An FcRn-dependent role for anti-flagellin immunoglobulin G in pathogenesis of colitis in mice

Abstract

Background & aims: The neonatal Fc receptor for immunoglobulin (Ig)G (FcRn) protects monomeric IgG from catabolism in parenchymal and hematopoietic cells during adult life. In dendritic cells, FcRn also promotes presentation of antigens in association with IgG. Because IgGs with anti-bacterial specificity are a hallmark of inflammatory bowel disease, we sought to determine their significance and relationship to FcRn expression in antigen-presenting cells, focusing on IgGs specific for flagellin.

Methods: Levels of circulating anti-flagellin IgG were induced in wild-type and FcRn(-/-) mice, followed by induction of colitis with dextran sodium sulfate (DSS). Bone marrow chimera models were used to localize the site of FcRn action.

Results: Wild-type mice that received anti-flagellin IgG exhibited more severe colitis following administration of DSS, compared with mice that received control IgG. Wild-type mice immunized with flagellin exhibited significantly more severe colitis in response to DSS administration than that observed in similarly treated FcRn(-/-) mice. In chimera studies, FcRn(-/-) mice given wild-type bone marrow and immunized with flagellin exhibited significantly more colitis than wild-type mice given FcRn(-/-) bone marrow and immunized with flagellin. Serum anti-flagellin IgG levels were similar in both sets of chimeric mice, consistent with the equal participation of hematopoietic and nonhematopoeitic cells in FcRn-mediated IgG protection.

Conclusions: Anti-bacterial IgG antibodies are involved in the pathogenesis of colitis; this pathway requires FcRn in antigen presenting cells, the major subset of hematopoietic cells that express FcRn.

Figure 1. Anti-Flagellin IgG is only detectable during the recovery phase of DSS colitis

A. Overview of the experimental protocol. B. Serum anti-flagellin IgG levels measured by ELISA increased 25 and 45 days after a 7 day DSS exposure. C. Total serum IgG levels measured by ELISA decreased slightly during the same period. The average values of eight mice are shown and all error bars denote standard error of the mean.

Figure 1. Anti-Flagellin IgG is only detectable during the recovery phase of DSS colitis

A. Overview of the experimental protocol. B. Serum anti-flagellin IgG levels measured by ELISA increased 25 and 45 days after a 7 day DSS exposure. C. Total serum IgG levels measured by ELISA decreased slightly during the same period. The average values of eight mice are shown and all error bars denote standard error of the mean.

Figure 1. Anti-Flagellin IgG is only detectable during the recovery phase of DSS colitis

A. Overview of the experimental protocol. B. Serum anti-flagellin IgG levels measured by ELISA increased 25 and 45 days after a 7 day DSS exposure. C. Total serum IgG levels measured by ELISA decreased slightly during the same period. The average values of eight mice are shown and all error bars denote standard error of the mean.

Figure 2. Anti-Flagellin IgG exacerbates colonic injury in DSS colitis

A. Overview of the experimental protocol. Wild-type C57Bl6 mice were used. B. Serum anti-flagellin IgG level was detectable only in mice that received anti-flagellin IgG. C. Total serum IgG levels were similar in both groups of mice. D. Body weight loss was more pronounced in wild type mice that received anti-flagellin IgG (n=8) during DSS colitis compared to mice that received control IgG (n=7). E. Histological score of colonic tissue was higher in mice injected with anti-flagellin IgG. All error bars denote standard error of the mean and asterisk indicates P<0.05.

Figure 2. Anti-Flagellin IgG exacerbates colonic injury in DSS colitis

A. Overview of the experimental protocol. Wild-type C57Bl6 mice were used. B. Serum anti-flagellin IgG level was detectable only in mice that received anti-flagellin IgG. C. Total serum IgG levels were similar in both groups of mice. D. Body weight loss was more pronounced in wild type mice that received anti-flagellin IgG (n=8) during DSS colitis compared to mice that received control IgG (n=7). E. Histological score of colonic tissue was higher in mice injected with anti-flagellin IgG. All error bars denote standard error of the mean and asterisk indicates P<0.05.

Figure 2. Anti-Flagellin IgG exacerbates colonic injury in DSS colitis

A. Overview of the experimental protocol. Wild-type C57Bl6 mice were used. B. Serum anti-flagellin IgG level was detectable only in mice that received anti-flagellin IgG. C. Total serum IgG levels were similar in both groups of mice. D. Body weight loss was more pronounced in wild type mice that received anti-flagellin IgG (n=8) during DSS colitis compared to mice that received control IgG (n=7). E. Histological score of colonic tissue was higher in mice injected with anti-flagellin IgG. All error bars denote standard error of the mean and asterisk indicates P<0.05.

Figure 2. Anti-Flagellin IgG exacerbates colonic injury in DSS colitis

A. Overview of the experimental protocol. Wild-type C57Bl6 mice were used. B. Serum anti-flagellin IgG level was detectable only in mice that received anti-flagellin IgG. C. Total serum IgG levels were similar in both groups of mice. D. Body weight loss was more pronounced in wild type mice that received anti-flagellin IgG (n=8) during DSS colitis compared to mice that received control IgG (n=7). E. Histological score of colonic tissue was higher in mice injected with anti-flagellin IgG. All error bars denote standard error of the mean and asterisk indicates P<0.05.

Figure 2. Anti-Flagellin IgG exacerbates colonic injury in DSS colitis

A. Overview of the experimental protocol. Wild-type C57Bl6 mice were used. B. Serum anti-flagellin IgG level was detectable only in mice that received anti-flagellin IgG. C. Total serum IgG levels were similar in both groups of mice. D. Body weight loss was more pronounced in wild type mice that received anti-flagellin IgG (n=8) during DSS colitis compared to mice that received control IgG (n=7). E. Histological score of colonic tissue was higher in mice injected with anti-flagellin IgG. All error bars denote standard error of the mean and asterisk indicates P<0.05.

Figure 3. FcRn deficient mice are protected from flagellin IgG mediated DSS colitis

A. Overview of the experimental protocol. B. After two rounds of flagellin immunization, serum anti-flagellin levels were increased in both wild type (n=8) and FcRn-deficient mice (n=8) during DSS colitis. C. Body weight loss was significantly more severe in wild type mice during DSS colitis. D. Body weight change was similar in wild type (n=6) and FcRn deficient mice (n=6) during DSS colitis after immunization with OVA. E. Body weight change was similar in wild type (n=6) and FcRn deficient mice (n=6) during DSS colitis after immunization with PBS. All error bars denote standard error of the mean and an asterisk indicates P<0.05.

Figure 3. FcRn deficient mice are protected from flagellin IgG mediated DSS colitis

A. Overview of the experimental protocol. B. After two rounds of flagellin immunization, serum anti-flagellin levels were increased in both wild type (n=8) and FcRn-deficient mice (n=8) during DSS colitis. C. Body weight loss was significantly more severe in wild type mice during DSS colitis. D. Body weight change was similar in wild type (n=6) and FcRn deficient mice (n=6) during DSS colitis after immunization with OVA. E. Body weight change was similar in wild type (n=6) and FcRn deficient mice (n=6) during DSS colitis after immunization with PBS. All error bars denote standard error of the mean and an asterisk indicates P<0.05.

Figure 3. FcRn deficient mice are protected from flagellin IgG mediated DSS colitis

A. Overview of the experimental protocol. B. After two rounds of flagellin immunization, serum anti-flagellin levels were increased in both wild type (n=8) and FcRn-deficient mice (n=8) during DSS colitis. C. Body weight loss was significantly more severe in wild type mice during DSS colitis. D. Body weight change was similar in wild type (n=6) and FcRn deficient mice (n=6) during DSS colitis after immunization with OVA. E. Body weight change was similar in wild type (n=6) and FcRn deficient mice (n=6) during DSS colitis after immunization with PBS. All error bars denote standard error of the mean and an asterisk indicates P<0.05.

Figure 3. FcRn deficient mice are protected from flagellin IgG mediated DSS colitis

A. Overview of the experimental protocol. B. After two rounds of flagellin immunization, serum anti-flagellin levels were increased in both wild type (n=8) and FcRn-deficient mice (n=8) during DSS colitis. C. Body weight loss was significantly more severe in wild type mice during DSS colitis. D. Body weight change was similar in wild type (n=6) and FcRn deficient mice (n=6) during DSS colitis after immunization with OVA. E. Body weight change was similar in wild type (n=6) and FcRn deficient mice (n=6) during DSS colitis after immunization with PBS. All error bars denote standard error of the mean and an asterisk indicates P<0.05.

Figure 3. FcRn deficient mice are protected from flagellin IgG mediated DSS colitis

A. Overview of the experimental protocol. B. After two rounds of flagellin immunization, serum anti-flagellin levels were increased in both wild type (n=8) and FcRn-deficient mice (n=8) during DSS colitis. C. Body weight loss was significantly more severe in wild type mice during DSS colitis. D. Body weight change was similar in wild type (n=6) and FcRn deficient mice (n=6) during DSS colitis after immunization with OVA. E. Body weight change was similar in wild type (n=6) and FcRn deficient mice (n=6) during DSS colitis after immunization with PBS. All error bars denote standard error of the mean and an asterisk indicates P<0.05.

Figure 4. Severity of DSS induced colitis is decreased in FcRn deficient mice

A. Representative histology of wild type and FcRn-deficient mice on day 14 of the DSS colitis. B. Histological scores of colonic tissue on day 14 of the DSS exposure was evaluated in a blinded fashion by a pathologist. C. Histology of colitis lesion in KO→WT and WT→KO chimeras immunized with either flagellin or PBS. D. Histological score of the colonic tissue KO→WT (n=8, filled columns) and WT→KO (n=7, open columns) chimera immunized with either flagellin or PBS.

Figure 4. Severity of DSS induced colitis is decreased in FcRn deficient mice

A. Representative histology of wild type and FcRn-deficient mice on day 14 of the DSS colitis. B. Histological scores of colonic tissue on day 14 of the DSS exposure was evaluated in a blinded fashion by a pathologist. C. Histology of colitis lesion in KO→WT and WT→KO chimeras immunized with either flagellin or PBS. D. Histological score of the colonic tissue KO→WT (n=8, filled columns) and WT→KO (n=7, open columns) chimera immunized with either flagellin or PBS.

Figure 4. Severity of DSS induced colitis is decreased in FcRn deficient mice

A. Representative histology of wild type and FcRn-deficient mice on day 14 of the DSS colitis. B. Histological scores of colonic tissue on day 14 of the DSS exposure was evaluated in a blinded fashion by a pathologist. C. Histology of colitis lesion in KO→WT and WT→KO chimeras immunized with either flagellin or PBS. D. Histological score of the colonic tissue KO→WT (n=8, filled columns) and WT→KO (n=7, open columns) chimera immunized with either flagellin or PBS.

Figure 4. Severity of DSS induced colitis is decreased in FcRn deficient mice

A. Representative histology of wild type and FcRn-deficient mice on day 14 of the DSS colitis. B. Histological scores of colonic tissue on day 14 of the DSS exposure was evaluated in a blinded fashion by a pathologist. C. Histology of colitis lesion in KO→WT and WT→KO chimeras immunized with either flagellin or PBS. D. Histological score of the colonic tissue KO→WT (n=8, filled columns) and WT→KO (n=7, open columns) chimera immunized with either flagellin or PBS.

Figure 5. Colitis provoked by anti-flagellin specific IgG is mediated by FcRn expressed in myeloid cells

A. Bone marrow chimeric mice were generated with wild-type and FcRn-deficient mice. Serum levels of the passively transferred rabbit IgG was followed daily in each group (n=4) by ELISA. B. Overview of the experimental protocol. C. The serum anti-flagellin IgG levels measured by ELISA were similar in FcRn KO→WT chimera (n=8) and WT→KO chimera (n=7). D. Body weight loss was significantly higher in WT→KO (n=7) and WT→WT (n=7) groups compared with the KO→WT (n=8) and KO→KO (n=4) groups during DSS colitis immunized with flagellin. E. The levels of inflammatory cytokines IL-12p70, IFN-γ and IL-10, expressed as pg per mg total protein, in colonic tissue homogenates of KO→WT (filled columns) and WT→KO chimera (open columns) were measured by ELISA. All error bars denote standard error of the mean and an asterisk indicates P<0.05.

Figure 5. Colitis provoked by anti-flagellin specific IgG is mediated by FcRn expressed in myeloid cells

A. Bone marrow chimeric mice were generated with wild-type and FcRn-deficient mice. Serum levels of the passively transferred rabbit IgG was followed daily in each group (n=4) by ELISA. B. Overview of the experimental protocol. C. The serum anti-flagellin IgG levels measured by ELISA were similar in FcRn KO→WT chimera (n=8) and WT→KO chimera (n=7). D. Body weight loss was significantly higher in WT→KO (n=7) and WT→WT (n=7) groups compared with the KO→WT (n=8) and KO→KO (n=4) groups during DSS colitis immunized with flagellin. E. The levels of inflammatory cytokines IL-12p70, IFN-γ and IL-10, expressed as pg per mg total protein, in colonic tissue homogenates of KO→WT (filled columns) and WT→KO chimera (open columns) were measured by ELISA. All error bars denote standard error of the mean and an asterisk indicates P<0.05.

Figure 5. Colitis provoked by anti-flagellin specific IgG is mediated by FcRn expressed in myeloid cells

A. Bone marrow chimeric mice were generated with wild-type and FcRn-deficient mice. Serum levels of the passively transferred rabbit IgG was followed daily in each group (n=4) by ELISA. B. Overview of the experimental protocol. C. The serum anti-flagellin IgG levels measured by ELISA were similar in FcRn KO→WT chimera (n=8) and WT→KO chimera (n=7). D. Body weight loss was significantly higher in WT→KO (n=7) and WT→WT (n=7) groups compared with the KO→WT (n=8) and KO→KO (n=4) groups during DSS colitis immunized with flagellin. E. The levels of inflammatory cytokines IL-12p70, IFN-γ and IL-10, expressed as pg per mg total protein, in colonic tissue homogenates of KO→WT (filled columns) and WT→KO chimera (open columns) were measured by ELISA. All error bars denote standard error of the mean and an asterisk indicates P<0.05.

Figure 5. Colitis provoked by anti-flagellin specific IgG is mediated by FcRn expressed in myeloid cells

A. Bone marrow chimeric mice were generated with wild-type and FcRn-deficient mice. Serum levels of the passively transferred rabbit IgG was followed daily in each group (n=4) by ELISA. B. Overview of the experimental protocol. C. The serum anti-flagellin IgG levels measured by ELISA were similar in FcRn KO→WT chimera (n=8) and WT→KO chimera (n=7). D. Body weight loss was significantly higher in WT→KO (n=7) and WT→WT (n=7) groups compared with the KO→WT (n=8) and KO→KO (n=4) groups during DSS colitis immunized with flagellin. E. The levels of inflammatory cytokines IL-12p70, IFN-γ and IL-10, expressed as pg per mg total protein, in colonic tissue homogenates of KO→WT (filled columns) and WT→KO chimera (open columns) were measured by ELISA. All error bars denote standard error of the mean and an asterisk indicates P<0.05.

Figure 5. Colitis provoked by anti-flagellin specific IgG is mediated by FcRn expressed in myeloid cells

A. Bone marrow chimeric mice were generated with wild-type and FcRn-deficient mice. Serum levels of the passively transferred rabbit IgG was followed daily in each group (n=4) by ELISA. B. Overview of the experimental protocol. C. The serum anti-flagellin IgG levels measured by ELISA were similar in FcRn KO→WT chimera (n=8) and WT→KO chimera (n=7). D. Body weight loss was significantly higher in WT→KO (n=7) and WT→WT (n=7) groups compared with the KO→WT (n=8) and KO→KO (n=4) groups during DSS colitis immunized with flagellin. E. The levels of inflammatory cytokines IL-12p70, IFN-γ and IL-10, expressed as pg per mg total protein, in colonic tissue homogenates of KO→WT (filled columns) and WT→KO chimera (open columns) were measured by ELISA. All error bars denote standard error of the mean and an asterisk indicates P<0.05.