RESUMEN
A systematic quantitative analysis of temporal changes in host and viral proteins throughout the course of a productive infection could provide dynamic insights into virus-host interaction. We developed a proteomic technique called "quantitative temporal viromics" (QTV), which employs multiplexed tandem-mass-tag-based mass spectrometry. Human cytomegalovirus (HCMV) is not only an important pathogen but a paradigm of viral immune evasion. QTV detailed how HCMV orchestrates the expression of >8,000 cellular proteins, including 1,200 cell-surface proteins to manipulate signaling pathways and counterintrinsic, innate, and adaptive immune defenses. QTV predicted natural killer and T cell ligands, as well as 29 viral proteins present at the cell surface, potential therapeutic targets. Temporal profiles of >80% of HCMV canonical genes and 14 noncanonical HCMV open reading frames were defined. QTV is a powerful method that can yield important insights into viral infection and is applicable to any virus with a robust in vitro model.
Asunto(s)
Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/virología , Citomegalovirus/fisiología , Interacciones Huésped-Patógeno , Proteómica , Virología/métodos , Humanos , Evasión Inmune , Células Asesinas Naturales/inmunología , Transducción de Señal , Linfocitos T/inmunología , Proteínas Virales/análisisRESUMEN
Human cytomegalovirus (HCMV) is a paradigm of pathogen immune evasion and sustains lifelong persistent infection in the face of exceptionally powerful host immune responses through the concerted action of multiple immune-evasins. These reduce NK cell activation by inhibiting ligands for activating receptors, expressing ligands for inhibitory receptors, or inhibiting synapse formation. However, these functions only inhibit direct interactions with the infected cell. To determine whether the virus also expresses soluble factors that could modulate NK function at a distance, we systematically screened all 170 HCMV canonical protein-coding genes. This revealed that UL4 encodes a secreted and heavily glycosylated protein (gpUL4) that is expressed with late-phase kinetics and is capable of inhibiting NK cell degranulation. Analyses of gpUL4 binding partners by mass spectrometry identified an interaction with TRAIL. gpUL4 bound TRAIL with picomolar affinity and prevented TRAIL from binding its receptor, thus acting as a TRAIL decoy receptor. TRAIL is found in both soluble and membrane-bound forms, with expression of the membrane-bound form strongly up-regulated on NK cells in response to interferon. gpUL4 inhibited apoptosis induced by soluble TRAIL, while also binding to the NK cell surface in a TRAIL-dependent manner, where it blocked NK cell degranulation and cytokine secretion. gpUL4 therefore acts as an immune-evasin by inhibiting both soluble and membrane-bound TRAIL and is a viral-encoded TRAIL decoy receptor. Interestingly, gpUL4 could also suppress NK responses to heterologous viruses, suggesting that it may act as a systemic virally encoded immunosuppressive agent.
Asunto(s)
Citomegalovirus , Células Asesinas Naturales , Humanos , Citomegalovirus/fisiología , Evasión Inmune , Glicoproteínas/metabolismo , ApoptosisRESUMEN
Human cytomegalovirus (HCMV) is a major human pathogen whose life-long persistence is enabled by its remarkable capacity to systematically subvert host immune defenses. In exploring the finding that HCMV infection up-regulates tumor necrosis factor receptor 2 (TNFR2), a ligand for the pro-inflammatory antiviral cytokine TNFα, we found that the underlying mechanism was due to targeting of the protease, A Disintegrin And Metalloproteinase 17 (ADAM17). ADAM17 is the prototype 'sheddase', a family of proteases that cleaves other membrane-bound proteins to release biologically active ectodomains into the supernatant. HCMV impaired ADAM17 surface expression through the action of two virally-encoded proteins in its UL/b' region, UL148 and UL148D. Proteomic plasma membrane profiling of cells infected with an HCMV double-deletion mutant for UL148 and UL148D with restored ADAM17 expression, combined with ADAM17 functional blockade, showed that HCMV stabilized the surface expression of 114 proteins (P < 0.05) in an ADAM17-dependent fashion. These included reported substrates of ADAM17 with established immunological functions such as TNFR2 and jagged1, but also numerous unreported host and viral targets, such as nectin1, UL8, and UL144. Regulation of TNFα-induced cytokine responses and NK inhibition during HCMV infection were dependent on this impairment of ADAM17. We therefore identify a viral immunoregulatory mechanism in which targeting a single sheddase enables broad regulation of multiple critical surface receptors, revealing a paradigm for viral-encoded immunomodulation.
Asunto(s)
Citomegalovirus , Factor de Necrosis Tumoral alfa , Humanos , Citomegalovirus/fisiología , Factor de Necrosis Tumoral alfa/metabolismo , Proteoma/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Proteómica , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Citocinas/metabolismo , Membrana Celular/metabolismo , Metaloproteasas/metabolismo , Proteína ADAM17/genética , Proteína ADAM17/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Virales/metabolismoRESUMEN
Human cytomegalovirus (HCMV) is an important human pathogen and a paradigm of viral immune evasion, targeting intrinsic, innate, and adaptive immunity. We have employed two orthogonal multiplexed tandem mass tag-based proteomic screens to identify host proteins down-regulated by viral factors expressed during the latest phases of viral infection. This approach revealed that the HIV-1 restriction factor Schlafen-11 (SLFN11) was degraded by the poorly characterized, late-expressed HCMV protein RL1, via recruitment of the Cullin4-RING E3 Ubiquitin Ligase (CRL4) complex. SLFN11 potently restricted HCMV infection, inhibiting the formation and spread of viral plaques. Overall, we show that a restriction factor previously thought only to inhibit RNA viruses additionally restricts HCMV. We define the mechanism of viral antagonism and also describe an important resource for revealing additional molecules of importance in antiviral innate immunity and viral immune evasion.
Asunto(s)
Infecciones por Citomegalovirus/inmunología , Citomegalovirus/inmunología , Evasión Inmune , Proteínas Nucleares/inmunología , Proteolisis , Proteínas del Envoltorio Viral/inmunología , Citomegalovirus/genética , Infecciones por Citomegalovirus/genética , Humanos , Proteínas Nucleares/genética , Complejos de Ubiquitina-Proteína Ligasa/genética , Complejos de Ubiquitina-Proteína Ligasa/inmunología , Proteínas del Envoltorio Viral/genéticaRESUMEN
The human cytomegalovirus (HCMV) pUS2 glycoprotein exploits the host's endoplasmic reticulum (ER)-associated degradation (ERAD) pathway to degrade major histocompatibility complex class I (MHC-I) and prevent antigen presentation. Beyond MHC-I, pUS2 has been shown to target a range of cellular proteins for degradation, preventing their cell surface expression. Here we have identified a novel pUS2 target, ER-resident protein lectin mannose binding 2 like (LMAN2L). pUS2 expression was both necessary and sufficient for the downregulation of LMAN2L, which was dependent on the cellular E3 ligase TRC8. Given the hypothesized role of LMAN2L in the trafficking of glycoproteins, we employed proteomic plasma membrane profiling to measure LMAN2L-dependent changes at the cell surface. A known pUS2 target, integrin alpha-6 (ITGA6), was downregulated from the surface of LMAN2L-deficient cells, but not other integrins. Overall, these results suggest a novel strategy of pUS2-mediated protein degradation whereby pUS2 targets LMAN2L to impair trafficking of ITGA6. Given that pUS2 can directly target other integrins, we propose that this single viral protein may exhibit both direct and indirect mechanisms to downregulate key cell surface molecules.
Asunto(s)
Citomegalovirus , Retículo Endoplásmico , Proteínas del Envoltorio Viral , Proteínas Virales , Humanos , Citomegalovirus/genética , Citomegalovirus/metabolismo , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/virología , Proteínas Virales/metabolismo , Proteínas Virales/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteolisis , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Lectinas de Unión a Manosa/metabolismo , Lectinas de Unión a Manosa/genética , Degradación Asociada con el Retículo Endoplásmico , Interacciones Huésped-Patógeno , Membrana Celular/metabolismo , Membrana Celular/virologíaRESUMEN
Fibrosis in response to tissue damage or persistent inflammation is a pathological hallmark of many chronic degenerative diseases. By using a model of acute peritoneal inflammation, we have examined how repeated inflammatory activation promotes fibrotic tissue injury. In this context, fibrosis was strictly dependent on interleukin-6 (IL-6). Repeat inflammation induced IL-6-mediated T helper 1 (Th1) cell effector commitment and the emergence of STAT1 (signal transducer and activator of transcription-1) activity within the peritoneal membrane. Fibrosis was not observed in mice lacking interferon-γ (IFN-γ), STAT1, or RAG-1. Here, IFN-γ and STAT1 signaling disrupted the turnover of extracellular matrix by metalloproteases. Whereas IL-6-deficient mice resisted fibrosis, transfer of polarized Th1 cells or inhibition of MMP activity reversed this outcome. Thus, IL-6 causes compromised tissue repair by shifting acute inflammation into a more chronic profibrotic state through induction of Th1 cell responses as a consequence of recurrent inflammation.
Asunto(s)
Interleucina-6/metabolismo , Peritoneo/patología , Peritonitis/genética , Peritonitis/patología , Células TH1/inmunología , Enfermedad Aguda , Traslado Adoptivo , Animales , Células Cultivadas , Enfermedad Crónica , Modelos Animales de Enfermedad , Matriz Extracelular/inmunología , Retroalimentación Fisiológica , Fibrosis , Humanos , Interferón gamma/genética , Interferón gamma/metabolismo , Interleucina-6/genética , Interleucina-6/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Transducción de Señal , Células TH1/trasplanteRESUMEN
The heterodimeric cytokine interleukin 27 (IL-27) signals through the IL-27Rα subunit of its receptor, combined with gp130, a common receptor chain used by several cytokines, including IL-6. Notably, the IL-27 subunits p28 (IL-27p28) and EBI3 are not always expressed together, which suggests that they may have unique functions. Here we show that IL-27p28, independently of EBI3, antagonized cytokine signaling through gp130 and IL-6-mediated production of IL-17 and IL-10. Similarly, the ability to generate antibody responses was dependent on the activity of gp130-signaling cytokines. Mice transgenic for expression of IL-27p28 showed a substantial defect in the formation of germinal centers and antibody production. Thus, IL-27p28, as a natural antagonist of gp130-mediated signaling, may be useful as a therapeutic for managing inflammation mediated by cytokines that signal through gp130.
Asunto(s)
Receptor gp130 de Citocinas/metabolismo , Interleucinas/metabolismo , Transducción de Señal/inmunología , Animales , Formación de Anticuerpos/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Separación Celular , Receptor gp130 de Citocinas/inmunología , Citocinas/inmunología , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Inmunohistoquímica , Interleucinas/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Antígenos de Histocompatibilidad Menor , Receptores de Citocinas/inmunología , Receptores de Citocinas/metabolismoRESUMEN
CD58 is an adhesion molecule that is known to play a critical role in costimulation of effector cells and is intrinsic to immune synapse structure. Herein, we describe a virally encoded gene that inhibits CD58 surface expression. Human cytomegalovirus (HCMV) UL148 was necessary and sufficient to promote intracellular retention of CD58 during HCMV infection. Blocking studies with antagonistic anti-CD58 mAb and an HCMV UL148 deletion mutant (HCMV∆UL148) with restored CD58 expression demonstrated that the CD2/CD58 axis was essential for the recognition of HCMV-infected targets by CD8+ HCMV-specific cytotoxic T lymphocytes (CTLs). Further, challenge of peripheral blood mononuclear cells ex vivo with HCMV∆UL148 increased both CTL and natural killer (NK) cell degranulation against HCMV-infected cells, including NK-driven antibody-dependent cellular cytotoxicity, showing that UL148 is a modulator of the function of multiple effector cell subsets. Our data stress the effect of HCMV immune evasion functions on shaping the immune response, highlighting the capacity for their potential use in modulating immunity during the development of anti-HCMV vaccines and HCMV-based vaccine vectors.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Infecciones por Citomegalovirus/inmunología , Citomegalovirus/inmunología , Evasión Inmune , Inmunidad Celular , Células Asesinas Naturales/inmunología , Proteínas Virales de Fusión/inmunología , Linfocitos T CD8-positivos/patología , Línea Celular Transformada , Citomegalovirus/genética , Infecciones por Citomegalovirus/genética , Infecciones por Citomegalovirus/patología , Humanos , Células Asesinas Naturales/patología , Proteínas Virales de Fusión/genéticaRESUMEN
Molluscum contagiosum virus (MCV) is a common cause of benign skin lesions in young children and currently the only endemic human poxvirus. Following the infection of primary keratinocytes in the epidermis, MCV induces the proliferation of infected cells and this results in the production of wart-like growths. Full productive infection is observed only after the infected cells differentiate. During this prolonged replication cycle the virus must avoid elimination by the host immune system. We therefore sought to investigate the function of the two major histocompatibility complex class-I-related genes encoded by the MCV genes mc033 and mc080. Following insertion into a replication-deficient adenovirus vector, codon-optimized versions of mc033 and mc080 were expressed as endoglycosidase-sensitive glycoproteins that localized primarily in the endoplasmic reticulum. MC080, but not MC033, downregulated cell-surface expression of endogenous classical human leucocyte antigen (HLA) class I and non-classical HLA-E by a transporter associated with antigen processing (TAP)-independent mechanism. MC080 exhibited a capacity to inhibit or activate NK cells in autologous assays in a donor-specific manner. MC080 consistently inhibited antigen-specific T cells being activated by peptide-pulsed targets. We therefore propose that MC080 acts to promote evasion of HLA-I-restricted cytotoxic T cells.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Regulación hacia Abajo/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Evasión Inmune/inmunología , Células Asesinas Naturales/inmunología , Virus del Molusco Contagioso/inmunología , Presentación de Antígeno/inmunología , Línea Celular , Retículo Endoplásmico/inmunología , Interacciones Huésped-Patógeno/inmunología , Humanos , Queratinocitos/inmunología , Linfocitos T Citotóxicos/inmunología , Proteínas Virales/inmunologíaRESUMEN
Vascular endothelial growth factor (VEGF) is implicated in the peritoneal membrane remodeling that limits ultrafiltration in patients on peritoneal dialysis (PD). Although the exact mechanism of VEGF induction in PD is unclear, VEGF concentrations in drained dialysate correlate with IL-6 levels, suggesting a link between these cytokines. Human peritoneal mesothelial cells (HPMCs), the main source of IL-6 and VEGF in the peritoneum, do not bear the cognate IL-6 receptor and are thus unable to respond to classic IL-6 receptor signaling. Here, we investigated whether VEGF release by HPMCs is controlled by IL-6 in combination with its soluble receptor (IL-6 trans-signaling). Although treatment with either IL-6 or soluble IL-6 receptor (sIL-6R) alone had no effect on VEGF production, stimulation of HPMCs with IL-6 in combination with sIL-6R promoted VEGF expression and secretion through a transcriptional mechanism involving STAT3 and SP4. Conditioned medium from HPMCs cultured with IL-6 and sIL-6R promoted angiogenic endothelial tube formation, which could be blocked by silencing SP4. In vivo, induction of peritoneal inflammation in wild-type and IL-6-deficient mice showed IL-6 involvement in the control of Sp4 and Vegf expression and new vessel formation, confirming the role of IL-6 trans-signaling in these processes. Taken together, these findings identify a novel mechanism linking IL-6 trans-signaling and angiogenesis in the peritoneal membrane.
Asunto(s)
Interleucina-6/fisiología , Neovascularización Patológica , Peritoneo/irrigación sanguínea , Peritonitis/etiología , Receptores de Interleucina-6/fisiología , Transducción de Señal , Animales , Ratones , Factor A de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
UNLABELLED: Clinical human cytomegalovirus (HCMV) strains invariably mutate when propagatedin vitro Mutations in gene RL13 are selected in all cell types, whereas in fibroblasts mutants in the UL128 locus (UL128L; genes UL128, UL130, and UL131A) are also selected. In addition, sporadic mutations are selected elsewhere in the genome in all cell types. We sought to investigate conditions under which HCMV can be propagated without incurring genetic defects. Bacterial artificial chromosomes (BACs) provide a stable, genetically defined source of viral genome. Viruses were generated from BACs containing the genomes of strains TR, TB40, FIX, and Merlin, as well as from Merlin-BAC recombinants containing variant nucleotides in UL128L from TB40-BAC4 or FIX-BAC. Propagation of viruses derived from TR-BAC, TB40-BAC4, and FIX-BAC in either fibroblast or epithelial cells was associated with the generation of defects around the prokaryotic vector, which is retained in the unique short (US) region of viruses. This was not observed for Merlin-BAC, from which the vector is excised in derived viruses; however, propagation in epithelial cells was consistently associated with mutations in the unique longb' (UL/b') region, all impacting on gene UL141. Viruses derived from Merlin-BAC in fibroblasts had mutations in UL128L, but mutations occurred less frequently with recombinants containing UL128L nucleotides from TB40-BAC4 or FIX-BAC. Viruses derived from a Merlin-BAC derivative in which RL13 and UL128L were either mutated or repressed were remarkably stable in fibroblasts. Thus, HCMV containing a wild-type gene complement can be generatedin vitroby deriving virus from a self-excising BAC in fibroblasts and repressing RL13 and UL128L. IMPORTANCE: Researchers should aim to study viruses that accurately represent the causative agents of disease. This is problematic for HCMV because clinical strains mutate rapidly when propagatedin vitro, becoming less cell associated, altered in tropism, more susceptible to natural killer cells, and less pathogenic. Following isolation from clinical material, HCMV genomes can be stabilized by cloning into bacterial artificial chromosomes (BACs), and then virus is regenerated by DNA transfection. However, mutations can occur not only during isolation prior to BAC cloning but also when virus is regenerated. We have identified conditions under which BAC-derived viruses containing an intact, wild-type genome can be propagatedin vitrowith minimal risk of mutants being selected, enabling studies of viruses expressing the gene complement of a clinical strain. However, even under these optimized conditions, sporadic mutations can occur, highlighting the advisability of sequencing the HCMV stocks used in experiments.
Asunto(s)
Cromosomas Artificiales Bacterianos , Citomegalovirus/crecimiento & desarrollo , Cultivo de Virus/métodos , Línea Celular , Citomegalovirus/genética , Células Epiteliales , Fibroblastos , Genes Virales , Genoma Viral , Inestabilidad Genómica , Humanos , Técnicas In Vitro , Glicoproteínas de Membrana/genética , Proteínas del Envoltorio Viral/genéticaRESUMEN
NKG2D plays a major role in controlling immune responses through the regulation of natural killer (NK) cells, αß and γδ T-cell function. This activating receptor recognizes eight distinct ligands (the MHC Class I polypeptide-related sequences (MIC) A andB, and UL16-binding proteins (ULBP)1-6) induced by cellular stress to promote recognition cells perturbed by malignant transformation or microbial infection. Studies into human cytomegalovirus (HCMV) have aided both the identification and characterization of NKG2D ligands (NKG2DLs). HCMV immediate early (IE) gene up regulates NKGDLs, and we now describe the differential activation of ULBP2 and MICA/B by IE1 and IE2 respectively. Despite activation by IE functions, HCMV effectively suppressed cell surface expression of NKGDLs through both the early and late phases of infection. The immune evasion functions UL16, UL142, and microRNA(miR)-UL112 are known to target NKG2DLs. While infection with a UL16 deletion mutant caused the expected increase in MICB and ULBP2 cell surface expression, deletion of UL142 did not have a similar impact on its target, MICA. We therefore performed a systematic screen of the viral genome to search of addition functions that targeted MICA. US18 and US20 were identified as novel NK cell evasion functions capable of acting independently to promote MICA degradation by lysosomal degradation. The most dramatic effect on MICA expression was achieved when US18 and US20 acted in concert. US18 and US20 are the first members of the US12 gene family to have been assigned a function. The US12 family has 10 members encoded sequentially through US12-US21; a genetic arrangement, which is suggestive of an 'accordion' expansion of an ancestral gene in response to a selective pressure. This expansion must have be an ancient event as the whole family is conserved across simian cytomegaloviruses from old world monkeys. The evolutionary benefit bestowed by the combinatorial effect of US18 and US20 on MICA may have contributed to sustaining the US12 gene family.
Asunto(s)
Citomegalovirus , Antígenos de Histocompatibilidad Clase I/metabolismo , Evasión Inmune , Células Asesinas Naturales/inmunología , Lisosomas/metabolismo , Proteolisis , Proteínas Virales/fisiología , Adulto , Proteínas Bacterianas/metabolismo , Células Cultivadas , Citomegalovirus/inmunología , Citomegalovirus/patogenicidad , Inhibidores Enzimáticos/farmacología , Humanos , Evasión Inmune/efectos de los fármacos , Células Asesinas Naturales/efectos de los fármacos , Leupeptinas/farmacología , Proteínas Luminiscentes/metabolismo , Lisosomas/efectos de los fármacos , Macrólidos/farmacología , Subfamilia K de Receptores Similares a Lectina de Células NK/fisiología , Proteolisis/efectos de los fármacos , Proteínas Recombinantes/metabolismoRESUMEN
In celebrating the 60th anniversary of the first isolation of human cytomegalovirus (HCMV), we reflect on the merits and limitations of the viral strains currently being used to develop urgently needed treatments. HCMV research has been dependent for decades on the high-passage strains AD169 and Towne, heavily exploiting their capacity to replicate efficiently in fibroblasts. However, the genetic integrity of these strains is so severely compromised that great caution needs to be exercised when considering their past and future use. It is now evident that wild-type HCMV strains are not readily propagated in vitro. HCMV mutants are rapidly selected during isolation in fibroblasts, reproducibly affecting gene RL13, the UL128 locus (which includes genes UL128, UL130 and UL131A) and often the U(L)/b' region. As a result, the virus becomes less cell associated, altered in tropism and less pathogenic. This problem is not restricted to high-passage strains, as even low-passage strains can harbour biologically significant mutations. Cloning and manipulation of the HCMV genome as a bacterial artificial chromosome (BAC) offers a means of working with stable, genetically defined strains. To this end, the low-passage strain Merlin genome was cloned as a BAC and sequentially repaired to match the viral sequence in the original clinical sample from which Merlin was derived. Restoration of UL128L to wild type was detrimental to growth in fibroblasts, whereas restoration of RL13 impaired growth in all cell types tested. Stable propagation of phenotypically wild-type virus could be achieved only by placing both regions under conditional expression. In addition to the development of these tools, the Merlin transcriptome and proteome have been characterized in unparalleled detail. Although Merlin may be representative of the clinical agent, high-throughput whole-genome deep sequencing studies have highlighted the remarkable high level of interstrain variation present in circulating virus. There is a need to develop systems capable of addressing the significance of this diversity, free from the confounding effects of genetic changes associated with in vitro adaptation. The generation of a set of BAC clones, each containing the genome of a different HCMV strain repaired to match the sequence in the clinical sample, would provide a pathway to address the biological and clinical effects of natural variation in wild-type HCMV.
Asunto(s)
Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/virología , Citomegalovirus/fisiología , Animales , Citomegalovirus/clasificación , Evolución Molecular , Regulación Viral de la Expresión Génica , Genes Virales , Variación Genética , Genoma Viral , Humanos , Mutación , Selección Genética , Biología de SistemasRESUMEN
Interleukin (IL)-6 has become a major target for clinical intervention in various autoimmune conditions. Here, drugs including the humanized anti-IL-6 receptor (IL-6R) antibody tocilizumab emphasize the clinical importance of IL-6 in driving disease and poor patient outcomes. During the course of this review, we will outline the biology surrounding IL-6 and discuss the impact of IL-6 in renal disease and the clinical complications associated with renal replacement therapies and transplantation. We will also consider the merit of IL-6 measurement as a prognostic indicator and provide a clinical perspective on IL-6-blocking therapies in renal disease.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Interleucina-6/antagonistas & inhibidores , Enfermedades Renales/tratamiento farmacológico , Receptores de Interleucina-6/antagonistas & inhibidores , Humanos , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , PronósticoRESUMEN
Human cytomegalovirus (HCMV) is known to evade extrinsic pro-apoptotic pathways not only by downregulating cell surface expression of the death receptors TNFR1, TRAIL receptor 1 (TNFRSF10A) and TRAIL receptor 2 (TNFRSF10B), but also by impeding downstream signalling events. Fas (CD95/APO-1/TNFRSF6) also plays a prominent role in apoptotic clearance of virus-infected cells, so its fate in HCMV-infected cells needs to be addressed. Here, we show that cell surface expression of Fas was suppressed in HCMV-infected fibroblasts from 24 h onwards through the late phase of productive infection, and was dependent on de novo virus-encoded gene expression but not virus DNA replication. Significant levels of the fully glycosylated (endoglycosidase-H-resistant) Fas were retained within HCMV-infected cells throughout the infection within intracellular membranous structures. HCMV infection provided cells with a high level of protection against Fas-mediated apoptosis. Downregulation of Fas was observed with HCMV strains AD169, FIX, Merlin and TB40.
Asunto(s)
Citomegalovirus/fisiología , Interacciones Huésped-Patógeno , Evasión Inmune , Receptor fas/antagonistas & inhibidores , Receptor fas/inmunología , Células Cultivadas , Fibroblastos/virología , HumanosRESUMEN
Balancing the generation of immune responses capable of controlling virus replication with those causing immunopathology is critical for the survival of the host and resolution of influenza-induced inflammation. Based on the capacity of interleukin-6 (IL-6) to govern both optimal T-cell responses and inflammatory resolution, we hypothesised that IL-6 plays an important role in maintaining this balance. Comparison of innate and adaptive immune responses in influenza-infected wild-type control and IL-6-deficient mice revealed striking differences in virus clearance, lung immunopathology and generation of heterosubtypic immunity. Mice lacking IL-6 displayed a profound defect in their ability to mount an anti-viral T-cell response. Failure to adequately control virus was further associated with an enhanced infiltration of inflammatory monocytes into the lung and an elevated production of the pro-inflammatory cytokines, IFN-α and TNF-α. These events were associated with severe lung damage, characterised by profound vascular leakage and death. Our data highlight an essential role for IL-6 in orchestrating anti-viral immunity through an ability to limit inflammation, promote protective adaptive immune responses and prevent fatal immunopathology.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Virus de la Influenza A/fisiología , Interleucina-6/inmunología , Infecciones por Orthomyxoviridae/inmunología , Neumonía Viral/inmunología , Animales , Linfocitos T CD4-Positivos/virología , Movimiento Celular/genética , Células Cultivadas , Citocinas/metabolismo , Femenino , Mediadores de Inflamación/metabolismo , Interleucina-6/genética , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infecciones por Orthomyxoviridae/genética , Neumonía Viral/genética , Neumonía Viral/patología , Carga Viral/genética , Replicación Viral/genéticaRESUMEN
Human cytomegalovirus (HCMV) is an important human pathogen that regulates host immunity and hijacks host compartments, including lysosomes, to assemble virions. We combined a quantitative proteomic analysis of HCMV infection with a database of proteins involved in vacuolar acidification, revealing Dmx-like protein-1 (DMXL1) as the only protein that acidifies vacuoles yet is degraded by HCMV. Systematic comparison of viral deletion mutants reveals the uncharacterized 7 kDa US33A protein as necessary and sufficient for DMXL1 degradation, which occurs via recruitment of the E3 ubiquitin ligase Kip1 ubiquitination-promoting complex (KPC). US33A-mediated DMXL1 degradation inhibits lysosome acidification and autophagic cargo degradation. Formation of the virion assembly compartment, which requires lysosomes, occurs significantly later with US33A-expressing virus infection, with reduced viral replication. These data thus identify a viral strategy for cellular remodeling, with the potential to employ US33A in therapies for viral infection or rheumatic conditions, in which inhibition of lysosome acidification can attenuate disease.
Asunto(s)
Citomegalovirus , Proteómica , Humanos , Citomegalovirus/fisiología , Ensamble de Virus , Replicación Viral , Proteínas , Autofagia , Lisosomas , Concentración de Iones de HidrógenoRESUMEN
The ability to control HCV with IFN-α-based treatments provides an opportunity in humans to study how the rate of viral clearance in vivo impinges on the development of antiviral responses. Ex vivo (IFN-γ-producing) and cultured antiviral CD4(+) T cells, serum cytokines, and viral loads were measured repeatedly in a cohort of chronically HCV-infected subjects (n = 33) receiving IFN-α. Rapid control of virus indicated by an increased calculated rate of virus clearance, occurred in those subjects demonstrating absent/minimal T-cell responses (p < 0.0006). Surprisingly, in subjects who demonstrated the most robust T-cell responses (and reduced serum IL-10), there was actually a reduced rate of early virus clearance. A subsequent analysis of NK-cell function in available subjects (n = 8) revealed an inverse correlation between pretreatment NK-cell expression of NKp46 and the potential to upregulate cytotoxic function on exposure to IFN-α (p < 0.004), as well as the subsequent measured rate of viral clearance (p = 0.045). Thus, the CD4(+) T-cell response during IFN-α treatment appears to be shaped by the rate of innate virus suppression. These data suggest that individuals who respond most effectively to immune intervention may be most in need of subsequent vaccination to prevent reinfection.
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Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Hepacivirus/inmunología , Hepatitis C Crónica/tratamiento farmacológico , Hepatitis C Crónica/inmunología , Interferón-alfa/inmunología , Interferón-alfa/uso terapéutico , Adulto , Anciano , Alanina Transaminasa/inmunología , Procesos de Crecimiento Celular/efectos de los fármacos , Estudios de Cohortes , Femenino , Hepatitis C Crónica/virología , Humanos , Interleucina-10/sangre , Interleucina-10/inmunología , Células Asesinas Naturales/inmunología , Masculino , Persona de Mediana Edad , Receptor 1 Gatillante de la Citotoxidad Natural/inmunología , Resultado del Tratamiento , Carga Viral/inmunologíaRESUMEN
Introduction: The antigen presentation molecule MHC class I related protein-1 (MR1) is best characterized by its ability to present bacterially derived metabolites of vitamin B2 biosynthesis to mucosal-associated invariant T-cells (MAIT cells). Methods: Through in vitro human cytomegalovirus (HCMV) infection in the presence of MR1 ligand we investigate the modulation of MR1 expression. Using coimmunoprecipitation, mass spectrometry, expression by recombinant adenovirus and HCMV deletion mutants we investigate HCMV gpUS9 and its family members as potential regulators of MR1 expression. The functional consequences of MR1 modulation by HCMV infection are explored in coculture activation assays with either Jurkat cells engineered to express the MAIT cell TCR or primary MAIT cells. MR1 dependence in these activation assays is established by addition of MR1 neutralizing antibody and CRISPR/Cas-9 mediated MR1 knockout. Results: Here we demonstrate that HCMV infection efficiently suppresses MR1 surface expression and reduces total MR1 protein levels. Expression of the viral glycoprotein gpUS9 in isolation could reduce both cell surface and total MR1 levels, with analysis of a specific US9 HCMV deletion mutant suggesting that the virus can target MR1 using multiple mechanisms. Functional assays with primary MAIT cells demonstrated the ability of HCMV infection to inhibit bacterially driven, MR1-dependent activation using both neutralizing antibodies and engineered MR1 knockout cells. Discussion: This study identifies a strategy encoded by HCMV to disrupt the MR1:MAIT cell axis. This immune axis is less well characterized in the context of viral infection. HCMV encodes hundreds of proteins, some of which regulate the expression of antigen presentation molecules. However the ability of this virus to regulate the MR1:MAIT TCR axis has not been studied in detail.
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Células T Invariantes Asociadas a Mucosa , Humanos , Antígenos de Histocompatibilidad Clase I , Citomegalovirus/metabolismo , Antígenos de Histocompatibilidad Menor , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
Antibodies capable of neutralizing SARS-CoV-2 are well studied, but Fc receptor-dependent antibody activities that can also significantly impact the course of infection have not been studied in such depth. Since most SARS-CoV-2 vaccines induce only anti-spike antibodies, here we investigated spike-specific antibody-dependent cellular cytotoxicity (ADCC). Vaccination produced antibodies that weakly induced ADCC; however, antibodies from individuals who were infected prior to vaccination (hybrid immunity) elicited strong anti-spike ADCC. Quantitative and qualitative aspects of humoral immunity contributed to this capability, with infection skewing IgG antibody production toward S2, vaccination skewing toward S1, and hybrid immunity evoking strong responses against both domains. A combination of antibodies targeting both spike domains support strong antibody-dependent NK cell activation, with 3 regions of antibody reactivity outside the receptor-binding domain (RBD) corresponding with potent anti-spike ADCC. Consequently, ADCC induced by hybrid immunity with ancestral antigen was conserved against variants containing neutralization escape mutations in the RBD. Induction of antibodies recognizing a broad range of spike epitopes and eliciting strong and durable ADCC may partially explain why hybrid immunity provides superior protection against infection and disease compared with vaccination alone, and it demonstrates that spike-only subunit vaccines would benefit from strategies that induce combined anti-S1 and anti-S2 antibody responses.