine) or rectally administered (for colon) FITC-conjugated dextran. The result showed that vancomycin treatment significantly decreased the permeability of small intestine (Fig. 7A) and had a trend to decrease colonic permeability (Fig. S4A). Neomycin, on the other hand, did not change intestinal permeability (Fig. S4B). In addition, vancomycin significantly increased the epithelial expression of barrier-forming tight junction transcripts Occludin, ZO-1 (Fig. 7B), Cldn1 and Cldn3 (Fig. S4C), whereas the transcript level of pore-forming tight junction protein Cldn2 did not change with vancomycin treatment (data now shown). While the intestinal epithelium appeared intact regardless of treatment, significantly less inflammation was observed with vancomycin treatment (Fig. 7C). Further studies that directly measured LPS in the circulation indicated that vancomycin indeed significantly decreased the serum level of LPS (Fig. 7D). This is consistent with significantly reduced bacterial translocation to MLN with vancomycin treatment (Fig. 7E). Together, these results suggest that vancomycin may attenuate lupus-like disease in MRL/lpr mice by reducing the “leakiness” of the gut epithelium and preventing the translocation of LPS and/or LPS-containing bacteria from the gut lumen to the circulation.