RESUMEN
BACKGROUND: Inflammatory visceral pain is endogenously controlled by enkephalins locally released by mucosal CD4+ T lymphocytes in mice. The present study aimed at identifying opioid receptor(s) expressed on nociceptive sensory nerves involved in this peripheral opioid-mediated analgesia. METHODS: The peripheral analgesia associated with the accumulation of CD4+ T lymphocytes within the inflamed colonic mucosa was assessed in conditional knockout mice specifically deleted for either of the two opioid receptors for enkephalins (i.e., µ (MOR) and δ (DOR) receptors) in Nav1.8-expressing sensory neurons in the dextran sulfate sodium (DSS)-induced colitis model. RESULTS: Endogenous analgesia is lost in conditional knockout mice for DOR, but not MOR at the later phase of the DSS-induced colitis. The absence of either of the opioid receptors on sensory nerves had no impact on both the colitis severity and the rate of T lymphocytes infiltrating the inflamed colonic mucosa. CONCLUSION: The key role of DOR on primary afferents in relieving intestinal inflammatory pain opens new therapeutic opportunities for peripherally restricted DOR analgesics to avoid most of the side effects associated with MOR-targeting drugs used in intestinal disorders.
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Colitis/metabolismo , Mucosa Intestinal/metabolismo , Nociceptores/metabolismo , Receptores Opioides delta/metabolismo , Dolor Visceral/metabolismo , Analgesia , Animales , Colitis/genética , Modelos Animales de Enfermedad , Inflamación/genética , Inflamación/metabolismo , Ratones , Ratones Noqueados , Receptores Opioides delta/genética , Dolor Visceral/genéticaRESUMEN
OBJECTIVE: Data from clinical research suggest that certain probiotic bacterial strains have the potential to modulate colonic inflammation. Nonetheless, these data differ between studies due to the probiotic bacterial strains used and the poor knowledge of their mechanisms of action. DESIGN: By mass-spectrometry, we identified and quantified free long chain fatty acids (LCFAs) in probiotics and assessed the effect of one of them in mouse colitis. RESULTS: Among all the LCFAs quantified by mass spectrometry in Escherichia coli Nissle 1917 (EcN), a probiotic used for the treatment of multiple intestinal disorders, the concentration of 3-hydroxyoctadecaenoic acid (C18-3OH) was increased in EcN compared with other E. coli strains tested. Oral administration of C18-3OH decreased colitis induced by dextran sulfate sodium in mice. To determine whether other bacteria composing the microbiota are able to produce C18-3OH, we targeted the gut microbiota of mice with prebiotic fructooligosaccharides (FOS). The anti-inflammatory properties of FOS were associated with an increase in colonic C18-3OH concentration. Microbiota analyses revealed that the concentration of C18-3OH was correlated with an increase in the abundance in Allobaculum, Holdemanella and Parabacteroides. In culture, Holdemanella biformis produced high concentration of C18-3OH. Finally, using TR-FRET binding assay and gene expression analysis, we demonstrated that the C18-3OH is an agonist of peroxisome proliferator activated receptor gamma. CONCLUSION: The production of C18-3OH by bacteria could be one of the mechanisms implicated in the anti-inflammatory properties of probiotics. The production of LCFA-3OH by bacteria could be implicated in the microbiota/host interactions.
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Colitis/tratamiento farmacológico , Mucosa Intestinal/metabolismo , PPAR gamma/metabolismo , Estearatos/metabolismo , Estearatos/uso terapéutico , Animales , Bacteroidetes , Células CACO-2 , Permeabilidad de la Membrana Celular , Quimiocina CXCL1/genética , Colitis/inducido químicamente , Colitis/metabolismo , Sulfato de Dextran , Células Epiteliales/fisiología , Escherichia coli/metabolismo , Firmicutes/metabolismo , Microbioma Gastrointestinal/fisiología , Expresión Génica/efectos de los fármacos , Humanos , Interleucina-1beta/genética , Espectrometría de Masas , Ratones , Oligosacáridos/farmacología , PPAR gamma/genética , Proteínas Asociadas a Pancreatitis/genética , Permeabilidad , Ganglios Linfáticos Agregados , Prebióticos , Probióticos/química , Estearatos/análisis , Proteína de la Zonula Occludens-1/genéticaRESUMEN
Mucosal CD4+ T lymphocytes display a potent opioid-mediated analgesic activity in interleukin (IL)-10 knockout mouse model of inflammatory bowel diseases (IBD). Considering that endogenous opioids may also exhibit anti-inflammatory activities in the periphery, we examined the consequences of a peripheral opioid receptor blockade by naloxone-methiodide, a general opioid receptor antagonist unable to cross the blood-brain barrier, on the development of piroxicam-accelerated colitis in IL-10-deficient (IL-10-/-) mice. Here, we show that IL-10-deficient mice treated with piroxicam exhibited significant alterations of the intestinal barrier function, including permeability, inflammation-related bioactive lipid mediators, and mucosal CD4+ T lymphocyte subsets. Opioid receptor antagonization in the periphery had virtually no effect on colitis severity but significantly worsened epithelial cell apoptosis and intestinal permeability. Thus, although the endogenous opioid tone is not sufficient to reduce the severity of colitis significantly, it substantially contributes to the protection of the physical integrity of the epithelial barrier.
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Colitis/metabolismo , Interleucina-10/genética , Mucosa Intestinal/efectos de los fármacos , Naloxona/análogos & derivados , Antagonistas de Narcóticos/administración & dosificación , Piroxicam/farmacología , Receptores Opioides/metabolismo , Animales , Antiinflamatorios/farmacología , Antiinflamatorios no Esteroideos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Linfocitos T CD4-Positivos/efectos de los fármacos , Colitis/inducido químicamente , Colitis/genética , Colitis/patología , Citocinas/genética , Citocinas/metabolismo , Células Epiteliales/efectos de los fármacos , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Interleucina-10/metabolismo , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Naloxona/farmacología , Permeabilidad/efectos de los fármacos , Compuestos de Amonio Cuaternario/farmacología , Índice de Severidad de la EnfermedadRESUMEN
Inside the human host, Leishmania infection starts with phagocytosis of infective promastigotes by macrophages. In order to survive, Leishmania has developed several strategies to manipulate macrophage functions. Among these strategies, Leishmania as a source of bioactive lipids has been poorly explored. Herein, we assessed the biosynthesis of polyunsaturated fatty acid metabolites by infective and noninfective stages of Leishmania and further explored the role of these metabolites in macrophage polarization. The concentration of docosahexaenoic acid metabolites, precursors of proresolving lipid mediators, was increased in the infective stage of the parasite compared with the noninfective stage, and cytochrome P450-like proteins were shown to be implicated in the biosynthesis of these metabolites. The treatment of macrophages with lipids extracted from the infective forms of the parasite led to M2 macrophage polarization and blocked the differentiation into the M1 phenotype induced by IFN-γ. In conclusion, Leishmania polyunsaturated fatty acid metabolites, produced by cytochrome P450-like protein activity, are implicated in parasite/host interactions by promoting the polarization of macrophages into a proresolving M2 phenotype.
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Ácidos Grasos Insaturados/metabolismo , Interacciones Huésped-Parásitos , Leishmania/fisiología , Animales , Células CHO , Cricetulus , Leishmania/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Macrófagos/parasitología , Masculino , Ratones , Ratones Endogámicos C57BL , FenotipoRESUMEN
The Focal adhesion kinase (FAK) is a ubiquitous cytoplasmic tyrosine-kinase promoting tumor progression and metastasis processes by acting in cancer cells and their tumor microenvironment partners. FAK overexpression in primary colon tumors and their metastasis is associated to poor colorectal cancer (CRC) patients' outcome. Eight FAK mRNA alternative splice variants have been described and contribute to additional level of FAK activity regulation, some of them corresponding to overactivated FAK isoforms. To date, FAK mRNA alternative splice variants expression and implication in CRC processes remain unknown. Here, using different human CRC cells lines displaying differential invasive capacities in an in vivo murine model recapitulating the different steps of CRC development from primary tumors to liver and lung metastasis, we identified three out of the eight mRNA variants (namely FAK0 , FAK28 and FAK6 ) differentially expressed along the CRC process and the tumor sites. Our results highlight an association between FAK0 and FAK6 expressions and the metastatic potential of the most aggressive cell lines HT29 and HCT116, suggesting that FAK0 and FAK6 could represent aggressiveness markers in CRC. Our findings also suggest a more specific role for FAK28 in the interactions between the tumors cells and their microenvironment. In conclusion, targeting FAK0 , the common form of FAK, might not be a good strategy based on the numerous roles of this kinase in physiological processes. In contrast, FAK6 or FAK28 splice variants, or their corresponding protein isoforms, may putatively represent future therapeutic target candidates in the development of CRC primary tumors and metastasis.
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Empalme Alternativo , Neoplasias Colorrectales/patología , Quinasa 1 de Adhesión Focal/genética , Neoplasias Hepáticas/secundario , Neoplasias Pulmonares/secundario , Animales , Biomarcadores de Tumor/genética , Neoplasias Colorrectales/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Células HT29 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Invasividad Neoplásica , Trasplante de Neoplasias , Isoformas de ARN/genética , Regulación hacia ArribaRESUMEN
BACKGROUND: T cell-derived opioids play a key role in the control of inflammatory pain. However, the nature of opioids produced by T cells is still matter of debate in mice. Whereas ß-endorphin has been found in T lymphocytes by using antibody-based methods, messenger RNA (mRNA) quantification shows mainly mRNA encoding for enkephalins. The objective of the study is to elucidate the nature of T cell-derived opioids responsible for analgesia and clarify discrepancy of the results at the protein and genetic levels. METHODS: CD4(+) T lymphocytes were isolated from wild-type and enkephalin-deficient mice. mRNA encoding for ß-endorphin and enkephalin was quantified by RT-qPCR. The binding of commercially available polyclonal anti-endorphin antibodies to lymphocytes from wild-type or enkephalin knockout mice was assessed by cytofluorometry. Opioid-mediated analgesic properties of T lymphocytes from wild-type and enkephalin-deficient mice were compared in a model of inflammation-induced somatic pain by measuring sensitivity to mechanical stimuli using calibrated von Frey filaments. RESULTS: CD4(+) T lymphocytes expressed high level of mRNA encoding for enkephalins but not for ß-endorphin in mice. Anti-ß-endorphin polyclonal IgG antibodies are specific for ß-endorphin but cross-react with enkephalins. Anti-ß-endorphin polyclonal antibodies bound to wild-type but not enkephalin-deficient CD4(+) T lymphocytes. Endogenous regulation of inflammatory pain by wild-type T lymphocytes was completely abolished when T lymphocytes were deficient in enkephalins. Pain behavior of immune-deficient (i.e., without B and T lymphocytes) mice was superimposable to that of mice transferred with enkephalin-deficient lymphocytes. CONCLUSIONS: Rabbit polyclonal anti-ß-endorphin serum IgG bind to CD4(+) T lymphocytes because of their cross-reactivity towards enkephalins. Thus, staining of T lymphocytes by anti-ß-endorphin polyclonal IgG reported in most of studies in mice is because of their binding to enkephalins. In mice, CD4(+) T lymphocytes completely lose their analgesic opioid-mediated activity when lacking enkephalins.
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Analgesia/métodos , Linfocitos T CD4-Positivos/metabolismo , Encefalinas/metabolismo , Dimensión del Dolor/métodos , Dolor/metabolismo , Dolor/prevención & control , Secuencia de Aminoácidos , Animales , Linfocitos T CD4-Positivos/inmunología , Encefalinas/genética , Encefalinas/inmunología , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Dolor/inmunología , Conejos , Distribución AleatoriaRESUMEN
BACKGROUND & AIMS: A dysregulated response of CD4(+) T cells against the microbiota contributes to the development of inflammatory bowel disease. Effector CD4(+) T cells, generated in response to microbe-derived antigens, can reduce somatic inflammatory pain through the local release of opioids. We investigated whether colitogenic CD4(+) T cells that accumulate in the inflamed colon also produce opioids and are able to counteract inflammation-induced visceral pain in mice. METHODS: Colitis was induced via transfer of naive CD4(+)CD45RB(high) T cells to immune-deficient mice or by administration of dextran sulfate sodium. Mice without colitis were used as controls. Samples of colon tissue were collected, and production of opioids by immune cells from inflamed intestine was assessed by quantitative polymerase chain reaction and cytofluorometry analyses. The role of intestinal opioid tone in inflammation-induced visceral hypersensitivity was assessed by colorectal distention. RESULTS: In mice with T cell- or dextran sulfate sodium-induced colitis, colitogenic CD4(+) T cells (T-helper 1 and Th17 cells) accumulated in the inflamed intestine and expressed a high level of endogenous opioids. In contrast, macrophages and epithelial cells did not express opioids; opioid synthesis in the myenteric plexus was not altered on induction of inflammation. In mice with colitis, the local release of opioids by colitogenic CD4(+) T cells led to significant reduction of inflammation-associated visceral hypersensitivity. CONCLUSIONS: In mice, colitogenic Th1 and Th17 cells promote intestinal inflammation and colonic tissue damage but have simultaneous opioid-mediated analgesic activity, thereby reducing abdominal pain.
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Colitis/inmunología , Colon/inmunología , Plexo Mientérico/inmunología , Péptidos Opioides/inmunología , Células TH1/inmunología , Células Th17/inmunología , Dolor Visceral/inmunología , Animales , Colitis/inducido químicamente , Colitis/patología , Colon/inervación , Colon/patología , Sulfato de Dextran/efectos adversos , Modelos Animales de Enfermedad , Inmunidad Mucosa , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Plexo Mientérico/fisiología , Péptidos Opioides/fisiologíaRESUMEN
Pain is an inherent component of inflammation often accompanying immune response. A large spectrum of molecules released within the inflamed tissue induces pain by stimulating primary afferent neurons in situ. Activity of primary sensitive fibers can be counteracted by local opioid release by leukocytes. In this study, we investigated the endogenous regulation of CFA-induced inflammatory pain in the context of adaptive T cell immune response. The nociceptive response to mechanical stimuli was studied using von Frey filaments in mice immunized with OVA in CFA. The nociceptive response of nude versus wild-type mice was dramatically increased, demonstrating T cell deficiency associated with increased pain sensitivity. Based on adoptive transfer experiments of OVA-specific CD4(+) T lymphocytes into nude mice, we show that Ag-specific activated, but not resting T lymphocytes are responsible for the spontaneous relief of inflammation-induced pain following Ag challenge. The analgesia was dependent on opioid release by Ag-primed CD4(+) T lymphocytes at the inflammatory site. Indeed, T cell-mediated analgesia was inhibited by local injection of an opioid receptor antagonist, unable to cross the blood-brain barrier. Notably, we found opioid precursor mRNA to be >7-fold increased in Ag-specific activated CD4(+) T lymphocytes, as compared with resting T lymphocytes in vivo. Taken together, our results show that CD4(+) T lymphocytes acquire antinociceptive effector properties when specifically primed by Ag and point out analgesia as a property linked to the effector phase of adaptive T cell response.
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Inmunidad Adaptativa/inmunología , Linfocitos T CD4-Positivos/inmunología , Péptidos Opioides/inmunología , Dolor/inmunología , Traslado Adoptivo , Animales , Separación Celular , Citometría de Flujo , Inmunohistoquímica , Inflamación/complicaciones , Activación de Linfocitos/inmunología , Ratones , Ratones Desnudos , Dolor/etiología , Reacción en Cadena de la PolimerasaRESUMEN
Opioid-dependent immune-mediated analgesic effects have been broadly reported upon inflammation. In preclinical mouse models of intestinal inflammatory diseases, the local release of enkephalins (endogenous opioids) by colitogenic T lymphocytes alleviate inflammation-induced pain by down-modulating gut-innervating nociceptor activation in periphery. In this study, we wondered whether this immune cell-derived enkephalin-mediated regulation of the nociceptor activity also operates under steady state conditions. Here, we show that chimeric mice engrafted with enkephalin-deficient bone marrow cells exhibit not only visceral hypersensitivity but also an increase in both epithelial paracellular and transcellular permeability, an alteration of the microbial topography resulting in increased bacteria-epithelium interactions and a higher frequency of IgA-producing plasma cells in Peyer's patches. All these alterations of the intestinal homeostasis are associated with an anxiety-like behavior despite the absence of an overt inflammation as observed in patients with irritable bowel syndrome. Thus, our results show that immune cell-derived enkephalins play a pivotal role in maintaining gut homeostasis and normal behavior in mice. Because a defect in the mucosal opioid system remarkably mimics some major clinical symptoms of the irritable bowel syndrome, its identification might help to stratify subgroups of patients.
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Síndrome del Colon Irritable , Humanos , Animales , Ratones , Analgésicos Opioides , Encefalinas/genética , Inflamación , DolorRESUMEN
Current analgesic treatments for Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS) are limited. Here, we propose a novel antinociceptive strategy exploiting the opioid-mediated analgesic properties of T lymphocytes to relieve from bladder pain. In a chronic model of IC/BPS in rats, we show that a secondary T cell response against intravesically administered ovalbumin prevents from visceral pain in OVA-primed animals. The analgesic effect is associated with the recruitment of T lymphocytes within the inflamed mucosa and is reversed by naloxone-methiodide, a peripheral opioid receptor antagonist. Similarly, intravesical instillation of BCG or tetanus toxoid antigens in vaccinated rats protects from pain in the same model. We show opioid-dependent analgesic properties of local vaccine antigen recall in a preclinical rat model of chronic cystitis. Since BCG bladder instillation is regularly used in humans (as anticancer therapy), our results open it as a new therapeutic positioning for a pain management indication for IC/BPS patients.
RESUMEN
BACKGROUND AND AIMS: Intestinal epithelial cells [IECs] from inflammatory bowel disease [IBD] patients exhibit an excessive induction of endoplasmic reticulum stress [ER stress] linked to altered intestinal barrier function and inflammation. Colonic tissues and the luminal content of IBD patients are also characterized by increased serine protease activity. The possible link between ER stress and serine protease activity in colitis-associated epithelial dysfunctions is unknown. We aimed to study the association between ER stress and serine protease activity in enterocytes and its impact on intestinal functions. METHODS: The impact of ER stress induced by Thapsigargin on serine protease secretion was studied using either human intestinal cell lines or organoids. Moreover, treating human intestinal cells with protease-activated receptor antagonists allowed us to investigate ER stress-resulting molecular mechanisms that induce proteolytic activity and alter intestinal epithelial cell biology. RESULTS: Colonic biopsies from IBD patients exhibited increased epithelial trypsin-like activity associated with elevated ER stress. Induction of ER stress in human intestinal epithelial cells displayed enhanced apical trypsin-like activity. ER stress-induced increased trypsin activity destabilized intestinal barrier function by increasing permeability and by controlling inflammatory mediators such as C-X-C chemokine ligand 8 [CXCL8]. The deleterious impact of ER stress-associated trypsin activity was specifically dependent on the activation of protease-activated receptors 2 and 4. CONCLUSIONS: Excessive ER stress in IECs caused an increased release of trypsin activity that, in turn, altered intestinal barrier function, promoting the development of inflammatory process.
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Colitis Ulcerosa/patología , Enfermedad de Crohn/patología , Estrés del Retículo Endoplásmico/fisiología , Enterocitos/fisiología , Absorción Intestinal/fisiología , Tripsina/metabolismo , Técnicas de Cultivo de Célula , Línea Celular , Colitis Ulcerosa/etiología , Colitis Ulcerosa/metabolismo , Enfermedad de Crohn/etiología , Enfermedad de Crohn/metabolismo , Humanos , Organoides , TapsigarginaRESUMEN
BACKGROUND: The opioid-mediated analgesic activity of mucosal CD4+ T lymphocytes in colitis has been reported in immunocompetent mice so far. Here, we investigated whether CD4+ T lymphocytes alleviate from inflammation-induced abdominal pain in mice with defective immune regulation. METHODS: Endogenous control of visceral pain by opioids locally produced in inflamed mucosa was assessed in IL-10-deficient mice. KEY RESULTS: CD4+ T lymphocytes but not F4/80+ macrophages isolated from the lamina propria of IL-10-deficient mice with colitis express enkephalin-containing opioid peptides as assessed by cytofluorometry. Colitis in IL-10-/- mice was not associated with abdominal pain. Intraperitoneal injection of naloxone-methiodide, a peripheral opioid receptor antagonist, induced abdominal hypersensitivity in IL-10-/- mice with colitis. CONCLUSION AND INFERENCES: Opioid-mediated analgesic activity of mucosal T lymphocytes remains operating in IL-10-/- mice with impaired immune regulation. The data suggest that endogenous T cell-derived opioids might reduce inflammation-induced abdominal pain in inflammatory bowel diseases associated with homozygous "loss of function mutations" in interleukin-10.
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Linfocitos T CD4-Positivos/inmunología , Interleucina-10/deficiencia , Mucosa Intestinal/inmunología , Péptidos Opioides/inmunología , Dolor Visceral/inmunología , Animales , Colitis/complicaciones , Colitis/inmunología , Inflamación/complicaciones , Inflamación/inmunología , Mucosa Intestinal/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Dolor Visceral/etiologíaRESUMEN
Background & Aims: Irritable bowel syndrome (IBS) is a multifactorial disease arising from a complex interplay between genetic predisposition and environmental influences. To date, environmental triggers are not well known. Aluminum is commonly present in food, notably by its use as food additive. We investigated the effects of aluminum ingestion in rodent models of visceral hypersensitivity, and the mechanisms involved. Methods: Visceral hypersensitivity was recorded by colorectal distension in rats administered with oral low doses of aluminum. Inflammation was analyzed in the colon of aluminum-treated rats by quantitative PCR for cytokine expression and by immunohistochemistry for immune cells quantification. Involvement of mast cells in the aluminum-induced hypersensitivity was determined by cromoglycate administration of rats and in mast cell-deficient mice (KitW-sh/W-sh). Proteinase-activated receptor-2 (PAR2) activation in response to aluminum was evaluated and its implication in aluminum-induced hypersensitivity was assessed in PAR2 knockout mice. Results: Orally administered low-dose aluminum induced visceral hypersensitivity in rats and mice. Visceral pain induced by aluminum persisted over time even after cessation of treatment, reappeared and was amplified when treatment resumed. As observed in humans, female animals were more sensitive than males. Major mediators of nociception were up-regulated in the colon by aluminum. Activation of mast cells and PAR2 were required for aluminum-induced hypersensitivity. Conclusions: These findings indicate that oral exposure to aluminum at human dietary level reproduces clinical and molecular features of IBS, highlighting a new pathway of prevention and treatment of visceral pain in some susceptible patients.
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Aluminio/toxicidad , Colon/patología , Hipersensibilidad/patología , Recto/patología , Administración Oral , Aluminio/administración & dosificación , Animales , Colon/efectos de los fármacos , Femenino , Inflamación/patología , Masculino , Mastocitos/efectos de los fármacos , Mastocitos/inmunología , Ratones Endogámicos C57BL , Ratones Noqueados , Nocicepción/efectos de los fármacos , Ratas Sprague-Dawley , Receptor PAR-2/metabolismo , Recto/efectos de los fármacos , Dolor Visceral/metabolismo , Dolor Visceral/patologíaRESUMEN
BACKGROUND: Endogenous opioids, including enkephalins, are fundamental regulators of pain. In inflammatory conditions, the local release of opioids by leukocytes at the inflammatory site inhibits nociceptor firing, thereby inducing analgesia. Accordingly, in chronic intestinal Th1/Th17-associated inflammation, enkephalins released by colitogenic CD4+ T lymphocytes relieve inflammation-induced visceral pain. The present study aims to investigate whether mucosal T-cell-derived enkephalins also exhibit a potent anti-inflammatory activity as described for exogenous opioid drugs in Th1/Th17-associated colitis. METHODS: The anti-inflammatory effects of endogenous opioids were investigated in both Th1/Th17-associated (transfer of CD4+CD45RBhigh T lymphocytes) and Th2-associated (oxazolone) colitis models in mice. Inflammation-induced colonic damage and CD4+ T cell subsets were compared in mice treated or not treated with naloxone methiodide, a peripheral antagonist of opioid receptors. The anti-inflammatory activity of T-cell-derived enkephalins was further estimated by comparison of colitis severity in immunodeficient mice into which naïve CD4+CD45RBhigh T lymphocytes originating from wild-type or enkephalin-knockout mice had been transferred. RESULTS: Peripheral opioid receptor blockade increases the severity of Th1/Th17-induced colitis and attenuates Th2 oxazolone colitis. The opposite effects of naloxone methiodide treatment in these two models of intestinal inflammation are dependent on the potency of endogenous opioids to promote a Th2-type immune response. Accordingly, the transfer of enkephalin-deficient CD4+CD45RBhigh T lymphocytes into immunodeficient mice exacerbates inflammation-induced colonic injury. CONCLUSIONS: Endogenous opioids, including T-cell-derived enkephalins, promote a Th2-type immune response, which, depending on the context, may either attenuate (Th1/Th17-associated) or aggravate (Th2-associated) intestinal inflammation.
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Colitis/inmunología , Encefalinas/inmunología , Células TH1/inmunología , Células Th17/inmunología , Animales , Linfocitos T CD4-Positivos/trasplante , Colitis/tratamiento farmacológico , Inmunidad Mucosa , Transfusión de Linfocitos , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Naloxona/uso terapéutico , Subgrupos de Linfocitos T/inmunología , Dolor Visceral/inmunologíaRESUMEN
T lymphocytes play a pivotal role in endogenous regulation of inflammatory visceral pain. The analgesic activity of T lymphocytes is dependent on their production of opioids, a property acquired on antigen activation. Accordingly, we investigated whether an active recruitment of T lymphocytes within inflamed colon mucosa via a local vaccinal strategy may counteract inflammation-induced visceral pain in mice. Mice were immunized against ovalbumin (OVA). One month after immunization, colitis was induced by adding 3% (wt/vol) dextran sulfate sodium into drinking water containing either cognate antigen OVA or control antigen bovine serum albumin for 5 days. Noncolitis OVA-primed mice were used as controls. Visceral sensitivity was then determined by colorectal distension. Oral administration of OVA but not bovine serum albumin significantly reduced dextran sulfate sodium-induced abdominal pain without increasing colitis severity in OVA-primed mice. Analgesia was dependent on local release of enkephalins by effector anti-OVA T lymphocytes infiltrating the inflamed mucosa. The experiments were reproduced with the bacillus Calmette-Guerin vaccine as antigen. Similarly, inflammatory visceral pain was dramatically alleviated in mice vaccinated against bacillus Calmette-Guerin and then locally administered with live Mycobacterium bovis. Together, these results show that the induction of a secondary adaptive immune response against vaccine antigens in inflamed mucosa may constitute a safe noninvasive strategy to relieve from visceral inflammatory pain.
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Linfocitos T CD4-Positivos/fisiología , Colitis/complicaciones , Colitis/etiología , Inmunización/efectos adversos , Membrana Mucosa/patología , Dolor Visceral , Animales , Antígenos CD11/genética , Antígenos CD11/metabolismo , Colitis/patología , Modelos Animales de Enfermedad , Encefalinas/deficiencia , Encefalinas/genética , Encefalinas/farmacología , Adyuvante de Freund/toxicidad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Ovalbúmina/efectos adversos , Precursores de Proteínas/deficiencia , Precursores de Proteínas/genética , Estadísticas no Paramétricas , Dolor Visceral/etiología , Dolor Visceral/inmunología , Dolor Visceral/patologíaRESUMEN
A number of studies have been dedicated to estimate the consequences on immunity of the clinical use of opioids by focusing on mitogen-induced polyclonal proliferation of T cells from blood or spleen. Here we examined, under physiological conditions, the contribution of endogenous opioids in the development of a CD4(+) T cell response within draining lymph nodes. We show in OVA-primed DO11.10 mice that delta-opioid receptors were up-regulated upon T cell activation in vivo and that opioid receptor neutralization increased the number of specific anti-OVA T lymphocytes without promoting their capacity to proliferate. The sensitivity to Fas-mediated apoptosis of T lymphocytes and the synthesis of homeostatic lymphoid chemokines were not either affected suggesting that opioids operate mainly before the entry of T lymphocytes into lymph nodes.
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Linfocitos T CD4-Positivos/inmunología , Proliferación Celular , Ganglios Linfáticos/citología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Opioides/fisiología , Animales , Linfocitos T CD4-Positivos/fisiología , Muerte Celular/efectos de los fármacos , Muerte Celular/inmunología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Femenino , Citometría de Flujo/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Genes MHC Clase II/genética , Inmunoglobulina G/farmacología , Ganglios Linfáticos/inmunología , Activación de Linfocitos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Naltrexona/farmacología , Antagonistas de Narcóticos/farmacología , Ovalbúmina/inmunología , Receptores de Antígenos de Linfocitos T/efectos de los fármacos , Receptores de Antígenos de Linfocitos T/genética , Receptor fas/inmunologíaRESUMEN
Effector CD4(+) T lymphocytes generated in response to antigens produce endogenous opioids. Thus, in addition to their critical role in host defenses against pathogens, effector CD4(+) T lymphocytes contribute to relieving inflammatory pain. In this study, we investigated mechanisms of opioid release by antigen-experienced effector CD4(+) T cells that leave draining lymph nodes and come back into the inflammatory site. Effector antigen-primed CD4(+) T lymphocytes generated in vitro were intravenously injected into nude mice previously immunized with either cognate or irrelevant antigens in complete Freund adjuvant (CFA). CFA-induced mechanical hyperalgesia was only reduced in mice immunized with cognate antigen. Thus, antinociceptive activity of effector CD4(+) T cells requires the presence of the antigen for which they are specific within the inflammatory site. Accordingly, analgesia was inhibited by neutralizing cognate T cell receptor-mediated interaction between effector CD4(+) T lymphocytes and antigen-presenting cells at the site of inflammation. Analgesia was observed by transferring effector CD4(+) T lymphocytes with Th1 or Th2 phenotype, suggesting that antinociceptive activity is a fundamental property of effector CD4(+) T lymphocytes irrespective of their effector functions. Based on the use of agonists and antagonists selective for each of the opioid receptor subclasses, we showed that analgesia induced by T cell-derived opioids is elicited via activation of δ-type opioid receptors in the periphery. Thus, the antinociceptive activity is a fundamental property associated with the effector phase of adaptive immunity, which is driven by recognition of the cognate antigen by effector CD4(+) T lymphocytes at the inflammatory site.
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Analgesia/métodos , Linfocitos T CD4-Positivos/metabolismo , Péptidos Opioides/metabolismo , Dolor/inmunología , Inmunidad Adaptativa , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Células Cultivadas , Encefalinas/sangre , Encefalinas/genética , Encefalinas/metabolismo , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Péptidos Opioides/sangre , Péptidos Opioides/genética , Dolor/metabolismo , Dolor/patología , Precursores de Proteínas/sangre , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismoRESUMEN
Endogenous opioid peptides mainly produced by neurons are also released by immune cells. They bind to mu- (mu-opioid receptor, MOR), delta-, and kappa-opioid receptors. On the basis of studies on mice showing that MOR is the main mediator of the deleterious effects of opioids on immunity, we wondered whether MOR, absent under normal conditions, is expressed in some pathological situations such as lymphomas. mRNA expression for all three opioid receptors was examined in lymph node biopsy samples from patients with non-Hodgkin's B-cell and T-cell lymphomas. We found that MOR and one of its ligands (enkephalin) are simultaneously expressed almost exclusively in lymph nodes from patients with Sézary cutaneous T cell lymphoma. As MOR was undetectable in circulating malignant T lymphocytes and in normal immune cells, we hypothesized that tumor-released cytokines might induce MOR expression in non-neoplastic lymph node cells. The correlation between mRNA levels of MOR and interleukin-13 (IL-13) within lymph nodes from Sézary patients led us to investigate the ability of IL-13 to upregulate MOR expression in normal immune cell subsets. We found that IL-13 upregulates MOR in activated Langerhans cells. Thus, our data suggest that, under pathological conditions, IL-13 overexpression might allow immune-derived endogenous opioids to down-modulate immune response.
Asunto(s)
Interleucina-13/genética , Receptores Opioides mu/genética , Síndrome de Sézary/inmunología , Síndrome de Sézary/fisiopatología , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/fisiopatología , Biopsia , Linfocitos T CD4-Positivos/patología , Linfocitos T CD4-Positivos/fisiología , Linfocitos T CD8-positivos/patología , Linfocitos T CD8-positivos/fisiología , Células Cultivadas , Células Dendríticas/patología , Células Dendríticas/fisiología , Expresión Génica/inmunología , Humanos , Tolerancia Inmunológica/fisiología , Interleucina-13/inmunología , Células de Langerhans/patología , Células de Langerhans/fisiología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Monocitos/patología , Monocitos/fisiología , ARN Mensajero/metabolismo , Receptores Opioides mu/inmunología , Síndrome de Sézary/patología , Neoplasias Cutáneas/patologíaRESUMEN
Fas (CD95)-induced hepatocyte apoptosis and cytotoxic activity of neutrophils infiltrating the injured liver are two major events leading to hepatitis. Because it has been reported that opioids, via a direct interaction, sensitize splenocytes to Fas-mediated apoptosis by upregulating Fas messenger RNA (mRNA) and modulated neutrophil activity, we assumed that opioids may participate in the pathophysiology of hepatitis. Using the hepatitis model induced by agonistic anti-Fas antibody in mice, we showed that opioid receptor blockade reduced liver damage and consequently increased the survival rate of animals when the antagonist naltrexone was injected simultaneously or prior to antibody administration. Treatment of mice with morphine enhanced mortality. Naloxone methiodide-a selective peripheral opioid antagonist-had a protective effect, but the absence of opioid receptors in the liver, together with lack of morphine effect in Fas-induced apoptosis of primary cultured hepatocytes, ruled out a direct effect of opioids on hepatocytes. In addition, the neutralization of opioid activity by naltrexone did not modify Fas mRNA expression in the liver as assessed with real-time quantitative polymerase chain reaction. Injured livers were infiltrated by neutrophils, but granulocyte-depleted mice were not protected against the enhancing apoptotic effect of morphine. In conclusion, opioid receptor blockade improves the resistance of mice to Fas-induced hepatitis via a peripheral mechanism that does not involve a down-modulation of Fas mRNA in hepatocytes nor a decrease in proinflammatory activity of neutrophils.
Asunto(s)
Hepatitis/etiología , Hepatitis/prevención & control , Naloxona/análogos & derivados , Antagonistas de Narcóticos , Receptor fas/metabolismo , Animales , Anticuerpos/farmacología , Células Cultivadas , Femenino , Hepatitis/mortalidad , Hepatitis/patología , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Endogámicos BALB C , Morfina/farmacología , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Narcóticos/farmacología , Compuestos de Amonio Cuaternario , ARN Mensajero/metabolismo , Receptor fas/genética , Receptor fas/inmunologíaRESUMEN
We previously observed the presence of anti-human mu-opioid-receptor (anti-hMOR) autoantibodies in IgG pools prepared from several thousand healthy blood donors. These autoantibodies behaved agonistically because of their ability to bind to the first and third extracellular loops of the receptor. In this study, we found that each healthy donor's serum contained anti-hMOR IgG autoantibodies with a specific activity against both the first and the third extracellular loops of the receptor. Because of the inability of IgG to cross the blood-brain barrier, we investigated the effects of the expression of anti-hMOR autoantibodies on immune cells. In analogy to studies of the effects of morphine, we investigated the ability of antibodies to sensitize splenocytes to Fas (CD95)-mediated apoptosis. We took advantage of the high sequence homology between murine MOR and hMOR extracellular loops to estimate the effect on murine splenocytes of anti-hMOR antibodies raised by immunizing mice. Splenocytes from mice injected with Chinese hamster ovary (CHO) cells expressing MOR were sensitized to Fas-mediated apoptosis, whereas those from mice injected with CHO cells or phosphate-buffered saline were not. Similar sensitization to Fas-mediated apoptosis was observed in splenocytes from mice undergoing passive transfer either with IgG from mice previously immunized against CHO cells expressing MOR or with IgG directed against the first and third extracellular loops of the receptor. Together, our data show that anti-MOR autoantibodies are commonly expressed in healthy humans and could participate in the control of lymphocyte homeostasis by promoting Fas-mediated apoptosis.