RESUMO
Natalizumab, a humanized monoclonal antibody (mAb) against α4-integrin, reduces the number of dendritic cells (DC) in cerebral perivascular spaces in multiple sclerosis (MS). Selective deletion of α4-integrin in CD11c+ cells should curtail their migration to the central nervous system (CNS) and ameliorate experimental autoimmune encephalomyelitis (EAE). We generated CD11c.Cre+/-ITGA4fl/fl C57BL/6 mice to selectively delete α4-integrin in CD11c+ cells. Active immunization and adoptive transfer EAE models were employed and compared with WT controls. Multiparameter flow cytometry was utilized to immunophenotype leukocyte subsets. Single-cell RNA sequencing was used to profile individual cells. α4-Integrin expression by CD11c+ cells was significantly reduced in primary and secondary lymphoid organs in CD11c.Cre+/-ITGA4fl/fl mice. In active EAE, a delayed disease onset was observed in CD11c.Cre+/-ITGA4fl/fl mice, during which CD11c+CD88+ cells were sequestered in the blood. Upon clinical EAE onset, CD11c+CD88+ cells appeared in the CNS and expressed CD317+ In adoptive transfer experiments, CD11c.Cre+/-ITGA4fl/fl mice had ameliorated clinical disease phenotype associated with significantly diminished numbers of CNS CD11c+CD88+CD317+ cells. In human cerebrospinal fluid from subjects with neuroinflammation, microglia-like cells display coincident expression of ITGAX (CD11c), C5AR1 (CD88), and BST2 (CD317). In mice, we show that only activated, but not naïve microglia expressed CD11c, CD88, and CD317. Finally, anti-CD317 treatment prior to clinical EAE substantially enhanced recovery in mice.
Assuntos
Antígenos CD/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Integrina alfa4/metabolismo , Células Mieloides/metabolismo , Animais , Apresentação de Antígeno , Células Cultivadas , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/metabolismo , Feminino , Humanos , Masculino , Camundongos , Microglia/metabolismoRESUMO
For over 70 years experimental autoimmune encephalomyelitis (EAE) has been induced with myelin autoantigens emulsified in complete Freund's adjuvant (CFA) which has significant side effects such as pain, inflammation, and tissue necrosis at the injection site. ß-1,3-d-glucan particles (GPs) are hollow microcapsules prepared from Saccharomyces cerevisiae cell walls that induce potent Th17 cell responses without causing strong injection site tissue reactions. We evaluated the potential of GPs complexed with neuroantigens to induce EAE while avoiding undesirable side effects. GPs loaded with myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) or proteolipid protein 139-151 (PLP139-151) peptides effectively induced EAE in C57BL/6 mice and SJL mice. Disease severity, CNS pathology and immune responses were comparable between GP- and CFA-immunized mice. Importantly, injection with GPs resulted in significantly decreased inflammation compared with CFA. We posit that use of GPs provides an alternative means for inducing EAE that results in comparable disease, but less discomfort to animals.
Assuntos
Adjuvantes Imunológicos/metabolismo , Cápsulas/metabolismo , Parede Celular/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Esclerose Múltipla/imunologia , Proteoglicanas/metabolismo , Saccharomyces cerevisiae/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Adjuvante de Freund , Humanos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Proteína Proteolipídica de Mielina/imunologia , Glicoproteína Mielina-Oligodendrócito/imunologia , Fragmentos de Peptídeos/imunologia , Proteoglicanas/imunologia , Células Th17/imunologiaRESUMO
Multiple sclerosis (MS) is a neuroinflammatory disorder characterized by autoimmune-mediated inflammatory lesions in CNS leading to myelin damage and axonal loss. MS is a heterogenous disease with variable and unpredictable disease course. Due to its complex nature, MS is difficult to diagnose and responses to specific treatments may vary between individuals. Therefore, there is an indisputable need for biomarkers for early diagnosis, prediction of disease exacerbations, monitoring the progression of disease, and for measuring responses to therapy. Genomic and proteomic studies have sought to understand the molecular basis of MS and find biomarker candidates. Advances in next-generation sequencing and mass-spectrometry techniques have yielded an unprecedented amount of genomic and proteomic data; yet, translation of the results into the clinic has been underwhelming. This has prompted the development of novel data science techniques for exploring these large datasets to identify biologically relevant relationships and ultimately point towards useful biomarkers. Herein we discuss optimization of omics study designs, advances in the generation of omics data, and systems biology approaches aimed at improving biomarker discovery and translation to the clinic for MS.
Assuntos
Biomarcadores/análise , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Genômica/métodos , Humanos , Proteômica/métodos , Biologia de Sistemas/métodosRESUMO
While it was long held that T cells were the primary mediators of multiple sclerosis (MS) pathogenesis, the beneficial effects observed in response to treatment with Rituximab (RTX), a monoclonal antibody (mAb) targeting CD20, shed light on a key contributor to MS that had been previously underappreciated: B cells. This has been reaffirmed by results from clinical trials testing the efficacy of subsequently developed B cell-depleting mAbs targeting CD20 as well as studies revisiting the effects of previous disease-modifying therapies (DMTs) on B cell subsets thought to modulate disease severity. In this review, we summarize current knowledge regarding the complex roles of B cells in MS pathogenesis and current and potential future B cell-directed therapies.
Assuntos
Subpopulações de Linfócitos B/imunologia , Esclerose Múltipla/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos CD20/imunologia , Humanos , Rituximab/imunologiaRESUMO
The human autoimmune disease-associated HLA alleles HLA-DR2b (DRB1*1501) and HLA-DR4 (DRB1*0401) are strongly linked to increased susceptibility for multiple sclerosis (MS) and rheumatoid arthritis (RA), respectively. The underlying mechanisms are not fully understood, but these MHC alleles may shape the repertoire of pathogenic T cells via central tolerance. The transcription factor autoimmune regulator (AIRE) promotes central T cell tolerance via ectopic expression of tissue-specific antigens (TSAs). Aire deficiency in humans causes autoimmune polyendocrinopathy syndrome type 1 (APS1), and Aire knockout mice (Aire-/-) develop spontaneous autoimmune pathology characterized by multi-organ lymphocytic infiltrates. Here, we asked whether impaired TSAs gene expression in the absence of Aire promoted spontaneous MS- or RA-like autoimmune pathology in the context of human HLA alleles in HLA-DR2b or HLA-DR4 transgenic (tg) mice. The results show that reduced TSAs gene expression in the thymus of Aire-deficient HLA-DR2b or HLA-DR4 tg mice corresponded to mild spontaneous inflammatory infiltrates in salivary glands, liver, and pancreas. Moreover, Aire-deficiency modestly enhanced experimental autoimmune encephalomyelitis (EAE) in HLA-DR tg mice, but the animals did not show signs of spontaneous neuroinflammation or arthritis. No significant changes were observed in CD4+ T cell numbers, T cell receptor (TCR) distribution, regulatory T cells (Treg), or antigen-induced cytokine production. Abrogating Treg function by treatment with anti-CTLA-4 or anti-CD25 mAb in Aire-deficient HLA-DR tg mice did not trigger EAE or other autoimmune pathology. Our results suggest a redundant role for Aire in maintaining immune tolerance in the context of autoimmune disease-associated human HLA alleles.
Assuntos
Doenças Autoimunes/imunologia , Encefalomielite Autoimune Experimental/imunologia , Antígeno HLA-DR2/imunologia , Antígeno HLA-DR4/imunologia , Fatores de Transcrição/imunologia , Animais , Antígenos/imunologia , Antígenos/metabolismo , Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Doenças Autoimunes/genética , Doenças Autoimunes/metabolismo , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/metabolismo , Antígeno HLA-DR2/genética , Antígeno HLA-DR2/metabolismo , Antígeno HLA-DR4/genética , Antígeno HLA-DR4/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Esclerose Múltipla/metabolismo , Especificidade de Órgãos/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína AIRERESUMO
Evidence has suggested both a pathogenic and a protective role for the proinflammatory cytokine IFN-γ in experimental autoimmune encephalomyelitis (EAE). However, the mechanisms underlying the protective role of IFN-γ in EAE have not been fully resolved, particularly in the context of CNS antigen-presenting cells (APCs). In this study we examined the role of IFN-γ in myelin antigen uptake by CNS APCs during EAE. We found that myelin antigen colocalization with APCs was decreased substantially and that EAE was significantly more severe and showed a chronic-progressive course in IFN-γ knockout (IFN-γ-/-) or IFN-γ receptor knockout (IFN-γR-/-) mice as compared with WT animals. IFN-γ was a critical regulator of phagocytic/activating receptors on CNS APCs. Importantly, "free" myelin debris and lipid peroxidation activity at CNS lesions was increased in mice lacking IFN-γ signaling. Treatment with N-acetyl-l-cysteine, a potent antioxidant, abolished lipid peroxidation activity and ameliorated EAE in IFN-γ-signaling-deficient mice. Taken together the data suggest a protective role for IFN-γ in EAE by regulating the removal of myelin debris by CNS APCs and thereby limiting the substrate available for the generation of neurotoxic lipid peroxidation products.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Interferon gama/imunologia , Peroxidação de Lipídeos/imunologia , Bainha de Mielina/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Apoptose/genética , Apoptose/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/metabolismo , Citometria de Fluxo , Interferon gama/deficiência , Interferon gama/genética , Ativação Linfocitária/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Bainha de Mielina/metabolismo , Fagocitose/genética , Fagocitose/imunologia , Receptores de Interferon/deficiência , Receptores de Interferon/genética , Receptores de Interferon/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptor de Interferon gamaRESUMO
BACKGROUND: Identifying disease correlated features early before large number of molecules are impacted by disease progression with significant abundance change is very advantageous to biologists for developing early disease diagnosis biomarkers. Disease correlated features have relatively low level of abundance change at early stages. Finding them using existing bioinformatic tools in high throughput data is a challenging task since the technology suffers from limited dynamic range and significant noise. Most existing biomarker discovery algorithms can only detect molecules with high abundance changes, frequently missing early disease diagnostic markers. RESULTS: We present a new statistic called early response index (ERI) to prioritize disease correlated molecules as potential early biomarkers. Instead of classification accuracy, ERI measures the average classification accuracy improvement attainable by a feature when it is united with other counterparts for classification. ERI is more sensitive to abundance changes than other ranking statistics. We have shown that ERI significantly outperforms SAM and Localfdr in detecting early responding molecules in a proteomics study of a mouse model of multiple sclerosis. Importantly, ERI was able to detect many disease relevant proteins before those algorithms detect them at a later time point. CONCLUSIONS: ERI method is more sensitive for significant feature detection during early stage of disease development. It potentially has a higher specificity for biomarker discovery, and can be used to identify critical time frame for disease intervention.
Assuntos
Biomarcadores/metabolismo , Esclerose Múltipla/diagnóstico , Proteômica/métodos , Algoritmos , Animais , Sistema Nervoso Central/metabolismo , Diagnóstico Precoce , Camundongos , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Proteoma/metabolismo , Fatores de TempoRESUMO
CD4(+) T helper (Th) cells are critical for proper immune cell homeostasis and host defense, but are also major contributors to pathology of autoimmune and inflammatory diseases. Since the discovery of the Th1/Th2 dichotomy, many additional Th subsets were discovered, each with a unique cytokine profile, functional properties, and presumed role in autoimmune tissue pathology. This includes Th1, Th2, Th17, Th22, Th9, and Treg cells which are characterized by specific cytokine profiles. Cytokines produced by these Th subsets play a critical role in immune cell differentiation, effector subset commitment, and in directing the effector response. Cytokines are often categorized into proinflammatory and anti-inflammatory cytokines and linked to Th subsets expressing them. This article reviews the different Th subsets in terms of cytokine profiles, how these cytokines influence and shape the immune response, and their relative roles in promoting pathology in autoimmune and inflammatory diseases. Furthermore, we will discuss whether Th cell pathogenicity can be defined solely based on their cytokine profiles and whether rigid definition of a Th cell subset by its cytokine profile is helpful.
Assuntos
Doenças Autoimunes/imunologia , Citocinas/imunologia , Inflamação/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T CD4-Positivos/imunologia , Humanos , Esclerose Múltipla/imunologia , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Células Th17/imunologia , Células Th2/imunologiaRESUMO
Induction of experimental autoimmune encephalomyelitis (EAE) in susceptible animals requires reactivation of encephalitogenic CD4(+) T cells by APCs in the CNS. However, it has remained unresolved from where APCs in the CNS acquire myelin Ag for T cell activation and under which conditions, that is, whether only during EAE or also in the naive CNS. In this study, we investigated the kinetics of myelin Ag uptake by CNS APCs during EAE and in the naive CNS. Our results show that during EAE CX3CR1(+)CD11b(+) microglia were the first APCs in the CNS to contain myelin Ag upon induction of disease, albeit in very small numbers. Dendritic cells (DCs) arrived in the CNS in sizable numbers significantly later (day 5 postimmunization), without detectable myelin Ag, but acquired it by day 7 postimmunization. Furthermore, a sharp increase in neuroantigen-containing DCs coincided with the onset of EAE symptoms. Importantly, in naive mice a low but consistent number of microglia contained myelin Ag, suggesting release by oligodendrocytes under steady state conditions. Although microglia isolated from naive brain and spinal cord did not elicit a strong CD4(+) T cell response in vitro, myelin Ag-containing microglia may still play a local role in modulating encephalitogenic CD4(+) T cell responses in early EAE prior to the arrival of other professional APCs, such as DCs. Finally, newly arriving DCs in the CNS not yet loaded with myelin Ag before the onset of EAE may be a potential therapeutic target.
Assuntos
Antígenos/metabolismo , Encéfalo/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Proteína Proteolipídica de Mielina/farmacocinética , Glicoproteína Mielina-Oligodendrócito/farmacocinética , Células Mieloides/metabolismo , Fragmentos de Peptídeos/farmacocinética , Medula Espinal/metabolismo , Transferência Adotiva , Animais , Apresentação de Antígeno , Antígenos/imunologia , Autoanticorpos/biossíntese , Autoanticorpos/imunologia , Encéfalo/imunologia , Movimento Celular , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Adjuvante de Freund , Imageamento Tridimensional , Imunização , Injeções Intraperitoneais , Injeções Subcutâneas , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Mutantes Neurológicos , Camundongos Transgênicos , Microglia/metabolismo , Microscopia Confocal , Proteína Básica da Mielina/análise , Proteína Básica da Mielina/imunologia , Proteína Proteolipídica de Mielina/imunologia , Glicoproteína Mielina-Oligodendrócito/imunologia , Fragmentos de Peptídeos/imunologia , Toxina Pertussis , Medula Espinal/imunologiaRESUMO
Fractalkine, a chemokine anchored to neurons or peripheral endothelial cells, serves as an adhesion molecule or as a soluble chemoattractant. Fractalkine binds CX3CR1 on microglia and circulating monocytes, dendritic cells, and NK cells. The aim of this study is to determine the role of CX3CR1 in the trafficking and function of myeloid cells to the CNS during experimental autoimmune encephalomyelitis (EAE). Our results show that, in models of active EAE, Cx3cr1(-/-) mice exhibited more severe neurologic deficiencies. Bone marrow chimeric mice confirmed that CX3CR1 deficiency in bone marrow enhanced EAE severity. Notably, CX3CR1 deficiency was associated with an increased accumulation of CD115(+)Ly6C(-)CD11c(+) dendritic cells into EAE-affected brains that correlated with enhanced demyelination and neuronal damage. Furthermore, higher IFN-γ and IL-17 levels were detected in cerebellar and spinal cord tissues of CX3CR1-deficient mice. Analyses of peripheral responses during disease initiation revealed a higher frequency of IFN-γ- and IL-17-producing T cells in lymphoid tissues of CX3CR1-deficient as well as enhanced T cell proliferation induced by CX3CR1-deficient dendritic cells. In addition, adoptive transfer of myelin oligodendrocyte glycoprotein35-55-reactive wild-type T cells induced substantially more severe EAE in CX3CR1-deficient recipients when compared with wild-type recipients. Collectively, the data demonstrate that besides its role in chemoattraction, CX3CR1 is a key regulator of myeloid cell activation contributing to the establishment of adaptive immune responses.
Assuntos
Autoimunidade , Encefalomielite Autoimune Experimental/imunologia , Inflamação/imunologia , Células Mieloides/metabolismo , Receptores de Quimiocinas/metabolismo , Receptores de Citocinas/metabolismo , Receptores de HIV/metabolismo , Imunidade Adaptativa , Animais , Antígenos Ly/genética , Antígenos Ly/metabolismo , Células da Medula Óssea , Antígeno CD11c/genética , Antígeno CD11c/metabolismo , Receptor 1 de Quimiocina CX3C , Proliferação de Células , Sistema Nervoso Central/citologia , Quimera , Doenças Desmielinizantes/genética , Células Dendríticas/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Interferon gama/metabolismo , Interleucina-1/metabolismo , Interleucina-17/metabolismo , Ativação Linfocitária/imunologia , Tecido Linfoide/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Glicoproteína Mielina-Oligodendrócito/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Receptores de Quimiocinas/deficiência , Receptores de Quimiocinas/genética , Receptores de Citocinas/imunologia , Receptores de HIV/imunologia , Linfócitos T/metabolismoRESUMO
The strong association of HLA-DR2b (DRB1*1501) with multiple sclerosis (MS) suggests this molecule as prime target for specific immunotherapy. Inhibition of HLA-DR2b-restricted myelin-specific T cells has the potential to selectively prevent CNS pathology mediated by these MHC molecules without undesired global immunosuppression. In this study, we report development of a highly selective small molecule inhibitor of peptide binding and presentation by HLA-DR2b. PV-267, the candidate molecule used in these studies, inhibited cytokine production and proliferation of myelin-specific HLA-DR2b-restricted T cells. PV-267 had no significant effect on T cell responses mediated by other MHC class II molecules, including HLA-DR1, -DR4, or -DR9. Importantly, PV-267 did not induce nonspecific immune activation of human PBMC. Lastly, PV-267 showed treatment efficacy both in preventing experimental autoimmune encephalomyelitis and in treating established disease. The results suggest that blocking the MS-associated HLA-DR2b allele with small molecule inhibitors may be a promising therapeutic strategy for the treatment of MS.
Assuntos
Encefalomielite Autoimune Experimental/prevenção & controle , Encefalomielite Autoimune Experimental/terapia , Antígeno HLA-DR2/metabolismo , Linfócitos T/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Encefalomielite Autoimune Experimental/imunologia , Antígeno HLA-DR2/efeitos dos fármacos , Antígeno HLA-DR2/imunologia , Humanos , Leucócitos Mononucleares/imunologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Transgênicos , Esclerose Múltipla/imunologia , Esclerose Múltipla/terapia , Proteína Básica da Mielina , Peptídeos/farmacologia , Linfócitos T/efeitos dos fármacosRESUMO
Multiple sclerosis (MS) and neuromyelitis optica (NMO) are the most prevalent neuroinflammatory diseases of the central nervous system (CNS). The immunological cascade of these disorders is complex, and the exact spatial and temporal role of different immune cells is not fully understood. Although MS has been considered for many years to be primarily T cell driven, it is well established that B cells and the humoral immune response play an important role in its pathogenesis. This has long been evident from laboratory findings that include the presence of oligoclonal bands in the CSF. In NMO, the importance of the humoral immune system appears even more obvious as evidenced by pathogenic antibodies against aquaporin 4 (AQP4). Besides their capacity to mature into antibody-producing plasma cells, B cells are potent antigen-presenting cells to T lymphocytes and they can provide soluble factors for cell activation and differentiation to other immune-competent cells. In MS and NMO, there are substantial data from clinical trials that B cell depletion with CD20-directed agents is effective and relatively safe. Plasma cells, which produce antibodies against molecular targets expressed by the host, but which also provide humoral immune responses against pathogens, are not targeted by anti-CD20 therapies. Therefore, the depletion of CD19-expressing cells would offer potential advantages with regard to efficacy, but potentially higher risks with regard to infectious complications. This review will outline the rationale for CD19 as a molecular target in CNS autoimmunity. The current stage of drug development is illustrated. Potential safety concerns will be discussed.
Assuntos
Antígenos CD19/metabolismo , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/imunologia , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/fisiologia , HumanosRESUMO
TLR signaling is critical for early host defense against pathogens, but the contributions of mast cell TLR-mediated mechanisms and subsequent effector functions during pulmonary infection are largely unknown. We have previously demonstrated that mast cells, through the production of IL-4, effectively control Francisella tularensis replication. In this study, the highly human virulent strain of F. tularensis SCHU S4 and the live vaccine strain were used to investigate the contribution of mast cell/TLR regulation of Francisella. Mast cells required TLR2 for effective bacterial killing, regulation of the hydrolytic enzyme cathepsin L, and for coordination and trafficking of MHC class II and lysosomal-associated membrane protein 2. Infected TLR2(-/-) mast cells, in contrast to wild-type and TLR4(-/-) cells, lacked detectable IL-4 and displayed increased cell death with a 2-3 log increase of F. tularensis replication, but could be rescued with rIL-4 treatment. Importantly, MHC class II and lysosomal-associated membrane protein 2 localization with labeled F. tularensis in the lungs was greater in wild-type than in TLR2(-/-) mice. These results provide evidence for the important effector contribution of mast cells and TLR2-mediated signaling on early innate processes in the lung following pulmonary F. tularensis infection and provide additional insight into possible mechanisms by which intracellular pathogens modulate respiratory immune defenses.
Assuntos
Francisella tularensis/crescimento & desenvolvimento , Francisella tularensis/imunologia , Mastócitos/imunologia , Mastócitos/metabolismo , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/deficiência , Receptor 2 Toll-Like/fisiologia , Animais , Morte Celular/genética , Morte Celular/imunologia , Interleucina-4/deficiência , Mastócitos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transporte Proteico/genética , Transporte Proteico/imunologia , Transdução de Sinais/genética , Receptor 4 Toll-Like/fisiologia , Tularemia/imunologia , Tularemia/microbiologia , Tularemia/prevenção & controleRESUMO
A number of heterologous enzymes have been investigated for cancer treatment and other therapeutic applications; however, immunogenicity issues have limited their clinical utility. Here, a new approach has been created for heterologous enzyme deimmunization whereby combinatorial saturation mutagenesis is coupled with a screening strategy that capitalizes on the evolutionary biology concept of neutral drift, and combined with iterative computational prediction of T-cell epitopes to achieve extensive reengineering of a protein sequence for reduced MHC-II binding propensity without affecting catalytic and pharmacological properties. Escherichia coli L-asparaginase II (EcAII), the only nonhuman enzyme approved for repeated administration, is critical in treatment of childhood acute lymphoblastic leukemia (ALL), but elicits adverse antibody responses in a significant fraction of patients. The neutral drift screening of combinatorial saturation mutagenesis libraries at a total of 12 positions was used to isolate an EcAII variant containing eight amino acid substitutions within computationally predicted T-cell epitopes--of which four were nonconservative--while still exhibiting k(cat)/K(M) = 10(6) M(-1) s(-1) for L-Asn hydrolysis. Further, immunization of HLA-transgenic mice expressing the ALL-associated DRB1*0401 allele with the engineered variant resulted in significantly reduced T-cell responses and a 10-fold reduction in anti-EcAII IgG titers relative to the existing therapeutic. This significant reduction in the immunogenicity of EcAII may be clinically relevant for ALL treatment and illustrates the potential of employing neutral drift screens to achieve large jumps in sequence space as may be required for the deimmunization of heterologous proteins.
Assuntos
Asparaginase/imunologia , Epitopos de Linfócito T/imunologia , Proteínas de Escherichia coli/imunologia , Imunização/métodos , Animais , Asparaginase/química , Asparaginase/genética , Domínio Catalítico , Biologia Computacional/métodos , Evolução Molecular Direcionada , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Feminino , Citometria de Fluxo , Deriva Genética , Antígenos HLA-DR/genética , Antígenos HLA-DR/imunologia , Cadeias HLA-DRB1 , Humanos , Interferon gama/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Mutação , Biblioteca de Peptídeos , Estrutura Terciária de Proteína , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
23-Hydroxy ursolic acid (23-OH UA) is a potent atheroprotective and anti-obesogenic phytochemical, with anti-inflammatory and inflammation-resolving properties. In this study, we examined whether dietary 23-OH UA protects mice against the acute onset and progression of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS). Female C57BL/6 mice were fed either a defined low-calorie maintenance diet (MD) or an MD supplemented with 0.2% wgt/wgt 23-OH UA for 5 weeks prior to actively inducing EAE and during the 30 days post-immunization. We observed no difference in the onset of EAE between the groups of mice, but ataxia and EAE disease severity were suppressed by 52% and 48%, respectively, and disease incidence was reduced by over 49% in mice that received 23-OH UA in their diet. Furthermore, disease-associated weight loss was strikingly ameliorated in 23-OH UA-fed mice. ELISPOT analysis showed no significant differences in frequencies of T cells producing IL-17 or IFN-γ between 23-OH UA-fed mice and control mice, suggesting that 23-OH UA does not appear to regulate peripheral T cell responses. In summary, our findings in EAE mice strongly suggest that dietary 23-OH UA may represent an effective oral adjunct therapy for the prevention and treatment of relapsing-remitting MS.
Assuntos
Encefalomielite Autoimune Experimental , Esclerose Múltipla , Feminino , Camundongos , Animais , Encefalomielite Autoimune Experimental/tratamento farmacológico , Esclerose Múltipla/tratamento farmacológico , Ácido Ursólico , Modelos Animais de Doenças , Incidência , Camundongos Endogâmicos C57BL , Suplementos NutricionaisRESUMO
At one time considered a possible form of neuromyelitis optica (NMO) spectrum disorder (NMOSD), it is now accepted that myelin oligodendrocyte glycoprotein (MOG) antibody (Ab)-associated disorder (MOGAD) is a distinct entity from either NMO or multiple sclerosis (MS) and represents a broad spectrum of clinical phenotypes. Whereas Abs targeting aquaporin-4 (AQP4) in NMO are pathogenic, the extent that anti-MOG Abs contribute to CNS damage in MOGAD is unclear. Both AQP4-specific Abs in NMO and MOG-specific Abs in MOGAD are predominantly IgG1, a T cell-dependent immunoglobulin (Ig) subclass. Key insights in neuroimmunology and MOGAD pathogenesis have been learned from MOG experimental autoimmune encephalomyelitis (EAE), described 2 decades before the term MOGAD was introduced. MOG-specific T cells are required in MOG EAE, and while anti-MOG Abs can exacerbate EAE and CNS demyelination, those Abs are neither necessary nor sufficient to cause EAE. Knowledge regarding the spectrum of MOGAD clinical and radiologic presentations is advancing rapidly, yet our grasp of MOGAD pathogenesis is incomplete. Understanding both the humoral and cellular immunology of MOGAD has implications for diagnosis, treatment, and prognosis.
Assuntos
Autoanticorpos , Glicoproteína Mielina-Oligodendrócito , Neuromielite Óptica , Glicoproteína Mielina-Oligodendrócito/imunologia , Humanos , Animais , Autoanticorpos/imunologia , Neuromielite Óptica/imunologia , Autoimunidade/imunologia , Encefalomielite Autoimune Experimental/imunologia , Doenças Autoimunes Desmielinizantes do Sistema Nervoso Central/imunologiaRESUMO
It is known that certain human leukocyte antigen (HLA) genes are associated with autoimmune central nervous system (CNS) diseases, such as multiple sclerosis (MS), but their exact role in disease susceptibility and etiopathogenesis remains unclear. The best studied HLA-associated autoimmune CNS disease is MS, and thus will be the primary focus of this review. Other HLA-associated autoimmune CNS diseases, such as autoimmune encephalitis and neuromyelitis optica will be discussed. The lack of animal models to accurately capture the complex human autoimmune response remains a major challenge. HLA transgenic (tg) mice provide researchers with powerful tools to investigate the underlying mechanisms promoting susceptibility and progression of HLA-associated autoimmune CNS diseases, as well as for elucidating the myelin epitopes potentially targeted by T cells in autoimmune disease patients. We will discuss the potential role(s) of autoimmune disease-associated HLA alleles in autoimmune CNS diseases and highlight information provided by studies using HLA tg mice to investigate the underlying pathological mechanisms and opportunities to use these models for development of novel therapies.
Assuntos
Modelos Animais de Doenças , Antígenos HLA , Camundongos Transgênicos , Animais , Camundongos , Humanos , Antígenos HLA/genética , Antígenos HLA/imunologia , Esclerose Múltipla/imunologia , Esclerose Múltipla/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/genética , Neuromielite Óptica/imunologia , Neuromielite Óptica/genética , Doenças do Sistema Nervoso Central/imunologia , Doenças do Sistema Nervoso Central/genéticaRESUMO
Ab-mediated blockade of the adhesion molecule VLA-4 has been shown to ameliorate disease in human multiple sclerosis patients and experimental autoimmune encephalomyelitis (EAE) animal models. We wanted to determine whether anti-VLA-4 Ab treatment affected the function and persistence of autoreactive T cells in mice with EAE. Unexpectedly, we observed a high level of mortality in anti-VLA-4 mAb (PS/2)-treated mice with actively induced EAE despite decreased disease severity. Investigation of the underlying mechanism showed that injection of PS/2 mAb in combination with pertussis toxin resulted in anaphylaxis and mortality. Furthermore, the data showed that CD4(+) T cells were required for this effect and suggested a role for IL-1ß and TNF-α in the underlying pathology. The results reveal a previously not appreciated deleterious effect of anti-VLA-4 Ab treatment in combination with exposure to pertussis toxin.
Assuntos
Anafilaxia/imunologia , Anafilaxia/mortalidade , Anticorpos Monoclonais/administração & dosagem , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/mortalidade , Integrina alfa4beta1/imunologia , Toxina Pertussis/administração & dosagem , Anafilaxia/genética , Animais , Anticorpos Bloqueadores/administração & dosagem , Combinação de Medicamentos , Encefalomielite Autoimune Experimental/genética , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Proteínas da Mielina , Glicoproteína Associada a Mielina/administração & dosagem , Glicoproteína Associada a Mielina/imunologia , Glicoproteína Mielina-Oligodendrócito , Análise de Sobrevida , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/deficiência , Fator de Necrose Tumoral alfa/genéticaRESUMO
We hypothesized that quantitative MS/MS-based proteomics at multiple time points, incorporating immunoenrichment prior to rapid microwave and magnetic (IM(2) ) sample preparation, might enable correlation of the relative expression of CD47 and other low abundance proteins to disease progression in the experimental autoimmune encephalomyelitis (EAE) animal model of multiple sclerosis. To test our hypothesis, anti-CD47 antibodies were used to enrich for low abundance CD47 prior to microwave and magnetic proteomics in EAE. Decoding protein expression at each time point, with CD47-immunoenriched samples and targeted proteomic analysis, enabled peptides from the low abundance proteins to be precisely quantified throughout disease progression, including: CD47: 86-99, corresponding to the "marker of self" overexpressed by myelin that prevents phagocytosis, or "cellular devouring," by microglia and macrophages; myelin basic protein: 223-228, corresponding to myelin basic protein; and migration inhibitory factor: 79-87, corresponding to a proinflammatory cytokine that inhibits macrophage migration. While validation in a larger cohort is underway, we conclude that IM(2) proteomics is a rapid method to precisely quantify peptides from CD47 and other low abundance proteins throughout disease progression in EAE. This is likely due to improvements in selectivity and sensitivity, necessary to partially overcome masking of low abundance proteins by high abundance proteins and improve dynamic range.
Assuntos
Antígeno CD47/análise , Encefalomielite Autoimune Experimental/metabolismo , Imunoensaio/métodos , Proteoma/análise , Proteômica/métodos , Sequência de Aminoácidos , Análise de Variância , Animais , Química Encefálica , Antígeno CD47/química , Antígeno CD47/metabolismo , Modelos Animais de Doenças , Feminino , Magnetismo , Camundongos , Camundongos Endogâmicos C57BL , Micro-Ondas , Dados de Sequência Molecular , Esclerose Múltipla/metabolismoRESUMO
We hypothesized that quantitative MS/MS-based proteomics at multiple time points, incorporating rapid microwave and magnetic (M(2) ) sample preparation, could enable relative protein expression to be correlated to disease progression in the experimental autoimmune encephalomyelitis (EAE) animal model of multiple sclerosis. To test our hypothesis, microwave-assisted reduction/alkylation/digestion of proteins from brain tissue lysates bound to C8 magnetic beads and microwave-assisted isobaric chemical labeling were performed of released peptides, in 90 s prior to unbiased proteomic analysis. Disease progression in EAE was assessed by scoring clinical EAE disease severity and confirmed by histopathologic evaluation for central nervous system inflammation. Decoding the expression of 283 top-ranked proteins (p <0.05) at each time point relative to their expression at the peak of disease, from a total of 1191 proteins observed in four technical replicates, revealed a strong statistical correlation to EAE disease score, particularly for the following four proteins that closely mirror disease progression: 14-3-3ε (p = 3.4E-6); GPI (p = 2.1E-5); PLP1 (p = 8.0E-4); PRX1 (p = 1.7E-4). These results were confirmed by Western blotting, signaling pathway analysis, and hierarchical clustering of EAE risk groups. While validation in a larger cohort is underway, we conclude that M(2) proteomics is a rapid method to quantify putative prognostic/predictive protein biomarkers and therapeutic targets of disease progression in the EAE animal model of multiple sclerosis.