Neutrophils and Ly6C^hi monocytes collaborate in generating an optimal cytokine response that protects against pulmonary <i>Legionella pneumophila</i> infection

<div><p>Early responses mounted by both tissue-resident and recruited innate immune cells are essential for host defense against bacterial pathogens. In particular, both neutrophils and Ly6C^hi monocytes are rapidly recruited to sites of infection. While neutrophils and monocytes produce bactericidal molecules, such as reactive nitrogen and oxygen species, both cell types are also capable of synthesizing overlapping sets of cytokines important for host defense. Whether neutrophils and monocytes perform redundant or non-redundant functions in the generation of anti-microbial cytokine responses remains elusive. Here, we sought to define the contributions of neutrophils and Ly6C^hi monocytes to cytokine production and host defense during pulmonary infection with <i>Legionella pneumophila</i>, responsible for the severe pneumonia Legionnaires’ disease. We found that both neutrophils and monocytes are critical for host defense against <i>L</i>. <i>pneumophila</i>. Both monocytes and neutrophils contribute to maximal IL-12 and IFNγ responses, and monocytes are also required for TNF production. Moreover, natural killer (NK) cells, NKT cells, and γδ T cells are sources of IFNγ, and monocytes direct IFNγ production by these cell types. Thus, neutrophils and monocytes cooperate in eliciting an optimal cytokine response that promotes effective control of bacterial infection.</p></div

Exogenously administered IL-12 partially restores bacterial control and IFNγ production in mice lacking neutrophils and/or monocytes following infection.

<p>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 Δ<i>flaA L</i>. <i>pneumophila</i> 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.</p

Ly6C^hi monocytes are required for control of pulmonary <i>L</i>. <i>pneumophila</i> infection.

<p>B6 or <i>Ccr2</i>^-/- mice were infected with Δ<i>flaA L</i>. <i>pneumophila</i>. (A) Representative flow cytometry plots of lung cells from B6 or <i>Ccr2</i>^-/- mice 24 hours post-infection, with gates drawn around neutrophils and Ly6C^hi cells. Ly6C^hi monocytes (MCs) were further defined as CD11b⁺ cells. Total numbers of Ly6C^hi MCs (B), neutrophils (C), and DCs (D) in the lung were quantified at 24 and 48 hours post-infection. (E) <i>L</i>. <i>pneumophila</i> 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.</p

Gr-1⁺ cells are required for TNF and IL-12 production early during pulmonary infection with <i>L</i>. <i>pneumophila</i>.

<p>B6 mice treated with isotype control (ISO) or anti-Gr-1 (α-Gr-1) antibody were infected with Δ<i>flaA L</i>. <i>pneumophila</i>. 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.</p

Neutrophils are required for maximal IL-12 production early during pulmonary infection with <i>L</i>. <i>pneumophila</i>.

<p>B6 mice treated with either isotype control (ISO) or anti-Ly6G (α-Ly6G) antibody were infected with <i>ΔflaA L</i>. <i>pneumophila</i>. 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.</p

Anti-Ly6G-mediated depletion of neutrophils impairs control of pulmonary <i>L</i>. <i>pneumophila</i> infection.

<p>B6 mice treated with isotype control (ISO) or anti-Ly6G (α-Ly6G) antibody were infected with Δ<i>flaA L</i>. <i>pneumophila</i>. 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 Ly6C^hi MCs (C) in the lung were quantified at 24 and 72 hours post-infection. (D) <i>L</i>. <i>pneumophila</i> 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.</p

Monocytes and DCs produce IL-12, and monocytes and neutrophils are required for IFNγ production during pulmonary <i>L</i>. <i>pneumophila</i> infection.

<p>(A & B) B6 mice were uninfected (naïve) or infected with Δ<i>flaA L</i>. <i>pneumophila</i> (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 Ly6C^hi 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 Δ<i>flaA L</i>. <i>pneumophila</i>-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 <i>Ccr2</i>^-/- 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.</p

Ly6C^hi monocytes are required for maximal TNF and IL-12 production during pulmonary <i>L</i>. <i>pneumophila</i> infection.

<p>B6 or <i>Ccr2</i>^-/- mice were infected with Δ<i>flaA L</i>. <i>pneumophila</i>. 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.</p

Caspase-11 activation is independent of ASC and NLRC4.

<p>(<b>A</b>) Unprimed B6, <i>Casp1^−/−Casp11^−/−</i>, or <i>Asc^−/−Nlrc4^−/−</i> BMDMs were infected with WT <i>L. pneumophila</i> (WT Lp), Δ<i>dotA</i> Lp, Δ<i>flaA</i> Lp, or PBS (mock infection) for 20 hours, and levels of IL-1α and IL-1β in the supernatants were measured by ELISA. Graphs show the mean ± SEM of triplicate wells. (<b>B</b>) Unprimed B6, <i>Casp1^−/−Casp11^−/−</i>, or <i>Asc^−/−Nlrc4^−/−</i> BMDMs were infected with WT Lp, Δ<i>dotA</i> Lp, Δ<i>flaA</i> Lp, or PBS (mock infection) for 20 hours or treated with LPS+ATP for 1 hour. Levels of processed caspase-1 (casp-1 p10) and caspase-11 (casp-11 p26) in the supernatants, and pro-caspase-1, pro-caspase-11, and β-actin (loading control) in the cell lysates were determined by immunoblot analysis. (<b>C</b>) 8–12 week old B6 and <i>Asc^−/−</i> mice were infected intranasally with either 1×10⁶ Δ<i>flaA</i> Lp or PBS. Bronchoalveolar lavage fluid (BALF) was collected 24 hours post-infection, and levels of IL-1α and IL-1β were measured by ELISA. Graphs show the mean ± SEM of 9 mice per group. Dashed line represents the limit of detection. Data are representative of three independent experiments (A,B) or are displayed as the pooled results of two independent experiments (C). *** is p<0.001 by two-way ANOVA with Bonferroni post-test. ** is p<0.01 by unpaired t-test. NS is not significant.</p

IL-1α and IL-1β control bacterial burden and neutrophil recruitment <i>in vivo</i>.

<p>(<b>A</b>) 8–12 week old B6 or <i>Il1r1^−/−</i> mice were infected with 1×10⁶ Δ<i>flaA L. pneumophila</i> intranasally (IN). Lungs were plated to quantify CFU per gram. Graph shows the mean ± SEM of three or four infected mice per group. Dashed line represents the limit of detection. (<b>B</b> and <b>C</b>) B6 or <i>Il1r1^−/−</i> mice were infected with 1×10⁶ Δ<i>flaA</i> Lp IN. 24 hours post-infection, bronchoalveolar lavage fluid (BALF) was collected and the percentage of neutrophils in the BALF was quantified by flow cytometry. Percentages are reported as the frequency of live cells in the BALF. (B) Representative flow cytometry plots showing the percentage of Gr-1⁺Ly6G⁺ neutrophils. (C) Graph showing the percentage of neutrophils. Each point represents an individual mouse and lines indicate the mean of 4 mice per group. (<b>D, E</b>, and <b>F</b>) B6 mice were injected intraperitoneally (IP) with either PBS, 100 µg isotype control antibody (iso), 100 µg anti-IL-1α antibody, 100 µg anti-IL-1β antibody, or 100 µg each of anti-IL-1α and anti-IL-1β (anti-IL-1α/β) 16 hours before infection. The mice were then intranasally infected with either 1×10⁶ Δ<i>flaA</i> Lp or mock infected with PBS. (D and E) 24 hours post-infection, BALF was collected and flow cytometry was performed to quantify the percentage of neutrophils. (D) Representative flow cytometry plots showing the percentage of Gr-1⁺Ly6G⁺ neutrophils. (E) Graph showing the percentage of neutrophils. Each point represents an individual mouse, lines indicate the mean of 8 mice per group, and error bars represent SEM. Shown are the pooled results of two independent experiments. (F) 72 hours post-infection, the lungs were plated to quantify CFU per gram. Each point represents an individual mouse. Line indicates the mean of 4 infected mice per group with error bars representing SEM. *** is p<0.001 by one-way ANOVA with Tukey post-test or unpaired t-test (C). **is p<0.01 and *is p<0.05 by unpaired t-test. NS is not significant.</p