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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

IntroductionAnandamide (AEA) is an endocannabinoid that has recently been recognized as a regulator of various inflammatory diseases as well as cancer. While AEA was thought to predominantly engage cannabinoid (CB) receptors,recent findings suggest that,given its protective anti-inflammatory role in pathological conditions,anandamide may engage not only CB receptors.MethodsIn this study,we studied the role of exogenous AEA in a mouse AirPouch model of acute inflammation by examining immune cell infiltrates by flow cytometry. Human primary immune cells were used to validate findings towards immune cell activation and migration by flow cytometry and bead-based ELISA.ResultsWe found that AEA decreases the acute infiltration of myeloid cells including granulocytes and monocytes into the inflamed area,but unexpectedly increases the number of T cells at the site of inflammation. This was related to AEA signaling through nuclear receptor subfamily 4A (NR4A) transcription factors rather than CB receptors. Exploring regulatory mechanisms in the human system,we found that AEA broadly inhibits the migratory capacity of immune cells,arguing for blocked emigration of T cells from the inflamed tissue. Taking a closer look at the impact of AEA on T cells revealed that AEA profoundly alters the activation and exhaustion status of CD4+ T and CD8+ T cells,thereby strongly inhibiting TH17 responses,while not altering TH1 differentiation.DiscussionThese data suggest that AEA has the potential to block chronic inflammation without influencing crucial anti-viral and anti-microbial immune defense mechanisms,and may therefore be an attractive molecule to interfere with the establishment of chronic inflammation. View Publication

SummaryHere,we present a protocol to generate craniofacial cartilage organoids from human stem cells via neural crest stem cells (NCSCs). We describe steps for inducing human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) to form NCSCs using sequential treatments of small molecules and growth factors and isolating NCSCs by magnetic bead sorting. We then detail procedures for defining conditions where NCSCs migrate together and self-organize into craniofacial cartilage organoids. Recapitulating craniofacial chondrogenesis will facilitate craniofacial reconstruction and disease modeling.For complete details on the use and execution of this protocol,please refer to Foltz et al.1 Graphical abstract Highlights•Protocol for inducing hESCs or iPSCs to form neural crest stem cells (NCSCs)•Steps for differentiating NCSCs into craniofacial cartilage organoids•Instructions for preparing appropriate media and conditions for differentiation•Guidance for assessing changes in cell and organoid morphology during differentiation Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here,we present a protocol to generate craniofacial cartilage organoids from human stem cells via neural crest stem cells (NCSCs). We describe steps for inducing human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) to form NCSCs using sequential treatments of small molecules and growth factors and isolating NCSCs by magnetic bead sorting. We then detail procedures for defining conditions where NCSCs migrate together and self-organize into craniofacial cartilage organoids. Recapitulating craniofacial chondrogenesis will facilitate craniofacial reconstruction and disease modeling. View Publication

IntroductionThe role of miRNAs in regulating variable molecular functions has been sought by scientists for its promising utility in regulating the immune response and,hence,in treating various diseases. In hepatocellular carcinoma (HCC) specifically,a reduction in the number and efficiency of circulating and intrahepatic natural killer (NK) cells has been reported. Our project aims to investigate the role of miR-216a-3p in the regulation of NK cell cytotoxicity,especially since it plays a tumor suppressor role in the context of HCC.MethodsTo achieve our aim,we isolated NK cells from the whole blood of 86 patients with HCC and 23 healthy controls. We assessed the expression profile of miR-216a-3p in NK cells of patients and controls. Furthermore,we induced the expression of miR-216a-3p in NK cells isolated from healthy controls,followed by measuring the release of interferon-gamma (IFN-γ),tumor necrosis factor-alpha (TNF-α),perforins (PRF) and granzyme B (GrB) using ELISA as well as NK cells cytolytic activity against Huh7 cells using lactate dehydrogenase (LDH) cytotoxicity assay. After that,we performed an in silico analysis to understand the mechanistic regulation imposed by miR-216a-3p on NK cells to study its impact on one of its potential downstream targets.ResultsOur results have indicated that miR-216a-3p has higher expression in NK cells of patients with HCC,and simulating this elevated expression pattern via forcing miR-216a-3p expression in normal NK cells has negatively impacted the release of TNF- α,IFN- γ,GrB,and PRF. Consequently,a decrease in cell cytolysis was observed. Our in silico analysis revealed that the predicted downstream targets of miR-216a-3p are enriched in the FOXO-signaling pathway. Among those targets is FOXO-1,which has been reported to play a role in NK cell maturation. Thus,we evaluated FOXO-1 expression upon mimicking miR-216a-3p in control NK cells that showed significant downregulation of FOXO-1 on both RNA and protein levels.ConclusionIn conclusion,we report miR-216-3p as a negative regulator of NK cell cytotoxicity. View Publication

The persistence of HIV-1 latency reservoirs in CD4+ T cells is a significant obstacle for curing HIV-1. Shock-and-kill strategies,which aim to reactivate latent HIV-1 followed by cytotoxic clearance,have shown limited success in vivo due to insufficient efficacy of latency reversal agents (LRAs) and off-target effects. Natural killer (NK) cells,with their ability to mediate cytotoxicity independent of antigen specificity,offer a promising avenue for enhancing the shock-and-kill approach. Previously,we observed that pan-caspase inhibitors induce NK cells to secrete an LRA in vitro. Here,we aimed to identify this LRA using a targeted proteomic approach. We identified lymphotoxin-α (LTα) as the key LRA secreted by NK cells following pan-caspase inhibitor treatment. LTα was shown to significantly induce HIV-1 LTR promoter activity,a hallmark of viral reactivation. Neutralization of LTα effectively abolished the observed LRA activity,confirming its central role. Moreover,cytokine-primed but not resting human primary NK cells exhibited LRA activity that could be neutralized with LTα neutralizing antibodies. Finally,pan-caspase inhibitor treatment did not decrease the ability of the cytokine-primed NK cells to kill target cells. These findings demonstrate that cytokine-primed NK cells,through LTα secretion,can effectively reactivate latent HIV-1 following pan-caspase inhibitor treatment,without compromising NK cell cytotoxicity. This highlights a potential enhancement strategy utilizing NK cells for shock-and-kill approaches in HIV-1 cure research. View Publication

Post-transcriptional modifications to RNA,which comprise the epitranscriptome,play important roles in RNA metabolism,gene regulation,and human disease,including viral pathogenesis. Modifications to the RNA viral genome and transcripts of human immunodeficiency virus 1 (HIV-1) have been reported and investigated in the context of virus and host biology. However,the diversity of experimental approaches used has made clear correlations across studies,as well as the significance of the HIV-1 epitranscriptome in biology and disease,difficult to assess. Therefore,we established a reference HIV-1 epitranscriptome. We sequenced the model NL4–3 HIV-1 genome from infected primary CD4+ T cells and the Jurkat cell line using the latest nanopore chemistry,optimized RNA preparation methods,and the most current and readily available base-calling algorithms. A highly reproducible sense and a preliminary antisense HIV-1 epitranscriptome were created,where N6-methyladenosine (m6A),5-methylcytosine (m5C),pseudouridine (psi),inosine,and 2’-O-methyl (Nm) modifications could be identified by rapid multiplexed base-calling. We observed that sequence and neighboring modification contexts induced modification miscalling,which could be corrected with synthetic HIV-1 RNA fragments. We validated m6A modification sites with STM2457,a small molecule inhibitor of methyltransferase-like 3 (METTL3). We find that modifications are quite stable under combination antiretroviral therapy (cART) treatment,in primary CD4+ T cells,and in HIV-1 virions. Sequencing samples from people living with HIV (PLWH) revealed conservation of m6A modifications. However,analysis of spliced transcript variants suggests transcript-dependent modification levels. Our approach and reference data offer a straightforward benchmark that can be adopted to help advance rigor,reproducibility,and uniformity across HIV-1 epitranscriptomics studies. They also provide a roadmap for the creation of reference epitranscriptomes for many other viruses or pathogens. View Publication