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. Author manuscript; available in PMC: 2012 Dec 23.
Published in final edited form as: Immunity. 2011 Dec 23;35(6):997–1009. doi: 10.1016/j.immuni.2011.10.018

Figure 3.

Figure 3

Mucosal immunity to an autologous bacterial challenge is mediated by B-cells and Th17 cells. (A) C57BL/6 mice were immunized intranasally with 20 µg of heat-killed K. Pneumoniae-43816 (ATCC) (serotype K2) at Day 0 and Day 7. 4 weeks after the 2nd immunization, immunized mice and naïve unimmunized mice were infected with 104 live K. Pneumoniae-43816 (ATCC) (serotype K2) and sacrificed 24h after. Lung burden and systemic dissemination were determined by lung (left panel) and spleen (right panel) CFUs. Each group has an N>20 from 4 independent experiments. (B) 4 week after 2nd immunization, immunized mice and naïve mice were infected with 104 live K. Pneumoniae-43816 (ATCC) (serotype K2) and sacrificed 24h after. A group of immunized mice also received i.p. rat IgG or 1A8 24 hours and 2 hours prior to infection. A group of immunized mice received neutralizing antibody against IL-17A and/or IL-22 i.t. immediately prior to infection. Bacterial burdens were determined by lung CFU (left panel) and spleen CFU (middle panel) and neutrophil recruitment was determined by flow cytometry on Bronchoalveolar lavage cells (right panel). Each group has an N=5. (C) Rag2−/−Il2rg−/− mice were immunized intranasally with 20 µg K. Pneumoniae-43816 (ATCC) (serotype K2) at Day 0 and Day 7. Four weeks after the 2nd immunization, immunized mice and naïve unimmunized mice were infected with 104 live K. Pneumoniae-43816 (ATCC) (serotype K2) and sacrificed 24h after. Lung burden and systemic dissemination were determined by lung (left panel) and spleen (right panel) CFUs. Each group has 4–5 mice. (D) C57BL/6 mice were immunized intranasally with 20 µg heat-killed K. Pneumoniae-43816 (ATCC) (serotype K2) at Day 0 and Day 7. Four weeks after the 2nd immunization, immunized mice and naïve unimmunized mice were infected with 104 live K. Pneumoniae-43816 (ATCC) (serotype K2) and sacrificed 24h after. Anti- K. Pneumoniae-43816 (ATCC) (serotype K2) IgG and IgA in the serum and lung homogenate from naïve uninfected, infected naïve unimmunized and immunized mice were measured by ELISA. (E) C57BL/6 mice were immunized intranasally with 20 µg heat-killed K. Pneumoniae-43816 (ATCC) (serotype K2) at Day 0 and Day 7 and sacrificed on Day14. flow cytometry sorted B220+ cells from immunized mice lung were adoptively transferred into Rag2−/− Il2rg−/− mice. Rag2−/−Il2rg−/− mice receiving PBS or B-cells from immunized mice were infected with 104 live K. Pneumoniae-43816 (ATCC) (serotype K2) and sacrificed 24h later. Lung burden and systemic dissemination were determined by lung (left panel) and spleen (right panel) CFUs. Each group has N=4 mice. (F) Ighm−/− mice were immunized intranasally with 20 µg heat-killed K. Pneumoniae-43816 (ATCC) (serotype K2) at Day 0 and Day 7. 4 weeks after the 2nd immunization, immunized Ighm−/− mice and naïve-unimmunized Ighm−/− mice were infected with 104 live K. Pneumoniae-43816 (ATCC) (serotype K2) and sacrificed 24h after. A group of immunized mice also received i.p. rat IgG or 1A8 24h and 2h prior to infection. A group of immunized mice received neutralizing antibody against IL-17A i.t. immediately prior to infection. Bacterial burden were determined by lung CFU (left panel) and spleen CFU (right panel). Data are from two independent experiments with 2–4 mice in each group.