RESUMO
Importance: High-intensity, aerobically prepared fecal microbiota transplantation (FMT) has demonstrated efficacy in treating active ulcerative colitis (UC). FMT protocols involving anaerobic stool processing methods may enhance microbial viability and allow efficacy with a lower treatment intensity. Objective: To assess the efficacy of a short duration of FMT therapy to induce remission in UC using anaerobically prepared stool. Design, Setting, and Participants: A total of 73 adults with mild to moderately active UC were enrolled in a multicenter, randomized, double-blind clinical trial in 3 Australian tertiary referral centers between June 2013 and June 2016, with 12-month follow-up until June 2017. Interventions: Patients were randomized to receive either anaerobically prepared pooled donor FMT (n = 38) or autologous FMT (n = 35) via colonoscopy followed by 2 enemas over 7 days. Open-label therapy was offered to autologous FMT participants at 8 weeks and they were followed up for 12 months. Main Outcomes and Measures: The primary outcome was steroid-free remission of UC, defined as a total Mayo score of ≤2 with an endoscopic Mayo score of 1 or less at week 8. Total Mayo score ranges from 0 to 12 (0 = no disease and 12 = most severe disease). Steroid-free remission of UC was reassessed at 12 months. Secondary clinical outcomes included adverse events. Results: Among 73 patients who were randomized (mean age, 39 years; women, 33 [45%]), 69 (95%) completed the trial. The primary outcome was achieved in 12 of the 38 participants (32%) receiving pooled donor FMT compared with 3 of the 35 (9%) receiving autologous FMT (difference, 23% [95% CI, 4%-42%]; odds ratio, 5.0 [95% CI, 1.2-20.1]; P = .03). Five of the 12 participants (42%) who achieved the primary end point at week 8 following donor FMT maintained remission at 12 months. There were 3 serious adverse events in the donor FMT group and 2 in the autologous FMT group. Conclusions and Relevance: In this preliminary study of adults with mild to moderate UC, 1-week treatment with anaerobically prepared donor FMT compared with autologous FMT resulted in a higher likelihood of remission at 8 weeks. Further research is needed to assess longer-term maintenance of remission and safety. Trial Registration: anzctr.org.au Identifier: ACTRN12613000236796.
Assuntos
Colite Ulcerativa/terapia , Transplante de Microbiota Fecal , Adulto , Anaerobiose , Colonoscopia , Método Duplo-Cego , Enema , Transplante de Microbiota Fecal/efeitos adversos , Transplante de Microbiota Fecal/métodos , Feminino , Microbioma Gastrointestinal , Humanos , Masculino , Metaboloma , Pessoa de Meia-Idade , Indução de Remissão/métodos , Inquéritos e Questionários , Transplante Autólogo , Transplante Homólogo , Adulto JovemRESUMO
Inflammatory bowel disease (IBD) is characterized by chronic remitting and relapsing inflammation of the lower gastrointestinal tract. The etiology underlying IBD remains unknown, but it is thought to involve a hypersensitive immune response to environmental antigens, including the microbiota. Diagnosis and monitoring of IBD is heavily reliant on endoscopy, which is invasive and does not provide information regarding specific mediators. This review describes recent developments in imaging of IBD with a focus on positron emission tomography (PET) and single-photon emission computed tomography (SPECT) of inflammatory mediators, and how these developments may be applied to the microbiota.
Assuntos
Citocinas/análise , Imunoglobulina G/análise , Doenças Inflamatórias Intestinais/diagnóstico por imagem , Integrinas/análise , Intestinos/diagnóstico por imagem , Animais , Citocinas/genética , Citocinas/imunologia , Modelos Animais de Doenças , Endoscopia Gastrointestinal , Fluordesoxiglucose F18/farmacocinética , Microbioma Gastrointestinal/imunologia , Expressão Gênica , Humanos , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/microbiologia , Integrinas/genética , Integrinas/imunologia , Intestinos/imunologia , Intestinos/microbiologia , Camundongos , Tomografia por Emissão de Pósitrons , Coelhos , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
OBJECTIVE: Little is understood regarding how disease progression alters immune and sensory nerve function in colitis. We investigated how acute colitis chronically alters immune recruitment and the impact this has on re-activated colitis. To understand the impact of disease progress on sensory systems we investigated the mechanisms underlying altered colonic neuro-immune interactions after acute colitis. DESIGN: Inflammation was compared in mouse models of health, acute tri-nitrobenzene sulphonic acid (TNBS) colitis, Remission and Reactivated colitis. Cytokine concentrations were compared by ELISA in-situ and in explanted colon tissue. Colonic infiltration by CD11b/F4-80 macrophage, CD4 THELPER (TH) and CD8 TCYTOTOXIC (TC) and α4ß7 expression on mesenteric lymph node (MLN) TH and TC was determined by flow cytometry. Cytokine and effector receptor mRNA expression was determined on colo-rectal afferent neurons and the mechanisms underlying cytokinergic effects on high-threshold colo-rectal afferent function were investigated using electrophysiology. RESULTS: Colonic damage, MPO activity, macrophage infiltration, IL-1ß and IL-6 concentrations were lower in Reactivated compared to Acute colitis. TH infiltration and α4ß7 expression on TH MLN was increased in Remission but not Acute colitis. IFN-γ concentrations, TH infiltration and α4ß7 expression on TH and TC MLN increased in Reactivated compared to Acute colitis. Reactivated explants secreted more IL-1ß and IL-6 than Acute explants. IL-6 and TNF-α inhibited colo-rectal afferent mechanosensitivity in Remission mice via a BKCa dependent mechanism. CONCLUSIONS: Acute colitis persistently alters immune responses and afferent nerve signalling pathways to successive episodes of colitis. These findings highlight the complexity of viscero-sensory neuro-immune interactions in painful remitting and relapsing diseases.
Assuntos
Colite/imunologia , Colo/metabolismo , Inflamação/imunologia , Neurônios Aferentes/imunologia , Doença Aguda , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Opioids are one of the most prescribed drug classes for treating acute pain. However, chronic use is often associated with tolerance as well as debilitating side effects, including nausea and dependence, which are mediated by the central nervous system, as well as constipation emerging from effects on the enteric nervous system. These gastrointestinal (GI) side effects limit the usefulness of opioids in treating pain in many patients. Understanding the mechanism(s) of action of opioids on the nervous system that shows clinical benefit as well as those that have unwanted effects is critical for the improvement of opioid drugs. The opioidergic system comprises three classical receptors (µ, δ, κ) and a nonclassical receptor (nociceptin), and each of these receptors is expressed to varying extents by the enteric and intestinal extrinsic sensory afferent nerves. The purpose of this review is to discuss the role that the opioidergic system has on enteric and extrinsic afferent nerves in the lower GI tract in health and diseases of the lower GI tract, particularly inflammatory bowel disease and irritable bowel syndrome, and the implications of opioid treatment on clinical outcomes. Consideration is also given to emerging developments in our understanding of the immune system as a novel source of endogenous opioids and the mechanisms underlying opioid tolerance, including the potential influence of opioid receptor splice variants and heteromeric complexes.
Assuntos
Analgésicos Opioides/farmacologia , Gastroenteropatias/tratamento farmacológico , Trato Gastrointestinal Inferior/inervação , Receptores Opioides/metabolismo , Analgesia , HumanosRESUMO
Glycoprotein 130 (gp130) is the signal transducing receptor subunit for cytokines of the interleukin-6 (IL-6) family, and it is expressed in a multitude of cell types of the immune and nervous system. IL-6-like cytokines are not only key regulators of innate immunity and inflammation but are also essential factors for the differentiation and development of the somatosensory system. Mice with a null mutation of gp130 in primary nociceptive afferents (SNS-gp130(-/-)) are largely protected from hypersensitivity to mechanical stimuli in mouse models of pathological pain. Therefore, we set out to investigate how neuronal gp130 regulates mechanonociception. SNS-gp130(-/-) mice revealed reduced mechanosensitivity to high mechanical forces in the von Frey assay in vivo, and this was associated with a reduced sensitivity of nociceptive primary afferents in vitro. Together with these findings, transient receptor potential ankyrin 1 (TRPA1) mRNA expression was significantly reduced in DRG from SNS-gp130(-/-) mice. This was also reflected by a reduced number of neurons responding with calcium transients to TRPA1 agonists in primary DRG cultures. Downregulation of Trpa1 expression was predominantly discovered in nonpeptidergic neurons, with the deficit becoming evident during stages of early postnatal development. Regulation of Trpa1 mRNA expression levels downstream of gp130 involved the classical Janus kinase family-signal transducer and activator of transcription pathway. Our results closely link proinflammatory cytokines to the expression of TRPA1, both of which have been shown to contribute to hypersensitive pain states. We suggest that gp130 has an essential role in mechanonociception and in the regulation of TRPA1 expression.
Assuntos
Receptor gp130 de Citocina/deficiência , Regulação para Baixo/genética , Deleção de Genes , Mecanorreceptores/metabolismo , Células Receptoras Sensoriais/metabolismo , Canais de Potencial de Receptor Transitório/antagonistas & inibidores , Canais de Potencial de Receptor Transitório/genética , Animais , Células Cultivadas , Receptor gp130 de Citocina/antagonistas & inibidores , Receptor gp130 de Citocina/genética , Feminino , Masculino , Camundongos , Camundongos Knockout , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , Transdução de Sinais/genética , Canal de Cátion TRPA1 , Canais de Potencial de Receptor Transitório/biossínteseRESUMO
BACKGROUND & AIMS: Linaclotide is a minimally absorbed agonist of guanylate cyclase-C (GUCY2C or GC-C) that reduces symptoms associated with irritable bowel syndrome with constipation (IBS-C). Little is known about the mechanism by which linaclotide reduces abdominal pain in patients with IBS-C. METHODS: We determined the effects of linaclotide on colonic sensory afferents in healthy mice and those with chronic visceral hypersensitivity. We assessed pain transmission by measuring activation of dorsal horn neurons in the spinal cord in response to noxious colorectal distention. Levels of Gucy2c messenger RNA were measured in tissues from mice using quantitative reverse transcription polymerase chain reaction and in situ hybridization. We used human intestinal cell lines to measure release of cyclic guanosine-3',5'-monophosphate (cGMP) by linaclotide. We performed a post-hoc analysis of data from a phase III, double-blind, parallel-group study in which 805 patients with IBS-C were randomly assigned to groups given an oral placebo or 290 µg linaclotide once daily for 26 weeks. We quantified changes in IBS-C symptoms, including abdominal pain. RESULTS: In mice, linaclotide inhibited colonic nociceptors with greater efficacy during chronic visceral hypersensitivity. Intra-colonic administration of linaclotide reduced signaling of noxious colorectal distention to the spinal cord. The colonic mucosa, but not neurons, was found to express linaclotide's target, GC-C. The downstream effector of GC-C, cGMP, was released after administration of linaclotide and also inhibited nociceptors. The effects of linaclotide were lost in Gucy2c(-/-) mice and prevented by inhibiting cGMP transporters or removing the mucosa. During 26 weeks of linaclotide administration, a significantly greater percentage of patients (70%) had at least a 30% reduction in abdominal pain compared with patients given placebo (50%). CONCLUSIONS: We have identified an analgesic mechanism of linaclotide: it activates GC-C expressed on mucosal epithelial cells, resulting in the production and release of cGMP. This extracellular cGMP acts on and inhibits nociceptors, thereby reducing nociception. We also found that linaclotide reduces chronic abdominal pain in patients with IBS-C.
Assuntos
Dor Abdominal/prevenção & controle , Colo/inervação , GMP Cíclico/fisiologia , Guanilato Ciclase/fisiologia , Nociceptores/efeitos dos fármacos , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Dor Abdominal/induzido quimicamente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Células CACO-2 , Linhagem Celular , Colo/efeitos dos fármacos , Colo/patologia , Modelos Animais de Doenças , Método Duplo-Cego , Feminino , Humanos , Síndrome do Intestino Irritável/induzido quimicamente , Síndrome do Intestino Irritável/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Peptídeos Natriuréticos/farmacologia , Nociceptores/fisiologia , Receptores do Fator Natriurético Atrial/fisiologia , Receptores de Enterotoxina , Receptores Acoplados a Guanilato Ciclase/fisiologia , Receptores de Peptídeos/fisiologia , Resultado do Tratamento , Ácido Trinitrobenzenossulfônico/efeitos adversosRESUMO
Alterations in the neuro-immune axis contribute toward viscerosensory nerve sensitivity and symptoms in Irritable Bowel Syndrome (IBS). Inhibitory factors secreted from immune cells inhibit colo-rectal afferents in health, and loss of this inhibition may lead to hypersensitivity and symptoms. We aimed to determine the immune cell type(s) responsible for opioid secretion in humans and whether this is altered in patients with IBS. The ß-endorphin content of specific immune cell lineages in peripheral blood and colonic mucosal biopsies were compared between healthy subjects (HS) and IBS patients. Peripheral blood mononuclear cell (PBMC) supernatants from HS and IBS patients were applied to colo-rectal sensory afferent endings in mice with post-inflammatory chronic visceral hypersensitivity (CVH). ß-Endorphin was identified predominantly in monocyte/macrophages relative to T or B cells in human PBMC and colonic lamina propria. Monocyte derived ß-endorphin levels and colonic macrophage numbers were lower in IBS patients than healthy subjects. PBMC supernatants from healthy subjects had greater inhibitory effects on colo-rectal afferent mechanosensitivity than those from IBS patients. The inhibitory effects of PBMC supernatants were more prominent in CVH mice compared to healthy mice due to an increase in µ-opioid receptor expression in dorsal root ganglia neurons in CVH mice. Monocyte/macrophages are the predominant immune cell type responsible for ß-endorphin secretion in humans. IBS patients have lower monocyte derived ß-endorphin levels than healthy subjects, causing less inhibition of colonic afferent endings. Consequently, altered immune function contributes toward visceral hypersensitivity in IBS.
Assuntos
Colo/imunologia , Síndrome do Intestino Irritável/imunologia , Leucócitos Mononucleares/metabolismo , Células Receptoras Sensoriais/imunologia , beta-Endorfina/metabolismo , Adulto , Animais , Colo/metabolismo , Colo/fisiopatologia , Feminino , Humanos , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiopatologia , Síndrome do Intestino Irritável/metabolismo , Síndrome do Intestino Irritável/fisiopatologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Mastócitos/imunologia , Mastócitos/metabolismo , Camundongos , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/metabolismo , Células Receptoras Sensoriais/metabolismoRESUMO
OBJECTIVE: The gut is a major site of contact between immune and sensory systems and evidence suggests that patients with irritable bowel syndrome (IBS) have immune dysfunction. Here we show how this dysfunction differs between major IBS subgroups and how immunocytes communicate with sensory nerves. DESIGN: Peripheral blood mononuclear cell supernatants from 20 diarrhoea predominant IBS (D-IBS) patients, 15 constipation predominant IBS (C-IBS) patients and 36 healthy subjects were applied to mouse colonic sensory nerves and effects on mechanosensitivity assessed. Cytokine/chemokine concentration in the supernatants was assessed by proteomic analysis and correlated with abdominal symptoms, and expression of cytokine receptors evaluated in colonic dorsal root ganglia neurons. We then determined the effects of specific cytokines on colonic afferents. RESULTS: D-IBS supernatants caused mechanical hypersensitivity of mouse colonic afferent endings, which was reduced by infliximab. C-IBS supernatants did not, but occasionally elevated basal discharge. Supernatants of healthy subjects inhibited afferent mechanosensitivity via an opioidergic mechanism. Several cytokines were elevated in IBS supernatants, and levels correlated with pain frequency and intensity in patients. Visceral afferents expressed receptors for four cytokines: IL-1ß, IL-6, IL-10 and TNF-α. TNF-α most effectively caused mechanical hypersensitivity which was blocked by a transient receptor potential channel TRPA1 antagonist. IL-1ß elevated basal firing, and this was lost after tetrodotoxin blockade of sodium channels. CONCLUSIONS: Distinct patterns of immune dysfunction and interaction with sensory pathways occur in different patient groups and through different intracellular pathways. Our results indicate IBS patient subgroups would benefit from selective targeting of the immune system.
Assuntos
Síndrome do Intestino Irritável/imunologia , Neuroimunomodulação/fisiologia , Adulto , Animais , Estudos de Casos e Controles , Células Cultivadas , Colo/imunologia , Colo/inervação , Constipação Intestinal/etiologia , Constipação Intestinal/imunologia , Meios de Cultivo Condicionados/farmacologia , Citocinas/biossíntese , Diarreia/etiologia , Diarreia/imunologia , Feminino , Gânglios Espinais/imunologia , Humanos , Síndrome do Intestino Irritável/complicações , Síndrome do Intestino Irritável/fisiopatologia , Leucócitos Mononucleares/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Neuroimunomodulação/imunologia , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/fisiologia , Dor/etiologia , Dor/imunologia , Receptores de Citocinas/metabolismo , beta-Endorfina/metabolismoRESUMO
Irritable bowel syndrome (IBS) is a functional disorder of the gastrointestinal (GI) tract characterized by pain or discomfort from the lower abdominal region, which is associated with altered bowel habit. Despite its prevalence, there is currently a lack of effective treatment options for patients. IBS has long been considered as a neurological condition resulting from alterations in the brain gut axis, but immunological alterations are increasingly reported in IBS patients, consistent with the hypothesis that there is a chronic, but low-grade, immune activation. Mediators released by immune cells act to either dampen or amplify the activity of GI nerves. Release of a number of these mediators correlates with symptoms of IBS, highlighting the importance of interactions between the immune and the nervous systems. Investigation of the role of microbiota in these interactions is in its early stages, but may provide many answers regarding the mechanisms underlying activation of the immune system in IBS. Identifying what the key changes in the GI immune system are in IBS and how these changes modulate viscerosensory nervous function is essential for the development of novel therapies for the underlying disorder.
Assuntos
Imunidade Adaptativa , Colo/inervação , Imunidade Inata , Síndrome do Intestino Irritável/imunologia , Neuroimunomodulação , Células Apresentadoras de Antígenos/imunologia , Linfócitos B/imunologia , Trato Gastrointestinal/microbiologia , Humanos , Síndrome do Intestino Irritável/microbiologia , Mastócitos/imunologia , Metagenoma , Linfócitos T/imunologiaAssuntos
Interleucina-6 , Pâncreas , Dor Abdominal , Humanos , Interleucinas , Neoplasias Pancreáticas , Células de SchwannRESUMO
The mechanosensory role of TRPA1 and its contribution to mechanical hypersensitivity in sensory neurons remains enigmatic. We elucidated this role by recording mechanically activated currents in conjunction with TRPA1 over- and under-expression and selective pharmacology. First, we established that TRPA1 transcript, protein and functional expression are more abundant in smaller-diameter neurons than larger-diameter neurons, allowing comparison of two different neuronal populations. Utilising whole cell patch clamping, we applied calibrated displacements to neurites of dorsal root ganglion (DRG) neurons in short-term culture and recorded mechanically activated currents termed intermediately (IAMCs), rapidly (RAMCs) or slowly adapting (SAMCs). Trpa1 deletion (/) significantly reduced maximum IAMC amplitude by 43% in small-diameter neurons compared with wild-type (+/+) neurons. All other mechanically activated currents in small- and large-diameter Trpa1−/− neurons were unaltered. Seventy-three per cent of Trpa1+/+ small-diameter neurons responding to the TRPA1 agonist allyl-isothiocyanate (AITC) displayed IAMCs to neurite displacement, which were significantly enhanced after AITC addition. The TRPA1 antagonist HC-030031 significantly decreased Trpa1+/+ IAMC amplitudes, but only in AITC responsive neurons. Using a transfection system we also showed TRPA1 over-expression in Trpa1+/+ small-diameter neurons increases IAMC amplitude, an effect reversed by HC-030031. Furthermore, TRPA1 introduction into Trpa1−/− small-diameter neurons restored IAMC amplitudes to Trpa1+/+ levels, which was subsequently reversed by HC-030031. In summary our data demonstrate TRPA1 makes a contribution to normal mechanosensation in a specific subset of DRG neurons. Furthermore, they also provide new evidence illustrating mechanisms by which sensitisation or over-expression of TRPA1 enhances nociceptor mechanosensitivity. Overall, these findings suggest TRPA1 has the capacity to tune neuronal mechanosensitivity depending on its degree of activation or expression.
Assuntos
Potenciais de Ação/fisiologia , Gânglios Espinais/fisiologia , Hiperalgesia/fisiopatologia , Células Receptoras Sensoriais/fisiologia , Canais de Potencial de Receptor Transitório/fisiologia , Acetanilidas/farmacologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/genética , Animais , Células Cultivadas , Gânglios Espinais/efeitos dos fármacos , Isotiocianatos/farmacologia , Mecanotransdução Celular/efeitos dos fármacos , Mecanotransdução Celular/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas de Patch-Clamp/métodos , Purinas/farmacologia , Células Receptoras Sensoriais/efeitos dos fármacos , Canal de Cátion TRPA1 , Canais de Potencial de Receptor Transitório/agonistas , Canais de Potencial de Receptor Transitório/antagonistas & inibidoresRESUMO
Gastrointestinal inflammation is a hallmark of highly prevalent disorders, including cancer treatment-induced mucositis and ulcerative colitis. These disorders cause debilitating symptoms, have a significant impact on quality of life, and are poorly managed. The activation of toll-like receptor 4 (TLR4) has been proposed to have a major influence on the inflammatory signalling pathways of the intestinal tract. Inhibition of TLR4 has been postulated as an effective way to treat intestinal inflammation. However, there are a limited number of studies looking into the potential of TLR4 antagonism as a therapeutic approach for intestinal inflammation. This review surveyed available literature and reported on the in vitro, ex vivo and in vivo effects of TLR4 antagonism on different models of intestinal inflammation. Of the studies reviewed, evidence suggests that there is indeed potential for TLR4 antagonists to treat inflammation, although only a limited number of studies have investigated treating intestinal inflammation with TLR4 antagonists directly. These results warrant further research into the effect of TLR4 antagonists in the intestinal tract.
Assuntos
Anti-Inflamatórios/farmacologia , Fármacos Gastrointestinais/farmacologia , Doenças Inflamatórias Intestinais/tratamento farmacológico , Intestinos/efeitos dos fármacos , Receptor 4 Toll-Like/antagonistas & inibidores , Animais , Humanos , Inflamação , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND & AIMS: The transient receptor potential (TRP) channel family includes transducers of mechanical and chemical stimuli for visceral sensory neurons. TRP ankyrin 1 (TRPA1) is implicated in inflammatory pain; it interacts with G-protein-coupled receptors, but little is known about its role in the gastrointestinal (GI) tract. Sensory information from the GI tract is conducted via 5 afferent subtypes along 3 pathways. METHODS: Nodose and dorsal root ganglia whose neurons innnervate 3 different regions of the GI tract were analyzed from wild-type and TRPA1(-/-) mice using quantitative reverse-transcription polymerase chain reaction, retrograde labeling, and in situ hybridization. Distal colon sections were analyzed by immunohistochemistry. In vitro electrophysiology and pharmacology studies were performed, and colorectal distension and visceromotor responses were measured. Colitis was induced by administration of trinitrobenzene sulphonic acid. RESULTS: TRPA1 is required for normal mechano- and chemosensory function in specific subsets of vagal, splanchnic, and pelvic afferents. The behavioral responses to noxious colonic distension were substantially reduced in TRPA1(-/-) mice. TRPA1 agonists caused mechanical hypersensitivity, which increased in mice with colitis. Colonic afferents were activated by bradykinin and capsaicin, which mimic effects of tissue damage; wild-type and TRPA1(-/-) mice had similar direct responses to these 2 stimuli. After activation by bradykinin, wild-type afferents had increased mechanosensitivity, whereas, after capsaicin exposure, mechanosensitivity was reduced: these changes were absent in TRPA1(-/-) mice. No interaction between protease-activated receptor-2 and TRPA1 was evident. CONCLUSIONS: These findings demonstrate a previously unrecognized role for TRPA1 in normal and inflamed mechanosensory function and nociception within the viscera.
Assuntos
Colite/metabolismo , Colo/metabolismo , Gânglios Espinais/metabolismo , Hiperalgesia/metabolismo , Mecanotransdução Celular , Gânglio Nodoso/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Potenciais de Ação , Vias Aferentes/metabolismo , Vias Aferentes/fisiopatologia , Animais , Bradicinina/farmacologia , Capsaicina/farmacologia , Colite/induzido quimicamente , Colite/fisiopatologia , Colo/efeitos dos fármacos , Colo/inervação , Colo/fisiopatologia , Modelos Animais de Doenças , Feminino , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/fisiopatologia , Hiperalgesia/induzido quimicamente , Hiperalgesia/fisiopatologia , Imuno-Histoquímica , Hibridização In Situ , Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiopatologia , Masculino , Mecanotransdução Celular/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas de Rastreamento Neuroanatômico , Gânglio Nodoso/efeitos dos fármacos , Gânglio Nodoso/fisiopatologia , Medição da Dor , Pelve/inervação , Pressão , RNA Mensageiro/metabolismo , Receptor PAR-2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Nervos Esplâncnicos/metabolismo , Nervos Esplâncnicos/fisiopatologia , Estimulação Química , Canal de Cátion TRPA1 , Canais de Potencial de Receptor Transitório/deficiência , Canais de Potencial de Receptor Transitório/genética , Ácido TrinitrobenzenossulfônicoRESUMO
Intestinal fibrosis is a common complication of inflammatory bowel disease but remains difficult to detect. Matrix metalloproteases (MMPs) have key roles in fibrosis and are therefore potential targets for fibrosis detection. We determined whether immunoPET of F(ab')2 antibody fragments targeting MMPs detects colitis induced colonic fibrosis. Mice were administered 2% dextran sulfate sodium treated water for 1 cycle (inflamed) or 3 cycles (fibrotic), or were untreated (control). Colonic and kidney collagen, innate cytokine, MMPs and fecal MPO concentrations were analyzed by multiplex/ELISA. α-pro-MMP-9 F(ab')2 fragments were engineered and conjugated to 89Zr for PET imaging, ex-vivo Cherenkov analysis and bio-distribution. Colonic innate cytokine concentrations and fecal myeloperoxidase were increased in inflamed mice but not fibrotic mice, while collagen concentrations were increased in fibrotic mice. MMPs were increased in inflamed mice, but only pro-MMP-9 remained increased in fibrotic mice. 89Zr-pro-MMP-9 F(ab')2 uptake was increased in the intestine but also in the kidney of fibrotic mice, where collagen and pro-MMP-9 concentrations were increased. 89Zr-pro-MMP-9 F(ab')2 detects colitis induced intestinal fibrosis and associated kidney fibrosis.
Assuntos
Colite/diagnóstico por imagem , Colo/diagnóstico por imagem , Precursores Enzimáticos/genética , Fragmentos Fab das Imunoglobulinas/química , Rim/diagnóstico por imagem , Metaloproteinase 9 da Matriz/genética , Animais , Colite/induzido quimicamente , Colite/genética , Colite/metabolismo , Colágeno/genética , Colágeno/metabolismo , Colo/metabolismo , Colo/patologia , Precursores Enzimáticos/metabolismo , Fezes/química , Fibrose , Expressão Gênica , Fragmentos Fab das Imunoglobulinas/metabolismo , Rim/metabolismo , Rim/patologia , Imageamento por Ressonância Magnética , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Peroxidase/genética , Peroxidase/metabolismo , Tomografia por Emissão de Pósitrons , Radioisótopos/química , Dodecilsulfato de Sódio/administração & dosagem , Zircônio/químicaRESUMO
BACKGROUND & AIMS: Although there are many candidates as molecular mechanotransducers, so far there has been no evidence for molecular specialization of visceral afferents. Here, we show that colonic afferents express a specific molecular transducer that underlies their specialized mechanosensory function: the transient receptor potential channel, vanilloid 4 (TRPV4). METHODS: We found TRPV4 mRNA is highly enriched in colonic sensory neurons compared with other visceral and somatic sensory neurons. TRPV4 protein was found in colonic nerve fibers from patients with inflammatory bowel disease, and it colocalized in a subset of fibers with the sensory neuropeptide CGRP in mice. We characterized the responses of 8 subtypes of vagal, splanchnic, and pelvic mechanoreceptors. RESULTS: Mechanosensory responses of colonic serosal and mesenteric afferents were enhanced by a TRPV4 agonist and dramatically reduced by targeted deletion of TRPV4 or by a TRP antagonist. Other subtypes of vagal and pelvic afferents, by contrast, were unaffected by these interventions. The behavioral responses to noxious colonic distention were also substantially reduced in mice lacking TRPV4. CONCLUSIONS: These data indicate that TRPV4 contributes to mechanically evoked visceral pain, with relevance to human disease. In view of its distribution in favor of specific populations of visceral afferents, we propose that TRPV4 may present a selective novel target for the reduction of visceral pain, which is an important opportunity in the absence of current treatments.
Assuntos
Colo/inervação , Sistema Nervoso Entérico/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Mecanotransdução Celular , Dor/etiologia , Sensação , Canais de Cátion TRPV/metabolismo , Vias Aferentes/metabolismo , Animais , Comportamento Animal , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Cateterismo , Sistema Nervoso Entérico/fisiopatologia , Feminino , Humanos , Doenças Inflamatórias Intestinais/complicações , Doenças Inflamatórias Intestinais/fisiopatologia , Masculino , Mecanorreceptores/metabolismo , Camundongos , Camundongos Knockout , Modelos Animais , Neurônios Aferentes/metabolismo , Dor/metabolismo , Dor/fisiopatologia , Medição da Dor , Limiar da Dor , RNA Mensageiro/metabolismo , Canais de Cátion TRPV/deficiência , Canais de Cátion TRPV/genéticaRESUMO
Inflammatory bowel disease (IBD) is a chronic relapsing and remitting inflammatory disease of the gastrointestinal tract. The diagnosis and monitoring of IBD are reliant on endoscopy, which is invasive and does not provide information on specific mediators. Symptom flare in IBD is associated with increased activation of innate immune pathways. Immuno-PET approaches have previously demonstrated the ability to detect colitis; however, a direct comparison of antibodies targeted to innate immune mediators and cells has not been done. We aimed to compare immuno-PET of antibodies to IL-1ß and CD11b against standard 18F-FDG and MRI approaches to detect colonic inflammation. Methods: Colonic concentrations of IL-1ß and myeloperoxidase were determined by ELISA, and colonic infiltration by CD11b-positive CD3-negative innate immune cells was determined by flow cytometry and compared between healthy and dextran sodium sulphate-treated colitic mice. PET of 89Zr-lα-IL-1ß, 89Zr-α-CD11b, and 18F-FDG was compared by volume-of-interest analysis and with MRI by region-of-interest analysis. Imaging results were confirmed by ex vivo biodistribution analysis. Results: Colonic inflammation was associated with impaired colonic epithelial barrier permeability, increased colonic IL-1ß and myeloperoxidase concentrations, and increased CD11b-positive CD3-negative innate immune cell infiltration into the colon. 89Zr-α-IL-1ß and 89Zr-α-CD11b immuno-PET detected colonic inflammation, as did 18F-FDG, and all PET tracers were more sensitive than MRI. Although 18F-FDG volumes of interest correlated with colitis severity and a strong trend was observed with 89Zr-α-IL-1ß, no correlation was observed for 89Zr-α-CD11b or MRI. 89Zr-α-IL-1ß was distributed mainly to the gastrointestinal tract, whereas 89Zr-α-CD11b was distributed to more tissue types. Conclusion: Immuno-PET using antibodies directed to innate immune markers detected colonic inflammation, with 89Zr-α-IL-1ß providing a more tissue-specific signal than 89Zr-α-CD11b. Development of these technologies for human subjects will potentially provide a less invasive approach than endoscopy for diagnosing and monitoring IBD.
Assuntos
Antígeno CD11b/metabolismo , Colite/diagnóstico por imagem , Imunidade Inata , Interleucina-1beta/metabolismo , Tomografia por Emissão de Pósitrons , Animais , Transporte Biológico , Biomarcadores/metabolismo , Antígeno CD11b/imunologia , Colite/imunologia , Colite/metabolismo , Modelos Animais de Doenças , Fluordesoxiglucose F18 , Imunoconjugados/imunologia , Imunoconjugados/metabolismo , Imunoconjugados/farmacocinética , Inflamação/diagnóstico por imagem , Interleucina-1beta/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Radioisótopos , Distribuição Tecidual , ZircônioRESUMO
BACKGROUND: Inflammatory bowel disease (IBD) has a remitting and relapsing disease course; however, relatively little is understood regarding how inflammatory damage in acute colitis influences the microbiota, epithelial barrier, and immune function in subsequent colitis. METHODS: Mice were administered trinitrobenzene sulphonic acid (TNBS) via enema, and inflammation was assessed 2 days (d2) or 28 days (d28) later. Colitis was reactivated in some mice by re-treating at 28 days with TNBS and assessing 2 days later (d30). Epithelial responsiveness to secretagogues, microbiota composition, colonic infiltration, and immune activation was compared between all groups. RESULTS: At day 28, the distal colon had healed, mucosa was restored, and innate immune response had subsided, but colonic transepithelial transport (P = 0.048), regulatory T-cell (TREG) infiltration (P = 0.014), adherent microbiota composition (P = 0.0081), and responsiveness of stimulated innate immune bone marrow cells (P < 0.0001 for IL-1ß) differed relative to health. Two days after subsequent instillation of TNBS (d30 mice), the effects on inflammatory damage (P < 0.0001), paracellular permeability (P < 0.0001), and innate immune infiltration (P < 0.0001 for Ly6C+ Ly6G- macrophages) were reduced relative to d2 colitis. However, TREG infiltration was increased (P < 0.0001), and the responsiveness of stimulated T cells in the mesenteric lymph nodes shifted from pro-inflammatory at d2 to immune-suppressive at d30 (P < 0.0001 for IL-10). These effects were observed despite similar colonic microbiota composition and degradation of the mucosal layer between d2 and d30. CONCLUSIONS: Collectively, these results indicate that acute colitis chronically alters epithelial barrier function and both innate and adaptive immune responses. These effects reduce the consequences of a subsequent colitis event, warranting longitudinal studies in human IBD subjects.
Assuntos
Imunidade Adaptativa/imunologia , Permeabilidade da Membrana Celular/imunologia , Colite/complicações , Epitélio/patologia , Tolerância Imunológica/imunologia , Imunidade Inata/imunologia , Inflamação/etiologia , Doença Aguda , Animais , Colite/induzido quimicamente , Colite/patologia , Modelos Animais de Doenças , Epitélio/imunologia , Epitélio/lesões , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ácido Trinitrobenzenossulfônico/toxicidadeRESUMO
BACKGROUND: Inflammatory Bowel Disease (IBD) is characterized by overt inflammation of the intestine and is typically accompanied by symptoms of bloody diarrhea, abdominal pain and cramping. The Colonic Migrating Motor Complex (CMMC) directs the movement of colonic luminal contents over long distances. The tri-nitrobenzene sulphonic acid (TNBS) model of colitis causes inflammatory damage to enteric nerves, however it remains to be determined whether these changes translate to functional outcomes in CMMC activity. We aimed to visualize innate immune cell infiltration into the colon using two-photon laser scanning intra-vital microscopy, and to determine whether CMMC activity is altered in the tri-nitro benzene sulphonic (TNBS) model of colitis. METHODS: Epithelial barrier permeability was compared between TNBS treated and healthy control mice in-vitro and in-vivo. Innate immune activation was determined by ELISA, flow cytometry and by 2-photon intravital microscopy. The effects of TNBS treatment and IL-1ß on CMMC function were determined using a specialized organ bath. RESULTS: TNBS colitis increased epithelial barrier permeability in-vitro and in-vivo. Colonic IL-1ß concentrations, colonic and systemic CD11b+ cell infiltration, and the number of migrating CD11b+ cells on colonic blood vessels were all increased in TNBS treated mice relative to controls. CMMC frequency and amplitude were inhibited in the distal and mid colon of TNBS treated mice. CMMC activity was not altered by superfusion with IL-1ß. CONCLUSIONS: TNBS colitis damages the epithelial barrier and increases innate immune cell activation in the colon and systemically. Innate cell migration into the colon is readily identifiable by two-photon intra-vital microscopy. CMMC are inhibited by inflammation, but this is not due to direct effects of IL-1ß.