The interaction between the commensal microbiota and the immune system do not only influences functions that affect immune system homeostasis but also play a role in the development of disease. This interaction is widely known as the microbe-host crosstalk and it involves the interaction of several pathways that contribute to a healthy gut microenvironment. An imbalance in intestinal bacteria, enterocytes and cells from the local immune system is known to lead to overstimulation of the immune system, thereby contributing to the development of several autoimmune diseases. In the case of Multiple Sclerosis (MS), recent studies have shown that the microbiota plays an active role in influencing the local immune system of the gut-associated lymphoid tissue (GALT) but the exact mechanism behind this interaction remains largely unknown. In this study, we explore the effect that epithelial MyD88 signals have in the microbial-host crosstalk and its possible influence in Experimental Autoimmune Encephalomyelitis (EAE) development. Intestinal epithelial cells (IECs) specific MyD88 knock-out mice (MyD88?IEC mice) were assessed for changes in their intestinal homeostasis as well as profiled for their cytokine production and microbiota composition. Epithelial MyD88 signals have been observed to play an important role in the development of EAE as a more severe course of the disease was observed in these mice when compared to control littermates. Likewise, a higher incidence in the development of spontaneous EAE was observed when crossing MyD88?IEC mice with Opticospinal EAE (OSE) mice. The phenotype observed in these mice could be attributed to a dampened Th2 local response, featuring a slight reduction in IL-4 and IL-10 cytokines that is accompanied with a significant reduction in small intestine lamina propria (SiLPL) FoxP3+ Treg cells. On the other hand, alterations in the local Th1/Th17 response were not observed in MyD88?IEC steadystate mice. In this work, we could exclude a potential translocation of gut bacteria into the gut tissues of MyD88?IEC as we failed to detect the presence of 16S rRNA in peripheral organs. In addition, no significant changes in the overall microbiota composition that could hint to a dysbiosis in MyD88?IEC mice were observed when sequencing the V3/V4 regions of 16S RNA genes. Furthermore, frequencies of fecal IgA in MyD88?IEC mice did not differ from those of control mice, discarding the possibility of an impaired IgAmediated immune response in gut tissues. Having explored different mechanisms to explain the exacerbated course and increased incidence of EAE in MyD88?IEC mice, we propose an alternative theory. We theorize that the slight increase in the gut SUMMARY 4 permeability observed in MyD88?IEC mice in this work could allow local dendritic cells (DCs) to sample a higher number of commensal bacterial products from the luminal bacteria. Local DCs could in turn activate B and T cells within the Peyers Patches; therefore, unleashing a pro-inflammatory response that cannot be controlled by local FoxP3+ Treg cells as MyD88?IEC mice were found to have a significant decrease in FoxP3+ Tregs cells within the SiLPL. This, combined with other possible potential alterations in the gut homeostasis (such as AMP production, tight-junction expression profile and defects in the mucus layer of small intestine and colon) could offer an explanation to the phenotype observed when studying EAE development in MyD88ΔIEC mmice. In summary, the lack IEC-MyD88 dependent signals are shown to significantly reduce the frequencies of local FoxP3+ Tregs in the intestinal lamina propria of the small intestine of MyD88ΔIEC m mice, a finding that might contribute to the increased EAE severity observed in these mice. However, these signals appear not to influence fecal IgA frequencies, overall microbiota composition and local gut Th1/Th17 immune response in MyD88ΔIEC m steady-state mice.