Lactobacillus spp. act in synergy to attenuate splenomegaly and lymphadenopathy in lupus-prone MRL/lpr mice.

Commensal bacteria and the immune system have a close and strong relationship that maintains a balance to control inflammation. Alterations of the microbiota, known as dysbiosis, can direct reactivity to self-antigens not only in the intestinal mucosa but also at the systemic level. Our laboratory previously reported gut dysbiosis, particularly lower abundance of bacteria in the family Lactobacillaceae, in lupus-prone MRL/lpr mice, a model of systemic autoimmunity. Restoring the microbiota with a mix of 5 different Lactobacillus species (spp.), L. reuteri, L. oris, L. johnsonii, L. gasseri and L. rhamnosus, attenuated lupus-liked clinical signs, including splenomegaly and lymphadenopathy. However, our understanding of the mechanism was limited. In this study, we first investigated the effects of individual species. Surprisingly, none of the species individually recapitulated the benefits of the mix. Instead, Lactobacillus spp. acted synergistically to attenuate splenomegaly and renal lymphadenopathy through secreted factors and a CX3CR1-dependent mechanism. Interestingly, oral administration of MRS broth exerted the same benefits likely through increasing the relative abundance of endogenous Lactobacillus spp. Mechanistically, we found increased percentages of FOXP3-negative type 1 regulatory T cells with administration of the mix in both spleen and mesenteric lymph nodes. In addition, oral gavage of Lactobacillus spp. decreased the percentage of central memory T cells while increasing that of effector memory T cells in the lymphoid organs. Furthermore, a decreased percentage of double negative T cells was observed in the spleen with the mix. These results suggest that Lactobacillus spp. might act on T cells to attenuate splenomegaly and lymphadenopathy. Together, this study advances our understanding of how Lactobacillus spp. attenuate lupus in MRL/lpr mice. The synergistic action of these bacteria suggests that multiple probiotic bacteria in combination may dampen systemic autoimmunity and benefit lupus patients.

Phenotypic Drift in Lupus-Prone MRL/lpr Mice: Potential Roles of MicroRNAs and Gut Microbiota.

MRL/lpr mice have been extensively used as a murine model of lupus. Disease progression in MRL/lpr mice can differ among animal facilities, suggesting a role for environmental factors. We noted a phenotypic drift of our in-house colony, which was the progeny of mice obtained from The Jackson Laboratory (JAX; stocking number 000485), that involved attenuated glomerulonephritis, increased splenomegaly, and reduced lymphadenopathy. To validate our in-house mice as a model of lupus, we compared these mice with those newly obtained from JAX, which were confirmed to be genetically identical to our in-house mice. Surprisingly, the new JAX mice exhibited a similar phenotypic drift, most notably the attenuation of glomerulonephritis. Interestingly, our in-house colony differed from JAX mice in body weight and kidney size (both sexes), as well as in splenic size, germinal center formation, and level of anti-dsDNA auto-IgG in the circulation (male only). In addition, we noted differential expression of microRNA (miR)-21 and miR-183 that might explain the splenic differences in males. Furthermore, the composition of gut microbiota was different between in-house and new JAX mice at early time points, which might explain some of the renal differences (e.g., kidney size). However, we could not identify the reason for attenuated glomerulonephritis, a shared phenotypic drift between the two colonies. It is likely that this was due to certain changes of environmental factors present in both JAX and our facilities. Taken together, these results suggest a significant phenotypic drift in MRL/lpr mice in both colonies that may require strain recovery from cryopreservation.

STAT3-miR-17/20 signalling axis plays a critical role in attenuating myocardial infarction following rapamycin treatment in diabetic mice.

AIMS: Deregulation of mTOR (mammalian target of rapamycin) signalling occurs in diabetes, which exacerbates injury following myocardial infarction (MI). We therefore investigated the infarct-limiting effect of chronic treatment with rapamycin (RAPA, mTOR inhibitor) in diabetic mice following myocardial ischaemia/reperfusion (I/R) injury and delineated the potential protective mechanism. METHODS AND RESULTS: Adult male diabetic (db/db) or wild-type (WT) (C57) mice were treated with RAPA (0.25 mg/kg/day, intraperitoneal) or vehicle (5% DMSO) for 28 days. The hearts from treated mice were subjected to global I/R in Langendorff mode. Cardiomyocytes, isolated from treated mice, were subjected to simulated ischaemia/reoxygenation (SI/RO) to assess necrosis and apoptosis. Myocardial infarct size was increased in diabetic heart following I/R as compared to WT. Likewise, enhanced necrosis and apoptosis were observed in isolated cardiomyocytes of diabetic mice following SI/RO. Treatment with RAPA reduced infarct size as well as cardiomyocyte necrosis and apoptosis of diabetes and WT mice. RAPA increased STAT3 phosphorylation and miRNA-17/20a expression in diabetic hearts. In addition, RAPA restored AKT phosphorylation (target of mTORC2) but suppressed S6 phosphorylation (target of mTORC1) following I/R injury. RAPA-induced cardioprotection against I/R injury as well as the induction of miR-17/20a and AKT phosphorylation were abolished in cardiac-specific STAT3-deficient diabetic mice, without alteration of S6 phosphorylation. The infarct-limiting effect of RAPA was obliterated in cardiac-specific miRNA-17-92-deficient diabetic mice. The post-I/R restoration of phosphorylation of STAT3 and AKT with RAPA were also abolished in miRNA-17-92-deficient diabetic mice. Additionally, RAPA suppressed the pro-apoptotic prolyl hydroxylase (Egln3/PHD3), a target of miRNA-17/20a in diabetic hearts, which was abrogated in miRNA-17-92-deficient diabetic mice. CONCLUSION: Induction of STAT3-miRNA-17-92 signalling axis plays a critical role in attenuating MI in RAPA-treated diabetic mice. Our study indicates that chronic treatment with RAPA might be a promising pharmacological intervention for attenuating MI and improving prognosis in diabetic patients.

Gut Microbiota and Bacterial DNA Suppress Autoimmunity by Stimulating Regulatory B Cells in a Murine Model of Lupus.

Autoimmune diseases, such as systemic lupus erythematosus, are characterized by excessive inflammation in response to self-antigens. Loss of appropriate immunoregulatory mechanisms contribute to disease exacerbation. We previously showed the suppressive effect of vancomycin treatment during the "active-disease" stage of lupus. In this study, we sought to understand the effect of the same treatment given before disease onset. To develop a model in which to test the regulatory role of the gut microbiota in modifying autoimmunity, we treated lupus-prone mice with vancomycin in the period before disease development (3-8 weeks of age). We found that administration of vancomycin to female MRL/lpr mice early, only during the pre-disease period but not from 3 to 15 weeks of age, led to disease exacerbation. Early vancomycin administration also reduced splenic regulatory B (Breg) cell numbers, as well as reduced circulating IL-10 and IL-35 in 8-week old mice. Further, we found that during the pre-disease period, administration of activated IL-10 producing Breg cells to mice treated with vancomycin suppressed lupus initiation, and that bacterial DNA from the gut microbiota was an inducer of Breg function. Oral gavage of bacterial DNA to mice treated with vancomycin increased Breg cells in the spleen and mesenteric lymph node at 8 weeks of age and reduced autoimmune disease severity at 15 weeks. This work suggests that a form of oral tolerance induced by bacterial DNA-mediated expansion of Breg cells suppress disease onset in the autoimmune-prone MRL/lpr mouse model. Future studies are warranted to further define the mechanism behind bacterial DNA promoting Breg cells.