Nicotinamide mononucleotide impacts HIV-1 infection by modulating immune activation in T lymphocytes and humanized mice.

BACKGROUND: HIV-1-associated immune activation drives CD4+ T cell depletion and the development of acquired immunodeficiency syndrome. We aimed to determine the role of nicotinamide mononucleotide (NMN), the direct precursor of nicotinamide adenine dinucleotide (NAD) co-enzyme, in CD4+ T cell modulation during HIV-1 infection. METHODS: We examined HIV-1 integrated DNA or transcribed RNA, intracellular p24 protein, and T cell activation markers in CD4+ T cells including in vitro HIV-1-infected cells, reactivated patient-derived cells, and in HIV-1-infected humanized mice, under NMN treatment. RNA-seq and CyTOF analyses were used for investigating the effect of NMN on CD4+ T cells. FINDINGS: We found that NMN increased the intracellular NAD amount, resulting in suppressed HIV-1 p24 production and proliferation in infected CD4+ T cells, especially in activated CD25+CD4+ T cells. NMN also inhibited CD25 expression on reactivated resting CD4+ T cells derived from cART-treated people living with HIV-1 (PLWH). In HIV-1-infected humanized mice, the frequency of CD4+ T cells was reconstituted significantly by combined cART and NMN treatment as compared with cART or NMN alone, which correlated with suppressed hyperactivation of CD4+ T cells. INTERPRETATION: Our results highlight the suppressive role of NMN in CD4+ T cell activation during HIV-1 infection. It warrants future clinical investigation of NMN as a potential treatment in combination with cART in PLWH. FUNDING: This work was supported by the Hong Kong Research Grants Council Theme-Based Research Scheme (T11-706/18-N), University Research Committee of The University of Hong Kong, the Collaborative Research with GeneHarbor (Hong Kong) Biotechnologies Limited and National Key R&D Program of China (Grant2021YFC2301900).

A follow-up study: 6-year cART-free virologic control of rhesus macaques after PD-1-based DNA vaccination against pathogenic SHIVSF162P3CN challenge.

IMPORTANCE: Efficient strategies for HIV-1 cART-free virologic control are critical for ending the AIDS pandemic. The essential role of effector-memory CD8+ T cells in controlling viremia and eliminating virus-infected cells has made them a promising target for vaccine development. It has been previously reported that PD-1-based DNA vaccination was effective in inducing polyfunctional effector-memory CD8+ T cells for AIDS virus control for 2 years in rhesus monkeys. This follow-up study extends the findings and shows that a viremia-free period of over 6 years was detected in two monkeys immunized with PD-1-based DNA vaccine against pathogenic SHIVSF162P3CN infection in the absence of antiretroviral therapy. Long-term vaccine-induced memory T cell responses were detected. Our results warrant the clinical trials of PD-1-based DNA vaccines for achieving HIV-1 cART-free virologic control used either alone or in combination with other biomedical interventions.

Transforming Growth Factor ß Signaling Promotes HIV-1 Infection in Activated and Resting Memory CD4+ T Cells.

Understanding the facilitator of HIV-1 infection and subsequent latency establishment may aid the discovery of potential therapeutic targets. Here, we report the elevation of plasma transforming growth factor ß (TGF-ß) during acute HIV-1 infection among men who have sex with men (MSM). Using a serum-free in vitro system, we further delineated the role of TGF-ß signaling in mediating HIV-1 infection of activated and resting memory CD4+ T cells. TGF-ß could upregulate both the frequency and expression of the HIV-1 coreceptor CCR5, thereby augmenting CCR5-tropic viral infection of resting and activated memory CD4+ T cells via Smad3 activation. The production of live HIV-1JR-FL upon infection and reactivation was increased in TGF-ß-treated resting memory CD4+ T cells without increasing CD4 expression or inducing T cell activation. The expression of CCR7, a central memory T cell marker that serves as a chemokine receptor to facilitate T cell trafficking into lymphoid organs, was also elevated on TGF-ß-treated resting and activated memory CD4+ T cells. Moreover, the expression of CXCR3, a chemokine receptor recently reported to facilitate CCR5-tropic HIV-1 infection, was increased on resting and activated memory CD4+ T cells upon TGF-ß treatment. These findings were coherent with the observation that ex vivo CCR5 and CXCR3 expression on total resting and resting memory CD4+ T cells in combination antiretroviral therapy (cART)-naive and cART-treated patients were higher than in healthy individuals. Overall, the study demonstrated that TGF-ß upregulation induced by acute HIV-1 infection might promote latency reservoir establishment by increasing infected resting memory CD4+ T cells and lymphoid organ homing of infected central memory CD4+ T cells. Therefore, TGF-ß blockade may serve as a potential supplementary regimen for HIV-1 functional cure by reducing viral latency. IMPORTANCE Incomplete eradication of HIV-1 latency reservoirs remains the major hurdle in achieving a complete HIV/AIDS cure. Dissecting the facilitator of latency reservoir establishment may aid the discovery of druggable targets for HIV-1 cure. This study showed that the T cell immunomodulatory cytokine TGF-ß was upregulated during the acute phase of infection. Using an in vitro serum-free system, we specifically delineated that TGF-ß promoted HIV-1 infection of both resting and activated memory CD4+ T cells via the induction of host CCR5 coreceptor. Moreover, TGF-ß-upregulated CCR7 or CXCR3 might promote HIV-1 latent infection by facilitating lymphoid homing or IP-10-mediated viral entry and DNA integration, respectively. Infected resting and central memory CD4+ T cells are important latency reservoirs. Increased infection of these cells mediated by TGF-ß will promote latency reservoir establishment during early infection. This study, therefore, highlighted the potential use of TGF-ß blockade as a supplementary regimen with cART in acute patients to reduce viral latency.

Mitochondrial regulation of acute extrafollicular B-cell responses to COVID-19 severity.

BACKGROUND: Patients with COVID-19 display a broad spectrum of manifestations from asymptomatic to life-threatening disease with dysregulated immune responses. Mechanisms underlying the detrimental immune responses and disease severity remain elusive. METHODS: We investigated a total of 137 APs infected with SARS-CoV-2. Patients were divided into mild and severe patient groups based on their requirement of oxygen supplementation. All blood samples from APs were collected within three weeks after symptom onset. Freshly isolated PBMCs were investigated for B cell subsets, their homing potential, activation state, mitochondrial functionality and proliferative response. Plasma samples were tested for cytokine concentration, and titer of Nabs, RBD-, S1-, SSA/Ro- and dsDNA-specific IgG. RESULTS: While critically ill patients displayed predominantly extrafollicular B cell activation with elevated inflammation, mild patients counteracted the disease through the timely induction of mitochondrial dysfunction in B cells within the first week post symptom onset. Rapidly increased mitochondrial dysfunction, which was caused by infection-induced excessive intracellular calcium accumulation, suppressed excessive extrafollicular responses, leading to increased neutralizing potency index and decreased inflammatory cytokine production. Patients who received prior COVID-19 vaccines before infection displayed significantly decreased extrafollicular B cell responses and mild disease. CONCLUSION: Our results reveal an immune mechanism that controls SARS-CoV-2-induced detrimental B cell responses and COVID-19 severity, which may have implications for viral pathogenesis, therapeutic interventions and vaccine development.

Imaging and analysis on the interaction between human antigen-pulsed Vδ2 T cells and antigen-specific CD4 T cells.

This protocol describes how to visualize surface protein-protein co-localization across a cell-cell interface between antigen-presenting γδ-T cells and CD4 T cells. By consolidating immunofluorescence assay, confocal microscopy and 3D imaging analysis, it enables assessment of interaction between cell surface proteins such as Δ42PD1 and TLR4 between co-cultured γδ-T and CD4 T cells. This protocol can be applied to study a surface protein of interest and its potential interaction with a target cell/protein at the cell-cell interface. For complete details on the use and execution of this profile, please refer to Mo et al. (2020).

Mitochondrial Dysfunction Associates With Acute T Lymphocytopenia and Impaired Functionality in COVID-19 Patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection results in rapid T lymphocytopenia and functional impairment of T cells. The underlying mechanism, however, remains incompletely understood. In this study, we focused on characterizing the phenotype and kinetics of T-cell subsets with mitochondrial dysfunction (MD) by multicolor flow cytometry and investigating the association between MD and T-cell functionality. While 73.9% of study subjects displayed clinical lymphocytopenia upon hospital admission, a significant reduction of CD4 or CD8 T-cell frequency was found in all asymptomatic, symptomatic, and convalescent cases. CD4 and CD8 T cells with increased MD were found in both asymptomatic and symptomatic patients within the first week of symptom onset. Lower proportion of memory CD8 T cell with MD was found in severe patients than in mild ones at the stage of disease progression. Critically, the frequency of T cells with MD in symptomatic patients was preferentially associated with CD4 T-cell loss and CD8 T-cell hyperactivation, respectively. Patients bearing effector memory CD4 and CD8 T cells with the phenotype of high MD exhibited poorer T-cell responses upon either phorbol 12-myristate-13-acetate (PMA)/ionomycin or SARS-CoV-2 peptide stimulation than those with low MD. Our findings demonstrated an MD-associated mechanism underlying SARS-CoV-2-induced T lymphocytopenia and functional impairment during the acute phase of infection.

Δ42PD1-TLR4 Augments γδ-T Cell Activation of the Transitional Memory Subset of CD4+ T Cells.

TLR ligands can contribute to T cell immune responses by indirectly stimulating antigen presentation and cytokines and directly serving as co-stimulatory signals. We have previously reported that the human endogenous surface protein, Δ42PD1, is expressed primarily on (Vγ9)Vδ2 cells and can interact with TLR4. Since Vδ2 cells possess antigen presentation capacity, we sought to further characterize if the Δ42PD1-TLR4 interaction has a role in stimulating T cell responses. In this study, we found that stimulation of Vδ2 cells not only upregulated Δ42PD1 expression but also increased MHC class II molecules necessary for the antigen presentation. In a mixed leukocyte reaction assay, upregulation of Δ42PD1 on Vδ2 cells elevated subsequent T cell proliferation. Furthermore, the interaction between Δ42PD1-TLR4 augments Vδ2 cell stimulation of autologous CMV pp65-or TT-specific CD4+ T cell proliferation and IFN-γ responses, which was specifically and significantly reduced by blocking the Δ42PD1-TLR4 interaction. Furthermore, confocal microscopy analysis confirmed the interaction between Δ42PD1+HLA-DR+Vδ2 cells and TLR4+CD4 T cells. Interestingly, the subset of CD4+ T cells expressing TLR4 appears to be PD-1+ CD45RO+CD45RA+ transitional memory T cells and responded to Δ42PD1+HLA-DR+Vδ2 cells. Overall, this study demonstrated an important biological role of Δ42PD1 protein exhibited by Vδ2 antigen-presenting cells in augmenting T cell activation through TLR4, which may serve as an additional co-stimulatory signal.

Viruses harness YxxØ motif to interact with host AP2M1 for replication: A vulnerable broad-spectrum antiviral target.

Targeting a universal host protein exploited by most viruses would be a game-changing strategy that offers broad-spectrum solution and rapid pandemic control including the current COVID-19. Here, we found a common YxxØ-motif of multiple viruses that exploits host AP2M1 for intracellular trafficking. A library chemical, N-(p-amylcinnamoyl)anthranilic acid (ACA), was identified to interrupt AP2M1-virus interaction and exhibit potent antiviral efficacy against a number of viruses in vitro and in vivo, including the influenza A viruses (IAVs), Zika virus (ZIKV), human immunodeficiency virus, and coronaviruses including MERS-CoV and SARS-CoV-2. YxxØ mutation, AP2M1 depletion, or disruption by ACA causes incorrect localization of viral proteins, which is exemplified by the failure of nuclear import of IAV nucleoprotein and diminished endoplasmic reticulum localization of ZIKV-NS3 and enterovirus-A71-2C proteins, thereby suppressing viral replication. Our study reveals an evolutionarily conserved mechanism of protein-protein interaction between host and virus that can serve as a broad-spectrum antiviral target.

Acute SARS-CoV-2 Infection Impairs Dendritic Cell and T Cell Responses.

The SARS-CoV-2 pandemic has resulted in millions of infections, yet the role of host immune responses in early COVID-19 pathogenesis remains unclear. By investigating 17 acute and 24 convalescent patients, we found that acute SARS-CoV-2 infection resulted in broad immune cell reduction including T, natural killer, monocyte, and dendritic cells (DCs). DCs were significantly reduced with functional impairment, and ratios of conventional DCs to plasmacytoid DCs were increased among acute severe patients. Besides lymphocytopenia, although neutralizing antibodies were rapidly and abundantly generated in patients, there were delayed receptor binding domain (RBD)- and nucleocapsid protein (NP)-specific T cell responses during the first 3 weeks after symptoms onset. Moreover, acute RBD- and NP-specific T cell responses included relatively more CD4 T cells than CD8 T cells. Our findings provided evidence that impaired DCs, together with timely inverted strong antibody but weak CD8 T cell responses, could contribute to acute COVID-19 pathogenesis and have implications for vaccine development.

Schisandra sphenanthera extract (Wuzhi Tablet) protects against chronic-binge and acute alcohol-induced liver injury by regulating the NRF2-ARE pathway in mice.

Alcohol abuse leads to alcoholic liver disease and no effective therapy is currently available. Wuzhi Tablet (WZ), a preparation of extract from Schisandra sphenanthera that is a traditional hepato-protective herb, exerted a significant protective effect against acetaminophen-induced liver injury in our recent studies, but whether WZ can alleviate alcohol-induced toxicity remains unclear. This study aimed to investigate the contribution of WZ to alcohol-induced liver injury by using chronic-binge and acute models of alcohol feeding. The activities of ALT and AST in serum were assessed as well as the level of GSH and the activity of SOD in the liver. The expression of CYP2E1 and proteins in the NRF2-ARE signaling pathway including NRF2, GCLC, GCLM, HO-1 were measured, and the effect of WZ on NRF2 transcriptional activity was determined. We found that both models resulted in liver steatosis accompanied by increased transaminase activities, but that liver injury was significantly attenuated by WZ. WZ administration also inhibited CYP2E1 expression induced by alcohol, and elevated the level of GSH and the activity of SOD in the liver. Moreover, the NRF2-ARE signaling pathway was activated by WZ and the target genes were all upregulated. Furthermore, WZ significantly activated NRF2 transcriptional activity. Collectively, our study demonstrates that WZ protected against alcohol-induced liver injury by reducing oxidative stress and improving antioxidant defense, possibly by activating the NRF2-ARE pathway.

Gut-homing Δ42PD1+Vδ2 T cells promote innate mucosal damage via TLR4 during acute HIV type 1 infection.

The innate immune cells underlying mucosal inflammatory responses and damage during acute HIV-1 infection remain incompletely understood. Here, we report a Vδ2 subset of gut-homing γδ T cells with significantly upregulated Δ42PD1 (a PD1 isoform) in acute (~20%) HIV-1 patients compared to chronic HIV-1 patients (~11%) and healthy controls (~2%). The frequency of Δ42PD1+Vδ2 cells correlates positively with plasma levels of pro-inflammatory cytokines and fatty-acid-binding protein before detectable lipopolysaccharide in acute patients. The expression of Δ42PD1 can be induced by in vitro HIV-1 infection and is accompanied by high co-expression of gut-homing receptors CCR9/CD103. To investigate the role of Δ42PD1+Vδ2 cells in vivo, they were adoptively transferred into autologous humanized mice, resulting in small intestinal inflammatory damage, probably due to the interaction of Δ42PD1 with its cognate receptor Toll-like receptor 4 (TLR4). In addition, blockade of Δ42PD1 or TLR4 successfully reduced the cytokine effect induced by Δ42PD1+Vδ2 cells in vitro, as well as the mucosal pathological effect in humanized mice. Our findings have therefore uncovered a Δ42PD1-TLR4 pathway exhibited by virus-induced gut-homing Vδ2 cells that may contribute to innate immune activation and intestinal pathogenesis during acute HIV-1 infection. Δ42PD1+Vδ2 cells may serve as a target for the investigation of diseases with mucosal inflammation.

Effect of Ginkgo Biloba Extract on the Pharmacokinetics and Metabolism of Clopidogrel in Rats.

Ginkgo biloba extract (GBE), a traditional herbal product used worldwide as both medicine and supplement, is often supplied with clopidogrel for the treatment of cerebrovascular diseases. The aim of the current study was to explore the effect of GBE on the metabolism and pharmacokinetics of clopidogrel. The in vitro study using rat liver microsomes revealed that GBE significantly induced the conversion of clopidogrel into its active metabolite. The effect of GBE on the pharmacokinetics of clopidogrel was also investigated in vivo. Compared to rats without GBE pretreatment, administration of 4 mg/kg, 20 mg/kg, and 100 mg/kg of GBE significantly decreased the Cmax and the AUC0-∞ of clopidogrel in a dose-dependent manner. As expected, pretreatment of high dose GBE significantly increased the Cmax and AUC0-∞ of the clopidogrel active metabolite. However, no marked change was observed following medium and low dose of GBE, suggesting that the biotransformation of clopidogrel was altered differently by high dose of GBE. Our study suggested that the awareness of the potential herb-drug interactions between GBE and clopidogrel should be increased in clinical practice. Copyright © 2016 John Wiley & Sons, Ltd.

Graphene/Ionic liquid composite films and ion exchange.

Wettability of graphene is adjusted by the formation of various ionic surfaces combining ionic liquid (IL) self-assembly with ion exchange. The functionalized ILs were designed and synthesized with the goal of obtaining adjustable wettability. The wettability of the graphene surface bearing various anions was measured systematically. The effect of solvent systems on ion exchange ratios on the graphene surface has also been investigated. Meanwhile, the mechanical properties of the graphene/IL composite films were investigated on a nanometer scale. The elasticity and adhesion behavior of the thin film was determined with respected to the indentation deformation by colloid probe nanoindentation method. The results indicate that anions played an important role in determining graphene/IL composite film properties. In addition, surface wetting and mechanics can be quantitatively determined according to the counter-anions on the surface. This study might suggest an alternate way for quantity detection of surface ions by surface force.

Induced marine fungus Chondrostereum sp. as a means of producing new sesquiterpenoids chondrosterins I and J by using glycerol as the carbon source.

Chondrostereum sp., a marine fungus isolated from a soft coral Sarcophyton tortuosum, can yield hirsutane framework sesquiterpenoids. However, the metabolites profiles vary dramatically with the composition change of the culture media. This fungus was cultured in a liquid medium containing glycerol as the carbon source, and two new metabolites, chondrosterins I and J (1 and 2), were obtained. Their structures were elucidated primarily based on MS, NMR and X-ray single-crystal diffraction data. By comparison with the known hirsutane sesquiterpenoids, chondrosterins I and J have unique structural features, including a methyl was migrated from C-2 to C-6, and the methyl at C-3 was carboxylated. Compound 2 exhibited potent cytotoxic activities against the cancer cell lines CNE-1 and CNE-2 with the IC50 values of 1.32 and 0.56 µM.

Fabrication of novel graphene-fullerene hybrid lubricating films based on self-assembly for MEMS applications.

The novel graphene-C60 hybrid films have been fabricated successfully on silicon surfaces by a multistep self-assembly process, and showed synergistic effects beyond individual performance in micro/nano-tribological behaviors. It is expected that the graphene-C60 hybrid films may find wide applications as high performance lubricating films in MEMS.

Preparation and mechanics of nanotextures on adapting a low adhesive surface using local oxidation nanolithography.

This paper describes an application for atomic force microscopy to the fabrication of nanotextures with various features on a GaAs surface by local oxidation nanolithography (LON). By controlling the geometrical shapes and surface coverage of the nanotexture, the surface adhesion can be adjusted to a low adhesive surface. The influence of environmental conditions, such as relative humidity and temperature on adhesion behavior, was studied. An optic heater was employed to minimize thermal effect on an atomic force microscope (AFM) cantilever and PbZrTiO3 scanner. In our study, AFM is used for both fabrication and characterization. LON allows the fabricated nanotextures to be altered in situ without the need to change masks or repeat the entire fabrication process. Furthermore, the nanoadhesion characterization of nanotextures on a GaAs surface was investigated with a colloidal probe method.

Preparation and characterization of ultrathin dual-layer ionic liquid lubrication film assembled on silica surfaces.

A novel ultrathin dual-layer film, which contained both bonded and mobile phases in ionic liquids (ILs) layer, was fabricated successfully on a silicon substrate modified by a self-assembled monolayer (SAM). The formation and surface properties of the films were analyzed using ellipsometer, water contact angle meter, attenuated total reflectance Fourier transform infrared spectroscopy, multi-functional X-ray photoelectron spectroscopy, and atomic force microscope. Meanwhile, the adhesive and nanotribological behaviors of the films were evaluated by a homemade colloidal probe. A ball-on-plate tribometer was used to evaluate the microtribological performances of the films. Compared with the single-layer ILs film deposited directly on the silicon surface, the as-prepared dual-layer film shows the improved tribological properties, which is attributed to the special chemical structure and outstanding physical properties of the dual-layer film, i.e., the strong adhesion between bonded phase of ILs and silicon substrate via the chemical bonding with SAM, the interlinked hydrogen bonds among the molecules, and two-phase structure composed of steady bonded phase with load-carrying capacity and flowable mobile phase with self-replenishment property.

Fabrication and adhesion of biomimetic nanotextures fabricated by local oxidation nanolithography.

Functional surfaces with biomimetic nanotexture have aroused much interest because of their great advantages in applications. The nanometer-sized biomimetic textures are fabricated using current induced local anodic oxide (LAO) method. By controlling pulsed bias voltage, pulsewidth and relative humidity, the dimensions of biomimetic textures can be precisely controlled. In our study, an atomic force microscopy (AFM) is used for both fabrication and characterization. Conductive AFM allows fabrication process of biomimetic nanotexture without the need to change masks or repeat entire fabrication process. Furthermore, the adhesive characterization of the biomimetic nanotexture was investigated by a colloidal probe.

Nanotribological properties of precision-controlled regular nanotexture on H-passivated Si surface by current-induced local anodic oxidation.

Nano-sized textures resulted from localized electrochemical oxidation by using atomic force microscopy (AFM) were fabricated on H-passivated Si surface. In this paper, the fabrication and nanotribological properties of nanotexture by local anodic oxidation (LAO) on H-passivated Si surface are presented. A special attention is paid to find the relation between the size of oxide nanotexture and operational parameters such as tip-sample pulsed bias voltage, pulsewidth, and relative humidity to fabricate oxide nanotexture. The nanotribological properties were investigated by a colloidal probe. The results indicate that the nanotextures exhibited low adhesion and greatly reduced friction force at nanometer scale.

Precise positioning of lubricant on a surface using the local anodic oxide method.

This letter describes a new method for the precise positioning of a lubricant on a surface. The nanometer-sized patterns are first fabricated using the atomic force microscopy (AFM)-based local anodic oxide (LAO) method. Multiply alkylated cyclopentanes (MACs) serving as lubricant matrix layers on nanopatterns of silicon dioxide are fabricated. By controlling the velocity of pull-off and solution conditions, we selectively immobilize MACs on the patterned areas using the dip-coating method. In our study, AFM is used for both fabrication and characterization. AFM-LAO allows the fabricated patterns to be altered in situ without the need to change masks or repeat the entire fabrication process. Furthermore, the nanotribological characterization of lubricant matrix layers on the nanopatterns was investigated with a colloidal probe.