Response Of Human Cells Research Articles

Ambrisentan reverses the prosenescent and anti-autophagic effect of high glucose on human pulmonary artery endothelial cells exposed to hypoxia

Abstract Background Cardiovascular (CV) comorbidities (systemic hypertension, hyperlipidemia, obesity, diabetes mellitus, peripheral arterial disease, coronary artery disease) are associated with reduced treatment response to specific drugs for group 1 pulmonary arterial hypertension (PAH). Often this epidemiological evidence is the result of misdiagnosis of group 2 PH associated with left heart disease, but it can also be explained as the intrinsic loss of the ability of pulmonary endothelial cells to respond to specific antiremodeling drugs. Aims To evaluate the impact of high glucose and hyperosmolar stress on the responsiveness of human pulmonary artery endothelial cells (hPAECs) to the endothelin receptor antagonist ambrisentan in terms of senescence, autophagic activity, viability and expression of some microRNAs involved in pulmonary arterial remodeling. Methods We incubated hPAECs with high glucose (HG, 30.5 mM) or high mannitol (HM, 25 mM), with/without ambrisentan (0.02 nM) in normoxia (N) or hypoxia (H) for 24 h and tested them for cell viability (MTT assay), protein and miRNA levels, autophagy and senescence (β-Gal assay) in vitro. Results H induced cell death as compared to N (752 ± 44 vs 701 ± 45 Abs units, p = 0.03). H reduced autophagy (Figure 1A-C) and induced senescence (Figure 1D), an effect further potentiated by HG and HM. Treatment with A in N and H significantly reversed the effect of HG but not HM on autophagy (Figure 1A-C) and senescence (Figure 1D). H increased the abundance of antiapoptotic miR124-3, compared to N in vehicle (V)-treated hPAEC (p=0.008) (Figure 2A), and induced a similar effect on antiapoptotic and proliferative miR191-3p, although not statistically significant (Figure 2B). In N, A potentiated the expression of both miR124-3p (p=0.007) and miR191-3p (p=0.02) in HG-treated hPAEC, and only miR191-3p in HM-treated hPAEC (p=0.01). A induced a similar effect on miR191-3p in hPAEC exposed to H+HG (p=0.02), with a non-statistically significant trend on miR124-3p. Conclusions In hPAEC exposed to H, A retains its pro-autophagic and antisenescent effects in an in vitro model mimicking diabetes. miR124-3p and miR191-3p may act as biomarkers of disease and treatment response to specific drugs in patients with PAH.Figure 1:autophagy and senescenceFigure 2:miRNA expression

Interaction of RECQL4 with poly(ADP-ribose) is critical for the DNA double-strand break response in human cells.

To overcome genotoxicity, cells have evolved powerful and effective mechanisms to detect and respond to DNA lesions. RecQ Like Helicase-4 (RECQL4) plays a vital role in DNA damage responses. RECQL4 is recruited to DNA double-strand break (DSB) sites in a poly(ADP-ribosyl)ation (PARylation)-dependent manner, but the mechanism and significance of this process remain unclear. Here, we showed that the domain of RECQL4 recruited to DSBs in a PARylation-dependent manner directly interacts with poly(ADP-ribose) (PAR) and contains a PAR-binding motif (PBM). By replacing this PBM with a PBM of hnRNPA2 or its mutated form, we demonstrated that the PBM in RECQL4 is required for PARylation-dependent recruitment and the roles of RECQL4 in the DSB response. These results suggest that the direct interaction of RECQL4 with PAR is critical for proper cellular response to DSBs and provide insights to understand PARylation-dependent control of the DSB response and cancer therapeutics using PARylation inhibitors.

Segatella clades adopt distinct roles within a single individual’s gut

Segatella is a prevalent genus within individuals’ gut microbiomes worldwide, especially in non-Western populations. Although metagenomic assembly and genome isolation have shed light on its genetic diversity, the lack of available isolates from this genus has resulted in a limited understanding of how members’ genetic diversity translates into phenotypic diversity. Within the confines of a single gut microbiome, we have isolated 63 strains from diverse lineages of Segatella. We performed comparative analyses that exposed differences in cellular morphologies, preferences in polysaccharide utilization, yield of short-chain fatty acids, and antibiotic resistance across isolates. We further show that exposure to Segatella isolates either evokes strong or muted transcriptional responses in human intestinal epithelial cells. Our study exposes large phenotypic differences within related Segatella isolates, extending this to host-microbe interactions.

TRIM7 knockdown protects against LPS-induced autophagy, ferroptosis, and inflammatory responses in human bronchial epithelial cells.

Asthma is one of the most common respiratory diseases in pediatric department. Several asthma-associated events including inflammatory responses, autophagy, and ferroptosis have been identified as typical pathological processes. TRIM7 is a member of TRIM proteins family associated with several types of diseases. Nevertheless, its role in asthma is still elusive. The current research showed that TRIM7 was involved in the pathogenesis of asthma mainly by regulating the Akt signaling pathway. In detail, we found that TRIM7 was highly expressed in patients with asthma and in an in vitro model of asthma. The following analysis indicated that TRIM7 knockdown attenuated the expression and secretion of inflammatory cytokines including TNF-α, IL-1β and IL-6 in lipopolysaccharide (LPS)-exposed human bronchial epithelial cells (HBECs). Meanwhile, knockdown of TRIM7 exerted inhibitory effects on LPS-induced autophagy and ferroptosis. Further mechanistic studies showed that TRIM7 knockdown inhibited LPS-induced activation of Akt pathway, while overexpression of Akt attenuated the inhibitory effects of TRIM7 knockdown on LPS-exposed HBECs. Collectively, we reported here that TRIM7 knockdown inhibited LPS-induced autophagy, ferroptosis, and inflammatory cytokine secretion in HBECs via regulating the Akt pathway, providing a new insight into the strategies for improving asthma treatments.

Effect of CAT gene polymorphism on sensitivity of human lymphocytes to genotoxic effect of organochlorine pesticide in vitro

Introduction. Over recent decades, toxicogenetic studies have focused on the issues of genome instability under the action of genotoxicants, taking into account biomarkers of sensitivity. The question about the genotoxic potential of chlorpyrifos remains open, since both positive and negative effects have been revealed in various tests. The aim of the study is the investigation of sensitivity of donor peripheral blood lymphocytes to chlorpyrifos in vitro and evaluation of the contribution of polymorphism of antioxidant defense system genes (CAT (rs1001179), SOD2 (rs4880)) to the response of human cells to the action of genotoxicant. Materials and methods. The DNA damaging effect of chlorpyrifos was assessed on lymphocytes from fifty two donors using DNA-comet assay with metabolic activation (+S9) and without it (–S9). The study of cytotoxic effects of chlorpyrifos on human lymphocytes was carried out using an automatic fluorescent cell analyzer ADAMII LS. Results. Chlorpyrifos had a pronounced cytotoxic effect on lymphocytes in most donors in the absence of metabolic activation system. With increasing concentration of the pesticide in the medium and time of cultivation, the viability of lymphocytes decreased, and the proportion of cells in late apoptosis and necrosis increased. Positive genotoxic effects were found on the cells of 33 donors (-S9). In the presence of the S9, mild but statistically significant effects were detected only on cells from 2 donors. % DNA values in the comet tail after exposure to the pesticide varied for cells from different donors. In the absence of metabolic activation, a statistically significant increase in the level of DNA damage was found in cells of individuals with genotype AA (homozygote for the minor allele) for the CAT G262A catalase gene (rs1001179), compared with homozygote for the dominant GG allele. Limitations. The genotoxicity of chlorpyryfos was studied in vitro. Conclusion. The results of the study shown cytotoxic and genotoxic effects of chlorpyrifos. The sensitivity of lymphocytes from different donors to the pesticide was found to be significantly different. The association of the level of DNA damage under exposure of chlorpyrifos in vitro with the G262A polymorphism of the catalase gene was found. The research also confirms the possibility of using a model test-system with peripheral blood lymphocytes to assess the potential genetic risk for humans and to study the contribution of gene polymorphism to individual sensitivity to the action of genotoxicants.

Frequency-Dependent Antioxidant Responses in HT-1080 Human Fibrosarcoma Cells Exposed to Weak Radio Frequency Fields.

This study explores the complex relationship between radio frequency (RF) exposure and cancer cells, focusing on the HT-1080 human fibrosarcoma cell line. We investigated the modulation of reactive oxygen species (ROS) and key antioxidant enzymes, including superoxide dismutase (SOD), peroxidase, and glutathione (GSH), as well as mitochondrial superoxide levels and cell viability. Exposure to RF fields in the 2-5 MHz range at very weak intensities (20 nT) over 4 days resulted in distinct, frequency-specific cellular effects. Significant increases in SOD and GSH levels were observed at 4 and 4.5 MHz, accompanied by reduced mitochondrial superoxide levels and enhanced cell viability, suggesting improved mitochondrial function. In contrast, lower frequencies like 2.5 MHz induced oxidative stress, evidenced by GSH depletion and increased mitochondrial superoxide levels. The findings demonstrate that cancer cells exhibit frequency-specific sensitivity to RF fields even at intensities significantly below current safety standards, highlighting the need to reassess exposure limits. Additionally, our analysis of the radical pair mechanism (RPM) offers deeper insight into RF-induced cellular responses. The modulation of ROS and antioxidant enzyme activities is significant for cancer treatment and has broader implications for age-related diseases, where oxidative stress is a central factor in cellular degeneration. The findings propose that RF fields may serve as a therapeutic tool to selectively modulate oxidative stress and mitochondrial function in cancer cells, with antioxidants playing a key role in mitigating potential adverse effects.

MRNA-based influenza vaccine expands breadth of B cell response in humans.

Eliciting broad and durable antibody responses against rapidly evolving pathogens like influenza viruses remains a formidable challenge. The germinal center (GC) reaction enables the immune system to generate broad, high-affinity, and durable antibody responses to vaccination. mRNA-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines induce persistent GC B cell responses in humans. Whether an mRNA-based influenza vaccine could induce a superior GC response in humans compared to the conventional inactivated influenza virus vaccine remains unclear. We assessed B cell responses in peripheral blood and draining lymph nodes in cohorts receiving the inactivated or mRNA-based quadrivalent seasonal influenza vaccine. Participants receiving the mRNA-based vaccine produced more robust plasmablast responses and higher antibody titers to H1N1 and H3N2 influenza A viruses and comparable antibody titers against influenza B virus strains. Importantly, mRNA-based vaccination stimulated robust recall B cell responses characterized by sustained GC reactions that lasted at least 26 weeks post-vaccination in three of six participants analyzed. In addition to promoting the maturation of responding B cell clones, these sustained GC reactions resulted in enhanced engagement of low-frequency pre-existing memory B cells, expanding the landscape of vaccine-elicited B cell clones. This translated to expansion of the serological repertoire and increased breadth of serum antibody responses. These findings reveal an important role for the induction of persistent GC responses to influenza vaccination in humans to broaden the repertoire of vaccine-induced antibodies.

MERS-CoV-nsp5 expression in human epithelial BEAS 2b cells attenuates type I interferon production by inhibiting IRF3 nuclear translocation

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is an enveloped, positive-sense RNA virus that emerged in 2012, causing sporadic cases and localized outbreaks of severe respiratory illness with high fatality rates. A characteristic feature of the immune response to MERS-CoV infection is low type I IFN induction, despite its importance in viral clearance. The non-structural proteins (nsps) of other coronaviruses have been shown to block IFN production. However, the role of nsp5 from MERS-CoV in IFN induction of human respiratory cells is unclear. In this study, we elucidated the role of MERS-CoV-nsp5, the viral main protease, in modulating the host’s antiviral responses in human bronchial epithelial BEAS 2b cells. We found that overexpression of MERS-CoV-nsp5 had a dose-dependent inhibitory effect on IFN-β promoter activation and cytokine production induced by HMW-poly(I:C). It also suppressed IFN-β promoter activation triggered by overexpression of key components in the RIG-I-like receptor (RLR) pathway, including RIG-I, MAVS, IKK-ε and IRF3. Moreover, the overexpression of MERS-CoV-nsp5 did not impair expression or phosphorylation of IRF3, but suppressed the nuclear translocation of IRF3. Further investigation revealed that MERS-CoV-nsp5 specifically interacted with IRF3. Using docking and molecular dynamic (MD) simulations, we also found that amino acids on MERS-CoV-nsp5, IRF3, and KPNA4 may participate in protein-protein interactions. Additionally, we uncovered protein conformations that mask the nuclear localization signal (NLS) regions of IRF3 and KPNA4 when interacting with MERS-CoV-nsp5, suggesting a mechanism by which this viral protein blocks IRF3 nuclear translocation. Of note, the IFN-β expression was restored after administration of protease inhibitors targeting nsp5, indicating this suppression of IFN-β production was dependent on the enzyme activity of nsp5. Collectively, our findings elucidate a mechanism by which MERS-CoV-nsp5 disrupts the host’s innate antiviral immunity and thus provides insights into viral pathogenesis.

Acetamiprid elicits oxidative stress, pro-inflammatory response, and cellular proliferation in human bronchial epithelial cells in vitro and in silico: alleviative implications of the mixture of heat-killed Lactobacillus strains

BackgroundAcetamiprid (ACE), a neonicotinoid insecticide, has been extensively used to control pests in agricultural and industrial environments. It has been reported that ACE is detrimental to the lungs. Nevertheless, the extent to which the activation of oxidative stress, inflammation, and cellular proliferation contributes to the pulmonary toxicity induced by ACE exposure remains insufficiently understood. This study explored the mechanism of toxicological consequences after ACE exposure in bronchial epithelial cells (BEAS-2B cells). The research also examined the potential ameliorative effects of the mixture of heat-killed Lactobacillus delbrueckii and Lactobacillus fermentum (HKL) on the toxicities of ACE.ResultsFollowing 14 days of exposure to ACE at 0.5 and 1 μM, oxidative stress was induced, as evidenced by the decreased levels of reduced glutathione, catalase, glutathione peroxidase, and superoxide dismutase, along with increased levels of malondialdehyde. Also, ACE exposure results in overexpression and raised protein levels of the IL-25, NF-κB1, NF-κB2, IL-33, TSLP, and NF-κB target genes, which induce inflammatory responses. In addition, ACE boosted Ki-67-positive BEAS-2B cells. The molecular docking of ACE with target genes and their proteins demonstrated a potent binding affinity, further supported by the presence of hydrophobic contacts, electrostatic interactions, and hydrogen bonds. The post-treatment of HKL following the ACE (1 μM) exhibited its antioxidant, anti-inflammatory, and antiproliferative activities in suppressing ACE-induced toxicity.ConclusionsOur research revealed that ACE toxicity in BEAS-2B cells is caused by driving oxidative stress, pro-inflammatory response, and cellular proliferation. This study would give us a strategy to alleviate ACE-induced lung impairment by heat-killed probiotic supplements. As a result, dietary supplements that contain these microorganisms may potentially be beneficial in countries with high levels of pesticide contamination.

Hypoxia-Induced Inflammation in In Vitro Model of Human Blood-Brain Barrier: Modulatory Effects of the Olfactory Ensheathing Cell-Conditioned Medium.

Hypoxia compromises the integrity of the blood-brain barrier (BBB) and increases its permeability, thereby inducing inflammation. Olfactory ensheathing cells (OECs) garnered considerable interest due to their neuroregenerative and anti-inflammatory properties. Here, we aimed to investigate the potential modulatory effects of OEC-conditioned medium (OEC-CM) on the response of human brain microvascular endothelial cells (HBMECs), constituting the BBB, when exposed to hypoxia. HBMECs were utilized to establish the in vitro BBB model. OECs were isolated from mouse olfactory bulbs, and OEC-CM was collected after 48h of culture. The effect of OEC-CM treatment on the HBMEC viability was evaluated under both normoxic and hypoxic conditions at 6h, 24h, and 30h. Western blot and immunostaining techniques were employed to assess NF-κB/phospho-NF-κB expression. HIF-1α, VEGF-A, and cPLA2 mRNA expression levels were quantified using digital PCR. ELISA assays were performed to measure PGE2, VEGF-A, IL-8 secretion, and cPLA2 specific activity. The in vitro formation of HBMEC capillary-like structures was examined using a three-dimensional matrix system. OEC-CM attenuated pro-inflammatory responses and mitigated the HIF-1α/VEGFA signaling pathway activation in HBMECs under hypoxic condition. Hypoxia-induced damage of the BBB can be mitigated by novel therapeutic strategies harnessing OEC potential.

A Novel Chimeric Oncolytic Virus Mediates a Multifaceted Cellular Immune Response in a Syngeneic B16 Melanoma Model

Background/Objectives: Oncolytic virotherapy is a promising approach in cancer immunotherapy. We have previously described a recombinant hybrid oncolytic virus (OV), VSV-NDV, which has a favorable safety profile and therapeutic immunogenicity, leading to direct oncolysis, abscopal effects, and prolonged survival in syngeneic in vivo tumor models. While OVs are known to mediate systemic anti-tumor immune responses, the detailed characterization of local and systemic immune responses to fusogenic oncolytic virotherapy remains unexplored. Methods and Results: We analyzed immune cell compartments in the spleen, blood, tumor-draining lymph nodes (TDLNs), and tumors over the course of VSV-NDV therapy in a bilateral syngeneic melanoma mouse model. Our results revealed significant local infiltration and activation of T lymphocytes in tumors and globally in the blood and spleen. Notably, in vivo CD8+ T cell depletion led to complete abrogation of the tumor response, highlighting the crucial role of T cells in promoting the therapeutic effects of oncolytic VSV-NDV. In vitro co-culture experiments enabled the interrogation of human immune cell responses to VSV-NDV-mediated oncolysis. Human peripheral blood mononuclear cells (PBMCs) were efficiently stimulated by exposure to VSV-NDV-infected cancer cells, which recapitulates the in vivo murine findings. Conclusions: Taken together, these data characterize a broad anti-tumor immune cell response to oncolytic VSV-NDV therapy and suggest that CD8+ T cells play a decisive role in therapeutic outcome, which supports the further development of this chimeric vector as a multimechanistic immunotherapy for solid cancers.

The cytotoxic activities of the major diterpene extracted from Salvia multicaulis (Bardakosh) are mediated by the regulation of heat-shock response and fatty acid metabolism pathways in human leukemia cells

BackgroundLeukemia is one of the most lethal cancers worldwide and represents the sixth-leading cause of cancer deaths. The results of leukemia treatment have not been as positive as desired, and recurrence is common. PurposeThus, there is an urgent requirement for the development of new therapeutic drugs. Salvia multicaulis (Bardakosh) is a widespread species that contains multiple phytochemical components with anti-cancer activities. MethodsWe isolated and characterized the major diterpene candesalvone B methyl ester from S. multicaulis and investigated its action as a cytotoxic agent towards sensitive and drug-resistant leukemia cells by the resazurin reduction assay. Additionally, the targeted genes and the affected molecular mechanisms attributed to the potent cytotoxic activities were discovered by transcriptome-wide mRNA expression profiling. The targets predicted to be regulated by candesalvone B methyl ester in each cell line were confirmed by qRT-PCR, molecular docking, microscale thermophoresis, and western blotting. Moreover, cell cycle distribution and apoptosis were analyzed by flow cytometry. ResultsCandesalvone B methyl ester was cytotoxic with IC50 values of 20.95 ± 0.15 µM against CCRF-CEM cells and 4.13 ± 0.10 µM against multidrug-resistant CEM/ADR5000 leukemia cells. The pathway enrichment analysis disclosed that candesalvone B methyl ester could regulate the heat-shock response signaling pathway via targeting heat shock factor 1 (HSF1) in CCRF-CEM cells and ELOVL fatty acid elongase 5 (ELOVL5) controls the fatty acid metabolism pathway in CEM/ADR5000 cells. Microscale thermophoresis showed the binding of candesalvone B methyl ester with HSF1 and ELOVL5, confirming the results of molecular docking analysis. Down-regulation of both HSF1 and ELOVL5 by candesalvone B methyl ester as detected by both western blotting and RT-qPCR was related to the reversal of drug resistance in the leukemia cells. Furthermore, candesalvone B methyl ester increased the arrest in the sub-G1 phase of the cell cycle in a dose-dependent manner from 1.3 % to 32.3 % with concomitant induction of apoptosis up to 29.0 % in CCRF-CEM leukemic cells upon inhibition of HSF1. ConclusionCandesalvone B methyl ester isolated from S. multicaulis exerted cytotoxicity by affecting apoptosis, cell division, and modulation of expression levels of genes contributing to the heat stress signaling and fatty acid metabolism pathways that could relieve drug resistance of leukemia cells.

Dynamic responses of human lung innate and adaptive immune cells highlight the roles of genes at asthma risk loci.

The lung is a unique immunological niche with diverse immune cell types. The effects of stimulation through innate and adaptive immune receptors on human lung immune cells has largely been extrapolated from studies of blood immune cells. While multiple immune cell types and many genes have been implicated as contributing to asthma, the dynamics of these in human lung immune cells following activation will yield insights into asthma pathogenesis and lung immunity more broadly. Human lung immune cells from 6 donors were isolated. Mixed leukocytes were treated separately with lipopolysaccharide (LPS), F(ab)2-anti-human-IgM/IgG + IL4 and anti-CD3/CD28 for 4 and 18 hours and underwent single cell RNA sequencing (scRNAseq). Lung immune cell types were annotated, and gene expression compared across conditions. Genes at prior asthma-associated genetic loci were characterized across cell types, treatments and timepoints. Expression of non-classical class II genes associated with asthma, HLA-DQA2 and HLA-DQB2, and their protein products was characterized with immunohistochemistry. We characterized gene expression in 116,697 lung immune cells. Cell-, treatment-, and timepoint-specific effects on gene expression were detected in all lung immune cell populations. Correlation of gene expression between lung and blood lymphocyte populations decreased following stimulation. Among the genes that were differentially expressed, 97 receptor:ligand pairs had changes with treatments. 96.0% of genes at asthma risk loci demonstrated differential expression in at least one cell type and at least one treatment. B cells were the cell type with the highest expression of HLA-DQA2 and HLA-DQB2 which increased with anti-IgM/IgG treatment and the HLA-DQB2 protein was identified in lung B cells from a donor with asthma. Human lung immune activation elicits a broad range of cellular responses that deviate from those of blood immune cells and are relevant to asthma. Lung B cells expressing HLA-DQA2 and HLA-DQB2 appear to be involved in a novel antigen presentation pathway that contributes to asthma risk.

Comparative Analysis of the Aspergillus fumigatus Cell Wall Modification and Ensuing Human Dendritic Cell Responses by β-(1,3)-Glucan Synthase Inhibitors-Caspofungin and Enfumafungin.

Caspofungin, a lipopeptide, is an antifungal drug that belong to the class of echinocandin. It inhibits fungal cell wall β-(1,3)-glucan synthase activity and is the second-line of drug for invasive aspergillosis, a fatal infection caused mainly by Aspergillus fumigatus. On the other hand, Enfumafungin is a natural triterpene glycoside also with a β-(1,3)-glucan synthase inhibitory activity and reported to have antifungal potential. In the present study, we compared the growth as well as modifications in the A. fumigatus cell wall upon treatment with Caspofungin or Enfumafungin, consequentially their immunomodulatory capacity on human dendritic cells. Caspofungin initially inhibited the growth of A. fumigatus, but the effect was lost over time. By contrast, Enfumafungin inhibited this fungal growth for the duration investigated. Both Caspofungin and Enfumafungin caused a decrease in the cell wall β-(1,3)-glucan content with a compensatory increase in the chitin, and to a minor extent they also affected cell wall galactose content. Treatment with these two antifungals did not result in the exposure of β-(1,3)-glucan on A. fumigatus mycelial surface. Enzymatic digestion suggested a modification of β-(1,3)-glucan structure, specifically its branching, upon Enfumafungin treatment. While there was no difference in the immunostimulatory capacity of antifungal treated A. fumigatus conidia, alkali soluble-fractions from Caspofungin treated mycelia weakly stimulated the dendritic cells, possibly due to an increased content of immunosuppressive polysaccharide galactosaminogalactan. Overall, we demonstrate a novel mechanism that Enfumafungin not only inhibits β-(1,3)-glucan synthase activity, but also causes modifications in the structure of β-(1,3)-glucan in the A. fumigatus cell wall.

A combination of four Toxoplasma gondii nuclear-targeted effectors protects against interferon gamma-driven human host cell death.

In both mice and humans, Type II interferon gamma (IFNγ) is crucial for the regulation of Toxoplasma gondii (T. gondii) infection, during acute or chronic phases. To thwart this defense, T. gondii secretes protein effectors hindering the host's immune response. For example, T. gondii relies on the MYR translocon complex to deploy soluble dense granule effectors (GRAs) into the host cell cytosol or nucleus. Recent genome-wide loss-of-function screens in IFNγ-primed primary human fibroblasts identified MYR translocon components as crucial for parasite resistance against IFNγ-driven vacuole clearance. However, these screens did not pinpoint specific MYR-dependent GRA proteins responsible for IFNγ signaling blockade, suggesting potential functional redundancy. Our study reveals that T. gondii depends on the MYR translocon complex to prevent parasite premature egress and host cell death in human cells stimulated with IFNγ post-infection, a unique phenotype observed in various human cell lines but not in murine cells. Intriguingly, inhibiting parasite egress did not prevent host cell death, indicating this mechanism is distinct from those described previously. Genome-wide loss-of-function screens uncovered TgIST, GRA16, GRA24, and GRA28 as effectors necessary for a complete block of IFNγ response. GRA24 and GRA28 directly influenced IFNγ-driven transcription, GRA24's action depended on its interaction with p38 MAPK, while GRA28 disrupted histone acetyltransferase activity of CBP/p300. Given the intricate nature of the immune response to T. gondii, it appears that the parasite has evolved equally elaborate mechanisms to subvert IFNγ signaling, extending beyond direct interference with the JAK/STAT1 pathway, to encompass other signaling pathways as well.IMPORTANCEToxoplasma gondii, an intracellular parasite, affects nearly one-third of the global human population, posing significant risks for immunocompromised patients and infants infected in utero. In murine models, the core mechanisms of IFNγ-mediated immunity against T. gondii are consistently preserved, showcasing a remarkable conservation of immune defense mechanisms. In humans, the recognized restriction mechanisms vary among cell types, lacking a universally applicable mechanism. This difference underscores a significant variation in the genes employed by T. gondii to shield itself against the IFNγ response in human vs murine cells. Here, we identified a specific combination of four parasite-secreted effectors deployed into the host cell nucleus, disrupting IFNγ signaling. This disruption is crucial in preventing premature egress of the parasite and host cell death. Notably, this phenotype is exclusive to human cells, highlighting the intricate and unique mechanisms T. gondii employs to modulate host responses in the human cellular environment.

CRISPRi with barcoded expression reporters dissects regulatory networks in human cells.

Genome-wide CRISPR screens have emerged as powerful tools for uncovering the genetic underpinnings of diverse biological processes. Incisive screens often depend on directly measuring molecular phenotypes, such as regulated gene expression changes, provoked by CRISPR-mediated genetic perturbations. Here, we provide quantitative measurements of transcriptional responses in human cells across genome-scale perturbation libraries by coupling CRISPR interference (CRISPRi) with barcoded expression reporter sequencing (CiBER-seq). To enable CiBER-seq in mammalian cells, we optimize the integration of highly complex, barcoded sgRNA libraries into a defined genomic context. CiBER-seq profiling of a nuclear factor kappa B (NF-κB) reporter delineates the canonical signaling cascade linking the transmembrane TNF-alpha receptor to inflammatory gene activation and highlights cell-type-specific factors in this response. Importantly, CiBER-seq relies solely on bulk RNA sequencing to capture the regulatory circuit driving this rapid transcriptional response. Our work demonstrates the accuracy of CiBER-seq and its potential for dissecting genetic networks in mammalian cells with superior time resolution.

Repeated radon exposure induced ATM kinase-mediated DNA damage response and protective autophagy in mice and human bronchial epithelial cells.

Radon ( 222 Rn) is a naturally occurring radioactive gas that has been closely linked with the development of lung cancer. In this study, we investigated the radon-induced DNA strand breaks, a critical event in lung carcinogenesis, and the corresponding DNA damage response (DDR) in mice and human bronchial epithelial (BEAS-2B) cells. Biomarkers of DNA double-strand breaks (DSBs), DNA repair response to DSBs, ataxia-telangiectasia mutated (ATM) kinase, autophagy, and a cell apoptosis signaling pathway as well as cell-cycle arrest and the rate of apoptosis were determined in mouse lung and BEAS-2B cells after radon exposure. Repeated radon exposure induced DSBs indicated by the increasing expressions of γ-Histone 2AX (H2AX) protein and H2AX gene in a time and dose-dependent manner. Additionally, a panel of ATM-dependent repair cascades [i.e. non-homologous DNA end joining (NHEJ), cell-cycle arrest and the p38 mitogen activated protein kinase (p38MAPK)/Bax apoptosis signaling pathway] as well as the autophagy process were activated. Inhibition of autophagy by 3-methyladenine pre-treatment partially reversed the expression of NHEJ-related genes induced by radon exposure in BEAS-2B cells. The findings demonstrated that long-term exposure to radon gas induced DNA lesions in the form of DSBs and a series of ATM-dependent DDR pathways. Activation of the ATM-mediated autophagy may provide a protective and pro-survival effect on radon-induced DSBs.

P21-66 Soot particles induce cellular stress, inflammation and remodelling responses in human respiratory cells over time

P21-66 Soot particles induce cellular stress, inflammation and remodelling responses in human respiratory cells over time

TLR10 (CD290) Is a Regulator of Immune Responses in Human Plasmacytoid Dendritic Cells.

TLRs are the most thoroughly studied group of pattern-recognition receptors that play a central role in innate immunity. Among them, TLR10 (CD290) remains the only TLR family member without a known ligand and clearly defined functions. One major impediment to studying TLR10 is its absence in mice. A recent study on TLR10 knock-in mice demonstrated its intrinsic inhibitory role in B cells, indicating that TLR10 is a potential drug target in autoimmune diseases. In this study, we interrogated the expression and function of TLR10 in human plasmacytoid dendritic cells (pDCs). We have seen that primary human pDCs, B cells, and monocytes constitutively express TLR10. Upon preincubation with an anti-TLR10 Ab, production of cytokines in pDCs was downregulated in response to stimulation with DNA and RNA viruses. Upon further investigation into the possible mechanism, we documented phosphorylation of STAT3 upon Ab-mediated engagement of TLR10. This leads to the induction of inhibitory molecule suppressor of cytokine signaling 3 (SOCS3) expression. We have also documented the inhibition of nuclear translocation of transcription factor IFN regulatory factor 7 (IRF7) in pDCs following TLR10 engagement. Our data provide the (to our knowledge) first evidence that TLR10 is constitutively expressed on the surface of human pDCs and works as a regulator of their innate response. Our findings indicate the potential of harnessing the function of pDCs by Ab-mediated targeting of TLR10 that may open a new therapeutic avenue for autoimmune disorders.

An alveolus lung-on-a-chip model of Mycobacterium fortuitum lung infection.

Lung disease due to non-tuberculous mycobacteria (NTM) is rising in incidence. While both two dimensional cell culture and animal models exist for NTM infections, a major knowledge gap is the early responses of human alveolar and innate immune cells to NTM within the human alveolar microenvironment. Here we describe development of a humanized, three-dimensional, alveolus lung-on-a-chip (ALoC) model of Mycobacterium fortuitum lung infection that incorporates only primary human cells such as pulmonary vascular endothelial cells in a vascular channel, and type I and II alveolar cells and monocyte-derived macrophages in an alveolar channel along an air-liquid interface. M. fortuitum introduced into the alveolar channel primarily infected macrophages, with rare bacteria inside alveolar cells. Bulk-RNA sequencing of infected chips revealed marked upregulation of transcripts for cytokines, chemokines and secreted protease inhibitors (SERPINs). Our results demonstrate how a humanized ALoC system can identify critical early immune and epithelial responses to M. fortuitum infection. We envision potential application of the ALoC to other NTM and for studies of new antibiotics.