Altered Immunoglobulin Repertoire and Decreased IgA Somatic Hypermutation in the Gut during Chronic HIV-1 Infection.

Humoral immune perturbations contribute to pathogenic outcomes in persons with HIV-1 infection (PWH). Gut barrier dysfunction in PWH is associated with microbial translocation and alterations in microbial communities (dysbiosis), and IgA, the most abundant immunoglobulin (Ig) isotype in the gut, is involved in gut homeostasis by interacting with the microbiome. We determined the impact of HIV-1 infection on the antibody repertoire in the gastrointestinal tract by comparing Ig gene utilization and somatic hypermutation (SHM) in colon biopsies from PWH (n = 19) versus age and sex-matched controls (n = 13). We correlated these Ig parameters with clinical, immunological, microbiome and virological data. Gene signatures of enhanced B cell activation were accompanied by skewed frequencies of multiple Ig Variable genes in PWH. PWH showed decreased frequencies of SHM in IgA and possibly IgG, with a substantial loss of highly mutated IgA sequences. The decline in IgA SHM in PWH correlated with gut CD4+ T cell loss and inversely correlated with mucosal inflammation and microbial translocation. Diminished gut IgA SHM in PWH was driven by transversion mutations at A or T deoxynucleotides, suggesting a defect not at the AID/APOBEC3 deamination step but at later stages of IgA SHM. These results expand our understanding of humoral immune perturbations in PWH that could have important implications in understanding mucosal immune defects in individuals with chronic HIV-1 infection. IMPORTANCE The gut is a major site of early HIV-1 replication and pathogenesis. Extensive CD4+ T cell depletion in this compartment results in a compromised epithelial barrier that facilitates the translocation of microbes into the underlying lamina propria and systemic circulation, resulting in chronic immune activation. To date, the consequences of microbial translocation on the mucosal humoral immune response (or vice versa) remains poorly integrated into the panoply of mucosal immune defects in PWH. We utilized next-generation sequencing approaches to profile the Ab repertoire and ascertain frequencies of somatic hypermutation in colon biopsies from antiretroviral therapy-naive PWH versus controls. Our findings identify perturbations in the Ab repertoire of PWH that could contribute to development or maintenance of dysbiosis. Moreover, IgA mutations significantly decreased in PWH and this was associated with adverse clinical outcomes. These data may provide insight into the mechanisms underlying impaired Ab-dependent gut homeostasis during chronic HIV-1 infection.

Gut Bacteria Induce Granzyme B Expression in Human Colonic ILC3s In Vitro in an IL-15-Dependent Manner.

Group 3 innate lymphoid cells (ILC3s) in the gut mucosa have long been thought to be noncytotoxic lymphocytes that are critical for homeostasis of intestinal epithelial cells through secretion of IL-22. Recent work using human tonsillar cells demonstrated that ILC3s exposed to exogenous inflammatory cytokines for a long period of time acquired expression of granzyme B, suggesting that under pathological conditions ILC3s may become cytotoxic. We hypothesized that inflammation associated with bacterial exposure might trigger granzyme B expression in gut ILC3s. To test this, we exposed human colon lamina propria mononuclear cells to a panel of enteric bacteria. We found that the Gram-negative commensal and pathogenic bacteria induced granzyme B expression in a subset of ILC3s that were distinct from IL-22-producing ILC3s. A fraction of granzyme B+ ILC3s coexpressed the cytolytic protein perforin. Granzyme B expression was mediated, in part, by IL-15 produced upon exposure to bacteria. ILC3s coexpressing all three IL-15R subunits (IL15Rα/ß/γ) increased following bacterial stimulation, potentially allowing for cis presentation of IL-15 during bacterial exposure. Additionally, a large frequency of colonic myeloid dendritic cells expressed IL-15Rα, implicating myeloid dendritic cells in trans presentation of IL-15 to ILC3s. Tonsillar ILC3s minimally expressed granzyme B when exposed to the same bacteria or to rIL-15. Overall, these data establish the novel, to our knowledge, finding that human colonic ILC3s can express granzyme B in response to a subset of enteric bacteria through a process mediated by IL-15. These observations raise new questions about the multifunctional role of human gut ILC3s.

Qualitative Differences Between the IFNα subtypes and IFNß Influence Chronic Mucosal HIV-1 Pathogenesis.

The Type I Interferons (IFN-Is) are innate antiviral cytokines that include 12 different IFNα subtypes and IFNß that signal through the IFN-I receptor (IFNAR), inducing hundreds of IFN-stimulated genes (ISGs) that comprise the 'interferome'. Quantitative differences in IFNAR binding correlate with antiviral activity, but whether IFN-Is exhibit qualitative differences remains controversial. Moreover, the IFN-I response is protective during acute HIV-1 infection, but likely pathogenic during the chronic stages. To gain a deeper understanding of the IFN-I response, we compared the interferomes of IFNα subtypes dominantly-expressed in HIV-1-exposed plasmacytoid dendritic cells (1, 2, 5, 8 and 14) and IFNß in the earliest cellular targets of HIV-1 infection. Primary gut CD4 T cells from 3 donors were treated for 18 hours ex vivo with individual IFN-Is normalized for IFNAR signaling strength. Of 1,969 IFN-regulated genes, 246 'core ISGs' were induced by all IFN-Is tested. However, many IFN-regulated genes were not shared between the IFNα subtypes despite similar induction of canonical antiviral ISGs such as ISG15, RSAD2 and MX1, formally demonstrating qualitative differences between the IFNα subtypes. Notably, IFNß induced a broader interferome than the individual IFNα subtypes. Since IFNß, and not IFNα, is upregulated during chronic HIV-1 infection in the gut, we compared core ISGs and IFNß-specific ISGs from colon pinch biopsies of HIV-1-uninfected (n = 13) versus age- and gender-matched, antiretroviral-therapy naïve persons with HIV-1 (PWH; n = 19). Core ISGs linked to inflammation, T cell activation and immune exhaustion were elevated in PWH, positively correlated with plasma lipopolysaccharide (LPS) levels and gut IFNß levels, and negatively correlated with gut CD4 T cell frequencies. In sharp contrast, IFNß-specific ISGs linked to protein translation and anti-inflammatory responses were significantly downregulated in PWH, negatively correlated with gut IFNß and LPS, and positively correlated with plasma IL6 and gut CD4 T cell frequencies. Our findings reveal qualitative differences in interferome induction by diverse IFN-Is and suggest potential mechanisms for how IFNß may drive HIV-1 pathogenesis in the gut.

Gut Innate Immunity and HIV Pathogenesis.

PURPOSE OF REVIEW: In the gastro-intestinal tract, the complex network of multiple innate cell populations play critical roles not only as a first line of defense against invading pathogens and in driving adaptive immune responses but also in maintaining intestinal homeostasis. Here, we describe the roles of various innate immune cell populations in gut immunity and detail studies investigating the impact of acute and chronic HIV infection on these cell populations. RECENT FINDINGS: Alterations in frequencies, phenotype and/or function of innate lymphoid cells, dendritic cells, macrophages, neutrophils, and innate-like T cells have been reported in people with HIV (PWH), with many of these features persisting despite anti-retroviral therapy and virological suppression. Dysregulated gut innate immunity in PWH is a feature of gut pathogenesis. A greater understanding of the mechanisms driving impairment in the multiple different gut innate immune cell populations and the downstream consequences of an altered innate immune response on host defense and gut homeostasis in PWH is needed to develop more effective HIV treatments and cure strategies.

Reduced immune-regulatory molecule expression on human colonic memory CD4 T cells in older adults.

BACKGROUND: The etiology of the low-level chronic inflammatory state associated with aging is likely multifactorial, but a number of animal and human studies have implicated a functional decline of the gastrointestinal immune system as a potential driver. Gut tissue-resident memory T cells play critical roles in mediating protective immunity and in maintaining gut homeostasis, yet few studies have investigated the effect of aging on human gut T cell immunity. To determine if aging impacted CD4 T cell immunity in the human large intestine, we utilized multi-color flow cytometry to measure colonic lamina propria (LP) CD4 T cell frequencies and immune-modulatory marker expression in younger (mean ± SEM: 38 ± 1.5 yrs) and older (77 ± 1.6 yrs) adults. To determine cellular specificity, we evaluated colon LP CD8 T cell frequency and phenotype in the same donors. To probe tissue specificity, we evaluated the same panel of markers in peripheral blood (PB) CD4 T cells in a separate cohort of similarly aged persons. RESULTS: Frequencies of colonic CD4 T cells as a fraction of total LP mononuclear cells were higher in older persons whereas absolute numbers of colonic LP CD4 T cells per gram of tissue were similar in both age groups. LP CD4 T cells from older versus younger persons exhibited reduced CTLA-4, PD-1 and Ki67 expression. Levels of Bcl-2, CD57, CD25 and percentages of activated CD38+HLA-DR+ CD4 T cells were similar in both age groups. In memory PB CD4 T cells, older age was only associated with increased CD57 expression. Significant age effects for LP CD8 T cells were only observed for CTLA-4 expression, with lower levels of expression observed on cells from older adults. CONCLUSIONS: Greater age was associated with reduced expression of the co-inhibitory receptors CTLA-4 and PD-1 on LP CD4 T cells. Colonic LP CD8 T cells from older persons also displayed reduced CTLA-4 expression. These age-associated profiles were not observed in older PB memory CD4 T cells. The decline in co-inhibitory receptor expression on colonic LP T cells may contribute to local and systemic inflammation via a reduced ability to limit ongoing T cell responses to enteric microbial challenge.

Spectroscopic Investigation of Cysteamine Dioxygenase.

Thiol dioxygenases are mononuclear non-heme FeII-dependent metalloenzymes that initiate the oxidative catabolism of thiol-containing substrates to their respective sulfinates. Cysteine dioxygenase (CDO), the best characterized mammalian thiol dioxygenase, contains a three-histidine (3-His) coordination environment rather than the 2-His-1-carboxylate facial triad seen in most mononuclear non-heme FeII enzymes. A similar 3-His active site is found in the bacterial thiol dioxygenase 3-mercaptopropionate dioxygenase (MDO), which converts 3-mercaptopropionate into 3-sulfinopropionic acid as part of the bacterial sulfur metabolism pathway. In this study, we have investigated the active site geometric and electronic structures of a third non-heme FeII-dependent thiol dioxygenase, cysteamine dioxygenase (ADO), by using a spectroscopic approach. Although a 3-His facial triad had previously been implicated on the basis of sequence alignment and site-directed mutagenesis studies, little is currently known about the active site environment of ADO. Our magnetic circular dichroism and electron paramagnetic resonance data provide compelling evidence that ADO features a 3-His facial triad, like CDO and MDO. Despite this similar coordination environment, spectroscopic results obtained for ADO incubated with various substrate analogues are distinct from those obtained for the other FeII-dependent thiol dioxygenases. This finding suggests that the secondary coordination sphere of ADO is distinct from those of CDO and MDO, demonstrating the significant role that secondary-sphere residues play in dictating substrate specificity.

The transcriptome of HIV-1 infected intestinal CD4+ T cells exposed to enteric bacteria.

Global transcriptome studies can help pinpoint key cellular pathways exploited by viruses to replicate and cause pathogenesis. Previous data showed that laboratory-adapted HIV-1 triggers significant gene expression changes in CD4+ T cell lines and mitogen-activated CD4+ T cells from peripheral blood. However, HIV-1 primarily targets mucosal compartments during acute infection in vivo. Moreover, early HIV-1 infection causes extensive depletion of CD4+ T cells in the gastrointestinal tract that herald persistent inflammation due to the translocation of enteric microbes to the systemic circulation. Here, we profiled the transcriptome of primary intestinal CD4+ T cells infected ex vivo with transmitted/founder (TF) HIV-1. Infections were performed in the presence or absence of Prevotella stercorea, a gut microbe enriched in the mucosa of HIV-1-infected individuals that enhanced both TF HIV-1 replication and CD4+ T cell death ex vivo. In the absence of bacteria, HIV-1 triggered a cellular shutdown response involving the downregulation of HIV-1 reactome genes, while perturbing genes linked to OX40, PPAR and FOXO3 signaling. However, in the presence of bacteria, HIV-1 did not perturb these gene sets or pathways. Instead, HIV-1 enhanced granzyme expression and Th17 cell function, inhibited G1/S cell cycle checkpoint genes and triggered downstream cell death pathways in microbe-exposed gut CD4+ T cells. To gain insights on these differential effects, we profiled the gene expression landscape of HIV-1-uninfected gut CD4+ T cells exposed to bacteria. Microbial exposure upregulated genes involved in cellular proliferation, MAPK activation, Th17 cell differentiation and type I interferon signaling. Our findings reveal that microbial exposure influenced how HIV-1 altered the gut CD4+ T cell transcriptome, with potential consequences for HIV-1 susceptibility, cell survival and inflammation. The HIV-1- and microbe-altered pathways unraveled here may serve as a molecular blueprint to gain basic insights in mucosal HIV-1 pathogenesis.

Inside Out: HIV, the Gut Microbiome, and the Mucosal Immune System.

The components of the human gut microbiome have been found to influence a broad array of pathologic conditions ranging from heart disease to diabetes and even to cancer. HIV infection upsets the delicate balance in the normal host-microbe interaction both through alterations in the taxonomic composition of gut microbial communities as well as through disruption of the normal host response mechanisms. In this article we review the current methods of gut microbiome analysis and the resulting data regarding how HIV infection might change the balance of commensal bacteria in the gut. Additionally, we cover the various effects gut microbes have on host immune homeostasis and the preliminary but intriguing data on how HIV disrupts those mechanisms. Finally, we briefly describe some of the important biomolecules produced by gut microbiota and the role that they may play in maintaining host immune homeostasis with and without HIV infection.

The Role of Mixed Amine/Amide Ligation in Nickel Superoxide Dismutase.

Superoxide dismutases (SODs) utilize a ping-pong mechanism in which a redox-active metal cycles between oxidized and reduced forms that differ by one electron to catalyze the disproportionation of superoxide to dioxygen and hydrogen peroxide. Nickel-dependent SOD (NiSOD) is a unique biological solution for controlling superoxide levels. This enzyme relies on the use of cysteinate ligands to bring the Ni(III/II) redox couple into the range required for catalysis (∼300 mV vs. NHE). The use of cysteine thiolates, which are not found in any other SOD, is a curious choice because of their well-known oxidation by peroxide and dioxygen. The NiSOD active site cysteinate ligands are resistant to oxidation, and prior studies of synthetic and computational models point to the backbone N-donors in the active site (the N-terminal amine and the amide N atom of Cys2) as being involved in stabilizing the cysteines to oxidation. To test the role of the backbone N-donors, we have constructed a variant of NiSOD wherein an alanine residue was added to the N-terminus (Ala0-NiSOD), effectively altering the amine ligand to an amide. X-ray absorption, electronic absorption, and magnetic circular dichroism (MCD) spectroscopic analyses of as-isolated Ala0-NiSOD coupled with density functional theory (DFT) geometry optimized models that were evaluated on the basis of the spectroscopic data within the framework of DFT and time-dependent DFT computations are consistent with a diamagnetic Ni(II) site with two cysteinate, one His1 amide, and one Cys2 amidate ligands. The variant protein is catalytically inactive, has an altered electronic absorption spectrum associated with the nickel site, and is sensitive to oxidation. Mass spectrometric analysis of the protein exposed to air shows the presence of a mixture of oxidation products, the principal ones being a disulfide, a bis-sulfenate, and a bis-sulfinate derived from the active site cysteine ligands. Details of the electronic structure of the Ni(III) site available from the DFT calculations point to subtle changes in the unpaired spin density on the S-donors as being responsible for the altered sensitivity of Ala0-NiSOD to O2.

Interferon-α Subtypes in an Ex Vivo Model of Acute HIV-1 Infection: Expression, Potency and Effector Mechanisms.

HIV-1 is transmitted primarily across mucosal surfaces and rapidly spreads within the intestinal mucosa during acute infection. The type I interferons (IFNs) likely serve as a first line of defense, but the relative expression and antiviral properties of the 12 IFNα subtypes against HIV-1 infection of mucosal tissues remain unknown. Here, we evaluated the expression of all IFNα subtypes in HIV-1-exposed plasmacytoid dendritic cells by next-generation sequencing. We then determined the relative antiviral potency of each IFNα subtype ex vivo using the human intestinal Lamina Propria Aggregate Culture model. IFNα subtype transcripts from the centromeric half of the IFNA gene complex were highly expressed in pDCs following HIV-1 exposure. There was an inverse relationship between IFNA subtype expression and potency. IFNα8, IFNα6 and IFNα14 were the most potent in restricting HIV-1 infection. IFNα2, the clinically-approved subtype, and IFNα1 were both highly expressed but exhibited relatively weak antiviral activity. The relative potencies correlated with binding affinity to the type I IFN receptor and the induction levels of HIV-1 restriction factors Mx2 and Tetherin/BST-2 but not APOBEC3G, F and D. However, despite the lack of APOBEC3 transcriptional induction, the higher relative potency of IFNα8 and IFNα14 correlated with stronger inhibition of virion infectivity, which is linked to deaminase-independent APOBEC3 restriction activity. By contrast, both potent (IFNα8) and weak (IFNα1) subtypes significantly induced HIV-1 GG-to-AG hypermutation. The results unravel non-redundant functions of the IFNα subtypes against HIV-1 infection, with strong implications for HIV-1 mucosal immunity, viral evolution and IFNα-based functional cure strategies.

Electronic Structure of Anilinopyridinate-Supported Ru25+ Paddlewheel Compounds.

The electronic structures of the diruthenium compounds Ru2(ap)4Cl (1, ap = 2-anilinopyridinate) and Ru2(ap)4OTf (2) were investigated with UV-vis, resonance Raman, and magnetic circular dichroism (MCD) spectroscopies; SQUID magnetometry; and density functional theory (DFT) calculations. Both compounds have quartet spin ground states with large axial zero-field splitting of ∼60 cm-1 that is characteristic of Ru25+ compounds having a (π*, δ*)3 electron configuration and a Ru-Ru bond order of ∼2.5. Two major visible absorption features are observed at ∼770 and 430 nm in the electronic spectra, the assignments of which have previously been ambiguous. Both bands have significant charge-transfer character with some contributions from d → d transitions. MCD spectra were measured to enable the identification of d → d transitions that are not easily observable by UV-vis spectroscopy. In this way, we are able to identify bands due to δ → δ* and δ → π* transitions at ∼16 100 and 11 200-12 300 cm-1, respectively, the latter band being sensitive to the π-donating character of the axial ligand. The Ru-Ru stretches are coupled with pyridine rocking motions and give rise to observed resonance Raman peaks at ∼350 and 420 cm-1, respectively.

Enhancement of HIV-1 infection and intestinal CD4+ T cell depletion ex vivo by gut microbes altered during chronic HIV-1 infection.

BACKGROUND: Early HIV-1 infection is characterized by high levels of HIV-1 replication and substantial CD4 T cell depletion in the intestinal mucosa, intestinal epithelial barrier breakdown, and microbial translocation. HIV-1-induced disruption of intestinal homeostasis has also been associated with changes in the intestinal microbiome that are linked to mucosal and systemic immune activation. In this study, we investigated the impact of representative bacterial species that were altered in the colonic mucosa of viremic HIV-1 infected individuals (HIV-altered mucosal bacteria; HAMB) on intestinal CD4 T cell function, infection by HIV-1, and survival in vitro. Lamina propria (LP) mononuclear cells were infected with CCR5-tropic HIV-1BaL or mock infected, exposed to high (3 gram-negative) or low (2 gram-positive) abundance HAMB or control gram-negative Escherichia coli and levels of productive HIV-1 infection and CD4 T cell depletion assessed. HAMB-associated changes in LP CD4 T cell activation, proliferation and HIV-1 co-receptor expression were also evaluated. RESULTS: The majority of HAMB increased HIV-1 infection and depletion of LP CD4 T cells, but gram-negative HAMB enhanced CD4 T cell infection to a greater degree than gram-positive HAMB. Most gram-negative HAMB enhanced T cell infection to levels similar to that induced by gram-negative E. coli despite lower induction of T cell activation and proliferation by HAMB. Both gram-negative HAMB and E. coli significantly increased expression of HIV-1 co-receptor CCR5 on LP CD4 T cells. Lipopolysaccharide, a gram-negative bacteria cell wall component, up-regulated CCR5 expression on LP CD4 T cells whereas gram-positive cell wall lipoteichoic acid did not. Upregulation of CCR5 by gram-negative HAMB was largely abrogated in CD4 T cell-enriched cultures suggesting an indirect mode of stimulation. CONCLUSIONS: Gram-negative commensal bacteria that are altered in abundance in the colonic mucosa of HIV-1 infected individuals have the capacity to enhance CCR5-tropic HIV-1 productive infection and depletion of LP CD4 T cells in vitro. Enhanced infection appears to be primarily mediated indirectly through increased expression of CCR5 on LP CD4 T cells without concomitant large scale T cell activation. This represents a novel mechanism potentially linking intestinal dysbiosis to HIV-1 mucosal pathogenesis.

Accessing Ni(III)-thiolate versus Ni(II)-thiyl bonding in a family of Ni-N2S2 synthetic models of NiSOD.

Superoxide dismutase (SOD) catalyzes the disproportionation of superoxide (O2(• -)) into H2O2 and O2(g) by toggling through different oxidation states of a first-row transition metal ion at its active site. Ni-containing SODs (NiSODs) are a distinct class of this family of metalloenzymes due to the unusual coordination sphere that is comprised of mixed N/S-ligands from peptide-N and cysteine-S donor atoms. A central goal of our research is to understand the factors that govern reactive oxygen species (ROS) stability of the Ni-S(Cys) bond in NiSOD utilizing a synthetic model approach. In light of the reactivity of metal-coordinated thiolates to ROS, several hypotheses have been proffered and include the coordination of His1-Nδ to the Ni(II) and Ni(III) forms of NiSOD, as well as hydrogen bonding or full protonation of a coordinated S(Cys). In this work, we present NiSOD analogues of the general formula [Ni(N2S)(SR')](-), providing a variable location (SR' = aryl thiolate) in the N2S2 basal plane coordination sphere where we have introduced o-amino and/or electron-withdrawing groups to intercept an oxidized Ni species. The synthesis, structure, and properties of the NiSOD model complexes (Et4N)[Ni(nmp)(SPh-o-NH2)] (2), (Et4N)[Ni(nmp)(SPh-o-NH2-p-CF3)] (3), (Et4N)[Ni(nmp)(SPh-p-NH2)] (4), and (Et4N)[Ni(nmp)(SPh-p-CF3)] (5) (nmp(2-) = dianion of N-(2-mercaptoethyl)picolinamide) are reported. NiSOD model complexes with amino groups positioned ortho to the aryl-S in SR' (2 and 3) afford oxidized species (2(ox) and 3(ox)) that are best described as a resonance hybrid between Ni(III)-SR and Ni(II)-(•)SR based on ultraviolet-visible (UV-vis), magnetic circular dichroism (MCD), and electron paramagnetic resonance (EPR) spectroscopies, as well as density functional theory (DFT) calculations. The results presented here, demonstrating the high percentage of S(3p) character in the highest occupied molecular orbital (HOMO) of the four-coordinate reduced form of NiSOD (NiSODred), suggest that the transition from NiSODred to the five-coordinate oxidized form of NiSOD (NiSODox) may go through a four-coordinate Ni-(•)S(Cys) (NiSODox-Hisoff) that is stabilized by coordination to Ni(II).

Microbial exposure alters HIV-1-induced mucosal CD4+ T cell death pathways Ex vivo.

BACKGROUND: Early HIV-1 infection causes massive CD4+ T cell death in the gut and translocation of bacteria into the circulation. However, the programmed cell death (PCD) pathways used by HIV-1 to kill CD4+ T cells in the gut, and the impact of microbial exposure on T cell loss, remain unclear. Understanding mucosal HIV-1 triggered PCD could be advanced by an ex vivo system involving lamina propria mononuclear cells (LPMCs). We therefore modeled the interactions of gut LPMCs, CCR5-tropic HIV-1 and a commensal gut bacterial species, Escherichia coli. In this Lamina Propria Aggregate Culture (LPAC) model, LPMCs were infected with HIV-1BaL by spinoculation and cultured in the presence or absence of heat killed E.coli. CD4+ T cell numbers derived from flow cytometry and viable cell counts were reported relative to mock infection. Viable cells were identified by viability dye exclusion (AqVi), and intracellular HIV-1 Gag p24 protein was used to identify infected cells. Annexin V and AqVi were used to identify apoptotic versus necrotic cells. Caspase-1 and Caspase-3 activities were blocked using specific inhibitors YVAD and DEVD, respectively. RESULTS: CD4+ T cell depletion following HIV-1 infection was reproducibly observed by 6 days post infection (dpi). Depletion at 6 dpi strongly correlated with infection frequency at 4 dpi, was significantly blocked by Efavirenz treatment, and was primarily driven by p24-negative cells that were predominantly necrotic. HIV-1 infection significantly induced CD4+ T-cell intrinsic Caspase-1 activity, whereas Caspase-1 inhibition, but not Caspase-3 inhibition, significantly blocked CD4+ T cell depletion. Exposure to E.coli enhanced HIV-1 infection and CD4+ T depletion, and significantly increased the number of apoptotic p24+ cells. Notably, CD4+ T cell depletion in the presence of E.coli was partially blocked by Caspase-3, but not by Caspase-1 inhibition. CONCLUSIONS: In the LPAC model, HIV-1 induced Caspase-1 mediated pyroptosis in bystander CD4+ T cells, but microbial exposure shifted the PCD mechanism toward apoptosis of productively infected T cells. These results suggest that mucosal CD4+ T cell death pathways may be altered in HIV-infected individuals after gut barrier function is compromised, with potential consequences for mucosal inflammation, viral dissemination and systemic immune activation.

Increased Escherichia coli-induced interleukin-23 production by CD16+ monocytes correlates with systemic immune activation in untreated HIV-1-infected individuals.

The level of microbial translocation from the intestine is increased in HIV-1 infection. Proinflammatory cytokine production by peripheral antigen-presenting cells in response to translocated microbes or microbial products may contribute to systemic immune activation, a hallmark of HIV-1 infection. We investigated the cytokine responses of peripheral blood myeloid dendritic cells (mDCs) and monocytes to in vitro stimulation with commensal enteric Escherichia coli in peripheral blood mononuclear cells (PBMC) from untreated HIV-1-infected subjects and from uninfected controls. Levels of interleukin 23 (IL-23) produced by PBMC from HIV-1-infected subjects in response to E. coli stimulation were significantly higher than those produced by PBMC from uninfected subjects. IL-23 was produced primarily by CD16(+) monocytes. This subset of monocytes was increased in frequency and expressed higher levels of Toll-like receptor 4 (TLR4) in HIV-1-infected individuals than in controls. Blocking TLR4 on total CD14(+) monocytes reduced IL-23 production in response to E. coli stimulation. Levels of soluble CD27, an indicator of systemic immune activation, were elevated in HIV-1-infected subjects and were associated with the percentage of CD16(+) monocytes and the induction of IL-23 by E. coli, providing a link between these parameters and systemic inflammation. Taken together, these results suggest that IL-23 produced by CD16(+) monocytes in response to microbial stimulation may contribute to systemic immune activation in HIV-1-infected individuals.

HIV-1 infection of human intestinal lamina propria CD4+ T cells in vitro is enhanced by exposure to commensal Escherichia coli.

Microbial translocation has been linked to systemic immune activation in HIV-1 disease, yet mechanisms by which microbes may contribute to HIV-associated intestinal pathogenesis are poorly understood. Importantly, our understanding of the impact of translocating commensal intestinal bacteria on mucosal-associated T cell responses in the context of ongoing viral replication that occurs early in HIV-1 infection is limited. We previously identified commensal Escherichia coli-reactive Th1 and Th17 cells in normal human intestinal lamina propria (LP). In this article, we established an ex vivo assay to investigate the interactions between Th cell subsets in primary human LP mononuclear cells (LPMCs), commensal E. coli, and CCR5-tropic HIV-1(Bal). Addition of heat-killed E. coli to HIV-1-exposed LPMCs resulted in increases in HIV-1 replication, CD4 T cell activation and infection, and IL-17 and IFN-γ production. Conversely, purified LPS derived from commensal E. coli did not enhance CD4 T cell infection. E. coli exposure induced greater proliferation of LPMC Th17 than Th1 cells. Th17 cells were more permissive to infection than Th1 cells in HIV-1-exposed LPMC cultures, and Th17 cell infection frequencies significantly increased in the presence of E. coli. The E. coli-associated enhancement of infection was dependent on the presence of CD11c(+) LP dendritic cells and, in part, on MHC class II-restricted Ag presentation. These results highlight a potential role for translocating microbes in impacting mucosal HIV-1 pathogenesis during early infection by increasing HIV-1 replication and infection of intestinal Th1 and Th17 cells.

Endogenous retroelement expression in the gut microenvironment of people living with HIV-1.

BACKGROUND: Endogenous retroelements (EREs), including human endogenous retroviruses (HERVs) and long interspersed nuclear elements (LINEs), comprise almost half of the human genome. Our previous studies of the interferome in the gut suggest potential mechanisms regarding how IFNb may drive HIV-1 gut pathogenesis. As ERE activity is suggested to partake in type 1 immune responses and is incredibly sensitive to viral infections, we sought to elucidate underlying interactions between ERE expression and gut dynamics in people living with HIV-1 (PLWH). METHODS: ERE expression profiles from bulk RNA sequencing of colon biopsies and PBMC were compared between a cohort of PLWH not on antiretroviral therapy (ART) and uninfected controls. FINDINGS: 59 EREs were differentially expressed in the colon of PLWH when compared to uninfected controls (padj <0.05 and FC ≤ -1 or ≥ 1) [Wald's Test]. Of these 59, 12 EREs were downregulated in PLWH and 47 were upregulated. Colon expression of the ERE loci LTR19_12p13.31 and L1FLnI_1q23.1s showed significant correlations with certain gut immune cell subset frequencies in the colon. Furthermore L1FLnI_1q23.1s showed a significant upregulation in peripheral blood mononuclear cells (PBMCs) of PLWH when compared to uninfected controls suggesting a common mechanism of differential ERE expression in the colon and PBMC. INTERPRETATION: ERE activity has been largely understudied in genomic characterizations of human pathologies. We show that the activity of certain EREs in the colon of PLWH is deregulated, supporting our hypotheses that their underlying activity could function as (bio)markers and potential mediators of pathogenesis in HIV-1 reservoirs. FUNDING: US NIH grants NCI CA260691 (DFN) and NIAID UM1AI164559 (DFN).

Effects of a Nutrition Education Programme in Stage IV Cardiac Rehabilitation Patients: A 3-Arm Randomised Controlled Trial.

This trial examined the influence of two nutrition intervention programmes on health-related and nutritional intake indices pertinent to cardiovascular disease compared to usual care in stage IV cardiac rehabilitation patients. Sixty-six patients were recruited from the Heartbeat North-West cardiac rehabilitation centre in Preston (United Kingdom). Patients were randomly assigned to Usual care, Biggest loser or Nutrition education arms. Usual care undertook their usual two exercise classes per week without nutritional support, Biggest loser underwent weekly education sessions focussing on weight loss using the themes within the British Heart Foundation 'So You Want to Lose Weight for Good' guidelines, and Nutrition education followed the same themes as Biggest loser but in a more focussed manner informed by patient focus groups. In total, this was a 12-week trial in which patients spent 6 weeks engaged in their allocated intervention arm, after which all participants switched to Usual care. The primary outcome was systolic blood pressure, and secondary measures of anthropometric, blood biomarker, nutritional knowledge (via the Mediterranean Diet Assessment Tool) and nutritional intake indices were measured at baseline, 6 weeks, and 12 weeks (follow-up). Intention-to-treat analyses revealed no significant alterations in the primary outcome (Usual care: baseline = 130.45 mmHg, 6 weeks = 127.83 mmHg, and follow-up = 126.35 mmHg, Biggest loser: baseline = 133.50 mmHg, 6 weeks = 123.06 mmHg, and follow-up = 135.22 mmHg, or Nutrition education: baseline = 135.23 mmHg, 6 weeks = 129.20 mmHg, and follow-up = 126.26 mmHg) between arms. However, at 6 weeks, improvements in triglycerides were significantly greater in Usual care (baseline = 1.61 mmol/L and 6 weeks = 1.43 mmol/L) group compared to Nutrition education (baseline = 1.70 mmol/L and 6 weeks = 2.21 mmol/L). Improvements in nutrition knowledge were significantly greater in Nutrition education (baseline = 8.48, 6 weeks = 9.77, and follow-up = 10.07) compared to Usual care (baseline = 7.71, 6 weeks = 8.00, and follow-up = 8.00) and Biggest loser (baseline = 7.71, 6 weeks = 8.73, and follow-up = 8.36). At 6 weeks, the Biggest loser group (baseline = 4.75 g and 6 weeks = 3.83 g) exhibited significantly greater improvements in salt intake compared to Usual care (baseline = 4.87 g and 6 weeks = 4.28 g) and Nutrition education (baseline = 19.25 g and 6 weeks = 16.78 g) in saturated fatty acids compared to Usual care (baseline = 20.26 g and 6 weeks = 21.34 g). This trial shows the effectiveness of nutritional interventions for improving nutritional knowledge and dietary practices in stage IV cardiac rehabilitation, but the mechanisms and longer-term effects of increased triglyceride levels in the Nutrition education group requires further exploration.

Health Benefits of Montmorency Tart Cherry Juice Supplementation in Adults with Mild to Moderate Ulcerative Colitis: A Protocol for a Placebo Randomized Controlled Trial.

Ulcerative colitis, characterized by its relapsing and remissive nature, negatively affects perception, body image, and overall quality of life. The associated financial burden underscores the need for alternative treatment approaches with fewer side effects, alongside pharmaceutical interventions. Montmorency tart cherries, rich in anthocyanins, have emerged as a potential natural anti-inflammatory agent for ulcerative colitis. This manuscript outlines the study protocol for a randomized placebo-controlled trial investigating the effects of Montmorency tart cherry in individuals with ulcerative colitis. The trial aims to recruit 40 participants with mild to moderate disease activity randomly assign them to either a Montmorency tart cherry or placebo group. The intervention will span 6 weeks, with baseline and 6-week assessments. The primary outcome measure is the Inflammatory Bowel Disease Quality of Life Questionnaire. Secondary outcomes include other health-related questionnaires and biological indices. Statistical analysis will adhere to an intention-to-treat approach using linear mixed effect models. Ethical approval has been obtained from the University of Hertfordshire (cLMS/SF/UH/05240), and the trial has been registered as a clinical trial (NCT05486507). The trial findings will be disseminated through a peer-reviewed publication in a scientific journal.

Microbiota-dependent indole production is required for the development of collagen-induced arthritis.

Altered tryptophan catabolism has been identified in inflammatory diseases like rheumatoid arthritis (RA) and spondyloarthritis (SpA), but the causal mechanisms linking tryptophan metabolites to disease are unknown. Using the collagen-induced arthritis (CIA) model we identify alterations in tryptophan metabolism, and specifically indole, that correlate with disease. We demonstrate that both bacteria and dietary tryptophan are required for disease, and indole supplementation is sufficient to induce disease in their absence. When mice with CIA on a low-tryptophan diet were supplemented with indole, we observed significant increases in serum IL-6, TNF, and IL-1ß; splenic RORγt+CD4+ T cells and ex vivo collagen-stimulated IL-17 production; and a pattern of anti-collagen antibody isotype switching and glycosylation that corresponded with increased complement fixation. IL-23 neutralization reduced disease severity in indole-induced CIA. Finally, exposure of human colon lymphocytes to indole increased expression of genes involved in IL-17 signaling and plasma cell activation. Altogether, we propose a mechanism by which intestinal dysbiosis during inflammatory arthritis results in altered tryptophan catabolism, leading to indole stimulation of arthritis development. Blockade of indole generation may present a novel therapeutic pathway for RA and SpA.