Fig 1.
Anti-Gr-1-mediated depletion of neutrophils and Ly6Chi monocytes impairs control of pulmonary L. pneumophila infection.
B6 mice treated with isotype control (ISO) or anti-Gr-1 (α-Gr-1) antibody were infected with ΔflaA L. pneumophila. (A) Representative flow cytometry plots of lung cells from ISO or anti-Gr-1 treated mice at 24 hours (left) and 72 hours (right) post-infection, with gates drawn around Ly6Ghi neutrophils and Ly6Chi cells. Ly6Chi monocytes (MCs) were further defined as CD11b+, Ly6Chi cells. Total numbers of neutrophils (B), MCs (C), and DCs (D) in the lung were quantified at 24 and 72 hours post-infection. (E) L. pneumophila CFUs in the lung were enumerated at 24 and 72 hours post-infection. Data shown are the pooled results of 2 independent experiments with 3 or 4 mice per group per experiment. * is p<0.05, *** is p<0.001 and **** is p<0.0001 by unpaired t-test. NS is not significant. Dashed line represents the limit of detection.
Fig 2.
Gr-1+ cells are required for TNF and IL-12 production early during pulmonary infection with L. pneumophila.
B6 mice treated with isotype control (ISO) or anti-Gr-1 (α-Gr-1) antibody were infected with ΔflaA L. pneumophila. Levels of IL-1α (A), IL-1β (B), IL-6 (C), TNF (D), IL-18 (E), IL-12p70 (F), IL-12p40 (G), IL-4 (H), and IL-10 (I) in the BALF at 24 and 72 hours post-infection were quantified by ELISA. Data shown are the pooled results of 2 independent experiments with 3 or 4 mice per group per experiment. * is p<0.05, ** is p<0.01, and *** is p<0.001 by unpaired t-test. NS is not significant. Dashed line represents the limit of detection.
Fig 3.
Anti-Ly6G-mediated depletion of neutrophils impairs control of pulmonary L. pneumophila infection.
B6 mice treated with isotype control (ISO) or anti-Ly6G (α-Ly6G) antibody were infected with ΔflaA L. pneumophila. A) Representative flow cytometry plots of lung cells from ISO or anti-Ly6G treated mice at 24 hours post-infection, with gates drawn around neutrophils and MCs. Total numbers of neutrophils (B) and Ly6Chi MCs (C) in the lung were quantified at 24 and 72 hours post-infection. (D) L. pneumophila CFUs in the lung were enumerated at 24 and 72 hours post-infection. Data shown are the pooled results of 4 independent experiments with 3 or 4 mice per group per experiment. * is p<0.05 and *** is p<0.001 by unpaired t-test. NS is not significant. Dashed line represents the limit of detection.
Fig 4.
Neutrophils are required for maximal IL-12 production early during pulmonary infection with L. pneumophila.
B6 mice treated with either isotype control (ISO) or anti-Ly6G (α-Ly6G) antibody were infected with ΔflaA L. pneumophila. Levels of IL-1α (A), IL-1β (B), IL-6 (C), TNF (D), IL-18 (E), IL-12p70 (F), and IL-12p40 (G) in the BALF at 24 and 72 hours post-infection were quantified by ELISA. Data shown are the pooled results of 4 independent experiments with 3 or 4 mice per group per experiment. * is p<0.05, ** is p<0.01, and *** is p<0.001 by unpaired t-test. NS is not significant. Dashed line represents the limit of detection.
Fig 5.
Ly6Chi monocytes are required for control of pulmonary L. pneumophila infection.
B6 or Ccr2-/- mice were infected with ΔflaA L. pneumophila. (A) Representative flow cytometry plots of lung cells from B6 or Ccr2-/- mice 24 hours post-infection, with gates drawn around neutrophils and Ly6Chi cells. Ly6Chi monocytes (MCs) were further defined as CD11b+ cells. Total numbers of Ly6Chi MCs (B), neutrophils (C), and DCs (D) in the lung were quantified at 24 and 48 hours post-infection. (E) L. pneumophila CFUs in the lung were enumerated at 24, 48, and 96 hours post-infection. Data shown are the pooled results of 3 to 5 independent experiments per time point with 3 or 4 mice per group per experiment. * is p<0.05 and *** is p<0.001 by unpaired t-test. NS is not significant. Dashed line represents the limit of detection.
Fig 6.
Ly6Chi monocytes are required for maximal TNF and IL-12 production during pulmonary L. pneumophila infection.
B6 or Ccr2-/- mice were infected with ΔflaA L. pneumophila. Levels of IL-1α (A), IL-1β (B), IL-6 (C), TNF (D), IL-18 (E), IL-12p70 (F), and IL-12p40 (G) in the BALF at 24 and 48 hours post-infection were quantified by ELISA. Data shown are the pooled results of 3 to 5 independent experiments per time point with 3 or 4 mice per group per experiment. * is p<0.05 and *** is p<0.001 by unpaired t-test. NS is not significant. Dashed line shows the limit of detection.
Fig 7.
Monocytes and DCs produce IL-12, and monocytes and neutrophils are required for IFNγ production during pulmonary L. pneumophila infection.
(A & B) B6 mice were uninfected (naïve) or infected with ΔflaA L. pneumophila (Lp-infected). Intracellular cytokine staining for IL-12p40 was performed on lung cells. Representative flow cytometry plots and graphs show the total numbers of IL-12p40-expressing MCs (A), and DCs (B), in the lung at 24 hours post-infection. (C & D) IL-12p40-YFP reporter mice (YET40) or B6 mice were uninfected (naïve) or infected with Lp. Representative flow cytometry plots and graphs show the total numbers of YFP-expressing Ly6Chi MCs (C), and DCs (D) in the lung at 48 hours post-infection, with YFP gates drawn based on MCs and DCs from B6 mice infected with Lp. IFNγ was quantified by ELISA at 24, 48, or 72 hours post-infection in the BALF of ΔflaA L. pneumophila-infected B6 mice treated with isotype control (ISO) or anti-Gr-1 (α-Gr-1) antibody (E), infected B6 mice treated with ISO or anti-Ly6G (α-Ly6G) antibody (F), or infected B6 or Ccr2-/- mice (G). Data shown are the pooled results of 3 (A-B), 2 (C-D), or 2 to 5 (E-G) independent experiments with 2 to 7 mice per group per experiment. * is p<0.05, ** is p<0.01 and *** is p<0.001 by unpaired t-test. NS is not significant. Dashed line shows the limit of detection.
Fig 8.
Exogenously administered IL-12 partially restores bacterial control and IFNγ production in mice lacking neutrophils and/or monocytes following infection.
B6 mice treated with isotype control (ISO) or anti-Gr-1 (α-Gr-1) antibody (A), Ccr2-/- mice (B), or B6 mice treated with isotype control or anti-Ly6G (α-Ly6G) antibody (C) were infected with ΔflaA L. pneumophila and given 500ng of recombinant IL-12 (rIL-12) intranasally. CFUs were enumerated and IFNγ levels in BALF were measured 72 hours post-infection. Data shown are the pooled results of 2–3 independent experiments with 3 or 4 mice per group per experiment. * is p<0.05, ** is p<0.01 and *** is p<0.001 by unpaired t-test. NS is not significant.
Fig 9.
NK cells, NKT cells, and γδ T cells produce IFNγ, and monocytes are required for NK cells and γδ T cells to produce IFNγ during pulmonary L. pneumophila infection.
Representative flow cytometry plots and graphs showing the total number of IFNγ+ NK cells (A & B), IFNγ+ NKT cells (C & D), and IFNγ+ γδ T cells (E & F) in the lungs of uninfected (naïve) or ΔflaA L. pneumophila-infected (Lp-infected) B6 or Ccr2-/- mice at 24 hours post-infection. Data shown are the pooled results of 2 independent experiments with 2 to 7 mice per group per experiment. ** is p<0.01 and *** is p<0.001 by unpaired t-test. NS is not significant. Dashed line represents the limit of detection.