IRF8 deficiency induces the transcriptional, functional, and epigenetic reprogramming of cDC1 into the cDC2 lineage.

Conventional dendritic cells (cDCs) consist of two major functionally and phenotypically distinct subsets, cDC1 and cDC2, whose development is dependent on distinct sets of transcription factors. Interferon regulatory factor 8 (IRF8) is required at multiple stages of cDC1 development, but its role in committed cDC1 remains unclear. Here, we used Xcr1-cre to delete Irf8 in committed cDC1 and demonstrate that Irf8 is required for maintaining the identity of cDC1. In the absence of Irf8, committed cDC1 acquired the transcriptional, functional, and chromatin accessibility properties of cDC2. This conversion was independent of Irf4 and was associated with the decreased accessibility of putative IRF8, Batf3, and composite AP-1-IRF (AICE)-binding elements, together with increased accessibility of cDC2-associated transcription-factor-binding elements. Thus, IRF8 expression by committed cDC1 is required for preventing their conversion into cDC2-like cells.

A self-sustaining layer of early-life-origin B cells drives steady-state IgA responses in the adult gut.

The adult immune system consists of cells that emerged at various times during ontogeny. We aimed to define the relationship between developmental origin and composition of the adult B cell pool during unperturbed hematopoiesis. Lineage tracing stratified murine adult B cells based on the timing of output, revealing that a substantial portion originated within a restricted neonatal window. In addition to B-1a cells, early-life time-stamped B cells included clonally interrelated IgA plasma cells in the gut and bone marrow. These were actively maintained by B cell memory within gut chronic germinal centers and contained commensal microbiota reactivity. Neonatal rotavirus infection recruited recurrent IgA clones that were distinct from those arising by infection with the same antigen in adults. Finally, gut IgA plasma cells arose from the same hematopoietic progenitors as B-1a cells during ontogeny. Thus, a complex layer of neonatally imprinted B cells confer unique antibody responses later in life.

Immune Profiling of Human Gut-Associated Lymphoid Tissue Identifies a Role for Isolated Lymphoid Follicles in Priming of Region-Specific Immunity.

The intestine contains some of the most diverse and complex immune compartments in the body. Here we describe a method for isolating human gut-associated lymphoid tissues (GALTs) that allows unprecedented profiling of the adaptive immune system in submucosal and mucosal isolated lymphoid follicles (SM-ILFs and M-ILFs, respectively) as well as in GALT-free intestinal lamina propria (LP). SM-ILF and M-ILF showed distinct patterns of distribution along the length of the intestine, were linked to the systemic circulation through MAdCAM-1+ high endothelial venules and efferent lymphatics, and had immune profiles consistent with immune-inductive sites. IgA sequencing analysis indicated that human ILFs are sites where intestinal adaptive immune responses are initiated in an anatomically restricted manner. Our findings position ILFs as key inductive hubs for regional immunity in the human intestine, and the methods presented will allow future assessment of these compartments in health and disease.

Identification and characterization of murine glycoprotein 2-expressing intestinal dendritic cells.

The intestinal lamina propria (LP) contains distinct subsets of classical dendritic cells (cDC), each playing key non-redundant roles in intestinal immune homeostasis. Here, we show that glycoprotein 2 (GP2), a GPI-anchored protein and receptor for bacterial type-I fimbriae, is selectively expressed by CD103+CD11b+ cDC in the murine small intestine (SI). GP2 expression was induced on CD103+CD11b+ cDC within the SI-LP and was regulated by IRF4, TGFßR1- and retinoic acid signalling. Mice selectively lacking Gp2 on CD103+CD11b+ cDC (huLang-Cre.gp2fl/fl mice) had normal numbers and proportions of innate and adaptive immune cells in the SI-LP suggesting that GP2 expression by CD103+CD11b+ cDC is not required for intestinal immune homoeostasis.

Some news from the unknown soldier, the Peyer's patch macrophage.

In mammals, macrophages (MF) are present in virtually all tissues where they serve many different functions linked primarily to the maintenance of homeostasis, innate defense against pathogens, tissue repair and metabolism. Although some of these functions appear common to all tissues, others are specific to the homing tissue. Thus, MF become adapted to perform particular functions in a given tissue. Accordingly, MF express common markers but also sets of tissue-specific markers linked to dedicated functions. One of the largest pool of MF in the body lines up the wall of the gut. Located in the small intestine, Peyer's patches (PP) are primary antigen sampling and mucosal immune response inductive sites. Surprisingly, although markers of intestinal MF, such as F4/80, have been identified more than 30 years ago, MF of PP escaped any kind of phenotypic description and remained "unknown" for decades. In absence of MF identification, the characterization of the PP mononuclear phagocyte system (MPS) functions has been impaired. However, taking into account that PP are privileged sites of entry for pathogens, it is important to understand how the latter are handled by and/or escape the PP MPS, especially MF, which role in killing invaders is well known. This review focuses on recent advances on the PP MPS, which have allowed, through new criteria of PP phagocyte subset identification, the characterization of PP MF origin, diversity, specificity, location and functions.

Intragranuloma Accumulation and Inflammatory Differentiation of Neutrophils Underlie Mycobacterial ESX-1-Dependent Immunopathology.

The conserved ESX-1 type VII secretion system is a major virulence determinant of pathogenic mycobacteria, including Mycobacterium tuberculosis and Mycobacterium marinum. ESX-1 is known to interact with infected macrophages, but its potential roles in regulating other host cells and immunopathology have remained largely unexplored. Using a murine M. marinum infection model, we identify neutrophils and Ly6C+MHCII+ monocytes as the main cellular reservoirs for the bacteria. We show that ESX-1 promotes intragranuloma accumulation of neutrophils and that neutrophils have a previously unrecognized required role in executing ESX-1-mediated pathology. To explore if ESX-1 also regulates the function of recruited neutrophils, we performed a single-cell RNA-sequencing analysis that indicated that ESX-1 drives newly recruited uninfected neutrophils into an inflammatory phenotype via an extrinsic mechanism. In contrast, monocytes restricted the accumulation of neutrophils and immunopathology, demonstrating a major host-protective function for monocytes specifically by suppressing ESX-1-dependent neutrophilic inflammation. Inducible nitric oxide synthase (iNOS) activity was required for the suppressive mechanism, and we identified Ly6C+MHCII+ monocytes as the main iNOS-expressing cell type in the infected tissue. These results suggest that ESX-1 mediates immunopathology by promoting neutrophil accumulation and phenotypic differentiation in the infected tissue, and they demonstrate an antagonistic interplay between monocytes and neutrophils by which monocytes suppress host-detrimental neutrophilic inflammation. IMPORTANCE The ESX-1 type VII secretion system is required for virulence of pathogenic mycobacteria, including Mycobacterium tuberculosis. ESX-1 interacts with infected macrophages, but its potential roles in regulating other host cells and immunopathology have remained largely unexplored. We demonstrate that ESX-1 promotes immunopathology by driving intragranuloma accumulation of neutrophils, which upon arrival adopt an inflammatory phenotype in an ESX-1-dependent manner. In contrast, monocytes limited the accumulation of neutrophils and neutrophil-mediated pathology via an iNOS-dependent mechanism, suggesting a major host-protective function for monocytes specifically by restricting ESX-1-dependent neutrophilic inflammation. These findings provide insight into how ESX-1 promotes disease, and they reveal an antagonistic functional relationship between monocytes and neutrophils that might regulate immunopathology not only in mycobacterial infection but also in other infections as well as in inflammatory conditions and cancer.

Peyer's patch phagocytes acquire specific transcriptional programs that influence their maturation and activation profiles.

Peyer's patches (PPs) are secondary lymphoid organs in contact with the external environment via the intestinal lumen, thus combining antigen sampling and immune response initiation sites. Therefore, they provide a unique opportunity to study the entire process of phagocyte differentiation and activation in vivo. Here, we deciphered the transcriptional and spatial landscape of PP phagocyte populations from their emergence in the tissue to their final maturation state at homeostasis and under stimulation. Activation of monocyte-derived Lysozyme-expressing dendritic cells (LysoDCs) differs from that of macrophages by their upregulation of conventional DC (cDC) signature genes such as Ccr7 and downregulation of typical monocyte-derived cell genes such as Cx3cr1. We identified gene sets that distinguish PP cDCs from the villus ones and from LysoDCs. We also identified key immature, early, intermediate, and late maturation markers of PP phagocytes. Finally, exploiting the ability of the PP interfollicular region to host both villous and subepithelial dome emigrated cDCs, we showed that the type of stimulus, the subset, but also the initial location of cDCs shape their activation profile and thus direct the immune response. Our study highlights the importance of targeting the right phagocyte subset at the right place and time to manipulate the immune response.

Differentiation Paths of Peyer's Patch LysoDCs Are Linked to Sampling Site Positioning, Migration, and T Cell Priming.

The monocyte-derived phagocytes termed LysoDCs are hallmarks of Peyer's patches, where their main function is to sample intestinal microorganisms. Here, we study their differentiation pathways in relation with their sampling, migratory, and T cell-priming abilities. Among four identified LysoDC differentiation stages displaying similar phagocytic activity, one is located in follicles, and the others reside in subepithelial domes (SED), where they proliferate and mature as they get closer to the epithelium. Mature LysoDCs but not macrophages express a gene set in common with conventional dendritic cells and prime naive helper T cells in vitro. At steady state, they do not migrate into naive T cell-enriched interfollicular regions (IFRs), but upon stimulation, they express the chemokine receptor CCR7 and migrate from SED to the IFR periphery, where they strongly interact with proliferative immune cells. Finally, we show that LysoDCs populate human Peyer's patches, strengthening their interest as targets for modulating intestinal immunity.

The Peyer's Patch Mononuclear Phagocyte System at Steady State and during Infection.

The gut represents a potential entry site for a wide range of pathogens including protozoa, bacteria, viruses, or fungi. Consequently, it is protected by one of the largest and most diversified population of immune cells of the body. Its surveillance requires the constant sampling of its encounters by dedicated sentinels composed of follicles and their associated epithelium located in specialized area. In the small intestine, Peyer's patches (PPs) are the most important of these mucosal immune response inductive sites. Through several mechanisms including transcytosis by specialized epithelial cells called M-cells, access to the gut lumen is facilitated in PPs. Although antigen sampling is critical to the initiation of the mucosal immune response, pathogens have evolved strategies to take advantage of this permissive gateway to enter the host and disseminate. It is, therefore, critical to decipher the mechanisms that underlie both host defense and pathogen subversive strategies in order to develop new mucosal-based therapeutic approaches. Whereas penetration of pathogens through M cells has been well described, their fate once they have reached the subepithelial dome (SED) remains less well understood. Nevertheless, it is clear that the mononuclear phagocyte system (MPS) plays a critical role in handling these pathogens. MPS members, including both dendritic cells and macrophages, are indeed strongly enriched in the SED, interact with M cells, and are necessary for antigen presentation to immune effector cells. This review focuses on recent advances, which have allowed distinguishing the different PP mononuclear phagocyte subsets. It gives an overview of their diversity, specificity, location, and functions. Interaction of PP phagocytes with the microbiota and the follicle-associated epithelium as well as PP infection studies are described in the light of these new criteria of PP phagocyte identification. Finally, known alterations affecting the different phagocyte subsets during PP stimulation or infection are discussed.

Gene expression profiling of the Peyer's patch mononuclear phagocyte system.

Peyer's patches (PPs) are primary inductive sites of mucosal immunity. The PP mononuclear phagocyte system, which encompasses both dendritic cells (DCs) and macrophages, is essential for the initiation of the mucosal immune response. We recently developed a method to isolate each mononuclear phagocyte subset of PP (Bonnardel et al., 2015). We performed a transcriptional analysis of three of these subsets: the CD11b(+) conventional DC, the lysozyme-expressing monocyte-derived DC termed LysoDC and the CD11c(hi) lysozyme-expressing macrophages. Here, we provide details of the gating strategy we used to isolate each phagocyte subset and show the quality controls and analysis associated with our gene array data deposited into Gene Expression Omnibus (GEO) under GSE65514.

Innate and adaptive immune functions of peyer's patch monocyte-derived cells.

Peyer's patches (PPs) are primary inductive sites of mucosal immunity. Defining PP mononuclear phagocyte system (MPS) is thus crucial to understand the initiation of mucosal immune response. We provide a comprehensive analysis of the phenotype, distribution, ontogeny, lifespan, function, and transcriptional profile of PP MPS. We show that monocytes give rise to macrophages and to lysozyme-expressing dendritic cells (LysoDCs), which are both involved in particulate antigen uptake, display strong innate antiviral and antibacterial gene signatures, and, upon TLR7 stimulation, secrete IL-6 and TNF, but neither IL-10 nor IFNγ. However, unlike macrophages, LysoDCs display a rapid renewal rate, strongly express genes of the MHCII presentation pathway, and prime naive helper T cells for IFNγ production. Our results show that monocytes differentiate locally into LysoDCs and macrophages, which display distinct features from their adjacent villus counterparts.