Proliferation drives quorum sensing of microbial products in human macrophage populations

Macrophages coordinate the initial host inflammatory response to tissue infection, as well as mediating the reparative phase, by producing growth factors that promote tissue repair. One model of this functional dichotomy is that peripherally recruited monocyte-derived macrophages drive acute inflammatory responses to infection, whereas tissue-resident macrophages are responsible for tissue repair. Alternatively, inflammation and repair may be inter-dependent molecular programs, such that both recruited and resident cells have equivalent capacity to contribute. Repeated exposure to pathogenic challenge results in innate tolerance, which may also alter the contributions of discrete macrophage populations to inflammation or repair. In this study a village model of tissue resident and recruited macrophages was created using induced pluripotent stem cell-derived macrophages and peripheral blood monocyte-derived macrophages, respectively. Population responses to repeated exposure to lipopolysaccharide were assessed with single-cell RNA sequencing and donors demultiplexed with Vireo. A subset of genes escaped classical tolerance programs in the iPSC, but not monocyte-derived macrophages, and this was associated with differences in their proliferative capacity. This suggests that targeting the proliferative resident macrophages would be most effective to limit inflammatory signaling.


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Multiple tissue-resident macrophage subsets have been described and defined by roles that are 82 specialized to their tissue of residency, such as bone resorption by osteoclasts 1 , neural pruning 83 by microglia 2 , lipid-associated macrophages 3 , stem cell licensing in bone marrow 4 and intestine 5 , 84 or iron recycling in splenic macrophages 6 . Differences between macrophage subsets have also 85 been described in the context of ontogeny (reviewed in 7 ), anatomical location, or molecular 86 phenotype 8 . Given the diverse roles of macrophages in tissue development, homeostasis, and in 87 host defense, there is considerable interest in understanding mechanisms that might target 88 macrophage subsets in tissue inflammation or repair.

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Functional differences between tissue-resident macrophages (TRM) and monocyte-derived 91 macrophages (MDM) recruited in response to inflammation are controversial. In some models, 92 such as brain, the resident macrophages (microglia) play a neuroprotective role, while recruited 93 MDM are thought to be the inflammatory mediator 9 . In contrast, others have described diverse 94 monocyte and macrophage subsets associated with age-related inflammation, cancer or 95 trauma 10 , as well as inflammatory phenotypes associated with differences in IRF8 signaling across varying levels of gene expression after LPS stimulation, suggesting the coexistence of macrophage subsets that could be propagated as subpopulations. In the second study, 110 heterogeneity in the timing of RelA recruitment to the TNF promoter in a RAW264.7 reporter line 111 was observed after LPS stimulation, but variability between cells could also be explained by 112 population density. These studies demonstrate that multiple mechanisms impact on the timing, 113 duration, and amplitude of macrophage activation, even in simple models receiving a highly 114 polarizing inflammatory signal, such as LPS.

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It is obvious that, in a tissue setting, macrophages will be integrating signals from the local 117 environment and it is difficult to deconvolute the influence of these signals from ontogeny over 118 the course of an inflammatory event. Tissue culture models offer an opportunity to address cell-   While others 19 have shown that PSCMs recognize and respond to LPS, it is not known whether 130 they can be tolerized on repeated exposure, which would more accurately mimic the regulatory 131 program seen after an infection than a single high dose pathogen exposure. We were also curious 132 to see how coordinated acute responses to LPS were across human macrophage populations, as 133 perhaps early versus late responders within a population could explain some of the previously 134 described heterogeneity in trained monocytes 20 . Here, we use single cell RNA-sequencing to 135 investigate population responses to high-dose LPS activation and then re-stimulation using a coculture, or village, model of PSCMs and MDMs, as models of tissue residency versus recruited

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Establishing a 'village' of macrophages to assess macrophage heterogeneity at the single cell 141 level.

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The transcriptional heterogeneity of macrophage subsets was evident even in the unstimulated 143 populations ( Figure 1). The use of a village model removed population density as a possible driver 144 of heterogeneity ( Figure 1A), as all the cells were exposed to the same local environment, 145 maintained in CSF-1 (C, control), or exposed to LPS for 2 hours (A, acute) or 18 hours (R, rest),   Table 2).