RESUMO
It is clear that IL-10 plays an essential role in maintaining homeostasis in the gut in response to the microbiome. However, it is unknown whether IL-10 also facilitates immune homeostasis at distal sites. To address this question, we asked whether splenic immune populations were altered in IL-10-deficient (Il10(-/-)) mice in which differences in animal husbandry history were associated with susceptibility to spontaneous enterocolitis that is microbiome dependent. The susceptible mice exhibited a significant increase in splenic macrophages, neutrophils, and marginal zone (MZ) B cells that was inhibited by IL-10 signaling in myeloid, but not B cells. The increase in macrophages was due to increased proliferation that correlated with a subsequent enhancement in MZ B cell differentiation. Cohousing and antibiotic treatment studies suggested that the alteration in immune homeostasis in the spleen was microbiome dependent. The 16S rRNA sequencing revealed that susceptible mice harbored a different microbiome with a significant increase in the abundance of the bacterial genus Helicobacter. The introduction of Helicobacter hepaticus to the gut of nonsusceptible mice was sufficient to drive macrophage expansion and MZ B cell development. Given that myeloid cells and MZ B cells are part of the first line of defense against blood-borne pathogens, their increase following a breach in the gut epithelial barrier would be protective. Thus, IL-10 is an essential gatekeeper that maintains immune homeostasis at distal sites that can become functionally imbalanced upon the introduction of specific pathogenic bacteria to the intestinal track.
Assuntos
Linfócitos B/imunologia , Disbiose/microbiologia , Microbioma Gastrointestinal/genética , Infecções por Helicobacter/imunologia , Helicobacter hepaticus/imunologia , Interleucina-10/genética , Animais , Linfócitos B/citologia , Sequência de Bases , Contagem de Células , Diferenciação Celular/imunologia , Proliferação de Células , DNA Bacteriano/genética , Enterocolite/imunologia , Enterocolite/microbiologia , Infecções por Helicobacter/microbiologia , Interleucina-10/imunologia , Ativação Linfocitária/imunologia , Tecido Linfoide/citologia , Tecido Linfoide/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/imunologia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Transdução de Sinais/imunologiaRESUMO
Oxidative stress is thought to contribute to disease pathogenesis in the central nervous system (CNS) disease multiple sclerosis (MS). Myeloperoxidase (MPO), a potent peroxidase that generates toxic radicals and oxidants, is increased in the CNS during MS. However, the exact mechanism whereby MPO drives MS pathology is not known. We addressed this question by inhibiting MPO in mice with experimental autoimmune encephalomyelitis (EAE) using our non-toxic MPO inhibitor N-acetyl lysyltyrosylcysteine amide (KYC). We found that therapeutic administration of KYC for 5 days starting at the peak of disease significantly attenuated EAE disease severity, reduced myeloid cell numbers and permeability of the blood-brain barrier. These data indicate that inhibition of MPO by KYC restores blood-brain barrier integrity thereby limiting migration of myeloid cells into the CNS that drive EAE pathogenesis. In addition, these observations indicate that KYC may be an effective therapeutic agent for the treatment of MS. We propose that during experimental autoimmune encephalomyelitis (EAE) onset macrophages and neutrophils migrate into the CNS and upon activation release myeloperoxidase (MPO) that promotes disruption of the blood-brain barrier (BBB) and disease progression. KYC restores BBB function by inhibiting MPO activity and in so doing ameliorates disease progression.
RESUMO
UNLABELLED: Gammaherpesviruses are ubiquitous pathogens that are associated with the development of B cell lymphomas. Gammaherpesviruses employ multiple mechanisms to transiently stimulate a broad, polyclonal germinal center reaction, an inherently mutagenic stage of B cell differentiation that is thought to be the primary target of malignant transformation in virus-driven lymphomagenesis. We found that this gammaherpesvirus-driven germinal center expansion was exaggerated and lost its transient nature in the absence of interferon-regulatory factor 1 (IRF-1), a transcription factor with antiviral and tumor suppressor functions. Uncontrolled and persistent expansion of germinal center B cells led to pathological changes in the spleens of chronically infected IRF-1-deficient animals. Additionally, we found decreased IRF-1 expression in cases of human posttransplant lymphoproliferative disorder, a malignant condition associated with gammaherpesvirus infection. The results of our study define an unappreciated role for IRF-1 in B cell biology and provide insight into the potential mechanism of gammaherpesvirus-driven lymphomagenesis. IMPORTANCE: Gammaherpesviruses establish lifelong infection in most adults and are associated with B cell lymphomas. While the infection is asymptomatic in many hosts, it is critical to identify individuals who may be at an increased risk of virus-induced cancer. Such identification is currently impossible, as the host risk factors that predispose individuals toward viral lymphomagenesis are poorly understood. The current study identifies interferon-regulatory factor 1 (IRF-1) to be one of such candidate host factors. Specifically, we found that IRF-1 enforces long-term suppression of an inherently mutagenic stage of B cell differentiation that gammaherpesviruses are thought to target for transformation. Correspondingly, in the absence of IRF-1, chronic gammaherpesvirus infection induced pathological changes in the spleens of infected animals. Further, we found decreased IRF-1 expression in human gammaherpesvirus-induced B cell malignancies.
Assuntos
Linfócitos B/imunologia , Linfócitos B/virologia , Transformação Celular Viral , Gammaherpesvirinae/imunologia , Centro Germinativo/imunologia , Interações Hospedeiro-Patógeno , Fator Regulador 1 de Interferon/metabolismo , Animais , Centro Germinativo/virologia , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias , Baço/imunologia , Baço/patologia , Baço/virologiaRESUMO
Although classically B cells are known to play important roles in immune protection via humoral immunity, recently their regulatory mechanisms have been best appreciated in the context of autoimmunity. Several studies have identified different subsets of regulatory B cells that vary not only in their phenotype but also in their mechanism of action. Although the best-studied mechanism of B-cell immune regulation is IL-10 production, other IL-10-independent mechanisms have been proposed. These include maintenance of CD4(+)Foxp3(+) regulatory T cells; production of transforming growth factor-ß, IL-35, IgM or adenosine or expression of PD-L1 (programmed death 1 ligand 1) or FasL (Fas ligand). Given that B-cell-targeted therapy is being increasingly used in the clinic, a complete understanding of the mechanisms whereby B cells regulate inflammation associated with specific diseases is required for designing safe and effective immunotherapies targeting B cells.
Assuntos
Linfócitos B Reguladores/imunologia , Linhagem da Célula/imunologia , Encefalomielite Autoimune Experimental/imunologia , Doença Enxerto-Hospedeiro/imunologia , Interleucinas/imunologia , Animais , Linfócitos B Reguladores/patologia , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , Proteína Ligante Fas/genética , Proteína Ligante Fas/imunologia , Regulação da Expressão Gênica/imunologia , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/patologia , Humanos , Interleucina-10 , Interleucinas/genética , Camundongos , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/imunologiaRESUMO
R-Ras is a member of the Ras superfamily of small GTPases, which are regulators of various cellular processes, including adhesion, survival, proliferation, trafficking, and cytokine production. R-Ras is expressed by immune cells and has been shown to modulate dendritic cell (DC) function in vitro and has been associated with liver autoimmunity. We used Rras-deficient mice to study the mechanism whereby R-Ras contributes to autoimmunity using experimental autoimmune encephalomyelitis (EAE), a mouse model of the CNS autoimmune disease multiple sclerosis. We found that a lack of R-Ras in peripheral immune cells resulted in attenuated EAE disease. Further investigation revealed that, during EAE, absence of R-Ras promoted the formation of MHC II(low) DC concomitant with a significant increase in proliferation of natural regulatory T cells, resulting in an increase in their cell numbers in the periphery. Our study suggests a novel role for R-Ras in promoting autoimmunity through negative regulation of natural regulatory T cell numbers by inhibiting the development of MHCII(low) DC with tolerogenic potential.
Assuntos
Células Dendríticas/imunologia , Encefalomielite Autoimune Experimental/imunologia , Tolerância Imunológica , Esclerose Múltipla/imunologia , Linfócitos T Reguladores/imunologia , Proteínas ras/imunologia , Animais , Células Dendríticas/patologia , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Camundongos , Camundongos Knockout , Esclerose Múltipla/genética , Esclerose Múltipla/patologia , Linfócitos T Reguladores/patologia , Proteínas ras/genéticaRESUMO
The composition of the microbiome in health and disease has only recently become a major research focus. Although it is clear that an imbalance or dysbiosis in the microbiota is associated with disease, its interrelatedness to disease penetrance is largely unknown. Inflammatory bowel disease (IBD) is an excellent disease in which to explore these questions because of the extensive genetic studies identifying disease susceptibility loci and the ability to easily sample the intestinal microbiota in IBD patients due to the accessibility of stool samples. In addition, mouse models of IBD have contributed to our understanding of the interrelatedness of the gut microbiota and genes associated with IBD. The power of the mouse studies is that multiple colitis models exist that can be used in combination with genetically modified mice that harbour deficiencies in IBD susceptibility genes. Collectively, these studies revealed that bacterial dysbiosis does occur in human IBD and in mouse colitis models. In addition, with an emphasis on immune genes, the mouse studies provided evidence that specific immune regulatory proteins associated with IBD influence the gut microbiota in a manner consistent with disease penetrance. In this review, we will discuss studies in both humans and mice that demonstrate the impact of immunodeficiences in interleukin-10, interleukin-17, nucleotide-binding oligomerization domain (NOD) 2, NOD-like receptor proteins 3 and 6, Toll-like receptor or IgA have on the interrelatedness between the composition of the gut microbiota and disease penetrance of IBD and its mouse models.
Assuntos
Colite/imunologia , Colite/microbiologia , Disbiose/imunologia , Microbioma Gastrointestinal/imunologia , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/microbiologia , Animais , Modelos Animais de Doenças , Disbiose/microbiologia , Humanos , Deficiência de IgA/imunologia , Deficiência de IgA/microbiologia , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/microbiologia , Interleucina-10/deficiência , Interleucina-10/imunologia , Camundongos , Modelos Imunológicos , Proteínas Adaptadoras de Sinalização NOD/deficiência , Proteínas Adaptadoras de Sinalização NOD/imunologia , Penetrância , Células Th17/imunologia , Receptores Toll-Like/imunologiaRESUMO
B cells are important for the regulation of autoimmune responses. In experimental autoimmune encephalomyelitis (EAE), B cells are required for spontaneous recovery in acute models. Production of IL-10 by regulatory B cells has been shown to modulate the severity EAE and other autoimmune diseases. Previously, we suggested that B cells regulated the number of CD4(+)Foxp3(+) T regulatory cells (Treg) in the CNS during EAE. Because Treg suppress autoimmune responses, we asked whether B cells control autoimmunity by maintenance of Treg numbers. B cell deficiency achieved either genetically (µMT) or by depletion with anti-CD20 resulted in a significant reduction in the number of peripheral but not thymic Treg. Adoptive transfer of WT B cells into µMT mice restored both Treg numbers and recovery from EAE. When we investigated the mechanism whereby B cells induce the proliferation of Treg and EAE recovery, we found that glucocorticoid-induced TNF ligand, but not IL-10, expression by B cells was required. Of clinical significance is the finding that anti-CD20 depletion of B cells accelerated spontaneous EAE and colitis. Our results demonstrate that B cells play a major role in immune tolerance required for the prevention of autoimmunity by maintenance of Treg via their expression of glucocorticoid-induced TNFR ligand.
Assuntos
Autoimunidade/imunologia , Linfócitos B/imunologia , Encefalomielite Autoimune Experimental/imunologia , Linfócitos T Reguladores/imunologia , Fatores de Necrose Tumoral/fisiologia , Transferência Adotiva , Animais , Anticorpos Monoclonais Murinos/farmacologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/transplante , Antígenos B7/imunologia , Técnicas de Cocultura , Homeostase , Interleucina-10/fisiologia , Contagem de Linfócitos , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Rituximab , Baço/imunologia , Linfócitos T Reguladores/efeitos dos fármacosRESUMO
The endocannabinoid system has emerged as an important regulator of immune responses, with the cannabinoid receptor 2 (CB2) and its principle ligand 2-archidonoylglycerol playing a major role. How CB2 regulates B cell functions is not clear, even though they express the highest levels of CB2 among immune cell subsets. In this study, we show that CB2-deficient mice have a significant reduction in the absolute number of marginal zone (MZ) B cells and their immediate precursor, transitional-2 MZ precursor. The loss of MZ lineage cells in CB2(-/-) mice was shown to be B cell intrinsic using bone marrow chimeras and was not due to a developmental or functional defect as determined by B cell phenotype, proliferation, and Ig production. Furthermore, CB2(-/-) B cells were similar to wild type in their apoptosis, cell turnover, and BCR and Notch-2 signaling. We then demonstrated that CB2(-/-) MZ lineage B cells were less efficient at homing to the MZ and that their subsequent retention was also regulated by CB2. CB2(-/-) mice immunized with T-independent Ags produced significantly less Ag-specific IgM. This study demonstrates that CB2 positively regulates T-independent immune responses by controlling the localization and positioning of MZ lineage cells to the MZ.
Assuntos
Linfócitos B/imunologia , Linhagem da Célula , Movimento Celular , Quimiotaxia de Leucócito/imunologia , Receptor CB2 de Canabinoide/imunologia , Baço/imunologia , Animais , Linfócitos B/citologia , Separação Celular , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunofluorescência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Baço/citologiaRESUMO
B-cell IgD Low (BDL) B cells have been shown to promote immunological tolerance by inducing proliferation of CD4+Foxp3+ T-regulatory cells (Treg) in a glucocorticoid-induced tumor necrosis factor receptor-related protein ligand (GITRL, Tnfsf18)-dependent manner. BDL cells constitute a small subset of splenic B lymphocytes that, in mice, are characterized by the B220+IgMintCD21intCD23+CD93-IgDlow/- cell surface expression profile. In this chapter, we show the flow cytometry gating strategy developed to identify and purify BDL. In addition, we describe an in vitro assay and two in vivo assays to assess BDL regulatory activity by quantitating Treg expansion/proliferation and indicate how they can be used in mouse models of disease. Collectively, these methods are useful to track and quantitate BDL and Treg numbers and assess their regulatory activity in inflammatory disease models.
Assuntos
Linfócitos B Reguladores/imunologia , Citometria de Fluxo/métodos , Imunoglobulina D/isolamento & purificação , Animais , Linfócitos B Reguladores/metabolismo , Linfócitos T CD4-Positivos/imunologia , Feminino , Fatores de Transcrição Forkhead/metabolismo , Tolerância Imunológica , Imunoglobulina D/imunologia , Imunoglobulina D/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Receptores do Fator de Necrose Tumoral/metabolismo , Baço/citologia , Linfócitos T Reguladores/imunologiaRESUMO
Drug development in oncology commonly exploits the tools of molecular biology to gain therapeutic benefit through reprograming of cellular responses. In immuno-oncology (IO) the aim is to direct the patient's own immune system to fight cancer. After remarkable successes of antibodies targeting PD1/PD-L1 and CTLA4 receptors in targeted patient populations, the focus of further development has shifted toward combination therapies. However, the current drug-development approach of exploiting a vast number of possible combination targets and dosing regimens has proven to be challenging and is arguably inefficient. In particular, the unprecedented number of clinical trials testing different combinations may no longer be sustainable by the population of available patients. Further development in IO requires a step change in selection and validation of candidate therapies to decrease development attrition rate and limit the number of clinical trials. Quantitative systems pharmacology (QSP) proposes to tackle this challenge through mechanistic modeling and simulation. Compounds' pharmacokinetics, target binding, and mechanisms of action as well as existing knowledge on the underlying tumor and immune system biology are described by quantitative, dynamic models aiming to predict clinical results for novel combinations. Here, we review the current QSP approaches, the legacy of mathematical models available to quantitative clinical pharmacologists describing interaction between tumor and immune system, and the recent development of IO QSP platform models. We argue that QSP and virtual patients can be integrated as a new tool in existing IO drug development approaches to increase the efficiency and effectiveness of the search for novel combination therapies.
Assuntos
Alergia e Imunologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Desenvolvimento de Medicamentos , Inibidores de Checkpoint Imunológico/uso terapêutico , Oncologia , Simulação de Dinâmica Molecular , Neoplasias/tratamento farmacológico , Biologia de Sistemas , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Simulação por Computador , Humanos , Inibidores de Checkpoint Imunológico/efeitos adversos , Inibidores de Checkpoint Imunológico/farmacocinética , Modelos Imunológicos , Terapia de Alvo Molecular , Neoplasias/imunologia , Neoplasias/metabolismo , Microambiente TumoralRESUMO
A number of different B cell subsets have been shown to exhibit regulatory activity using a variety of mechanisms to attenuate inflammatory diseases. Here we show, using anti-CD20-mediated partial B cell depletion in mice, that a population of mature B cells distinguishable by IgDlow/- expression maintains tolerance by, at least in part, promoting CD4+Foxp3+ regulatory T cell homeostatic expansion via glucocorticoid-induced tumor necrosis factor receptor ligand, or GITRL. Cell surface phenotyping, transcriptome analysis and developmental study data show that B cells expressing IgD at a low level (BDL) are a novel population of mature B cells that emerge in the spleen from the transitional-2 stage paralleling the differentiation of follicular B cells. The cell surface phenotype and regulatory function of BDL are highly suggestive that they are a new B cell subset. Human splenic and peripheral blood IgDlow/- B cells also exhibit BDL regulatory activity, rendering them of therapeutic interest.
Assuntos
Subpopulações de Linfócitos B/imunologia , Dermatite de Contato/imunologia , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Tolerância Imunológica , Linfócitos T Reguladores/imunologia , Animais , Subpopulações de Linfócitos B/metabolismo , Separação Celular/métodos , Células Cultivadas , Técnicas de Cocultura , Modelos Animais de Doenças , Citometria de Fluxo/métodos , Perfilação da Expressão Gênica , Voluntários Saudáveis , Humanos , Imunoglobulina D/metabolismo , Leucócitos Mononucleares , Camundongos , Camundongos Endogâmicos C57BL , Oxazolona/imunologia , Baço/citologia , Baço/crescimento & desenvolvimento , Baço/imunologia , Linfócitos T Reguladores/metabolismo , Fatores de Necrose Tumoral/imunologia , Fatores de Necrose Tumoral/metabolismoRESUMO
In the past two decades it has become clear that in addition to antigen presentation and antibody production B cells play prominent roles in immune regulation. While B cell-derived IL-10 has garnered much attention, B cells also effectively regulate inflammation by a variety of IL-10-independent mechanisms. B cell regulation has been studied in both autoimmune and inflammatory diseases. While collectively called regulatory B cells (Breg), no definitive phenotype has emerged for B cells with regulatory potential. This has made their study challenging and thus unique B cell regulatory mechanisms have emerged in a disease-dependent manner. Thus to harness the therapeutic potential of Breg, further studies are needed to understand how they emerge and are induced to evoke their regulatory activities.
RESUMO
Porin of Shigella dysenteriae type 1 increased the mRNA levels for Toll-like receptor (TLR) 2 and TLR6 by 1.5- and 2.9-fold respectively, of peritoneal cavity B-1a and B-1b cells, implicating that coexpression of TLR2 and TLR6 is essential as a combinatorial repertoire for recognition of porin by the B-1 cells. Among the two key TLRs, TLR2 and TLR4, which are primarily responsible for recognizing majority of the bacterial products, TLR2 and not TLR4, participates in porin recognition. TLR2 got increased on both the B-1 cell populations whereas the TLR4 expression remained unaffected. Besides TLRs, mRNA for MyD88, an effector molecule associated with TLR-mediated response was enhanced by 1.8-fold that suggests of its involvement in the activity of porin. Both of the B-1 cell populations expressed strongly the mRNA for NF-kappaB in the presence of porin, that was 2.4-fold more than untreated control, conforming to the earlier finding that coexpression of TLR2 and TLR6, resulted in robust NF-kappaB activation for signaling. Porin treatment of B-1 cell populations of C57BL/6 mice, and C3H/HeJ mice in particular, selectively up-regulated the expression of the costimulatory molecules. CD80 expression got enhanced on the B-1a cells whereas CD86 got solely expressed on B-1b cells. Porin-induced cell surface expression of IgM and IgA on B-1 cell populations from C57BL/6 mice. The IgA-generating capacity, hallmark of mucosal immune response, was confirmed with B-1 cells of C3H/HeJ, the lipopolysaccharide non-responder mouse, in response to the protein. The porin-mediated induction of IgA was augmented by interleukin-6 on B-1a and B-1b cells, by 2.4- and 2.6-fold, respectively. The IgA expressed on both B-1a and B-1b cell surfaces after 72 h of culture was found to bind to the 38 kDa monomer of porin confirming it to be anti-porin IgA antibody.
Assuntos
Antígenos CD/biossíntese , Antígeno B7-1/biossíntese , Imunoglobulina A/biossíntese , Glicoproteínas de Membrana/biossíntese , Cavidade Peritoneal/citologia , Porinas/farmacologia , Receptores de Superfície Celular/fisiologia , Animais , Reações Antígeno-Anticorpo , Antígeno B7-2 , Imunoglobulina A/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos , NF-kappa B/genética , NF-kappa B/metabolismo , Porinas/imunologia , RNA Mensageiro/biossíntese , Receptores de Superfície Celular/genética , Shigella dysenteriae/química , Shigella dysenteriae/imunologia , Receptor 2 Toll-Like , Receptor 6 Toll-Like , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologiaRESUMO
The signaling lymphocyte activation molecule (SLAM) family plays important roles in adaptive immune responses. Herein, we evaluated whether the SLAM family member 2B4 (CD244) plays a role in immune cell development, homeostasis and antibody responses. We found that the splenic cellularity in Cd244-/- mice was significantly reduced due to a reduction in both CD4 T cells and follicular (Fo) B cells; whereas, the number of peritoneal cavity B cells was increased. These findings led us to examine whether 2B4 modulates B cell immune responses. When we examined T-dependent B cell responses, while there was no difference in the kinetics or magnitude of the antigen-specific IgM and IgG1 antibody response there was a reduction in bone marrow (BM) memory, but not plasma cells in Cd244-/- mice. When we evaluated T-independent immune responses, we found that antigen-specific IgM and IgG3 were elevated in the serum following immunization. These data indicate that 2B4 dampens T-independent B cell responses due to a reduction in peritoneal cavity B cells, but has minimal impact on T-dependent B cell responses.
Assuntos
Formação de Anticorpos/imunologia , Antígenos CD/imunologia , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Cavidade Peritoneal/fisiologia , Receptores Imunológicos/imunologia , Animais , Medula Óssea/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Família de Moléculas de Sinalização da Ativação LinfocitáriaRESUMO
The monomeric and oligomeric forms of Vibrio cholerae hemolysin (HlyA), a membrane damaging toxin that forms transmembrane pentameric diffusion channels in target eukaryotic membrane, show a pronounced difference in protease susceptibility, presumably due to masking of sensitive peptide bonds during oligomerization. In this work, we examined if resistance of a protein to proteolytic processing affects the expression of costimulatory molecules, CD80 and CD86, on macrophage exposed to the same antigen. The murine peritoneal cavity macrophages expressed both CD80 and CD86 after 24 h of incubation with HlyA monomer but failed to express the costimulatory molecules when treated with the HlyA oligomer. The expression of CD80 molecule on macrophage after 48 h by the HlyA oligomer that failed to express the costimulatory molecules after 24 h indicates that proteolytic processing plays a decisive role in expression of CD80 and CD86 on cell surface.
Assuntos
Antígenos CD/imunologia , Antígeno B7-1/imunologia , Proteínas Hemolisinas/imunologia , Macrófagos/imunologia , Glicoproteínas de Membrana/imunologia , Vibrio cholerae/imunologia , Animais , Antígeno B7-2 , Proteínas de Bactérias , Proteínas Hemolisinas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Pronase/metabolismoRESUMO
Sera of patients convalescing from shigellosis reacted strongly and specifically with the 38,000 Da monomer of porin of Shigella dysenteriae type 1. Since human, the only natural host of S. dysenteriae type 1, recognized the protein through humoral immune response, it is of great significance to study the surface-exposed outer membrane antigen as an adjuvant. Porin treatment of CD11b+ peritoneal cavity (PerC) MPhi of BALB/c mouse was found to up-regulate CD80 on cell surface and had no effect on CD86 expression. The surface expression of CD80 got increased by 1.6-fold in the presence of gamma interferon (IFN-gamma) supporting selective regulation of the B7-1 (CD80) member of the B7 family. MPhi released 7.25 pg of interleukin-12 (IL-12) in the presence of porin. The protein in combination with IFN-gamma augmented profoundly the release of IL-12 by 2.6-fold. Porin-mediated induction of IL-12 release would therefore influence Th1-type response, known to be preferentially triggered due to up-regulation of CD80 expression. Treatment of PerC MPhi by the protein showed an increase of mRNA for both Toll-like receptor (TLR)2 and myeloid differentiation factor 88 (MyD88) by 2- and 2.3-fold respectively, emphasizing that TLR2 is essential for recognition of S. dysenteriae type 1 porin. Understanding the mechanism of adjuvanticity of porin of S. dysenteriae type 1 is a necessary step towards the development of a better adjuvant against shigellosis.
Assuntos
Antígenos de Diferenciação/metabolismo , Antígeno B7-1/biossíntese , Disenteria Bacilar/metabolismo , Interleucina-12/metabolismo , Glicoproteínas de Membrana/metabolismo , Porinas/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Imunológicos/metabolismo , Shigella dysenteriae/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Adjuvantes Imunológicos , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Diferenciação/genética , Western Blotting , Disenteria Bacilar/imunologia , Feminino , Humanos , Macrófagos Peritoneais , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Fator 88 de Diferenciação Mieloide , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Superfície Celular/genética , Receptores Imunológicos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Shigella dysenteriae/genética , Shigella dysenteriae/imunologia , Receptor 2 Toll-Like , Receptores Toll-Like , Regulação para CimaRESUMO
B cells are thought to play a pathogenic role in multiple sclerosis (MS), an autoimmune disease affecting the central nervous system (CNS). This idea is supported by the reduction of disease in MS patients undergoing antibody-mediated B cell depletion therapy. In contrast, in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, B cells have been shown to play a regulatory role. This is suggestive of a dual role for B cells in CNS autoimmunity. It is possible that a critical balance between the pathogenic and regulatory populations of B cells might be involved in the manifestation of the disease. Although in mice, different B cell subsets have been shown to exert immunoregulation through varied mechanisms, the phenotype of regulatory B cells in humans and factors affecting their function are not well known. Also, the origin and development of regulatory B cells is not known. It is important to thoroughly identify the different populations of B cells that might be involved in suppressing CNS autoimmunity, their mode of function and factors that regulate their immunosuppressive properties for using regulatory B cells as a therapy for MS. Here we present methods to study the phenotype and mechanisms of immune suppression by B cells in different mouse models of EAE.
Assuntos
Linfócitos B Reguladores/imunologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/imunologia , Transferência Adotiva , Sequência de Aminoácidos , Animais , Linfócitos B Reguladores/citologia , Linfócitos B Reguladores/patologia , Separação Celular/métodos , Sistema Nervoso Central/citologia , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/patologia , Técnicas de Cocultura/métodos , Citocinas/análise , Citocinas/imunologia , Encefalomielite Autoimune Experimental/patologia , Sistema Imunitário/citologia , Imunização , Imunofenotipagem/métodos , Camundongos , Dados de Sequência Molecular , Peptídeos/administração & dosagem , Peptídeos/química , Peptídeos/imunologia , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
The CD45 congenic marker system is a highly utilized technique to track hematopoietic cells following bone marrow transplantation (BMT), with CD45.1 and CD45.2 being efficiently distinguished by flow cytometry. During the analysis of control mixed BM chimera mice in which lethally irradiated recipients were transplanted with an equal number of BM cells from WT CD45.1 and WT CD45.2 mice, we observed an unequal reconstitution of specific B cell subsets in the bone marrow (BM), lymph node (LN) and spleen. Specifically, in the BM and LN, there was an increase in the percentage of CD45.2 mature B cells. In the spleen, an increase in the percentage of CD45.2 transitional (T) 1 and T2 cells was observed. In contrast, the percentage of splenic CD45.1 marginal zone (MZ) B cells was significantly increased. When we compared the percentage of B cell subsets in unmanipulated WT CD45.1 and WT CD45.2 mice, we found that WT CD45.2 mice had significantly more LN B cells while WT CD45.1 mice exhibited an increase in MZ B cells. These data indicate that the alteration in the ratio of CD45.1 and CD45.2 B cell subsets in mixed chimera mice is a cell-intrinsic effect. Thus whenever the CD45 congenic system is used to track two genetically distinct populations of immune cells WT chimeras must be generated to allow normalization of the experimental data to avoid the reporting of unintentionally skewed data.
Assuntos
Subpopulações de Linfócitos B/citologia , Células da Medula Óssea/citologia , Antígenos Comuns de Leucócito/genética , Alelos , Animais , Medula Óssea , Transplante de Medula Óssea , Quimera , Antígenos Comuns de Leucócito/biossíntese , CamundongosRESUMO
The cannabinoid receptor 2 (CB2) has been reported to modulate B cell functions including migration, proliferation and isotype class switching. Since these processes are required for the generation of the germinal center (GC) and antigen-specific plasma and memory cells following immunization with a T-dependent antigen, CB2 has the capacity to alter the quality and magnitude of T-dependent immune responses. To address this question, we immunized WT and CB2(-/-) mice with the T-dependent antigen 4-hydroxy-3-nitrophenylacetyl (NP)-chicken-gamma-globulin (CGG) and measured GC B cell formation and the generation of antigen-specific B cells and serum immunoglobulin (Ig). While there was a significant reduction in the number of splenic GC B cells in CB2(-/-) mice early in the response there was no detectable difference in the number of NP-specific IgM and IgG1 plasma cells. There was also no difference in NP-specific IgM and class switched IgG1 in the serum. In addition, we found no defect in the homing of plasma cells to the bone marrow (BM) and affinity maturation, although memory B cell cells in the spleen were reduced in CB2(-/-) mice. CB2-deficient mice also generated similar levels of antigen-specific IgM and IgG in the serum as WT following immunization with sheep red blood cells (sRBC). This study demonstrates that although CB2 plays a role in promoting GC and memory B cell formation/maintenance in the spleen, it is dispensable on all immune cell types required for the generation of antigen-specific IgM and IgG in T-dependent immune responses.
Assuntos
Antígenos/imunologia , Linfócitos B/imunologia , Centro Germinativo/imunologia , Imunoglobulina G/metabolismo , Imunoglobulina M/metabolismo , Memória Imunológica/imunologia , Receptor CB2 de Canabinoide/fisiologia , Animais , Linfócitos B/metabolismo , Diferenciação Celular , Proliferação de Células , Centro Germinativo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , OvinosRESUMO
MS (multiple sclerosis) is the most prevalent autoimmune disease of the CNS (central nervous system) historically characterized as an inflammatory and demyelinating disease. More recently, extensive neuronal pathology has lead to its classification as a neurodegenerative disease as well. While the immune system initiates the autoimmune response it remains unclear how it orchestrates neuronal damage. In our previous studies, using in vitro cultured embryonic neurons, we demonstrated that MBP (myelin basic protein)-specific encephalitogenic CD4 T-cells induce early neuronal damage. In an extension of those studies, here we show that polarized CD4 Th1 and Th17 cells as wells as CD8 T-cells and NK (natural killer) cells induce microtubule destabilization within neurites in a contact-independent manner. Owing to the cytotoxic potential of these immune cells, we isolated the luminal components of lytic granules and determined that they were sufficient to drive microtubule destabilization. Since lytic granules contain cytolytic proteins, we determined that the induction of microtubule destabilization occurred prior to signs of apoptosis. Furthermore, we determined that microtubule destabilization was largely restricted to axons, sparing dendrites. This study demonstrated that lymphocytes with cytolytic activity have the capacity to directly drive MAD (microtubule axonal destabilization) in a bystander manner that is independent of neuronal death.