Acellular Bordetella pertussis vaccine enhances mucosal interleukin-10 production, induces apoptosis of activated Th1 cells and attenuates colitis in Galphai2-deficient mice

Abstract

Mice deficient for the inhibitory G protein subunit alpha2 (Galphai2(-/-)) spontaneously develop a progressive inflammatory bowel disease resembling ulcerative colitis, and have a T helper 1 (Th1)-dominated immune response prior to onset of colitis, which is further augmented after the onset of disease. The present study was performed to investigate whether the Galphai2(-/-) mice were able to down-regulate the Th1-dominated inflammatory mucosal immune response and/or induce an anti-inflammatory Th2/T regulatory response and thereby diminish the severity of colitis following treatment with acellular Bordetella pertussis vaccine. The acellular vaccine against B. pertussis, the causative agent of whooping cough, has been demonstrated to induce a Th2-mediated response in both man and mice. We therefore treated Galphai2(-/-) mice intraperitoneally with a three-component acellular B. pertussis vaccine. The treated Galphai2(-/-) mice showed significantly increased interleukin (IL)-10 production in intestinal tissue, associated with significantly reduced colitis and decreased mortality, compared to untreated Galphai2(-/-) mice. The attenuation of colitis in Galphai2(-/-) mice was due, at least partly, to the B. pertussis surface antigen filamentous haemagglutinin (FHA), which almost completely inhibited proliferation of CD4(+) T cells and stimulated apoptosis of activated CD4(+) T helper 1 cells. In conclusion, the three-component acellular B. pertussis vaccine containing filamentous haemagglutinin increases the production of IL-10 in the intestinal mucosa, induces apoptosis of activated Th1 cells and attenuates colitis in Galphai2(-/-) mice.

Fig. 1

Decreased mortality and attenuated colitis in inhibitory G-protein α2 subunit (Gαi2^−/−) mice treated with three-component Bordetella pertussis vaccine. Gαi2^−/− mice were given 5 µg B. pertussis three-component vaccine at the age of 6, 10 and 14 weeks of age, and untreated Gαi2^−/− mice were used as controls. (a) The numbers of mice that died due to severe colitis up to 19 weeks of age were recorded. Results are pooled from two independent experiments with a total of 12 mice in each group. (b) Blinded clinical evaluation of colitis in the large intestine from three-component B. pertussis vaccine treated and untreated Gαi2^−/− mice at 19 weeks of age. The clinical colitis was assessed as declared in Materials and methods, rating the inflammation score from 1 to 5. Results shown are from two independent experiments with a total of 12 mice in each group at the start of the experiment. Each dot represents one mouse and the median for each group is denoted. (c) Blinded histopathological evaluation of colitis in the large intestine from three-component B. pertussis vaccine treated and untreated Gαi2^−/− mice at 19 weeks of age. The histopathological intensity of colonic inflammation was analysed according to previous established criteria, rating the inflammation score from 1 to 5. Results shown are from two independent experiments with a total of 12 mice in each group at the start of the experiment, although showing histological score of only nine animals in the untreated group as three mice died (indicated in the figure by a cross) due to severe colitis during the study, and were not included in the histological evaluation. Each dot represents one mouse and the median for each group is denoted.

Fig. 2

Increased spontaneous production of interleukin (IL-10) but not interferon (IFN)-γ in small and large intestinal mucosa of Bordetella pertussis vaccine-treated inhibitory G-protein α2 subunit (Gαi2^−/−) mice at 19 weeks of age. Mice were given three doses of 5 µg B. pertussis three-component vaccine at the age of 6, 10 and 14 weeks of age, and were killed at 19 weeks of age; 50 mg intestinal tissue was cultured for 24 h, and the spontaneous production of IL-10 and IFN-γ was determined by enzyme-linked immunosorbent assay (ELISA). Results are pooled from two separate experiments and shown as mean ± s.d. from 12 mice in each group.

Fig. 3

Significantly decreased serum IgG2a/IgG1 ratio from inhibitory G-protein α2 subunit (Gαi2^−/−) mice treated with three-component Bordetella pertussis vaccine. Gαi2^−/− mice were given 5 µg B. pertussis three-component vaccine at the age of 6, 10 and 14 weeks of age and untreated Gαi2^−/− mice were used as controls, and the serum IgG subclasses were determined by enzyme-linked immunosorbent assay (ELISA) analysis at 19 weeks of age. Results are shown as the ratio of serum IgG2a : IgG1 and are based on two separate experiments and shown as mean ± s.d. from 12 B. pertussis treated mice and nine untreated mice. Absolute values for IgG1 were 2768 ± 1156 µg/ml in B. pertussis-treated Gαi2^−/− mice compared to 1391 ± 1980 µg/ml in untreated Gαi2^−/− mice. In wild-type mice IgG1 levels were 7816 ± 2314 µg/ml and 1013 ± 869 µg/ml in B. pertussis and untreated mice, respectively. For IgG2a the absolute values were 8068 ± 2785 µg/ml in B. pertussis-treated and 8523 ± 3738 µg/ml in untreated Gαi2^−/− mice, 9764 ± 3433 µg/ml in B. pertussis treated and 3711 ± 1611 µg/ml in untreated wild-type mice.

Fig. 4

Decreased T lymphocyte proliferation and increased levels of interleukin (IL)-2 in the presence of filamentous haemagglutinin (FHA). (a) Splenocytes from inhibitory G-protein αi2 subunit (Gαi2^−/−) or wild-type mice were cultured in the presence of α-CD3 together with the Bordetella pertussis surface antigens FHA (5 µg/ml) or pertactin (PRN) (5 µg/ml) for 72 h with the addition of 1 µCi [³H]TdR for the final 6 h of culture to determine proliferative responses. (b) Splenocytes from Gαi2^−/− or wild-type mice were cultured in the presence of α-CD3 together with the B. pertussis surface antigens FHA (5 µg/ml) or PRN (5 µg/ml) and the supernatants were collected after 24 h of culture and the spontaneous production of IL-2 was determined by enzyme-linked immunosorbent assay (ELISA). Results are shown as mean ± s.d. of individual mice from one representative experiment of two giving similar results, both with four mice in each group.

Fig. 5

Filamentous haemagglutinin (FHA) induces increased apoptosis of activated CD4⁺ T cells. Inhibitory G-protein αi2 subunit deficient (Gαi2^−/−) mice were given 5 µg FHA i.p. once and untreated Gαi2^−/− mice were used as controls. Mice were killed 48 h after treatment and spleen cells were isolated and double-stained with either annexin-V fluorescein isothiocyanate (FITC) and CD4 phycoerythrin (PE) for detection of apoptotic CD4⁺ T cells (a), PI and CD4 PE for detection of necrotic CD4⁺ T cells (b), annexin-V FITC and CD44 PE for detection of apoptotic CD44^hi cells (c) or PI and CD44 PE for detection of necrotic CD44^hi cells (d).