Differential gene expression of leaflet tissue in chronic ovine functional tricuspid regurgitation.

OBJECTIVES: Severe functional tricuspid regurgitation (FTR) is associated with subvalvular remodelling, but leaflet tissue alterations may also contribute. We set out to investigate molecular mechanisms driving leaflet remodelling in chronic ovine FTR. METHODS: Thirteen adult sheep (55 ± 4kg) underwent left thoracotomy, epicardial echocardiography, and pulmonary artery banding (PAB) to induce right heart failure and FTR. After 16 weeks, 13 banded (FTR) and 12 control (CTL) animals underwent median sternotomy for epicardial echocardiography and were subsequently sacrificed with each tricuspid leaflet tissue harvested for RNA-seq and histology. RESULTS: After 16 weeks, 7 animals developed severe, 2 moderate, and 4 mild tricuspid regurgitation (TR). Relative to CTL, FTR animals had increased PAP, TR, tricuspid annular diameter, and right atrial volume, while tricuspid annular plane systolic excursion (TAPSE) and RV fractional area change decreased. FTR leaflets exhibited altered constituents and an increase in cellularity. RNA-seq identified 85 significantly differentially expressed genes (DEG) with 17, 53, and 127 within the anterior, posterior, and septal leaflets respectively. RRM2, PRG4, and CXCL8 (IL-8) were identified as DEGs across all leaflets and CXCL8 was differentially expressed between FTR severity grades. RRM2, PRG4, and CXCL8 significantly correlated with TAPSE, and this correlation was consistent regardless of the anatomical location of the leaflet. CONCLUSIONS: PAB in our ovine model resulted in RV failure and FTR. Leaflet RNA-seq identified several DEGs, specifically RRM2, PRG4, and CXCL8, with known roles in tissue remodelling. These data along with an overall increase in leaflet cellularity suggest tricuspid leaflets actively remodel in FTR.

Utilization of a novel human hepatocyte-endothelial cell coculture model to determine differential toxicities of pyrrolizidine alkaloid food contaminants.

Pyrrolizidine alkaloids (PA) are comprised of a family of hundreds of metabolites, produced by plants as a mechanism to protect against herbivory. Upon ingestion and metabolism, dehydropyrrolizidine alkaloids are formed, which are known to generate DNA adducts and subsequently double-strand DNA breaks. Within the liver, the most sensitive cell type to PA exposure is the sinusoidal endothelial cell, as evidenced by the generation of veno-occlusive disease in the human population. PAs are a common crop contaminant and have been regulated by some agencies, using the precautionary principle; each equally potent and genotoxic. Therefore, as a proof of principle we have established a human in vitro coculture model system, utilizing the metabolically active HepaRG hepatocyte and the SK-Hep-1 endothelial cell, to determine differential potencies of different PAs commonly found in crops and food products, notably cell death, targeting of endothelial cells, and genotoxicity comparing the micronucleus assay versus γH2AX assay. Our results demonstrate differential potencies of the PAs used, which encompass three esterification states (monoester, cyclic diester, and open-chain diester). The results suggest that a more nuanced approach to the regulation of PAs may be more appropriate in the regulatory decision-making process.

When the weight of evidence does not weigh enough: EFSA's draft scientific opinion on BPA.

In November of 2021, the European Food Safety Authority (EFSA) released a draft scientific opinion on bisphenol A (BPA) exposure and health outcomes released to the public. EFSA concluded that the most sensitive outcome category to BPA exposure is the immune system. In this scientific opinion, EFSA utilized a weight of evidence approach to conclude that it is likely that BPA exposure promotes the development of TH17 cell-mediated atopic respiratory disease (eg, wheezing, rhinitis and asthma). Here, we present a dissenting analysis to that put forward in the draft EFSA scientific opinion and raise concerns about the studies and EFSA's interpretation of data that were used to arrive at their conclusion.

Valvular complex and tissue remodelling in ovine functional tricuspid regurgitation.

OBJECTIVES: Pathophysiology of function tricuspid regurgitation (FTR) is incompletely understood. We set out to comprehensively evaluate geometric and tissue remodelling of the tricuspid valve complex in ovine FTR. METHODS: Twenty adult sheep underwent left thoracotomy and pulmonary artery banding (PAB) to induce right heart pressure overload and FTR. After 8 weeks, 17 surviving animals and 10 healthy controls (CTL) underwent sternotomy, echocardiography and implantation of sonomicrometry crystals on right ventricle and tricuspid valvular apparatus. Haemodynamic and sonomicrometry data were acquired in all animals after weaning from cardiopulmonary bypass. Leaflet tissue was harvested for pentachrome histologic analysis and biomechanical testing. RESULTS: Animal weight was 62 ± 5 and 63 ± 3 kg for CTL and PAB, respectively (P = 0.6). At terminal procedure, systolic pulmonary artery pressure was 22 ± 3 and 40 ± 7 mmHg for CTL and PAB, respectively (P = 0.0001). The mean TR grade (+0-4) was 0.8 ± 0.4 and 3.2 ± 1.2 (P = 0.0001) for control and banded animals, respectively. Right ventricle volume (126 ± 13 vs 172 ± 34 ml, P = 0.0019), tricuspid annular area (651 ± 109 vs 865 ± 247 mm2, P = 0.037) and area between papillary muscle tips (162 ± 51 vs 302 ± 75 mm2, P = 0.001) increased substantially while systolic excursion of anterior leaflet decreased significantly (23.8 ± 6.1° vs 7.4 ± 4.5°, P = 0.001) with banding. Total leaflet surface area increased from 806 ± 94 to 953 ± 148 mm2 (P = 0.009), and leaflets became thicker and stiffer. CONCLUSIONS: Detailed analysis of the tricuspid valve complex revealed significant ventricular, annular, subvalvular and leaflet remodelling to be associated with ovine functional tricuspid regurgitation. Durable surgical repair of severe FTR may require a multi-level approach to the valvular apparatus.

Establishment of a point of departure for CBD hepatotoxicity employing human HepaRG spheroids.

The United States Food and Drug Administration recently approved the use of Cannabis sativa derived cannabidiol (CBD) in the treatment of Dravet Syndrome and Lennox-Gastaut Syndrome, under the trade name, Epidiolex. In double-blinded, placebo-controlled clinical trials, elevated ALT levels were observed in some patients, but these findings could not be uncoupled from the confounds of potential drug-drug interactions with co-administration of valproate and clobazam. Given the uncertainty of the potential hepatatoxic effects of CBD, the objective of the present study was to determine a point of departure for CBD, using human HepaRG spheroid cultures, followed by transcriptomic benchmark dose analysis. Treatment of HepaRG spheroids with CBD for 24 and 72 h, resulted in EC50 concentrations for cytotoxicity of 86.27 µM and 58.04 µM, respectively. Subsequent transcriptomic analysis at these timepoints demonstrated little alteration of gene and pathway data sets at a CBD concentration at or below 10 µM. Although this current analysis was conducted using liver cells, interestingly the findings at 72 h post CBD treatment showed suppression of many genes more commonly associated with immune regulation. Indeed, the immune system is a well-established target for CBD based on immune function assays. Collectively, in the present studies a point of departure was derived using transcriptomic changes produced by CBD in a human cell-based model system, which has been shown to accurately translate to human hepatotoxicity modeling.

CCR5 and Biological Complexity: The Need for Data Integration and Educational Materials to Address Genetic/Biological Reductionism at the Interface of Ethical, Legal, and Social Implications.

In the age of genomics, public understanding of complex scientific knowledge is critical. To combat reductionistic views, it is necessary to generate and organize educational material and data that keep pace with advances in genomics. The view that CCR5 is solely the receptor for HIV gave rise to demand to remove the gene in patients to create host HIV resistance, underestimating the broader roles and complex genetic inheritance of CCR5. A program aimed at providing research projects to undergraduates, known as CODE, has been expanded to build educational material for genes such as CCR5 in a rapid approach, exposing students and trainees to large bioinformatics databases and previous experiments for broader data to challenge commitment to biological reductionism. Our students organize expression databases, query environmental responses, assess genetic factors, generate protein models/dynamics, and profile evolutionary insights into a protein such as CCR5. The knowledgebase generated in the initiative opens the door for public educational information and tools (molecular videos, 3D printed models, and handouts), classroom materials, and strategy for future genetic ideas that can be distributed in formal, semiformal, and informal educational environments. This work highlights that many factors are missing from the reductionist view of CCR5, including the role of missense variants or expression of CCR5 with neurological phenotypes and the role of CCR5 and the delta32 variant in complex critical care patients with sepsis. When connected to genomic stories in the news, these tools offer critically needed Ethical, Legal, and Social Implication (ELSI) education to combat biological reductionism.

Inhibition of the Myocardin-Related Transcription Factor Pathway Increases Efficacy of Trametinib in NRAS-Mutant Melanoma Cell Lines.

The Ras/MEK/ERK pathway has been the primary focus of targeted therapies in melanoma; it is aberrantly activated in almost 80% of human cutaneous melanomas (≈50% BRAFV600 mutations and ≈30% NRAS mutations). While drugs targeting the MAPK pathway have yielded success in BRAFV600 mutant melanoma patients, such therapies have been ineffective in patients with NRAS mutant melanomas in part due to their cytostatic effects and primary resistance. Here, we demonstrate that increased Rho/MRTF-pathway activation correlates with high intrinsic resistance to the MEK inhibitor, trametinib, in a panel of NRAS mutant melanoma cell lines. A combination of trametinib with the Rho/MRTF-pathway inhibitor, CCG-222740, synergistically reduced cell viability in NRAS mutant melanoma cell lines in vitro. Furthermore, the combination of CCG-222740 with trametinib induced apoptosis and reduced clonogenicity in SK-Mel-147 cells, which are highly resistant to trametinib. These findings suggest a role of the Rho/MRTF-pathway in intrinsic trametinib resistance in a subset of NRAS mutant melanoma cell lines and highlight the therapeutic potential of concurrently targeting the Rho/MRTF-pathway and MEK in NRAS mutant melanomas.

The MDM2 inhibitor CGM097 combined with the BET inhibitor OTX015 induces cell death and inhibits tumor growth in models of neuroblastoma.

BACKGROUND: Neuroblastoma (NB) is the most common extracranial solid tumor in infants and children, with amplification of the oncogene MYCN being a hallmark of high-risk disease and poor prognosis. Although less frequent, overexpression of MYC is similarly an indicator of poor prognosis. Most NB tumors initially respond to chemotherapy, however, most will relapse, resulting in chemoresistant disease. After relapse, there is growing evidence of p53 inactivation. MYC/MYCN and MDM2 have been shown to interact and contribute to NB growth and disease progression. MDM2 inhibitors and Bromodomain and Extra-Terminal domain (BET) inhibitors have both shown promise in treating NB by increasing the expression of p53 and decreasing MYC/MYCN expression, respectively. Our study focuses on the combined treatment of a MDM2 inhibitor (CGM097) with a BET inhibitor (OTX015) in neuroblastoma. METHODS: Two p53 wild-type and two p53 mutant established neuroblastoma cells lines were used to test this combination. Ray design assays were used to test whether this combination was synergistically cytotoxic to NB cells. Western blots were performed to check signaling pathways of interest after drug treatment. IncuCyte imaging and flow cytometry were utilized to quantify the apoptotic and cytostatic effects of these drugs on NB cells. In vivo studies were carried out to test the antitumor effect of this combination in a living host. RESULTS: The combination of CGM097 and OTX015 resulted in p53 activation, decreased expression of MYC family proteins and a subsequent synergistic increase in NB cell death. CONCLUSION: This study warrants further investigation into the combination of MDM2 inhibitors and BET inhibitors for the treatment in NB.

Chronic low-level cadmium exposure in rats affects cytokine production by activated T cells.

Cadmium is a toxic metal and common environmental contaminant. Chronic cadmium exposure results in kidney, bone, reproductive, and immune toxicity as well as cancer. Cadmium induces splenomegaly and affects the adaptive immune system, but specific effects vary depending on the dose, model, and endpoint. This study investigates the effects of subchronic, oral, and low-dose cadmium exposure (32 ppm cadmium chloride in drinking water for 10 weeks) on the rat immune system, focusing on T cell function. Cadmium-exposed animals demonstrated slight increases in the spleen-to-body weight ratios, and decreases in overall splenic cell numbers and markers of oxidative stress. The relative ratios of splenic cell populations remained similar, except for modest increases in regulatory T cells in the cadmium-exposed animals. Cadmium exposure also significantly increased the production of IFNγ, a pro-inflammatory cytokine, and IL-10, a cytokine produced by multiple T cell subsets that typically inhibits IFNγ expression, by activated T cells. The increase in IFNγ and IL-10 suggests that cadmium exposure may affect multiple T cell subsets. Collectively, this study suggests that subchronic, low-dose cadmium exposure impacts both immune cell function and cellularity, and may enhance inflammatory responses.

Differential effects of the Nrf2 activators tBHQ and CDDO-Im on the early events of T cell activation.

We previously demonstrated that activation of the transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2) promotes CD4+ Th2 differentiation. In the current study, we assessed the role of Nrf2 in early events following T cell activation. The Nrf2 activators, tBHQ (tert-butylhydroquinone) and CDDO-Im (the imidazolide derivative of the triterpenoid CDDO), were used in conjunction with splenocytes derived from wild-type and Nrf2-null mice to distinguish between Nrf2-specific and off-target effects. CDDO-Im inhibited early IFNγ production in a largely Nrf2-dependent manner. In contrast, tBHQ and CDDO-Im had little effect on expression of CD25 or CD69. Furthermore, tBHQ inhibited GM-CSF and IL-2 production in both wild-type and Nrf2-null T cells, suggesting this effect is Nrf2-independent. Conversely, CDDO-Im caused a concentration-dependent increase in IL-2 secretion in wild-type, but not Nrf2-null, splenocytes, suggesting that Nrf2 promotes IL-2 production. Interestingly, both compounds inhibit NFκB DNA binding, where the suppression by tBHQ is Nrf2-independent and CDDO-Im is Nrf2-dependent. Surprisingly, as compared to wild-type splenocytes, Nrf2-null splenocytes showed lower nuclear accumulation of c-Jun, a member of the AP-1 family of transcription factors, which have been shown to drive multiple immune genes, including IL-2. Both Nrf2 activators caused a Nrf2-dependent trend toward increased nuclear accumulation of c-Jun. These data suggest that modulation of cytokine secretion by tBHQ likely involves multiple pathways, including AP-1, NFκB, and Nrf2. Overall, the data suggest that Nrf2 activation inhibits secretion of the Th1 cytokine IFNγ, and increases early production of IL-2, which has been shown to promote Th2 differentiation, and may support the later occurrence of Th2 polarization.

Inhibition of early T cell cytokine production by arsenic trioxide occurs independently of Nrf2.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a stress-activated transcription factor that induces a variety of cytoprotective genes. Nrf2 also mediates immunosuppressive effects in multiple inflammatory models. Upon activation, Nrf2 dissociates from its repressor protein, Keap1, and translocates to the nucleus where it induces Nrf2 target genes. The Nrf2-Keap1 interaction is disrupted by the environmental toxicant and chemotherapeutic agent arsenic trioxide (ATO). The purpose of the present study was to determine the effects of ATO on early events of T cell activation and the role of Nrf2 in those effects. The Nrf2 target genes Hmox-1, Nqo-1, and Gclc were all upregulated by ATO (1-2 µM) in splenocytes derived from wild-type, but not Nrf2-null, mice, suggesting that Nrf2 is activated by ATO in splenocytes. ATO also inhibited IFNγ, IL-2, and GM-CSF mRNA and protein production in wild-type splenocytes activated with the T cell activator, anti-CD3/anti-CD28. However, ATO also decreased production of these cytokines in activated splenocytes from Nrf2-null mice, suggesting the inhibition is independent of Nrf2. Interestingly, ATO inhibited TNFα protein secretion, but not mRNA expression, in activated splenocytes suggesting the inhibition is due to post-transcriptional modification. In addition, c-Fos DNA binding was significantly diminished by ATO in wild-type and Nrf2-null splenocytes activated with anti-CD3/anti-CD28, consistent with the observed inhibition of cytokine production by ATO. Collectively, this study suggests that although ATO activates Nrf2 in splenocytes, inhibition of early T cell cytokine production by ATO occurs independently of Nrf2 and may instead be due to impaired AP-1 DNA binding.

Nrf2-Dependent and -Independent Effects of tert-Butylhydroquinone, CDDO-Im, and H2O2 in Human Jurkat T Cells as Determined by CRISPR/Cas9 Gene Editing.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a stress-activated transcription factor activated by stimuli such as electrophilic compounds and other reactive xenobiotics. Previously, we have shown that the commonly used food additive and Nrf2 activator tert-butylhydroquinone (tBHQ) suppresses interleukin-2 (IL-2) production, CD25 expression, and NFκB activity in human Jurkat T cells. The purpose of the current studies was to determine whether these effects were dependent upon Nrf2 by developing a human Nrf2-null T cell model using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 technology. The current studies show that suppression of CD25 expression by tBHQ is partially dependent on Nrf2, whereas inhibition of IL-2 secretion is largely Nrf2-independent. Interestingly, tBHQ inhibited NFκB activation in an Nrf2-independent manner. This was an unexpected finding since Nrf2 inhibits NFκB activation in other models. These results led us to investigate another more potent Nrf2 activator, the synthetic triterpenoid 1[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im). Treatment of wild-type and Nrf2-null Jurkat T cells with CDDO-Im resulted in an Nrf2-dependent suppression of IL-2. Furthermore, susceptibility to reactive oxygen species was significantly enhanced in the Nrf2-null clones as determined by decreased mitochondrial membrane potential and cell viability. Importantly, this study is the first to describe the generation of a human Nrf2-null model, which is likely to have multiple applications in immunology and cancer biology. Collectively, this study demonstrates a role for Nrf2 in the effects of CDDO-Im on CD25 and IL-2 expression, whereas the effect of tBHQ on these parameters is complex and likely involves modulation of multiple stress-activated transcription factors, including NFκB and Nrf2.

The Nrf2 activator tBHQ inhibits T cell activation of primary human CD4 T cells.

The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates a battery of antioxidant, detoxification, and cell stress genes. It is activated by oxidative stress and a number of exogenous compounds, one of which is tert-butylhydroquinone (tBHQ), a widely used food preservative. Nrf2 modulates immune responses in numerous rodent models of inflammation, but its effects on human immune cells are not well characterized. The purpose of these studies was to evaluate the effects of the Nrf2 activator tBHQ on early events of T cell activation in primary human cells. Treatment with tBHQ induced mRNA expression of the Nrf2 target genes HMOX-1, GCLC, and NQO1, and also increased NRF2 mRNA expression, albeit to a lesser extent than the other target genes. tBHQ decreased production of the cytokines IL-2 and IFN-γ at both the protein and mRNA levels after stimulation with anti-CD3/anti-CD28 in human peripheral blood mononuclear cells and to an even greater extent in isolated CD4 T cells. Likewise, tBHQ decreased induction of CD25 and CD69 in peripheral blood mononuclear cells (PBMCs) and this decrease was even more marked in isolated CD4 T cells. In addition, tBHQ inhibited induction of NFκB DNA binding in anti-CD3/anti-CD28-activated PBMCs. Collectively, these data suggest that tBHQ inhibits activation of primary human CD4 T cells, which correlates with activation of Nrf2 and inhibition of NFκB DNA binding. Although these studies suggest the food additive tBHQ negatively impacts T cell activation, further studies will be needed to fully elucidate the effect of tBHQ on human immune responses.

The Nrf2 activator, tBHQ, differentially affects early events following stimulation of Jurkat cells.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that is activated by cellular stresses, such as oxidative compounds. After activation, Nrf2 induces transcription of its target genes, many of which have cytoprotective functions. Previously, we have shown that activation of Nrf2 by tert-butylhydroquinone (tBHQ) skews murine CD4⁺ T-cell differentiation. Although the role of Nrf2 in murine T cells is somewhat characterized, it is largely uncharacterized in human T cells. Therefore, the aim of the current studies was to characterize the effects of the Nrf2 activator, tBHQ, on the early events of human CD4⁺ T-cell activation. Pretreatment of Jurkat T cells with tBHQ, prior to activation with anti-CD3/anti-CD28, diminished the production of interleukin-2 (IL-2) at both the transcript and protein levels. Similarly, the expression of CD25 also diminished, albeit to a lesser degree than IL-2, after pretreatment with tBHQ. The decrease in IL-2 production was not due to decreased nuclear translocation of c-fos or c-jun. Although tBHQ caused both a delay and a decrease in Ca²âº influx in activated Jurkat cells, no decrease in nuclear factor of activated T cells (NFAT) DNA binding or transcriptional activity was observed. In contrast to NFAT, tBHQ significantly decreased NFκB transcriptional activity. Collectively, our studies show that the Nrf2 activator, tBHQ, inhibits IL-2 and CD25 expression, which correlates with decreased NFκB transcriptional activity in activated Jurkat cells. Overall, our studies suggest that Nrf2 represents a novel mechanism for the regulation of both human and mouse T cell function.