RESUMO
Renal cell carcinoma (RCC) is the deadliest form of urological cancer and is projected to be the fourth most common neoplasm in the USA in males by 2040. In addition to the current poor prognosis with 5-year survival rates hardly reaching 15%, the prevalence of resistance to currently available systemic therapies has also established an urgent need to develop new treatment regimen(s) for advanced RCC. Interferon-stimulated gene 15 (ISG15) is the first identified ubiquitin-like modifier and has been intensively studied for its central role in innate immunity against intracellular pathogens. However, in this study, we identified ISG15 as a novel tumor-associated antigen and prognostic marker in RCC. Further, we therapeutically targeted elevated ISG15 expression by means of a Listeria monocytogenes (Lm)-based vaccine, designated Lm-LLO-ISG15, in both subcutaneous and orthotopic RCC mouse models. Treatment with Lm-LLO-ISG15 resulted in an influx of tumor-infiltrating effector T cells and significant anti-tumor efficacy in both subcutaneous and orthotopic RCC tumor models. Treatment with Lm-LLO-ISG15 also generated a robust interferon-gamma response and attracted a larger pool of polyfunctional T cells into the tumor microenvironment. Importantly, the therapeutic efficacy of Lm-LLO-ISG15 in RCC is comparable to that of anti-PD-1 and sunitinib, the current frontline therapies for RCC patients. Collectively, our work illustrates that targeting ISG15 in RCC with a CTL-based immunotherapy such as Lm-LLO-ISG15 is a promising and potentially translatable therapeutic strategy to enhance survival in RCC patients.
Assuntos
Antígenos de Neoplasias , Vacinas Anticâncer , Carcinoma de Células Renais , Citocinas , Neoplasias Renais , Listeria monocytogenes , Ubiquitinas , Animais , Humanos , Masculino , Camundongos , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/terapia , Linhagem Celular Tumoral , Citocinas/antagonistas & inibidores , Citocinas/imunologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias Renais/imunologia , Neoplasias Renais/terapia , Listeria monocytogenes/imunologia , Linfócitos do Interstício Tumoral/imunologia , Camundongos Endogâmicos BALB C , Células NIH 3T3 , Sunitinibe/uso terapêutico , Linfócitos T/imunologia , Ubiquitinas/antagonistas & inibidores , Ubiquitinas/imunologia , Microambiente Tumoral/imunologiaRESUMO
RIG-I-like receptors (RLRs) are involved in the discrimination of self versus non-self via the recognition of double-stranded RNA (dsRNA). Emerging evidence suggests that immunostimulatory dsRNAs are ubiquitously expressed but are disrupted or sequestered by cellular RNA binding proteins (RBPs). TDP-43 is an RBP associated with multiple neurological disorders and is essential for cell viability. Here, we demonstrate that TDP-43 regulates the accumulation of immunostimulatory dsRNA. The immunostimulatory RNA is identified as RNA polymerase III transcripts, including 7SL and Alu retrotransposons, and we demonstrate that the RNA-binding activity of TDP-43 is required to prevent immune stimulation. The dsRNAs activate a RIG-I-dependent interferon (IFN) response, which promotes necroptosis. Genetic inactivation of the RLR-pathway rescues the interferon-mediated cell death associated with loss of TDP-43. Collectively, our study describes a role for TDP-43 in preventing the accumulation of endogenous immunostimulatory dsRNAs and uncovers an intricate relationship between the control of cellular gene expression and IFN-mediated cell death.
Assuntos
Proteína DEAD-box 58/genética , Proteínas de Ligação a DNA/genética , Herpesvirus Humano 8/genética , Necroptose/genética , RNA de Cadeia Dupla/genética , Receptores Imunológicos/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Elementos Alu , Linhagem Celular Tumoral , Sobrevivência Celular , Citocinas/genética , Citocinas/imunologia , Proteína DEAD-box 58/antagonistas & inibidores , Proteína DEAD-box 58/imunologia , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/imunologia , Células Epiteliais/imunologia , Células Epiteliais/virologia , Regulação da Expressão Gênica , Células HEK293 , Herpesvirus Humano 8/crescimento & desenvolvimento , Herpesvirus Humano 8/imunologia , Humanos , Imunização , Interferons/genética , Interferons/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Necroptose/imunologia , Neurônios/imunologia , Neurônios/virologia , RNA Polimerase III/genética , RNA Polimerase III/imunologia , RNA de Cadeia Dupla/imunologia , RNA Mensageiro/genética , RNA Mensageiro/imunologia , RNA Citoplasmático Pequeno/genética , RNA Citoplasmático Pequeno/imunologia , RNA Viral/genética , RNA Viral/imunologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/imunologia , Partícula de Reconhecimento de Sinal/genética , Partícula de Reconhecimento de Sinal/imunologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Ubiquitinas/genética , Ubiquitinas/imunologiaRESUMO
The IFN stimulated gene 15 (ISG15) encodes a 15-kDa ubiquitin-like protein, that is induced by type I IFNs and is conjugated to the bulk of newly synthesized polypeptides at the ribosome. ISG15 functions as an antiviral molecule possibly by being covalently conjugated to viral proteins and disturbing virus particle assembly. Here, we have investigated the effect of ISGylation on degradation and antigen presentation of viral and cellular proteins. ISGylation did not induce proteasomal degradation of bulk ISG15 target proteins neither after overexpressing ISG15 nor after induction by IFN-ß. The MHC class I cell surface expression of splenocytes derived from ISG15-deficient mice or mice lacking the catalytic activity of the major de-ISGylating enzyme USP18 was unaltered as compared to WT mice. Fusion of ubiquitin or FAT10 to the long-lived nucleoprotein (NP) of lymphocytic choriomeningitis virus accelerated the proteasomal degradation of NP while fusion to ISG15 did not detectably speed up NP degradation. Nevertheless, MHC-I restricted presentation of two epitopes of NP were markedly enhanced when it was fused to ISG15 similarly to fusion with ubiquitin or FAT10. Thus, we provide evidence that ISG15 can enhance the presentation of antigens on MHC-I most likely by promoting co-translational antigen processing.
Assuntos
Apresentação de Antígeno/imunologia , Citocinas/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Ubiquitinas/imunologia , Animais , Citocinas/deficiência , Citocinas/genética , Citocinas/metabolismo , Células HEK293 , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Vírus da Coriomeningite Linfocítica/genética , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/imunologia , Proteínas do Nucleocapsídeo/metabolismo , Complexo de Endopeptidases do Proteassoma/imunologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Modificação Traducional de Proteínas/imunologia , Proteólise , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Ubiquitina Tiolesterase/deficiência , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/imunologia , Ubiquitinas/deficiência , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMO
Human interferon-stimulated gene 15 (ISG15) is a 15-kDa ubiquitin-like protein that can be detected as either free ISG15 or covalently associated with its target proteins through a process termed ISGylation. Interestingly, extracellular free ISG15 has been proposed as a cytokinelike protein, whereas ISGylation is a posttranslational modification. ISG15 is a small protein with implications in some biological processes and pathologies that include cancer. This review highlights the findings of both free ISG15 and protein ISGylation involved in several molecular pathways, emerging as central elements in some cancer types.
Assuntos
Citocinas/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias/genética , Processamento de Proteína Pós-Traducional , Ubiquitina Tiolesterase/genética , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitinas/genética , Citocinas/química , Citocinas/imunologia , Regulação Neoplásica da Expressão Gênica , Humanos , Imunidade Inata , Interferon-alfa/genética , Interferon-alfa/imunologia , Interferon beta/genética , Interferon beta/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Modelos Moleculares , Neoplasias/imunologia , Neoplasias/patologia , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Ubiquitina Tiolesterase/imunologia , Enzimas de Conjugação de Ubiquitina/imunologia , Ubiquitinação , Ubiquitinas/química , Ubiquitinas/imunologiaAssuntos
Betacoronavirus/genética , Cisteína Endopeptidases/genética , Interações Hospedeiro-Patógeno/genética , Interferon-alfa/genética , Interferon beta/genética , Transdução de Sinais/genética , Proteínas não Estruturais Virais/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Betacoronavirus/imunologia , Linhagem Celular Tumoral , Proteases 3C de Coronavírus , Cisteína Endopeptidases/imunologia , Citocinas/genética , Citocinas/imunologia , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/imunologia , Células Epiteliais/imunologia , Células Epiteliais/virologia , Regulação da Expressão Gênica , Hepatócitos/imunologia , Hepatócitos/virologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Interferon-alfa/imunologia , Interferon beta/imunologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Receptores Imunológicos , SARS-CoV-2 , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/imunologia , Transdução de Sinais/imunologia , Ubiquitinas/genética , Ubiquitinas/imunologia , Proteínas não Estruturais Virais/imunologiaRESUMO
As a member of DExD/H-box helicase family, DDX41 (DEAD box polypeptide 41) acts as an intracellular DNA sensor that induces type I IFN expression in mammals. Fish DDX41 shares some similar properties with the mammalian counterparts. In this study, a DDX41 orthologous gene from grass carp (Ctenopharyngodon idellus) (CiDDX41) was cloned and characterized. The ORF of CiDDX41 encodes a polypeptide of 614 amino acids. Multiple alignments showed that DDX41 is highly conserved among different species. Phylogenetic tree analysis revealed that CiDDX41 shares a high degree of homology with Sinocyclocheilus rhinocerous DDX41. CiDDX41 is highly expressed in kidney, intestines, liver and spleen. Their expressions are up-regulated more obviously after the treatment with GCRV. Over-expression of CiDDX41 in CIK cells increases the transcription level of grass carp IFN I and ISG15. On the contrary, knockdown of CiDDX41 inhibits the IFN I and ISG15 transcription. Moreover, a part of CiDDX41 translocates from the nuclear to cytoplasm to interact with grass carp STING (CiSTING). In CIK cells, overexpression of CiDDX41 and CiSTING can promote the phosphorylation and nuclear-cytoplasm translocation of grass carp IRF7 (CiIRF7) and then acutely up-regulate the IFN I and ISG15 expression. However, the knockdown of CiDDX41 inhibits the phosphorylation IRF7. Taken together, all these results above suggested that CiDDX41 performs as an activator for innate immune through STING-IRF7 mediated signaling pathway.
Assuntos
Carpas/genética , Carpas/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Citocinas/química , Citocinas/genética , Citocinas/imunologia , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/imunologia , Proteínas de Peixes/química , Perfilação da Expressão Gênica/veterinária , Interferon Tipo I/química , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Filogenia , Alinhamento de Sequência/veterinária , Ubiquitinas/química , Ubiquitinas/genética , Ubiquitinas/imunologiaRESUMO
The intestinal organoid culture system is a pathbreaking working model for investigating pathogen-host interactions in the intestines. However, due to the limitations of the first generation of intestinal organoids, basal-out structure and growth in Matrigel, most pathogens can rarely attach to the apical membrane directly and hardly initiate infection. In this study, we first developed a next-generation porcine intestinal organoid culture system, characterized by an apical membrane on the surface, called apical-out. To investigate the infectivity and antiviral immune responses of this apical-out porcine intestinal organoid, a swine enteric virus, transmissible gastroenteritis virus (TGEV), was employed to inoculate the culture system. Both reverse transcription-quantitative PCR (RT-qPCR) and immunofluorescence assay (IFA) analysis demonstrated that TGEV replicated in the apical-out porcine intestinal organoid culture system. Additionally, our results illustrated that TGEV infection significantly upregulated the expression levels of alpha interferon (IFN-α), IFN-λ1, interferon-stimulated gene 15 (ISG15), ISG58, tumor necrosis factor alpha (TNF-α), and interleukin 6 (IL-6) in this culture system. Hence, we successfully developed a porcine intestinal apical-out organoid culture system, which will facilitate the investigation of pathogen-host interactions in pig intestines.IMPORTANCE Intestinal organoids are a newly developed culture system for investigating pathogen-host interactions. Intestinal organoid models have been widely used since their development, because the results obtained from this type of culture model better represent physiological conditions than those from well-established cell lines. The three-dimensional (3D) porcine intestinal organoid model was reported in 2018 and 2019 for the investigation of intestinal pathogens. However, those organoid culture models were basal-out intestinal organoids, which are not suitable for porcine enteric virus research because they invade the intestines via the apical side of epithelial cells on villi. In this study, we developed a porcine apical-out intestinal organoid culture system and verified its infectivity, type I and type III interferon (IFN) antiviral responses, and inflammatory responses following infection by a swine enteric virus. Our results imply that this apical-out porcine intestinal organoid culture system is an ideal model for the investigation of interactions between swine enteric viruses and the intestines.
Assuntos
Células Epiteliais/imunologia , Gastroenterite Suína Transmissível/imunologia , Interações Hospedeiro-Patógeno/imunologia , Mucosa Intestinal/imunologia , Organoides/imunologia , Vírus da Gastroenterite Transmissível/imunologia , Animais , Bioensaio , Quimiocina CXCL10/genética , Quimiocina CXCL10/imunologia , Células Epiteliais/patologia , Células Epiteliais/virologia , Gastroenterite Suína Transmissível/genética , Gastroenterite Suína Transmissível/virologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Interferon-alfa/genética , Interferon-alfa/imunologia , Interferons/genética , Interferons/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Mucosa Intestinal/patologia , Mucosa Intestinal/virologia , Organoides/patologia , Organoides/virologia , Suínos , Vírus da Gastroenterite Transmissível/crescimento & desenvolvimento , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Ubiquitinas/genética , Ubiquitinas/imunologia , Replicação ViralRESUMO
Type I interferons (IFN), which activate many IFN-stimulated genes (ISG), are known to regulate tumorigenesis. However, little is known regarding how various ISGs coordinate with one another in developing antitumor effects. Here, we report that the ISG UBA7 is a tumor suppressor in breast cancer. UBA7 encodes an enzyme that catalyzes the covalent conjugation of the ubiquitin-like protein product of another ISG (ISG15) to cellular proteins in a process known as "ISGylation." ISGylation of other ISGs, including STAT1 and STAT2, synergistically facilitates production of chemokine-receptor ligands to attract cytotoxic T cells. These gene-activation events are further linked to clustering and nuclear relocalization of STAT1/2 within IFN-induced promyelocytic leukemia (PML) bodies. Importantly, this coordinated ISG-ISGylation network plays a central role in suppressing murine breast cancer growth and metastasis, which parallels improved survival in patients with breast cancer. These findings reveal a cooperative IFN-inducible gene network in orchestrating a tumor-suppressive microenvironment. SIGNIFICANCE: We report a highly cooperative ISG network, in which UBA7-mediated ISGylation facilitates clustering of transcription factors and activates an antitumor gene-expression program. These findings provide mechanistic insights into immune evasion in breast cancer associated with UBA7 loss, emphasizing the importance of a functional ISG-ISGylation network in tumor suppression.This article is highlighted in the In This Issue feature, p. 327.
Assuntos
Neoplasias da Mama/genética , Interferon Tipo I/genética , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT2/genética , Enzimas Ativadoras de Ubiquitina/genética , Animais , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Redes Reguladoras de Genes/imunologia , Humanos , Camundongos , Linfócitos T/imunologia , Fatores de Transcrição/genética , Ubiquitinas/genética , Ubiquitinas/imunologiaRESUMO
Interferon-stimulated gene 15 (ISG15) is known to be involved in tumor progression. We previously reported that ISG15 expressed on nasopharyngeal carcinoma (NPC) cells and related to poor prognosis of patients with NPC. We further observed that ISG15 can be secreted by NPC cell and expressed on the macrophages in situ. However, the role of ISG15 in tumor-associated macrophages (TAMs) remains poorly understood. In the present study, we found that ISG15 treatment induces macrophages with M2-like phenotype, and the enhancement of NPC cell migration and tumorigenicity. Mechanically, ISG15-induced M2-like phenotype is dependent on the interaction with its receptor, LFA-1, and engagement of SRC family kinase (SFK) signal, and the subsequent secretion of CCL18. Blocking LFA-1, or SRC signal with small molecular inhibitors, or neutralizing with anti-CCL18 antibody can impede the activation of LFA-1-SFK-CCL18 axis in ISG15-treated macrophages. Clinically, ISG15+ CD163+ TAMs related to impaired survival of patients and advanced tumor stage of NPC. Furthermore, we found ISG15+ CD163+ macrophages inhibited antitumor CD8+ cells responses in NPC. Together, our findings suggested tumor cell-secreted ISG15, which acted as a tumor microenvironmental factor, induces M2-like phenotype, promoting tumor progression and suppression of cytotoxic T lymphocyte response.
Assuntos
Movimento Celular/imunologia , Citocinas/imunologia , Tolerância Imunológica , Macrófagos/imunologia , Proteínas de Neoplasias/imunologia , Neoplasias/imunologia , Ubiquitinas/imunologia , Adulto , Idoso , Feminino , Humanos , Macrófagos/patologia , Masculino , Pessoa de Meia-Idade , Neoplasias/patologiaRESUMO
Upon detection of viral infections, cells activate the expression of type I interferons (IFNs) and pro-inflammatory cytokines to control viral dissemination. As part of their antiviral response, cells also trigger the translational shutoff response which prevents translation of viral mRNAs and cellular mRNAs in a non-selective manner. Intriguingly, mRNAs encoding for antiviral factors bypass this translational shutoff, suggesting the presence of additional regulatory mechanisms enabling expression of the self-defence genes. Here, we identified the dsRNA binding protein ILF3 as an essential host factor required for efficient translation of the central antiviral cytokine, IFNB1, and a subset of interferon-stimulated genes. By combining polysome profiling and next-generation sequencing, ILF3 was also found to be necessary to establish the dsRNA-induced transcriptional and translational programs. We propose a central role for the host factor ILF3 in enhancing expression of the antiviral defence mRNAs in cellular conditions where cap-dependent translation is compromised.
Assuntos
Interferon beta/genética , Proteínas do Fator Nuclear 90/genética , Biossíntese de Proteínas , RNA de Cadeia Dupla/genética , RNA Mensageiro/genética , RNA Viral/genética , Células A549 , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/imunologia , Quimiocina CCL5/genética , Quimiocina CCL5/imunologia , Quimiocina CXCL10/genética , Quimiocina CXCL10/imunologia , Citocinas/genética , Citocinas/imunologia , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/imunologia , Regulação da Expressão Gênica , Células HeLa , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Interferon beta/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Proteínas do Fator Nuclear 90/imunologia , Poli I-C/farmacologia , Polirribossomos/efeitos dos fármacos , Polirribossomos/genética , Polirribossomos/imunologia , RNA de Cadeia Dupla/antagonistas & inibidores , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/imunologia , RNA Viral/antagonistas & inibidores , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Receptores Imunológicos , Transdução de Sinais , Ubiquitinas/genética , Ubiquitinas/imunologia , Replicação ViralRESUMO
Long noncoding RNAs (lncRNAs) are single-stranded RNA molecules longer than 200 nt that regulate many cellular processes. MicroRNA 155 host gene (MIR155HG) encodes the microRNA (miR)-155 that regulates various signalling pathways of innate and adaptive immune responses against viral infections. MIR155HG also encodes a lncRNA that we call lncRNA-155. Here, we observed that expression of lncRNA-155 was markedly upregulated during influenza A virus (IAV) infection both in vitro (several cell lines) and in vivo (mouse model). Interestingly, robust expression of lncRNA-155 was also induced by infections with several other viruses. Disruption of lncRNA-155 expression in A549 cells diminished the antiviral innate immunity against IAV. Furthermore, knockout of lncRNA-155 in mice significantly increased IAV replication and virulence in the animals. In contrast, overexpression of lncRNA-155 in human cells suppressed IAV replication, suggesting that lncRNA-155 is involved in host antiviral innate immunity induced by IAV infection. Moreover, we found that lncRNA-155 had a profound effect on expression of protein tyrosine phosphatase 1B (PTP1B) during the infection with IAV. Inhibition of PTP1B by lncRNA-155 resulted in higher production of interferon-beta (IFN-ß) and several critical interferon-stimulated genes (ISGs). Together, these observations reveal that MIR155HG derived lncRNA-155 can be induced by IAV, which modulates host innate immunity during the virus infection via regulation of PTP1B-mediated interferon response.
Assuntos
Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Vírus da Influenza A/imunologia , MicroRNAs/genética , Infecções por Orthomyxoviridae/genética , RNA Longo não Codificante/genética , Células A549 , Animais , Citocinas/genética , Citocinas/imunologia , Regulação da Expressão Gênica , Células HEK293 , Interações Hospedeiro-Patógeno/genética , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/crescimento & desenvolvimento , Vírus da Influenza A/patogenicidade , Influenza Humana/genética , Influenza Humana/imunologia , Influenza Humana/virologia , Helicase IFIH1 Induzida por Interferon/genética , Helicase IFIH1 Induzida por Interferon/imunologia , Interferon beta/genética , Interferon beta/imunologia , Interferon gama/genética , Interferon gama/imunologia , Camundongos , MicroRNAs/antagonistas & inibidores , MicroRNAs/imunologia , NF-kappa B/genética , NF-kappa B/imunologia , Células NIH 3T3 , Oligorribonucleotídeos/genética , Oligorribonucleotídeos/metabolismo , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/mortalidade , Infecções por Orthomyxoviridae/virologia , Células RAW 264.7 , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/imunologia , Transdução de Sinais , Análise de Sobrevida , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/imunologia , Ubiquitinas/genética , Ubiquitinas/imunologia , Replicação Viral/genética , Replicação Viral/imunologiaRESUMO
The 20S immunoproteasome (IP) is an interferon(IFN)-γ - and tumor necrosis factor (TNF) -inducible variant of the 20S constitutive proteasome (CP) in which all its peptidolytically active subunits ß1, ß2, and ß5 are replaced by their cytokine inducible homologues ß1i (LMP2), ß2i (MECL-1), and ß5i (LMP7). These subunit replacements alter the cleavage specificity of the proteasome and the spectrum of proteasome-generated peptide ligands of MHC class I molecules. In addition to antigen processing, the IP has recently been shown to serve unique functions in the generation of pro-inflammatory T helper cell subtypes and cytokines as well as in the pathogenesis of autoimmune diseases, but the mechanistic involvement of the IP in these processes has remained elusive. In this study we investigated whether the IP differs from the CP in the interaction with two IFN-γ/TNF inducible factors: the 11S proteasome regulator PA28αß and the ubiquitin-like modifier FAT10 (ubiquitin D). Using thermophoresis, we determined the affinity of PA28αß for the CP and IP to be 12.2nM +/- 2.8nM and 15.3nM +/- 2.7nM, respectively, which is virtually identical. Also the activation of the peptidolytic activities of the IP and CP by PA28αß did not differ. For FAT10 we determined the degradation kinetics in cycloheximide chase experiments in cells expressing almost exclusively IP or CP as well as in IFN-γ stimulated and unstimulated cells and found no differences between the degradation rates. Taken together, we conclude that neither differences in the binding strength to, nor activation by PA28αß, nor a difference in the rate of FAT10-mediated degradation can account for distinct functional capabilities of the IP as compared to the CP.
Assuntos
Complexo de Endopeptidases do Proteassoma/imunologia , Ubiquitinas/imunologia , Animais , Apresentação de Antígeno/imunologia , Doenças Autoimunes/imunologia , Citocinas/imunologia , Citoplasma/imunologia , Células HEK293 , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Interferon gama/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Peptídeos/imunologia , Proteólise , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
Previously, a spontaneous 88-amino-acid (aa) deletion in nsp2 was associated with cell-adaptation of porcine reproductive and respiratory syndrome virus (PRRSV) strain JXM100, which arose during passaging of the highly pathogenic PRRSV (HP-PRRSV) strain JX143 in MARC-145 cells. Here, to elucidate the biological role of this deletion, we specifically deleted the region of a cDNA clone of HP-PRRSV strain JX143 (pJX143) corresponding to these 88 amino acids. The effect of the deletion on virus replication in cultured cells and transcriptional activation of inflammatory cytokines and chemokines in pulmonary alveolar macrophages (PAMs) was examined. Mutant virus with the 88-aa deletion in nsp2 (rJX143-D88) had faster growth kinetics and produced larger plaques in MARC-145 cells than the parental virus (rJX143), suggesting that the deletion enhanced virus replication in MARC-145 cells. In contrast, the overall yield of rJX143 was almost 1 log higher than that of rJX143-D88, suggesting that the 88-aa deletion in nsp2 decreased the production of infectious viruses in PAMs. Infection with the mutant virus with the 88-aa deletion resulted in increased mRNA expression of type I interferon (IFN-α and IFN-ß) and chemokines genes. In addition, the mRNA expression of antiviral genes (ISG15, ISG54 and PKR) regulated by the IFN response was upregulated in PAMs infected with the mutant virus rJX143-D88. Our results demonstrate that virus-specific host immunity can be enhanced by modifying certain nsp2 epitope regions. These findings provide important insights for understanding virus pathogenesis and development of future vaccines.
Assuntos
Sequência de Aminoácidos , Cisteína Endopeptidases/genética , Interações Hospedeiro-Patógeno , Macrófagos Alveolares/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Deleção de Sequência , Replicação Viral/genética , Animais , Linhagem Celular , Células Cultivadas , Quimiocinas/genética , Quimiocinas/imunologia , Chlorocebus aethiops , Cisteína Endopeptidases/imunologia , Células Epiteliais/imunologia , Células Epiteliais/virologia , Regulação da Expressão Gênica , Interferon-alfa/genética , Interferon-alfa/imunologia , Interferon beta/genética , Interferon beta/imunologia , Macrófagos Alveolares/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/crescimento & desenvolvimento , Vírus da Síndrome Respiratória e Reprodutiva Suína/patogenicidade , Transdução de Sinais , Suínos , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia , Ubiquitinas/genética , Ubiquitinas/imunologia , eIF-2 Quinase/genética , eIF-2 Quinase/imunologiaRESUMO
Chlamydia trachomatis (CT) and the Chlamydophila species (CS) Chlamydophila pneumoniae (CPn), and Chlamydophila psittaci (CPs) are suggested to induce autoantibodies causative of several human autoimmune disorders like rheumatoid arthritis and systemic lupus erythematosus (SLE). The aim of the present study was therefore to identify cellular protein interaction partners with antisera to CT (α-CT) or CS (α-CS) and to identify functional consequences of such interaction in vitro. As detected with a commercial first trimester human prenatal brain multiprotein array (hEXselect, Engine, Germany), the most frequent interaction partner with both α-CT and α-CS was the ribosomal small subunit protein RPS27a. This could be confirmed by Western blot analysis with a recombinant RPS27a sample. In addition, immunocytochemistry with both antisera in the human choroid plexus papilloma cell line HIBCPP revealed a granular cytoplasmic staining, and Western blot analysis with whole-cell protein samples of HIBCPP cells revealed both antisera to label protein bands of different molecular weights and intensity. By 2D Western blot analysis and mass spectrometry, one of the protein spots interacting with α-CT could be identified as the RPS27a. Finally, two different methods for the detection of protein synthesis activity, the SUnSET technique and an HPG fluorescence assay revealed both antisera to cause reduced translational activity in HIBCPP cells. Together with previous findings of RPS27a as an autoimmune target in a mouse model of systemic lupus erythematosus (SLE), these results suggest that infections with CT and/or CS could induce SLE-associated immune modifications. However, direct evidence for a pathogenic role of these interactions for SLE demands further investigations.
Assuntos
Artrite Reumatoide/imunologia , Autoantígenos/metabolismo , Infecções por Chlamydia/imunologia , Chlamydia trachomatis/imunologia , Infecções por Chlamydophila/imunologia , Chlamydophila pneumoniae/imunologia , Chlamydophila psittaci/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Papiloma do Plexo Corióideo/imunologia , Proteínas Ribossômicas/metabolismo , Ubiquitinas/metabolismo , Animais , Artrite Reumatoide/microbiologia , Autoanticorpos/metabolismo , Autoantígenos/imunologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Soros Imunes/metabolismo , Lúpus Eritematoso Sistêmico/microbiologia , Camundongos , Papiloma do Plexo Corióideo/microbiologia , Gravidez , Ligação Proteica , Biossíntese de Proteínas/imunologia , Proteínas Ribossômicas/imunologia , Ubiquitinas/imunologiaRESUMO
Hepatitis E virus (HEV), a single-stranded positive-sense RNA virus, generally causes self-limiting acute viral hepatitis, although chronic HEV infection has recently become a significant clinical problem in immunocompromised individuals, especially in solid-organ transplant recipients. Innate immunity, via the type I interferon (IFN) response, plays an important role during the initial stages of a viral infection. IFN-stimulated gene 15 (ISG15), an IFN-induced ubiquitin-like protein, is known to have an immunomodulatory role and can have a direct antiviral effect on a wide spectrum of virus families. In the present study, we investigated the antiviral effect as well as the potential immunomodulatory role of ISG15 during HEV replication. The results revealed that HEV induced high levels of ISG15 production both in vitro (Huh7-S10-3 liver cells) and in vivo (liver tissues from HEV-infected pigs); however, ISG15 is not required for virus replication. We also demonstrated that ISG15 silencing potentiates enhanced type I IFN-mediated signaling, resulting in an increase in the type I IFN-mediated antiviral effect during HEV replication. This observed enhanced type I IFN signaling correlated with an increase in IFN-stimulated gene expression levels during HEV replication. Furthermore, we showed that PKR and OAS1 played important roles in the ISG15-mediated type I IFN sensitivity of HEV. Taken together, the results from this study suggest that ISG15 plays an important immunomodulatory role and regulates HEV sensitivity to exogenous type I IFN.IMPORTANCE Hepatitis E virus (HEV) infection typically causes self-limiting acute viral hepatitis. However, chronic HEV infection has recently become a significant clinical problem in immunocompromised patients. Pegylated interferon (IFN) has been used to treat chronic HEV infection in solid-organ transplant patients with some success. However, the mechanism behind the type I IFN-mediated antiviral effect against HEV remains unclear. This report demonstrates that ISG15 induced by HEV replication in Huh7-S10-3 human liver cells plays an immunomodulatory role by negatively regulating type I IFN signaling and, thus, HEV sensitivity to type I IFN. Our results also show that PKR and OAS1 play important roles in the ISG15-mediated type I IFN sensitivity of HEV.
Assuntos
Citocinas/imunologia , Vírus da Hepatite E/crescimento & desenvolvimento , Hepatite E/imunologia , Interferon-alfa/imunologia , Ubiquitinas/imunologia , Replicação Viral/imunologia , 2',5'-Oligoadenilato Sintetase/metabolismo , Animais , Linhagem Celular Tumoral , Citocinas/genética , Hepatite E/virologia , Vírus da Hepatite E/genética , Vírus da Hepatite E/imunologia , Hepatócitos/imunologia , Hepatócitos/virologia , Humanos , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de Sinais/imunologia , Suínos , Ubiquitinas/genética , Replicação Viral/genética , eIF-2 Quinase/metabolismoRESUMO
ISG15 is a ubiquitin-like type I IFN-stimulated protein of 15 kDa and is one of the most prominently expressed proteins in viral infections. ISG15 is widely known to be involved in a process called ISGylation, where it binds to over 150 targets from a variety of classes of proteins including central immune signaling pathways such as those mediated by NFκB, JNK, and IRF-3. However, ISG15 also exists in a free form that can act intra- or extracellularly. In vitro and in vivo evidences suggest that free ISG15 play different roles in several cellular processes, from cancer and defense against viral infections to activation of immune cells such as lymphocytes, monocytes, and NK cells. This review discusses the roles of free intracellular and secreted ISG15 approaching questions yet to be answered about the mechanism of action of this protein.
Assuntos
Infecções Bacterianas/imunologia , Citocinas/imunologia , Interferon gama/imunologia , Transdução de Sinais/imunologia , Ubiquitinas/imunologia , Viroses/imunologia , Infecções Bacterianas/genética , Infecções Bacterianas/microbiologia , Citocinas/genética , Regulação da Expressão Gênica , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Interferon gama/genética , Monócitos/imunologia , Monócitos/microbiologia , Monócitos/virologia , Neutrófilos/imunologia , Neutrófilos/microbiologia , Neutrófilos/virologia , Linfócitos T/imunologia , Linfócitos T/microbiologia , Linfócitos T/virologia , Ubiquitinas/genética , Viroses/genética , Viroses/virologia , eIF-2 Quinase/genética , eIF-2 Quinase/imunologiaRESUMO
The Bowman-Birk inhibitor (BBI), a soybean-derived protease inhibitor, is known to have anti-inflammatory effect in both in vitro and in vivo systems. Macrophages play a key role in inflammation and immune activation, which is implicated in HIV disease progression. Here, we investigated the effect of BBI on HIV infection of peripheral blood monocyte-derived macrophages. We demonstrated that BBI could potently inhibit HIV replication in macrophages without cytotoxicity. Investigation of the mechanism(s) of BBI action on HIV showed that BBI induced the expression of IFN-ß and multiple IFN stimulated genes (ISGs), including Myxovirus resistance protein 2 (Mx2), 2',5'-oligoadenylate synthetase (OAS-1), Virus inhibitory protein (viperin), ISG15 and ISG56. BBI treatment of macrophages also increased the expression of several known HIV restriction factors, including APOBEC3F, APOBEC3G and tetherin. Furthermore, BBI enhanced the phosphorylation of IRF3, a key regulator of IFN-ß. The inhibition of IFN-ß pathway by the neutralization antibody to type I IFN receptor (Anti-IFNAR) abolished BBI-mediated induction of the anti-HIV factors and inhibition of HIV in macrophages. These findings that BBI could activate IFN-ß-mediated signaling pathway, initialize the intracellular innate immunity in macrophages and potently inhibit HIV at multiple steps of viral replication cycle indicate the necessity to further investigate BBI as an alternative and cost-effective anti-HIV natural product.
Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Interferon beta/antagonistas & inibidores , Macrófagos/imunologia , Inibidor da Tripsina de Soja de Bowman-Birk/farmacologia , Replicação Viral/efeitos dos fármacos , 2',5'-Oligoadenilato Sintetase/genética , 2',5'-Oligoadenilato Sintetase/imunologia , Desaminase APOBEC-3G/genética , Desaminase APOBEC-3G/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Anticorpos Neutralizantes/farmacologia , Células Cultivadas , Citocinas/genética , Citocinas/imunologia , Citosina Desaminase/genética , Citosina Desaminase/imunologia , Regulação da Expressão Gênica , HIV-1/genética , HIV-1/crescimento & desenvolvimento , Interações Hospedeiro-Patógeno/genética , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Interferon beta/genética , Interferon beta/imunologia , Macrófagos/metabolismo , Macrófagos/virologia , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/imunologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Proteínas/genética , Proteínas/imunologia , Proteínas de Ligação a RNA , Receptor de Interferon alfa e beta/antagonistas & inibidores , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/imunologia , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia , Ubiquitinas/genética , Ubiquitinas/imunologiaRESUMO
The genetic regulatory network controlling the innate immune system is well understood in many species. However, the role of the epigenetic mechanisms underlying the expression of immunoregulatory genes is less clear, especially in livestock species. Histone H3 lysine 9 dimethylation (H3K9me2) is an epigenetic modification associated with transcriptional silencing within the euchromatin regions. Euchromatic histone-lysine N-methyltransferase 2 (EHMT2; also known as G9a) is a crucial enzyme responsible for regulating the dynamics of this epigenetic modification. It has been shown that histone modifications play a role in regulating type I interferon (IFN) response. In the present study, we investigated the role of EHMT2 in the epigenetic regulation of bovine antiviral innate immunity and explored its therapeutic potential against viral infections. We evaluated the effects of pharmacological and RNAi-mediated inhibition of EHMT2 on the transcription of IFN-ß and other IFN-inducible antiviral genes, as well as its effect on foot-and-mouth disease virus (FMDV) and vesicular stomatitis virus (VSV) replication in bovine cells. We show that treatment of primary bovine cells with the synthetic EHMT2 inhibitor (UNC0638) either before or shortly after virus infection resulted in a significant increase in transcript levels of bovine IFN-ß (boIFN-ß; 300-fold) and other IFN-inducible genes, including IFN-stimulated gene 15 (ISG-15), myxovirus resistance 1 (Mx-1), Mx-2, RIG-I, 2',5'-oligoadenylate synthetase 1 (OAS-1), and protein kinase R (PKR). Expression of these factors correlated with a significant decrease in VSV and FMDV viral titers. Our data confirm the involvement of EHMT2 in the epigenetic regulation of boIFN-ß and demonstrate the activation of a general antiviral state after EHMT2 inhibition.
Assuntos
Epigênese Genética , Vírus da Febre Aftosa/efeitos dos fármacos , Antígenos de Histocompatibilidade/imunologia , Histona-Lisina N-Metiltransferase/imunologia , Interferon beta/imunologia , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , 2',5'-Oligoadenilato Sintetase/genética , 2',5'-Oligoadenilato Sintetase/imunologia , Animais , Bovinos , Linhagem Celular , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/imunologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/virologia , Eucromatina/química , Eucromatina/efeitos dos fármacos , Eucromatina/metabolismo , Feto , Fibroblastos/efeitos dos fármacos , Fibroblastos/imunologia , Fibroblastos/virologia , Vírus da Febre Aftosa/crescimento & desenvolvimento , Vírus da Febre Aftosa/imunologia , Antígenos de Histocompatibilidade/genética , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/genética , Imunidade Inata , Interferon beta/farmacologia , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/imunologia , Poli I-C/farmacologia , Cultura Primária de Células , Quinazolinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/imunologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transcrição Gênica , Ubiquitinas/genética , Ubiquitinas/imunologia , Vírus da Estomatite Vesicular Indiana/crescimento & desenvolvimento , Vírus da Estomatite Vesicular Indiana/imunologia , eIF-2 Quinase/genética , eIF-2 Quinase/imunologiaRESUMO
Given the multifactorial nature of action of type I interferon (IFN) in HIV-1 infection and the need to firmly establish the action of key components of IFN pathways, we compared the IFN stimulated gene (ISG)15 expression with that of other well-characterized ISGs, evaluating its relationship with immunosuppressive factors regulating T-cell response in HIV-1 patients. PBMC from 225 subjects were included: healthy donors (n=30), naïve (n=93) and HAART treated HIV-1 subjects (n=102). Levels of ISG15-mRNA, ISG56-mRNA, APOBEC3G/3F-mRNA, TRAIL-mRNA, IDO-mRNA, proviral load andISG15 (rs15842 and rs1921) SNPs were evaluated by using TaqMan assays. We found that ISG15, ISG56, APOBEC3G/3F levels were increased in untreated HIV-1 patients compared to healthy donors, being ISG15 the highest ISG expressed. The amount of ISG15 correlated with viral load and with CD4+ T cell counts whereas no relationship was found between all ISGs analyzed and proviral load or HIV-1 tropism. ISG15 expression was reduced following long-term antiretroviral therapy. In addition, ISG15 levels were correlated with those of TRAIL and IDO in HIV-1 viremic patients. Lastly, ISG15 SNPs had no influence on ISG15 levels. We demonstrates that ISG15 is elevated in viremic HIV-1 patients and is associated with high TRAIL and IDO levels.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Citocinas/genética , Infecções por HIV/genética , Indolamina-Pirrol 2,3,-Dioxigenase/genética , RNA Mensageiro/genética , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ubiquitinas/genética , Viremia/genética , Desaminase APOBEC-3G , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Terapia Antirretroviral de Alta Atividade , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/virologia , Estudos de Casos e Controles , Citidina Desaminase/genética , Citidina Desaminase/imunologia , Citocinas/imunologia , Citosina Desaminase/genética , Citosina Desaminase/imunologia , Feminino , Regulação da Expressão Gênica , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/imunologia , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/imunologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/imunologia , Proteínas de Ligação a RNA , Transdução de Sinais , Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia , Ubiquitinas/imunologia , Carga Viral/efeitos dos fármacos , Tropismo Viral , Viremia/tratamento farmacológico , Viremia/imunologia , Viremia/virologiaRESUMO
ISG15 is an ubiquitin-like protein induced by type I interferon associated with antiviral activity. ISG15 is also secreted and known to function as an immunomodulatory molecule. However, ISG15's role in influencing the adaptive CD8 T-cell responses has not been studied. Here, we demonstrate the efficacy of ISG15 as a vaccine adjuvant, inducing human papilloma virus (HPV) E7-specific IFNγ responses as well as the percentage of polyfunctional, cytolytic, and effector CD8 T-cell responses. Vaccination with ISG15 conferred remarkable control and/or regression of established HPV-associated tumor-bearing mice. T-cell depletion coupled with adoptive transfer experiments revealed that ISG15 protective efficacy was CD8 T-cell mediated. Importantly, we demonstrate that ISG15 vaccine-induced responses could be generated independent of ISGylation, suggesting that responses were mostly influenced by free ISG15. Our results provide more insight into the immunomodulatory properties of ISG15 and its potential to serve as an effective immune adjuvant in a therapeutic tumor or infectious disease setting.