Phagocytes, including neutrophils and macrophages, have been suggested to function in a cooperative way in the initial phase of inflammatory responses, but their interaction and integration in the resolution of inflammation and tissue repair remain unclear. Here we show that neutrophils have crucial functions in liver repair by promoting the phenotypic conversion of pro-inflammatory Ly6C hi CX 3 CR1 lo monocytes/macrophages to pro-resolving Ly6C lo CX 3 CR1 hi macrophages. Intriguingly, reactive oxygen species (ROS), expressed predominantly by neutrophils, are important mediators that trigger this phenotypic conversion to promote liver repair. Moreover, this conversion is prevented by the depletion of neutrophils via anti-Ly6G antibody, genetic deficiency of granulocyte colony-stimulating factor, or genetic deficiency of NADPH oxidase 2 (Nox2). By contrast, adoptive transfer of WT rather than Nox2 −/− neutrophils rescues the impaired phenotypic conversion of macrophages in neutrophil-depleted mice. Our findings thus identify an intricate cooperation between neutrophils and macrophages that orchestrate resolution of inflammation and tissue repair.

Macrophages have been suggested to contribute to constructing a cancer stem cell (CSC) niche. However, whether and how macrophages regulate the activity of CSCs through juxtacrine signaling are poorly understood. Here we report LSECtin, a transmembrane protein highly expressed on tumor-associated macrophages (TAMs), enhances stemness of breast cancer cells (BCCs). We identified BTN3A3, a B7 family member with previously unknown functions as the receptor for LSECtin on BCCs responsible for stemness-promoting effect of LSECtin. In mice bearing human tumor xenografts, either macrophage-specific ablation of LSECtin or silencing of BTN3A3 in BCCs decreased CSC frequency and tumor growth. Admixture of LSECtin-positive macrophages increased the tumorigenic activity of BCCs dependent on BTN3A3. Disruption of the LSECtin-BTN3A3 axis with BTN3A3-Fc or anti-BTN3A3 mAb has a therapeutic effect on breast cancer. These findings define a juxtacrine signaling mechanism by which TAMs promote cancer stemness. Targeting this axis in the CSC niche may provide potential therapies to breast cancer.

Macrophages are highly heterogeneous immune cells that fulfill tissue-specific functions. Tissue-derived signals play a critical role in determining macrophage heterogeneity. However, these signals remain largely unknown. BMP receptor ALK1 is well-known for its role in blood vessel formation; however, its role within the immune system has never been revealed. Here, we found that BMP9/BMP10-ALK1 signaling controlled the identity and self-renewal of Kupffer cells (KCs) through a Smad4-dependent pathway. In contrast, ALK1 was dispensable for the maintenance of macrophages located in the lung, kidney, spleen and brain. Following ALK1 deletion, KCs were lost over time and were replaced by monocyte-derived macrophages. These hepatic macrophages displayed significantly reduced expression of the complement receptor VSIG4 and alterations in immune zonation and morphology, which is important for the tissue-specialized function of KCs. Furthermore, we found that this signaling pathway was important for KC-mediated Listeria monocytogenes capture, as the loss of ALK1 and Smad4 led to a failure of bacterial capture and overwhelming disseminated infections. Thus, ALK1 signaling instructs a tissue-specific phenotype that allows KCs to protect the host from systemic bacterial dissemination. environment in maintaining the integrity of KC-mediated intravascular defenses.Activin receptor-like kinase 1 (ALK1, ACVRL1) is a type I receptor of the TGFβ receptor superfamily with two ligands, BMP9 and BMP10 (15). ALK1 is predominantly expressed in endothelial cells and plays a critical role in regulating developmental and pathological angiogenesis (16). However, unlike TGFβR2 signaling, the role of ALK1 in the immune system has not been reported to date. Here, we found that BMP9/BMP10-ALK1 signaling controlled the specific gene expression program and survival of KCs through a Smad4-dependent pathway. Functionally, the loss of ALK1 resulted in impaired capture of Listeria monocytogenes and overwhelming disseminated infections. Taken together, our data revealed a previously unappreciated role of ALK1 signaling in maintaining KC homeostasis and function. ResultsLoss of Alk1, rather than Tgfbr2, Alk2 and Alk3, leads to an altered phenotype of KCsRecently, Clec4F was identified as a specific surface marker for Kupffer cells (5). To specifically target KCs, we first generated Clec4f Cre/DTR mice (hereafter referred to as Clec4f Cre ), in which an expression cassette encoding an internal ribosomal entry site (IRES), the Cre enzyme, a self-cleaving 2A peptide and the human DTR was inserted into the 3' untranslated region of the Clec4f gene. Clec4f Cre mice were crossed with a conditional reporter strain (R26-tdTomato) to determine the efficiency and specificity of Cre-mediated recombination using flow cytometry. In the liver, the Clec4f Cre strain efficiently recombined in CD64 + F4/80 + KCs (>90%), and almost all tdTomato-positive cells were KCs (Supplemental Figure 1, A and B, and Supplemental Figure 2A). Furthermore, tdTomato expression was no...

Macrophages are a heterogeneous population of immune cells that play central roles in a broad range of biological processes, including the resolution of inflammation. Although diverse macrophage subpopulations have been identified, the characterization and functional specialization of certain macrophage subsets in inflamed tissues remain unclear. Here we uncovered a key role of specific macrophage subsets in tissue repair using proteomics, bioinformatics and functional analysis. We isolated two hepatic monocyte-derived macrophage subpopulations: Ly6C hi CX 3 CR1 lo macrophages and Ly6C lo CX 3 CR1 hi macrophages during distinct phases of acute liver injury and employed label-free proteomics approach to profile the proteome of these cells. We found that the endocytosis- and apoptotic cell clearance-related proteins were specifically enriched in Ly6C lo CX 3 CR1 hi macrophages at the resolution phase. Intriguingly, 12/15-lipoxygenase (Alox15), the most strongly up-regulated protein in Ly6C lo CX 3 CR1 hi macrophages, was identified as a specific marker for these macrophages. In co-culture systems, Ly6C lo CX 3 CR1 hi macrophages specifically induced hepatocyte proliferation. Furthermore, selective depletion of this population in CD11b-diphtheria toxin receptor mice significantly delayed liver repair. Overall, our studies shed light on the functional specialization of distinct macrophage subsets from different phases in the resolution of inflammation.

IntroductionFull-thickness skin wound healing remains a serious undertaking for patients. While stem cell-derived exosomes have been proposed as a potential therapeutic approach, the underlying mechanism of action has yet to be fully elucidated. The current study aimed to investigate the impact of exosomes derived from human umbilical cord mesenchymal stem cells (hucMSC-Exosomes) on the single-cell transcriptome of neutrophils and macrophages in the context of wound healing.MethodsUtilizing single-cell RNA sequencing, the transcriptomic diversity of neutrophils and macrophages was analyzed in order to predict the cellular fate of these immune cells under the influence of hucMSC-Exosomes and to identify alterations of ligand-receptor interactions that may influence the wound microenvironment. The validity of the findings obtained from this analysis was subsequently corroborated by immunofluorescence, ELISA, and qRT-PCR. Neutrophil origins were characterized based on RNA velocity profiles.ResultsThe expression of RETNLG and SLC2A3 was associated with migrating neutrophils, while BCL2A1B was linked to proliferating neutrophils. The hucMSC-Exosomes group exhibited significantly higher levels of M1 macrophages (215 vs 76, p < 0.00001), M2 macrophages (1231 vs 670, p < 0.00001), and neutrophils (930 vs 157, p < 0.00001) when compared to control group. Additionally, it was observed that hucMSC-Exosomes elicit alterations in the differentiation trajectories of macrophages towards more anti-inflammatory phenotypes, concomitant with changes in ligand-receptor interactions, thereby facilitating healing.DiscussionThis study has revealed the transcriptomic heterogeneity of neutrophils and macrophages in the context of skin wound repair following hucMSC-Exosomes interventions, providing a deeper understanding of cellular responses to hucMSC-Exosomes, a rising target of wound healing intervention.