RESUMEN
Multiple sclerosis is an autoimmune disease that is caused by the interplay of genetic, particularly the HLA-DR15 haplotype, and environmental risk factors. How these etiologic factors contribute to generating an autoreactive CD4+ T cell repertoire is not clear. Here, we demonstrate that self-reactivity, defined as "autoproliferation" of peripheral Th1 cells, is elevated in patients carrying the HLA-DR15 haplotype. Autoproliferation is mediated by memory B cells in a HLA-DR-dependent manner. Depletion of B cells in vitro and therapeutically in vivo by anti-CD20 effectively reduces T cell autoproliferation. T cell receptor deep sequencing showed that in vitro autoproliferating T cells are enriched for brain-homing T cells. Using an unbiased epitope discovery approach, we identified RASGRP2 as target autoantigen that is expressed in the brain and B cells. These findings will be instrumental to address important questions regarding pathogenic B-T cell interactions in multiple sclerosis and possibly also to develop novel therapies.
Asunto(s)
Linfocitos B/patología , Subtipos Serológicos HLA-DR/inmunología , Esclerosis Múltiple/inmunología , Autoantígenos/inmunología , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/fisiopatología , Linfocitos B/metabolismo , Encéfalo/patología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/fisiología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Subtipos Serológicos HLA-DR/genética , Humanos , Esclerosis Múltiple/genética , Esclerosis Múltiple/fisiopatología , Receptores de Antígenos de Linfocitos T , Células TH1/fisiologíaRESUMEN
OBJECTIVE: Specific human leucocyte antigen (HLA) alleles are not only associated with higher risk to develop multiple sclerosis (MS) and other autoimmune diseases, but also with the severity of various viral and bacterial infections. Here, we analyzed the most specific biomarker for MS, that is, the polyspecific intrathecal IgG antibody production against measles, rubella, and varicella zoster virus (MRZ reaction), for possible HLA associations in MS. METHODS: We assessed MRZ reaction from 184 Swiss patients with MS and clinically isolated syndrome (CIS) and 89 Swiss non-MS/non-CIS control patients, and performed HLA sequence-based typing, to check for associations of positive MRZ reaction with the most prevalent HLA alleles. We used a cohort of 176 Swedish MS/CIS patients to replicate significant findings. RESULTS: Whereas positive MRZ reaction showed a prevalence of 38.0% in MS/CIS patients, it was highly specific (97.7%) for MS/CIS. We identified HLA-DRB1*15:01 and other tightly linked alleles of the HLA-DR15 haplotype as the strongest HLA-encoded risk factors for a positive MRZ reaction in Swiss MS/CIS (odds ratio [OR], 3.90, 95% confidence interval [CI] 2.05-7.46, padjusted = 0.0004) and replicated these findings in Swedish MS/CIS patients (OR 2.18, 95%-CI 1.16-4.02, padjusted = 0.028). In addition, female MS/CIS patients had a significantly higher probability for a positive MRZ reaction than male patients in both cohorts combined (padjusted <0.005). INTERPRETATION: HLA-DRB1*15:01, the strongest genetic risk factor for MS, and female sex, 1 of the most prominent demographic risk factors for developing MS, predispose in MS/CIS patients for a positive MRZ reaction, the most specific CSF biomarker for MS. ANN NEUROL 2024;95:1112-1126.
Asunto(s)
Inmunoglobulina G , Esclerosis Múltiple , Humanos , Femenino , Masculino , Esclerosis Múltiple/genética , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/líquido cefalorraquídeo , Inmunoglobulina G/sangre , Adulto , Persona de Mediana Edad , Herpesvirus Humano 3/inmunología , Herpesvirus Humano 3/genética , Cadenas HLA-DRB1/genética , Suecia/epidemiología , Estudios de Cohortes , Adulto Joven , Virus de la Rubéola/genética , Virus de la Rubéola/inmunología , Antígenos HLA/genética , Anticuerpos Antivirales/líquido cefalorraquídeo , Anticuerpos Antivirales/sangre , Alelos , Suiza/epidemiologíaRESUMEN
Epstein-Barr virus (EBV) infection has been advocated as a prerequisite for developing multiple sclerosis (MS) and possibly the propagation of the disease. However, the precise mechanisms for such influences are still unclear. A large-scale study investigating the host genetics of EBV serology and related clinical manifestations, such as infectious mononucleosis (IM), may help us better understand the role of EBV in MS pathogenesis. This study evaluates the host genetic factors that influence serological response against EBV and history of IM and cross-evaluates them with MS risk and genetic susceptibility in the Swedish population. Plasma IgG antibody levels against EBV nuclear antigen-1 (EBNA-1, truncated=aa[325-641], peptide=aa[385-420]) and viral capsid antigen p18 (VCAp18) were measured using bead-based multiplex serology for 8744 MS cases and 7229 population-matched controls. The MS risk association for high/low EBV antibody levels and history of IM was compared to relevant clinical measures along with sex, age at sampling, and associated HLA allele variants. Genome-wide and HLA allele association analyses were also performed to identify genetic risk factors for EBV antibody response and IM history. Higher antibody levels against VCAp18 (OR=1.74, 95% CI=1.60-1.88) and EBNA-1, particularly the peptide (OR=3.13, 95% CI=2.93-3.35), were associated with an increased risk for MS. The risk increased with higher anti-EBNA-1 IgG levels up to twelve times the reference risk. We also identified several independent HLA haplotypes associated with EBV serology overlapping with known MS risk alleles (e.g., DRB1*15:01). Although there were several candidates, no variants outside the HLA region reached genome-wide significance. Cumulative HLA risk for anti-EBNA-1 IgG levels, particularly the peptide fragment, was strongly associated with MS. In contrast, the genetic risk for high anti-VCAp18 IgG levels was not as strongly associated with MS risk. IM history was not associated with class II HLA genes but negatively associated with A*02:01, which is protective against MS. Our findings emphasize that the risk association between anti-EBNA-1 IgG levels and MS may be partly due to overlapping HLA associations. Additionally, the increasing MS risk with increasing anti-EBNA-1 levels would be consistent with a pathogenic role of the EBNA-1 immune response, perhaps through molecular mimicry. Given that high anti-EBNA-1 antibodies may reflect a poorly controlled T-cell defense against the virus, our findings would be consistent with DRB1*15:01 being a poor class II antigen in the immune defense against EBV. Lastly, the difference in genetic control of IM supports the independent roles of EBNA-1 and IM in MS susceptibility.
RESUMEN
Recent studies have highlighted the important role of B cells in the pathogenesis of multiple sclerosis (MS). B cell activating factor (BAFF) and A proliferation inducing ligand (APRIL) play a major role in B cell survival and homeostasis. Here, we studied the association of BAFF and APRIL with B cell immune markers in MS and following B cell depletion and repopulation. We found that BAFF but not APRIL was significantly higher in plasma in untreated MS compared to controls. BAFF increased after rituximab treatment and decreased again during repopulation displaying an inverse correlation with B cell numbers, and more specifically switched memory B cell numbers. Cerebrospinal fluid BAFF inversely correlated with IgG index. BAFF displayed an inverse association to anti-EBV-CA antibodies. In summary, our study identified immune cells and factors that might regulate or be regulated by BAFF and APRIL levels in MS, and during B cell depletion and repopulation.
Asunto(s)
Esclerosis Múltiple , Humanos , Factor Activador de Células B , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral , Rituximab/uso terapéutico , Linfocitos B/patología , Interleucina-4RESUMEN
BACKGROUND AND PURPOSE: Mechanisms behind hypogammaglobulinaemia during rituximab treatment are poorly understood. METHODS: In this register-based multi-centre retrospective cohort study of multiple sclerosis (MS) patients in Sweden, 2745 patients from six participating Swedish MS centres were identified via the Swedish MS registry and included between 14 March 2008 and 25 January 2021. The exposure was treatment with at least one dose of rituximab for MS or clinically isolated syndrome, including data on treatment duration and doses. The degree of yearly decrease in immunoglobulin G (IgG) and immunoglobulin M (IgM) levels was evaluated. RESULTS: The mean decrease in IgG was 0.27 (95% confidence interval 0.17-0.36) g/L per year on rituximab treatment, slightly less in older patients, and without significant difference between sexes. IgG or IgM below the lower limit of normal (<6.7 or <0.27 g/L) was observed in 8.8% and 8.3% of patients, respectively, as nadir measurements. Six out of 2745 patients (0.2%) developed severe hypogammaglobulinaemia (IgG below 4.0 g/L) during the study period. Time on rituximab and accumulated dose were the main predictors for IgG decrease. Previous treatment with fingolimod and natalizumab, but not teriflunomide, dimethyl fumarate, interferons or glatiramer acetate, were significantly associated with lower baseline IgG levels by 0.80-1.03 g/L, compared with treatment-naïve patients. Switching from dimethyl fumarate or interferons was associated with an additional IgG decline of 0.14-0.19 g/L per year, compared to untreated. CONCLUSIONS: Accumulated dose and time on rituximab treatment are associated with a modest but significant decline in immunoglobulin levels. Previous MS therapies may influence additional IgG decline.
Asunto(s)
Agammaglobulinemia , Factores Inmunológicos , Esclerosis Múltiple , Rituximab , Humanos , Suecia , Femenino , Masculino , Agammaglobulinemia/inducido químicamente , Agammaglobulinemia/sangre , Rituximab/efectos adversos , Rituximab/uso terapéutico , Adulto , Persona de Mediana Edad , Factores Inmunológicos/efectos adversos , Factores Inmunológicos/administración & dosificación , Esclerosis Múltiple/tratamiento farmacológico , Estudios Retrospectivos , Sistema de Registros , Estudios de Cohortes , Inmunoglobulina G/sangreRESUMEN
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease affecting the central nervous system (CNS). Small non-coding RNAs (sncRNAs) and, in particular, microRNAs (miRNAs) have frequently been associated with MS. Here, we performed a comprehensive analysis of all classes of sncRNAs in matching samples of peripheral blood mononuclear cells (PBMCs), plasma, cerebrospinal fluid (CSF) cells, and cell-free CSF from relapsing-remitting (RRMS, n = 12 in relapse and n = 11 in remission) patients, secondary progressive (SPMS, n = 6) MS patients, and noninflammatory and inflammatory neurological disease controls (NINDC, n = 11; INDC, n = 5). We show widespread changes in miRNAs and sncRNA-derived fragments of small nuclear, nucleolar, and transfer RNAs. In CSF cells, 133 out of 133 and 115 out of 117 differentially expressed sncRNAs were increased in RRMS relapse compared to remission and RRMS compared to NINDC, respectively. In contrast, 65 out of 67 differentially expressed PBMC sncRNAs were decreased in RRMS compared to NINDC. The striking contrast between the periphery and CNS suggests that sncRNA-mediated mechanisms, including alternative splicing, RNA degradation, and mRNA translation, regulate the transcriptome of pathogenic cells primarily in the CNS target organ.
Asunto(s)
Esclerosis Múltiple/genética , Esclerosis Múltiple/inmunología , Transcriptoma/genética , Adulto , Femenino , Expresión Génica/genética , Perfilación de la Expresión Génica/métodos , Humanos , Leucocitos/metabolismo , Leucocitos Mononucleares/metabolismo , Masculino , MicroARNs/sangre , MicroARNs/líquido cefalorraquídeo , MicroARNs/genética , Persona de Mediana Edad , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple Crónica Progresiva/genética , Esclerosis Múltiple Recurrente-Remitente/genética , Recurrencia Local de Neoplasia/metabolismo , ARN Pequeño no Traducido/sangre , ARN Pequeño no Traducido/líquido cefalorraquídeo , ARN Pequeño no Traducido/genéticaRESUMEN
Most of the variation in outcome following severe traumatic brain injury (TBI) remains unexplained by currently recognized prognostic factors. Neuroinflammation may account for some of this difference. We hypothesized that TBI generated variable autoantibody responses between individuals that would contribute to outcome. We developed a custom protein microarray to detect autoantibodies to both CNS and systemic Ags in serum from the acute-phase (the first 7 d), late (6-12 mo), and long-term (6-13 y) intervals after TBI in human patients. We identified two distinct patterns of immune response to TBI. The first was a broad response to the majority of Ags tested, predominantly IgM mediated in the acute phase, then IgG dominant at late and long-term time points. The second was responses to specific Ags, most frequently myelin-associated glycopeptide (MAG), which persisted for several months post-TBI but then subsequently resolved. Exploratory analyses suggested that patients with a greater acute IgM response experienced worse outcomes than predicted from current known risk factors, suggesting a direct or indirect role in worsening outcome. Furthermore, late persistence of anti-MAG IgM autoantibodies correlated with raised serum neurofilament light concentrations at these time points, suggesting an association with ongoing neurodegeneration over the first year postinjury. Our results show that autoantibody production occurs in some individuals following TBI, can persist for many years, and is associated with worse patient outcome. The complexity of responses means that conventional approaches based on measuring responses to single antigenic targets may be misleading.
Asunto(s)
Autoanticuerpos/inmunología , Lesiones Traumáticas del Encéfalo/inmunología , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
BACKGROUND AND PURPOSE: Recent findings document a blunted humoral response to SARS-CoV-2 vaccination in patients on anti-CD20 treatment. Although most patients develop a cellular response, it is still important to identify predictors of seroconversion to optimize vaccine responses. METHODS: We determined antibody responses after SARS-CoV-2 vaccination in a real-world cohort of multiple sclerosis patients (n = 94) treated with anti-CD20, mainly rituximab, with variable treatment duration (median = 2.9, range = 0.4-9.6 years) and time from last anti-CD20 infusion to vaccination (median = 190, range = 60-1032 days). RESULTS: We find that presence of B cells and/or rituximab in blood predict seroconversion better than time since last infusion. Using multiple logistic regression, presence of >0.5% B cells increased probability of seroconversion with an odds ratio (OR) of 5.0 (95% confidence interval [CI] = 1.0-28.1, p = 0.055), whereas the corresponding OR for ≥6 months since last infusion was 1.45 (95% CI = 0.20-10.15, p = 0.705). In contrast, detectable rituximab levels were negatively associated with seroconversion (OR = 0.05, 95% CI = 0.002-0.392, p = 0.012). Furthermore, naïve and memory IgG+ B cells correlated with antibody levels. Although retreatment with rituximab at 4 weeks or more after booster depleted spike-specific B cells, it did not noticeably affect the rate of decline in antibody titers. Interferon-γ and/or interleukin-13 T-cell responses to the spike S1 domain were observed in most patients, but with no correlation to spike antibody levels. CONCLUSIONS: These findings are relevant for providing individualized guidance to patients and planning of vaccination schemes, in turn optimizing benefit-risk with anti-CD20.
Asunto(s)
Linfocitos B , Vacunas contra la COVID-19 , COVID-19 , Esclerosis Múltiple , Anticuerpos Antivirales , Linfocitos B/citología , COVID-19/prevención & control , Vacunas contra la COVID-19/administración & dosificación , Humanos , Inmunoglobulina G , Interferón gamma , Interleucina-13 , Esclerosis Múltiple/tratamiento farmacológico , Rituximab/farmacocinética , Rituximab/uso terapéutico , SARS-CoV-2 , Vacunación , Eficacia de las VacunasRESUMEN
BACKGROUND: Severe traumatic brain injury (TBI) is associated with blood-brain barrier (BBB) disruption and a subsequent neuroinflammatory process. We aimed to perform a multiplex screening of brain enriched and inflammatory proteins in blood and cerebrospinal fluid (CSF) in order to study their role in BBB disruption, neuroinflammation and long-term functional outcome in TBI patients and healthy controls. METHODS: We conducted a prospective, observational study on 90 severe TBI patients and 15 control subjects. Clinical outcome data, Glasgow Outcome Score, was collected after 6-12 months. We utilized a suspension bead antibody array analyzed on a FlexMap 3D Luminex platform to characterize 177 unique proteins in matched CSF and serum samples. In addition, we assessed BBB disruption using the CSF-serum albumin quotient (QA), and performed Apolipoprotein E-genotyping as the latter has been linked to BBB function in the absence of trauma. We employed pathway-, cluster-, and proportional odds regression analyses. Key findings were validated in blood samples from an independent TBI cohort. RESULTS: TBI patients had an upregulation of structural CNS and neuroinflammatory pathways in both CSF and serum. In total, 114 proteins correlated with QA, among which the top-correlated proteins were complement proteins. A cluster analysis revealed protein levels to be strongly associated with BBB integrity, but not carriage of the Apolipoprotein E4-variant. Among cluster-derived proteins, innate immune pathways were upregulated. Forty unique proteins emanated as novel independent predictors of clinical outcome, that individually explained ~ 10% additional model variance. Among proteins significantly different between TBI patients with intact or disrupted BBB, complement C9 in CSF (p = 0.014, ΔR2 = 7.4%) and complement factor B in serum (p = 0.003, ΔR2 = 9.2%) were independent outcome predictors also following step-down modelling. CONCLUSIONS: This represents the largest concomitant CSF and serum proteomic profiling study so far reported in TBI, providing substantial support to the notion that neuroinflammatory markers, including complement activation, predicts BBB disruption and long-term outcome. Individual proteins identified here could potentially serve to refine current biomarker modelling or represent novel treatment targets in severe TBI.
Asunto(s)
Barrera Hematoencefálica/anomalías , Lesiones Traumáticas del Encéfalo/complicaciones , Líquido Cefalorraquídeo/metabolismo , Proteómica , Adulto , Biomarcadores/sangre , Biomarcadores/líquido cefalorraquídeo , Barrera Hematoencefálica/metabolismo , Lesiones Traumáticas del Encéfalo/sangre , Lesiones Traumáticas del Encéfalo/fisiopatología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , SueciaRESUMEN
Besides its vital role in immunity, the complement system also contributes to the shaping of the synaptic circuitry of the brain. We recently described that soluble Complement Receptor 2 (sCR2) is part of the nerve injury response in rodents. We here study CR2 in context of multiple sclerosis (MS) and explore the molecular effects of CR2 on C3 activation. Significant increases in sCR2 levels were evident in cerebrospinal fluid (CSF) from both patients with relapsing-remitting MS (n=33; 6.2ng/mL) and secondary-progressive MS (n=9; 7.0ng/mL) as compared to controls (n=18; 4.1ng/mL). Furthermore, CSF sCR2 levels correlated significantly both with CSF C3 and C1q as well as to a disease severity measure. In vitro, sCR2 inhibited the cleavage and down regulation of C3b to iC3b, suggesting that it exerts a modulatory role in complement activation downstream of C3. These results propose a novel function for CR2/sCR2 in human neuroinflammatory conditions.
Asunto(s)
Complemento C3/inmunología , Esclerosis Múltiple Recurrente-Remitente/inmunología , Esclerosis Múltiple/inmunología , Receptores de Complemento 3d/inmunología , Adulto , Activación de Complemento/inmunología , Complemento C1q/líquido cefalorraquídeo , Complemento C1q/inmunología , Complemento C3/líquido cefalorraquídeo , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Modelos Lineales , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/líquido cefalorraquídeo , Esclerosis Múltiple/patología , Esclerosis Múltiple Recurrente-Remitente/líquido cefalorraquídeo , Esclerosis Múltiple Recurrente-Remitente/patología , Índice de Severidad de la Enfermedad , Adulto JovenRESUMEN
The complement system is activated in a wide spectrum of CNS diseases and is suggested to play a role in degenerative phenomena such as elimination of synaptic terminals. Still, little is known of mechanisms regulating complement activation in the CNS. Loss of synaptic terminals in the spinal cord after an experimental nerve injury is increased in the inbred DA strain compared with the PVG strain and is associated with expression of the upstream complement components C1q and C3, in the absence of membrane attack complex activation and neutrophil infiltration. To further dissect pathways regulating complement expression, we performed genome-wide expression profiling and linkage analysis in a large F2(DA × PVG) intercross, which identified quantitative trait loci regulating expression of C1qa, C1qb, C3, and C9. Unlike C1qa, C1qb, and C9, which all displayed distinct coregulation with different cis-regulated C-type lectins, C3 was regulated in a coexpression network immediately downstream of butyrylcholinesterase. Butyrylcholinesterase hydrolyses acetylcholine, which exerts immunoregulatory effects partly through TNF-α pathways. Accordingly, increased C3, but not C1q, expression was demonstrated in rat and mouse glia following TNF-α stimulation, which was abrogated in a dose-dependent manner by acetylcholine. These findings demonstrate new pathways regulating CNS complement expression using unbiased mapping in an experimental in vivo system. A direct link between cholinergic activity and complement activation is supported by in vitro experiments. The identification of distinct pathways subjected to regulation by naturally occurring genetic variability is of relevance for the understanding of disease mechanisms in neurologic conditions characterized by neuronal injury and complement activation.
Asunto(s)
Sistema Nervioso Central/metabolismo , Fibras Colinérgicas/fisiología , Activación de Complemento , Complemento C3/biosíntesis , Regulación de la Expresión Génica/inmunología , Redes Reguladoras de Genes , Acetilcolina/farmacología , Acetilcolina/fisiología , Animales , Animales Congénicos , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Lesiones Encefálicas/inmunología , Lesiones Encefálicas/fisiopatología , Butirilcolinesterasa/fisiología , Células Cultivadas , Sistema Nervioso Central/química , Sistema Nervioso Central/patología , Complemento C1q/biosíntesis , Complemento C1q/genética , Complemento C3/genética , Desnervación , Factores de Transcripción Forkhead/metabolismo , Ligamiento Genético , Estudio de Asociación del Genoma Completo , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Microglía/metabolismo , Sitios de Carácter Cuantitativo , Ratas , Rizotomía , Organismos Libres de Patógenos Específicos , Raíces Nerviosas Espinales/cirugía , Sinaptofisina/análisis , Factor de Necrosis Tumoral alfa/farmacología , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
BACKGROUND: Activation of the complement system has been implicated in both acute and chronic states of neurodegeneration. However, a detailed understanding of this complex network of interacting components is still lacking. METHODS: Large-scale global expression profiling in a rat F2(DAxPVG) intercross identified a strong cis-regulatory influence on the local expression of complement receptor 2 (Cr2) in the spinal cord after ventral root avulsion (VRA). Expression of Cr2 in the spinal cord was studied in a separate cohort of DA and PVG rats at different time-points after VRA, and also following sciatic nerve transection (SNT) in the same strains. Consequently, Cr2 (-/-) mice and Wt controls were used to further explore the role of Cr2 in the spinal cord following SNT. The in vivo experiments were complemented by astrocyte and microglia cell cultures. RESULTS: Expression of Cr2 in naïve spinal cord was low but strongly up regulated at 5-7 days after both VRA and SNT. Levels of Cr2 expression, as well as astrocyte activation, was higher in PVG rats than DA rats following both VRA and SNT. Subsequent in vitro studies proposed astrocytes as the main source of Cr2 expression. A functional role for Cr2 is suggested by the finding that transgenic mice lacking Cr2 displayed increased loss of synaptic nerve terminals following nerve injury. We also detected increased levels of soluble CR2 (sCR2) in the cerebrospinal fluid of rats following VRA. CONCLUSIONS: These results demonstrate that local expression of Cr2 in the central nervous system is part of the axotomy reaction and is suggested to modulate subsequent complement mediated effects.
Asunto(s)
Receptores de Complemento 3d/metabolismo , Médula Espinal/metabolismo , Raíces Nerviosas Espinales/lesiones , Raíces Nerviosas Espinales/patología , Regulación hacia Arriba/fisiología , Análisis de Varianza , Animales , Antígenos CD/metabolismo , Astrocitos/metabolismo , Antígeno CD11b/metabolismo , Células Cultivadas , Lateralidad Funcional , Redes Reguladoras de Genes , Proteína Ácida Fibrilar de la Glía/metabolismo , Ratones Transgénicos , Análisis por Micromatrices , Microglía/metabolismo , ARN Mensajero/metabolismo , Ratas , Receptores de Complemento 3d/genética , Neuropatía Ciática/metabolismo , Neuropatía Ciática/patología , Sinaptofisina/metabolismoRESUMEN
INTRODUCTION: Neuropathic pain is believed to be influenced in part by inflammatory processes. In this study we examined the effect of variability in the C-type lectin gene cluster (Aplec) on the development of neuropathic pain-like behavior after ligation of the L5 spinal nerve in the inbred DA and the congenic Aplec strains, which carries seven C-type lectin genes originating from the PVG strain. RESULTS: While both strains displayed neuropathic pain behavior early after injury, the Aplec strain remained sensitive throughout the whole study period. Analyses of several mRNA transcripts revealed that the expression of Interleukin-1ß, Substance P and Cathepsin S were more up-regulated in the dorsal part of the spinal cord of Aplec rats compared to DA, indicating a stronger inflammatory response. This notion was supported by flow cytometric analysis revealing increased infiltration of activated macrophages into the spinal cord. In addition, macrophages from the Aplec strain stimulated in vitro displayed higher expression of inflammatory cytokines compared to DA cells. Finally, we bred a recombinant congenic strain (R11R6) comprising only four of the seven Aplec genes, which displayed similar clinical and immune phenotypes as the Aplec strain. CONCLUSION: We here for the first time demonstrate that C-type lectins, a family of innate immune receptors with largely unknown functions in the nervous system, are involved in regulation of inflammation and development of neuropathic pain behavior after nerve injury. Further experimental and clinical studies are needed to dissect the underlying mechanisms more in detail as well as any possible relevance for human conditions.
Asunto(s)
Lectinas Tipo C/genética , Neuralgia/metabolismo , Traumatismos de los Nervios Periféricos/metabolismo , Animales , Catepsinas/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Variación Genética , Inflamación , Interleucina-1beta/metabolismo , Masculino , Modelos Genéticos , Familia de Multigenes , Neuralgia/terapia , Neuropéptidos/metabolismo , Fenotipo , ARN Mensajero/metabolismo , Ratas , Receptores de Interleucina-8A/metabolismo , Transducción de Señal , Sustancia P/metabolismoRESUMEN
BACKGROUND: C-type lectin (CLEC) receptors are important for initiating and shaping immune responses; however, their role in inflammatory reactions in the central nervous system after traumatic injuries is not known. The antigen-presenting lectin-like receptor gene complex (Aplec) contains a few CLEC genes, which differ genetically among inbred rat strains. It was originally thought to be a region that regulates susceptibility to autoimmune arthritis, autoimmune neuroinflammation and infection. METHODS: The inbred rat strains DA and PVG differ substantially in degree of spinal cord motor neuron death following ventral root avulsion (VRA), which is a reproducible model of localized nerve root injury. A large F2 (DAxPVG) intercross was bred and genotyped after which global expressional profiling was performed on spinal cords from F2 rats subjected to VRA. A congenic strain, Aplec, created by transferring a small PVG segment containing only seven genes, all C-type lectins, ontoDA background, was used for further experiments together with the parental strains. RESULTS: Global expressional profiling of F2 (DAxPVG) spinal cords after VRA and genome-wide eQTL mapping identified a strong cis-regulated difference in the expression of Clec4a3 (Dcir3), a C-type lectin gene that is a part of the Aplec cluster. Second, we demonstrate significantly improved motor neuron survival and also increased T-cell infiltration into the spinal cord of congenic rats carrying Aplec from PVG on DA background compared to the parental DA strain. In vitro studies demonstrate that the Aplec genes are expressed on microglia and upregulated upon inflammatory stimuli. However, there were no differences in expression of general microglial activation markers between Aplec and parental DA rats, suggesting that the Aplec genes are involved in the signaling events rather than the primary activation of microglia occurring upon nerve root injury. CONCLUSIONS: In summary, we demonstrate that a genetic variation in Aplec occurring among inbred strains regulates both survival of axotomized motor neurons and the degree of lymphocyte infiltration. These results demonstrate a hitherto unknown role for CLECs for intercellular communication that occurs after damage to the nervous system, which is relevant for neuronal survival.
Asunto(s)
Lectinas Tipo C/genética , Neuronas Motoras/fisiología , Familia de Multigenes/genética , Radiculopatía/genética , Radiculopatía/patología , Linfocitos T/fisiología , Animales , Animales Congénicos , Presentación de Antígeno , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Astrocitos/metabolismo , Recuento de Células , Supervivencia Celular/fisiología , Células Cultivadas , Femenino , Citometría de Flujo , Inmunohistoquímica , Lectinas Tipo C/metabolismo , Análisis por Micromatrices , Microglía/metabolismo , Proteínas de la Mielina/metabolismo , Oligodendroglía/metabolismo , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Raíces Nerviosas Espinales/patologíaRESUMEN
Increasing evidence suggests that genetic background affects outcome of traumatic brain injuries (TBI). Still, there is limited detailed knowledge on what pathways/processes are affected by genetic heterogeneity. The inbred rat strains DA and PVG differ in neuronal survival following TBI. We here carried out global expressional profiling to identify differentially regulated pathways governing the response to an experimental controlled brain contusion injury. One of the most differentially regulated molecular networks concerned immune cell trafficking. Subsequent characterization of the involved cells using flow cytometry demonstrated greater infiltration of neutrophils and monocytes, as well as a higher degree of microglia activation in DA compared to PVG rats. In addition, DA rats displayed a higher number of NK cells and a higher ratio of CD161bright compared to CD161dim NK cells. Local expression of complement pathway molecules such as C1 and C3 was higher in DA and both the key complement component C3 and membrane-attack complex (MAC) could be demonstrated on axons and nerve cells. A stronger activation of the complement system in DA was associated with higher cerebrospinal fluid levels of neurofilament-light, a biomarker for nerve/axonal injury. In summary, we demonstrate substantial differences between DA and PVG rats in activation of inflammatory pathways; in particular, immune cell influx and complement activation associated with neuronal/axonal injury after TBI. These findings suggest genetic influences acting on inflammatory activation to be of importance in TBI and motivate further efforts using experimental forward genetics to identify genes/pathways that affect outcome.
Asunto(s)
Lesiones Encefálicas , Activación de Complemento , Leucocitos , ARN Mensajero/análisis , Ratas Endogámicas , Animales , Lesiones Encefálicas/genética , Lesiones Encefálicas/inmunología , Movimiento Celular/genética , Activación de Complemento/genética , Activación de Complemento/inmunología , Complemento C1q/genética , Complemento C1q/inmunología , Complemento C3/genética , Complemento C3/inmunología , Complejo de Ataque a Membrana del Sistema Complemento/genética , Complejo de Ataque a Membrana del Sistema Complemento/inmunología , Proteínas del Sistema Complemento/genética , Proteínas del Sistema Complemento/inmunología , Citocinas/genética , Citocinas/inmunología , Perfilación de la Expresión Génica , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Leucocitos/citología , Leucocitos/inmunología , Microglía/citología , Microglía/inmunología , Monocitos/citología , Monocitos/inmunología , Subfamilia B de Receptores Similares a Lectina de Células NK/genética , Subfamilia B de Receptores Similares a Lectina de Células NK/inmunología , Neutrófilos/citología , Neutrófilos/inmunología , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Ratas Endogámicas/genética , Ratas Endogámicas/inmunologíaRESUMEN
BACKGROUND AND OBJECTIVES: B cell-depleting therapies are highly effective in relapsing-remitting multiple sclerosis (RRMS) but are associated with increased infection risk and blunted humoral vaccination responses. Extension of dosing intervals may mitigate such negative effects, but its consequences on MS disease activity are yet to be ascertained. The objective of this study was to determine clinical and neuroradiologic disease activity, as well as B-cell repopulation dynamics, after implementation of extended rituximab dosing in RRMS. METHODS: We conducted a prospective observational study in a specialized-care, single-center setting, including patients with RRMS participating in the COMBAT-MS and MultipleMS observational drug trials, who had received at least 2 courses of rituximab (median follow-up 4.2 years, range 0.1-8.9 years). Using Cox regression, hazard ratios (HRs) of clinical relapse and/or contrast-enhancing lesions on MRI were calculated in relation to time since last dose of rituximab. RESULTS: A total of 3,904 dose intervals were accumulated in 718 patients and stratified into 4 intervals: <8, ≥8 to 12, ≥12 to 18, and ≥18 months. We identified 24 relapses of which 20 occurred within 8 months since previous infusion and 4 with intervals over 8 months. HRs for relapse when comparing ≥8 to 12, ≥12 to 18, and ≥18 months with <8 months since last dose were 0.28 (95% CI 0.04-2.10), 0.38 (95% CI 0.05-2.94), and 0.89 (95% CI 0.20-4.04), respectively, and thus nonsignificant. Neuroradiologic outcomes mirrored relapse rates. Dynamics of total B-cell reconstitution varied considerably, but median total B-cell counts reached lower level of normal after 12 months and median memory B-cell counts after 16 months. DISCUSSION: In this prospective cohort of rituximab-treated patients with RRMS exposed to extended dosing intervals, we could not detect a relation between clinical or neuroradiologic disease activity and time since last infusion. Total B- and memory B-cell repopulation kinetics varied considerably. These findings, relevant for assessing risk-mitigation strategies with anti-CD20 therapies in RRMS, suggest that relapse risk remains low with extended infusion intervals. Further studies are needed to investigate the relation between B-cell repopulation dynamics and adverse event risks associated with B-cell depletion.
Asunto(s)
Esclerosis Múltiple Recurrente-Remitente , Esclerosis Múltiple , Humanos , Rituximab/efectos adversos , Esclerosis Múltiple/tratamiento farmacológico , Estudios Prospectivos , Factores Inmunológicos/efectos adversos , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Esclerosis Múltiple Recurrente-Remitente/inducido químicamente , Recurrencia , Enfermedad CrónicaRESUMEN
BACKGROUND: Nitric oxide is a key mediator of post-traumatic inflammation in the brain. We examined the expressions of iNOS, nNOS, and eNOS in inbred DA and PVGa rat strains where DA is susceptible to autoimmune neuroinflammation and PVGa-resistant. METHODS: Parietal contusions using a weight drop model were produced in five rats per genotype. After 24 h, the brains were removed and analyzed using a range of immunohistochemical methods. RESULTS: PVGa presented significantly increased iNOS expression in infiltrating inflammatory cells in the perilesional area compared to DA (p < 0.05). The amount of w3/13-positive infiltrating inflammatory cells did not differ between strains. eNOS and nNOS expression did not differ between strains. iNOS-positive cells coexpressed neuronal (NeuN), macrophage (ED-1), and leucocyte (w3/13) markers. MnSOD was significantly increased in PVGa (p < 0.05). 3-Nitrotyrosine, a measure of peroxynitrite levels, and fluoro-jade stained neuronal degeneration, did not differ between strains. CONCLUSIONS: Two inbred rat strains with genetically determined differences in susceptibility to develop autoimmune disease displayed different levels of the inflammatory and anti-inflammatory mediators iNOS and MnSOD, indicating genetic regulation. Interestingly, the increased levels of iNOS did not lead to elevated expression of the neuronal cell-death marker fluoro-jade. The increased iNOS expression was correlated with increased expression of superoxide scavenger MnSOD. Excessive peroxynitrite formation was probably prevented by limitation of available superoxide. Subsequently, the higher expression of potentially deleterious iNOS in PVGa did not result in increased neuronal death.
Asunto(s)
Lesiones Encefálicas/enzimología , Lesiones Encefálicas/patología , Predisposición Genética a la Enfermedad/genética , Inflamación/enzimología , Inflamación/patología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Animales , Lesiones Encefálicas/genética , Modelos Animales de Enfermedad , Genotipo , Inflamación/genética , Masculino , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa de Tipo II/genética , Ratas , Ratas Endogámicas , Transducción de Señal/genética , Transducción de Señal/fisiología , Especificidad de la EspecieRESUMEN
OBJECTIVE: Multiple sclerosis (MS) is a neuroinflammatory disease where immune cells cross the blood-brain barrier (BBB) into the central nervous system (CNS). What predisposes these immune cells to cross the BBB is still unknown. Here, we examine the possibility that genomic rearrangements could predisposespecific immune cells in the peripheral blood to cross the BBB and form sub-populations of cells involved in the inflammatory process in the CNS. METHODS: We compared copy number variations in paired peripheral blood mononuclear cells (PBMCs) and cerebrospinal fluid (CSF) cells from MS patients. Thereafter, using next generation sequencing, we studied the T-cell receptor beta (TRB) locus rearrangements and profiled the αß T cell repertoire in peripheral CD4+ and CD8+ T cells and in the CSF. RESULTS: We identified deletions in the T-cell receptor alpha/delta (TRA/D), gamma (TRG), and TRB loci in CSF cells compared to PBMCs. Further characterization revealed diversity of the TRB locus which was used to describe the character and clonal expansion of T cells in the CNS. T-cell repertoire profiling from either side of the BBB concluded that the most frequent clones in the CSF samples are unique to an individual. Furthermore, we observed a difference in the proportion of expanded T-cell clones when comparing samples from MS patients in relapse and remission with opposite trends in CSF and peripheral blood. INTERPRETATION: This study provides a characterization of the T cells in the CSF and might indicate a role of expanded clones in MS pathogenicity.
Asunto(s)
Esclerosis Múltiple , Barrera Hematoencefálica , Linfocitos T CD8-positivos , Variaciones en el Número de Copia de ADN , Humanos , Leucocitos Mononucleares , Receptores de Antígenos de Linfocitos TRESUMEN
Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS), in which pathological T cells, likely autoimmune, play a key role. Despite its central importance, the autoantigen repertoire remains largely uncharacterized. Using a novel in vitro antigen delivery method combined with the Human Protein Atlas library, we screened for T cell autoreactivity against 63 CNS-expressed proteins. We identified four previously unreported autoantigens in MS: fatty acid-binding protein 7, prokineticin-2, reticulon-3, and synaptosomal-associated protein 91, which were verified to induce interferon-γ responses in MS in two cohorts. Autoreactive profiles were heterogeneous, and reactivity to several autoantigens was MS-selective. Autoreactive T cells were predominantly CD4+ and human leukocyte antigen-DR restricted. Mouse immunization induced antigen-specific responses and CNS leukocyte infiltration. This represents one of the largest systematic efforts to date in the search for MS autoantigens, demonstrates the heterogeneity of autoreactive profiles, and highlights promising targets for future diagnostic tools and immunomodulatory therapies in MS.
RESUMEN
Genetic regulation of autoimmune neuroinflammation is a well known phenomenon, but genetic influences on inflammation following traumatic nerve injuries have received little attention. In this study we examined the inflammatory response in a rat traumatic brain injury (TBI) model, with a particular focus on major histocompatibility class II (MHC II) presentation, in two inbred rat strains that have been extensively characterized in experimental autoimmune encephalomyelitis (EAE); DA and PVG. In addition, MHC and Vra4 congenic strains on these backgrounds were studied to give information on MHC and non-MHC gene contribution. Thus, allelic differences in Vra4, harboring the Ciita gene, was found to regulate expression of the invariant chain at the mRNA level, with a much smaller effect exerted by the MHC locus itself. Notably, however, at the protein level the MHC congenic PVG-RT1(av1) strain displayed much stronger MHCII(+) presentation, as shown both by immunolabeling and flow cytometry, than the PVG strain, dwarfing the effect of Ciita. The PVG-RT1(av1) strain had significantly more T-cell influx than both DA and PVG, suggesting regulation both by MHC and non-MHC genes. Finally, in terms of outcome, the EAE susceptible DA strain displayed a significantly smaller resulting lesion volume than the resistant PVG-RT1(av1) strain. These results provide additional support for a role of adaptive immune response after neurotrauma and demonstrate that outcome is significantly affected by host genetic factors.