Figure 4. TLR4 Signaling Is Required for Induction of Microbiota-Specific IgG.

(A) Serum concentrations of IgG against fecal bacteria in age-matched WT, Tlr2^−/−, Tlr2^−/−Tlr4^−/−, Nod1^−/−Nod2^−/−, and Nlrp3^−/− mice (n = 4–8 per genotype).

(B) Serum concentrations of IgG, IgA, and IgM against fecal bacteria in age-matched and cohoused WT and Tlr2^−/−Tlr4^−/− mice (n = 8–10 per genotype).

(C) Immunoblotting for IgG in sera from age-matched WT and Tlr2^−/−Tlr4^−/− mice that bound to gram-negative C. rodentium (CR), Salmonella (ST), E. coli (EC), and K. pneumoniae (KP). Coomassie-stained SDS-PAGE gels loaded with lysates are shown.

(D) Serum concentrations of IgG, IgA, and IgM against fecal bacteria in age-matched and co-housed WT, Myd88^−/−, Trif^−/−, and Myd88^−/−Trif^−/− mice.

(E) Survival of WT and Tlr2^−/−Tlr4^−/− mice and of Tlr2^−/−Tlr4^−/− mice after treatment of 2.5% DSS for 7 days (WT n = 9; Tlr2^−/−Tlr4^−/− n = 7).

(F) Survival of WT and Tlr2^−/−Tlr4^−/− mice and of Tlr2^−/−Tlr4^−/− mice that were administered 400 mg of purified serum IgG from WT or QM mice 24 hr prior to i.p. infection with ECM6L4 (WT n = 5; Tlr2^−/−Tlr4^−/− n= 6; Tlr2^−/−Tlr4^−/− +WT IgG n = 6; Tlr2^−/−Tlr4^−/− +QM IgG n = 5).

(G) Serum concentrations of IgG, IgA, and IgM against fecal bacteria in co-housed bone marrow chimeras that were transplanted with J_H^−/−, WT+ J_H^−/−, or Tlr2^−/− Tlr4^−/− + J_H^−/− bone marrow cells.

(H) Peritoneal B1 and B2 cells, and splenic marginal zone (MZ) and follicular (FO) B cells from WT or Tlr2^−/−Tlr4^−/− mice were stimulated ex vivo with heat-killed fecal bacteria for 72 hr before analysis by ELISpot to determine the number of cells secreting IgG that recognized fecal bacteria. Data represent two to three independent experiments. Error bars indicate SD. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S3.