RESUMO
The role of CD8+ T cells in SARS-CoV-2 pathogenesis or mRNA-LNP vaccine-induced protection from lethal COVID-19 is unclear. Using mouse-adapted SARS-CoV-2 virus (MA30) in C57BL/6 mice, we show that CD8+ T cells are unnecessary for the intrinsic resistance of female or the susceptibility of male mice to lethal SARS-CoV-2 infection. Also, mice immunized with a di-proline prefusion-stabilized full-length SARS-CoV-2 Spike (S-2P) mRNA-LNP vaccine, which induces Spike-specific antibodies and CD8+ T cells specific for the Spike-derived VNFNFNGL peptide, are protected from SARS-CoV-2 infection-induced lethality and weight loss, while mice vaccinated with mRNA-LNPs encoding only VNFNFNGL are protected from lethality but not weight loss. CD8+ T cell depletion ablates protection in VNFNFNGL but not in S-2P mRNA-LNP-vaccinated mice. Therefore, mRNA-LNP vaccine-induced CD8+ T cells are dispensable when protective antibodies are present but essential for survival in their absence. Hence, vaccine-induced CD8+ T cells may be critical to protect against SARS-CoV-2 variants that mutate epitopes targeted by protective antibodies.
Assuntos
Anticorpos Antivirais , Linfócitos T CD8-Positivos , Vacinas contra COVID-19 , COVID-19 , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Animais , Linfócitos T CD8-Positivos/imunologia , Camundongos , SARS-CoV-2/imunologia , COVID-19/imunologia , COVID-19/prevenção & controle , Feminino , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Vacinas contra COVID-19/imunologia , Masculino , Anticorpos Antivirais/imunologia , Camundongos Endogâmicos C57BL , Humanos , Modelos Animais de DoençasRESUMO
Receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like pseudokinase (MLKL) are proteins that are critical for necroptosis, a mechanism of programmed cell death that is both activated when apoptosis is inhibited and thought to be antiviral. Here, we investigated the role of RIPK3 and MLKL in controlling the Orthopoxvirus ectromelia virus (ECTV), a natural pathogen of the mouse. We found that C57BL/6 (B6) mice deficient in RIPK3 (Ripk3-/-) or MLKL (Mlkl-/-) were as susceptible as wild-type (WT) B6 mice to ECTV lethality after low-dose intraperitoneal infection and were as resistant as WT B6 mice after ECTV infection through the natural footpad route. Additionally, after footpad infection, Mlkl-/- mice, but not Ripk3-/- mice, endured lower viral titers than WT mice in the draining lymph node (dLN) at three days postinfection and in the spleen or in the liver at seven days postinfection. Despite the improved viral control, Mlkl-/- mice did not differ from WT mice in the expression of interferons or interferon-stimulated genes or in the recruitment of natural killer (NK) cells and inflammatory monocytes (iMOs) to the dLN. Additionally, the CD8 T-cell responses in Mlkl-/- and WT mice were similar, even though in the dLNs of Mlkl-/- mice, professional antigen-presenting cells were more heavily infected. Finally, the histopathology in the livers of Mlkl-/- and WT mice at 7 dpi did not differ. Thus, the mechanism of the increased virus control by Mlkl-/- mice remains to be defined. IMPORTANCE The molecules RIPK3 and MLKL are required for necroptotic cell death, which is widely thought of as an antiviral mechanism. Here we show that C57BL/6 (B6) mice deficient in RIPK3 or MLKL are as susceptible as WT B6 mice to ECTV lethality after a low-dose intraperitoneal infection and are as resistant as WT B6 mice after ECTV infection through the natural footpad route. Mice deficient in MLKL are more efficient than WT mice at controlling virus loads in various organs. This improved viral control is not due to enhanced interferon, natural killer cell, or CD8 T-cell responses. Overall, the data indicate that deficiencies in the molecules that are critical to necroptosis do not necessarily result in worse outcomes following viral infection and may improve virus control.
Assuntos
Ectromelia Infecciosa , Animais , Camundongos , Vírus da Ectromelia , Ectromelia Infecciosa/imunologia , Interferons/metabolismo , Camundongos Endogâmicos C57BL , Necroptose/imunologia , Proteínas Quinases/genética , Proteínas Quinases/imunologia , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/imunologiaRESUMO
Type I interferons (IFN-I) are antiviral cytokines that signal through the ubiquitous IFN-I receptor (IFNAR). Following footpad infection with ectromelia virus (ECTV), a mouse-specific pathogen, C57BL/6 (B6) mice survive without disease, while B6 mice broadly deficient in IFNAR succumb rapidly. We now show that for survival to ECTV, only hematopoietic cells require IFNAR expression. Survival to ECTV specifically requires IFNAR in both natural killer (NK) cells and monocytes. However, intrinsic IFNAR signaling is not essential for adaptive immune cell responses or to directly protect non-hematopoietic cells such as hepatocytes, which are principal ECTV targets. Mechanistically, IFNAR-deficient NK cells have reduced cytolytic function, while lack of IFNAR in monocytes dampens IFN-I production and hastens virus dissemination. Thus, during a pathogenic viral infection, IFN-I coordinates innate immunity by stimulating monocytes in a positive feedback loop and by inducing NK cell cytolytic function.
Assuntos
Vírus da Ectromelia/imunologia , Ectromelia Infecciosa/imunologia , Receptor de Interferon alfa e beta/metabolismo , Transdução de Sinais , Animais , Citocinas/imunologia , Resistência à Doença , Ectromelia Infecciosa/virologia , Feminino , Hepatócitos/imunologia , Hepatócitos/virologia , Imunidade Inata , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Monócitos/virologia , Receptor de Interferon alfa e beta/genéticaRESUMO
Cytotoxic CD4 T lymphocytes (CD4-CTL) are important in antiviral immunity. For example, we have previously shown that in mice, CD4-CTL are important to control ectromelia virus (ECTV) infection. How viral infections induce CD4-CTL responses remains incompletely understood. We demonstrate here that not only ECTV but also vaccinia virus and lymphocytic choriomeningitis virus induce CD4-CTL, though the response to ECTV is stronger. Using ECTV, we also demonstrate that in contrast to CD8-CTL, CD4-CTL differentiation requires constant virus replication and ceases once the virus is controlled. We also show that major histocompatibility complex class II molecules on CD11c+ cells are required for CD4-CTL differentiation and for mousepox resistance. Transcriptional analysis indicated that antiviral CD4-CTL and noncytolytic T helper 1 (Th1) CD4 T cells have similar transcriptional profiles, suggesting that CD4-CTL are terminally differentiated classical Th1 cells. Interestingly, CD4-CTL and classical Th1 cells expressed similar mRNA levels of the transcription factors ThPOK and GATA-3, necessary for CD4 T cell linage commitment, and Runx3, required for CD8 T cell development and effector function. However, at the protein level, CD4-CTL had higher levels of the three transcription factors, suggesting that further posttranscriptional regulation is required for CD4-CTL differentiation. Finally, CRISPR/Cas9-mediated deletion of Runx3 in CD4 T cells inhibited CD4-CTL but not classical Th1 cell differentiation in response to ECTV infection. These results further our understanding of the mechanisms of CD4-CTL differentiation during viral infection and the role of posttranscriptionally regulated Runx3 in this process. IMPORTANCE While it is well established that cytotoxic CD4 T cells (CD4-CTLs) directly contribute to viral clearance, it remains unclear how CD4-CTL are induced. We now show that CD4-CTLs require sustained antigen presentation and are induced by CD11c-expressing antigen-presenting cells. Moreover, we show that CD4-CTLs are derived from the terminal differentiation of classical T helper 1 (Th1) subset of CD4 cells. Compared to Th1 cells, CD4-CTLs upregulate protein levels of the transcription factors ThPOK, Runx3, and GATA-3 posttranscriptionally. Deletion of Runx3 in differentiated CD4 T cells prevents induction of CD4-CTLs but not classical Th1 cells. These results advance our knowledge of how CD4-CTLs are induced during viral infection.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Ectromelia Infecciosa/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Células Th1/imunologia , Viroses/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Antígenos CD11/análise , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Citotoxicidade Imunológica , Vírus da Ectromelia/fisiologia , Ectromelia Infecciosa/virologia , Antígenos de Histocompatibilidade Classe II/análise , Fígado/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Baço/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Citotóxicos/metabolismo , Células Th1/metabolismo , Transcriptoma , Replicação ViralRESUMO
Chronic viral infections. like those of humans with cytomegalovirus, human immunodeficiency virus (even when under antiretroviral therapy), and hepatitis C virus or those of mice with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), result in immune dysfunction that predisposes the host to severe infections with unrelated pathogens. It is known that C57BL/6 (B6) mice are resistant to mousepox, a lethal disease caused by the orthopoxvirus ectromelia virus (ECTV), and that this resistance requires natural killer (NK) cells and other immune cells. We show that most B6 mice chronically infected with CL13 succumb to mousepox but that most of those that recovered from acute infection with the LCMV Armstrong (Arm) strain survive. We also show that B6 mice chronically infected with CL13 and those that recovered from Arm infection have a reduced frequency and a reduced number of NK cells. However, at steady state, NK cells in mice that have recovered from Arm infection mature normally and, in response to ECTV, get activated, become more mature, proliferate, and increase their cytotoxicity in vivo Conversely, in mice chronically infected with CL13, NK cells are immature and residually activated, and following ECTV infection, they do not mature, proliferate, or increase their cytotoxicity. Given the well-established importance of NK cells in resistance to mousepox, these data suggest that the NK cell dysfunction caused by CL13 persistence may contribute to the susceptibility of CL13-infected mice to mousepox. Whether chronic infections similarly affect NK cells in humans should be explored.IMPORTANCE Infection of adult mice with the clone 13 (CL13) strain of lymphocytic choriomeningitis virus (LCMV) is extensively used as a model of chronic infection. In this paper, we show that mice chronically infected with CL13 succumb to challenge with ectromelia virus (ECTV; the agent of mousepox) and that natural killer (NK) cells in CL13-infected mice are reduced in numbers and have an immature and partially activated phenotype but do respond to ECTV. These data may provide additional clues why humans chronically infected with certain pathogens are less resistant to viral diseases.
Assuntos
Vírus da Ectromelia/imunologia , Ectromelia Infecciosa/imunologia , Células Matadoras Naturais/imunologia , Coriomeningite Linfocítica/imunologia , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Vírus da Coriomeningite Linfocítica/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
It is well established that chronic viral infections can cause immune suppression, resulting in increased susceptibility to other infectious diseases. However, the effects of chronic viral infection on T-cell responses and vaccination against highly pathogenic viruses are not well understood. We have recently shown that C57BL/6 (B6) mice lose their natural resistance to wild-type (WT) ectromelia virus (ECTV) when chronically infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13). Here we compared the T-cell response to ECTV in previously immunologically naive mice that were chronically infected with CL13 or that were convalescent from acute infection with the Armstrong (Arm) strain of LCMV. Our results show that mice that were chronically infected with CL13 but not those that had recovered from Arm infection have highly defective ECTV-specific CD8+ and CD4+ T-cell responses to WT ECTV. These defects are at least partly due to the chronic infection environment. In contrast to mice infected with WT ECTV, mice chronically infected with CL13 survived without signs of disease when infected with ECTV-Δ036, a mutant ECTV strain that is highly attenuated. Strikingly, mice chronically infected with CL13 mounted a strong CD8+ T-cell response to ECTV-Δ036 and survived without signs of disease after a subsequent challenge with WT ECTV. Our work suggests that enhanced susceptibility to acute viral infections in chronically infected individuals can be partly due to poor T-cell responses but that sufficient T-cell function can be recovered and resistance to acute infection can be restored by immunization with highly attenuated vaccines.IMPORTANCE Chronic viral infections may result in immunosuppression and enhanced susceptibility to infections with other pathogens. For example, we have recently shown that mice chronically infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13) are highly susceptible to mousepox, a disease that is caused by ectromelia virus and that is the mouse homolog of human smallpox. Here we show chronic CL13 infection severely disrupts the expansion, proliferation, activation, and cytotoxicity of T cells in response due at least in part to the suppressive effects of the chronic infection milieu. Notably, despite this profound immunodeficiency, mice chronically infected with CL13 could be protected by vaccination with a highly attenuated variant of ECTV. These results demonstrate that protective vaccination of immunosuppressed individuals is possible, provided that proper immunization tools are used.
Assuntos
Ectromelia Infecciosa/imunologia , Imunidade Inata/imunologia , Imunização , Coriomeningite Linfocítica/imunologia , Linfócitos T/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Modelos Animais de Doenças , Vírus da Ectromelia/imunologia , Feminino , Humanos , Tolerância Imunológica , Memória Imunológica , Vírus da Coriomeningite Linfocítica/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , VacinaçãoRESUMO
Numerous attempts to produce antiviral vaccines by harnessing memory CD8 T cells have failed. A barrier to progress is that we do not know what makes an Ag a viable target of protective CD8 T cell memory. We found that in mice susceptible to lethal mousepox (the mouse homolog of human smallpox), a dendritic cell vaccine that induced memory CD8 T cells fully protected mice when the infecting virus produced Ag in large quantities and with rapid kinetics. Protection did not occur when the Ag was produced in low amounts, even with rapid kinetics, and protection was only partial when the Ag was produced in large quantities but with slow kinetics. Hence, the amount and timing of Ag expression appear to be key determinants of memory CD8 T cell antiviral protective immunity. These findings may have important implications for vaccine design.
Assuntos
Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Memória Imunológica/imunologia , Animais , Células Dendríticas/imunologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Varíola/imunologia , Vaccinia virus/imunologiaRESUMO
Intracellular serpins are proposed to inactivate proteases released from lysosome-related organelles into the host cell interior, preventing cell death. Serpinb9 opposes the immune cytotoxic protease, granzyme B, and in a number of settings protects cells against granzyme B-mediated cell death. Using a knockout mouse line engineered to express green fluorescent protein under the serpbinb9 promoter, we demonstrate that serpinb9 is vital for host survival during Ectromelia virus infection by maintaining both mature natural killer NK) cells, and activated CD8+ T cells. Serpinb9 expression parallels granzyme B expression within both populations during infection. Maturing serpinb9-null NK cells exhibit higher levels of granzyme B-mediated apoptosis during infection; hence there are fewer mature NK cells, and these cells also have lower cytotoxic potential. Thus the serpinb9-granzyme B axis is important for homeostasis of both major cytotoxic effector cell populations.
Assuntos
Granzimas/antagonistas & inibidores , Células Matadoras Naturais/imunologia , Proteínas de Membrana/farmacologia , Infecções por Poxviridae/imunologia , Poxviridae/imunologia , Serpinas/farmacologia , Animais , Morte Celular , Sobrevivência Celular , Homeostase , Humanos , Espaço Intracelular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
The interstitial fluids in tissues are constantly drained into the lymph nodes (LNs) as lymph through afferent lymphatic vessels and from LNs into the blood through efferent lymphatics. LNs are strategically positioned and have the appropriate cellular composition to serve as sites of adaptive immune initiation against invading pathogens. However, for lymph-borne viruses, which disseminate from the entry site to other tissues through the lymphatic system, immune cells in the draining LN (dLN) also play critical roles in curbing systemic viral dissemination during primary and secondary infections. Lymph-borne viruses in tissues can be transported to dLNs as free virions in the lymph or within infected cells. Regardless of the entry mechanism, infected myeloid antigen-presenting cells, including various subtypes of dendritic cells, inflammatory monocytes, and macrophages, play a critical role in initiating the innate immune response within the dLN. This innate immune response involves cellular crosstalk between infected and bystander innate immune cells that ultimately produce type I interferons (IFN-Is) and other cytokines and recruit inflammatory monocytes and natural killer (NK) cells. IFN-I and NK cell cytotoxicity can restrict systemic viral spread during primary infections and prevent serious disease. Additionally, the memory CD8+ T-cells that reside or rapidly migrate to the dLN can contribute to disease prevention during secondary viral infections. This review explores the intricate innate immune responses orchestrated within dLNs that contain primary viral infections and the role of memory CD8+ T-cells following secondary infection or CD8+ T-cell vaccination.
RESUMO
Because of the rapid mutations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an effective vaccine against SARS-CoV-2 variants is needed to prevent coronavirus disease 2019 (COVID-19). T cells, in addition to neutralizing antibodies, are an important component of naturally acquired protective immunity, and a number of studies have shown that T cells induced by natural infection or vaccination contribute significantly to protection against several viral infections including SARS-CoV-2. However, it has never been tested whether a T cell-inducing vaccine can provide significant protection against SARS-CoV-2 infection in the absence of preexisting antibodies. In this study, we designed and evaluated lipid nanoparticle (LNP) formulated mRNA vaccines that induce only T cell responses or both T cell and neutralizing antibody responses by using two mRNAs. One mRNA encodes SARS-CoV-2 Omicron Spike protein in prefusion conformation for induction of neutralizing antibodies. The other mRNA encodes over one hundred T cell epitopes (multi-T cell epitope or MTE) derived from non-Spike but conserved regions of the SARS-CoV-2. We show immunization with MTE mRNA alone protected mice from lethal challenge with the SARS-CoV-2 Delta variant or a mouse-adapted virus MA30. Immunization with both mRNAs induced the best protection with the lowest viral titer in the lung. These results demonstrate that induction of T cell responses, in the absence of preexisting antibodies, is sufficient to confer protection against severe disease, and that a vaccine containing mRNAs encoding both the Spike and MTE could be further developed as a universal SARS-CoV-2 vaccine.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Animais , Humanos , Camundongos , COVID-19/prevenção & controle , SARS-CoV-2 , Anticorpos Neutralizantes , Epitopos de Linfócito T , RNA Mensageiro/genéticaRESUMO
Genetically modified (GM) mice are essential tools in biomedical research. Traditional methods for generating GM mice are expensive and require specialized personnel and equipment. The use of clustered regularly interspaced short palindromic repeats (CRISPR) coupled with improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) has highly increased the feasibility of producing GM mice in research laboratories. However, genetic modification in inbred mouse strains of interest such as C57BL/6 (B6) is still challenging because of their low fertility and embryo fragility. We have successfully generated multiple novel GM mouse strains in the B6 background while attempting to optimize i-GONAD. We found that i-GONAD reduced the litter size in superovulated pregnant females but did not impact pregnancy rates. Natural mating or low-hormone dose did not increase the low fertility rate observed in superovulated B6 females. However, diet enrichment had a positive effect on pregnancy success. We also optimized breeding conditions to increase the survival of small litters by co-housing i-GONAD-treated pregnant B6 females with synchronized pregnant FVB/NJ companion mothers. Thus, GM mice generation was increased by an enriched diet and shared pup rearing with highly fertile females such as FVB/NJ. In the present study, we generated 16 GM mice using a CRISPR/Cas system to target individual and multiple loci simultaneously or consecutively. We also compared homology-directed repair efficiency using different methods for LoxP insertion for conditional knockout mouse production. We found that a two-step serial LoxP insertion, in which each LoxP sequence was inserted individually in different i-GONAD procedures, was a low-risk high-efficiency method for generating floxed mice.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Gravidez , Feminino , Humanos , Camundongos , Animais , Edição de Genes/métodos , Sistemas CRISPR-Cas/genética , Camundongos Endogâmicos C57BL , Oviductos , Camundongos Knockout , GônadasRESUMO
Ectromelia virus (ECTV) is a natural pathogen of mice that causes mousepox, and many of its genes have been implicated in the modulation of host immune responses. Serine protease inhibitor 2 (SPI-2) is one of these putative ECTV host response modifier proteins. SPI-2 is conserved across orthopoxviruses, but results defining its mechanism of action and in vivo function are lacking or contradictory. We studied the role of SPI-2 in mousepox by deleting the SPI-2 gene or its serine protease inhibitor reactive site. We found that SPI-2 does not affect viral replication or cell-intrinsic apoptosis pathways, since mutant viruses replicate in vitro as efficiently as wild-type virus. However, in the absence of SPI-2 protein, ECTV is attenuated in mousepox-susceptible mice, resulting in lower viral loads in the liver, decreased spleen pathology, and substantially improved host survival. This attenuation correlates with more effective immune responses in the absence of SPI-2, including an earlier serum gamma interferon (IFN-γ) response, raised serum interleukin 18 (IL-18), increased numbers of granzyme B(+) CD8(+) T cells, and, most notably, increased numbers and activation of NK cells. Both virus attenuation and the improved immune responses associated with SPI-2 deletion from ECTV are lost when mice are depleted of NK cells. Consequently, SPI-2 renders mousepox lethal in susceptible strains by preventing protective NK cell defenses.
Assuntos
Vírus da Ectromelia/patogenicidade , Ectromelia Infecciosa/mortalidade , Interações Hospedeiro-Patógeno , Células Matadoras Naturais/imunologia , Serpinas/metabolismo , Proteínas Virais/metabolismo , Fatores de Virulência/metabolismo , Animais , Vírus da Ectromelia/genética , Vírus da Ectromelia/imunologia , Ectromelia Infecciosa/virologia , Deleção de Genes , Interferon gama/metabolismo , Interleucina-18/metabolismo , Fígado/virologia , Subpopulações de Linfócitos/química , Subpopulações de Linfócitos/imunologia , Camundongos , Serpinas/genética , Baço/patologia , Análise de Sobrevida , Carga Viral , Proteínas Virais/genética , Replicação ViralRESUMO
Inflammatory monocytes (iMOs) and B cells are the main targets of the poxvirus ectromelia virus (ECTV) in the lymph nodes of mice and play distinct roles in surviving the infection. Infected and bystander iMOs control ECTV's systemic spread, preventing early death, while B cells make antibodies that eliminate ECTV. Our work demonstrates that within an infected animal that survives ECTV infection, intrinsic and bystander infection of iMOs and B cells differentially control the transcription of genes important for immune cell function and, perhaps, cell identity. Bystander cells upregulate metabolism, antigen presentation, and interferon-stimulated genes. Infected cells downregulate many cell-type-specific genes and upregulate transcripts typical of non-immune cells. Bystander (Bys) and infected (Inf) iMOs non-redundantly contribute to the cytokine milieu and the interferon response. Furthermore, we uncover how type I interferon (IFN-I) or IFN-γ signaling differentially regulates immune pathways in Inf and Bys iMOs and that, at steady state, IFN-I primes iMOs for rapid IFN-I production and antigen presentation.
Assuntos
Vírus da Ectromelia , Ectromelia Infecciosa , Interferon Tipo I , Poxviridae , Animais , Camundongos , Monócitos , AntiviraisRESUMO
IFN-ß has been the treatment for multiple sclerosis (MS) for almost three decades, but understanding the mechanisms underlying its beneficial effects remains incomplete. We have shown that MS patients have increased numbers of GM-CSF+ Th cells in circulation, and that IFN-ß therapy reduces their numbers. GM-CSF expression by myelin-specific Th cells is essential for the development of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. These findings suggested that IFN-ß therapy may function via suppression of GM-CSF production by Th cells. In the current study, we elucidated a feedback loop between monocytes and Th cells that amplifies autoimmune neuroinflammation, and found that IFN-ß therapy ameliorates central nervous system (CNS) autoimmunity by inhibiting this proinflammatory loop. IFN-ß suppressed GM-CSF production in Th cells indirectly by acting on monocytes, and IFN-ß signaling in monocytes was required for EAE suppression. IFN-ß increased IL-10 expression by monocytes, and IL-10 was required for the suppressive effects of IFN-ß. IFN-ß treatment suppressed IL-1ß expression by monocytes in the CNS of mice with EAE. GM-CSF from Th cells induced IL-1ß production by monocytes, and, in a positive feedback loop, IL-1ß augmented GM-CSF production by Th cells. In addition to GM-CSF, TNF and FASL expression by Th cells was also necessary for IL-1ß production by monocyte. IFN-ß inhibited GM-CSF, TNF, and FASL expression by Th cells to suppress IL-1ß secretion by monocytes. Overall, our study describes a positive feedback loop involving several Th cell- and monocyte-derived molecules, and IFN-ß actions on monocytes disrupting this proinflammatory loop.
Assuntos
Autoimunidade , Comunicação Celular , Interferon beta/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Autoimunidade/efeitos dos fármacos , Comunicação Celular/genética , Comunicação Celular/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças/imunologia , Encefalomielite Autoimune Experimental/etiologia , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Interferon beta/farmacologia , Camundongos , Camundongos Knockout , Monócitos/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/efeitos dos fármacosRESUMO
It is known that aging decreases natural resistance to viral diseases due to dysfunctional innate and adaptive immune responses, but the nature of these dysfunctions, particularly in regard to innate immunity, is not well understood. We have previously shown that C57BL/6J (B6) mice lose their natural resistance to footpad infection with ectromelia virus (ECTV) due to impaired maturation and recruitment of natural killer (NK) cells to the draining popliteal lymph node (dLN). More recently, we have also shown that in young B6 mice infected with ECTV, the recruitment of NK cells is dependent on a complex cascade whereby migratory dendritic cells (mDCs) traffic from the skin to the dLN, where they produce CCL2 and CCL7 to recruit inflammatory monocytes (iMOs). In the dLN, mDCs also upregulate NKG2D ligands to induce interferon gamma (IFN-γ) expression by group 1 innate lymphoid cells (G1-ILCs), mostly NK in cells but also some ILC1. In response to the IFN-γ, the incoming uninfected iMOs secret CXCL9 to recruit the critical NK cells. Here, we show that in aged B6 mice, the trafficking of mDCs to the dLN in response to ECTV is decreased, resulting in impaired IFN-γ expression by G1-ILCs, reduced accumulation of iMOs, and attenuated CXCL9 production by iMOs, which likely contributes to decrease in NK cell recruitment. Together, these data indicate that defects in the mDC response to viral infection during aging result in a reduced innate immune response in the dLN and contribute to increased susceptibility to viral disease in the aged.
Assuntos
Células Dendríticas/metabolismo , Vírus da Ectromelia/imunologia , Imunidade Inata/imunologia , Linfonodos/metabolismo , Envelhecimento , Animais , CamundongosRESUMO
Mousepox is caused by the orthopoxvirus ectromelia virus (ECTV), and is thought to be transmitted via skin abrasions. We studied the ECTV virulence factor N1 following subcutaneous infection of mousepox-susceptible BALB/c mice. In this model, ECTV lacking N1L gene was attenuated more than 1000-fold compared with wild-type virus and replication was profoundly reduced as early as four days after infection. However, in contrast to data from an intranasal model, N1 protein was not required for virus dissemination. Further, neither T cell nor cytokine responses were enhanced in the absence of N1. Together with the early timing of reduced virus titres, this suggests that in a cutaneous model, N1 exerts its function at the level of infected cells or in the inhibition of the very earliest effectors of innate immunity.
Assuntos
Vírus da Ectromelia/fisiologia , Ectromelia Infecciosa/virologia , Proteínas Virais/genética , Animais , Interações Hospedeiro-Patógeno , Camundongos , Carga Viral , Proteínas Virais/metabolismo , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Replicação ViralRESUMO
Recombinant poxviruses are important tools for research and some are candidate vaccines. To make these viruses a simple, small vector that can be used to engineer multiple strains of vaccinia virus and other model poxviruses, including ectromelia virus is of value. Here a set of plasmids and methods for making these viruses that uses an enhanced green fluorescent protein-blasticidin resistance (GFP-bsd) fusion gene as a transient selectable marker are described. This gene is smaller than any of the bi-functional selection markers used previously. The versatility of the method across different poxviruses is demonstrated by engineering changes into multiple loci of the WR and Modified Vaccinia Ankara (MVA) strains of vaccinia virus and also ectromelia virus. Finally, a set of vaccinia virus sequences for directing homologous recombination that are very highly conserved was designed and tested. These sequences allow a single plasmid to be used to insert a transgene into multiple strains of the virus.
Assuntos
Vírus da Ectromelia/genética , Engenharia Genética/métodos , Vetores Genéticos , Seleção Genética , Coloração e Rotulagem/métodos , Vaccinia virus/genética , Virologia/métodos , Aminoidrolases/genética , Antivirais/farmacologia , Farmacorresistência Viral , Proteínas de Fluorescência Verde/genética , Nucleosídeos/farmacologia , Proteínas Recombinantes de Fusão/genéticaRESUMO
In 2004, an outbreak of HCPS in Brazil made hantaviruses a national threat to the rural and urban population. During this outbreak, 164 cases were reported, and 18.3% of them occurred in the Federal District. In this study, hantavirus genomic sequences were amplified from seven patients who resided in Central Brazil and then sequenced and compared to other hantavirus sequences. The complete S segment sequence, which is 1847 bases long and potentially encodes the 428 amino acid nucleocapsid protein, was determined for one patient. Moreover, a 700 base-pair sequence of the S segment was obtained from two other patients, and we analyzed M segment sequences from all samples. It can be inferred by both identity and phylogenetic analysis that the sequences obtained are highly related to Araraquara variant and Maciel virus. Phylogenetic results show that hantaviruses isolated in Central Brazil can be divided into two monophyletic groups: one group that clusters with Araraquara variant and the other group that includes the complete S segment sequence obtained in this study. Therefore, we propose the name Paranoa for this variant that co-exists with the Araraquara-like hantavirus in Central Brazil.