Infected Peripheral Blood Mononuclear Cells Research Articles

LncRNA SNHG16 Inhibits Intracellular M. tuberculosis Growth Involving Cathelicidin Pathway, Autophagy, and Effector Cytokines Production.

Long noncoding small nucleolar RNA (LncRNA) host gene 16 (SNHG16) is associated with certain diseases, including cancers. However, its role and mechanism in Mycobacterium tuberculosis (Mtb) infection remain unclear. Here, we demonstrated that SNHG16 expression levels were suppressed in peripheral blood mononuclear cells (PBMCs) and CD14+ monocytes of tuberculosis (TB) patients. SNHG16 was up-regulated by acute Mtb infection of PBMCs from healthy control (HC) subjects. Such TB suppression of SNHG16 was consistent with an immunosuppressive-like state driven by IL-10 signaling as seen in TB patients. Notably, SNHG16 limited Mtb growth in macrophages/monocytes through autophagy and vitamin D receptor (VDR)-dependent cathelicidin (CAMP) antimicrobial pathways. Concurrently, SNHG16 was highly expressed in lymphocytes, including CD8+ and Vγ2 Vδ2 T-cell subsets in HCs. SNHG16 overexpression in lymphocytes allowed them to control Mtb infection in macrophages, and SNHG16 epigenetically increased the expression of anti-Mtb effector cytokines in lymphocytes by developing more accessible chromatin states in gene loci encoding IFN-γ, TNF-α, and Granzyme B. Furthermore, the adoptive transfer of SNHG16-overexpressing human PBMCs into Mtb-infected SCID mice conferred protective immunity against Mtb infection. Thus, SNHG16 drove the induction of pleiotropic effector functions that inhibited intracellular Mtb growth in vitro and in vivo, serving as an immunotherapy target in TB.

Diacerein reduces inflammasome activation and SARS-CoV-2 virus replication: a proof-of-concept translational study.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is linked to high mortality, primarily through an intense inflammatory response. Diacerein has emerged as a potential therapy for COVID-19 due to its potential impact in decreasing the inflammasome activation and coronavirus replication. This study aims to explore diacerein's influence in inhibiting both viral replication and the inflammatory response after SARS-CoV-2 infection. Human peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers and infected in vitro with SARS-CoV-2. Additionally, we carried out a pilot randomized, double-blind, placebo-controlled study with 14 participants allocated to diacerein (n = 7) or placebo (n = 7) therapies every 12h for 10 days. The primary endpoint was change in plasma markers of inflammasome activation (NLRP3, caspase-1, and gasdermin-D). In vitro protocols have shown that rhein, diacerein's primary metabolite, decreased IL-1β secretion caused by SARS-CoV-2 infection in human PBMCs (p < 0.05), and suppressed viral replication when administered either before or after the virus incubation (p < 0.05). This later effect was, at least partially, attributed to its inhibitory effect on 3-chymotrypsin-like protease (SARS-CoV-2 3CLpro) and papain-like protease in the SARS-CoV-2 (SARS-CoV-2 PLpro) virus and in the phosphorylation of proteins related cytoskeleton network (p < 0.05). Diacerein-treated COVID-19 patients presented a smaller area under the curve for NLRP3, caspase-1 and GSDM-D measured on days 2, 5, and 10 after hospitalization compared to those receiving a placebo (p < 0.05). The indicated mechanisms of action of diacerein/rhein can reduce viral replication and mitigate the inflammatory response related to SARS-CoV-2. These findings are preliminary and require confirmation in clinical trials.

Tucaresol: A Clinical Stage Oral Candidate Drug with Two Distinct Antiviral Mechanisms

Globally, approximately 39 million people are living with Human Immunodeficiency Virus, HIV, arising from approximately 86 million infections since this epidemic began in 1981. However, the number of HIV infections is unevenly distributed with two thirds of global infections confined to Sub-Saharan Africa. Due to viral drug resistance, the most effective treatment requires a triple drug combination thereby adding to the complexity and cost of therapy. As such, many people living with HIV or at risk of infection do not have access to prevention or treatment of this potentially fatal disease. There is no cure for HIV [1]. Tucaresol is an orally active clinical stage drug which functions as a host targeted antiviral agent by protection or reconstitution of CD4+ T helper immune cells. We report herein that Tucaresol also displays in-vitro activity against HIV in infected human peripheral blood mononuclear cells. Although this in-vitro antiviral activity is not potent, the excellent safety profile and bioavailability of Tucaresol, along with its low Molecular Weight, support attainment of relevant drug concentrations in man to achieve significant in-vivo activity. This is demonstrated by previously reported stabilization of viremia in a prior proof of concept phase 1b/2a HIV clinical trial [2]. It is possible that the significant in-vivo activity of Tucaresol arises from synergy between co-stimulation of CD4+ T helper cells and the direct activity against virally infected cells. A pan in-vitro viral screen of Tucaresol further revealed a weak, direct antiviral activity against human herpes virus 6B, human papillomavirus 11, measles virus and hepatitis B virus.

Ex vivo host transcriptomics during Cryptococcus neoformans, Cryptococcus gattii, and Candida albicans infection of PBMCs from South African volunteers.

Cryptococcus neoformans, Cryptococcus gattii and Candida albicans are opportunistic fungal pathogens associated with infections in immunocompromised hosts. Cryptococcal meningitis (CM) is the leading fungal cause of HIV-related deaths globally, with the majority occurring in Africa. The human immune response to C. albicans infection has been studied extensively in large genomics studies whereas cryptococcal infections, despite their severity, are comparatively understudied. Here we investigated the transcriptional response of immune cells after in vitro stimulation with in vitro C. neoformans, C. gattii and C. albicans infection of peripheral blood mononuclear cells (PBMCs) collected from healthy South African volunteers. We found a lower transcriptional response to cryptococcal stimuli compared to C. albicans and unique expression signatures from all three fungal stimuli. This work provides a starting point for further studies comparing the transcriptional signature of CM in immunocompromised patients, with the goal of identifying biomarkers of disease severity and possible novel treatment targets.

HIV Infection Drives Foam Cell Formation via NLRP3 Inflammasome Activation.

Persistent immune activation is linked to an increased risk of cardiovascular disease (CVD) in people with HIV (PWH) on antiretroviral therapy (ART). The NLRP3 inflammasome may contribute to elevated CVD risk in PWH. This study utilized peripheral blood mononuclear cells (PBMCs) from 25 PWH and 25 HIV-negative controls, as well as HIV in vitro infections. Transcriptional changes were analyzed using RNAseq and pathway analysis. Our results showed that in vitro HIV infection of macrophages and PBMCs from PWH had increased foam cell formation and expression of the NLRP3 inflammasome components and downstream cytokines (caspase-1, IL-1β, and IL-18), which was reduced with inhibition of NLRP3 activity using MCC950. Transcriptomic analysis revealed an increased expression of multiple genes involved in lipid metabolism, cholesterol storage, coronary microcirculation disorders, ischemic events, and monocyte/macrophage differentiation and function with HIV infection and oxLDL treatment. HIV infection and NLRP3 activation increased foam cell formation and expression of proinflammatory cytokines, providing insights into the mechanisms underlying HIV-associated atherogenesis. This study suggests that HIV itself may contribute to increased CVD risk in PWH. Understanding the involvement of the inflammasome pathway in HIV atherosclerosis can help identify potential therapeutic targets to mitigate cardiovascular risks in PWH.

Anti-Leishmania major activity of Calotropis procera extract by increasing ROS production and upregulating TNF-α, IFN-γ and iNOS mRNA expression under in vitro conditions

BackgroundLeishmaniasis, caused by protozoan parasites of the genus Leishmania, is a neglected tropical disease with 700,000 to 1,000,000 global new cases annually. Adverse effects associated with expense, long-term treatment and drug resistance have made conventional therapies unfavorable, encouraging the search for alternative drugs based on plant products. In this study, the effect of Calotropis procera (Asclepiadaceae) extract against viability of promastigotes and amastigotes of Leishmania major was evaluated in vitro.MethodsThe extract from the leaves of C. procera seedlings was prepared using a methanol maceration method. The colorimetric cell viability 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the growth-inhibitory effect of the extract on promastigotes. The level of reactive oxygen species (ROS) in promastigote cultures was determined after treatment with the extract using the 2',7'-dichlorofluorescein diacetate (DCFH-DA) method and compared with untreated cultures (control). After exposure to the extract the expression levels of tumor necrosis factor-α (TNF-α), interferon gamma (IFN-γ) and inducible nitric oxide synthase (iNOS) genes were determined and compared to control in peripheral blood mononuclear cells (PBMCs) infected with L. major.ResultsBased on the MTT assay, the C. procera extract significantly reduced the proliferation of L. major promastigotes with IC50 values of 377.28 and 222.44 μg/mL for 24 and 72 h, respectively (p < 0.01). After treatment with 222.44 and 377.28 μg/mL of C. procera extract, ROS production in L. major promastigote cultures increased 1.2- to 1.65-fold and 2- to 4-fold compared to the control, respectively (p < 0.05). C. procera extract induced significant increases in gene expression of TNF-α (2.76–14.83 fold), IFN-γ (25.63–threefold) and iNOS (16.32–3.97 fold) in infected PBMCs compared to control (p < 0.01).ConclusionsOn the basis of its anti-leishmanial activity, C. procera can be considered as a promising new plant source for the potential treatment of leishmaniasis.

Enterovirus E infects bovine peripheral blood mononuclear cells. Implications for pathogenesis?

Enterovirus E (EV-E) is a common viral pathogen endemic in cattle worldwide. Little is known, however, about its potential interactions with bovine immune cells. The EV-E-permissiveness of bovine peripheral blood mononuclear cells (PBMCs) was evaluated. The infectious titres of extracellular virus were measured and the intracellular viral RNA levels were determined by reverse transcription quantitative PCR after cell inoculation. The effects of EV-E on cell viability and proliferative response were investigated with a methyl thiazolyl tetrazolium bromide reduction assay, the percentages of main lymphocyte subsets and oxidative burst activity of blood phagocytes were determined with flow cytometry, and pro-inflammatory cytokine secretion was measured with an ELISA. Enterovirus E productively infected bovine PBMCs. The highest infectious dose of EV-E decreased cell viability and T-cell proliferation. All of the tested doses of virus inhibited the proliferation of high responding to lipopolysaccharide B cells and stimulated the secretion of interleukin 1β, interleukin 6 and tumour necrosis factor α pro-inflammatory cytokines. Interactions of EV-E with bovine immune cells may indicate potential evasion mechanisms of the virus. There is also a risk that an infection with this virus can predispose the organism to secondary infections, especially bacterial ones.

HIV-1 Restriction in Systemic Lupus Erythematosus Involves Iron Homeostasis

INTRODUCTION: Over the past two decades, there has been a growing emphasis on host restriction factors. However, a comprehensive understanding of their role in modulating HIV-1 infection in vivo requires further detailed investigation. Intracellular iron levels can affect HIV-1 replication by influencing expression of host cell viral restriction factors. Systemic Lupus Erythematosus (SLE) is characterized by upregulation of interferon response and the coexistence of SLE and HIV infection is rare. In lupus nephritis, deregulation of proteins involved in iron homeostasis have also been identified, with hepcidin suggested to play a role in disease severity . Hif1α, a protein regulated by hypoxia and under iron depletion, and reported to block HIV transcription, is upregulated in SLE. High levels of IL-10 and IL-16 which inhibits HIV infection are upregulated in SLE. APOBECs and oxysterols and Interferon Regulating Factors (IRFs) are also known to be upregulated in SLE. The current study is aimed to investigate ex vivo HIV-1 infection in peripheral blood mononuclear cells (PBMCs) derived from individuals SLE and identify factors that contributed to this inhibition. METHODS: We utilized replication deficient VSVg-pseudotype HIV-1 and replication competent HIV-IIIB to infect SLE PBMCs. Real-time PCR was conducted to determine the upregulation of genes involved in HIV-1 replication. RNA-seq of PBMCs were done to identify altered restriction factors. RESULTS: We investigated one round of HIV-1 infection in peripheral blood mononuclear cells (PBMCs) obtained from individuals with Systemic Lupus Erythematosus (SLE) and compared them to control PBMCs. We found that the HIV-1 infection in SLE-derived PBMCs was significantly reduced, as indicated by lower mRNA levels of Gag and Nef, two essential viral proteins. Our ongoing study, which focused on the inhibition of HIV-1 replication ex vivo in Sickle Cell Disease (SCD), revealed interesting findings. RNA sequencing of PBMCs from individuals with SCD showed an upregulation of APOBEC3A, APOBEC3B, and CH25H, which are known restriction factors against HIV-1. Given these intriguing results, we decided to analyze the RNA expression of these restriction factors in SLE-derived PBMCs. Surprisingly, we found a significant increase in the mRNA levels of APOBEC3A, APOBEC3B, and CH25H in SLE-derived PBMCs as well. This suggests that the enhanced expression of these restriction factors could be contributing to the reduced HIV-1 infection observed in SLE-derived PBMCs. Additionally, we explored the RNA expression of HIF-1α and TFR1, which are iron-regulated factors, and found that their mRNA levels were upregulated in SLE-derived PBMCs. Furthermore, we observed an upregulation of interferon regulatory factors, including IL-10, IFN-β, and IRF-7, which are involved in immune responses and antiviral defense mechanisms. Interestingly IRF-7 was upregulated in SCD PBMCs too. DISSUCION: The ongoing study in our lab on SCD and the study reported here are known for deregulated iron metabolism. Interestingly on one hand PBMCs derived from both inhibit HIV-1 and on other have some common restriction factors, iron regulated genes and IRF upregulated. Iron-regulated factors, such as TFR1, HIF-1α and ferroportin have been associated with antiviral effects. Modulating the expression or activity of these factors could influence HIV infection. CONCLUSIONs: Taken together, our findings indicate that SLE-derived PBMCs exhibit a distinct gene expression profile that may confer protection against HIV-1 infection. The upregulation of APOBEC3A, APOBEC3B, CH25H, HIF-1α, TFR1, and interferon regulatory factors could collectively contribute to the observed reduction in HIV-1 replication in SLE-derived PBMCs. Further investigation is needed to fully understand the mechanisms underlying these observations and their implications for HIV-1 pathogenesis under altered intracellular iron levels influencing HIV-1 replication by modulating host cell factors. Acknowledgments This work was supported by NIH Research Grants 1R01HL125005, 5U54MD007597, P30AI087714 and P50AI150476. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

MiR-150 regulates the Leishmania infantum parasitic load and granzyme B levels in peripheral blood mononuclear cells of dogs with canine visceral leishmaniosis

Leishmania infantum causes visceral leishmaniosis, a neglected tropical disease that can modulate the host immune response by altering the expression of small non-coding RNAs called microRNAs (miRNAs). Some miRNAs are differentially expressed in peripheral blood mononuclear cells (PBMCs) of dogs with canine visceral leishmaniosis (CanL), like the down-regulated miR-150. Even though miR-150 is negatively correlated with L. infantum parasitic load, it is unclear if miR-150 directly affects L. infantum parasitic load and (if so) how this miRNA would contribute to infection. Here, we isolated PBMCs from 14 naturally infected dogs (CanL group) and six healthy dogs (Control group) and treated them in vitro with miR-150 mimic or inhibitor. We measured L. infantum parasitic load using qPCR and compared treatments. We also measured miR-150 in silico predicted target protein levels (STAT1, TNF-α, HDAC8, and GZMB) using flow cytometry or enzyme-linked immunosorbent assays. Increasing miR-150 activity diminished L. infantum parasitic load in CanL PBMCs. We also found that inhibition of miR-150 reduced GZMB (granzyme B) levels. These findings demonstrate that miR-150 plays an important role in L. infantum infection in canine PBMCs, and they merit further studies aiming at drug development.

Discovering common pathogenic processes between COVID-19 and HFRS by integrating RNA-seq differential expression analysis with machine learning.

Zoonotic virus spillover in human hosts including outbreaks of Hantavirus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) imposes a serious impact on the quality of life of patients. Recent studies provide a shred of evidence that patients with Hantavirus-caused hemorrhagic fever with renal syndrome (HFRS) are at risk of contracting SARS-CoV-2. Both RNA viruses shared a higher degree of clinical features similarity including dry cough, high fever, shortness of breath, and certain reported cases with multiple organ failure. However, there is currently no validated treatment option to tackle this global concern. This study is attributed to the identification of common genes and perturbed pathways by combining differential expression analysis with bioinformatics and machine learning approaches. Initially, the transcriptomic data of hantavirus-infected peripheral blood mononuclear cells (PBMCs) and SARS-CoV-2 infected PBMCs were analyzed through differential gene expression analysis for identification of common differentially expressed genes (DEGs). The functional annotation by enrichment analysis of common genes demonstrated immune and inflammatory response biological processes enriched by DEGs. The protein-protein interaction (PPI) network of DEGs was then constructed and six genes named RAD51, ALDH1A1, UBA52, CUL3, GADD45B, and CDKN1A were identified as the commonly dysregulated hub genes among HFRS and COVID-19. Later, the classification performance of these hub genes were evaluated using Random Forest (RF), Poisson Linear Discriminant Analysis (PLDA), Voom-based Nearest Shrunken Centroids (voomNSC), and Support Vector Machine (SVM) classifiers which demonstrated accuracy >70%, suggesting the biomarker potential of the hub genes. To our knowledge, this is the first study that unveiled biological processes and pathways commonly dysregulated in HFRS and COVID-19, which could be in the next future used for the design of personalized treatment to prevent the linked attacks of COVID-19 and HFRS.

Abstract 697: Extracellular vesicles (EV) - mediators of therapeutic vaccination? In vivo and in vitro characterization of EVs generated after infection of human and murine cells with therapeutic poxviruses

Abstract Virus-based therapies show promise in inducing systemic anti-tumor immunity. Over the past years, modified poxviruses were developed to induce a sustained antitumoral immune response. Several studies confirm this hypothesis and report induction of T cell response against the antigen targeted by these vaccines. We previously reported MVA induced T-cell response against HPV oncoviral antigens E6 and E7 in HPV anogenital cancers (NCT03260023) and against private tumor neoantigen in ovarian and head and neck cancer. Despite these promising results the molecular and cellular mechanisms involved in vaccine response remains to be elucidated. In this work, we inquired whether EVs, well known for mediating intercellular communication, play a role in these therapeutic vaccination process. We first asked whether poxviral infection of human primary cells modifies their EV secretion properties. To this end, peripheral blood mononuclear cells (PBMCs) from healthy donors were infected with poxviral vectors, cell culture supernatants were harvested, and different isolation methods were compared for their ability to separate EVs from viral particles: ultracentrifugation, immunocapture, density gradient, velocity gradient, size exclusion chromatography and filtration. EVs size distribution and concentration were determined by nanoparticle tracking analysis (NTA) and the presence of infectious viral particles was titrated by plaque assays. Our data showed that 0.1µm filtration allowed the complete separation of EVs from infectious viral particles. All other methods showed co-isolation of EVs and infectious viral particles. Using this method, we demonstrate that infection of PBMCs with the poxviruses VACV, MVA and PCPV significantly increased the amount of secreted EVs. Next, we investigated the molecular content of EVs isolated from MVA-infected PBMCs by mass spectrometry. We observed differences in the abundance of at least 57 human proteins between EVs from infected and non-infected cells. In addition, several viral proteins, including virus-encoded payloads were detected in EVs from infected cells. We are now confirming those data with flow cytometry to detect virus-encoded payloads like tumor antigens, antigen-presenting molecules, and EV markers such as tetraspanins. The RNA content of EVs from infected and non-infected cells will be compared. Finally, EVs were isolated from murine dendritic cells infected with therapeutic pox vaccines. We are now evaluating the effects of local and systemic application of EVs in murine tumor models. Citation Format: Lucas Walther, Caroline Tosch, Jules Deforges, Christine Carapito, Magali Rompais, Marie-Christine Claudepierre, Nathalie Silvestre, Kaïdre Bendjama, Eric Quemeneur, Jacky Goetz, Vincent Hyenne, Karola Rittner. Extracellular vesicles (EV) - mediators of therapeutic vaccination? In vivo and in vitro characterization of EVs generated after infection of human and murine cells with therapeutic poxviruses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 697.

An Efficient Humanized Mouse Model for Oral Anti-Retroviral Administration.

HIV anti-retrovirals (ARVs) have vastly improved the life expectancy of people living with HIV (PLWH). However, toxic effects attributed to long-term ARV use also contribute to HIV-related co-morbidities such as heart disease, bone loss and HIV-associated neurocognitive disorders (HAND). Unfortunately, mouse models used to study the effects of ARVs on viral suppression, toxicity and HIV latency/tissue reservoirs have not been widely established. Here, we demonstrate an effective mouse model utilizing immune-compromised mice, reconstituted with infected human peripheral blood mononuclear cell (PBMCs). ARVs areincorporated into mouse chow and administered daily with combination ARV regimens includingAtripla (efavirenz, tenofovir disoproxil fumarate, and emtricitabine) and Triumeq (abacavir, dolutegravir and lamivudine). This model measures HIV-infected human cell trafficking, and ARV penetration throughout most relevant HIV organs and plasma, with a large amount of trafficking to the secondary lymphoid organs. Furthermore, the HIV viral load within each organ and the plasma was reduced in ARV treated vs. untreated control. Overall, we have demonstrated a mouse model that is relatively easy and affordable to establish and utilize to study ARVs' effect on various tissues, including the co-morbid conditions associated with PLWH, such as HAND, and other toxic effects.

Development of a flow cytometry method for detection of bovine multi-cytokines

This study aims to develop a method to detect bovine multi-cytokines based on flow cytometry. Previously we have prepared and screened monoclonal antibodies against bovine cytokines IFN-γ, IL-2, TNF-α, IP-10 and MCP-1. These bovine cytokine monoclonal antibodies were fluorescently labeled, and the combination of antibody and cell surface molecules were used to develop the method for detecting bovine multi-cytokines. Subsequently, the developed method was used to determine the cytokine expression profile of Mycobacterium bovis BCG infected bovine peripheral blood mononuclear cells in vitro, and evaluate the cytokine expression level of peripheral blood CD4+ T cells of tuberculosis-positive cattle. The bovine multi-cytokine flow cytometry detection method can effectively determine the cytokine expression of BCG-infected bovine peripheral blood T lymphocytes. Among them, the expression levels of IFN-γ, IL-2, and TNF-α continue to increase after 40 hours of infection, while the expression levels of IP-10 and MCP-1 decreased. The combined detection of IFN-γ, IL-2, and TNF-α on CD4+ T lymphocytes in peripheral blood of cattle can effectively distinguish tuberculosis-positive and tuberculosis-negative samples. This method may facilitate evaluating the level of cellular immune response after bovine pathogen infection and vaccine injection.

Cysteamine/Cystamine Exert Anti-Mycobacterium abscessus Activity Alone or in Combination with Amikacin.

Host-directed therapies are emerging as a promising tool in the curing of difficult-to-treat infections, such as those caused by drug-resistant bacteria. In this study, we aim to test the potential activity of the FDA- and EMA-approved drugs cysteamine and cystamine against Mycobacterium abscessus. In human macrophages (differentiated THP-1 cells), these drugs restricted M. abscessus growth similar to that achieved by amikacin. Here, we use the human ex vivo granuloma-like structures (GLS) model of infection with the M. abscessus rough (MAB-R) and smooth (MAB-S) variants to study the activity of new therapies against M. abscessus. We demonstrate that cysteamine and cystamine show a decrease in the number of total GLSs per well in the MAB-S and MAB-R infected human peripheral blood mononuclear cells (PBMCs). Furthermore, combined administration of cysteamine or cystamine with amikacin resulted in enhanced activity against the two M. abscessus morpho variants compared to treatment with amikacin only. Treatment with cysteamine and cystamine was more effective in reducing GLS size and bacterial load during MAB-S infection compared with MAB-R infection. Moreover, treatment with these two drugs drastically quenched the exuberant proinflammatory response triggered by the MAB-R variant. These findings showing the activity of cysteamine and cystamine against the R and S M. abscessus morphotypes support the use of these drugs as novel host-directed therapies against M. abscessus infections.

Transcriptome analysis of PBMCs isolated from piglets treated with a miR-124 sponge construct identified miR124/IQGAP2/Rho GTPase as a target pathway support Salmonella Typhimurium infection.

miR-124 is a significantly up-regulated miRNA in peripheral blood collected from piglets infected with Salmonella Typhimurium, suggesting that it may play an important role in Salmonella pathogenesis. This study focused on the transcriptomic analysis of peripheral blood mononuclear cells (PBMCs) isolated from miR-124 sponge and Salmonella Typhimurium-treated piglets, and trying to investigate the function of miR-124 in Salmonella infection. The transcriptome profiling analysis revealed that 2778 genes in miR-124 sponge + Salmonella Typhimurium treatment versus control, 2271 genes in Salmonella Typhimurium treatment versus control, and 1301 genes in miR-124 sponge + Salmonella Typhimurium versus Salmonella Typhimurium treatment, were differentially expressed, respectively (FDR < 0.05 and fold change > 2.0). Pathway analysis indicated that the MAPK signaling pathway, Ribosome pathway, and T-cell receptor signaling pathway were the most significantly enriched pathway in differentially expressed genes between miR-124 sponge + Salmonella Typhimurium and Salmonella Typhimurium along treatment (FDR < 0.05). Reporter assays and electrophoretic mobility shift assays showed that miR-124 is a crucial regulatory factor that targets IQ motif containing GTPase-activating protein 2 (IQGAP2). Cell culture experiment indicated that miR-124 attenuated the Salmonella Typhimurium-mediated activation of CDC42 and RAC1 (P < 0.05). Cultured PBMCs treated with miR-124 and IQGAP2-siRNA had higher intracellular Salmonella count than control samples, particularly 12h post-infection (P < 0.05). Immunofluorescence analysis revealed that miR-124 treatment reduced the percentage of LAMP-1-positive phagosomes. The miR-124 could be an important regulator for IQGAP2/Rho GTPase pathway in Salmonella Typhimurium-infected PBMCs, and this pathway could be a target for Salmonella that support its infection in PBMCs in piglets.

Increased HIV-1 infection in PBMCs treated in vitro with menstrual cycle phase hormones or medroxyprogesterone acetate likely occurs via different mechanisms.

Both luteal phase progesterone (P4) levels and use of the intramuscular (IM) injectable progestin-only contraceptive depo-medroxyprogesterone acetate (DMPA-IM) have been linked to increased S/HIV acquisition in animal, clinical and in vitro models. Several plausible mechanisms could explain MPA-induced HIV-1 acquisition while those for the luteal phase are underexplored. Peripheral blood mononuclear cells (PBMCs) were treated with P4 and estrogen at concentrations mimicking the luteal phase, follicular phase or with levels of MPA mimicking peak serum levels in DMPA-IM users. Cells were infected with an R5-tropic infectious molecular clone and HIV-1 infection was measured. A role for the glucocorticoid receptor (GR) was investigated using the GR/PR antagonist RU486. CCR5 protein levels and activation status, assessed by levels of the activation marker CD69, were measured by flow cytometry after treatment in vitro and in PBMCs from naturally-cycling women or DMPA-IM users. Both MPA and luteal phase hormones significantly increased HIV-1 infection in vitro. However, MPA but not luteal phase hormones increased the CD4+/CD8+ T cell ratio, CCR5 protein expression on CD4+ T cells and increased expression of the activation marker CD69. The GR is involved in MPA-induced, but not luteal phase hormone-induced increased HIV-1 infection. In DMPA-IM users, the frequency of CCR5-expressing CD3+ and CD8+ cells was higher than for women in the luteal phase. MPA increases HIV-1 infection in a manner different from that of luteal phase hormones, most likely involving the GR and at least in part changes in the frequency and/or expression of CCR5 and CD69.

Transcriptome reprogramming of Epstein-Barr virus infected epithelial and B cells reveals distinct host-virus interaction profiles

Epstein-Barr virus (EBV) is an opportunistic pathogen that can manifest itself as a potential contributor to human diseases years after primary infection, specifically in lymphoid and epithelial cell malignancies in immune-competent and immune-compromised hosts. The virus shuttles between B cells and epithelial cells during its infection cycle, facilitating its persistence and transmission in humans. While EBV efficiently infects and transforms B-lymphocytes, epithelial cells are not as susceptible to transformation in vitro. We utilized a 3D platform for culturing normal oral keratinocyte cells (NOKs) using Matrigel for greater insights into the molecular interactions between EBV and infected cells. We determined the transcriptome of EBV infected NOKs and peripheral blood mononuclear cells (PBMCs) for 7 and 15 days. LMPs (−1, −2A, and −2B) and EBNAs (−1, −2, −3A, −3B and −3C) were detected in all samples, and lytic gene expression was significantly higher in NOKs than PBMCs. We identified over 2000 cellular genes that were differentially expressed (P-value<0.05). Gene ontology (GO) and pathway analyses significantly identified pathways related to collagen-activation, chemokine signaling, immune response, metabolism, and antiviral responses. We also identified significant changes in metalloproteases and genes encoding chemotactic ligands and cell surface molecules. C-X-C chemokine receptor type 4 (CXCR4) was dramatically downregulated in PBMCs and upregulated in NOKs. However, MMP1 was significantly downregulated in NOKs and upregulated in PBMCs. Therefore, multiple pathways contribute to distinct pathologies associated with EBV infection in epithelial and B cells, and MMP1 and CXCR4 are critical molecules involved in regulation of latent and lytic states linked to viral associated diseases.

A Phenotypic Screen Identifies Potent DPP9 Inhibitors Capable of Killing HIV-1 Infected Cells.

Although current antiretroviral therapy can control HIV-1 replication and prevent disease progression, it is not curative. Identifying mechanisms that can lead to eradication of persistent viral reservoirs in people living with HIV-1 (PLWH) remains an outstanding challenge to achieving cure. Utilizing a phenotypic screen, we identified a novel chemical class capable of killing HIV-1 infected peripheral blood mononuclear cells. Tool compounds ICeD-1 and ICeD-2 ("inducer of cell death-1 and 2"), optimized for potency and selectivity from screening hits, were used to deconvolute the mechanism of action using a combination of chemoproteomic, biochemical, pharmacological, and genetic approaches. We determined that these compounds function by modulating dipeptidyl peptidase 9 (DPP9) and activating the caspase recruitment domain family member 8 (CARD8) inflammasome. Efficacy of ICeD-1 and ICeD-2 was dependent on HIV-1 protease activity and synergistic with efavirenz, which promotes premature activation of HIV-1 protease at high concentrations in infected cells. This in vitro synergy lowers the efficacious cell kill concentration of efavirenz to a clinically relevant dose at concentrations of ICeD-1 or ICeD-2 that do not result in complete DPP9 inhibition. These results suggest engagement of the pyroptotic pathway as a potential approach to eliminate HIV-1 infected cells.

Viral infection‐related gene upregulation in monocytes in children with signs of β‐cell autoimmunity

ObjectiveThe pathogenesis of type 1 diabetes (T1D) is associated with genetic predisposition and immunological changes during presymptomatic disease. Differences in immune cell subset numbers and phenotypes between T1D patients and healthy controls have been described; however, the role and function of these changes in the pathogenesis is still unclear. Here we aimed to analyze the transcriptomic landscapes of peripheral blood mononuclear cells (PBMCs) during presymptomatic disease.MethodsTranscriptomic differences in PBMCs were compared between cases positive for islet autoantibodies and autoantibody negative controls (9 case–control pairs) and further in monocytes and lymphocytes separately in autoantibody positive subjects and control subjects (25 case–control pairs).ResultsNo significant differential expression was found in either data set. However, when gene set enrichment analysis was performed, the gene sets “defence response to virus” (FDR <0.001, ranking 2), “response to virus” (FDR <0.001, ranking 3) and “response to type I interferon” (FDR = 0.002, ranking 12) were enriched in the upregulated genes among PBMCs in cases. Upon further analysis, this was also seen in monocytes in cases (FDR = 0.01, ranking 2; FDR = 0.04, ranking 3 and FDR = 0.02, ranking 1, respectively) but not in lymphocytes.ConclusionGene set enrichment analysis of children with T1D‐associated autoimmunity revealed changes in pathways relevant for virus infection in PBMCs, particularly in monocytes. Virus infections have been repeatedly implicated in the pathogenesis of T1D. These results support the viral hypothesis by suggesting altered immune activation of viral immune pathways in monocytes during diabetes.

Estradiol inhibits HIV-1BaL infection and induces CFL1 expression in peripheral blood mononuclear cells and endocervical mucosa

An inhibitory effect of estradiol (E2) on HIV-1 infection was suggested by several reports. We previously identified increased gene expression of actin-binding protein cofilin 1 (CFL1) in endocervix in the E2-dominated proliferative phase of the menstrual cycle. Actin cytoskeleton has an integral role in establishing and spreading HIV-1 infection. Herein, we studied in vitro effects of E2 on HIV-1 infection and on CFL1 expression to gain insight into the mechanism of HIV-1 inhibition by E2. E2 dose-dependently inhibited HIV-1BaL infection in peripheral blood mononuclear cells (PBMCs) and endocervix. In PBMCs and endocervix, E2 increased protein expression of total CFL1 and phosphorylated CFL1 (pCFL1) and pCFL1/CFL1 ratios. LIMKi3, a LIM kinase 1 and 2 inhibitor, abrogated the phenotype and restored infection in both PBMCs and endocervix; inhibited E2-induced expression of total CFL1, pCFL1; and decreased pCFL1/CFL1 ratios. Knockdown of CFL1 in PBMCs also abrogated the phenotype and partially restored infection. Additional analysis of soluble mediators revealed decreased concentrations of pro-inflammatory chemokines CXCL10 and CCL5 in infected tissues incubated with E2. Our results suggest a link between E2-mediated anti-HIV-1 activity and expression of CFL1 in PBMCs and endocervical mucosa. The data support exploration of cytoskeletal signaling pathway targets for the development of prevention strategies against HIV-1.