Post-resolution macrophages shape long-term tissue immunity and integrity in a mouse model of pneumococcal pneumonia

Resolving inflammation is thought to return the affected tissue back to homoeostasis but recent evidence supports a non-linear model of resolution involving a phase of prolonged immune activity. Here we show that within days following resolution of Streptococcus pneumoniae-triggered lung inflammation, there is an influx of antigen specific lymphocytes with a memory and tissue-resident phenotype as well as macrophages bearing alveolar or interstitial phenotype. The transcriptome of these macrophages shows enrichment of genes associated with prostaglandin biosynthesis and genes that drive T cell chemotaxis and differentiation. Therapeutic depletion of post-resolution macrophages, inhibition of prostaglandin E2 (PGE2) synthesis or treatment with an EP4 antagonist, MF498, reduce numbers of lung CD4+/CD44+/CD62L+ and CD4+/CD44+/CD62L-/CD27+ T cells as well as their expression of the α-integrin, CD103. The T cells fail to reappear and reactivate upon secondary challenge for up to six weeks following primary infection. Concomitantly, EP4 antagonism through MF498 causes accumulation of lung macrophages and marked tissue fibrosis. Our study thus shows that PGE2 signalling, predominantly via EP4, plays an important role during the second wave of immune activity following resolution of inflammation. This secondary immune activation drives local tissue-resident T cell development while limiting tissue injury


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Experiments were blinded where practical -Moreover in all cases blinded and unblinded, we routinely take several steps to avoid bias, including (1) mice are housed in cages of 4 mice per cage allowing for social interaction.(2) To avoid circadian rhythm variances, experiments are scheduled at the same time each day.

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Cell abundance in post-sort populations was determined using the gating strategy in Figure 2.A.On alveolar macrophages accounted for ~10% of CD45+ cells.Of the remaining CD64+, F4/80+ and MerTK+ macrophages; Lyve1+MHC-IIaccounted for 30%, Lyve1-MHC-II+ accounted for 20%, and Lyve-MHC-II-accounted for 50%.In other flow cytometry experiments absolute cell number was calculated using CountBrightTM Absolute Counting Beads (ThermoFisher Scientific) and the gating strategies described in the paper.Number of cells is normalised to tissue weight.

Gating strategy
Myeloid cells: Immune cells were identified as CD45+ following exclusion of debris and doublets.Alveolar macrophages were identified as SiglecF+ and CD11bint.Neutrophils were identified as SSC-Ahigh and Ly6G+ cells.MHC-II+ CD64+ cells were further divided into CD11b+ CD11c-interstitial macrophages and CD11c+ dendritic cells.MHC-II-monocytes were subdivided into two populations: Ly6Chi monocytes and Ly6Clo monocytes.Gating shown in supplementary information.T cells: B lymphocytes were identified as CD19+.NK Cells were identified as NK1.1+ cells.All T lymphocytes were identified as CD3+ cells and were further divided into CD4+ T Cells and CD8+ T Cells.Both CD4+ and CD8+ T cells were divided into Naïve T Cells, effector memory T Cells and central Memory T Cells based on their differing expression profile of CD44 and CD62L.Memory T Cells were then further divided into CD62L+, CD27+ TCM, CD62L-CD27+ TEME and CD62L-CD27-TEML.These populations were further analysed using the resident memory markers CD103, CD49a and CD69 as well as the intracellular cytokines IL-17, IFNy and TNFa.Gating shown in supplementary information.
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