CD11c identifies microbiota and EGR2-dependent MHCII+ serous cavity macrophages with sexually dimorphic fate in mice.

The murine serous cavities contain a rare and enigmatic population of short-lived F4/80lo MHCII+ macrophages but what regulates their development, survival, and fate is unclear. Here, we show that mature F4/80lo MHCII+ peritoneal macrophages arise after birth, but that this occurs largely independently of colonization by microbiota. Rather, microbiota specifically regulate development of a subpopulation of CD11c+ cells that express the immunoregulatory cytokine RELM-α, are reliant on the transcription factor EGR2, and develop independently of the growth factor CSF1. Furthermore, we demonstrate that intrinsic expression of RELM-α, a signature marker shared by CD11c+ and CD11c- F4/80lo MHCII+ cavity macrophages, regulates survival and differentiation of these cells in the peritoneal cavity in a sex-specific manner. Thus, we identify a previously unappreciated diversity in serous cavity F4/80lo MHCII+ macrophages that is regulated by microbiota, and describe a novel sex and site-specific function for RELM-α in regulating macrophage endurance that reveals the unique survival challenge presented to monocyte-derived macrophages by the female peritoneal environment.

Tissue-based IL-10 signalling in helminth infection limits IFNγ expression and promotes the intestinal Th2 response.

Type 2 immunity is activated in response to both allergens and helminth infection. It can be detrimental or beneficial, and there is a pressing need to better understand its regulation. The immunosuppressive cytokine IL-10 is known as a T helper 2 (Th2) effector molecule, but it is currently unclear whether IL-10 dampens or promotes Th2 differentiation during infection. Here we show that helminth infection in mice elicits IL-10 expression in both the intestinal lamina propria and the draining mesenteric lymph node, with higher expression in the infected tissue. In vitro, exogenous IL-10 enhanced Th2 differentiation in isolated CD4+ T cells, increasing expression of GATA3 and production of IL-5 and IL-13. The ability of IL-10 to amplify the Th2 response coincided with its suppression of IFNγ expression and in vivo we found that, in intestinal helminth infection, IL-10 receptor expression was higher on Th1 cells in the small intestine than on Th2 cells in the same tissue, or on any Th cell in the draining lymph node. In vivo blockade of IL-10 signalling during helminth infection resulted in an expansion of IFNγ+ and Tbet+ Th1 cells in the small intestine and a coincident decrease in IL-13, IL-5 and GATA3 expression by intestinal T cells. These changes in Th2 cytokines correlated with reduced expression of type 2 effector molecules, such as RELMα, and increased parasite egg production. Together our data indicate that IL-10 signalling promotes Th2 differentiation during helminth infection at least in part by regulating competing Th1 cells in the infected tissue.

Targeted Delivery of Narrow-Spectrum Protein Antibiotics to the Lower Gastrointestinal Tract in a Murine Model of Escherichia coli Colonization.

Bacteriocins are narrow-spectrum protein antibiotics that could potentially be used to engineer the human gut microbiota. However, technologies for targeted delivery of proteins to the lower gastrointestinal (GI) tract in preclinical animal models are currently lacking. In this work, we have developed methods for the microencapsulation of Escherichia coli targeting bacteriocins, colicin E9 and Ia, in a pH responsive formulation to allow their targeted delivery and controlled release in an in vivo murine model of E. coli colonization. Membrane emulsification was used to produce a water-in-oil emulsion with the water-soluble polymer subsequently cross-linked to produce hydrogel microcapsules. The microcapsule fabrication process allowed control of the size of the drug delivery system and a near 100% yield of the encapsulated therapeutic cargo. pH-triggered release of the encapsulated colicins was achieved using a widely available pH-responsive anionic copolymer in combination with alginate biopolymers. In vivo experiments using a murine E. coli intestinal colonization model demonstrated that oral delivery of the encapsulated colicins resulted in a significant decrease in intestinal colonization and reduction in E. coli shedding in the feces of the animals. Employing controlled release drug delivery systems such as that described here is essential to enable delivery of new protein therapeutics or other biological interventions for testing within small animal models of infection. Such approaches may have considerable value for the future development of strategies to engineer the human gut microbiota, which is central to health and disease.

Monocytes mediate Salmonella Typhimurium-induced tumor growth inhibition in a mouse melanoma model.

The use of bacteria as an alternative cancer therapy has been reinvestigated in recent years. SL7207: an auxotrophic Salmonella enterica serovar Typhimurium aroA mutant with immune-stimulatory potential has proven a promising strain for this purpose. Here, we show that systemic administration of SL7207 induces melanoma tumor growth arrest in vivo, with greater survival of the SL7207-treated group compared to control PBS-treated mice. Administration of SL7207 is accompanied by a change in the immune phenotype of the tumor-infiltrating cells toward pro-inflammatory, with expression of the TH 1 cytokines IFN-γ, TNF-α, and IL-12 significantly increased. Interestingly, Ly6C+ MHCII+ monocytes were recruited to the tumors following SL7207 treatment and were pro-inflammatory. Accordingly, the abrogation of these infiltrating monocytes using clodronate liposomes prevented SL7207-induced tumor growth inhibition. These data demonstrate a previously unappreciated role for infiltrating inflammatory monocytes underlying bacterial-mediated tumor growth inhibition. This information highlights a possible novel role for monocytes in controlling tumor growth, contributing to our understanding of the immune responses required for successful immunotherapy of cancer.

The mannose receptor (CD206) identifies a population of colonic macrophages in health and inflammatory bowel disease.

To understand the contribution of mononuclear phagocytes (MNP), which include monocyte-derived intestinal macrophages, to the pathogenesis of inflammatory bowel disease (IBD), it is necessary to identify functionally-different MNP populations. We aimed to characterise intestinal macrophage populations in patients with IBD. We developed 12-parameter flow cytometry protocols to identify and human intestinal MNPs. We used these protocols to purify and characterize colonic macrophages from colonic tissue from patients with Crohn's disease (CD), ulcerative colitis (UC), or non-inflamed controls, in a cross-sectional study. We identify macrophage populations (CD45+CD64+ HLA-DR+) and describe two distinct subsets, differentiated by their expression of the mannose receptor, CD206. CD206+ macrophages expressed markers consistent with a mature phenotype: high levels of CD68 and CD163, higher transcription of IL-10 and lower expression of TREM1. CD206- macrophages appear to be less mature, with features more similar to their monocytic precursors. We identified and purified macrophage populations from human colon. These appear to be derived from a monocytic precursor with high CCR2 and low CD206 expression. As these cells mature, they acquire expression of IL-10, CD206, CD63, and CD168. Targeting the newly recruited monocyte-derived cells may represent a fruitful avenue to ameliorate chronic inflammation in IBD.

Antibiotic-induced disturbances of the gut microbiota result in accelerated breast tumor growth.

The gut microbiota's function in regulating health has seen it linked to disease progression in several cancers. However, there is limited research detailing its influence in breast cancer (BrCa). This study found that antibiotic-induced perturbation of the gut microbiota significantly increases tumor progression in multiple BrCa mouse models. Metagenomics highlights the common loss of several bacterial species following antibiotic administration. One such bacteria, Faecalibaculum rodentium, rescued this increased tumor growth. Single-cell transcriptomics identified an increased number of cells with a stromal signature in tumors, and subsequent histology revealed an increased abundance of mast cells in the tumor stromal regions. We show that administration of a mast cell stabilizer, cromolyn, rescues increased tumor growth in antibiotic treated animals but has no influence on tumors from control cohorts. These findings highlight that BrCa-microbiota interactions are different from other cancers studied to date and suggest new research avenues for therapy development.

Mucosal CD8 T Cell Responses Are Shaped by Batf3-DC After Foodborne Listeria monocytogenes Infection.

While immune responses have been rigorously examined after intravenous Listeria monocytogenes (Lm) infection, less is understood about its dissemination from the intestines or the induction of adaptive immunity after more physiologic models of foodborne infection. Consequently, this study focused on early events in the intestinal mucosa and draining mesenteric lymph nodes (MLN) using foodborne infection of mice with Lm modified to invade murine intestinal epithelium (InlAMLm). InlAMLm trafficked intracellularly from the intestines to the MLN and were associated with Batf3-independent dendritic cells (DC) in the lymphatics. Consistent with this, InlAMLm initially disseminated from the gut to the MLN normally in Batf3-/- mice. Activated migratory DC accumulated in the MLN by 3 days post-infection and surrounded foci of InlAMLm. At this time Batf3-/- mice displayed reduced InlAMLm burdens, implicating cDC1 in maximal bacterial accumulation in the MLN. Batf3-/- mice also exhibited profound defects in the induction and gut-homing of InlAMLm-specific effector CD8 T cells. Restoration of pathogen burden did not rescue antigen-specific CD8 T cell responses in Batf3-/- mice, indicating a critical role for Batf3 in generating anti-InlAMLm immunity following foodborne infection. Collectively, these data suggest that DC play diverse, dynamic roles in the early events following foodborne InlAMLm infection and in driving the establishment of intestinal Lm-specific effector T cells.

Regulatory T cells control the dynamic and site-specific polarization of total CD4 T cells following Salmonella infection.

FoxP3+ regulatory T cells (Tregs) control inflammation and maintain mucosal homeostasis, but their functions during infection are poorly understood. Th1, Th2, and Th17 cells can be identified by master transcription factors (TFs) T-bet, GATA3, and RORγT; Tregs also express these TFs. While T-bet+ Tregs can selectively suppress Th1 cells, it is unclear whether distinct Treg populations can alter Th bias. To address this, we used Salmonella enterica serotype Typhimurium to induce nonlethal colitis. Following infection, we observed an early colonic Th17 response within total CD4 T cells, followed by a Th1 bias. The early Th17 response, which contains both Salmonella-specific and non-Salmonella-specific cells, parallels an increase in T-bet+ Tregs. Later, Th1 cells and RORγT+ Tregs dominate. This reciprocal dynamic may indicate that Tregs selectively suppress Th cells, shaping the immune response. Treg depletion 1-2 days post-infection shifted the early Th17 response to a Th1 bias; however, Treg depletion 6-7 days post-infection abrogated the Th1 bias. Thus, Tregs are necessary for the early Th17 response, and for a maximal Th1 response later. These data show that Tregs shape the overall tissue CD4 T cell response and highlight the potential for subpopulations of Tregs to be used in targeted therapeutic approaches.

Isolation and functional characterisation of lamina propria leukocytes from helminth-infected, murine small intestine.

The use of helminth infections as tools to understand the type 2 immune response is a well-established technique and important to many areas of immunological research. The phenotype and function of immune cell populations at the site of infection is a key determinant of pathogen clearance. However, infections with helminths such as the murine nematode Heligomosmoides polygryrus cause increased mucus production and thickening of the intestinal wall, which can result in extensive cell death when isolating and analysing cells from the lamina propria (LP). Populations of larger immune cells such as macrophages and dendritic cells are often trapped within mucus or dying tissues. Here we describe an optimised protocol for isolating LP leukocytes from the small intestine of H.polygyrus -infected mice, and we demonstrate phenotypic and functional identification of myeloid and CD4+ T cell subsets using cytokine staining and flow cytometry. Our protocol may provide a useful experimental method for the immunological analysis of the affected tissue site during helminth infections.

Exposure to the antimicrobial peptide LL-37 produces dendritic cells optimized for immunotherapy.

Immunization of patients with autologous, ex vivo matured dendritic cell (DC) preparations, in order to prime antitumor T-cell responses, is the focus of intense research. Despite progress and approval of clinical approaches, significant enhancement of these personalized immunotherapies is urgently needed to improve efficacy. We show that immunotherapeutic murine and human DC, generated in the presence of the antimicrobial host defense peptide LL-37, have dramatically enhanced expansion and differentiation of cells with key features of the critical CD103+/CD141+ DC subsets, including enhanced cross-presentation and co-stimulatory capacity, and upregulation of CCR7 with improved migratory capacity. These LL-37-DC enhanced proliferation, activation and cytokine production by CD8+ (but not CD4+) T cells in vitro and in vivo. Critically, tumor antigen-presenting LL-37-DC increased migration of primed, activated CD8+ T cells into established squamous cell carcinomas in mice, and resulted in tumor regression. This advance therefore has the potential to dramatically enhance DC immunotherapy protocols.

Salmonella enterica Serovar Typhimurium Travels to Mesenteric Lymph Nodes Both with Host Cells and Autonomously.

Salmonella infection is a globally important cause of gastroenteritis and systemic disease and is a useful tool to study immune responses in the intestine. Although mechanisms leading to immune responses against Salmonella have been extensively studied, questions remain about how bacteria travel from the intestinal mucosa to the mesenteric lymph nodes (MLN), a key site for Ag presentation. In this study, we used a mouse model of infection with Salmonella enterica serovar Typhimurium (STM) to identify changes in intestinal immune cells induced during early infection. We then used fluorescently labeled STM to identify interactions with immune cells from the site of infection through migration in lymph to the MLN. We show that viable STM can be carried in the lymph by any subset of migrating dendritic cells but not by macrophages. Moreover, approximately half of the STM in lymph are not associated with cells at all and travel autonomously. Within the MLN, STM associates with dendritic cells and B cells but predominantly with MLN-resident macrophages. In conclusion, we describe the routes used by STM to spread systemically in the period immediately postinfection. This deeper understanding of the infection process could open new avenues for controlling it.

Antibiotics induce sustained dysregulation of intestinal T cell immunity by perturbing macrophage homeostasis.

Macrophages in the healthy intestine are highly specialized and usually respond to the gut microbiota without provoking an inflammatory response. A breakdown in this tolerance leads to inflammatory bowel disease (IBD), but the mechanisms by which intestinal macrophages normally become conditioned to promote microbial tolerance are unclear. Strong epidemiological evidence linking disruption of the gut microbiota by antibiotic use early in life to IBD indicates an important role for the gut microbiota in modulating intestinal immunity. Here, we show that antibiotic use causes intestinal macrophages to become hyperresponsive to bacterial stimulation, producing excess inflammatory cytokines. Re-exposure of antibiotic-treated mice to conventional microbiota induced a long-term, macrophage-dependent increase in inflammatory T helper 1 (TH1) responses in the colon and sustained dysbiosis. The consequences of this dysregulated macrophage activity for T cell function were demonstrated by increased susceptibility to infections requiring TH17 and TH2 responses for clearance (bacterial Citrobacter rodentium and helminth Trichuris muris infections), corresponding with increased inflammation. Short-chain fatty acids (SCFAs) were depleted during antibiotic administration; supplementation of antibiotics with the SCFA butyrate restored the characteristic hyporesponsiveness of intestinal macrophages and prevented T cell dysfunction. Butyrate altered the metabolic behavior of macrophages to increase oxidative phosphorylation and also promoted alternative macrophage activation. In summary, the gut microbiota is essential to maintain macrophage-dependent intestinal immune homeostasis, mediated by SCFA-dependent pathways. Oral antibiotics disrupt this process to promote sustained T cell-mediated dysfunction and increased susceptibility to infections, highlighting important implications of repeated broad-spectrum antibiotic use.

The Transcription Factor ZEB2 Is Required to Maintain the Tissue-Specific Identities of Macrophages.

Heterogeneity between different macrophage populations has become a defining feature of this lineage. However, the conserved factors defining macrophages remain largely unknown. The transcription factor ZEB2 is best described for its role in epithelial to mesenchymal transition; however, its role within the immune system is only now being elucidated. We show here that Zeb2 expression is a conserved feature of macrophages. Using Clec4f-cre, Itgax-cre, and Fcgr1-cre mice to target five different macrophage populations, we found that loss of ZEB2 resulted in macrophage disappearance from the tissues, coupled with their subsequent replenishment from bone-marrow precursors in open niches. Mechanistically, we found that ZEB2 functioned to maintain the tissue-specific identities of macrophages. In Kupffer cells, ZEB2 achieved this by regulating expression of the transcription factor LXRα, removal of which recapitulated the loss of Kupffer cell identity and disappearance. Thus, ZEB2 expression is required in macrophages to preserve their tissue-specific identities.

Isolation and Identification of Conventional Dendritic Cell Subsets from the Intestine of Mice and Men.

The identification of conventional dendritic cells (cDCs) in the intestinal mucosa has been hampered by the difficulties associated with isolating cells from the intestine and by the fact that overlapping markers have made it complicated to discriminate them accurately from other intestinal mononuclear phagocytes such as macrophages (MFs). Here we detail the protocols we have developed to isolate live leukocytes from both murine and human small and large intestines and describe reliable strategies which can be used to identify bona fide cDCs in such preparations.

Ankylosing spondylitis patients display altered dendritic cell and T cell populations that implicate pathogenic roles for the IL-23 cytokine axis and intestinal inflammation.

OBJECTIVE: AS is a systemic inflammatory disease of the SpA family. Polymorphisms at loci including HLA-B27, IL-23R and ERAP-1 directly implicate immune mechanisms in AS pathogenesis. Previously, in an SpA model, we identified HLA-B27-mediated effects on dendritic cells that promoted disease-associated Th17 cells. Here we extend these studies to AS patients using deep immunophenotyping of candidate pathogenic cell populations. The aim of our study was to functionally characterize the immune populations mediating AS pathology. METHODS: Using 11-parameter flow cytometry, we characterized the phenotype and functions of lymphocyte and myeloid cells from peripheral blood, and the synovial phenotype of AS patients and age-matched healthy controls. RESULTS: Significantly fewer circulating CD1c-expressing dendritic cells were observed in AS patients, offset by an increase in CD14(-) CD16(+) mononuclear cells. Ex vivo functional analysis revealed that this latter population induced CCR6 expression and promoted secretion of IL-1ß and IL-6 when co-cultured with naive CD4(+) T cells. Additionally, systemic inflammation in AS patients significantly correlated with increased proportions of activated CCR9(+) CD4(+) T cells. CONCLUSION: CD14(-) CD16(+) mononuclear cells may contribute to AS by promoting Th17 responses, and antigen-presenting cells of mucosal origin are likely to contribute to systemic inflammation in AS.

Characterization of conventional and atypical receptors for the chemokine CCL2 on mouse leukocytes.

Chemokine-directed leukocyte migration is crucial for effective immune and inflammatory responses. Conventional chemokine receptors (cCKRs) directly control cell movement; atypical chemokine receptors (ACKRs) regulate coexpressed cCKRs; and both cCKRs and ACKRs internalize chemokines to limit their abundance in vivo, a process referred to as scavenging. A leukocyte's migratory and chemokine-scavenging potential is determined by which cCKRs and ACKRs it expresses, and by the ligand specificity, signaling properties, and chemokine internalization capacity of these receptors. Most chemokines can bind at least one cCKR and one ACKR. CCL2 can bind to CCR2 (a cCKR) and two ACKRs (ACKR1 and ACKR2). In this study, by using fluorescent CCL2 uptake to label cells bearing functional CCL2 receptors, we have defined the expression profile, scavenging activity, and ligand specificity of CCL2 receptors on mouse leukocytes. We show that qualitative and quantitative differences in the expression of CCR2 and ACKR2 endow individual leukocyte subsets with distinctive CCL2 receptor profiles and CCL2-scavenging capacities. We reveal that some cells, including plasmacytoid dendritic cells, can express both CCR2 and ACKR2; that Ly6C(high) monocytes have particularly strong CCL2-scavenging potential in vitro and in vivo; and that CCR2 is a much more effective CCL2 scavenger than ACKR2. We confirm the unique, overlapping, ligand specificities of CCR2 and ACKR2 and, unexpectedly, find that cell context influences the interaction of CCL7 and CCL12 with CCR2. Fluorescent chemokine uptake assays were instrumental in providing these novel insights into CCL2 receptor biology, and the sensitivity, specificity, and versatility of these assays are discussed.

Intestinal macrophages and dendritic cells: what's the difference?

Mononuclear phagocytes (MPs) in the murine intestine, comprising dendritic cells (DCs) and macrophages (Mϕs), perform disparate yet complementary immunological functions. Functional analyses of these distinct MP subsets have been complicated by the substantial overlap in their surface phenotypes. Here, we review recent findings that have enabled more accurate definition of these MP subsets. We discuss these recent advances in the context of the current understanding of the functions of DCs and Mϕs in the maintenance of intestinal homeostasis, and how their functions may alter when homeostasis is disrupted.

Hyporesponsiveness of intestinal dendritic cells to TLR stimulation is limited to TLR4.

Dendritic cells (DCs) are crucial to intestinal immune regulation because of their roles in inducing protective immunity against pathogens while maintaining tolerance to commensal bacteria. Nonetheless, relatively little is known about intestinal DC responsiveness to innate immune stimuli via TLRs. We have previously shown that DCs migrating from the rat intestine in lymph (iLDCs) are hyporesponsive to LPS stimulation, thus possibly preventing harmful immune responses being induced to commensal flora. In this study, to understand how iLDC function is regulated by innate immune stimuli, we have characterized the expression and function of TLRs in iLDCs isolated from the thoracic duct lymph of mesenteric lymphadenectomized rats and compared these with DCs grown from bone marrow in the presence of Flt3 ligand. We show that iLDCs express mRNAs for all TLRs, but express significantly less TLR4 mRNA than bone marrow-derived DCs. Functionally, iLDCs could be activated by TLR agonists representing intestinal pathogen-associated molecular patterns, with the important exception of the TLR4 agonist LPS. Furthermore, we show that DCs in the intestinal wall interact directly with noninvasive bacteria (Bacillus subtilis spores), leading to an increase in the output of activated iLDCs into lymph, and that DCs containing spores are activated selectively. These data highlight a functional difference between TLR4 and other TLRs. As iLDCs can respond to TLR stimulation in vitro, there must be other mechanisms that prevent their activation by commensal bacteria under steady-state conditions.

New insights into the roles of dendritic cells in intestinal immunity and tolerance.

Dendritic cells (DCs) play a critical key role in the initiation of immune responses to pathogens. Paradoxically, they also prevent potentially damaging immune responses being directed against the multitude of harmless antigens, to which the body is exposed daily. These roles are particularly important in the intestine, where only a single layer of epithelial cells provides a barrier against billions of commensal microorganisms, pathogens, and food antigens, over a huge surface area. In the intestine, therefore, DCs are required to perform their dual roles very efficiently to protect the body from the dual threats of invading pathogens and unwanted inflammatory reactions. In this review, we first describe the biology of DCs and their interactions with other cells types, paying particular attention to intestinal DCs. We, then, examine the ways in which this biology may become misdirected, resulting in inflammatory bowel disease. Finally, we discuss how DCs potentiate immune responses against viral, bacterial, parasitic infections, and their importance in the pathogenesis of prion diseases. We, therefore, provide an overview of the complex cellular interactions that affect intestinal DCs and control the balance between immunity and tolerance.

Regulation of intestinal immunity: effects of the oral adjuvant Escherichia coli heat-labile enterotoxin on migrating dendritic cells.

Escherichia coli heat-labile enterotoxin (Etx) is an oral adjuvant in mice. We show that this is also true for rats. To understand this adjuvant activity we examined lymph dendritic cells (DC) migrating from the intestine to mesenteric lymph nodes (MLN) in animals fed Etx. These DC can prime antigen-specific antibody responses. We show that in rats the small intestine contains 7-24 million DC and 8 x 10(5 )of these migrate to MLN each day. Surprisingly, Etx does not stimulate increased migration of lymph DC. However, oral Etx affects the activation, antigen transport and localization of migratory DC. Specifically, expression of CD25 increases on the CD172a(high) subset of lymph DC. Oral Etx also increases the number of CD172a(high) lymph DC containing co-administered ovalbumin. CD172a(high) lymph DC treated with Etx in vitro, or purified from the lymph of animals fed Etx, stimulate stronger proliferative responses from primed T cells. Etx also directs more of the CD172a(high) lymph DC into the central region of the MLN T cell areas. This change in DC localization is associated with an increase in the expression of CCR7. These data help advance our understanding of the role of DC in initiating mucosal immune responses in vivo.