RESUMO
OBJECTIVES: We have previously reported using gene-deficient mice that the interleukin (IL)-23p19 subunit is required for the development of innate immune-driven arthritic pain and disease. We aimed to explore here, using a number of in vivo approaches, how the IL-23p19 subunit can mechanistically control arthritic pain and disease in a T- and B- lymphocyte-independent manner. METHODS: We used the zymosan-induced arthritis (ZIA) model in wild-type and Il23p19-/- mice, by a radiation chimera approach, and by single cell RNAseq and qPCR analyses, to identify the IL23p19-expressing and IL-23-responding cell type(s) in the inflamed joints. This model was also utilized to investigate the efficacy of IL-23p19 subunit blockade with a neutralizing monoclonal antibody (mAb). A novel IL-23-driven arthritis model was established, allowing the identification of putative downstream mediators of IL-23 in the control of pain and disease. Pain and arthritis were assessed by relative static weight distribution and histology, respectively. RESULTS: We present evidence that (i) IL-23p19+ non-bone marrow-derived macrophages are required for the development of ZIA pain and disease, (ii) prophylactic and therapeutic blockade of the IL-23p19 subunit ameliorate ZIA pain and disease and (iii) systemically administered IL-23 can induce arthritic pain and disease in a manner dependent on TNF, GM-CSF, CCL17 and cyclooxygenase activity, but independently of lymphocytes, CGRP, NGF and substance P. CONCLUSIONS: The data presented should aid IL-23 targeting both in the choice of inflammatory disease to be treated and the design of clinical trials.
Assuntos
Artrite Experimental , Camundongos Endogâmicos C57BL , Camundongos Knockout , Animais , Artrite Experimental/imunologia , Artrite Experimental/patologia , Camundongos , Interleucina-23/metabolismo , Interleucina-23/imunologia , Subunidade p19 da Interleucina-23/imunologia , Subunidade p19 da Interleucina-23/antagonistas & inibidores , Subunidade p19 da Interleucina-23/genética , Dor/etiologia , Zimosan , Masculino , Inflamação/imunologia , Inflamação/metabolismoRESUMO
IL-23 is a double-subunit cytokine that plays an important role in shaping the immune response. IL-23 was found to be associated with several autoinflammatory diseases by generating sustained inflammatory loops that lead to tissue damage. Antibody neutralization of IL-23 was proven to be effective in ameliorating associated diseases. However, antibodies as large proteins have limited tissue penetration and tend to elicit anti-drug antibodies. Additionally, anti-IL-23 antibodies target only one subunit of IL-23 leaving the other one unneutralized. Here, we attempted to isolate a recycling single domain antibody by phage display. One of IL-23 subunits, p19, was expressed in E. coli fused to Gamillus protein to stabilize the α-helix-only p19. To remove Gamillus binders, two biopanning methods were investigated, first, preselection with Gamillus and second, challenge with IL-23 then on the subsequent round challenge with p19-Gam. The isolation of calcium-dependent and pH-dependent recycling binders was performed with EDTA and citrate buffers respectively. Both methods of panning failed to isolate high-affinity and specific p19 recycling binders, while from the second panning method, a high affinity and specific p19 standard binder, namely H11, was successfully isolated. H11 significantly inhibited the gene expression of IL-17 and IL-22 in IL-23-challenged PBMCs indicating H11 specificity and neutralizing ability for IL-23. The new binder due to its small size can overcome antibodies limitations, also, it can be further engineered in the future for antigen clearance such as fusing it to cell penetrating peptides, granting H11 the ability to clear excess IL-23 and enhancing its potential therapeutic effect.
Assuntos
Subunidade p19 da Interleucina-23 , Anticorpos de Domínio Único , Humanos , Subunidade p19 da Interleucina-23/imunologia , Subunidade p19 da Interleucina-23/genética , Anticorpos de Domínio Único/imunologia , Inflamação/imunologia , Técnicas de Visualização da Superfície Celular , Escherichia coli/genética , Interleucina-23/imunologia , Interleucina-23/metabolismo , Biblioteca de PeptídeosRESUMO
HLA-B27 is a major risk factor for spondyloarthritis (SpA), yet the underlying mechanisms remain unclear. HLA-B27 misfolding-induced IL-23, which is mediated by endoplasmic reticulum (ER) stress has been hypothesized to drive SpA pathogenesis. Expression of HLA-B27 and human ß2m (hß2m) in rats (HLA-B27-Tg) recapitulates key SpA features including gut inflammation. Here we determined whether deleting the transcription factor CHOP (Ddit3-/-), which mediates ER-stress induced IL-23, affects gut inflammation in HLA-B27-Tg animals. ER stress-mediated Il23a overexpression was abolished in CHOP-deficient macrophages. Although CHOP-deficiency also reduced Il23a expression in immune cells isolated from the colon of B27+ rats, Il17a levels were not affected, and gut inflammation was not reduced. Rather, transcriptome analysis revealed increased expression of pro-inflammatory genes, including Il1a, Ifng and Tnf in HLA-B27-Tg colon tissue in the absence of CHOP, which was accompanied by higher histological Z-scores. RNAScope localized Il17a mRNA to the lamina propria of the HLA-B27-Tg rats and revealed similar co-localization with Cd3e (CD3) in the presence and absence of CHOP. This demonstrates that CHOP-deficiency does not improve, but rather exacerbates gut inflammation in HLA-B27-Tg rats, indicating that HLA-B27 is not promoting gut disease through ER stress-induced IL-23. Hence, CHOP may protect rats from more severe HLA-B27-induced gut inflammation.
Assuntos
Colite , Estresse do Retículo Endoplasmático , Antígeno HLA-B27 , Espondilartrite , Fator de Transcrição CHOP , Animais , Antígeno HLA-B27/genética , Antígeno HLA-B27/metabolismo , Fator de Transcrição CHOP/metabolismo , Fator de Transcrição CHOP/genética , Colite/metabolismo , Colite/genética , Colite/induzido quimicamente , Colite/patologia , Ratos , Espondilartrite/metabolismo , Espondilartrite/patologia , Espondilartrite/genética , Modelos Animais de Doenças , Interleucina-23/metabolismo , Interleucina-23/genética , Humanos , Subunidade p19 da Interleucina-23/genética , Subunidade p19 da Interleucina-23/metabolismo , Ratos Transgênicos , Interleucina-17/metabolismo , Interleucina-17/genética , Colo/patologia , Colo/metabolismo , Macrófagos/metabolismo , Macrófagos/imunologiaRESUMO
The interleukin-12 (IL-12) family is a class of heterodimeric cytokines that play crucial roles in pro-inflammatory and pro-stimulatory responses. Although some IL-12 and IL-23 paralogues have been found in fish, their functional activity in fish remains poorly understood. In this study, Pf_IL-12p35a/b, Pf_IL-23p19 and Pf_IL-12p40a/b/c genes were cloned from yellow catfish (Pelteobagrus fulvidraco), four α-helices were found in Pf_IL-12p35a/b and Pf_IL-23p19. The transcripts of these six genes were relatively high in mucus and immune tissues of healthy individuals, and in gill leukocytes. Following Edwardsiella ictaluri infection, Pf_IL-12p35a/b and Pf_IL-23p19 mRNAs were induced in brain and kidney (or head kidney), Pf_IL-12p40a mRNA was induced in gill, and Pf_IL-12p40b/c mRNAs were induced in brain and liver (or skin). The mRNA expression of these genes in PBLs was induced by phytohaemagglutinin (PHA) and polyinosinic-polycytidylic acid (poly I:C), while lipopolysaccharides (LPS) induced the mRNA expression of Pf_IL-12p35a and Pf_IL-12p40b/c in PBLs. After stimulation with recombinant (r) Pf_IL-12 and rPf_IL-23 subunit proteins, either alone or in combination, mRNA expression patterns of genes related to T helper cell development exhibited distinct differences. The results suggest that Pf_IL-12 and Pf_IL-23 subunits may play important roles in regulating immune responses to pathogens and T helper cell development.
Assuntos
Peixes-Gato , Infecções por Enterobacteriaceae , Doenças dos Peixes , Proteínas de Peixes , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Imunidade Inata , Subunidade p40 da Interleucina-12 , Animais , Peixes-Gato/genética , Peixes-Gato/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Proteínas de Peixes/química , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/veterinária , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Subunidade p40 da Interleucina-12/genética , Subunidade p40 da Interleucina-12/imunologia , Perfilação da Expressão Gênica/veterinária , Imunidade Inata/genética , Edwardsiella ictaluri/fisiologia , Subunidade p35 da Interleucina-12/genética , Subunidade p35 da Interleucina-12/imunologia , Filogenia , Sequência de Aminoácidos , Alinhamento de Sequência/veterinária , Subunidade p19 da Interleucina-23/genética , Subunidade p19 da Interleucina-23/imunologia , Poli I-C/farmacologiaRESUMO
INTRODUCTION: Optimizing treatment with biological agents is an ideal goal for patients with ulcerative colitis (UC). Recent data suggest that mucosal inflammation patterns and serum cytokine profiles differ between patients who respond and those who do not. Ustekinumab, a monoclonal antibody targeting the p40 subunit of interleukin (IL)-12 and IL-23, has shown promise, but predicting treatment response remains a challenge. We aimed to identify prognostic markers of response to ustekinumab in patients with active UC, utilizing information from their mucosal transcriptome. METHODS: We performed a prospective observational study of 36 UC patients initiating treatment with ustekinumab. Colonic mucosal biopsies were obtained before treatment initiation for a gene expression analysis using a microarray panel of 84 inflammatory genes. A differential gene expression analysis (DGEA), correlation analysis, and network centrality analysis on co-expression networks were performed to identify potential biomarkers. Additionally, machine learning (ML) models were employed to predict treatment response based on gene expression data. RESULTS: Seven genes, including BCL6, CXCL5, and FASLG, were significantly upregulated, while IL23A and IL23R were downregulated in non-responders compared to responders. The co-expression analysis revealed distinct patterns between responders and non-responders, with key genes like BCL6 and CRP highlighted in responders and CCL11 and CCL22 in non-responders. The ML algorithms demonstrated a high predictive power, emphasizing the significance of the IL23R, IL23A, and BCL6 genes. CONCLUSIONS: Our study identifies potential biomarkers associated with ustekinumab response in UC patients, shedding light on its underlying mechanisms and variability in treatment outcomes. Integrating transcriptomic approaches, including gene expression analyses and ML, offers valuable insights for personalized treatment strategies and highlights avenues for further research to enhance therapeutic outcomes for patients with UC.
Assuntos
Colite Ulcerativa , Biologia Computacional , Ustekinumab , Humanos , Ustekinumab/uso terapêutico , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/genética , Masculino , Feminino , Biologia Computacional/métodos , Adulto , Pessoa de Meia-Idade , Resultado do Tratamento , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Estudos Prospectivos , Transcriptoma , Perfilação da Expressão Gênica/métodos , Subunidade p19 da Interleucina-23/genética , Subunidade p19 da Interleucina-23/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Mucosa Intestinal/efeitos dos fármacos , Aprendizado de Máquina , PrognósticoRESUMO
Interleukin (IL)-23 is implicated in the pathogenesis of several inflammatory diseases and is usually linked with helper T cell (Th17) biology. However, there is some data linking IL-23 with innate immune biology in such diseases. We therefore examined the effects of IL-23p19 genetic deletion and/or neutralization on in vitro macrophage activation and in an innate immune-driven peritonitis model. We report that endogenous IL-23 was required for maximal macrophage activation by zymosan as determined by pro-inflammatory cytokine production, including a dramatic upregulation of granulocyte-colony stimulating factor (G-CSF). Furthermore, both IL-23p19 genetic deletion and neutralization in zymosan-induced peritonitis (ZIP) led to a specific reduction in the neutrophil numbers, as well as a reduction in the G-CSF levels in exudate fluids. We conclude that endogenous IL-23 can contribute significantly to macrophage activation during an inflammatory response, mostly likely via an autocrine/paracrine mechanism; of note, endogenous IL-23 can directly up-regulate macrophage G-CSF expression, which in turn is likely to contribute to the regulation of IL-23-dependent neutrophil number and function during an inflammatory response, with potential significance for IL-23 targeting particularly in neutrophil-associated inflammatory diseases.
Assuntos
Inflamação , Interleucina-23 , Células Mieloides , Neutrófilos , Zimosan , Animais , Inflamação/metabolismo , Inflamação/imunologia , Interleucina-23/metabolismo , Camundongos , Neutrófilos/metabolismo , Neutrófilos/imunologia , Células Mieloides/metabolismo , Peritonite/metabolismo , Peritonite/imunologia , Camundongos Endogâmicos C57BL , Fator Estimulador de Colônias de Granulócitos/metabolismo , Ativação de Macrófagos , Macrófagos/metabolismo , Macrófagos/imunologia , Subunidade p19 da Interleucina-23/metabolismo , Subunidade p19 da Interleucina-23/genética , Camundongos KnockoutRESUMO
OBJECTIVE: Interleukin-23 (IL-23) is a crucial cytokine implicated in chronic inflammation and autoimmunity, associated with various diseases such as psoriasis, psoriatic arthritis, and systemic lupus erythematosus (SLE). This study aimed to create and characterize a transgenic mouse model overexpressing human IL-23A (TghIL-23A), providing a valuable tool for investigating the pathogenic role of human IL-23A and evaluating the efficacy of anti-human IL-23A therapeutics. METHODS: TghIL-23A mice were generated via microinjection of CBA × C57BL/6 zygotes with a fragment of the human IL23A gene, flanked by its 5'-regulatory sequences and the 3' untranslated region of human ß-globin. The TghIL-23A pathology was assessed through hematologic and biochemic analyses, cytokine and antinuclear antibody detection, and histopathologic examination of skin and renal tissues. The response to the anti-human IL-23A therapeutic agent guselkumab was evaluated in groups of eight mixed-sex mice receiving subcutaneous treatment twice weekly for 10 weeks using clinical, biomarker, and histopathologic readouts. RESULTS: TghIL-23A mice exhibited interactions between human IL-23A and mouse IL-23/IL-12p40 and developed a chronic multiorgan autoimmune disease marked by proteinuria, anti-double-stranded DNA antibodies, severe inflammatory lesions in the skin, and milder phenotypes in the kidneys and lungs. The TghIL-23A pathologic features exhibited significant similarities to those observed in human patients with SLE, and they were reversed following guselkumab treatment. CONCLUSION: We have generated and characterized a novel genetic mouse model of SLE, providing proof-of-concept for the etiopathogenic role of human IL-23A. This new model has a normal life span and integrates several characteristics of the human disease's complexity and chronicity, making it an attractive preclinical tool for studying IL-23-dependent pathogenic mechanisms and assessing the efficacy of anti-human IL-23A or modeled disease-related therapeutics.
Assuntos
Modelos Animais de Doenças , Subunidade p19 da Interleucina-23 , Lúpus Eritematoso Sistêmico , Camundongos Transgênicos , Animais , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Camundongos , Subunidade p19 da Interleucina-23/genética , Subunidade p19 da Interleucina-23/imunologia , Humanos , Feminino , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Camundongos Endogâmicos C57BL , Masculino , Anticorpos Antinucleares/imunologia , Camundongos Endogâmicos CBARESUMO
The interleukin-23 (IL-23)/IL-17 immune axis has been linked to the pathology of psoriasis, but how this axis contributes to skin inflammation in this disease remains unclear. We measured inflammatory cytokines associated with the IL-23/IL-17 immune axis in the serum of patients with psoriasis using enzyme-linked immunosorbent assays. Psoriasis was induced in male C57BL/6J mice using imiquimod (IMQ) cream, and animals received intraperitoneal injections of recombinant mouse anti-IL-23A or anti-IL-17A antibodies for 7 days. The potential effects of the IL-23/IL-17 immune axis on skin inflammation were assessed based on pathology scoring, hematoxylin-eosin staining of skin samples, and quantitation of inflammatory cytokines. Western blotting was used to evaluate levels of the following factors in skin: ACT1, TRAF6, TAK1, NF-κB, and pNF-κB. The serum of psoriasis patients showed elevated levels of several cytokines involved in the IL-23/IL-17 immune axis: IL-2, IL-4, IL-8, IL-12, IL-17, IL-22, IL-23, and interferon-γ. Levels of IL-23p19 and IL-17 were increased in serum and skin of IMQ-treated mice, while ACT1, TRAF6, TAK1, NF-κB, and pNF-κB were upregulated in the skin. A large proportion of NF-κB p65 localized in nucleus of involucrin+ cells in the epidermis and in F4/80+ cells of the dermis of psoriatic lesional skin. Treating these animals with anti-IL-23 or anti-IL-17 antibodies improved pathological score and immune imbalance, mitigated skin inflammation and downregulated ACT1, TRAF6, TAK1, NF-κB, and pNF-κB in skin. Our results suggest that skin inflammation mediated by the IL-23/IL-17 immune axis in psoriasis involves activation of the ACT1/TRAF6/TAK1/NF-κB pathway in keratinocytes and macrophage.
Assuntos
Imiquimode , Interleucina-17 , Interleucina-23 , NF-kappa B , Psoríase , Animais , Masculino , Camundongos , Citocinas/metabolismo , Modelos Animais de Doenças , Imiquimode/efeitos adversos , Inflamação/patologia , Interleucina-23/genética , Subunidade p19 da Interleucina-23/genética , Subunidade p19 da Interleucina-23/metabolismo , Queratinócitos/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , NF-kappa B/genética , NF-kappa B/metabolismo , Psoríase/patologia , Pele/patologia , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Interleucina-17/metabolismoRESUMO
CONTEXT: Teprotumumab, an IGF-I receptor (IGF-IR) inhibitor, is effective in thyroid-associated ophthalmopathy (TAO). The drug can modulate induction by TSH of IL-6 and IL-8 in CD34+â fibrocytes and their putative derivatives, CD34+â orbital fibroblasts (CD34+â OF). Fibrocytes express multiple thyroid autoantigens and cytokines implicated in TAO, which are downregulated by Slit2. Inflammation and disordered hyaluronan (HA) accumulation occur in TAO. Whether teprotumumab alters these processes directly in fibrocytes/CD34+â OF remains uncertain. OBJECTIVE: Determine teprotumumab effects on expression/synthesis of several TAO-relevant molecules in fibrocytes and GD-OF. DESIGN/SETTING/PARTICIPANTS: Patients with TAO and healthy donors were recruited from an academic endocrine and oculoplastic practice. MAIN OUTCOME MEASURES: Real-time PCR, specific immunoassays. RESULTS: Teprotumumab attenuates basal and TSH-inducible autoimmune regulator protein, thyroglobulin, sodium iodide symporter, thyroperoxidase, IL-10, and B-cell activating factor levels in fibrocytes. It downregulates IL-23p19 expression/induction while enhancing IL-12p35, intracellular and secreted IL-1 receptor antagonists, and Slit2. These effects are mirrored by linsitinib. HA production is marginally enhanced by teprotumumab, the consequence of enhanced HAS2 expression. CONCLUSION: Teprotumumab affects specific gene expression in fibrocytes and GD-OF in a target-specific, nonmonolithic manner, whereas IGF-IR control of these cells appears complex. The current results suggest that the drug may act on cytokine expression and HA production systemically and locally, within the TAO orbit. These findings extend our insights into the mechanisms through which IGF-IR inhibition might elicit clinical responses in TAO, including a potential role of Slit2 in attenuating inflammation and tissue remodeling.
Assuntos
Oftalmopatia de Graves , Anticorpos Monoclonais Humanizados , Autoantígenos/metabolismo , Fator Ativador de Células B/genética , Fator Ativador de Células B/metabolismo , Fator Ativador de Células B/farmacologia , Células Cultivadas , Fibroblastos/metabolismo , Expressão Gênica , Oftalmopatia de Graves/tratamento farmacológico , Oftalmopatia de Graves/genética , Humanos , Ácido Hialurônico/metabolismo , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/metabolismo , Interleucina-10/metabolismo , Subunidade p35 da Interleucina-12/genética , Subunidade p35 da Interleucina-12/metabolismo , Subunidade p35 da Interleucina-12/farmacologia , Subunidade p19 da Interleucina-23/genética , Subunidade p19 da Interleucina-23/metabolismo , Subunidade p19 da Interleucina-23/farmacologia , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Órbita/metabolismo , Receptor IGF Tipo 1/genética , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Tireoglobulina/genética , Tireotropina/metabolismoRESUMO
Interleukin (IL) 23 (p19/p40) plays a critical role in the pathogenesis of psoriasis and is upregulated in psoriasis skin lesions. In clinical practice, anti-IL-23Ap19 antibodies are highly effective against psoriasis. IL-39 (p19/ Epstein-Barr virus-induced (EBI) 3), a newly discovered cytokine in 2015, shares the p19 subunit with IL-23. Anti-IL-23Ap19 antibodies may bind to IL-39; also, the cytokine may contribute to the pathogenesis of psoriasis. To investigate IL23Ap19- and/or EBI3-including cytokines in psoriatic keratinocytes, we analyzed IL-23Ap19 and EBI3 expressions in psoriasis skin lesions, using immunohistochemistry and normal human epidermal keratinocytes (NHEKs) stimulated with inflammatory cytokines, using quantitative real-time polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and liquid chromatography-electrospray tandem mass spectrometry (LC-Ms/Ms). Immunohistochemical analysis showed that IL-23Ap19 and EBI3 expressions were upregulated in the psoriasis skin lesions. In vitro, these expressions were synergistically induced by the triple combination of tumor necrosis factor (TNF)-α, IL-17A, and interferon (IFN)-γ, and suppressed by dexamethasone, vitamin D3, and acitretin. In ELISA and LC-Ms/Ms analyses, keratinocyte-derived IL-23Ap19 and EBI3, but not heterodimeric forms, were detected with humanized anti-IL-23Ap19 monoclonal antibodies, tildrakizumab, and anti-EBI3 antibodies, respectively. Psoriatic keratinocytes may express IL-23Ap19 and EBI3 proteins in a monomer or homopolymer, such as homodimer or homotrimer.
Assuntos
Subunidade p19 da Interleucina-23/metabolismo , Interleucinas/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Psoríase/imunologia , Regulação para Cima , Anticorpos Monoclonais Humanizados/farmacologia , Linhagem Celular , Cromatografia Líquida , Citocinas/genética , Citocinas/metabolismo , Humanos , Subunidade p19 da Interleucina-23/genética , Interleucinas/genética , Queratinócitos/imunologia , Antígenos de Histocompatibilidade Menor/genética , Psoríase/genética , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Regulação para Cima/efeitos dos fármacosRESUMO
C-reactive protein (CRP) is an acute-phase protein in humans that is produced in high quantities by the liver upon infection and under inflammatory conditions. Although CRP is commonly used as a marker of inflammation, CRP can also directly contribute to inflammation by eliciting pro-inflammatory cytokine production by immune cells. Since CRP is highly elevated in serum under inflammatory conditions, we have studied the CRP-induced cytokine profile of human monocytes, one of the main innate immune cell populations in blood. We identified that CRP is relatively unique in its capacity to induce production of the pro-inflammatory cytokine IL-23, which was in stark contrast to a wide panel of pattern recognition receptor (PRR) ligands. We show that CRP-induced IL-23 production was mediated at the level of gene transcription, since CRP particularly promoted gene transcription of IL23A (encoding IL-23p19) instead of IL12A (encoding IL-12p35), while PRR ligands induce the opposite response. Interestingly, when CRP stimulation was combined with PRR ligand stimulation, as for example, occurs in the context of sepsis, IL-23 production by monocytes was strongly reduced. Combined, these data identify CRP as a unique individual ligand to induce IL-23 production by monocytes, which may contribute to shaping systemic immune responses under inflammatory conditions.
Assuntos
Proteína C-Reativa/metabolismo , Subunidade p19 da Interleucina-23/metabolismo , Monócitos/metabolismo , Células Cultivadas , Humanos , Subunidade p19 da Interleucina-23/genética , RNA Mensageiro/genética , Ativação TranscricionalRESUMO
Melanoma differentiation associated gene-9 (MDA-9), Syntenin-1, or syndecan binding protein is a differentially regulated prometastatic gene with elevated expression in advanced stages of melanoma. MDA-9/Syntenin expression positively associates with advanced disease stage in multiple histologically distinct cancers and negatively correlates with patient survival and response to chemotherapy. MDA-9/Syntenin is a highly conserved PDZ-domain scaffold protein, robustly expressed in a spectrum of diverse cancer cell lines and clinical samples. PDZ domains interact with a number of proteins, many of which are critical regulators of signaling cascades in cancer. Knockdown of MDA-9/Syntenin decreases cancer cell metastasis, sensitizing these cells to radiation. Genetic silencing of MDA-9/Syntenin or treatment with a pharmacological inhibitor of the PDZ1 domain, PDZ1i, also activates the immune system to kill cancer cells. Additionally, suppression of MDA-9/Syntenin deregulates myeloid-derived suppressor cell differentiation via the STAT3/interleukin (IL)-1ß pathway, which concomitantly promotes activation of cytotoxic T lymphocytes. Biologically, PDZ1i treatment decreases metastatic nodule formation in the lungs, resulting in significantly fewer invasive cancer cells. In summary, our observations indicate that MDA-9/Syntenin provides a direct therapeutic target for mitigating aggressive breast cancer and a small-molecule inhibitor, PDZ1i, provides a promising reagent for inhibiting advanced breast cancer pathogenesis.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Interleucina-1beta/genética , Neoplasias Pulmonares/tratamento farmacológico , Oxidiazóis/farmacologia , Pirimidinas/farmacologia , Sinteninas/genética , Animais , Antineoplásicos/síntese química , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Quimiocina CCL11/genética , Quimiocina CCL11/imunologia , Quimiocina CCL17/genética , Quimiocina CCL17/imunologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-1alfa/genética , Interleucina-1alfa/imunologia , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/imunologia , Subunidade p19 da Interleucina-23/genética , Subunidade p19 da Interleucina-23/imunologia , Interleucina-5/genética , Interleucina-5/imunologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos BALB C , Oxidiazóis/síntese química , Pirimidinas/síntese química , Transdução de Sinais , Sinteninas/antagonistas & inibidores , Sinteninas/imunologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/patologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Among various cytokines, interleukin (IL)-12 family cytokines have very unique characteristics in that they are composed of two distinct subunits and these subunits are shared with each other. IL-23, one of the IL-12 family cytokines, consists of p19 and p40 subunits, is mainly produced by antigen-presenting cells, and plays a critical role in the expansion and maintenance of pathogenic helper CD4+ T (Th)17 cells. Since we initially found that p19 is secreted in the culture supernatant of activated CD4+ T cells, we have further investigated the role of p19. p19 was revealed to associate with CD5 antigen-like (CD5L), which is a repressor of Th17 pathogenicity and is highly expressed in non-pathogenic Th17 cells, to form a composite p19/CD5L. This p19/CD5L was shown to activate STAT5 and enhance the differentiation into granulocyte macrophage colony-stimulating factor (GM-CSF)-producing CD4+ T cells. Both CD4+ T cell-specific conditional p19-deficient mice and complete CD5L-deficient mice showed significantly alleviated experimental autoimmune encephalomyelitis (EAE) with reduced frequency of GM-CSF+CD4+ T cells. During the course of EAE, the serum level of p19/CD5L, but not CD5L, correlated highly with the clinical symptoms. Thus, the composite p19/CD5L is a possible novel heterodimeric cytokine that contributes to EAE development with GM-CSF up-regulation.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Antígenos CD5/genética , Encefalomielite Autoimune Experimental/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Subunidade p19 da Interleucina-23/genética , Receptores Depuradores/genética , Animais , Células Apresentadoras de Antígenos/imunologia , Proteínas Reguladoras de Apoptose/imunologia , Linfócitos T CD4-Positivos/imunologia , Antígenos CD5/imunologia , Antígenos CD5/ultraestrutura , Dimerização , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Humanos , Subunidade p19 da Interleucina-23/imunologia , Subunidade p19 da Interleucina-23/ultraestrutura , Camundongos , Receptores Depuradores/imunologia , Células Th1/imunologia , Células Th17/imunologiaRESUMO
OBJECTIVE: ZAP-70W163C BALB/c (SKG) mice develop reactive arthritis (ReA) following infection with Chlamydia muridarum. Since intracellular pathogens enhance their replicative fitness in stressed host cells, we examined how myeloid cells infected with C muridarum drive arthritis. METHODS: SKG, Il17a-deficient SKG, and BALB/c female mice were infected with C muridarum or C muridarum luciferase in the genitals. C muridarum dissemination was assessed by in vivo imaging or genomic DNA amplification. Macrophages were depleted using clodronate liposomes. Anti-tumor necrosis factor (anti-TNF) and anti-interleukin-23p19 (anti-IL-23p19) were administered after infection or arthritis onset. Gene expression of Hspa5, Tgtp1, Il23a, Il17a, Il12b, and Tnf was compared in SKG mice and BALB/c mice. RESULTS: One week following infection with C muridarum, macrophages and neutrophils were observed to have infiltrated the uteri of mice and were also shown to have carried C muridarum DNA to the spleen. C muridarum load was higher in SKG mice than in BALB/c mice. Macrophage depletion was shown to reduce C muridarum load and prevent development of arthritis. Compared with BALB/c mice, expression of Il23a and Il17a was increased in the uterine and splenic neutrophils of SKG mice. The presence of anti-IL-23p19 during infection or Il17a deficiency suppressed arthritis. Tnf was overexpressed in the joints of SKG mice within 1 week postinfection, and persisted beyond the first week. TNF inhibition during infection or at arthritis onset suppressed the development of arthritis. Levels of endoplasmic reticulum stress were constitutively increased in the joints of SKG mice but were induced, in conjunction with immunity-related GTPase, by C muridarum infection in the uterus. CONCLUSION: C muridarum load is higher in SKG mice than in BALB/c mice. Whereas proinflammatory IL-23 produced by neutrophils contributes to the initiation of C muridarum-mediated ReA, macrophage depletion reduces C muridarum dissemination to other tissues, tissue burden, and the development of arthritis. TNF inhibition was also shown to suppress arthritis development. Our data suggest that enhanced bacterial dissemination in macrophages of SKG mice drives the TNF production needed for persistent arthritis.
Assuntos
Artrite Reativa/imunologia , Infecções por Chlamydia/imunologia , Subunidade p19 da Interleucina-23/imunologia , Interleucina-23/imunologia , Macrófagos/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Artrite Experimental/genética , Artrite Reativa/genética , Chlamydia muridarum , Chaperona BiP do Retículo Endoplasmático , Feminino , Perfilação da Expressão Gênica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/imunologia , Subunidade p40 da Interleucina-12/genética , Subunidade p40 da Interleucina-12/imunologia , Interleucina-17/genética , Interleucina-17/imunologia , Subunidade p19 da Interleucina-23/genética , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/imunologia , Fator de Necrose Tumoral alfa/genética , Proteína-Tirosina Quinase ZAP-70/genéticaRESUMO
OBJECTIVE: Dysregulated immune responses are the cause of IBDs. Studies in mice and humans suggest a central role of interleukin (IL)-23-producing mononuclear phagocytes in disease pathogenesis. Mechanistic insights into the regulation of IL-23 are prerequisite for selective IL-23 targeting therapies as part of personalised medicine. DESIGN: We performed transcriptomic analysis to investigate IL-23 expression in human mononuclear phagocytes and peripheral blood mononuclear cells. We investigated the regulation of IL-23 expression and used single-cell RNA sequencing to derive a transcriptomic signature of hyperinflammatory monocytes. Using gene network correlation analysis, we deconvolved this signature into components associated with homeostasis and inflammation in patient biopsy samples. RESULTS: We characterised monocyte subsets of healthy individuals and patients with IBD that express IL-23. We identified autosensing and paracrine sensing of IL-1α/IL-1ß and IL-10 as key cytokines that control IL-23-producing monocytes. Whereas Mendelian genetic defects in IL-10 receptor signalling induced IL-23 secretion after lipopolysaccharide stimulation, whole bacteria exposure induced IL-23 production in controls via acquired IL-10 signalling resistance. We found a transcriptional signature of IL-23-producing inflammatory monocytes that predicted both disease and resistance to antitumour necrosis factor (TNF) therapy and differentiated that from an IL-23-associated lymphocyte differentiation signature that was present in homeostasis and in disease. CONCLUSION: Our work identifies IL-10 and IL-1 as critical regulators of monocyte IL-23 production. We differentiate homeostatic IL-23 production from hyperinflammation-associated IL-23 production in patients with severe ulcerating active Crohn's disease and anti-TNF treatment non-responsiveness. Altogether, we identify subgroups of patients with IBD that might benefit from IL-23p19 and/or IL-1α/IL-1ß-targeting therapies upstream of IL-23.
Assuntos
Resistência a Medicamentos/genética , Doenças Inflamatórias Intestinais/genética , Interleucina-10/genética , Subunidade p19 da Interleucina-23/biossíntese , Subunidade p19 da Interleucina-23/genética , Monócitos/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Comunicação Autócrina , Células Cultivadas , Feminino , Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Homeostase/genética , Humanos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Interleucina-10/metabolismo , Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Lipopolissacarídeos , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Comunicação Parácrina , Receptores de Interleucina-10/antagonistas & inibidores , Receptores de Interleucina-10/metabolismo , Transdução de Sinais/genética , Transcriptoma , Fator de Necrose Tumoral alfa/efeitos adversos , Adulto JovemRESUMO
Members of the IL-6/IL-12 cytokine family are critical regulators of innate and adaptive immunity and have emerged as key players controlling inflammatory and autoimmune disorders. This cytokine family comprises of IL-12, IL-23, IL-27, and IL-35, each consisting of distinct α- and ß-cytokine subunits that form heterodimers. A new member of this family, IL-39, was identified in the murine species and was shown to consist of the IL-23p19 and Epstein-Barr Virus-induced 3 (EBI3) subunits. Subsequently, it was shown that IL-39 was implicated in the immunopathogenesis of murine experimental lupus erythematosus. The existence of IL-39 in the human system has yet to be confirmed. Based on the clinical success of IL-23p19 neutralizing approaches in moderate-to-severe psoriasis, anti-IL-23p19 antibodies in the clinic may not only neutralize IL-23, but additionally IL-39, implying that IL-39 might also contribute to the pathogenesis of psoriasis. It is therefore pivotal to demonstrate IL-39 expression and to characterize its function in the human system. In this study, we provided evidence for the existence of secreted heterodimeric p19 and EBI3 complexes in supernatants originating from p19 and EBI3 transfected HEK293FT cells. We attempted to detect IL-39 expression from stimulated human primary B cells, human keratinocytes and in vitro polarized human macrophages. Whereas, the expression of p19 and EBI3 mRNA was elevated, we failed to detect p19 and EBI3 heterodimers. Functional assays were conducted with conditioned media containing human IL-39 or with a human recombinant IL-39 Fc protein. Immune cells targeted by IL-39 in mouse, such as neutrophils and PBMCs, did not respond to human IL-39 stimulation and IL-39 failed to activate STAT3 in a reporter cell line. These results suggest that, while the secretion of p19/EBI3 complexes can be forced in human cells, it is secreted below the lower quantity of detection or it has no functional role.
Assuntos
Imunidade Adaptativa/genética , Imunidade Inata/genética , Subunidade p19 da Interleucina-23/genética , Interleucinas/genética , Antígenos de Histocompatibilidade Menor/genética , Receptores de Citocinas/genética , Animais , Humanos , Subunidade p19 da Interleucina-23/imunologia , Interleucinas/imunologia , Camundongos , Antígenos de Histocompatibilidade Menor/imunologia , Neutrófilos/imunologia , Receptores de Citocinas/imunologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologiaRESUMO
The calcium-sensing receptor (CaSR) is a ubiquitously expressed multifunctional G protein-coupled receptor. Several studies reported that the CaSR plays an anti-inflammatory and anti-tumorigenic role in the intestine, and that it is down-regulated during colorectal carcinogenesis. We hypothesized that positive allosteric CaSR modulators (type II calcimimetics) selectively targeting the intestinal cells could be used for the treatment of intestinal pathologies. Therefore, the aim of this study was to determine the effect of pharmacological stimulation of CaSR on gene expression in vitro and on tumor growth in vivo. We stably transduced two colon cancer cell lines (HT29 and Caco2) with lentiviral vectors containing either the CaSR fused to GFP or GFP only. Using RNA sequencing, RT-qPCR experiments and ELISA, we determined that CaSR over-expression itself had generally little effect on gene expression in these cells. However, treatment with 1 µM of the calcimimetic NPS R-568 increased the expression of pro-inflammatory factors such as IL-23α and IL-8 and reduced the transcription of various differentiation markers in the cells over-expressing the CaSR. In vivo, neither the presence of the CaSR nor p.o. treatment of the animals with the calcimimetic cinacalcet affected tumor growth, tumor cell proliferation or tumor vascularization of murine HT29 xenografts. In summary, CaSR stimulation in CaSR over-expressing cells enhanced the expression of inflammatory markers in vitro, but was not able to repress colorectal cancer tumorigenicity in vivo. These findings suggest potential pro-inflammatory effects of the CaSR and type II calcimimetics in the intestine.
Assuntos
Calcimiméticos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Receptores de Detecção de Cálcio/genética , Receptores Acoplados a Proteínas G/genética , Animais , Células CACO-2 , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Xenoenxertos , Humanos , Subunidade p19 da Interleucina-23/genética , Interleucina-8/genética , Camundongos , Fenetilaminas/farmacologia , Propilaminas/farmacologiaRESUMO
Indoleamine 2,3-dioxygenase 1 (IDO1) is an enzyme known to suppress immune responses, and several reports have showed that it is associated with psoriasis. IDO2 is an isoform of IDO1, recently identified as a catalytic enzyme in the tryptophan-kynurenine pathway, which is expressed in dendritic cells and monocytes. The expression of IDO2 in immune cells suggests that IDO2 may contribute to immune functions. However, the role of IDO2 in the pathogenesis of psoriasis remains unclear. In this study, to elucidate the role of IDO2 in psoriasis, we assessed imiquimod (IMQ)-induced psoriasis-like dermatitis in IDO2 knockout (KO) mice. Skin inflammation, evaluated by scoring erythema, scaling, and ear thickness, was significantly worse in the IDO2 KO mice than in the wild-type (WT) mice. The mRNA expression levels of TNF-α, IL-23p19, and IL-17A, key cytokines involved in the development of psoriasis, were also increased in the IDO2 KO mice. Furthermore, immunohistochemistry revealed that the number of Ki67-positive cells in the epidermis and CD4-, CD8-, and IL-17-positive lymphocytes infiltrating the dermis were significantly increased in the IDO2 KO mice. These results suggest that IDO2 might decrease IL-17 expression, thereby resulting in the suppression of skin inflammation in IMQ-induced psoriasis-like dermatitis.
Assuntos
Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Inflamação/metabolismo , Psoríase/metabolismo , Pele/metabolismo , Animais , Citocinas/genética , Citocinas/metabolismo , Dermatite/genética , Dermatite/metabolismo , Feminino , Regulação da Expressão Gênica , Imiquimode , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Inflamação/genética , Interleucina-17/genética , Interleucina-17/metabolismo , Subunidade p19 da Interleucina-23/genética , Subunidade p19 da Interleucina-23/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Psoríase/induzido quimicamente , Psoríase/genética , Pele/patologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Interleukin (IL)-12 and IL-23 belong to the IL-12 type family and are composite cytokines, consisting of the common ß subunit p40 and the specific cytokine α subunit p35 and p19, respectively. IL-12 signals via the IL-12Rß1·IL-12Rß2 receptor complex, and IL-23 uses also IL-12Rß1 but engages IL-23R as second receptor. Importantly, binding of IL-12 and IL-23 to IL-12Rß1 is mediated by p40, and binding to IL-12Rß2 and IL-23R is mediated by p35 and p19, respectively. Previously, we have identified a W157A substitution at site 3 of murine IL-23p19 that abrogates binding to murine IL-23R. Here, we demonstrate that the analogous Y185R site 3 substitution in murine and Y189R site 3 substitution in human IL-12p35 abolishes binding to IL-12Rß2 in a cross-species manner. Although Trp157 is conserved between murine and human IL-23p19 (Trp156 in the human ortholog), the site 3 W156A substitution in hIL-23p19 did not affect signaling of cells expressing human IL-12Rß1 and IL-23R, suggesting that the interface of murine IL-23p19 required for binding to IL-23R is different from that in the human ortholog. Hence, we introduced additional hIL-23p19 substitutions within its binding interface to hIL-23R and found that the combined site 3 substitutions of W156A and L160E, which become buried at the complex interface, disrupt binding of hIL-23p19 to hIL-23R. In summary, we have identified substitutions in IL-12p35 and IL-23p19 that disrupt binding to their cognate receptors IL-12Rß2 and IL-23R in a murine/human cross-species manner.
Assuntos
Subunidade p40 da Interleucina-12 , Subunidade p19 da Interleucina-23 , Receptores de Interleucina-12 , Receptores de Interleucina , Substituição de Aminoácidos , Animais , Células CHO , Células COS , Chlorocebus aethiops , Cricetulus , Células HEK293 , Humanos , Subunidade p40 da Interleucina-12/química , Subunidade p40 da Interleucina-12/genética , Subunidade p40 da Interleucina-12/metabolismo , Subunidade p19 da Interleucina-23/química , Subunidade p19 da Interleucina-23/genética , Subunidade p19 da Interleucina-23/metabolismo , Camundongos , Mutação de Sentido Incorreto , Ligação Proteica , Receptores de Interleucina/química , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Receptores de Interleucina-12/química , Receptores de Interleucina-12/genética , Receptores de Interleucina-12/metabolismoRESUMO
Interleukin 23 (IL-23) triggers pathogenic features in pro-inflammatory, IL-17-secreting T cells (Th17 and Tγδ17) that play a key role in the development of inflammatory diseases. However, the IL-23 signaling cascade remains largely undefined. Here, we used quantitative phosphoproteomics to characterize IL-23 signaling in primary murine Th17 cells. We quantified 6,888 phosphorylation sites in Th17 cells and found 168 phosphorylations regulated upon IL-23 stimulation. IL-23 increased the phosphorylation of the myosin regulatory light chain (RLC), an actomyosin contractibility marker, in Th17 and Tγδ17 cells. IL-23-induced RLC phosphorylation required Janus kinase 2 (JAK2) and Rho-associated protein kinase (ROCK) catalytic activity, and further study of the IL-23/ROCK connection revealed an unexpected role of IL-23 in the migration of Tγδ17 and Th17 cells through ROCK activation. In addition, pharmacological inhibition of ROCK reduced Tγδ17 recruitment to inflamed skin upon challenge with inflammatory agent Imiquimod. This work (i) provides new insights into phosphorylation networks that control Th17 cells, (ii) widely expands the current knowledge on IL-23 signaling, and (iii) contributes to the increasing list of immune cells subsets characterized by global phosphoproteomic approaches.