Effects of gut microbiota-derived products on cardiovascular complications of systemic lupus erythematosus

Abstract

In this thesis, we embark on a comprehensive exploration of the multifaceted interactions between microbiota and SLE. Through a meticulous examination of existing literature, coupled with original research endeavors, we aim to delineate the mechanistic underpinnings that govern this intricate relationship. By elucidating the key players and pathways involved, we seek to pave the way for novel therapeutic approaches that harness the potential of gut microbiota and its metabolites in the management of SLE and its consequent cardiovascular complications. Therefore, the general objective of this thesis was to analyse the role of gut microbiota and its bacterial by-products (TMAO, SCFA) in the development of cardiovascular complications in SLE. To achieve this aim we performed several experiments: I) Role of gut microbiota in the development of hypertension in a TLR-7- dependent lupus mouse model Female BALB/cJRj mice were randomly assigned to four experimental groups: an untreated control (CTR), a group treated with the TLR-7 agonist imiquimod (IMQ), IMQ-treated with vancomycin, and IMQ-treated with a cocktail of broad-spectrum antibiotics. We carried out faecal microbiota transplant (FMT) from donor CTR or IMQ mice to recipient IMQ or CTR animals, respectively. Vancomycin inhibited the increase in blood pressure, improved kidney injury, endothelial function and oxidative stress, and reduced T helper (Th)17 infiltration in aortas from IMQtreated mice. The rise in blood pressure and vascular complications present in IMQ mice were also observed in the CTR mice recipients of IMQ microbiota. Reduced relative populations of Sutterella and Anaerovibrio were associated to high blood pressure in our animals, which were increased after stool transplantation of healthy microbiota to IMQ mice. The reduced endothelium-dependent vasodilator responses to acetylcholine induced by IMQ microbiota were normalized after interleukin-17 neutralization. In conclusion, gut microbiota plays a role in the TLR7- driven increase in Th17 cell, endothelial dysfunction, vascular inflammation and hypertension. The vascular changes induced by IMQ microbiota were initiated by Th17 infiltrating the vasculature. II) Role of TMAO in SLE development and cardiovascular complications in a lupus mouse model induced by TLR-7 activation Female BALB/c mice were randomly divided into 4 groups: untreated control mice, control mice treated with the trimethylamine lyase inhibitor 3,3-dimethyl-1- butanol (DMB), IMQ mice, and IMQ mice treated with DMB. The DMB-treated groups were administered in their drinking water for 8 weeks. Treatment with DMB reduced plasma levels of TMAO in mice with IMQ-induced lupus. DMB prevents the development of hypertension, reduces disease progression (plasma levels of antidsDNA autoantibodies, splenomegaly and proteinuria), polarization of T lymphocytes towards Th17/Th1 in secondary lymph organs and improves endothelial function in mice with IMQ-induced lupus. The deleterious vascular effects caused by TMAO appear to be associated with an increase in vascular oxidative stress generated by increased NADPH oxidase activity, derived in part from the vascular infiltration of Th17/Th1 lymphocytes, and reduced NRF-2-driven antioxidant defense. In conclusion, our findings identify the bacterial-derived TMAO as a regulator of immune system allowing the development of autoimmunity and endothelial dysfunction in SLE mice. III) Effects of SCFA on cardiovascular complications in TLR-7-induced lupus mouse model SCFA and dietary fibers rich in resistant starch (RS) or inulin-type fructans (ITF) effectively prevented the development of hypertension and cardiac hypertrophy. Additionally, these treatments improved aortic relaxation and mitigated vascular oxidative stress. Both SCFA treatments also contributed to the maintenance of colonic integrity, reduced endotoxemia, and decreased the proportion of Th-17 cells in MLN, blood, and aorta in TLR7-induced SLE mice. The observed changes in MLNs were correlated with increased levels of GPR43 mRNA in mice treated with acetate and increased GPR41 levels along with decreased histone deacetylase (HDAC)-3 levels in mice treated with butyrate. Notably, the effects attributed to acetate, but not butyrate, were nullified when co-administered with the GPR43 antagonist GLPG-0974. T cell priming and differentiation into Th17 cells in MLNs, as well as increased Th17 cell infiltration, were linked to aortic endothelial dysfunction and hypertension subsequent to the transfer of faecal microbiota from IMQ-treated mice to germ-free (GF) mice. These effects were counteracted in GF mice through treatment with either acetate or butyrate. To conclude, these findings underscore the potential of SCFA consumption in averting hypertension by restoring balance to the interplay between the gut, immune system, and vascular wall in TLR-7-driven SLE.

IV) Role of dietary fiber intake in the raise of BP in NZBWF1 mice Female NZBWF1 (SLE) mice were treated with dietary fibers rich in resistant-starch (RS) or inulin-type frutans (ITF). In addition, inoculation of faecal microbiota from these experimental groups to recipient normotensive female C57Bl/6J germ-free (GF) mice was performed. Both fiber treatments, especially RS, prevented the development of hypertension, renal injury, improved the aortic relaxation induced by acetylcholine, and the vascular oxidative stress. RS and ITF treatments increased the proportion of acetate-, and butyrate-producing bacteria, respectively, improved colonic inflammation and integrity, endotoxemia, and decreased Th17 proportion in MLN, blood, and aorta in SLE mice. However, disease activity (splenomegaly and anti-ds-DNA) was unaffected by both fibers. T cell priming and Th17 differentiation in MLNs and increased Th17 infiltration was linked to aortic endothelial dysfunction and hypertension after inoculation of faecal microbiota from SLE mice to GF mice, without changes in proteinuria and autoimmunity. All these effects were lower in GF mice after faecal inoculation from fiber treated SLE mice. In conclusion, these findings support that fiber consumption prevented the development of hypertension by rebalancing of dysfunctional gutimmune system-vascular wall axis in SLE. All of these findings suggest that gut microbiota modulation based on dietary interventions could be a novel alternative for the prevention of SLE-linked cardiovascular complications.