Unfolded Protein Response Genes Research Articles

Protein disulfide isomerase-9 interacts with the lumenal region of the transmembrane endoplasmic reticulum stress sensor kinase, IRE1, to modulate the unfolded protein response in Arabidopsis

Environmental stressors disrupt secretory protein folding and proteostasis in the endoplasmic reticulum (ER), leading to ER stress. The unfolded protein response (UPR) senses ER stress and restores proteostasis by increasing the expression of ER-resident protein folding chaperones, such as protein disulfide isomerases (PDIs). In plants, the transmembrane ER stress sensor kinase, IRE1, activates the UPR by unconventionally splicing the mRNA encoding the bZIP60 transcription factor, triggering UPR gene transcription. The induced PDIs catalyze disulfide-based polypeptide folding to restore the folding capacity in the ER; however, the substrates with which PDIs interact are largely unknown. Here, we demonstrate that the Arabidopsis PDI-M subfamily member, PDI9, modulates the UPR through interaction with IRE1. This PDI9–IRE1 interaction was largely dependent on Cys63 in the first dithiol redox active domain of PDI9, and Cys233 and Cys107 in the ER lumenal domain of IRE1A and IRE1B, respectively. In vitro and in vivo, PDI9 coimmunoprecipitated with IRE1A and IRE1B. Moreover, the PDI9:RFP and Green Fluorescence Protein (GFP):IRE1 fusions exhibited strong interactions as measured by fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) when coexpressed in mesophyll protoplasts. The UPR-responsive PDI9 promoter:mCherry reporter and the UPR-dependent splicing of the bZIP60 intron from the mRNA of the 35S::bZIP60-intron:GFP reporter were both significantly induced in the pdi9 mutants, indicating a derepression and hyperactivation of UPR. The inductions of both reporters were substantially attenuated in the ire1a–ire1b mutant. We propose a model in which PDI9 modulates the UPR through two competing activities: secretory protein folding and via interaction with IRE1 to maintain proteostasis in plants.

Protein quality control systems in the endoplasmic reticulum and the cytosol coordinately prevent alachlor-induced proteotoxic stress in Saccharomyces cerevisiae

Alachlor, a widely used chloroacetanilide herbicide for controlling annual grasses in crops, has been reported to rapidly trigger protein denaturation and aggregation in the eukaryotic model organism Saccharomyces cerevisiae. Therefore, this study aimed to uncover cellular mechanisms involved in preventing alachlor-induced proteotoxicity. The findings reveal that the ubiquitin-proteasome system (UPS) plays a crucial role in eliminating alachlor-denatured proteins by tagging them with polyubiquitin for subsequent proteasomal degradation. Exposure to alachlor rapidly induced an inhibition of proteasome activity by 90 % within 30 min. The molecular docking analysis suggests that this inhibition likely results from the binding of alachlor to β subunits within the catalytic core of the proteasome. Notably, our data suggest that nascent proteins in the endoplasmic reticulum (ER) are the primary targets of alachlor. Consequently, the unfolded protein response (UPR), responsible for coping with aberrant proteins in the ER, becomes activated within 1 h of alachlor treatment, leading to the splicing of HAC1 mRNA into the active transcription activator Hac1p and the upregulation of UPR gene expression. These findings underscore the critical roles of the protein quality control systems UPS and UPR in mitigating alachlor-induced proteotoxicity by degrading alachlor-denatured proteins and enhancing the protein folding capacity of the ER.

Boosting wound healing in diabetic rats: The role of nicotinamide riboside and resveratrol in UPR modulation and pyroptosis inhibition

BackgroundDiabetes-related skin ulcers provide a substantial therapeutic issue, sometimes leading to amputation, needing immediate practical treatments for efficient wound care. While the exact mechanisms are unknown, pyroptosis and deregulation of the unfolded protein response (UPR) are known to exacerbate inflammation. Nicotinamide Riboside (NR) and Resveratrol (RV), which are known for their Nicotinamide adenine dinucleotide (NAD+) boosting and anti-inflammatory properties, are being studied as potential treatments. The purpose of this study was to shed light on the underlying molecular mechanisms and explore the medical application of NR and RV in diabetic wound healing. Methods54 male Sprague-Dawley rats divided into control, diabetic (DM), Gel Base, DM-NR, DM-RV, and DM-NR + RV. Rats were orally administered 50 mg/kg/day of RV and 300 mg/kg/day of NR for 5 weeks. Following diabetes induction, their wounds were topically treated with 5 % NR and RV gel for 15 days. The wound closure rate, body weight, and serum lipid profiles were examined. Gene expression study evaluated UPR and pyroptosis-related genes (BIP, PERK, ATF6, IRE1α, sXBP1, CHOP, NLRP3, caspase-1, NFκB, and IL1-β) in wound tissues, alongside histological assessment of cellular changes. ResultsNR and RV treatments greatly enhanced wound healing. Molecular investigation demonstrated UPR and pyroptosis marker modifications, suggesting UPR balance and anti-inflammatory effects. Histological investigation demonstrated decreased inflammation and increased re-epithelialization. The combination of NR and RV therapy had better results than either treatment alone. ConclusionThis study shows that NR and RV have therapeutic promise in treating diabetic wounds by addressing UPR dysregulation, and pyroptosis. The combination therapy is a viable strategy to improving the healing process, providing a multimodal intervention for diabetic skin ulcers. These findings pave the way for additional investigation and possible therapeutic applications, giving hope for better outcomes in diabetic wound care.

Abstract 5371: Multimodal CRISPR activation screens reveal new mechanisms to sensitize cancer cells to targeted T cell mediated elimination

Abstract Cell autonomous resistance mechanisms can protect cancer cells from targeted T cell elimination, resulting in resistance to a broad range of immunotherapies. High-throughput gene knockout and gene inhibition screens have been instrumental in identifying key drivers of such mechanisms, yet often uncovering gene activity that is essential but not sufficient for response. Here we set out to leverage gene activation as an orthogonal approach to identify novel regulators of the cancer-immune interface. First, we performed CRISPR activation screens in melanoma cells co-cultured with antigen-specific primary CD8+ T cells. Our findings revealed a collection of novel genes that substantially sensitize melanoma cells to targeted T cell-driven elimination when activated, including DNA damage and unfolded protein response genes (e.g., ETS1, TSPYL2), Wnt ligands (e.g., WNT3A, WNT1), and others that are deleterious only in combination with targeting T cells. Among the resistance genes we found regulators of insulin signaling (e.g., HGF, TCF7L2) and numerous glycoproteins (e.g., PDPN, CD44). Second, we validated top novel hits in 2D and in 3D spheroid models, using optical readouts to investigate their impact on cancer cell elimination and cancer-T-cell spatial dynamics. Third, we performed a Perturb-seq screen to stratify dozens of hits into functional groups based on the transcriptional readouts in the cancer cells. Lastly, we developed an in situ Perturb-Seq method, where we combine high-plex spatial transcriptomic readouts at subcellular resolution with in situ detection of genetically modified cells, and applied it to track the impact of the (de)sensitizing perturbations not only on the cancer cells themselves, but also on T cell recruitment and suppression. Taken together, our study provides a catalog of immune evasion mechanisms and an array of novel targets, demonstrating that synthetic gene activation can diminish immune evasion and open novel avenues for development of agonist-based and RNA-based interventions. Citation Format: Reece V. Akana, Chang Sun, YoungMin Kim, Yuxin Cai, Subin Kim, Jeehyun Yoe, Olivia Laveroni, Livnat Jerby-Arnon. Multimodal CRISPR activation screens reveal new mechanisms to sensitize cancer cells to targeted T cell mediated elimination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5371.

Mapping Interindividual Variability of Toxicodynamics Using High-Throughput Transcriptomics and Primary Human Hepatocytes from Fifty Donors.

Understanding the variability across the human population with respect to toxicodynamic responses after exposure to chemicals, such as environmental toxicants or drugs, is essential to define safety factors for risk assessment to protect the entire population. Activation of cellular stress response pathways are early adverse outcome pathway (AOP) key events of chemical-induced toxicity and would elucidate the estimation of population variability of toxicodynamic responses. We aimed to map the variability in cellular stress response activation in a large panel of primary human hepatocyte (PHH) donors to aid in the quantification of toxicodynamic interindividual variability to derive safety uncertainty factors. High-throughput transcriptomics of over 8,000 samples in total was performed covering a panel of 50 individual PHH donors upon 8 to 24 h exposure to broad concentration ranges of four different toxicological relevant stimuli: tunicamycin for the unfolded protein response (UPR), diethyl maleate for the oxidative stress response (OSR), cisplatin for the DNA damage response (DDR), and tumor necrosis factor alpha () for signaling. Using a population mixed-effect framework, the distribution of benchmark concentrations (BMCs) and maximum fold change were modeled to evaluate the influence of PHH donor panel size on the correct estimation of interindividual variability for the various stimuli. Transcriptome mapping allowed the investigation of the interindividual variability in concentration-dependent stress response activation, where the average of BMCs had a maximum difference of 864-, 13-, 13-, and 259-fold between different PHHs for UPR, OSR, DDR, and signaling-related genes, respectively. Population modeling revealed that small PHH panel sizes systematically underestimated the variance and gave low probabilities in estimating the correct human population variance. Estimated toxicodynamic variability factors of stress response activation in PHHs based on this dataset ranged between 1.6 and 6.3. Overall, by combining high-throughput transcriptomics and population modeling, improved understanding of interindividual variability in chemical-induced activation of toxicity relevant stress pathways across the human population using a large panel of plated cryopreserved PHHs was established, thereby contributing toward increasing the confidence of in vitro-based prediction of adverse responses, in particular hepatotoxicity. https://doi.org/10.1289/EHP11891.

SlCathB2 as a negative regulator mediates a novel regulatory pathway upon high-temperature stress response in tomato.

High-temperature stress (HS) is a major abiotic stress that affects the yield and quality of plants. Cathepsin B-like protease 2 (CathB2) has been reported to play a role in developmental processes and stress response, but its involvement in HS response has not been identified. Here, overexpression, virus-induced gene silencing (VIGS)and RNA-sequencing analysis were performed to uncover the functional characteristics of SlCathB2-1 and SlCathB2-2 genes for HS response in tomato. The results showed that overexpression of SlCathB2-1 and SlCathB2-2 resulted in reduced heat tolerance of tomato to HS while silencing the genes resulted in enhanced heat tolerance. RNA-sequencing analysis revealed that the heat shock proteins (HSPs) exhibited higher expression in WT than in SlCathB2-1 and SlCathB2-2 overexpression lines. Furthermore, the possible molecular regulation mechanism underlying SlCathB2-1 and SlCathB2-2-mediated response to HS was investigated. We found that SlCathB2-1 and SlCathB2-2 negatively regulated antioxidant capacity by regulating a set of genes involved in antioxidant defence and reactive oxygen species (ROS) signal transduction. We also demonstrated that SlCathB2-1 and SlCathB2-2 positively regulated ER-stress-induced PCD (ERSID) by regulating unfolded protein response (UPR) gene expression. Furthermore, SlCathB2-1 and SlCathB2-2 interacting with proteasome subunit beta type-4 (PBA4) was identified in the ERSID pathway using yeast two-hybrid (Y2H) analysis and bimolecular fluorescence complementation (BiFC) screening. Overall, the study identified both SlCathB2-1 and SlCathB2-2 as new negative regulators to HS and presented a new HS response pathway. This provided the foundation for the construction of heat-tolerant molecular mechanisms and breeding strategies aiming to improve the thermotolerance of tomato plants.

Expression of key unfolded protein response genes predicts patient survival and an immunosuppressive microenvironment in glioblastoma

Abstract Background Dysregulation of cellular processes related to protein folding and trafficking leads to the accumulation of misfolded proteins in the endoplasmic reticulum (ER), triggering ER stress. Cells cope with ER stress by activating the unfolded protein response (UPR), a signaling pathway that has been implicated in a variety of diseases, including cancer. However, the role of the UPR in cancer initiation and progression is still unclear. Methods Here we used bulk and single cell RNA sequencing data to investigate ER stress-related gene expression in glioblastoma, as well as the impact key UPR genes have on patient survival. Results ER stress-related genes are highly expressed in both cancer cells and tumor-associated macrophages, with evidence of high intra- and inter-tumor heterogeneity. High expression of the UPR-related genes HSPA5, P4HB, and PDIA4 was identified as risk factors while high MAPK8 (JNK1) expression was identified as a protective factor in glioblastoma patients, indicating UPR genes have prognostic potential in this cancer type. Finally, expression of XBP1 and MAPK8, two key downstream targets of the ER sentinel IRE1α, correlates with the presence of immune cell types associated with immunosuppression and a worse patient outcome. This suggests that the expression of these genes is associated with an immunosuppressive tumor microenvironment and uncover a potential link between stress response pathways, tumor microenvironment and glioblastoma patient survival. Conclusions We performed a comprehensive transcriptional characterization of the unfolded protein response in glioblastoma patients and identified UPR-related genes associated with glioblastoma patient survival, providing potential prognostic and predictive biomarkers as well as promising targets for developing new therapeutic interventions in glioblastoma treatment.

PERK modulation, with GSK2606414, Sephin1 or salubrinal, failed to produce therapeutic benefits in the SOD1G93A mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) has been linked to overactivity of the protein kinase RNA-like ER kinase (PERK) branch of the unfolded protein response (UPR) pathway, both in ALS patients and mouse models. However, attempts to pharmacologically modulate PERK for therapeutic benefit have yielded inconsistent and often conflicting results. This study sought to address these discrepancies by comprehensively evaluating three commonly used, CNS-penetrant, PERK modulators (GSK2606414, salubrinal, and Sephin1) in the same experimental models, with the goal of assessing the viability of targeting the PERK pathway as a therapeutic strategy for ALS. To achieve this goal, a tunicamycin-challenge assay was developed using wild-type mice to monitor changes in liver UPR gene expression in response to PERK pathway modulation. Subsequently, multiple dosing regimens of each PERK modulator were tested in standardized, well-powered, gender-matched, and litter-matched survival efficacy studies using the SOD1G93A mouse model of ALS. The alpha-2-adrenergic receptor agonist clonidine was also tested to elucidate the results obtained from the Sephin1, and of the previously reported guanabenz studies, by comparing the effects of presence or absence of α-2 agonism. The results revealed that targeting PERK may not be an ideal approach for ALS treatment. Inhibiting PERK with GSK2606414 or activating it with salubrinal did not confer therapeutic benefits. While Sephin1 showed some promising therapeutic effects, it appears that these outcomes were mediated through PERK-independent mechanisms. Clonidine also produced some favorable therapeutic effects, which were unexpected and not linked to the UPR. In conclusion, this study highlights the challenges of pharmacologically targeting PERK for therapeutic purposes in the SOD1G93A mouse model and suggests that exploring other targets within, and outside, the UPR may be more promising avenues for ALS treatment.

Mitochondrial Unfolded Protein Response Gene Clpp Is Required for Oocyte Function and Female Fertility.

Mitochondrial unfolded protein stress response (mtUPR) plays a critical role in regulating cellular and metabolic stress response and helps maintain protein homeostasis. Caseinolytic peptidase P (CLPP) is one of the key regulators of mtUPR and promotes unfolded protein degradation. Previous studies demonstrated that global deletion of Clpp resulted in female infertility, whereas no impairment was found in the mouse model with targeted deletion of Clpp in cumulus/granulosa cells. These results suggest the need to delineate the function of Clpp in oocytes. In this study, we aimed to further explore the role of mtUPR in female reproductive competence and senescence using a mouse model. Oocyte-specific targeted deletion of Clpp in mice resulted in female subfertility associated with metabolic and functional abnormalities in oocytes, thus highlighting the importance of CLPP-mediated protein homeostasis in oocyte competence and reproductive function.

MUCOSAL SINGLE-CELL PROFILING OF CROHN’S-LIKE DISEASE OF THE POUCH REVEALS COMMON PATHOGENICS AND THERAPEUTIC TARGETS

Abstract BACKGROUND & AIMS After restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) for ulcerative colitis (UC), a subset of patients develop Crohn’s-like disease of the pouch (CDP), an inflammatory condition that affects both the ileoanal pouch and extra-pouch organs including pre-pouch ileum. The cellular and molecular identities of CDP are unknown, and its diagnosis and treatment remain challenging. To define the pathophysiology of CDP, we examined mucosal cells from patients with and without CDP using single cell analyses. METHODS Endoscopic samples from the pouch and pre-pouch ileum of 50 patients with an IPAA were collected for single-cell RNA sequencing (scRNA-seq) or mass cytometry (or CyTOF). We analyzed immune and non-immune cells from pouch and ileal tissues of patients with normal pouch/ileum and CDP (n = 5 or 6) using scRNA-seq. CyTOF was performed on mucosal immune cells from independent cohorts of patients with normal pouch/ileum, CDP, and pouchitis (n = 9 or 10). Mucosal samples from patients with familial adenomatous polyposis (FAP) after pouch formation were also analyzed by CyTOF. Some scRNA-seq and CyTOF findings were independently validated using immunohistochemistry (n = 6 or 8). RESULTS We revealed distinct immune and non-immune cell clusters in normal pouch and pre-pouch ileum. Colitogenic immune cells expanded in normal UC pouch compared to normal FAP pouch. Compared to normal pouch/pre-pouch ileum, CDP tissues exhibited expanded TCR clonotypes, elevated Th17 signaling, and diminished T cell exhaustion markers. Elevated frequencies of plasma cells, inflammatory fibroblasts and monocytes were noted in CDP pouch and ileum (Fig 1; only pouch data were shown). CDP also harbored elevated Th17-inducing cytokines such as IL23, IL1B, and IL6 produced by myeloid cells. ScRNA-seq and CyTOF identified increased CD14+TREM1+ pathogenic monocytes in both pouch and pre-pouch ileum of CDP. Ligand-receptor interactions further uncovered an essential role of proinflammatory myeloid cells in the pathogenesis of CDP. In addition, pouch and pre-pouch ileum of CDP showed prominent activation of the unfolded protein response (UPR) across all major cell compartments (Fig 2), which was not present in UC, CD or pouchitis based on integrated analysis of published databases. CONCLUSIONS CDP demonstrates altered immune and non-immune cell populations/states in the pouch and pre-pouch ileum. CDP represents a distinct entity of inflammatory bowel disease with extensive UPR activation, a unique feature that may serve as novel diagnostic markers and therapeutic targets using ER stress-alleviating agents including chemical chaperones. Fig 1 CDP reshapes immune and non-immune landscapes in pouch and pre-pouch ileum. (A) UMAP visualization of all cells from the pouch and ileum of CLDP and normal controls. (B) Diversity of epithelial cells, stromal cells, myeloid cells, T cells, innate lymphoid cells (ILCs), B cells and plasma cells in CLDP vs. normal controls in the pouch estimated by scRNA-seq (n = 5 or 6). *p<0.01. Fig 2 Extensive UPR activation in CDP tissues. (A) UPR gene expression in major cell compartments in CDP and normal controls, highlighted genes including CALR, XBP1, and SERP1 were upregulated in all 5 cell compartments. (B) ER stress/UPR pathways are enriched in CDP. (C) IHC of UPR proteins XBP1 and HSPA5/BiP.

Plant extracts modulate cellular stress to inhibit replication of mouse Coronavirus MHV-A59

The Covid-19 infection outbreak led to a global epidemic, and although several vaccines have been developed, the appearance of mutations has allowed the virus to evade the immune response. Added to this is the existing risk of the appearance of new emerging viruses. Therefore, it is necessary to explore novel antiviral therapies. Here, we investigate the potential in vitro of plant extracts to modulate cellular stress and inhibit murine hepatitis virus (MHV)-A59 replication. L929 cells were treated with P2Et (Caesalpinia spinosa) and Anamu SC (Petiveria alliacea) plant extracts during infection and virus production, ROS (reactive oxygen species), UPR (unfolded protein response), and autophagy were assessed. P2Et inhibited virus replication and attenuated both ROS production and UPR activation induced during infection. In contrast, the sustained presence of Anamu SC during viral adsorption and replication was required to inhibit viral infection, tending to induce pro-oxidant effects, and increasing UPR gene expression. Notably, the loss of the PERK protein resulted in a slight decrease in virus yield, suggesting a potential involvement of this UPR pathway during replication. Intriguingly, both extracts either maintained or increased the calreticulin surface exposure induced during infection. In conclusion, our findings highlight the development of antiviral natural plant extracts that differentially modulate cellular stress.

Heterologous expression of influenza haemagglutinin leads to early and transient activation of the unfolded protein response in Nicotiana benthamiana.

The unfolded protein response (UPR) allows cells to cope with endoplasmic reticulum (ER) stress induced by accumulation of misfolded proteins in the ER. Due to its sensitivity to Agrobacterium tumefaciens, the model plant Nicotiana benthamiana is widely employed for transient expression of recombinant proteins of biopharmaceutical interest, including antibodies and virus surface proteins used for vaccine production. As such, study of the plant UPR is of practical significance, since enforced expression of complex secreted proteins often results in ER stress. After 6 days of expression, we recently reported that influenza haemagglutinin H5 induces accumulation of UPR proteins. Since up-regulation of corresponding UPR genes was not detected at this time, accumulation of UPR proteins was hypothesized to be independent of transcriptional induction, or associated with early but transient UPR gene up-regulation. Using time course sampling, we here show that H5 expression does result in early and transient activation of the UPR, as inferred from unconventional splicing of NbbZIP60 transcripts and induction of UPR genes with varied functions. Transient nature of H5-induced UPR suggests that this response was sufficient to cope with ER stress provoked by expression of the secreted protein, as opposed to an antibody that triggered stronger and more sustained UPR activation. As up-regulation of defence genes responding to H5 expression was detected after the peak of UPR activation and correlated with high increase in H5 protein accumulation, we hypothesize that these immune responses, rather than the UPR, were responsible for onset of the necrotic symptoms on H5-expressing leaves.

Single Cell RNA Sequencing of Sequential Samples before and after BCMA-Directed CAR-T Reveal Features Associated with Non-Durable Response, Exhausted T-Cells and Decreased Expression of Genes Encoding Key Surface Targets in Particular in Patients with Extramedullary Disease

Introduction Single cell sequencing confers a unique opportunity to study the complex interplay between the malignant plasma cells (PCs) and the host tumor microenvironment (TME) within the bone marrow niche. Pre-treatment factors associated with the achievement of durable responses from anti-B-cell maturation antigen (BCMA) directed CART cell therapy remain poorly understood. Herein, we performed a longitudinal single cell analysis to identify factors associated with response, or therapeutic failure, to FDA approved BCMA-directed CART cell therapy. Methods We performed single-cell RNA sequencing (scRNA-seq) to comprehensively characterize both the cellular and molecular properties of malignant plasma cells and TME. Separated CD138+ and CD138- components were universally encapsulated within hours of pre-CART baseline (BL) and day 90 post treatment with commercial CAR-T using 10x Genomics. Libraries were sequenced separately on an Illumina NovaSeq 6000 instrument. Cell Ranger pipelines with default parameters were used to trim reads, align, count unique molecular identifiers, and call cells. Downstream processing was carried out using Seurat. Batch effects were corrected using canonical correlation analysis (CCA) integration. After quality control for feature counts and mitochondrial gene expression, there were a total of 110,923 single cells, including 48,150 plasma cells (PC) and 62,773 TME cells. In addition, we integrated these data with an internal repository of bulk RNA sequencing (RNAseq) (N=844 CD138+ MM cells) to evaluate co-regulation of genes (Renatino-Canevarolo et al, ASH 2022). Patients were split into two broad groups, durable (DR) vs non-durable response (NDR) based on PFS cutoff of 9 months. Given its negative impact on outcomes, we also examined differences between those with or without extra-medullary disease (EMD). Results Median PFS for the total cohort of N=13 at the time of data cutoff was 332 days (range 279-569) for DR and 28 days (12-197) for NDR. Malignant cells were identified based on inferred copy number alterations and comparison to benign plasma cell controls from sequenced healthy donors. Single-cell expression profiles of plasma cells are primarily clustered by patient, whereas expression of non-plasma cells largely clustered by cell types. In DR, expression of BL malignant PCs revealed enriched expression of genes encoding several known surface markers, including FCRL5, CD38, TACI and BCMA, and genes associated with a more plasma-cell-like phenotype, such as XBP1, whereas EEF2 and CD48/SLAMF2 were increased in BL PCs from NDR.Pathway analysis of DR vs NDR revealed upregulated activity genes involved in the interleukin-6 pathway, interferon γ and oxidative phosphorylation (padj <0.1). In contrast, MYC pathways and unfolded protein response genes (padj < 0.1) were significantly upregulated in NDR ( Figure 1). Analysis of the BL TME illustrated fewer CD4 naïve, CD4 effector memory (EM), T effector memory RA-positive (TEMRA) cells and higher CD8 central memory (CM) cells in NDR vs DR patients. Exhaustion signatures were higher among BL CD4 and CD8 cells pre-CART in NDR relative to DR, and in D90 samples this was also more pronounced among CD4 cells in NDR vs DR samples. Comparing D90 to BL, DR patients had fewer Tregs, increased numbers of trafficking monocytes and more proliferating CD8 T-cells. Finally, looking at patients with EMD, differences in surface target gene expression were noted with decreased expression of BCMA, TACI, FCRL5, and CD38 observed, although CD44 and Ly9 expression appeared equivalent or enriched in those with known EMD (Figure 2). Interrogation of bulk RNAseq revealed that several of these genes, including BCMA, PRDM1/Blimp-1, XBP1, CD138 are co-regulated in a common geneset. This geneset was significantly downregulated in patients with NDR vs DR. Conclusion These data highlight the pre-treatment features of both tumor and T-cell that can pre-dispose real-world patients with RRMM to early relapse after anti-BCMA CAR-T, notably pre-existing T-cell exhaustion, and the presence of highly proliferative plasma cells with underlying resistance mechanisms, including downregulation of several known immunotherapy targets, especially in those with co-existing EMD. Ciara L Freeman, Xiaohong Zhao & Mark B Meads contributed equally

Stress-induced β cell early senescence confers protection against type 1 diabetes

During the progression of type 1 diabetes (T1D), β cells are exposed to significant stress and, therefore, require adaptive responses to survive. The adaptive mechanisms that can preserve β cell function and survival in the face of autoimmunity remain unclear. Here, we show that the deletion ofthe unfolded protein response (UPR) genes Atf6α or Ire1αinβ cellsof non-obese diabetic (NOD) mice prior to insulitis generates a p21-driven early senescence phenotype and alters the β cell secretome that significantly enhances the leukemia inhibitory factor-mediated recruitment of M2 macrophages to islets. Consequently, M2 macrophages promote anti-inflammatory responses and immune surveillance that cause the resolution of islet inflammation, the removal of terminally senesced β cells, the reduction of β cell apoptosis, and protection against T1D. We further demonstrate that the p21-mediated early senescence signature is conserved in the residual β cells of T1D patients. Our findings reveal a previously unrecognized link between β cell UPR and senescence that, if leveraged, may represent a novel preventive strategy for T1D.

Abstract 18021: Environmental Circadian Desynchronization and Metabolic Stress Drive Heart Failure With Preserved Ejection Fraction (HFpEF)

Background: The pathogenic mechanisms underlying HFpEF (Heart Failure with preserved Ejection Fraction), which accounts for 50% of heart failure, remain unclear. Elevated endoplasmic reticulum (ER) stress has been implicated in HFpEF. Circadian disruption (CD) as seen in shift workers has been associated with an excess risk for chronic age-related disorders, including cardiovascular disease. Currently, the role of CD in HFpEF is not known. Hypothesis: The dual 'hits' of chronic metabolic stress and circadian disruption lead to increased ER stress, impaired unfolded protein response (UPR), and adverse cardiac remodeling resulting in HFpEF. Methods: High fat diet (HFD) fed C57BL6 male mice were subjected to chronic shifted light-dark (LD) cycles mimicking shift work (Shift), with appropriate controls. Metabolic (glucose tolerance, insulin sensitivity, body fat) and cardiac function parameters (systolic and diastolic function by ECHO, MRI, and terminal PV-loops) were collected. Cardiac tissue was analyzed using qPCR, western blot, bulk RNA-seq, spatial transcriptomics, proteomics, and histology. For in vitro studies, Bmal1 was deleted in H9C2 cells using CRISPR/Cas9. Results: ‘HS’ mice (HFD & Shift) had the most insulin resistance, glucose intolerance, and obesity, compared to ‘HR’ (HFD & Regular LD), ‘CS’ (Normal Chow & Shift) and ‘CR’ (Chow and Regular LD) groups. ECHO and MRI revealed preserved EF in all groups, with diastolic impairment in both HS and HR (↑MV E/E’, ↓MV E/A, and ↓LV diastolic strain) groups, HS>HR. Only ‘HS’ mice showed ↓cardiac compliance (↑β in EDPVR) and ↑lung weight suggestive of HF consistent with HFpEF. Gene and protein expression revealed dysregulation of numerous UPR pathway molecules (Atf6, Chop, Bip, Perk, and others) in HS mice. Mechanistically, Bmal1 deletion in CMs resulted in similar dysregulation of UPR genes. HS mouse hearts showed increased fibrosis, and spatial transcriptomics displayed enrichment of ATF4-related pathways in CMs and activated fibroblasts. Conclusions: The dual hit of metabolic and circadian stress induces HFpEF, which cannot be recapitulated by only one stressor. Our results identify previously unrecognized roles of UPR perturbation in driving HFpEF under circadian and metabolic stress.

Cloning, expression and functional characterization of recombinant tumor necrosis factor α1 (TNFα1) from white crucian carp in gut immune regulation

TNFα is one of important cytokines belonging to TNF superfamily, which can exhibit a pleiotropic effect in immune modulation, homeostasis as well as pathogenesis. However, its immunoregulatory function on mucosal immunity in fish gut are still unclear. In this study, we aimed to investigated the immunoregulatory role of TNFα1 in midgut of white crucian carp (WCC). WCC-TNFα1 sequence and its deduced structure were firstly identified in WCC. Then, tissue-specific analysis revealed that high-level WCC-TNFα1 expression was detected in gill. After Aeromonas hydrophila and lipopolysaccharide (LPS) stimulated, increased trends of WCC-TNFα1 expressions were detected in immune-related tissues and cultured fish cells, respectively. WCC anal-intubated with WCC-TNFα1 fusion protein showed the increased levels of edema and fuzzy appearance in impaired villi, along with atrophy and reduction of goblet cells (GC). Moreover, the expression levels of tight junction (TJ) genes and mucin genes were consistently lower than those of the control (P < 0.05). WCC-TNFα1 treatment could sharply decrease antioxidant status in midgut, while the expression levels of caspase (CASP) genes, unfolded protein response (UPR) genes and redox response genes increased dramatically. Our results suggested that WCC-TNFα1 could exhibit a detrimental effect on antioxidant and mucosal immune regulation in midgut of WCC.

A bacterial pathogen induces developmental slowing by high reactive oxygen species and mitochondrial dysfunction in Caenorhabditis elegans

Host-pathogen interactions are complex by nature, and the host developmental stage increases this complexity. By utilizing Caenorhabditis elegans larvae as the host and the bacterium Pseudomonas aeruginosa as the pathogen, we investigated how a developing organism copes with pathogenic stress. By screening 36 P.aeruginosa isolates, we found that the CF18 strain causes a severe but reversible developmental delay via induction of reactive oxygen species (ROS) and mitochondrial dysfunction. While the larvae upregulate mitophagy, antimicrobial, and detoxification genes, mitochondrial unfolded protein response (UPRmt) genes are repressed. Either antioxidant or iron supplementation rescues the phenotypes. We examined the virulence factors of CF18 via transposon mutagenesis and RNA sequencing (RNA-seq). We found that non-phenazine toxins that are regulated by quorum sensing (QS) and the GacA/S system are responsible for developmental slowing. This study highlights the importance of ROS levels and mitochondrial health as determinants of developmental rate and how pathogens can attack these important features.

Toxic effects of copper on duck cerebrum: a crucial role of oxidative stress and endoplasmic reticulum quality control.

To study the effects of Cu overload on ER quality control in duck cerebrums, 144 ducks were treated with 8 mg/kg, 100 mg/kg, 200 mg/kg and 400 mg/kg Cu added in the feed for 45 days. From histopathological examination, we found that excessive Cu increased the amount of microglia and disintegrated neuron, decreased the number of Nissl bodies, perturbed nerve fibers in duck cerebrums. Cu poisoning also increased Cu, H2O2, T-SOD, and MDA levels, decreased Fe and CAT contents in duck cerebrums. Furthermore, Cu treatment upregulated the mRNA levels of the unfolded protein response genes (PERK, ATF6, and IRE1), ER-associated degradation genes (CNX, Derlin1, and Derlin2), autophagy genes (ATG5, ATG7, ATG10, Beclin1, LC3A, LC3B, and P62), and heat shock response genes (Hsp70 and Hsp90) in duck cerebrums; elevated the protein levels of p-PERK, CNX, SEL1L, Beclin1, P62, and LC3BII/LC3BI in duck cerebrums; increased the numbers of SEL1L and LC3B puncta in duck cerebrums. Thus, our data showed that excessive Cu could cause histopathological damage to duck cerebrums, disrupt the balance of the trace elements, induce oxidative stress and activation of ER quality control, thereby resulting in duck cerebrums damage.

SMALLER TRICHOMES WITH VARIABLE BRANCHES (SVB) and its homolog SVBL act downstream of transcription factor NAC089 and function redundantly in Arabidopsis unfolded protein response.

The unfolded protein response (UPR) is a cellular mechanism that alleviates endoplasmic reticulum stress to maintain protein homeostasis. Although SMALLER TRICHOMES WITH VARIABLE BRANCHES (SVB) is characterized as an emerging UPR factor downstream of the IRE-bZIP60 pathway, whether its homologs participate in the plant UPR remains unknown. Here, we showed that an SVB homolog, SVB-like (SVBL), functions redundantly with SVB in endoplasmic reticulum stress tolerance. The svb-1 svbl-1 double mutant showed a hypersensitivity phenotype and had higher UPR gene expression under endoplasmic reticulum stress than single mutants and the wild type. SVB responded to endoplasmic reticulum stress by accumulating in the root epidermis and phloem cells, but SVBL did not. Ectopic expression of the UPR factor NAC089 up-regulated both SVB and SVBL genes, suggesting that SVB and SVBL work downstream of NAC089. Thus, SVB and SVBL play distinct roles that are modulated by the common upstream regulator NAC089 to cope with endoplasmic reticulum stress in Arabidopsis.

Triggering of Endoplasmic Reticulum Stress by Tannic Acid Inhibits the Proliferation and Migration of Colorectal Cancer Cells.

Due to the pivotal role of endoplasmic reticulum (ER) stress in cancers, interfering with its function can cause the accumulation of unfolded proteins, which ultimately leads to the activation of the unfolded protein response (UPR) signaling pathway and apoptosis. Therefore, the use of plant compounds such as tannic acid with UPR-inducing properties can be proposed as a possible treatment method for cancer. In this study, we investigated the effect of tannic acid on cell migration, colony formation, growth, and UPR-induced apoptosis in the SW48 colorectal cancer cell line. The MTT assay was performed to investigate the cytotoxic effect of tannic acid. We performed the qPCR method to elucidate the effect of tannic acid on the expression of Bim, MMP-9, Bcl-xL, cyclin D1, CHOP, and ATF4 genes. We also used the colony formation and migration experiments to investigate the effect of this compound on the colony formation and migration ability of tumor cells. Finally, we used Hoechst staining to measure cell apoptosis. Tannic acid inhibited the cell survival, clonogenic, and migration of colon cancer cells. This compound increased the expression of ER stress-mediated UPR genes, ATF4 and CHOP. Moreover; tannic acid increased the expression of pro-apoptotic proteins like Bim, while at the same time causing a sharp decline in the expression of anti-apoptotic protein Bcl-xL. A decline in MMP-9 expression confirmed the anti-metastatic role of this compound. Taken together, tannic acid can induce apoptosis via ER stress-mediated UPR pathway, and has a suppressive effect on cell viability, growth, migration, colony formation, and metastasis, suggesting it may be a potential drug in colorectal cancer treatment.