RESUMO
Therapeutic IL-12 has demonstrated the ability to reduce local immune suppression in preclinical models, but clinical development has been limited by severe inflammation-related adverse events with systemic administration. Here, we show that potent immunologic tumor control of established syngeneic carcinomas can be achieved by i.t. administration of a tumor-targeted IL-12 antibody fusion protein (NHS-rmIL-12) using sufficiently low doses to avoid systemic toxicity. Single-cell transcriptomic analysis and ex vivo functional assays of NHS-rmIL-12-treated tumors revealed reinvigoration and enhanced proliferation of exhausted CD8+ T lymphocytes, induction of Th1 immunity, and a decrease in Treg number and suppressive capacity. Similarly, myeloid cells transitioned toward inflammatory phenotypes and displayed reduced suppressive capacity. Cell type-specific IL-12 receptor-KO BM chimera studies revealed that therapeutic modulation of both lymphoid and myeloid cells is required for maximum treatment effect and tumor cure. Study of single-cell data sets from human head and neck carcinomas revealed IL-12 receptor expression patterns similar to those observed in murine tumors. These results describing the diverse mechanisms underlying tumor-directed IL-12-induced antitumor immunity provide the preclinical rationale for the clinical study of i.t. NHS-IL-12.
Assuntos
Carcinoma , Interleucina-12 , Animais , Linfócitos T CD8-Positivos , Interleucina-12/genética , Interleucina-12/metabolismo , Camundongos , Receptores de Interleucina-12/genética , Receptores de Interleucina-12/metabolismo , Linfócitos T ReguladoresRESUMO
Interleukin (IL)-12 and IL-23 are composite cytokines consisting of p35/p40 and p19/p40, respectively, which signal via the common IL-12 receptor ß1 (IL-12Rß1) and the cytokine-specific receptors IL-12Rß2 and IL-23R. Previous data showed that the p40 component interacts with IL-12Rß1, whereas p19 and p35 subunits solely bind to IL-23R and IL-12Rß2, resulting in tetrameric signaling complexes. In the absence of p19 and p35, p40 forms homodimers and may induce signaling via IL-12Rß1 homodimers. The critical amino acids of p19 and p35 required for binding to IL-23R and IL-12Rß2 are known, and two regions of p40 critical for binding to IL-12Rß1 have recently been identified. In order to characterize the involvement of the N-terminal region of p40 in binding to IL-12Rß1, we generated deletion variants of the p40-p19 fusion cytokine. We found that an N-terminal deletion variant missing amino acids M23 to P39 failed to induce IL-23-dependent signaling and did not bind to IL-12Rß1, whereas binding to IL-23R was maintained. Amino acid replacements showed that p40W37K largely abolished IL-23-induced signal transduction and binding to IL-12Rß1, but not binding to IL-23R. Combining p40W37K with D36K and T38K mutations eliminated the biological activity of IL-23. Finally, homodimeric p40D36K/W37K/T38K did not interact with IL-12Rß1, indicating binding of homodimeric p40 to IL-12Rß1 is comparable to the interaction of IL-23/IL-12 and IL-12Rß1. In summary, we have defined D36, W37, and T38 as hotspot amino acids for the interaction of IL-12/IL-23 p40 with IL-12Rß1. Structural insights into cytokine-cytokine receptor binding are important to develop novel therapeutic strategies.
Assuntos
Subunidade p40 da Interleucina-12 , Multimerização Proteica , Receptores de Interleucina-12 , Transdução de Sinais , Animais , Células CHO , Cricetulus , Células HEK293 , Humanos , Subunidade p40 da Interleucina-12/química , Subunidade p40 da Interleucina-12/genética , Subunidade p40 da Interleucina-12/metabolismo , Camundongos , Ligação Proteica , Receptores de Interleucina-12/química , Receptores de Interleucina-12/genética , Receptores de Interleucina-12/metabolismo , TriptofanoRESUMO
The receptors for IL-35, IL-12Rß2 and gp130, have been implicated in the inflammatory pathophysiology of autoimmune diseases. In this study, we set out to investigate the serum IL-35 levels and the surface levels of IL-12Rß2 and gp130 in CD3+CD4+, CD3+CD4â and CD3âCD4â lymphocyte subpopulations in systemic lupus erythematosus (SLE) patients (n=50) versus healthy controls (n=50). The potential T cell subsets associated with gp130 transcript (i.e. IL6ST) expression in CD4+ T cells of SLE patients was also examined in publicly-available gene expression profiling (GEP) datasets. Here, we report that serum IL-35 levels were significantly higher in SLE patients than healthy controls (p=0.038) but it was not associated with SLEDAI-2K scores. The proportions of IL-12Rß2+ and gp130+ cells in SLE patients did not differ significantly with those of healthy controls in all lymphocyte subpopulations investigated. Essentially, higher SLEDAI-2K scores were positively correlated with increased proportion of gp130+ cells, but not IL-12Rß2+ cells, on CD3+CD4+ T cells (r=0.425, p=0.002, q=0.016). Gene Set Enrichment Analysis (GSEA) of a GEP dataset of CD4+ T cells isolated from SLE patients (n=8; GSE4588) showed that IL6ST expression was positively associated with genes upregulated in CD4+ T cells vs myeloid or B cells (q<0.001). In an independent GEP dataset of CD4+ T cells isolated from SLE patients (n=9; GSE1057), IL6ST expression was induced upon anti-CD3 stimulation, and that Treg, TCM and CCR7+ T cells gene sets were significantly enriched (q<0.05) by genes highly correlated with IL6ST expression (n=92 genes; r>0.75 with IL6ST expression) upon anti-CD3 stimulation in these SLE patients. In conclusion, gp130 signaling in CD3+CD4+ T cell subsets may contribute to increased disease activity in SLE patients, and it represents a promising therapeutic target for inhibition in the disease.
Assuntos
Receptor gp130 de Citocina/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Subpopulações de Linfócitos T/imunologia , Adulto , Complexo CD3 , Antígenos CD4 , Feminino , Citometria de Fluxo , Humanos , Interleucinas/sangue , Interleucinas/metabolismo , Masculino , Pessoa de Meia-Idade , Receptores CCR7/metabolismo , Receptores de Interleucina-12/metabolismo , Índice de Gravidade de Doença , Linfócitos T ReguladoresRESUMO
Pathogenic conversion of Th17 cells into multifunctional helper T cells or Th1 cells contributes to the pathogenesis of autoimmune diseases; however, the mechanism regulating the plasticity of Th17 cells remains unclear. Here, we found that Th17 cells expressed latent TGF-ß1 in a manner dependent on autocrine TGF-ß1. By employing IL-17-producing cell-specific Tgfb1 conditional knockout and fate-mapping systems, we demonstrated that TGF-ß1-deficient Th17 cells are relatively susceptible to becoming IFN-γ producers through IL-12Rß2 and IL-27Rα upregulation. TGF-ß1-deficient Th17 cells exacerbated tissue inflammation compared to TGF-ß1-sufficient Th17 cells in adoptive transfer models of experimental autoimmune encephalomyelitis and colitis. Thus, TGF-ß1 production by Th17 cells provides an essential autocrine signal for maintaining the stability and regulating the pathogenicity of Th17 cells in vivo.
Assuntos
Autoimunidade , Imunomodulação , Células Th17/imunologia , Células Th17/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Comunicação Autócrina , Biomarcadores , Colite/etiologia , Colite/metabolismo , Colite/patologia , Suscetibilidade a Doenças , Encefalomielite Autoimune Experimental/etiologia , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Regulação da Expressão Gênica , Imunofenotipagem , Contagem de Linfócitos , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Receptores de Interleucina-12/genética , Receptores de Interleucina-12/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Fator de Crescimento Transformador beta1/genéticaRESUMO
Interleukin-35 (IL-35) regulates immune cell function in inflammation, infection, cancer, and autoimmune diseases. However, the modulatory activity of IL-35 exerted on T cells is not fully understood in Kawasaki disease. For this purpose, the present study included 28 patients with Kawasaki disease and 16 healthy controls. The mRNA levels of IL-35 receptor subunits, including IL-12Rß2 and gp130, were determined by conducting real-time PCR. CD4+ and CD8+ T cells were enriched, and stimulated with recombinant human IL-35. The influence of IL-35 on transcription factors and cytokine secretion by CD4+ T cells was assessed by performing real-time PCR and ELISA. The modulatory activity of IL-35 on CD8+ T cells was investigated by measuring target cell death, perforin/granzyme B secretion, and immune checkpoint molecule expression. IL-12Rß2 and gp130 mRNA levels were comparable in CD4+ and CD8+ T cells between patients with Kawasaki disease and controls. Patients with Kawasaki disease showed stronger Th1, Th17, and Th22 responses, but weaker Treg response compared with controls. IL-35 stimulation suppressed Th1, Th17, and Th22 responses but enhanced Treg response. Patients with Kawasaki disease showed elevated CD8+ T cell-induced cytotoxicity. IL-35 stimulation inhibited CD8+ T cell-induced target cell death. The downregulation of IFN-γ expression and perforin/granzyme B secretion, and the upregulation of PD-1, CTLA-4, and LAG-3 expression following IL-35 stimulation were responsible for decreased CD8+ T cell-induced cytotoxicity. IL-35 may play a pivotal immunosuppressive role in T cell function, which may be involved in the protective mechanism against inflammation in Kawasaki disease.
Assuntos
Interleucinas/imunologia , Síndrome de Linfonodos Mucocutâneos/imunologia , Linfócitos T/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Criança , Pré-Escolar , Receptor gp130 de Citocina/genética , Receptor gp130 de Citocina/metabolismo , Citocinas/metabolismo , Citotoxicidade Imunológica , Feminino , Granzimas/metabolismo , Humanos , Proteínas de Checkpoint Imunológico/metabolismo , Masculino , Perforina/metabolismo , Receptores de Interleucina-12/genética , Receptores de Interleucina-12/metabolismoRESUMO
Interleukin-12 (IL-12) and IL-23 are heterodimeric cytokines that are produced by antigen-presenting cells to regulate the activation and differentiation of lymphocytes, and they share IL-12Rß1 as a receptor signaling subunit. We present a crystal structure of the quaternary IL-23 (IL-23p19/p40)/IL-23R/IL-12Rß1 complex, together with cryoelectron microscopy (cryo-EM) maps of the complete IL-12 (IL-12p35/p40)/IL-12Rß2/IL-12Rß1 and IL-23 receptor (IL-23R) complexes, which reveal "non-canonical" topologies where IL-12Rß1 directly engages the common p40 subunit. We targeted the shared IL-12Rß1/p40 interface to design a panel of IL-12 partial agonists that preserved interferon gamma (IFNγ) induction by CD8+ T cells but impaired cytokine production from natural killer (NK) cells in vitro. These cell-biased properties were recapitulated in vivo, where IL-12 partial agonists elicited anti-tumor immunity to MC-38 murine adenocarcinoma absent the NK-cell-mediated toxicity seen with wild-type IL-12. Thus, the structural mechanism of receptor sharing used by IL-12 family cytokines provides a protein interface blueprint for tuning this cytokine axis for therapeutics.
Assuntos
Interleucina-12/química , Interleucina-12/metabolismo , Células Matadoras Naturais/metabolismo , Receptores de Interleucina/química , Receptores de Interleucina/metabolismo , Linfócitos T/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Microscopia Crioeletrônica , Cristalografia por Raios X , Epitopos/imunologia , Feminino , Células HEK293 , Humanos , Imunidade , Interleucina-12/agonistas , Subunidade p40 da Interleucina-12/química , Subunidade p40 da Interleucina-12/metabolismo , Camundongos Endogâmicos C57BL , Modelos Moleculares , Neoplasias/imunologia , Neoplasias/patologia , Estrutura Quaternária de Proteína , Receptores de Interleucina/ultraestrutura , Receptores de Interleucina-12/metabolismo , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
This study aimed to explore the expressions of interleukin-12 (IL-12) and its receptors IL-23R and IL12RB2 in patients with lumbar disc herniation (LDH) before and after treatment and their relationship with clinical efficacy. A total of 172 LDH patients undergoing surgical treatment in Wuhan Third Hospital, Tongren Hospital of Wuhan University were enrolled as the study group, and 170 healthy subjects as the control group. 5 mL of fasting venous blood was taken before surgery (T0), 1 d (T1), 3 d (T2), 5 d (T3) and 7 d (T4) after treatment respectively. The concentrations of IL-12, IL-23R and IL12RB2 in the two groups were detected, and the correlation between them and the treatment duration and clinical efficacy was analyzed. The study group showed significantly higher serum IL-12, IL-23R and IL12RB2 than the control group before treatment (P < 0.001). In the study group, IL-12, IL-23R and IL-12RB2 were the lowest at T4 (P < 0.001), followed by T3 (P < 0.001). There was no significant difference in IL-23R at T1 and T0 (P > 0.050), and in IL12RB2 at T1 and T2 (P > 0.050). Spearman rank correlation showed that IL-12, IL-23R, IL12RB2 were negatively correlated with treatment duration in the study group (P < 0.001), and were positively correlated with clinical efficacy (P < 0.001). In conclusion, the concentrations of serum IL-12, IL-23R and IL12RB2 in LDH patients are significantly higher than those in normal controls. Moreover, the concentrations are closely related to the rehabilitation of patients and are expected to become therapeutic targets for LDH.
Assuntos
Interleucina-12/metabolismo , Degeneração do Disco Intervertebral/metabolismo , Deslocamento do Disco Intervertebral/metabolismo , Vértebras Lombares/patologia , Receptores de Interleucina-12/metabolismo , Idoso , Feminino , Humanos , Interleucina-12/sangue , Degeneração do Disco Intervertebral/sangue , Deslocamento do Disco Intervertebral/sangue , Masculino , Pessoa de Meia-Idade , Receptores de Interleucina-12/sangue , Resultado do TratamentoRESUMO
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
Transforming growth factor beta 1 (TGF-ß1) is an immunosuppresive cytokine that plays an essential role in immune homeostasis. It is well known that regulatory T (Treg) cells express TGF-ß1; however, the role of autocrine TGF-ß1 in the development, function, and stability of Treg cells remains poorly understood. We found that Treg cell-derived TGF-ß1 was not required for the development of thymic Treg cells in mice, but played a role in the expression of latency-associated peptide and optimal suppression of naïve T cell proliferation in vitro. Moreover, the frequency of Treg cells was significantly reduced in the mesenteric lymph nodes of the Treg cell-specific TGF-ß1-deficient mice, which was associated with increased frequency of IFN-γ-producers among Treg cells. TGF-ß1-deficient Treg cells were more prone to express IFN-γ than TGF-ß1-sufficient Treg cells in a dendritic cell-mediated stimulation in vitro as well as in an adoptive transfer study in vivo. Mechanistically, TGF-ß1-deficient Treg cells expressed higher levels of Il12rb2 and were more sensitive to IL-12-induced conversion into IFN-γ-producing Treg cells or IFN-γ-producing exTreg cells than TGF-ß1-sufficient Treg cells. Our findings demonstrate that autocrine TGF-ß1 plays a critical role in the optimal suppressive activity and stability of Treg cells by downregulating IL-12R on Treg cells.
Assuntos
Regulação para Baixo , Fatores de Transcrição Forkhead/metabolismo , Receptores de Interleucina-12/metabolismo , Linfócitos T Reguladores/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Comunicação Autócrina , Fatores de Transcrição Forkhead/genética , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Camundongos Transgênicos , Receptores de Interleucina-12/genéticaRESUMO
BACKGROUND: Oncogenesis rewires signaling networks to confer a fitness advantage to malignant cells. For instance, the B16F0 melanoma cell model creates a cytokine sink for Interleukin-12 (IL-12) to deprive neighboring cells of this important anti-tumor immune signal. While a cytokine sink provides an indirect fitness advantage, does IL-12 provide an intrinsic advantage to B16F0 cells? METHODS: Acute in vitro viability assays were used to compare the cytotoxic effect of imatinib on a melanoma cell line of spontaneous origin (B16F0) with a normal melanocyte cell line (Melan-A) in the presence of IL-12. The results were analyzed using a mathematical model coupled with a Markov Chain Monte Carlo approach to obtain a posterior distribution in the parameters that quantified the biological effect of imatinib and IL-12. Intracellular signaling responses to IL-12 were compared using flow cytometry in 2D6 cells, a cell model for canonical signaling, and B16F0 cells, where potential non-canonical signaling occurs. Bayes Factors were used to select among competing signaling mechanisms that were formulated as mathematical models. Analysis of single cell RNAseq data from human melanoma patients was used to explore generalizability. RESULTS: Functionally, IL-12 enhanced the survival of B16F0 cells but not normal Melan-A melanocytes that were challenged with a cytotoxic agent. Interestingly, the ratio of IL-12 receptor components (IL12RB2:IL12RB1) was increased in B16F0 cells. A similar pattern was observed in human melanoma. To identify a mechanism, we assayed the phosphorylation of proteins involved in canonical IL-12 signaling, STAT4, and cell survival, Akt. In contrast to T cells that exhibited a canonical response to IL-12 by phosphorylating STAT4, IL-12 stimulation of B16F0 cells predominantly phosphorylated Akt. Mechanistically, the differential response in B16F0 cells is explained by both ligand-dependent and ligand-independent aspects to initiate PI3K-AKT signaling upon IL12RB2 homodimerization. Namely, IL-12 promotes IL12RB2 homodimerization with low affinity and IL12RB2 overexpression promotes homodimerization via molecular crowding on the plasma membrane. CONCLUSIONS: The data suggest that B16F0 cells shifted the intracellular response to IL-12 from engaging immune surveillance to favoring cell survival. Identifying how signaling networks are rewired in model systems of spontaneous origin can inspire therapeutic strategies in humans. Interleukin-12 is a key cytokine that promotes anti-tumor immunity, as it is secreted by antigen presenting cells to activate Natural Killer cells and T cells present within the tumor microenvironment. Thinking of cancer as an evolutionary process implies that an immunosuppressive tumor microenvironment could arise during oncogenesis by interfering with endogenous anti-tumor immune signals, like IL-12. Previously, we found that B16F0 cells, a cell line derived from a spontaneous melanoma, interrupts this secreted heterocellular signal by sequestering IL-12, which provides an indirect fitness advantage. Normally, IL-12 signals via a receptor comprised of two components, IL12RB1 and IL12RB2, that are expressed in a 1:1 ratio and activates STAT4 as a downstream effector. Here, we report that B16F0 cells gain an intrinsic advantage by rewiring the canonical response to IL-12 to instead initiate PI3K-AKT signaling, which promotes cell survival. The data suggest a model where overexpressing one component of the IL-12 receptor, IL12RB2, enables melanoma cells to shift the functional response via both IL-12-mediated and molecular crowding-based IL12RB2 homodimerization. To explore the generalizability of these results, we also found that the expression of IL12RB2:IL12RB1 is similarly skewed in human melanoma based on transcriptional profiles of melanoma cells and tumor-infiltrating lymphocytes. Additional file 6: Video abstract. (MP4 600 kb).
Assuntos
Sobrevivência Celular , Interleucina-12/metabolismo , Melanoma Experimental/imunologia , Receptores de Interleucina-12/metabolismo , Animais , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias CutâneasRESUMO
OBJECTIVE: The role of Th1 and Th17 lymphocyte responses in human infection and sepsis of elderly patients has yet to be clarified. DESIGN: A prospective observational study of patients with sepsis, infection only and healthy controls. SETTING: The acute medical wards and intensive care units in a 1000 bed university hospital. PATIENTS: 32 patients with sepsis, 20 patients with infection, and 20 healthy controls. Patients and controls were older than 65 years of age. Patients with recognised underlying immune compromise were excluded. METHODS: Phenotype, differentiation status and cytokine production by T lymphocytes were determined by flow cytometry. MEASUREMENTS: The differentiation states of circulating CD3+, CD4+, and CD8+ T cells were characterised as naive (CD45RA+, CD197+), central memory (CD45RA-, CD197+), effector memory (CD45RA-, CD197-), or terminally differentated (CD45RA+, CD197-). Expression of IL-12 and IL-23 receptors, and the transcription factors T-bet and RORγt, was analysed in circulating T lymphocytes. Expression of interferon- γ and IL-17A were analysed following stimulation in vitro. RESULTS: CD4+ T cells from patients with infection predominantly expressed effector-memory or terminally differentiated phenotypes but CD4+ T cells from patients with severe sepsis predominantly expressed naive phenotypes (p<0.0001). CD4+ T cells expressing IL-23 receptor were lower in patients with sepsis compared to patients with infection alone (p = 0.007). RORγt expression by CD4+ T cells was less frequent in patients with sepsis (p<0.001), whereas T-bet expressing CD8+ T cells that do not express RORγt was lower in the sepsis patients. HLA-DR expression by monocytes was lower in patients with sepsis. In septic patients fewer monocytes expressed IL-23. CONCLUSION: Persistent failure of T cell activation was observed in patients with sepsis. Sepsis was associated with attenuated CD8+Th1 and CD4+Th17 based lymphocyte response.
Assuntos
Hospitalização , Infecções/imunologia , Infecções/terapia , Sepse/imunologia , Sepse/terapia , Células Th1/patologia , Células Th17/patologia , Idoso , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Estudos de Casos e Controles , Feminino , Regulação da Expressão Gênica , Humanos , Infecções/metabolismo , Masculino , Receptores de Interleucina/metabolismo , Receptores de Interleucina-12/metabolismo , Sepse/metabolismo , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Mendelian susceptibility to mycobacterial disease (MSMD) is characterized by increased susceptibility to weakly virulent mycobacteria (Bacillus Calmette-Guérin [BCG] vaccines and environmental mycobacteria), Mycobacterium tuberculosis, Candida spp. and Salmonella spp. The aim of this study is to evaluate clinical features and immunological findings of MSMD patients with interleukin 12 receptor beta 1 (IL12Rß1) deficiency. METHODS: Among 117 screened patients with BCG infection following vaccination, 23 suspected MSMD subjects were recruited to this study by the exclusion of severe combined immunodeficiencies and chronic granulomatous diseases. Flow cytometric assessment for surface expression of IL12Rß1 was performed. Moreover, the clinical and immunological data from the patients was evaluated. RESULTS: A significant decrease (less than 1%) in the surface expression of IL12Rß1 was reported in six cases which showed a significant increase in the count of lymphocytes (p=0.009) and CD8+ T cells (p=0.008) as compared to MSMD subjects with normal expression of surface IL12Rß1. The frequency of disseminated BCGosis (50% vs. 20%, p=0.29), recurrent infection (83.3% vs. 40%, p=0.14) and salmonellosis (33.3% vs. 0.0%, p=0.07) was higher in IL12Rß1 deficient subjects than IL12Rß1 sufficient individuals. CONCLUSION: MSMD patients with childhood onset of mycobacteriosis (mostly after BCG vaccination) and recurrent salmonellosis could be evaluated for IL12Rß1 expression with flow cytometry for punctual diagnosis.
Assuntos
Herpes Simples/imunologia , Síndromes de Imunodeficiência/imunologia , Mutação/genética , Infecções por Mycobacterium não Tuberculosas/imunologia , Mycobacterium bovis/imunologia , Receptores de Interleucina-12/genética , Simplexvirus/fisiologia , Adolescente , Criança , Pré-Escolar , Feminino , Predisposição Genética para Doença , Herpes Simples/genética , Humanos , Síndromes de Imunodeficiência/genética , Lactente , Masculino , Infecções por Mycobacterium não Tuberculosas/genética , Estudos Prospectivos , Receptores de Interleucina-12/metabolismoRESUMO
BACKGROUND: Systemic inflammation induced by sterile or infectious insults is associated with an enhanced susceptibility to life-threatening opportunistic, mostly bacterial, infections due to unknown pathogenesis. Natural killer (NK) cells contribute to the defence against bacterial infections through the release of Interferon (IFN) γ in response to Interleukin (IL) 12. Considering the relevance of NK cells in the immune defence we investigated whether the function of NK cells is disturbed in patients suffering from serious systemic inflammation. METHODS: NK cells from severely injured patients were analysed from the first day after the initial inflammatory insult until the day of discharge in terms of IL-12 receptor signalling and IFN-γ synthesis. FINDINGS: During systemic inflammation, the expression of the IL-12 receptor ß2 chain, phosphorylation of signal transducer and activation 4, and IFN-γ production on/in NK cells was impaired upon exposure to Staphylococcus aureus. The profound suppression of NK cells developed within 24â¯h after the initial insult and persisted for several weeks. NK cells displayed signs of exhaustion. Extrinsic changes were mediated by the early and long-lasting presence of growth/differentiation factor (GDF) 15 in the circulation that signalled through the transforming growth factor ß receptor I and activated Smad1/5. Moreover, the concentration of GDF-15 in the serum inversely correlated with the IL-12 receptor ß2 expression on NK cells and was enhanced in patients who later acquired septic complications. INTERPRETATION: GDF-15 is associated with the development of NK cell dysfunction during systemic inflammation and might represent a novel target to prevent nosocomial infections. FUND: The study was supported by the Department of Orthopaedics and Trauma Surgery, University Hospital Essen.
Assuntos
Antígeno CD56/metabolismo , Infecção Hospitalar/etiologia , Infecção Hospitalar/metabolismo , Fator 15 de Diferenciação de Crescimento/sangue , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Adulto , Biomarcadores , Comorbidade , Infecção Hospitalar/sangue , Infecção Hospitalar/diagnóstico , Feminino , Humanos , Imunofenotipagem , Mediadores da Inflamação/metabolismo , Interferon gama/metabolismo , Interleucina-12/metabolismo , Masculino , Pessoa de Meia-Idade , Fosforilação , Receptores de Interleucina-12/metabolismo , Fator de Transcrição STAT4/metabolismo , Índice de Gravidade de Doença , Transdução de Sinais , Síndrome de Resposta Inflamatória Sistêmica/sangue , Síndrome de Resposta Inflamatória Sistêmica/etiologia , Síndrome de Resposta Inflamatória Sistêmica/metabolismoRESUMO
TYK2 is a JAK family member that functions downstream of multiple cytokine receptors. Genome wide association studies have linked a SNP (rs34536443) within TYK2 encoding a Proline to Alanine substitution at amino acid 1104, to protection from multiple autoimmune diseases including systemic lupus erythematosus (SLE) and multiple sclerosis (MS). The protective role of this SNP in autoimmune pathogenesis, however, remains incompletely understood. Here we found that T follicular helper (Tfh) cells, switched memory B cells, and IFNAR signaling were decreased in healthy individuals that expressed the protective variant TYK2A1104 (TYK2P ). To study this variant in vivo, we developed a knock-in murine model of this allele. Murine Tyk2P expressing T cells homozygous for the protective allele, but not cells heterozygous for this change, manifest decreased IL-12 receptor signaling, important for Tfh lineage commitment. Further, homozygous Tyk2P T cells exhibited diminished in vitro Th1 skewing. Surprisingly, despite these signaling changes, in vivo formation of Tfh and GC B cells was unaffected in two models of T cell dependent immune responses and in two alternative SLE models. TYK2 is also activated downstream of IL-23 receptor engagement. Here, we found that Tyk2P expressing T cells had reduced IL-23 dependent signaling as well as a diminished ability to skew toward Th17 in vitro. Consistent with these findings, homozygous, but not heterozygous, Tyk2P mice were fully protected in a murine model of MS. Homozygous Tyk2P mice had fewer infiltrating CD4+ T cells within the CNS. Most strikingly, homozygous mice had a decreased proportion of IL-17+/IFNγ+, double positive, pathogenic CD4+ T cells in both the draining lymph nodes (LN) and CNS. Thus, in an autoimmune model, such as EAE, impacted by both altered Th1 and Th17 signaling, the Tyk2P allele can effectively shield animals from disease. Taken together, our findings suggest that TYK2P diminishes IL-12, IL-23, and IFN I signaling and that its protective effect is most likely manifest in the setting of autoimmune triggers that concurrently dysregulate at least two of these important signaling cascades.
Assuntos
Autoimunidade/imunologia , TYK2 Quinase/imunologia , Células Th1/metabolismo , Células Th17/imunologia , Adulto , Animais , Linfócitos B/imunologia , Encefalomielite Autoimune Experimental/imunologia , Feminino , Técnicas de Introdução de Genes , Humanos , Interferon Tipo I/metabolismo , Interleucina-12/metabolismo , Interleucina-23/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Esclerose Múltipla/imunologia , Polimorfismo de Nucleotídeo Único , Receptores de Interleucina-12/metabolismo , TYK2 Quinase/genética , Adulto JovemRESUMO
OBJECTIVE: To investigate the characteristics of circulating natural killer (NK) cells and their interferon (IFN)-γ-producing ability in adult-onset Still disease (AOSD). METHODS: Peripheral blood mononuclear cells were obtained from 22 patients in the acute phase of AOSD (acute AOSD); 7 of the 22 patients after treatment (remission AOSD), and 11 healthy controls (HC). NK cells and their IFN-γ expression levels were analyzed by flow cytometry. Additionally, the cytokine receptors of interleukin (IL)-12, IL-15, and IL-18 on NK cells were also evaluated. RESULTS: The frequency of NK cells was significantly lower in acute AOSD than in HC. NK cell counts significantly increased in remission AOSD. Expression of IL-12 and IL-15 receptors on NK cells was significantly increased in acute AOSD, whereas that of IL-18 receptor indicated no significant difference among 3 groups. IFN-γ expression in NK cells was significantly higher in acute AOSD than in HC, and significantly decreased in remission AOSD. The absolute number of NK cells and IFN-γ-expressing NK cells revealed an inverse correlation with serum ferritin levels in acute AOSD. In 2 distinct subsets of NK cells, CD56dim NK cells significantly exhibited higher IFN-γ expression than CD56bright NK cells in acute AOSD. CONCLUSION: In acute AOSD, NK cells displayed lower proportion, whereas they had higher ability for IFN-γ production than in HC; moreover, upregulation of IL-12 and IL-15 receptors on NK cells may promote IFN-γ production. In addition, disease activity may be implicated in regulating the number of NK cells and IFN-γ-expressing NK cells in AOSD.
Assuntos
Interferon gama/metabolismo , Células Matadoras Naturais/imunologia , Doença de Still de Início Tardio/imunologia , Doença Aguda , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígeno CD56/metabolismo , Feminino , Ferritinas/sangue , Citometria de Fluxo , Humanos , Células Matadoras Naturais/metabolismo , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Receptores de Interleucina-12/metabolismo , Receptores de Interleucina-15/metabolismo , Doença de Still de Início Tardio/sangueRESUMO
Human IL12RB1 is an autosomal gene that is essential for mycobacterial disease resistance and T cell differentiation. Using primary human tissue and PBMCs, we demonstrate that lung and T cell IL12RB1 expression is allele-biased, and the extent to which cells express one IL12RB1 allele is unaffected by activation. Furthermore following its expression the IL12RB1 pre-mRNA is processed into either IL12RB1 Isoform 1 (IL12Rß1, a positive regulator of IL12 responsiveness) or IL12RB1 Isoform 2 (a protein of heretofore unknown function). T cells choice to process pre-mRNA into Isoform 1 or Isoform 2 is controlled by intragenic competition of IL12RB1 exon 9-10 splicing with IL12RB1 exon 9b splicing, as well as an IL12RB1 exon 9b-associated polyadenylation site. Heterogeneous nuclear ribonucleoprotein H (hnRNP H) binds near the regulated polyadenylation site, but is not required for exon 9b polyadenylation. Finally, microRNA-mediated knockdown experiments demonstrated that IL12RB1 Isoform 2 promotes T cell IL12 responses. Collectively, our data support a model wherein tissue expression of human IL12RB1 is allele-biased and produces an hnRNP H-bound pre-mRNA, the processing of which generates a novel IL12 response regulator.
Assuntos
Alelos , Interleucina-12/genética , Splicing de RNA , Receptores de Interleucina-12/genética , Células Cultivadas , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/metabolismo , Humanos , Interleucina-12/metabolismo , Células Jurkat , Pulmão/metabolismo , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Interleucina-12/metabolismo , Linfócitos T/metabolismoRESUMO
Cytokines of the Interleukin (IL)-12 family, consisting of IL-12, IL-23, IL-27 and IL-35, are important regulators in (chronic) inflammatory disorders such as rheumatoid arthritis and multiple sclerosis, but also in cardiovascular diseases. Cytokines of the IL-12 family consist of two subunits and are known for their regulatory functions in the immunologic response, more specifically in the regulation and differentiation of T-helper (Th) cells such as Th1 and Th17 cells. Binding of these cytokines to its specific heterodimeric receptor results in the activation of the JAK-STAT signaling. Despite similarities in structure, the members of the IL-12 family have diverse, both pro- and anti-inflammatory, effects and functions. Because of the pro-inflammatory effects of IL-12 cytokine family members on immune responses, the IL-12 cytokines have been implicated in the development and progression of cardiovascular diseases such as atherosclerosis, but also in acute cardiovascular syndromes such as myocardial infarction and stroke. For example, patients suffering from cardiovascular disease display increased blood levels of IL-12, IL-23 and IL-27, while decreased IL-35 levels have been linked to a lower cardiovascular risk. In this review, we aim to highlight the current understandings of the IL-12 cytokine family and its specific family members to cardiovascular diseases, including both clinical and experimental studies. We will also discuss the potential of these cytokines as a biomarker in acute cardiovascular syndromes.
Assuntos
Doenças Cardiovasculares/metabolismo , Interleucina-12/metabolismo , Receptores de Interleucina-12/metabolismo , Animais , Doenças Cardiovasculares/terapia , Progressão da Doença , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/terapia , Interleucina-23/metabolismo , Interleucina-27/metabolismo , Interleucinas/metabolismo , Modelos Animais , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/terapia , Transdução de Sinais/genética , Células Th1/imunologia , Células Th17/imunologiaRESUMO
BACKGROUND/AIMS: This study aimed to pathologically elucidate the roles of interleukin-12 receptor (IL-12R) ß2 and interleukin-23 receptor (IL-23R) expression in tumor cells and tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment and to determine their combined effect on prognosis of laryngeal cancer (LC). METHODS: The tumor-cell expression scores and TIL positivity ratiosof IL-12Rß2 and IL-23R in matched LC and normal laryngeal tissue samples from 61 LC patients were measured via immunohistochemistry (IHC). We adopted a linear regression model to analyze the correlation between IL-12Rß2 and IL-23R expression in tumor cells and TIL ratios. TheKaplan-Meier log-rank test and Cox regression hazard ratios were used to analyze survival. RESULTS: LC tumor cells had a higher IL-12Rß2 expression and TIL ratio than IL-23R expression and TIL ratio. The significant correlations between IL-12Rß2 and IL-23R expression and TIL ratios were identified in LC tissues, particularly in well-differentiated LC. Furthermore, either high tumor cell IL-12Rß2 or low IL-23R expression had better survival than its corresponding low or high expression, respectively. Similar results did for IL-12Rß2 ratio and IL-23R ratio. Finally, patients with both high IL-12Rß2 and low IL-23R had the best prognosis among any other combined groups with both gene expression (HR, 0.1; 95% CI, 0.0-0.8). Likewise, patients with positive ratios of high IL-12Rß2 and low IL-23R TILs had the best survival (HR, 0.1; 95% CI, 0.0-0.4). CONCLUSION: IL-12Rß2 and IL-23R create a homeostasis within the tumor cells and TILs, and this homeostasis affects prognosis. While the intrinsic mechanisms of epigenetic immunoediting for IL-12Rß2 and IL-23R remain unknown, additional larger and functional studies are warranted for validation.
Assuntos
Neoplasias Laríngeas/patologia , Receptores de Interleucina-12/metabolismo , Receptores de Interleucina/metabolismo , Feminino , Humanos , Estimativa de Kaplan-Meier , Neoplasias Laríngeas/metabolismo , Neoplasias Laríngeas/mortalidade , Modelos Lineares , Linfócitos do Interstício Tumoral/citologia , Linfócitos do Interstício Tumoral/metabolismo , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Modelos de Riscos ProporcionaisRESUMO
Rheumatoid arthritis (RA) is a type of chronic systemic inflammatory disease; it has a very complicated pathogenesis, and multiple pathological changes are implicated. Traditional Chinese medicine (TCM) like Tripterygium wilfordii Hook. F. or Sinomenium acutum (Thunb.) Rehd et Wils. has been extensively used for centuries in the treatment of arthritic diseases and been reported effective for relieving the severity of RA. Hei-Gu-Teng Zhuifenghuoluo granule (HGT) which contains Periploca forrestii Schltr., Sinomenium acutum (Thunb.) Rehd et Wils., and Lysimachia paridiformis Franch. var. stenophylla Franch. was a representative natural rattan herb formula for the treatment of RA in China, but the mechanism has not been elucidated. This study aimed at exploring the mechanism of HGT on RA using the bioinformatics analysis with in vivo and in vitro experiment validation. The potential action mechanism was first investigated by bioinformatics analysis via Ingenuity Pathway Analysis (IPA) software. After that, we use experimental validation such as collagen-induced arthritis (CIA) mice model in vivo and U937 cell model in vitro. The bioinformatics results suggested that HGT may have anti-inflammatory characteristic on RA and IL-12 signaling pathway could be the potential key trigger. In vivo experiments demonstrated that HGT ameliorated the symptoms in CIA mice and decreased the production of inflammatory cytokines in both mice ankle joints and serum. Furthermore, HGT effectively inhibited the activation of IL-12R and STAT4 on IL-12 signaling pathway. In vitro experiments showed that HGT inhibited the production of IL-12R and STAT4 induced by IL-12 in lipopolysaccharide- (LPS-) stimulated U937 cells. Moreover, IL-12R knockdown was able to interfere with the inhibition effects of HGT on the production of these cytokines. Our results confirmed the anti-inflammatory property of HGT, which was attributed to its inhibition on IL-12 signaling pathway.
Assuntos
Anti-Inflamatórios/farmacologia , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Interleucina-12/metabolismo , Animais , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos DBA , Periploca/química , Primulaceae/química , Distribuição Aleatória , Receptores de Interleucina-12/metabolismo , Fator de Transcrição STAT4/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sinomenium/química , Células U937RESUMO
Interleukin-35 (IL-35) is implicated in tumorigenesis, but its exact impact on intrahepatic cholangiocarcinoma (ICC) is not clear. The aim of the present study was to explore the specific effect of IL-35 on patient prognosis. Additionally, we formulated an effective prognostic nomogram for ICC patients after curative resection. Immunohistochemistry was applied to explore IL-35 expression as well as IL-35 receptor (IL-35R) in 102 ICC patients. Results showed that IL-35 was highly expressed in ICC tumor tissues and was positively associated with lymph node metastasis (LNM), TNM stage and vascular invasion and was an independent prognostic factor for patients' overall survival (OS) and recurrence-free survival (RFS). High expression of IL-35R (gp130 and IL-12Rß2) was also observed in ICC cancer tissues, but only gp130 was an independent prognostic factor for OS and RFS and was indispensable in IL-35-mediated ICC clinical prognosis. The nomogram comprising carcinoembryonic antigen, LNM, IL-35 and gp130 expression achieved better predictive accuracy compared with TNM stage for OS. Our data support that high IL-35 expression correlates with ICC aggressiveness and emerges as a valuable biomarker for evaluating ICC progression and prognosis in clinical work.