Detection and Isolation of Dendritic Cells Using Lewis X‑Functionalized Magnetic Nanoparticles

dendritic cell (DC)-specific intracellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) is a receptor found on DCs that recognizes antigens bearing mannose-rich or fucosylated glycans, including Lewis X (Le^X). Here, we report the fabrication of magnetic nanoparticles coated with multivalent Le^X glycans using Cu (I)-catalyzed azide–alkyne cycloaddition. The resulting nanoparticles are selective and biocompatible, serving as a highly efficient tool for DC detection and enrichment

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

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

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