Lipoxin A 4 /FPR2 signaling mitigates ferroptosis of alveolar epithelial cells via NRF2-dependent pathway during lung ischemia-reperfusion injury.

BACKGROUND: Post-lung transplantation (LTx) injury can involve sterile inflammation due to ischemia-reperfusion injury (IRI). We investigated the cell-specific role of ferroptosis (excessive iron-mediated cell death) in mediating lung IRI and determined if specialized pro-resolving mediators such as Lipoxin A4 (LxA 4 ) can protect against ferroptosis in lung IRI. METHODS: Single-cell RNA sequencing of lung tissue from post-LTx patients was analyzed. Lung IRI was evaluated in C57BL/6 (WT), formyl peptide receptor 2 knockout ( Fpr2 -/- ) and nuclear factor erythroid 2-related factor 2 knockout ( Nrf2 -/- ) mice using a hilar-ligation model with or without LxA 4 administration. Furthermore, the protective efficacy of LxA 4 was evaluated employing a murine orthotopic LTx model and in vitro studies using alveolar type II epithelial (ATII) cells. RESULTS: Differential expression of ferroptosis-related genes was observed in post-LTx patient samples compared to healthy controls. A significant increase in the levels of oxidized lipids and reduction in the levels of intact lipids were observed in mice subjected to IRI compared to shams. Furthermore, pharmacological inhibition of ferroptosis with liproxstatin-1 mitigated lung IRI and lung dysfunction. Importantly, LxA 4 treatment attenuated pulmonary dysfunction, ferroptosis and inflammation in WT mice subjected to lung IRI, but not in Fpr2 -/- or Nrf2 -/- mice, after IRI. In the murine LTx model, LxA 4 treatment increased PaO 2 levels and attenuated lung IRI. Mechanistically, LxA 4 -mediated protection involves increase in NRF2 activation and glutathione concentration as well as decrease in MDA levels in ATII cells. CONCLUSIONS: LxA 4 /FPR2 signaling on ATII cells mitigates ferroptosis via NRF2 activation and protects against lung IRI.

Resolvin D2/GPR18 signaling enhances monocytic myeloid-derived suppressor cell function to mitigate abdominal aortic aneurysm formation.

Abdominal aortic aneurysm (AAA) formation is a chronic vascular pathology characterized by inflammation, leukocyte infiltration and vascular remodeling. The aim of this study was to delineate the protective role of Resolvin D2 (RvD2), a bioactive isoform of specialized proresolving lipid mediators, via G-protein coupled receptor 18 (GPR18) receptor signaling in attenuating AAAs. Importantly, RvD2 and GPR18 levels were significantly decreased in aortic tissue of AAA patients compared with controls. Furthermore, using an established murine model of AAA in C57BL/6 (WT) mice, we observed that treatment with RvD2 significantly attenuated aortic diameter, pro-inflammatory cytokine production, immune cell infiltration (neutrophils and macrophages), elastic fiber disruption and increased smooth muscle cell α-actin expression as well as increased TGF-ß2 and IL-10 expressions compared to untreated mice. Moreover, the RvD2-mediated protection from vascular remodeling and AAA formation was blocked when mice were previously treated with siRNA for GPR18 signifying the importance of RvD2/GPR18 signaling in vascular inflammation. Mechanistically, RvD2-mediated protection significantly enhanced infiltration and activation of monocytic myeloid-derived suppressor cells (M-MDSCs) by increasing TGF-ß2 and IL-10 secretions that mitigated smooth muscle cell activation in a GPR18-dependent manner to attenuate aortic inflammation and vascular remodeling via this intercellular crosstalk. Collectively, this study demonstrates RvD2 treatment induces an expansion of myeloid-lineage committed progenitors, such as M-MDSCs, and activates GPR18-dependent signaling to enhance TGF-ß2 and IL-10 secretion that contributes to resolution of aortic inflammation and remodeling during AAA formation.

MerTK-dependent efferocytosis by monocytic-MDSCs mediates resolution of post-lung transplant injury.

Rationale: Patients with end stage lung diseases require lung transplantation (LTx) that can be impeded by ischemia-reperfusion injury (IRI) leading to subsequent chronic lung allograft dysfunction (CLAD) and inadequate outcomes. Objectives: We examined the undefined role of MerTK (receptor Mer tyrosine kinase) on monocytic myeloid-derived suppressor cells (M-MDSCs) in efferocytosis (phagocytosis of apoptotic cells) to facilitate resolution of lung IRI. Methods: Single-cell RNA sequencing of lung tissue and BAL from post-LTx patients was analyzed. Murine lung hilar ligation and allogeneic orthotopic LTx models of IRI were used with Balb/c (WT), cebpb -/- (MDSC-deficient), Mertk -/- or MerTK-CR (cleavage resistant) mice. Lung function, IRI (inflammatory cytokine and myeloperoxidase expression, immunohistology for neutrophil infiltration), and flow cytometry of lung tissue for efferocytosis of apoptotic neutrophils were assessed in mice. Measurements and Main Results: A significant downregulation in MerTK-related efferocytosis genes in M-MDSC populations of CLAD patients compared to healthy subjects was observed. In the murine IRI model, significant increase in M-MDSCs, MerTK expression and efferocytosis was observed in WT mice during resolution phase that was absent in cebpb -/- Land Mertk -/- mice. Adoptive transfer of M-MDSCs in cebpb -/- mice significantly attenuated lung dysfunction, and inflammation leading to resolution of IRI. Additionally, in a preclinical murine orthotopic LTx model, increases in M-MDSCs were associated with resolution of lung IRI in the transplant recipients. In vitro studies demonstrated the ability of M-MDSCs to efferocytose apoptotic neutrophils in a MerTK-dependent manner. Conclusions: Our results suggest that MerTK-dependent efferocytosis by M-MDSCs can significantly contribute to the resolution of post-LTx IRI.

Evaluation of programmed cell death ligand 1 expression in a contemporary cohort of penile squamous cell carcinoma and its correlation with clinicopathologic and survival parameters: A study of 134 patients.

OBJECTIVES: Penile squamous cell carcinomas (PCs) are rare malignancies with a dismal prognosis in a metastatic setting; therefore, novel immunotherapeutic modalities are an unmet need. One such modality is the immune checkpoint molecule programmed cell death ligand 1 (PD-L1). We sought to analyze PD-L1 expression and its correlation with various clinicopathologic parameters in a contemporary cohort of 134 patients with PC. METHODS: A cohort of 134 patients with PC was studied for PD-L1 immunohistochemistry. The PD-L1 expression was evaluated using a combined proportion score with a cutoff of 1 or higher to define positivity. The results were correlated with various clinicopathologic parameters. RESULTS: Overall, 77 (57%) patients had positive PD-L1 expression. Significantly high PD-L1 expression was observed in high-grade tumors (P = .006). We found that 37% of human papillomavirus (HPV)-associated subtypes and 73% of other histotype tumors expressed PD-L1, while 63% of HPV-associated tumors and 27% of other histotype tumors did not (odds ratio, 1.35; P = .002 when compared for HPV-associated groups vs all others). Similarly, PD-L1-positive tumors had a 3.61-times higher chance of being node positive than PD-L1-negative tumors (P = .0009). In addition, PD-L1 high-positive tumors had a 5-times higher chance of being p16ink4a negative than PD-L1 low-positive tumors (P = .004). The PD-L1-positive tumors had a lower overall survival and cancer-specific survival than PD-L1-negative tumors. CONCLUSIONS: Overall, PD-L1 expression is associated with high-grade and metastatic tumors. Lower PD-L1 expression is observed more frequently in HPV-associated (warty or basaloid) subtypes than in other, predominantly HPV-independent types. As a result, PD-L1 positivity, including higher expression, portends lower overall and cancer-specific survival. These data provide a rational for further investigating PD-L1-based immunotherapeutics in PC.

The common TMEM173 HAQ, AQ alleles rescue CD4 T cellpenia, restore T-regs, and prevent SAVI (N153S) inflammatory disease in mice.

The significance of STING (encoded by the TMEM173 gene), in tissue inflammation and cancer immunotherapy has been increasingly recognized. Intriguingly, common human STING alleles R71H-G230A-R293Q (HAQ) and G230A-R293Q (AQ) are carried by ~60% of East Asians and ~40% of Africans, respectively. Here, we examine the modulatory effects of HAQ, AQ alleles on STING-associated vasculopathy with onset in infancy (SAVI), an autosomal dominant, fatal inflammatory disease caused by gain-of-function human STING mutations. CD4 T cellpenia is evident in SAVI patients and mouse models. Using STING knock-in mice expressing common human STING alleles HAQ, AQ, and Q293, we found that HAQ, AQ, and Q293 splenocytes resist STING-mediated cell death ex vivo, establishing a critical role of STING residue 293 in cell death. The HAQ/SAVI(N153S) and AQ/SAVI(N153S) mice did not have CD4 T cellpenia. The HAQ/SAVI(N153S), AQ/SAVI(N153S) mice have more (~10-fold, ~20-fold, respectively) T-regs than WT/SAVI(N153S) mice. Remarkably, while they have comparable TBK1, IRF3, and NFκB activation as the WT/SAVI, the AQ/SAVI mice have no tissue inflammation, regular body weight, and normal lifespan. We propose that STING activation promotes tissue inflammation by depleting T-regs cells in vivo. Billions of modern humans have the dominant HAQ, AQ alleles. STING research and STING-targeting immunotherapy should consider TMEM173 heterogeneity in humans.

Effect of Scaling and Root Planing in Conjunction with Antimicrobial Therapy on Glycated Hemoglobin Levels in Type 2 Diabetes Mellitus Patients.

Background: Scaling and root planing with systemic doxycycline had an effect on blood glucose levels in type 2 diabetes patients, according to the results of a glycosylated hemoglobin assay. The trial was conducted to determine whether periodontal therapy affected blood sugar regulation as a result of this finding. Methods: A total of 60 people with type 2 diabetes (diabetes mellitus not caused by insulin deficiency) were split into three groups. Group I (control group) consists of patients who are only taking an oral anti-diabetic medication. Patients on an oral anti-diabetic drug plus scaling and root planing make up group II. Patients on an oral anti-diabetic drug, scaling, root planing, and doxycycline constitute Group III. The patients were re-evaluated for glycated hemoglobin levels and periodontal status using the clinical parameters of gingival index by Loe and Silness and clinical attachment level at baseline, 30th, 60th, and 90th day. Results: Compared with group II, the findings showed a statistically significant decrease in glycated hemoglobin values (p =0.001) and improvements in clinical attachment level (p =0.022, 0.05) and gingival index (p = 0.009.,01) in group III. In the control group (group I), no such finding was made. The average gingival index, however, increased statistically significantly (p = 0.032, 0.05). Conclusion: Various studies have been showing the benefits of non-surgical periodontal therapy as a benefit for improving the overall glycemic status of the patient. Non-surgical mechanical periodontal therapy combined with systemic administration of antimicrobials such as doxycycline enhances the metabolic status of type 2 diabetes mellitus patients better than non-surgical mechanical periodontal therapy alone.

Sex differences in specialized pro-resolving lipid mediators and their receptors in abdominal aortic aneurysms.

Objective: In this study, we tested the hypothesis that endogenous expression of specialized pro-resolving lipid mediators (SPMs) that facilitate the resolution of inflammation, specifically Resolvin D1and -D2, as well as Maresin1 (MaR1), can impact abdominal aortic aneurysm (AAA) formation and progression in a sex-specific manner. Methods: SPM expression was quantified in aortic tissue from human AAA samples and from a murine in vivo AAA model via liquid chromatography-tandem mass spectrometry. mRNA expression for SPM receptors FPR2, LGR6, and GPR18 were quantified by real-time polymerase chain reaction. A Student t test with nonparametric Mann-Whitney or Wilcoxon test was used for pair-wise comparisons of groups. One-way analysis of variance after post hoc Tukey test was used to determine the differences among multiple comparative groups. Results: Human aortic tissue analysis revealed a significant decrease in RvD1 levels in male AAAs compared with controls, whereas FPR2 and LGR6 receptor expressions were downregulated in male AAAs compared with male controls. In vivo studies of elastase-treated mice showed higher levels of RvD2 and MaR1 as well as the SPM precursors, omega-3 fatty acids DHA and EPA, in aortic tissue from males compared with females. FPR2 expression was increased in elastase-treated females compared with males. Conclusions: Our findings demonstrate that specific differences in SPMs and their associated G-protein coupled receptors exist between sexes. These results indicate the relevance of SPM-mediated signaling pathways in sex differences impacting the pathogenesis of AAAs.

Pharmacologic inhibition by spironolactone attenuates experimental abdominal aortic aneurysms.

Background: Abdominal aortic aneurysms (AAA) are characterized by vascular inflammation and remodeling that can lead to aortic rupture resulting in significant mortality. Pannexin-1 channels on endothelial cells (ECs) can modulate ATP secretion to regulate the pathogenesis of AAA formation. Our hypothesis focused on potential of spironolactone to inhibit EC-mediated ATP release for the mitigation of AAA formation. Methods: A topical elastase AAA model was used initially in C57BL/6 (wild-type; WT) male mice. Mice were administered either a vehicle control (saline) or spironolactone and analyzed on day 14. In a second chronic AAA model, mice were subjected to elastase and ß-aminopropionitrile (BAPN) treatment with/without administration of spironolactone to pre-formed aneurysms starting on day 14 and analyzed on day 28. Aortic diameter was evaluated by video micrometry and aortic tissue was analyzed for cytokine expression and histology. ATP measurement and matrix metalloproteinase (MMP2) activity was evaluated in aortic tissue on days 14 or -28. In vitro studies were performed to evaluate the crosstalk between aortic ECs with macrophages or smooth muscle cells. Results: In the elastase AAA model, spironolactone treatment displayed a significant decrease in aortic diameter compared to elastase-treated controls on day 14. A significant increase in smooth muscle α-actin expression as well as decrease in elastic fiber disruption and immune cell (macrophages and neutrophils) infiltration was observed in mice treated with spironolactone compared to saline-treated controls. Spironolactone treatment also significantly mitigated pro-inflammatory cytokine expression, MMP2 activity and ATP content in aortic tissue compared to controls. Moreover, in the chronic AAA model, spironolactone treatment of pre-formed aneurysms significantly attenuated vascular inflammation and remodeling to attenuate the progression of AAAs compared to controls. Mechanistically, in vitro data demonstrated that spironolactone treatment attenuates extracellular ATP release from endothelial cells to mitigate macrophage activation (IL-1ß and HMGB1 expression) and smooth muscle cell-dependent vascular remodeling (MMP2 activity). Conclusion: These results demonstrate that spironolactone can mitigate aortic inflammation and remodeling to attenuate AAA formation as well as decrease growth of pre-formed aneurysms via inhibition of EC-dependent ATP release. Therefore, this study implicates a therapeutic application of spironolactone in the treatment of AAAs.

Resolution of post-lung transplant ischemia-reperfusion injury is modulated via Resolvin D1-FPR2 and Maresin 1-LGR6 signaling.

BACKGROUND: Dysregulation of inflammation-resolution pathways leads to postlung transplant (LTx) ischemia-reperfusion (IR) injury and allograft dysfunction. Our hypothesis is that combined treatment with specialized pro-resolving lipid mediators, that is, Resolvin D1 (RvD1) and Maresin-1 (MaR1), enhances inflammation-resolution of lung IR injury. METHODS: Expression of RvD1 and MaR1 was analyzed in bronchoalveolar lavage (BAL) fluid of patients on days 0, 1, and 7 post-LTx. Lung IR injury was evaluated in C57BL/6 (WT), FPR2-/-, and LGR6 siRNA treated mice using a hilar-ligation model with or without administration with RvD1 and/or MaR1. A donation after circulatory death and murine orthotopic lung transplantation model was used to evaluate the protection by RvD1 and MaR1 against lung IR injury. In vitro studies analyzed alveolar macrophages and type II epithelial cell activation after treatment with RvD1 or MaR1. RESULTS: RvD1 and MaR1 expressions in BAL from post-LTx patients was significantly increased on day 7 compared to days 0 and 1. Concomitant RvD1 and MaR1 treatment significantly mitigated early pulmonary inflammation and lung IR injury in WT mice, which was regulated via FPR2 and LGR6 receptors. In the murine orthotopic donation after cardiac death LTx model, RvD1 and MaR1 treatments significantly attenuated lung IR injury and increased PaO2 levels compared to saline-treated controls. Mechanistically, RvD1/FPR2 signaling on alveolar macrophages attenuated HMGB1 and TNF-α secretion and upregulated uptake of macrophage-dependent apoptotic neutrophils (efferocytosis), whereas MaR1/LGR6 signaling mitigated CXCL1 secretion by epithelial cells. CONCLUSIONS: Bioactive proresolving lipid mediator-dependent signaling that is, RvD1/FPR2 and MaR1/LGR6- offers a novel therapeutic strategy in post-LTx injury.

Resolution of inflammation via RvD1/FPR2 signaling mitigates Nox2 activation and ferroptosis of macrophages in experimental abdominal aortic aneurysms.

Abdominal aortic aneurysm (AAA) formation is characterized by inflammation, leukocyte infiltration, and vascular remodeling. Resolvin D1 (RvD1) is derived from ω-3 polyunsaturated fatty acids and is involved in the resolution phase of chronic inflammatory diseases. The aim of this study was to decipher the protective role of RvD1 via formyl peptide receptor 2 (FPR2) receptor signaling in attenuating abdominal aortic aneurysms (AAA). The elastase-treatment model of AAA in C57BL/6 (WT) mice and human AAA tissue was used to confirm our hypotheses. Elastase-treated FPR2-/- mice had a significant increase in aortic diameter, proinflammatory cytokine production, immune cell infiltration (macrophages and neutrophils), elastic fiber disruption, and decrease in smooth muscle cell α-actin expression compared to elastase-treated WT mice. RvD1 treatment attenuated AAA formation, aortic inflammation, and vascular remodeling in WT mice, but not in FPR2-/- mice. Importantly, human AAA tissue demonstrated significantly decreased FPR2 mRNA expression compared to non-aneurysm human aortas. Mechanistically, RvD1/FPR2 signaling mitigated p47phox phosphorylation and prevented hallmarks of ferroptosis, such as lipid peroxidation and Nrf2 translocation, thereby attenuating HMGB1 secretion. Collectively, this study demonstrates RvD1-mediated immunomodulation of FPR2 signaling on macrophages to mitigate ferroptosis and HMGB1 release, leading to resolution of aortic inflammation and remodeling during AAA pathogenesis.

Iron availability enhances the cellular energetics of aerobic Escherichia coli cultures while upregulating anaerobic respiratory chains.

Aerobic Escherichia coli growth at restricted iron concentrations (≤ 1.75 ± 0.04 µM) is characterized by lower biomass yield, higher acetate accumulation and higher activation of the siderophore iron-acquisition systems. Although iron homeostasis in E. coli has been studied intensively, previous studies focused only on understanding the regulation of the iron import systems and the iron-requiring enzymes. Here, the effect of iron availability on the energy metabolism of E. coli has been investigated. It was established that aerobic cultures growing under limiting iron conditions showed lower ATP yield per glucose, lower growth rate and lower TCA cycle activity and respiration, at the same time as increased glucose consumption, acetate and pyruvate accumulation, practically mimicking microaerobic growth. However, at excess iron, independent of oxygen availability, the cultures showed high cellular energetics (5.8 ATP/mol of glucose) by using pathways requiring iron-rich complex proteins found in the TCA cycle and respiratory chain. In conditions of iron excess, some iron-requiring terminal reductases of the respiratory chain, that were thought to function only under anaerobiosis, were used by the E. coli, when in aerobic conditions, to maintain high respiratory activity. This allowed it to produce more biomass and more reactive oxygen species that were controlled by the higher activity of the antioxidant defenses (SOD, peroxidase and catalase) and the iron-sulfur cluster repair systems.

Endothelial pannexin-1 channels modulate macrophage and smooth muscle cell activation in abdominal aortic aneurysm formation.

Pannexin-1 (Panx1) channels have been shown to regulate leukocyte trafficking and tissue inflammation but the mechanism of Panx1 in chronic vascular diseases like abdominal aortic aneurysms (AAA) is unknown. Here we demonstrate that Panx1 on endothelial cells, but not smooth muscle cells, orchestrate a cascade of signaling events to mediate vascular inflammation and remodeling. Mechanistically, Panx1 on endothelial cells acts as a conduit for ATP release that stimulates macrophage activation via P2X7 receptors and mitochondrial DNA release to increase IL-1ß and HMGB1 secretion. Secondly, Panx1 signaling regulates smooth muscle cell-dependent intracellular Ca2+ release and vascular remodeling via P2Y2 receptors. Panx1 blockade using probenecid markedly inhibits leukocyte transmigration, aortic inflammation and remodeling to mitigate AAA formation. Panx1 expression is upregulated in human AAAs and retrospective clinical data demonstrated reduced mortality in aortic aneurysm patients treated with Panx1 inhibitors. Collectively, these data identify Panx1 signaling as a contributory mechanism of AAA formation.

Mesenchymal Stem Cells Alter MicroRNA Expression and Attenuate Thoracic Aortic Aneurysm Formation.

BACKGROUND: Thoracic aortic aneurysms (TAA) are a progressive disease characterized by inflammation, smooth muscle cell activation and matrix degradation. We hypothesized that mesenchymal stem cells (MSCs) can immunomodulate vascular inflammation and remodeling via altered microRNA (miRNAs) expression profile to attenuate TAA formation. MATERIALS AND METHODS: C57BL/6 mice underwent topical elastase application to form descending TAAs. Mice were also treated with MSCs on days 1 and 5 and aortas were analyzed on day 14 for aortic diameter. Cytokine array was performed in aortic tissue and total RNA was tagged and hybridized for miRNAs microarray analysis. Immunohistochemistry was performed for elastin degradation and leukocyte infiltration. RESULTS: Treatment with MSCs significantly attenuated aortic diameter and TAA formation compared to untreated mice. MSC administration also attenuated T-cell, neutrophil and macrophage infiltration and prevented elastic degradation to mitigate vascular remodeling. MSC treatment also attenuated aortic inflammation by decreasing proinflammatory cytokines (CXCL13, IL-27, CXCL12 and RANTES) and upregulating anti-inflammatory interleukin-10 expression in aortic tissue of elastase-treated mice. TAA formation demonstrated activation of specific miRNAs that are associated with aortic inflammation and vascular remodeling. Our results also demonstrated that MSCs modulate a different set of miRNAs that are associated with decrease leukocyte infiltration and vascular inflammation to attenuate the aortic diameter and TAA formation. CONCLUSIONS: These results indicate that MSCs immunomodulate specific miRNAs that are associated with modulating hallmarks of aortic inflammation and vascular remodeling of aortic aneurysms. Targeted therapies designed using MSCs and miRNAs have the potential to regulate the growth and development of TAAs.

In vivo reprogramming of pathogenic lung TNFR2+ cDC2s by IFNß inhibits HDM-induced asthma.

Asthma is a common inflammatory lung disease with no known cure. Previously, we uncovered a lung TNFR2+ conventional DC2 subset (cDC2s) that induces regulatory T cells (Tregs) maintaining lung tolerance at steady state but promotes TH2 response during house dust mite (HDM)-induced asthma. Lung IFNß is essential for TNFR2+ cDC2s-mediated lung tolerance. Here, we showed that exogenous IFNß reprogrammed TH2-promoting pathogenic TNFR2+ cDC2s back to tolerogenic DCs, alleviating eosinophilic asthma and preventing asthma exacerbation. Mechanistically, inhaled IFNß, not IFNα, activated ERK2 signaling in pathogenic lung TNFR2+ cDC2s, leading to enhanced fatty acid oxidation (FAO) and lung Treg induction. Last, human IFNß reprogrammed pathogenic human lung TNFR2+ cDC2s from patients with emphysema ex vivo. Thus, we identified an IFNß-specific ERK2-FAO pathway that might be harnessed for DC therapy.

Maresin 1 activates LGR6 signaling to inhibit smooth muscle cell activation and attenuate murine abdominal aortic aneurysm formation.

The specialized pro-resolving lipid mediator maresin 1 (MaR1) is involved in the resolution phase of tissue inflammation. It was hypothesized that exogenous administration of MaR1 would attenuate abdominal aortic aneurysm (AAA) growth in a cytokine-dependent manner via LGR6 receptor signaling and macrophage-dependent efferocytosis of smooth muscle cells (SMCs). AAAs were induced in C57BL/6 wild-type (WT) mice and smooth muscle cell specific TGF-ß2 receptor knockout (SMC-TGFßr2-/- ) mice using a topical elastase AAA model. MaR1 treatment significantly attenuated AAA growth as well as increased aortic SMC α-actin and TGF-ß2 expressions in WT mice, but not SMC-TGFßr2-/- mice, compared to vehicle-treated mice. In vivo inhibition of LGR6 receptors obliterated MaR1-dependent protection in AAA formation and SMC α-actin expression. Furthermore, MaR1 upregulated macrophage-dependent efferocytosis of apoptotic SMCs in murine aortic tissue during AAA formation. In vitro studies demonstrate that MaR1-LGR6 interaction upregulates TGF-ß2 expression and decreases MMP2 activity during crosstalk of macrophage-apoptotic SMCs. In summary, these results demonstrate that MaR1 activates LGR6 receptors to upregulate macrophage-dependent efferocytosis, increases TGF-ß expression, preserves aortic wall remodeling and attenuate AAA formation. Therefore, this study demonstrates the potential of MaR1-LGR6-mediated mitigation of vascular remodeling through increased efferocytosis of apoptotic SMCs via TGF-ß2 to attenuate AAA formation.

Knockout of the caspase 8-associated protein 2 gene improves recombinant protein expression in HEK293 cells through up-regulation of the cyclin-dependent kinase inhibitor 2A gene.

Cell lines used in bioproduction are routinely engineered to improve their production efficiency. Numerous strategies, such as random mutagenesis, RNA interference screens, and transcriptome analyses have been employed to identify effective engineering targets. A genome-wide small interfering RNA screen previously identified the CASP8AP2 gene as a potential engineering target for improved expression of recombinant protein in the HEK293 cell line. Here, we validate the CASP8AP2 gene as an engineering target in HEK293 cells by knocking it out using CRISPR/Cas9 genome editing and assessing the effect of its knockout on recombinant protein expression, cell growth, cell viability, and overall gene expression. HEK293 cells lacking CASP8AP2 showed a seven-fold increase in specific expression of recombinant luciferase and a 2.5-fold increase in specific expression of recombinant SEAP, without significantly affecting cell growth and viability. Transcriptome analysis revealed that the deregulation of the cell cycle, specifically the upregulation of the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene, contributed to the improvement in recombinant protein expression in CASP8AP2 deficient cells. The results validate the CASP8AP2 gene is a viable engineering target for improved recombinant protein expression in the HEK293 cell line.

MicroRNA-206 antagomiR‒enriched extracellular vesicles attenuate lung ischemia‒reperfusion injury through CXCL1 regulation in alveolar epithelial cells.

BACKGROUND: Our hypothesis is that the immunomodulatory capacities of mesenchymal stem cell‒derived extracellular vesicles (EVs) can be enhanced by specific microRNAs (miRNAs) to effectively attenuate post-transplant lung ischemia‒reperfusion (IR) injury. METHODS: The expression of miR-206 was analyzed in bronchoalveolar lavage (BAL) fluid of patients on Days 0 and 1 after lung transplantation. Lung IR injury was evaluated in C57BL/6 mice using a left lung hilar-ligation model with or without treatment with EVs or antagomiR-206‒enriched EVs. Murine lung tissue was used for miRNA microarray hybridization analysis, and cytokine expression, lung injury, and edema were evaluated. A donation after circulatory death and murine orthotopic lung transplantation model was used to evaluate the protection by enriched EVs against lung IR injury. In vitro studies analyzed type II epithelial cell activation after coculturing with EVs. RESULTS: A significant upregulation of miR-206 was observed in the BAL fluid of patients on Day 1 after lung transplantation compared with Day 0 and in murine lungs after IR injury compared with sham. Treatment with antagomiR-206‒enriched EVs attenuated lung dysfunction, injury, and edema compared with treatment with EVs alone after murine lung IR injury. Enriched EVs reduced lung injury and neutrophil infiltration as well as improved allograft oxygenation after murine orthotopic lung transplantation. Enriched EVs significantly decreased proinflammatory cytokines, especially epithelial cell‒dependent CXCL1 expression, in the in vivo and in vitro IR injury models. CONCLUSIONS: EVs can be used as biomimetic nanovehicles for protective immunomodulation by enriching them with antagomiR-206 to mitigate epithelial cell activation and neutrophil infiltration in the lungs after IR injury.

A novel knock out strategy to enhance recombinant protein expression in Escherichia coli.

BACKGROUND: The expression of recombinant proteins triggers a stress response which downregulates key metabolic pathway genes leading to a decline in cellular health and feedback inhibition of both growth and protein expression. Instead of individually upregulating these downregulated genes or improving transcription rates by better vector design, an innovative strategy would be to block this stress response thereby ensuring a sustained level of protein expression. RESULTS: We postulated that the genes which are commonly up-regulated post induction may play the role of signalling messengers in mounting the cellular stress response. We identified those genes which have no known downstream regulatees and created knock outs which were then tested for GFP expression. Many of these knock outs showed significantly higher expression levels which was also sustained for longer periods. The highest product yield (Yp/x) was observed in a BW25113ΔcysJ knock out (Yp/x 0.57) and BW25113ΔelaA (Yp/x 0.49), whereas the Yp/x of the control W3110 strain was 0.08 and BW25113 was 0.16. Double knock out combinations were then created from the ten best performing single knock outs leading to a further enhancement in expression levels. Out of 45 double knock outs created, BW25113ΔelaAΔyhbC (Yp/x 0.7) and BW25113ΔcysJΔyhbC (Yp/x 0.64) showed the highest increase in product yield compared to the single gene mutant strains. We confirmed the improved performance of these knock outs by testing and obtaining higher levels of recombinant asparaginase expression, a system better suited for analysing sustained expression since it gets exported to the extracellular medium. CONCLUSION: Creating key knock outs to block the CSR and enhance expression is a radically different strategy that can be synergistically combined with traditional methods of improving protein yields thus helping in the design of superior host platforms for protein expression.