ABSTRACT
Proinflammatory cytokine production following infection with severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) is associated with poor clinical outcomes. Like SARS CoV-1, SARS CoV-2 enters host cells via its spike protein, which attaches to angiotensin-converting enzyme 2 (ACE2). As SARS CoV-1 spike protein is reported to induce cytokine production, we hypothesized that this pathway could be a shared mechanism underlying pathogenic immune responses. We herein compared the capabilities of Middle East Respiratory Syndrome (MERS), SARS CoV-1 and SARS CoV-2 spike proteins to induce cytokine expression in human peripheral blood mononuclear cells (PBMC). We observed that only specific commercial lots of SARS CoV-2 induce cytokine production. Surprisingly, recombinant SARS CoV-2 spike proteins from different vendors and batches exhibited different patterns of cytokine induction, and these activities were not inhibited by blockade of spike protein-ACE2 binding using either soluble ACE2 or neutralizing anti-S1 antibody. Moreover, commercial spike protein reagents contained varying levels of lipopolysaccharide (LPS), which correlated directly with their abilities to induce cytokine production. The LPS inhibitor, polymyxin B, blocked this cytokine induction activity. In addition, SARS CoV-2 spike protein avidly bound soluble LPS in vitro, rendering it a cytokine inducer. These results not only suggest caution in monitoring the purity of SARS CoV-2 spike protein reagents, but they indicate the possibility that interactions of SARS CoV-2 spike protein with LPS from commensal bacteria in virally infected mucosal tissues could promote pathogenic inflammatory cytokine production.
Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Cytokines/metabolism , Leukocytes, Mononuclear/metabolism , Lipopolysaccharides/pharmacology , Models, Biological , Spike Glycoprotein, Coronavirus/pharmacology , Healthy Volunteers , Humans , In Vitro Techniques , Leukocytes, Mononuclear/drug effectsABSTRACT
Optical biosensors based on scattered-light measurements are being developed for rapid and label-free detection of single virions captured from body fluids. Highly controlled, stable, and non-biohazardous reference materials producing virus-like signals are valuable tools to calibrate, evaluate, and refine the performance of these new optical biosensing methods. To date, spherical polymer nanoparticles have been the only non-biological reference materials employed with scattered-light biosensing techniques. However, pathogens like filoviruses, including the Ebola virus, are far from spherical and their shape strongly affects scattered-light signals. Using electron beam lithography, we fabricated nanostructures resembling individual filamentous virions attached to a biosensing substrate (silicon wafer overlaid with silicon oxide film) and characterized their dimensions with scanning electron and atomic force microscopes. To assess the relevance of these nanostructures, we compared their signals across the visible spectrum to signals recorded from Ebola virus-like particles which exhibit characteristic filamentous morphology. We demonstrate the highly stable nature of our nanostructures and use them to obtain new insights into the relationship between virion dimensions and scattered-light signal.
Subject(s)
Biosensing Techniques , Ebolavirus/isolation & purification , Nanostructures/chemistry , Body Fluids/virology , Ebolavirus/pathogenicity , Electrons , Filoviridae/isolation & purification , Filoviridae/pathogenicity , Hemorrhagic Fever, Ebola/diagnosis , Hemorrhagic Fever, Ebola/virology , Humans , Microscopy, Atomic Force , Nanotechnology/methods , Polymers/chemistryABSTRACT
We have elucidated a putative mechanism for the host resistance against HIV-1 infection of primary human monocyte-derived macrophages (MDM) stimulated with lipopolysaccharide (LPS). We show that LPS-activated MDM both inhibited HIV-1 entry into the cells and were refractory to post-entry productive viral replication. LPS-treated cells were virtually negative for mature virions as revealed by transmission electron microscopy. LPS activation of MDM markedly enhanced the expression of heme oxygenase-1 (HO-1), a potent inducible cytoprotective enzyme. Increased HO-1 expression was accompanied by elevated production of macrophage inflammatory chemokines (MIP1α and MIP1ß) by LPS-activated MDM, significantly decreased surface chemokine receptor-5 (CCR-5) expression, and substantially reduced virus replication. Treatment of cells with HO-1 inhibitor SnPP IX (tin protoporphyrin IX) attenuated the LPS-mediated responses, HIV-1 replication and secretion of MIP1α, MIP1ß, and LD78ß chemokines with little change in surface CCR-5 expression. These results identify a novel role for HO-1 in the modulation of host immune response against HIV infection of MDM.
Subject(s)
Chemokines/metabolism , HIV Infections/immunology , HIV Infections/prevention & control , HIV-1/pathogenicity , Heme Oxygenase-1/biosynthesis , Lipopolysaccharides/pharmacology , Macrophage Activation/immunology , Receptors, Chemokine/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/biosynthesis , Chemokines/biosynthesis , HIV Infections/enzymology , HIV-1/immunology , Heme Oxygenase-1/physiology , Host-Pathogen Interactions/immunology , Humans , Macrophages/enzymology , Macrophages/immunology , Macrophages/virologyABSTRACT
Ebolavirus is a hemorrhagic fever virus associated with high mortality. Although much has been learned about the viral lifecycle and pathogenesis, many questions remain about virus entry. We recently showed that binding of the receptor binding region (RBR) of the ebolavirus glycoprotein (GP) and infection by GP pseudovirions increase on cell adhesion independently of mRNA or protein synthesis. One model to explain these observations is that, on cell adhesion, an RBR binding partner translocates from an intracellular vesicle to the cell surface. Here, we provide evidence for this model by showing that suspension 293F cells contain an RBR binding site within a membrane-bound compartment associated with the trans-Golgi network and microtubule-organizing center. Consistently, trafficking of the RBR binding partner to the cell surface depends on microtubules, and the RBR binding partner is internalized when adherent cells are placed in suspension. Based on these observations, we reexamined the claim that lymphocytes, which are critical for ebolavirus pathogenesis, are refractory to infection because they lack an RBR binding partner. We found that both cultured and primary human lymphocytes (in suspension) contain an intracellular pool of an RBR binding partner. Moreover, we identified two adherent primate lymphocytic cell lines that bind RBR at their surface and strikingly, support GP-mediated entry and infection. In summary, our results reveal a mode of determining viral entry by a membrane-trafficking event that translocates an RBR binding partner to the cell surface, and they suggest that this process may be operative in cells important for ebolavirus pathogenesis (e.g., lymphocytes and macrophages).
Subject(s)
Ebolavirus/physiology , Ebolavirus/pathogenicity , Lymphocytes/physiology , Lymphocytes/virology , Viral Envelope Proteins/physiology , Virus Internalization , B-Lymphocytes/physiology , B-Lymphocytes/virology , Binding Sites , Cell Adhesion/physiology , Cell Line , Cell Membrane/physiology , Cell Membrane/virology , Host-Pathogen Interactions/physiology , Humans , In Vitro Techniques , Jurkat Cells , Microtubule-Associated Proteins/physiology , Plant Proteins/physiology , Receptors, Virus/physiology , Viral Envelope Proteins/chemistry , trans-Golgi Network/physiologyABSTRACT
Human immunodeficiency virus type 2 (HIV-2) is known to be less pathogenic than HIV-1. However, the mechanism(s) underlying the decreased HIV-2 pathogenicity is not fully understood. Herein, we report that ß-chemokine CCL2 expression was increased in HIV-1-infected human monocyte-derived macrophages (MDM) but decreased in HIV-2-infected MDM when compared to uninfected MDM. Inhibition of CCL2 expression following HIV-2 infection occurred at both protein and mRNA levels. By microarray analysis, quantitative PCR, and Western blotting, we identified that Signal Transducer and Activator of Transcription 1 (STAT1), a critical transcription factor for inducing CCL2 gene expression, was also reduced in HIV-2-infected MDM. Blockade of STAT1 in HIV-infected MDM using a STAT1 inhibitor significantly reduced the production of CCL2. In contrast, transduction of STAT1-expressing pseudo-retrovirus restored CCL2 production in HIV-2-infected MDM. These findings support the concept that CCL2 inhibition in HIV-2-infected MDM is meditated by reduction of STAT1. Furthermore, we showed that STAT1 reduction in HIV-2-infected MDM was regulated by the CUL2/RBX1 ubiquitin E3 ligase complex-dependent proteasome pathway. Knockdown of CUL2 or RBX1 restored the expression of STAT1 and CCL2 in HIV-2-infected MDM. Taken together, our findings suggest that differential regulation of the STAT1-CCL2 axis may be one of the mechanisms underlying the different pathogenicity observed for HIV-1 and HIV-2.
Subject(s)
Chemokine CCL2 , HIV Infections , HIV-1 , HIV-2 , Humans , Cells, Cultured , Gene Expression Regulation , HIV Seropositivity , HIV-1/genetics , HIV-2/genetics , Macrophages , Virulence , Virus Replication , Chemokine CCL2/metabolism , HIV Infections/metabolism , HIV Infections/virologyABSTRACT
BACKGROUND: The role of disulfide bond remodeling in HIV-1 infection is well described, but the process still remains incompletely characterized. At present, the data have been predominantly obtained using established cell lines and/or CXCR4-tropic laboratory-adapted virus strains. There is also ambiguity about which disulfide isomerases/reductases play a major role in HIV-1 entry, as protein disulfide isomerase (PDI) and/or thioredoxin (Trx) have emerged as the two enzymes most often implicated in this process. RESULTS: We have extended our previous findings and those of others by focusing on CCR5-using HIV-1 strains and their natural targets--primary human macrophages and CD4+ T lymphocytes. We found that the nonspecific thiol/disulfide exchange inhibitor, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), significantly reduced HIV-1 entry and infection in cell lines, human monocyte-derived macrophages (MDM), and also phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC). Subsequent studies were performed using specific anti-PDI or Trx monoclonal antibodies (mAb) in HIV-1 envelope pseudotyped and wild type (wt) virus infection systems. Although human donor-to-donor variability was observed as expected, Trx appeared to play a greater role than PDI in HIV-1 infection of MDM. In contrast, PDI, but not Trx, was predominantly involved in HIV-1 entry and infection of the CD4+/CCR5+ T cell line, PM-1, and PHA-stimulated primary human T lymphocytes. Intriguingly, both PDI and Trx were present on the surface of MDM, PM-1 and PHA-stimulated CD4+ T cells. However, considerably lower levels of Trx were detected on freshly isolated CD4+ lymphocytes, compared to PHA-stimulated cells. CONCLUSIONS: Our findings clearly demonstrate the role of thiol/disulfide exchange in HIV-1 entry in primary T lymphocytes and MDM. They also establish a cell-type specificity regarding the involvement of particular disulfide isomerases/reductases in this process and may provide an explanation for differences among previously published studies. More importantly, from an in vivo perspective, the preferential utilization of PDI may be relevant to the HIV-1 entry and establishment of virus reservoirs in resting CD4+ cells, while the elevated levels of Trx reported in the chronic stages of HIV-1 infection may facilitate the virus entry in macrophages and help to sustain high viremia during the decline of T lymphocytes.
Subject(s)
Disulfides/metabolism , HIV-1/physiology , Protein Disulfide-Isomerases/metabolism , Viral Tropism , Virus Internalization , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , CD4 Antigens/metabolism , Cell Line , Dithionitrobenzoic Acid/pharmacology , HIV-1/drug effects , Humans , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/virology , Macrophages/metabolism , Macrophages/virology , Protein Disulfide-Isomerases/immunology , Receptors, CCR5/metabolism , Viral Tropism/drug effects , Virus Internalization/drug effectsABSTRACT
An increasing number of Investigational New Drug (IND) applications for therapeutic monoclonal antibodies (mAbs) have been submitted to US FDA over the past several years. Monoclonal antibodies and related products are under development for a wide range of indications. In addition, the diversity of antibody-related products is increasing including IgG2/IgG4 subclasses and engineered Fc regions to enhance or reduce antibody effector functionality. Recent findings highlight the need to more fully characterize these products and their activity. Advances in product characterization tools, immunogenicity assessments, and other bioanalytical assays can be used to better understand product performance and facilitate development.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Immunoglobulin Fc Fragments/immunology , Immunoglobulin Isotypes/immunology , Animals , Antibodies, Monoclonal/adverse effects , Cytokines/metabolism , Humans , Immunoglobulin Fc Fragments/therapeutic use , Immunoglobulin Isotypes/therapeutic use , Protein EngineeringABSTRACT
Very little is known about disease transmission via the gut microbiome. We hypothesized that certain inflammatory features could be transmitted via the gut microbiome and tested this hypothesis using an animal model of inflammatory diseases. Twelve-week-old healthy C57 Bl/6 and Germ-Free (GF) female and male mice were fecal matter transplanted (FMT) under anaerobic conditions with TNFΔARE-/+ donors exhibiting spontaneous Rheumatoid Arthritis (RA) and Inflammatory Bowel Disease (IBD) or with conventional healthy mice control donors. The gut microbiome analysis was performed using 16S rRNA sequencing amplification and bioinformatics analysis with the HIVE bioinformatics platform. Histology, immunohistochemistry, ELISA Multiplex analysis, and flow cytometry were conducted to confirm the inflammatory transmission status. We observed RA and IBD features transmitted in the GF mice cohort, with gut tissue disruption, cartilage alteration, elevated inflammatory mediators in the tissues, activation of CD4/CD8+ T cells, and colonization and transmission of the gut microbiome similar to the donors' profile. We did not observe a change or transmission when conventional healthy mice were FMT with TNFΔARE-/+ donors, suggesting that a healthy microbiome might withstand an unhealthy transplant. These findings show the potential involvement of the gut microbiome in inflammatory diseases. We identified a cluster of bacteria playing a role in this mechanism.
ABSTRACT
The particularly unique composition of the gut microbiota has the potential to influence the health or disease status of animal and human hosts. Altering the homeostasis of the host-bacteria could lead to changes in gut flora that result in disease or activation of a specific immunological response, which could explain the variations observed in patient responses to current therapies. A standardized model is crucial for studying the influence of the gut microbiota on therapeutic modalities. A step by step mouse model and sterility management system that compares a control strain of C57BL/6 mice to the established C57BL/6 germ-free (GF) strain has been developed. The GF BL/6 mouse phenotype is well established, and the anatomical differences between the GF and control mice were evident in this model. This method could be applied to research studies investigating the microbiome impact, the response to various therapies, or disease transfer via fecal transplants. A standardized sterility maintenance method is crucial in this context.
ABSTRACT
Macrophages are one of the first and also a major site of filovirus replication and, in addition, are a source of multiple cytokines, presumed to play a critical role in the pathogenesis of the viral infection. Some of these cytokines are known to induce macrophage phenotypic changes in vitro, but how macrophage polarization may affect the cell susceptibility to filovirus entry remains largely unstudied. We generated different macrophage subsets using cytokine pre-treatment and subsequently tested their ability to fuse with beta-lactamase containing virus-like particles (VLP), pseudotyped with the surface glycoprotein of Ebola virus (EBOV) or the glycoproteins of other clinically relevant filovirus species. We found that pre-incubation of primary human monocyte-derived macrophages (MDM) with interleukin-10 (IL-10) significantly enhanced filovirus entry into cells obtained from multiple healthy donors, and the IL-10 effect was preserved in the presence of pro-inflammatory cytokines found to be elevated during EBOV disease. In contrast, fusion of IL-10-treated macrophages with influenza hemagglutinin/neuraminidase pseudotyped VLPs was unchanged or slightly reduced. Importantly, our in vitro data showing enhanced virus entry are consistent with the correlation established between elevated serum IL-10 and increased mortality in filovirus infected patients and also reveal a novel mechanism that may account for the IL-10-mediated increase in filovirus pathogenicity.
Subject(s)
Cytokines/pharmacology , Filoviridae/physiology , Macrophages/drug effects , Virus Internalization/drug effects , Cells, Cultured , Ebolavirus/physiology , Filoviridae Infections/immunology , Filoviridae Infections/virology , Humans , Interleukin-10/pharmacology , Macrophages/virology , Membrane Fusion/drug effects , Viral Envelope Proteins/metabolismABSTRACT
BACKGROUND: Female hormones are known to play an important role in predisposition for many infectious diseases. Recent work suggests there are gender effects in HIV/AIDS progression. Here we ask whether the sex steroid hormone beta-estradiol affects the replication of HIV-1 or the efficacy of a common anti-retroviral drug, Stavudine (D4T). RESULTS: Human PBL were infected with HIV-1 in the presence or absence of combinations of sex steroid hormones and the anti-retroviral drug, D4T. After seven days in culture, viral supernatants were assayed for HIV-1 p24 protein. beta-estradiol resulted in a modest inhibition of HIV-1 replication of approximately 26%. However, 2 nM beta-estradiol increased the amount of HIV-1 replication in the presence of 50 nM D4T from a baseline of 33% (+/- SE = 5.4) to 74% (+/- SE = 5.4) of control virus levels in the absence of drug. Both results were statistically highly significant (p < 0.001). beta-estradiol did not increase the replication of a D4T-resistant strain of HIV in the presence of D4T. The effects were unlikely to be due to general cell inhibition or toxicity because these concentrations of drug and hormone cause no cytotoxicity in PBL as measured by trypan blue exclusion. CONCLUSION: beta-estradiol inhibited both HIV-1 replication in primary human PBL and the antiretroviral efficacy of D4T in PBL cultures. To optimize antiretroviral drug therapy, it may be necessary to monitor patient hormonal status.
Subject(s)
Anti-HIV Agents/pharmacology , Estradiol/pharmacology , Gonadal Steroid Hormones/pharmacology , HIV-1/drug effects , Stavudine/pharmacology , Cells, Cultured , Culture Media/chemistry , Drug Interactions , Female , HIV Core Protein p24/biosynthesis , HIV-1/growth & development , Humans , Lymphocytes/virology , Virus Replication/drug effectsABSTRACT
Binding of HIV-1 envelope glycoprotein (Env) to its cellular receptors elicits a variety of signaling events, including the activation of select tyrosine kinases. To evaluate the potential role of such signaling, we examined the effects of the tyrosine kinase inhibitor, genistein, on HIV-1 entry and infection of human macrophages using a variety of assays. Without altering cell viability, cell surface expression of CD4 and CCR5 or their abilities to interact with Env, genistein inhibited infection of macrophages by reporter gene-encoding, beta-lactamase containing, or wild type virions, as well as Env-mediated cell-fusion. The observation that genistein blocked virus infection if applied before, during or immediately after the infection period, but not 24h later; coupled with a more pronounced inhibition of infection in the reporter gene assays as compared to both beta-lactamase and p24 particle entry assays, imply that genistein exerts its inhibitory effects on both entry and early post-entry steps. These findings suggest that other exploitable targets, or steps, of the HIV-1 infection process may exist and could serve as additional opportunities for the development of new therapeutics.
Subject(s)
Genistein/pharmacology , HIV Infections/virology , HIV-1/drug effects , Protein-Tyrosine Kinases/antagonists & inhibitors , Cells, Cultured , Dose-Response Relationship, Drug , HIV Envelope Protein gp120/drug effects , HIV Envelope Protein gp120/metabolism , HIV-1/physiology , Humans , Macrophages/virology , Membrane Fusion/drug effects , Protein-Tyrosine Kinases/metabolism , Time Factors , Tumor Necrosis Factor-alpha/drug effects , Tumor Necrosis Factor-alpha/metabolism , Virus Replication/drug effectsABSTRACT
We demonstrated recently that selective side-chain modification of functional cysteine-rich (Tat(21-40)) and arginine-rich (Tat(53-68)) domains of the HIV-1 Tat protein blocks pathogenic activities of these peptides while retaining their immunological characteristics. In the present study, we have synthesized a multiple-peptide conjugate system comprising modified Tat(21-40) and Tat(53-68) peptides (HIV-1-Tat-MPC). Immunization of mice with this highly homogeneous 10.7 kDa HIV-1-Tat-MPC synthetic construct induced an effective immune response in mice. The antibodies generated against HIV-1-Tat-MPC efficiently suppressed Tat-induced viral replication and significantly reduced HIV-associated cytopathic effects in human monocytes. These results indicate that epitope-specific antibodies directed against functional sites of Tat protein using non-pathogenic peptides inhibit HIV pathogenesis. The HIV-1-Tat-MPC, therefore, has potential for the development of a safe, effective, and economical therapeutic vaccine to reduce the progression of HIV infection.
Subject(s)
Gene Products, tat/immunology , HIV Antibodies/biosynthesis , HIV-1/immunology , AIDS Vaccines/immunology , Amino Acid Sequence , Animals , Cells, Cultured , Epitopes/chemistry , Epitopes/genetics , Epitopes/immunology , Female , Gene Products, tat/chemistry , Gene Products, tat/genetics , HIV Infections/immunology , HIV Infections/prevention & control , HIV Infections/virology , HIV-1/genetics , HIV-1/physiology , Humans , Immunization , Mice , Mice, Inbred BALB C , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/immunology , Vaccines, Conjugate/immunology , Virus Replication , tat Gene Products, Human Immunodeficiency VirusABSTRACT
We report a novel mechanism, involving up-regulation of the interleukin (IL)-7 cytokine receptor, by which human immunodeficiency virus (HIV) enhances its own production in monocyte-derived macrophages (MDM) in vitro. HIV-1 infection or treatment of MDM cultures with exogenous HIV-1 Tat(86) protein up-regulates the IL-7 receptor (IL-7R) alpha-chain at the levels of steady-state RNA, protein, and functional IL-7R on the cell surface (as measured by ligand-induced receptor signaling). This IL-7R up-regulation is associated with increased amounts of HIV-1 virions in the supernatants of infected MDM cultures treated with exogenous IL-7 cytokine. The overall effect of IL-7 stimulation on HIV replication in MDM culture supernatants is typically in the range of one log and greater. The results are consistent with a model in which HIV infection produces the Tat protein, which in turn up-regulates IL-7R in a paracrine manner. This results in increased IL-7R signaling in response to the IL-7 cytokine, which ultimately promotes early events in HIV replication, including binding/entry and possibly other steps prior to reverse transcription. The results suggest that the effects of IL-7 on HIV replication in MDM should be considered when analyzing and designing clinical trials involving treatment of patients with IL-7 or Tat vaccines.
Subject(s)
Gene Products, tat/physiology , HIV-1/physiology , Interleukin-7/physiology , Macrophages/virology , Models, Biological , Virus Replication/physiology , Cells, Cultured/drug effects , Cells, Cultured/metabolism , Cells, Cultured/virology , Genes, tat , HIV Reverse Transcriptase/metabolism , Humans , Interleukin-7/adverse effects , Interleukin-7/pharmacology , Macrophages/drug effects , Macrophages/metabolism , Paracrine Communication , STAT3 Transcription Factor/metabolism , Virion , Virus Replication/drug effects , tat Gene Products, Human Immunodeficiency VirusABSTRACT
Extracellular Tat protein of HIV-1 activates virus replication in HIV-infected cells and induces a variety of host factors in the uninfected cells, some of which play a critical role in the progression of HIV infection. The cysteine-rich and arginine-rich basic domains represent key components of the HIV-Tat protein for pathogenic effects of the full-length Tat protein and, therefore, could be ideal candidates for the development of a therapeutic AIDS vaccine. The present study describes selective modifications of the side-chain functional groups of cysteine and arginine amino acids of these HIV-Tat peptides to minimize the pathogenic effects of these peptides while maintaining natural peptide linkages. Modification of cysteine by introducing either a methyl or t-butyl group in the free sulfhydryl group and replacing the guanidine group with a urea linkage in the side chain of arginine in the cysteine-rich and arginine-rich Tat peptide sequences completely blocked the ability of these peptides to induce HIV replication, chemokine receptor CCR-5 expression, and NF-kappaB activity in monocytes. Such modifications also inhibited angiogenesis and migration of Kaposi's sarcoma cells normally induced by Tat peptides. Such chemical modifications of the cysteine-rich and arginine-rich peptides did not affect their reactivity with antibodies against the full-length Tat protein. With an estimated 40 million HIV-positive individuals worldwide and approximately 4 million new infections emerging every year, a synthetic subunit HIV-Tat vaccine comprised of functionally inactive Tat domains could provide a safe, effective, and economical therapeutic vaccine to reduce the progression of HIV disease.
Subject(s)
Arginine/chemistry , Cysteine/chemistry , Gene Products, tat/chemistry , Gene Products, tat/pharmacology , HIV Infections/pathology , HIV-1/pathogenicity , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Cell Line , Cell Line, Tumor , DNA Replication , Enzyme Activation , Gene Products, tat/immunology , HIV Infections/drug therapy , Humans , Molecular Structure , NF-kappa B , Neovascularization, Pathologic , Peptide Fragments/immunology , Receptors, CCR5/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/pharmacology , tat Gene Products, Human Immunodeficiency VirusABSTRACT
Osteoarticular diseases, such as arthritis or osteoarthritis, are multifactorial diseases with an underlying genetic etiology that are challenging to study. Genome-Wide Association studies (GWAS) have identified several genetic loci associated with these diseases. Epigenetics is a complex mechanism of chromatin and gene modulation through DNA methylation, histone deacetylation or microRNA, which might contribute to the inheritability of disease. Some of these mechanisms have been studied for decades in other diseases or as part of the aging process, where epigenetic changes seem to play an important role. With the implementation of better technological tools, such as the Illumina next generation sequencing, altered methylation of DNA has been linked to articular diseases and these mechanisms have been shown to regulate metalloprotease (MMP) expression and cartilage matrix integrity. Some miRNA have also been identified and more extensively characterized, such as delineation of the role played by miR-140 in chondrogenesis, followed by the discovery of numerous miRNA potentially involved in the epigenetic regulation of osteoarthritic disease. Histone deacetylases have long been linked to aging, particularly with respect to the Sirtuin family with Sirt1 as the major player. Because aging is the major risk factor for osteoarthritis, the involvement of Sirtuins in the etiology of osteoarthritis has been suggested and investigated. All of these fine regulations together shed new light on cartilage disease pathophysiology. We present in this short review an update of the role of these pathways in articular diseases.
Subject(s)
Arthritis/genetics , Cartilage Diseases/genetics , Epigenesis, Genetic/genetics , Aging/genetics , Aging/physiology , DNA Methylation/genetics , Histone Deacetylases/genetics , Histones/metabolism , Humans , MicroRNAs/geneticsSubject(s)
Gastrointestinal Microbiome , Tumor Necrosis Factor Inhibitors , Animals , Dysbiosis , Humans , MiceABSTRACT
Recent advances in our understanding of the cellular and molecular mechanisms of HIV-1 entry provide the basis for novel therapeutic strategies that prevent viral penetration of the target cell-membrane, while reducing detrimental virus and treatment effects on cells and prolonging virion exposure to immune defenses. A number of potential sites for therapeutic intervention become accessible during the narrow window between virus attachment and the subsequent fusion of viral envelope with the cell membrane. Initial approaches considered for prevention of HIV-1 entry included the use of natural ligands, small-molecule inhibitors and/or monoclonal antibodies, which could interfere with gp120-CD4 and/or gp120-CXCR4/CCR5 interaction. Others avenues pursued were the use of agents that interfere with the conformational changes of gp120 or gp41 leading to subsequent fusion of viral and cellular membranes. More recently, strategies have emerged that involve inhibition of thiol/disulfide oxidoreductases (factors which facilitate Env transition from an inactive to a fusion-competent conformation) to block redox exchange, thereby impeding the entry process. This review provides a summary of the cellular and viral factors mediating the HIV-1 entry process, with an emphasis on novel therapeutics targeting Env-receptor/coreceptor interaction, Env conformational change and the membrane fusion process.
Subject(s)
HIV Fusion Inhibitors/therapeutic use , HIV Infections/drug therapy , HIV-1/drug effects , Membrane Fusion/drug effects , Receptors, HIV/drug effects , Antibodies, Monoclonal/therapeutic use , Antibodies, Viral/therapeutic use , Cell Membrane/metabolism , Cell Membrane/virology , HIV Envelope Protein gp120/immunology , HIV Envelope Protein gp120/metabolism , HIV Envelope Protein gp41/immunology , HIV Envelope Protein gp41/metabolism , HIV Infections/virology , HIV-1/physiology , Humans , Protein Conformation , Protein Disulfide Reductase (Glutathione)/antagonists & inhibitors , Receptors, HIV/immunologyABSTRACT
Macrophage colony-stimulating factor (M-CSF) enhances the susceptibility of macrophages to infection with HIV-1, in part by increasing the expression of CD4 and CCR5. Human monocyte-derived macrophages (MDMs) infected in vitro with HIV-1 endogenously produce M-CSF, with kinetics paralleling virus replication, which can lead to enhanced spreading of the infection. AZT and ritonavir both inhibit HIV replication, but their impact on M-CSF production by HIV-infected human MDMs is unknown. The dose response and kinetics of virus replication in the presence of AZT and ritonavir were determined for HIV-infected MDMs from HIV-seronegative donors. Harvested supernatants were monitored for reverse transcriptase activity, M-CSF production, and HIV proteins. Our data suggest that threshold levels of HIV replication must occur before maximum M-CSF production is induced. Addition of AZT or ritonavir before or after establishment of productive HIV infection dramatically reduces virus replication and M-CSF production by human MDMs. However, ongoing virus replication and M-CSF production are slow to return to baseline levels after addition of AZT or ritonavir, suggesting that HIV replication and virion release from infected macrophages continue long after initiation of antiretroviral therapy. Our results suggest that, in human macrophages, HIV-1 replication and M-CSF production are inextricably linked, such that inhibition of one leads to a concomitant reduction of the other. Low-level HIV replication and M-CSF release during ongoing antiretroviral therapies may facilitate the survival and maintenance of infected macrophages and suggests that additional therapies targeting M-CSF may be critical for elimination of macrophage reservoirs.
Subject(s)
Anti-HIV Agents/pharmacology , HIV-1/drug effects , Macrophage Colony-Stimulating Factor/biosynthesis , Macrophages/drug effects , Virus Replication/drug effects , Zidovudine/pharmacology , HIV Infections/virology , HIV-1/physiology , Humans , Macrophages/metabolism , Macrophages/virology , Ritonavir/pharmacologyABSTRACT
Platelet factor 4 (CXCL4), a member of the CXC chemokine subfamily released in high amounts by activated platelets, has been identified as a monocyte survival factor that induces monocyte differentiation into macrophages. Although CXCL4 has been shown to have biological effects unique to chemokines, nothing is known about the role of CXCL4-derived human macrophages or CXCL4 in human immunodeficiency virus (HIV) disease. In this study, CXCL4-derived macrophages are compared with macrophage-colony stimulating factor (M-CSF)-derived macrophages for their ability to support HIV-1 replication. We show that CXCL4-derived macrophages can be infected with macrophage-tropic HIV-1 that uses either CC-chemokine receptor 5 (CCR5) or CXC-chemokine receptor 4 (CXCR4) as a co-receptor for viral entry. We also find that M-CSF and the chemokines, monocyte chemoattractant protein 1 (MCP-1; CCL2) and macrophage-inflammatory-protein-1-alpha (MIP-1alpha; CCL3) are produced upon R5- and X4-tropic HIV-1 replication in both M-CSF- and CXCL4-derived human macrophages. In addition, CXCL4 added to M-CSF-derived macrophages after virus adsorption and maintained throughout the infection enhances HIV-1 replication. We thus propose a novel role for CXCL4 in HIV disease.