Conventional Housing Conditions Attenuate the Development of Experimental Autoimmune Encephalomyelitis

BACKGROUND: The etiology of multiple sclerosis (MS) has remained unclear, but a causative contribution of factors outside the central nervous system (CNS) is conceivable. It was recently suggested that gut bacteria trigger the activation of CNS-reactive T cells and the development of demyelinative disease. METHODS: C57BL/6 (B6) mice were kept either under specific pathogen free or conventional housing conditions, immunized with the myelin basic protein (MBP)-proteolipid protein (PLP) fusion protein MP4 and the development of EAE was clinically monitored. The germinal center size of the Peyer's patches was determined by immunohistochemistry in addition to the level of total IgG secretion which was assessed by ELISPOT. ELISPOT assays were also used to measure MP4-specific T cell and B cell responses in the Peyer's patches and the spleen. Ear swelling assays were performed to determine the extent of delayed-type hypersensitivity reactions in specific pathogen free and conventionally housed mice. RESULTS: In B6 mice that were actively immunized with MP4 and kept under conventional housing conditions clinical disease was significantly attenuated compared to specific pathogen free mice. Conventionally housed mice displayed increased levels of IgG secretion in the Peyer's patches, while the germinal center formation in the gut and the MP4-specific TH17 response in the spleen were diminished after immunization. Accordingly, these mice displayed an attenuated delayed type hypersensitivity (DTH) reaction in ear swelling assays. CONCLUSIONS: The data corroborate the notion that housing conditions play a substantial role in the induction of murine EAE and suggest that the presence of gut bacteria might be associated with a decreased immune response to antigens of lower affinity. This concept could be of importance for MS and calls for caution when considering the therapeutic approach to treat patients with antibiotics.

Attenuated MP4-specific IL-17 production in conventionally housed mice.

<p>Mice were housed either under conventional (n = 10) or SPF (n = 13) conditions and actively immunized with 200 µg MP4. On days 9–15 after immunization ELISPOT assays were performed to measure the MP4-specific secretion of IFN-γ and IL-17 in the spleen. Data are shown for individual mice that are represented by circles. The mean values are shown by the bars.</p

Attenuated DTH reaction in conventionally housed mice.

<p>Mice were housed either under conventional or SPF conditions and actively immunized with 200 µg MP4. On days 22–25 after immunization mice were challenged by intradermal ear injections with either 10 µg of MP4 or the irrelevant antigen OVA. After 24 h ear biopsies were taken and the extent of ear swelling (A–E) and cellular infiltration were determined (F–J) in each group. In the conventionally housed group n = 4 MP4- and n = 4 OVA-treated ears were analyzed. In the SPF group n = 5 MP4- and n = 6 OVA-treated ears were analyzed.</p

Increased IgG secretion in Peyer’s patches of mice kept under conventional conditions.

<p>Mice were housed either under conventional (n = 6) or SPF (n = 4) conditions and actively immunized with 200 µg MP4. On days 23–30 after immunization ELISPOT assays were performed to measure the extent of total IgG secretion in the Peyer’s patches. A, Number of spots per 10⁶ plated cells derived from the Peyer’s patches, the mesenteric or inguinal lymph nodes (LN). B, Representative ELISPOT well images showing medium control and total IgG responses in SPF and conventionally housed mice.</p

Increased germinal center reactions in Peyer’s patches after immunization in SPF housed mice.

<p>Mice were housed either under conventional or SPF conditions and actively immunized with 200 µg MP4. On days 23–30 after immunization the germinal center size was studied histologically on Ki67-stained sections taken from the Peyer’s patches of n = 6 conventionally and n = 6 SPF housed mice. In addition n = 7 conventionally and n = 9 SPF housed unimmunized control mice were studied. A, Germinal center size per Peyer’s patch area shown as mean + SD in SPF mice. B, Germinal center size per Peyer’s patch area shown as mean + SD in conventionally housed mice. C–F, Representative images of Ki67 IHC in the different groups.</p

MP4-specific production of IgG, IL-17 and IFN-γ in the gut.

<p>Mice were housed either under conventional or SPF conditions and actively immunized with 200 µg MP4. On days 23–30 after immunization ELISPOT assays were performed to measure the MP4-specific B cell response in the Peyer’s patches (n = 14 mice in each group). On days 9–15 after immunization ELISPOT assays were performed to measure the MP4-specific secretion of IL-17 (n = 19 SPF and n = 13 conventionally housed mice) and IFN-γ (n = 6 SPF versus n = 3 conventionally housed mice) in the Peyer’s patches. Data are shown for individual mice represented by circles. The mean values are shown by the bars.</p