Co-stimulation with opposing macrophage polarization cues leads to orthogonal secretion programs in individual cells

Abstract

14Macrophages are innate immune cells that contribute to fighting infections, tissue repair, and 15 maintaining tissue homeostasis. To enable such functional diversity, macrophages resolve 16 potentially conflicting cues in the microenvironment via mechanisms that remain unclear. Here, 17we used single-cell RNA sequencing to explore how individual macrophages respond when co-18 stimulated with the inflammatory stimuli, LPS+IFN-γ, and the resolving cytokine, IL-4. We found 19 that co-stimulated macrophages displayed a distinct global transcriptional program. However, 20 variable negative cross-regulation between some LPS+IFN-γ-and IL-4-specific genes resulted in 21 significant cell-to-cell heterogeneity in transcription. Interestingly, negative cross-regulation led 22to mutually exclusive expression of the T-cell-polarizing cytokines Il6 and Il12b versus the IL-4-23 associated factors Arg1 and Chil3 in single co-stimulated macrophages, and single-cell secretion 24 measurements showed that these specialized functions were maintained for at least 48 hours. 25Overall, our study suggests that increasing functional diversity in the population is one strategy 26 macrophages use to respond to conflicting environmental cues. 27 LPS+IFN-γ or the IL-4 transcriptional program in response to co-stimulation. 131 132 Co-stimulation with LPS+IFN-γ and IL-4 induces transcriptional cross-regulation that 133 varies substantially across individual cells 134We next focused on how co-stimulation with LPS+IFN-γ and IL-4 affected the expression 135 of genes uniquely upregulated by either LPS+IFN-γ or IL-4 alone ( Fig. 2a), which we refer to as 136 the core gene programs. For both LPS+IFN-γ-and IL-4-induced genes, co-stimulation with the 137 other cue caused both transcriptional upregulation and inhibition in a subset of core genes 138 belonging to each program, consistent with our own and previously reported observations 14 . 139Among the LPS+IFN-γ-induced core genes, 87 were inhibited by co-stimulation and 97 were 140 augmented by co-stimulation, while among the IL-4-induced core genes, 196 were inhibited and 141 16 were augmented by co-stimulation (Fig. 2b). 142We compared observations from our single-cell dataset to population-level RT-qPCR data 514

A data-driven computational model enables integrative and mechanistic characterization of dynamic macrophage polarization

“…This simulation-derived phenomenon of partial response due to cell-level macrophage heterogeneity could have important implications for therapies that aim to treat diseases by modulating macrophages in the microenvironment (e.g., targeting tumor-associated macrophages to treat cancer), as the remaining macrophages that are unresponsive to targeted therapies may still possess sufficient functions to drive disease progression and hamper the overall treatment efficacies, and this proposed idea is supported by a recent study that examined macrophage-targeted therapies in colon cancer ( Zhang et al., 2020 ). Furthermore, based on the simulation of our virtual macrophage population, we find that a small portion of cells under control condition (no external stimulation) can already exhibit M1-or M2-like phenotypes, whereas most cells are at unpolarized states ( Figure 7 F), and this phenomenon has also been confirmed in vitro by recent single-cell studies ( Muñoz-Rojas et al., 2020 ; Li et al., 2019 ). In summary, the simulations presented here suggest that our systems-level model can be exploited in highly flexible and efficient ways to enable in silico investigation of macrophage polarization from the single-cell perspective, while offering extra mechanistic insights regarding its temporal, dose-dependent, and quantitative features.…”

“…to describe the differential phenotypic activation of iNOS (inducible nitric oxide synthase) and arginase pathways observed in strain-selected macrophages in response to certain stimuli in vitro , mirroring the classical T helper type-1/type-2 dichotomy ( Mills et al., 2000 ). Over the years, accumulating evidence especially a number of single-cell profiling studies have suggested that the M1 (or canonically activated) and M2 (or alternatively activated) macrophages rather represent two extremes in the entire spectrum of macrophage polarization and activation, and that in vivo macrophage phenotypes in health and disease tend to be more continuous and less exclusive: the M1 (and M1-like) macrophages are generally associated with microbicidal, pro-inflammatory, and tumoricidal activities, whereas the M2 (and M2-like) macrophages are shown to possess reparative, immune-suppressive, and angiogenic functions ( Xue et al., 2014 ; Sica and Mantovani, 2012 ; Mosser and Edwards, 2008 ; Li et al., 2019 ; Muñoz-Rojas et al., 2020 ). Therefore, given the high plasticity and context-dependent functions of macrophages as well as their significant presence and infiltration in pathological tissues observed clinically in many major human diseases, therapeutic strategies that are designed to alter the pathology-driven macrophage polarization have been widely explored and tested in cancer ( Cassetta and Pollard, 2018 ), cardiovascular ( Barrett, 2020 ; Peet et al., 2020 ; Lindsey et al., 2016 ) and metabolic diseases ( Kazankov et al., 2019 ), central nervous system disorders ( Mammana et al., 2018 ), and autoimmune diseases ( Ma et al., 2019 ).…”

Single-cell atlas of epithelial and stromal cell heterogeneity by lobe and strain in the mouse prostate

“…In stark contrast, the ventral lobe has a lower, in some cases undetectable, amount of mast cells and T cells relative to the other lobes (Figure 4D). We assessed macrophage polarization by performing gene signature analysis using gene sets generated from two independent studies (GEO ID: GSE38705 and GEO ID: GSE161125) of stimulated and unstimulated bone marrow-derived macrophages collected from mice [49,50]. Gene signature analysis showed a progression of M0 to M1 polarization in macrophages, with a small population of M2 polarized cells.…”

“…The GSE38705 M1 gene set contains all genes with Log2 FC < −1 and Benjamini & Hochberg FDR-adjusted p-value < 0.05. The GSE161125 M0, GSE161125 M1, and GSE161125 M2 gene sets were derived from a scRNA-seq dataset of bone marrow-derived macrophages collected from wild-type C57BL/6J mice (GEO ID: GSE161125) [50]. Macrophages in culture media (M0, N = 1914 cells), culture media containing bacterial lipopolysaccharide and interferon-gamma (M1, N = 1815 cells), or culture media containing in interleukin 4 (M2, N = 1474 cells) were analyzed in Seurat (version 3.1.5).…”

Macrophage polarization in hypoxia and ischemia/reperfusion: Insights into the role of energetic metabolism

“…(3) Various signals in an organism and cellular environment induce functional diversity of the macrophages. 6,57 Smith et al 57 studied the macrophage response to a combined M1 and M2 activation triggered either simultaneously or sequentially. They showed that simultaneous action of LPS, IFN-γ, IL-4, and IL-13 induces both M1 marker, CD86, and M2 marker, CD206.…”