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1.
Arthritis Res Ther ; 26(1): 120, 2024 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-38867295

RESUMEN

BACKGROUND: Kinases are intracellular signalling mediators and key to sustaining the inflammatory process in rheumatoid arthritis (RA). Oral inhibitors of Janus Kinase family (JAKs) are widely used in RA, while inhibitors of other kinase families e.g. phosphoinositide 3-kinase (PI3K) are under development. Most current biomarker platforms quantify mRNA/protein levels, but give no direct information on whether proteins are active/inactive. Phosphoproteome analysis has the potential to measure specific enzyme activation status at tissue level. METHODS: We validated the feasibility of phosphoproteome and total proteome analysis on 8 pre-treatment synovial biopsies from treatment-naive RA patients using label-free mass spectrometry, to identify active cell signalling pathways in synovial tissue which might explain failure to respond to RA therapeutics. RESULTS: Differential expression analysis and functional enrichment revealed clear separation of phosphoproteome and proteome profiles between lymphoid and myeloid RA pathotypes. Abundance of specific phosphosites was associated with the degree of inflammatory state. The lymphoid pathotype was enriched with lymphoproliferative signalling phosphosites, including Mammalian Target Of Rapamycin (MTOR) signalling, whereas the myeloid pathotype was associated with Mitogen-Activated Protein Kinase (MAPK) and CDK mediated signalling. This analysis also highlighted novel kinases not previously linked to RA, such as Protein Kinase, DNA-Activated, Catalytic Subunit (PRKDC) in the myeloid pathotype. Several phosphosites correlated with clinical features, such as Disease-Activity-Score (DAS)-28, suggesting that phosphosite analysis has potential for identifying novel biomarkers at tissue-level of disease severity and prognosis. CONCLUSIONS: Specific phosphoproteome/proteome signatures delineate RA pathotypes and may have clinical utility for stratifying patients for personalised medicine in RA.


Asunto(s)
Artritis Reumatoide , Fosfoproteínas , Proteómica , Transducción de Señal , Membrana Sinovial , Humanos , Artritis Reumatoide/metabolismo , Membrana Sinovial/metabolismo , Transducción de Señal/fisiología , Proteómica/métodos , Femenino , Fosfoproteínas/metabolismo , Fosfoproteínas/análisis , Persona de Mediana Edad , Masculino , Adulto , Anciano , Proteoma/análisis , Proteoma/metabolismo
2.
Osteoarthritis Cartilage ; 32(2): 166-176, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-37984558

RESUMEN

OBJECTIVES: Osteoarthritis (OA) is a debilitating and heterogeneous condition, characterized by various levels of articular cartilage degradation, osteophytes formation, and synovial inflammation. Multiple evidences suggest that synovitis may appear early in the disease development and correlates with disease severity and pain, therefore representing a relevant therapeutic target. In a typical synovitis-driven joint disease, namely rheumatoid arthritis (RA), several pathotypes have been described by our group and associated with clinical phenotypes, disease progression, and response to therapy. However, whether these pathotypes can be also observed in the OA synovium is currently unknown. METHODS: Here, using histological approaches combined with semi-quantitative scoring and quantitative digital image analyses, we comparatively characterize the immune cell infiltration in a large cohort of OA and RA synovial tissue samples collected at the time of total joint replacement. RESULTS: We demonstrate that OA synovium can be categorized also into three pathotypes and characterized by disease- and stage-specific features. Moreover, we revealed that pathotypes specifically reflect distinct levels of peripheral inflammation. CONCLUSIONS: In this study, we provide a novel and relevant pathological classification of OA synovial inflammation. Further studies investigating synovial molecular pathology in OA may contribute to the development of disease-modifying therapies.


Asunto(s)
Artritis Reumatoide , Osteoartritis , Sinovitis , Humanos , Osteoartritis/metabolismo , Artritis Reumatoide/metabolismo , Membrana Sinovial/metabolismo , Sinovitis/patología , Inflamación/metabolismo
3.
Front Med (Lausanne) ; 9: 1013660, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36465908

RESUMEN

Follicular dendritic cells (FDCs) fundamentally contribute to the formation of synovial ectopic lymphoid-like structures in rheumatoid arthritis (RA) which is associated with poor clinical prognosis. Despite this critical role, regulation of FDC development in the RA synovium and its correlation with synovial pathotype differentiation remained largely unknown. Here, we demonstrate that CNA.42+ FDCs distinctively express the pericyte/fibroblast-associated markers PDGFR-ß, NG2, and Thy-1 in the synovial perivascular space but not in established follicles. In addition, synovial RNA-Seq analysis revealed that expression of the perivascular FDC markers was strongly correlated with PDGF-BB and fibroid synovitis, whereas TNF-α/LT-ß was significantly associated with lymphoid synovitis and expression of CR1, CR2, and FcγRIIB characteristic of mature FDCs in lymphoid follicles. Moreover, PDGF-BB induced CNA.42+ FDC differentiation and CXCL13 secretion from NG2+ synovial pericytes, and together with TNF-α/LT-ß conversely regulated early and late FDC differentiation genes in unsorted RA synovial fibroblasts (RASF) and this was confirmed in flow sorted stromal cell subsets. Furthermore, RASF TNF-αR expression was upregulated by TNF-α/LT-ß and PDGF-BB; and TNF-α/LT-ß-activated RASF retained ICs and induced B cell activation in in vitro germinal center reactions typical of FDCs. Additionally, FDCs trapped peptidyl citrulline, and strongly correlated with IL-6 expression, and plasma cell, B cell, and T cell infiltration of the RA synovium. Moreover, synovial FDCs were significantly associated with RA disease activity and radiographic features of tissue damage. To the best of our knowledge, this is the first report describing the reciprocal interaction between PDGF-BB and TNF-α/LT-ß in synovial FDC development and evolution of RA histological pathotypes. Selective targeting of this interplay could inhibit FDC differentiation and potentially ameliorate RA in clinically severe and drug-resistant patients.

4.
Nat Med ; 28(6): 1256-1268, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35589854

RESUMEN

Patients with rheumatoid arthritis (RA) receive highly targeted biologic therapies without previous knowledge of target expression levels in the diseased tissue. Approximately 40% of patients do not respond to individual biologic therapies and 5-20% are refractory to all. In a biopsy-based, precision-medicine, randomized clinical trial in RA (R4RA; n = 164), patients with low/absent synovial B cell molecular signature had a lower response to rituximab (anti-CD20 monoclonal antibody) compared with that to tocilizumab (anti-IL6R monoclonal antibody) although the exact mechanisms of response/nonresponse remain to be established. Here, in-depth histological/molecular analyses of R4RA synovial biopsies identify humoral immune response gene signatures associated with response to rituximab and tocilizumab, and a stromal/fibroblast signature in patients refractory to all medications. Post-treatment changes in synovial gene expression and cell infiltration highlighted divergent effects of rituximab and tocilizumab relating to differing response/nonresponse mechanisms. Using ten-by-tenfold nested cross-validation, we developed machine learning algorithms predictive of response to rituximab (area under the curve (AUC) = 0.74), tocilizumab (AUC = 0.68) and, notably, multidrug resistance (AUC = 0.69). This study supports the notion that disease endotypes, driven by diverse molecular pathology pathways in the diseased tissue, determine diverse clinical and treatment-response phenotypes. It also highlights the importance of integration of molecular pathology signatures into clinical algorithms to optimize the future use of existing medications and inform the development of new drugs for refractory patients.


Asunto(s)
Antirreumáticos , Artritis Reumatoide , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Antirreumáticos/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/genética , Biomarcadores/análisis , Biopsia , Humanos , Rituximab/uso terapéutico
5.
Front Immunol ; 12: 706737, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34594326

RESUMEN

Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease resulting from the inflammatory infiltration of exocrine glands, mainly salivary and lacrimal glands, leading to secretory dysfunction and serious complications including debilitating fatigue, systemic autoimmunity, and lymphoma. Like other autoimmune disorders, a strong interferon (IFN) signature is present among subsets of pSS patients, suggesting the involvement of innate immunity in pSS pathogenesis. NCR3/NKp30 is a natural killer (NK) cell-specific activating receptor regulating the cross talk between NK and dendritic cells including type II IFN secretion upon NK-cell activation. A genetic association between single-nucleotide polymorphisms (SNPs) in the NCR3/NKp30 promoter gene and a higher susceptibility for pSS has been previously described, with pSS patients most frequently carrying the major allele variant associated with a higher NKp30 transcript and IFN-γ release as a consequence of the receptor engagement. In the present study, we combined RNA-sequencing and histology from pSS salivary gland biopsies to better characterize NKp30 (NCR3) and its ligand B7/H6 (NCR3LG1) in pSS salivary gland tissues. Levels of NCR3/NKp30 were significantly increased both in salivary glands and in circulating NK cells of pSS patients compared with sicca controls, especially in salivary glands with organized ectopic lymphoid structures. In line with this observation, a strong correlation between NCR3/NKp30 levels and salivary gland infiltrating immune cells (CD3, CD20) was found. Furthermore, NCR3/NKp30 levels also correlated with higher IFN-γ, Perforin, and Granzyme-B expression in pSS SGs with organized ectopic lymphoid structures, suggesting an activation state of NK cells infiltrating SG tissue. Of note, NKp30+ NK cells accumulated at the border of the inflammatory foci, while the NKp30 ligand, B7/H6, is shown to be expressed mainly by ductal epithelial cells in pSS salivary glands. Finally, immunomodulatory treatment, such as the B-cell depleting agent rituximab, known to reduce the infiltration of immune cells in pSS SGs, prevented the upregulation of NCR3/NKp30 within the glands.


Asunto(s)
Receptor 3 Gatillante de la Citotoxidad Natural/metabolismo , Glándulas Salivales/inmunología , Síndrome de Sjögren/inmunología , Estructuras Linfoides Terciarias/inmunología , Humanos , Factores Inmunológicos/uso terapéutico , Células Asesinas Naturales/inmunología , Rituximab/uso terapéutico , Glándulas Salivales/efectos de los fármacos , Glándulas Salivales/metabolismo , Síndrome de Sjögren/tratamiento farmacológico , Síndrome de Sjögren/metabolismo , Regulación hacia Arriba
6.
Front Immunol ; 12: 640070, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33679801

RESUMEN

Biologic drugs, especially anti-TNF, are considered as the gold standard therapy in rheumatoid arthritis. However, non-uniform efficacy, incidence of infections, and high costs are major concerns. Novel tissue-specific agents may overcome the current limitations of systemic administration, providing improved potency, and safety. We developed a bispecific antibody (BsAb), combining human arthritic joint targeting, via the synovial-specific single-chain variable fragment (scFv)-A7 antibody, and TNFα neutralization, via the scFv-anti-TNFα of adalimumab, with the binding/blocking capacity comparable to adalimumab -immunoglobulin G (IgG). Tissue-targeting capacity of the BsAb was confirmed on the human arthritic synovium in vitro and in a synovium xenograft Severe combined immune deficient (SCID) mouse model. Peak graft accumulation occurred at 48 h after injection with sustained levels over adalimumab-IgG for 7 days and increased therapeutic effect, efficiently decreasing tissue cellularity, and markers of inflammation with higher potency compared to the standard treatment. This study provides the first description of a BsAb capable of drug delivery, specifically to the disease tissue, and a strong evidence of improved therapeutic effect on the human arthritic synovium, with applications to other existing biologics.


Asunto(s)
Anticuerpos Biespecíficos/inmunología , Artritis Reumatoide/inmunología , Membrana Sinovial/inmunología , Inhibidores del Factor de Necrosis Tumoral/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Adalimumab/inmunología , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunoglobulina G/inmunología , Inmunoterapia/métodos , Inflamación/inmunología , Masculino , Ratones , Ratones SCID , Anticuerpos de Cadena Única/inmunología
7.
J Invest Dermatol ; 141(2): 404-414.e6, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-32682912

RESUMEN

Nonmelanoma skin cancer such as cutaneous squamous cell carcinoma (cSCC) is the most common form of cancer and can occur as a consequence of DNA damage to the epithelium by UVR or chemical carcinogens. There is growing evidence that the complement system is involved in cancer immune surveillance; however, its role in cSCC remains unclear. Here, we show that complement genes are expressed in tissue from patients with cSCC, and C3 activation fragments are present in cSCC biopsies, indicating complement activation. Using a range of complement-deficient mice in a two-stage mouse model of chemically-induced cSCC, where a subclinical dose of 7,12-dimethylbenz[a]anthracene causes oncogenic mutations in epithelial cells and 12-O-tetradecanoylphorbol-13-acetate promotes the outgrowth of these cells, we found that C3-deficient mice displayed a significantly reduced tumor burden, whereas an opposite phenotype was observed in mice lacking C5aR1, C5aR2, and C3a receptor. In addition, in mice unable to form the membrane attack complex, the tumor progression was unaltered. C3 deficiency did not affect the cancer response to 7,12-dimethylbenz[a]anthracene treatment alone but reduced the epidermal hyperplasia during 12-O-tetradecanoylphorbol-13-acetate-induced inflammation. Collectively, these data indicate that C3 drives tumorigenesis during chronic skin inflammation, independently of the downstream generation of C5a or membrane attack complex.


Asunto(s)
Carcinoma de Células Escamosas/inmunología , Complemento C3/metabolismo , Neoplasias Experimentales/inmunología , Neoplasias Cutáneas/inmunología , 9,10-Dimetil-1,2-benzantraceno/administración & dosificación , 9,10-Dimetil-1,2-benzantraceno/toxicidad , Animales , Carcinógenos/administración & dosificación , Carcinógenos/toxicidad , Carcinoma de Células Escamosas/inducido químicamente , Carcinoma de Células Escamosas/patología , Activación de Complemento/genética , Activación de Complemento/inmunología , Complemento C3/genética , Complemento C5/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Neoplasias Experimentales/sangre , Neoplasias Experimentales/inducido químicamente , Neoplasias Experimentales/patología , Receptor de Anafilatoxina C5a/genética , Receptor de Anafilatoxina C5a/metabolismo , Receptores de Complemento/genética , Receptores de Complemento/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Piel/efectos de los fármacos , Piel/inmunología , Piel/patología , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/patología , Escape del Tumor
8.
Ann Rheum Dis ; 79(12): 1588-1599, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-32963045

RESUMEN

OBJECTIVES: To explore the relevance of T-follicular-helper (Tfh) and pathogenic peripheral-helper T-cells (Tph) in promoting ectopic lymphoid structures (ELS) and B-cell mucosa-associated lymphoid tissue (MALT) lymphomas (MALT-L) in Sjögren's syndrome (SS) patients. METHODS: Salivary gland (SG) biopsies with matched peripheral blood were collected from four centres across the European Union. Transcriptomic (microarray and quantitative PCR) analysis, FACS T-cell immunophenotyping with intracellular cytokine detection, multicolor immune-fluorescence microscopy and in situ hybridisation were performed to characterise lesional and circulating Tfh and Tph-cells. SG-organ cultures were used to investigate functionally the blockade of T-cell costimulatory pathways on key proinflammatory cytokine production. RESULTS: Transcriptomic analysis in SG identified Tfh-signature, interleukin-21 (IL-21) and the inducible T-cell co-stimulator (ICOS) costimulatory pathway as the most upregulated genes in ELS+SS patients, with parotid MALT-L displaying a 400-folds increase in IL-21 mRNA. Peripheral CD4+CXC-motif chemokine receptor 5 (CXCR5)+programmed cell death protein 1 (PD1)+ICOS+ Tfh-like cells were significantly expanded in ELS+SS patients, were the main producers of IL-21, and closely correlated with circulating IgG and reduced complement C4. In the SG, lesional CD4+CD45RO+ICOS+PD1+ cells selectively infiltrated ELS+ tissues and were aberrantly expanded in parotid MALT-L. In ELS+SG and MALT-L parotids, conventional CXCR5+CD4+PD1+ICOS+Foxp3- Tfh-cells and a uniquely expanded population of CXCR5-CD4+PD1hiICOS+Foxp3- Tph-cells displayed frequent IL-21/interferon-γ double-production but poor IL-17 expression. Finally, ICOS blockade in ex vivo SG-organ cultures significantly reduced the production of IL-21 and inflammatory cytokines IL-6, IL-8 and tumour necrosis factor-α (TNF-α). CONCLUSIONS: Overall, these findings highlight Tfh and Tph-cells, IL-21 and the ICOS costimulatory pathway as key pathogenic players in SS immunopathology and exploitable therapeutic targets in SS.


Asunto(s)
Coristoma/inmunología , Centro Germinal , Linfoma de Células B de la Zona Marginal/inmunología , Enfermedades de las Glándulas Salivales/inmunología , Síndrome de Sjögren/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Adulto , Anciano , Coristoma/etiología , Coristoma/patología , Femenino , Humanos , Inmunofenotipificación , Proteína Coestimuladora de Linfocitos T Inducibles/inmunología , Interleucinas/inmunología , Linfoma de Células B de la Zona Marginal/etiología , Linfoma de Células B de la Zona Marginal/patología , Masculino , Persona de Mediana Edad , Enfermedades de las Glándulas Salivales/patología , Síndrome de Sjögren/complicaciones , Síndrome de Sjögren/patología , Células T Auxiliares Foliculares/inmunología
9.
J Autoimmun ; 105: 102297, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31277965

RESUMEN

The mechanisms underlying the transition of rheumatoid arthritis (RA) systemic autoimmunity to the joints remain largely unknown. Here, we demonstrate that macrophages in the secondary lymphoid organs (SLOs) and synovial ectopic lymphoid-like structures (ELSs) express peptidylarginine deiminase 4 (PAD4) in murine collagen induced arthritis (CIA) and synovial biopsies from RA patients. Moreover, peptidyl citrulline colocalized with macrophages in SLOs and ELSs, and depletion of macrophages in CIA decreased lymphoid tissue citrullination and serum anti-citrullinated protein/peptide antibody (ACPA) levels. Furthermore, PAD was released from activated murine and RA synovial tissue and fluid (SF) macrophages which functionally deiminated extracellular proteins/peptides in vitro. Additionally, activated murine and SF macrophages displayed macrophage extracellular trap formation (METosis) and release of intracellular citrullinated histones. Moreover, presentation of citrullinated proteins induced ACPA production in vitro. Thus, lymphoid tissue macrophages contribute to self-antigen citrullination and ACPA production, indicating that their selective targeting would potentially ameliorate citrullination-dependent autoimmune disorders.


Asunto(s)
Artritis Experimental/inmunología , Autoanticuerpos/inmunología , Enfermedades Autoinmunes/inmunología , Citrulinación/inmunología , Trampas Extracelulares/inmunología , Macrófagos/inmunología , Arginina Deiminasa Proteína-Tipo 4/inmunología , Animales , Formación de Anticuerpos/inmunología , Artritis Reumatoide/inmunología , Autoantígenos/inmunología , Autoinmunidad/inmunología , Citrulina/inmunología , Histonas/inmunología , Masculino , Ratones , Ratones Endogámicos DBA , Líquido Sinovial/inmunología , Membrana Sinovial/inmunología
10.
J Leukoc Biol ; 105(3): 589-602, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30575993

RESUMEN

Salivary glands (SGs) represent a permissive site for several sialotropic viruses whose persistence is linked to the development of autoimmunity. Natural Killer (NK) cells play a key role in viral clearance but their involvement in viral infection control and in tertiary lymphoid structures (TLS) development within SGs is unknown. By using an inducible model of TLS in the SGs of wild-type C57BL/6 mice, induced by the local delivery of a replication-defective adenovirus (AdV), we demonstrated that circulating NK cells are rapidly recruited to SGs and highly enrich the early inflammatory infiltrate prior to TLS development. NK cells migrating to SGs in response to AdV infection up-regulate NKp46, undergo proliferation, acquire cytotoxic potential, produce Granzyme-B and IFN-γ, and reduce viral load in the acute phase of the infection. Nonetheless, the selective depletion of both circulating and infiltrating NK cells in AdV-infected mice neither affect the development and frequency of TLS nor the onset of autoimmunity. These data demonstrate that, upon local viral delivery of AdV, peripheral NK cells homing to SGs can exert an early control of the viral infection but are dispensable for the formation of TLS and breach of immunologic tolerance.


Asunto(s)
Adenoviridae/fisiología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/virología , Linfocitos/patología , Linfocitos/virología , Glándulas Salivales/patología , Glándulas Salivales/virología , Animales , Proliferación Celular , Femenino , Inmunidad Humoral , Ratones Endogámicos C57BL , Receptor 1 Gatillante de la Citotoxidad Natural/metabolismo
11.
Ann Rheum Dis ; 77(12): 1773-1781, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30127058

RESUMEN

OBJECTIVES: Mast cells (MCs) are involved in the pathogenesis of rheumatoid arthritis (RA). However, their contribution remains controversial. To establish their role in RA, we analysed their presence in the synovium of treatment-naïve patients with early RA and their association and functional relationship with histological features of synovitis. METHODS: Synovial tissue was obtained by ultrasound-guided biopsy from treatment-naïve patients with early RA (n=99). Immune cells (CD3/CD20/CD138/CD68) and their relationship with CD117+MCs in synovial tissue were analysed by immunohistochemistry (IHC) and immunofluorescence (IF). The functional involvement of MCs in ectopic lymphoid structures (ELS) was investigated in vitro, by coculturing MCs with naïve B cells and anticitrullinated protein antibodies (ACPA)-producing B cell clones, and in vivo in interleukin-27 receptor alpha (IL27ra)-deficient and control mice during antigen-induced arthritis (AIA). RESULTS: High synovial MC counts are associated with local and systemic inflammation, autoantibody positivity and high disease activity. IHC/IF showed that MCs reside at the outer border of lymphoid aggregates. Furthermore, human MCs promote the activation and differentiation of naïve B cells and induce the production of ACPA, mainly via contact-dependent interactions. In AIA, synovial MC numbers increase in IL27ra deficient mice, in association with ELS and worse disease activity. CONCLUSIONS: Synovial MCs identify early RA patients with a severe clinical form of synovitis characterised by the presence of ELS.


Asunto(s)
Artritis Reumatoide/inmunología , Autoanticuerpos/inmunología , Linfocitos B/inmunología , Mastocitos/inmunología , Sinovitis/inmunología , Animales , Artritis Experimental/inmunología , Femenino , Humanos , Masculino , Ratones , Estructuras Linfoides Terciarias/inmunología
12.
Kidney Int ; 89(4): 823-32, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26924054

RESUMEN

C3 glomerulopathy is a complement-mediated renal disease that is frequently associated with abnormalities in regulation of the complement alternative pathway. Mice with deficiency of factor H (Cfh(-/-)), a negative alternative pathway regulator, are an established experimental model of C3 glomerulopathy in which complement C3 fragments including iC3b accumulate along the glomerular basement membrane. Here we show that deficiency of complement receptor 3 (CR3), the main receptor for iC3b, enhances the severity of spontaneous renal disease in Cfh(-/-) mice. This effect was found to be dependent on CR3 expression on bone marrow-derived cells. CR3 also mediated renal protection outside the setting of factor H deficiency, as shown by the development of enhanced renal injury in CR3-deficient mice during accelerated nephrotoxic nephritis. The iC3b-CR3 interaction downregulated the proinflammatory cytokine response of both murine and human macrophages to lipopolysaccharide stimulation in vitro, suggesting that the protective effect of CR3 on glomerular injury was mediated via modulation of macrophage-derived proinflammatory cytokines. Thus, CR3 has a protective role in glomerulonephritis and suggests that pharmacologic potentiation of the macrophage CR3 interaction with iC3b could be therapeutically beneficial.


Asunto(s)
Complemento C3/metabolismo , Factor H de Complemento/deficiencia , Glomerulonefritis/metabolismo , Enfermedades Renales/metabolismo , Antígeno de Macrófago-1/metabolismo , Animales , Antígeno CD11b/genética , Factor H de Complemento/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Enfermedades por Deficiencia de Complemento Hereditario , Ratones Endogámicos C57BL , Células Mieloides/metabolismo
13.
Cell Rep ; 12(11): 1802-15, 2015 Sep 22.
Artículo en Inglés | MEDLINE | ID: mdl-26344769

RESUMEN

Monocytes are heterogeneous effector cells involved in the maintenance and restoration of tissue integrity. However, their response to hyperlipidemia remains poorly understood. Here, we report that in the presence of elevated levels of triglyceride-rich lipoproteins, induced by administration of poloxamer 407, the blood numbers of non-classical Ly6C/Gr1(low) monocytes drop, while the number of bone marrow progenitors remains similar. We observed an increased crawling and retention of the Gr1(low) monocytes at the endothelial interface and a marked accumulation of CD68(+) macrophages in several organs. Hypertriglyceridemia was accompanied by an increased expression of tissue, and plasma CCL4 and blood Gr1(low) monocyte depletion involved a pertussis-toxin-sensitive receptor axis. Collectively, these findings demonstrate that a triglyceride-rich environment can alter blood monocyte distribution, promoting the extravasation of Gr1(low) cells. The behavior of these cells in response to dyslipidemia highlights the significant impact that high levels of triglyceride-rich lipoproteins may have on innate immune cells.


Asunto(s)
Antígenos Ly/metabolismo , Lipoproteínas/metabolismo , Monocitos/metabolismo , Triglicéridos/metabolismo , Animales , Ratones , Ratones Endogámicos C57BL
14.
Immunology ; 144(1): 149-57, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25039245

RESUMEN

A role for complement, particularly the classical pathway, in the regulation of immune responses is well documented. Deficiencies in C1q or C4 predispose to autoimmunity, while deficiency in C3 affects the suppression of contact sensitization and generation of oral tolerance. Complement components including C3 have been shown to be required for both B-cell and T-cell priming. The mechanisms whereby complement can mediate these diverse regulatory effects are poorly understood. Our previous work, using the mouse minor histocompatibility (HY) model of skin graft rejection, showed that both C1q and C3 were required for the induction of tolerance following intranasal peptide administration. By comparing tolerance induction in wild-type C57BL/6 and C1q-, C3-, C4- and C5-deficient C57BL/6 female mice, we show here that the classical pathway components including C3 are required for tolerance induction, whereas C5 plays no role. C3-deficient mice failed to generate a functional regulatory T (Treg) -dendritic cell (DC) tolerogenic loop required for tolerance induction. This was related to the inability of C3-deficient DC to up-regulate the arginine-consuming enzyme, inducible nitric oxide synthase (Nos-2), in the presence of antigen-specific Treg cells and peptide, leading to reduced Treg cell generation. Our findings demonstrate that the classical pathway and C3 play a critical role in the peptide-mediated induction of tolerance to HY by modulating DC function.


Asunto(s)
Proteínas del Sistema Complemento/genética , Células Dendríticas/inmunología , Antígeno H-Y/inmunología , Tolerancia Inmunológica/efectos de los fármacos , Péptidos/farmacología , Linfocitos T Reguladores/inmunología , Administración Intranasal , Animales , Proteínas del Sistema Complemento/inmunología , Células Dendríticas/citología , Femenino , Antígeno H-Y/genética , Tolerancia Inmunológica/genética , Ratones , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/inmunología , Péptidos/inmunología , Linfocitos T Reguladores/citología
15.
Part Fibre Toxicol ; 11: 64, 2014 Nov 26.
Artículo en Inglés | MEDLINE | ID: mdl-25425420

RESUMEN

BACKGROUND: The complement system is a key component of innate immunity implicated in the neutralization and clearance of invading pathogens. Dextran coated superparamagnetic iron oxide (SPIO) nanoparticle is a promising magnetic resonance imaging (MRI) contrast agent. However, dextran SPIO has been associated with significant number of complement-related side effects in patients and some agents have been discontinued from clinical use (e.g., Feridex™). In order to improve the safety of these materials, the mechanisms of complement activation by dextran-coated SPIO and the differences between mice and humans need to be fully understood. METHODS: 20 kDa dextran coated SPIO nanoworms (SPIO NW) were synthesized using Molday precipitation procedure. In vitro measurements of C3 deposition on SPIO NW using sera genetically deficient for various components of the classical pathway (CP), lectin pathway (LP) or alternative pathway (AP) components were used to study mechanisms of mouse complement activation. In vitro measurements of fluid phase markers of complement activation C4d and Bb and the terminal pathway marker SC5b-C9 in normal and genetically deficient sera were used to study the mechanisms of human complement activation. Mouse data were analyzed by non-paired t-test, human data were analyzed by ANOVA followed by multiple comparisons with Student-Newman-Keuls test. RESULTS: In mouse sera, SPIO NW triggered the complement activation via the LP, whereas the AP contributes via the amplification loop. No involvement of the CP was observed. In human sera the LP together with the direct enhancement of the AP turnover was responsible for the complement activation. In two samples out of six healthy donors there was also a binding of anti-dextran antibodies and C1q, suggesting activation via the CP, but that did not affect the total level of C3 deposition on the particles. CONCLUSIONS: There were important differences and similarities in the complement activation by SPIO NW in mouse versus human sera. Understanding the mechanisms of immune recognition of nanoparticles in mouse and human systems has important preclinical and clinical implications and could help design more efficient and safe nano-formulations.


Asunto(s)
Activación de Complemento/efectos de los fármacos , Medios de Contraste/farmacología , Dextranos/farmacología , Adulto , Animales , Biomarcadores/sangre , Vía Alternativa del Complemento/efectos de los fármacos , Vía Clásica del Complemento/efectos de los fármacos , Lectina de Unión a Manosa de la Vía del Complemento/efectos de los fármacos , Proteínas del Sistema Complemento/genética , Proteínas del Sistema Complemento/metabolismo , Humanos , Nanopartículas de Magnetita , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Especificidad de la Especie , Propiedades de Superficie
16.
J Autoimmun ; 50: 107-22, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24491821

RESUMEN

Regulatory B cells (Breg) have attracted increasing attention for their roles in maintaining peripheral tolerance. Interleukin 33 (IL-33) is a recently identified IL-1 family member, which leads a double-life with both pro- and anti-inflammatory properties. We report here that peritoneal injection of IL-33 exacerbated inflammatory bowel disease in IL-10-deficient (IL-10(-/-)) mice, whereas IL-33-treated IL-10-sufficient (wild type) mice were protected from the disease induction. A phenotypically unconventional subset(s) (CD19(+)CD25(+)CD1d(hi)IgM(hi)CD5(-)CD23(-)Tim-1(-)) of IL-10 producing Breg-like cells (Breg(IL-33)) was identified responsible for the protection. We demonstrated further that Breg(IL-33) isolated from these mice could suppress immune effector cell expansion and functions and, upon adoptive transfer, effectively blocked the development of spontaneous colitis in IL-10(-/-) mice. Our findings indicate an essential protective role, hence therapeutic potential, of Breg(IL-33) against mucosal inflammatory disorders in the gut.


Asunto(s)
Linfocitos B Reguladores/inmunología , Colitis/inmunología , Mucosa Gástrica/efectos de los fármacos , Interleucina-10/inmunología , Interleucinas/farmacología , Traslado Adoptivo , Animales , Antígenos CD/genética , Antígenos CD/inmunología , Linfocitos B Reguladores/efectos de los fármacos , Linfocitos B Reguladores/trasplante , Colitis/genética , Colitis/patología , Femenino , Mucosa Gástrica/inmunología , Mucosa Gástrica/patología , Expresión Génica , Inyecciones Intraperitoneales , Interleucina-10/deficiencia , Interleucina-10/genética , Interleucina-33 , Interleucinas/inmunología , Activación de Linfocitos , Ratones , Ratones Noqueados
17.
Nat Commun ; 5: 3039, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24423728

RESUMEN

Tuned and distinct responses of macrophages and dendritic cells to Toll-like receptor 4 (TLR4) activation induced by lipopolysaccharide (LPS) underpin the balance between innate and adaptive immunity. However, the molecule(s) that confer these cell-type-specific LPS-induced effects remain poorly understood. Here we report that the integrin α(M) (CD11b) positively regulates LPS-induced signalling pathways selectively in myeloid dendritic cells but not in macrophages. In dendritic cells, which express lower levels of CD14 and TLR4 than macrophages, CD11b promotes MyD88-dependent and MyD88-independent signalling pathways. In particular, in dendritic cells CD11b facilitates LPS-induced TLR4 endocytosis and is required for the subsequent signalling in the endosomes. Consistent with this, CD11b deficiency dampens dendritic cell-mediated TLR4-triggered responses in vivo leading to impaired T-cell activation. Thus, by modulating the trafficking and signalling functions of TLR4 in a cell-type-specific manner CD11b fine tunes the balance between adaptive and innate immune responses initiated by LPS.


Asunto(s)
Antígeno CD11b/fisiología , Células Dendríticas/fisiología , Macrófagos/fisiología , Transducción de Señal/fisiología , Receptor Toll-Like 4/fisiología , Inmunidad Adaptativa/fisiología , Animales , Antígeno CD11b/genética , Células Cultivadas , Quimiocina CCL5/metabolismo , Citocinas/metabolismo , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Femenino , Inmunidad Innata/fisiología , Lipopolisacáridos/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/fisiología
18.
Proc Natl Acad Sci U S A ; 111(4): 1503-8, 2014 Jan 28.
Artículo en Inglés | MEDLINE | ID: mdl-24474777

RESUMEN

Apoptotic cells are a source of autoantigens and impairment of their removal contributes to the development of autoimmunity in C1q deficiency. However, the lack of complement component 3 (C3), the predominant complement opsonin, does not predispose to autoimmunity, suggesting a modifying role of C3 in disease pathogenesis. To explore this hypothesis, here we investigated the role of C3 in the T-cell response to apoptotic cell-associated antigens. By comparing the phagosome maturation and the subsequent MHC class II presentation of a peptide derived from the internalized cargo between C3-deficient or C3-sufficient dendritic cells, we found that C3 deficiency accelerated the fusion of the apoptotic cargo with lysosomes. As a result, C3 deficiency led to impaired antigen-specific T-cell proliferation in vitro and in vivo. Notably, preopsonization of the apoptotic cells with C3 activation fragments rectified the trafficking and T-cell stimulation defects. These data indicate that activated C3 may act as a "chaperone" in the intracellular processing of an apoptotic cargo and, thus, may modulate the T-cell response to self-antigens displayed on dying cells.


Asunto(s)
Apoptosis , Autoantígenos/inmunología , Complemento C3/metabolismo , Endocitosis , Proteínas Opsoninas/metabolismo , Linfocitos T/inmunología , Animales , Ratones , Ratones Noqueados , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunología
19.
PLoS One ; 8(2): e57082, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23451151

RESUMEN

The CD11b/CD18 integrin (complement receptor 3, CR3) is a surface receptor on monocytes, neutrophils, macrophages and dendritic cells that plays a crucial role in several immunological processes including leukocyte extravasation and phagocytosis. The minor allele of a non-synonymous CR3 polymorphism (rs1143679, conversation of arginine to histidine at position 77: R77H) represents one of the strongest genetic risk factor in human systemic lupus erythematosus, with heterozygosity (77R/H) being the most common disease associated genotype. Homozygosity for the 77H allele has been reported to reduce adhesion and phagocytosis in human monocytes and monocyte-derived macrophages, respectively, without affecting surface expression of CD11b. Herein we comprehensively assessed the influence of R77H on different CR3-mediated activities in monocytes, neutrophils, macrophages and dendritic cells. R77H did not alter surface expression of CD11b including its active form in any of these cell types. Using two different iC3b-coated targets we found that the uptake by heterozygous 77R/H macrophages, monocytes and neutrophils was significantly reduced compared to 77R/R cells. Allele-specific transduced immortalized macrophage cell lines demonstrated that the minor allele, 77H, was responsible for the impaired phagocytosis. R77H did not affect neutrophil adhesion, neutrophil transmigration in vivo or Toll-like receptor 7/8-mediated cytokine release by monocytes or dendritic cells with or without CR3 pre-engagement by iC3b-coated targets. Our findings demonstrate that the reduction in CR3-mediated phagocytosis associated with the 77H CD11b variant is not macrophage-restricted but demonstrable in other CR3-expressing professional phagocytic cells. The association between 77H and susceptibility to systemic lupus erythematosus most likely relates to impaired waste disposal, a key component of lupus pathogenesis.


Asunto(s)
Antígeno CD11b/inmunología , Lupus Eritematoso Sistémico/inmunología , Antígeno de Macrófago-1/fisiología , Células Mieloides/inmunología , Fagocitosis , Alelos , Animales , Secuencia de Bases , Antígeno CD11b/genética , Citocinas/metabolismo , Cartilla de ADN , Humanos , Antígeno de Macrófago-1/inmunología , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa
20.
Arthritis Rheum ; 64(8): 2707-18, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22392450

RESUMEN

OBJECTIVE: Although the accelerating effect of systemic lupus erythematosus (SLE) on atherosclerosis is well established, the underlying mechanisms are unknown. The aim of this study was to explore the hypothesis that lupus autoimmunity modulates the effect of hypercholesterolemia in driving arterial pathologic development. METHODS: Low-density lipoprotein receptor-deficient (Ldlr(-/-) ) mice were crossed with B6.129-Sle16 (Sle16)-congenic autoimmune mice to obtain Sle16. Ldlr(-/-) mice, which were compared with Ldlr(-/-) and Sle16 control mice. All mice were fed either a low-fat or high-fat diet. Groups of mice were compared, by strain and by diet group, for features of accelerated atherosclerosis and autoimmunity. RESULTS: Presence of the Sle16 locus significantly increased the extent of atherosclerosis in Ldlr(-/-) mice. Circulating C3 levels were significantly reduced in Sle16.Ldlr(-/-) mice compared to Ldlr(-/-) control mice and this was paralleled by a marked reduction in arterial lesion C3 deposition despite similar levels of IgG deposition between the groups. Increased numbers of apoptotic cells in plaques were observed in the high-fat-fed Sle16.Ldlr(-/-) mice, consistent with the observed defective clearance of cellular debris. After receiving the high-fat diet, Sle16.Ldlr(-/-) mice developed glomerulonephritis and displayed enhanced glomerular C3 deposition. CONCLUSION: These results indicate that accelerated atherosclerosis and renal inflammation in SLE are closely linked via immune complex formation and systemic complement depletion. However, whereas hyperlipidemia will enhance renal immune complex-mediated complement activation and the development of nephritis, accelerated atherosclerosis is, instead, related to complement depletion and a reduction in the uptake of apoptotic/necrotic debris. These results suggest that aggressive treatment of hyperlipidemia in patients with SLE may reduce the occurrence of lupus nephritis, as well as diminish the risk of accelerated atherosclerosis.


Asunto(s)
Aterosclerosis/fisiopatología , Complemento C3/fisiología , Glomerulonefritis/fisiopatología , Hiperlipidemias/fisiopatología , Lupus Eritematoso Sistémico/fisiopatología , Animales , Complejo Antígeno-Anticuerpo/metabolismo , Apoptosis/fisiología , Aterosclerosis/epidemiología , Aterosclerosis/patología , Comorbilidad , Grasas de la Dieta , Modelos Animales de Enfermedad , Femenino , Glomerulonefritis/epidemiología , Glomerulonefritis/patología , Hiperlipidemias/epidemiología , Hiperlipidemias/metabolismo , Inmunoglobulina G/metabolismo , Lupus Eritematoso Sistémico/epidemiología , Lupus Eritematoso Sistémico/metabolismo , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de LDL/deficiencia , Receptores de LDL/genética , Receptores de LDL/metabolismo
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