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1.
Sci Immunol ; 8(90): eadd5724, 2023 Dec 22.
Artículo en Inglés | MEDLINE | ID: mdl-38134242

RESUMEN

Tissue-resident CD8+ T cells (TRM) continuously scan peptide-MHC (pMHC) complexes in their organ of residence to intercept microbial invaders. Recent data showed that TRM lodged in exocrine glands scan tissue in the absence of any chemoattractant or adhesion receptor signaling, thus bypassing the requirement for canonical migration-promoting factors. The signals eliciting this noncanonical motility and its relevance for organ surveillance have remained unknown. Using mouse models of viral infections, we report that exocrine gland TRM autonomously generated front-to-back F-actin flow for locomotion, accompanied by high cortical actomyosin contractility, and leading-edge bleb formation. The distinctive mode of exocrine gland TRM locomotion was triggered by sensing physical confinement and was closely correlated with nuclear deformation, which acts as a mechanosensor via an arachidonic acid and Ca2+ signaling pathway. By contrast, naïve CD8+ T cells or TRM surveilling microbe-exposed epithelial barriers did not show mechanosensing capacity. Inhibition of nuclear mechanosensing disrupted exocrine gland TRM scanning and impaired their ability to intercept target cells. These findings indicate that confinement is sufficient to elicit autonomous T cell surveillance in glands with restricted chemokine expression and constitutes a scanning strategy that complements chemosensing-dependent migration.


Asunto(s)
Linfocitos T CD8-positivos , Virosis , Ratones , Animales , Linfocitos T CD8-positivos/metabolismo , Memoria Inmunológica , Glándulas Exocrinas , Transducción de Señal
2.
EMBO Rep ; 24(6): e56818, 2023 06 05.
Artículo en Inglés | MEDLINE | ID: mdl-37042686

RESUMEN

Immature dendritic cells (iDCs) migrate in microenvironments with distinct cell and extracellular matrix densities in vivo and contribute to HIV-1 dissemination and mounting of antiviral immune responses. Here, we find that, compared to standard 2D suspension cultures, 3D collagen as tissue-like environment alters iDC properties and their response to HIV-1 infection. iDCs adopt an elongated morphology with increased deformability in 3D collagen at unaltered activation, differentiation, cytokine secretion, or responsiveness to LPS. While 3D collagen reduces HIV-1 particle uptake by iDCs, fusion efficiency is increased to elevate productive infection rates due to elevated cell surface exposure of the HIV-1-binding receptor DC-SIGN. In contrast, 3D collagen reduces HIV transfer to CD4 T cells from iDCs. iDC adaptations to 3D collagen include increased pro-inflammatory cytokine production and reduced antiviral gene expression in response to HIV-1 infection. Adhesion to a 2D collagen matrix is sufficient to increase iDC deformability, DC-SIGN exposure, and permissivity to HIV-1 infection. Thus, mechano-physical cues of 2D and 3D tissue-like collagen environments regulate iDC function and shape divergent roles during HIV-1 infection.


Asunto(s)
Infecciones por VIH , VIH-1 , Humanos , Citocinas/metabolismo , Colágeno/metabolismo , Antivirales , Células Dendríticas
3.
Front Immunol ; 13: 982383, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36341455

RESUMEN

Naive B cells use the chemokine receptor CXCR5 to enter B cell follicles, where they scan CXCL13-expressing ICAM-1+ VCAM-1+ follicular dendritic cells (FDCs) for the presence of antigen. CXCL13-CXCR5-mediated motility is mainly driven by the Rac guanine exchange factor DOCK2, which contains a binding domain for phosphoinositide-3,4,5-triphosphate (PIP3) and other phospholipids. While p110δ, the catalytic subunit of the class IA phosphoinositide-3-kinase (PI3K) δ, contributes to CXCR5-mediated B cell migration, the precise interdependency of DOCK2, p110δ, or other PI3K family members during this process remains incompletely understood. Here, we combined in vitro chemotaxis assays and in vivo imaging to examine the contribution of these two factors during murine naïve B cell migration to CXCL13. Our data confirm that p110δ is the main catalytic subunit mediating PI3K-dependent migration downstream CXCR5, whereas it does not contribute to chemotaxis triggered by CXCR4 or CCR7, two other chemokine receptors expressed on naïve B cells. The contribution of p110δ activity to CXCR5-driven migration was complementary to that of DOCK2, and pharmacological or genetic interference with both pathways completely abrogated B cell chemotaxis to CXCL13. Intravital microscopy of control and gene-deficient B cells migrating on FDCs confirmed that lack of DOCK2 caused a profound migration defect, whereas p110δ contributed to cell speed and directionality. B cells lacking active p110δ also displayed defective adhesion to ICAM-1; yet, their migration impairment was maintained on ICAM-1-deficient FDCs. In sum, our data uncover two complementary signaling pathways mediated by DOCK2 and p110δ, which enable CXCR5-driven naïve B cell examination of FDCs.


Asunto(s)
Molécula 1 de Adhesión Intercelular , Fosfatidilinositol 3-Quinasas , Ratones , Animales , Molécula 1 de Adhesión Intercelular/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Receptores CXCR5/metabolismo , Transducción de Señal , Factores de Intercambio de Guanina Nucleótido/genética , Quimiotaxis de Leucocito , Receptores de Quimiocina , Fosfatidilinositoles , Proteínas Activadoras de GTPasa
4.
mBio ; 13(2): e0370521, 2022 04 26.
Artículo en Inglés | MEDLINE | ID: mdl-35229634

RESUMEN

Combinations of direct-acting antivirals are needed to minimize drug resistance mutations and stably suppress replication of RNA viruses. Currently, there are limited therapeutic options against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and testing of a number of drug regimens has led to conflicting results. Here, we show that cobicistat, which is an FDA-approved drug booster that blocks the activity of the drug-metabolizing proteins cytochrome P450-3As (CYP3As) and P-glycoprotein (P-gp), inhibits SARS-CoV-2 replication. Two independent cell-to-cell membrane fusion assays showed that the antiviral effect of cobicistat is exerted through inhibition of spike protein-mediated membrane fusion. In line with this, incubation with low-micromolar concentrations of cobicistat decreased viral replication in three different cell lines including cells of lung and gut origin. When cobicistat was used in combination with remdesivir, a synergistic effect on the inhibition of viral replication was observed in cell lines and in a primary human colon organoid. This was consistent with the effects of cobicistat on two of its known targets, CYP3A4 and P-gp, the silencing of which boosted the in vitro antiviral activity of remdesivir in a cobicistat-like manner. When administered in vivo to Syrian hamsters at a high dose, cobicistat decreased viral load and mitigated clinical progression. These data highlight cobicistat as a therapeutic candidate for treating SARS-CoV-2 infection and as a potential building block of combination therapies for COVID-19. IMPORTANCE The lack of effective antiviral treatments against SARS-CoV-2 is a significant limitation in the fight against the COVID-19 pandemic. Single-drug regimens have so far yielded limited results, indicating that combinations of antivirals might be required, as previously seen for other RNA viruses. Our work introduces the drug booster cobicistat, which is approved by the FDA and typically used to potentiate the effect of anti-HIV protease inhibitors, as a candidate inhibitor of SARS-CoV-2 replication. Beyond its direct activity as an antiviral, we show that cobicistat can enhance the effect of remdesivir, which was one of the first drugs proposed for treatment of SARS-CoV-2. Overall, the dual action of cobicistat as a direct antiviral and a drug booster can provide a new approach to design combination therapies and rescue the activity of compounds that are only partially effective in monotherapy.


Asunto(s)
Tratamiento Farmacológico de COVID-19 , Hepatitis C Crónica , Adenosina Monofosfato/análogos & derivados , Alanina/análogos & derivados , Animales , Antivirales/farmacología , Antivirales/uso terapéutico , Cobicistat , Cricetinae , Progresión de la Enfermedad , Humanos , Mesocricetus , Pandemias , SARS-CoV-2 , Carga Viral
5.
Cell Rep ; 38(7): 110387, 2022 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-35134331

RESUMEN

SARS-CoV-2 variants of concern (VOCs) display enhanced transmissibility and resistance to antibody neutralization. Comparing the early 2020 isolate EU-1 to the VOCs Alpha, Beta, and Gamma in mice transgenic for human ACE2 reveals that VOCs induce a broadened scope of symptoms, expand systemic infection to the gastrointestinal tract, elicit the depletion of natural killer cells, and trigger variant-specific cytokine production patterns. Gamma infections result in accelerated disease progression associated with increased immune activation and inflammation. All four SARS-CoV-2 variants induce pDC depletion in the lungs, paralleled by reduced interferon responses. Remarkably, VOCs also use the murine ACE2 receptor for infection to replicate in the lungs of wild-type animals, which induce cellular and innate immune responses that apparently curtail the spread of overt disease. VOCs thus display distinct intrinsic pathogenic properties with broadened tissue and host range. The enhanced pathogenicity of VOCs and their potential for reverse zoonotic transmission pose challenges to clinical and pandemic management.


Asunto(s)
COVID-19/virología , Modelos Animales de Enfermedad , SARS-CoV-2/fisiología , SARS-CoV-2/patogenicidad , Animales , COVID-19/inmunología , Citocinas/metabolismo , Especificidad del Huésped , Inmunidad Celular , Inmunidad Innata , Pulmón/inmunología , Pulmón/virología , Ratones , Especificidad de la Especie , Carga Viral , Tropismo Viral , Virulencia , Replicación Viral
6.
Mol Ther ; 30(5): 2005-2023, 2022 05 04.
Artículo en Inglés | MEDLINE | ID: mdl-35038579

RESUMEN

Despite rapid development and deployment of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), clinically relevant modalities to curb the pandemic by directly attacking the virus on a genetic level remain highly desirable and are urgently needed. Here we comprehensively illustrate the capacity of adeno-associated virus (AAV) vectors co-expressing a cocktail of three short hairpin RNAs (shRNAs; RNAi triggers) directed against the SARS-CoV-2 RdRp and N genes as versatile and effective antiviral agents. In cultured monkey cells and human gut organoids, our most potent vector, SAVIOR (SARS virus repressor), suppressed SARS-CoV-2 infection to background levels. Strikingly, in control experiments using single shRNAs, multiple SARS-CoV-2 escape mutants quickly emerged from infected cells within 24-48 h. Importantly, such adverse viral adaptation was fully prevented with the triple-shRNA AAV vector even during long-term cultivation. In addition, AAV-SAVIOR efficiently purged SARS-CoV-2 in a new model of chronically infected human intestinal cells. Finally, intranasal AAV-SAVIOR delivery using an AAV9 capsid moderately diminished viral loads and/or alleviated disease symptoms in hACE2-transgenic or wild-type mice infected with human or mouse SARS-CoV-2 strains, respectively. Our combinatorial and customizable AAV/RNAi vector complements ongoing global efforts to control the coronavirus disease 2019 (COVID-19) pandemic and holds great potential for clinical translation as an original and flexible preventive or therapeutic antiviral measure.


Asunto(s)
COVID-19 , SARS-CoV-2 , Animales , Antivirales , COVID-19/prevención & control , Dependovirus , Ratones , Pandemias , Interferencia de ARN , ARN Interferente Pequeño/genética , SARS-CoV-2/genética
7.
Cell Rep Med ; 2(6): 100317, 2021 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-34195682

RESUMEN

Polymorphonuclear neutrophils (PMNs), the most abundant white blood cells, are recruited rapidly to sites of infection to exert potent anti-microbial activity. Information regarding their role in infection with human immunodeficiency virus (HIV) is limited. Here we report that addition of PMNs to HIV-infected cultures of human tonsil tissue or peripheral blood mononuclear cells causes immediate and long-lasting suppression of HIV-1 spread and virus-induced depletion of CD4 T cells. This inhibition of HIV-1 spread strictly requires PMN contact with infected cells and is not mediated by soluble factors. 2-Photon (2PM) imaging visualized contacts of PMNs with HIV-1-infected CD4 T cells in tonsil tissue that do not result in lysis or uptake of infected cells. The anti-HIV activity of PMNs also does not involve degranulation, formation of neutrophil extracellular traps, or integrin-dependent cell communication. These results reveal that PMNs efficiently blunt HIV-1 replication in primary target cells and tissue by an unconventional mechanism.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , VIH-1/genética , Leucocitos Mononucleares/inmunología , Neutrófilos/inmunología , Tonsila Palatina/inmunología , Linfocitos T CD4-Positivos/virología , Comunicación Celular , Trampas Extracelulares , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/crecimiento & desarrollo , VIH-1/patogenicidad , Humanos , Integrinas/genética , Integrinas/inmunología , Leucocitos Mononucleares/virología , Neutrófilos/virología , Tonsila Palatina/citología , Cultivo Primario de Células , Carga Viral , Replicación Viral
8.
EMBO J ; 39(24): e105594, 2020 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-33146906

RESUMEN

Failures to produce neutralizing antibodies upon HIV-1 infection result in part from B-cell dysfunction due to unspecific B-cell activation. How HIV-1 affects antigen-specific B-cell functions remains elusive. Using an adoptive transfer mouse model and ex vivo HIV infection of human tonsil tissue, we found that expression of the HIV-1 pathogenesis factor NEF in CD4 T cells undermines their helper function and impairs cognate B-cell functions including mounting of efficient specific IgG responses. NEF interfered with T cell help via a specific protein interaction motif that prevents polarized cytokine secretion at the T-cell-B-cell immune synapse. This interference reduced B-cell activation and proliferation and thus disrupted germinal center formation and affinity maturation. These results identify NEF as a key component for HIV-mediated dysfunction of antigen-specific B cells. Therapeutic targeting of the identified molecular surface in NEF will facilitate host control of HIV infection.


Asunto(s)
Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Infecciones por VIH/inmunología , Animales , Células HEK293 , VIH-1 , Humanos , Evasión Inmune/inmunología , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL
9.
Sci Immunol ; 5(46)2020 04 03.
Artículo en Inglés | MEDLINE | ID: mdl-32245888

RESUMEN

It is well established that tissue macrophages and tissue-resident memory CD8+ T cells (TRM) play important roles for pathogen sensing and rapid protection of barrier tissues. In contrast, the mechanisms by which these two cell types cooperate for homeostatic organ surveillance after clearance of infections is poorly understood. Here, we used intravital imaging to show that TRM dynamically followed tissue macrophage topology in noninflamed murine submandibular salivary glands (SMGs). Depletion of tissue macrophages interfered with SMG TRM motility and caused a reduction of interepithelial T cell crossing. In the absence of macrophages, SMG TRM failed to cluster in response to local inflammatory chemokines. A detailed analysis of the SMG microarchitecture uncovered discontinuous attachment of tissue macrophages to neighboring epithelial cells, with occasional macrophage protrusions bridging adjacent acini and ducts. When dissecting the molecular mechanisms that drive homeostatic SMG TRM motility, we found that these cells exhibit a wide range of migration modes: In addition to chemokine- and adhesion receptor-driven motility, resting SMG TRM displayed a remarkable capacity for autonomous motility in the absence of chemoattractants and adhesive ligands. Autonomous SMG TRM motility was mediated by friction and insertion of protrusions into gaps offered by the surrounding microenvironment. In sum, SMG TRM display a unique continuum of migration modes, which are supported in vivo by tissue macrophages to allow homeostatic patrolling of the complex SMG architecture.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Homeostasis/inmunología , Macrófagos/inmunología , Glándulas Salivales/inmunología , Animales , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Especificidad de Órganos/inmunología , Encuestas y Cuestionarios
10.
J Virol ; 94(1)2019 12 12.
Artículo en Inglés | MEDLINE | ID: mdl-31597760

RESUMEN

HIV-1 Nef promotes virus spread and disease progression by altering host cell transport and signaling processes through interaction with multiple host cell proteins. The N-terminal region in HIV-1 Nef encompassing residues 12 to 39 has been implicated in many Nef activities, including disruption of CD4 T lymphocyte polarization and homing to lymph nodes, antagonism of SERINC5 restriction to virion infectivity, downregulation of cell surface CD4 and major histocompatibility complex class I (MHC-I), release of Nef-containing extracellular vesicles, and phosphorylation of Nef by recruitment of the Nef-associated kinase complex (NAKC). How this region mediates these pleiotropic functions is unclear. Characterization of a panel of alanine mutants spanning the N-terminal region to identify specific functional determinants revealed this region to be dispensable for effects of Nef from HIV-1 strain SF2 (HIV-1SF2Nef) on T cell actin organization and chemotaxis, retargeting of the host cell kinase Lck to the trans-Golgi network, and incorporation of Nef into extracellular vesicles. MHC-I downmodulation was specific to residue M20, and inhibition of T cell polarization by Nef required the integrity of the entire region. In contrast, downmodulation of cell surface CD4 and SERINC5 antagonism were mediated by a specific motif encompassing residues 32 to 39 that was also essential for efficient HIV replication in primary CD4 T lymphocytes. Finally, Nef phosphorylation via association with the NAKC was mediated by two EP repeats within residues 24 to 29 but was dispensable for other functions. These results identify the N-terminal region as a multifunctional interaction module for at least three different host cell ligands that mediate independent functions of HIV-1SF2Nef to facilitate immune evasion and virus spread.IMPORTANCE HIV-1 Nef critically determines virus spread and disease progression in infected individuals by acting as a protein interaction adaptor via incompletely defined mechanisms and ligands. Residues 12 to 39 near the N terminus of Nef have been described as an interaction platform for the Nef-associated kinase complex (NAKC) and were recently identified as essential determinants for a broad range of Nef activities. Here, we report a systematic mapping of this amino acid stretch that revealed the presence of three independent interaction motifs with specific ligands and activities. While downmodulation of cell surface MHC-I depends on M20, two EP repeats are the minimal binding site for the NAKC, and residues 32 to 39 mediate antagonism of the host cell restriction factor SERINC5 as well as downmodulation of cell surface CD4. These results reveal that the N-terminal region of HIV-1SF2Nef is a versatile and multifunctional protein interaction module that exerts essential functions of the pathogenicity factor via independent mechanisms.


Asunto(s)
VIH-1/genética , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/genética , Dominios Proteicos , Virión/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Células COS , Chlorocebus aethiops , Expresión Génica , Células HEK293 , VIH-1/metabolismo , Humanos , Evasión Inmune , Células Jurkat , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Mutación , Cultivo Primario de Células , Unión Proteica , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Virión/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/química , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo
11.
J Immunol ; 203(9): 2377-2387, 2019 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-31548330

RESUMEN

Flotillin-1 (Flot1) is an evolutionary conserved, ubiquitously expressed lipid raft-associated scaffolding protein. Migration of Flot1-deficient neutrophils is impaired because of a decrease in myosin II-mediated contractility. Flot1 also accumulates in the uropod of polarized T cells, suggesting an analogous role in T cell migration. In this study, we analyzed morphology and migration parameters of murine wild-type and Flot1-/- CD8+ T cells using in vitro assays and intravital two-photon microscopy of lymphoid and nonlymphoid tissues. Flot1-/- CD8+ T cells displayed significant alterations in cell shape and motility parameters in vivo but showed comparable homing to lymphoid organs and intact in vitro migration to chemokines. Furthermore, their clonal expansion and infiltration into nonlymphoid tissues during primary and secondary antiviral immune responses was comparable to wild-type CD8+ T cells. Taken together, Flot1 plays a detectable but unexpectedly minor role for CD8+ T cell behavior under physiological conditions.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Proteínas de la Membrana/fisiología , Animales , Linfocitos T CD8-positivos/fisiología , Movimiento Celular , Epidermis/inmunología , Femenino , Memoria Inmunológica , Activación de Linfocitos , Masculino , Microdominios de Membrana/fisiología , Ratones , Ratones Endogámicos C57BL
12.
J Immunol ; 201(9): 2731-2743, 2018 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-30257886

RESUMEN

HIV-1 Nef is a multifunctional protein that optimizes virus spread and promotes immune evasion of infected cells to accelerate disease progression in AIDS patients. As one of its activities, Nef reduces the motility of infected CD4+ T lymphocytes in confined space. In vivo, Nef restricts T lymphocyte homing to lymph nodes as it reduces the ability for extravasation at the diapedesis step. Effects of Nef on T lymphocyte motility are typically mediated by its ability to reduce actin remodeling. However, interference with diapedesis does not depend on residues in Nef required for inhibition of host cell actin dynamics. In search for an alternative mechanism by which Nef could alter T lymphocyte extravasation, we noted that the viral protein interferes with the polarization of primary human CD4+ T lymphocytes upon infection with HIV-1. Expression of Nef alone is sufficient to disrupt T cell polarization, and this effect is conserved among lentiviral Nef proteins. Nef acts by arresting the oscillation of CD4+ T cells between polarized and nonpolarized morphologies. Mapping studies identified the binding site for the Nef-associated kinase complex (NAKC) as critical determinant of this Nef activity and a NAKC-binding-deficient Nef variant fails to impair CD4+ T lymphocyte extravasation and homing to lymph nodes. These results thus imply the disruption of T lymphocyte polarity via its NAKC binding site as a novel mechanism by which lentiviral Nef proteins alter T lymphocyte migration in vivo.


Asunto(s)
Linfocitos T CD4-Positivos/virología , Polaridad Celular/inmunología , Quimiotaxis de Leucocito/inmunología , Migración Transendotelial y Transepitelial/inmunología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Animales , Sitios de Unión , Linfocitos T CD4-Positivos/inmunología , Humanos , Ganglios Linfáticos/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL
13.
J Exp Med ; 215(7): 1869-1890, 2018 07 02.
Artículo en Inglés | MEDLINE | ID: mdl-29875261

RESUMEN

T cells are actively scanning pMHC-presenting cells in lymphoid organs and nonlymphoid tissues (NLTs) with divergent topologies and confinement. How the T cell actomyosin cytoskeleton facilitates this task in distinct environments is incompletely understood. Here, we show that lack of Myosin IXb (Myo9b), a negative regulator of the small GTPase Rho, led to increased Rho-GTP levels and cell surface stiffness in primary T cells. Nonetheless, intravital imaging revealed robust motility of Myo9b-/- CD8+ T cells in lymphoid tissue and similar expansion and differentiation during immune responses. In contrast, accumulation of Myo9b-/- CD8+ T cells in NLTs was strongly impaired. Specifically, Myo9b was required for T cell crossing of basement membranes, such as those which are present between dermis and epidermis. As consequence, Myo9b-/- CD8+ T cells showed impaired control of skin infections. In sum, we show that Myo9b is critical for the CD8+ T cell adaptation from lymphoid to NLT surveillance and the establishment of protective tissue-resident T cell populations.


Asunto(s)
Linfocitos T CD8-positivos/metabolismo , Miosinas/metabolismo , Animales , Linfocitos T CD8-positivos/citología , Movimiento Celular , Polaridad Celular , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Epidermis/patología , Epidermis/virología , Matriz Extracelular/metabolismo , Inmunidad , Activación de Linfocitos/inmunología , Tejido Linfoide/metabolismo , Ratones Endogámicos C57BL , Miosinas/deficiencia , Receptores Mensajeros de Linfocitos/metabolismo , Proteínas de Unión al GTP rho/metabolismo
14.
J Vis Exp ; (135)2018 05 07.
Artículo en Inglés | MEDLINE | ID: mdl-29781999

RESUMEN

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, including cell biology, oncology, dentistry, and immunology. The SMG is an exocrine gland comprised of secretory epithelial cells, myofibroblasts, endothelial cells, nerves, and extracellular matrix. Dynamic cellular processes in the rat and mouse SMG have previously been imaged, mostly using inverted multi-photon microscope systems. Here, we describe a straightforward protocol for the surgical preparation and stabilization of the murine SMG in anesthetized mice for in vivo imaging with upright multi-photon microscope systems. We present representative intravital image sets of endogenous and adoptively transferred fluorescent cells, including the labeling of blood vessels or salivary ducts and second harmonic generation to visualize fibrillar collagen. In sum, our protocol allows for surgical preparation of mouse salivary glands in upright microscopy systems, which are commonly used for intravital imaging in the field of immunology.


Asunto(s)
Microscopía Intravital/métodos , Glándulas Salivales/diagnóstico por imagen , Glándula Submandibular/diagnóstico por imagen , Animales , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Glándulas Salivales/patología , Glándula Submandibular/patología
15.
J Virol ; 90(23): 10915-10927, 2016 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-27681140

RESUMEN

SERINC3 (serine incorporator 3) and SERINC5 are recently identified host cell inhibitors of HIV-1 particle infectivity that are counteracted by the viral pathogenesis factor Nef. Here we confirm that HIV-1 Nef, but not HIV-1 Vpu, antagonizes the particle infectivity restriction of SERINC5. SERINC5 antagonism occurred in parallel with other Nef activities, including cell surface receptor downregulation, trans-Golgi network targeting of Lck, and inhibition of host cell actin dynamics. Interaction motifs with host cell endocytic machinery and the Nef-associated kinase complex, as well as CD4 cytoplasmic tail/HIV-1 protease, were identified as essential Nef determinants for SERINC5 antagonism. Characterization of antagonism-deficient Nef mutants revealed that counteraction of SERINC5 occurs in the absence of retargeting of the restriction factor to intracellular compartments and reduction of SERINC5 cell surface density is insufficient for antagonism. Consistent with virion incorporation of SERINC5 being a prerequisite for its antiviral activity, the infectivity of HIV-1 particles produced in the absence of a SERINC5 antagonist decreased with increasing amounts of virion SERINC5. At low levels of SERINC5 expression, enhancement of virion infectivity by Nef was associated with reduced virion incorporation of SERINC5 and antagonism-defective Nef mutants failed to exclude SERINC5 from virions. However, at elevated levels of SERINC5 expression, Nef maintained infectious HIV particles, despite significant virion incorporation of the restriction factor. These results suggest that in addition to virion exclusion, Nef employs a cryptic mechanism to antagonize virion-associated SERINC5. The involvement of common determinants suggests that the antagonism of Nef to SERINC5 and the downregulation of cell surface CD4 by Nef involve related molecular mechanisms. IMPORTANCE: HIV-1 Nef critically determines virus spread and disease progression in infected individuals by incompletely defined mechanisms. SERINC3 and SERINC5 were recently identified as potent inhibitors of HIV particle infectivity whose antiviral activity is antagonized by HIV-1 Nef. To address the mechanism of SERINC5 antagonism, we identified four molecular determinants of Nef antagonism that are all linked to the mechanism by which Nef downregulates cell surface CD4. Functional characterization of these mutants revealed that endosomal targeting and cell surface downregulation of SERINC5 are dispensable and insufficient for antagonism, respectively. In contrast, virion exclusion and antagonism of SERINC5 were correlated; however, Nef was also able to enhance the infectivity of virions that incorporated robust levels of SERINC5. These results suggest that the antagonism of HIV-1 Nef to SERINC5 restriction of virion infectivity is mediated by a dual mechanism that is related to CD4 downregulation.


Asunto(s)
VIH-1/fisiología , VIH-1/patogenicidad , Proteínas de la Membrana/fisiología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/fisiología , Antígenos CD4/metabolismo , Regulación hacia Abajo , Genes Virales , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/genética , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Interacciones Huésped-Patógeno/fisiología , Humanos , Proteínas de la Membrana/genética , Mutación , Virión/genética , Virión/fisiología , Virulencia/genética , Virulencia/fisiología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética
16.
Virology ; 498: 44-56, 2016 11.
Artículo en Inglés | MEDLINE | ID: mdl-27560372

RESUMEN

The HIV-1 pathogenesis factor Nef interacts with numerous ligands to affect cellular vesicular transport, signal transduction and cytoskeletal dynamics. While most Nef functions depend on multivalent protein interaction motifs, disrupting actin dynamics requires a motif that specifically recruits the host kinase PAK2. An adjacent aspartate was recently predicted to mediate Nef-ß-catenin interactions. We report here that ß-catenin can be co-immunoprecipitated with Nef.GFP from Jurkat T cell lysates. This association is conserved among lentiviral Nef proteins but does not involve classical Nef protein interaction motifs, including the critical aspartate. While aspartate-to-alanine mutations impaired cell surface receptor downregulation and interference with actin dynamics and cell motility by HIV-1 NA7 Nef, analogous mutations did not affect HIV-1 SF2 Nef function. These allelic differences were determined by a proximal lysine/arginine polymorphism. These results emphasize differences between Nef alleles regarding the functional role of individual residues and underscore the need for allele-specific structure-function analyses.


Asunto(s)
Alelos , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/genética , Polimorfismo de Nucleótido Simple , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Línea Celular , Humanos , Modelos Moleculares , Mutación , Posición Específica de Matrices de Puntuación , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Receptores de Superficie Celular/metabolismo , beta Catenina/química , beta Catenina/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/química , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Quinasas p21 Activadas/química , Quinasas p21 Activadas/metabolismo
17.
FEBS Lett ; 590(13): 2014-26, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-26938770

RESUMEN

Intravital multiphoton imaging allows visualization of infections and pathogenic mechanisms within intact organs in their physiological context. Today, most organs of mice and rats are applicable to in vivo or ex vivo imaging, opening completely new avenues for many researchers. Advances in fluorescent labeling of pathogens and infected cells, as well as improved small animal models for human pathogens, led to the increased application of in vivo imaging in infectious diseases research in recent years. Here, we review the latest literature on intravital or ex vivo imaging of viral and bacterial infections and critically discuss requirements, benefits and drawbacks of applied animal models, labeling strategies, and imaged organs.


Asunto(s)
Infecciones Bacterianas/patología , Microscopía Intravital/métodos , Animales , Modelos Animales de Enfermedad , Humanos , Especificidad de Órganos , Coloración y Etiquetado
18.
Nat Commun ; 6: 8292, 2015 Sep 22.
Artículo en Inglés | MEDLINE | ID: mdl-26392213

RESUMEN

The overall composition of the mammalian intestinal microbiota varies between individuals: within each individual there are differences along the length of the intestinal tract related to host nutrition, intestinal motility and secretions. Mucus is a highly regenerative protective lubricant glycoprotein sheet secreted by host intestinal goblet cells; the inner mucus layer is nearly sterile. Here we show that the outer mucus of the large intestine forms a unique microbial niche with distinct communities, including bacteria without specialized mucolytic capability. Bacterial species present in the mucus show differential proliferation and resource utilization compared with the same species in the intestinal lumen, with high recovery of bioavailable iron and consumption of epithelial-derived carbon sources according to their genome-encoded metabolic repertoire. Functional competition for existence in this intimate layer is likely to be a major determinant of microbiota composition and microbial molecular exchange with the host.


Asunto(s)
Bacterias/clasificación , Bacterias/aislamiento & purificación , Mucosa Intestinal/microbiología , Animales , Bacterias/genética , Regulación Bacteriana de la Expresión Génica , Vida Libre de Gérmenes , Hierro/metabolismo , Ratones , Ratones Endogámicos C57BL , Familia de Multigenes , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Transcriptoma
19.
J Virol ; 88(24): 14241-57, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25275127

RESUMEN

UNLABELLED: HIV-1 Nef and Vpu are thought to optimize virus replication in the infected host, at least in part via their ability to interfere with vesicular host cell trafficking. Despite the use of distinct molecular mechanisms, Nef and Vpu share specificity for some molecules such as CD4 and major histocompatibility complex class I (MHC-I), while disruption of intracellular transport of the host cell restriction factor CD317/tetherin represents a specialized activity of Vpu not exerted by HIV-1 Nef. To establish a profile of host cell receptors whose intracellular transport is affected by Nef, Vpu, or both, we comprehensively analyzed the effect of these accessory viral proteins on cell surface receptor levels on A3.01 T lymphocytes. Thirty-six out of 105 detectable receptors were significantly downregulated by HIV-1 Nef, revealing a previously unappreciated scope with which HIV-1 Nef remodels the cell surface of infected cells. Remarkably, the effects of HIV-1 Vpu on host cell receptor exposure largely matched those of HIV-1 Nef in breadth and specificity (32 of 105, all also targeted by Nef), even though the magnitude was generally less pronounced. Of particular note, cell surface exposure of all members of the tetraspanin (TSPAN) protein family analyzed was reduced by both Nef and Vpu, and the viral proteins triggered the enrichment of TSPANs in a perinuclear area of the cell. While Vpu displayed significant colocalization and physical association with TSPANs, interactions of Nef with TSPANs were less robust. TSPANs thus emerge as a major target of deregulation in host cell vesicular transport by HIV-1 Nef and Vpu. The conservation of this activity in two independent accessory proteins suggests its importance for the spread of HIV-1 in the infected host. IMPORTANCE: In this paper, we define that HIV-1 Nef and Vpu display a surprising functional overlap and affect the cell surface exposure of a previously unexpected breadth of cellular receptors. Our analyses furthermore identify the tetraspanin protein family as a previously unrecognized target of Nef and Vpu activity. These findings have implications for the interpretation of effects detected for these accessory gene products on individual host cell receptors and illustrate the coevolution of Nef and Vpu function.


Asunto(s)
VIH-1/fisiología , Interacciones Huésped-Patógeno , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Receptores de Superficie Celular/biosíntesis , Tetraspaninas/biosíntesis , Proteínas Reguladoras y Accesorias Virales/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Línea Celular , Humanos , Linfocitos T/química , Linfocitos T/virología
20.
Proc Natl Acad Sci U S A ; 109(45): 18541-6, 2012 Nov 06.
Artículo en Inglés | MEDLINE | ID: mdl-23093676

RESUMEN

HIV-1 negative factor (Nef) elevates virus replication and contributes to immune evasion in vivo. As one of its established in vitro activities, Nef interferes with T-lymphocyte chemotaxis by reducing host cell actin dynamics. To explore Nef's influence on in vivo recirculation of T lymphocytes, we assessed lymph-node homing of Nef-expressing primary murine lymphocytes and found a drastic impairment in homing to peripheral lymph nodes. Intravital imaging and 3D immunofluorescence reconstruction of lymph nodes revealed that Nef potently impaired T-lymphocyte extravasation through high endothelial venules and reduced subsequent parenchymal motility. Ex vivo analyses of transendothelial migration revealed that Nef disrupted T-lymphocyte polarization and interfered with diapedesis and migration in the narrow subendothelial space. Consistently, Nef specifically affected T-lymphocyte motility modes used in dense environments that pose high physical barriers to migration. Mechanistically, inhibition of lymph node homing, subendothelial migration and cell polarization, but not diapedesis, depended on Nef's ability to inhibit host cell actin remodeling. Nef-mediated interference with in vivo recirculation of T lymphocytes may compromise T-cell help and thus represents an important mechanism for its function as a HIV pathogenicity factor.


Asunto(s)
Movimiento Celular/inmunología , Microambiente Celular/inmunología , VIH-1/metabolismo , Linfocitos T/citología , Linfocitos T/virología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Animales , Separación Celular , Células Cultivadas , Endotelio Vascular/metabolismo , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos C57BL , Porosidad , Estrés Mecánico , Transducción Genética , Migración Transendotelial y Transepitelial , Vénulas/metabolismo
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