Cooperation between Monocyte-Derived Cells and Lymphoid Cells in the Acute Response to a Bacterial Lung Pathogen

<div><p><i>Legionella pneumophila</i> is the causative agent of Legionnaires’ disease, a potentially fatal lung infection. Alveolar macrophages support intracellular replication of <i>L</i>. <i>pneumophila</i>, however the contributions of other immune cell types to bacterial killing during infection are unclear. Here, we used recently described methods to characterise the major inflammatory cells in lung after acute respiratory infection of mice with <i>L</i>. <i>pneumophila</i>. We observed that the numbers of alveolar macrophages rapidly decreased after infection coincident with a rapid infiltration of the lung by monocyte-derived cells (MC), which, together with neutrophils, became the dominant inflammatory cells associated with the bacteria. Using mice in which the ability of MC to infiltrate tissues is impaired it was found that MC were required for bacterial clearance and were the major source of IL12. IL12 was needed to induce IFNγ production by lymphoid cells including NK cells, memory T cells, NKT cells and γδ T cells. Memory T cells that produced IFNγ appeared to be circulating effector/memory T cells that infiltrated the lung after infection. IFNγ production by memory T cells was stimulated in an antigen-independent fashion and could effectively clear bacteria from the lung indicating that memory T cells are an important contributor to innate bacterial defence. We also determined that a major function of IFNγ was to stimulate bactericidal activity of MC. On the other hand, neutrophils did not require IFNγ to kill bacteria and alveolar macrophages remained poorly bactericidal even in the presence of IFNγ. This work has revealed a cooperative innate immune circuit between lymphoid cells and MC that combats acute <i>L</i>. <i>pneumophila</i> infection and defines a specific role for IFNγ in anti-bacterial immunity.</p></div

IFNγ production by memory T cells in the acute phase of the response to <i>L</i>. <i>pneumophila</i> is non-cognate and can contribute to bacterial clearance.

<p>A-C. C57BL/6 mice were injected with 10⁷ gBT-I.IFNγ-YFP T cells that had been stimulated <i>in vitro</i> with antigen. After 30 days mice were infected with <i>L</i>. <i>pneumophila</i>. A. Number of T cells derived from the recipient mice (Endogenous) and gBT-I T cells in lungs at the times shown. Cells were identified using congenic markers. Mean ± SEM is shown. n ≥ 10 for all groups and pooled from 4 separate experiments. B. Flow cytometric profiles of YFP expression of gBT-I.IFNγ-YFP T cells in lung and spleen at the days shown. Indicated gates set by analysis of endogenous cells. C. Proportion of gBT-I.IFNγ-YFP T cells in lung expressing YFP on the days shown. D. Experimental design to examine the effectiveness of IFNγ produced by memory T cells in <i>L</i>. <i>pneumophila</i> clearance. See text for further details. E. Results of experiment shown in ‘D’. WT, wild type C57BL/6 mice without transfer of T cells. IFNγ^-/-, IFNγ^-/- mice without transfer of T cells. IFNγ^-/- (+gBT-I), IFNγ^-/- mice that received IFNγ-sufficient gBT-I T cells. IFNγ^-/- (+gBT-I.IFNγ^-/-), IFNγ^-/- mice that received T cells from gBT-I.IFNγ^-/- mice. CFU/lung at day 3 after infection are shown. In C and E, each symbol represents data for one mouse. C, Data pooled from 4 separate experiments. D, Data pooled from 4 separate experiments. **. P < 0.01, ***. P < 0.005.</p

Optimal bactericidal activity of MC, but not neutrophils, is dependent upon IFNγ.

<p>C57BL/6 (A, B) or IFNγ^-/- (B) mice were infected with <i>L</i>. <i>pneumophila</i> and at 2 days after infection the indicated cell types were isolated, lysed and the lysates cultured on selective bacterial culture plates to determine the number of <i>L</i>. <i>pneumophila</i> CFU per 10⁴ cells in each cell population. In B, lines connect data points from individual experiments using the same bacterial inoculum. Each point represents the results from one experiment where lungs from 4–5 mice were pooled. *. P < 0.05. In A all comparisons to AM group. In B, the fold change refers to the ratio of the averages of the two groups.</p

An innate immune network in the acute response to a bacterial lung pathogen.

<p>We propose the following model for the role and interactions of phagocytic and lymphoid cells in the acute phase of <i>L</i>. <i>pneumophila</i> infection. <b><i>1</i>.</b> Tissue resident alveolar macrophages and conventional dendritic cells (not shown) are the first to engulf bacteria and produce inflammatory mediators such as cytokines and chemokines. Alveolar macrophages support replication of <i>L</i>. <i>pneumophila</i> but are also rapidly depleted, perhaps by cell death mechanisms, and may release inflammatory death-associated molecular patterns to potentiate the immune response. <b><i>2</i>.</b> Neutrophils flood into the lung in response to the inflammatory stimuli. Neutrophils engulf and kill bacteria from early stages of infection and don’t require activation by IFNγ. <b><i>3</i>.</b> Slightly after neutrophils, monocytes infiltrate and develop into monocyte-derived cells (MC) which then build to become the dominant phagocytic cell type. MC contribute to bacterial clearance by production of IL12 (<b><i>4</i></b>), which in turn stimulates IFNγ production by NK cells and various populations of memory T cells, NKT cells and γδ T cells. The different coloured cells labelled ‘Mem’ represent different types of memory T cells. (<b><i>5</i></b>). IFNγ is critical in stimulating bactericidal activity of MC (<b><i>6</i></b>), an activity that appears critical for optimal bacterial clearance.</p

Optimal bactericidal activity of MC, but not neutrophils, is dependent upon IFNγ.

<p>C57BL/6 (A, B) or IFNγ^-/- (B) mice were infected with <i>L</i>. <i>pneumophila</i> and at 2 days after infection the indicated cell types were isolated, lysed and the lysates cultured on selective bacterial culture plates to determine the number of <i>L</i>. <i>pneumophila</i> CFU per 10⁴ cells in each cell population. In B, lines connect data points from individual experiments using the same bacterial inoculum. Each point represents the results from one experiment where lungs from 4–5 mice were pooled. *. P < 0.05. In A all comparisons to AM group. In B, the fold change refers to the ratio of the averages of the two groups.</p

MC are required for optimal clearance of <i>L</i>. <i>pneumophila</i>.

<p>Wild type C57BL/6 or CCR2^-/- mice were infected with <i>L</i>. <i>pneumophila</i> and analysed for MC number in the lung (at day 2 in A and at day 3 and 5 in B) and <i>L</i>. <i>pneumophila</i> CFU in lung (C). In B and C mean ± SEM is shown. B, n ≥ 9 for all groups and pooled from ≥ 4 separate experiments. C, n ≥ 9 for all groups and pooled from ≥ 4 separate experiments. **. P < 0.01, ****. P < 0.001.</p

NK cells and memory T cells produce IFNγ in the acute response <i>L</i>. <i>pneumophila</i>.

<p>IFNγ-YFP reporter mice (GREAT mice, A-F), IFNγ-YFP.IL12p35^-/- (A,D,E) or C57BL/6 mice (G) were infected with <i>L</i>. <i>pneumophila</i> and analysed on day 2 (A, D-F) or as indicated (B, C, G). A. YFP fluorescence in the cell types indicated. NK cells were defined as NK1.1⁺NKp46⁺CD3^-, T cells as CD3⁺TCRβ⁺ or CD3⁺TCRγδ⁺ and NKT cells as TCRβ⁺ cells that stained with a CD1d-tetramer. Indicated gates set by analysis of wildtype mice. B-E. Enumeration of NK and T cells from IFNγ-YFP reporter mice (B, C) or the mouse strains indicated (D, E). F. CD62L and CD44 expression of all IFNγ-YFP⁺CD3⁺TCRβ⁺ cells. G. Enumeration of NK cells, CD44⁺CD3⁺TCRβ⁺ T cells (CD44⁺TCRβ⁺ cells, which includes conventional T and NKT cells) and γδ T cells. In A and F the numbers represent percentage of cells in the gate shown. In B, C, G. mean ± SEM is shown. B, n ≥ 4 for all groups and pooled from 4 separate experiments. C, n ≥ 4 for all groups and pooled from 5 separate experiments. In D, E, each dot refers to one mouse. D, n ≥ 6 for all groups and pooled from 2 separate experiments. E, n ≥ 6 for all groups and pooled from 2 separate experiments. G, n ≥ 7 for all groups and pooled from ≥ 3 separate experiments **. P < 0.01, ***. P < 0.005.</p

Neutrophils and MC are the dominant inflammatory phagocytic cells in lung following <i>L</i>. <i>pneumophila</i> infection.

<p>C57BL/6 mice were infected with <i>L</i>. <i>pneumophila</i> and CD45⁺ cells were analysed on day 2 after infection. A. Gating strategy to identify neutrophils (Neut), AM, MC and cDC. Upper panels, uninfected mice, Lower panels, infected mice. Dashed lines indicate the gated population is further analysed in adjacent panel. Expression of CD11b and Ly6G on CD11c^- cells allowed identification of neutrophils. AM, like DC are CD11c⁺, but also expressed Siglec F. Siglec F^-CD11c⁺ cells comprised MC and cDCs and these were separated by using Fcε receptor I and CD64 as well as CD11b and CD103, respectively. B. Enumeration of the number of cells per lung for the indicated cell types. C. Cells from <i>L</i>. <i>pneumophila</i>-infected mice were stained with a <i>L</i>. <i>pneumophila</i>-specific antibody (upper panels). Lower panels, isotype control. Numbers represent percentage of cells in the gate shown. Cells were identified using the strategy shown in ‘A’. D. Enumeration of cells per lung that stained with a <i>L</i>. <i>pneumophila</i> antibody. In B and D mean ± SEM is shown. B, n ≥ 14 for all groups and pooled from ≥ 4 separate experiments. D, n ≥ 11 for all groups and pooled from ≥ 4 separate experiments.</p

Distinct APC Subtypes Drive Spatially Segregated CD4⁺ and CD8⁺ T-Cell Effector Activity during Skin Infection with HSV-1

<div><p>Efficient infection control requires potent T-cell responses at sites of pathogen replication. However, the regulation of T-cell effector function <i>in situ</i> remains poorly understood. Here, we show key differences in the regulation of effector activity between CD4⁺ and CD8⁺ T-cells during skin infection with HSV-1. IFN-γ-producing CD4⁺ T cells disseminated widely throughout the skin and draining lymph nodes (LN), clearly exceeding the epithelial distribution of infectious virus. By contrast, IFN-γ-producing CD8⁺ T cells were only found within the infected epidermal layer of the skin and associated hair follicles. Mechanistically, while various subsets of lymphoid- and skin-derived dendritic cells (DC) elicited IFN-γ production by CD4⁺ T cells, CD8⁺ T cells responded exclusively to infected epidermal cells directly presenting viral antigen. Notably, uninfected cross-presenting DCs from both skin and LNs failed to trigger IFN-γ production by CD8⁺ T-cells. Thus, we describe a previously unappreciated complexity in the regulation of CD4⁺ and CD8⁺ T-cell effector activity that is subset-specific, microanatomically distinct and involves largely non-overlapping types of antigen-presenting cells (APC).</p></div

Distribution of IFN-γ⁺ T_EFF cells.

<p>Mice received naïve gBT-I or gDT-II cells prior to HSV-1 skin infection. (<b>A</b>) Quantification of T_EFF cells in epidermis (Epi) and dermis (Der) 5–10 days post-infection, tracked as CD45.2⁺CD45.1⁺ Vα2⁺ (gBT-I) or Vα3.2⁺ (gDT-II) cells (dispase digestion). Data pooled from <i>n</i> = 9–14 mice/time point. (<b>B</b>,<b>C</b>) Analysis of IFN-γ⁺ T_EFF cells in skin (dispase digestion) 5 days post-infection. **, <i>P</i><0.01; ns, not significant by Mann Whitney test; <i>n</i> = 5–6 mice/group from 3–4 experiments. (<b>D</b>) Analysis of IFN-γ⁺ T_EFF cells 5–10 days post-infection; <i>n</i> = 4–7 mice/time point from 1–4 experiments. (<b>E</b>,<b>F</b>) Analysis of IFN-γ⁺ T_EFF cells in skin (gBT-I, epidermis; gDT-II, total skin), brachial (brLN) and axillary (axLN) LNs and spleen (5 days post-infection in <b>E</b>, or as indicated in <b>F</b>); <i>n</i> = 2–7 mice/organ/time point from 1–4 experiments.</p