Recruitment Of Inflammatory Cells Research Articles

Understanding the Underlying Molecular Mechanisms of Meiotic Arrest during In Vitro Spermatogenesis in Rat Prepubertal Testicular Tissue.

In vitro spermatogenesis appears to be a promising approach to restore the fertility of childhood cancer survivors. The rat model has proven to be challenging, since germ cell maturation is arrested in organotypic cultures. Here, we report that, despite a meiotic entry, abnormal synaptonemal complexes were found in spermatocytes, and in vitro matured rat prepubertal testicular tissues displayed an immature phenotype. RNA-sequencing analyses highlighted up to 600 differentially expressed genes between in vitro and in vivo conditions, including genes involved in blood-testis barrier (BTB) formation and steroidogenesis. BTB integrity, the expression of two steroidogenic enzymes, and androgen receptors were indeed altered in vitro. Moreover, most of the top 10 predicted upstream regulators of deregulated genes were involved in inflammatory processes or immune cell recruitment. However, none of the three anti-inflammatory molecules tested in this study promoted meiotic progression. By analysing for the first time in vitro matured rat prepubertal testicular tissues at the molecular level, we uncovered the deregulation of several genes and revealed that defective BTB function, altered steroidogenic pathway, and probably inflammation, could be at the origin of meiotic arrest.

Hypothyroidism affects corneal homeostasis and wound healing in mice

PurposeThyroid hormones have a critical role in maintaining metabolic and physiological homeostasis. However, understanding of the possible effects of thyroid dysfunction on corneal homeostasis and the wound healing process is quite limited. To explore the influence of hypothyroidism on corneal homeostasis and the post-wound repair processes of the murine cornea. MethodsA hypothyroidism model was established by total thyroidectomy (TThy) in C57BL/6J mice. On day 10 after TThy, hypothyroidism was confirmed via thyronine (T3 and T4) and thyroid-stimulating hormone serum levels. We further assessed changes in corneal thickness, corneal sensitivity, sub-basal nerve density, and the corneal expression of thyroid hormone receptors. A corneal epithelial abrasion model was established via mechanical removal of a central epithelium 2 mm in diameter. Wound closure and recruitment of inflammatory cells (neutrophils and γδ T-cells) were evaluated. RNA-sequencing and gene set enrichment analysis were performed in injured corneas after abrasion. The effect of local T3 administration on corneal wound healing in thyroidectomized mice was also observed. ResultsCompared with sham-operated control mice, the TThy-treated mice showed the following: (1) a significant decrease in corneal epithelial thickness, sensitivity to external stimuli, and sub-basal nerve density, as well as an alteration in thyroid hormone receptor expression in the steady state; (2) delayed corneal wound repair and enhanced inflammatory response after corneal abrasion; (3) down-regulation of actin-skeleton and DNA replication pathways and up-regulation of inflammation-associated pathways in abraded corneas; and (4) significant restoration of delayed corneal wound repair and inhibition of excessive inflammation following topical T3 administration. ConclusionsWe conclude that deficient thyroid hormone secretion significantly affects corneal homeostasis and post-wound repair processes. Topical T3 administration might have a potential reversal effect on delayed corneal wound repair among hypothyroid individuals.

FC089: The Absence of Sting Prevents Peritoneal Damage in a Murine Model of Peritoneal Fibrosis

Abstract BACKGROUND AND AIMS Peritoneal dialysis (PD) is the current replacement therapy for end-stage renal disease (ESRD) patients until renal transplantation can be achieved. However, chronic exposure to non-physiological PD fluids (PDF) and occasional infection episodes may lead to peritoneal damage and membrane transport failure. Chronic inflammation, together with mesothelial-to-mesenchymal transition (MMT) and submesothelial fibrosis, is a hallmark of PDF-associated peritoneal damage whose intrinsic mechanisms are still expected to be disclosed [1–2]. Activation of the innate immunity is the cornerstone for infectious agents’ elimination, also enrolled in response to damage-associated molecular patterns (DAMPs) released from stressed or injured host cells. Leakage of DNA from stressed mitochondria and damaged genomic DNA may act as DAMPs that are recognized by cytosolic double-strand DNA sensors, like the stimulator of interferon gene (STING), to mount an inflammatory response [3]. Excessive activation of STING is involved in autoinflammatory, autoimmune, and increasingly related to several chronic inflammatory conditions [4]. Thus, this study aimed to investigate the role of STING in experimental peritoneal damage. METHOD We induced peritoneal fibrosis in wild-type and STING-KO mice by intraperitoneal administration of 0.1% chlorhexidine gluconate (CHX) daily for 4 weeks. No treated WT and STING-KO mice were used as control. Then, mice were euthanized, and peritoneal tissue and peritoneal lavage fluids were recollected for the following experiments. STING mRNA (Tmem173) and protein levels in control and CHX-treated wild-type were measured by RT-qPCR, western blot and immunohistochemistry. Peritoneal levels of the STING downstream proteins TBK1 and IRF3 and their phosphorylated isoforms were assayed by western blot. Peritoneal membrane thickness was measured in formalin-fixed paraffin-embedded tissues stained with Masson's trichrome stain. Fibronectin protein levels were evaluated by western blot and MMT, fibrosis, and inflammation markers were determined by RT-qPCR. Inflammatory cells present in peritoneal effluents were assayed by flow cytometry. RESULTS We found that in the peritoneum of CHX-treated wild-type mice both Tmem173 expression and STING protein levels were increased. Interestingly, STING-positive cells were located in areas of peritoneal thickness and immune infiltration. Moreover, the protein levels of IRF3, p-IRF3, TBK1 and p-TBK1 were increased, indicating that the STING pathway is activated in experimental peritoneal damage. In STING-KO mice, CHX-induced peritoneal damage was prevented. STING-KO mice under CHX treatment showed decreased peritoneal membrane thickness and attenuated CHX-induced expression of fibrosis and MMT markers, such as Tgb1, Snai1, Cdh2, Col1a1, Fn1 and fibronectin protein when compared with CHX-treated WT mice. The absence of STING also prevented the gene expression of cytokines (Il1b, Il6 and Ifng) and chemokines (Ccl5, Ccl2 and Ccl19) and macrophages infiltration into the peritoneum. Inflammatory cell recruitment into the peritoneal cavity was also decreased in STING-KO mice, including CD3+CD4+ lymphocytes and CD11b+F4/80+ macrophages. CONCLUSION In summary, STING absence prevents peritoneal membrane thickness, fibrosis, and inflammation induced by CHX in a mice model. These results suggest that STING plays a key role in peritoneal membrane injury. Therefore, STING may become a new potential therapeutic target in PD-associated peritoneal damage.

MO365: Single-Cell Profiling Analysis Reveals the Transcriptomic Landscape Of Kidneys in Patients With Acute Kidney Injury

Abstract BACKGROUND AND AIMS Acute kidney injury (AKI) is a common clinical syndrome associated with considerable mortality and healthcare costs. Up to now, the underlying pathological mechanisms of AKI are still unclear, and specific strategies for early diagnosis and treatment of AKI are lacking. In this research, single-cell RNA sequencing (scRNA-seq) was applied to kidney biopsies of patients with AKI to elucidate novel molecular mechanisms and intercellular communications. METHOD The transcriptomes of human kidney specimens obtained from subjects with AKI and renal transplant healthy living donors were defined by scRNA-seq technology. Differentially expressed genes (DEGs) and cell clusters of the kidney were determined. Gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed, and cell–cell crosstalk communication analysis among distince cell subtypes were conducted. RESULTS A total of 16 distinct cell clusters were determined in kidney from subjects of AKI and control. Proximal tubule cells of AKI had up-regulation of novel pro-apoptotic genes including USP47, RASSF4, EBAG9, IER3 and NUB1, which have not been reported previously. Further, proximal tubule cells highly expressed endoplasmic reticulum stress related genes (PDIA6, ATF6, HSPA5 and DNAJC3), autophagy related genes (S100A11, CLDN1, TMBIM6, RB1CC1 and VMP1) and genes enriched in RIG-1 signaling. PALM3 was overexpressed in proximal tubule cells of AKI at both mRNA and protein levels, which is involved in cell adhesion, TLR4 signaling and infammation response. DEGs overexpressed in other tubular cells were primarily enriched in NOD-like receptor signaling, estrogen signaling, interleukin-12-mediated signaling and IL-17 signaling. Glomerular cells such as EC upregulated DEGs participating in cell adhesion, apoptosis and chemotaxis. Overexpressed genes in kidney-resident immune cells including macrophages, NK-T cells, monocytes and dendritic cells suggested abnormal regulation related with leukocyte activation, chemotaxis, cell adhesion and complement activation. In addition, the ligand-receptor interactions analysis revealed cell–cell communication contributing to adhesion, recruitment and infiltration of inflammatory cells into the kidneys, and cytokine production in the process of AKI. CONCLUSION Together, this study reveals distinct cell-specific transcriptional landscapes of kidney in AKI patients, also altered signaling pathways and potential cell-cell crosstalk in development of AKI. These data may provide new insights into the pathogenesis and potential therapeutic strategies of AKI.

Abstract 220: Smad3 Regulates Smooth Muscle Cell Fate And Governs Adverse Remodeling And Calcification Of Atherosclerotic Plaque

Atherosclerotic plaques consist mostly of smooth muscle cells (SMC), and genes that influence SMC phenotype can modulate coronary artery disease (CAD) risk. Allelic variation at 15q22.33 has been identified by genome-wide association studies to modify the risk of CAD, and is associated with expression of SMAD3 in SMC , however the mechanism by which this gene modifies CAD risk remains poorly understood. SMC-specific deletion of Smad3 in a murine atherosclerosis model resulted in greater plaque burden, more positive remodeling, and increased vascular calcification. Single-cell transcriptomic analyses revealed that loss of Smad3 altered SMC transition cell state toward two fates: a novel SMC phenotype that governs both vascular remodeling and recruitment of inflammatory cells, as well as a chondromyocyte fate. The remodeling population was marked by uniquely high Mmp3 and Cxcl12 expression, and its appearance correlated with higher risk plaque features such as increased positive remodeling and macrophage content. Further, investigation of transcriptional mechanisms by which Smad3 alters SMC cell fate revealed novel roles for Hox and Sox transcription factors whose direct interaction with Smad3 regulate an extensive transcriptional program balancing remodeling and vascular extracellular matrix. These findings have significant implications for atherosclerotic and Mendelian aortic aneurysmal diseases. Together, these data suggest that Smad3 expression in SMC inhibits the emergence of specific SMC phenotypic transition cells that mediate adverse plaque features, including positive remodeling, monocyte recruitment, and vascular calcification.

Modeling Inflammatory Immune Cell Recruitment and Response on Human Colon Intestine-Chip

Abstract Objective Immune cell recruitment into tissues is an essential step in inflammatory responses. This occurs in a highly tissue- and stimulus-specific manner, which presents a significant challenge to modeling disease and testing therapeutics ex vivo. We previously developed an advanced primary human vascularized Colon Intestine-Chip model and showed that it recapitulates physiologic cell composition, morphology and barrier function. The goal of this study was to test the ability of this system to model inflammatory bowel disease (IBD)-like immune cell reactions ex vivo. Methods We perfused primary human peripheral blood mononuclear cells (PBMC) across the vascular channel in untreated ‘resting’ or TNFα/chemokine-treated ‘inflamed’ Colon Intestine-Chips. We analyzed total cell recruitment, inflammatory cytokine secretion and barrier function in the following 24–72 hours. Results We show that the perfused PBMC efficiently adhered and transmigrated to the epithelial channel in an inflammation-specific manner. This was followed by an accumulation of proinflammatory cytokines characteristic of IBD (e.g., INFγ, IL1β, IL18), as well as loss of barrier function, the hallmark feature of IBD. We further showed that 1) the recruited cells were strongly enriched in the ‘gut trophic’ α4β7+/CCR9+ PBMC subsets and 2) this recruitment could be blocked with the IBD therapeutic Entyvio, which targets the α4β7-MAdCAM-1 interaction. Conclusion Our findings indicate that our Colon Intestine-Chip can model inflammatory immune cell recruitment and in situ immune reactions that reflect key clinical correlates of IBD. This model may prove effective for development of new anti-inflammatory therapeutics for human intestinal diseases. Supported by Emulate Bio

A mast cell stabilizer attenuates pathological angiogenesis in a model of choroidal neovascularization

Age‐related macular degeneration (AMD) is one of the leading causes of irreversible blindness in the elderly worldwide. Wet AMD is characterized by pathologic choroidal neovascularization that invades the RPE and compromises retinal function. Although anti‐vascular endothelial growth factor (anti‐VEGF) therapy has been shown to improve symptoms of wet AMD in a large number of people, long‐term treatment is associated with the development of geographic atrophy. The development of new therapeutic alternatives to prevent the progression of exudative AMD is still necessary. Inflammation and deterioration of the RPE are considered to be the main sources of the pathogenesis of AMD. Recent studies have reported accumulation and degranulation of mast cells in the compromised choroid of patients with AMD. When activated, mast cells can secrete a variety of biologically active mediators, including inflammatory cytokines and proteolytic enzymes such as tryptase. However, the precise role of mast cells in the pathogenesis of AMD remains largely unknown. We are interested in how mast cells participate in the initiation of inflammation in the choroid which results in pathological choroidal neovascularization. We investigated the effect of a mast cell stabilizer on angiogenic response in the choroid. The angiogenic effect of a mast cell stabilizer, ketotifen fumarate, was evaluated in laser‐induced CNV model. Mice received intraperitoneal injections of ketotifen fumarate (5 and 25 mg/kg/d) or vehicle. CNV area and mononuclear phagocytes were determined respectively by using lectin staining and an anti–IBA‐1 antibody on RPE/choroid flat mounts. CNV was attenuated by ketotifen fumarate with no effect on inflammatory cell recruitment. Conditioned media from activated peritoneal mast cells exerted proangiogenic effects on vascular sprouting. Tryptase increased choroidal endothelial cell migration and choroidal vascular sprouting. The study supports possible modulation of mast cells as a therapeutic modality for CNV.

Cardamonin Antioxidant Effects Through the Modulation of Proteins and Genes Expressions in LPS‐Activated BV‐2 Microglial Cells

Neurodegeneration is characterized by slow and progressive loss of neuronal cells and synapses in different brain regions. These pathologic effects are the major feature of Alzheimer's disease (AD), leading to a decline in learning and memory function. The main hallmarks of AD are the aggregation and deposition of amyloid‐β peptides on the extracellular surface of neuronal cells. Hyperphosphorylation of tau protein and the formation of neurofibrillary tangles is another feature of AD. Neuroinflammation and oxidative stress follow the accumulation of amyloid‐β and neurofibrillary tangles. Continuous activation of microglia cells, known as the resident, innate immune cells in the brain, participates in producing numerous neurotoxic factors, including nitric oxide and reactive oxygen species (ROS), which induce neurodegeneration. Numerous natural compounds, such as cardamonin, a bioactive compound found in many plants, have been reported to exert multiple therapeutic properties, including anti‐cancer, anti‐inflammatory, antioxidant, antiviral antibiotic, antifungal, and antiallergic activities. This study examined the effects of cardamonin on LPS‐activated BV‐2 microglial cells. Results of this study showed that cardamonin concentrations of 6.25 to 50 µM reduced over 95% the release of NO in LPS‐activated BV‐2 cells, compared to the cells treated with LPS only (P<0.001). Moreover, cardamonin significantly decreased the cellular production of SOD 3‐fold (P < 0.05) and increased the levels of expression of CAT 2.5‐fold (P < 0.05) and glutathione 2‐fold (p < 0.05) in the LPS‐activated BV‐2 cells. In RT‐PCR assays, cardamonin increased the mRNA expression of CAT and decreased the levels of NOS2, the enzyme responsible for the production of NO and associated with inflammatory processes. Furthermore, cardamonin downregulated the mRNA expression of CCL5/RANTES (5‐fold), which promotes the recruitment and activation of inflammatory cells. Also, cardamonin downregulated SLC38A1 (3‐fold), whose expression is upregulated during oxidative stress, and TXNIP (2‐fold) that regulates thioredoxin, an important redox protein that controls levels of ROS in cells. The data show cardamonin efficacy in modulating proteins and genes involved in inflammation and the control of elevated intracellular levels of ROS. In conclusion, these findings suggest that cardamonin may have potential in the therapy of microglia‐derived neurodegeneration, helping to prevent or slow the progression of the disease in the CNS.

Early life exposure to Streptococcus pneumonia has sex-specific long-term impact on inflammatory responses

Abstract INTRODUCTION Lung microbiota plays a central role in health and disease development. Recently, recurrent viral infections in preschool age children were associated with lung microbiota carriage of Streptococcus pneumonia (SP). In addition, immune development and inflammatory responses have sexual dimorphism, but it is still controversial whether there is sex-specific microbiota composition. OBJECTVE Given that immune development occurs early in life, the objective of this project is to evaluate the consequences of early life lung microbiota alteration by SP exposure on inflammatory response later in life. METHODS Neonatal Brown Norway rats were exposed to SP via intranasal administration (sub inflammatory dose). Lung microbiota was assessed until weaning age (21 days old), using v3–v4 16S rRNA sequencing. At 8 weeks old (post-puberty), rats were infected with LPS and their inflammatory responses were assessed 24h post using bronchoalveolar lavage (BAL). RESULTS Early life exposure to SP reduced lung microbiota diversity and impacted its constitution until weaning age. Although control females exposed to LPS had higher inflammatory response than control males, early life exposure to SP significantly reduced BAL inflammatory cell recruitment in females but not in SP-males. Opposingly, in SP-males, the inflammatory response shifted from a neutrophilic response to a more eosinophilic inflammation. CONCLUSION Early life exposure to SP modulated both lung microbiota and immune responses later in life. The sex-specific immune modulation by lung microbiota composition in early life could have large implication for many inflammatory diseases presenting sexual bias. Supported by grants from Quebec Health Research Network and Fondation IUCPQ (Quebec, Canada).

Abstract 241: Nicotinamide Nucleotide Transhydrogenase Regulates Mitochondrial Function And Oxldl Induced Macrophage Endothelial Interaction.

Atherosclerosis is a chronic inflammatory disease effecting large and medium sized arteries that leads to the buildup of plaque that contributes to myocardial infraction, stroke, and other pathologies. Mounting evidence has identified mitochondrial reactive oxygen species (ROS) as a key contributor to plaque formation and progression. A inner mitochondrial membrane enzyme Nicotinamide nucleotide transhydrogenase (NNT) sustains NADPH pools required for the mitochondrial antioxidant systems. We have observed that the expression of NNT is decreased in severe human atherosclerotic plaques leading us to hypothesize that the loss of NNT contributes to increased mitochondrial ROS and drives the progression of atherosclerosis by enhancing endothelial and vascular dysfunction. Utilizing AAV/PCSK9 and HFD induced models of early and late-stage atherosclerosis, we found that NNT knockout (NNT KO) mice display significantly increased VCAM-1 expression in aortic endothelial cells during early atherosclerosis and significantly larger necrotic cores in late-stage atherosclerosis. Additionally, NNT KO mice displayed a significantly thinner fibrous cap in late-stage plaques. Similar results were observed in vitro where the loss of NNT in human aortic endothelial cells exacerbated oxLDL induced VCAM-1 expression that was associated with decreased mitochondrial NADPH levels, glutathione peroxidase (Gpx2) activity, and increased H 2 O 2 production. The observed increase in necrotic core area in NNT KO mice is suggestive of increased inflammatory cell recruitment to the plaque. Furthermore, bone marrow derived macrophages (BMDM) from NNT KO mice display decreased lipid content, poor lipid uptake and a predisposition to differentiate into a proinflammatory M1 phenotype. Based on these studies, we conclude that the loss of NNT contributes to unstable plaques with large necrotic cores, mitochondrial ROS driven endothelial dysfunction, increased inflammatory cell recruitment, and enhanced M1 macrophage activity. The novel relationship between vascular NNT expression and plaque severity in human samples implicates NNT as a novel clinical target in atherosclerosis.

Mechanisms Underlying Curcumin-Induced Neuroprotection in Cerebral Ischemia.

Ischemic stroke is the leading cause of death and disability worldwide, and restoring the blood flow to ischemic brain tissues is currently the main therapeutic strategy. However, reperfusion after brain ischemia leads to excessive reactive oxygen species production, inflammatory cell recruitment, the release of inflammatory mediators, cell death, mitochondrial dysfunction, endoplasmic reticulum stress, and blood–brain barrier damage; these pathological mechanisms will further aggravate brain tissue injury, ultimately affecting the recovery of neurological functions. It has attracted the attention of researchers to develop drugs with multitarget intervention effects for individuals with cerebral ischemia. A large number of studies have established that curcumin plays a significant neuroprotective role in cerebral ischemia via various mechanisms, including antioxidation, anti-inflammation, anti-apoptosis, protection of the blood–brain barrier, and restoration of mitochondrial function and structure, restoring cerebral circulation, reducing infarct volume, improving brain edema, promoting blood–brain barrier repair, and improving the neurological functions. Therefore, summarizing the results from the latest literature and identifying the potential mechanisms of action of curcumin in cerebral ischemia will serve as a basis and guidance for the clinical applications of curcumin in the future.

Smallpox vaccination induces a substantial increase in commensal skin bacteria that promote pathology and influence the host response.

Interactions between pathogens, host microbiota and the immune system influence many physiological and pathological processes. In the 20th century, widespread dermal vaccination with vaccinia virus (VACV) led to the eradication of smallpox but how VACV interacts with the microbiota and whether this influences the efficacy of vaccination are largely unknown. Here we report that intradermal vaccination with VACV induces a large increase in the number of commensal bacteria in infected tissue, which enhance recruitment of inflammatory cells, promote tissue damage and influence the host response. Treatment of vaccinated specific-pathogen-free (SPF) mice with antibiotic, or infection of genetically-matched germ-free (GF) animals caused smaller lesions without alteration in virus titre. Tissue damage correlated with enhanced neutrophil and T cell infiltration and levels of pro-inflammatory tissue cytokines and chemokines. One month after vaccination, GF and both groups of SPF mice had equal numbers of VACV-specific CD8+ T cells and were protected from disease induced by VACV challenge, despite lower levels of VACV-neutralising antibodies observed in GF animals. Thus, skin microbiota may provide an adjuvant-like stimulus during vaccination with VACV and influence the host response to vaccination.

Protection and Pathology in Leishmania braziliensis Infection.

Leishmania killing is mediated by IFN-γ-activated macrophages, but IFN-γ production and macrophage activation are insufficient to control L. braziliensis infection. In American tegumentary leishmaniasis (ATL), pathology results from an exaggerated inflammatory response. This report presents an overview of our contributions regarding ATL pathogenesis, highlighting future directions to improve the management of L. braziliensis infection. Monocytes and lymphocytes from individuals exposed to L. braziliensis but who do not develop CL, i.e., subclinical infection (SC), exhibit lower respiratory burst and IFN-γ production, yet more efficiently kill L. braziliensis. As vaccines aimed at inducing IL-12 and IFN-γ do not sufficiently prevent CL, the elucidation of how subjects with SC infection kill Leishmania may lead to new approaches to controlling ATL. While inflammation arising from the recruitment of inflammatory cells via chemokines induced by IFN-γ and TNF or IL-17 is observed and contributes to pathology, cytotoxic CD8+ T cells and NK cells play a key role in the pathogenesis of L. braziliensis infection. The increased transcription of genes related to inflammation and cytotoxicity, e.g., granzyme A, granzyme B, NLRP3 and IL-1β, has been documented in CL tissue samples. The release of products by killed cells leads to NLRP3 inflammasome activation, IL-1β production and additional damage to skin and mucosal tissues. The use of drugs that downmodulate the inflammatory response in combination with chemotherapy improves the ATL cure rate and decreases healing time.

Antiatherosclerotic Effects of CSL112 Mediated by Enhanced Cholesterol Efflux Capacity.

Approximately 12% of patients with acute myocardial infarction (AMI) experience a recurrent major adverse cardiovascular event within 1 year of their primary event, with most occurring within the first 90 days. Thus, there is a need for new therapeutic approaches that address this 90‐day post‐AMI high‐risk period. The formation and eventual rupture of atherosclerotic plaque that leads to AMI is elicited by the accumulation of cholesterol within the arterial intima. Cholesterol efflux, a mechanism by which cholesterol is removed from plaque, is predominantly mediated by apolipoprotein A‐I, which is rapidly lipidated to form high‐density lipoprotein in the circulation and has atheroprotective properties. In this review, we outline how cholesterol efflux dysfunction leads to atherosclerosis and vulnerable plaque formation, including inflammatory cell recruitment, foam cell formation, the development of a lipid/necrotic core, and degradation of the fibrous cap. CSL112, a human plasma‐derived apolipoprotein A‐I, is in phase 3 of clinical development and aims to reduce the risk of recurrent cardiovascular events in patients with AMI in the first 90 days after the index event by increasing cholesterol efflux. We summarize evidence from preclinical and clinical studies suggesting that restoration of cholesterol efflux by CSL112 can stabilize plaque by several anti‐inflammatory/immune‐regulatory processes. These effects occur rapidly and could stabilize vulnerable plaques in patients who have recently experienced an AMI, thereby reducing the risk of recurrent major adverse cardiovascular events in the high‐risk early post‐AMI period.

Protein restriction during pregnancy affects lung development and promotes oxidative stress and inflammation in C57 BL/6 mice offspring

ObjectivesThe present study aimed to evaluate the effects of maternal protein restriction during pregnancy on the lungs of 1-d and 31-d old offspring of C57BL/6 mice. MethodsThe C57BL/6 mice (8–10 wk) were used for breeding. After pregnancy confirmation, female mice were randomly divided into a control group (CG) receiving a standard diet (22% protein) and a protein-restriction group (PRG) receiving a low-protein diet (6% protein). In the low-protein diet, protein was replaced by carbohydrate. After parturition, female mice that received the low-protein diet were fed the standard diet. Male offspring were euthanized 1 d and 31 d after birth for subsequent analysis. We evaluated the effects of a protein-restricted diet during gestation in pulmonary organogenesis, lung oxidative stress, and pulmonary inflammatory response of the offspring. ResultsPRG mice 1 d after birth showed lower body and lung mass, length, relative mass, lung density, and erythrocyte count compared with CG mice. There was an increase in alveolar airspace density and a higher mean linear intercept (Lm), greater oxidative damage, and inflammation in PRG mice compared with CG mice. At 31 d after birth, PRG mice had lower body mass, length, and lung mass values compared with CG mice. PRG mice showed greater recruitment of inflammatory cells to the airways. In addition, there was increased collagen deposition in the lungs, altered inflammatory mediators, and greater oxidative damage compared with CG mice. ConclusionsProtein restriction during pregnancy reduces the body weight of offspring and promotes inflammation and oxidative stress, resulting in a simplification of the lung structure.

Smad3 regulates smooth muscle cell fate and mediates adverse remodeling and calcification of the atherosclerotic plaque.

Atherosclerotic plaques consist mostly of smooth muscle cells (SMC), and genes that influence SMC phenotype can modulate coronary artery disease (CAD) risk. Allelic variation at 15q22.33 has been identified by genome-wide association studies to modify the risk of CAD and is associated with the expression of SMAD3 in SMC. However, the mechanism by which this gene modifies CAD risk remains poorly understood. Here we show that SMC-specific deletion of Smad3 in a murine atherosclerosis model resulted in greater plaque burden, more outward remodelling and increased vascular calcification. Single-cell transcriptomic analyses revealed that loss of Smad3 altered SMC transition cell state toward two fates: a SMC phenotype that governs both vascular remodelling and recruitment of inflammatory cells, as well as a chondromyocyte fate. Together, the findings reveal that Smad3 expression in SMC inhibits the emergence of specific SMC phenotypic transition cells that mediate adverse plaque features, including outward remodelling, monocyte recruitment, and vascular calcification.

Inflammation and neutrophil extracellular traps in cerebral cavernous malformation

Cerebral Cavernous Malformation (CCM) is a brain vascular disease with various neurological symptoms. In this study, we describe the inflammatory profile in CCM and show for the first time the formation of neutrophil extracellular traps (NETs) in rodents and humans with CCM. Through RNA-seq analysis of cerebellum endothelial cells from wild-type mice and mice with an endothelial cell-specific ablation of the Ccm3 gene (Ccm3iECKO), we show that endothelial cells from Ccm3iECKO mice have an increased expression of inflammation-related genes. These genes encode proinflammatory cytokines and chemokines, as well as adhesion molecules, which promote recruitment of inflammatory and immune cells. Similarly, immunoassays showed elevated levels of these cytokines and chemokines in the cerebellum of the Ccm3iECKO mice. Consistently, both flow cytometry and immunofluorescence analysis showed infiltration of different subsets of leukocytes into the CCM lesions. Neutrophils, which are known to fight against infection through different strategies, including the formation of NETs, represented the leukocyte subset within the most pronounced increase in CCM. Here, we detected elevated levels of NETs in the blood and the deposition of NETs in the cerebral cavernomas of Ccm3iECKO mice. Degradation of NETs by DNase I treatment improved the vascular barrier. The deposition of NETs in the cavernomas of patients with CCM confirms the clinical relevance of NETs in CCM.

Key benefits of dexamethasone and antibody treatment in COVID-19 hamster models revealed by single-cell transcriptomics

For coronavirus disease 2019 (COVID-19), effective and well-understood treatment options are still scarce. Since vaccine efficacy is challenged by novel variants, short-lasting immunity, and vaccine hesitancy, understanding and optimizing therapeutic options remains essential.We aimed at better understanding the effects of two standard-of-care drugs, dexamethasone and anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies, on infection and host responses. By using two COVID-19 hamster models, pulmonary immune responses were analyzed to characterize effects of single or combinatorial treatments.Pulmonary viral burden was reduced by anti-SARS-CoV-2 antibody treatment and unaltered or increased by dexamethasone alone. Dexamethasone exhibited strong anti-inflammatory effects and prevented fulminant disease in a severe disease model. Combination therapy showed additive benefits with both anti-viral and anti-inflammatory potency. Bulk and single-cell transcriptomic analyses confirmed dampened inflammatory cell recruitment into lungs upon dexamethasone treatment and identified a specifically responsive subpopulation of neutrophils, thereby indicating a potential mechanism of action.Our analyses confirm the anti-inflammatory properties of dexamethasone and suggest possible mechanisms, validate anti-viral effects of anti-SARS-CoV-2 antibody treatment, and reveal synergistic effects of a combination therapy, thus informing more effective COVID-19 therapies.

Effect of Houpo-Mahuang Decoction on aggravated asthma induced by cigarette smoke and the expression of TRPA1 and tight junctions in mice

Ethnopharmacological relevanceCigarette smoke (CS) is a common environmental irritant and a risk factor for asthma, as it induces as well as aggravates asthmatic attacks. The injured airway epithelial tight junctions (TJs) aggravate asthma. CS can aggravate asthma by activating the transient receptor potential ankyrin A1 (TRPA1) channel and enhancing TJs destruction. Houpo Mahuang decoction (HPMHD) is a classic traditional Chinese prescription for the treatment of asthma. However, its underlying action mechanism is unclear. Aim of the studyThe present study aimed to evaluate the effect of HPMHD on the asthma phenotype and the regulation of TRPA1 and TJs in a CS-induced mouse model of aggravated asthma. Materials and methodsUnder optimized chromatographic and mass spectrometry conditions, the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) technique was used to detect and analyze the major chemical components of HPMHD. C57BL/6 female mice were randomly divided into seven groups, viz, normal saline (NS) group, ovalbumin (OVA) + CS group, dexamethasone group, HPMHD high-dose group and low-dose groups, n-butanol extract group, and ethyl acetate extract group, with 10 mice in each group. OVA sensitization and challenge, and CS exposure were used to establish the aggravated asthma model. As the main indices to evaluate the protective effect of HPMHD, the eosinophils count in peripheral blood, percentages of inflammatory cells classified and the levels of interleukin (IL)-4, IL-5, IL-13 in the bronchoalveolar lavage fluid (BALF), airway responsiveness enhanced pause (Penh), and changes in lung histopathology were determined and compared among the groups. The mRNA and protein expression of TRPA1 and TJs in lung tissue was also examined. ResultsUsing UPLC-QTOF-MS, the chemical components of HPMHD, including ephedrine, pseudoephedrine, laetrile, and amygdalin amide, were identified by 51 signal peaks. Compared with those in the NS group, the eosinophil number in the peripheral blood and the eosinophils and neutrophils percentages in BALF of the OVA + CS group were remarkably increased. Following the inhalation of 50 μl of acetylcholine chloride (ACH) at doses of 25 and 50 mg/mL, the Penh increased significantly (p < 0.01). Moreover, in the OVA + CS group, hematoxylin and eosin (H&E) staining of lung tissue showed a significant number of infiltrated inflammatory cells, increased mucus secretion in the lumen, damaged bronchial mucosa, increased thickness of tracheal wall, and increased score of lung damage (p < 0.01). The IL-4/5/13 levels were also remarkably increased (p < 0.01). The protein as well as gene expression of both ZO-1 and occludin decreased markedly in the lung tissue, while the expression of TRPA1 and claudin-2 was increased (p < 0.05, p < 0.01). Next, the OVA + CS group and the treatment groups were compared. The inflammatory cells, Penh value, and levels of IL-4/5/13 were significantly reduced, and less lung injury was observed in the treatment groups. The gene and protein levels of TRPA1 and TJs were corrected (p < 0.05, p < 0.01); the effects on the HPMHD high-dose and ethyl acetate extract groups were particularly remarkable. ConclusionsHPMHD reduced airway hyperresponsiveness, inflammatory cell recruitment and Th2 cytokine secretion in CS-induced aggravated asthma mice, in a manner potentially dependent on regulation of the expression of TRPA1 and TJ proteins. Both the n-butanol and ethyl acetate extracts contained the active ingredients, especially the ethyl acetate extract.

A potent myeloid response is rapidly activated in the lungs of premature Rhesus macaques exposed to intra-uterine inflammation

Up to 40% of preterm births are associated with histological chorioamnionitis (HCA), which leads to elevated levels of pro-inflammatory mediators and microbial products in the amniotic fluid, which come in contact with fetal lungs. Yet, fetal pulmonary immune responses to such exposure remain poorly characterized. To address this gap, we used our established HCA model, in which pregnant Rhesus macaques receive intraamniotic (IA) saline or LPS. IA LPS induced a potent and rapid myeloid cell response in fetal lungs, dominated by neutrophils and monocytes/macrophages. Infiltrating and resident myeloid cells exhibited transcriptional profiles consistent with exposure to TLR ligands, as well as cytokines, notably IL-1 and TNFα. Although simultaneous, in vivo blockade of IL-1 and TNFα signaling did not prevent the inflammatory cell recruitment, it blunted the lung overall inflammatory state reducing communication between, and activation of, infiltrating immune cells. Our data indicate that the fetal innate immune system can mount a rapid multi-faceted pulmonary immune response to in utero exposure to inflammation. These data provide mechanistic insights into the association between HCA and the postnatal lung morbidities of the premature infant and highlight therapeutic potential of inflammatory blockade in the fetus.