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We identified activin A receptor type I (ACVR1), a member of the TGF-ß superfamily, as a factor favoring acute myeloid leukemia (AML) growth and a new potential therapeutic target. ACVR1 is overexpressed in FLT3-mutated AML and inhibition of ACVR1 expression sensitized AML cells to FLT3 inhibitors. We developed a novel ACVR1 inhibitor, TP-0184, which selectively caused growth arrest in FLT3-mutated AML cell lines. Molecular docking and in vitro kinase assays revealed that TP-0184 binds to both ACVR1 and FLT3 with high affinity and inhibits FLT3/ACVR1 downstream signaling. Treatment with TP-0184 or in combination with BCL2 inhibitor, venetoclax dramatically inhibited leukemia growth in FLT3-mutated AML cell lines and patient-derived xenograft models in a dose-dependent manner. These findings suggest that ACVR1 is a novel biomarker and plays a role in AML resistance to FLT3 inhibitors and that FLT3/ACVR1 dual inhibitor TP-0184 is a novel potential therapeutic tool for AML with FLT3 mutations.
Assuntos
Leucemia Mieloide Aguda , Humanos , Simulação de Acoplamento Molecular , Mutação , Linhagem Celular Tumoral , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/uso terapêutico , Apoptose , Receptores de Ativinas Tipo I/genética , Receptores de Ativinas Tipo I/uso terapêuticoRESUMO
Purpose: The immune system's role in mediating the cytotoxic effects of chemoradiotherapy remains not completely understood. The integration of immunotherapies into treatment will require insight into features and timing of the immune microenvironment associated with treatment response. Here, we investigated the role of circulating neutrophils and tumor-associated myeloid cells (TSAMs) as potential agents and biomarkers for disease-related outcomes in locally advanced cervical cancer (LACC). Material and Methods: Hematologic parameters for two LACC patient cohorts, a retrospective clinical and a prospective translational cohort, were obtained at baseline, weekly during chemoradiotherapy for the retrospective cohort, biweekly during chemoradiotherapy for the prospective cohort, and at the first follow-up visit for both cohorts (mean 14.7 weeks, range 8.1-25.1 weeks for the prospective cohort and 5.3 weeks with a range of 2.7-9.0 weeks for the retrospective cohort). In both cohorts, baseline as well as mean and lowest on-treatment values for platelets, hemoglobin, absolute neutrophil count (ANC), and absolute lymphocyte count (ALC) were analyzed for correlations with disease-related outcomes. In the prospective cohort, circulating myeloid cells were isolated from peripheral blood mononuclear cells (PBMCs), and TSAMs were isolated from tumor tissue via a novel serial cytobrush sampling assay. The samples were analyzed by flow cytometry. Results: In both cohorts, the only hematologic parameter significantly associated with survival was elevated on-treatment mean ANC (mANC), which was associated with lower local failure-free and overall survival rates in the retrospective and prospective cohorts, respectively. mANC was not associated with a difference in distant metastases. CD11b+CD11c- TSAMs, which act as a surrogate marker for intratumoral neutrophils, steadily decreased during the course of chemoRT and nadier'd at week 5 of treatment. Conversely, circulating myeloid cells identified from PBMCs steadily increased through week 5 of treatment. Regression analysis confirmed an inverse relationship between circulating myeloid cells and TSAMs at this time point. Conclusions: These findings identify on-treatment mean neutrophil count as a predictor of disease-related outcomes, suggest that neutrophils contribute to chemoradiation treatment resistance, and demonstrate the importance of techniques to measure intratumoral immune activity.
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We describe a role of CD44-mediated signaling during host-defense against tuberculosis (TB) using a mouse model of TB and studies in M. tuberculosis (Mtb) infected human macrophage (MФ). Liposomes targeting CD44 using thioaptamers (CD44TA-LIP) were designed and tested as new vaccines to boost host immunity in TB. CD44TA-LIP enhanced killing of Mtb in human MФ, which correlated with an increased production of pro-inflammatory cytokines IL-1ß, TNF-α and IL-12. CD44TA-LIP activated MФ showed an enhanced MHC-II dependent antigen presentation to CD4 T-cells. Inhibition of cellular proliferation and cytoskeleton rearrangement pathways downstream of CD44 signaling abrogated CD44TA-LIP-induced antimicrobial effects. Blockade of inflammatory pathways also reduced antigen presentation by MФ and activation of CD4 T cells. Mtb infected MФ treated with CD44TA-LIP exhibited increased nitric oxide and HßD2 defensin peptide production. Among Mtb infected mice with increased lung and spleen loads of organisms, intranasal administration of CD44TA-LIP led to a ten-fold reduction of colony forming units of Mtb and elevated IFN-γ + CD4, effector, central and resident memory T cells. Biodistribution studies demonstrated that CD44TA-LIP preferentially accumulated in the lungs and were associated with CD11b + cells. CD44TA-LIP treated mice showed no weight loss or increased liver LDH levels. This study highlights the importance of CD44-mediated signaling in host-defense during TB and the therapeutic potential of CD44TA-LIP.
Assuntos
Anti-Infecciosos , Receptores de Hialuronatos/metabolismo , Mycobacterium tuberculosis , Nanopartículas , Tuberculose , Defensinas , Humanos , Interleucina-12 , Lipossomos , Antígeno de Macrófago 1 , Óxido Nítrico , Distribuição Tecidual , Tuberculose/tratamento farmacológico , Fator de Necrose Tumoral alfaRESUMO
Trichuriasis is one of the most common neglected tropical diseases of the world's poorest people. A recombinant vaccine composed of Tm-WAP49, an immunodominant antigen secreted by adult Trichuris stichocytes into the mucosa of the cecum to which the parasite attaches, is under development. The prototype is being evaluated in a mouse model of Trichuris muris infection, with the ultimate goal of producing a mucosal vaccine through intranasal delivery. Intranasal immunization of mice with Tm-WAP49 formulated with the adjuvant OCH, a truncated analog of alpha-GalCer with adjuvanticity to stimulate natural killer T cells (NKT) and mucosal immunity, induced significantly high levels of IgG and its subclasses (IgG1 and IgG2a) in immunized mice. This also resulted in a significant reduction of worm burden after challenge with T. muris-infective eggs. The addition of QS-21 adjuvant to this vaccine formulation further reduced worm counts. The improved protection from the dual-adjuvanted vaccine correlated with higher serum antibody responses (IgG, IgG1, IgG2a, IgA) as well as with the induction of antigen-specific IgA in the nasal mucosa. It was also associated with the robust cellular responses including functional subsets of CD4 T cells producing IL-4, and cytotoxic CD8 T cells expressing granzyme B. The worm reduction achieved by mucosal immunization was higher than that induced by subcutaneous immunization. Intranasal immunization also induced a significantly higher nasal mucosa-secreted antigen-specific IgA response, as well as higher functional cellular responses including CD4+IL4+ (Th1) and CD8+GnzB+ (Th2) T cells, and antigen-specific INFγ-producing T cells in both spleen and MLNs and antibody-producing B cells (CD19+B220+/B220+GL7+). Mucosal immunization further induced long-term T lymphocyte memory with increased central (CD62L+CD44+) and effector (CD62L-CD44+) memory subsets of both CD4 and CD8 T cells at 60 days after the last immunization. In summary, intranasal immunization with recombinant Tm-WAP49 protein induced strong protection versus murine trichuriasis. It represents a promising vaccination approach against intestinal nematodes.
Assuntos
Tricuríase/imunologia , Adjuvantes Imunológicos/farmacologia , Administração Intranasal , Animais , Formação de Anticorpos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Feminino , Imunidade Celular/efeitos dos fármacos , Imunidade nas Mucosas/imunologia , Imunização , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos AKR , Camundongos Endogâmicos BALB C , Mucosa/imunologia , Células Th1/imunologia , Trichuris/imunologia , Vacinação/métodos , Vacinas SintéticasRESUMO
Human papillomavirus (HPV) infection causes 600,000 new cancers worldwide each year. HPV-related cancers express the oncogenic proteins E6 and E7, which could serve as tumor-specific antigens. It is not known whether immunity to E6 and E7 evolves during chemoradiotherapy or affects survival. Using T cells from 2 HPV16+ patients, we conducted functional T-cell assays to identify candidate HPV-specific T cells and common T-cell receptor motifs, which we then analyzed across 86 patients with HPV-related cancers. The HPV-specific clones and E7-related T-cell receptor motifs expanded in the tumor microenvironment over the course of treatment, whereas non-HPV-specific T cells did not. In HPV16+ patients, improved recurrence-free survival was associated with HPV-responsive T-cell expansion during chemoradiotherapy.
Assuntos
Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Neoplasias do Colo do Útero , Quimiorradioterapia , Feminino , Papillomavirus Humano 16 , Humanos , Proteínas E7 de Papillomavirus , Prognóstico , Proteínas Repressoras , Linfócitos T , Microambiente TumoralRESUMO
Diversity of the gut microbiome is associated with higher response rates for cancer patients receiving immunotherapy but has not been investigated in patients receiving radiation therapy. Additionally, current studies investigating the gut microbiome and outcomes in cancer patients may not have adjusted for established risk factors. Here, we sought to determine if diversity and composition of the gut microbiome was independently associated with survival in cervical cancer patients receiving chemoradiation. Our study demonstrates that the diversity of gut microbiota is associated with a favorable response to chemoradiation. Additionally, compositional variation among patients correlated with short term and long-term survival. Short term survivor fecal samples were significantly enriched in Porphyromonas, Porphyromonadaceae, and Dialister, whereas long term survivor samples were significantly enriched in Escherichia Shigella, Enterobacteriaceae, and Enterobacteriales. Moreover, analysis of immune cells from cervical tumor brush samples by flow cytometry revealed that patients with a high microbiome diversity had increased tumor infiltration of CD4+ lymphocytes as well as activated subsets of CD4 cells expressing ki67+ and CD69+ over the course of radiation therapy. Modulation of the gut microbiota before chemoradiation might provide an alternative way to enhance treatment efficacy and improve treatment outcomes in cervical cancer patients.
Assuntos
Quimiorradioterapia , Microbioma Gastrointestinal , Intestinos/microbiologia , Neoplasias do Colo do Útero/terapia , Adulto , Idoso , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Quimiorradioterapia/efeitos adversos , Quimiorradioterapia/mortalidade , Feminino , Humanos , Antígeno Ki-67/metabolismo , Lectinas Tipo C/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Pessoa de Meia-Idade , Estudos Prospectivos , Fatores de Tempo , Resultado do Tratamento , Microambiente Tumoral , Neoplasias do Colo do Útero/imunologia , Neoplasias do Colo do Útero/microbiologia , Neoplasias do Colo do Útero/mortalidadeRESUMO
Studies have demonstrated that purported biofield therapy emitted from humans can inhibit the proliferation of cancer cells and suppress tumor growth in various cancers. We explored the effects of biofield therapy on tumor growth in the Lewis lung carcinoma and expanded mechanistic outcomes. We found biofield therapy did not inhibit tumor growth. However, the experimental (Ex) condition exposed tumors had a significantly higher percentage of necrosis (24.4 ± 6.8%) compared with that of the Control condition (6.5 ± 2.7%; P < .02) and cleaved caspase-3 positive cells were almost 2.3-fold higher (P < .05). Similarly, tumor-infiltrating lymphocytes profiling showed that CD8+/CD45+ immune cell population was significantly increased by 2.7-fold in Ex condition (P < .01) whereas the number of intratumoral FoxP3+/CD4+ (T-reg cells) was 30.4% lower than that of the Control group (P = .01), leading to a significant 3.1-fold increase in the ratio of CD8+/T-reg cells (P < .01). Additionally, there was a 51% lower level of strongly stained CD68+ cells (P < .01), 57.9% lower level of F4/80high/CD206+ (M2 macrophages; P < .02) and a significant 1.8-fold increase of the ratio of M1/M2 macrophages (P < .02). Furthermore, Ex exposure resulted in a 15% reduction of stem cell marker CD44 and a significant 33% reduction of SOX2 compared with that of the Controls (P < .02). The Ex group also engaged in almost 50% less movement throughout the session than the Controls. These findings suggest that exposure to purported biofields from a human is capable of enhancing cancer cell death, in part mediated through modification of the tumor microenvironment and stemness of tumor cells in mouse Lewis lung carcinoma model. Future research should focus on defining the optimal treatment duration, replication with different biofield therapists, and exploring the mechanisms of action.
Assuntos
Carcinoma , Neoplasias Pulmonares , Animais , Humanos , Pulmão , Neoplasias Pulmonares/terapia , Linfócitos do Interstício Tumoral , Camundongos , Microambiente TumoralRESUMO
Human papillomavirus (HPV)-induced cancers continue to affect millions of women around the world, and the five year survival rate under the current standard of care for these cancers is less than 60% in some demographics. Therefore there is still an unmet need to develop an effective therapy that can be easily administered to treat established HPV cervical cancer lesions. We sought to investigate the potential of an intranasal HPV peptide therapeutic vaccine incorporating the combination of α-Galactosylceramide (α-GalCer) and CpG-ODN adjuvants (TVAC) against established HPV genital tumors in a syngeneic C57BL/6J mouse model. We obtained evidence to show that TVAC, delivered by the mucosal intranasal route, induced high frequencies of antigen-specific CD8 T cells concurrent with significant reduction in the immunosuppressive regulatory T cells and myeloid derived suppressor cells in the tumor microenvironment (TME), correlating with sustained elimination of established HPV genital tumors in over 85% of mice. Inclusion of both the adjuvants in the vaccine was necessary for significant increase of antigen-specific CD8 T cells to the tumor and antitumor efficacy because vaccination incorporating either adjuvant alone was inefficient. These results strongly support the utility of the TVAC administered by needle-free intranasal route as a safe and effective strategy for the treatment of established genital HPV tumors.
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Therapy for intracerebral hemorrhage (ICH) remains elusive, in part dependent on the severity of the hemorrhage itself as well as multiple deleterious effects of blood and its breakdown products such as hemin and free iron. While oxidative injury and genomic damage have been seen following ICH, the details of this injury and implications remain unclear. Here, we discovered that, while free iron produced mostly reactive oxygen species (ROS)-related single-strand DNA breaks, hemin unexpectedly induced rapid and persistent nuclear and mitochondrial double-strand breaks (DSBs) in neuronal and endothelial cell genomes and in mouse brains following experimental ICH comparable to that seen with γ radiation and DNA-complexing chemotherapies. Potentially as a result of persistent DSBs and the DNA damage response, hemin also resulted in senescence phenotype in cultured neurons and endothelial cells. Subsequent resistance to ferroptosis reported in other senescent cell types was also observed here in neurons. While antioxidant therapy prevented senescence, cells became sensitized to ferroptosis. To address both senescence and resistance to ferroptosis, we synthesized a modified, catalytic, and rapidly internalized carbon nanomaterial, poly(ethylene glycol)-conjugated hydrophilic carbon clusters (PEG-HCC) by covalently bonding the iron chelator, deferoxamine (DEF). This multifunctional nanoparticle, DEF-HCC-PEG, protected cells from both senescence and ferroptosis and restored nuclear and mitochondrial genome integrity in vitro and in vivo. We thus describe a potential molecular mechanism of hemin/iron-induced toxicity in ICH that involves a rapid induction of DSBs, senescence, and the consequent resistance to ferroptosis and provide a mechanistic-based combinatorial therapeutic strategy.
Assuntos
Carbono/farmacologia , Hemorragia Cerebral/tratamento farmacológico , Nanopartículas/química , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Hemorragia Cerebral/genética , Hemorragia Cerebral/metabolismo , Quebras de DNA de Cadeia Simples/efeitos dos fármacos , Dano ao DNA , Desferroxamina/farmacologia , Hemina/antagonistas & inibidores , Hemina/farmacologia , Humanos , Ferro/farmacologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Polietilenoglicóis/farmacologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: The US is experiencing an epidemic of HPV+ oropharyngeal cancers (OPC), the rates and burden of which now exceed that for cervical cancer. Immunotherapy targeting programmed death 1 (PD-1) on tumor-infiltrating lymphocytes and/or its ligand PD-L1 on tumor cells, which was effective in several cancers has however, showed efficacy in only less than 15% of patients. METHODS: We used a preclinical HPV+ oral tumor model, mEER, consisting of mouse tonsil derived epithelial cells expressing HPV-16 E6 and E7 genes, along with the H-ras oncogene to test strategies for enhancing the efficacy of anti-PD-1 therapy. RESULTS: Monotherapy with PD-1 blocking antibody was ineffective against flank-implanted tumors, but induced regression in 54% of mice bearing orthotopic tongue tumors that correlated with higher CD8 T cell responses. Since the CD8+ T cells derived from tongue tumors also showed high levels of the immune checkpoint inhibitory receptor CTLA-4, we tested combination immunotherapy targeting both CTLA-4 and PD-1 together and observed 93.3% survival of mice bearing tumors in the tongue for the duration of our 100-day study. Protective immunity correlated with a significant decrease in immunosuppressive lymphoid and myeloid populations within the tumor microenvironment. Consistent with the reported capacity of interferon-driven PD-L1/PD-1 pathway induction to serve as a biomarker of response to PD-1 blockade, we observed elevated interferon signaling and significantly higher levels of PD-1/PD-L1 in tongue-implanted mEER tumors compared to those growing on the flank correlating with their preferential responsiveness to PD-1 blockade. More importantly, in a pseudometastasic mouse model bearing both flank and tongue tumors to represent metastatic disease, delivery of Stimulator of Interferon Induced Genes (STING) agonist into the flank tumors combined with systemic treatment with α-PD-1 and α-CTLA-4 antibodies resulted in sustained tumor regression in 71% of mice. In this case, productive abscopal anti-tumor immunity was associated with robust increases in the ratios of cytotoxic CD8+ T cells (CTL) versus regulatory T cells (Treg) and versus functional myeloid-derived suppressor cells (MDSC). CONCLUSIONS: These results support combining α-PD-1 therapy with induction of IFN-α/ß signaling via provision of STING agonist and/or through CTLA-4 blockade as potential treatment option for HNSCC patients, especially, those not responding to α-PD-1 monotherapy.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Bucais/tratamento farmacológico , Infecções por Papillomavirus/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Antígeno CTLA-4/antagonistas & inibidores , Antígeno CTLA-4/imunologia , Linhagem Celular Tumoral/transplante , Modelos Animais de Doenças , Ensaios de Seleção de Medicamentos Antitumorais , Células Epiteliais , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/imunologia , Humanos , Interferons/imunologia , Interferons/metabolismo , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Masculino , Proteínas de Membrana/agonistas , Proteínas de Membrana/imunologia , Camundongos , Neoplasias Bucais/genética , Neoplasias Bucais/imunologia , Neoplasias Bucais/virologia , Proteínas Oncogênicas Virais/genética , Tonsila Palatina/citologia , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/virologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Proteínas Repressoras/genética , Transdução de Sinais/imunologiaRESUMO
Cannabinoids are the most commonly abused illicit drugs worldwide. While cannabis can be beneficial for certain heath conditions, abuse of potent synthetic cannabinoids has been on the rise. Exposure to cannabinoids is also prevalent in women of child-bearing age and pregnant women. These compounds can cross the placental barrier and directly affect the fetus. They mediate their effects primarily through G-protein coupled cannabinoid receptors, CB1 and CB2. In addition to significant neurological effects, cannabinoids can trigger robust immunomodulation by altering cytokine levels, causing apoptosis of lymphoid cells and inducing suppressor cells of the immune system. Profound effects of cannabinoids on the immune system as discussed in this review, suggest that maternal exposure during pregnancy could lead to dysregulation of innate and adaptive immune system of developing fetus and offspring potentially leading to weakening of immune defenses against infections and cancer later in life. Emerging evidence also indicates the underlying role of epigenetic mechanisms causing long-lasting impact following cannabinoid exposure in utero.
Assuntos
Canabinoides/intoxicação , Desenvolvimento Fetal/efeitos dos fármacos , Sistema Imunitário/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal/imunologia , Imunidade Adaptativa/efeitos dos fármacos , Imunidade Adaptativa/imunologia , Canabinoides/química , Feminino , Desenvolvimento Fetal/imunologia , Humanos , Sistema Imunitário/embriologia , Sistema Imunitário/imunologia , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/imunologia , Estrutura Molecular , Gravidez , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamenteRESUMO
Endometriosis affects women of reproductive age via unclear immunological mechanism(s). Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of myeloid cells with potent immunosuppressive and angiogenic properties. Here, we found MDSCs significantly increased in the peripheral blood of patients with endometriosis and in the peritoneal cavity of a mouse model of surgically induced endometriosis. Majority of MDSCs were granulocytic, produced ROS, and arginase, and suppressed T-cell proliferation. Depletion of MDSCs by antiGr-1 antibody dramatically suppressed development of endometrial lesions in mice. The chemokines CXCL1, 2, and 5 were expressed at sites of lesion while MDSCs expressed CXCR-2. These CXC-chemokines promoted MDSC migration toward endometriotic implants both in vitro and in vivo. Also, CXCR2-deficient mice show significantly decreased MDSC induction, endometrial lesions, and angiogenesis. Importantly, adoptive transfer of MDSCs into CXCR2-KO mice restored endometriotic growth and angiogenesis. Together, this study demonstrates that MDSCs play a role in the pathogenesis of endometriosis and identifies a novel CXC-chemokine and receptor for the recruitment of MDSCs, thereby providing a potential target for endometriosis treatment.
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Indutores da Angiogênese/imunologia , Endometriose/imunologia , Endométrio/imunologia , Granulócitos/imunologia , Células Supressoras Mieloides/imunologia , Animais , Arginase/metabolismo , Células Cultivadas , Quimiocinas/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Endométrio/irrigação sanguínea , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espécies Reativas de Oxigênio/metabolismo , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismoRESUMO
Cannabidiol (CBD) is a natural nonpsychotropic cannabinoid from marijuana (Cannabis sativa) with anti-epileptic and anti-inflammatory properties. Effect of CBD on naive immune system is not precisely understood. In this study, we observed that administering CBD into naive mice triggers robust induction of CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSC) in the peritoneum, which expressed functional arginase 1, and potently suppressed T cell proliferation ex vivo. Furthermore, CBD-MDSC suppressed LPS-induced acute inflammatory response upon adoptive transfer in vivo. CBD-induced suppressor cells were comprised of CD11b(+)Ly6-G(+)Ly6-C(+) granulocytic and CD11b(+)Ly6-G(-)Ly6-C(+) monocytic subtypes, with monocytic MDSC exhibiting higher T cell-suppressive function. Induction of MDSC by CBD was markedly attenuated in Kit-mutant (Kit(W/W-v)) mast cell-deficient mice. MDSC response was reconstituted upon transfer of wild-type bone marrow-derived mast cells in Kit(W/W-v) mice, suggesting the key role of cKit (CD117) as well as mast cells. Moreover, mast cell activator compound 48/80 induced significant levels of MDSC in vivo. CBD administration in mice induced G-CSF, CXCL1, and M-CSF, but not GM-CSF. G-CSF was found to play a key role in MDSC mobilization inasmuch as neutralizing G-CSF caused a significant decrease in MDSC. Lastly, CBD enhanced the transcriptional activity of peroxisome proliferator-activated receptor γ in luciferase reporter assay, and PPAR-γ selective antagonist completely inhibited MDSC induction in vivo, suggesting its critical role. Together, the results suggest that CBD may induce activation of PPAR-γ in mast cells leading to secretion of G-CSF and consequent MDSC mobilization. CBD being a major component of Cannabis, our study indicates that marijuana may modulate or dysregulate the immune system by mobilizing MDSC.
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Anti-Inflamatórios/farmacologia , Canabidiol/farmacologia , Cannabis/metabolismo , Células Mieloides/imunologia , PPAR gama/genética , Animais , Arginase/biossíntese , Antígeno CD11b/metabolismo , Proliferação de Células/efeitos dos fármacos , Quimiocina CXCL1/biossíntese , Feminino , Fator Estimulador de Colônias de Granulócitos/biossíntese , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Fator Estimulador de Colônias de Macrófagos/biossíntese , Mastócitos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/metabolismo , PPAR gama/antagonistas & inibidores , Receptores de Quimiocinas/metabolismo , Linfócitos T/imunologia , Ativação Transcricional/genética , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
Inflammatory bowel disease (IBD), a chronic intestinal inflammatory condition that affects millions of people worldwide, results in high morbidity and exorbitant health-care costs. The critical features of both innate and adaptive immunity are to control inflammation and dysfunction in this equilibrium is believed to be the reason for the development of IBD. miR-155, a microRNA, is up-regulated in various inflammatory disease states, including IBD, and is a positive regulator of T-cell responses. To date, no reports have defined a function for miR-155 with regard to cellular responses in IBD. Using an acute experimental colitis model, we found that miR-155(-/-) mice, as compared to wild-type control mice, have decreased clinical scores, a reversal of colitis-associated pathogenesis, and reduced systemic and mucosal inflammatory cytokines. The increased frequency of CD4+ lymphocytes in the spleen and lamina propria with dextran sodium sulphate induction was decreased in miR-155(-/-) mice. Similarly, miR-155 deficiency abrogated the increased numbers of interferon-γ expressing CD4+ T cells typically observed in wild-type mice in this model. The frequency of systemic and mucosal T helper type 17-, CCR9-expressing CD4+ T cells was also reduced in miR-155(-/-) mice compared with control mice. These findings strongly support a role for miR-155 in facilitating pro-inflammatory cellular responses in this model of IBD. Loss of miR-155 also results in decreases in T helper type 1/type 17, CD11b+) and CD11c+ cells, which correlated with reduced clinical scores and severity of disease. miR-155 may serve as a potential therapeutic target for the treatment of IBD.
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Colite/genética , Colite/imunologia , MicroRNAs/genética , Células Th1/imunologia , Células Th1/metabolismo , Células Th17/imunologia , Células Th17/metabolismo , Animais , Peso Corporal , Antígeno CD11b/metabolismo , Antígeno CD11c/metabolismo , Colite/sangue , Colite/induzido quimicamente , Colite/patologia , Citocinas/sangue , Sulfato de Dextrana/efeitos adversos , Modelos Animais de Doenças , Feminino , Imunofenotipagem , Contagem de Linfócitos , Camundongos , Camundongos Knockout , Índice de Gravidade de DoençaRESUMO
Marijuana is one of the most abused drugs due to its psychotropic effects. Interestingly, it is also used for medicinal purposes. The main psychotropic component in marijuana, Δ(9)-tetrahydrocannabinol (THC), has also been shown to mediate potent anti-inflammatory properties. Whether the immunomodulatory activity of THC is mediated by epigenetic regulation has not been investigated previously. In this study, we employed ChIP-Seq technology to examine the in vivo effect of THC on global histone methylation in lymph node cells of mice immunized with a superantigen, staphylococcal enterotoxin B. We compared genome-wide histone H3 Lys-4, Lys-27, Lys-9, and Lys-36 trimethylation and histone H3 Lys-9 acetylation patterns in such cells exposed to THC or vehicle. Our results showed that THC treatment leads to the association of active histone modification signals to Th2 cytokine genes and suppressive modification signals to Th1 cytokine genes, indicating that such a mechanism may play a critical role in the THC-mediated switch from Th1 to Th2. At the global level, a significant portion of histone methylation and acetylation regions were altered by THC. However, the overall distribution of these histone methylation signals among the genomic features was not altered significantly by THC, suggesting that THC activates the expression of a subset of genes while suppressing the expression of another subset of genes through histone modification. Functional classification of these histone marker-associated genes showed that these differentially associated genes were involved in various cellular functions, from cell cycle regulation to metabolism, suggesting that THC had a pleiotropic effect on gene expression in immune cells. Altogether, the current study demonstrates for the first time that THC may modulate immune response through epigenetic regulation involving histone modifications.
Assuntos
Dronabinol/farmacologia , Enterotoxinas/imunologia , Histonas/metabolismo , Fatores Imunológicos/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Animais , Proliferação de Células/efeitos dos fármacos , Feminino , Marcadores Genéticos/genética , Genômica , Histonas/química , Linfonodos/imunologia , Metilação/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Linfócitos T/citologia , Transcriptoma/efeitos dos fármacos , Transcriptoma/imunologiaRESUMO
SCOPE: Cases of oral allergy syndrome following the ingestion of sapodilla plum (Manilkara zapota) have been reported rarely. As the causative allergens are not known, the main objective of this study was to identify and characterize the important allergens in sapodilla. METHODS AND RESULTS: Allergy to sapodilla was diagnosed by case history, skin prick test, and serum allergen-specific IgE. The allergen was detected by IgE immunoblotting, purified on SP-Sepharose and characterized by native/SDS-PAGE, IEF, MS, and amino acid composition. Several cases of allergy to sapodilla fruit were identified; majority of the sapodilla-allergic subjects (6/7) experienced typical oral allergy syndrome symptoms, and allergen-specific IgE to the purified protein was positive. The allergen has a pI ≥9.5 and high contents of arginine, threonine, glycine, and cysteine. Circular dichroism revealed a secondary structure rich in beta sheets/turns. Based on its N-terminal sequence of A-T-F-D-I-Q-N-N-C-X-Y-, the allergen (21 578 Da) was identified as a thaumatin-like protein by homology. CONCLUSION: The causative allergen in sapodilla plum has been identified and characterized as a highly basic thaumatin-like protein belonging to the pathogenesis-related protein (PR-5) family, which has been recognized as a new family of conserved, cross-reactive plant allergens.
Assuntos
Antígenos de Plantas/imunologia , Hipersensibilidade Alimentar/imunologia , Manilkara , Proteínas de Plantas/imunologia , Antígenos de Plantas/isolamento & purificação , Reações Cruzadas , Eletroforese em Gel de Poliacrilamida , Humanos , Imunoglobulina E , Homologia de Sequência de Aminoácidos , Testes CutâneosRESUMO
Endocannabinoids are lipid-signaling molecules found in the nervous system; however, their precise role in the periphery is unclear. In the current study, we observed that a single i.p. administration of AEA caused rapid induction of MDSCs. The MDSCs contained a mixture of granulocytic and monocytic subtypes and expressed Arg-1 and iNOS. The MDSCs suppressed T cell proliferation in vitro and used iNOS to mediate their effect. Moreover, adoptive transfer of MDSCs led to suppression of mBSA-induced DTH. Through the use of pharmacological inhibition, as well as genetic knockout mice, we found that the induction of MDSCs by AEA was CB1-dependent. The induction of MDSCs by AEA was reduced significantly in mast cell-deficient mice, while maintained in LPS-insensitive mice, showing that the induction of MDSCs by AEA was dependent, at least in part, on mast cells and independent of TLR4. Chemokine analysis of AEA- treated WT mice showed an early spike of MCP-1, which was decreased in Kit(W/W-sh) mice, showing a role of mast cells in the secretion of MCP-1 in response to AEA. Also, use of antibodies against MCP-1 or mice deficient in MCP-1 confirmed the role played by MCP-1. Interestingly, MCP-1 played a significant role in the induction of monocytic but not granulocytic MDSCs. Our studies demonstrate for the first time that endocannaboinids activate CB1 on mast cells to induce MCP-1, which facilitates recruitment of monocytic MDSCs.
Assuntos
Ácidos Araquidônicos/farmacologia , Quimiocina CCL2/fisiologia , Endocanabinoides/farmacologia , Mastócitos/fisiologia , Células Mieloides/efeitos dos fármacos , Alcamidas Poli-Insaturadas/farmacologia , Animais , Antígeno CD11b/análise , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/fisiologia , Receptor CB1 de Canabinoide/efeitos dos fármacos , Receptor CB1 de Canabinoide/fisiologia , Receptores de Quimiocinas/análiseRESUMO
Δ(9)-Tetrahydrocannabinol (THC), the major bioactive component of marijuana, has been shown to induce functional myeloid-derived suppressor cells (MDSCs) in vivo. Here, we studied the involvement of microRNA (miRNA) in this process. CD11b(+)Gr-1(+) MDSCs were purified from peritoneal exudates of mice administered with THC and used for genome-wide miRNA profiling. Expression of CD31 and Ki-67 confirmed that the THC-MDSCs were immature and proliferating. THC-induced MDSCs exhibited distinct miRNA expression signature relative to various myeloid cells and BM precursors. We identified 13 differentially expressed (>2-fold) miRNA in THC-MDSCs relative to control BM precursors. In silico target prediction for these miRNA and pathway analysis using multiple bioinformatics tools revealed significant overrepresentation of Gene Ontology clusters within hematopoiesis, myeloid cell differentiation, and regulation categories. Insulin-like growth factor 1 signaling involved in cell growth and proliferation, and myeloid differentiation pathways were among the most significantly enriched canonical pathways. Among the differentially expressed, miRNA-690 was highly overexpressed in THC-MDSCs (â¼16-fold). Transcription factor CCAAT/enhancer-binding protein α (C/EBPα) was identified as a potential functional target of miR-690. Supporting this, C/EBPα expression was attenuated in THC-MDSCs as compared with BM precursors and exhibited an inverse relation with miR-690. miR-690 knockdown using peptide nucleic acid-antagomiR was able to unblock and significantly increase C/EBPα expression establishing the functional link. Further, CD11b(+)Ly6G(+)Ly6C(+) and CD11b(+)Ly6G(-)Ly6C(+) purified subtypes showed high levels of miR-690 with attenuated C/EBPα expression. Moreover, EL-4 tumor-elicited MDSCs showed increased miR-690 expression. In conclusion, miRNA are significantly altered during the generation of functional MDSC from BM. Select miRNA such as miR-690 targeting genes involved in myeloid expansion and differentiation likely play crucial roles in this process and therefore in cannabinoid-induced immunosuppression.
Assuntos
Analgésicos não Narcóticos/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , Dronabinol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Tolerância Imunológica/efeitos dos fármacos , Células Mieloides/metabolismo , Animais , Antígenos de Diferenciação/biossíntese , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/imunologia , Células Cultivadas , Feminino , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Tolerância Imunológica/genética , Camundongos , Células Mieloides/citologia , Células Mieloides/imunologiaRESUMO
BACKGROUND: Interstitial cystitis (IC), more recently called painful bladder syndrome (PBS) is a complex disease associated with chronic bladder inflammation that primarily affects women. Its symptoms include frequent urinary urgency accompanied by discomfort or pain in the bladder and lower abdomen. In the United States, eight million people, mostly women, have IC/PBS. New evidence that autoimmune mechanisms are important in the pathogenesis of IC/PBS triggered interest. METHODOLOGY/PRINCIPAL FINDINGS: SWXJ mice immunized with a homogenate of similar mice's urinary bladders develop an autoimmune phenotype comparable to clinical IC with functional and histological alterations confined to the urinary bladder. Using the murine model of experimental autoimmune cystitis (EAC), we found that serum levels of CXCR3 ligand and local T helper type 1 (Th1) cytokine are elevated. Also, IFN-γ-inducible protein10 (CXCL10) blockade attenuated overall cystitis severity scores; reversed the development of IC; decreased local production of CXCR3 and its ligands, IFN-γ, and tumor necrosis factor-α (TNF-α); and lowered systemic levels of CXCR3 ligands. Urinary bladder CD4(+) T cells, mast cells, and neutrophils infiltrates were reduced following anti-CXCL10 antibody (Ab) treatment of mice. Anti-CXCL10 Ab treatment also reversed the upregulated level of CXCR3 ligand mRNA at urinary bladder sites. The decreased number and percentage of systemic CD4(+) T cells in EAC mice returned to normal after anti-CXCL10 Ab treatment. CONCLUSION/SIGNIFICANCE: Taken together, our findings provide important new information about the mechanisms underlying EAC pathogenesis, which has symptoms similar to those of IC/PBS. CXCL10 has the potential for use in developing new therapy for IC/PBS.
Assuntos
Anticorpos/uso terapêutico , Quimiocina CXCL10/metabolismo , Cistite Intersticial/tratamento farmacológico , Cistite/tratamento farmacológico , Animais , Quimiocina CXCL10/imunologia , Quimiocina CXCL11/metabolismo , Quimiocina CXCL9/metabolismo , Feminino , Interferon gama/metabolismo , Camundongos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Allergy to sapodilla (Manilkara zapota) fruit ingestion is rare. An independent study from our group has identified a basic thaumatin-like protein (TLP 2) as the major allergen. The present study was aimed at identifying and characterizing additional allergens from sapodilla. METHODS: Allergic subjects were identified by case history, skin prick test (SPT) and allergen-specific IgE. Sapodilla extract was fractionated using SP-Sepharose into 3 components (SP1, SP2 and SP3) which were analyzed by native/SDS-PAGE, IgE-immunoblot, isoelectric focusing (IEF) and N-terminal sequencing. The conserved regions of plant TLPs and the N-terminal sequence were used to design primers for PCR. RESULTS: SPT and ELISA confirmed a subject with oral allergy syndrome (OAS) to sapodilla and custard apple. Two proteins (26.9 and 24.5kDa; reducing conditions) were detected as allergens, of which the latter in SP2 has already been identified as basic TLP (TLP 2). The 26.9kDa protein present in SP1 was identified as an acidic TLP based on native PAGE, IEF and N-terminal sequencing. Presence of a basic ß-1,3-glucanase in SP3 was inferred by zymography. Sequence analysis of the genomic clone of the acidic TLP gene revealed that it is intronless and non-glycosylated. Evolutionary relatedness to olive, grape and kiwi fruit allergenic TLPs were inferred by phylogenetic analysis. CONCLUSIONS: An acidic TLP (TLP 1) was identified as a new allergen in sapodilla. TLP 1 is a single polypeptide (207 residues) belonging to the thaumatin family of the GH64-TLP-SF superfamily. Clinically, sapodilla should be considered in the list of fruits causing OAS.