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1.
Autophagy ; : 1-22, 2023 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-37978868

RESUMO

Macroautophagy/autophagy contributes to maladaptive kidney repair by inducing pro-fibrotic factors such as FGF2 (fibroblast growth factor 2), but the underlying mechanism remains elusive. Here, we show that EGR1 (early growth response 1) was induced in injured proximal tubules after ischemic acute kidney injury (AKI) and this induction was suppressed by autophagy deficiency in inducible, renal tubule-specific atg7 (autophagy related 7) knockout (iRT-atg7 KO) mice. In cultured proximal tubular cells, TGFB1 (transforming growth factor beta 1) induced EGR1 and this induction was also autophagy dependent. Egr1 knockdown in tubular cells reduced FGF2 expression during TGFB1 treatment, leading to less FGF2 secretion and decreased paracrine effects on fibroblasts. ChIP assay detected an increased binding of EGR1 to the Fgf2 gene promoter in TGFB1-treated tubular cells. Both Fgf2 and Egr1 transcription was inhibited by FGF2 neutralizing antibody, suggesting a positive feedback for EGR1-mediated FGF2 autoregulation. This feedback was confirmed using fgf2-deficient tubular cells and fgf2-deficient mice. Upstream of EGR1, autophagy deficiency in mice suppressed MAPK/ERK (mitogen-activated protein kinase) activation in post-ischemic renal tubules. This inhibition correlated with SQSTM1/p62 (sequestosome 1) aggregation and its sequestration of MAPK/ERK. SQSTM1/p62 interacted with MAPK/ERK and blocked its activation during TGFB1 treatment in autophagy-deficient tubular cells. Inhibition of MAPK/ERK suppressed EGR1 and FGF2 expression in maladaptive tubules, leading to the amelioration of renal fibrosis and improvement of renal function. These results suggest that autophagy activates MAPK/ERK in renal tubular cells, which induces EGR1 to transactivate FGF2. FGF2 is then secreted into the interstitium to stimulate fibroblasts for fibrogenesis.Abbreviation: 3-MA: 3-methyladenine; ACTA2/α-SMA: actin alpha 2, smooth muscle, aorta; ACTB/ß-actin: actin, beta; AKI: acute kidney injury; aa: amino acid; ATG/Atg: autophagy related; BUN: blood urea nitrogen; ChIP: chromatin immunoprecipitation; CKD: chronic kidney disease; CM: conditioned medium; COL1A1: collagen, type I, alpha 1; COL4A1: collagen, type IV, alpha 1; CQ: chloroquine; DBA: dolichos biflorus agglutinin; EGR1: early growth response 1; ELK1: ELK1, member of ETS oncogene family; FGF2: fibroblast growth factor 2; FN1: fibronectin 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HAVCR1/KIM-1: hepatitis A virus cellular receptor 1; IP: immunoprecipitation; LIR: LC3-interacting region; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MAP2K/MEK: mitogen-activated protein kinase kinase; MAPK: mitogen-activated protein kinase; NFKB: nuclear factor kappa B; PB1: Phox and Bem1; PFT: pifithrin α; PPIB/cyclophilin B: peptidylprolyl isomerase B; RT-qPCR: real time-quantitative PCR; SQSTM1/p62: sequestosome 1; TGFB1/TGF-ß1: transforming growth factor beta 1; VIM: vimentin.

2.
J Immunol ; 211(5): 853-861, 2023 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-37477694

RESUMO

APCs such as dendritic cells and macrophages play a pivotal role in mediating immune tolerance and restoring intestinal immune homeostasis by limiting inflammatory responses against commensal bacteria. However, cell-intrinsic molecular regulators critical for programming intestinal APCs to a regulatory state rather than an inflammatory state are unknown. In this study, we report that the transcription factor retinoid X receptor α (RXRα) signaling in CD11c+ APCs is essential for suppressing intestinal inflammation by imparting an anti-inflammatory phenotype. Using a mouse model of ulcerative colitis, we demonstrated that targeted deletion of RXRα in CD11c+ APCs in mice resulted in the loss of T cell homeostasis with enhanced intestinal inflammation and increased histopathological severity of colonic tissue. This was due to the increased production of proinflammatory cytokines that drive Th1/Th17 responses and decreased expression of immune-regulatory factors that promote regulatory T cell differentiation in the colon. Consistent with these findings, pharmacological activation of the RXRα pathway alleviated colitis severity in mice by suppressing the expression of inflammatory cytokines and limiting Th1/Th17 cell differentiation. These findings identify an essential role for RXRα in APCs in regulating intestinal immune homeostasis and inflammation. Thus, manipulating the RXRα pathway could provide novel opportunities for enhancing regulatory responses and dampening colonic inflammation.


Assuntos
Colite , Fatores de Transcrição , Animais , Camundongos , Colo , Citocinas/metabolismo , Homeostase , Inflamação , Mucosa Intestinal , Intestinos/patologia , Camundongos Endogâmicos C57BL , Receptor X Retinoide alfa , Fatores de Transcrição/metabolismo
3.
Lab Invest ; 103(3): 100009, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36925200

RESUMO

Kidney repair after injury involves the cross-talk of injured kidney tubules with interstitial fibroblasts and immune cells. Although tubular cells produce multiple cytokines, the role and regulation of specific cytokines in kidney repair are largely undefined. In this study, we detected the induction of fibroblast growth factor 2 (FGF2) in mouse kidneys after repeated low-dose cisplatin (RLDC) treatment and in RLDC-treated renal proximal tubule cells in vitro. We further detected FGF2 in the culture medium of RLDC-treated renal tubular cells but not in the medium of control cells, indicating that RLDC induces FGF2 expression and secretion. Compared with the medium of control cells, the medium of RLDC-treated renal tubular cells was twice as effective in promoting fibroblast proliferation. Remarkably, the proliferative effect of the RLDC-treated cell medium was diminished by FGF2-neutralizing antibodies. In addition, the RLDC-treated cell medium induced the expression of fibrosis-related proteins, which was partially suppressed by FGF2-neutralizing antibodies. In mice, FGF2 deficiency partially prevented RLDC-induced decline in kidney function, loss of kidney weight, renal fibrosis, and inflammation. Together, these results indicate that FGF2 is produced by renal tubular cells after kidney injury and acts as an important paracrine factor in maladaptive kidney repair and disease progression.


Assuntos
Cisplatino , Fator 2 de Crescimento de Fibroblastos , Camundongos , Animais , Fator 2 de Crescimento de Fibroblastos/metabolismo , Cisplatino/farmacologia , Rim/patologia , Túbulos Renais/metabolismo , Fibrose , Citocinas/metabolismo
4.
J Immunol ; 209(2): 368-378, 2022 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-35760519

RESUMO

Extraintestinal manifestations are common in inflammatory bowel disease and involve several organs, including the kidney. However, the mechanisms responsible for renal manifestation in inflammatory bowel disease are not known. In this study, we show that the Wnt-lipoprotein receptor-related proteins 5 and 6 (LRP5/6) signaling pathway in macrophages plays a critical role in regulating colitis-associated systemic inflammation and renal injury in a murine dextran sodium sulfate-induced colitis model. Conditional deletion of the Wnt coreceptors LRP5/6 in macrophages in mice results in enhanced susceptibility to dextran sodium sulfate colitis-induced systemic inflammation and acute kidney injury (AKI). Furthermore, our studies show that aggravated colitis-associated systemic inflammation and AKI observed in LRP5/6LysM mice are due to increased bacterial translocation to extraintestinal sites and microbiota-dependent increased proinflammatory cytokine levels in the kidney. Conversely, depletion of the gut microbiota mitigated colitis-associated systemic inflammation and AKI in LRP5/6LysM mice. Mechanistically, LRP5/6-deficient macrophages were hyperresponsive to TLR ligands and produced higher levels of proinflammatory cytokines, which are associated with increased activation of MAPKs. These results reveal how the Wnt-LRP5/6 signaling in macrophages controls colitis-induced systemic inflammation and AKI.


Assuntos
Injúria Renal Aguda , Colite , Microbioma Gastrointestinal , Doenças Inflamatórias Intestinais , Microbiota , Injúria Renal Aguda/metabolismo , Animais , Colite/induzido quimicamente , Citocinas/metabolismo , Sulfato de Dextrana/toxicidade , Inflamação/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Rim/metabolismo , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Via de Sinalização Wnt/genética
5.
J Immunol ; 207(5): 1428-1436, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34348977

RESUMO

Dendritic cells (DCs) are professional APCs that play a crucial role in initiating robust immune responses against invading pathogens while inducing regulatory responses to the body's tissues and commensal microorganisms. A breakdown of DC-mediated immunological tolerance leads to chronic inflammation and autoimmune disorders. However, cell-intrinsic molecular regulators that are critical for programming DCs to a regulatory state rather than to an inflammatory state are not known. In this study, we show that the activation of the TCF4 transcription factor in DCs is critical for controlling the magnitude of inflammatory responses and limiting neuroinflammation. DC-specific deletion of TCF4 in mice increased Th1/Th17 responses and exacerbated experimental autoimmune encephalomyelitis pathology. Mechanistically, loss of TCF4 in DCs led to heightened activation of p38 MAPK and increased levels of proinflammatory cytokines IL-6, IL-23, IL-1ß, TNF-α, and IL-12p40. Consistent with these findings, pharmacological blocking of p38 MAPK activation delayed experimental autoimmune encephalomyelitis onset and diminished CNS pathology in TCF4ΔDC mice. Thus, manipulation of the TCF4 pathway in DCs could provide novel opportunities for regulating chronic inflammation and represents a potential therapeutic approach to control autoimmune neuroinflammation.


Assuntos
Encefalomielite Autoimune Experimental , Células Th1 , Animais , Células Dendríticas , Camundongos , Camundongos Endogâmicos C57BL , Células Th17 , Fator de Transcrição 4
6.
Front Immunol ; 12: 691134, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34394085

RESUMO

For decades, lactate has been considered an innocuous bystander metabolite of cellular metabolism. However, emerging studies show that lactate acts as a complex immunomodulatory molecule that controls innate and adaptive immune cells' effector functions. Thus, recent advances point to lactate as an essential and novel signaling molecule that shapes innate and adaptive immune responses in the intestine and systemic sites. Here, we review these recent advances in the context of the pleiotropic effects of lactate in regulating diverse functions of immune cells in the tissue microenvironment and under pathological conditions.


Assuntos
Células Dendríticas/imunologia , Ácido Láctico/imunologia , Macrófagos/imunologia , Animais , Autoimunidade , Proteínas de Ciclo Celular/imunologia , Humanos , Imunomodulação , Infecções/imunologia , Doenças Inflamatórias Intestinais/imunologia , Transportadores de Ácidos Monocarboxílicos/imunologia , Neoplasias/imunologia , Receptores Acoplados a Proteínas G/imunologia
7.
Mol Cancer Res ; 19(9): 1486-1497, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34099522

RESUMO

DNA damage, induced by either chemical carcinogens or environmental pollutants, plays an important role in the initiation of colorectal cancer. DNA repair processes, however, are involved in both protecting against cancer formation, and also contributing to cancer development, by ensuring genomic integrity and promoting the efficient DNA repair in tumor cells, respectively. Although DNA repair pathways have been well exploited in the treatment of breast and ovarian cancers, the role of DNA repair processes and their therapeutic efficacy in colorectal cancer is yet to be appreciably explored. To understand the role of DNA repair, especially homologous recombination (HR), in chemical carcinogen-induced colorectal cancer growth, we unraveled the role of RAD51AP1 (RAD51-associated protein 1), a protein involved in HR, in genotoxic carcinogen (azoxymethane, AOM)-induced colorectal cancer. Although AOM treatment alone significantly increased RAD51AP1 expression, the combination of AOM and dextran sulfate sodium (DSS) treatment dramatically increased by several folds. RAD51AP1 expression is found in mouse colonic crypt and proliferating cells. RAD51AP1 expression is significantly increased in majority of human colorectal cancer tissues, including BRAF/KRAS mutant colorectal cancer, and associated with reduced treatment response and poor prognosis. Rad51ap1-deficient mice were protected against AOM/DSS-induced colorectal cancer. These observations were recapitulated in a genetically engineered mouse model of colorectal cancer (ApcMin /+ ). Furthermore, chemotherapy-resistant colorectal cancer is associated with increased RAD51AP1 expression. This phenomenon is associated with reduced cell proliferation and colorectal cancer stem cell (CRCSC) self-renewal. Overall, our studies provide evidence that RAD51AP1 could be a novel diagnostic marker for colorectal cancer and a potential therapeutic target for colorectal cancer prevention and treatment. IMPLICATIONS: This study provides first in vivo evidence that RAD51AP1 plays a critical role in colorectal cancer growth and drug resistance by regulating CRCSC self-renewal.


Assuntos
Autorrenovação Celular , Neoplasias Colorretais/tratamento farmacológico , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Resistencia a Medicamentos Antineoplásicos , Fluoruracila/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/fisiologia , Animais , Antimetabólitos Antineoplásicos/farmacologia , Apoptose , Estudos de Casos e Controles , Proliferação de Células , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Proteínas de Ligação a DNA/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Prognóstico , Proteínas de Ligação a RNA/genética , Taxa de Sobrevida , Células Tumorais Cultivadas
8.
Methods Mol Biol ; 2224: 133-146, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33606212

RESUMO

Crohn's disease (CD) and ulcerative colitis are two main clinically defined forms of chronic inflammatory bowel disease (IBD). Chronic intestinal inflammation is inextricably linked to colitis-associated colon carcinogenesis (CAC). Patients with ulcerative colitis (UC) and Crohn's disease (CD) have an increased risk of colon cancer. Our understanding of IBD and IBD-associated colon carcinogenesis depends largely on rodent models. AOM-DSS-induced colitis-associated colon cancer in mice is the most widely used and accepted model that can recapitulate the human IBD-associated colon cancer. Here, we have provided detailed protocols of this mouse model of experimentally induced chronic intestinal inflammation-associated colon cancer. We will also discuss the protocols for the isolation and analysis of inflammatory immune cells from the colon.


Assuntos
Colite Ulcerativa/patologia , Neoplasias Associadas a Colite/patologia , Neoplasias do Colo/patologia , Animais , Colite Ulcerativa/induzido quimicamente , Colo/patologia , Doença de Crohn/induzido quimicamente , Doença de Crohn/patologia , Sulfato de Dextrana/farmacologia , Modelos Animais de Doenças , Feminino , Doenças Inflamatórias Intestinais/induzido quimicamente , Doenças Inflamatórias Intestinais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL
9.
J Immunol ; 205(8): 2265-2275, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32917787

RESUMO

Loss of immune tolerance to gut microflora is inextricably linked to chronic intestinal inflammation and colitis-associated colorectal cancer (CAC). The LRP5/6 signaling cascade in APCs contributes to immune homeostasis in the gut, but whether this pathway in APCs protects against CAC is not known. In the current study, using a mouse model of CAC, we show that the LRP5/6-ß-catenin-IL-10 signaling axis in intestinal CD11c+ APCs protects mice from CAC by regulating the expression of tumor-promoting inflammatory factors in response to commensal flora. Genetic deletion of LRP5/6 in CD11c+ APCs in mice (LRP5/6ΔCD11c) resulted in enhanced susceptibility to CAC. This is due to a microbiota-dependent increased expression of proinflammatory factors and decreased expression of the immunosuppressive cytokine IL-10. This condition could be improved in LRP5/6ΔCD11c mice by depleting the gut flora, indicating the importance of LRP5/6 in mediating immune tolerance to the gut flora. Moreover, mechanistic studies show that LRP5/6 suppresses the expression of tumor-promoting inflammatory factors in CD11c+ APCs via the ß-catenin-IL-10 axis. Accordingly, conditional activation of ß-catenin specifically in CD11c+ APCs or in vivo administration of IL-10 protected LRP5/6ΔCD11c mice from CAC by suppressing the expression of inflammatory factors. In summary, in this study, we identify a key role for the LRP5/6-ß-catenin-IL-10 signaling pathway in intestinal APCs in resolving chronic intestinal inflammation and protecting against CAC in response to the commensal flora.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Colite/imunologia , Neoplasias do Colo/imunologia , Microbioma Gastrointestinal/imunologia , Interleucina-10/imunologia , Via de Sinalização Wnt/imunologia , beta Catenina/imunologia , Animais , Células Apresentadoras de Antígenos/patologia , Colite/complicações , Colite/genética , Colite/patologia , Neoplasias do Colo/etiologia , Neoplasias do Colo/genética , Neoplasias do Colo/prevenção & controle , Microbioma Gastrointestinal/genética , Interleucina-10/genética , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Via de Sinalização Wnt/genética , beta Catenina/genética
10.
PLoS Pathog ; 16(9): e1008842, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32898178

RESUMO

Signaling through retinoic acid inducible gene I (RIG-I) like receptors (RLRs) is tightly regulated, with activation occurring upon sensing of viral nucleic acids, and suppression mediated by negative regulators. Under homeostatic conditions aberrant activation of melanoma differentiation-associated protein-5 (MDA5) is prevented through editing of endogenous dsRNA by RNA editing enzyme Adenosine Deaminase Acting on RNA (ADAR1). In addition, ADAR1 is postulated to play pro-viral and antiviral roles during viral infections that are dependent or independent of RNA editing activity. Here, we investigated the importance of ADAR1 isoforms in modulating influenza A virus (IAV) replication and revealed the opposing roles for ADAR1 isoforms, with the nuclear p110 isoform restricting versus the cytoplasmic p150 isoform promoting IAV replication. Importantly, we demonstrate that p150 is critical for preventing sustained RIG-I signaling, as p150 deficient cells showed increased IFN-ß expression and apoptosis during IAV infection, independent of RNA editing activity. Taken together, the p150 isoform of ADAR1 is important for preventing sustained RIG-I induced IFN-ß expression and apoptosis during viral infection.


Assuntos
Adenosina Desaminase/metabolismo , Apoptose , Proteína DEAD-box 58/metabolismo , Vírus da Influenza A/fisiologia , Influenza Humana/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Replicação Viral , Células A549 , Adenosina Desaminase/genética , Proteína DEAD-box 58/genética , Células HEK293 , Humanos , Influenza Humana/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de Ligação a RNA/genética , Receptores Imunológicos
11.
Cancer Res ; 80(18): 3855-3866, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32665355

RESUMO

RAD51-associated protein 1 (RAD51AP1) plays an integral role in homologous recombination by activating RAD51 recombinase. Homologous recombination is essential for preserving genome integrity and RAD51AP1 is critical for D-loop formation, a key step in homologous recombination. Although RAD51AP1 is involved in maintaining genomic stability, recent studies have shown that RAD51AP1 expression is significantly upregulated in human cancers. However, the functional role of RAD51AP1 in tumor growth and the underlying molecular mechanism(s) by which RAD51AP1 regulates tumorigenesis have not been fully understood. Here, we use Rad51ap1-knockout mice in genetically engineered mouse models of breast cancer to unravel the role of RAD51AP1 in tumor growth and metastasis. RAD51AP1 gene transcript was increased in both luminal estrogen receptor-positive breast cancer and basal triple-negative breast cancer, which is associated with poor prognosis. Conversely, knockdown of RAD51AP1 (RADP51AP1 KD) in breast cancer cell lines reduced tumor growth. Rad51ap1-deficient mice were protected from oncogene-driven spontaneous mouse mammary tumor growth and associated lung metastasis. In vivo, limiting dilution studies provided evidence that Rad51ap1 plays a critical role in breast cancer stem cell (BCSC) self-renewal. RAD51AP1 KD improved chemotherapy and radiotherapy response by inhibiting BCSC self-renewal and associated pluripotency. Overall, our study provides genetic and biochemical evidences that RAD51AP1 is critical for tumor growth and metastasis by increasing BCSC self-renewal and may serve as a novel target for chemotherapy- and radiotherapy-resistant breast cancer. SIGNIFICANCE: This study provides in vivo evidence that RAD51AP1 plays a critical role in breast cancer growth and metastasis by regulating breast cancer stem cell self-renewal.


Assuntos
Neoplasias da Mama/patologia , Autorrenovação Celular/genética , Proteínas de Ligação a DNA/deficiência , Neoplasias Mamárias Animais/patologia , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/terapia , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Ativação Enzimática , Feminino , Humanos , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Neoplásicas , Proteínas de Ligação a RNA/genética , Rad51 Recombinase/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Regulação para Cima
12.
Front Immunol ; 11: 122, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32132993

RESUMO

Dendritic cells (DCs) control the strength and quality of antigen-specific adaptive immune responses. This is critical for launching a robust immunity against invading pathogens while maintaining a state of tolerance to self-antigens. However, this also represents a fundamental barrier to anti-tumor immune responses and cancer immunotherapy. DCs in the tumor microenvironment (TME) play a key role in this process. The factors in the TME and signaling networks that program DCs to a regulatory state are not fully understood. Recent advances point to novel mechanisms by which the canonical Wnt signaling cascade in DCs regulates immune suppression, and the same pathway in tumors is associated with the evasion of anti-tumor immunity. Here, we review these recent advances in the context of the pleiotropic effects of the Wnts in shaping anti-tumor immune responses by modulating DC functions. In addition, we will discuss how Wnt/ß-catenin pathway in DCs can be targeted for successful cancer immunotherapy.


Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Imunidade/imunologia , Microambiente Tumoral/imunologia , Microambiente Tumoral/fisiologia , Via de Sinalização Wnt/imunologia , Humanos , Imunoterapia , Neoplasias/imunologia , Transdução de Sinais/imunologia , beta Catenina/imunologia , beta Catenina/metabolismo
13.
J Virol ; 94(9)2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32075925

RESUMO

Seasonal influenza virus infections cause mild illness in healthy adults, as timely viral clearance is mediated by the functions of cytotoxic T cells. However, avian H5N1 influenza virus infections can result in prolonged and fatal illness across all age groups, which has been attributed to the overt and uncontrolled activation of host immune responses. Here, we investigate how excessive innate immune responses to H5N1 impair subsequent adaptive T cell responses in the lungs. Using recombinant H1N1 and H5N1 strains sharing 6 internal genes, we demonstrate that H5N1 (2:6) infection in mice causes higher stimulation and increased migration of lung dendritic cells to the draining lymph nodes, resulting in greater numbers of virus-specific T cells in the lungs. Despite robust T cell responses in the lungs, H5N1 (2:6)-infected mice showed inefficient and delayed viral clearance compared with H1N1-infected mice. In addition, we observed higher levels of inhibitory signals, including increased PD-1 and interleukin-10 (IL-10) expression by cytotoxic T cells in H5N1 (2:6)-infected mice, suggesting that delayed viral clearance of H5N1 (2:6) was due to the suppression of T cell functions in vivo Importantly, H5N1 (2:6)-infected mice displayed decreased numbers of tissue-resident memory T cells compared with H1N1-infected mice; however, despite the decreased number of tissue-resident memory T cells, H5N1 (2:6) was protected against a heterologous challenge from H3N2 virus (X31). Taken together, our study provides mechanistic insight for the prolonged viral replication and protracted illness observed in H5N1-infected patients.IMPORTANCE Influenza viruses cause upper respiratory tract infections in humans. In healthy adults, seasonal influenza virus infections result in mild disease. Occasionally, influenza viruses endemic in domestic birds can cause severe and fatal disease even in healthy individuals. In avian influenza virus-infected patients, the host immune system is activated in an uncontrolled manner and is unable to control infection in a timely fashion. In this study, we investigated why the immune system fails to effectively control a modified form of avian influenza virus. Our studies show that T cell functions important for clearing virally infected cells are impaired by higher negative regulatory signals during modified avian influenza virus infection. In addition, memory T cell numbers were decreased in modified avian influenza virus-infected mice. Our studies provide a possible mechanism for the severe and prolonged disease associated with avian influenza virus infections in humans.


Assuntos
Virus da Influenza A Subtipo H5N1/genética , Influenza Humana/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Aves , Humanos , Imunidade Inata/imunologia , Memória Imunológica/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Virus da Influenza A Subtipo H5N1/metabolismo , Vírus da Influenza A/fisiologia , Influenza Humana/metabolismo , Pulmão/virologia , Camundongos , Infecções por Orthomyxoviridae/virologia , Replicação Viral/genética
14.
Am J Physiol Lung Cell Mol Physiol ; 318(4): L750-L761, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32073894

RESUMO

The adaptive immune response could play a major role in the resolution of lung injury. Although regulatory T cells (Tregs) have been implicated in promoting the resolution of lung injury, therapeutic strategies to enhance Treg quantity and activity at the site of injury need further exploration. In the current study, Akt inhibition using triciribine (TCBN), given 48 h after lipopolysaccharide (LPS) administration, increased Tregs-promoted resolution of acute lung injury (ALI). TCBN treatment enhanced the resolution of LPS-induced ALI on day 7 by reducing pulmonary edema and neutrophil activity associated with an increased number of CD4+/FoxP3+/CD103+ and CTLA4+ effector Tregs, specifically in the injured lungs and not in the spleen. Treatment of EL-4 T-lymphocytes with two Akt inhibitors (TCBN and MK-2206) for 72 h resulted in increased FoxP3 expression in vitro. On the other end, Treg-specific PTEN knockout (PTENTreg KO) mice that have a higher Akt activity in its Tregs exhibited a significant impairment in ALI resolution, increased edema, and neutrophil activity associated with a reduced number of CD4+/FoxP3+/CD103+ and CTLA4+ effector Tregs as compared with the control group. In conclusion, our study identifies a potential target for the treatment of late-stage ALI by promoting resolution through effector Treg-mediated suppression of inflammation.


Assuntos
Lesão Pulmonar Aguda/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linfócitos T Reguladores/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Transferência Adotiva/métodos , Animais , Antígenos CD/metabolismo , Antígenos CD4/metabolismo , Modelos Animais de Doenças , Feminino , Fatores de Transcrição Forkhead/metabolismo , Cadeias alfa de Integrinas/metabolismo , Lipopolissacarídeos/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Edema Pulmonar/induzido quimicamente , Edema Pulmonar/metabolismo , Baço , Linfócitos T Reguladores/efeitos dos fármacos
15.
J Immunol ; 200(9): 3259-3268, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29602775

RESUMO

Aberrant Wnt/ß-catenin signaling occurs in several inflammatory diseases, including inflammatory bowel disease and inflammatory bowel disease-associated colon carcinogenesis. However, its role in shaping mucosal immune responses to commensals in the gut remains unknown. In this study, we investigated the importance of canonical Wnt signaling in CD11c+ APCs in controlling intestinal inflammation. Using a mouse model of ulcerative colitis, we demonstrated that canonical Wnt signaling in intestinal CD11c+ APCs controls intestinal inflammation by imparting an anti-inflammatory phenotype. Genetic deletion of Wnt coreceptors, low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) in CD11c+ APCs in LRP5/6ΔCD11c mice, resulted in enhanced intestinal inflammation with increased histopathological severity of colonic tissue. This was due to microbiota-dependent increased production of proinflammatory cytokines and decreased expression of immune-regulatory factors such as IL-10, retinoic acid, and IDO. Mechanistically, loss of LRP5/6-mediated signaling in CD11c+ APCs resulted in altered microflora and T cell homeostasis. Furthermore, our study demonstrates that conditional activation of ß-catenin in CD11c+ APCs in LRP5/6ΔCD11c mice resulted in reduced intestinal inflammation with decreased histopathological severity of colonic tissue. These results reveal a mechanism by which intestinal APCs control intestinal inflammation and immune homeostasis via the canonical Wnt-signaling pathway.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Microbioma Gastrointestinal/imunologia , Imunidade nas Mucosas/imunologia , Mucosa Intestinal/imunologia , Via de Sinalização Wnt/imunologia , Animais , Colite Ulcerativa/imunologia , Colite Ulcerativa/microbiologia , Colo/imunologia , Colo/microbiologia , Homeostase/imunologia , Inflamação/imunologia , Mucosa Intestinal/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
16.
J Immunol ; 200(5): 1781-1789, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29386257

RESUMO

At mucosal sites such as the intestine, the immune system launches robust immunity against invading pathogens while maintaining a state of tolerance to commensal flora and ingested food Ags. The molecular mechanisms underlying this phenomenon remain poorly understood. In this study, we report that signaling by GPR81, a receptor for lactate, in colonic dendritic cells and macrophages plays an important role in suppressing colonic inflammation and restoring colonic homeostasis. Genetic deletion of GPR81 in mice led to increased Th1/Th17 cell differentiation and reduced regulatory T cell differentiation, resulting in enhanced susceptibility to colonic inflammation. This was due to increased production of proinflammatory cytokines (IL-6, IL-1ß, and TNF-α) and decreased expression of immune regulatory factors (IL-10, retinoic acid, and IDO) by intestinal APCs lacking GPR81. Consistent with these findings, pharmacological activation of GPR81 decreased inflammatory cytokine expression and ameliorated colonic inflammation. Taken together, these findings identify a new and important role for the GPR81 signaling pathway in regulating immune tolerance and colonic inflammation. Thus, manipulation of the GPR81 pathway could provide novel opportunities for enhancing regulatory responses and treating colonic inflammation.


Assuntos
Colite/metabolismo , Homeostase/fisiologia , Ácido Láctico/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Th1/metabolismo
17.
Front Immunol ; 7: 460, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27833613

RESUMO

The Wnt/ß-catenin pathway is an evolutionarily conserved signaling pathway critical for several biological processes. An aberrant Wnt/ß-catenin signaling is linked to several human diseases. Emerging studies have highlighted the regulatory role of the Wnt/ß-catenin signaling pathway in normal physiological processes of parenchymal and hematopoietic cells. Recent studies have shown that the activation of Wnt/ß-catenin pathway in dendritic cells (DCs) play a critical role in mucosal tolerance and suppression of chronic autoimmune pathologies. Alternatively, tumors activate Wnt/ß-catenin pathway in DCs to induce immune tolerance and thereby evade antitumor immunity through suppression of effector T cell responses and promotion of regulatory T cell responses. Here, we review our work and current understanding of how Wnt/ß-catenin signaling in DCs shapes the immune response in cancer and autoimmunity and discuss how Wnt/ß-catenin pathway can be targeted for successful therapeutic interventions in various human diseases.

18.
PLoS Pathog ; 12(7): e1005754, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27438481

RESUMO

Retinoic acid inducible gene-I (RIG-I) is an innate RNA sensor that recognizes the influenza A virus (IAV) RNA genome and activates antiviral host responses. Here, we demonstrate that RIG-I signaling plays a crucial role in restricting IAV tropism and regulating host immune responses. Mice deficient in the RIG-I-MAVS pathway show defects in migratory dendritic cell (DC) activation, viral antigen presentation, and priming of CD8+ and CD4+ T cell responses during IAV infection. These defects result in decreased frequency of polyfunctional effector T cells and lowered protection against heterologous IAV challenge. In addition, our data show that RIG-I activation is essential for protecting epithelial cells and hematopoietic cells from IAV infection. These diverse effects of RIG-I signaling are likely imparted by the actions of type I interferon (IFN), as addition of exogenous type I IFN is sufficient to overcome the defects in antigen presentation by RIG-I deficient BMDC. Moreover, the in vivo T cell defects in RIG-I deficient mice can be overcome by the activation of MDA5 -MAVS via poly I:C treatment. Taken together, these findings demonstrate that RIG-I signaling through MAVS is critical for determining the quality of polyfunctional T cell responses against IAV and for providing protection against subsequent infection from heterologous or novel pandemic IAV strains.


Assuntos
Proteínas de Membrana/imunologia , Proteínas do Tecido Nervoso/imunologia , Infecções por Orthomyxoviridae/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Técnicas de Cocultura , Células Dendríticas/imunologia , Modelos Animais de Doenças , Citometria de Fluxo , Vírus da Influenza A/imunologia , Interferon Tipo I/biossíntese , Interferon Tipo I/imunologia , Ativação Linfocitária/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Reação em Cadeia da Polimerase , Receptores de Superfície Celular , Linfócitos T/metabolismo
19.
Oncoimmunology ; 5(4): e1115941, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27141399

RESUMO

The tumor microenvironment (TME) contains high levels of the Wnt family of ligands, and aberrant Wnt-signaling occurs in many tumors. Past studies have been directed toward how the Wnt signaling cascade regulates cancer development, progression and metastasis. However, its effects on host antitumor immunity remain unknown. In this report, we show that Wnts in the TME condition dendritic cells (DCs) to a regulatory state and suppress host antitumor immunity. DC-specific deletion of Wnt co-receptors low-density lipoprotein receptor-related protein 5 and 6 (LRP5/6) in mice markedly delayed tumor growth and enhanced host antitumor immunity. Mechanistically, loss of LRP5/6-mediated signaling in DCs resulted in enhanced effector T cell differentiation and decreased regulatory T cell differentiation. This was due to increased production of pro-inflammatory cytokines and decreased production of IL-10, TGF-ß1 and retinoic acid (RA). Likewise, pharmacological inhibition of the Wnts' interaction with its cognate co-receptors LRP5/6 and Frizzled (Fzd) receptors had similar effects on tumor growth and effector T cell responses. Moreover, blocking Wnt-signaling in DCs resulted in enhanced capture of tumor-associated antigens and efficient cross-priming of CD8+ T cells. Hence, blocking the Wnt pathway represents a potential therapeutic to overcome tumor-mediated immune suppression and augment antitumor immunity.

20.
Cancer Res ; 76(11): 3224-35, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27197203

RESUMO

Recently, impressive technical advancements have been made in the isolation and validation of mammary stem cells and cancer stem cells (CSC), but the signaling pathways that regulate stem cell self-renewal are largely unknown. Furthermore, CSCs are believed to contribute to chemo- and radioresistance. In this study, we used the MMTV-Neu-Tg mouse mammary tumor model to identify potential new strategies for eliminating CSCs. We found that both luminal progenitor and basal stem cells are susceptible to genetic and epigenetic modifications, which facilitate oncogenic transformation and tumorigenic potential. A combination of the DNMT inhibitor 5-azacytidine and the HDAC inhibitor butyrate markedly reduced CSC abundance and increased the overall survival in this mouse model. RNA-seq analysis of CSCs treated with 5-azacytidine plus butyrate provided evidence that inhibition of chromatin modifiers blocks growth-promoting signaling molecules such as RAD51AP1 and SPC25, which play key roles in DNA damage repair and kinetochore assembly. Moreover, RAD51AP1 and SPC25 were significantly overexpressed in human breast tumor tissues and were associated with reduced overall patient survival. In conclusion, our studies suggest that breast CSCs are intrinsically sensitive to genetic and epigenetic modifications and can therefore be significantly affected by epigenetic-based therapies, warranting further investigation of combined DNMT and HDAC inhibition in refractory or drug-resistant breast cancer. Cancer Res; 76(11); 3224-35. ©2016 AACR.


Assuntos
Azacitidina/farmacologia , Neoplasias da Mama/prevenção & controle , Carcinoma Basocelular/prevenção & controle , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Histona Desacetilase 1/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Animais , Antimetabólitos Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma Basocelular/metabolismo , Carcinoma Basocelular/patologia , Proliferação de Células , DNA (Citosina-5-)-Metiltransferase 1 , Quimioterapia Combinada , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas
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