RESUMEN
Vaccines can serve as essential tools to prevent bacterial diseases via the induction of long-lasting IgG responses. The efficacy of such vaccines depends on the effector mechanisms triggered by IgG. The complement system and Fc-gamma receptors (FcγRs) can potentially play a crucial role in IgG-mediated immunity against bacterial diseases. However, their relative importance in vivo is unclear, and has been the object of controversy and debate. In this brief study, we have used gene-targeted mice lacking either FcγRI, II, II and IV or the C3 complement component as well as a novel mouse strain lacking both C3 and FcγRs to conclusively show the essential role of complement in antibody-mediated host resistance to Salmonella enterica systemic infection. By comparing the effect of IgG2a antibodies against Salmonella O-antigen in gene-targeted mice, we demonstrate that the complement system is essential for the IgG-mediated reduction of bacterial numbers in the tissues.
Asunto(s)
Complemento C3/metabolismo , Antígenos O/inmunología , Receptores de IgG/metabolismo , Infecciones por Salmonella/inmunología , Vacunas contra la Salmonella/inmunología , Salmonella enterica/fisiología , Animales , Carga Bacteriana , Activación de Complemento , Complemento C3/genética , Humanos , Inmunidad Humoral , Inmunoglobulina G/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de IgG/genéticaRESUMEN
Deficiency of the cysteine protease inhibitor cystatin M/E (Cst6) in mice leads to disturbed epidermal cornification, impaired barrier function, and neonatal lethality. We report the rescue of the lethal skin phenotype of ichq (Cst6-deficient; Cst6-/-) mice by transgenic, epidermis-specific, reexpression of Cst6 under control of the human involucrin (INV) promoter. Rescued Tg(INV-Cst6)Cst6ichq/ichq mice survive the neonatal phase, but display severe eye pathology and alopecia after 4 mo. We observed keratitis and squamous metaplasia of the corneal epithelium, comparable to Cst6-/-Ctsl+/- mice, as we have reported in other studies. We found the INV promoter to be active in the hair follicle infundibulum; however, we did not observe Cst6 protein expression in the lower regions of the hair follicle in Tg(INV-Cst6)Cst6ichq/ichq mice. This result suggests that unrestricted activity of proteases is involved in disturbance of hair follicle biology, eventually leading to baldness. Using quenched activity-based probes, we identified mouse cathepsin B (CtsB), which is expressed in the lower regions of the hair follicle, as an additional target of mouse Cst6. These data suggest that Cst6 is necessary to control CtsB activity in hair follicle morphogenesis and highlight Cst6-controlled proteolytic pathways as targets for preventing hair loss.-Oortveld, M. A. W., van Vlijmen-Willems, I. M. J. J., Kersten, F. F. J., Cheng, T., Verdoes, M., van Erp, P. E. J., Verbeek, S., Reinheckel, T., Hendriks, W. J. A. J., Schalkwijk, J., Zeeuwen, P. L. J. M. Cathepsin B as a potential cystatin M/E target in the mouse hair follicle.
Asunto(s)
Catepsina B/metabolismo , Diferenciación Celular/fisiología , Cistatina M/metabolismo , Epidermis/metabolismo , Folículo Piloso/metabolismo , Alopecia/metabolismo , Animales , Catepsina L/metabolismo , Células Cultivadas , Cistatina M/deficiencia , Humanos , Ratones , Piel/metabolismoRESUMEN
The canonical Wnt signalling pathway has been implicated in organogenesis and self-renewal of essentially all stem cell systems. In vivo reporter systems are crucial to assess the role of Wnt signalling in the biology and pathology of stem cell systems. We set out to develop a Turquoise (TQ) fluorescent protein based Wnt reporter. We used a CRISPR-Cas9 approach to insert a TQ fluorescent protein encoding gene into the general Wnt target gene Axin2, thereby establishing a Wnt reporter mouse similar to previously generated Wnt reporter mice but with the mTurquoise2 gene instead of E. coli-ß-galactosidase (LacZ). The use of mTurquoise2 is especially important in organ systems in which cells need to a be alive for further experimentation such as in vitro activation or transplantation studies. We here report successful generation of Axin2-TQ mice and show that cells from these mice faithfully respond to Wnt signals. High Wnt signals were detected in the intestinal crypts, a classical Wnt signalling site in vivo, and by flow cytometry in the thymus. These mice are an improved tool to further elucidate the role of Wnt signalling in vivo.
Asunto(s)
Proteína Axina/metabolismo , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Vía de Señalización Wnt , Animales , Proteína Axina/genética , Sistemas CRISPR-Cas , Marcación de Gen/métodos , Proteínas Fluorescentes Verdes/metabolismo , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Timo/citología , Timo/metabolismoRESUMEN
The CD40/CD40L dyad is deemed to play a central role in several inflammatory processes, including atherosclerosis. As CD40 is overexpressed in atherosclerotic lesions, it constitutes a promising candidate for targeted imaging approaches. Here we describe the design of a novel, selective peptide ligand for CD40 by phage display. A synthetic peptide corresponding with the phage insert NP31 displayed nanomolar affinity for CD40. Affinity was further enhanced by mutimeric presentation of NP31. An essential 11-mer peptide motif was identified by truncation and alanine scan studies. Enriched phage selectively bound human CD40 and homed to inflammatory joints in a murine model of rheumatoid arthritis. NP31 ablated VEGF and IL-6 transcriptional activation and partially inhibited IL-6 production by CD40L-activated endothelial cells. Notably, NP31 did not only alter the biodistribution profile of a streptavidin scaffold but also markedly increased accumulation of the carrier in atherosclerotic aortic lesions of aged ApoE(-/-) mice in a CD40-dependent manner. This potent and selective peptide ligand has potential for targeted imaging and drug delivery approaches in CD40-dependent inflammatory disorders such as atherosclerosis.
Asunto(s)
Ligando de CD40/metabolismo , Biblioteca de Péptidos , Secuencia de Aminoácidos , Animales , Aorta/patología , Artritis Reumatoide/diagnóstico , Aterosclerosis/metabolismo , Aterosclerosis/patología , Ligando de CD40/genética , Línea Celular , Humanos , Inflamación , Ratones , Unión ProteicaRESUMEN
The ectodomain of matrix protein 2 (M2e) of influenza A virus is an attractive target for a universal influenza A vaccine: the M2e sequence is highly conserved across influenza virus subtypes, and induced humoral anti-M2e immunity protects against a lethal influenza virus challenge in animal models. Clinical phase I studies with M2e vaccine candidates have been completed. However, the in vivo mechanism of immune protection induced by M2e-carrier vaccination is unclear. Using passive immunization experiments in wild-type, FcRγ(-/-), FcγRI(-/-), FcγRIII(-/-), and (FcγRI, FcγRIII)(-/-) mice, we report in this study that Fc receptors are essential for anti-M2e IgG-mediated immune protection. M2e-specific IgG1 isotype Abs are shown to require functional FcγRIII for in vivo immune protection but other anti-M2e IgG isotypes can rescue FcγRIII(-/-) mice from a lethal challenge. Using a conditional cell depletion protocol, we also demonstrate that alveolar macrophages (AM) play a crucial role in humoral M2e-specific immune protection. Additionally, we show that adoptive transfer of wild-type AM into (FcγRI, FcγRIII)(-/-) mice restores protection by passively transferred anti-M2e IgG. We conclude that AM and Fc receptor-dependent elimination of influenza A virus-infected cells are essential for protection by anti-M2e IgG.
Asunto(s)
Inmunoglobulina G/metabolismo , Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Macrófagos Alveolares/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Dominios y Motivos de Interacción de Proteínas/inmunología , Receptores Fc/fisiología , Proteínas de la Matriz Viral/inmunología , Animales , Muerte Celular/genética , Muerte Celular/inmunología , Citotoxicidad Inmunológica , Modelos Animales de Enfermedad , Femenino , Inmunización Pasiva , Inmunoglobulina G/toxicidad , Virus de la Influenza A/genética , Vacunas contra la Influenza/genética , Vacunas contra la Influenza/uso terapéutico , Depleción Linfocítica/métodos , Macrófagos Alveolares/patología , Macrófagos Alveolares/virología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/patología , Dominios y Motivos de Interacción de Proteínas/genética , Receptores Fc/deficiencia , Receptores Fc/uso terapéutico , Receptores de IgG/deficiencia , Receptores de IgG/metabolismo , Receptores de IgG/fisiología , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/uso terapéuticoRESUMEN
Uptake of Leishmania major by dendritic cells (DCs) results in activation and interleukin (IL)-12 release. Infected DCs efficiently stimulate CD4- and CD8- T cells and vaccinate against leishmaniasis. In contrast, complement receptor 3-dependent phagocytosis of L. major by macrophages (MPhi) leads exclusively to MHC class II-restricted antigen presentation to primed, but not naive, T cells, and no IL-12 production. Herein, we demonstrate that uptake of L. major by DCs required parasite-reactive immunoglobulin (Ig)G and involved FcgammaRI and FcgammaRIII. In vivo, DC infiltration of L. major-infected skin lesions coincided with the appearance of antibodies in sera. Skin of infected B cell-deficient mice and Fcgamma-/- mice contained fewer parasite-infected DCs in vivo. Infected B cell-deficient mice as well as Fcgamma-/- mice (all on the C57BL/6 background) showed similarly increased disease susceptibility as assessed by lesion volumes and parasite burdens. The B cell-deficient mice displayed impaired T cell priming and dramatically reduced IFN-gamma production, and these deficits were normalized by infection with IgG-opsonized parasites. These data demonstrate that DC and MPhi use different receptors to recognize and ingest L. major with different outcomes, and indicate that B cell-derived, parasite-reactive IgG and DC FcgammaRI and FcgammaRIII are essential for optimal development of protective immunity.
Asunto(s)
Células Dendríticas/inmunología , Leishmania major/inmunología , Leishmaniasis Cutánea/prevención & control , Receptores de IgG/inmunología , Animales , Linfocitos B/inmunología , Células Cultivadas , Células Dendríticas/parasitología , Inmunoglobulina G/inmunología , Leishmania major/patogenicidad , Leishmaniasis Cutánea/inmunología , Leishmaniasis Cutánea/parasitología , Antígeno de Macrófago-1/inmunología , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , FagocitosisRESUMEN
Many patients with colorectal cancer will develop liver metastases, even after successful surgical removal of the primary tumor at a time at which no visible metastases are present. We previously demonstrated that surgery--although mandatory--paradoxically enhances the risk of developing liver metastases. Because Ab therapy has been acknowledged as a successful strategy to treat malignancies, we studied the potential of postoperative adjuvant Ab therapy to prevent outgrowth of liver metastases. Treatment with murine anti-gp75 (TA99) mAb completely prevented outgrowth of B16F10 liver metastases in over 90% of mice. Therapeutic efficacy was maintained in either C1q- or complement receptor 3-deficient mice but was completely abrogated in FcR gamma-chain knockout mice. This indicates that the classical complement pathway was not essential, but interaction with activatory Fc gammaR was necessary for successful therapy. TA99-treatment was still effective in Fc gammaRI(-/-), Fc gammaRIII(-/-), Fc gammaRI/III(-/-), and Fc gammaRI/II/III(-/-) mice, suggesting an important role for Fc gammaRIV. However, wild-type mice that were treated with TA99 Abs and an Fc gammaRIV blocking Ab (mAb 9E9) were protected against development of liver metastases as well. Only when both Fc gammaRI and Fc gammaRIV functions were simultaneously inhibited, TA99-mediated curative Ab treatment was abrogated, indicating functional redundancy between both IgG receptors in the liver. Furthermore, depletion of liver macrophages (Kupffer cells) reduced the efficacy of Ab therapy, supporting that Kupffer cells are involved as effector cells. Importantly, since Ab treatment almost completely prevented development of liver metastases, postoperative adjuvant Ab therapy may help to improve patient prognosis.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Neoplasias Hepáticas/prevención & control , Neoplasias Hepáticas/secundario , Receptores de IgG/inmunología , Animales , Neoplasias Colorrectales/patología , Citometría de Flujo , Macrófagos del Hígado/inmunología , Macrófagos del Hígado/metabolismo , Ratones , Ratones Noqueados , Receptores de IgG/metabolismoRESUMEN
Cytomegalovirus (CMV)-based vaccine vectors are promising vaccine platforms because they induce strong and long-lasting immune responses. Recently it has been shown that vaccination with a mouse CMV (MCMV) vector expressing the melanoma-specific antigen TRP2 (MCMV-TRP2) protects mice against outgrowth of TRP2-positive B16 melanoma tumors, and this protection was dependent on the induction of IgG antibodies. Here we demonstrate that, although mice lacking all receptors for the Fc part of IgG (FcγRs) develop normal IgG responses after MCMV-TRP2 vaccination, the protection against B16 melanoma was completely abrogated, indicating that FcγRs are indispensable in the downstream effector pathway of the polyclonal anti-TRP2 antibody response. By investigating compound FcγR-deficient mouse strains and by using immune cell type-specific cell ablation we show that the IgG antibody-mediated tumor protection elicited by MCMV-TRP2 mainly depends on FcγRI expression on macrophages, whereas FcγRIV plays only a modest role. Thus, tumor-specific antibody therapy might benefit from combination therapy that recruits FcγRI-expressing pro-inflammatory macrophages to the tumor micro-environment.
RESUMEN
Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease generally caused by reading frame disrupting mutations in the DMD gene resulting in loss of functional dystrophin protein. The reading frame can be restored by antisense oligonucleotide (AON)-mediated exon skipping, allowing production of internally deleted, but partially functional dystrophin proteins as found in the less severe Becker muscular dystrophy. Due to genetic variation between species, mouse models with mutations in the murine genes are of limited use to test and further optimize human specific AONs in vivo. To address this we have generated the del52hDMD/mdx mouse. This model carries both murine and human DMD genes. However, mouse dystrophin expression is abolished due to a stop mutation in exon 23, while the expression of human dystrophin is abolished due to a deletion of exon 52. The del52hDMD/mdx model, like mdx, shows signs of muscle dystrophy on a histological level and phenotypically mild functional impairment. Local administration of human specific vivo morpholinos induces exon skipping and dystrophin restoration in these mice. Depending on the number of mismatches, occasional skipping of the murine Dmd gene, albeit at low levels, could be observed. Unlike previous models, the del52hDMD/mdx model enables the in vivo analysis of human specific AONs targeting exon 51 or exon 53 on RNA and protein level and muscle quality and function. Therefore, it will be a valuable tool for optimizing human specific AONs and genome editing approaches for DMD.
Asunto(s)
Secuencia de Bases , Distrofina , Exones , Oligodesoxirribonucleótidos Antisentido , Eliminación de Secuencia , Animales , Evaluación Preclínica de Medicamentos , Distrofina/genética , Distrofina/metabolismo , Humanos , Ratones , Ratones Endogámicos mdx , Ratones Transgénicos , Distrofia Muscular de Duchenne/tratamiento farmacológico , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patología , Oligodesoxirribonucleótidos Antisentido/genética , Oligodesoxirribonucleótidos Antisentido/farmacologíaRESUMEN
Macrophages are considered essential mediators in multiple sclerosis (MS) pathogenesis, presumably through myelin phagocytosis and release of inflammatory mediators. Macrophages and microglia express activating Fcgamma receptors (FcgammaRI and FcgammaRIII), which depend on the FcRgamma chain for surface expression and signaling. In MS lesions, crosslinking of FcgammaR by immunoglobulins (IgG) directed against myelin may enhance myelin phagocytosis and inflammation. We studied the role of FcgammaR and anti-myelin antibodies in MOG35-55-induced experimental allergic encephalomyelitis (EAE) in C57BL/6 mice, a model of MS-like disease. Incidence and severity of EAE were similar in FcRy chain-/- (FcRgamma-/-) and wild-type (wt) mice, albeit with delayed onset in FcRgamma-/- mice. This demonstrates that the FcRy chain is not essential for induction of EAE, but that FcRgamma signaling may contribute to the preclinical phase. The role of FcgammaR in antibody-mediated demyelination was addressed by injection of anti-myelin antibodies (Z12 mAb) at onset of MOG35-55-induced EAE. Injection of Z12 mAb rapidly reduced survival time in both wt and FcRgamma-/- mice, demonstrating that antibody-mediated exacerbation of EAE is independent of the FcRgamma chain. Interestingly, Z12-induced exacerbation of inflammation and demyelination persisted longer in wt than FcRgamma-/- mice, suggesting that IgG-FcgammaR interactions may contribute to a sustained pathologic effect of anti-myelin antibodies in the CNS.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Vaina de Mielina/inmunología , Subunidades de Proteína/metabolismo , Receptores de IgG/metabolismo , Animales , Enfermedades Desmielinizantes/inmunología , Inflamación/inmunología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de la Mielina , Glicoproteína Asociada a Mielina/inmunología , Glicoproteína Mielina-Oligodendrócito , Subunidades de Proteína/genética , Receptores de IgG/genética , Tasa de SupervivenciaRESUMEN
Despite developments in targeted gene sequencing and whole-genome analysis techniques, the robust detection of all genetic variation, including structural variants, in and around genes of interest and in an allele-specific manner remains a challenge. Here we present targeted locus amplification (TLA), a strategy to selectively amplify and sequence entire genes on the basis of the crosslinking of physically proximal sequences. We show that, unlike other targeted re-sequencing methods, TLA works without detailed prior locus information, as one or a few primer pairs are sufficient for sequencing tens to hundreds of kilobases of surrounding DNA. This enables robust detection of single nucleotide variants, structural variants and gene fusions in clinically relevant genes, including BRCA1 and BRCA2, and enables haplotyping. We show that TLA can also be used to uncover insertion sites and sequences of integrated transgenes and viruses. TLA therefore promises to be a useful method in genetic research and diagnostics when comprehensive or allele-specific genetic information is needed.
Asunto(s)
Genómica/métodos , Haplotipos/genética , Modelos Genéticos , Técnicas de Amplificación de Ácido Nucleico/métodos , Análisis de Secuencia de ADN/métodos , Fusión Génica/genética , Genes BRCA1 , Genes BRCA2 , Sitios Genéticos/genética , Humanos , Neoplasias/genética , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
Duchenne muscular dystrophy (DMD) is a muscle-wasting disease in which muscle is continuously damaged, resulting in loss of muscle tissue and function. Antisense-mediated exon skipping is a promising therapeutic approach for DMD. This method uses sequence specific antisense oligonucleotides (AONs) to reframe disrupted dystrophin transcripts. As AONs function in a sequence specific manner, human specific AONs cannot be tested in the mdx mouse, which carries a mutation in the murine Dmd gene. We have previously generated a mouse model carrying the complete human DMD gene (hDMD mouse) integrated in the mouse genome to overcome this problem. However, as this is not a disease model, it cannot be used to study the effect of AON treatment on protein level and muscle function. Therefore, our long term goal is to generate deletions in the human DMD gene in a mouse carrying the hDMD gene in an mdx background. Towards this aim, we generated a male ES cell line carrying the hDMD gene while having the mdx point mutation. Inheritance of the hDMD gene by the ES cell was confirmed both on DNA and mRNA level. Quality control of the ES cells revealed that the pluripotency marker genes Oct-4 and Nanog are well expressed and that 85% of cells have 40 chromosomes. Germ line competence of this cell line has been confirmed, and 2 mice strains were derived from this cell line and crossed back on a C57BL6 background: hDMD/mdx and mdx(BL6).
RESUMEN
Antibodies play an important role in immunity to Salmonella enterica. Here we evaluated the requirement for Fcgamma receptors in host resistance to S. enterica using an in vivo model of systemic infection. We show that mice lacking FcgammaRI, II and III can control and clear a primary infection with S. enterica micro-organisms of low virulence, but are impaired in the expression of vaccine-induced acquired immunity to oral challenge with virulent bacteria. We also show that, in vivo, FcgammaRI, II, III(-/-) mice were able to mount efficient T-helper 1 type T-cell responses and antibody responses specific for S. enterica. The work indicates that targeting S. enterica to FcgammaR is needed for the expression of vaccine-induced acquired immunity, but is not essential for the engenderment of T- and B-cell immunity to the bacterium in vivo.
Asunto(s)
Receptores de IgG/inmunología , Infecciones por Salmonella/inmunología , Vacunas contra la Salmonella/inmunología , Salmonella typhimurium/crecimiento & desarrollo , Animales , Anticuerpos Antibacterianos/biosíntesis , Femenino , Inmunidad Celular , Hígado/inmunología , Hígado/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , Infecciones por Salmonella/microbiología , Infecciones por Salmonella/prevención & control , Salmonella typhimurium/inmunología , Salmonella typhimurium/patogenicidad , Bazo/inmunología , Bazo/microbiología , Subgrupos de Linfocitos T/inmunología , VirulenciaRESUMEN
Conventional silencing of many podocyte-specific genes in mice is associated with embryonic or perinatal lethality. Therefore, it would be of great importance to generate mouse models that allow the modification of genes that are expressed in podocytes at later stages of age. Herein is described a transgenic mouse with doxycycline-inducible podocyte-specific expression of Cre recombinase. For the generation of this binary system, a single transgenic construct that contained two separate genes was used: One encoding the optimized M2 version of the doxycycline-dependent transcription transactivator reverse tetracycline-controlled transcriptional activator (rtTA) under control of the human podocin (NPHS2) promoter and the other encoding the recombinase Cre under control of the rtTA/doxycycline-responsive minimal cytomegalovirus (CMV) Tet operator sequence 7 promotor. Microinjection of the JRC-CRE construct in fertilized oocytes from FVB/N mice resulted in 16 transgenic founders. Double-transgenic offspring from breeding of a selected founder with the Z/AP reporter mouse showed alkaline phosphatase staining only upon doxycycline administration and exclusively in podocytes. These data indicate that this new inducible Cre recombinase mouse line is an excellent tool in conditional, kidney glomerular podocyte-specific gene deletion in adult mice.
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Doxiciclina/farmacología , Integrasas/genética , Glomérulos Renales/metabolismo , Modelos Animales , Podocitos/metabolismo , Proteínas Virales/genética , Animales , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Integrasas/metabolismo , Ratones , Ratones Transgénicos , Fenotipo , ARN Mensajero/análisis , Sensibilidad y Especificidad , Proteínas Virales/metabolismoRESUMEN
Immune serum has a protective role against Salmonella infections in mice, domestic animals and humans. In this study, the effect of antibody on the interaction between murine macrophages and S. enterica serovar Typhimurium was examined. Detailed analysis at the single-cell level demonstrated that opsonization of the bacteria with immune serum enhanced bacterial uptake and altered bacterial distribution within individual phagocytic cells. Using gene-targeted mice deficient in individual Fc gamma receptors it was shown that immune serum enhanced bacterial internalization by macrophages via the high-affinity immunoglobulin G (IgG) receptor, Fc gamma receptor I. Exposure of murine macrophages to S. enterica serovar Typhimurium opsonized with immune serum resulted in increased production of superoxide, leading to enhanced antibacterial functions of the infected cells. However, opsonization of bacteria with immune serum did not increase either nitric oxide production in response to S. enterica serovar Typhimurium or fusion of phagosomes with lysosomes.
Asunto(s)
Sueros Inmunes/inmunología , Macrófagos/microbiología , Receptores de IgG/inmunología , Salmonelosis Animal/inmunología , Salmonella typhimurium/inmunología , Animales , Células de la Médula Ósea/microbiología , Femenino , Lisosomas/fisiología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Fagocitosis/inmunología , Fagosomas/fisiología , Especies de Nitrógeno Reactivo/biosíntesis , Especies Reactivas de Oxígeno/metabolismo , Salmonelosis Animal/metabolismoRESUMEN
Gene inactivation often leads to an embryonic-lethal phenotype. In focal diseases like renal cell carcinomas and polycystic kidney disease, somatic gene inactivation in subsets of cells is likely to occur at later stages. We generated a transgenic mouse line with an inducible form of Cre recombinase for conditional gene modifications in kidney epithelial cells. To this end a 1.4-kb promoter fragment of the kidney-specific cadherin gene (KspCad) was cloned upstream of a tamoxifen-inducible Cre recombinase (CreER(T2)) encoding sequence. Expression and activity of Cre was evaluated using reverse transcriptase polymerase chain reaction (RT-PCR) analysis and by crossbreeding to Z/EG reporter mice. One KspCad-CreER(T2) line showed kidney-specific Cre expression and mediated recombination upon tamoxifen treatment in Z/EG reporter mice. No reporter gene expression was detected in untreated animals or in extrarenal tissues upon treatment. Within the kidneys, enhanced green fluorescent protein (EGFP) fluorescence was observed in epithelial cells in several nephronic segments. In addition, the system successfully recombined a floxed Pkd1 gene.
Asunto(s)
Células Epiteliales , Marcación de Gen/métodos , Riñón/citología , Ratones Transgénicos , Tamoxifeno/farmacología , Animales , Cadherinas/genética , Expresión Génica , Genes Letales , Integrasas , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos/genética , Regiones Promotoras Genéticas , Canales Catiónicos TRPP , Tamoxifeno/administración & dosificaciónRESUMEN
OBJECTIVE: To determine the relationship between synovial inflammation and the concomitant occurrence of cartilage and bone erosion during conditions of variable inflammation using various Fcgamma receptor knockout (FcgammaR(-/-)) mice. METHODS: Antigen-induced arthritis (AIA) was introduced in the knee joints of various FcgammaR(-/-) mice and wild-type controls. Joint inflammation and cartilage and bone destruction levels were determined by histologic analysis. Cathepsin K, RANKL, and osteoprotegerin (OPG) levels were detected by immunolocalization. RESULTS: In FcgammaRIIb(-/-) mice, which lack the inhibiting Fcgamma receptor IIb, levels of joint inflammation and cartilage and bone destruction were significantly higher (infiltrate 93%, exudate 200%, cartilage 100%, bone 156%). AIA in mice lacking activating FcgammaR types I, III, and IV, but not FcgammaRIIb (FcR gamma-chain(-/-) mice), prevented cartilage destruction completely. In contrast, levels of bone erosion and joint inflammation were comparable with their wild-type controls. Of great interest, in arthritic mice lacking activating FcgammaR types I, II, and III, but not IV (FcgammaRI/II/III(-/-) mice), levels of joint inflammation were highly elevated (infiltrate and exudate, 100% and 188%, respectively). Cartilage destruction levels were decreased by 92%, whereas bone erosion was increased by 200%. Cathepsin K, a crucial mediator of osteoclasts, showed a strong correlation with the amount of inflammation but not with the amount of activating FcgammaR, which was low in osteoclasts. RANKL, but not OPG, levels were higher in the inflammatory cells of arthritic knee joints of FcgammaRI/II/III(-/-) mice versus wild-type mice. CONCLUSION: Activating FcgammaR are crucial in mediating cartilage destruction independently of joint inflammation. In contrast, FcgammaR are not directly involved in bone erosion. Indirectly, FcgammaR drive bone destruction by regulating joint inflammation.
Asunto(s)
Artritis Experimental/patología , Cartílago Articular/patología , Articulaciones/patología , Receptores de IgG/metabolismo , Animales , Complejo Antígeno-Anticuerpo/metabolismo , Artritis Experimental/metabolismo , Artritis Experimental/microbiología , Cartílago Articular/metabolismo , Catepsina K , Catepsinas/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Regulación de la Expresión Génica , Técnicas para Inmunoenzimas , Articulaciones/metabolismo , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Osteoclastos/patología , Osteoprotegerina/metabolismo , Ligando RANK/metabolismo , Ligando RANK/farmacología , ARN Mensajero/metabolismo , Receptores de IgG/genética , Sinovitis/metabolismo , Sinovitis/patologíaRESUMEN
Ca(v)2.1 (P/Q-type) voltage-gated calcium channels play an important role in neurotransmitter release at many brain synapses and at the neuromuscular junction. Mutations in the CACNA1A gene, encoding the pore forming alpha(1) subunit of Ca(v)2.1 channels, are associated with a wide spectrum of neurological disorders. Here we generated mice with a conditional, floxed, Cacna1a allele without any overt phenotype. Deletion of the floxed Cacna1a allele resulted in ataxia, dystonia, and lethality during the fourth week, a severe phenotype similar to conventional Ca(v)2.1 knockout mice. Although neurotransmitter release at the neuromuscular junction was not affected in the conditional mice, homozygous deletion of the floxed allele caused an ablation of Ca(v)2.1 channel-mediated neurotransmission that was accompanied by a compensatory upregulation of Ca(v)2.3 (R-type) channels at this synapse. Pharmacological inhibition of Ca(v)2.1 channels is possible, but the contributing cell-types and time windows relevant to the different Ca(v)2.1-related neurological disorders can only be reliably determined using Cacna1a conditional mice.
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Canales de Calcio Tipo P/genética , Canales de Calcio Tipo Q/genética , Silenciador del Gen , ARN/metabolismo , Animales , Northern Blotting , Canales de Calcio Tipo N , Cartilla de ADN , Electrofisiología , Componentes del Gen , Ratones , Ratones Transgénicos , Unión Neuromuscular/genética , Neurotransmisores/metabolismo , ARN/genética , Sinapsis/genéticaRESUMEN
Mucosal tolerance prevents the body from eliciting productive immune responses against harmless Ags that enter the body via the mucosae, and is mediated by the induction of regulatory T cells that differentiate in the mucosa-draining lymph nodes (LN) under defined conditions of Ag presentation. In this study, we show that mice deficient in FcgammaRIIB failed to develop mucosal tolerance to OVA, and demonstrate in vitro and in vivo a critical role for this receptor in modulating the Ag-presenting capacity of dendritic cells (DC). In vitro it was shown that absence of FcgammaRIIB under tolerogenic conditions led to increased IgG-induced release of inflammatory cytokines such as MCP-1, TNF-alpha, and IL-6 by bone marrow-derived DC, and increased their expression of costimulatory molecules, resulting in an altered immunogenic T cell response associated with increased IL-2 and IFN-gamma secretion. In vivo we could show enhanced LN-DC activation and increased numbers of Ag-specific IFN-gamma-producing T cells when FcgammaRIIB(-/-) mice were treated with OVA via the nasal mucosa, inferring that DC modulation by FcgammaRIIB directed the phenotype of the T cell response. Adoptive transfer of CD4(+) T cells from the spleen of FcgammaRIIB(-/-) mice to naive acceptor mice demonstrated that OVA-responding T cells failed to differentiate into regulatory T cells, explaining the lack of tolerance in these mice. Our findings demonstrate that signaling via FcgammaRIIB on DC, initiated by local IgG in the mucosa-draining LN, down-regulates DC activation induced by nasally applied Ag, resulting in those defined conditions of Ag presentation that lead to Tr induction and tolerance.
Asunto(s)
Antígenos CD/fisiología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Tolerancia Inmunológica , Mucosa Bucal/inmunología , Mucosa Bucal/metabolismo , Mucosa Nasal/inmunología , Mucosa Nasal/metabolismo , Receptores de IgG/fisiología , Administración Intranasal , Traslado Adoptivo , Animales , Presentación de Antígeno/genética , Presentación de Antígeno/inmunología , Antígenos CD/genética , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Células Cultivadas , Técnicas de Cocultivo , Células Dendríticas/patología , Regulación hacia Abajo/genética , Regulación hacia Abajo/inmunología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mucosa Nasal/patología , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Receptores de IgG/deficiencia , Receptores de IgG/genética , Linfocitos T/inmunología , Linfocitos T/trasplante , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patologíaRESUMEN
Studies of FcgammaRII-/- mice identified the inhibitory function of this receptor in joint inflammation and cartilage destruction induced with immune complexes (ICs). To extend our insight in the role of FcgammaRII in arthritis, we explored the role of FcgammaRII in the absence of activating receptors I and III using FcgammaRI/III-/- as well as FcgammaRI/II/III-/- mice. When antigen-induced arthritis (AIA) was elicited, which is a mixture of T cell and IC-driven inflammation, arthritis was almost absent at day 7 in FcgammaRI/III-/- mice. Remarkably, in FcgammaRI/II/III-/- mice, this model induced a tremendously increased arthritis as compared to wild-type controls. This implies that FcgammaRII regulates joint inflammation also in the absence of activating FcgammaRI and III. To confirm the IC specificity of this finding, similar studies were done with ICs or zymosan as arthritogenic stimuli. Strongly elevated inflammation was found in FcgammaRI/II/III-/- mice with IC but not with zymosan. Clearance studies identified accumulation of IgG in the knee joint in the absence of FcgammaRII. Moreover, macrophages expressing only FcgammaRII showed prominent endocytosis of preformed soluble ICs not different from controls. In total absence of FcgammaR (FcgammaRI/II/III-/-), macrophages completely failed to endocytose ICs. Although joint inflammation was much higher in AIA arthritic knee joints of FcgammaRI/II/III-/- and the inflammatory cells still expressed an inflammatory phenotype, severe cartilage destruction (MMP-mediated neoepitopes in the matrix and chondrocyte death) was completely prevented in contrast to the marked destruction which was observed in the wild-type. Our study indicates that FcgammaRII reduces joint inflammation in the absence of activating FcgammaR by promoting endocytosis and clearance of ICs from the joint. Infiltrating cells, which fail to express activating FcgammaR although they still become stimulated are no longer capable of inducing severe cartilage destruction.