RESUMEN
B cells interact with T follicular helper (Tfh) cells in germinal centers (GCs) to generate high-affinity antibodies. Much less is known about how cognate T-B-cell interactions influence Th cells that enter circulation and peripheral tissues. Therefore, we generated mice lacking MHC-II expressing B cells and, by thoracic duct cannulation, analyzed Th cells in the efferent lymph at defined intervals post-immunization. Focusing on gut-draining mesenteric lymph nodes (MLNs), we show that antigen-specific α4ß7+ gut-homing effector Th cells enter the circulation prior to CXCR5+PD-1+ Tfh-like cells. B cells appear to have no or limited impact on the early generation and egress of gut-homing Th cells but are critical for the subsequent appearance of Tfh-like cells that peak in the lymph before GCs have developed. At this stage, antigen-presenting B cells also reduce the proportion of α4ß7+ Th cells in the MLN and efferent lymph. Furthermore, cognate B-cell interaction drives a broad transcriptional program in Th cells, including IL-4 that is confined to the Tfh cell lineage. The IL-4-producing Tfh-like cells originate from Bcl6+ precursors in the LNs and have gut-homing capacity. Hence, B cells program the efferent lymph Th cell response within a limited window of time after antigenic challenge.
Asunto(s)
Interleucina-4 , Linfocitos T Colaboradores-Inductores , Animales , Linfocitos B , Centro Germinal , Ratones , Receptores CXCR5/genéticaRESUMEN
The metabolic syndrome has been demonstrated in gene deficient animals, e.g. db/db mice, to include a systemic inflammation leading to insulin resistance, obesity and type 2 diabetes (T2D). To determine the importance of inflammation in obesity and diabetes, in a normal non-genetically modified species, an intervention study with neutralizing anti-IL-20 antibodies was conducted in the spontaneous T2D model Psammomys obesus. All IL-20 receptor chains were expressed on protein level in the Psammomys obesus. Neutralization of IL-20 did not modulate blood glucose, HbA1c, insulin levels or lymphocyte numbers after five weeks treatment although a trend to reduced weight gain rate was observed upon anti-IL-20 treatment. Inhibition of IL-20 significantly increased the number of CD11bhigh/low cells and the CD11bGr-1int myeloid derived suppressor cells in the spleen. Importantly, although the number of M1-like monocytes remained unchanged the M1-like marker CD11c expression level was reduced on the cells upon anti-IL-20 treatment. Anti-IL-20 treatment reduced both TLR4 and CCR2b expression on the macrophages upon treatment. Further, a marked shift in the protein signature in the pancreatic tissue after anti-IL-20 treatment was observed including enhanced expression of CXCL12, TIMP-1 and IL-10 while IL-1ß, CXCL4, PEDF and ADAMTS1 were reduced. In conclusion, we describe for the first time the systemic immune response in the diabetic Psammomys obesus. Neutralizing IL-20 modulated the myeloid compartment, the adaptive immunity, and local expression of proteins in the diabetic pancreatic tissue as well as improved on weight gain and hence may place IL-20 as a cytokine to be considered in obesity.
Asunto(s)
Diabetes Mellitus Tipo 2/inmunología , Gerbillinae/inmunología , Inflamación/inmunología , Interleucinas/metabolismo , Macrófagos/inmunología , Células Supresoras de Origen Mieloide/inmunología , Obesidad/inmunología , Páncreas/inmunología , Animales , Animales Modificados Genéticamente , Anticuerpos Bloqueadores/uso terapéutico , Diabetes Mellitus Tipo 2/terapia , Humanos , Inflamación/terapia , Mediadores de Inflamación/metabolismo , Insulina/sangre , Interleucinas/inmunología , Macrófagos/efectos de los fármacos , Masculino , Ratones , Células Supresoras de Origen Mieloide/efectos de los fármacos , Obesidad/terapia , Páncreas/efectos de los fármacos , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Aumento de Peso/efectos de los fármacosRESUMEN
Macrophages are heterogeneous and can polarize into specific subsets, e.g. pro-inflammatory M1-like and re-modelling M2-like macrophages. To determine if peritoneal macrophages (PEMs) or bone marrow derived macrophages (BMDMs) resembled aortic macrophages from ApoE-/- mice, their M1/M2 phenotype, inflammatory status, and lipid metabolism signatures were compared. oxLDL accumulation was similar in PEMs and BMDMs. On protein expression level, BMDMs showed an M2-like CD206highCD11clow profile, while cholesterol loading led to enhanced CD11c expression and reduced MCP-1 secretion. In contrast, PEMs expressed low levels of CD206 and CD11c, and responded to cholesterol loading by increasing CD11c expression and MCP-1 secretion. mRNA expression of M1/M2 markers was higher in PEMS than BMDMs, while lipid metabolism genes were similarly expressed. Whole aorta flow cytometry showed an accumulation of M2-like CD206highCD11clow macrophages in advanced versus early atherosclerotic disease in ApoE-/- mice. In isolated lesions, mRNA levels of the M2 markers Socs2, CD206, Retnla, and IL4 were downregulated with increasing disease severity. Likewise, mRNA expression of lipid metabolism genes (SREBP2, ACSL1, SRB1, DGAT1, and cpt1a) was decreased in advanced versus early lesions. In conclusion, PEMs and BMDMs are phenotypically distinct and differ from macrophages in lesions with respect to expression of M1/M2 markers and lipid metabolism genes.
Asunto(s)
Células de la Médula Ósea/citología , Inflamación/patología , Lipoproteínas LDL/metabolismo , Macrófagos Peritoneales/citología , Animales , Femenino , Ratones , Ratones Endogámicos C57BLRESUMEN
The cytokine interleukin-1ß (IL-1ß) is known to stimulate proinflammatory immune responses and impair ß-cell function and viability, all critical events in the pathogenesis of type 1 diabetes (T1D). Here we evaluate the effect of SER140, a small peptide IL-1ß receptor antagonist, on diabetes progression and cellular pancreatic changes in female nonobese diabetic (NOD) mice. Eight weeks of treatment with SER140 reduced the incidence of diabetes by more than 50% compared with vehicle, decreased blood glucose, and increased plasma insulin. Additionally, SER140 changed the endocrine and immune cells dynamics in the NOD mouse pancreas. Together, the data suggest that SER140 treatment postpones the onset of diabetes in female NOD mice by interfering with IL-1ß activated pathways.
Asunto(s)
Diabetes Mellitus/prevención & control , Hipoglucemiantes/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Interleucina-1beta/metabolismo , Péptidos/farmacología , Receptores de Interleucina-1/antagonistas & inhibidores , Animales , Biomarcadores/sangre , Glucemia/efectos de los fármacos , Glucemia/metabolismo , Diabetes Mellitus/sangre , Diabetes Mellitus/patología , Femenino , Insulina/sangre , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Ratones Endogámicos NOD , Oligopéptidos/farmacología , Receptores de Interleucina-1/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de TiempoRESUMEN
Low grade inflammation is present in pre-clinical and human type 2 diabetes. In this process, several cytokines like IL-1ß and inflammatory cells like macrophages are activated and demonstrated to participate to the disease initiation and progression. IL-20 is a cytokine known to play non-redundant roles in progression of several inflammatory diseases. To address the therapeutic effect of inhibiting the IL-20 pathway in diabetes, diabetic db/db mice were treated with neutralizing anti-IL20 antibodies in vivo and both metabolic and inflammatory parameters were followed. Diabetic islets expressed the IL-20 cytokine and all IL-20 receptor components in elevated levels compared to resting non-diabetic islets. Islets were responsive to ex vivo IL-20 stimulation measured as SOCS induction and KC and IL-6 production. Neutralizing anti-IL20 treatment in vivo had no effect on HbA1c or weight although the slope of blood glucose increase was lowered. In contrast, anti-IL20 treatment significantly reduced the systemic low-grade inflammation and modulated the local pancreatic immunity. Significant reduction of the systemic IL-1ß and MCP-1 was demonstrated upon anti-IL20 treatment which was orchestrated with a reduced RANTES, IL-16 and IL-2 but increased TIMP-1, MCP-1 and IL-6 protein expression locally in the pancreas. Interestingly, anti-IL20 treatment induced an expansion of the myeloid suppressor CD11bGr1int macrophage while reducing the number of CD8 T cells. Taken together, anti-IL20 treatment showed moderate effects on metabolic parameters, but significantly altered the low grade local and systemic inflammation. Hence, future combination therapies with anti-IL20 may provide beneficial therapeutic effects in type 2 diabetes through a reduction of inflammation.
Asunto(s)
Anticuerpos Neutralizantes/farmacología , Diabetes Mellitus Tipo 2/prevención & control , Hemoglobina Glucada/metabolismo , Inflamación/prevención & control , Interleucinas/farmacología , Animales , Línea Celular Tumoral , Citocinas/sangre , Citocinas/inmunología , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/inmunología , Citometría de Flujo , Expresión Génica , Humanos , Inflamación/genética , Inflamación/inmunología , Interleucinas/genética , Interleucinas/inmunología , Masculino , Ratones Endogámicos C57BL , Páncreas/efectos de los fármacos , Páncreas/inmunología , Páncreas/metabolismo , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/efectos de los fármacos , Bazo/inmunología , Bazo/metabolismoRESUMEN
Development of long-lived humoral immunity is dependent on CXCR5-expressing T follicular helper (Tfh) cells, which develop concomitantly to effector Th cells that support cellular immunity. Conventional dendritic cells (cDCs) are critical APCs for initial priming of naive CD4(+) T cells but, importantly, also provide accessory signals that govern effector Th cell commitment. To define the accessory role of cDCs during the concurrent development of Tfh and effector Th1 cells, we performed high-dose Ag immunization in conjunction with the Th1-biased adjuvant polyinosinic:polycytidylic acid (pI:C). In the absence of cDCs, pI:C failed to induce Th1 cell commitment and IgG2c production. However, cDC depletion did not impair Tfh cell differentiation or germinal center formation, and long-lived IgG1 responses of unaltered affinity developed in mice lacking cDCs at the time point for immunization. Thus, cDCs are required for the pI:C-driven Th1 cell fate commitment but have no crucial accessory function in relation to Tfh cell differentiation.
Asunto(s)
Células Dendríticas/inmunología , Poli I-C/inmunología , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Diferenciación Celular/inmunología , Quimera/inmunología , Células Dendríticas/citología , Centro Germinal/inmunología , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/inmunología , Interferón gamma/biosíntesis , Depleción Linfocítica , Ratones , Ratones Endogámicos C57BL , Receptores CXCR5/biosíntesisRESUMEN
Diabetic nephropathy (DN) is a serious complication of longstanding diabetes affecting up to 30% of all diabetes patients and is the main cause of end-stage kidney disease globally. Current standard treatment e.g. ACE-inhibitors like enalapril merely offers a delay in the progression leading to DN. Herein, we describe in two preclinical models evidence to local effects on the inflammatory signatures after intervention treatment with enalapril which provides enhanced understanding of the mechanism of ACE inhibitors. Enalapril transiently reduced albuminuria in both the db/db and the STZ-induced DN models with established disease, without modulating the HbA1c%. Albuminuria was strongly associated with loss of leukocytes, particularly B cells, but also of sub-populations of macrophages and CD4(+) T cells. The remaining kidney macrophages were polarized into a M2-like sub-population with reduced surface expression of the M1-like macrophage marker CD11c and enhanced expression of galectin-3. Enalapril treatment counteracted the reduction of leukocytes in the diabetic kidney towards levels noted in the non-diabetic kidney. Particularly, a subset of macrophages was increased and a clear expansion of CD4(+) and CD8(+) T cells was observed. However, enalapril failed to modulate the B cell compartment. Interestingly, enalapril treatment resulted in a re-polarization of the macrophages towards a M1-like phenotype characterized by elevated levels of CD11c with moderate down-regulation of the M2 marker galectin-3. The data demonstrate that ACE inhibition in pre-clinical models of DN shows a transient beneficial effect on albuminuria which is unexpectedly associated with restoration of T cells and M1-like macrophages in the kidney.
Asunto(s)
Albuminuria/tratamiento farmacológico , Inhibidores de la Enzima Convertidora de Angiotensina/administración & dosificación , Diabetes Mellitus Experimental/tratamiento farmacológico , Nefropatías Diabéticas/tratamiento farmacológico , Enalapril/administración & dosificación , Riñón/efectos de los fármacos , Macrófagos/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Albuminuria/inmunología , Inhibidores de la Enzima Convertidora de Angiotensina/efectos adversos , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Antígeno CD11c/metabolismo , Diferenciación Celular/efectos de los fármacos , Diabetes Mellitus Experimental/inmunología , Nefropatías Diabéticas/inmunología , Modelos Animales de Enfermedad , Enalapril/efectos adversos , Galectina 3/metabolismo , Humanos , Riñón/fisiología , Recuento de Linfocitos , Macrófagos/inmunología , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Mutantes , Linfocitos T/inmunologíaRESUMEN
Low-grade inflammation, characterized by increased pro-inflammatory cytokine levels, is present in patients with obesity-linked insulin resistance, hyperglycemia and hyperlipidemia and considered to play a leading role to progression into type 2 diabetes (T2D). In adipose tissue in obese patients and in pancreatic islets in T2D patients cellular inflammation is present. However, the systemic leukocyte compartment and the circulating endothelial/precursor compartment in patients at risk to develop T2D has so far not been analyzed in detail. To address this, peripheral blood cells from a cohort of 20 subjects at risk to develop diabetes with normal to impaired glucose tolerance were analyzed by flow cytometry using a wide range of cellular markers and correlated to known metabolic risk factors for T2D i.e. fasting plasma glucose (FPG), 2 h plasma glucose (2 h PG), HbA1c, body mass index (BMI), homeostasis model assessment of ß-cell function (HOMA-B), homeostasis model assessment of insulin sensitivity (HOMA-IS) and fasting insulin (FI). The four highest ranked cell markers for each risk factor were identified by random forest analysis. In the cohort, a significant negative correlation between the number of TLR4(+) CD4 T cells and increased FPG was demonstrated. Similarly, with increased BMI the frequency of TLR4(+) B cells was significantly decreased, as was the frequency of IL-21R(+) CD4 T cells. Unlinked to metabolic risk factors, the frequency of regulatory T cells was reduced and TLR4(+) CD4 T cells were increased with age. Taken together, in this small cohort of subjects at risk to develop T2D, a modulation of the circulating immune cell pool was demonstrated to correlate with risk factors like FPG and BMI. This may provide novel insights into the inflammatory mechanisms involved in the progression to diabetes in subjects at risk.
Asunto(s)
Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/inmunología , Linfocitos/citología , Envejecimiento/inmunología , Glucemia/metabolismo , Recuento de Células , Ingestión de Alimentos , Células Progenitoras Endoteliales/citología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Monocitos/citología , Factores de Riesgo , Linfocitos T/citología , Linfocitos T/metabolismo , Receptor Toll-Like 4/metabolismoRESUMEN
Type 2 diabetes (T2D) is evolving into a global disease and patients have a systemic low-grade inflammation, yet the role of this inflammation is still not established. One plausible mechanism is enhanced expression and activity of the innate immune system. Therefore, we evaluated the expression and the function of the toll-like receptor 4 (TLR4) on pancreatic ß-cells in primary mouse islets and on the murine ß-cell line MIN6 in the presence or absence of macrophages. Diabetic islets have 40% fewer TLR4 positive ß-cells, but twice the number of TLR4 positive macrophages as compared to healthy islets. Healthy and diabetic islets respond to a TLR4 challenge with enhanced production of cytokines (5-10-fold), while the TLR4 negative ß-cell line MIN6 fails to produce cytokines. TLR4 stimulation induces ß-cell dysfunction in mouse islets, measured as reduced glucose stimulated insulin secretion. Diabetic macrophages from 4-months old mice have acquired a transient enhanced capacity to produce cytokines when stimulated with LPS. Interestingly, this is lost in 6-months old diabetic mice. TLR4 activation alone does not induce apoptosis in islets or MIN-6 cells. In contrast, macrophages mediate TLR4-dependent cell-contact dependent (3-fold) as well as cell-contact independent (2-fold) apoptosis of both islets and MIN-6 cells. Importantly, diabetic macrophages have a significantly enhanced capacity to induce ß-cell apoptosis compared to healthy macrophages. Taken together, the TLR4 responsiveness is elevated in the diabetic islets and mainly mediated by newly recruited macrophages. The TLR4 positive macrophages, in both a cell-contact dependent and independent manner, induce apoptosis of ß-cells in a TLR4 dependent fashion and TLR4 activation directly induces ß-cell dysfunction. Thus, targeting either the TLR4 pathway or the macrophages provides a novel attractive treatment regime for T2D.
Asunto(s)
Apoptosis/fisiología , Diabetes Mellitus Tipo 2/fisiopatología , Islotes Pancreáticos/metabolismo , Macrófagos/metabolismo , Receptor Toll-Like 4/metabolismo , Análisis de Varianza , Animales , Citocinas/metabolismo , Femenino , Citometría de Flujo , Inmunoensayo , Insulina/sangre , Mediciones Luminiscentes , Masculino , Ratones , Receptor Toll-Like 4/inmunologíaRESUMEN
Human T2D is characterized by a low-grade systemic inflammation, loss of ß-cells, and diminished insulin production. Local islet immunity is still poorly understood, and hence, we evaluated macrophage subpopulations in pancreatic islets in the well-established murine model of T2D, the db/db mouse. Already at 8 weeks of disease, on average, 12 macrophages were observed in the diabetic islets, whereas only two were recorded in the nondiabetic littermates. On a detailed level, the islet resident macrophages increased fourfold compared with nondiabetic littermates, whereas a pronounced recruitment (eightfold) of a novel subset of macrophages (CD68+F4/80-) was observed. The majority of the CD68+F4/80+ but only 40% of the CD68+F4/80- islet macrophages expressed CD11b. Both islet-derived macrophage subsets expressed moderate MHC-II, high galectin-3, and low CD80/CD86 levels, suggesting the cells to be macrophages rather than DCs. On a functional level, the vast majority of the macrophages in the diabetic islets was of the proinflammatory, M1-like phenotype. The systemic immunity in diabetic animals was characterized by a low-grade inflammation with elevated cytokine levels and increase of splenic cytokine, producing CD68+F4/80- macrophages. In late-stage diabetes, the cytokine signature changed toward a TGF-ß-dominated profile, coinciding with a significant increase of galectin-3-positive macrophages in the spleen. In summary, our results show that proinflammatory M1-like galectin-3+ CD80/CD86(low) macrophages invade diabetic islets. Moreover, the innate immunity matures in a diabetes-dependent manner from an initial proinflammatory toward a profibrotic phenotype, supporting the concept that T2D is an inflammatory disease.
Asunto(s)
Diabetes Mellitus Tipo 2/inmunología , Islotes Pancreáticos/inmunología , Macrófagos/inmunología , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Glucemia , Citocinas/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Humanos , Inmunofenotipificación , Mediadores de Inflamación/metabolismo , Insulina/biosíntesis , Insulina/deficiencia , Islotes Pancreáticos/patología , Macrófagos/metabolismo , Masculino , Ratones , FenotipoRESUMEN
Transferrin internalization via clathrin-mediated endocytosis and subsequent recycling after iron delivery has been extensively studied. Here we demonstrate a previously unrecognized parameter regulating this recycling, the binding of galectin-3 to particular glycoforms of transferrin. Two fractions of transferrin, separated by affinity chromatography based on their binding or not to galectin-3, are targeted to kinetically different endocytic pathways in HFL-1 cells expressing galectin-3 but not in SKBR3 cells lacking galectin-3; the SKBR3 cells, however, can acquire the ability to target these transferrin glycoforms differently after preloading with exogenously added galectin-3. In all, this study provides the first evidence of a functional role for transferrin glycans, in intracellular trafficking after uptake. Moreover, the galectin-3-bound glycoform increased in cancer, suggesting a pathophysiological regulation. These are novel aspects of transferrin cell biology, which has previously considered only a degree of iron loading, but not other forms of heterogeneity.
Asunto(s)
Endocitosis , Galectina 3/metabolismo , Transferrina/metabolismo , Adulto , Anciano , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Línea Celular , Femenino , Glicosilación , Humanos , Células MCF-7 , Persona de Mediana Edad , Polisacáridos/química , Unión Proteica , Conformación Proteica , Transporte de ProteínasRESUMEN
T follicular helper (Tfh) cells represent a recently defined CD4(+) T cell subset characterized by the expression of the chemokine receptor CXCR5 and an enhanced ability to support B cells to mount antibody responses. Here, we demonstrate that lymph-node-resident CXCR5(+) Tfh cells and gut-homing integrin alpha(4)beta(7)-expressing T helper cells are generated as separate subsets in the gut-draining mesenteric lymph nodes. Type I interferon signaling in dendritic cells and in nonhematopoietic cells selectively stimulates Tfh cell development in response to antigen in conjunction with Toll-like receptor (TLR)3 or TLR4 agonists. Consistent with this, the ability of dendritic cells to produce the cytokine IL-6, required for in vivo Tfh differentiation, and antibody affinity maturation are both reduced in absence of type I interferon signaling. Thus, our results identify type I interferon as a natural adjuvant that selectively supports the generation of lymph node resident Tfh cells.
Asunto(s)
Diferenciación Celular , Células Dendríticas/inmunología , Interferón Tipo I/inmunología , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Antígeno CD11c/inmunología , Linaje de la Célula , Integrinas/metabolismo , Interleucina-6/biosíntesis , Ganglios Linfáticos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor de Interferón alfa y beta/deficiencia , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/inmunología , Receptores CXCR5/metabolismo , Transducción de Señal , Linfocitos T Colaboradores-Inductores/metabolismoRESUMEN
The CC chemokine receptor (CCR)9 is expressed on the majority of small intestinal, but few colonic, T cells, whereas its ligand CCL25 is constitutively expressed by small intestinal epithelial cells. As such, CCR9/CCL25 have been proposed to play a central role in regulating small intestinal but not colonic immune responses and thus to organize regionalized immunity within the intestinal mucosa. Here, we demonstrate that CCL25 is expressed at reduced levels by epithelial cells in the distal compared with proximal small intestine, which correlated with less efficient CCR9-dependent effector CD8alphabeta+ T cell entry into the ileal epithelium. In vitro-generated alpha4beta7+ effector CD8alphabeta+ T cell entry into the lamina propria was less dependent on CCR9 than entry into the epithelium along the entire length of the small intestine and in particular in the ileum. CCR9-independent alpha4beta7+ effector CD8alphabeta+ T cell entry was pertussis toxin-sensitive, suggesting a role for additional Galpha(I)-linked G protein-coupled receptors. Finally, in vivo-primed effector CD8alphabeta+ T cells displayed regionalized differences in their entry to the small intestinal epithelium with enhanced CCR9-independent entry to the ileum. These results highlight a hitherto underappreciated compartmentalization of immune responses within the small intestine and have direct implications for targeting strategies aimed at regulating T cell localization to the small intestinal mucosa.