Background/Purpose: Intestinal microbiota dysbiosis has been implicated in the pathogenesis of autoimmune disease.  How the microbiota affects the peripheral immune system leading to the development of systemic autoimmune disease is elusive. Herein, we generated germ-free (GF) BXD2 mice, a complex lupus mouse model, to dissect the impact of microbiota on disease associated peripheral B and T cell signatures in these mice.

Methods: GF BXD2 mice were generated and maintained at UAB gnotobiotic core facility. Sera autoantibody levels were assessed by ELISA in special pathogen free (SPF) BXD2, GF BXD2, SPF B6, and GF B6 mice. The frequencies and absolute numbers of GC B (CD19⁺GL-7⁺Fas⁺), Tfh (CD4⁺PD1⁺ICOS⁺) and Treg (CD4⁺Foxp3⁺CD25⁺) in spleen were determined by FACS. Immunostaining and confocal imaging were applied to assess the immune architecture of the spleens from each strain of mice. Antibodies against bacterial lysates from the intestine were measured by western blot. Histology was performed to evaluate disease development in the kidney, lung, liver and intestine. The microbiome in 4 strains of mice were comprehensively assessed by 16S rRNA V4 gene sequencing. GC B (CD19⁺GL-7⁺Fas⁺) and Tfh (CD4⁺PD1⁺CXCR5⁺) cells from SPF BXD2 and GF BXD2 were FACS sorted for RNA-seq transcriptome analysis.

Results: Deep 16S rRNA V4 gene sequencing revealed two distinctive clusters of microbiome compositions between GF and SPF. Serum levels of IgG against bacteria lysates of cecal content from SPF or GF BXD2 mice were lower in GF BXD2 than in SPF BXD2 mice. Surprisingly, the GF status did not diminish sera levels of IgG or IgM autoantibodies against dsDNA, BiP, histone, rheumatoid factor (RF), MARCO, SR-AI, and SSA in BXD2 mice. The differences of frequency and absolute numbers of GC B cells, CD4⁺ T cells, Tfh cells were also undistinguishable between SPF BXD2 spleen and GF BXD2 mouse spleen although GF BXD2 mice exhibited a higher number of CD19⁺ B cells, compared to the SPF counterpart. Interestingly, upstream pathway analysis of the transcriptomes of Tfh and GC B cells indicated that the GF status has favored the programs of Tfh-IFNɣ over Tfh-IL-17.  There was also an increased IL10RA program in GF BXD2 Tfh cells and this was further supported by an increased follicular T-regulatory (Tfr) cells in GF BXD2 mice.  Immunofluorescent staining demonstrated similar spleen follicle structures and enlarged GCs in both SPF and GF BXD2 mice, compared to normal B6 mice. Histologic study in the organs that are most affected in lupus showed similar disease scores in kidney and lung between SPF and GF BXD2 mice.

Conclusion: This study provided the fundamental evidence that the commensal microbiome is not the main driver for the development of systemic autoimmune disease in BXD2 mice. Since the Tfh-IFNɣ/Tfh-IL-17 balance and the Tfr programs were altered in GF mice, the results suggest that certain immune phenotypes may be modulated through leaky intestine, yet they are not sufficient to alter the autoimmune responses in BXD2 mice. 

This work was supported by grants from VA grant (I01BX004049), NIH grants R01-AI-071110, R01 AI134023, and LRA Distinguished Innovator Award to J.D.M, the LRA Novel Research Award to H-C.H., and the P30-AR-048311 and the P30-AI-027767.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/intestinal-microbiota-alters-th1-th17-balance-but-is-dispensable-for-the-development-of-systemic-autoimmune-disease-in-bxd2-mice/