RESUMO
Rationale: Bronchopulmonary dysplasia (BPD) is a leading complication of preterm birth that affects infants born in the saccular stage of lung development at <32 weeks of gestation. Although the mechanisms driving BPD remain uncertain, exposure to hyperoxia is thought to contribute to disease pathogenesis.Objectives: To determine the effects of hyperoxia on epithelial-mesenchymal interactions and to define the mediators of activated Wnt/ß-catenin signaling after hyperoxia injury.Methods: Three hyperoxia models were used: A three-dimensional organotypic coculture using primary human lung cells, precision-cut lung slices (PCLS), and a murine in vivo hyperoxia model. Comparisons of normoxia- and hyperoxia-exposed samples were made by real-time quantitative PCR, RNA in situ hybridization, quantitative confocal microscopy, and lung morphometry.Measurements and Main Results: Examination of an array of Wnt ligands in the three-dimensional organotypic coculture revealed increased mesenchymal expression of WNT5A. Inhibition of Wnt5A abrogated the BPD transcriptomic phenotype induced by hyperoxia. In the PCLS model, Wnt5A inhibition improved alveolarization following hyperoxia exposure, and treatment with recombinant Wnt5a reproduced features of the BPD phenotype in PCLS cultured in normoxic conditions. Chemical inhibition of NF-κB with BAY11-7082 reduced Wnt5a expression in the PCLS hyperoxia model and in vivo mouse hyperoxia model, with improved alveolarization in the PCLS model.Conclusions: Increased mesenchymal Wnt5A during saccular-stage hyperoxia injury contributes to the impaired alveolarization and septal thickening observed in BPD. Precise targeting of Wnt5A may represent a potential therapeutic strategy for the treatment of BPD.
Assuntos
Células Epiteliais Alveolares/metabolismo , Fibroblastos/metabolismo , Hiperóxia/genética , Pulmão/metabolismo , Células-Tronco Mesenquimais/metabolismo , Proteína Wnt-5a/genética , Animais , Displasia Broncopulmonar , Técnicas de Cocultura , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Hiperóxia/metabolismo , Hibridização In Situ , Pulmão/crescimento & desenvolvimento , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Microscopia Confocal , NF-kappa B/antagonistas & inibidores , Nitrilas/farmacologia , Técnicas de Cultura de Órgãos , Reação em Cadeia da Polimerase em Tempo Real , Sulfonas/farmacologia , Proteína Wnt-5a/efeitos dos fármacos , Proteína Wnt-5a/metabolismoRESUMO
Monophosphoryl lipid A (MPLA) is a clinically used TLR4 agonist that has been found to drive nonspecific resistance to infection for up to 2 wk. However, the molecular mechanisms conferring protection are not well understood. In this study, we found that MPLA prompts resistance to infection, in part, by inducing a sustained and dynamic metabolic program in macrophages that supports improved pathogen clearance. Mice treated with MPLA had enhanced resistance to infection with Staphylococcus aureus and Candida albicans that was associated with augmented microbial clearance and organ protection. Tissue macrophages, which exhibited augmented phagocytosis and respiratory burst after MPLA treatment, were required for the beneficial effects of MPLA. Further analysis of the macrophage phenotype revealed that early TLR4-driven aerobic glycolysis was later coupled with mitochondrial biogenesis, enhanced malate shuttling, and increased mitochondrial ATP production. This metabolic program was initiated by overlapping and redundant contributions of MyD88- and TRIF-dependent signaling pathways as well as downstream mTOR activation. Blockade of mTOR signaling inhibited the development of the metabolic and functional macrophage phenotype and ablated MPLA-induced resistance to infection in vivo. Our findings reveal that MPLA drives macrophage metabolic reprogramming that evolves over a period of days to support a macrophage phenotype highly effective at mediating microbe clearance and that this results in nonspecific resistance to infection.
Assuntos
Macrófagos/metabolismo , Receptor 4 Toll-Like/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Candida albicans/efeitos dos fármacos , Candidíase/tratamento farmacológico , Candidíase/metabolismo , Glicólise/fisiologia , Lipídeo A/análogos & derivados , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/metabolismo , Transdução de Sinais/fisiologia , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismoRESUMO
OBJECTIVES: To determine whether synthetic phosphorylated hexa-acyl disaccharides provide antimicrobial protection in clinically relevant models of bacterial infection. DESIGN: Laboratory study. SETTING: University laboratory. SUBJECTS: BALB/c, C57BL/10J, and C57BL/10ScNJ mice. INTERVENTIONS: Mice were treated with lactated Ringer's (vehicle) solution, monophosphoryl lipid A, or phosphorylated hexa-acyl disaccharides at 48 and 24 hours prior to intraperitoneal Pseudomonas aeruginosa or IV Staphylococcus aureus infection. Leukocyte recruitment, cytokine production, and bacterial clearance were measured 6 hours after P. aeruginosa infection. In the systemic S. aureus infection model, one group of mice was monitored for 14-day survival and another for S. aureus tissue burden at 3 days postinfection. Duration of action for 3-deacyl 6-Acyl phosphorylated hexa-acyl disaccharide was determined at 3, 10, and 14 days using a model of intraperitoneal P. aeruginosa infection. Effect of 3-deacyl 6-Acyl phosphorylated hexa-acyl disaccharide on in vivo leukocyte phagocytosis and respiratory burst was examined. Leukocyte recruitment, cytokine production, and bacterial clearance were measured after P. aeruginosa infection in wild-type and toll-like receptor 4 knockout mice treated with 3-deacyl 6-Acyl phosphorylated hexa-acyl disaccharide or vehicle to assess receptor specificity. MEASUREMENTS AND MAIN RESULTS: During intraperitoneal P. aeruginosa infection, phosphorylated hexa-acyl disaccharides significantly attenuated infection-induced hypothermia, augmented leukocyte recruitment and bacterial clearance, and decreased cytokine production. At 3 days post S. aureus infection, bacterial burden in lungs, spleen, and kidneys was significantly decreased in mice treated with monophosphoryl lipid A or phosphorylated hexa-acyl disaccharides, which was associated with improved survival. Leukocyte phagocytosis and respiratory burst functions were enhanced after treatment with monophosphoryl lipid A or phosphorylated hexa-acyl disaccharides. A time course study showed that monophosphoryl lipid A- and 3-deacyl 6-Acyl phosphorylated hexa-acyl disaccharide-mediated protection against P. aeruginosa lasts for up to 10 days. Partial loss of augmented innate antimicrobial responses was observed in toll-like receptor 4 knockout mice treated with 3-deacyl 6-Acyl phosphorylated hexa-acyl disaccharide. CONCLUSIONS: Phosphorylated hexa-acyl disaccharides significantly augment resistance against clinically relevant Gram-negative and Gram-positive infections via enhanced leukocyte recruitment, phagocytosis, and respiratory burst functions of innate leukocytes. Improved antimicrobial protection persists for up to 10 days and is partially mediated through toll-like receptor 4.
Assuntos
Infecção Hospitalar/prevenção & controle , Citocinas/metabolismo , Dissacarídeos/farmacologia , Hexosaminidase A/farmacologia , Cavidade Peritoneal/fisiopatologia , Infecções Estafilocócicas/fisiopatologia , Análise de Variância , Animais , Western Blotting/métodos , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Cavidade Peritoneal/microbiologia , Distribuição Aleatória , Infecções Estafilocócicas/mortalidade , Estatísticas não Paramétricas , Taxa de SobrevidaRESUMO
The magnitude of the LPS-elicited cytokine response is commonly used to assess immune function in critically ill patients. A suppressed response, known as endotoxin tolerance, is associated with worse outcomes, yet endotoxin tolerance-inducing TLR4 ligands are known to protect animals from infection. Thus, it remains unknown whether the magnitude of the LPS-elicited cytokine response provides an accurate assessment of antimicrobial immunity. To address this, the ability of diverse TLR ligands to modify the LPS-elicited cytokine response and resistance to infection were assessed. Priming of mice with LPS, monophosphoryl lipid A (MPLA), or poly(I:C) significantly reduced plasma LPS-elicited proinflammatory cytokines, reflecting endotoxin tolerance, whereas CpG-ODN-primed mice showed augmented cytokine production. In contrast, LPS, MPLA, and CpG-ODN, but not poly(I:C), improved the host response to a Pseudomonas aeruginosa infection. Mice primed with protective TLR ligands, including CpG-ODN, showed reduced plasma cytokines during P. aeruginosa infection. The protection imparted by TLR ligands persisted for up to 15 d yet was independent of the adaptive immune system. In bone marrow-derived macrophages, protective TLR ligands induced a persistent metabolic phenotype characterized by elevated glycolysis and oxidative metabolism as well as augmented size, granularity, phagocytosis, and respiratory burst. Sustained augmentation of glycolysis in TLR-primed cells was dependent, in part, on hypoxia-inducible factor 1-α and was essential for increased phagocytosis. In conclusion, the magnitude of LPS-elicited cytokine production is not indicative of antimicrobial immunity after exposure to TLR ligands. Additionally, protective TLR ligands induce sustained augmentation of phagocyte metabolism and antimicrobial function.
Assuntos
Citocinas/imunologia , Lipopolissacarídeos/imunologia , Infecções por Pseudomonas/imunologia , Animais , Citocinas/biossíntese , Modelos Animais de Doenças , Citometria de Fluxo , Ligantes , Lipídeo A/análogos & derivados , Lipídeo A/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Poli I-C/imunologia , Pseudomonas aeruginosa , Receptores Toll-Like/antagonistas & inibidores , Receptores Toll-Like/imunologiaRESUMO
Interleukin 15 is essential for the development and differentiation of NK and memory CD8+ (mCD8+) T cells. Our laboratory previously showed that NK and CD8+ T lymphocytes facilitate the pathobiology of septic shock. However, factors that regulate NK and CD8+ T lymphocyte functions during sepsis are not well characterized. We hypothesized that IL-15 promotes the pathogenesis of sepsis by maintaining NK and mCD8+ T cell integrity. To test our hypothesis, the pathogenesis of sepsis was assessed in IL-15-deficient (IL-15 knockout, KO) mice. IL-15 KO mice showed improved survival, attenuated hypothermia, and less proinflammatory cytokine production during septic shock caused by cecal ligation and puncture or endotoxin-induced shock. Treatment with IL-15 superagonist (IL-15 SA, IL-15/IL-15Rα complex) regenerated NK and mCD8+ T cells and re-established mortality of IL-15 KO mice during septic shock. Preventing NK cell regeneration attenuated the restoration of mortality caused by IL-15 SA. If given immediately prior to septic challenge, IL-15-neutralizing IgG M96 failed to protect against septic shock. However, M96 caused NK cell depletion if given 4 d prior to septic challenge and conferred protection. IL-15 SA treatment amplified endotoxin shock, which was prevented by NK cell or IFN-γ depletion. IL-15 SA treatment also exacerbated septic shock caused by cecal ligation and puncture when given after the onset of sepsis. In conclusion, endogenous IL-15 does not directly augment the pathogenesis of sepsis but enables the development of septic shock by maintaining NK cell numbers and integrity. Exogenous IL-15 exacerbates the severity of sepsis by activating NK cells and facilitating IFN-γ production.
Assuntos
Interleucina-15/fisiologia , Células Matadoras Naturais/imunologia , Choque Séptico/etiologia , Animais , Feminino , Interferon gama/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Choque Séptico/imunologiaRESUMO
IL-15 is currently undergoing clinical trials to assess its efficacy for treatment of advanced cancers. The combination of IL-15 with soluble IL-15Rα generates a complex termed IL-15 superagonist (IL-15 SA) that possesses greater biological activity than IL-15 alone. IL-15 SA is considered an attractive antitumor and antiviral agent because of its ability to selectively expand NK and memory CD8(+) T (mCD8(+) T) lymphocytes. However, the adverse consequences of IL-15 SA treatment have not been defined. In this study, the effect of IL-15 SA on physiologic and immunologic functions of mice was evaluated. IL-15 SA caused dose- and time-dependent hypothermia, weight loss, liver injury, and mortality. NK (especially the proinflammatory NK subset), NKT, and mCD8(+) T cells were preferentially expanded in spleen and liver upon IL-15 SA treatment. IL-15 SA caused NK cell activation as indicated by increased CD69 expression and IFN-γ, perforin, and granzyme B production, whereas NKT and mCD8(+) T cells showed minimal, if any, activation. Cell depletion and adoptive transfer studies showed that the systemic toxicity of IL-15 SA was mediated by hyperproliferation of activated NK cells. Production of the proinflammatory cytokine IFN-γ, but not TNF-α or perforin, was essential to IL-15 SA-induced immunotoxicity. The toxicity and immunological alterations shown in this study are comparable to those reported in recent clinical trials of IL-15 in patients with refractory cancers and advance current knowledge by providing mechanistic insights into IL-15 SA-mediated immunotoxicity.
Assuntos
Citotoxicidade Imunológica/imunologia , Interferon gama/imunologia , Subunidade alfa de Receptor de Interleucina-15/imunologia , Interleucina-15/imunologia , Células Matadoras Naturais/imunologia , Animais , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos de Diferenciação de Linfócitos T/metabolismo , Temperatura Corporal/efeitos dos fármacos , Temperatura Corporal/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células/efeitos dos fármacos , Citotoxicidade Imunológica/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Citometria de Fluxo , Granzimas/imunologia , Granzimas/metabolismo , Humanos , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-15/metabolismo , Subunidade alfa de Receptor de Interleucina-15/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexos Multiproteicos/imunologia , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/farmacologia , Perforina/imunologia , Perforina/metabolismoRESUMO
Prior exposure to lipopolysaccharide (LPS) produces a reduced or "tolerant" inflammatory response to subsequent challenges with LPS, however the potent pro-inflammatory effects of LPS limit its clinical benefit. The adjuvant monophosphoryl lipid A (MPLA) is a weak toll-like receptor 4 (TLR4) agonist that induces negligible inflammation but retains potent immunomodulatory properties. We postulated that pre-treatment with MPLA would inhibit the inflammatory response of endothelial cells to secondary LPS challenge. Human umbilical vein endothelial cells (HUVECs), were exposed to MPLA (10 µg/ml), LPS (100 ng/ml) or vehicle control. HUVECs were then washed and maintained in culture for 24 h before being challenged with LPS (100 ng/ml). Supernatants were collected and examined for cytokine production in the presence or absence of siRNA inhibitors of critical TLR4 signalling proteins. Pre-treatment with MPLA attenuated interleukin (IL)-6 production to secondary LPS challenge to a similar degree as LPS. The application of myeloid differentiation primary response gene 88 (MyD88) siRNA dramatically reduced MPLA-induced tolerance while TIR-domain-containing adapter-inducing interferon-ß (TRIF) siRNA had no effect. The tolerant phenotype in endothelial cells was associated with reduced IκB kinase (IKK), p38 and c-Jun N-terminal kinase (JNK) phosphorylation and enhanced IL-1 receptor associated kinase-M (IRAK-M) expression for LPS-primed HUVECs, but less so in MPLA primed cells. Instead, MPLA-primed HUVECs demonstrated enhanced p-extracellular-signal-regulated kinase (ERK) phosphorylation. In contrast with leucocytes in which tolerance is largely TRIF-dependent, MyD88 signalling mediated endotoxin tolerance in endothelial cells. Most importantly, MPLA, a vaccine adjuvant with a wide therapeutic window, induced tolerance to LPS in endothelial cells.
Assuntos
Adjuvantes Imunológicos/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Lipídeo A/análogos & derivados , Fator 88 de Diferenciação Mieloide/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Tolerância Imunológica/efeitos dos fármacos , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Lipídeo A/farmacologia , Lipopolissacarídeos , Fosforilação/efeitos dos fármacosRESUMO
Exposure to pathogen-associated molecular patterns (PAMPs) induces an augmented, broad-spectrum antimicrobial response to subsequent infection, a phenomenon termed innate immune memory. This study examined the effects of treatment with ß-glucan, a fungus-derived dectin-1 ligand, or monophosphoryl lipid A (MPLA), a bacteria-derived Toll-like receptor 4 ligand, on innate immune memory with a focus on identifying common cellular and molecular pathways activated by these diverse PAMPs. Treatment with either PAMP prepared the innate immune system to respond more robustly to Pseudomonas aeruginosa infection in vivo by facilitating mobilization of innate leukocytes into blood, recruitment of leukocytes to the site of infection, augmentation of microbial clearance, and attenuation of cytokine production. Examination of macrophages ex vivo showed amplification of metabolism, phagocytosis, and respiratory burst after treatment with either agent, although MPLA more robustly augmented these activities and more effectively facilitated killing of bacteria. Both agents activated gene expression pathways in macrophages that control inflammation, antimicrobial functions, and protein synthesis and suppressed pathways regulating cell division. ß-glucan treatment minimally altered macrophage differential gene expression in response to lipopolysaccharide (LPS) challenge, whereas MPLA attenuated the magnitude of the LPS-induced transcriptional response, especially cytokine gene expression. These results show that ß-glucan and MPLA similarly augment the innate response to infection in vivo. Yet, MPLA more potently induces alterations in macrophage metabolism, antimicrobial functions, gene transcription and the response to LPS.
Assuntos
Anti-Infecciosos , beta-Glucanas , Lipopolissacarídeos/farmacologia , Moléculas com Motivos Associados a Patógenos , Imunidade Treinada , Ligantes , Citocinas , beta-Glucanas/farmacologia , Bactérias , Imunidade InataRESUMO
Fast-spiking interneurons (FSIs) can exert powerful control over striatal output, and deficits in this cell population have been observed in human patients with Tourette syndrome and rodent models of dystonia. However, a direct experimental test of striatal FSI involvement in motor control has never been performed. We applied a novel pharmacological approach to examine the behavioral consequences of selective FSI suppression in mouse striatum. IEM-1460, an inhibitor of GluA2-lacking AMPARs, selectively blocked synaptic excitation of FSIs but not striatal projection neurons. Infusion of IEM-1460 into the sensorimotor striatum reduced the firing rate of FSIs but not other cell populations, and elicited robust dystonia-like impairments. These results provide direct evidence that hypofunction of striatal FSIs can produce movement abnormalities, and suggest that they may represent a novel therapeutic target for the treatment of hyperkinetic movement disorders.
Assuntos
Potenciais de Ação/fisiologia , Corpo Estriado/patologia , Discinesias/etiologia , Discinesias/patologia , Interneurônios/fisiologia , Adamantano/efeitos adversos , Adamantano/análogos & derivados , Análise de Variância , Animais , Área Sob a Curva , Antagonistas Colinérgicos/efeitos adversos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Interações Medicamentosas , Antagonistas de Aminoácidos Excitatórios/efeitos adversos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Feminino , Lateralidade Funcional/efeitos dos fármacos , Lateralidade Funcional/fisiologia , Proteínas de Fluorescência Verde/genética , Interneurônios/classificação , Interneurônios/efeitos dos fármacos , Proteínas com Homeodomínio LIM/genética , Masculino , Mecamilamina/efeitos adversos , Camundongos , Camundongos Transgênicos , N-Metilaspartato/farmacologia , Proteínas do Tecido Nervoso/genética , Escopolamina/efeitos adversos , Fatores de Transcrição/genéticaRESUMO
Monophosphoryl lipid A (MPLA), a less toxic derivative of lipopolysaccharide (LPS), is employed as a vaccine adjuvant and is under investigation as a non-specific immunomodulator. However, the differential response of human leukocytes to MPLA and LPS has not been well characterized. The goal of this study was to compare the differential transcriptomic response of human blood to LPS and MPLA. Venous blood from human volunteers was stimulated with LPS, MPLA or vehicle. Gene expression was determined using microarray analysis. Among 21,103 probes profiled, 136 and 130 genes were differentially regulated by LPS or MPLA, respectively. Seventy four genes were up-regulated and 9 were down-regulated by both ligands. The remaining genes were differentially induced by either agent. Ingenuity Pathway Analysis predicted that LPS and MPLA share similar upstream regulators and have comparable effects on canonical pathways and cellular functions. However, some pro-inflammatory cytokine and inflammasome-associated transcripts were more strongly induced by LPS. In contrast, only the macrophage-regulating chemokine CCL7 was preferentially up-regulated by MPLA. In conclusion, LPS and MPLA induce similar transcriptional profiles. However, LPS more potently induces pro-inflammatory cytokine and inflammasome-linked transcripts. Thus, MPLA is a less potent activator of the pro-inflammatory response but retains effective immunomodulatory activity.
Assuntos
Adjuvantes Imunológicos/metabolismo , Leucócitos/efeitos dos fármacos , Lipídeo A/análogos & derivados , Lipopolissacarídeos/metabolismo , Receptor 4 Toll-Like/metabolismo , Transcriptoma , Voluntários Saudáveis , Humanos , Fatores Imunológicos/biossíntese , Lipídeo A/metabolismo , Análise em MicrossériesRESUMO
Treatment with the TLR4 agonist MPLA augments innate resistance to common bacterial pathogens. However, the cellular and molecular mechanisms by which MPLA augments innate immunocyte functions are not well characterized. This study examined the importance of MyD88- and TRIF-dependent signaling for leukocyte mobilization, recruitment, and activation following administration of MPLA. MPLA potently induced MyD88- and TRIF-dependent signaling. A single injection of MPLA caused rapid mobilization and recruitment of neutrophils, a response that was largely mediated by the chemokines CXCL1 and -2 and the hemopoietic factor G-CSF. Rapid neutrophil recruitment and chemokine production were regulated by both pathways although the MyD88-dependent pathway showed some predominance. In further studies, multiple injections of MPLA potently induced mobilization and recruitment of neutrophils and monocytes. Neutrophil recruitment after multiple injections of MPLA was reliant on MyD88-dependent signaling, but effective monocyte recruitment required activation of both pathways. MPLA treatment induced expansion of myeloid progenitors in bone marrow and upregulation of CD11b and shedding of L-selectin by neutrophils, all of which were attenuated in MyD88- and TRIF-deficient mice. These results show that MPLA-induced neutrophil and monocyte recruitment, expansion of bone marrow progenitors and augmentation of neutrophil adhesion molecule expression are regulated by both the MyD88- and TRIF-dependent pathways.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Imunidade Inata , Lipídeo A/análogos & derivados , Monócitos/imunologia , Fator 88 de Diferenciação Mieloide/fisiologia , Neutrófilos/imunologia , Receptor 4 Toll-Like/agonistas , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Animais , Antígeno CD11b/biossíntese , Antígeno CD11b/genética , Quimiocina CXCL1/fisiologia , Quimiocina CXCL2/fisiologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/fisiologia , Selectina L/metabolismo , Lipídeo A/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Fator 88 de Diferenciação Mieloide/deficiência , Mielopoese/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Receptores de Interleucina-8B/fisiologia , Transdução de Sinais , Receptor 4 Toll-Like/fisiologiaRESUMO
OBJECTIVE: Bruton's tyrosine kinase (BTK) is a B cell signaling protein that also contributes to innate immunity. BTK inhibitors prevent autoimmune arthritis but have off-target effects, and the mechanisms of protection remain unknown. We undertook these studies using genetic deletion to investigate the role of BTK in adaptive and innate immune responses that drive inflammatory arthritis. METHODS: BTK-deficient K/BxN mice were generated to study the role of BTK in a spontaneous model that requires both adaptive and innate immunity. The K/BxN serum-transfer model was used to bypass the adaptive system and elucidate the role of BTK in innate immune contributions to arthritis. RESULTS: BTK deficiency conferred disease protection to K/BxN mice, confirming outcomes of BTK inhibitors. B lymphocytes were profoundly reduced, more than in other models of BTK deficiency. Subset analysis revealed loss of B cells at all developmental stages. Germinal center B cells were also decreased, with downstream effects on numbers of follicular helper T cells and greatly reduced autoantibodies. In contrast, total IgG was only mildly decreased. Strikingly, and in contrast to small molecule inhibitors, BTK deficiency had no effect in the serum-transfer model of arthritis. CONCLUSION: BTK contributes to autoimmune arthritis primarily through its role in B cell signaling and not through innate immune components.