The skin is a site of constant dialogue between the immune system and commensal bacteria. Epi-2W to the back skin of adult C57Bl/6 mice resulted in long-term persistence of the strain (Physique 1A). Flow cytometric and histologic evaluation of Epi- 2W colonized skin failed to reveal evidence of tissue inflammation (Figures 1B and C), suggesting that a state of true commensalism was achieved. Physique 1 Antigen-specific recognition of commensal microbes across an intact skin hurdle To test whether commensal antigens were acknowledged across an intact skin hurdle, we assayed the 2W-specific immune response in mice colonized with Epi-2W. Adult animals were colonized with Epi-2W every three days for one week, and skin, skin-draining lymph nodes (SDLNs) and spleen were examined at day 10. In colonized animals we observed a significant increase in the frequency and absolute number of activated (CD44+) antigen-specific (2W MHC Class II tetramer-positive) CD4+ T cells in both SDLNs (Figures 1D and 1E) and spleen (Figures 1F and 1G). Taken buy 1427782-89-5 together, these data suggest that we have established a model that closely recapitulates normal cutaneous bacterial buy 1427782-89-5 commensalism, in that stable colonization occurs without resultant tissue inflammation. The results indicate that skin bacterial antigens were acknowledged by the adaptive immune system across an intact skin hurdle. These findings are consistent with buy 1427782-89-5 and build upon a recent report showing that skin commensal bacteria influence cutaneous immunity without causing tissue inflammation (Naik et al., 2015). Bacterial colonization during adult life does not establish tolerance We hypothesized that the adaptive immune system plays a role in mediating tolerance to skin commensal bacteria. To test this, we colonized the skin of 6 week-old adult mice with Epi-2W. Three to four weeks later, we challenged these mice alongside age-matched control animals (that were not colonized with Epi-2W) by applying Epi-2W in conjunction with light tape-stripping of the epidermis to minimally abrade the skin surface (Physique H2A). We rationalized that this approach was the most physiologically appropriate method of elucidating anti-commensal immune responses because it recapitulated increased exposure to commensal antigens in the setting of frequent incidental skin trauma (at the.g. abrasions, scratching), a mildly inflammatory context during which mechanisms of immune tolerance would need to be active. Pre-colonization with Epi-2W did not attenuate skin inflammation upon challenge. Ten days after challenge was initiated, both pre-colonized and control groups had equivalently increased histologic evidence of skin inflammation (Physique 2A) and comparative numbers and frequency of skin neutrophils (Physique 2B). Consistent with the above observations, pre-colonization did not alter the number of activated antigen-specific CD44+CD4+FoxP3neg effector T (Teff) cells in SDLNs (Physique 2C) nor did it preferentially enrich for antigen-specific Treg cells in either the SDLNs or skin (Figures 2D, 2E and S2W). These results suggest that initial colonization by a skin commensal during adult life is usually not sufficient to establish tolerance to commensal antigens. Physique 2 Colonization with commensal bacteria in adult mice does not establish immune tolerance Neonatal colonization is usually required to establish tolerance to skin commensal bacteria Because the host-commensal relationship is usually formed immediately after birth (Dominguez-Bello et al., 2010; Rotimi and Duerden, 1981), we hypothesized that mechanisms required to establish tolerance may be preferentially active during this period of time. To test this, we Rabbit Polyclonal to Tyrosine Hydroxylase employed the previously layed out experimental approach, but instead colonized mice early in the postnatal period. Neonatal mice were colonized with Epi-2W for one week beginning on postnatal day 7 and challenged alongside Epi- 2W na?ve age-matched animals 3C4 weeks later in adult life, using the skin abrasion technique previously described. In contrast to our observations with adult animals, mice colonized with Epi-2W in the neonatal period demonstrated markedly diminished histologic skin inflammation and substantially reduced neutrophilic infiltration upon challenge with Epi-2W (Figures 3A and 3B). This was associated with significantly fewer activated 2W-specific CD44+CD4+ Teff cells in the SDLNs (Figure.