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Ceramidas , Colitis , Interleucina-10 , Metabolismo de los Lípidos , Macrófagos , Transducción de Señal , Ceramidas/metabolismo , Ceramidas/genética , Interleucina-10/genética , Interleucina-10/metabolismo , Colitis/genética , Colitis/metabolismo , Colitis/patología , Macrófagos/metabolismo , Macrófagos/inmunología , Animales , Metabolismo de los Lípidos/genética , Transducción de Señal/genética , Ratones , Humanos , Homeostasis/genéticaRESUMEN
Monocytes, as well as downstream macrophages and dendritic cells, are essential players in the immune system, fulfilling key roles in homeostasis as well as in inflammatory conditions. Conventionally, driven by studies on reporter models, mouse monocytes are categorized into a classical and a non-classical subset based on their inversely correlated surface expression of Ly6C/CCR2 and CX3CR1. Here, we aimed to challenge this concept by antibody staining and reporter mouse models. Therefore, we took advantage of Cx3cr1GFP and Ccr2RFP reporter mice, in which the respective gene was replaced by a fluorescent reporter protein gene. We analyzed the expression of CX3CR1 and CCR2 by flow cytometry using several validated fluorochrome-coupled antibodies and compared them with the reporter gene signal in these reporter mouse strains. Although we were able to validate the specificity of the fluorochrome-coupled flow cytometry antibodies, mouse Ly6Chigh classical and Ly6Clow non-classical monocytes showed no differences in CX3CR1 expression levels in the peripheral blood and spleen when stained with these antibodies. On the contrary, in Cx3cr1GFP reporter mice, we were able to reproduce the inverse correlation of the CX3CR1 reporter gene signal and Ly6C surface expression. Furthermore, differential CCR2 surface expression correlating with the expression of Ly6C was observed by antibody staining, but not in Ccr2RFP reporter mice. In conclusion, our data suggest that phenotyping strategies for mouse monocyte subsets should be carefully selected. In accordance with the literature, the suitability of CX3CR1 antibody staining is limited, whereas for CCR2, caution should be applied when using reporter mice.
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Receptor 1 de Quimiocinas CX3C , Citometría de Flujo , Monocitos , Receptores CCR2 , Animales , Receptores CCR2/metabolismo , Receptores CCR2/genética , Monocitos/metabolismo , Receptor 1 de Quimiocinas CX3C/metabolismo , Receptor 1 de Quimiocinas CX3C/genética , Ratones , Anticuerpos/inmunología , Genes Reporteros , Fenotipo , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Fluorescentes Verdes/metabolismo , Antígenos Ly/metabolismo , Antígenos Ly/genéticaRESUMEN
Despite promising preclinical and earlier clinical data, a recent phase III trial on the anti-ß7 integrin antibody etrolizumab in Crohn's disease (CD) did not reach its primary endpoint. The mechanisms leading to this outcome are not well understood. Here we characterize the ß7+ T cell compartment from patients with CD in comparison to cells from individuals without inflammatory bowel disease. By flow cytometric, transcriptomic and functional profiling of circulating T cells, we find that triple-integrin-expressing (α4+ß7+ß1hi) T cells have the potential to home to the gut despite α4ß7 blockade and have a specific cytotoxic signature. A subset of triple-integrin-expressing cells readily acquires αE expression and could be co-stimulated via E-Cadherin-αEß7 interactions in vitro. Etrolizumab-s fails to block such αEß7 signalling at high levels of T cell stimulation. Consistently, in CD patients treated with etrolizumab, T cell activation correlates with cytotoxic signatures. Collectively, our findings might add one important piece to the puzzle to explain phase III trial results with etrolizumab, while they also highlight that αEß7 remains an interesting target for future therapeutic approaches in inflammatory bowel disease.
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Anticuerpos Monoclonales Humanizados , Enfermedades Inflamatorias del Intestino , Linfocitos T Citotóxicos , Humanos , Integrinas , CadherinasRESUMEN
OBJECTIVE: Mucosal T cells play a major role in inflammatory bowel disease (IBD). However, their immunometabolism during intestinal inflammation is poorly understood. Due to its impact on cellular metabolism and proinflammatory immune cell function, we here focus on the enzyme ATP citrate lyase (ACLY) in mucosal T cell immunometabolism and its relevance for IBD. DESIGN: ACLY expression and its immunometabolic impact on colitogenic T cell function were analysed in mucosal T cells from patients with IBD and in two experimental colitis models. RESULTS: ACLY was markedly expressed in colon tissue under steady-state conditions but was significantly downregulated in lamina propria mononuclear cells in experimental dextran sodium sulfate-induced colitis and in CD4+ and to a lesser extent in CD8+ T cells infiltrating the inflamed gut in patients with IBD. ACLY-deficient CD4+ T cells showed an impaired capacity to induce intestinal inflammation in a transfer colitis model as compared with wild-type T cells. Assessment of T cell immunometabolism revealed that ACLY deficiency dampened the production of IBD-relevant cytokines and impaired glycolytic ATP production but enriched metabolites involved in the biosynthesis of phospholipids and phosphatidylcholine. Interestingly, the short-chain fatty acid butyrate was identified as a potent suppressor of ACLY expression in T cells, while IL-36α and resolvin E1 induced ACLY levels. In a translational approach, in vivo administration of the butyrate prodrug tributyrin downregulated mucosal infiltration of ACLYhigh CD4+ T cells and ameliorated chronic colitis. CONCLUSION: ACLY controls mucosal T cell immunometabolism and experimental colitis. Therapeutic modulation of ACLY expression in T cells emerges as a novel strategy to promote the resolution of intestinal inflammation.
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Colitis , Enfermedades Inflamatorias del Intestino , Linfocitos Intraepiteliales , Humanos , Animales , Linfocitos Intraepiteliales/metabolismo , ATP Citrato (pro-S)-Liasa/metabolismo , Linfocitos T CD8-positivos/metabolismo , Colitis/metabolismo , Inflamación/metabolismo , Butiratos , Mucosa Intestinal/metabolismo , Sulfato de Dextran , Modelos Animales de EnfermedadRESUMEN
BACKGROUND AND AIMS: The G protein coupled receptor GPR15 is expressed on and functionally important for T cells homing to the large intestine. However, the precise mechanisms by which GPR15 controls gut homing have been unclear. Thus, we aimed to elucidate these mechanisms as well as to explore the potential of targeting GPR15 for interfering with T cell recruitment to the colon in inflammatory bowel disease [IBD]. METHODS: We used dynamic adhesion and transmigration assays, as well as a humanised in vivo model of intestinal cell trafficking, to study GPR15-dependent effects on gut homing. Moreover, we analysed GPR15 and integrin expression in patients with and without IBD, cross-sectionally and longitudinally. RESULTS: GPR15 controlled T cell adhesion to MAdCAM-1 and VCAM-1 upstream of α4ß7 and α4ß1 integrin, respectively. Consistently, high co-expression of these integrins with GPR15 was found on T cells from patients with IBD, and GPR15 also promoted T cell recruitment to the colon in humanised mice. Anti-GPR15 antibodies effectively blocked T cell gut homing in vitro and in vivo. In vitro data, as well as observations in a cohort of patients treated with vedolizumab, suggest that this might be more effective than inhibiting α4ß7. CONCLUSIONS: GPR15 seems to have a broad, but organ-selective, impact on T cell trafficking and is therefore a promising target for future therapy of IBD. Further studies are needed.
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Adhesión Celular , Enfermedades Inflamatorias del Intestino , Mucoproteínas , Receptores Acoplados a Proteínas G , Linfocitos T , Receptores Acoplados a Proteínas G/metabolismo , Animales , Humanos , Ratones , Linfocitos T/metabolismo , Linfocitos T/inmunología , Mucoproteínas/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Adhesión Celular/efectos de los fármacos , Integrinas/metabolismo , Movimiento Celular , Moléculas de Adhesión Celular/metabolismo , Colon/metabolismo , Anticuerpos Monoclonales Humanizados/farmacología , Integrina alfa4beta1/metabolismo , Inmunoglobulinas/metabolismo , Femenino , Receptores de PéptidosRESUMEN
IL-3 has been reported to be involved in various inflammatory disorders, but its role in inflammatory bowel disease (IBD) has not been addressed so far. Here, we determined IL-3 expression in samples from patients with IBD and studied the impact of Il3 or Il3r deficiency on T cell-dependent experimental colitis. We explored the mechanical, cytoskeletal and migratory properties of Il3r -/- and Il3r +/+ T cells using real-time deformability cytometry, atomic force microscopy, scanning electron microscopy, fluorescence recovery after photobleaching and in vitro and in vivo cell trafficking assays. We observed that, in patients with IBD, the levels of IL-3 in the inflamed mucosa were increased. In vivo, experimental chronic colitis on T cell transfer was exacerbated in the absence of Il-3 or Il-3r signalling. This was attributable to Il-3r signalling-induced changes in kinase phosphorylation and actin cytoskeleton structure, resulting in increased mechanical deformability and enhanced egress of Tregs from the inflamed colon mucosa. Similarly, IL-3 controlled mechanobiology in human Tregs and was associated with increased mucosal Treg abundance in patients with IBD. Collectively, our data reveal that IL-3 signaling exerts an important regulatory role at the interface of biophysical and migratory T cell features in intestinal inflammation and suggest that this might be an interesting target for future intervention.
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Colitis , Enfermedades Inflamatorias del Intestino , Humanos , Linfocitos T Reguladores , Receptores de Interleucina-3/metabolismo , Interleucina-3/metabolismo , Inflamación/metabolismo , Colitis/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Mucosa Intestinal/metabolismoRESUMEN
BACKGROUND AND AIMS: The anti-MAdCAM-1 antibody ontamalimab demonstrated efficacy in a phase II trial in ulcerative colitis and results of early terminated phase III trials are pending, but its precise mechanisms of action are still unclear. Thus, we explored the mechanisms of action of ontamalimab and compared it to the anti-α4ß7 antibody vedolizumab. METHODS: We studied MAdCAM-1 expression with RNA sequencing and immunohistochemistry. The mechanisms of action of ontamalimab were assessed with fluorescence microscopy, dynamic adhesion and rolling assays. We performed in vivo cell trafficking studies in mice and compared ontamalimab and vedolizumab surrogate [-s] antibodies in experimental models of colitis and wound healing. We analysed immune cell infiltration under anti-MAdCAM-1 and anti-α4ß7 treatment by single-cell transcriptomics and studied compensatory trafficking pathways. RESULTS: MAdCAM-1 expression was increased in active inflammatory bowel disease. Binding of ontamalimab to MAdCAM-1 induced the internalization of the complex. Functionally, ontamalimab blocked T cell adhesion similar to vedolizumab, but also inhibited L-selectin-dependent rolling of innate and adaptive immune cells. Despite conserved mechanisms in mice, the impact of ontamalimab-s and vedolizumab-s on experimental colitis and wound healing was similar. Single-cell RNA sequencing demonstrated enrichment of ontamalimab-s-treated lamina propria cells in specific clusters, and in vitro experiments indicated that redundant adhesion pathways are active in these cells. CONCLUSIONS: Ontamalimab has unique and broader mechanisms of action compared to vedolizumab. However, this seems to be compensated for by redundant cell trafficking circuits and leads to similar preclinical efficacy of anti-α4ß7 and anti-MAdCAM-1 treatment. These results will be important for the interpretation of pending phase III data.
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Colitis Ulcerosa , Enfermedades Inflamatorias del Intestino , Animales , Ratones , Fármacos Gastrointestinales/farmacología , Fármacos Gastrointestinales/uso terapéutico , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Colitis Ulcerosa/tratamiento farmacológico , Inflamación/tratamiento farmacológico , IntegrinasRESUMEN
BACKGROUND: Closing mucosal defects to reach mucosal healing is an important goal of therapy in inflammatory bowel disease (IBD). Among other cells, monocyte-derived macrophages are centrally involved in such intestinal wound healing. We had previously demonstrated that the anti-α4ß7 integrin antibody vedolizumab blocks the recruitment of non-classical monocytes as biased progenitors of wound healing macrophages to the gut and delays wound healing. However, although important for the interpretation of disappointing results in recent phase III trials in IBD, the effects of the anti-ß7 antibody etrolizumab on wound healing are unclear so far. METHODS: We analyzed the expression of etrolizumab targets on human and mouse monocyte subsets by flow cytometry and assessed their function in adhesion and homing assays. We explored wound-associated monocyte recruitment dynamics with multi-photon microscopy and compared the effects of etrolizumab and vedolizumab surrogate (-s) antibodies on experimental wound healing and wound-associated macrophage abundance. Finally, we investigated wound healing macrophage signatures in the large intestinal transcriptome of patients with Crohn's disease treated with etrolizumab. RESULTS: Human and mouse non-classical monocytes expressed more αEß7 integrin than classical monocytes and were a target of etrolizumab-s, which blocked non-classical monocyte adhesion to MAdCAM-1 and E-Cadherin as well as gut homing in vivo. Intestinal wound healing was delayed on treatment with etrolizumab-s along with a reduction of peri-lesional wound healing macrophages. Wound healing macrophage signatures in the colon of patients with Crohn's disease were substantially down-regulated on treatment with etrolizumab, but not with placebo. CONCLUSIONS: Combined blockade of αEß7 and α4ß7 with etrolizumab seems to exceed the effect of anti-α4ß7 treatment on intestinal wound healing, which might help to inform further investigations to understand the recent observations in the etrolizumab phase III trial program.
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Fármacos Gastrointestinales , Enfermedades Inflamatorias del Intestino , Integrinas , Macrófagos , Cicatrización de Heridas , Animales , Humanos , Ratones , Enfermedad de Crohn/tratamiento farmacológico , Enfermedad de Crohn/inmunología , Enfermedad de Crohn/patología , Fármacos Gastrointestinales/inmunología , Fármacos Gastrointestinales/farmacología , Fármacos Gastrointestinales/uso terapéutico , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/patología , Integrinas/antagonistas & inhibidores , Integrinas/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/patología , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/patología , Cicatrización de Heridas/efectos de los fármacos , Cicatrización de Heridas/inmunologíaRESUMEN
OBJECTIVE: Increased apoptotic shedding has been linked to intestinal barrier dysfunction and development of inflammatory bowel diseases (IBD). In contrast, physiological cell shedding allows the renewal of the epithelial monolayer without compromising the barrier function. Here, we investigated the role of live cell extrusion in epithelial barrier alterations in IBD. DESIGN: Taking advantage of conditional GGTase and RAC1 knockout mice in intestinal epithelial cells (Pggt1b iΔIEC and Rac1 iΔIEC mice), intravital microscopy, immunostaining, mechanobiology, organoid techniques and RNA sequencing, we analysed cell shedding alterations within the intestinal epithelium. Moreover, we examined human gut tissue and intestinal organoids from patients with IBD for cell shedding alterations and RAC1 function. RESULTS: Epithelial Pggt1b deletion led to cytoskeleton rearrangement and tight junction redistribution, causing cell overcrowding due to arresting of cell shedding that finally resulted in epithelial leakage and spontaneous mucosal inflammation in the small and to a lesser extent in the large intestine. Both in vivo and in vitro studies (knockout mice, organoids) identified RAC1 as a GGTase target critically involved in prenylation-dependent cytoskeleton dynamics, cell mechanics and epithelial cell shedding. Moreover, inflamed areas of gut tissue from patients with IBD exhibited funnel-like structures, signs of arrested cell shedding and impaired RAC1 function. RAC1 inhibition in human intestinal organoids caused actin alterations compatible with arresting of cell shedding. CONCLUSION: Impaired epithelial RAC1 function causes cell overcrowding and epithelial leakage thus inducing chronic intestinal inflammation. Epithelial RAC1 emerges as key regulator of cytoskeletal dynamics, cell mechanics and intestinal cell shedding. Modulation of RAC1 might be exploited for restoration of epithelial integrity in the gut of patients with IBD.
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Citoesqueleto , Enfermedades Inflamatorias del Intestino , Animales , Humanos , Ratones , Células Epiteliales , Inflamación , Enfermedades Inflamatorias del Intestino/genética , Mucosa Intestinal/fisiología , Ratones Noqueados , Proteína de Unión al GTP rac1RESUMEN
Inflammatory bowel diseases are medically intractable and require constant therapy in many cases. While a growing number of biologicals and small molecules is available for treatment, a substantial portion of patients experiences primary non-response to these compounds and head-to-head evidence for therapy selection is scarce. Thus, approaches to predict treatment success in individual patients are a huge unmet need. We had previously suggested that the expression and function of α4ß7 integrin on T cells in the peripheral blood correlate to outcomes of therapy with the anti-α4ß7 integrin antibody vedolizumab. Here, we conducted a translational multicenter trial to prospectively evaluate this hypothesis. In a cohort of 89 patients with inflammatory bowel disease undergoing regular therapy with vedolizumab, lower baseline expression of α4ß7 was associated with short-term clinical response. Consistently, low α4ß7 expression in patients achieving remission predicted sustained remission in week 30. Moreover, high dynamic adhesion of CD4+ T cells to MAdCAM-1 and high reduction of adhesion by vedolizumab in vitro at baseline were associated with clinical remission. These data substantiate the potential of α4ß7 integrin function and expression to forecast outcomes of vedolizumab therapy. Further translational efforts are necessary to improve the performance of the assays and to implement the concept in clinical practice.
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Fármacos Gastrointestinales , Enfermedades Inflamatorias del Intestino , Humanos , Fármacos Gastrointestinales/uso terapéutico , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Anticuerpos Monoclonales Humanizados/uso terapéutico , Integrinas/metabolismoRESUMEN
The successful treatment of advanced colorectal cancer disease still represents an insufficiently solved clinical challenge, which is further complicated by the fact that the majority of malignant colon tumors show only relatively low immunogenicity and therefore have only limited responsiveness to immunotherapeutic approaches, such as, for instance, the use of checkpoint inhibitors. As it has been well established over the past two decades that the local tumor microenvironment and, in particular, the quantity, quality, and activation status of intratumoral immune cells critically influence the clinical prognosis of patients diagnosed with colorectal cancer and their individual benefits from immunotherapy, the enhancement of the intratumoral accumulation of cytolytic effector T lymphocytes and other cellular mediators of the antitumor immune response has emerged as a targeted objective. For the future identification and clinical validation of novel therapeutic target structures, it will thus be essential to further decipher the molecular mechanisms and cellular interactions in the intestinal tumor microenvironment, which are crucially involved in immune cell recruitment and activation. In this context, our review article aims at providing an overview of the key chemokines and cytokines whose presence in the tumor micromilieu relevantly modulates the numeric composition and antitumor capacity of tumor-infiltrating lymphocytes.
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BACKGROUND: Blocking immune cell gut homing via α4ß7 integrin with the monoclonal antibody vedolizumab is an established therapeutic strategy in inflammatory bowel disease. However, despite promising preclinical and phase 2 clinical data, the anti-ß7 antibody etrolizumab yielded disappointing results in a large phase 3 trial program in UC. Mechanistic explanations are still lacking. We have recently shown that vedolizumab is associated with residual homing of regulatory T (Treg) cells in a certain exposure range and aimed to investigate whether a similar mechanism applies for etrolizumab. METHODS: We used flow cytometry, competitive dynamic adhesion, and transmigration assays to assess binding of the etrolizumab surrogate (etrolizumab-s) antibody FIB504 to Treg and effector T cells (Teff) and to explore the impact on cell trafficking. RESULTS: We observed only minimal differences in the binding of etrolizumab-s to Treg and Teff cells. Dynamic adhesion and transmigration of Treg and Teff cells was not substantially differentially affected at relevant concentrations. The ß1+ and PI16+ Treg cells were only resistant to etrolizumab-s at low concentrations. CONCLUSIONS: Etrolizumab does not seem to induce notable residual trafficking of Treg cells. Thus, the Teff overweight in the inflamed gut might persist despite reduced overall T cell recruitment. This might be one piece of the puzzle to explain recent clinical results in phase 3.
The efficacy of etrolizumab in phase 3 was disappointing. Our data suggest that, unlike vedolizumab, etrolizumab does not induce relevant residual trafficking of regulatory T cells. This might be one part of the explanation for recent observations in clinical trials.
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Colitis Ulcerosa , Enfermedades Inflamatorias del Intestino , Humanos , Fármacos Gastrointestinales/uso terapéutico , Linfocitos T Reguladores/metabolismo , Colitis Ulcerosa/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/tratamiento farmacológicoRESUMEN
Background: In Crohn's disease and ulcerative colitis, the anti-α4ß7 integrin antibody vedolizumab has demonstrated efficacy in phase III trials and has been successfully used under real-world conditions. Occasionally, it has also been used in other forms of inflammatory bowel disease (IBD) such as microscopic colitis (MC). However, the mechanisms of vedolizumab in MC have not been studied to date. Therefore, we aimed to investigate the expression and functional role of gut-homing integrins and in particular α4ß7 integrin in a cohort study in MC. Methods: We studied the expression of gut homing integrins on T cells from patients with MC and healthy controls by flow cytometry. To investigate the function of α4ß7 integrin in MC and the potential of vedolizumab to block it, we used dynamic adhesion assays and transmigrations assays. Moreover, we describe two clinical cases of MC patients treated with vedolizumab. Results: A specific profile of gut homing markers can be found on T cells from patients with MC. α4ß7 integrin functionally leads to firm adhesion to MAdCAM-1 and supports transmigration. Vedolizumab is able to block both processes. In two cases of MC, we observed reduced clinical symptoms and histologic improvement upon therapy with vedolizumab. Conclusion: Our data suggest that α4ß7 mediates gut homing of T cells also in MC and that, on single cell level, vedolizumab blocks the function of α4ß7 in MC. Thus, we provide mechanistic evidence supporting vedolizumab as promising therapeutic option for MC.
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OBJECTIVES: The anti-α4ß7 integrin antibody vedolizumab (VDZ) is successfully used for the treatment of inflammatory bowel diseases. However, only a subgroup of patients respond to therapy. VDZ is administered at a fixed dose, leading to a wide range of serum concentrations in patients. Previous work from our group showed a dose-dependent preferential binding of VDZ to effector compared with regulatory CD4 + T cells. Therefore, we aimed to determine the dose-dependent binding profile of VDZ to other leukocyte subsets. METHODS: We characterized α4ß7 integrin expression on CD8 + T cells, CD19 + B cells, CD14 + monocytes, natural killer cells, and eosinophils from patients with inflammatory bowel disease and healthy controls. We studied the binding of VDZ to these cells at different concentrations and investigated the functional consequences for dynamic adhesion and transmigration in vitro . RESULTS: The expression of α4ß7 differed between the analyzed leukocyte subsets and was significantly higher on eosinophils from inflammatory bowel disease patients compared with controls. Almost all α4ß7-expressing cells from these subsets were bound by VDZ at a concentration of 10 µg/mL. Dynamic cell adhesion was significantly impaired in all subsets, but there were no dose-dependent differences in the inhibition of adhesion. DISCUSSION: Our data suggest that α4ß7-expressing CD8 + T cells, CD19 + B cells, CD14 + monocytes, natural killer cells, and eosinophils are a target of VDZ. However, there do not seem to be concentration-dependent differences, regarding the effects on these cells in the clinically relevant range. Thus, the reported exposure-efficacy characteristic of VDZ can probably mainly be attributed to CD4 + T-cell subsets.
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Anticuerpos Monoclonales Humanizados , Enfermedades Inflamatorias del Intestino , Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Humanos , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Integrinas/metabolismo , Integrinas/uso terapéutico , Leucocitos/metabolismoRESUMEN
Generalized pustular psoriasis is a severe psoriatic subtype characterized by epidermal neutrophil infiltration. Although variants in IL36RN and MPO have been shown to affect immune cells, a systematic analysis of neutrophils and PBMC subsets and their differential gene expression dependent on MPO genotypes was not performed yet. We assessed the transcriptomes of MPO-deficient patients using single-cell RNA sequencing of PBMCs and RNA sequencing of neutrophils in a stable disease state. Cell-type annotation by multimodal reference mapping of single-cell RNA-sequencing data was verified by flow cytometry of surface and intracellular markers; the proportions of CD4+ cytotoxic T lymphocytes and other CD4+ effector cells were increased in generalized pustular psoriasis, whereas the frequencies of naïve CD4+ T cells were significantly lower. The expression of FGFBP2 marking CD4+ cytotoxic T lymphocytes and CD8+ effector memory T cells was elevated in patients with generalized pustular psoriasis with disease-contributing variants compared with that in noncarriers (P = 0.0015). In neutrophils, differentially expressed genes were significantly enriched in genes of the classical complement activation pathway. Future studies assessing affected cell types and pathways will show their contribution to generalized pustular psoriasis's pathogenesis and indicate whether findings can be transferred to the acute epidermal situation and whether depletion or inactivation of CD4+ cytotoxic T lymphocytes may be a reasonable therapeutic approach.
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Peroxidasa , Psoriasis , Enfermedades Cutáneas Vesiculoampollosas , Transcriptoma , Enfermedad Aguda , Linfocitos T CD4-Positivos/patología , Enfermedad Crónica , Humanos , Leucocitos Mononucleares/patología , Peroxidasa/deficiencia , Psoriasis/patología , Enfermedades Cutáneas Vesiculoampollosas/patología , Linfocitos T CitotóxicosRESUMEN
Introduction: Macrophages play an important role in intestinal wound healing. However, the trajectories from circulating monocytes to gut macrophages are incompletely understood. Methods: Taking advantage of mice depleted for non-classical monocytes due to deficiency for the transcription factor Nr4a1, we addressed the relevance of non-classical monocytes for large intestinal wound healing using flow cytometry, in vivo wound healing assays and immunofluorescence. Results: We show that wound healing in Nr4a1-deficient mice is substantially delayed and associated with reduced peri-lesional presence of macrophages with a wound healing phenotype. Discussion: Our data suggest that non-classical monocytes are biased towards wound healing macrophages. These insights might help to understand, how targeting monocyte recruitment to the intestine can be used to modulate intestinal macrophage functions.
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Macrófagos , Monocitos , Ratones , Animales , Cicatrización de Heridas , Intestino Grueso , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genéticaRESUMEN
OBJECTIVE: The anti-α4ß7 integrin antibody vedolizumab is administered at a fixed dose for the treatment of IBDs. This leads to a wide range of serum concentrations in patients and previous studies had suggested that highest exposure levels are associated with suboptimal clinical response. We aimed to determine the mechanisms underlying these non-linear exposure-efficacy characteristics of vedolizumab. DESIGN: We characterised over 500 samples from more than 300 subjects. We studied the binding of vedolizumab to T cells and investigated the functional consequences for dynamic adhesion, transmigration, gut homing and free binding sites in vivo. Employing single-cell RNA sequencing, we characterised α4ß7 integrin-expressing T cell populations 'resistant' to vedolizumab and validated our findings in vitro and in samples from vedolizumab-treated patients with IBD. We also correlated our findings with a post-hoc analysis of the Gemini II and III studies. RESULTS: Regulatory T (TReg) cells exhibited a right-shifted vedolizumab binding profile compared with effector T (TEff) cells. Consistently, in a certain concentration range, the residual adhesion, transmigration, homing of and availability of functional α4ß7 on TReg cells in vivo was higher than that of/on TEff cells. We identified a vedolizumab-'resistant' α4ß7-expressing ß1+PI16+ TReg cell subset with pronounced regulatory properties as the substrate for this effect. Our observations correlated with exposure-efficacy data from Gemini II and III trials. CONCLUSION: Completely blocking TEff cell trafficking with vedolizumab, while simultaneously permitting residual homing of powerful TReg cells in an optimal 'therapeutic window' based on target exposure levels might be a strategy to optimise treatment outcomes in patients with IBD.
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Fármacos Gastrointestinales , Enfermedades Inflamatorias del Intestino , Anticuerpos Monoclonales Humanizados , Proteínas Portadoras , Fármacos Gastrointestinales/farmacología , Fármacos Gastrointestinales/uso terapéutico , Glicoproteínas/metabolismo , Glicoproteínas/uso terapéutico , Humanos , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Integrinas , Linfocitos T Reguladores/metabolismoRESUMEN
BACKGROUND: The anti-α4ß7 integrin antibody vedolizumab is an established therapeutic option for the treatment of inflammatory bowel disease (IBD). It has also been successfully used in patients with chronic antibiotic-refractory pouchitis following proctocolectomey with ileal pouch-anal anastomosis. However, the expression and function of gut-homing markers as well as strategies to predict the response to vedolizumab in pouchitis are understudied so far. METHODS: We used flow cytometry and dynamic adhesion assays to study the expression and function of gut-homing integrins on T cells from patients with pouchitis and controls as well as longitudinally during therapy of pouchitis with vedolizumab. Moreover, we describe clinical effects of vedolizumab in a cohort of patients with pouchitis. RESULTS: T cells from patients with pouchitis express a specific profile of gut-homing integrins. Integrin α4ß7 on T cells from patients with pouchitis mediates adhesion to mucosal addressin cell adhesion molecule (MAdCAM)-1, which can be blocked by vedolizumab in vitro. Vedolizumab efficiently treats pouchitis in a portion of patients and response correlates with dynamic adhesion profiles to MAdCAM-1. CONCLUSION: Our data suggest that T cell trafficking seems to be important for the pathogenesis of pouchitis and support the therapeutic use of vedolizumab. Integrin function might serve as a biomarker to predict response to vedolizumab.