RESUMO
BACKGROUND: Psoriasis is an inflammatory, IL-17-driven skin disease in which autoantigen-induced CD8+ T cells have been identified as pathogenic drivers. OBJECTIVE: Our study focused on comprehensively characterizing the phenotypic variation of CD8+ T cells in psoriatic lesions. METHODS: We used single-cell RNA sequencing to compare CD8+ T-cell transcriptomic heterogeneity between psoriatic and healthy skin. RESULTS: We identified 11 transcriptionally diverse CD8+ T-cell subsets in psoriatic and healthy skin. Among several inflammatory subsets enriched in psoriatic skin, we observed 2 Tc17 cell subsets that were metabolically divergent, were developmentally related, and expressed CXCL13, which we found to be a biomarker of psoriasis severity and which achieved comparable or greater accuracy than IL17A in a support vector machine classifier of psoriasis and healthy transcriptomes. Despite high coinhibitory receptor expression in the Tc17 cell clusters, a comparison of these cells with melanoma-infiltrating CD8+ T cells revealed upregulated cytokine, cytolytic, and metabolic transcriptional activity in the psoriatic cells that differed from an exhaustion program. CONCLUSION: Using high-resolution single-cell profiling in tissue, we have uncovered the diverse landscape of CD8+ T cells in psoriatic and healthy skin, including 2 nonexhausted Tc17 cell subsets associated with disease severity.
Assuntos
Autoimunidade , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Psoríase/etiologia , Psoríase/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Estudos de Casos e Controles , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Memória Imunológica , Imunofenotipagem , Interleucina-17/biossíntese , Neoplasias/genética , Neoplasias/imunologia , Análise de Célula ÚnicaRESUMO
Cathepsin S (CatS) is a cysteine protease found in lysosomes of hematopoietic and microglial cells and in secreted form in the extracellular space. While CatS has been shown to contribute significantly to neuropathic pain, the precise mechanisms remain unclear. In this report, we describe JNJ-39641160, a novel non-covalent, potent, selective and orally-available CatS inhibitor that is peripherally restricted (non-CNS penetrant) and may represent an innovative class of immunosuppressive and analgesic compounds and tools useful toward investigating peripheral mechanisms of CatS in neuropathic pain. In C57BL/6 mice, JNJ-39641160 dose-dependently blocked the proteolysis of the invariant chain, and inhibited both T-cell activation and antibody production to a vaccine antigen. In the spared nerve injury (SNI) model of chronic neuropathic pain, in which T-cell activation has previously been demonstrated to be a prerequisite for the development of pain hypersensitivity, JNJ-39641160 fully reversed tactile allodynia in wild-type mice but was completely ineffective in the same model in CatS knockout mice (which exhibited a delayed onset in allodynia). By contrast, in the acute mild thermal injury (MTI) model, JNJ-39641160 only weakly attenuated allodynia at the highest dose tested. These findings support the hypothesis that blockade of peripheral CatS alone is sufficient to fully reverse allodynia following peripheral nerve injury and suggest that the mechanism of action likely involves interruption of T-cell activation and peripheral cytokine release. In addition, they provide important insights toward the development of selective CatS inhibitors for the treatment of neuropathic pain in humans.
Assuntos
Analgésicos/uso terapêutico , Catepsinas/antagonistas & inibidores , Hiperalgesia/tratamento farmacológico , Imunossupressores/uso terapêutico , Neuralgia/tratamento farmacológico , Traumatismos dos Nervos Periféricos/tratamento farmacológico , Inibidores de Proteases/uso terapêutico , Analgésicos/farmacocinética , Analgésicos/farmacologia , Animais , Encéfalo/metabolismo , Catepsinas/genética , Catepsinas/metabolismo , Linhagem Celular , Citocinas/imunologia , Temperatura Alta , Humanos , Hiperalgesia/imunologia , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Imunossupressores/farmacocinética , Imunossupressores/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuralgia/imunologia , Traumatismos dos Nervos Periféricos/imunologia , Inibidores de Proteases/farmacocinética , Inibidores de Proteases/farmacologia , Nervo Isquiático/lesões , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Toxoide Tetânico/administração & dosagem , TatoRESUMO
Phosphoinositide 3-kinases, delta (PI3Kδ) and gamma (PI3Kγ) are enriched in immune cells and regulate the development and function of innate and adaptive immunity. Dual PI3Kδγ inhibitors are considered high value targets for their potential to treat a variety of immune-mediated diseases, but their discovery has been challenging. Here we describe the preclinical pharmacology of HM5023507, an orally active dual inhibitor of δγ isoforms in immune signaling. HM5023507 inhibited PI3Kδ and PI3Kγ isoforms with greater than 100-fold selectivity against PI3Kα and PI3Kß in recombinant enzymatic assays and in primary human immune cells with an exquisite selectivity against other targets. HM5023507 attenuated the PI3Kδ/γ signaling in human basophils (IC50: 42/340 nmol/L; selectivity ratio ~1:8). HM5023507 attenuated the activation and function of human B and T cells, Th17 differentiation of CD4 T cells in the blood of healthy donors and rheumatoid arthritis patients, and cytokine and IgG production in human T and B cell cocultures, in vitro. Orally dosed HM5023507 attenuated PI3K δ/γ-mediated immune signaling in the rat in a dose-related manner. In addition, HM5023507 inhibited semiestablished collagen-induced arthritic inflammation in the rats (ED50 of 0.25mg/kg, p.o. BID or 0.5 mg/kg, QD, AUC: 1422 ng/mL*h), improved histopathology- and micro-computed tomography (µCT)-based indices of joint damage, bone destruction, and attenuated the levels of anti-collagen antibody, with an overall anti-inflammatory profile matching that of a TNFα neutralizing antibody. The PI3K δγ inhibitory profile of HM5023507 and its selectivity make it a useful tool to further delineate immunobiology of dual PI3K δγ targeting.
Assuntos
Linfócitos B/citologia , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Inibidores Enzimáticos/administração & dosagem , Linfócitos T/citologia , Administração Oral , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Cultura Primária de Células , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
Rheumatoid arthritis (RA) is an autoimmune disease that primarily affects the synovial joints and can lead to bone erosion and cartilage damage. One hallmark of RA is anticitrullinated protein autoantibodies (ACPA) and memory citrulline-specific B-cells, which have been implicated in RA pathogenesis. While depletion of B-cells with Rituximab improves clinical responses in RA patients, this treatment strategy leaves patients susceptible to infections. Here we use of Siglec-engaging Tolerance-inducing Antigenic Liposomes (STALs) to selectively target the citrulline-specific B-cells. ACPA production from purified human RA patients' B-cells in vitro was achieved through a set of stimulation conditions, which includes the following: BAFF, anti-CD40, IL-21, and LPS. In vivo generation of citrulline specific B-cells and ACPA production was accomplished by antigenic liposomes consisting of monophosphoryl lipid A (MPLA) and a cyclic citrullinated peptide (CCP) administered to SJL/J mice. We show that STALs that codisplay a high affinity CD22 glycan ligand and synthetic citrullinated antigen (CCP STALs) can prevent ACPA production from RA patients' memory B-cells in vitro. These CCP STALs were also effective in inducing tolerance to citrullinated antigens in SJL/J mice. The results demonstrate that tolerization of the B-cells responsible for ACPA can be achieved by exploiting the inhibitory receptor CD22 with high-affinity glycan ligands. Such a treatment strategy could be beneficial in the treatment of RA.
Assuntos
Artrite Reumatoide/metabolismo , Autoanticorpos/imunologia , Linfócitos B/imunologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Animais , Estudos de Casos e Controles , Citrulina/metabolismo , Humanos , Memória Imunológica , CamundongosRESUMO
The follicular helper T cell (TFH) are established regulators of germinal center (GC) B cells, whether TFH have pathogenic potential independent of B cells is unknown. Based on in vitro TFH cell differentiation, in vivo T cell transfer animal colitis model, and intestinal tissues of inflammatory bowel disease (IBD) patients, TFH and its functions in colitis development were analyzed by FACS, ChIP, ChIP-sequencing, WB, ELISA and PCR. Herein we demonstrate that intestinal tissues of patients and colon tissues obtained from Rag1-/- recipients of naïve CD4+ T cells with colitis, each over-express TFH-associated gene products. Adoptive transfer of naïve Bcl6-/- CD4+ T cells into Rag1-/- recipient mice abrogated development of colitis and limited TFH differentiation in vivo, demonstrating a mechanistic link. In contrast, T cell deficiency of interferon regulatory factor 8 (IRF8) resulted in augmentation of TFH induction in vitro and in vivo. Functional studies showed that adoptive transfer of IRF8 deficient CD4+ T cells into Rag1-/- recipients exacerbated colitis development associated with increased gut TFH-related gene expression, while Irf8-/-/Bcl6-/- CD4+ T cells abrogated colitis, together indicating that IRF8-regulated TFH can directly cause colon inflammation. Molecular analyses revealed that IRF8 suppresses TFH differentiation by inhibiting transcription and transactivation of the TF IRF4, which is also known to be essential for TFH induction. Our documentation showed that IRF8-regulated TFH can function as B-cell-independent, pathogenic, mediators of colitis suggests that targeting TFH could be effective for treatment of IBD.
Assuntos
Linfócitos B/imunologia , Colite/imunologia , Colo/metabolismo , Doença de Crohn/imunologia , Centro Germinativo/imunologia , Fatores Reguladores de Interferon/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Transferência Adotiva , Animais , Células Cultivadas , Colite/genética , Colo/patologia , Doença de Crohn/genética , Modelos Animais de Doenças , Humanos , Fatores Reguladores de Interferon/genética , Ativação Linfocitária , Camundongos , Camundongos Knockout , Comunicação Parácrina , Proteínas Proto-Oncogênicas c-bcl-6/genética , Linfócitos T Auxiliares-Indutores/transplanteRESUMO
CD22, a sialic acid-binding Ig-type lectin (Siglec) family member, is an inhibitory coreceptor of the BCR with established roles in health and disease. The restricted expression pattern of CD22 on B cells and most B cell lymphomas has made CD22 a therapeutic target for B cell-mediated diseases. Models to better understand how in vivo targeting of CD22 translates to human disease are needed. In this article, we report the development of a transgenic mouse expressing human CD22 (hCD22) in B cells and assess its ability to functionally substitute for murine CD22 (mCD22) for regulation of BCR signaling, Ab responses, homing, and tolerance. Expression of hCD22 on transgenic murine B cells is comparable to expression on human primary B cells, and it colocalizes with mCD22 on the cell surface. Murine B cells expressing only hCD22 have identical calcium (Ca2+) flux responses to anti-IgM as mCD22-expressing wild-type B cells. Furthermore, hCD22 transgenic mice on an mCD22-/- background have restored levels of marginal zone B cells and Ab responses compared with deficiencies observed in CD22-/- mice. Consistent with these observations, hCD22 transgenic mice develop normal humoral responses in a peanut allergy oral sensitization model. Homing of B cells to Peyer's patches was partially rescued by expression of hCD22 compared with CD22-/- B cells, although not to wild-type levels. Notably, Siglec-engaging antigenic liposomes formulated with an hCD22 ligand were shown to prevent B cell activation, increase cell death, and induce tolerance in vivo. This hCD22 transgenic mouse will be a valuable model for investigating the function of hCD22 and preclinical studies targeting hCD22.
Assuntos
Linfócitos B/imunologia , Hipersensibilidade a Amendoim/imunologia , Nódulos Linfáticos Agregados/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Transdução de Sinais/imunologia , Animais , Linfócitos B/patologia , Modelos Animais de Doenças , Humanos , Ativação Linfocitária/genética , Camundongos , Camundongos Transgênicos , Hipersensibilidade a Amendoim/genética , Hipersensibilidade a Amendoim/patologia , Nódulos Linfáticos Agregados/patologia , Receptores de Antígenos de Linfócitos B/genética , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Transdução de Sinais/genéticaRESUMO
RORγt and RORα are transcription factors of the RAR-related orphan nuclear receptor (ROR) family. They are expressed in Th17 cells and have been suggested to play a role in Th17 differentiation. Although RORγt signature genes have been characterized in mouse Th17 cells, detailed information on its transcriptional control in human Th17 cells is limited and even less is known about RORα signature genes which have not been reported in either human or mouse T cells. In this study, global gene expression of human CD4 T cells activated under Th17 skewing conditions was profiled by RNA sequencing. RORγt and RORα signature genes were identified in these Th17 cells treated with specific siRNAs to knock down RORγt or RORα expression. We have generated selective small molecule RORγt modulators and they were also utilized as pharmacological tools in RORγt signature gene identification. Our results showed that RORγt controlled the expression of a very selective number of genes in Th17 cells and most of them were regulated by RORα as well albeit a weaker influence. Key Th17 genes including IL-17A, IL-17F, IL-23R, CCL20 and CCR6 were shown to be regulated by both RORγt and RORα. Our results demonstrated an overlapping role of RORγt and RORα in human Th17 cell differentiation through regulation of a defined common set of Th17 genes. RORγt as a drug target for treatment of Th17 mediated autoimmune diseases such as psoriasis has been demonstrated recently in clinical trials. Our results suggest that RORα could be involved in same disease mechanisms and gene signatures identified in this report could be valuable biomarkers for tracking the pharmacodynamic effects of compounds that modulate RORγt or RORα activities in patients.
Assuntos
Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Células Th17/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genes Reporter , Humanos , Concentração Inibidora 50 , Leucócitos Mononucleares/citologia , Ativação Linfocitária , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/metabolismo , Células Th1/citologiaRESUMO
Kv1.3 is a voltage-gated potassium channel expressed on T cells that plays an important role in T cell activation. Previous studies have shown that blocking Kv1.3 channels in human T cells during activation results in reduced calcium entry, cytokine production, and proliferation. The aim of the present study was to further explore the effects of Kv1.3 blockers on the response of different human T cell subsets under various stimulation conditions. Our studies show that, unlike the immune suppressor cyclosporine A, the inhibitory effect of Kv1.3 blockers was partial and stimulation strength dependent, with reduced inhibitory efficacy on T cells under strengthened anti-CD3/CD28 stimulations. T cell responses to allergens including house dust mites and ragweed were partially reduced by Kv1.3 blockers. The effect of Kv1.3 inhibition was dependent on T cell subsets, with stronger effects on CCR7- effector memory compared to CCR7+ central memory CD4 T cells. Calcium entry studies also revealed a population of CD4 T cells resistant to Kv1.3 blockade. Activation of CD4 T cells was accompanied with an increase in Kv1.3 currents but Kv1.3 transcripts were found to be reduced, suggesting a posttranscriptional mechanism in the regulation of Kv1.3 activities. In summary, Kv1.3 blockers inhibit T cell activation in a manner that is highly dependent on the T cell identity and stimulation strength, These findings suggest that Kv1.3 blockers inhibit T cells in a unique, conditional manner, further refining our understanding of the therapeutic potential of Kv1.3 blockers.
Assuntos
Canal de Potássio Kv1.3/antagonistas & inibidores , Ativação Linfocitária , Bloqueadores dos Canais de Potássio/farmacologia , Subpopulações de Linfócitos T , Linfócitos T/imunologia , Perfilação da Expressão Gênica , Humanos , Canal de Potássio Kv1.3/genética , Canal de Potássio Kv1.3/metabolismo , Técnicas de Patch-ClampRESUMO
The IL-23/IL-17 pathway is implicated in autoimmune diseases, particularly psoriasis, where biologics targeting IL-23 and IL-17 have shown significant clinical efficacy. Retinoid-related orphan nuclear receptor gamma t (RORγt) is required for Th17 differentiation and IL-17 production in adaptive and innate immune cells. We identified JNJ-54271074, a potent and highly-selective RORγt inverse agonist, which dose-dependently inhibited RORγt-driven transcription, decreased co-activator binding and promoted interaction with co-repressor protein. This compound selectively blocked Th17 differentiation, significantly reduced IL-17A production from memory T cells, and decreased IL-17A- and IL-22-producing human and murine γδ and NKT cells. In a murine collagen-induced arthritis model, JNJ-54271074 dose-dependently suppressed joint inflammation. Furthermore, JNJ-54271074 suppressed IL-17A production in human PBMC from rheumatoid arthritis patients. RORγt-deficient mice showed decreased IL-23-induced psoriasis-like skin inflammation and cytokine gene expression, consistent with dose-dependent inhibition in wild-type mice through oral dosing of JNJ-54271074. In a translational model of human psoriatic epidermal cells and skin-homing T cells, JNJ-54271074 selectively inhibited streptococcus extract-induced IL-17A and IL-17F. JNJ-54271074 is thus a potent, selective RORγt modulator with therapeutic potential in IL-23/IL-17 mediated autoimmune diseases.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Peptídeos Cíclicos/administração & dosagem , Psoríase/tratamento farmacológico , Células Th17/efeitos dos fármacos , Administração Oral , Animais , Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-17/metabolismo , Interleucinas/metabolismo , Camundongos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Peptídeos Cíclicos/farmacologia , Psoríase/genética , Psoríase/metabolismo , Células Th17/citologia , Células Th17/metabolismo , Transcrição Gênica , Interleucina 22RESUMO
The RAR-related orphan receptor gamma t (RORγt) is a nuclear receptor required for generating IL-17-producing CD4(+) Th17 T cells, which are essential in host defense and may play key pathogenic roles in autoimmune diseases. Oxysterols elicit profound effects on immune and inflammatory responses as well as on cholesterol and lipid metabolism. Here, we describe the identification of several naturally occurring oxysterols as RORγt agonists. The most potent and selective activator for RORγt is 7ß, 27-dihydroxycholesterol (7ß, 27-OHC). We show that these oxysterols reverse the inhibitory effect of an RORγt antagonist, ursolic acid, in RORγ- or RORγt-dependent cell-based reporter assays. These ligands bind directly to recombinant RORγ ligand binding domain (LBD), promote recruitment of a coactivator peptide, and reduce binding of a corepressor peptide to RORγ LBD. In primary cells, 7ß, 27-OHC and 7α, 27-OHC enhance the differentiation of murine and human IL-17-producing Th17 cells in an RORγt-dependent manner. Importantly, we showed that Th17, but not Th1 cells, preferentially produce these two oxysterols. In vivo, administration of 7ß, 27-OHC in mice enhanced IL-17 production. Mice deficient in CYP27A1, a key enzyme in generating these oxysterols, showed significant reduction of IL-17-producing cells, including CD4(+) and γδ(+) T cells, similar to the deficiency observed in RORγt knockout mice. Our results reveal a previously unknown mechanism for selected oxysterols as immune modulators and a direct role for CYP27A1 in generating these RORγt agonist ligands, which we propose as RORγt endogenous ligands, driving both innate and adaptive IL-17-dependent immune responses.
Assuntos
Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Esteróis/farmacologia , Células Th17/citologia , Animais , Diferenciação Celular , Colestanotriol 26-Mono-Oxigenase/metabolismo , Interleucina-17/biossíntese , Ligantes , Camundongos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Esteróis/metabolismoRESUMO
Ion channels are an attractive class of drug targets, but progress in developing inhibitors for therapeutic use has been limited largely due to challenges in identifying subtype selective small molecules. Animal venoms provide an alternative source of ion channel modulators, and the venoms of several species, such as scorpions, spiders and snails, are known to be rich sources of ion channel modulating peptides. Importantly, these peptides often bind to hyper-variable extracellular loops, creating the potential for subtype selectivity rarely achieved with small molecules. We have engineered scorpion venom peptides and incorporated them in fusion proteins to generate highly potent and selective Kv1.3 inhibitors with long in vivo half-lives. Kv1.3 has been reported to play a role in human T cell activation, and therefore, these Kv1.3 inhibitor fusion proteins may have potential for the treatment of autoimmune diseases. Our results support an emerging approach to generating subtype selective therapeutic ion channel inhibitors.
Assuntos
Proteínas de Artrópodes/farmacologia , Canal de Potássio Kv1.3/antagonistas & inibidores , Ativação Linfocitária/efeitos dos fármacos , Peptídeos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Engenharia de Proteínas , Venenos de Escorpião/farmacologia , Linfócitos T/metabolismo , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Células CHO , Cricetinae , Cricetulus , Sistemas de Liberação de Medicamentos , Meia-Vida , Humanos , Canal de Potássio Kv1.3/genética , Canal de Potássio Kv1.3/metabolismo , Peptídeos/química , Peptídeos/genética , Bloqueadores dos Canais de Potássio/química , Ratos , Venenos de Escorpião/química , Venenos de Escorpião/genéticaRESUMO
To demonstrate the benefits of RNA-Seq over microarray in transcriptome profiling, both RNA-Seq and microarray analyses were performed on RNA samples from a human T cell activation experiment. In contrast to other reports, our analyses focused on the difference, rather than similarity, between RNA-Seq and microarray technologies in transcriptome profiling. A comparison of data sets derived from RNA-Seq and Affymetrix platforms using the same set of samples showed a high correlation between gene expression profiles generated by the two platforms. However, it also demonstrated that RNA-Seq was superior in detecting low abundance transcripts, differentiating biologically critical isoforms, and allowing the identification of genetic variants. RNA-Seq also demonstrated a broader dynamic range than microarray, which allowed for the detection of more differentially expressed genes with higher fold-change. Analysis of the two datasets also showed the benefit derived from avoidance of technical issues inherent to microarray probe performance such as cross-hybridization, non-specific hybridization and limited detection range of individual probes. Because RNA-Seq does not rely on a pre-designed complement sequence detection probe, it is devoid of issues associated with probe redundancy and annotation, which simplified interpretation of the data. Despite the superior benefits of RNA-Seq, microarrays are still the more common choice of researchers when conducting transcriptional profiling experiments. This is likely because RNA-Seq sequencing technology is new to most researchers, more expensive than microarray, data storage is more challenging and analysis is more complex. We expect that once these barriers are overcome, the RNA-Seq platform will become the predominant tool for transcriptome analysis.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Perfilação da Expressão Gênica , Transcriptoma , Análise de Variância , Sequência de Bases , Células Cultivadas , Humanos , Ativação Linfocitária , Anotação de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Análise de Sequência de RNARESUMO
Phosphatidylinositol-3-kinase gamma (PI3Kγ) is a leukocyte-specific lipid kinase with signaling function downstream of G protein-coupled receptors to regulate cell trafficking, but its role in T cells remains unclear. To investigate the requirement of PI3Kγ kinase activity in T-cell function, we studied T cells from PI3Kγ kinase-dead knock-in (PI3Kγ(KD/KD)) mice expressing the kinase-inactive PI3Kγ protein. We show that CD4(+) and CD8(+) T cells from PI3Kγ(KD/KD) mice exhibit impaired TCR/CD28-mediated activation that could not be rescued by exogenous IL-2. The defects in proliferation and cytokine production were also evident in naïve and memory T cells. Analysis of signaling events in activated PI3Kγ(KD/KD) T cells revealed a reduction in phosphorylation of protein kinase B (AKT) and ERK1/2, a decrease in lipid raft formation, and a delay in cell cycle progression. Furthermore, PI3Kγ(KD/KD) CD4(+) T cells displayed compromised differentiation toward Th1, Th2, Th17, and induced Treg cells. PI3Kγ(KD/KD) mice also exhibited an impaired response to immunization and a reduced delayed-type hypersensitivity to Ag challenge. These findings indicate that PI3Kγ kinase activity is required for optimal T-cell activation and differentiation, as well as for mounting an efficient T cell-mediated immune response. The results suggest that PI3Kγ kinase inhibitors could be beneficial in reducing the undesirable immune response in autoimmune diseases.
Assuntos
Diferenciação Celular/imunologia , Classe Ib de Fosfatidilinositol 3-Quinase/imunologia , Ativação Linfocitária/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Antígenos/imunologia , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Diferenciação Celular/genética , Classe Ib de Fosfatidilinositol 3-Quinase/genética , Inibidores Enzimáticos/uso terapêutico , Hipersensibilidade Tardia/genética , Hipersensibilidade Tardia/imunologia , Hipersensibilidade Tardia/patologia , Ativação Linfocitária/genética , Microdomínios da Membrana/genética , Microdomínios da Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/imunologia , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/genética , Fosforilação/imunologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologiaRESUMO
Phosphoinositide 3-kinase gamma (PI3Kγ) kinase activity is important for its signaling functions in T cell development, activation, differentiation, and trafficking. Protection of PI3Kγ knockout mice from disease in multiple autoimmune models suggests that targeting PI3Kγ alone, or in combination with PI3Kδ, could be a promising approach to disease therapy.
Assuntos
Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , Linfócitos T/metabolismo , Animais , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/metabolismo , Modelos Animais de Doenças , Camundongos , Camundongos Knockout , Terapia de Alvo Molecular , Linfócitos T/enzimologiaRESUMO
OBJECTIVE: Antagonism of the histamine H4 receptor (H4R) has been shown to be anti-inflammatory in a number of preclinical disease models, however the exact mechanisms behind this are still being uncovered. In vitro, the receptor interacts with TLR and impacts inflammatory mediator production from a number of different cell types. Here it is shown that this interaction also occurs in vivo. MATERIALS AND METHODS: Wild-type and H4R deficient BALB/c mice received an i.p. injection of LPS in PBS in conjunction with p.o. JNJ 7777120 or JNJ 28307474 (H4R antagonists). Two hours later blood was collected and TNF was measured. RESULTS: Two different H4R antagonists inhibited LPS-induced TNF production in mice and this production was also reduced in H4R-deficient mice. The TNF mRNA analysis showed that the major source of the cytokine was the liver and not blood, and that the H4R antagonist only reduced the expression levels in the liver. Depletion or inactivation of macrophages reduced the TNF levels and eliminated the H4R sensitivity. Treatment with an H4R antagonist also reduced LPS-induced liver injury and blocked LPS-enhanced lung inflammation in mice. CONCLUSION: The data support an interaction between H4R and TLR activation in vivo that can drive inflammatory responses.
Assuntos
Antagonistas dos Receptores Histamínicos/farmacologia , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Fator de Necrose Tumoral alfa/sangue , Alérgenos , Animais , Asma/induzido quimicamente , Asma/tratamento farmacológico , Asma/imunologia , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/imunologia , Feminino , Humanos , Indóis/farmacologia , Interleucina-13/imunologia , Células de Kupffer/metabolismo , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Ovalbumina , Piperazinas/farmacologia , Receptores Acoplados a Proteínas G/fisiologia , Receptores Histamínicos/fisiologia , Receptores Histamínicos H4 , Fator de Necrose Tumoral alfa/genéticaRESUMO
Susceptibility to rheumatoid arthritis (RA) is associated with HLA-DRB1 alleles coding a 5-amino acid sequence motif called the shared epitope (SE). To explore the potential mechanisms that lead to RA susceptibility, we analyze the in vitro effect of peptides bearing different HLA-DR4 sequences on human peripheral blood-derived cells. Three 15-mer peptides were used: 65-79*0401 (an HLA-DRB1*04:01-coded sequence carrying the SE motif, QKRAA); 65-79*0402 (an HLA-DRB1*04:02-coded sequence carrying a SE-negative motif, DERAA); 65-79*0403 (an HLA-DRB1*04:03-coded sequence carrying a SE-negative motif, QRRAE). We found that CD4 TH17 cells are regulated by peptide treatment with gender bias. In male-derived T cells, all peptide treatments significantly reduced TH17 cell differentiation in vitro when compared to no peptide treatment, and to female samples. TH17 differentiation in samples not treated with peptides, either in the presence or absence of TH17-polarizing cytokines, was higher in males than in females; however, in unfractionated PBMC after treatment with TH17 polarizing cytokines, IL-17A positive cells were more abundant in females than in males. In addition, SE-positive females showed a significantly higher percentage of IL-17A-positive cells compared to SE-negative females. In conclusion, donor's SE status and gender may both influence TH17 immune polarization.
RESUMO
PI3Kδ is a lipid kinase of the PI3K class IA family involved in early signaling events of leukocytes responding to a wide variety of stimuli. The leukocyte specificity of PI3Kδ is defined by its expression, whereas its signaling function is via the production of phosphoinositide 3,4,5-triphosphates at the proximity of activated receptors for recruiting other signaling molecules. The importance of PI3Kδ in B cell development and function is most apparent, and its role in other leukocyte cell types can be easily demonstrated as well. PI3Kδ participates in the development, activation and migration of T cells and NK cells. The role of PI3Kδ in myeloid cell activities, such as inflammation driven cell infiltration, neutrophil oxidative burst, immune complex mediated macrophage activation, as well as mast cell maturation and degranulation, has been well illustrated in various studies. As a result of the broad effects of PI3Kδ in leukocyte functions, the disruption of PI3Kδ expression or activity leads to decreased inflammatory and immune responses in vivo. The protective role of PI3Kδ inactivation in animal models of arthritis, asthma or obstructive respiratory diseases has been demonstrated. These findings suggest the potential efficacy achievable with PI3Kδ inhibitors in the treatment of autoimmune and respiratory diseases.
Assuntos
Leucócitos/fisiologia , Fosfatidilinositol 3-Quinase/metabolismo , Animais , Asma/enzimologia , Doenças Autoimunes/enzimologia , Linfócitos B/enzimologia , Linfócitos B/fisiologia , Humanos , Hipersensibilidade/enzimologia , Imunidade Celular , Células Matadoras Naturais/enzimologia , Células Matadoras Naturais/fisiologia , Leucócitos/enzimologia , Células Mieloides/enzimologia , Células Mieloides/fisiologia , Doença Pulmonar Obstrutiva Crônica/enzimologia , Transdução de Sinais , Linfócitos T/enzimologia , Linfócitos T/fisiologiaRESUMO
Phosphoinositide 3-kinase gamma (PI3Kgamma) is a lipid kinase in leukocytes that generates phosphatidylinositol 3,4,5-trisphosphate to recruit and activate downstream signaling molecules. Distinct from other members in the PI3K family, PI3Kgamma is activated by G-protein coupled-receptors responding to chemotactic ligands. PI3Kgamma plays an important role in migration of both myeloid and lymphoid cells. It is also required for other leukocyte functions such as neutrophil oxidative burst, T cell proliferation and mast degranulation. Mice with PI3Kgamma inactivated by genetic or pharmacological approaches are protected from disease development in a number of inflammation and autoimmune disease models. The function of PI3Kgamma depends on its kinase activity and therefore it has been suggested by many reports that small molecules inhibiting its kinase activity could be promising for the treatment of inflammation and autoimmune diseases. Over the last five years, a number of pharmaceutical companies have reported a wide variety of PI3Kgamma inhibitors, of which several x-ray crystal structures with PI3Kgamma have been elucidated. The structural characteristics and selectivity profiles of these inhibitors, in particular thiazolidinones and 2-aminoheterocycles, and those disclosed in related patent applications are summarized in this review.
Assuntos
Doenças Autoimunes/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Tiazolidinedionas/farmacologia , Animais , Classe Ib de Fosfatidilinositol 3-Quinase , Inibidores Enzimáticos/uso terapêutico , Humanos , Inflamação , Isoenzimas/antagonistas & inibidores , Patentes como Assunto , Transdução de Sinais , Relação Estrutura-Atividade , Especificidade por Substrato , Tiazolidinedionas/químicaRESUMO
The innate immune system is equipped with sensitive and efficient machineries to provide an immediate, first line defense against infections. Toll-like receptors (TLRs) detect pathogens and the IL-1 receptor (IL-1R) family enables cells to quickly respond to inflammatory cytokines by mounting an efficient protective response. Interleukin-1 receptor activated kinases (IRAKs) are key mediators in the signaling pathways of TLRs/IL-1Rs. By means of their kinase and adaptor functions, IRAKs initiate a cascade of signaling events eventually leading to induction of inflammatory target gene expression. Due to this pivotal role, IRAK function is also highly regulated via multiple mechanisms. In this review, we focus on IRAK1, the earliest known and yet the most interesting member of this family. An overview on its structure, function and biology is given, with emphasis on the different novel mechanisms that regulate IRAK1 function. We also highlight several unresolved questions in this field and evaluate the potential of IRAK1 as a target for therapeutic intervention.
Assuntos
Imunidade Inata/imunologia , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Transdução de Sinais , Animais , Doenças Autoimunes/enzimologia , Humanos , Quinases Associadas a Receptores de Interleucina-1/química , Quinases Associadas a Receptores de Interleucina-1/genética , Receptores Imunológicos/imunologia , UbiquitinaçãoRESUMO
Inhibition of the p38 map kinase pathway has been shown to be beneficial in the treatment of inflammatory diseases. The first class of potent p38 kinase inhibitors was the pyridinylimidazole compounds from SKB. Since then several pyridinylimidazole-based compounds have been shown to inhibit activated p38 kinase in vitro and in vivo. We have developed a novel series of pyridinylimidazole-based compounds, which potently inhibit the p38 pathway by binding to unactivated p38 kinase and only weakly inhibiting activated p38 kinase activity in vitro.