RESUMO
The lysosomal enzyme ß-glucuronidase (Gusb) is a key regulator of Lyme-associated and K/B×N-induced arthritis severity. The luminal enzymes present in lysosomes provide essential catabolic functions for the homeostatic degradation of a variety of macromolecules. In addition to this essential catabolic function, lysosomes play important roles in the inflammatory response following infection. Secretory lysosomes and related vesicles can participate in the inflammatory response through fusion with the plasma membrane and release of bioactive contents into the extracellular milieu. In this study, we show that GUSB hypomorphism potentiates lysosomal exocytosis following inflammatory stimulation. This leads to elevated secretion of lysosomal contents, including glycosaminoglycans, lysosomal hydrolases, and matrix metalloproteinase 9, a known modulator of Lyme arthritis severity. This mechanistic insight led us to test the efficacy of rapamycin, a drug known to suppress lysosomal exocytosis. Both Lyme and K/B×N-associated arthritis were suppressed by this treatment concurrent with reduced lysosomal release.
Assuntos
Glucuronidase/metabolismo , Doença de Lyme/metabolismo , Lisossomos/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Animais , Membrana Celular/metabolismo , Modelos Animais de Doenças , Exocitose/efeitos dos fármacos , Exocitose/imunologia , Glucuronidase/deficiência , Glucuronidase/genética , Imunossupressores/farmacologia , Doença de Lyme/tratamento farmacológico , Doença de Lyme/genética , Doença de Lyme/patologia , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Camundongos , Camundongos Knockout , Modelos Biológicos , Transporte Proteico , Sirolimo/farmacologiaRESUMO
MicroRNAs have been shown to be important regulators of inflammatory and immune responses and are implicated in several immune disorders including systemic lupus erythematosus and rheumatoid arthritis, but their role in Lyme borreliosis remains unknown. We performed a microarray screen for expression of miRNAs in joint tissue from three mouse strains infected with Borrelia burgdorferi. This screen identified upregulation of miR-146a, a key negative regulator of NF-κB signaling, in all three strains, suggesting it plays an important role in the in vivo response to B. burgdorferi. Infection of B6 miR-146a-/- mice with B. burgdorferi revealed a critical nonredundant role of miR-146a in modulating Lyme arthritis without compromising host immune response or heart inflammation. The impact of miR-146a was specifically localized to the joint, and did not impact lesion development or inflammation in the heart. Furthermore, B6 miR-146a-/- mice had elevated levels of NF-κB-regulated products in joint tissue and serum late in infection. Flow cytometry analysis of various lineages isolated from infected joint tissue of mice showed that myeloid cell infiltration was significantly greater in B6 miR-146a-/- mice, compared to B6, during B. burgdorferi infection. Using bone marrow-derived macrophages, we found that TRAF6, a known target of miR-146a involved in NF-κB activation, was dysregulated in resting and B. burgdorferi-stimulated B6 miR-146a-/- macrophages, and corresponded to elevated IL-1ß, IL-6 and CXCL1 production. This dysregulated protein production was also observed in macrophages treated with IL-10 prior to B. burgdorferi stimulation. Peritoneal macrophages from B6 miR-146a-/- mice also showed enhanced phagocytosis of B. burgdorferi. Together, these data show that miR-146a-mediated regulation of TRAF6 and NF-κB, and downstream targets such as IL-1ß, IL-6 and CXCL1, are critical for modulation of Lyme arthritis during chronic infection with B. burgdorferi.
Assuntos
Artrite Infecciosa/genética , Borrelia burgdorferi/imunologia , Doença de Lyme/imunologia , MicroRNAs/genética , Miocardite/genética , Animais , Artrite Infecciosa/microbiologia , Borrelia burgdorferi/patogenicidade , Quimiocina CXCL1/imunologia , Regulação da Expressão Gênica/genética , Inflamação/imunologia , Mediadores da Inflamação/imunologia , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-1beta/imunologia , Interleucina-6/imunologia , Doença de Lyme/genética , Doença de Lyme/patologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocardite/microbiologia , NF-kappa B/genética , NF-kappa B/imunologia , Fagocitose/genética , Fagocitose/imunologia , Transdução de Sinais/imunologia , Fator 6 Associado a Receptor de TNF/biossíntese , Fator 6 Associado a Receptor de TNF/imunologiaRESUMO
The protein Zfp318 is expressed during the transition of naive B cells from an immature to mature state. To evaluate its role in mature B cell functions, a conditional gene deficiency in Zfp318 was created and deleted in bone marrow lineages via Vav-Cre. B cell development was minimally altered in the absence of the protein, although transitional 2 (T2) B cell populations were depressed in the absence of Zfp318. Intriguingly, the analysis of IgM and IgD expression by maturing and mature naive B cells demonstrated an elevated level of IgM gene products and a virtual loss of IgD products. Transcriptome analysis of Zfp318-deficient B cells revealed that only two gene products showed altered expression in the absence of Zfp318 (Ighd and Sva), demonstrating a remarkable specificity of Zfp318 action. In the absence of Zfp318, Ighm/Ighd transcripts, which would normally encode IgM and IgD from heterogeneous nuclear RNA transcripts via alternative splicing, lack intron and exon sequences from the IgD (Ighd)-encoding region. This finding indicates that Zfp318, in a novel manner, functions by repressing recognition of the transcriptional termination site at the 3' end of the terminal IgM-encoding exon, allowing for synthesis of the complete Ighm/Ighd heterogeneous nuclear RNA.
Assuntos
Proteínas de Ligação a DNA/imunologia , Regulação da Expressão Gênica/imunologia , Loci Gênicos/imunologia , Imunoglobulina D/imunologia , Imunoglobulina M/imunologia , Terminação da Transcrição Genética/imunologia , Animais , Proteínas de Ligação a DNA/genética , Éxons/imunologia , Regulação da Expressão Gênica/genética , Imunoglobulina D/genética , Imunoglobulina M/genética , Camundongos , Camundongos TransgênicosRESUMO
Localized upregulation of type I IFN was previously implicated in development of Borrelia burgdorferi-induced arthritis in C3H mice, and was remarkable due to its absence in the mildly arthritic C57BL/6 (B6) mice. Independently, forward genetics analysis identified a quantitative trait locus on Chr4, termed B. burgdorferi-associated locus 1 (Bbaa1), that regulates Lyme arthritis severity and includes the 15 type I IFN genes. Involvement of Bbaa1 in arthritis development was confirmed in B6 mice congenic for the C3H allele of Bbaa1 (B6.C3-Bbaa1), which developed more severe Lyme arthritis and K/B×N model of rheumatoid arthritis (RA) than did parental B6 mice. Administration of a type I IFN receptor blocking mAb reduced the severity of both Lyme arthritis and RA in B6.C3-Bbaa1 mice, formally linking genetic elements within Bbaa1 to pathological production of type I IFN. Bone marrow-derived macrophages from Bbaa1 congenic mice implicated this locus as a regulator of type I IFN induction and downstream target gene expression. Bbaa1-mediated regulation of IFN-inducible genes was upstream of IFN receptor-dependent amplification; however, the overall magnitude of the response was dependent on autocrine/paracrine responses to IFN-ß. In addition, the Bbaa1 locus modulated the functional phenotype ascribed to bone marrow-derived macrophages: the B6 allele promoted expression of M2 markers, whereas the C3H allele promoted induction of M1 responses. This report identifies a genetic locus physically and functionally linked to type I IFN that contributes to the pathogenesis of both Lyme and RA.
Assuntos
Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Borrelia burgdorferi/imunologia , Interferon Tipo I/metabolismo , Doença de Lyme/genética , Doença de Lyme/metabolismo , Locos de Características Quantitativas , Alelos , Animais , Artrite Reumatoide/imunologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Fatores Reguladores de Interferon/genética , Interferon Tipo I/farmacologia , Doença de Lyme/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose/genética , Fagocitose/imunologia , Fenótipo , Receptor de Interferon alfa e beta/antagonistas & inibidores , Receptor de Interferon alfa e beta/genética , Ativação TranscricionalRESUMO
Transcriptional regulation of gene expression is a key component of orchestrating proper immune cell development and function. One strategy for maintaining these transcriptional programs has been the evolution of transcription factor families with members possessing overlapping functions. Using the germ line deletion of Snai2 combined with the hematopoietic specific deletion of Snai3, we report that these factors function redundantly to preserve the development of B and T cells. Such animals display severe lymphopenia, alopecia and dermatitis as well as profound autoimmunity manifested by the production of high levels of autoantibodies as early as 3 weeks of age and die by 30 days after birth. Autoantibodies included both IgM and IgG isotypes and were reactive against cytoplasmic and membranous components. A regulatory T cell defect contributed to the autoimmune response in that adoptive transfer of wild type regulatory T cells alleviated symptoms of autoimmunity. Additionally, transplantation of Snai2/Snai3 double deficient bone marrow into Snai2 sufficient Rag2(-/-) recipients resulted in autoantibody generation. The results demonstrated that appropriate expression of Snai2 and Snai3 in cells of hematopoietic derivation plays an important role in development and maintenance of immune tolerance.
Assuntos
Autoimunidade/imunologia , Deleção de Genes , Fatores de Transcrição/imunologia , Dedos de Zinco/imunologia , Células 3T3 , Animais , Autoanticorpos/imunologia , Autoimunidade/genética , Linfócitos B/imunologia , Linfócitos B/metabolismo , Western Blotting , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Expressão Gênica/imunologia , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia de Fluorescência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição da Família Snail , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Dedos de Zinco/genéticaRESUMO
Follicular dendritic cells (FDCs) and complement receptor (Cr)1 and complement receptor (Cr)2 are important for the generation of humoral immunity. Cr1/2 expression on B cells and FDCs was shown to provide a secondary signal for B cell activation, to facilitate transport of Ag in immune follicles, and to enhance retention of immune complexes by FDCs. We show in this study that murine B cells predominantly express the Cr2 product from the Cr2 gene, whereas FDCs almost exclusively express the Cr1 isoform generated from the Cr2 gene. To define the specific role of Cr1, we created an animal that maintains normal cell-restricted expression of Cr2 but does not express Cr1. Cr1-deficient (Cr1KO) mice develop normal B1 and B2 immature and mature B cell subsets and have normal levels of naive serum Abs but altered levels of natural Abs. Immunization of the Cr1KO animal demonstrates deficient Ab responses to T-dependent, but not T-independent, Ags. Germinal centers from the immunized Cr1KO animal possess a deficiency in activated B cells, similar to that seen for animals lacking both Cr1 and Cr2 or C3. Finally, animals lacking only Cr1 respond similarly to wild-type animals to infections with Streptococcus pneumoniae, a pathogen to which animals lacking C3 or both Cr1 and Cr2 are particularly sensitive. Altogether, these data suggest that the production of Cr1, primarily by FDCs, is critical in the generation of appropriately activated B cells of the germinal center and the generation of mature Ab responses.
Assuntos
Subpopulações de Linfócitos B/imunologia , Centro Germinativo/imunologia , Imunoglobulina M/biossíntese , Ativação Linfocitária/imunologia , Receptores de Complemento 3b/genética , Receptores de Complemento 3d/biossíntese , Subpopulações de Linfócitos T/imunologia , Animais , Subpopulações de Linfócitos B/metabolismo , Células Dendríticas Foliculares/imunologia , Células Dendríticas Foliculares/metabolismo , Feminino , Regulação da Expressão Gênica/imunologia , Centro Germinativo/citologia , Imunoglobulina M/genética , Ativação Linfocitária/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Receptores de Complemento 3b/deficiência , Receptores de Complemento 3b/imunologia , Receptores de Complemento 3d/genética , Receptores de Complemento 3d/imunologia , Streptococcus pneumoniae/imunologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/microbiologiaRESUMO
The Mef2 family transcriptional regulator Mef2c (myocyte enhancer factor 2c) is highly expressed in maturing bone marrow and peripheral mature B-cells. To evaluate the role of this transcription factor in B-cell development, we generated a B-cell-specific conditional deletion of Mef2c using the Mb-1-Cre transgene that is expressed during the early stages of immunoglobulin rearrangement. Young mice possessing this defect demonstrated a significant impairment in B-cell numbers in bone marrow and spleen. This phenotype was evident in all B-cell subsets; however, as the animals mature, the deficit in the peripheral mature B-cell compartments was overcome. The absence of Mef2c in mature B-cells led to unique CD23+ and CD23- subsets that were evident in Mef2c knockout primary samples as well as Mef2c-deficient cultured, differentiated B-cells. Genome-wide expression analysis of immature and mature B-cells lacking Mef2c indicated altered expression for a number of key regulatory proteins for B-cell function including Ciita, CD23, Cr1/Cr2 and Tnfsf4. Chromatin immunoprecipitation analysis confirmed Mef2c binding to the promoters of these genes indicating a direct link between the presence (or absence) of Mef2c and altered transcriptional control in mature B-cells.
Assuntos
Linfócitos B/citologia , Linfócitos B/metabolismo , Medula Óssea/metabolismo , Regulação da Expressão Gênica/genética , Transcrição Gênica/genética , Animais , Linfócitos B/imunologia , Fatores de Transcrição MEF2/deficiência , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Camundongos , Camundongos Endogâmicos , Transativadores/genéticaRESUMO
IL-10 is a nonredundant inflammatory modulator that suppresses arthritis development in Borrelia burgdorferi-infected mice. Infected C57BL/6 (B6) IL-10(-/-) mice were previously found to have a prolonged IFN-inducible response in joint tissue. Infection of B6 IL-10 reporter mice identified macrophages and CD4(+) T cells as the primary sources of IL-10 in the infected joint tissue, suggesting that early local production of IL-10 dampened the proarthritic IFN response. Treatment of B6 IL-10(-/-) mice with anti-IFN-γ reduced the increase in arthritis severity and suppressed IFN-inducible transcripts to wild-type levels, thereby linking dysregulation of IFN-γ to disease in the B6 IL-10(-/-) mouse. Arthritis in B6 IL-10(-/-) mice was associated with elevated numbers of NK cell, NKT cell, α/ß T cell, and macrophage infiltration of the infected joint. FACS lineage sorting revealed NK cells and CD4(+) T cells as sources of IFN-γ in the joint tissue of B6 IL-10(-/-) mice. These findings suggest the presence of a positive-feedback loop in the joint tissue of infected B6 IL-10(-/-) mice, in which production of inflammatory chemokines, infiltration of IFN-γ-producing cells, and additional production of inflammatory cytokines result in arthritis. This mechanism of arthritis is in contrast to that seen in C3H/He mice, in which arthritis development is linked to transient production of type I IFN and develops independently of IFN-γ. Due to the sustained IFN response driven by NK cells and T cells, we propose the B6 IL-10(-/-) mouse as a potential model to study the persistent arthritis observed in some human Lyme disease patients.
Assuntos
Movimento Celular/imunologia , Inflamação/imunologia , Interferon gama/farmacologia , Interleucina-10/biossíntese , Doença de Lyme/imunologia , Animais , Borrelia burgdorferi , Retroalimentação Fisiológica , Humanos , Interferon gama/imunologia , Interleucina-10/deficiência , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Knockout , Linfócitos T/imunologiaRESUMO
The complement system has been shown to facilitate peripheral prion pathogenesis. Mice lacking complement receptors CD21/35 partially resist terminal prion disease when infected i.p. with mouse-adapted scrapie prions. Chronic wasting disease (CWD) is an emerging prion disease of captive and free-ranging cervid populations that, similar to scrapie, has been shown to involve the immune system, which probably contributes to their relatively facile horizontal and environmental transmission. In this study, we show that mice overexpressing the cervid prion protein and susceptible to CWD (Tg(cerPrP)5037 mice) but lack CD21/35 expression completely resist clinical CWD upon peripheral infection. CD21/35-deficient Tg5037 mice exhibit greatly impaired splenic prion accumulation and replication throughout disease, similar to CD21/35-deficient murine prion protein mice infected with mouse scrapie. TgA5037;CD21/35(-/-) mice exhibited little or no neuropathology and deposition of misfolded, protease-resistant prion protein associated with CWD. CD21/35 translocate to lipid rafts and mediates a strong germinal center response to prion infection that we propose provides the optimal environment for prion accumulation and replication. We further propose a potential role for CD21/35 in selecting prion quasi-species present in prion strains that may exhibit differential zoonotic potential compared with the parental strains.
Assuntos
Receptores de Complemento 3b/deficiência , Receptores de Complemento 3b/genética , Receptores de Complemento 3d/deficiência , Receptores de Complemento 3d/genética , Receptores de Complemento/deficiência , Receptores de Complemento/genética , Doença de Emaciação Crônica/imunologia , Doença de Emaciação Crônica/prevenção & controle , Animais , Cervos , Modelos Animais de Doenças , Técnicas de Inativação de Genes/métodos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Doenças Priônicas/imunologia , Doenças Priônicas/mortalidade , Doenças Priônicas/prevenção & controle , Doença de Emaciação Crônica/genéticaRESUMO
Localized elevation in type I IFN has been uniquely linked to the severe Lyme arthritis that develops in C3H mice infected with the spirochete Borrelia burgdorferi. In this study, the dynamic interactions that result in generation of these responses were further examined in C3H mice carrying the type I IFN receptor gene ablation, which effectively blocks all autocrine/paracrine signaling crucial to induction of downstream effectors. Reciprocal radiation chimeras between C3H and IFNAR1â»/â» mice implicated both radiation-sensitive and radiation-resistant cells of the joint tissue in the proarthritic induction of type I IFN. Ex vivo analysis of cells from the naive joint revealed CD45⺠cells residing in the tissue to be uniquely capable of initiating the type I IFN response to B. burgdorferi. Type I IFN responses were analyzed in real time by lineage sorting of cells from infected joint tissue. This demonstrated that myeloid cells, endothelial cells, and fibroblasts were responsible for propagating the robust IFN response, which peaked at day 7 postinfection and rapidly resolved. Endothelial cells and fibroblasts were the dominant sources of IFN signature transcripts in the joint tissue. Fibroblasts were also the major early source of chemokines associated with polymorphonuclear leukocyte and monocyte/macrophage infiltration, thus providing a focal point for arthritis development. These findings suggest joint-localized interactions among related and unrelated stromal, endothelial, and myeloid cell lineages that may be broadly applicable to understanding the pathogeneses of diseases associated with type I IFN signature, including systemic lupus erythematosus and some rheumatoid arthritides.
Assuntos
Artrite Experimental/imunologia , Fibroblastos/imunologia , Interferon Tipo I/biossíntese , Doença de Lyme/imunologia , Células Mieloides/imunologia , Regulação para Cima/imunologia , Animais , Articulação do Tornozelo/imunologia , Articulação do Tornozelo/microbiologia , Articulação do Tornozelo/patologia , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Borrelia burgdorferi/imunologia , Borrelia burgdorferi/patogenicidade , Células Cultivadas , Quimiocinas/biossíntese , Quimiocinas/genética , Fibroblastos/metabolismo , Fibroblastos/patologia , Interferon Tipo I/deficiência , Interferon Tipo I/genética , Doença de Lyme/metabolismo , Doença de Lyme/patologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/metabolismo , Células Mieloides/patologia , Índice de Gravidade de Doença , Transdução de Sinais/imunologia , Transcrição Gênica/imunologia , Regulação para Cima/genéticaRESUMO
The altered expression of transcription factors in hematopoietic stem cells and their subsequent lineages can alter the development of lymphoid and myeloid lineages. The role of the transcriptional repressor Snai3 protein in the derivation of cells of the hemato-poietic system was investigated. Snai3 is expressed in terminal T-cell and myeloid lineages, therefore, we chose to determine if expressing Snai3 in the early stages of hematopoietic development would influence cell-lineage determination. Expression of Snai3 by retroviral transduction of hematopoietic stem cells using bone marrow chimera studies demonstrated a block in lymphoid-cell development and enhanced expansion of myeloid-lineage cells. Analysis of Snai3-expressing hematopoietic precursor cells showed normal numbers of immature cells, but a block in the development of cells committed to lymphoid lineages. These data indicate that the overexpression of Snai3 does alter bone marrow cell development and that the identification of genes whose expression is altered by the presence of Snai3 would aid in our understanding of these developmental pathways.
Assuntos
Regulação da Expressão Gênica/imunologia , Linfócitos/imunologia , Células Mieloides/imunologia , Proteínas Repressoras/imunologia , Fatores de Transcrição/imunologia , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/fisiologia , Regulação da Expressão Gênica/genética , Linfócitos/metabolismo , Camundongos , Camundongos Transgênicos , Células Mieloides/metabolismo , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Fatores de Transcrição da Família Snail , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Transdução GenéticaRESUMO
The capacity of immature B cells of the spleen and bone marrow to differentiate in vitro into cells representing mature end stage cells was investigated using B-cell activating factor belonging to the TNF family (BAFF) and Notch pathway activators. Immature splenic and bone marrow B cells were found, in the presence of both of these activators, to mature into cells with follicular mature (FM) and marginal zone (MZ) cell phenotypes. Such cells were functionally responsive to B-cell-specific activation. The derivation in vitro of cells with an MZ phenotype was more robust from CD23(-) populations than CD23(+) immature/transitional B cells, suggesting a direct immature/T1 B cell to MZ cell differentiation pathway. Transcript analysis of the in vitro-derived B-cell populations demonstrated expression profiles similar to maturing B cells in vivo. FACS-purified populations of B220(+)CD19(+)CD21(-)CD23(-) cells from bone marrow of 2-wk-old mice gave rise to populations of CD21(+)CD23(-) cells with MZ cell phenotypes as well as CD21(+)CD23(+) cells with FM cell phenotypes in percentages similar to those found in vivo. These data suggest that the commitment to an MZ and FM B cell phenotype is set prior to immature B-cell release from the marrow.
Assuntos
Fator Ativador de Células B/farmacologia , Linfócitos B/citologia , Células da Medula Óssea/efeitos dos fármacos , Linfopoese/efeitos dos fármacos , Células-Tronco Multipotentes/efeitos dos fármacos , Baço/citologia , Animais , Células da Medula Óssea/citologia , Técnicas de Cultura de Células , Linhagem da Célula , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Imunofenotipagem , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Multipotentes/citologia , Receptores Notch/fisiologiaRESUMO
Complement component C3 has established roles in both innate and adaptive immune responses. C3 cleavage products function in B cell activation through the complement receptors CD21/35. Phenotypes of Ab production between CD21/35(-/-) and C3(-/-) mice are not always congruent, implicating additional roles for C3 in B cell responses. To further characterize complement and complement receptors, we have identified a role for C3 in the regulation of CR3 on splenic B cells. Splenic B2 cells are not defined as expressing CR3, yet the analysis of splenic B cells from C3(-/-) animals demonstrate cell surface expression of CR3. B cells from both wild-type (WT) and C3(-/-) animals express CR3/CD11b/Itgam (integrin alpha M) gene transcripts although the level of such transcripts is 2- to 3-fold higher in B cells from the C3(-/-) animal vs WT cells. C3(-/-) and WT animals have similar B cell subpopulations with identical CR3 expression on B220(-) cells from the spleen, marrow, and lymph nodes. The C3-deficient environment is responsible for altered CR3 expression as WT splenic B cells transferred into C3(-/-) animals expressed cell surface CR3 within 48 h while transfer of C3(-/-) splenic B cells into WT animals depressed surface expression of CR3. Furthermore, transfer of C3-producing splenic macrophages into C3(-/-) mice depressed CR3 expression by resident B cells. These data suggest a role for C3 in influencing the level of expression of CR3 by modulating the transcript levels encoding the CD11b alpha integrin protein.
Assuntos
Linfócitos B/metabolismo , Complemento C3/fisiologia , Regulação da Expressão Gênica/imunologia , Antígeno de Macrófago 1/genética , Baço/citologia , Animais , Antígeno CD11b/genética , Camundongos , Camundongos Knockout , RNA Mensageiro , Receptores de Complemento/genéticaRESUMO
We recently discovered a critical role for type I interferon (IFN) in the development of murine Lyme arthritis. Borrelia burgdorferi-mediated induction of IFN-responsive genes by bone marrow-derived macrophages (BMDMs) was dependent upon a functional type I IFN receptor but independent of Toll-like receptor 2 (TLR2), TLR4, TLR9, and the adapter molecule MyD88. We now demonstrate that induction of the IFN transcriptional profile in B. burgdorferi-stimulated BMDMs occurs independently of the adapter TRIF and of the cytoplasmic sensor NOD2. In contrast, B. burgdorferi-induced transcription of these genes was dependent upon a rapid STAT1 feedback amplification pathway. IFN profile gene transcription was IRF3 dependent but did not utilize B. burgdorferi-derived DNA or DNase-sensitive ligands. Instead, IFN-responsive gene expression could be induced by B. burgdorferi-derived RNA. Interferon regulatory factor 3 (IRF3)-dependent IFN profile gene transcription was also induced by sonicated bacteria, by the lipoprotein OspA, and by factors released into the BSKII medium during culture of B. burgdorferi. The IFN-stimulatory activity of B. burgdorferi culture supernatants was not destroyed by nuclease treatment. Nuclease digestion also had no effect on IFN profile induction mediated by sonicated B. burgdorferi. Thus, B. burgdorferi-derived RNA, OspA, and non-nucleic acid ligands present in both sonicated bacteria and B. burgdorferi culture medium contribute to type I IFN-responsive gene induction. These findings suggest that B. burgdorferi invasion of joint tissue and the resultant type I IFN induction associated with Lyme arthritis development may involve multiple triggering ligands.
Assuntos
Borrelia burgdorferi/fisiologia , Fator Regulador 3 de Interferon/fisiologia , Interferon Tipo I/biossíntese , Doença de Lyme/microbiologia , Animais , Borrelia burgdorferi/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Genes Bacterianos/genética , Genes Bacterianos/fisiologia , Interferon Tipo I/fisiologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Proteína Adaptadora de Sinalização NOD2/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT1/imunologia , Fator de Transcrição STAT1/fisiologia , Transcrição Gênica/genética , Transcrição Gênica/fisiologiaRESUMO
Gene expression analysis previously revealed a robust IFN-responsive gene induction profile that was selectively up-regulated in Borrelia burgdorferi-infected C3H mice at 1 wk postinfection. This profile was correlated with arthritis development, as it was absent from infected, mildly arthritic C57BL/6 mice. In this report we now demonstrate that profile induction in infected C3H scid mice occurs independently of B or T lymphocyte infiltration in the joint tissue. Additionally, type I IFN receptor-blocking Abs, but not anti-IFN-gamma Abs, dramatically reduced arthritis, revealing a critical but previously unappreciated role for type I IFN in Lyme arthritis development. Certain examined IFN-inducible transcripts were also significantly diminished within joint tissue of mice treated with anti-IFNAR1, whereas expression of other IFN-responsive genes was more markedly altered by anti-IFN-gamma treatment. These data indicate that induction of the entire IFN profile is not necessary for arthritis development. These findings further tie early type I IFN induction to Lyme arthritis development, a connection not previously made. Bone marrow-derived macrophages readily induced IFN-responsive genes following B. burgdorferi stimulation, and this expression required a functional type I IFN receptor. Strikingly, induction of these genes was independent of TLRs 2,4, and 9 and of the adapter molecule MyD88. These data demonstrate that the extracellular pathogen B. burgdorferi uses a previously unidentified receptor and a pathway traditionally associated with viruses and intracellular bacteria to initiate transcription of type I IFN and IFN-responsive genes and to initiate arthritis development.
Assuntos
Borrelia burgdorferi/imunologia , Interferon Tipo I/fisiologia , Doença de Lyme/imunologia , Doença de Lyme/microbiologia , Animais , Anticorpos Bloqueadores/administração & dosagem , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/patologia , Feminino , Perfilação da Expressão Gênica , Imunidade Inata/genética , Interferon Tipo I/biossíntese , Interferon Tipo I/deficiência , Doença de Lyme/metabolismo , Doença de Lyme/terapia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Receptor de Interferon alfa e beta/antagonistas & inibidores , Receptor de Interferon alfa e beta/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
The observation that Borrelia burgdorferi-induced arthritis is severe in C3H mice and milder in C57BL/6 (B6) mice has allowed a forward genetics approach for the identification of genetic elements that regulate the arthritis response. Quantitative trait loci (QTL) on five chromosomes (Chr) were identified previously in segregating crosses between C3H and B6 mice and collectively designated B. burgdorferi arthritis-associated (Bbaa) QTL. Reciprocal interval-specific congenic lines (ISCL) that encompass Bbaa1, Bbaa2-Bbaa3, Bbaa4, Bbaa6, and Bbaa12 on Chr 4, 5, 11, 12, and 1, respectively, have now been generated. Bidirectional transfer of the arthritis severity phenotype in association with Bbaa2-Bbaa3 and Bbaa4 was observed, and unidirectional transfer with the B6 allele of Bbaa6 was noted. These findings confirm the existence of polymorphic loci within Bbaa2-Bbaa3, Bbaa4, and Bbaa6 that regulate the severity of B. burgdorferi-induced arthritis. ISCL were used to assess the regulation of a previously identified interferon transcriptional profile associated with severe disease in C3H mice. The regulation of this transcriptional signature was found to be independent of penetrant Bbaa QTL, both in joint tissues and in isolated macrophages. These results clearly demonstrate the utility of forward genetics for the discovery of novel genes and pathways involved in the regulation of the severity of Lyme arthritis and predict the involvement of regulatory elements not evident from other experimental approaches.
Assuntos
Borrelia burgdorferi/patogenicidade , Cromossomos , Doença de Lyme/genética , Doença de Lyme/patologia , Locos de Características Quantitativas , Animais , Tornozelo/patologia , Cruzamentos Genéticos , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , FenótipoRESUMO
C57BL/6 mice deficient in TLR2 develop more severe arthritis following infection with Borrelia burgdorferi than do wild-type C57BL/6 mice, and this increase is suppressed by the simultaneous presence of the scid mutation. This suggested a requirement for lymphocytes in the development of subacute Lyme arthritis in TLR2(-/-) mice, a feature not commonly associated with this arthritis. The increased pathology of B. burgdorferi-infected TLR2(-/-) mice was also accompanied by an increase in mononuclear cell infiltration. In this study, T cells were found to be responsible for the increase in mononuclear cells in infected TLR2(-/-) C3H mice. Accordingly, transcripts for the IFN-inducible T cell chemokines, CXCL9 and CXCL10, were greatly enhanced in joint tissue from TLR2(-/-) mice, as were transcripts for a prototypical IFN-inducible gene IFN-gamma-induced GTPase (igtp). Treatment of murine synovial cells with sonicated B. burgdorferi resulted in induction of transcripts for chemokines and other IFN-inducible genes, irrespective of the presence of TLR2. The presence of T lymphocytes greatly enhanced the transcriptional response of synovial cells. These results suggest that the increased inflammatory cell infiltration in TLR2(-/-) C3H mice is the result of localized overproduction of T cell attracting chemokines.
Assuntos
Borrelia burgdorferi/imunologia , Doença de Lyme/imunologia , Doença de Lyme/patologia , Linfócitos T/imunologia , Receptor 2 Toll-Like/imunologia , Animais , Articulação do Tornozelo/imunologia , Articulação do Tornozelo/patologia , Quimiocina CXCL10/biossíntese , Quimiocina CXCL9/biossíntese , GTP Fosfo-Hidrolases/biossíntese , Camundongos , Camundongos Endogâmicos C3H , Camundongos Knockout , Receptor 2 Toll-Like/deficiênciaRESUMO
The TNF family member BAFF serves to promote the survival and differentiation of maturing splenic B cells. The major receptor for BAFF (BAFF-R) is expressed by the transition 2, marginal zone and follicular, mature conventional B-2 cell populations; functional BAFF/BAFF-R signaling is required for T1 to T2 cell B cell maturation. Induced expression of CD23 and CD21 is also coincident with the T1 to T2 maturation stage. A key question we address in this report is if BAFF signaling directly induces CD21 and CD23 gene transcription and expression at this B cell transition point, or if their expression is simply coincident with B cell maturation and differentiation. We present data that supports the contention that BAFF does not preferentially induce the expression of CD23 or CD21 at the T1 to T2 transition, nor does exogenous BAFF lead to preferential increased expression of these proteins/genes in mature B cell populations. The analysis of LPS-induced splenic B cells from BAFF-R defective (A/WySnJ) mice did not show the preferential induction of expression of CD21 or CD23 that might have been expected if NF-kappaB-p52 protein was lacking due to insufficient BAFF-R signaling in cells bearing this mutation. Indeed, chromatin immunoprecipitation analysis demonstrated stable NF-kappaB-p52 complexes on CD21 and CD23 genes obtained from both wild type and A/WySnJ B cells. FACS analysis of splenic B cells from 1-, 2-, 3- and 6-week-old A/WySnJ mice demonstrated a block in differentiation (thus reducing overall B cell numbers) resulting in a failure of such cells to express CD21 but allowing for the expression level of CD23 per cell to reach levels approaching wild type. We have dubbed this CD23(HI)CD21(LO) subset as the T1b transition B cell. These data support the recognized role of BAFF as promoting the survival and differentiation of splenic B cells but do not support a model of BAFF signaling directly inducing the expression of the CD21 and CD23 proteins via translocation of NF-kappaB-p52 species.
Assuntos
Fator Ativador de Células B/metabolismo , Regulação da Expressão Gênica , Receptores de Complemento 3d/genética , Receptores de Complemento 3d/metabolismo , Receptores de IgE/genética , Receptores de IgE/metabolismo , Envelhecimento/efeitos dos fármacos , Animais , Fator Ativador de Células B/deficiência , Fator Ativador de Células B/farmacologia , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Subunidade p52 de NF-kappa B/metabolismo , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Reguladoras de Ácido Nucleico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Baço/citologia , Baço/metabolismoAssuntos
Anticorpos Monoclonais/efeitos adversos , Ensaios Clínicos como Assunto/efeitos adversos , Ensaios Clínicos como Assunto/normas , Hipersensibilidade a Drogas/diagnóstico , Hipersensibilidade a Drogas/prevenção & controle , Anafilaxia/induzido quimicamente , Anafilaxia/diagnóstico , Anafilaxia/prevenção & controle , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados , HumanosRESUMO
The mouse Pactolus and CD18 genes are highly conserved paralogues. The expression patterns of these genes are diverse in that most cells of hematopoietic lineage express CD18, but Pactolus is only expressed by maturing neutrophils. The minimal promoters of these two genes are homologous, including the conservation of two tandem PU.1-binding sites upstream of the transcriptional start site. To define the means by which these two structurally similar but functionally distinct promoters operate, a series of reporter assays, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation analyses, were performed. Transfection of Pactolus constructs into mouse macrophages, which do not express Pactolus, defined a negative control element within the first 100 base pairs. The presence of this negative regulatory site, distinct from the PU.1-binding site, was confirmed by EMSA oligonucleotide competition and gene reporter assays of Pactolus/CD18 chimeric constructs. Although PU.1 binding can be detected on Pactolus and CD18 minimal promoter segments with EMSA, only the CD18 promoter shows PU.1 binding in vivo, suggesting that the negative regulatory protein may block PU.1 from binding to the Pactolus promoter, thus inhibiting transcription of the gene. Sequence analysis of the negative control region in the Pactolus promoter suggested potential control by Snail and/or Smad families of transcription regulators. EMSA supershift analysis with antibodies against these proteins, using extracts from macrophages and mucosal mast cells, identified specific binding of Smuc to the promoter element, including a Smuc/PU.1/DNA trimeric complex. These data implicate Smuc as blocking Pactolus transcription in cells expressing PU.1 (and CD18) but not Pactolus.