Microbial energy metabolism fuels an intestinal macrophage niche in solitary isolated lymphoid tissues through purinergic signaling.

Maintaining macrophage (MΦ) heterogeneity is critical to ensure intestinal tissue homeostasis and host defense. The gut microbiota and host factors are thought to synergistically guide intestinal MΦ development, although the exact nature, regulation, and location of such collaboration remain unclear. Here, we report that microbial biochemical energy metabolism promotes colony-stimulating factor 2 (CSF2) production by group 3 innate lymphoid cells (ILC3s) within solitary isolated lymphoid tissues (SILTs) in a cell-extrinsic, NLRP3/P2X7R-dependent fashion in the steady state. Tissue-infiltrating monocytes accumulating around SILTs followed a spatially constrained, distinct developmental trajectory into SILT-associated MΦs (SAMs). CSF2 regulated the mitochondrial membrane potential and reactive oxygen species production of SAMs and contributed to the antimicrobial defense against enteric bacterial infections. Collectively, these findings identify SILTs and CSF2-producing ILC3s as a microanatomic niche for intestinal MΦ development and functional programming fueled by the integration of commensal microbial energy metabolism.

Tissue-Dependent Adaptations and Functions of Innate Lymphoid Cells.

Tissue-resident immune cells reside in distinct niches across organs, where they contribute to tissue homeostasis and rapidly respond to perturbations in the local microenvironment. Innate lymphoid cells (ILCs) are a family of innate immune cells that regulate immune and tissue homeostasis. Across anatomical locations throughout the body, ILCs adopt tissue-specific fates, differing from circulating ILC populations. Adaptations of ILCs to microenvironmental changes have been documented in several inflammatory contexts, including obesity, asthma, and inflammatory bowel disease. While our understanding of ILC functions within tissues have predominantly been based on mouse studies, development of advanced single cell platforms to study tissue-resident ILCs in humans and emerging patient-based data is providing new insights into this lymphocyte family. Within this review, we discuss current concepts of ILC fate and function, exploring tissue-specific functions of ILCs and their contribution to health and disease across organ systems.

NLRP1B and NLRP3 Control the Host Response following Colonization with the Commensal Protist Tritrichomonas musculis.

Commensal intestinal protozoa, unlike their pathogenic relatives, are neglected members of the mammalian microbiome. These microbes have a significant impact on the host's intestinal immune homeostasis, typically by elevating anti-microbial host defense. Tritrichomonas musculis, a protozoan gut commensal, strengthens the intestinal host defense against enteric Salmonella infections through Asc- and Il1r1-dependent Th1 and Th17 cell activation. However, the underlying inflammasomes mediating this effect remain unknown. In this study, we report that colonization with T. musculis results in an increase in luminal extracellular ATP that is followed by increased caspase activity, higher cell death, elevated levels of IL-1ß, and increased numbers of IL-18 receptor-expressing Th1 and Th17 cells in the colon. Mice deficient in either Nlrp1b or Nlrp3 failed to display these protozoan-driven immune changes and lost resistance to enteric Salmonella infections even in the presence of T. musculis These findings demonstrate that T. musculis-mediated host protection requires sensors of extracellular and intracellular ATP to confer resistance to enteric Salmonella infections.

Beyond Immunity: Underappreciated Functions of Intestinal Macrophages.

The gastrointestinal tract hosts the largest compartment of macrophages in the body, where they serve as mediators of host defense and immunity. Seeded in the complex tissue-environment of the gut, an array of both hematopoietic and non-hematopoietic cells forms their immediate neighborhood. Emerging data demonstrate that the functional diversity of intestinal macrophages reaches beyond classical immunity and includes underappreciated non-immune functions. In this review, we discuss recent advances in research on intestinal macrophage heterogeneity, with a particular focus on how non-immune functions of macrophages impact tissue homeostasis and function. We delve into the strategic localization of distinct gut macrophage populations, describe the potential factors that regulate their identity and functional heterogeneity within these locations, and provide open questions that we hope will inspire research dedicated to elucidating a holistic view on macrophage-tissue cell interactions in the body's largest mucosal organ.

Isolation of mononuclear phagocytes from the mouse gut.

The intestinal tract is home to trillions of microbes that make up the gut microbiota and is a major source of environmental antigens that can be derived from food, commensal microorganisms, and potential pathogens. Amidst this complex environment, myeloid cells, including macrophages (MPs) and dendritic cells (DCs), are key immunological sentinels that locally maintain both tissue and immune homeostasis. Recent research has revealed substantial functional and developmental heterogeneity within the intestinal DC and MP compartments, with evidence pointing to their regulation by the microbiota. DCs are classically divided into three subsets based on their CD103 and CD11b expression: CD103+CD11b-(XCR1+) cDC1s, CD103+CD11b+ cDC2s, and CD103-CD11b+ cDC2s. Meanwhile, mature gut MPs have recently been classified by their expression of Tim-4 and CD4 into a long-lived, self-maintaining Tim-4+CD4+ population and short-lived, monocyte-derived Tim-4-CD4+ and Tim-4-CD4- populations. In this chapter, we provide experimental procedures to classify and isolate these myeloid subsets from the murine intestinal lamina propria for functional characterization.

Rapid isolation of mouse ILCs from murine intestinal tissues.

Tissue-resident immune cells like innate lymphoid cells (ILCs) are regulators of local immunity and tissue homeostasis. Similar to Natural Killer (NK) cells, ILCs express germline-encoded natural cytotoxicity receptors (NCRs) that facilitate the rapid execution of effector functions. Recent advances using transgenic animal models have further uncovered the developmental, transcriptional, epigenetic, and functional differences between members of the ILC family. Isolation of ILCs, which are particularly enriched in non-lymphoid tissues, can often be challenging and time consuming. Here, we provide a simple and rapid protocol for the isolation of NK cells and ILCs from murine intestinal tissues. This protocol is suitable for Fluorescence Activated Cell Sorting (FACS) and intracellular analysis of cytokine and transcription factor expression using flow and mass cytometry.

ILC2 Activation by Protozoan Commensal Microbes.

Group 2 innate lymphoid cells (ILC2s) are a member of the ILC family and are involved in protective and pathogenic type 2 responses. Recent research has highlighted their involvement in modulating tissue and immune homeostasis during health and disease and has uncovered critical signaling circuits. While interactions of ILC2s with the bacterial microbiome are rather sparse, other microbial members of our microbiome, including helminths and protozoans, reveal new and exciting mechanisms of tissue regulation by ILC2s. Here we summarize the current field on ILC2 activation by the tissue and immune environment and highlight particularly new intriguing pathways of ILC2 regulation by protozoan commensals in the intestinal tract.