Search

Since the discovery of HIV-1, progress has been made in deciphering the viral replication cycle and mechanisms of host-pathogen interactions that has facilitated the implementation of effective antiretroviral therapies (ARTs). Major barriers to HIV-1 remission/cure include the persistence of viral reservoirs (VRs) in long-lived CD4

HIV reservoirs persist in gut-homing CD4+ T cells of people living with HIV and receiving antiretroviral therapy, but the antigenic specificity of such reservoirs remains poorly documented. The imprinting for gut homing is mediated by retinoic acid (RA), a vitamin A–derived metabolite produced by dendritic cells (DCs) exhibiting RA-synthesizing (RALDH) activity. RALDH activity in DCs can be induced by TLR2 ligands, such as bacterial peptidoglycans and fungal zymosan. Thus, we hypothesized that bacterial/fungal pathogens triggering RALDH activity in DCs fuel HIV reservoir establishment/outgrowth in pathogen-reactive CD4+ T cells. Our results demonstrate that DCs derived from intermediate/nonclassical CD16+ compared with classical CD16− monocytes exhibited superior RALDH activity and higher capacity to transmit HIV infection to autologous Staphylococcus aureus–reactive T cells. Exposure of total monocyte-derived DCs (MDDCs) to S. aureus lysates as well as TLR2 (zymosan and heat-killed preparation of Listeria monocytogenes) and TLR4 (LPS) agonists but not CMV lysates resulted in a robust upregulation of RALDH activity. MDDCs loaded with S. aureus or zymosan induced the proliferation of T cells with a CCR5+integrin β7+CCR6+ phenotype and efficiently transmitted HIV infection to these T cells via RALDH/RA–dependent mechanisms. Finally, S. aureus– and zymosan-reactive CD4+ T cells of antiretroviral therapy-treated people living with HIV carried replication-competent integrated HIV-DNA, as demonstrated by an MDDC-based viral outgrowth assay. Together, these results support a model in which bacterial/fungal pathogens in the gut promote RALDH activity in MDDCs, especially in CD16+ MDDCs, and subsequently imprint CD4+ T cells with gut-homing potential and HIV permissiveness. Thus, nonviral pathogens play key roles in fueling HIV reservoir establishment/outgrowth via RALDH/RA–dependent mechanisms that may be therapeutically targeted.

Background Increased intestinal barrier permeability and subsequent gut microbial translocation are significant contributors to inflammatory non-AIDS comorbidities in people living with HIV (PLWH). Evidence in animal models have shown that markers of intestinal permeability and microbial translocation vary over the course of the day and are affected by food intake and circadian rhythms. However, daily variations of these markers are not characterized yet in PLWH. Herein, we assessed the variation of these markers over 24 h in PLWH receiving antiretroviral therapy (ART) in a well-controlled environment. Methods As in Canada, PLWH are predominantly men and the majority of them are now over 50 years old, we selected 11 men over 50 receiving ART with undetectable viremia for more than 3 years in this pilot study. Blood samples were collected every 4 h over 24 h before snacks/meals from 8:00 in the morning to 8:00 the next day. All participants consumed similar meals at set times, and had a comparable amount of sleep, physical exercise and light exposure. Plasma levels of bacterial lipopolysaccharide (LPS) and fungal (1→3)-β-D-Glucan (BDG) translocation markers, along with markers of intestinal damage fatty acid binding protein (I-FABP) and regenerating islet-derived protein-3α (REG3α) were assessed by ELISA or the fungitell assay. Results Participants had a median age of 57 years old (range 50 to 63). Plasma levels of BDG and REG3α did not vary significantly over the course of the study. In contrast, a significant increase of LPS was detected between 12:00 and 16:00 (Z-score: − 1.15 ± 0.18 vs 0.16 ± 0.15, p = 0.02), and between 12:00 and 24:00 (− 1.15 ± 0.18 vs 0.89 ± 0.26, p Conclusions Conversely to the fungal translocation marker BDG and the gut damage marker REG3α, time of blood collection matters for the proper evaluation for LPS and I-FABP as markers for the risk of inflammatory non-AIDS co-morbidities. These insights are instrumental for orienting clinical investigations in PLWH.

Significance HIV-1 infects CD4 T cells, and, among these, T helper 17 (Th17) cells are known to be particularly permissive for virus replication. The infection of Th17 cells is critical for AIDS pathogenesis and viral persistence. It is, however, not clear why these cells are highly permissive to HIV-1. We found that Th17 cell permissiveness depends on the expression of the hormone receptor RORC2, which is the master transcriptional regulator of Th17 cell differentiation. We identify RORC2 as a cell-specific host-dependency factor that can be targeted by small molecules. Our results suggest that RORC2 may be a cell-specific target to mitigate the loss of Th17 cells as a consequence of their preferential HIV-1 infection., Among CD4+ T cells, T helper 17 (Th17) cells are particularly susceptible to HIV-1 infection and are depleted from mucosal sites, which causes damage to the gut barrier, resulting in a microbial translocation-induced systemic inflammation, a hallmark of disease progression. Furthermore, a proportion of latently infected Th17 cells persist long term in the gastrointestinal lymphatic tract where a low-level HIV-1 transcription is observed. This residual viremia contributes to chronic immune activation. Thus, Th17 cells are key players in HIV pathogenesis and viral persistence. It is, however, unclear why these cells are highly susceptible to HIV-1 infection. Th17 cell differentiation depends on the expression of the master transcriptional regulator RORC2, a retinoic acid-related nuclear hormone receptor that regulates specific transcriptional programs by binding to promoter/enhancer DNA. Here, we report that RORC2 is a key host cofactor for HIV replication in Th17 cells. We found that specific inhibitors that bind to the RORC2 ligand-binding domain reduced HIV replication in CD4+ T cells. The depletion of RORC2 inhibited HIV-1 infection, whereas its overexpression enhanced it. RORC2 was also found to promote HIV-1 gene expression by binding to the nuclear receptor responsive element in the HIV-1 long terminal repeats (LTR). In treated HIV-1 patients, RORC2+ CD4 T cells contained more proviral DNA than RORC2− cells. Pharmacological inhibition of RORC2 potently reduced HIV-1 outgrowth in CD4+ T cells from antiretroviral-treated patients. Altogether, these results provide an explanation as to why Th17 cells are highly susceptible to HIV-1 infection and suggest that RORC2 may be a cell-specific target for HIV-1 therapy.

Summary The crosstalk between intestinal epithelial cells (IECs) and Th17-polarized CD4+ T cells is critical for mucosal homeostasis, with HIV-1 causing significant alterations in people living with HIV (PLWH) despite antiretroviral therapy (ART). In a model of IEC and T cell co-cultures, we investigated the effects of IL-17A, the Th17 hallmark cytokine, on IEC ability to promote de novo HIV infection and viral reservoir reactivation. Our results demonstrate that IL-17A acts in synergy with TNF to boost IEC production of CCL20, a Th17-attractant chemokine, and promote HIV trans-infection of CD4+ T cells and viral outgrowth from reservoir cells of ART-treated PLWH. Importantly, the Illumina RNA-sequencing revealed an IL-17A-mediated pro-inflammatory and pro-viral molecular signature, including a decreased expression of type I interferon (IFN-I)-induced HIV restriction factors. These findings point to the deleterious features of IL-17A and raise awareness for caution when designing therapies aimed at restoring the paucity of mucosal Th17 cells in ART-treated PLWH., Graphical abstract, Highlights • IL-17A acts in synergy with TNF to enhance CCL20 production in IEC exposed to HIV • IL-17A/TNF-activated IEC efficiently promote HIV trans-infection of CD4+ T cells • IL-17A reprograms IEC to boost HIV outgrowth from CD4+ T cells of ART-treated PLWH • IL-17A decreases the expression of IFN-I-induced HIV restriction factors in IEC, Immunology; Immune response; Virology

Among CD4+ T-cells, T helper 17 (Th17) cells are particularly susceptible to HIV-1 infection and are depleted from mucosal sites, which causes damage to the gut barrier resulting in microbial translocation-induced systemic inflammation, a hallmark of disease progression. Furthermore, a proportion of latently infected Th17 cells persist long-term in the gastro-intestinal lymphatic tract, where low-level HIV-1 transcription is observed. This residual viremia contributes to chronic immune activation. Thus, Th17 cells are key players in HIV pathogenesis and viral persistence, however it is unclear why these cells are highly susceptible to HIV-1 infection. Th17 cell differentiation depends on expression of the master transcriptional regulator RORC2, a retinoic acid-related nuclear hormone receptor that regulates specific transcriptional programs by binding to promoter/enhancer DNA. Here, we report that RORC2 is a key host-cofactor for HIV replication in Th17 cells. We found that specific inhibitors that bind to the RORC2 ligand-binding domain reduced HIV replication in CD4+ T-cells. Depletion of RORC2 inhibited HIV-1 infection, whereas RORC2 overexpression enhanced it. RORC2 was found to promote HIV-1 gene expression. Chromatin immune precipitation revealed that RORC2 binds to the nuclear receptor responsive element (NRRE) in the HIV-1 LTR. In treated HIV-1 patients, RORC2+ CD4 T cells contained more proviral DNA than RORC2− cells. Pharmacological inhibition of RORC2 potently reduced HIV-1 outgrowth in CD4+ T-cells from antiretroviral-treated patients. Altogether, these results provide a new explanation as to why Th17 cells are highly susceptible to HIV-1 infection and point to RORC2 as a cell-specific target for HIV-1 therapy.Significance statementHIV-1 infects CD4 T cells and among these, Th17 cells are known to be particularly permissive for virus replication. Infection of Th17 cells is critical for AIDS pathogenesis and viral persistence, however it is not clear why these cells are highly permissive to HIV-1. We found that Th17 cell permissiveness depends on expression of the hormone receptor RORC2, which is the master transcriptional regulator of Th17 cell differentiation. We identify RORC2 as a new, cell-specific host-dependency factor that can be targeted by small molecules. Our results point to RORC2 as a cell-specific target for HIV-1 therapy, an entirely new concept in the field, and suggest HIV-1 might have evolved to exploit RORC2 to promote its own persistence.

Background: Chronic inflammation and residual HIV transcription persist in people living with HIV (PLWH) receiving antiretroviral therapy (ART), thus increasing the risk of developing non-AIDS co-morbidities. The mechanistic target of rapamycin (mTOR) is a key regulator of cellular metabolism and HIV transcription, and therefore represents an interesting novel therapeutic target. Methods: The LILAC pilot clinical trial, performed on non-diabetic ART-treated PLWH with CD4+/CD8+ T-cell ratios

HIV reservoirs persist in gut-homing CD4

Background People with HIV (PWH) taking antiretroviral therapy (ART) may experience weight gain, dyslipidemia, increased risk of non-AIDS comorbidities, and long-term alteration of the gut microbiota. Both low CD4/CD8 ratio and chronic inflammation have been associated with changes in the gut microbiota of PWH. The antidiabetic drug metformin has been shown to improve gut microbiota composition while decreasing weight and inflammation in diabetes and polycystic ovary syndrome. Nevertheless, it remains unknown whether metformin may benefit PWH receiving ART, especially those with a low CD4/CD8 ratio. Methods In the Lilac pilot trial, we recruited 23 nondiabetic PWH receiving ART for more than 2 years with a low CD4/CD8 ratio (, The ability to keep an HIV elite controller status is not permanent. We showed that, independently to protective MHC-I HLA alleles, elevated level of anti-CMV IgG is linked to CD4 T-cell decay in a Canadian cohort of elite controllers.

The persistence of replication-competent HIV reservoirs in people living with HIV (PLWH) receiving antiretroviral therapy (ART) is a barrier to cure. Therefore, their accurate quantification is essential for evaluating the efficacy of new therapeutic interventions and orienting the decision to interrupt ART. Quantitative viral outgrowth assays (QVOAs) represent the “gold standard” for measuring the size of replication-competent HIV reservoirs. However, they require large numbers of cells and are technically challenging. This justifies the need for the development of novel simplified methods adapted for small biological samples. Herein, we sought to simplify the viral outgrowth procedure (VOP) by (i) using memory CD4+ T-cells, documented to be enriched in HIV reservoirs (ii) optimizing cell-culture conditions, and (iii) supplementing with all-trans retinoic acid (ATRA), a positive regulator of HIV replication. Memory CD4+ T-cells were sorted from the peripheral blood of ART-treated (HIV+ART; n = 14) and untreated (HIV+; n = 5) PLWH. The VOP was first performed with one original replicate of 1 × 106 cells/well in 48-well plates. Cells were stimulated via CD3/CD28 for 3 days, washed to remove residual CD3/CD28 Abs, split every 3 days for optimal cell density, and cultured in the presence or the absence of ATRA for 12 days. Soluble and intracellular HIV-p24 levels were quantified by ELISA and flow cytometry, respectively. Optimal cell-culture density achieved by splitting improved HIV outgrowth detection. ATRA promoted superior/accelerated detection of replication-competent HIV in all HIV+ART individuals tested, including those with low/undetectable viral outgrowth in the absence of ATRA. Finally, this VOP was used to design a simplified ATRA-based QVOA by including 4 and 6 original replicates of 1 × 106 cells/well in 48-well plates and 2 × 105 cells/well in 96-well plates, respectively. Consistently, the number of infectious units per million cells (IUPM) was significantly increased in the presence of ATRA. In conclusion, we demonstrate that memory CD4+ T-cell splitting for optimal density in culture and ATRA supplementation significantly improved the efficacy of HIV outgrowth in a simplified ATRA-based QVOA performed in the absence of feeder/target cells or indicator cell lines.

The frequency and functions of Th17-polarized CCR6 + RORyt + CD4 + T cells are rapidly compromised upon HIV infection and are not restored with long-term viral suppressive antiretroviral therapy (ART). In line with this, Th17 cells represent selective HIV-1 infection targets mainly at mucosal sites, with long-lived Th17 subsets carrying replication-competent HIV-DNA during ART. Therefore, novel Th17-specific therapeutic interventions are needed as a supplement of ART to reach the goal of HIV remission/cure. Th17 cells express high levels of peroxisome proliferator-activated receptor gamma (PPARy), a transcriptional factor that represses the transcription of the HIV provirus and the rorc gene, which encodes for the Th17-specific master regulator RORyt/RORC2. Thus, we hypothesized that the pharmacological inhibition of PPARy will facilitate HIV reservoir reactivation while enhancing Th17 effector functions. Consistent with this prediction, the PPARy antagonist T0070907 significantly increased HIV transcription (cell-associated HIV-RNA) and RORyt-mediated Th17 effector functions (IL-17A). Unexpectedly, the PPARy antagonism limited HIV outgrowth from cells of ART-treated people living with HIV (PLWH), as well as HIV replication in vitro . Mechanistically, PPARy inhibition in CCR6 + CD4 + T cells induced the upregulation of transcripts linked to Th17-polarisation (RORyt, STAT3, BCL6 IL-17A/F, IL-21) and HIV transcription (NCOA1-3, CDK9, HTATIP2). Interestingly, several transcripts involved in HIV-restriction were upregulated (Caveolin-1, TRIM22, TRIM5α, BST2, miR-29), whereas HIV permissiveness transcripts were downregulated (CCR5, furin), consistent with the decrease in HIV outgrowth/replication. Finally, PPARy inhibition increased intracellular HIV-p24 expression and prevented BST-2 downregulation on infected T cells, suggesting that progeny virion release is restricted by BST-2-dependent mechanisms. These results provide a strong rationale for considering PPARy antagonism as a novel strategy for HIV-reservoir purging and restoring Th17-mediated mucosal immunity in ART-treated PLWH.

e24018 Background: Sarcopenia is associated with decreased treatment tolerance and overall survival in aggressive large B-cell lymphoma, the most common non-Hodgkin lymphoma (NHL) in the geriatric population. The impact of sarcopenia on event-free survival (EFS) in older adults with an aggressive B-cell NHL remains unclear. Our primary endpoint was to evaluate the association between sarcopenia and 6-month EFS in adults aged 65 and older diagnosed with an aggressive B-cell NHL. Methods: We performed a retrospective cohort study of adults 65 years or older diagnosed with an aggressive B-cell NHL between July 1st 2014 and June 30th 2018 who completed 1st line chemotherapy in an academic center. Sarcopenia was defined as the lowest tertile of psoas muscle index (PMI). Psoas muscle area was measured on a single axial slice at L4 using CoreSlicer software, and divided by height squared. EFS defined as no death, relapse or hospitalisation, was compared between sarcopenic and non-sarcopenic individuals using Kaplan-Meier curves and multivariable Cox regression. Results: In our cohort of 62 older adults, median age was 74 (interquartile range (IQR): 69-78) and median PMI was 7.2 cm2/m2 (IQR: 5.9-8.4). Twenty patients were sarcopenic with a median PMI of 5.5 (4.8-6.3) cm2/m2. Most patients (56%) received full dose chemotherapy. Among patients who received an attenuated dose of chemotherapy, the percentage of patients with sarcopenia was similar to the one of patients receiving full dose chemotherapy (39% vs 24%, p=0.200). After controlling for age, sex, ECOG, cardiac disease, creatinine, LDH and hemoglobin levels, sarcopenia was associated with a lower EFS (HR 3.5, 95%CI: 1.1-11, p=0.032). Although sarcopenia was not associated with the choice of an attenuated chemotherapy intensity, when stratifying for chemotherapy intensity, there was a trend in favor of decreased EFS with sarcopenia in the full dose group (p=0.095), which did not reach statistical significance. Conclusions: Sarcopenia is associated with a lower 6-month EFS in older adults who completed first line chemotherapy. These individuals may benefit from pre-chemotherapy geriatric evaluation and tighter follow-up.

Crohn's disease (CD) and ulcerative colitis (UC) are the two major forms of inflammatory bowel disease (IBD). These idiopathic and chronic diseases result from inflammation of the gastrointestinal tract and are mainly mediated by the immune system. Genome wide association studies link genes of the IL-12 and IL-23 biology to both CD and UC susceptibility. IL-12 and IL-23 cytokines share a functional subunit, p40, and their respective receptors also share a functional subunit, IL-12Rβ1. However, clinical trials targeting p40, and thus inhibiting both IL-12 and IL-23 pathways, provided mitigated effects on IBD, suggesting context dependent effects for each cytokine. In addition to IL-12 and IL-23, genetic deficiencies in IL-10 also result in severe IBD pathology. We generated various mouse models to determine how IL-12 or IL-23 interacts with IL-10 in IBD pathology. Whereas defects in both IL-10 and IL-12R do not impact the severity of the Dextran Sulfate Sodium (DSS)-induced colitis, combined deficiencies in both IL-10 and IL-23R aggravate the disease. In contrast to DSS-induced colitis, defects in IL-12R and IL-23R both protect from the spontaneous colitis observed in IL10-/- mice. Together, these studies exemplify the complexity of genetic and environmental interactions for identifying biological pathways predictive of pathological inflammatory processes.