Immune and metabolic defects were observed in germ-free (GF) mice due to lack of microbiota. GF mice produce extremely decreased amounts of SCFAs and increased amounts of indigestible oligosaccharide 22. GF and newborn mice exhibit biased Th2 immune response. When microbes are exposed to intestine at an early age, they modulate Th2-biased immune response which in turn stimulate differentiation of T helper cells e.g. Th1, Th17, regulatory T (Treg) cells. Treg cells are a crucial part of the adaptive immune system which controls inflammatory responses to maintain cellular homeostasis.23It was reported that drinking water supplemented with acetate, propionate, and butyrate increases the number of colonic Tregs as well as accumulation of G-protein coupled receptor: GPR43 dependent colonic Tregs in germ-free mice 5. Moreover, for proper development and function of the immune system gut microbiota-derived Tryptophan (Trp) metabolites are very important. Indole is a by-product of Tryptophan (Trp) catabolism, which decreases bacterial intestinal epithelial cell (IEC) adhesion, motility, biofilm formation, and pathogen chemotaxis for enterohemorrhagic E. coli. Indole also induces host IEC barrier integrity, expression of anti-inflammatory IL-10 and inhibits inflammatory TNF-?- mediated IL-8 and NF-?B signalling. In a germ-free mice study it was reported that during dextran sodium sulfate colitis, oral indole therapy alleviated GI pathology, mortality and weight loss which confirmed functions of indole 24.Different immune cells for instance T-cells, macrophages, dendritic cells express cytokines like Interleukin-10 (IL-10) which is an anti-inflammatory cytokine. IL-10 plays an important role in intestinal mucosal immunity regulation as IL-10 deficient mice showed a spontaneous development of intestinal inflammation unless reared in a germ-free condition 25.It was observed that commensal microbiota stimulate intestinal macrophages via up-regulating pro-IL 1? activity which recruits neutrophil for pathogen eradication in a GF and conventionally raised mouse model. Another study showed that neutrophil count and macrophage function e.g. phagocytosis, microbicidal activities including phagocytic superoxide anion production is lower in GF mice 26. Autoimmune diseases like rheumatoid arthritis, IBD have been found associated with alteration in commensal microbiota. In IBD patients, a low number of Firmicutes and Bacteroidetes and high number of Proteobacteria have been observed. A research group observed that colonization of GF mice with Bacteroides fragilis stimulates immune system via a bacterial polysaccharide (PSA) mediated pathway as well as restores Th1 and Th2 balance 26.