Goblet cell-produced retinoic acid suppresses CD86 expression and IL-12 production in bone marrow-derived cells.
ABSTRACT: Conjunctival goblet cell loss in ocular surface diseases is accompanied by increased number of interleukin-12 (IL-12)-producing antigen-presenting cells (APCs) and increased interferon-? (IFN-?) expression. This study tested the hypothesis that mouse conjunctival goblet cells produce biologically active retinoic acid (RA) that suppresses CD86 expression and IL-12 production by myeloid cells. We found that conditioned media from cultured conjunctival goblet cells (CjCM) suppressed stimulated CD86 expression, NF-?B p65 activation and IL-12 and IFN-? production in unstimulated and lipopolysaccharide-stimulated cultured bone marrow-derived cells (BMDCs) containing a mixed population of APCs. Goblet cell-conditioned, ovalbumin-loaded APCs suppressed IFN-? production and increased IL-13 production in co-cultured OTII cells. The goblet cell suppressive activity is due in part to their ability to synthesize RA from retinol. Conjunctival goblet cells had greater expression of aldehyde dehydrogenases Aldh1a1 and a3 and ALDEFLUOR activity than cornea epithelium lacking goblet cells. The conditioning activity was lost in goblet cells treated with an ALDH inhibitor, and a retinoid receptor alpha antagonist blocked the suppressive effects of CjCM on IL-12 production. Similar to RA, CjCM increased expression of suppressor of cytokine signaling 3 (SOCS3) in BMDCs. SOCS3 silencing reversed the IL-12-suppressive effects of CjCM. Our findings indicate that conjunctival goblet cells are capable of synthesizing RA from retinol secreted by the lacrimal gland into tears that can condition APCs. Evidence suggests goblet cell RA may function in maintaining conjunctival immune tolerance and loss of conjunctival goblet cells may contribute to increased Th1 priming in dry eye.
Project description:Intestinal epithelial cells condition tolerogenic properties in DCs. Aqueous-deficient dry eye is associated with goblet cell (GC) loss and increased IFN-? expression in the conjunctiva. We hypothesized that loss of GCs reduces tolerance-inducing properties of antigen presenting cells (APCs) in the conjunctiva and draining nodes. Mice lacking the SAM pointed domain containing ETS transcription factor (Spdef) that is required for GC differentiation had an increased frequency of macrophages in the conjunctiva and CD11b+CD11c+ DCs in the conjunctiva and draining nodes, and these cells had greater IL-12 expression than WT mice. Conditioned media from cultured WT conjunctival GCs suppressed LPS-induced IL-12 production by conjunctival APCs. OVA antigen-specific OTII CD4+ T cells primed by Spdef-KO draining lymph node APCs showed greater proliferation, lower frequency of Foxp3+, increased frequency of IFN-?+ and IL-17+ cells, and greater IFN-? production than those primed by WT APCs. The immune tolerance to OVA antigen topically applied to the conjunctiva measured by cutaneous delayed type hypersensitivity (DTH) reaction, OVA-specific T cell proliferation, Foxp3 induction, and IFN-? production observed in WT mice was lost in the Spdef-KO mice. We concluded that conjunctival GCs condition tolerogenic properties in APCs that suppress IL-12 production and Th1 polarization.
Project description:This study investigated the relationship between clinical severity and percentage of conjunctival antigen-presenting cells (APCs) in Sjögren's syndrome (SS)-associated keratoconjunctivitis sicca (KCS). KCS clinical severity was based on symptom severity, tear volume, tear break-up time, and ocular surface dye staining. Conjunctival goblet cell density (GCD) was measured in periodic acid Schiff (PAS)-stained membranes. Conjunctival cells obtained by impression cytology were used for flow cytometry to measure percentages of CD45?HLA-DR? APCs and mature CD11c?CD86? dendritic cells (DCs). Compared to normal conjunctiva, the percentages of HLA-DR? and CD11c?CD86? cells were higher in the conjunctiva of the KCS group (p < 0.05). The percentage of CD45?HLA-DR? cells positively correlated with clinical severity (r = 0.71, p < 0.05) and negatively correlated with GCD (r = -0.61, p < 0.05). Clinical severity also negatively correlated with GCD (r = -0.54, p < 0.05). These findings indicate that a higher percentage of APCs and mature DCs in the conjunctiva is associated with more severe KCS in SS. These APCs may contribute to the generation of the pathogenic Th1 cells that cause goblet cell loss in KCS.
Project description:Purpose: To investigate the mechanism for developing dry eye disease in the Pinkie mouse strain with a loss of function RXR mutation. Methods: Measures of dry eye disease were assessed in the cornea and conjunctiva. Expression profiling by single-cell RNA sequencing (scRNA-seq)was performed to compare gene expression in conjunctival immune cells. Conjunctival immune cells were immunophenotyped by flow cytometry and confocal microscopy. Activity of RXR ligand 9-cis retinoic acid (RA) was evaluated in cultured monocytes and T cells. Results: Compared to wild type (WT) C57BL/6, Pinkie has increased signs of dry eye disease, including corneal barrier disruption, conjunctival cornification and goblet cell loss, and corneal vascularization, opacification, and ulceration with aging. scRNA-seq of conjunctival immune cells identified T cells as the predominant IL-17 expressing population in both strains and there is a 4-fold increased percentage of T cells in Pinkie. Compared to WT, significantly increased expression of IL-17a and IL-17f in conventional T cells and IL-17f in T cells was found in Pinkie. Flow cytometry and immunostaining revealed an increased number of IL-17+ T cells in Pinkie. Tear concentration of the IL-17 inducer IL-23 is significantly higher in Pinkie. 9-cis RA treatment suppresses stimulated IL-17 production by T and stimulatory activity of monocyte supernatant on T cell IL-17 production. Compared to WT bone marrow chimeras, Pinkie chimeras have increased IL-17+ T cells in the conjunctiva after desiccating stress and anti-IL-17 treatment suppresses dry eye induced corneal MMP-9 production/activity and conjunctival goblet cell loss. Conclusion: These findings indicate that RXR suppresses generation of dry eye disease inducing T17 cells in the conjunctiva and identifies RXR as a potential therapeutic target in dry eye. Overall design: There are two experiments for which we performed Nano string RNA analysis: Experiment#1 : We compare the cornea from C57B6 control mice with Pinkie mice strain have a normal cornea or Pinkie mice with ulcerated cornea. Experiment#2 : In this experiment we compared the control monocytes with monocytes either treated with LPS alone or both LPS and 9CisRA.
Project description:To use human limbal explants as an alternative source for generating conjunctival epithelium and to determine the effect of interleukin-13 (IL-13) on goblet cell number, mucin expression, and stemness. Human limbal explants prepared from 17 corneoscleral rims were cultured with or without IL-13 (IL-13+ and IL-13-, respectively) and followed up to passage 2 (primary culture [P0]-P2). Cells were characterized by alcian blue/periodic acid-Schiff (AB/PAS) staining (goblet cells); immunofluorescent staining for p63? (progenitor cells), Ki-67 (proliferation), MUC5AC (mucin, goblet cells), and keratin 7 (K7, conjunctival epithelial and goblet cells); and by quantitative real-time polymerase chain reaction for expression of the p63? (TP63), MUC5AC, MUC4 (conjunctival mucins), K3, K12 (corneal epithelial cells), and K7 genes. Clonogenic ability was determined by colony-forming efficiency (CFE) assay. Using limbal explants, we generated epithelium with conjunctival phenotype and high viability in P0, P1, and P2 cultures under IL-13+ and IL-13- conditions, i.e., epithelium with strong K7 positivity, high K7 and MUC4 expression and the presence of goblet cells (AB/PAS and MUC5AC positivity; MUC5AC expression). p63? positivity was similar in IL-13+ and IL-13- cultures and was decreased in P2 cultures; however, there was increased TP63 expression in the presence of IL-13 (especially in the P1 cultures). Similarly, IL-13 increased proliferative activity in P1 cultures and significantly promoted P0 and P1 culture CFE. IL-13 did not increase goblet cell number in the P0-P2 cultures, nor did it influence MUC5AC and MUC4 expression. By harvesting unattached cells on day 1 of P1 we obtained goblet cell rich subpopulation showing AB/PAS, MUC5AC, and K7 positivity, but with no growth potential. In conclusion, limbal explants were successfully used to develop conjunctival epithelium with the presence of putative stem and goblet cells and with the ability to preserve the stemness of P0 and P1 cultures under IL-13 influence.
Project description:To investigate the effects of IL-13 on goblet cell proliferation, differentiation, and expression of mucin and immunomodulatory genes.Explants were excised from the conjunctiva of young C57BL/6 mice. Cultures received 200 ?L per week of either Keratinocyte media (KSFM) or KSFM supplemented with 10 ng/mL IL-13 and were incubated for 3 (D3), 7 (D7), or 14 (D14) days. Subsequently, cell proliferation was assessed or cultures were immunostained, collected for dot blot, or for reverse transcription (RT) and quantitative real-time PCR (qPCR) or for RT-PCR gene array.The cultured conjunctival epithelium expressed goblet cell associated keratin 7 and mucins MUC5AC and MUC2 and when stimulated with IL-13 showed increased proliferation at D3 and D7 (P < 0.05) compared with control. MUC5AC expression was increased in the IL-13-treated group at D3 and D14 (P < 0.05). IL-13-treated cultures showed increased chemokine ligand 26 (CCL26), chloride channel calcium activated channel 3 (CLCA3), fas ligand (FasL), and Relm-? at D7. All conjunctival cultures expressed MUC2, and its expression was decreased at D3 (P < 0.05) and increased at D14 (P < 0.05) with IL-13 treatment.This study demonstrated that conjunctival goblet cells are IL-13 responsive cells that produce factors known to maintain epithelial barrier, stimulate mucin production, and modulate immune response in nonocular mucosa when treated with IL-13. The functional significance of IL-13-stimulated factors remains to be determined.
Project description:Hantavirus infection reduces antiviral defenses, increases regulatory responses, and causes persistent infection in rodent hosts. To address whether hantaviruses alter the maturation and functional activity of antigen presenting cells (APCs), rat bone marrow-derived dendritic cells (BMDCs) and macrophages (BMDMs) were generated and infected with Seoul virus (SEOV) or stimulated with TLR ligands. SEOV infected both DCs and macrophages, but copies of viral RNA, viral antigen, and infectious virus titers were higher in macrophages. The expression of MHCII and CD80, production of IL-6, IL-10, and TNF-alpha, and expression of Ifnbeta were attenuated in SEOV-infected APCs. Stimulation of APCs with poly I:C prior to SEOV infection increased the expression of activation markers and production of inflammatory cytokines and suppressed SEOV replication. Infection of APCs with SEOV suppressed LPS-induced activation and innate immune responses. Hantaviruses reduce the innate immune response potential of APCs derived from a natural host, which may influence persistence of these zoonotic viruses in the environment.
Project description:Following initial invasion of Schistosoma mansoni cercariae, schistosomula reside in the skin for several days during which they can interact with the dermal immune system. While murine experiments have indicated that exposure to radiation-attenuated (RA) cercariae can generate protective immunity which is initiated in the skin stage, contrasting non-attenuated cercariae, such data is missing for the human model. Since murine skin does not form a reliable marker for immune responses in human skin, we used human skin explants to study the interaction with non-attenuated and RA cercariae with dermal innate antigen presenting cells (APCs) and the subsequent immunological responses. We exposed human skin explants to cercariae and visualized their invasion in real time (initial 30 min) using novel imaging technologies. Subsequently, we studied dermal immune responses and found an enhanced production of regulatory cytokine interleukin (IL)-10, pro-inflammatory cytokine IL-6 and macrophage inflammatory protein (MIP)-1? within 3 days of exposure. Analysis of dermal dendritic cells (DDCs) for their phenotype revealed an increased expression of immune modulators programmed death ligand (PD-L) 1 and 2, and increased IL-10 production. Ex vivo primed DDCs suppress Th1 polarization of naïve T-cells and increase T-cell IL-10 production, indicating their regulatory potential. These immune responses were absent or decreased after exposure to RA parasites. Using transwells, we show that direct contact between APCs and cercariae is required to induce their regulatory phenotype. To the best of our knowledge this is the first study that attempts to provide insight in the human dermal S. mansoni cercariae invasion and subsequent immune responses comparing non-attenuated with RA parasites. We reveal that cercariae induce a predominantly regulatory immune response whereas RA cercariae fail to achieve this. This initial understanding of the dermal immune suppressive capacity of S. mansoni cercariae in humans provides a first step toward the development of an effective schistosome vaccine.
Project description:NKT cells have been shown to promote peripheral tolerance in a number of model systems, yet the processes by which they exert their regulatory effects remain poorly understood. Here, we show that soluble factors secreted by human NKT cells instruct human peripheral blood monocytes to differentiate into myeloid APCs that have suppressive properties. NKT instructed monocytes acquired a cell surface phenotype resembling myeloid DCs. However, whereas control DCs that were generated by culturing monocytes with recombinant GM-CSF and IL-4 had a proinflammatory phenotype characterized by the production of IL-12 with little IL-10, NKT-instructed APCs showed the opposite cytokine production profile of high IL-10 with little or no IL-12. The control DCs efficiently stimulated peripheral blood T cell IFN-gamma secretion and proliferation, whereas NKT-instructed APCs silenced these T cell responses. Exposure to NKT cell factors had a dominant effect on the functional properties of the DCs, since DCs differentiated by recombinant GM-CSF and IL-4 in the presence of NKT cell factors inhibited T cell responses. To confirm their noninflammatory effects, NKT-instructed APCs were tested in an in vivo assay that depends on the activation of antigen-specific human T cells. Control DCs promoted substantial tissue inflammation; however, despite a marked neutrophilic infiltrate, there was little edema in the presence of NKT-instructed APCs, suggesting the inflammatory cascade was held in check. These results point to a novel pathway initiated by NKT cells that can contribute to the regulation of human antigen-specific Th1 responses.
Project description:Goblet cell numbers decrease within the conjunctival epithelium in drying and cicatrizing ocular surface diseases. Factors regulating goblet cell differentiation in conjunctival epithelium are unknown. Recent data indicate that the transcription factor SAM-pointed domain epithelial-specific transcription factor (Spdef) is essential for goblet cell differentiation in tracheobronchial and gastrointestinal epithelium of mice. Using Spdef(-/-) mice, we determined that Spdef is required for conjunctival goblet cell differentiation and that Spdef(-/-) mice, which lack conjunctival goblet cells, have significantly increased corneal surface fluorescein staining and tear volume, a phenotype consistent with dry eye. Microarray analysis of conjunctival epithelium in Spdef(-/-) mice revealed down-regulation of goblet cell-specific genes (Muc5ac, Tff1, Gcnt3). Up-regulated genes included epithelial cell differentiation/keratinization genes (Sprr2h, Tgm1) and proinflammatory genes (Il1-?, Il-1?, Tnf-?), all of which are up-regulated in dry eye. Interestingly, four Wnt pathway genes were down-regulated. SPDEF expression was significantly decreased in the conjunctival epithelium of Sjögren syndrome patients with dry eye and decreased goblet cell mucin expression. These data demonstrate that Spdef is required for conjunctival goblet cell differentiation and down-regulation of SPDEF may play a role in human dry eye with goblet cell loss. Spdef(-/-) mice have an ocular surface phenotype similar to that in moderate dry eye, providing a new, more convenient model for the disease.
Project description:Goblet cell numbers decrease within the conjunctival epithelium in drying and cicatrizing ocular surface diseases. Factors regulating goblet cell differentiation in conjunctival epithelium are unknown. Recent data indicate that the transcription factor SAM-pointed domain epithelial-specific transcription factor (Spdef) is essential for goblet cell differentiation in tracheobronchial and gastrointestinal epithelium of mice. Using Spdef -/- mice, we determined that Spdef is required for conjunctival goblet cell differentiation and that Spdef -/- mice, which lack conjunctival goblet cells, have significantly increased corneal surface fluorescein staining and tear volume, a phenotype consistent with dry eye. Microarray analysis of conjunctival epithelium in Spdef -/- mice identified 43 signficantly upregulated genes and 110 signficantly downregulated genes in the conjunctival epithelium of Spdef -/- mice compared to that of Spdef +/+ control mice (3 fold change, p<0.01). Downregulated genes of particular interested included goblet cell-specific genes (Muc5ac, Tff1, Gcnt3). Upregulated genes included epithelial cell differentiation/keratinization genes (Sprr2h, Tgm1) and pro-inflammatory genes (Il1-Î±, Il-1Î², Tnf-Î±), all of which are upregulated in dry eye. Interestingly, four Wnt pathway genes were downregulated. Conjunctival epithelium of Spdef +/+ and Spdef -/- mice was collected by laser capture microdissection for RNA extraction and hybridization on Affymetrix microarrays to determine if gene expression patterns in the conjunctival epithelium of Spdef -/- mice is altered compared to that of Spdef +/+ mice.