Expression of factor V by resident macrophages boosts host defense in the peritoneal cavity

Macrophages resident in different organs express distinct genes, but understanding how this diversity fits into tissue-specific features is limited. Here, we show that selective expression of coagulation factor V (FV) by resident peritoneal macrophages in mice promotes bacterial clearance in the peritoneal cavity and serves to facilitate the well-known but poorly understood macrophage disappearance reaction. Intravital imaging revealed that resident macrophages were nonadherent in peritoneal fluid during homeostasis. Bacterial entry into the peritoneum acutely induced macrophage adherence and associated bacterial phagocytosis. However, optimal control of bacterial expansion in the peritoneum also required expression of FV by the macrophages to form local clots that effectively brought macrophages and bacteria in proximity and out of the fluid phase. Thus, acute cellular adhesion and resident macrophage-induced coagulation operate independently and cooperatively to meet the challenges of a unique, open tissue environment. These events collectively account for the macrophage disappearance reaction in the peritoneal cavity

Single-cell RNA-seq analysis of human CSF microglia and myeloid cells in neuroinflammation

OBJECTIVE: To identify and characterize myeloid cell populations within the CSF of patients with MS and anti-myelin oligodendrocyte glycoprotein (MOG) disorder by high-resolution single-cell gene expression analysis. METHODS: Single-cell RNA sequencing (scRNA-seq) was used to profile individual cells of CSF and blood from 2 subjects with relapsing-remitting MS (RRMS) and one with anti-MOG disorder. Publicly available scRNA-seq data from the blood and CSF of 2 subjects with HIV were also analyzed. An informatics pipeline was used to cluster cell populations by transcriptomic profiling. Based on gene expression by CSF myeloid cells, a flow cytometry panel was devised to examine myeloid cell populations from the CSF of 11 additional subjects, including individuals with RRMS, anti-MOG disorder, and control subjects without inflammatory demyelination. RESULTS: Common myeloid populations were identified within the CSF of subjects with RRMS, anti-MOG disorder, and HIV. These included monocytes, conventional and plasmacytoid dendritic cells, and cells with a transcriptomic signature matching microglia. Microglia could be discriminated from other myeloid cell populations in the CSF by flow cytometry. CONCLUSIONS: High-resolution single-cell gene expression analysis clearly distinguishes distinct myeloid cell types present within the CSF of subjects with neuroinflammation. A population of microglia exists within the human CSF, which is detectable by surface protein expression. The function of these cells during immunity and disease requires further investigation

L-plastin is essential for alveolar macrophage production and control of pulmonary pneumococcal infection

We report that mice deficient for the hematopoietic-specific, actin-bundling protein L-plastin (LPL) succumb rapidly to intratracheal pneumococcal infection. The increased susceptibility of LPL(−/−) mice to pulmonary pneumococcal challenge correlated with reduced numbers of alveolar macrophages, consistent with a critical role for this cell type in the immediate response to pneumococcal infection. LPL(−/−) mice demonstrated a very early clearance defect, with an almost 10-fold-higher bacterial burden in the bronchoalveolar lavage fluid 3 h following infection. Clearance of pneumococci from the alveolar space in LPL(−/−) mice was defective compared to that in Rag1(−/−) mice, which lack all B and T lymphocytes, indicating that innate immunity is defective in LPL(−/−) mice. We did not identify defects in neutrophil or monocyte recruitment or in the production of inflammatory cytokines or chemokines that would explain the early clearance defect. However, efficient alveolar macrophage regeneration following irradiation required LPL. We thus identify LPL as being key to alveolar macrophage development and essential to an effective antipneumococcal response. Further analysis of LPL(−/−) mice will illuminate critical regulators of the generation of alveolar macrophages and, thus, effective pulmonary innate immunity

Batf3-Dependent CD11blow/− Peripheral Dendritic Cells Are GM-CSF-Independent and Are Not Required for Th Cell Priming after Subcutaneous Immunization

Dendritic cells (DCs) subsets differ in precursor cell of origin, functional properties, requirements for growth factors, and dependence on transcription factors. Lymphoid-tissue resident CD8α+ conventional DCs (cDCs) and CD11blow/−CD103+ non-lymphoid DCs are developmentally related, each being dependent on FMS-like tyrosine kinase 3 ligand (Flt3L), and requiring the transcription factors Batf3, Irf8, and Id2 for development. It was recently suggested that granulocyte/macrophage colony stimulating factor (GM-CSF) was required for the development of dermal CD11blow/−Langerin+CD103+ DCs, and that this dermal DC subset was required for priming autoreactive T cells in experimental autoimmune encephalitis (EAE). Here, we compared development of peripheral tissue DCs and susceptibility to EAE in GM-CSF receptor deficient (Csf2rb−/−) and Batf3−/− mice. We find that Batf3-dependent dermal CD11blow/−Langerin+ DCs do develop in Csf2rb−/− mice, but that they express reduced, but not absent, levels of CD103. Further, Batf3−/− mice lacking all peripheral CD11blow/− DCs show robust Th cell priming after subcutaneous immunization and are susceptible to EAE. Our results suggest that defective T effector priming and resistance to EAE exhibited by Csf2rb−/− mice does not result from the absence of dermal CD11blow/−Langerin+CD103+ DCs

Bhlhe40 controls cytokine production by T cells and is essential for pathogenicity in autoimmune neuroinflammation

T(H)1 and T(H)17 cells mediate neuroinflammation in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Pathogenic T(H) cells in EAE must produce the pro-inflammatory cytokine granulocyte-macrophage colony stimulating factor (GM-CSF). T(H) cell pathogenicity in EAE is also regulated by cell-intrinsic production of the immunosuppressive cytokine interleukin 10 (IL-10). Here, we demonstrate that mice deficient for the basic helix-loop-helix (bHLH) transcription factor Bhlhe40 (Bhlhe40(−/−)) are resistant to the induction of EAE. Bhlhe40 is required in vivo in a T cell-intrinsic manner, where it positively regulates the production of GM-CSF and negatively regulates the production of IL-10. In vitro, GM-CSF secretion is selectively abrogated in polarized Bhlhe40(−/−) T(H)1 and T(H)17 cells, and these cells show increased production of IL-10. Blockade of IL-10 receptor in Bhlhe40(−/−) mice renders them susceptible to EAE. These findings identify Bhlhe40 as a critical regulator of autoreactive T cell pathogenicity

Bhlhe40 is an essential repressor of IL-10 during Mycobacterium tuberculosis infection

The cytokine IL-10 antagonizes pathways that contro

BHLHE40 regulates the T-cell effector function required for tumor microenvironment remodeling and immune checkpoint therapy efficacy

Immune checkpoint therapy (ICT) using antibody blockade of programmed cell death protein 1 (PD-1) or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) can provoke T cell-dependent antitumor activity that generates durable clinical responses in some patients. The epigenetic and transcriptional features that T cells require for efficacious ICT remain to be fully elucidated. Herein, we report that anti-PD-1 and anti-CTLA-4 ICT induce upregulation of the transcription factor BHLHE40 in tumor antigen-specific CD8+ and CD4+ T cells and that T cells require BHLHE40 for effective ICT in mice bearing immune-edited tumors. Single-cell RNA sequencing of intratumoral immune cells in BHLHE40-deficient mice revealed differential ICT-induced immune cell remodeling. The BHLHE40-dependent gene expression changes indicated dysregulated metabolism, NF-κB signaling, and IFNγ response within certain subpopulations of CD4+ and CD8+ T cells. Intratumoral CD4+ and CD8+ T cells from BHLHE40-deficient mice exhibited higher expression of the inhibitory receptor gene Tigit and displayed alterations in expression of genes encoding chemokines/chemokine receptors and granzyme family members. Mice lacking BHLHE40 had reduced ICT-driven IFNγ production by CD4+ and CD8+ T cells and defects in ICT-induced remodeling of macrophages from a CX3CR1+CD206+ subpopulation to an iNOS+ subpopulation that is typically observed during effective ICT. Although both anti-PD-1 and anti-CTLA-4 ICT in BHLHE40-deficient mice led to the same outcome-tumor outgrowth-several BHLHE40-dependent alterations were specific to the ICT that was used. Our results reveal a crucial role for BHLHE40 in effective ICT and suggest that BHLHE40 may be a predictive or prognostic biomarker for ICT efficacy and a potential therapeutic target