RESUMEN
Upon its mucosal transmission, HIV type 1 (HIV-1) rapidly targets genital antigen-presenting Langerhans cells (LCs), which subsequently transfer infectious virus to CD4+ T cells. We previously described an inhibitory neuroimmune cross talk, whereby calcitonin gene-related peptide (CGRP), a neuropeptide secreted by peripheral pain-sensing nociceptor neurons innervating all mucosal epithelia and associating with LCs, strongly inhibits HIV-1 transfer. As nociceptors secret CGRP following the activation of their Ca2+ ion channel transient receptor potential vanilloid 1 (TRPV1), and as we reported that LCs secret low levels of CGRP, we investigated whether LCs express functional TRPV1. We found that human LCs expressed mRNA and protein of TRPV1, which was functional and induced Ca2+ influx following activation with TRPV1 agonists, including capsaicin (CP). The treatment of LCs with TRPV1 agonists also increased CGRP secretion, reaching its anti-HIV-1 inhibitory concentrations. Accordingly, CP pretreatment significantly inhibited LCs-mediated HIV-1 transfer to CD4+ T cells, which was abrogated by both TRPV1 and CGRP receptor antagonists. Like CGRP, CP-induced inhibition of HIV-1 transfer was mediated via increased CCL3 secretion and HIV-1 degradation. CP also inhibited direct CD4+ T cells HIV-1 infection, but in CGRP-independent manners. Finally, pretreatment of inner foreskin tissue explants with CP markedly increased CGRP and CCL3 secretion, and upon subsequent polarized exposure to HIV-1, inhibited an increase in LC-T cell conjugate formation and consequently T cell infection. Our results reveal that TRPV1 activation in human LCs and CD4+ T cells inhibits mucosal HIV-1 infection, via CGRP-dependent/independent mechanisms. Formulations containing TRPV1 agonists, already approved for pain relief, could hence be useful against HIV-1.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina , Infecciones por VIH , Humanos , Péptido Relacionado con Gen de Calcitonina/farmacología , Linfocitos T/metabolismo , Células de Langerhans/metabolismo , Membrana Mucosa/metabolismo , Capsaicina/farmacología , Dolor/metabolismo , Infecciones por VIH/metabolismo , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismoRESUMEN
Herpes simplex virus (HSV) is widespread globally, with both HSV-1 and HSV-2 responsible for genital herpes. During sexual transmission, HSV targets epithelial cells, sensory peripheral pain neurons secreting the mucosal neuropeptide calcitonin gene-related peptide (CGRP), and mucosal immune cells including Langerhans cells (LCs). We previously described a neuro-immune crosstalk, whereby CGRP inhibits LCs-mediated human immunodeficiency virus type 1 (HIV-1) transmission. Herein, to further explore CGRP-mediated anti-viral function, we investigated whether CGRP affects LCs infection with HSV. We found that both HSV-1 and HSV-2 primary isolates productively infect monocyte-derived LCs (MDLCs) and inner foreskin LCs. Moreover, CGRP significantly inhibits infection with both HSV subtypes of MDLCs and langerinhigh, but not langerinlow, inner foreskin LCs. For HSV-1, infection is mediated via the HSV-1-specific entry receptor 3-O sulfated heparan sulfate (3-OS HS) in a pH-depended manner, and CGRP down-regulates 3-OS HS surface expression, as well as abrogates pH dependency. For HSV-2, infection involves langerin-mediated endocytosis in a pH-independent manner, and CGRP up-regulates surface expression of atypical langerin double-trimer oligomers. Our results show that CGRP inhibits mucosal HSV infection by differentially modulating subtype-specific entry receptors and mechanisms in human LCs. CGRP could turn out useful for prevention of LCs-mediated HSV infection and HSV/HIV-1 co-infection.
Asunto(s)
Infecciones por VIH , Herpes Simple , Herpesvirus Humano 1 , Péptido Relacionado con Gen de Calcitonina/metabolismo , Péptido Relacionado con Gen de Calcitonina/farmacología , Infecciones por VIH/metabolismo , Herpesvirus Humano 2 , Humanos , Células de LangerhansRESUMEN
HIV reservoirs in tissues are poorly understood and their establishment largely depends on the nature of tissues that interact with the virus. In this chapter, we will describe in vitro and ex vivo models of human urethral mucosal macrophages used in the investigation of the establishment and maintenance of tissue HIV reservoirs. In addition, we will describe how macrophage latent HIV infection was assessed in these models by reverting a nonproductive state of infection back into a productive state. Consequently, infectious particles are released to the macrophage extracellular milieu and detected by adapted viral outgrowth assays. Altogether, these approaches provide invaluable tools for the investigation on tissue-specific pathways that HIV-1 employs to reach host cells and form reservoirs in the genital mucosa. These models will contribute to the development of an efficient and targeted prophylaxis against HIV and of a HIV cure.
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Infecciones por VIH , Latencia del Virus , Linfocitos T CD4-Positivos , Genitales Masculinos , Humanos , Masculino , Modelos Teóricos , Células MieloidesRESUMEN
Background: The vasodilator neuropeptide calcitonin gene-related peptide (CGRP) plays both detrimental and protective roles in different pathologies. CGRP is also an essential component of the neuro-immune dialogue between nociceptors and mucosal immune cells. We previously discovered that CGRP is endowed with anti-viral activity and strongly inhibits human immunodeficiency virus type 1 (HIV-1) infection, by suppressing Langerhans cells (LCs)-mediated HIV-1 trans-infection in-vitro and mucosal HIV-1 transmission ex-vivo. This inhibition is mediated via activation of the CGRP receptor non-canonical NFκB/STAT4 signaling pathway that induces a variety of cooperative mechanisms. These include CGRP-mediated increase in the expression of the LC-specific pathogen recognition C-type lectin langerin and decrease in LC-T-cell conjugates formation. The clinical utility of CGRP and modalities of CGRP receptor activation, for inhibition of mucosal HIV-1 transmission, remain elusive. Methods: We tested the capacity of CGRP to inhibit HIV-1 infection in-vivo in humanized mice. We further compared the anti-HIV-1 activities of full-length native CGRP, its metabolically stable analogue SAX, and several CGRP peptide fragments containing its binding C-terminal and activating N-terminal regions. These agonists were evaluated for their capacity to inhibit LCs-mediated HIV-1 trans-infection in-vitro and mucosal HIV-1 transmission in human mucosal tissues ex-vivo. Results: A single CGRP intravaginal topical treatment of humanized mice, followed by HIV-1 vaginal challenge, transiently restricts the increase in HIV-1 plasma viral loads but maintains long-lasting higher CD4+ T-cell counts. Similarly to CGRP, SAX inhibits LCs-mediated HIV-1 trans-infection in-vitro, but with lower potency. This inhibition is mediated via CGRP receptor activation, leading to increased expression of both langerin and STAT4 in LCs. In contrast, several N-terminal and N+C-terminal bivalent CGRP peptide fragments fail to increase langerin and STAT4, and accordingly lack anti-HIV-1 activities. Finally, like CGRP, treatment of human inner foreskin tissue explants with SAX, followed by polarized inoculation with cell-associated HIV-1, completely blocks formation of LC-T-cell conjugates and HIV-1 infection of T-cells. Conclusion: Our results show that CGRP receptor activation by full-length CGRP or SAX is required for efficient inhibition of LCs-mediated mucosal HIV-1 transmission. These findings suggest that formulations containing CGRP, SAX and/or their optimized agonists/analogues could be harnessed for HIV-1 prevention.
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Péptido Relacionado con Gen de Calcitonina/farmacología , Infecciones por VIH/prevención & control , Fragmentos de Péptidos/farmacología , Linfocitos T/efectos de los fármacos , Animales , Péptido Relacionado con Gen de Calcitonina/uso terapéutico , Dipéptidos/farmacología , Modelos Animales de Enfermedad , Femenino , Células HEK293 , Infecciones por VIH/diagnóstico , Infecciones por VIH/transmisión , Infecciones por VIH/virología , VIH-1/aislamiento & purificación , VIH-1/patogenicidad , Voluntarios Sanos , Humanos , Ratones , Membrana Mucosa/efectos de los fármacos , Membrana Mucosa/inmunología , Membrana Mucosa/virología , Fragmentos de Péptidos/uso terapéutico , Cultivo Primario de Células , Quinazolinas/farmacología , Linfocitos T/inmunología , Linfocitos T/virología , Técnicas de Cultivo de TejidosRESUMEN
Human immunodeficiency virus type 1 (HIV-1) eradication is prevented by the establishment on infection of cellular HIV-1 reservoirs that are not fully characterized, especially in genital mucosal tissues (the main HIV-1 entry portal on sexual transmission). Here, we show, using penile tissues from HIV-1-infected individuals under suppressive combination antiretroviral therapy, that urethral macrophages contain integrated HIV-1 DNA, RNA, proteins and intact virions in virus-containing compartment-like structures, whereas viral components remain undetectable in urethral T cells. Moreover, urethral cells specifically release replication-competent infectious HIV-1 following reactivation with the macrophage activator lipopolysaccharide, while the T-cell activator phytohaemagglutinin is ineffective. HIV-1 urethral reservoirs localize preferentially in a subset of polarized macrophages that highly expresses the interleukin-1 receptor, CD206 and interleukin-4 receptor, but not CD163. To our knowledge, these results are the first evidence that human urethral tissue macrophages constitute a principal HIV-1 reservoir. Such findings are determinant for therapeutic strategies aimed at HIV-1 eradication.
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Antirretrovirales/administración & dosificación , Reservorios de Enfermedades/virología , Infecciones por VIH/tratamiento farmacológico , VIH-1/fisiología , Macrófagos/virología , Uretra/virología , Adulto , Linfocitos T CD4-Positivos/virología , Femenino , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/genética , VIH-1/aislamiento & purificación , Humanos , Masculino , Persona de Mediana Edad , ARN Viral/genética , Replicación Viral/efectos de los fármacosRESUMEN
HIV-1 mucosal transmission in genital epithelia occurs through infection of Langerhans cells and subsequent transinfection of CD4+ T cells. We previously reported that the vasodilator neuropeptide calcitonin gene-related peptide (CGRP), secreted upon activation of sensory peripheral neurons that innervate all mucosal epithelia, significantly inhibits transinfection. To investigate the association between CGRP and HIV-1 during infection, we evaluated circulating CGRP levels in HIV-1-infected patients. Plasma was obtained from combination antiretroviral therapy (cART)-naive or cART-treated patients with primary/acute (PHI) or chronic (CHI) HIV-1 infection, as well as from individuals who naturally control HIV-1 infection, namely exposed seronegatives (ESNs), elite controllers (ECs), and long-term nonprogressors (LTNPs). CGRP plasma levels were measured using an enzyme immunoassay. Compared with healthy HIV-1-negative controls, CGRP plasma levels significantly decreased in PHI patients and even further in CHI patients, but remained unchanged in ESNs, ECs, and LTNPs. Moreover, CGRP plasma levels were restored to baseline upon cART in both PHI and CHI. Finally, CGRP plasma levels directly correlated with CD4+ T cell counts and inversely with viral loads. Altogether, CGRP could serve as a novel diagnostic plasma biomarker for progression of HIV-1 infection. Moreover, administration of CGRP to cART-naive HIV-1-infected patients, to compensate for CGRP decline, could help controlling on-going HIV-1 infection.
Asunto(s)
Biomarcadores/sangre , Péptido Relacionado con Gen de Calcitonina/sangre , Progresión de la Enfermedad , Infecciones por VIH/diagnóstico , Infecciones por VIH/patología , Plasma/química , Recuento de Linfocito CD4 , Humanos , Técnicas para Inmunoenzimas , Carga ViralRESUMEN
During sexual intercourse, HIV-1 crosses epithelial barriers composing the genital mucosa, a poorly understood feature that requires an HIV-1-infected cell vectoring efficient mucosal HIV-1 entry. Therefore, urethral mucosa comprising a polarized epithelium and a stroma composed of fibroblasts and macrophages were reconstructed in vitro. Using this system, we demonstrate by live imaging that efficient HIV-1 transmission to stromal macrophages depends on cell-mediated transfer of the virus through virological synapses formed between HIV-1-infected CD4+ T cells and the epithelial cell mucosal surface. We visualized HIV-1 translocation through mucosal epithelial cells via transcytosis in regions where virological synapses occurred. In turn, interleukin-13 is secreted and HIV-1 targets macrophages, which develop a latent state of infection reversed by lipopolysaccharide (LPS) activation. The live observation of virological synapse formation reported herein is key in the design of vaccines and antiretroviral therapies aimed at blocking HIV-1 access to cellular reservoirs in genital mucosa.
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Linfocitos T CD4-Positivos/inmunología , VIH-1/fisiología , Imagenología Tridimensional , Sinapsis Inmunológicas/virología , Macrófagos/virología , Adulto , Epitelio/virología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Humanos , Sinapsis Inmunológicas/metabolismo , Macrófagos/patología , Macrófagos/ultraestructura , Masculino , Modelos Biológicos , Membrana Mucosa/virología , Células del Estroma/patología , Células del Estroma/ultraestructura , Células del Estroma/virología , Uretra/patología , Virión/metabolismo , Virión/ultraestructuraRESUMEN
The neuroimmune dialogue between peripheral neurons and Langerhans cells (LCs) within mucosal epithelia protects against incoming pathogens. LCs rapidly internalize human immunodeficiency virus type 1 (HIV-1) upon its sexual transmission and then trans-infect CD4+ T cells. We recently found that the neuropeptide calcitonin gene-related peptide (CGRP), secreted mucosally from peripheral neurons, inhibits LC-mediated HIV-1 trans-infection. In this study, we investigated the mechanism of CGRP-induced inhibition, focusing on HIV-1 degradation in LCs and its interplay with trans-infection. We first show that HIV-1 degradation occurs in endolysosomes in untreated LCs, and functionally blocking such degradation with lysosomotropic agents results in increased trans-infection. We demonstrate that CGRP acts via its cognate receptor and at a viral postentry step to induce faster HIV-1 degradation, but without affecting the kinetics of endolysosomal degradation. We reveal that unexpectedly, CGRP shifts HIV-1 degradation from endolysosomes toward the proteasome, providing the first evidence for functional HIV-1 proteasomal degradation in LCs. Such efficient proteasomal degradation significantly inhibits the first phase of trans-infection, and proteasomal, but not endolysosomal, inhibitors abrogate CGRP-induced inhibition. Together, our results establish that CGRP controls the HIV-1 degradation mode in LCs. The presence of endogenous CGRP within innervated mucosal tissues, especially during the sexual response, to which CGRP contributes, suggests that HIV-1 proteasomal degradation predominates in vivo Hence, proteasomal, rather than endolysosomal, HIV-1 degradation in LCs should be enhanced clinically to effectively restrict HIV-1 trans-infection.IMPORTANCE During sexual transmission, HIV-1 is internalized and degraded in LCs, the resident antigen-presenting cells in mucosal epithelia. Yet during trans-infection, infectious virions escaping degradation are transferred to CD4+ T cells, the principal HIV-1 targets. We previously found that the neuroimmune dialogue between LCs and peripheral neurons, innervating mucosal epithelia, significantly inhibits trans-infection via the action of the secreted neuropeptide CGRP on LCs. In this study, we investigated whether CGRP-induced inhibition of trans-infection is linked to CGRP-controlled HIV-1 degradation in LCs. We show that in untreated LCs, HIV-1 is functionally degraded in endolysosomes. In sharp contrast, we reveal that in CGRP-treated LCs, HIV-1 is diverted toward and degraded via another cytosolic protein degradative pathway, namely, the proteasome. These results establish that CGRP regulates HIV-1 degradation in LCs. As CGRP contributes to the sexual response and present within mucosal epithelia, HIV-1 proteasomal degradation in LCs might predominate in vivo and should be enhanced clinically.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina/metabolismo , VIH-1/fisiología , Células de Langerhans/virología , Complejo de la Endopetidasa Proteasomal/metabolismo , Linfocitos T CD4-Positivos/virología , Péptido Relacionado con Gen de Calcitonina/farmacología , Células Cultivadas , VIH-1/efectos de los fármacos , Humanos , Células de Langerhans/efectos de los fármacos , Células de Langerhans/metabolismo , Leucocitos Mononucleares/virología , Lisosomas/metabolismo , Lisosomas/virología , Membrana Mucosa/metabolismoRESUMEN
The human penis is a main portal of entry for numerous pathogens, and vaccines able to control resulting infections locally are highly desirable. However, in contrast to the gastrointestinal or vaginal mucosa, the penile immune system and mechanisms inducing a penile immune response remain elusive. In this descriptive study, using multiparametric flow cytometry and immunohistochemistry, we characterized mucosal immune cells such as B, T, and natural killer (NK) cells from the urethra, fossa, and glans of human adult penile tissues. We show that memory B lymphocytes and CD138+ plasma cells are detected in all penile compartments. CD4+ and CD8+ T lymphocytes reside in the epithelium and lamina propria of the penile regions and have mostly a resting memory phenotype. All penile regions contain CD56dim NK cells surface expressing the natural cytotoxicity receptor NKp44 and the antibody-dependent cell cytotoxicity receptor CD16. These cells are also able to spontaneously secrete pro- and anti-inflammatory cytokines, such as IL-17 and IL-22. Finally, CCR10 is the main homing receptor detected in these penile cells although, together with CCR3, CCR6, and CCR9, their expression level differs between penile compartments. Unlike antigen-presenting cells which type differ between penile regions as we reported earlier, urethral, fossa, and glans content in immune B, T, and NK cells is comparable. However, median values per each analysis suggest that the glans, containing higher number and more activated NK cells together with higher number of terminally differentiate effector CD8+ T cells, is a superior effector site than the urethra and the fossa. Thus, the human penis is an immunologically active tissue containing the cellular machinery required to induce and produce a specific and effective response against mucosal pathogens. It can therefore be considered as a classic mucosal effector site, a feature that must be taken into account for the elaboration of efficient strategies, including vaccines, against sexually transmitted infections.
RESUMEN
TNFα is a very potent and pleiotropic pro-inflammatory cytokine, essential to the immune system for eradicating cancer and microorganisms, and to the nervous system, for brain development and ongoing function. Yet, excess and/or chronic TNFα secretion causes massive tissue damage in autoimmune, inflammatory and neurological diseases and injuries. Therefore, many patients with autoimmune/inflammatory diseases receive anti-TNFα medications. TNFα is secreted primarily by CD4(+) T cells, macrophages, monocytes, neutrophils and NK cells, mainly after immune stimulation. Yet, the cause for the pathologically high and chronic TNFα secretion is unknown. Can blocking of a particular ion channel in T cells induce by itself TNFα secretion? Such phenomenon was never revealed or even hypothesized. In this interdisciplinary study we discovered that: (1) normal human T cells express Kv1.1 voltage-gated potassium channel mRNA, and the Kv1.1 membrane-anchored protein channel; (2) Kv1.1 is expressed in most CD4(+)CD3(+) helper T cells (mean CD4(+)CD3(+)Kv1.1(+) T cells of 7 healthy subjects: 53.09 ± 22.17 %), but not in CD8(+)CD3(+) cytotoxic T cells (mean CD8(+)CD3(+)Kv1.1(+) T cells: 4.12 ± 3.04 %); (3) electrophysiological whole-cell recordings in normal human T cells revealed Kv currents; (4) Dendrotoxin-K (DTX-K), a highly selective Kv1.1 blocker derived from snake toxin, increases the rate of rise and decay of Kv currents in both resting and activated T cells, without affecting the peak current; (5) DTX-K by itself induces robust TNFα production and secretion by normal human T cells, without elevating IFNγ, IL-4 and IL-10; (6) intact Ca(2+) channels are required for DTX-induced TNFα secretion; (7) selective anti-Kv1.1 antibodies also induce by themselves TNFα secretion; (8) DTX-K activates NFκB in normal human T cells via the unique non-canonical-pathway; (9) injection of Kv1.1-blocked human T cells to SCID mice, causes recruitment of resident mouse cells into the liver, alike reported after TNFα injection into the brain. Based on our discoveries we speculate that abnormally blocked Kv1.1 in T cells (and other immune cells?), due to either anti-Kv1.1 autoimmune antibodies, or Kv1.1-blocking toxins alike DTX-K, or Kv1.1-blocking genetic mutations, may be responsible for the chronic/excessive TNFα in autoimmune/inflammatory diseases. Independently, we also hypothesize that selective block of Kv1.1 in CD4(+) T cells of patients with cancer or chronic infectious diseases could be therapeutic, since it may: a. augment beneficial secretion and delivery of TNFα to the disease-affected sites; b. induce recruitment and extravasation of curative immune cells and factors; c. improve accessibility of drugs to the brain and few peripheral organs thanks to TNFα-induced increased permeability of organ's barriers.
Asunto(s)
Enfermedades Autoinmunes , Linfocitos T CD4-Positivos/metabolismo , Inflamación , Canal de Potasio Kv.1.1/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/metabolismo , Western Blotting , Linfocitos T CD4-Positivos/inmunología , Ensayo de Cambio de Movilidad Electroforética , Femenino , Humanos , Inmunohistoquímica , Inflamación/inmunología , Inflamación/metabolismo , Masculino , Ratones , Ratones SCID , FN-kappa B/inmunología , FN-kappa B/metabolismo , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa , Transducción de Señal/inmunologíaRESUMEN
Glutamate is the most important excitatory neurotransmitter of the nervous system, critically needed for the brain's development and function. Glutamate has also a signaling role in peripheral organs. Herein, we discuss glutamate receptors (GluRs) and glutamate-induced direct effects on human T cells. T cells are the most important cells of the adaptive immune system, crucially needed for eradication of all infectious organisms and cancer. Normal, cancer and autoimmune human T cells express functional ionotropic and metabotropic GluRs. Different GluR subtypes are expressed in different T cell subtypes, and in resting vs. activated T cells. Glutamate by itself, at low physiological 10(-8)M to 10(-5)M concentrations and via its several types of GluRs, activates many key T cell functions in normal human T cells, among them adhesion, migration, proliferation, intracellular Ca(2+) fluxes, outward K(+) currents and more. Glutamate also protects activated T cells from antigen-induced apoptotic cell death. By doing all that, glutamate can improve substantially the function and survival of resting and activated human T cells. Yet, glutamate's direct effects on T cells depend dramatically on its concentration and might be inhibitory at excess pathological 10(-3)M glutamate concentrations. The effects of glutamate on T cells also depend on the specific GluRs types expressed on the target T cells, the T cell's type and subtype, the T cell's resting or activated state, and the presence or absence of other simultaneous stimuli besides glutamate. Glutamate also seems to play an active role in T cell diseases. For example, glutamate at several concentrations induces or enhances significantly very important functions of human T-leukemia and T-lymphoma cells, among them adhesion to the extracellular matrix, migration, in vivo engraftment into solid organs, and the production and secretion of the cancer-associated matrix metalloproteinase MMP-9 and its inducer CD147. Glutamate induces all these effects via activation of GluRs highly expressed in human T-leukemia and T-lymphoma cells. Glutamate also affects T cell-mediated autoimmune diseases. With regards to multiple sclerosis (MS), GluR3 is highly expressed in T cells of MS patients, and upregulated significantly during relapse and when there is neurological evidence of disease activity. Moreover, glutamate or AMPA (10(-8)M to 10(-5)M) enhances the proliferation of autoreactive T cells of MS patients in response to myelin proteins. Thus, glutamate may play an active role in MS. Glutamate and its receptors also seem to be involved in autoimmune rheumatoid arthritis and systemic lupus erythematosus. Finally, T cells can produce and release glutamate that in turn affects other cells, and during the contact between T cells and dendritic cells, the latter cells release glutamate that has potent effects on the T cells. Together, these evidences show that glutamate has very potent effects on normal, and also on cancer and autoimmune pathological T cells. Moreover, these evidences suggest that glutamate and glutamate-receptor agonists might be used for inducing and boosting beneficial T cell functions, for example, T cell activity against cancer and infectious organisms, and that glutamate-receptor antagonists might be used for preventing glutamate-induced activating effects on detrimental autoimmune and cancerous T cells.
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Ácido Glutámico/metabolismo , Receptores de Glutamato/metabolismo , Linfocitos T/fisiología , Animales , Autoinmunidad/fisiología , Humanos , Leucemia/fisiopatología , Linfoma/fisiopatología , Neuronas/fisiología , Neurotransmisores/metabolismoRESUMEN
OBJECTIVE: Anti-GluR3B antibodies (GluR3B Ab's), directed against peptide B/aa372-395 of GluR3 subunit of glutamate/AMPA receptors, are found in â¼35% of epilepsy patients, activate glutamate/AMPA receptors, evoke ion currents, kill neurons and damage the brain. We recently found that GluR3B Ab's also associate with neurological/psychiatric/behavioral abnormalities in epilepsy patients. Here we asked if GluR3B Ab's could be produced in DBA/2J mice, and also modulate seizure threshold and/or cause behavioral/motor impairments in these mice. METHODS: DBA/2J mice were immunized with the GluR3B peptide in Complete Freund's Adjuvant (CFA), or with controls: ovalbumin (OVA), CFA, or phosphate-buffer saline (PBS). GluR3B Ab's and OVA Ab's were tested. Seizures were induced in all mice by the chemoconvulsant pentylenetetrazole (PTZ) at three time points, each time with less PTZ to avoid non-specific death. Behavior was examined in Open-Field, RotaRod and Grip tests. RESULTS: GluR3B Ab's were produced only in GluR3B-immunized mice, while OVA Ab's were produced only in OVA-immunized mice, showing high Ab's specificity. In GluR3B Ab's negative mice, seizure severity scores and percentages of animals developing generalized seizures declined in response to decreasing PTZ doses. In contrast, both parameters remained unchanged/high in the GluR3B Ab's positive mice, showing that these mice were more susceptible to seizures. The seizure scores associated significantly with the GluR3B Ab's levels. GluR3B Ab's positive mice were also more anxious in Open-Field test, fell faster in RotaRod test, and fell more in Grip test, compared to all the control mice. CONCLUSIONS: GluR3B Ab's are produced in DBA/2J mice, facilitate seizures and induce behavioral/motor impairments. This animal model can therefore serve for studying autoimmune epilepsy and abnormal behavior mediated by pathogenic anti-GluR3B Ab's.
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Autoanticuerpos/biosíntesis , Conducta Animal/fisiología , Receptores AMPA/inmunología , Convulsiones/inmunología , Animales , Ratones , Ratones Endogámicos DBA , Convulsiones/metabolismoRESUMEN
Antibodies (Ab's) to glutamate receptors, directed specifically against AMPA receptors subunit 3 peptide B (i.e. GluR3 amino acids 372-395), named GluR3B Ab's, can by themselves activate GluR3-containing glutamate/AMPA receptors, evoke ion currents via the receptor's ion channel, kill neurons and damage the brain. Herein we first tested 14 consecutive epilepsy patients and 10 healthy controls, and found that 7 (50%) patients had GluR3B Ab's. Second, in 71 other consecutive epilepsy patients (20 generalized epilepsy, 51 partial epilepsy) and 49 controls, we found that 17 (24%) patients had GluR3B Ab's, of which 8 had generalized and 9 partial epilepsy. We then studied 41 epilepsy patients: 21 patients with GluR3B Ab's and 20 without such Ab's (pooled of both tests without biased selection), for possible association of GluR3B Ab's with disease severity and/or neurobehavioral/cognitive comorbidities. Of the 21 patients with GluR3B Ab's, 6 had symptomatic, 7 cryptogenic, and 8 idiopathic epilepsy. Of the 20 patients without GluR3B Ab's, 16 had idiopathic etiology, and 4 nonidiopathic epilepsy. We found that among the 21 patients with GluR3B Ab's, 19 patients (90%) had learning problems, 16 (76%) attention problems, and 15 (71%) psychiatric problems. In contrast, among the 20 patients without GluR3B Ab's, only 6 (30%) had learning problems (p<0.0001), 5 (25%) attention problems (p=0.0017), and 2 (10%) psychiatric problems (p<0.0001). These findings suggest either that neurobehavioral abnormalities occur more frequently in epilepsy patients already having GluR3B Ab's, and may be due to them, or that GluR3B Ab's are more frequent in patients already having neurobehavioral abnormalities.
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Anticuerpos/sangre , Trastornos del Conocimiento/sangre , Epilepsia/sangre , Trastornos Mentales/sangre , Receptores AMPA/inmunología , Adolescente , Adulto , Estudios de Casos y Controles , Niño , Preescolar , Trastornos del Conocimiento/complicaciones , Trastornos del Conocimiento/epidemiología , Epilepsia/complicaciones , Epilepsia/epidemiología , Femenino , Humanos , Lactante , Masculino , Trastornos Mentales/complicaciones , Trastornos Mentales/epidemiología , Estudios Retrospectivos , Adulto JovenRESUMEN
Upon its mucosal entry, human immunodeficiency virus type 1 (HIV-1) is internalized by Langerhans cells (LCs) in stratified epithelia and transferred locally to T cells. In such epithelia, LCs are in direct contact with peripheral neurons secreting calcitonin gene-related peptide (CGRP). Although CGRP has immunomodulatory effects on LC functions, its potential influence on the interactions between LCs and HIV-1 is unknown. We show that CGRP acts via its receptor expressed by LCs and interferes with multiple steps of LC-mediated HIV-1 transmission. CGRP increases langerin expression, decreases selected integrins, and activates NF-κB, resulting in decreased HIV-1 intracellular content, limited formation of LC-T cell conjugates, and elevated secretion of the CCR5-binding chemokine CCL3/MIP-1α. These mechanisms cooperate to efficiently inhibit HIV-1 transfer from LCs to T cells and T cell infection. In vivo, HIV-1 infection decreases CGRP plasma levels in both vaginally SHIV-challenged macaques and HIV-1-infected individuals. CGRP plasma levels return to baseline after highly active antiretroviral therapy. Our results reveal a novel path by which a peripheral neuropeptide acts at the molecular and cellular levels to limit mucosal HIV-1 transmission and suggest that CGRP receptor agonists might be used therapeutically against HIV-1.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina/farmacología , VIH-1/efectos de los fármacos , VIH-1/fisiología , Células de Langerhans/virología , Animales , Antígenos CD/metabolismo , Péptido Relacionado con Gen de Calcitonina/sangre , Adhesión Celular/efectos de los fármacos , Quimiocina CCL3/metabolismo , Femenino , Humanos , Células de Langerhans/metabolismo , Lectinas Tipo C/metabolismo , Macaca mulatta/virología , Masculino , Lectinas de Unión a Manosa/metabolismo , Membrana Mucosa/efectos de los fármacos , Membrana Mucosa/virología , Virus de la Inmunodeficiencia de los Simios , Linfocitos T/efectos de los fármacos , Linfocitos T/virología , Replicación Viral/efectos de los fármacosRESUMEN
Male circumcision reduces acquisition of HIV-1 by 60%. Hence, the foreskin is an HIV-1 entry portal during sexual transmission. We recently reported that efficient HIV-1 transmission occurs following 1 h of polarized exposure of the inner, but not outer, foreskin to HIV-1-infected cells, but not to cell-free virus. At this early time point, Langerhans cells (LCs) and T-cells within the inner foreskin epidermis are the first cells targeted by the virus. To gain in-depth insight into the molecular mechanisms governing inner foreskin HIV-1 entry, foreskin explants were inoculated with HIV-1-infeceted cells for 4 h. The chemokine/cytokine milieu secreted by the foreskin tissue, and resulting modifications in density and spatial distribution of T-cells and LCs, were then investigated. Our studies show that in the inner foreskin, inoculation with HIV-1-infected cells induces increased CCL5/RANTES (1.63-fold) and decreased CCL20/MIP-3-alpha (0.62-fold) secretion. Elevated CCL5/RANTES mediates recruitment of T-cells from the dermis into the epidermis, which is blocked by a neutralizing CCL5/RANTES Ab. In parallel, HIV-1-infected cells mediate a bi-phasic modification in the spatial distribution of epidermal LCs: attraction to the apical surface at 1 h, followed by migration back towards the basement membrane later on at 4 h, in correlation with reduced CCL20/MIP-3-alpha at this time point. T-cell recruitment fuels the continuous formation of LC-T-cell conjugates, permitting the transfer of HIV-1 captured by LCs. Together, these results reveal that HIV-1 induces a dynamic process of immune cells relocation in the inner foreskin that is associated with specific chemokines secretion, which favors efficient HIV-1 entry at this site.
Asunto(s)
Quimiocina CCL5/biosíntesis , Prepucio/virología , Infecciones por VIH/inmunología , VIH-1/fisiología , Células de Langerhans/inmunología , Linfocitos T/inmunología , Internalización del Virus , Comunicación Celular/inmunología , Comunicación Celular/fisiología , Movimiento Celular , Células Cultivadas , Quimiocina CCL20/biosíntesis , Quimiocina CCL5/inmunología , Circuncisión Masculina , Prepucio/inmunología , Infecciones por VIH/transmisión , Infecciones por VIH/virología , Humanos , Células de Langerhans/metabolismo , Células de Langerhans/virología , Activación de Linfocitos , Masculino , Linfocitos T/metabolismo , Linfocitos T/virologíaRESUMEN
Human immunodeficiency virus (HIV)-1 is mainly transmitted mucosally during sexual intercourse. We therefore evaluated the protective efficacy of a vaccine active at mucosal sites. Macaca mulatta monkeys were immunized via both the intramuscular and intranasal routes with an HIV-1 vaccine made of gp41-subunit antigens grafted on virosomes, a safe delivery carrier approved in humans with self-adjuvant properties. Six months after 13 vaginal challenges with simian-HIV (SHIV)-SF162P3, four out of five vaccinated animals remained virus-negative, and the fifth was only transiently infected. None of the five animals seroconverted to p27gag-SIV. In contrast, all 6 placebo-vaccinated animals became infected and seroconverted. All protected animals showed gp41-specific vaginal IgAs with HIV-1 transcytosis-blocking properties and vaginal IgGs with neutralizing and/or antibody-dependent cellular-cytotoxicity activities. In contrast, plasma IgGs totally lacked virus-neutralizing activity. The protection observed challenges the paradigm whereby circulating antiviral antibodies are required for protection against HIV-1 infection and may serve in designing a human vaccine against HIV-1-AIDS.
Asunto(s)
Vacunas contra el SIDA/administración & dosificación , Anticuerpos Anti-VIH/biosíntesis , Proteína gp41 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Inmunización , Macaca mulatta/inmunología , Fragmentos de Péptidos/inmunología , Vagina/inmunología , Virosomas/inmunología , Vacunas contra el SIDA/inmunología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Citotoxicidad Celular Dependiente de Anticuerpos , Sitios de Unión , Femenino , Anticuerpos Anti-VIH/inmunología , Proteína gp41 de Envoltorio del VIH/administración & dosificación , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , Infecciones por VIH/transmisión , Seropositividad para VIH , Datos de Secuencia Molecular , Fragmentos de Péptidos/administración & dosificación , Transcitosis , Viremia/inmunología , Viremia/prevención & control , Viremia/transmisión , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/análisisRESUMEN
HIV-1 is mainly a sexually transmitted infection, and epithelial surfaces covering genital mucosa are the primary site of HIV-1 transmission. Although male circumcision was reported to reduce male acquisition of HIV-1 by 60%, the initial mechanisms of HIV-1 transmission in the male genitals remain elusive. We established two novel models of the adult human foreskin epithelium that allowed for polarized infection via the mucosal pole with either HIV-1-infected cells that are present in all secretions vectorizing HIV-1 or cell-free HIV-1. Efficient HIV-1 transmission occurs following 1 hr of polarized exposure of the inner, but not outer, foreskin to mononuclear cells highly infected with HIV-1, but not to cell-free virus. HIV-1-infected cells form viral synapses with apical foreskin keratinocytes, leading to polarized budding of HIV-1, which is rapidly internalized by Langerhans cells (LCs) in the inner foreskin. In turn, LCs form conjugates with T-cells, thereby transferring HIV-1. Seminal plasma from HIV-negative men mixed with cervico-vaginal secretions from HIV-positive women, which mimics the in-vivo mixture of these genital fluids during woman-to-man HIV-1 sexual transmission, decreases HIV-1 infection at the foreskin. Our results rationalize at the cellular level the apparent protective outcome of circumcision against HIV-1 acquisition by men.
Asunto(s)
Prepucio/anatomía & histología , Prepucio/virología , Genitales Masculinos/virología , Infecciones por VIH/transmisión , Infecciones por VIH/virología , VIH-1/fisiología , Células Epiteliales/virología , Femenino , Humanos , Queratinocitos/virología , Células de Langerhans/virología , Masculino , Membrana Mucosa/anatomía & histología , Membrana Mucosa/virología , Semen , Vagina/metabolismoRESUMEN
Glutamate is the major excitatory neurotransmitter of the nervous system. We previously found that glutamate activates normal human T-cells, inducing their adhesion and chemotaxis, via its glutamate receptors of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype 3 (GluR3) expressed in these cells. Here, we discovered that human T-leukemia (Jurkat) and cutaneous sezary T-lymphoma (HuT-78) cells also express high levels of GluR3. Furthermore, glutamate (10 nM) elevates CD147/EMMPRIN, a cancer-associated matrix metalloproteinases (MMPs) inducer, promoting spread of many tumors. Glutamate-induced CD147 elevation in both cancerous and normal human T-cells was mimicked by AMPA (glutamate/AMPA-receptor agonist) and blocked by CNQX (glutamate/AMPA-receptor antagonist). Importantly, glutamate also increased gelatinase MMP-9 secretion by T-lymphoma. Finally, ex vivo pre-treatment of T-leukemia with glutamate enhanced their subsequent in vivo engraftment into chick embryo liver and chorioallantoic membrane. Together, these findings reveal that glutamate elevates cancer associated proteins and activity in T-cell cancers and by doing so may facilitate their growth and spread, especially to and within the nervous system. If so, glutamate receptors in T-cell malignancies should be blocked.
Asunto(s)
Basigina/metabolismo , Ácido Glutámico/farmacología , Metaloproteinasa 9 de la Matriz/metabolismo , Receptores AMPA/metabolismo , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Animales , Línea Celular Tumoral , Células Cultivadas , Embrión de Pollo , Membrana Corioalantoides/metabolismo , Membrana Corioalantoides/cirugía , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Supervivencia de Injerto/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Células Jurkat , Leucemia de Células T/genética , Leucemia de Células T/metabolismo , Leucemia de Células T/patología , Trasplante de Neoplasias/métodos , Reacción en Cadena de la Polimerasa , Trasplante HeterólogoRESUMEN
Glutamate is the major excitatory CNS neurotransmitter. Glutamate receptor autoantibodies have now been called to our attention, as they are found in many patients with epilepsy, systemic lupus erythematosus (SLE) and encephalitis, and can unquestionably cause brain damage. AMPA GluR3 autoantibodies have been found thus far in 27% of patients with different epilepsies, while NMDA NR2A or NR2B autoantibodies, some of which cross-react with double-stranded DNA, have been detected in 30% of SLE patients, with or without neuropsychiatric impairments. NR2 autoantibodies were also found in patients with epilepsy (33%), encephalitis and stroke. NR2 and GluR3 autoantibodies do not cross-react in patients with epilepsy. Human and animal studies show that both types of glutamate receptor autoantibodies can certainly damage the brain. GluR3 autoantibodies bind to neurons, possess a unique ability to activate their glutamate-receptor antigen, and cause neuronal death (either by excitotoxicity or by complement fixation independent of receptor activation), multiple brain damage and neurobehavioral/cognitive impairments. In animal models (mice, rats or rabbits) GluR3 autoantibodies may cause seizures, augment their severity or modulate their threshold. NR2/dsDNA autoantibodies, once present in the CNS, can bind and subsequently kill hippocampal and cortical neurons by an excitotoxic complement-independent mechanism. Herein, we discuss epilepsy, autoimmune epilepsy, SLE and neuropsychiatric SLE in general; summarize the up-to-date in vivo and in vitro evidence concerning the presence of glutamate receptor autoantibodies in human diseases; discuss the activity and pathogenicity of different glutamate receptor autoantibodies; and end with our conclusions, recommendations and suggested future directions.
Asunto(s)
Autoanticuerpos/sangre , Lesiones Encefálicas/etiología , Encefalitis , Epilepsia/complicaciones , Lupus Eritematoso Sistémico , Receptores de Glutamato/inmunología , Animales , Encefalitis/sangre , Encefalitis/complicaciones , Encefalitis/inmunología , Epilepsia/sangre , Epilepsia/inmunología , Humanos , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/complicaciones , Lupus Eritematoso Sistémico/inmunologíaRESUMEN
OBJECTIVE: The early steps of HIV entry into intact vaginal mucosa still need to be clarified. Here we investigated how HIV translocated across the vaginal pluristratified epithelium, either by transcytosis or by uptake in Langerhans cells. METHODS: Using human primary fibroblasts and vaginal epithelial cells, we developed an in-vitro model of vaginal mucosa in which Langerhans cells could also be integrated. Owing to the absence of T lymphocytes and macrophages, we specifically studied the role of Langerhans cells in HIV transmission and the transcytosis of cell-associated HIV. RESULTS: Our model has a normal mucosal tissue architecture and Langerhans cells were efficiently integrated within the pluristratified epithelium. In addition, tight junction proteins' expression, high transepithelium resistance and low fluorescein isothiocyanate-BSA passage confirmed the integrity and impermeability of the reconstruction. Furthermore, we showed that human Langerhans cells also expressed tight junction proteins. Then, we demonstrated that neither transcellular nor intercellular transport of free infectious virus released by R5-infected or X4-infected peripheral blood mononuclear cells inoculated apically occured in the vaginal mucosa, irrespective to the presence of Langerhans cells. CONCLUSION: For the first time, we documented that, within 4 h following contact with HIV-infected cells, translocation of free HIV particles across a pluristratified mucosa is not detectable and that, in this context, it seemed that Langerhans cells do not increase HIV transmission. Moreover, we provided a useful model for the development of strategies preventing HIV entry into the female genital tract, especially for testing the efficiency of various microbicides.