Ccrl2-centred immune-related lncRNA-mRNA co-expression network revealed the local skin immune activation mechanism of moxibustion on adjuvant arthritis mice.

BACKGROUND: Moxibustion is an important external therapy of traditional medicine that operates on some acupoints on the skin and is usually used for immune-related diseases. However, whether the immune function of the skin, especially the immune-related lncRNAs, contributes to the mechanism of moxibustion remains unclear. METHODS: Adjuvant arthritis (AA) was induced by injection of Complete Freund's adjuvant (CFA) into the right hind paw of mice. Moxibustion was administered on the Zusanli (ST36) acupoint for 3 weeks. The alteration of foot volume and cytokine concentration in serum was used to evaluate the anti-inflammation effect of moxibustion. CD83 expression in the local skin of ST36 was measured by immunofluorescence staining. Transcriptome RNA sequencing (RNA-seq) and lncRNA-mRNA network analysis were performed to construct a moxibustion-induced Immune-related lncRNA-mRNA co-expression network. qRT-PCR was used to validate the RNA-seq data. RESULTS: Moxibustion at ST36 relieved the foot swelling, decreased the TNF-α and IL-1ß concentrations in serum, and obviously increased the CD83 expression at the local skin of ST36. A total of 548 differentially expressed lncRNAs and 520 linked mRNAs were screened out. The significantly and predominately enriched Go term was inflammatory and immune response, and the main pathways related to inflammatory and immune responses include Toll-like receptor, cytokine-cytokine receptor, and MAPK signaling. The immune-related lncRNA-mRNA co-expression network showed 88 lncRNAs and 36 mRNAs, and Ccrl2 is the central hub of this network. CONCLUSION: Local immune activation is significantly triggered by moxibustion in ST36 of AA mice. The Ccrl2-centered immune-related lncRNA-mRNA co-expression network would be a promising target for decoding the mechanism of moxibustion for immune-related diseases.

Vasoactive intestinal peptide promotes host defense against enteric pathogens by modulating the recruitment of group 3 innate lymphoid cells.

Group 3 innate lymphoid cells (ILC3s) control the formation of intestinal lymphoid tissues and play key roles in intestinal defense. They express neuropeptide vasoactive intestinal peptide (VIP) receptor 2 (VPAC2), through which VIP modulates their function, but whether VIP exerts other effects on ILC3 remains unclear. We show that VIP promotes ILC3 recruitment to the intestine through VPAC1 independent of the microbiota or adaptive immunity. VIP is also required for postnatal formation of lymphoid tissues as well as the maintenance of local populations of retinoic acid (RA)-producing dendritic cells, with RA up-regulating gut-homing receptor CCR9 expression by ILC3s. Correspondingly, mice deficient in VIP or VPAC1 suffer a paucity of intestinal ILC3s along with impaired production of the cytokine IL-22, rendering them highly susceptible to the enteric pathogen Citrobacter rodentium This heightened susceptibility to C. rodentium infection was ameliorated by RA supplementation, adoptive transfer of ILC3s, or by recombinant IL-22. Thus, VIP regulates the recruitment of intestinal ILC3s and formation of postnatal intestinal lymphoid tissues, offering protection against enteric pathogens.

High-Dose Neonatal Vitamin A Supplementation to Bangladeshi Infants Increases the Percentage of CCR9-Positive Treg Cells in Infants with Lower Birthweight in Early Infancy, and Decreases Plasma sCD14 Concentration and the Prevalence of Vitamin A Deficiency at Two Years of Age.

BACKGROUND: Vitamin A (VA) stores are low in early infancy and may impair development of the immune system. OBJECTIVE: This study determined if neonatal VA supplementation (VAS) affects the following: 1) development of regulatory T (Treg) cells; 2) chemokine receptor 9 (CCR9) expression, which directs mucosal targeting of immune cells; and 3) systemic endotoxin exposure as indicated by changed plasma concentrations of soluble CD14 (sCD14). Secondarily, VA status, growth, and systemic inflammation were investigated. METHODS: In total, 306 Bangladeshi infants were randomly assigned to receive 50,000 IU VA or placebo (PL) within 48 h of birth, and immune function was assessed at 6 wk, 15 wk, and 2 y. Primary outcomes included the following: 1) peripheral blood Treg cells; 2) percentage of Treg, T, and B cells expressing CCR9; and 3) plasma sCD14. Secondary outcomes included the following: 4) VA status measured using the modified relative dose-response (MRDR) test and plasma retinol; 5) infant growth; and 6) plasma C-reactive protein (CRP). Statistical analysis identified group differences and interactions with sex and birthweight. RESULTS: VAS increased (P = 0.004) the percentage of CCR9+ Treg cells (13.2 ± 1.37%) relative to PL (9.17 ± 1.15%) in children below the median birthweight but had the opposite effect (P = 0.04) in those with higher birthweight (VA, 9.13 ± 0.89; PL, 12.1 ± 1.31%) at 6 and 15 wk (values are combined mean ± SE). VAS decreased (P = 0.003) plasma sCD14 (1.56 ± 0.025 mg/L) relative to PL (1.67 ± 0.032 mg/L) and decreased (P = 0.034) the prevalence of VA deficiency (2.3%) relative to PL (9.2%) at 2 y. CONCLUSIONS: Neonatal VAS enhanced mucosal targeting of Treg cells in low-birthweight infants. The decreased systemic exposure to endotoxin and improved VA status at 2 y may have been due to VA-mediated improvements in gut development resulting in improved barrier function and nutrient absorption. This trial was registered at clinicaltrials.gov as NCT01583972 and NCT02027610.

Effects of supplementation with ß-carotene on the growth performance and intestinal mucosal barriers in layer-type cockerels.

ß-carotene is a robust modulator of mucosal barriers, and it can amplify the immunoglobulin A (IgA) response via the retinoic acid (RA)-mediated pathway. We investigated the influence of ß-carotene on intestinal barriers in layer-type cockerels. In this study, ß-carotene has a positive influence on growth performance and intestinal morphology. ß-carotene remarkably enhanced serum secretory immunoglobulin A (sIgA) levels, jejunal mucosal sIgA, and IgA concentrations. ß-Carotene significantly enhanced mRNA expression levels of IgA, CC chemokine receptor-9 (CCR9), polymeric immunoglobulin receptor (pIgR), and retinoic acid receptor α (RARα) in the ileal tissues and pIgR in the jejunal tissues. ß-Carotene improves mRNA expression of intestinal barrier-related proteins including: mucin-2 (MUC-2), zonula occludens-2 (ZO-2), occludins (OCLN), and zonula occludens-1 (ZO-1) in the ileal tissues. Moreover, ß-carotene decreased the levels of Escherichia coli and elevates the levels of Lactobacillus. The results indicate that ß-carotene can promote growth performance and contribute to the gradual development of intestinal barriers in Hyline Brown chicks. This study enriches our knowledge about the effects of ß-carotene on intestinal barrier and highlights a theoretical basis of ß-carotene application in the poultry industry.

Retinoic acid elicits a coordinated expression of gut homing markers on T lymphocytes of Zambian men receiving oral Vivotif, but not Rotarix, Dukoral or OPVERO vaccines.

All-trans retinoic acid (ATRA) up-regulates, in laboratory animals, the expression of the gut homing markers α4ß7 integrin and CCR9 on lymphocytes, increasing their gut tropism. Here, we show that, in healthy adult volunteers, ATRA induced an increase of these gut homing markers on T cells in vivo in a time dependent manner. The coordinated increase of α4ß7 and CCR9 by ATRA was seen in 57% (12/21) of volunteers and only when given together with an oral Vivotif vaccine. When this coordinated response to ATRA and Vivotif vaccine was present, it was strongly correlated with the gut immunoglobulin A (IgA) specific response to vaccine LPS (ρ = 0.82; P = 0.02). Using RNA-Seq analysis of whole blood transcription, patients receiving ATRA and Vivotif in conjunction showed transcriptomic changes in immune-related pathways, particularly including interferon α/ß signaling pathway, membrane-ECM interactions and immune hubs. These results suggest that exogenous ATRA can be used to manipulate responses to a subclass of oral vaccines, so far limited to a live attenuated Vivotif vaccine.

In-silico guided discovery of novel CCR9 antagonists.

Antagonism of CCR9 is a promising mechanism for treatment of inflammatory bowel disease, including ulcerative colitis and Crohn's disease. There is limited experimental data on CCR9 and its ligands, complicating efforts to identify new small molecule antagonists. We present here results of a successful virtual screening and rational hit-to-lead campaign that led to the discovery and initial optimization of novel CCR9 antagonists. This work uses a novel data fusion strategy to integrate the output of multiple computational tools, such as 2D similarity search, shape similarity, pharmacophore searching, and molecular docking, as well as the identification and incorporation of privileged chemokine fragments. The application of various ranking strategies, which combined consensus and parallel selection methods to achieve a balance of enrichment and novelty, resulted in 198 virtual screening hits in total, with an overall hit rate of 18%. Several hits were developed into early leads through targeted synthesis and purchase of analogs.

The atypical receptor CCRL2 is required for CXCR2-dependent neutrophil recruitment and tissue damage.

CCRL2 is a 7-transmembrane domain receptor that shares structural and functional similarities with the family of atypical chemokine receptors (ACKRs). CCRL2 is upregulated by inflammatory signals and, unlike other ACKRs, it is not a chemoattractant-scavenging receptor, does not activate ß-arrestins, and is widely expressed by many leukocyte subsets. Therefore, the biological role of CCRL2 in immunity is still unclear. We report that CCRL2-deficient mice have a defect in neutrophil recruitment and are protected in 2 models of inflammatory arthritis. In vitro, CCRL2 was found to constitutively form homodimers and heterodimers with CXCR2, a main neutrophil chemotactic receptor. By heterodimerization, CCRL2 could regulate membrane expression and promote CXCR2 functions, including the activation of ß2-integrins. Therefore, upregulation of CCRL2 observed under inflammatory conditions is functional to finely tune CXCR2-mediated neutrophil recruitment at sites of inflammation.

Lower levels of vitamin A are associated with increased gastrointestinal graft-versus-host disease in children.

Vitamin A promotes development of mucosal tolerance and enhances differentiation of regulatory T cells. Vitamin A deficiency impairs epithelial integrity, increasing intestinal permeability. We hypothesized that higher vitamin A levels would reduce the risk of graft-versus-host disease (GVHD) through reduced gastrointestinal (GI) permeability, reduced mucosal injury, and reduced lymphocyte homing to the gut. We tested this hypothesis in a cohort study of 114 consecutive patients undergoing allogeneic stem cell transplant. Free vitamin A levels were measured in plasma at day 30 posttransplant. GI GVHD was increased in patients with vitamin A levels below the median (38% vs 12.4% at 100 days, P = .0008), as was treatment-related mortality (17.7% vs 7.4% at 1 year, P = .03). Bloodstream infections were increased in patients with vitamin A levels below the median (24% vs 8% at 1 year, P = .03), supporting our hypothesis of increased intestinal permeability. The GI mucosal intestinal fatty acid-binding protein was decreased after transplant, confirming mucosal injury, but was not correlated with vitamin A levels, indicating that vitamin A did not protect against mucosal injury. Expression of the gut homing receptor CCR9 on T-effector memory cells 30 days after transplant was increased in children with vitamin A levels below the median (r = -0.34, P = .03). Taken together, these data support our hypothesis that low levels of vitamin A actively promote GI GVHD and are not simply a marker of poor nutritional status or a sicker patient. Vitamin A supplementation might improve transplant outcomes.

Biarylsulfonamide CCR9 inhibitors for inflammatory bowel disease.

Inflammatory bowel disease, including Crohn's disease and ulcerative colitis, affects millions of people worldwide. CCR9 has been shown to be a key chemokine receptor mediating the local inflammatory responses in the GI tract. The CCR9 inhibitor Vercirnon advanced to phase 3 clinical trials, but carries several liabilities which we sought to improve.

Glutamine supplementation attenuates expressions of adhesion molecules and chemokine receptors on T cells in a murine model of acute colitis.

BACKGROUND: Migration of T cells into the colon plays a major role in the pathogenesis in inflammatory bowel disease. This study investigated the effects of glutamine (Gln) supplementation on chemokine receptors and adhesion molecules expressed by T cells in mice with dextran sulfate sodium- (DSS-) induced colitis. METHODS: C57BL/6 mice were fed either a standard diet or a Gln diet replacing 25% of the total nitrogen. After being fed the diets for 5 days, half of the mice from both groups were given 1.5% DSS in drinking water to induce colitis. Mice were killed after 5 days of DSS exposure. RESULTS: DSS colitis resulted in higher expression levels of P-selectin glycoprotein ligand- (PSGL-) 1, leukocyte function-associated antigen- (LFA-) 1, and C-C chemokine receptor type 9 (CCR9) by T helper (Th) and cytotoxic T (Tc) cells, and mRNA levels of endothelial adhesion molecules in colons were upregulated. Gln supplementation decreased expressions of PSGL-1, LFA-1, and CCR9 by Th cells. Colonic gene expressions of endothelial adhesion molecules were also lower in Gln-colitis mice. Histological finding showed that colon infiltrating Th cells were less in the DSS group with Gln administration. CONCLUSIONS: Gln supplementation may ameliorate the inflammation of colitis possibly via suppression of T cell migration.

Enhancement of recombinant adenovirus vaccine-induced primary but not secondary systemic and mucosal immune responses by all-trans retinoic acid.

Vaccination is an important tool for enhancing immune responses against mucosal pathogens. Intramuscularly administered adenovirus (Ad) vectors have been demonstrated to be strong inducers of both systemic and mucosal immune responses. Further enhancement of immune responses following Ad vaccination is highly desirable. All-trans retinoic acid (ATRA), a biologically active vitamin A metabolite, has been explored as an adjuvant for primary immune responses following vaccination. In this study, we investigated the effect of ATRA on a heterologous Ad prime boost regimen. ATRA co-administration during priming increased mucosal and systemic antibody responses as well as mucosal but not systemic CD8(+) T cell responses. However, this effect was no longer apparent after boosting regardless of whether ATRA was administered at the time of priming, at the time of boosting, or at both immunizations. Our findings confirm ATRA as an adjuvant for primary immune responses and suggest that the adjuvant effect does not extend to secondary immune responses.

Quercetin suppresses MIP-1α-induced adipose inflammation by downregulating its receptors CCR1/CCR5 and inhibiting inflammatory signaling.

Obesity-induced inflammation is characterized by recruitment of adipose tissue macrophages that release inflammatory cytokines and chemokines. MIP-1α (macrophage inflammatory protein 1α)/CCL3, a CC chemokine, induces monocyte/macrophage infiltration and thus is implicated in obesity-induced adipose inflammation. Quercetin has been shown to modulate obesity-induced inflammation, but the mechanism of its action remains unclear. Here we demonstrate that quercetin decreases MIP-1α release from adipocytes and macrophages and from cocultured adipocytes/macrophages; it also opposes MIP-1α-induced macrophage infiltration and activation. The inhibitory action of quercetin on the MIP-1α-induced inflammatory responses of macrophages is mediated by downregulation of CCR1/CCR5, and inhibition of activation of JNK, p38 mitogen-activated-protein kinase (MAPK), and IKK as well as IκBα degradation. These findings suggest that quercetin may be a useful agent against obesity-induced adipose tissue inflammation.

High-dose vitamin D3 supplementation is a requisite for modulation of skin-homing markers on regulatory T cells in HIV-infected patients.

Vitamin D(3) is known to have an effect on the immune function. We investigated the immunomodulatory capability of vitamin D(3) in HIV-infected patients and studied the expression of chemokine receptors on regulatory T cells (Treg). Vitamin D(3)-deficient HIV-1-seropositive subjects were treated with cholecalciferol (vitamin D(3)) at a dose of 800 IU daily for 3 months (n=9) or 25,000 IU weekly for 2 months (n=7). Peripheral blood mononuclear cells (PBMCs) were isolated and analyzed for skin-homing (CCR4 and CCR10) and gut-homing (CCR9 and integrin α(4)ß(7)) marker expression on Treg, by flow cytometry, before and after supplementation. Serum 25(OH)D(3) and parathyroid hormone (PTH) levels were determined at baseline and after the treatment period. Weekly doses of 25,000 IU cholecalciferol effectively achieved the optimal target serum 25(OH)D(3) concentration of >75 nmol/liter (30 ng/ml) in HIV-infected patients. High-dose cholecalciferol supplementation differentially influenced skin-homing markers on Treg with an increased level of CCR10 expression and while a reduction in CCR4 expression level was observed together with a lower percentage of Treg expressing CCR4. For both dosing regimens, there were no significant differences in the expression of gut-homing markers, CCR9, and integrin α(4)ß(7). High-dose vitamin D(3) supplementation is needed to reverse vitamin D(3) deficiency in HIV-infected individuals and this results in modulation of skin-homing markers but not gut-homing markers expression on Treg. At a standard dose of 800 IU/day, vitamin D(3) is not effective in achieving an optimal 25(OH)D(3) concentration in patients with an underlying T cell dysfunction and is unable to exert any immunomodulatory effects.

Retinoic acid as a vaccine adjuvant enhances CD8+ T cell response and mucosal protection from viral challenge.

Vaccine-induced memory T cells localized at mucosal sites can provide rapid protection from viral infection. All-trans-retinoic acid (ATRA) has been shown to act physiologically to induce the expression of gut-homing receptors on lymphocytes. We tested whether the administration of exogenous ATRA during a systemic vaccination of mice could enhance the generation of mucosal CD8(+) T cell immunity, which might represent a strategy for establishing better protection from viral infection via mucosal routes. ATRA induced the expression of CCR9 and α4ß7 on both mouse and human CD8(+) T cells activated in vitro. The administration of ATRA to mice during in vivo priming with a replication-defective recombinant adenovirus vector expressing the lymphocytic choriomeningitis virus glycoprotein (LCMVgp) (Ad5gp) increased numbers of both effector and memory T cells in intestinal mucosal tissues and showed higher frequencies of systemic central memory-like T cells that exhibited enhanced proliferation during boosting immunization with recombinant modified vaccinia virus Ankara expressing LCMVgp (MVAgp). Mice that received ATRA during Ad5gp vaccination were more resistant to intravaginal challenge by recombinant vaccinia virus expressing LCMVgp (VVgp), reflecting in part stronger T cell recall responses in situ. Thus, ATRA appears to be useful as an adjuvant during vaccination to increase memory T cell responses and protection from viral infection at mucosal sites and may facilitate the development of more effective vaccines against mucosally transmitted pathogens such as HIV.

Specific killing of CCR9 high-expressing acute T lymphocytic leukemia cells by CCL25 fused with PE38 toxin.

We have previously demonstrated that CCR9 plays a pivotal role in drug resistance and invasion in human acute T-lymphocytic leukemia (T-ALL). In this study, we investigated whether the MOLT4 cells, which naturally express CCR9 at high levels, can be successfully killed by the specific ligand, CCL25 fused to Pseudomonas exotoxin 38 (PE38) toxin. Our results demonstrated that CCL25-PE38 was able to specifically kill MOLT4 cells via apoptosis induction, and suppress the growth of CCR9(+) tumors. This work shows that CCR9 high-expressing human T-ALL cells can be successfully killed by delivering PE38 toxin fused to the ligand CCL25.

Lemon grass (Cymbopogon citratus) ameliorates murine spontaneous ileitis by decreasing lymphocyte recruitment to the inflamed intestine.

OBJECTIVE: Aberrant leukocyte migration has been implicated in the pathogenesis of inflammatory bowel disease (IBD). Lemon grass is a natural herb that contains citral, which suppresses lymphocyte expression of gut homing molecules by inhibiting retinoic acid formation. We therefore hypothesized that lemon grass intake could ameliorate excess migration of leukocytes to the inflamed intestine in chronic ileitis. METHODS: Migration of fluorescence-labeled T cells to microvessels in the ileal mucosa of SAMP1/Yit mice was monitored using intravital microscopy. In some mice, lemon grass solution was administered for two weeks. For evaluation of the effects on chronic ileitis, mice were treated with lemon grass for 26 weeks. RESULTS: Surface expression of beta7 and CCR9 on T lymphocytes was stronger in SAMP1/Yit mice than in AKR/J mice. Lemon grass treatment attenuated the surface expression of beta7-integrin and CCR9. The number of adherent lymphocytes to microvessels in chronic inflamed ileum was significantly few when lymphocytes were isolated from lemon grass treated mice. Long-term lemon grass treatment improved ileitis in SAMP1/Yit mice, which was assessed by body weight, histological changes and the infiltration of beta7-positive cells. CONCLUSION: Lemon grass ameliorated ileitis through decreasing lymphocyte migration by inhibiting beta7-expression, suggesting its therapeutic usefulness for IBD.

In vitro induction of mucosa-type dendritic cells by all-trans retinoic acid.

Efficient induction of mucosal immunity usually employs nasal or oral vaccination while parenteral immunization generally is ineffective at generating mucosal immune responses. This relates to the unique ability of resident mucosal dendritic cells (DC) to induce IgA switching and to imprint mucosa-specific homing receptors on lymphocytes. Based on the well-established plasticity of the DC system, this study sought to investigate whether peripheral DC could be modulated toward "mucosa-type" DC by treatment with immunomodulatory, and therefore potentially adjuvant-like, factors. In this study, we show that monocyte-derived DCs pretreated with the vitamin A derivative all-trans retinoic acid (RA) indeed acquired several attributes characteristic of mucosal DC: secretion of TGF-beta and IL-6 and the capacity to augment mucosal homing receptor expression and IgA responses in cocultured lymphocytes. Addition of a TGF-beta-neutralizing Ab to cocultures significantly inhibited alpha4beta7 integrin, but not CCR9 mRNA expression by the lymphocytes. Both alpha4beta7 integrin and CCR9 mRNA expression, but not IgA production, were suppressed in the presence of a RA receptor antagonist. None of the observed effects on the lymphocytes were influenced by citral, a retinal dehydrogenase inhibitor, arguing against a role for de novo-synthesized RA. Collectively, our findings identified a novel role for RA as a mucosal immune modulator targeting DC. Our results further demonstrate that DC can act as efficient carriers of RA at least in vitro. Consequently, RA targeting of DC shows potential for promoting vaccine-induced mucosal immune responses via a parenteral route of immunization.

CCR9A and CCR9B: two receptors for the chemokine CCL25/TECK/Ck beta-15 that differ in their sensitivities to ligand.

We isolated cDNAs for a chemokine receptor-related protein having the database designation GPR-9-6. Two classes of cDNAs were identified from mRNAs that arose by alternative splicing and that encode receptors that we refer to as CCR9A and CCR9B. CCR9A is predicted to contain 12 additional amino acids at its N terminus as compared with CCR9B. Cells transfected with cDNAs for CCR9A and CCR9B responded to the chemokine CC chemokine ligand 25 (CCL25)/thymus-expressed chemokine (TECK)/chemokine beta-15 (CK beta-15) in assays for both calcium flux and chemotaxis. No other chemokines tested produced responses specific for the cDNA-transfected cells. mRNA for CCR9A/B is expressed predominantly in the thymus, coincident with the expression of CCL25, and highest expression for CCR9A/B among thymocyte subsets was found in CD4+CD8+ cells. mRNAs encoding the A and B forms of the receptor were expressed at a ratio of approximately 10:1 in immortalized T cell lines, in PBMC, and in diverse populations of thymocytes. The EC50 of CCL25 for CCR9A was lower than that for CCR9B, and CCR9A was desensitized by doses of CCL25 that failed to silence CCR9B. CCR9 is the first example of a chemokine receptor in which alternative mRNA splicing leads to proteins of differing activities, providing a mechanism for extending the range of concentrations over which a cell can respond to increments in the concentration of ligand. The study of CCR9A and CCR9B should enhance our understanding of the role of the chemokine system in T cell biology, particularly during the stages of thymocyte development.