RESUMEN
BACKGROUND AND AIMS: Contact with distinct microbiota early in life has been shown to educate the mucosal immune system, hence providing protection against immune-mediated diseases. However, the impact of early versus late colonization with regard to the development of the intestinal macrophage compartment has not been studied so far. METHODS: Germ-free mice were colonized with specific-pathogen-free [SPF] microbiota at the age of 5 weeks. The ileal and colonic macrophage compartment were analysed by immunohistochemistry, flow cytometry, and RNA sequencing 1 and 5 weeks after colonization and in age-matched SPF mice, which had had contact with microbiota since birth. To evaluate the functional differences, dextran sulfate sodium [DSS]-induced colitis was induced, and barrier function analyses were undertaken. RESULTS: Germ-free mice were characterized by an atrophied intestinal wall and a profoundly reduced number of ileal macrophages. Strikingly, morphological restoration of the intestine occurred within the first week after colonization. In contrast, ileal macrophages required 5 weeks for complete restoration, whereas colonic macrophages were numerically unaffected. However, following DSS exposure, the presence of microbiota was a prerequisite for colonic macrophage infiltration. One week after colonization, mild colonic inflammation was observed, paralleled by a reduced inflammatory response after DSS treatment, in comparison with SPF mice. This attenuated inflammation was paralleled by a lack of TNFα production of LPS-stimulated colonic macrophages from SPF and colonized mice, suggesting desensitization of colonized mice by the colonization itself. CONCLUSIONS: This study provides the first data indicating that after colonization of adult mice, the numeric, phenotypic, and functional restoration of the macrophage compartment requires the presence of intestinal microbiota and is time dependent.
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Microbioma Gastrointestinal , Íleon/inmunología , Macrófagos/fisiología , Factores de Edad , Animales , Colitis/inducido químicamente , Colitis/inmunología , Colitis/microbiología , Colitis/patología , Sulfato de Dextran/farmacología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Microbioma Gastrointestinal/genética , Microbioma Gastrointestinal/inmunología , Vida Libre de Gérmenes , Íleon/citología , Íleon/microbiología , Íleon/patología , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Masculino , Ratones , Ratones Endogámicos C3H , ARN Ribosómico 16S/genética , Organismos Libres de Patógenos EspecíficosRESUMEN
Tubular cells recruit monocytic cells in inflammatory tubulointerstitial kidney diseases. The cell-cell communication that establishes pro- or anti-inflammatory activities is mainly influenced by cytokines, reactive oxygen species, nitric oxide, and phagocytosis. Key proteins orchestrating these processes such as cold-shock proteins linked with chemoattraction and cell maturation have been identified. The prototypic member of the cold-shock protein family, Y-box binding protein (YB)-1, governs specific phenotypic alterations in monocytic cells and was explored in the present study. Following tubulointerstitial injury by unilateral ureteral obstruction, increased inflammatory cell infiltration and tubular cell CCL5 expression was found in conditional Ybx1 knockout animals with specific depletion in monocytes/macrophages (YB-1ΔLysM). Furthermore, YB-1ΔLysM mice exhibit enhanced tissue damage, myofibroblast activation, and fibrosis. To investigate relevant molecular mechanism(s), we utilized bone marrow-derived macrophage cultures and found that YB-1-deficient macrophages display defects in cell polarization and function, including reduced proliferation and nitric oxide production, loss of phagocytic activity, and failure to upregulate IL-10 and CCL5 expression in response to inflammatory stimuli. Co-culture with primary tubular cells confirmed these findings. Thus, monocytic YB-1 has prominent and distinct roles for cellular feed-forward crosstalk and resolution of inflammatory processes by its ability to regulate cell differentiation and cytokine/chemokine synthesis.
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Diferenciación Celular , Proteínas de Unión al ADN/metabolismo , Túbulos Renales/patología , Monocitos/patología , Nefritis Intersticial/patología , Animales , Comunicación Celular , Quimiocina CCL5/metabolismo , Técnicas de Cocultivo , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Fibrosis , Humanos , Interleucina-10/metabolismo , Túbulos Renales/citología , Macrófagos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/metabolismo , Miofibroblastos/metabolismo , Miofibroblastos/patología , Cultivo Primario de CélulasRESUMEN
BACKGROUND: During the inflammation which occurs following nerve damage, macrophages are recruited to the site of injury. Phenotypic diversity is a hallmark of the macrophage lineage and includes pro-inflammatory M1 and anti-inflammatory M2 populations. Our aim in this study was to investigate the ability of polarized M0, M1, and M2 macrophages to secrete opioid peptides and to examine their relative contribution to the modulation of neuropathic pain. METHODS: Mouse bone marrow-derived cells were cultured as unstimulated M0 macrophages or were stimulated into an M1 phenotype using lipopolysaccharide and interferon-γ or into an M2 phenotype using interleukin-4. The macrophage phenotypes were verified using flow cytometry for surface marker analysis and cytokine bead array for cytokine profile assessment. Opioid peptide levels were measured by radioimmunoassay and enzyme immunoassay. As a model of neuropathic pain, a chronic constriction injury (CCI) of the sciatic nerve was employed. Polarized M0, M1, and M2 macrophages (5 × 105 cells) were injected perineurally twice, on days 14 and 15 following CCI or sham surgery. Mechanical and heat sensitivity were measured using the von Frey and Hargreaves tests, respectively. To track the injected macrophages, we also transferred fluorescently stained polarized cells and analyzed the surface marker profile of endogenous and injected cells in the nerves ex vivo. RESULTS: Compared to M0 and M1 cells, M2 macrophages contained and released higher amounts of opioid peptides, including Met-enkephalin, dynorphin A (1-17), and ß-endorphin. M2 cells transferred perineurally at the nerve injury site reduced mechanical, but not heat hypersensitivity following the second injection. The analgesic effect was reversed by the perineurally applied opioid receptor antagonist naloxone methiodide. M2 cells did not affect sensitivity following sham surgery. Neither M0 nor M1 cells altered mechanical and heat sensitivity in CCI or sham-operated animals. Tracing the fluorescently labeled M0, M1, and M2 cells ex vivo showed that they remained in the nerve and preserved their phenotype. CONCLUSIONS: Perineural transplantation of M2 macrophages resulted in opioid-mediated amelioration of neuropathy-induced mechanical hypersensitivity, while M1 macrophages did not exacerbate pain. Therefore, rather than focusing on macrophage-induced pain generation, promoting opioid-mediated M2 actions may be more relevant for pain control.
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Traslado Adoptivo/métodos , Citocinas/metabolismo , Macrófagos/fisiología , Neuralgia/inmunología , Neuralgia/patología , Péptidos Opioides/metabolismo , Aciltransferasas/metabolismo , Animales , Polaridad Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Dinorfinas/metabolismo , Citometría de Flujo , Antígenos de Histocompatibilidad Clase II/metabolismo , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatología , Lipopolisacáridos/farmacología , Macrófagos/clasificación , Macrófagos/efectos de los fármacos , Macrófagos/trasplante , Masculino , Ratones , Ratones Endogámicos C57BL , Umbral del Dolor/fisiología , Estimulación Física , betaendorfina/metabolismoRESUMEN
The intestinal microbiota is involved in many physiological processes and it is increasingly recognized that differences in community composition can influence the outcome of a variety of murine models used in biomedical research. In an effort to describe and account for the variation in intestinal microbiota composition across the animal facilities of participating members of the DFG Priority Program 1656 "Intestinal Microbiota", we performed a survey of C57BL/6J mice from 21 different mouse rooms/facilities located at 13 different institutions across Germany. Fresh feces was sampled from five mice per room/facility using standardized procedures, followed by extraction and 16S rRNA gene profiling (V1-V2 region, Illumina MiSeq) at both the DNA and RNA (reverse transcribed to cDNA) level. In order to determine the variables contributing to bacterial community differences, we collected detailed questionnaires of animal husbandry practices and incorporated this information into our analyses. We identified considerable variation in a number of descriptive aspects including the proportions of major phyla, alpha- and beta diversity, all of which displayed significant associations to specific aspects of husbandry. Salient findings include a reduction in alpha diversity with the use of irradiated chow, an increase in inter-individual variability (beta diversity) with respect to barrier access and open cages and an increase in bacterial community divergence with time since importing from a vendor. We further observe a high degree of facility-level individuality, which is likely due to each facility harboring its own unique combination of multiple varying attributes of animal husbandry. While it is important to account and control for such differences between facilities, the documentation of such diversity may also serve as a valuable future resource for investigating the origins of microbial-driven host phenotypes.
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Crianza de Animales Domésticos/métodos , Heces/microbiología , Microbioma Gastrointestinal , Animales , Análisis por Conglomerados , ADN Ribosómico/química , ADN Ribosómico/genética , Alemania , Masculino , Ratones Endogámicos C57BL , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Encuestas y CuestionariosRESUMEN
BACKGROUND: Macrophages are key players in inflammatory bowel diseases (IBD). This study aimed to determine site-specific effects of defined macrophage subtypes on the integrity of the intestinal epithelial barrier. METHODS: Macrophage subtypes in situ in intestinal specimens of patients with IBD were visualized by immunohistochemistry. In vitro polarization of human peripheral CD14 cells yielded M1 or M2 macrophages. The influence of primary monocytes or macrophage subtypes on epithelial barrier integrity was analyzed by transepithelial resistance measurements, Western blot analysis, confocal laser scanning microscopy, and cytometric bead array in a coculture model of primary human macrophages and layers of intestinal epithelial cell lines. RESULTS: The lamina propria of the inflamed intestine in patients with IBD, predominantly in Crohn's disease, is massively infiltrated by CD68 cells also positive for inducible nitric oxide synthase and tumor necrosis factor (TNF) α. The presence of M1 macrophage shifted the balance in the local macrophage compartment towards a proinflammatory state. In the coculture model, monocytes and M1 macrophages reduced transepithelial resistance as a marker for epithelial barrier integrity. The mechanisms for paracellular leakage included intracellular relocalization of tight junction proteins like claudin-2 and epithelial cell apoptosis. Determined by specific cytokine blockade, M1 macrophages exerted their deleterious effect mainly through TNF-α, whereas monocyte-mediated damage was driven by the inflammasome effector cytokines, interleukin-1ß and interleukin-18. CONCLUSIONS: Lamina propria monocytes and M1 macrophages invading intestinal tissues directly contribute to disrupting the epithelial barrier through deregulation of tight junction proteins and induction of epithelial cell apoptosis, thus driving intestinal inflammation in IBD.
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Enfermedades Inflamatorias del Intestino/etiología , Intestinos/patología , Macrófagos/citología , Monocitos/citología , Adolescente , Adulto , Anciano , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Apoptosis , Claudina-2/metabolismo , Citocinas/metabolismo , Células Epiteliales/fisiología , Humanos , Inflamasomas/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Receptores de Lipopolisacáridos , Macrófagos/metabolismo , Persona de Mediana Edad , Monocitos/metabolismo , Óxido Nítrico Sintasa/metabolismo , Migración Transendotelial y Transepitelial/fisiología , Adulto JovenRESUMEN
PURPOSE OF REVIEW: This review summarizes current knowledge on the contribution of mesenteric adipose tissue in intestinal inflammation. We will describe the cellular and humoral characteristics of creeping fat, their potential impact for Crohn's disease and propose a working model for the critical interplay between the creeping fat and the inflamed intestine. RECENT FINDINGS: Creeping fat can be distinguished from healthy adipose tissue by its distinctively small adipocytes, by a specific microenvironment defined by high levels of adipokines and by a dominant immune cell infiltration. In Crohn's disease transmural inflammation facilitates increased bacterial translocation into the creeping fat. Translocalizing antigens can directly activate (pre)adipocytes via innate receptors. Adipocyte-derived mediators modulate phenotype and function of innate and adaptive immune cells. Activated (pre)adipocytes and adipokine-modulated immune cells might support a degree of inflammatory activation within the creeping fat that allows competent immune defense against exogenous factors while preventing systemic inflammation. SUMMARY: Fat tissue as an active organ in health and disease has been ignored for too long. The last few years of research provided evidence for the complex metabolic and immunological functions of adipose tissue. On the basis of the available data, creeping fat in Crohn's disease exerts a protective function by a localized anti-inflammatory effect, thus preventing a systemic inflammatory response.
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Adipoquinas/metabolismo , Tejido Adiposo/metabolismo , Mediadores de Inflamación/metabolismo , Inflamación/inmunología , Enfermedades Inflamatorias del Intestino/inmunología , Mucosa Intestinal/inmunología , Metabolismo de los Lípidos/inmunología , Mesenterio/inmunología , Biomarcadores/metabolismo , Humanos , Inflamación/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Mucosa Intestinal/metabolismo , Leptina/metabolismo , Mesenterio/metabolismo , Receptores de Adiponectina/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores de Leptina/metabolismo , Resistina/metabolismoRESUMEN
Over the last decade it became broadly recognized that adipokines and thus the fat tissue compartment exert a regulatory function on the immune system. Our own group described the pro-inflammatory function of the adipokine leptin within intestinal inflammation in a variety of animal models. Following-up on this initial work, the aim was to reveal stimuli and mechanisms involved in the activation of the fat tissue compartment and the subsequent release of adipokines and other mediators paralleled by the infiltration of immune cells. This review will summarize the current literature on the possible role of the mesenteric fat tissue in intestinal inflammation with a focus on Crohn's disease (CD). CD is of particular interest in this context since the transmural intestinal inflammation has been associated with a characteristic hypertrophy of the mesenteric fat, a phenomenon called "creeping fat." The review will address three consecutive questions: (i) What is inducing adipocyte activation, (ii) which factors are released after activation and what are the consequences for the local fat tissue compartment and infiltrating cells; (iii) do the answers generated before allow for an explanation of the role of the mesenteric fat tissue within intestinal inflammation? With this review we will provide a working model indicating a close interaction in between bacterial translocation, activation of the adipocytes, and subsequent direction of the infiltrating immune cells. In summary, the models system mesenteric fat indicates a unique way how adipocytes can directly interact with the immune system.
RESUMEN
OBJECTIVE: The creeping fat in Crohn's disease (CD) is infiltrated by macrophages; local adipokine levels are increased. This study aimed to link these observations to define a role for macrophages in the pathology of human CD. METHODS: Human peripheral blood CD14 cells were polarised in vitro into M1 and M2 macrophages. The effects on adipokine receptors, phenotypic surface markers, cytokines and chemokines were assessed after treatment with leptin and adiponectin. Immunohistochemistry visualised macrophage subtypes in samples of mesenteric fat tissue from patients with CD. The chemotactic potential of secreted macrophage products was determined by T cell migration and chemokine production in vitro. RESULTS: Although both adipokines altered the phenotype and function of M1 and M2 macrophages, M2 macrophages were more susceptible. M1 responded to leptin by increased cytokine production, but the stronger effect was seen in M2 macrophages with high expression of interleukin (IL)-10, IL-6 and tumour necrosis factor α. Adiponectin exerted similar effects and led to upregulated mannose receptor expression by M2 macrophages. Large macrophage numbers within the mesenteric fat tissue of patients with CD comprise a unique infiltration predominantly of M2 macrophages, leading to an IL-10-rich environment. While leptin increased the potency of both subtypes to attract CD3 T cells, adiponectin only affected M2 macrophages. CONCLUSION: The adipocyte-dependent microenvironment within the creeping fat of patients with CD modulates the local macrophage compartment to a preference for the M2 subtype. The findings in this study with human cells suggest a protective role for the mesenteric fat in CD in terms of an enveloping barrier with the potential to limit intestinal inflammation.
Asunto(s)
Adipocitos/patología , Adiponectina/farmacología , Enfermedad de Crohn/patología , Leptina/farmacología , Macrófagos/efectos de los fármacos , Antígeno B7-1/genética , Antígeno B7-1/metabolismo , Biomarcadores/metabolismo , Quimiotaxis de Leucocito , Enfermedad de Crohn/metabolismo , Citocinas/metabolismo , Humanos , Inmunohistoquímica , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Macrófagos/metabolismo , Receptor de Manosa , Lectinas de Unión a Manosa/genética , Lectinas de Unión a Manosa/metabolismo , Mesenterio/patología , Fagocitosis , Reacción en Cadena de la Polimerasa , Receptores de Adiponectina/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores de Leptina/metabolismo , Linfocitos T/fisiologíaRESUMEN
Until a decade ago, fat tissue had been exclusively considered as an endocrine organ. The emerging functional characterization of adipokines as well as adipocytes and preadipocytes suggested for the first time a close link between the endocrine and the immune system. This is emphasized by the changes of the expression pattern of adipokines when the fat tissue is adjacent to inflamed sites. In addition, adipokines are capable of regulating adaptive and acquired immune responses. Remarkably, adipocytes express functional pattern recognition receptors and can consequently respond to bacterial and viral antigens. This seems to be highly relevant for intestinal inflammation and here in particular transmural inflammation where bacteria or bacterial antigens translocalize into the mesenteric fat tissue. Besides phagocytosis of these antigens, adipocytes as well as preadipocytes can be activated resulting in a release of adipokines and chemokines mediating the infiltration of immune cells thus allowing for an immune response. Recent data suggest that the adipokine milieu of the fat tissue closely regulates the polarization of infiltrating immune cells. This is of increasing interest since the pattern of infiltrating cells allows for a characterization of the underlying disease. Thus, in obesity pro-inflammatory M1 macrophages dominate this site. Remarkably, in colorectal carcinoma the presence of M1 and M2 macrophages represents a prognostic marker for the disease course. In conclusion, the visceral fat tissue represents a complex organ with multifaceted function linking the endocrine and the immune system.
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Grasa Intraabdominal/metabolismo , Adipocitos/metabolismo , Adipoquinas/metabolismo , Diverticulitis del Colon/patología , HumanosRESUMEN
Autoimmune gastritis is a common autoimmune disorder characterized by chronic inflammatory cell infiltrates, atrophy of the corpus and fundus, and the occurrence of autoantibodies to parietal cell antigen. In CCR7-deficient mice, autoimmune gastritis developed spontaneously and was accompanied by metaplasia of the gastric mucosa and by the formation of tertiary lymphoid organs at gastric mucosal sites. T cells of CCR7-deficient mice showed an activated phenotype in the gastric mucosa, mesenteric lymph nodes, and peripheral blood. In addition, elevated serum IgG levels specific to gastric parietal cell antigen were detected. Because the role of organized lymphocytic aggregates at this inflammatory site is not completely understood, we first analyzed the cellular requirements for the formation of these structures. Autoreactive CD4(+) T cells were pivotal for tertiary lymphoid follicle formation, most likely in cooperation with dendritic cells, macrophages, and B cells. Second, we analyzed the necessity of secondary lymph nodes and tertiary lymphoid organs for the development of autoimmune gastritis using CCR7 single- and CCR7/lymphotoxin α double-deficient mice. Strikingly, manifestation of autoimmune gastritis was observed in the absence of secondary lymph nodes and preceded the development of tertiary lymphoid organs. Taken together, these findings identify an inflammatory process where gastric autoreactive T cells independent of organized tertiary lymphoid organs and classic lymph nodes can induce and maintain autoimmune gastritis.
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Enfermedades Autoinmunes/patología , Gastritis/patología , Receptores CCR7/genética , Animales , Células de la Médula Ósea/citología , Antígeno CD11c/biosíntesis , Linfocitos T CD4-Positivos/citología , Células Dendríticas/citología , Citometría de Flujo/métodos , Mucosa Gástrica/metabolismo , Concentración de Iones de Hidrógeno , Inmunoglobulina G/química , Interferón gamma/metabolismo , Interleucina-17/metabolismo , Leucocitos Mononucleares/citología , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Many identified and yet unknown factors contribute to the pathogenesis of inflammatory bowel disease (IBD). The genome-wide association studies clearly support the earlier developed concept that IBD occurs in genetically predisposed individuals who are exposed to distinct environmental factors, which together result in dysregulation of the mucosal immune system. Thus, the majority of previous studies have focused on the immune response within the intestinal wall. The present review aims to emphasize the contribution of three extraluminal structures to this inflammatory process, namely the mesenteric fat tissue, the lymphatics and the microvasculature. Broadening our view across the intestinal wall will not only facilitate our understanding of the disease, but will also us to identify future therapeutic targets.
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Enfermedades Inflamatorias del Intestino/etiología , Enfermedades Inflamatorias del Intestino/fisiopatología , Adipocitos/inmunología , Adipoquinas/inmunología , Tejido Adiposo/citología , Tejido Adiposo/inmunología , Tejido Adiposo/patología , Humanos , Inmunidad Innata/inmunología , Inflamación/patología , Inflamación/fisiopatología , Enfermedades Inflamatorias del Intestino/patología , Sistema Linfático/anatomía & histología , Sistema Linfático/inmunología , Mesenterio/anatomía & histología , Mesenterio/inmunología , Mesenterio/patología , Microcirculación/inmunologíaRESUMEN
Besides being mandatory in the metabolic system, adipokines like leptin directly affect immunity. Leptin was found to be necessary in T helper 1 (Th1)-dependent inflammatory processes, whereas effects on Th2 cells are rarely understood. Here, we focused on leptin in T-helper cell polarization and in Th2-mediated intestinal inflammation in vivo. The induction of cytokine-producing Th1 or Th2 cells from naive CD4(+) T cells under polarizing conditions in vitro was generally decreased in cells from leptin-deficient ob/ob mice compared with wild-type mice. To explore the in vivo relevance of leptin in Th2-mediated inflammation, the model of oxazolone-induced colitis was employed in wild-type, ob/ob, and leptin-reconstituted ob/ob mice. Ob/ob mice were protected, whereas wild-type and leptin-reconstituted ob/ob mice developed colitis. The disease severity went in parallel with local production of the Th2 cytokine IL-13. A possible explanation for the protection of ob/ob mice in Th1- as well as in Th2-dependent inflammation is provided by a decreased expression of the key transcription factors for Th1 and Th2 polarization, T-bet and GATA-3, in naive ob/ob T cells. In conclusion, these results support the regulatory function of the adipokine leptin within T-cell polarization and thus in the acquired immune system and support the concept that there is a close interaction with the endocrine system.
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Linfocitos T CD4-Positivos/fisiología , Polaridad Celular/efectos de los fármacos , Leptina/fisiología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Linfocitos T CD4-Positivos/metabolismo , Polaridad Celular/genética , Polaridad Celular/inmunología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Femenino , Factor de Transcripción GATA3/genética , Factor de Transcripción GATA3/metabolismo , Gastroenteritis/genética , Gastroenteritis/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Leptina/genética , Leptina/metabolismo , Leptina/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Obesos , Receptores de Leptina/genética , Receptores de Leptina/metabolismo , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismoRESUMEN
Translocation of bacteria into the mesenteric fat during intestinal inflammation and the expression of functional TLR1-9 in murine preadipocytes and adipocytes suggest an active role for these cells in innate immunity. The present study focuses on nucleotide oligomerization domains 1 and 2 representing intracellular pattern recognition receptors that sense motifs derived from bacterial peptidoglycans. On mRNA level nucleotide oligomerization domain 1 was found to be constitutively expressed in the preadipocyte cell line 3T3L1 and in primary preadipocytes isolated from murine mesenteric fat, while nucleotide oligomerization domain 2 was only weakly expressed by these cells. Treatment with lactyl-tetra-diaminopimelic acid, muramyl dipeptide, LPS, IL-1beta, and TNF-alpha did not affect cellular nucleotide oligomerization domain 1 mRNA amounts. Except muramyl dipeptide, all factors significantly increased nucleotide oligomerization domain 2 mRNA in mesenteric fat preadipocytes after 4 h. However, specific stimulation of nucleotide oligomerization domain 1 induced IL-6 synthesis in preadipocytes from wild-type or TLR2/4-deficient mice. Confirming nucleotide oligomerization domain 1 specificity, transfection of nucleotide oligomerization domain 1-specific small interfering RNA significantly blocked the effect of lactyl-tetra-diaminopimelic acid on IL-6 production. With specific inhibitors and a NF-kappaB reporter plasmid, nucleotide oligomerization domain 1-mediated activation of NF-kappaB was shown to be responsible for the induction of IL-6 in preadipocytes. In addition, expression of functional nucleotide oligomerization domain 1 could be confirmed in primary human preadipocytes. In summary, we here identified preadipocytes as a novel cell population expressing nucleotide oligomerization domains 1 and 2. Not regulated on transcriptional level, nucleotide oligomerization domain 1 in preadipocytes serves as a sensor for bacterial degradation products and triggers proinflammatory effector responses. Thus, our results further strengthen the allocation of the mesenteric fat and especially of preadipocytes to the innate immune system.
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Adipocitos/inmunología , Regulación de la Expresión Génica , Inmunidad Innata , Proteína Adaptadora de Señalización NOD1/genética , Proteína Adaptadora de Señalización NOD2/genética , Células 3T3-L1 , Adipocitos/química , Animales , Infecciones Bacterianas/inmunología , Humanos , Mesenterio , Ratones , Proteína Adaptadora de Señalización NOD1/fisiología , Proteína Adaptadora de Señalización NOD2/fisiología , ARN Mensajero/análisisRESUMEN
BACKGROUND: Gut bacteria trigger colitis in animal models and are suspected to aggravate inflammatory bowel diseases. We have recently reported that Escherichia coli accumulates in murine ileitis and exacerbates small intestinal inflammation via Toll-like receptor (TLR) signaling. METHODOLOGY AND PRINCIPAL FINDINGS: Because knowledge on shifts in the intestinal microflora during colitis is limited, we performed a global survey of the colon flora of C57BL/10 wild-type (wt), TLR2(-/-), TLR4(-/-), and TLR2/4(-/-) mice treated for seven days with 3.5% dextrane-sulfate-sodium (DSS). As compared to wt animals, TLR2(-/-), TLR4(-/-), and TLR2/4(-/-) mice displayed reduced macroscopic signs of acute colitis and the amelioration of inflammation was associated with reduced IFN-gamma levels in mesenteric lymph nodes, lower amounts of neutrophils, and less FOXP3-positive T-cells in the colon in situ. During acute colitis E. coli increased in wt and TLR-deficient mice (P<0.05), but the final numbers reached were significantly lower in TLR2(-/-), TLR4(-/-) and TLR2/4(-/-) animals, as compared to wt controls (P<0.01). Concentrations of Bacteroides/ Prevotella spp., and enterococci did not increase during colitis, but their numbers were significantly reduced in the colon of DSS-treated TLR2/4(-/-) animals (P<0.01). Numbers of lactobacilli and clostridia remained unaffected by colitis, irrespective of the TLR-genotype of mice. Culture-independent molecular analyses confirmed the microflora shifts towards enterobacteria during colitis and showed that the gut flora composition was similar in both, healthy wt and TLR-deficient animals. CONCLUSIONS AND SIGNIFICANCE: DSS-induced colitis is characterized by a shift in the intestinal microflora towards pro-inflammatory Gram-negative bacteria. Bacterial products exacerbate acute inflammation via TLR2- and TLR4-signaling and direct the recruitment of neutrophils and regulatory T-cells to intestinal sites. E. coli may serve as a biomarker for colitis severity and DSS-induced barrier damage seems to be a valuable model to further identify bacterial factors involved in maintaining intestinal homeostasis and to test therapeutic interventions based upon anti-TLR strategies.
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Infecciones Bacterianas/fisiopatología , Colitis/microbiología , Colitis/fisiopatología , Colon/microbiología , Inflamación/microbiología , Receptor Toll-Like 2/fisiología , Receptor Toll-Like 4/fisiología , Enfermedad Aguda , Animales , Infecciones Bacterianas/complicaciones , Infecciones Bacterianas/genética , Infecciones Bacterianas/patología , Colitis/patología , Colitis/prevención & control , Colon/inmunología , Humanos , Inflamación/complicaciones , Inflamación/genética , Inflamación/prevención & control , Enfermedades Inflamatorias del Intestino/complicaciones , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/microbiología , Ratones , Ratones Endogámicos C57BL , Neutrófilos/fisiología , Transducción de Señal , Receptor Toll-Like 2/deficiencia , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 4/deficiencia , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunologíaRESUMEN
Leptin, an adipokine mainly produced by adipocytes, has been well characterized with regard to its regulatory function on immune cells. Thus the question occurred of how adipocytes and preadipocytes interact with the immune system and whether or not this communication is regulated by leptin. With the present study we evaluated the Toll-like receptor (TLR) expression and TLR ligand-specific activation of murine preadipocytes and adipocytes in the presence [wild type (WT), 3T3L1] or absence of leptin (ob/ob) or leptin signaling (db/db). The ob/ob as well as db/db adipocytes and preadipocytes were characterized by a significant up-regulation of TLR1 to -9 expression when compared with WT cells. In WT preadipocytes the TLR responsiveness increased during maturation to adipocytes; however, stimulation of ob/ob and db/db cells resulted in a 10- to 20-fold higher interleukin-6 production. Signaling studies revealed, in addition to the increased TLR expression, alterations in the phosphoinositide 3 kinase signaling cascade in ob/ob and db/db cells as an explanation for this increased responsiveness. In conclusion, the present study indicates the expression and responsiveness of TLR1 to -9 in murine preadipocytes as well as adipocytes, both of which are strongly regulated by the adipokine leptin. In summary, these data further emphasize the role of fat tissue in the immune system.
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Adipocitos/metabolismo , Leptina/metabolismo , Células Madre/metabolismo , Receptores Toll-Like/metabolismo , Adipocitos/citología , Androstadienos/metabolismo , Animales , Células Cultivadas , Femenino , Interleucina-6/metabolismo , Lipopolisacáridos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Leptina , Transducción de Señal/fisiología , Células Madre/citología , Receptores Toll-Like/genética , WortmaninaRESUMEN
BACKGROUND: IgA nephropathy (IgAN) is characterized by mesangial deposition of polymeric IgA (pIgA). In IgAN, mucosal pIgA production is reduced while systemic production is increased, making the latter the likely source of mesangial pIgA, and suggesting a displacement of pIgA-producing cells from mucosal to systemic sites. Upon activation, lymphocytes migrate through the circulation up-regulating homing receptors (HR) which direct their return to appropriate effector locations. We investigated the HR expression of T-cell subsets in IgAN, healthy adults and membranous nephropathy (MN). METHODS: Peripheral blood cells were labelled for CD3, CD4 and CD8, and for L-selectin (naive cells), integrin alpha4beta1 (systemically homing cells) and integrin alpha4beta7 (mucosally homing cells) and analysed by flow immunocytometry. RESULTS: In IgAN, CD3 T cells displayed reduced L-selectin and increased alpha4beta1hi expression, with no difference in alpha4beta7. No abnormality of T-cell HR expression was found in MN. Both IgAN and healthy adults maintained their patterns of T-cell HR expression when studied again at a later time point, and the changes in IgAN were entirely accounted for by the CD4 T-cell subset with CD8 HR expression being normal. CONCLUSIONS: The consistently reduced L-selectin expression by CD4 T cells indicates increased activation of this subset in IgAN. These activated cells express alpha4beta1 rather than alpha4beta7, and therefore home to systemic effector sites. CD4 T cells regulate antibody production, including IgA. As pIgA is overproduced in systemic sites in IgAN, we hypothesize that these activated systemic homing CD4 T cells may direct the aberrant systemic pIgA production observed in IgAN.
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Glomerulonefritis por IGA/metabolismo , Receptores Mensajeros de Linfocitos/metabolismo , Adulto , Complejo CD3/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Femenino , Citometría de Flujo , Humanos , Integrina alfa4beta1/metabolismo , Integrina beta1/metabolismo , Selectina L/metabolismo , Masculino , Persona de Mediana Edad , Subgrupos de Linfocitos T/metabolismo , Factores de TiempoRESUMEN
Inhibitors of histone deacetylases (HDAC) are being studied for their antiproliferative effects in preclinical cancer trials. Recent studies suggest an anti-inflammatory role for this class of compounds. Because inflammatory bowel disease is associated with an increased risk of malignancies, agents with antiproliferative and anti-inflammatory properties would be of therapeutic interest. HDAC inhibitors from various classes were selected and evaluated for their in vitro capacity to suppress cytokine production and to induce apoptosis and histone acetylation. Valproic acid (VPA) and suberyolanilide hydroxamic acid (SAHA) were chosen for further studies in dextran sulfate sodium- and trinitrobenzene sulfonic acid-induced colitis in mice. In vitro, inhibition of HDAC resulted in a dose-dependent suppression of cytokine synthesis and apoptosis induction requiring higher concentrations of HDAC inhibitors for apoptosis induction compared with cytokine inhibition. Oral administration of either VPA or SAHA reduced disease severity in dextran sulfate sodium-induced colitis. The macroscopic and histologic reduction of disease severity was associated with a marked suppression of colonic proinflammatory cytokines. In parallel to the beneficial effect observed, a dose-dependent increase in histone 3 acetylation at the site of inflammation was shown under VPA treatment. Furthermore, SAHA as well as VPA treatment resulted in amelioration of trinitrobenzene sulfonic acid-induced colitis, which was associated with an increase of apoptosis of lamina propria lymphocytes. Inhibitors of HDAC reveal strong protective effects in different models of experimental colitis by inducing apoptosis and suppressing proinflammatory cytokines, thereby representing a promising class of compounds for clinical studies in human inflammatory bowel disease.
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Colitis/tratamiento farmacológico , Colitis/metabolismo , Histonas/química , Histonas/metabolismo , Acetilación , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Antiinflamatorios no Esteroideos/farmacología , Apoptosis/efectos de los fármacos , Colitis/etiología , Colitis/patología , Citocinas/biosíntesis , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/farmacología , Femenino , Inhibidores de Histona Desacetilasas , Humanos , Ácidos Hidroxámicos/administración & dosificación , Ácidos Hidroxámicos/farmacología , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ácido Valproico/administración & dosificación , Ácido Valproico/farmacología , VorinostatRESUMEN
Leptin-deficient (ob/ob) mice are resistant in different models of autoimmunity and inflammation, suggesting that leptin regulates immunity and inflammation. To investigate whether leptin deficiency modulates the spontaneous intestinal inflammation observed in interleukin (IL)-10-deficient mice, double IL-10- and leptin-deficient [IL-10 knockout (KO) ob/ob] mice were generated and compared with single IL-10 KO mice for colitis severity. Body weight in IL-10 KO ob/ob mice was significantly reduced compared with that of ob/ob mice. However, when compared with wild-type or IL-10 KO mice, IL-10 KO ob/ob mice were still markedly obese. IL-10 KO and IL-10 KO ob/ob mice developed colitis with a comparable time-course and severity in terms of macroscopic and histologic scores. Likewise, production of interferon-gamma, IL-6, and IL-13 from colon cultures and splenocytes did not differ among these two groups. Conversely, rates of apoptosis were higher in lamina propria lymphocytes obtained from the colon of IL-10 KO ob/ob compared with IL-10 KO mice. In conclusion, although leptin deficiency has been associated with resistance in models of autoimmunity and inflammation induced by exogenous stimuli, leptin appears not to play a significant role in the spontaneous colitis of IL-10 KO mice, although it modulates survival of intestinal lymphocytes.
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Colitis/etiología , Interleucina-10/fisiología , Mucosa Intestinal/inmunología , Leptina/fisiología , Animales , Apoptosis , Peso Corporal , División Celular , Células Cultivadas , Colitis/patología , Colon/inmunología , Colon/metabolismo , Femenino , Interferón gamma/metabolismo , Interleucina-10/genética , Interleucina-13/metabolismo , Interleucina-6/metabolismo , Mucosa Intestinal/patología , Leptina/genética , Linfocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Obesos , Obesidad/complicaciones , Obesidad/genética , Obesidad/metabolismo , Bazo/inmunología , Bazo/metabolismoRESUMEN
Borna disease in rats represents an experimental model to study the immunopathological role of T cells in central nervous system disease. Adoptive transfer experiments were performed to investigate homing properties of T cells that infiltrate the brains of infected animals. Lymphocytes isolated from the brains of diseased rats were labelled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) and transferred into immunosuppressed infected recipients. In recipient rats displaying neurological disease, labeled lymphocytes were demonstrated in the vicinity of brain cell lesions, suggesting that the neuronal destruction was dependent on the presence of transferred lymphocytes. Furthermore, the presence of virus-specific cytotoxic T cells was scrutinized in secondary lymphatic tissue and the functional activity of lymphocytes isolated from spleens, cervical lymph nodes, and mesenteric lymph nodes of infected animals was tested immediately after isolation and after in vitro restimulation. The data presented here indicate that precursors of Borna disease virus (BDV)-specific CD8(+) T cells are present and cytotoxic activity was demonstrated after in vitro cocultivation with infected cells in cervical lymph nodes and spleens but not in mesenteric lymphoid tissue. Adoptive transfer of in vitro restimulated T cells induced alterations in BDV-infected, immunosuppressed rats that resemble the well-defined clinical symptoms and neuropathology of Borna disease. This report provides for the first time formal evidence that virus-specific cytotoxic T cells are primed in the periphery after BDV infection, a disease that exclusively manifests itself in the central nervous system.