RESUMO
Mammalian α-defensins are a family of abundant effector peptides of the mucosal innate immune system. Although primarily considered to be antimicrobial, α-defensins can increase rather than block infection by certain prominent bacterial and viral pathogens in cell culture and in vivo . We have shown previously that exposure of mouse and human adenoviruses (HAdVs) to α-defensins is able to overcome competitive inhibitors that block cell binding, leading us to hypothesize a defensin-mediated binding mechanism that is independent of known viral receptors. To test this hypothesis, we used genetic approaches to demonstrate that none of several primary receptors nor integrin co-receptors are needed for human α-defensin-mediated binding of HAdV to cells; however, infection remains integrin dependent. Thus, our studies have revealed a novel pathway for HAdV binding to cells that bypasses viral primary receptors. We speculate that this pathway functions in parallel with receptor-mediated entry and contributes to α-defensin-enhanced infection of susceptible cells. Remarkably, we also found that in the presence of α-defensins, HAdV tropism is expanded to non-susceptible cells, even when viruses are exposed to a mixture of both susceptible and non-susceptible cells. Therefore, we propose that in the presence of sufficient concentrations of α-defensins, such as in the lung or gut, integrin expression rather than primary receptor expression will dictate HAdV tropism in vivo . In summary, α-defensins may contribute to tissue tropism not only through the neutralization of susceptible viruses but also by allowing certain defensin-resistant viruses to bind to cells independently of previously described mechanisms. Author Summary: In this study, we demonstrate a novel mechanism for binding of human adenoviruses (HAdVs) to cells that is dependent upon interactions with α-defensin host defense peptides but is independent of known viral receptors and co-receptors. To block normal receptor-mediated HAdV infection, we made genetic changes to both host cells and HAdVs. Under these conditions, α-defensins restored cell binding; however, infection still required the function of HAdV integrin co-receptors. This was true for multiple types of HAdVs that use different primary receptors and for cells that are either naturally devoid of HAdV receptors or were engineered to be receptor deficient. These observations suggest that in the presence of concentrations of α-defensins that would be found naturally in the lung or intestine, there are two parallel pathways for HAdV binding to cells that converge on integrins for productive infection. Moreover, these binding pathways function independently, and both operate in mixed culture. Thus, we have found that viruses can co-opt host defense molecules to expand their tropism.
RESUMO
Mammalian α-defensins are a family of abundant effector peptides of the mucosal innate immune system. Although primarily considered to be antimicrobial, α-defensins can increase rather than block infection by certain prominent bacterial and viral pathogens in cell culture and in vivo. We have shown previously that exposure of mouse and human adenoviruses (HAdVs) to α-defensins is able to overcome competitive inhibitors that block cell binding, leading us to hypothesize a defensin-mediated binding mechanism that is independent of known viral receptors. To test this hypothesis, we used genetic approaches to demonstrate that none of several primary receptors nor integrin co-receptors are needed for human α-defensin-mediated binding of HAdV to cells; however, infection remains integrin dependent. Thus, our studies have revealed a novel pathway for HAdV binding to cells that bypasses viral primary receptors. We speculate that this pathway functions in parallel with receptor-mediated entry and contributes to α-defensin-enhanced infection of susceptible cells. Remarkably, we also found that in the presence of α-defensins, HAdV tropism is expanded to non-susceptible cells, even when viruses are exposed to a mixture of both susceptible and non-susceptible cells. Therefore, we propose that in the presence of sufficient concentrations of α-defensins, such as in the lung or gut, integrin expression rather than primary receptor expression will dictate HAdV tropism in vivo. In summary, α-defensins may contribute to tissue tropism not only through the neutralization of susceptible viruses but also by allowing certain defensin-resistant viruses to bind to cells independently of previously described mechanisms.
Assuntos
Adenovírus Humanos , Tropismo Viral , alfa-Defensinas , alfa-Defensinas/metabolismo , Humanos , Adenovírus Humanos/fisiologia , Adenovírus Humanos/metabolismo , Animais , Camundongos , Infecções por Adenovirus Humanos/metabolismo , Infecções por Adenovirus Humanos/virologia , Receptores Virais/metabolismo , Internalização do VírusRESUMO
Adeno-associated viruses (AAVs) are being developed as gene therapy vectors due to their low pathogenicity and tissue tropism properties. However, the efficacy of these vectors is impeded by interactions with the host immune system. One potential immune barrier to vector transduction is innate immune host defense peptides, such as alpha-defensins, which are potent antiviral agents against other nonenveloped viruses. To investigate the interaction between AAVs and alpha-defensins, we utilized two closely related AAV serotypes, AAV1 and AAV6. Although their capsids differ by only six residues, these two serotypes exhibit markedly different tissue tropisms and transduction efficiencies. Using two abundant human alpha-defensins, enteric human defensin 5 (HD5) and myeloid human neutrophil peptide 1 (HNP1), we found both serotype-specific and defensin-specific effects on AAV infection. AAV6 infection was uniformly neutralized by both defensins at low micromolar concentrations; however, inhibition of AAV1 infection was profoundly influenced by the timing of defensin exposure to the virus relative to viral attachment to the cell. Remarkably, these differences in the defensin-dependent infection phenotype between the viruses are completely dictated by the identity of a single, surface-exposed amino acid (position 531) that varies between the two serotypes. These findings reveal a determinant for defensin activity against a virus with unprecedented precision. Furthermore, they provide a rationale for the investigation of other AAV serotypes not only to understand the mechanism of neutralization of defensins against AAVs but also to design more efficient vectors. IMPORTANCE The ability of adeno-associated viruses (AAVs) to infect and deliver genetic material to a range of cell types makes them favorable gene therapy vectors. However, AAV vectors encounter a wide variety of host immune factors throughout the body, which can impede efficient gene delivery. One such group of factors is the alpha-defensins, which are a key component of the innate immune system that can directly block viral infection. By studying the impact that alpha-defensins have on AAV infection, we found that two similar AAV serotypes (AAV1 and AAV6) have different sensitivities to inhibition. We also identified a single amino acid (position 531) that differs between the two AAV serotypes and is responsible for mediating their defensin sensitivity. By investigating the effects that host immune factors have on AAV infection, more efficient vectors may be developed to evade intervention by the immune system prior to gene delivery.
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Dependovirus , Vetores Genéticos , alfa-Defensinas , Humanos , alfa-Defensinas/metabolismo , Aminoácidos/metabolismo , Dependovirus/imunologia , Dependovirus/fisiologia , Terapia GenéticaRESUMO
INTRODUCTION: Patient-centred care and care coordination are each key priority areas for delivering high quality healthcare. However, the intersection between these two concepts is poorly characterised. We theorise that greater advancements in healthcare quality could be realised when care is organised in a way that aligns with patients' preferences, needs and values across every level of the healthcare system. There is currently no published review that describes the intersection of patient-centred care and care coordination. We will undertake a scoping review that will be foundational to the development of a conceptual framework for patient-centred care coordination that integrates and synthesises the overlap between these two concepts and describe how it manifests across levels of the healthcare system. METHODS AND ANALYSIS: A multidisciplinary team of reviewers will conduct a scoping review of published and grey literature to identify and synthesise key concepts at the intersection of patient-centred care and care coordination, following Preferred Reporting Items for Systematic Reviews and Meta-analyses extension for Scoping Reviews guidance for scoping reviews. Databases we will use in our search include PubMed, CINAHL, Embase, Social Sciences Abstracts, Nursing and Allied Health Premium, Health and Medical Collection, and PsycINFO. Articles will be included that are English-language; published during or after 2001; describe a theory, conceptual model, theoretical framework or definition that addresses both patient-centred care and care coordination. Articles will be excluded if they do not address the intersection of patient-centred care and care coordination; discuss a patient-centred medical home without discussion on patient-centred care concepts; or discuss a paediatric, inpatient or palliative care setting. A data extraction template will facilitate qualitative thematic analysis and findings will be synthesised into a conceptual framework. ETHICS AND DISSEMINATION: This work does not require ethics approval. A preliminary framework will be presented to a group of patient stakeholders for refinement before dissemination through a peer-reviewed journal and conference presentations.
Assuntos
Enfermagem de Cuidados Paliativos na Terminalidade da Vida , Assistência Centrada no Paciente , Humanos , Cuidados Paliativos , Preferência do Paciente , Qualidade da Assistência à Saúde , Literatura de Revisão como Assunto , Revisões Sistemáticas como AssuntoRESUMO
The intestinal epithelium comprises the body's largest surface exposed to viruses. Additionally, the gut epithelium hosts a large population of intraepithelial T lymphocytes, or IELs, although their role in resistance against viral infections remains elusive. By fate-mapping T cells recruited to the murine intestine, we observed an accumulation of newly recruited CD4+ T cells after infection with murine norovirus CR6 and adenovirus type-2 (AdV), but not reovirus. CR6- and AdV-recruited intraepithelial CD4+ T cells co-expressed Ly6A and chemokine receptor CCR9, exhibited T helper 1 and cytotoxic profiles, and conferred protection against AdV in vivo and in an organoid model in an IFN-γ-dependent manner. Ablation of the T cell receptor (TCR) or the transcription factor ThPOK in CD4+ T cells prior to AdV infection prevented viral control, while TCR ablation during infection did not impact viral clearance. These results uncover a protective role for intraepithelial Ly6A+CCR9+CD4+ T cells against enteric adenovirus.
Assuntos
Intestino Delgado , Viroses , Animais , Antígenos Ly , Linfócitos T CD4-Positivos , Mucosa Intestinal , Proteínas de Membrana , Camundongos , Receptores de QuimiocinasRESUMO
Fecal-oral pathogens encounter constitutively expressed enteric alpha-defensins in the intestine during replication and transmission. Alpha-defensins can be potently antiviral and antibacterial; however, their primary sequences, the number of isoforms, and their activity against specific microorganisms often vary greatly between species, reflecting adaptation to species-specific pathogens. Therefore, alpha-defensins might influence not only microbial evolution and tissue tropism within a host but also species tropism and zoonotic potential. To investigate these concepts, we generated a panel of enteric and myeloid alpha-defensins from humans, rhesus macaques, and mice and tested their activity against group A rotaviruses, an important enteric viral pathogen of humans and animals. Rotaviral adaptation to the rhesus macaque correlated with resistance to rhesus enteric, but not myeloid, alpha-defensins and sensitivity to human alpha-defensins. While mouse rotaviral infection was increased in the presence of mouse enteric alpha-defensins, two prominent genotypes of human rotaviruses were differentially sensitive to human enteric alpha-defensins. Furthermore, the effects of cross-species alpha-defensins on human and mouse rotaviruses did not follow an obvious pattern. Thus, exposure to alpha-defensins may have shaped the evolution of some, but not all, rotaviruses. We then used a genetic approach to identify the viral attachment and penetration protein, VP4, as a determinant of alpha-defensin sensitivity. Our results provide a foundation for future studies of the VP4-dependent mechanism of defensin neutralization, highlight the species-specific activities of alpha-defensins, and focus future efforts on a broader range of rotaviruses that differ in VP4 to uncover the potential for enteric alpha-defensins to influence species tropism. IMPORTANCE Rotavirus is a leading cause of severe diarrhea in young children. Like other fecal-oral pathogens, rotaviruses encounter abundant, constitutively expressed defensins in the small intestine. These peptides are a vital part of the vertebrate innate immune system. By investigating the impact that defensins from multiple species have on the infectivity of different strains of rotavirus, we show that some rotaviral infections can be inhibited by defensins. We also found that some, but not all, rotaviruses may have evolved resistance to defensins in the intestine of their host species, and some even appropriate defensins to increase their infectivity. Because rotaviruses infect a broad range of animals and rotaviral infections are highly prevalent in children, identifying immune defenses against infection and how they vary across species and among viral genotypes is important for our understanding of the evolution, transmission, and zoonotic potential of these viruses as well as the improvement of vaccines.
Assuntos
Infecções por Rotavirus , Rotavirus , alfa-Defensinas , Animais , Humanos , Intestino Delgado/imunologia , Intestino Delgado/virologia , Macaca mulatta , Camundongos , Rotavirus/efeitos dos fármacos , Rotavirus/genética , Infecções por Rotavirus/fisiopatologia , Infecções por Rotavirus/virologia , Proteínas Estruturais Virais/metabolismo , alfa-Defensinas/genética , alfa-Defensinas/metabolismo , alfa-Defensinas/farmacologiaRESUMO
Intracellular pathogens alter their host cells' mechanics to promote dissemination through tissues. Conversely, host cells may respond to the presence of pathogens by altering their mechanics to limit infection. Here, we monitored epithelial cell monolayers infected with intracellular bacterial pathogens, Listeria monocytogenes or Rickettsia parkeri, over days. Under conditions in which these pathogens trigger innate immune signaling through NF-κB and use actin-based motility to spread non-lytically intercellularly, we found that infected cell domains formed three-dimensional mounds. These mounds resulted from uninfected cells moving toward the infection site, collectively squeezing the softer and less contractile infected cells upward and ejecting them from the monolayer. Bacteria in mounds were less able to spread laterally in the monolayer, limiting the growth of the infection focus, while extruded infected cells underwent cell death. Thus, the coordinated forceful action of uninfected cells actively eliminates large domains of infected cells, consistent with this collective cell response representing an innate immunity-driven process.
Assuntos
Competição entre as Células , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Imunidade Inata , Listeria monocytogenes/fisiologia , Listeriose/imunologia , Listeriose/microbiologia , Transdução de Sinais , Actomiosina/metabolismo , Animais , Apoptose , Fenômenos Biomecânicos , Adesão Celular , Linhagem Celular , Simulação por Computador , Cães , Interações Hospedeiro-Patógeno , Humanos , Junções Intercelulares/metabolismo , Terapia a Laser , Listeriose/genética , Células Madin Darby de Rim Canino , NF-kappa B/metabolismo , Imagem com Lapso de Tempo , Transcrição GênicaRESUMO
Enteric alpha-defensins are potent effectors of innate immunity that are abundantly expressed in the small intestine. Certain enteric bacteria and viruses are resistant to defensins and even appropriate them to enhance infection despite neutralization of closely related microbes. We therefore hypothesized that defensins impose selective pressure during fecal-oral transmission. Upon passaging a defensin-sensitive serotype of adenovirus in the presence of a human defensin, mutations in the major capsid protein hexon accumulated. In contrast, prior studies identified the vertex proteins as important determinants of defensin antiviral activity. Infection and biochemical assays suggest that a balance between increased cell binding and a downstream block in intracellular trafficking mediated by defensin interactions with all of the major capsid proteins dictates the outcome of infection. These results extensively revise our understanding of the interplay between defensins and non-enveloped viruses. Furthermore, they provide a feasible rationale for defensins shaping viral evolution, resulting in differences in infection phenotypes of closely related viruses.
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Infecções por Adenoviridae/virologia , Adenoviridae/genética , Antivirais/metabolismo , Proteínas do Capsídeo/genética , alfa-Defensinas/metabolismo , Células A549 , Adenoviridae/imunologia , Evolução Molecular , Humanos , Imunidade Inata , Intestino Delgado/imunologia , Intestino Delgado/virologia , Modelos Moleculares , Mutação , SorogrupoRESUMO
Recent studies have determined that inflammasome signaling plays an important role in driving intestinal epithelial cell (IEC) responses to bacterial infections, such as Salmonella enterica serovar Typhimurium. There are two primary inflammasome pathways, canonical (involving caspase-1) and noncanonical (involving caspase-4 and -5 in humans and caspase-11 in mice). Prior studies identified the canonical inflammasome as the major pathway leading to interleukin-18 (IL-18) release and restriction of S Typhimurium replication in the mouse cecum. In contrast, the human C2Bbe1 colorectal carcinoma cell line expresses little caspase-1 but instead utilizes caspase-4 to respond to S Typhimurium infection. Intestinal enteroid culture has enabled long-term propagation of untransformed IECs from multiple species, including mouse and human. Capitalizing on this technology, we used a genetic approach to directly compare the relative importance of different inflammatory caspases in untransformed mouse and human IECs and transformed human IECs upon S Typhimurium infection in vitro We show that caspase-1 is important for restricting intracellular S Typhimurium replication and initiating IL-18 secretion in mouse IECs but is dispensable in human IECs. In contrast, restriction of intracellular S Typhimurium and production of IL-18 are dependent on caspase-4 in both transformed and untransformed human IECs. Notably, cytosolic replication in untransformed cells from both species was less pronounced than in transformed human cells, suggesting that transformation may impact additional pathways that restrict S Typhimurium replication. Taken together, these data highlight the differences between mouse and human IECs and the utility of studying transformed and untransformed cells in parallel.
Assuntos
Inflamassomos/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Infecções por Salmonella/metabolismo , Infecções por Salmonella/microbiologia , Salmonella enterica/fisiologia , Animais , Biomarcadores , Caspases/metabolismo , Linhagem Celular , Citocinas/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Humanos , Mucosa Intestinal/patologia , Camundongos , Infecções por Salmonella/genéticaRESUMO
During DNA virus infections, detection of cytosolic DNA by the cGAS-STING pathway leads to activation of IFN-ß. Kaposi's Sarcoma Herpesvirus (KSHV), an oncogenic DNA virus, is the etiological agent of Kaposi's Sarcoma, an endothelial cell (EC)-based tumor. To investigate the role of STING during KSHV infection of primary ECs we identified a primary lymphatic EC sample that is defective for STING activation and we also knocked out STING in blood ECs. Ablation of STING in EC does not increase susceptibility to KSHV latent infection nor does it increase KSHV spread after lytic reactivation indicating STING signaling does not restrict KSHV. In contrast, STING ablation increases Adenovirus spread at low MOI, but STING is dispensable for blocking replication. These experiments reveal that the importance of STING depends on the DNA virus and that STING appears more important for restricting spread to bystander cells than for inhibition of viral replication.
Assuntos
Células Endoteliais/virologia , Infecções por Herpesviridae/metabolismo , Infecções por Herpesviridae/virologia , Herpesvirus Humano 8/fisiologia , Proteínas de Membrana/metabolismo , DNA Viral , Suscetibilidade a Doenças , Humanos , Imunidade Inata , Nucleotidiltransferases/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Replicação ViralRESUMO
BACKGROUND: Human papillomavirus (HPV) is linked to nearly all cases of cervical cancer. Despite available vaccines, a deeper understanding of the immune response to HPV is needed. Human α-defensin 5 (HD5), an innate immune effector peptide, blocks infection of multiple sero-types of HPV, including high-risk HPV16. While a common mechanism of α-defensin anti-viral activity against nonenveloped viruses such as HPV has emerged, there is limited understanding of how α-defensins bind to viral capsids to block infection. METHODS: We have used cryo-electron microscopy (cryoEM), mass spectrometry (MS) crosslinking and differential lysine modification studies, and molecular dynamics (MD) simulations to probe the interaction of HPV16 pseudovirions (PsVs) with HD5. RESULTS: CryoEM single particle reconstruction did not reveal HD5 density on the capsid surface. Rather, increased density was observed under the capsid shell in the presence of HD5. MS studies indicate that HD5 binds near the L1 and L2 capsid proteins and specifically near the C-terminal region of L1. MD simulations indicate that favorable electrostatic interactions can be formed between HD5 and the L1 C-terminal tail. CONCLUSIONS: A model is presented for how HD5 affects HPV16 structure and cell entry. In this model, HD5 binds to disordered regions of L1 and L2 protruding from the icosahedrally ordered capsid. HD5 acts to cement interactions between L1 and L2 and leads to a closer association of the L2/genome core with the L1 capsid. This model provides a structural rationale for our prior observation that HD5 interferes with the separation of L1 from the L2/genome complex during cell entry.
RESUMO
In this issue of Cell Host & Microbe, Bottermann et al. (2019) reveal that complement component C4 inhibits adenovirus by inactivating the virus capsid through mechanisms requiring antibody engagement, but not late-acting complement pathways. This antiviral function likely broadly impacts non-enveloped viruses and may help illuminate the process of virus disassembly.
Assuntos
Capsídeo , Complemento C4 , Proteínas do Capsídeo , Proteínas do Sistema Complemento , VírionRESUMO
Demonstrated improvements in patient outcomes will facilitate the clinical implementation of pharmacogenetic testing. Using the association between solute carrier organic anion transporter family member 1B1 (SLCO1B1) and statin-associated muscle symptoms (SAMSs) as a model, we conducted a systematic review of patient outcomes after delivery of SLCO1B1 results. Using PubMed and Embase searches through December 19, 2017, we identified 37 eligible records reporting preliminary or final outcomes, including six studies delivering only SLCO1B1 results and five large healthcare system-based implementation projects of multipharmacogene panels. Two small trials have demonstrated at least short-term improvements in low-density lipoprotein cholesterol after SLCO1B1 testing among previously statin intolerant patients. Evidence from large implementation projects suggests that SLCO1B1 results may change prescribing patterns for some high-risk patients. No study has reported improvements in SAMSs or cardiovascular events or tracked the economic outcomes of SLCO1B1 testing. Ongoing studies should collect and report outcomes relevant to pharmacogenetics stakeholders.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Inibidores de Hidroximetilglutaril-CoA Redutases , Transportador 1 de Ânion Orgânico Específico do Fígado/genética , Doenças Musculares , Testes Farmacogenômicos/métodos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/genética , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/prevenção & controle , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/efeitos adversos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacocinética , Doenças Musculares/induzido quimicamente , Doenças Musculares/genética , Doenças Musculares/prevenção & controle , FarmacogenéticaRESUMO
Murine adenovirus 2 (MAdV-2) infects cells of the mouse gastrointestinal tract. Like human adenoviruses, it is a member of the genus Mastadenovirus, family Adenoviridae. The MAdV-2 genome has a single fibre gene that expresses a 787 residue-long protein. Through analogy to other adenovirus fibre proteins, it is expected that the carboxy-terminal virus-distal head domain of the fibre is responsible for binding to the host cell, although the natural receptor is unknown. The putative head domain has little sequence identity to adenovirus fibres of known structure. In this report, we present high-resolution crystal structures of the carboxy-terminal part of the MAdV-2 fibre. The structures reveal a domain with the typical adenovirus fibre head topology and a domain containing two triple ß-spiral repeats of the shaft domain. Through glycan microarray profiling, saturation transfer difference nuclear magnetic resonance spectroscopy, isothermal titration calorimetry and site-directed mutagenesis, we show that the fibre specifically binds to the monosaccharide N-acetylglucosamine (GlcNAc). The crystal structure of the complex reveals that GlcNAc binds between the AB and CD loops at the top of each of the three monomers of the MAdV-2 fibre head. However, infection competition assays show that soluble GlcNAc monosaccharide and natural GlcNAc-containing polymers do not inhibit infection by MAdV-2. Furthermore, site-directed mutation of the GlcNAc-binding residues does not prevent the inhibition of infection by soluble fibre protein. On the other hand, we show that the MAdV-2 fibre protein binds GlcNAc-containing mucin glycans, which suggests that the MAdV-2 fibre protein may play a role in viral mucin penetration in the mouse gut.
Assuntos
Acetilglucosamina/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Domínios Proteicos , Receptores Virais/metabolismo , Animais , Cristalografia por Raios X , Camundongos , Ligação Proteica , Conformação ProteicaRESUMO
Murine norovirus (MNV) is an RNA virus that can prove lethal in mice with impaired innate immunity. We found that MNV-4 infection of Stat1-/- mice was not lethal, but produced a 100% penetrant, previously undescribed lymphatic phenotype characterized by chronic-active lymphangitis with hepatitis, splenitis, and chronic cecal and colonic inflammation. Lesion pathogenesis progressed from early ileal enteritis and regional dilated lymphatics to lymphangitis, granulomatous changes in the liver and spleen, and, ultimately, typhlocolitis. Lesion development was neither affected by antibiotics nor reproduced by infection with another enteric RNA virus, rotavirus. MNV-4 infection in Stat1-/- mice decreased expression of vascular endothelial growth factor (Vegf) receptor 3, Vegf-c, and Vegf-d and increased interferon (Ifn)-γ, tumor necrosis factor-α, and inducible nitric oxide synthase. However, anti-IFN-γ and anti-tumor necrosis factor-α antibody treatment did not attenuate the histologic lesions. Studies in Ifnαßγr-/- mice suggested that canonical signaling via interferon receptors did not cause MNV-4-induced disease. Infected Stat1-/- mice had increased STAT3 phosphorylation and expressed many STAT3-regulated genes, consistent with our findings of increased myeloid cell subsets and serum granulocyte colony-stimulating factor, which are also associated with increased STAT3 activity. In conclusion, in Stat1-/- mice, MNV-4 induces lymphatic lesions similar to those seen in Crohn disease as well as hepatitis, splenitis, and typhlocolitis. MNV-4-infected Stat1-/- mice may be a useful model to study mechanistic associations between viral infections, lymphatic dysfunction, and intestinal inflammation in a genetically susceptible host.
Assuntos
Infecções por Caliciviridae/complicações , Colite/patologia , Intestinos/patologia , Fígado/patologia , Linfangite/patologia , Fator de Transcrição STAT1/fisiologia , Baço/patologia , Animais , Infecções por Caliciviridae/virologia , Colite/metabolismo , Colite/virologia , Feminino , Interferons/metabolismo , Intestinos/virologia , Fígado/metabolismo , Fígado/virologia , Linfangite/metabolismo , Linfangite/virologia , Camundongos , Camundongos Knockout , Norovirus/isolamento & purificação , Transdução de Sinais , Baço/metabolismo , Baço/virologiaRESUMO
Human adenoviruses (HAdV) are significant human pathogens. Although only a subset of HAdV serotypes commonly cause gastroenteritis in humans, most HAdV species replicate in the gastrointestinal tract. Knowledge of the complex interaction between HAdVs and the human intestinal epithelium has been limited by the lack of a suitable cell culture system containing relevant cell types. Recently, this need has been met by the stable and prolonged cultivation of primary intestinal epithelial cells as enteroids. Human enteroids have been used to reveal novel and interesting aspects of rotavirus, norovirus, and enterovirus replication, prompting us to explore their suitability for HAdV culture. We found that both prototype strains and clinical isolates of enteric and nonenteric HAdVs productively replicate in human enteroids. HAdV-5p, a respiratory pathogen, and HAdV-41p, an enteric pathogen, are both sensitive to type I and III interferons in human enteroid monolayers but not A549 cells. Interestingly, HAdV-5p, but not HAdV-41p, preferentially infected goblet cells. And, HAdV-5p but not HAdV-41p was potently neutralized by the enteric human alpha-defensin HD5. These studies highlight new facets of HAdV biology that are uniquely revealed by primary intestinal epithelial cell culture.IMPORTANCE Enteric adenoviruses are a significant cause of childhood gastroenteritis worldwide, yet our understanding of their unique biology is limited. Here we report robust replication of both prototype and clinical isolates of enteric and respiratory human adenoviruses in enteroids, a primary intestinal cell culture system. Recent studies have shown that other fastidious enteric viruses replicate in human enteroids. Therefore, human enteroids may provide a unified platform for culturing enteric viruses, potentially enabling isolation of a greater diversity of viruses from patients. Moreover, both the ability of interferon to restrict respiratory and enteric adenoviruses and a surprising preference of a respiratory serotype for goblet cells demonstrate the power of this culture system to uncover aspects of adenovirus biology that were previously unattainable with standard cell lines.
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Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/isolamento & purificação , Células Caliciformes/virologia , Intestino Delgado/virologia , Replicação Viral , Infecções por Adenovirus Humanos/tratamento farmacológico , Infecções por Adenovirus Humanos/imunologia , Adulto , Antivirais/farmacologia , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/imunologia , Humanos , Interferons/farmacologia , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/imunologiaRESUMO
α, ß, and θ defensins are effectors of the innate immune system with potent antibacterial, antiviral, and antifungal activity. Defensins have direct antiviral activity in cell culture, with varied mechanisms for individual viruses, although some common themes have emerged. In addition, defensins have potent immunomodulatory activity that can alter innate and adaptive immune responses to viral infection. In some cases, there is evidence for paradoxical escape from defensin neutralization or enhancement of viral infection. The direct and indirect activities of defensins have led to their development as therapeutics and vaccine components. The major area of investigation that continues to lag is the connection between the effects of defensins in cell culture models and viral pathogenesis in vivo. Model systems to study defensin biology, including more physiologic models designed to bridge this gap, are also discussed.
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Defensinas/metabolismo , Viroses/imunologia , alfa-Defensinas/metabolismo , beta-Defensinas/metabolismo , Adenoviridae/efeitos dos fármacos , Adenoviridae/patogenicidade , Animais , Antivirais/farmacologia , Defensinas/genética , Defensinas/farmacologia , Defensinas/uso terapêutico , HIV/efeitos dos fármacos , HIV/patogenicidade , Herpesviridae/efeitos dos fármacos , Herpesviridae/patogenicidade , Humanos , Imunidade Inata , Imunomodulação , Camundongos , Papillomaviridae/efeitos dos fármacos , Papillomaviridae/patogenicidade , Viroses/tratamento farmacológico , alfa-Defensinas/genética , alfa-Defensinas/farmacologia , alfa-Defensinas/uso terapêutico , beta-Defensinas/genética , beta-Defensinas/farmacologia , beta-Defensinas/uso terapêuticoRESUMO
The small intestinal epithelium produces numerous antimicrobial peptides and proteins, including abundant enteric α-defensins. Although they most commonly function as potent antivirals in cell culture, enteric α-defensins have also been shown to enhance some viral infections in vitro. Efforts to determine the physiologic relevance of enhanced infection have been limited by the absence of a suitable cell culture system. To address this issue, here we use primary stem cell-derived small intestinal enteroids to examine the impact of naturally secreted α-defensins on infection by the enteric mouse pathogen, mouse adenovirus 2 (MAdV-2). MAdV-2 infection was increased when enteroids were inoculated across an α-defensin gradient in a manner that mimics oral infection but not when α-defensin levels were absent or bypassed through other routes of inoculation. This increased infection was a result of receptor-independent binding of virus to the cell surface. The enteroid experiments accurately predicted increased MAdV-2 shedding in the feces of wild type mice compared to mice lacking functional α-defensins. Thus, our studies have shown that viral infection enhanced by enteric α-defensins may reflect the evolution of some viruses to utilize these host proteins to promote their own infection.
Assuntos
Infecções por Adenoviridae/virologia , Adenoviridae/fisiologia , Intestino Delgado/metabolismo , alfa-Defensinas/metabolismo , Adenoviridae/genética , Animais , Feminino , Interações Hospedeiro-Patógeno , Humanos , Intestino Delgado/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Eliminação de Partículas Virais , alfa-Defensinas/genéticaRESUMO
α-Defensins are an important class of abundant innate immune effectors that are potently antiviral against a number of nonenveloped viral pathogens; however, a common mechanism to explain their ability to block infection by these unrelated viruses is lacking. We previously found that human defensin 5 (HD5) blocks a critical host-mediated proteolytic processing step required for human papillomavirus (HPV) infection. Here, we show that bypassing the requirement for this cleavage failed to abrogate HD5 inhibition. Instead, HD5 altered HPV trafficking in the cell. In the presence of an inhibitory concentration of HD5, HPV was internalized and reached the early endosome. The internalized capsid became permeable to antibodies and proteases; however, HD5 prevented dissociation of the viral capsid from the genome, reduced viral trafficking to the trans-Golgi network, redirected the incoming viral particle to the lysosome, and accelerated the degradation of internalized capsid proteins. This mechanism is equivalent to the mechanism by which HD5 inhibits human adenovirus. Thus, our data support capsid stabilization and redirection to the lysosome during infection as a general antiviral mechanism of α-defensins against nonenveloped viruses. IMPORTANCE: Although the antiviral activity of α-defensins against enveloped viruses can be largely explained by interference with receptor binding and fusion, a common mechanism for inhibition of nonenveloped viruses remains elusive. In studies of a prominent human α-defensin that is expressed in the gut and in the male and female genitourinary tract, we discovered striking parallels between the mechanisms of inhibition of HPV and human adenovirus infection. Thus, detailed studies of the impact of α-defensins on the intracellular trafficking of two disparate viruses support a general mechanism of α-defensin antiviral activity against nonenveloped viruses.
Assuntos
Proteínas do Capsídeo/metabolismo , Papillomavirus Humano 16/imunologia , Lisossomos/metabolismo , alfa-Defensinas/metabolismo , Linhagem Celular , Humanos , ProteóliseRESUMO
α-defensins are abundant antimicrobial peptides with broad, potent antibacterial, antifungal, and antiviral activities in vitro. Although their contribution to host defense against bacteria in vivo has been demonstrated, comparable studies of their antiviral activity in vivo are lacking. Using a mouse model deficient in activated α-defensins in the small intestine, we show that Paneth cell α-defensins protect mice from oral infection by a pathogenic virus, mouse adenovirus 1 (MAdV-1). Survival differences between mouse genotypes are lost upon parenteral MAdV-1 infection, strongly implicating a role for intestinal defenses in attenuating pathogenesis. Although differences in α-defensin expression impact the composition of the ileal commensal bacterial population, depletion studies using broad-spectrum antibiotics revealed no effect of the microbiota on α-defensin-dependent viral pathogenesis. Moreover, despite the sensitivity of MAdV-1 infection to α-defensin neutralization in cell culture, we observed no barrier effect due to Paneth cell α-defensin activation on the kinetics and magnitude of MAdV-1 dissemination to the brain. Rather, a protective neutralizing antibody response was delayed in the absence of α-defensins. This effect was specific to oral viral infection, because antibody responses to parenteral or mucosal ovalbumin exposure were not affected by α-defensin deficiency. Thus, α-defensins play an important role as adjuvants in antiviral immunity in vivo that is distinct from their direct antiviral activity observed in cell culture.