CX3CR1 defines functionally distinct intestinal mononuclear phagocyte subsets which maintain their respective functions during homeostatic and inflammatory conditions

Intestinal mononuclear phagocytes (iMNP) are critically involved in mucosal immunity and tissue homeostasis. Two major non-overlapping populations of iMNP have been identified in mice. CD103(+) iMNP represent a migratory population capable of inducing tolerogenic responses, whereas CX3CR1(+) iMNP are resident cells with disease-promoting potential. CX3CR1(+) iMNP can further be subdivided based on differential expression of CX3CR1. Using CX3CR1(GFP/+) ×RAG2(-/-) mice, we demonstrate that CX3CR1(hi) and CX3CR1(lo) iMNP clearly differ with respect to their morphological and functional properties. Compared with CX3CR1(hi) iMNP, CX3CR1(lo) iMNP are polarised towards pro-inflammatory responses already under homeostatic conditions. During a CD4(+) T-cell-induced colitis, CX3CR1(lo) cells accumulate in the inflamed mucosa and upregulate the expression of pro-inflammatory cytokines and triggering receptor expressed on myeloid cells-1 (TREM-1). In contrast, CX3CR1(hi) iMNP retain their non-inflammatory profile even during intestinal inflammation. These findings identify two functionally distinct iMNP subsets based on differential expression of CX3CR1 and indicate an unanticipated stability of iMNP.     

CX3CR1-deficiency is associated with increased severity of disease in experimental autoimmune uveitis

The role of CX3CR1 in regulating the function of monocytes and microglia was examined in mice in which CX3CR1 had been replaced by green fluorescent protein (GFP). Induction of experimental autoimmune uveitis (EAU) in these mice resulted in increased disease severity at day 23 postimmunization with uveitogenic peptide when compared with CX3CR1-positive mice and increased apoptosis of neuronal cells in the inner nuclear layer. Resident microglia within the retina were activated equally as EAU developed in mice with or without CX3CR1, as determined by changes in morphology, suggesting that the microglial cell response did not account for the differences. Although the inflammatory infiltrate had increased in mice without CX3CR1 at day 23 postimmunization, the percentage of natural killer cells in the infiltrate was not changed in these mice. Similarly, increased disease severity at this stage was not associated with an overall increased percentage of macrophages in the retinal inflammatory infiltrate or in increased activation of these cells. The increased recruitment of monocytes to the retina in response to EAU induction in CX3CR1^GFP/GFP mice compared with CX3CR1^GFP/+ mice was not reflected in increased migration away from vessels, leading to marked clustering of GFP⁺ cells around veins and venules in these mice. It is possible that this monocyte/macrophage clustering leads to the increased severity of disease seen in the mice by focusing and so intensifying the inflammatory response.     

Interleukin-17/interleukin-17 receptor axis elicits intestinal neutrophil migration,restrains gut dysbiosis and lipopolysaccharide translocation in high-fat diet-induced metabolic syndrome model

Sound evidence supports a role for interleukin-17 (IL-17) -producing γδ T cells and IL-17-producing helper T (Th17) cells in intestinal homeostasis, especially in intestinal barrier integrity. In the present study, we aimed to evaluate the role of IL-17 cytokine in the regulation of intestinal immunity and obesity-induced metabolic syndrome (MetS) in an experimental murine model. C57BL/6 wild-type (WT) mice and mice lacking the IL-17 cytokine receptor (IL-17RA^−/−) were fed either a control diet (CD) or a high-fat diet (HFD) for 9 weeks. Our data demonstrate that IL-17RA^−/− mice are protected against obesity, but develop hyperglycemia, hyperinsulinemia and insulin resistance. In parallel, HFD-fed IL-17RA^−/− mice display intense inflammation in the ileum compared with WT mice on the HFD. IL-17RA^−/− mice fed the HFD exhibit impaired neutrophil migration to the intestinal mucosa and reduced gene expression of the CXCL-1 chemokine and CXCR-2 receptor in the ileum. Interestingly, the populations of neutrophils (CD11b⁺ Ly6G⁺) and anti-inflammatory macrophages (CD11b⁺ CX3CR1⁺) are increased in the mesenteric lymph nodes of these mice. IL-17RA^−/− mice on the HFD also display increased commensal bacterial translocation into the bloodstream and elevated lipopolysaccharide (LPS) levels in the visceral adipose tissue (VAT). Metagenomic analysis of bacterial 16S gene revealed increased Proteobacteria and Bacteroidetes phyla, the main representatives of Gram-negative bacteria, and reduced Akkermansia muciniphila in the fecal samples of IL-17RA^−/− mice fed the HFD. Together, these data indicate that the IL-17/IL-17R axis drives intestinal neutrophil migration, limits gut dysbiosis and attenuates LPS translocation to VAT, resulting in protection to MetS.     

The impact of chemokine receptor CX3CR1 deficiency during respiratory infections with Mycobacterium tuberculosis or Francisella tularensis

Recruitment of immune cells to infection sites is a critical component of the host response to pathogens. This process is facilitated partly through interactions of chemokines with cognate receptors. Here, we examine the importance of fractalkine (CX3CL1) receptor, CX3CR1, which regulates function and trafficking of macrophages and dendritic cells, in the host''s ability to control respiratory infections with Mycobacterium tuberculosis or Francisella tularensis. Following low-dose aerosol challenge with M. tuberculosis, CX3CR1^−/− mice were no more susceptible to infection than wild-type C57BL/6 mice as measured by organ burden and survival time. Similarly, following inhalation of F. tularensis, CX3CR1^−/− mice displayed similar organ burdens to wild-type mice. CX3CR1^−/− mice had increased recruitment of monocytes and neutrophils in the lung; however, this did not result in increased abundance of infected monocytes or neutrophils. We conclude that CX3CR1-deficiency affects immune-cell recruitment; however, loss of CX3CR1 alone does not render the host more susceptible to M. tuberculosis or F. tularensis.     

CX3CL1 up-regulation is associated with recruitment of CX3CR1+ mononuclear phagocytes and T lymphocytes in the lungs during cigarette smoke-induced emphysema

CX3CR1 is expressed on monocytes, dendritic cells, macrophages, subsets of T lymphocytes, and natural killer cells and functions in diverse capacities such as leukocyte adhesion, migration, and cell survival on ligand binding. Expression of the CX3CL1 gene, whose expression product is the sole ligand for CX3CR1, is up-regulated in human lungs with chronic cigarette smoke-induced obstructive lung disease. At present, it is unknown whether CX3CL1 up-regulation is associated with the recruitment and accumulation of immune cells that express CX3CR1. We show that mice chronically exposed to cigarette smoke up-regulate CX3CL1 gene expression, which is associated with an influx of CX3CR1⁺ cells in the lungs. The increase in CX3CR1⁺ cells is primarily comprised of macrophages and T lymphocytes and is associated with the development of emphysema. In alveolar macrophages, cigarette smoke exposure increased the expression of both CX3CR1 and CX3CL1 genes. The inducibility of CX3CR1 expression was not solely dependent on a chronic stimulus because lipopolysaccharide up-regulated CX3CR1 in RAW264.7 cells in vitro and in mononuclear phagocytes in vivo. Our findings suggest a mechanism by which macrophages amplify and promote CX3CR1⁺ cell accumulation within the lungs during both acute and chronic inflammatory stress. We suggest that one function of the CX3CR1-CX3CL1 pathway is to recruit and sustain divergent immune cell populations implicated in the pathogenesis of cigarette smoke-induced emphysema.     

Resident and pro-inflammatory macrophages in the colon represent alternative context-dependent fates of the same Ly6Chi monocyte precursors

Macrophages (mϕ) are essential for intestinal homeostasis and the pathology of inflammatory bowel disease (IBD), but it is unclear whether discrete mϕ populations carry out these distinct functions or if resident mϕ change during inflammation. We show here that most resident mϕ in resting mouse colon express very high levels of CX3CR1, are avidly phagocytic and MHCII^hi, but are resistant to Toll-like receptor (TLR) stimulation, produce interleukin 10 constitutively, and express CD163 and CD206. A smaller population of CX3CR1^int cells is present in resting colon and it expands during experimental colitis. Ly6C^hiCCR2⁺ monocytes can give rise to all mϕ subsets in both healthy and inflamed colon and we show that the CX3CR1^int pool represents a continuum in which newly arrived, recently divided monocytes develop into resident CX3CR1^hi mϕ. This process is arrested during experimental colitis, resulting in the accumulation of TLR-responsive pro-inflammatory mϕ. Phenotypic analysis of human intestinal mϕ indicates that analogous processes occur in the normal and Crohn’s disease ileum. These studies show for the first time that resident and inflammatory mϕ in the intestine represent alternative differentiation outcomes of the same precursor and targeting these events could offer routes for therapeutic intervention in IBD.     

Inflammasome activation has an important role in the development of spontaneous colitis

Inflammatory bowel disease (IBD) is characterized for dysregulated intestinal inflammation. Conflicting reports have shown that activation of inflammasome could promote or decrease intestinal inflammation in an acute colitis model, whereas the involvement of inflammasome activation in chronic colitis is poorly understood. In this study, we investigated the role of inflammasome activation in the development of chronic intestinal inflammation by utilizing interleukin-10 (IL-10) knockout (KO) mouse as an animal model, which develops chronic colitis resembling human IBD. We demonstrate the causative link between inflammasome activation and the development of chronic intestinal inflammation. Our results show that mature IL-1β protein levels were significantly increased in all colon sections from IL-10-deficient mice compared with that of wild-type mice. We found that inhibition of inflammasome activities with IL-1 receptor antagonist or caspase-1 inhibitors suppressed IL-1β and IL-17 production from inflamed colon explants. Furthermore, blocking inflammasome activation with caspase-1 inhibitor in vivo significantly ameliorated the spontaneous colitis in IL-10 KO mice. Taken together, these observations demonstrate that inflammasome activation promotes the development of chronic intestinal inflammation.     

Differential expression of the chemokine receptors CX3CR1 and CCR1 by microglia and macrophages in myelin-oligodendrocyte-glycoprotein-induced experimental autoimmune encephalomyelitis

Chemokines are important for the recruitment of immune cells into sites of inflammation. To better understand their functional roles during inflammation we have here studied the in vivo expression of receptors for the chemokines CCL3/CCL5/CCL7 (MIP-1alpha/RANTES/MCP-3) and CX3CL1 (fractalkine), CCR1 and CX3CR1, respectively, in rat myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis. Combined in situ hybridization and immunohistochemistry demonstrated intensely upregulated CCR1 mRNA expression in early, actively demyelinating plaques, whereas CX3CR1 displayed a more generalized expression pattern. CX3CR1 mRNA expressing cells were identified as microglia on the basis of their cellular morphology and positive GSA/B4 lectin staining. In contrast, CCR1 mRNA was preferentially expressed by ED1+ GSA/B4+ macrophages. The notion of differential chemokine receptor expression in microglia and monocyte-derived macrophages was corroborated at the protein level by extraction and flow cytometric sorting of cells infiltrating the spinal cord using gating for the surface markers CD45, ED-2 and CD11b. These observations suggest a differential receptor expression between microglia and monocyte-derived macrophages and that mainly the latter cell type is responsible for active demyelination. This has great relevance for the possibility of therapeutic intervention in demyelinating diseases such as multiple sclerosis, for example by targeting signaling events leading to monocyte recruitment.     

CX3CL1 expression in the conjunctiva is involved in immune cell trafficking during toxic ocular surface inflammation

Inappropriate expression of the chemokine CX3CL1 is reportedly known to act on inflammatory conditions in extraocular immune diseases. We studied the expression and effects of CX3CL1 in human patients, cultured human conjunctival cells, and transgenic mice exposed to benzalkonium chloride (BAC), a commonly used preservative in ophthalmic medications despite its proinflammatory properties, to determine whether CX3CL1 is involved in conjunctival inflammation. We report that CX3CL1 expression is increased in the conjunctiva of patients receiving BAC-containing medication, and correlates with clinical inflammation. BAC enhances the production of CX3CL1 in a conjunctival epithelial cell line, through the tumor-necrosis factor-α pathway, which attracts specific leukocyte subsets. In vivo, BAC-induced macrophage infiltration and subsequent inflammation of the conjunctiva is decreased in CX3CR1-deficient mice as compared with CX3CR1^+/+ controls. This translational study opens new avenue to investigate ocular surface disorders by focusing on chemokine-related inflammation and immune cell trafficking in the ocular conjunctival mucosa.     

Human intestinal pro-inflammatory CD11chighCCR2+CX3CR1+ macrophages,but not their tolerogenic CD11c−CCR2−CX3CR1− counterparts,are expanded in inflammatory bowel disease

Although macrophages (Mϕ) maintain intestinal immune homoeostasis, there is not much available information about their subset composition, phenotype and function in the human setting. Human intestinal Mϕ (CD45⁺HLA-DR⁺CD14⁺CD64⁺) can be divided into subsets based on the expression of CD11c, CCR2 and CX3CR1. Monocyte-like cells can be identified as CD11c^highCCR2⁺CX3CR1⁺ cells, a phenotype also shared by circulating CD14⁺ monocytes. On the contrary, their Mϕ-like tissue-resident counterparts display a CD11c⁻CCR2⁻CX3CR1⁻ phenotype. CD11c^high monocyte-like cells produced IL-1β, both in resting conditions and after LPS stimulation, while CD11c⁻ Mϕ-like cells produced IL-10. CD11c^high pro-inflammatory monocyte-like cells, but not the others, were increased in the inflamed colon from patients with inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis. Tolerogenic IL-10-producing CD11c⁻ Mϕ-like cells were generated from monocytes following mucosal conditioning. Finally, the colonic mucosa recruited circulating CD14⁺ monocytes in a CCR2-dependent manner, being such capacity expanded in IBD. Mϕ subsets represent, therefore, transition stages from newly arrived pro-inflammatory monocyte-like cells (CD11c^highCCR2⁺CX3CR1⁺) into tolerogenic tissue-resident (CD11c⁻CCR2⁻CX3CR1⁻) Mϕ-like cells as reflected by the mucosal capacity to recruit circulating monocytes and induce CD11c⁻ Mϕ. The process is nevertheless dysregulated in IBD, where there is an increased migration and accumulation of pro-inflammatory CD11c^high monocyte-like cells.     

Recruitment of CD16+ monocytes into synovial tissues is mediated by fractalkine and CX3CR1 in rheumatoid arthritis patients

CD16+ monocytes, identified as a minor population of monocytes in human peripheral blood, have been implicated in several inflammatory diseases, including rheumatoid arthritis (RA). Fractalkine (FKN, CX3CL1), a member of the CX3 C subfamily, is induced by pro-inflammatory cytokines, while a receptor for FKN, CX3CR1, is capable of mediating both leukocyte migration and firm adhesion. Here, we investigated the role of FKN and CX3CR1 in activation of CD16+ monocytes and their recruitment into synovial tissues in RA patients. High levels of soluble FKN were detected in the synovial fluid and sera of RA patients. Circulating CD16+ monocytes showed a higher level of CX3CR1 expression than CD16- monocytes in both RA patients and healthy subjects. High level expression of CX3CR1 was also seen in CD16+ monocytes localized to the lining layer in RA synovial tissue. In the in vitro culture experiments, IL-10 induced CX3CR1 expression on the surface of monocytes, and TNFalpha induced membrane-bound FKN as well as soluble FKN expression in synovial fibroblasts. Moreover, soluble FKN was capable of inducing IL-1beta and IL-6 by activated monocytes. These results suggest that FKN might preferentially mediate migration and recruitment of CD16+ monocytes, and might contribute to synovial tissue inflammation.     

Development of intestinal inflammation in double IL-10- and leptin-deficient mice

Leptin-deficient (ob/ob) mice are resistant in different models of autoimmunity and inflammation, suggesting that leptin regulates immunity and inflammation. To investigate whether leptin deficiency modulates the spontaneous intestinal inflammation observed in interleukin (IL)-10-deficient mice, double IL-10- and leptin-deficient [IL-10 knockout (KO) ob/ob] mice were generated and compared with single IL-10 KO mice for colitis severity. Body weight in IL-10 KO ob/ob mice was significantly reduced compared with that of ob/ob mice. However, when compared with wild-type or IL-10 KO mice, IL-10 KO ob/ob mice were still markedly obese. IL-10 KO and IL-10 KO ob/ob mice developed colitis with a comparable time-course and severity in terms of macroscopic and histologic scores. Likewise, production of interferon-gamma, IL-6, and IL-13 from colon cultures and splenocytes did not differ among these two groups. Conversely, rates of apoptosis were higher in lamina propria lymphocytes obtained from the colon of IL-10 KO ob/ob compared with IL-10 KO mice. In conclusion, although leptin deficiency has been associated with resistance in models of autoimmunity and inflammation induced by exogenous stimuli, leptin appears not to play a significant role in the spontaneous colitis of IL-10 KO mice, although it modulates survival of intestinal lymphocytes.     

Macrophages are critical to the maintenance of IL-13-dependent lung inflammation and fibrosis

The roles of macrophages in type 2-driven inflammation and fibrosis remain unclear. Here, using CD11b-diphtheria toxin receptor (DTR) transgenic mice and three models of interleukin 13 (IL-13)-dependent inflammation, fibrosis, and immunity, we show that CD11b⁺ F4/80⁺ Ly6C⁺ macrophages are required for the maintenance of type 2 immunity within affected tissues but not secondary lymphoid organs. Direct depletion of macrophages during the maintenance or resolution phases of secondary Schistosoma mansoni egg-induced granuloma formation caused a profound decrease in inflammation, fibrosis, and type 2 gene expression. Additional studies with CD11c-DTR and CD11b/CD11c-DTR double-transgenic mice suggested that macrophages but not dendritic cells were critical. Mechanistically, macrophage depletion impaired effector CD4⁺ T helper type 2 (Th2) cell homing and activation within the inflamed lung. Depletion of CD11b⁺ F4/80⁺ Ly6C⁺ macrophages similarly reduced house dust mite-induced allergic lung inflammation and suppressed IL-13-dependent immunity to the nematode parasite Nippostrongylus brasiliensis. Consequently, therapeutic strategies targeting macrophages offer a novel approach to ameliorate established type 2 inflammatory diseases.     

Interleukin-34 accelerates intrauterine adhesions progress related to CX3CR1+ monocytes/macrophages

Intrauterine adhesions (IUA) are characterized by endometrial fibrosis and impose a great challenge for female reproduction. IL-34 is profoundly involved in various fibrotic diseases through regulating the survival, proliferation, and differentiation of monocytes/macrophages. However, it remains unclear how IL-34 regulates monocytes/macrophages in context of IUA. Here, we showed that the expression level of IL-34 and the amount of CX3CR1+ monocytes/macrophages were significantly increased in endometrial tissues of IUA patients. IL-34 promoted the differentiation of monocytes/macrophages, which express CX3CR1 via CSF-1R/P13K/Akt pathway in vitro. Moreover, IL-34-induced CX3CR1+ monocytes/macrophages promoted the differentiation of endometrial stromal cells into myofibroblasts. Of note, IL-34 caused endometrial fibrosis and increased the amount of CX3CR1+ monocytes/macrophages in endometrial tissues in vivo. IL-34 modulated endometrial fibrosis by regulating monocytes/macrophages since the elimination of endometrial monocytes/macrophages significantly suppressed the profibrotic function of IL-34. Finally, blocking of IL-34 in the LPS-IUA model resulted in the improvement of endometrial fibrosis and decreased number of CX3CR1+ monocytes/macrophages. Our studies uncover the novel mechanism of interaction between IL-34-induced CX3CR1+ monocytes/macrophages and endometrial stromal cells in endometrial fibrosis pathogenesis, and highlight IL-34 as a critical target for treating IUA.     

CD226 deficiency exacerbated intestinal immune dysregulation in mice with dinitrochlorobenzene-induced atopic dermatitis

Intestinal mucosal immunity plays a pivotal role in host defence. In this study, we found that cluster of differentiation 226 (CD226) gene knockout (KO) led to more severe atopic dermatitis (AD)-related skin pathologies and bowel abnormalities in a 2,4-dinitrochlorobenzene (DNCB)-induced AD-like mouse model. Following DNCB administration, the expression of CD226 was elevated in intestinal mucosal tissues, including group 3 innate lymphoid cells (ILC3s) and CD4⁺ T cells of Peyer's patches (PPs). CD226 deficiency led to an overactive intestinal immune response in the AD-like mice, as evidenced by increased inflammation and Th1/Th2-related cytokine levels as well as increased Paneth cell numbers and antimicrobial peptide (AMP) expression, which was likely due to the higher interleukin (IL)-22 production in the lamina propria. Additionally, CD226 deficiency increased the production of IL-4 and IL-17 in mesenteric lymph nodes as well as the number of PPs and expression of immunoglobulin (Ig) A in B cells. Moreover, insufficient expression of CD226 affected the characterization of intraepithelial and lamina propria lymphocytes in the intestinal mucosa. Finally, the number of PPs was increased in CD4⁺ T cell-specific CD226 KO and regulatory T (Treg) cell-specific CD226 KO mice; thus, loss of CD226 in Treg cells resulted in impaired Treg cell-suppressive function. Therefore, our findings indicate that CD226 deficiency alters intestinal immune functionality in inflammatory diseases.     

Neither Dectin-2 nor the Mannose Receptor Is Required for Resistance to Coccidioides immitis in Mice

We investigated the roles of the mannose receptor (MR) and Dectin-2 in resistance to pulmonary coccidioidomycosis in C57BL/6 (B6) mice and in the interaction of myeloid cells with spherules, using B6 mice with targeted mutations in Mrc1 and Clec4n. Spherules are the tissue form of Coccidioides, and we determined that the MR on bone marrow-derived dendritic cells (BMDC) was important for recognition of spherules (formalin-killed spherules [FKS]) and for secretion of interleukin 10 (IL-10) and proinflammatory cytokines in response to FKS by both elicited macrophages and BMDC. Infected MR knockout (KO) mice produced more IL-10 in their lungs than did B6 mice, and MR KO mice also made more protective Th-17 cytokines. In contrast to the MR, Dectin-2 was not required for recognition of FKS by BMDC or for the production of cytokines by BMDC in response to FKS. However, Dectin-2 KO was required for stimulation of elicited peritoneal macrophages. Despite that, lung cytokine levels were not significantly different in Dectin-2 KO mice and B6 mice 14 days after infection, except for IL-1β, which was higher in Dectin-2 KO lungs. Although both Dectin-2^−/− and MR^−/− myeloid cells had reduced proinflammatory cytokine responses to FKS in vitro, neither MR nor Dectin-2 deficiency reduced the resistance of B6 mice to pulmonary coccidioidomycosis.     

Vitamin D and the vitamin D receptor are critical for control of the innate immune response to colonic injury

Background

The active form of vitamin D (1,25(OH)₂D₃) has been shown to inhibit development of inflammatory bowel disease (IBD) in IL-10 KO mice. Here, the role of the vitamin D receptor (VDR) and 1,25(OH)₂D₃in acute experimental IBD was probed.

Results

VDR KO mice were extremely sensitive to dextran sodium sulfate (DSS) and there was increased mortality of the VDR KO mice at doses of DSS that only caused a mild form of colitis in wildtype (WT) mice. DSS colitis in the VDR KO mice was accompanied by high colonic expression of TNF-α, IL-1 α, IL-1β, IL-12, IFN-γ, IL-10, MIP-1α and KC. DSS concentrations as low as 0.5% were enough to induce bleeding, ulceration and weight loss in VDR KO mice. VDR KO mice failed to recover following the removal of DSS, while WT mice showed signs of recovery within 5 days of DSS removal. The early mortality of DSS treated VDR KO mice was likely due to perforation of the bowel and resulting endotoxemia. VDR KO mice were hyper-responsive to exogenously injected LPS and cultures of the peritoneal exudates of moribund DSS treated VDR KO mice were positive for bacterial growth. 1,25(OH)₂D₃in the diet or rectally decreased the severity and extent of DSS-induced inflammation in WT mice.

Conclusion

The data point to a critical role for the VDR and 1,25(OH)₂D₃in control of innate immunity and the response of the colon to chemical injury.