Intestinal CX3CR1 Research Articles

Salmonella manipulates macrophage migration via SteC-mediated myosin light chain activation to penetrate the gut-vascular barrier

The intestinal pathogen Salmonella enterica rapidly enters the bloodstream after the invasion of intestinal epithelial cells, but how Salmonella breaks through the gut-vascular barrier is largely unknown. Here, we report that Salmonella enters the bloodstream through intestinal CX3CR1+ macrophages during early infection. Mechanistically, Salmonella induces the migration/invasion properties of macrophages in a manner dependent on host cell actin and on the pathogen effector SteC. SteC recruits host myosin light chain protein Myl12a and phosphorylates its Ser19 and Thr20 residues. Myl12a phosphorylation results in actin rearrangement, and enhanced migration and invasion of macrophages. SteC is able to utilize a wide range of NTPs other than ATP to phosphorylate Myl12a. We further solved the crystal structure of SteC, which suggests an atypical dimerization-mediated catalytic mechanism. Finally, in vivo data show that SteC-mediated cytoskeleton manipulation is crucial for Salmonella breaching the gut vascular barrier and spreading to target organs.

Neonatal Intestinal Macrophages Promote Microvascular Development through an IGF‐1‐dependent Mechanism and Lack of Macrophage‐derived IGF‐1 Predisposes Newborn Mice to Necrotizing Enterocolitis

Defective intestinal microvascular development has been previously shown to play a role in necrotizing enterocolitis (NEC) pathogenesis. Decreased serum levels of IGF-1 in premature infants are associated with a higher risk of developing NEC and we recently found that serum and intestinal tissue IGF-1 level are decreased in a neonatal mouse NEC model. In the embryo, macrophages have been reported to promote angiogenesis. Here we hypothesize that neonatal intestinal macrophages promote endothelial cell proliferation and angiogenesis by producing IGF-1, thus protecting against NEC development. For this purpose, we first examined the role of IGF-1 on intestinal endothelial cell sprouting in vitro using IGF-1 and IGF-1R inhibitor and on intestinal endothelial cell proliferation in vivo using IGF-1R inhibitor. Secondly, using mice with IGF-1-deficient-macrophages and IGF-1-sufficient littermates, we assessed whether macrophage-derived IGF-1 plays a role in perinatal microvascular development by examining endothelial cell proliferation in vivo and endothelial cell sprouting in vitro in Matrigel co-cultures. Finally, we examined whether macrophage-derived IGF-1 protects against NEC. We found that, neonatal intestinal CX3CR1+ macrophages were essential for small intestinal endothelial cells to sprout in Matrigel, which process was further enhanced by exogenous IGF-1 and inhibited by the selective IGF-1R inhibitor picropodophyllin (PPP). Intestinal endothelial cell proliferation was also inhibited by PPP in vivo, to a similar extent as observed during NEC development. Furthermore, neonatal IGF-1-deficient macrophages significantly decreased pro-angiogenic activity in vitro. Mice with IGF-1-deficient-macrophages had less robust intestinal endothelial proliferation and decreased intestinal VEGF protein expression in vivo. Lastly, pups with IGF-1-deficient macrophages had increased incidence of severe NEC-like intestinal injury and mortality. In conclusion, neonatal intestinal macrophages are critical for promoting intestinal microvascular growth and for protecting pups from NEC development via IGF-1 production.

Perivascular localization of macrophages in the intestinal mucosa is regulated by Nr4a1 and the microbiome

While the ontogeny and recruitment of the intestinal monocyte/macrophage lineage has been studied extensively, their precise localization and function has been overlooked. Here we show by imaging the murine small and large intestines in steady-state that intestinal CX3CR1+ macrophages form an interdigitated network intimately adherent to the entire mucosal lamina propria vasculature. The macrophages form contacts with each other, which are disrupted in the absence of microbiome, monocyte recruitment (Ccr2−/−), or monocyte conversion (Nr4a1−/−). In dysbiosis, gaps exist between the perivascular macrophages correlating with increased bacterial translocation from the lamina propria into the bloodstream. The recruitment of monocytes and conversion to macrophages during intestinal injury is also dependent upon CCR2, Nr4a1 and the microbiome. These findings demonstrate a relationship between microbiome and the maturation of lamina propria perivascular macrophages into a tight anatomical barrier that might function to prevent bacterial translocation. These cells are also critical for emergency vascular repair.

Sensing Fungal Dysbiosis by Gut-Resident CX3CR1+ Mononuclear Phagocytes Aggravates Allergic Airway Disease

Abstract Sensing of the gut microbiota, including fungi, regulates mucosal immunity. Whether fungal sensing in the gut can influence immunity at other body sites is unknown. Here we show that fluconazole-induced gut fungal dysbiosis has persistent effects on allergic airway disease in a house dust mite challenge model. Mice with a defined community of bacteria, but lacking intestinal fungi were not susceptible to fluconazole-induced dysbiosis, while colonization with a fungal mixture recapitulated the detrimental effects. Gut-resident mononuclear phagocytes (MNPs) expressing the fractalkine receptor CX3CR1 were essential for the effect of gut fungal dysbiosis on peripheral immunity. However, how mycobiota influence immunity in gut distal sites is not well understood. We developed protocols for gut-targeted depletion of phagocytes to investigate the influence of fungi on gut-lung crosstalk. Depletion of CX3CR1+ MNPs or selective inhibition of Syk signaling downstream of fungal sensing in these cells ameliorated lung allergy. These results indicate that disruption of intestinal fungal communities can mediate gut-lung-directed immune crosstalk and aggravate disease severity through fungal sensing by gut-resident CX3CR1+ MNPs. Highlights Gut fungal dysbiosis persistently aggravates allergic airway disease (AAD) in mice. Gut colonization by commensal fungi is both required and sufficient to aggravate AAD in a mycobiome-free mouse model. Intestinal CX3CR1+ mononuclear phagocytes (MNPs) are essential for the systemic effects of gut fungal dysbiosis on AAD. Inhibition of Syk-mediated fungal sensing in intestinal CX3CR1+ MNPs ameliorates AAD.

GPR31-dependent dendrite protrusion of intestinal CX3CR1+ cells by bacterial metabolites.

Small intestinal mononuclear cells that express CX3CR1 (CX3CR1+ cells) regulate immune responses1-5. CX3CR1+ cells take up luminal antigens by protruding their dendrites into the lumen1-4,6. However, it remains unclear how dendrite protrusion by CX3CR1+ cells is induced in the intestine. Here we show in mice that the bacterial metabolites pyruvic acid and lactic acid induce dendrite protrusion via GPR31 in CX3CR1+ cells. Mice that lack GPR31, which was highly and selectively expressed in intestinal CX3CR1+ cells, showed defective dendrite protrusions of CX3CR1+ cells in the small intestine. A methanol-soluble fraction of the small intestinal contents of specific-pathogen-free mice, but not germ-free mice, induced dendrite extension of intestinal CX3CR1+ cells in vitro. We purified a GPR31-activating fraction, and identified lactic acid. Both lactic acid and pyruvic acid induced dendrite extension of CX3CR1+ cells of wild-type mice, but not of Gpr31b-/- mice. Oral administration of lactate and pyruvate enhanced dendrite protrusion of CX3CR1+ cells in the small intestine of wild-type mice, but not in that of Gpr31b-/- mice. Furthermore, wild-type mice treated with lactate or pyruvate showed an enhanced immune response and high resistance to intestinal Salmonella infection. These findings demonstrate that lactate and pyruvate, which are produced in the intestinal lumen in a bacteria-dependent manner, contribute to enhanced immune responses by inducing GPR31-mediated dendrite protrusion of intestinal CX3CR1+ cells.

CX3CR1+ Macrophages and CD8+ T Cells Control Intestinal IgA Production.

Secretory IgA is a key host defense mechanism that controls the intestinal microbiota. We investigated the role of CD11c+CX3CR1+CD64+ macrophages in IgA production in the intestine. Intestinal CX3CR1+ macrophages directly induced IgA secretion by B cells. Ag delivery to lamina propria (LP) CX3CR1+ macrophages specifically induced intestinal IgA production. The induction of IgA by CX3CR1+ macrophages required BAFF, a proliferation-inducing ligand, and TNF-α, but was surprisingly independent of TLR-mediated microbial recognition and retinoic acid signaling. IgA secretion by CX3CR1+ macrophages was enhanced by LP CD8+ T cells through the secretion of IL-9 and IL-13. CX3CR1+ macrophages and CD8+ T cells induced IgA production by B cells independently of mesenteric lymph nodes and Peyer patches. Our data reveal a previously unrecognized cellular circuitry in which LP CX3CR1+ macrophages, B cells, and CD8+ T cells coordinate the protective Ig secretion in the small intestine upon peripheral Ag delivery.

Critical role for the microbiota in limiting inflammatory intestinal T cell responses

Abstract Efficient functioning of the intestine and intestinal immune system depends on interactions between the host and microbiota. Breakdown in these interactions leads to reduced ability to respond to intestinal pathogens along with increased intestinal inflammation which together can lead to pathologic inflammatory conditions such as inflammatory bowel disease. While a number of intestinal immune cells are likely regulated by the microbiota, we have found critical regulation of intestinal antigen presenting cells (APCs) expressing the chemokine receptor, CX3CR1. CX3CR1+ APCs are able to secrete both pro- and anti-inflammatory cytokines and are capable of mediating intestinal protection or pathology. Using in vivo models, we identified a regulatory loop whereby intestinal CX3CR1+ APCs reduced induction of Th1 cells against intestinal pathogens and promoted generation of regulatory T cells against soluble food antigens and the microbiota itself. This regulatory response depends on anti-inflammatory cytokine IL-10 production by CX3CR1+ APCs. Disruption of the intestinal microbiota switched the functional capacity of CX3CR1+ APCs so they now drove inflammatory Th1 responses and mediated pathology in a model of colitis. Mechanistically, we determined that epithelial attachment by intestinal microbes was required to turn on anti-inflammatory functions by CX3CR1+ APCs. Our results identify a cellular mechanism by which the microbiota limits intestinal inflammation and promotes homeostasis. Our study demonstrates the potential therapeutic benefits of microbiota and host immune cell manipulation with the goal of amplifying or inhibiting intestinal T cell responses.

Heme-oxygenase-1 Production by Intestinal CX3CR1+ Macrophages Helps to Resolve Inflammation and Prevents Carcinogenesis.

CX3CR1+ macrophages in the intestinal lamina propria contribute to gut homeostasis through the immunomodulatory interleukin IL10, but there is little knowledge on how these cells or the CX3CR1 receptor may affect colorectal carcinogenesis. In this study, we show that CX3CR1-deficient mice fail to resolve gut inflammation despite high production of IL10 and have increased colitis and adenomatous polyps in chemical and genetic models of colon carcinogenesis. Mechanistically, CX3CL1-mediated engagement of the CX3CR1 receptor induced upregulation of heme-oxygenase-1 (HMOX-1), an antioxidant and anti-inflammatory enzyme. CX3CR1-deficient mice exhibited significantly lower expression of HMOX-1 in their adenomatous colon tissues. Combining LPS and CX3CL1 displayed a strong synergistic effect in vitro, but HMOX-1 levels were significantly lower in KO macrophages. Cohousing of wild-type and CX3CR1-/- mice during the AOM/DSS treatment attenuated disease severity in CX3CR1-/- mice, indicating the importance of the microbiome, but did not fully reinstate HMOX-1 levels and did not abolish polyp formation. In contrast, pharmacologic induction of HMOX-1 in vivo by cobalt protoporphyrin-IX treatment eradicated intestinal inflammation and fully protected KO mice from carcinogenesis. Taken together, our results establish an essential role for the receptor CX3CR1 in gut macrophages in resolving inflammation in the intestine, where it helps protects against colitis-associated cancer by regulating HMOX-1 expression. Cancer Res; 77(16); 4472-85. ©2017 AACR.

CX3CR1+ Cell-Mediated Salmonella Exclusion Protects the Intestinal Mucosa during the Initial Stage of Infection.

During Salmonella Typhimurium infection, intestinal CX3CR1+ cells can either extend transepithelial cellular processes to sample luminal bacteria or, very early after infection, migrate into the intestinal lumen to capture bacteria. However, until now, the biological relevance of the intraluminal migration of CX3CR1+ cells remained to be determined. We addressed this by using a combination of mouse strains differing in their ability to carry out CX3CR1-mediated sampling and intraluminal migration. We observed that the number of S. Typhimurium traversing the epithelium did not differ between sampling-competent/migration-competent C57BL/6 and sampling-deficient/migration-competent BALB/c mice. In contrast, in sampling-deficient/migration-deficient CX3CR1-/- mice the numbers of S. Typhimurium penetrating the epithelium were significantly higher. However, in these mice the number of invading S. Typhimurium was significantly reduced after the adoptive transfer of CX3CR1+ cells directly into the intestinal lumen, consistent with intraluminal CX3CR1+ cells preventing S. Typhimurium from infecting the host. This interpretation was also supported by a higher bacterial fecal load in CX3CR1+/gfp compared with CX3CR1gfp/gfp mice following oral infection. Furthermore, by using real-time in vivo imaging we observed that CX3CR1+ cells migrated into the lumen moving through paracellular channels within the epithelium. Also, we reported that the absence of CX3CR1-mediated sampling did not affect Ab responses to a noninvasive S. Typhimurium strain that specifically targeted the CX3CR1-mediated entry route. These data showed that the rapidly deployed CX3CR1+ cell-based mechanism of immune exclusion is a defense mechanism against pathogens that complements the mucous and secretory IgA Ab-mediated system in the protection of intestinal mucosal surface.

Role of CX3CR1+ cell in the protection of the intestinal mucosa

During infection intestinal CX3CR1+ cells can either extend transepithelial cellular processes to sample luminal bacteria or, very early after infection migrate into the intestinal lumen to capture bacteria. However, up to date, the biological relevance of the intraluminal migration of CX3CR1+ cells remained to be determined. We addressed this by using a combination of mouse strains differing in their ability to carry out CX3CR1-mediated sampling and intraluminal migration. We observed that, the number of S. Typhimurium traversing the epithelium did not differ between sampling-competent/migration-competent C57BL/6 and sampling-deficient/migration-competent Balb/c mice. By contrast, in sampling-deficient/migration-deficient CX3CR1-/- mice the numbers of S. Typhimurium penetrating the epithelium were significantly higher. However, in these mice the number of invading S. Typhimurium was significantly reduced after the adoptive transfer of CX3CR1+ cells directly into the intestinal lumen, consistent with intraluminal CX3CR1+ cells preventing S. Typhimurium from infecting the host. This interpretation was also supported by a higher bacterial faecal load in CX3CR1+/gfp compared to CX3CR1gfp/gfp mice following oral infection. Furthermore, by using real time in vivo imaging we observed that CX3CR1+ cells migrated into the lumen moving through paracellular channels within the epithelium. Also, we reported that the absence of CX3CR1-mediated sampling did not affect antibody responses to a non-invasive S. Typhimurium strain that specifically targeted the CX3CR1-mediated entry route. These data showed that the rapidly deployed CX3CR1+ cell-based mechanism of immune-exclusion is a defence mechanism against pathogens that complements the mucous and secretory (s)IgA antibody-mediated system in the protection of intestinal mucosal surface.

Intestinal resident CX3CR1+ cells support gut immune system by helping production of IgA from B cells

Abstract Intestinal resident CX3CR1+ cells are known to sample neighboring or circulatory antigens and present them to CD8+ T cells via cross-presentation. Here we show that intestinal CX3CR1+ cells co-cultured with B cells can induce production of IgA. Unlike intestinal CD103+ DCs, CX3CR1+ cells are independent of retinoic acid and signals from commensal microbiota to induce production of IgA. CX3CR1+ cells express BAFF, APRIL and TNF-α which is required for IgA class-switching of B cells. Intravenous injection of soluble OVA in mice can promote production of IgG and IgA in the serum and feces. Intestinal CD103+ DCs are known to present to CD4+ T cells, however CX3CR1+cells present antigens to CD8+ T cells via cross-presentation. Addition of OT-I cells further enhances production of IgA of B cells co-cultured with CX3CR1+cells. Intestinal CX3CR1+cells are tissue resident cells and do not migrate to the MLN. CCR7 deficiency or FTY720 treatment does not affect the production of intestinal IgA after intravenous injection of OVA in mice. These results suggested that intestinal CX3CR1+cells have an important role in maintaining homeostasis of the intestinal immune system.

MicroRNA-223 Regulates the Differentiation and Function of Intestinal Dendritic Cells and Macrophages by Targeting C/EBPβ.

Dendritic cells (DCs) and macrophages play important roles in maintaining intestinal homeostasis. However, the molecular mechanisms that regulate the differentiation and responses of intestinal DCs and macrophages remain poorly understood. Here, we have identified microRNA miR-223 as a key molecule for regulating these processes. Deficiency of miR-223 led to a significantly decreased number of intestinal CX3CR1(hi) macrophages at steady state. Both intestinal CX3CR1(hi) macrophages and CD103(+) conventional DCs (cDCs) in miR-223-deficient mice exhibited a strong pro-inflammatory phenotype. Moreover, miR-223-deficient monocytes gave rise to more monocyte-derived DCs (moDCs) and produced more pro-inflammatory cytokines upon stimulation. Using a mouse model of colitis, we demonstrated that the miR-223 deficiency resulted in more severe colitis. Target gene analysis further identified that the effects of miR-223 on DCs and macrophages were mediated by directly targeting C/EBPβ. Taken together, our study identifies a role for miR-223 as a critical regulator of intestinal homeostasis.

Macrophage-Restricted Interleukin-10 Receptor Deficiency, but Not IL-10 Deficiency, Causes Severe Spontaneous Colitis

Interleukin-10 (IL-10) is a pleiotropic anti-inflammatory cytokine produced and sensed by most hematopoietic cells. Genome-wide association studies and experimental animal models point at a central role of the IL-10 axis in inflammatory bowel diseases. Here we investigated the importance of intestinal macrophage production of IL-10 and their IL-10 exposure, as well as the existence of an IL-10-based autocrine regulatory loop in the gut. Specifically, we generated mice harboring IL-10 or IL-10 receptor (IL-10Rα) mutations in intestinal lamina propria-resident chemokine receptor CX3CR1-expressing macrophages. We found macrophage-derived IL-10 dispensable for gut homeostasis and maintenance of colonic T regulatory cells. In contrast, loss of IL-10 receptor expression impaired the critical conditioning of these monocyte-derived macrophages and resulted in spontaneous development of severe colitis. Collectively, our results highlight IL-10 as a critical homeostatic macrophage-conditioning agent in the colon and define intestinal CX3CR1(hi) macrophages as a decisive factor that determines gut health or inflammation.

Mind the gap

Intestinal CX3CR1+ macrophages can transfer processed antigen to CD103+ DCs through gap junctions for the induction of oral tolerance.

Intestinal CX3CR1+ macrophages induce and maintain unique CD103+ regulatory T cells (P3206)

Abstract In the gut, there are tremendous antigens existed such as food proteins, commensal bacteria and even self-antigens. For this reason, maintenance of intestinal tolerance is important to regulate excessive inflammations. Recently, Foxp3+ Tregs are required to maintain intestinal tolerance. However, their phenotypes and functions in immune regulation are heterogeneous, thus it is required to find which subpopulations of Tregs in the gut suppress inflammatory cells effectively to prevent inflammatory diseases. Here we found that majority of the Tregs in the small intestinal lamina propria highly express CD103 molecule on their surface compared to Tregs in other tissues such as thymus, spleen, mesenteric lymph nodes and colon. We next investigated which antigen presenting cells from small intestinal lamina propria can induce CD103+ Tregs in vitro. We found that intestinal CX3CR1+ macrophages, which produce immunoregulatory cytokine IL-10 highly, induce CD103+ Tregs more efficiently, but not dendritic cells. These CD103+ Tregs had effect on reducing occurrence of diarrhea in food allergy model. Further, intestinal CX3CR1+ macrophages induced helios+ Tregs which have been recently suggested to be stable Tregs, under inflammatory stimulations. These findings suggest that CD103+ Tregs induced by intestinal CX3CR1+ macrophages raise the possibility of improvement in therapeutic potential of Tregs for inflammatory diseases such as graft versus host diseases and autoimmune diseases.

Intestinal dendritic cells survey circulatory antigens prior to induction of regulatory CD8α/β T cells (106.2)

Abstract Background: Antigens in the intestine transition from fenestrated capillaries through the lamina propria into the draining lymphatics, but whether or how this process controls mucosal immune responses is unclear. CX3CR1+ DCs are uniquely positioned for the recognition of environmental and circulatory antigens in the lamina propria as they interact with both the epithelium and with the capillary vessel system in the lamina propria. Results: We demonstrate that small intestinal CX3CR1+ dendritic cells (DCs) acquire and process both circulatory and luminal antigens in the lamina propria. Through cross-presentation of antigen, CX3CR1+ DCs induce differentiation of IL-10 secreting CD8α/β T regulatory cells, that is dependent upon Programmed Death ligand 1/2 and its counter-structure PD1 of CD8 T cells. Differentiated CD8+ T regulatory cells represent a subset of intraepithelial lymphocytes (IELs) which express the specific adhesion molecule CD103. The CD8+ Tregs inhibit antigen specific CD4+ T cell activation through IL-10 secretion and prevent CD45RBhigh CD4 T cell-mediated small intestinal inflammation. Conclusion: Our finding shows that the lamina propria CX3CR1+ DCs facilitate the surveillance of circulatory antigens and act as a conduit for the processing of self- and intestinally- absorbed antigens leading to the induction of CD8+T regulatory cells, which govern tolerance in the mucosal immune system.

The Heme Oxygenase System: Update 2005

The Heme Oxygenase System: Update 2005