RESUMO
The cytosolic protein Sharpin is a component of the linear ubiquitin chain assembly complex, which regulates NF-κB signaling in response to specific ligands, such as TNF-α. Its inactivating mutation in chronic proliferative dermatitis mutation (Cpdm) mice causes multiorgan inflammation, yet this phenotype is not transferable into wild-type mice by hematopoietic stem cell transfer. Recent evidence demonstrated that Cpdm mice additionally display low bone mass, and that this osteopenia is corrected by Tnf deletion. Because the cellular mechanism underlying this pathology, however, was still undefined, we performed a thorough skeletal phenotyping of Cpdm mice on the basis of nondecalcified histology and cellular and dynamic histomorphometry. We show that the trabecular and cortical osteopenia in Cpdm mice is solely explained by impaired bone formation, whereas osteoclastogenesis is unaffected. Consistently, Cpdm primary calvarial cells display reduced osteogenic capacity ex vivo, and the same was observed with CD11b(-) bone marrow cells. Unexpectedly, short-term treatment of these cultures with TNF-α did not reveal an impaired molecular response in the absence of Sharpin. Instead, genome-wide and gene-specific expression analyses revealed that Cpdm mesenchymal cells display increased responsiveness toward TNF-α-induced expression of specific cytokines, such as CXCL5, IL-1ß, and IL-6. Therefore, our data not only demonstrate that the skeletal defects of Cpdm mice are specifically caused by impaired differentiation of osteoprogenitor cells, they also suggest that increased cytokine expression in mesenchymal bone marrow cells contributes to the inflammatory phenotype of Cpdm mice.
Assuntos
Células da Medula Óssea/imunologia , Proteínas de Transporte/imunologia , Diferenciação Celular/imunologia , Células-Tronco Mesenquimais/imunologia , Osteogênese/imunologia , Animais , Células da Medula Óssea/patologia , Proteínas de Transporte/genética , Diferenciação Celular/genética , Citocinas/genética , Citocinas/imunologia , Dermatite/genética , Dermatite/imunologia , Dermatite/patologia , Peptídeos e Proteínas de Sinalização Intracelular , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Mutantes , Osteogênese/genéticaRESUMO
Alterations in bone remodeling are a major public health issue, as therapeutic options for widespread bone disorders such as osteoporosis and tumor-induced osteolysis are still limited. Therefore, a detailed understanding of the regulatory mechanism governing bone cell differentiation in health and disease are of utmost clinical importance. Here we report a novel function of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), a member of the immunoglobulin superfamily involved in inflammation and tumorigenesis, in the physiologic regulation of bone remodeling. Assessing the expression of all members of the murine Ceacam family in bone tissue and marrow, we found CEACAM1 and CEACAM10 to be differentially expressed in both bone-forming osteoblasts and bone-resorbing osteoclasts. While Ceacam10-deficient mice displayed no alteration in structural bone parameters, static histomorphometry demonstrated a reduced trabecular bone mass in mice lacking CEACAM1. Furthermore, cellular and dynamic histomorphometry revealed an increased osteoclast formation in Ceacam1-deficient mice, while osteoblast parameters and the bone formation rate remained unchanged. In line with these findings, we detected accelerated osteoclastogenesis in Ceacam1-deficient bone marrow cells, while osteoblast differentiation, as determined by mineralization and alkaline phosphatase assays, was not affected. Therefore, our results provide in vivo and in vitro evidence for a physiologic role of CEACAM1 in the regulation of osteoclastogenesis.
Assuntos
Moléculas de Adesão Celular/deficiência , Osteoclastos/citologia , Animais , Antígenos CD/genética , Medula Óssea/metabolismo , Remodelação Óssea , Moléculas de Adesão Celular/genética , Linhagem Celular , Regulação da Expressão Gênica , Camundongos , Fatores de Transcrição NFATC/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese , FenótipoRESUMO
OBJECTIVE: Previously, we demonstrated the relevance for endothelial carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) expression in collateral formation. However, a proarteriogenic role for CEACAM1(+) myeloid cells is unknown. Here, we investigated the contribution of CEACAM1(+) myeloid cells on collateral formation. METHODS AND RESULTS: Collateral growth and vascular remodeling were analyzed in CEACAM1-competent and CEACAM1 null mice after femoral artery ligation in hindlimb ischemia. Reperfusion of the adductor muscles was evaluated by Laser Doppler measurements and microcomputed tomography imaging. In CEACAM1 null mice, poor reperfusion and reduced collateral formation were observed, accompanied by reduction in arterial diameters. Using flow cytometry, we identified an increase of the muscle-resident CD11b(+)/granulocyte receptor-1+ (Gr-1+) population in CEACAM1 null mice only, pointing toward a CEACAM1-dependent functional deviation. Direct and reciprocal bone marrow transplantations between CEACAM1-competent and CEACAM1 null mice, and antibody-mediated depletion of the CD11b(+)/Gr-1(+) population, confirmed the requirement of CEACAM1 expression on the CD11b(+)/Gr-1(+) population for reestablishment of perfusion after arterial occlusion. CONCLUSIONS: CEACAM1 expression on CD11b(+)/Gr-1(+) myeloid cells is a prerequisite for adequate collateral formation.
Assuntos
Antígeno CD11b/metabolismo , Antígeno Carcinoembrionário/metabolismo , Circulação Colateral , Isquemia/metabolismo , Músculo Esquelético/irrigação sanguínea , Células Mieloides/metabolismo , Neovascularização Fisiológica , Receptores de Quimiocinas/metabolismo , Animais , Transplante de Medula Óssea , Antígeno Carcinoembrionário/genética , Modelos Animais de Doenças , Citometria de Fluxo , Membro Posterior , Isquemia/diagnóstico por imagem , Isquemia/genética , Isquemia/fisiopatologia , Fluxometria por Laser-Doppler , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/imunologia , Células Mieloides/transplante , Fluxo Sanguíneo Regional , Fatores de Tempo , Microtomografia por Raio-XRESUMO
Interleukin-33 (IL-33) is the most recently identified member of the IL-1 family of cytokines, which is primarily known for its proinflammatory functions. We have previously reported that IL-33 is expressed by bone-forming osteoblasts, and that administration of recombinant IL-33 to bone marrow cultures inhibits their differentiation into bone-resorbing osteoclasts. Likewise, while the inhibitory effect of IL-33 on osteoclast differentiation was fully abolished in cultures lacking the IL-33 receptor ST2, mice lacking ST2 displayed low bone mass caused by increased osteoclastogenesis. Although these data suggested a physiological role of IL-33 as an inhibitor of bone resorption, direct in vivo evidence supporting such a function was still missing. Here we describe the generation and bone histomorphometric analysis of a transgenic mouse model (Col1a1-Il33) over-expressing IL-33 specifically in osteoblasts. While we did not observe differences in osteoblast number and bone formation between wildtype and Col1a1-Il33 mice, the number of osteoclasts was significantly reduced compared to wildtype littermates in two independent transgenic lines. Since we did not observe quantitative differences in the populations of eosinophils, neutrophils, basophils or M2-macrophages from the bone marrow of wildtype and Col1a1-Il33 mice, our data demonstrate that an inhibition of osteoclastogenesis is one of the major physiological functions of IL-33, at least in mice.
Assuntos
Interleucinas/fisiologia , Osteoblastos/fisiologia , Animais , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , Interleucina-33 , Interleucinas/genética , Camundongos , Camundongos Transgênicos , Osteoblastos/citologia , TransgenesRESUMO
Since the hematopoetic system is located within the bone marrow, it is not surprising that recent evidence has demonstrated the existence of molecular interactions between bone and immune cells. While interleukin 1 (IL-1) and IL-18, two cytokines of the IL-1 family, have been shown to regulate differentiation and activity of bone cells, the role of IL-33, another IL-1 family member, has not been addressed yet. Since we observed that the expression of IL-33 increases during osteoblast differentiation, we analyzed its possible influence on bone formation and observed that IL-33 did not affect matrix mineralization but enhanced the expression of Tnfsf11, the gene encoding RANKL. This finding led us to analyze the skeletal phenotype of Il1rl1-deficient mice, which lack the IL-33 receptor ST2. Unexpectedly, these mice displayed normal bone formation but increased bone resorption, thereby resulting in low trabecular bone mass. Since this finding suggested a negative influence of IL-33 on osteoclastogenesis, we next analyzed osteoclast differentiation from bone marrow precursor cells and observed that IL-33 completely abolished the generation of TRACP(+) multinucleated osteoclasts, even in the presence of RANKL and macrophage colony-stimulating factor (M-CSF). Although our molecular studies revealed that IL-33 treatment of bone marrow cells caused a shift toward other hematopoetic lineages, we further observed a direct negative influence of IL-33 on the osteoclastogenic differentiation of RAW264.7 macrophages, where IL-33 repressed the expression of Nfatc1, which encodes one of the key transciption factors of osteoclast differentiation. Taken together, these findings have uncovered a previously unknown function of IL-33 as an inhibitor of bone resorption.
Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular/fisiologia , Interleucinas/fisiologia , Osteoblastos/metabolismo , Osteoclastos/citologia , Fosfatase Ácida/metabolismo , Animais , Basófilos/citologia , Basófilos/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Reabsorção Óssea/metabolismo , Osso e Ossos/patologia , Calcitriol/farmacologia , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Células Cultivadas , Eosinófilos/citologia , Eosinófilos/metabolismo , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-33 , Interleucinas/farmacologia , Isoenzimas/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteoclastos/patologia , Ligante RANK/genética , Ligante RANK/farmacologia , Receptores de Interleucina/genética , Células Estromais/citologia , Células Estromais/fisiologia , Fosfatase Ácida Resistente a TartaratoRESUMO
Local inflammation during cutaneous leishmaniasis is accompanied by accumulation of CD11b(+) cells at the site of the infection. A functional role for these monocytic cells in local angiogenesis in leishmaniasis has not been described so far. Here, we show that CD11b(+) cells express high levels of the myeloid differentiation antigen carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). In experimental cutaneous leishmaniasis in C57BL/6 wild-type (B6.WT) and B6.Ceacam1(-/-) mice, we found that only B6.Ceacam1(-/-) mice develop edemas and exhibit impairment of both hemangiogenesis and lymphangiogenesis. Because CEACAM1 expression correlates with functional angiogenesis, we further analyzed the role of the CD11b(+) population. In B6.Ceacam1(-/-) mice, we found systemic reduction of Ly-6C(high)/CD11b(high) monocyte precursors. To investigate whether CEACAM1(+) myeloid cells are causally related to efficient angiogenesis, we used reverse bone marrow transplants (BMTs) to restore CEACAM1(+) or CEACAM1(-) bone marrow in B6.Ceacam1(-/-) or B6.WT recipients, respectively. We found that angiogenesis was restored by CEACAM1(+) BMT only. In addition, we observed reduced morphogenic potential of inflammatory cells in Matrigel implants in CEACAM1(-) backgrounds or after systemic depletion of CD11b(high) macrophages. Taken together, we show for the first time that CEACAM1(+) myeloid cells are crucial for angiogenesis in inflammation.
Assuntos
Antígeno Carcinoembrionário/análise , Inflamação/fisiopatologia , Leishmaniose Cutânea/fisiopatologia , Células Mieloides/fisiologia , Neovascularização Patológica/fisiopatologia , Animais , Anticorpos Antiprotozoários/biossíntese , Transplante de Medula Óssea , Antígeno CD11b/análise , Antígeno Carcinoembrionário/biossíntese , Antígeno Carcinoembrionário/genética , Colágeno , Combinação de Medicamentos , Edema/etiologia , Edema/patologia , Glicoproteínas/biossíntese , Imunidade Celular , Implantes Experimentais , Inflamação/etiologia , Inflamação/imunologia , Interferon gama/biossíntese , Laminina , Leishmania major/imunologia , Leishmaniose Cutânea/complicações , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/patologia , Vasos Linfáticos/metabolismo , Macrófagos/parasitologia , Macrófagos/fisiologia , Proteínas de Membrana Transportadoras , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/química , Células Mieloides/classificação , Neovascularização Patológica/patologia , Proteoglicanas , Quimera por Radiação , Células Th1/imunologiaRESUMO
BACKGROUND: Recent clinical trials, epidemiological studies and animal experiments have suggested that probiotics may help suppress the development of allergic responses. OBJECTIVE: To investigate whether the application of the probiotic Escherichia coli strain Nissle 1917 (EcN) protects mice from developing ovalbumin (OVA)-specific T helper-2 responses in the airways. METHODS: OVA-specific Th2 responses were induced by 2 intraperitoneal (i.p.) injections with OVA/alum followed by 1 intranasal (i.n.) challenge with OVA. EcN was given orally during the entire sensitization and challenge period, together with OVA/alum during the i.p. sensitizations, or i.n. before or during the airway challenge with OVA. RESULTS: We found that when the bacteria were given together with OVA/alum airway eosinophilia, airway hyper-reactivity, goblet cell metaplasia and IL-5 levels in the bronchoalveolar lavage and mediastinal lymph node cell cultures were reduced. This effect was associated with increased numbers of IFN-gamma producing T helper-1 cells and IFN-gamma levels in the airways and strongly increased OVA-specific IgG(2a) titers in the serum. The suppressive effect on airway eosinophilia was dependent on IFN-gamma but not TLR-4. Applying EcN i.n. or orally did not reduce the development of allergen-specific Th2 responses. CONCLUSIONS: Our results suggest that EcN can inhibit the development of allergic responses when the bacteria are present at the site of Th2 cell priming and that this immunomodulatory effect is due to a shift from Th2 to Th1 response. The data support the hypothesis that probiotics may help reduce allergic responses and that EcN may also be used as adjuvant therapy to induce allergen-specific Th1 responses.
Assuntos
Hiper-Reatividade Brônquica/prevenção & controle , Células Dendríticas/imunologia , Escherichia coli/imunologia , Hipersensibilidade/imunologia , Probióticos/uso terapêutico , Células Th2/imunologia , Adjuvantes Imunológicos/farmacologia , Administração Intranasal , Administração Oral , Alérgenos/imunologia , Compostos de Alúmen/farmacologia , Animais , Hiper-Reatividade Brônquica/imunologia , Líquido da Lavagem Broncoalveolar/imunologia , Líquido da Lavagem Broncoalveolar/microbiologia , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Eosinofilia/imunologia , Eosinofilia/metabolismo , Eosinofilia/microbiologia , Feminino , Células Caliciformes/imunologia , Células Caliciformes/patologia , Hipersensibilidade/metabolismo , Hipersensibilidade/microbiologia , Hipersensibilidade/prevenção & controle , Interferon gama/biossíntese , Interferon gama/imunologia , Interleucina-5/biossíntese , Interleucina-5/imunologia , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th1/microbiologia , Células Th2/metabolismo , Células Th2/microbiologiaRESUMO
The biological role of Langerin+ dendritic cells (DCs) such as Langerhans cells and a subset of dermal DCs (dDCs) in adaptive immunity against cutaneous pathogens remains enigmatic. Thus, we analyzed the impact of Langerin+ DCs in adaptive T cell-mediated immunity toward Leishmania major parasites in a Lang-DTR mouse model that allows conditional diphtheria toxin (DT)-induced ablation of Langerin+ DCs in vivo. For the first time, infection experiments with DT-treated Lang-DTR mice revealed that proliferation of L. major-specific CD8+ T cells is significantly reduced during the early phase of the immune response following depletion of Langerin+ DCs. Consequently, the total number of activated CD8+ T cells within the draining lymph node and at the site of infection is diminished. Furthermore, we show that the impaired CD8+ T cell response is due to the absence of Langerin+ dDCs and not Langerhans cells. Nevertheless, the CD4+ T cell response is not altered and the infection is cleared as effectively in DT-treated Lang-DTR mice as in control mice. This clearly demonstrates that Langerin+ DCs are, in general, dispensable for an efficient adaptive immune response against L. major parasites. Thus, we propose a novel concept that, in the experimental model of leishmaniasis, priming of CD4+ T cells is mediated by Langerin- dDCs, whereas Langerin+ dDCs are involved in early priming of CD8+ T cells.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/parasitologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/parasitologia , Células Dendríticas/classificação , Células Dendríticas/parasitologia , Leishmaniose Cutânea/imunologia , Ativação Linfocitária/imunologia , Animais , Antígenos de Superfície/genética , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Células Dendríticas/imunologia , Epiderme/imunologia , Epiderme/metabolismo , Epiderme/parasitologia , Feminino , Técnicas de Introdução de Genes/métodos , Imunidade Inata , Células de Langerhans/imunologia , Células de Langerhans/metabolismo , Células de Langerhans/parasitologia , Lectinas Tipo C/genética , Leishmania major/imunologia , Linfonodos/imunologia , Linfonodos/metabolismo , Linfonodos/parasitologia , Linfonodos/patologia , Ativação Linfocitária/genética , Lectinas de Ligação a Manose/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
The transmembrane glycoprotein CD83 has been described as a specific maturation marker for dendritic cells and several lines of evidence suggest that CD83 regulates thymic T cell maturation as well as peripheral T cell activation. Here we show for the first time that CD83 is involved also in the regulation of B cell function. CD83 is up-regulated on activated B cells in vivo, specifically in the draining lymph nodes of Leishmania major-infected mice. The ubiquitous transgenic (Tg) expression of CD83 interferes with Leishmania-specific T cell-dependent and with T cell-independent antibody production. This defect is restricted to the B cell population since the antigen-specific T cell response of CD83Tg mice to L. major infection is unchanged. The defective immunoglobulin (Ig) response is due to Tg expression of CD83 on the B cells because wild-type B cells display normal antigen-specific responses in CD83Tg hosts and CD83Tg B cells do not respond to immunization in a mixed wild-type/CD83Tg bone marrow chimera. Finally, the treatment of non-Tg C57BL/6 mice with anti-CD83 mAb induces a dramatic increase in the antigen-specific IgG response to immunization, thus demonstrating a regulatory role for naturally induced CD83 on wild-type B cells.
Assuntos
Antígenos CD/fisiologia , Linfócitos B/imunologia , Imunoglobulinas/fisiologia , Glicoproteínas de Membrana/fisiologia , Animais , Anticorpos Antiprotozoários/biossíntese , Antígenos CD/biossíntese , Antígenos CD/genética , Linfócitos B/metabolismo , Feminino , Imunoglobulinas/biossíntese , Imunoglobulinas/genética , Leishmania major/imunologia , Leishmaniose Cutânea/imunologia , Ativação Linfocitária/genética , Masculino , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Regulação para Cima/imunologia , Antígeno CD83RESUMO
We analysed the effects of murine polyomavirus-like particles (PLPs) on bone marrow-derived dendritic cells (BMDCs) and T cells in vitro. BMDCs activated with PLPs up-regulated CD40, CD80, CD86 and major histocompatibility complex (MHC) class II surface markers and produced proinflammatory cytokines. Chimeric PLPs [expressing the ovalbumin (OVA)-peptides OVA(257-264) or OVA(323-339)], but not wildtype PLPs, activated OVA-specific CD8 T cells and OVA-specific CD4 T cells, respectively, indicating both MHC class I and II presentation of the peptides by antigen-presenting cells. Our results suggest that PLPs may be used as vaccine adjuvants priming dendritic cells to induce potent T cell responses.
Assuntos
Células Dendríticas/imunologia , Ativação Linfocitária , Polyomavirus/imunologia , Linfócitos T/imunologia , Adjuvantes Imunológicos , Animais , Apresentação de Antígeno , Antígenos CD/imunologia , Citocinas/imunologia , Citocinas/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe II/imunologia , Camundongos , Camundongos Endogâmicos C57BL , OvalbuminaRESUMO
Experimental leishmaniasis represents a suitable model to analyze Th1-type associated immunity. In C57BL/6 mice healing of leishmaniasis correlates with activation of Th1 cells. Recently, it could be demonstrated that dermal dendritic cells rather than epidermal Langerhans cells are responsible for the activation of Th1 cells after infection, indicating a necessary reconsideration of the role of Langerhans cells. In our current work, epidermal application of Leishmania-antigen prior to infection resulted in an atypical course of disease that is characterized by an impaired Leishmania-specific Th1 response. Consequently, these mice cannot manage an efficient elimination of the parasites at the site of infection. These data point to the activation of immunomodulatory effects by epidermal incorporation of antigen.