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Background:A primary barrier to curing HIV is the HIV reservoir. The leading infectious cause of death worldwide for people living with HIV is tuberculosis (TB),but we do not know how TB impacts the HIV reservoir.Methods:Participants in identification and validation cohorts were selected from previously enrolled studies at Groupe Haïtien d'Étude du Sarcome de Kaposi et des Infections Opportunistes (GHESKIO) in Port au Prince,Haiti. Intact and non-intact proviral DNA were quantified using droplet digital PCR of peripheral blood mononuclear cell (PBMC)-derived CD4+ T cells. Kruskal-Wallis tests were used to compare medians with tobit regression for censoring.Results:In the identification cohort,we found that people living with HIV with a history of active pulmonary TB (n=19) had higher levels of intact provirus than people living with HIV without a history of active TB (n=47) (median 762; IQR,183-1173 vs 117; IQR,24-279 intact provirus per million CD4,respectively; P=0.0001). This difference also was seen in the validation cohort (n=31),(median 102; IQR,0-737 vs 0; IQR,0-24.5 intact provirus per million CD4,P=0.03) for TB vs no-TB history groups,respectively. The frequencies of CD4+ T cells with any detectable proviral fragment was directly proportional to the levels of interleukin-1 beta (r=0.524,P= 0.0025) and interleukin-2 (r=0.622,P=0.0002).Conclusions:People living with HIV with a history of active pulmonary TB have more HIV pro-virus in their circulating CD4+ T cells,even years after TB cure. We need to characterize which CD4+ T cells are harboring intact provirus to consider the impact of T cell-targeting HIV cure interventions for people living in TB-endemic areas. View Publication

Neutrophil dysfunction is a form of immune suppression in patients with β-thalassemia (Beta-thal),although data on this are limited. In this study,blood from patients and healthy volunteers was analyzed. Flow cytometry analysis demonstrated an increase in immature neutrophils (CD16− CD62L+) and aged (senescent) neutrophils (CD16+ CD62L−) in Beta-thal patients compared to healthy volunteers. The Beta-thal neutrophils demonstrated less prominent chemotaxis and phagocytosis than healthy neutrophils at the baseline. With phorbol myristate acetate (PMA) or lipopolysaccharide (LPS) stimulations,some of the indicators,including the flow cytometry markers (CD11b,CD62L,CD66b,CD63,apoptosis,and reactive oxygen species) and neutrophil extracellular traps (NETs; detected by anti-citrullinated histone 3 immunofluorescence),were lower than the control. Additionally,low-density neutrophils (LDNs),which are found in the peripheral blood mononuclear cell (PBMC) fraction,were observed in Beta-thal patients but not in the control group. The expression of CD11b,CD66b,CD63,arginase I,and ROS in LDNs was higher than the regular normal-density neutrophils (NDNs). The proliferation rate of CD3+ T cells isolated from the PBMC fraction of healthy volunteers was higher than that of the cells from patients with Beta-thal. The incubation of red blood cell (RBC) lysate plus ferric ions with healthy NDNs transformed the NDNs into the aged neutrophils (decreased CD62L) and LDNs. In conclusion,iron overload induces neutrophil diversity along with some dysfunctions. View Publication

The development and progression of chronic lymphocytic leukemia (CLL) depend on genetic abnormalities and on the immunosuppressive microenvironment. We have explored the possibility that genetic drivers might be responsible for the immune cell dysregulation that shapes the protumor microenvironment. We performed a transcriptome analysis of coding and non-coding RNAs (ncRNAs) during leukemia progression in the Rag2−/−γc−/− MEC1-based xenotransplantation model. The DLEU2/miR-16 locus was found downmodulated in monocytes/macrophages of leukemic mice. To validate the role of this cluster in the tumor immune microenvironment,we generated a mouse model that simultaneously mimics the overexpression of hTCL1 and the germline deletion of the minimal deleted region (MDR) encoding the DLEU2/miR-15a/miR-16-1 cluster. This model provides an innovative and faster CLL system where monocyte differentiation and macrophage polarization are exacerbated,and T-cells are dysfunctional. MDR deletion inversely correlates with the levels of predicted target proteins including BCL2 and PD1/PD-L1 on murine CLL cells and immune cells. The inverse correlation of miR-15a/miR-16-1 with target proteins has been confirmed on patient-derived immune cells. Forced expression of miR-16-1 interferes with monocyte differentiation into tumor-associated macrophages,indicating that selected ncRNAs drive the protumor phenotype of non-malignant immune cells. View Publication

Introduction: The maintenance of intestinal homeostasis depends on a complex interaction between the immune system,intestinal epithelial barrier,and microbiota. Alteration in one of these components could lead to the development of inflammatory bowel diseases (IBD). Variants within the autophagy gene ATG16L1 have been implicated in susceptibility and severity of Crohn's disease (CD). Individuals carrying the risk ATG16L1 T300A variant have higher caspase 3-dependent degradation of ATG16L1 resulting in impaired autophagy and increased cellular stress. ATG16L1-deficiency induces enhanced IL-1β secretion in dendritic cells in response to bacterial infection. Infection of ATG16L1-deficient mice with a persistent strain of murine norovirus renders these mice highly susceptible to dextran sulfate sodium colitis. Moreover,persistent norovirus infection leads to intestinal virus specific CD8+ T cells responses. Both Toll-like receptor 7 (TLR7),which recognizes single-stranded RNA viruses,and ATG16L1,which facilitates the delivery of viral nucleic acids to the autolysosome endosome,are required for anti-viral immune responses. Results and discussion: However,the role of the enteric virome in IBD is still poorly understood. Here,we investigate the role of TLR7 and ATG16L1 in intestinal homeostasis and inflammation. At steady state,Tlr7-/- mice have a significant increase in large intestinal lamina propria (LP) granzyme B+ tissue-resident memory CD8+ T (TRM) cells compared to WT mice,reminiscent of persistent norovirus infection. Deletion of Atg16l1 in myeloid (Atg16l1ΔLyz2 ) or dendritic cells (Atg16l1ΔCd11c ) leads to a similar increase of LP TRM. Furthermore,Tlr7-/- and Atg16l1ΔCd11c mice were more susceptible to dextran sulfate sodium colitis with an increase in disease activity index,histoscore,and increased secretion of IFN-γ and TNF-α. Treatment of Atg16l1ΔCd11c mice with the TLR7 agonist Imiquimod attenuated colonic inflammation in these mice. Our data demonstrate that ATG16L1-deficiency in myeloid and dendritic cells leads to an increase in LP TRM and consequently to increased susceptibility to colitis by impairing the recognition of enteric viruses by TLR7. Conclusion: In conclusion,the convergence of ATG16L1 and TLR7 signaling pathways plays an important role in the immune response to intestinal viruses. Our data suggest that activation of the TLR7 signaling pathway could be an attractive therapeutic target for CD patients with ATG16L1 risk variants. View Publication

Inflammatory bowel disease (IBD),encompassing Crohn’s disease (CD) and ulcerative colitis (UC),is a chronic inflammatory condition of the gut affecting both adults and children. Neutrophil extracellular traps (NETs) are structures released by activated neutrophils,potentially contributing to tissue damage in various diseases. This study aimed to explore the presence and role of NETs in pediatric IBD. We compared intestinal biopsies and peripheral blood from 20 pediatric IBD patients (UC and CD) to controls. Biopsy staining and techniques for neutrophil activation were used to assess neutrophil infiltration and NET formation. We also measured the enzymatic activity of key NET proteins and evaluated NET formation in UC patients in remission. Both UC and CD biopsies showed significantly higher levels of neutrophils and NETs compared to controls (p < 0.01),with UC exhibiting the strongest association. Peripheral blood neutrophils from UC patients at diagnosis displayed increased NET formation compared to controls and CD patients. Interestingly,NET formation normalized in UC patients following remission-inducing treatment. This pilot study suggests a potential role for NETs in pediatric IBD,particularly UC. These findings warrant further investigation into the mechanisms of NET involvement and the potential for targeting NET formation as a therapeutic strategy. View Publication

Background/Objectives: Asthma is a chronic inflammatory disorder of the airways affecting over 10% of the global population. It is characterized by airway inflammation,mucus hypersecretion,and bronchial hyperresponsiveness,driven predominantly by type 2 helper T cells (Th2) and type 2 innate lymphoid cells (ILC2s) in a subset of patients. However,a significant portion of asthmatic individuals present with “type 2-low” asthma that is often refractory to standard inhaled corticosteroid (ICS) therapy. Therefore,developing innovative therapeutic strategies has become essential. Recent studies have highlighted cannabidiol (CBD) as a promising anti-inflammatory agent capable of modulating immune responses. This study investigates the therapeutic potential of a high-CBD extract (CBD-X) in asthma. Methods: We evaluated the effects of CBD-X on cells involved in asthma pathogenesis using primary human Th2 cells,neutrophils,and asthma mouse model. Results: Our findings indicate that CBD-X extract inhibits Th2 differentiation and reduces the secretion of IL-5 and IL-13,which are crucial cytokines in asthma. Additionally,CBD-X significantly reduces pro-inflammatory cytokines IL-8 and IL-6 in neutrophils and impairs their migration,a critical step in airway inflammation. In a murine asthma model,CBD-X administration led to marked downregulation of IgE and pro-asthmatic cytokines,along with reduced leukocyte,eosinophil,and neutrophil infiltration in lung tissues. Conclusions: These results suggest that CBD-X extract could offer a novel and complementary approach to managing both type 2-high and type 2-low asthma by targeting key inflammatory pathways and modulating immune cell behavior. View Publication

Acquired immunodeficiency syndrome (AIDS) occurs when HIV depletes CD4+ helper T cells. Some patients develop AIDS slowly or not at all,and are termed long-term non-progressors (LTNP),and while mutations in the HIV-1 Viral Protein R (vpr) gene such as R77Q are associated with LTNP,mechanisms for this correlation are unclear. This study examines the induction of apoptosis,cell cycle arrest,and pro-inflammatory cytokine release in the HUT78 T cell line following infection with replication-competent wild-type strain NL4-3,the R77Q mutant,or a vpr Null mutant. Our results show a significant enhancement of apoptosis and G2 cell cycle arrest in HUT78 cells infected with R77Q,but not with WT NL4-3 or the vpr Null strain. Conversely,HUT78 cells infected with the WT virus show higher levels of necrosis. We also detected lower TNF and IL-6 release after infection with R77Q vs. WT. The apoptotic phenotype was also seen in the CEM cell line and in primary CD4+ T cells. Protein expression of the R77Q vpr variant was low compared to WT vpr,but expression levels alone cannot explain these phenotypes because the Null virus did not show apoptosis or G2 arrest. These results suggest that R77Q triggers a non-inflammatory apoptotic pathway that attenuates inflammation,possibly contributing to LTNP. View Publication

Elevated neutrophil counts in broncho-alveolar lavage (BAL) fluids of lung transplant (LTx) patients with chronic lung allograft dysfunction (CLAD) are associated with disease pathology. However,phenotypical characteristics of these cells remained largely unknown. Moreover,despite enhanced levels of the most potent human neutrophil-attracting chemokine CXCL8 in BAL fluid,no discrimination had been made between natural NH2-terminally truncated CXCL8 proteoforms,which exhibit up to 30-fold differences in biological activity. Therefore,we aimed to characterize the neutrophil maturation and activation state,as well as proteolytic activation of CXCL8,in BAL fluids and peripheral blood of LTx patients with CLAD or infection and stable LTx recipients. Flow cytometry and microscopy revealed a high diversity in neutrophil maturity in blood and BAL fluid,ranging from immature band to hypersegmented aged cells. In contrast,the activation phenotype of neutrophils in BAL fluid was remarkably homogeneous. The highly potentiated NH2-terminally truncated proteoforms CXCL8(6–77),CXCL8(8–77) and CXCL8(9–77),but also the partially inactivated CXCL8(10–77),were detected in BAL fluids of CLAD and infected LTx patients,as well as in COVID-19 and influenza patient cohorts by tandem mass spectrometry. Moreover,the most potent proteoform CXCL8(9–77) specifically correlated with the neutrophil counts in the LTx BAL fluids. Finally,rapid proteolysis of CXCL8 in BAL fluids could be inhibited by a combination of serine and metalloprotease inhibitors. In conclusion,proteolytic activation of CXCL8 promotes neutrophilic inflammation in LTx patients. Therefore,application of protease inhibitors may hold pharmacological promise for reducing excessive neutrophil-mediated inflammation and collateral tissue damage in the lungs. View Publication

Eosinophils,a type of granulocyte derived from myeloid precursors in the bone marrow,are distinguished by their cytoplasmic granules. They play crucial roles in immunoregulation,tissue homeostasis,and host defense,while also contributing to the pathogenesis of various inflammatory diseases. Although long non-coding RNAs (lncRNAs) are known to be involved in eosinophilic conditions,their specific expression and functions within eosinophils have not been thoroughly investigated,largely due to the reliance on tissue homogenates. In an effort to address this gap,we analyzed publicly available high-throughput RNA sequencing data to identify lncRNAs associated with eosinophilic conditions. Among the identified lncRNAs,ITGB2 antisense RNA 1 (ITGB2-AS1) was significantly downregulated in blood eosinophils from patients with hypereosinophilia. To further explore its role in eosinophil biology,we generated a stable ITGB2-AS1 knockdown in the HL-60 cell line. Interestingly,ITGB2-AS1 deficiency led to impaired eosinophil differentiation,as evidenced by a reduction in cytoplasmic granules and decreased expression of key eosinophil granule proteins,including eosinophil peroxidase (EPX) and major basic protein-1 (MBP-1). Additionally,ITGB2-AS1-deficient cells exhibited compromised eosinophil effector functions,with reduced degranulation and impaired production of reactive oxygen species (ROS). These findings suggest that ITGB2-AS1 plays a pivotal role in eosinophil differentiation and function,positioning it as a novel regulator in eosinophil biology. View Publication

Pathobionts have evolved many strategies to coexist with the host,but how immune evasion mechanisms contribute to the difficulty of developing vaccines against pathobionts is unclear. Meanwhile,Staphylococcus aureus (SA) has resisted human vaccine development to date. Here we show that prior SA exposure induces non-protective CD4+ T cell imprints,leading to the blunting of protective IsdB vaccine responses. Mechanistically,these SA-experienced CD4+ T cells express IL-10,which is further amplified by vaccination and impedes vaccine protection by binding with IL-10Rα on CD4+ T cell and inhibit IL-17A production. IL-10 also mediates cross-suppression of IsdB and sdrE multi-antigen vaccine. By contrast,the inefficiency of SA IsdB,IsdA and MntC vaccines can be overcome by co-treatment with adjuvants that promote IL-17A and IFN-γ responses. We thus propose that IL-10 secreting,SA-experienced CD4+ T cell imprints represent a staphylococcal immune escaping mechanism that needs to be taken into consideration for future vaccine development. Mechanisms of inefficient vaccine protection against pathobionts such as S. aureus (SA) are still unclear. Here the authors show that prior SA exposure induces non-protective CD4+ T cells,which impair IsdB vaccine protection by IL-10 secretion and IL-17A suppression,whereas IL-17A promoting adjuvant CAF01 overcomes this dilemma. View Publication

The lungs of people with cystic fibrosis (PwCF) are characterized by recurrent bacterial infections and inflammation. Infections in cystic fibrosis (CF) are left unresolved despite excessive neutrophil infiltration. The role of CFTR in neutrophils is not fully understood. In this study,we aimed to assess which antimicrobial functions are directly impaired by loss of CFTR function in neutrophils. In order to do so,we used a specific inhibitor of CFTR ion channel activity,inh-172. CF neutrophils from PwCF harboring severe CFTR mutations were additionally isolated to further discern CFTR-specific functional defects. We evaluated phagocytosis,reactive oxygen species (ROS) production,neutrophil elastase (NE) and myeloperoxidase (MPO) exocytosis and bacterial killing. The inh-172 model identified decreased acidification of the phagosome,increased bacterial survival and decreased ROS production upon stimulation. In PwCF neutrophils,we observed reduced degranulation of both NE and MPO. When co-culturing neutrophils with CF sputum supernatant and airway epithelial cells,the extent of phagocytosis was reduced,underscoring the importance of recreating an inflammatory environment as seen in PwCF lungs to model immune responses in vitro. Despite low CFTR expression in blood neutrophils,functional defects were found in inh-172-treated and CF neutrophils. The inh-172 model disregards donor variability and allows pinpointing neutrophil functions directly impaired by dysfunctional CFTR.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-024-82535-z. View Publication

IntroductionUpon infection,T cell-driven B cell responses in GC reactions induce memory B cells and antibody-secreting cells that secrete protective antibodies. How formation of specifically long-lived plasma cells is regulated via the interplay between specific B and CD4+ T cells is not well understood. Generally,antibody levels decline over time after clearance of the primary infection.MethodIn this study,convalescent individuals with stable RBD antibody levels (n=14,“sustainers”) were compared with donors (n=13) with the greatest antibody decline from a cohort of 132. To investigate the role of the cellular immune compartment in the maintenance of antibody levels,SARS-CoV-2-specific responses at 4 to 6 weeks post-mild COVID-19 infection were characterized using deep immune profiling.ResultsBoth groups had similar frequencies of total SARS-CoV-2-specific B and CD4+ T cells. Sustainers had fewer Spike-specific IgG+ memory B cells early after infection and increased neutralizing capacity of RBD antibodies over time,unlike the declining group. However,declining IgG titers correlated with lower frequency of Spike-specific CD4+ T cells.ConclusionThese data suggest that “sustainers” have unique dynamics of GC reactions,yield different outputs of terminally differentiating cells,and improve the quality of protective antibodies over time. This study helps identify factors controlling formation of long-lived PC and sustained antibody responses. View Publication