RESUMEN
The major barrier to an HIV cure is the HIV reservoir: latently-infected cells that persist despite effective antiretroviral therapy (ART). There have been few cohort-based studies evaluating host genomic or transcriptomic predictors of the HIV reservoir. We performed host RNA sequencing and HIV reservoir quantification (total DNA [tDNA], unspliced RNA [usRNA], intact DNA) from peripheral CD4+ T cells from 191 ART-suppressed people with HIV (PWH). After adjusting for nadir CD4+ count, timing of ART initiation, and genetic ancestry, we identified two host genes for which higher expression was significantly associated with smaller total DNA viral reservoir size, P3H3 and NBL1, both known tumor suppressor genes. We then identified 17 host genes for which lower expression was associated with higher residual transcription (HIV usRNA). These included novel associations with membrane channel (KCNJ2, GJB2), inflammasome (IL1A, CSF3, TNFAIP5, TNFAIP6, TNFAIP9, CXCL3, CXCL10), and innate immunity (TLR7) genes (FDR-adjusted q<0.05). Gene set enrichment analyses further identified significant associations of HIV usRNA with TLR4/microbial translocation (q = 0.006), IL-1/NRLP3 inflammasome (q = 0.008), and IL-10 (q = 0.037) signaling. Protein validation assays using ELISA and multiplex cytokine assays supported these observed inverse host gene correlations, with P3H3, IL-10, and TNF-α protein associations achieving statistical significance (p<0.05). Plasma IL-10 was also significantly inversely associated with HIV DNA (p = 0.016). HIV intact DNA was not associated with differential host gene expression, although this may have been due to a large number of undetectable values in our study. To our knowledge, this is the largest host transcriptomic study of the HIV reservoir. Our findings suggest that host gene expression may vary in response to the transcriptionally active reservoir and that changes in cellular proliferation genes may influence the size of the HIV reservoir. These findings add important data to the limited host genetic HIV reservoir studies to date.
Asunto(s)
Infecciones por VIH , VIH-1 , Humanos , Interleucina-10 , Inflamasomas , VIH-1/genética , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/genética , Linfocitos T CD4-Positivos , Inmunidad Innata/genética , Genes Supresores de Tumor , Expresión Génica , ADN , Carga ViralRESUMEN
Mycoplasma genitalium is a sexually transmitted bacterial pathogen that causes urogenital disease in men and women. M. genitalium infections can persist for months to years and can ascend to the upper reproductive tract in women where it is associated with serious sequelae including pelvic inflammatory disease, tubal factor infertility, and preterm birth. An animal model is needed to understand immune evasion strategies that allow persistence, mechanisms of ascending infection, and factors associated with clearance. In earlier studies, we determined that pig-tailed macaques are susceptible to cervical infection; however, not all primates were successfully infected, persistence varied between animals, and ascension to the upper reproductive tract was not observed after 4 or 8 weeks of follow-up. Building on our previous findings, we refined our inoculation methods to increase infection rates, extended observation to 18 weeks, and comprehensively sampled the upper reproductive tract to detect ascending infection. With these improvements, we established infection in all (3/3) primates inoculated with M. genitalium and demonstrated lower tract persistence for 16 to 18 weeks. Ascension to the upper reproductive tract at endpoint was observed in two out of three primates. All three primates developed serum and local antibodies reacting primarily to the MgpB and MgpC adherence proteins. Elevated genital polymorphonuclear leukocytes (PMNs) and inflammatory cytokines and chemokines, erythema of the ectocervix in one primate, and histologic evidence of vaginitis and endocervicitis in two primates suggest a mild to moderate inflammatory response to infection. This model will be valuable to understand the natural history of M. genitalium infection including mechanisms of persistence, immune evasion, and ascension to the upper reproductive tract.
Asunto(s)
Infecciones por Mycoplasma , Mycoplasma genitalium , Nacimiento Prematuro , Infecciones del Sistema Genital , Animales , Femenino , Humanos , Recién Nacido , Macaca nemestrina , Infecciones por Mycoplasma/microbiologíaRESUMEN
[This corrects the article DOI: 10.3389/fmicb.2020.574054.].
RESUMEN
Zika virus (ZIKV) is transmitted to people by bite of an infected mosquito and by sexual contact. ZIKV infects primary genital epithelial cells, the same cells targeted by herpes simplex virus 2 (HSV-2). HSV-2 seroprevalence is high in areas where ZIKV is endemic, but it is unknown whether HSV-2 increases the risk for ZIKV infection. Here, we found that pre-infecting female genital tract epithelial cells with HSV-2 leads to enhanced binding of ZIKV virions. This effect did not require active replication by HSV-2, implying that the effect results from the immune response to HSV-2 exposure or to viral genes expressed early in the HSV-2 lifecycle. Treating cells with toll-like receptor-3 ligand poly-I:C also lead to enhanced binding by ZIKV, which was inhibited by the JAK-STAT pathway inhibitor ruxolitinib. Blocking or knocking down the well-studied ZIKV receptor AXL did not prevent binding of ZIKV to epithelial cells, nor prevent enhanced binding in the presence of HSV-2 infection. Blocking the α5 integrin receptor did not prevent ZIKV binding to cells either. Overall, our results indicate that ZIKV binding to genital epithelial cells is not mediated entirely by a canonical receptor, but likely occurs through redundant pathways that may involve lectin receptors and glycosaminoglycans. Our studies may pave the way to new interventions that interrupt the synergism between herpes and Zika viruses.
RESUMEN
Sexual Zika virus (ZIKV) transmission from men to women occurs less frequently than the often-detected high viral loads in semen would suggest, but worries that this transmission route predisposes to fetal damage in pregnant women remain. To better understand sexual ZIKV pathogenesis, we studied the permissiveness of the human female genital tract to infection and the effect of semen on this process. ZIKV replicates in vaginal tissues and primary epithelial cells from the vagina, ectocervix, and endocervix and induces an innate immune response, but also continues to replicate without cytopathic effect. Infection of genital cells and tissues is strongly inhibited by extracellular vesicles (EV) in semen at physiological vesicle-to-virus ratios. Liposomes with the same composition as semen EVs also impair infection, indicating that the EV's lipid fraction, rather than their protein or RNA cargo, is responsible for this anti-viral effect. Thus, EVs in semen potently restrict ZIKV transmission, but the virus propagates well once infection in the recipient mucosa has been established.
RESUMEN
Pharmacological HIV-1 reactivation to reverse latent infection has been extensively studied. However, HIV-1 reactivation also occurs naturally, as evidenced by occasional low-level viremia ("viral blips") during antiretroviral treatment (ART). Clarifying where blips originate from and how they happen could provide clues to stimulate latency reversal more effectively and safely or to prevent viral rebound following ART cessation. We studied HIV-1 reactivation in the female genital tract, a dynamic anatomical target for HIV-1 infection throughout all disease stages. We found that primary endocervical epithelial cells from several women reactivated HIV-1 from latently infected T cells. The endocervical cells' HIV-1 reactivation capacity further increased upon Toll-like receptor 3 stimulation with poly(I·C) double-stranded RNA or infection with herpes simplex virus 2 (HSV-2). Notably, acyclovir did not eliminate HSV-2-induced HIV-1 reactivation. While endocervical epithelial cells secreted large amounts of several cytokines and chemokines, especially tumor necrosis factor alpha (TNF-α), CCL3, CCL4, and CCL20, their HIV-1 reactivation capacity was almost completely blocked by TNF-α neutralization alone. Thus, immunosurveillance activities by columnar epithelial cells in the endocervix can cause endogenous HIV-1 reactivation, which may contribute to viral blips during ART or rebound following ART interruption.IMPORTANCE A reason that there is no universal cure for HIV-1 is that the virus can hide in the genome of infected cells in the form of latent proviral DNA. This hidden provirus is protected from antiviral drugs until it eventually reactivates to produce new virions. It is not well understood where in the body or how this reactivation occurs. We studied HIV-1 reactivation in the female genital tract, which is often the portal of HIV-1 entry and which remains a site of infection throughout the disease. We found that the columnar epithelial cells lining the endocervix, the lower part of the uterus, are particularly effective in reactivating HIV-1 from infected T cells. This activity was enhanced by certain microbial stimuli, including herpes simplex virus 2, and blocked by antibodies against the inflammatory cytokine TNF-α. Avoiding HIV-1 reactivation could be important for maintaining a functional HIV-1 cure when antiviral therapy is stopped.
Asunto(s)
VIH-1/fisiología , Activación Viral/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Aciclovir/farmacología , Antirretrovirales/uso terapéutico , Antivirales/farmacología , Linfocitos T CD4-Positivos/virología , Línea Celular , Cuello del Útero/patología , Células Epiteliales/patología , Femenino , Regulación Viral de la Expresión Génica/efectos de los fármacos , Infecciones por VIH/virología , Seropositividad para VIH/tratamiento farmacológico , VIH-1/patogenicidad , Humanos , Cultivo Primario de Células , Viremia/tratamiento farmacológico , Latencia del Virus/efectos de los fármacos , Replicación Viral/fisiologíaRESUMEN
Polymorphisms in the human leukocyte antigen (HLA) class I genes can cause the rejection of pluripotent stem cell (PSC)-derived products in allogeneic recipients. Disruption of the Beta-2 Microglobulin (B2M) gene eliminates surface expression of all class I molecules, but leaves the cells vulnerable to lysis by natural killer (NK) cells. Here we show that this 'missing-self' response can be prevented by forced expression of minimally polymorphic HLA-E molecules. We use adeno-associated virus (AAV)-mediated gene editing to knock in HLA-E genes at the B2M locus in human PSCs in a manner that confers inducible, regulated, surface expression of HLA-E single-chain dimers (fused to B2M) or trimers (fused to B2M and a peptide antigen), without surface expression of HLA-A, B or C. These HLA-engineered PSCs and their differentiated derivatives are not recognized as allogeneic by CD8+ T cells, do not bind anti-HLA antibodies and are resistant to NK-mediated lysis. Our approach provides a potential source of universal donor cells for applications where the differentiated derivatives lack HLA class II expression.
Asunto(s)
Antígenos HLA/inmunología , Células Asesinas Naturales/inmunología , Células Madre Pluripotentes/inmunología , Trasplantes/inmunología , Animales , Femenino , Rechazo de Injerto/inmunología , Antígenos HLA/química , Antígenos HLA/genética , Humanos , Ratones , Células Madre Pluripotentes/química , Células Madre Pluripotentes/citología , Trasplantes/química , Trasplantes/citologíaRESUMEN
T-cell expression levels of CC chemokine receptor 5 (CCR5) are a critical determinant of HIV/AIDS susceptibility, and manifest wide variations (i) between T-cell subsets and among individuals and (ii) in T-cell activation-induced increases in expression levels. We demonstrate that a unifying mechanism for this variation is differences in constitutive and T-cell activation-induced DNA methylation status of CCR5 cis-regulatory regions (cis-regions). Commencing at an evolutionarily conserved CpG (CpG -41), CCR5 cis-regions manifest lower vs. higher methylation in T cells with higher vs. lower CCR5 levels (memory vs. naïve T cells) and in memory T cells with higher vs. lower CCR5 levels. HIV-related and in vitro induced T-cell activation is associated with demethylation of these cis-regions. CCR5 haplotypes associated with increased vs. decreased gene/surface expression levels and HIV/AIDS susceptibility magnify vs. dampen T-cell activation-associated demethylation. Methylation status of CCR5 intron 2 explains a larger proportion of the variation in CCR5 levels than genotype or T-cell activation. The ancestral, protective CCR5-HHA haplotype bears a polymorphism at CpG -41 that is (i) specific to southern Africa, (ii) abrogates binding of the transcription factor CREB1 to this cis-region, and (iii) exhibits a trend for overrepresentation in persons with reduced susceptibility to HIV and disease progression. Genotypes lacking the CCR5-Δ32 mutation but with hypermethylated cis-regions have CCR5 levels similar to genotypes heterozygous for CCR5-Δ32. In HIV-infected individuals, CCR5 cis-regions remain demethylated, despite restoration of CD4+ counts (≥800 cells per mm(3)) with antiretroviral therapy. Thus, methylation content of CCR5 cis-regions is a central epigenetic determinant of T-cell CCR5 levels, and possibly HIV-related outcomes.
Asunto(s)
Epigénesis Genética , VIH-1/metabolismo , Activación de Linfocitos , Receptores CCR5/metabolismo , Receptores Virales/metabolismo , Linfocitos T/inmunología , Metilación de ADN , Humanos , Receptores CCR5/genéticaRESUMEN
The clinical use of human pluripotent stem cells and their derivatives is limited by the rejection of transplanted cells due to differences in their human leukocyte antigen (HLA) genes. This has led to the proposed use of histocompatible, patient-specific stem cells; however, the preparation of many different stem cell lines for clinical use is a daunting task. Here, we develop two distinct genetic engineering approaches that address this problem. First, we use a combination of gene targeting and mitotic recombination to derive HLA-homozygous embryonic stem cell (ESC) subclones from an HLA-heterozygous parental line. A small bank of HLA-homozygous stem cells with common haplotypes would match a significant proportion of the population. Second, we derive HLA class I-negative cells by targeted disruption of both alleles of the Beta-2 Microglobulin (B2M) gene in ESCs. Mixed leukocyte reactions and peptide-specific HLA-restricted CD8(+) T cell responses were reduced in class I-negative cells that had undergone differentiation in embryoid bodies. These B2M(-/-) ESCs could act as universal donor cells in applications where the transplanted cells do not express HLA class II genes. Both approaches used adeno-associated virus (AAV) vectors for efficient gene targeting in the absence of potentially genotoxic nucleases, and produced pluripotent, transgene-free cell lines.
Asunto(s)
Antígenos HLA/genética , Células Madre Pluripotentes/citología , Alelos , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular , Línea Celular , Células Cultivadas , Dependovirus/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Marcación de Gen , Ingeniería Genética , Vectores Genéticos , Antígenos HLA/metabolismo , Haplotipos , Histocompatibilidad/genética , Homocigoto , Humanos , Células Madre Pluripotentes/metabolismo , Recombinación Genética , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismoRESUMEN
This study was performed to provide insight into the regulatory role of angiotensin II and arterial pressure on the activity of antioxidant enzymes and oxidative stress generation in the hypertensive kidney from an experimental animal model of renovascular hypertension. Aortic coarcted and sham-operated rats received vehicle, losartan or minoxidil in their drinking water. After 7 d of treatment rats were sacrificed; hypertensive kidneys were excised, and the NAD(P)H oxidase subunits expression, TBARS production, glutathione level and the activity of heme oxygenase-1 and classical antioxidant enzymes, were evaluated. Losartan administration significantly reduced oxidative stress generation decreasing NAD(P)H oxidase expression, independently of the drop in arterial pressure. On the other hand, antioxidant enzymes were regulated by arterial pressure and they were not implicated in kidney protection against oxidative damage. Findings here reported strongly suggest that clinical therapeutics with the Ang II type 1 receptor blocker prevents oxidative stress generation and may attenuate the kidney oxidative damage in the renovascular hypertension. We hypothesize that the pathway followed by the Ang II blocker to achieve this renoprotection, though independent of the primary antioxidant enzymatic system, depends on NAD(P)H oxidase downregulation.
Asunto(s)
Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Presión Sanguínea/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Riñón/efectos de los fármacos , Riñón/metabolismo , Losartán/farmacología , NADPH Oxidasas/metabolismo , Animales , Western Blotting , Glutatión/metabolismo , Hemo-Oxigenasa 1/metabolismo , Peróxido de Hidrógeno/metabolismo , Hipertensión Renovascular/tratamiento farmacológico , Técnicas In Vitro , Peroxidación de Lípido/efectos de los fármacos , Masculino , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
Fasciculins are peptides isolated from mamba (Dendroaspis) venoms which exert their toxic action by inhibiting acetylcholinesterase (AChE). They contain a characteristic triple stranded antiparallel beta-sheet formed by residues 22-27, 34-39 and 48-53. A chimeric peptide named Fas-C, encompassing most of these sequences was synthesized using SPPS/Boc-chemistry and characterized chemically, structurally and functionally. Fas-C has two disulfide bridges, formed sequentially using dual cysteine protection. SDS-PAGE patterns, HPLC profiles and MS proved the peptide identity. Circular dichroism indicated the presence of 13.6% and 41.6% of beta-sheet and beta-turn, respectively, comparable to values observed in the native toxin. An inhibitory effect on eel AChE was displayed by the peptide (Ki71.6 +/- 18.3 microM), although not reaching the affinity level of the parent native toxin (Ki 0.3 nM). It is confirmed that the principal binding region of fasciculin to AChE resides within loop II.