Lactobacillus spp. attenuate LN

When comparing the bacterial composition in the gut microbiota of lupus-prone lpr mice vs. MRL control mice, we found that female lpr mice had a significantly lower abundance of Lactobacillales in the gut microbiota than MRL controls at 5 weeks of age and prior to the onset of lupus-like disease (Additional file 1: Figure S1A). However, it was unclear whether the change was a cause or result of disease initiation. Therefore, we performed reciprocal cecal microbiota transplantation experiments from MRL to lpr mice (Additional file 1: Figure S1B) and vice versa. While the disease in MRL mice did not change after the transfer of cecal content from lpr mice (data not shown), MRL-to-lpr cecal transplantation led to significantly reduced production of autoantibodies against double-stranded (ds) DNA from the lower gastrointestinal tract (Additional file 1: Figure S1C). Since the gut microbiota of young MRL mice contained a higher abundance of Lactobacillales than lpr mice, we sought to determine if the decrease in disease could be due to the elevated Lactobacillales in lpr mice that were transferred from MRL mice upon cecal transplantation. Indeed, lpr mice receiving MRL cecal content had more abundant Lactobacillales in the gut microbiota than untreated controls (Additional file 1: Figure S1D), suggesting a positive correlation between a higher abundance of gut-colonized Lactobacillales and improved lupus symptoms.

The bacterial order Lactobacillales includes Lactobacillus spp. that are known as beneficial bacteria. We thus examined the effect of these beneficial bacteria on lpr mice by directly inoculating freshly cultured Lactobacillus isolates (Additional file 1: Figure S1E). We used a mixture of 5 Lactobacillus strains—Lactobacillus oris, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus johnsonii, and Lactobacillus gasseri. Different Lactobacillus strains have been reported to exert different immunological functions [14, 15]. Among the 5 strains, all except L. oris are known to colonize the gut. To improve engraftment of Lactobacillus spp., we pre-treated the mice with ampicillin, neomycin, vancomycin, and metronidazole for 2 days, followed by 2 days of resting to allow for excretion of the antibiotics prior to Lactobacillus treatment. The brief antibiotic treatment at the time of weaning did not change the disease severity (Additional file 1: Figures S1F and S1G). We found that weekly gavages of Lactobacillus spp. significantly increased the relative abundance of Lactobacillales in the gut microbiota at weeks 5 and 7 (Fig. 1a and Additional file 2: Table S1), significantly reduced the level of autoantibodies in the circulation (Fig. 1b), and significantly decreased proteinuria (Fig. 1c) and renal pathology scores (Fig. 1d). The spleen and MLN weights were not changed (Additional file 1: Figure S1H). Importantly, Lactobacillus treatment significantly increased the survival of female lpr mice (Fig. 1e). It is noteworthy that Lactobacillus treatment was given starting from 3 weeks of age and before disease establishment. When given after the onset of lupus disease, Lactobacillus treatment had a trend to reduce lupus disease, but the difference was not statistically significant (data not shown). These results suggest that the introduction of more “good” bacteria in the gut microbiota—in this case, Lactobacillus spp.—may be able to prevent disease progression in lupus-prone mice. This supports the notion that gut microbiota can directly control LN. How the increase of Lactobacilli in the gut affects disease pathogenesis in the kidney, which is extraintestinal, was unknown. Therefore, we next sought to identify potential “messengers” that transduced the disease-modulating signal from the gut to the kidney.

Fig. 1 Lactobacillus spp. protect female lpr mice from LN. a Time-dependent changes of fecal microbiota upon PBS or Lactobacillus (Lacto) treatment (n = 4 mice per group). b Level of anti-dsDNA IgG in the blood of 10-week-old mice (n = 7 mice per group; **P < 0.01). c Level of proteinuria over time (n = 7 mice per group; paired t test; *P < 0.05). d Renal histopathology at 14 weeks of age (n = 7 mice per group; chi-square test; *P < 0.05). Left: PAS-stained kidney sections; bar equals 100 μm. e Survival rate (n = 10 mice per group; chi-square test; ****P < 0.0001) Full size image

A “leaky” gut in lupus-prone mice

While 5 Lactobacillus strains were inoculated, we found by using 16S ribosomal RNA gene sequencing that, unexpectedly, two bacterial species accounted for >99% of the order Lactobacillales regardless of treatment status. The species were L. reuteri and an uncultured Lactobacillus sp. (Fig. 2a). The same phenomenon was observed for MRL mice (data not shown). This suggests that L. reuteri and the uncultured Lactobacillus sp. accounted for most of the observed effects. As L. reuteri is known to enhance the epithelial barrier function of the gut [16, 17], we measured the level of endotoxin in the blood, and found it to be significantly higher in lpr mice compared to the age-matched MRL controls (Fig. 2b). Interestingly, increasing colonization of Lactobacillales in the gut significantly decreased endotoxemia in lpr mice (Fig. 2c). These results suggest that the gut of lpr mice may be “leaky” and allow bacterial components (e.g., lipopolysaccharide, or LPS/endotoxin) to enter the blood stream. L. reuteri and the uncultured Lactobacillus sp., on the other hand, may be able to correct the leakiness. To test if the gut barrier was leaky in lpr mice, we gavaged them with FITC-dextran and found significantly more FITC-dextran in the blood compared to MRL mice. When we treated the lpr mice with Lactobacillus spp., the levels of FITC-dextran in the circulation significantly decreased (Fig. 2d).

Fig. 2 Lactobacillus spp. restore gut mucosal barrier function in female lpr mice. a Percentage of Lactobacillus strains in the order Lactobacillales (n = 4 per group). b Level of endotoxin in the blood of 6-week-old lpr mice (n = 6 mice per group; **P < 0.01). c Level of endotoxin in the blood of 10-week-old lpr mice with or without Lactobacillus treatment (n = 6 or 7 mice per group; *P < 0.05). d Level of FITC-dextran diffused to the blood (n = 5 or 7 mice per group; *P < 0.05). e Transcript levels of tight junction proteins and IL-18 in intestinal epithelial cells of 14-week-old lpr mice (n = 7 mice per group; **P < 0.01, ***P < 0.005). f Immunohistochemical stains of ZO-1 (green) in the ileum or colon. Nuclear stain (DAPI) is shown in blue. Bar equals 75 μm. g Transcript levels of IAP genes in the epithelium (n = 7 mice per group; **P < 0.01). h Immunohistochemical stains of IAP (green) in the ileum. Bar equals 75 μm Full size image

Two mucus layers cover the epithelial cells in the lower gastrointestinal tract [18]. Underneath the mucus layers, permeability of the intestinal epithelium is controlled by functions of tight junction proteins [19]. To determine whether lpr mice had alterations in epithelial cell junctions, we isolated intestinal epithelial cells and measured the level of tight junction protein transcripts. We found that treatment with Lactobacillus spp. significantly increased the expression of barrier-forming junction transcripts (ZO1, occludin, and Cldn1) without affecting the level of pore-forming junction transcript Cldn2 (Fig. 2e), suggesting enhanced barrier function of the intestinal epithelium with a higher abundance of Lactobacillales in the gut microbiota. Immunohistochemical analysis confirmed that the level of ZO-1 was increased by Lactobacillus treatment in both the ileum and the colon (Fig. 2f). We also found that epithelial expression of IL-18, a cytokine important for tissue repair [20] and limiting colonic T-helper 17 cell (Th17) differentiation [21], was significantly enhanced with Lactobacillus treatment (Fig. 2e). Interestingly, IL-18 can also be detrimental and promote inflammation in lpr mice [22]. We found that unlike epithelial expression, the level of IL-18 produced by MLN was significantly decreased by Lactobacillus treatment (data not shown). It is likely that Lactobacillus spp. can attenuate lupus disease through modulating the production of IL-18 from epithelial vs. immune cells.

In addition to strengthening intestinal mucosal barrier function, L. reuteri and the uncultured Lactobacillus sp. may also enhance LPS clearance by increasing the expression of intestinal alkaline phosphatase (IAP). IAP is a brush border enzyme expressed on the microvillus membranes of enterocytes [23] that can dephosphorylate LPS, leading to a 100-fold reduction in LPS toxicity [24]. In our studies, the epithelial expression of IAP (Alppl2 and Alpi) was significantly upregulated after Lactobacillus treatment in lpr mice compared to the controls (Fig. 2g). The upregulation of IAP was confirmed with immunohistochemical analysis (Fig. 2h). Interestingly, IAP has been reported to support the growth of Gram-positive bacteria [25], which may explain the increase of Bifidobacteria in Lactobacillus-treated mice (Fig. 1a). Bifidobacteria can also promote gut epithelial integrity by strengthening tight junctions [26]. Together, these results suggest that gut microbiota can restore intestinal mucosal barrier function that is compromised in lupus-prone lpr mice.

Control of gut inflammation in lupus

With an enhanced gut mucosal barrier, fewer bacteria are able to translocate across the intestinal epithelium leading to reduced activation and migration of CX3CR1+ and/or CD103+ antigen-presenting cells (APCs) to the draining lymph nodes of the lower intestinal tract [27,28,29]. The decrease in APC migration may decrease the activation of CD4± T cells. Indeed, we found significantly decreased levels of Cx3cr1 and Itgae (a subunit of CD103) specifically in the MLN with Lactobacillus treatment (Additional file 1: Figures S2A and S2B) suggesting that L. reuteri and the uncultured Lactobacillus sp. may reduce the migration of APC to the MLN. We next determined whether the activation of T cells was affected by the decrease of APC in the MLN. Upon activation, MLN T cells upregulate integrin α4β7 and chemokine receptor CCR9 for homing to the gut mucosa [30]. We found that Lactobacillus treatment significantly reduced the expression of both Itga4 and Ccr9 in the MLN (Additional file 1: Figures S2B and S2C), suggesting decreased activation of T cells. Consistent with this observation, migration of T cells to the intestinal lamina propria was reduced after mice were treated with the Lactobacillus spp. (Additional file 1: Figure S2D).

Among many pro-inflammatory cytokines produced by activated APC and T cells, IL-6 is known to promote antibody production from B cells [31] and suppress Treg cells [32], which are important for lupus progression in lpr mice [33,34,35]. We measured the transcript level of Il-6 in the MLN vs. spleen and found that it was significantly reduced by Lactobacillus treatment specifically in the MLN (Fig. 3a). CD4+CD8− T cells appeared to be a source of IL-6 in the MLN of lpr mice (Fig. 3b). As decreased IL-6 would theoretically allow for differentiation of Treg cells [32], we next evaluated the levels of TGFβ and IL-10. Both cytokines were significantly increased at the transcriptional level in the MLN, but not the spleen, with Lactobacillus treatment (Fig. 3c), suggesting gut-specific immunosuppression. The serum TGFβ level was also significantly enhanced with the treatment (Fig. 3d), while the level of IL-6 in the circulation did not change (data not shown). Importantly, the induction of IL-10 with more Lactobacillales in the gut microbiota was not only in the MLN, but also systemic (Fig. 3e), suggesting that L. reuteri and the uncultured Lactobacillus sp. may exert a global anti-inflammatory function in lpr mice through inducing IL-10 in the gut. Indeed, we also observed a significant elevation of IL-10 transcript levels in the kidney of lpr mice with Lactobacillus treatment compared to untreated controls (Fig. 3f). Further analysis of MLN cells revealed that most IL-10-producing cells in the gut were CD4+Foxp3− type 1 regulatory T (Tr1) cells (Fig. 3g). This observation is consistent with published results on IL-10-producing Tr1 cells in lpr mice [36]. Together, these results suggest that gut microbiota can promote an anti-inflammatory environment in the gut of lupus-prone mice, leading to induction of IL-10 that enters the circulation to provide systemic immunosuppression.

Fig. 3 Control of intestinal inflammation by gut microbiota in female lpr mice. a Transcript level of IL-6 in the spleen (SP) and MLN (n = 7 mice per group; **P < 0.01). b Percentage of IL-6-expressing cells in the MLN (n = 7 mice per group; **P < 0.01). c Transcript levels of TGFβ and IL-10 (n = 7 mice per group; *P < 0.05, ***P < 0.005). d Serum level of TGFβ (n = 7 mice per group; **P < 0.01). e Serum level of IL-10 (n = 7 mice per group; *P < 0.05). f Transcript level of IL-10 in the kidney (n = 7 mice per group; **P < 0.01). g FACS analysis of IL-10-expressing Tr1 cells in the MLN. Percentages of Tr1 cells in CD4+CD8− cells are shown (n = 7 mice per group; **P < 0.01) Full size image

Control of renal inflammation in lupus

IL-10 can inhibit kidney disease in lpr mice through preventing IFNγ-mediated production of IgG2a, a major immune deposit in the kidney of these mice [37]. We found that Lactobacillus treatment significantly reduced the level of IgG2a in the blood (Fig. 4a) and its deposition in the kidney (Fig. 4b). This suggests that IgG2a may act as another “messenger” (in addition to IL-10) to transduce the disease-modulating signal from the gut to the kidney. The levels of IgG1 and total IgG did not change with the treatment (data not shown). Interestingly, the level of IgA was reduced by Lactobacillus treatment in the circulation (Fig. 4c), suggesting a potential effect of L. reuteri and the uncultured Lactobacillus sp. on class-switched antibodies. Indeed, the expression level of Aicda, whose gene product mediates class switch recombination [38], was significantly lower in the MLN of lpr mice treated with Lactobacillus spp. (Fig. 4d). The change of IgA did not appear to be related to attenuation of LN, as it was not detectable in the kidney.

Fig. 4 Control of renal inflammation by gut microbiota in female lpr mice. a Serum level of IgG2a (n = 7 mice per group; ***P < 0.005). b Immunohistochemical stains of IgG2a (green) in the kidney. Bar equals 75 μm. Pathological scores are shown on the right (n = 4 mice per group; **P < 0.01). c Serum level of IgA (n = 7 or 8 mice per group; *P < 0.05). d Transcript level of Aicda (n = 7 mice per group; *P < 0.05). e Percentages of T cells and subpopulations in the kidney. f Percentage of CD4+Foxp3+ Treg cells in the kidney. Absolute Treg cell numbers are shown on the right (n = 7 mice per group; *P < 0.05). g FACS analysis of IL-17-producing CD4+ cells and percentage of RORγT+Tbet+ pathogenic Th17 cells in the kidney. Absolute pathogenic Th17 cell numbers are shown on the right (n = 7 mice per group; **P < 0.01) Full size image

Different immune cell populations, including T, B, neutrophils, dendritic cells, and macrophages, have been demonstrated to infiltrate in the kidney with LN. To determine how Lactobacillus treatment affects immune cell migration to renal tissue, we evaluated various immune cell populations and found marked influx of CD3+ T cells, particularly CD8+ T cells, into the kidney of Lactobacillus-treated lpr mice (Fig. 4e). As CD8+ T cells are generally considered protective in lupus [39,40,41], it would suggest that renal infiltration of these cells exerts a suppressive effect on the development of LN. In addition, the number of Foxp3+ Treg cells significantly increased (Fig. 4f), while that of pathogenic Th17 cells significantly decreased (Fig. 4g), with Lactobacillus treatment. Together, these results suggest that gut microbiota may attenuate LN by limiting renal deposition of IgG2a and skewing the Treg-Th17 balance in the kidney towards Treg.

Sex hormones and gut microbiota cooperatively regulate LN

SLE is a female-biased disease with women getting disease nearly 9:1 over men. The results shown so far were obtained from female mice. However, in lpr mice, both sexes get LN similarly. To investigate whether sex hormones and gut microbiota cooperatively regulate LN in lpr mice, we treated male mice with the same Lactobacillus strains after mock or castration surgery (Additional file 1: Figure S3A). Bacterial profiling showed that Lactobacillus treatment increased the gut colonization of Lactobacillales in both mock and castrated mice (Additional file 1: Figure S3B and Additional file 3: Table S2). Strikingly, Lactobacillus treatment significantly decreased proteinuria (Fig. 5a) and renal pathology (Fig. 5b) only in the castrated mice but not the intact animals, suggesting a possible role of androgenic hormones in suppressing the effects of Lactobacillus spp. The level of anti-double-stranded DNA (anti-dsDNA) IgG was not changed with Lactobacillus treatment (Additional file 1: Figure S3C). However, the total weight of lymph nodes (including mesenteric, renal, inguinal, lumbar, superficial, axillary/brachial, mediastinal lymph nodes) increased after mice were castrated, an effect reversed by Lactobacillus treatment (Additional file 1: Figure S3D). In addition, increasing gut colonization of Lactobacillales significantly decreased the serum levels of IgG2a and IgA in castrated male mice, but not in the mice receiving mock surgery (Fig. 5c). The decrease of IgA appears to have originated from the colon (Additional file 1: Figure S3E), where the majority of Lactobacillus spp. (in terms of total number) resided [42]. Importantly, we found that unlike mice receiving in mock surgery, Lactobacillus treatment significantly increased the transcript levels of TGFβ and IL-10 in the MLN in castrated male lpr mice (Fig. 5d). Lactobacillus treatment also significantly increased circulating IL-10 in castrated animals only (Fig. 5e). Together, these results suggest that Lactobacillus treatment was not effective in intact male lpr mice, while the response of castrated males to Lactobacillus treatment parallels that of female lpr mice.

Fig. 5 Sex hormones and gut microbiota cooperatively regulate LN. a Proteinuria after surgery (Mock vs. Castr/castration) and treatment (PBS vs. Lacto) of male lpr mice (n = 5 mice per group; Mann-Whitney test; ***P < 0.005). b Renal histopathology (n = 5 mice per group; Mann-Whitney test; *P < 0.05). Left: PAS-stained kidney sections; bar equals 100 μm. c Serum levels of IgG2a and IgA (n = 5 mice per group; *P < 0.05). d Transcript levels of TGFβ and IL-10 in the MLN (n = 5 mice per group; #P < 0.1, *P < 0.05). e Serum level of IL-10 (n = 5 mice per group; ***P < 0.005). f Levels of testosterone and luteinizing hormone (LH) in the blood (n = 3 mice per group; *P < 0.05). g Negative correlation between serum IL-10 and the ratio of LH to testosterone Full size image

As testis is the only source of testosterone in mice, castration surgery completely removed the male hormone regardless of Lactobacillus treatment (Fig. 5f). We then measured two hormones regulated by testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH). Both are known to be repressed by testosterone [43,44,45]. As anticipated, castration surgery increased the levels of LH and FSH when the mice were not treated with Lactobacilli (Fig. 5f and Additional file 1: Figure S3F). However, Lactobacillus treatment significantly decreased the serum level of LH, bringing it back to the level where testosterone was still present. We took the ratio of LH to testosterone and found it to be negatively correlated with serum IL-10 level (Fig. 5g). Whether LH directly affects IL-10, or vice versa, requires further investigation. Together, these results suggest that gut microbiota control LN in lpr mice in a sex hormone-dependent manner. To determine the effect of Lactobacilli on sex differences, in future studies, we will transfer the cecal contents of young females to male mice to determine whether the interaction between sex hormones and Lactobacillus treatment is required for the observed changes in autoimmune response and/or disease phenotype.