NOS Expression Research Articles

P124 Severe psoriasiform phenotype in CARMIL2 deficiency and successful therapy with dupilumab

Abstract A 22-year-patient with consanguineous parents presented with a diagnosis of severe psoriasis with erythroderma and recurrent episodes of fever that had been present since childhood. Neither systemic treatment with methotrexate, ciclosporin A, acitretin, apremilast nor tildrakizumab had led to a significant improvement. Clinically, there was persistent erythroderma with coarse lamellar scaling on the entire integument and pronounced lichenification. Histopathologically, the skin biopsy revealed regular acanthosis, raised papillary tips with inflammatory infiltrate, slight para- and hyperkeratosis, hypogranulosis and a strong expression of NOS2 and CCL27. Immunophenotypically, a reduced number of CD4+ and CD8+ memory cells in line with increased numbers of naïve T cells was found. The proportion of FOXP3+ Treg cells was also reduced and there was an increased number of naive B cells and insufficient conversion into memory B cells. Whole exome sequencing identified a novel homozygous frameshift mutation (c.1894del) in the CARMIL2 gene. CARMIL2 deficiency is a recently described autosomal recessive inherited combined immunodeficiency that impairs T- and B-cell activation and maturation through dysfunctional CD28-mediated costimulation and cell motility through impaired cytoskeletal functions. In addition to various skin manifestations, ranging from seborrheic, atopic or psoriasiform dermatitis and an increased susceptibility to bacterial, mycotic and viral skin infections, the majority of affected individuals present with gastrointestinal symptoms, respiratory infections and the occurrence of EBV+-smooth muscle tumours (SMTs). In our patient, the start of treatment with the monoclonal antibody dupilumab, which binds IL-4Rα and inhibits the signalling of IL-4 and IL-13, led to an impressive long-term improvement in the skin condition. Haematopoietic stem cell transplantation could be a therapeutic option for the long-term control of extracutaneous manifestations but requires control of skin inflammation. In refractory psoriasiform manifestations in combination with susceptibility to infection, gastrointestinal inflammation or EBV+-SMTs, CARMIL2 deficiency should be considered in the differential diagnosis. Our case expands the phenotypic spectrum of a newly discovered primary immunodeficiency and proves for the first time that dupilumab may be an effective treatment option for severe psoriasiform cutaneous manifestations in CARMIL2 deficiency.

Inhibitory effect of Zhujing Pill on myopia progression: Mechanistic insights based on metabonomics and network pharmacology.

This study endeavored to uncover the mechanisms by which Zhujing pill (ZJP) slows myopia progression. We employed biometric analyses to track diopter and axial length changes in guinea pigs with negative lens-induced myopia (LIM). Through integrating metabonomics and network pharmacology, we aimed to predict the anti-myopic targets and active ingredients of ZJP. Subsequent analysis, including real-time fluorescent quantitative PCR (qPCR) and Western blotting (WB), assessed the expression levels of CHRNA7, LPCAT1, and NOS2 in retinal tissues. Our findings demonstrate that ZJP significantly mitigates diopter increase and axial elongation in LIM guinea pigs. Metabonomic analysis revealed significant changes in 13 serum metabolites, with ZJP reversing the expression of 5 key metabolites. By integrating metabonomics with network pharmacology, we identified core targets of ZJP against myopia and constructed a compound-gene-disease-metabolite network. The expressions of LPCAT1 and CHRNA7 were found to decrease in the LIM group but increase with ZJP treatment, whereas NOS2 expression showed the opposite pattern. This investigation provides the first evidence of ZJP's multifaceted effectiveness in managing myopia, highlighting its impact on multiple components, targets, and pathways, including the novel involvement of LPCAT1 and CHRNA7 in myopia pathogenesis.

Growth Hormone-Releasing Peptide 2 May Be Associated With Decreased M1 Macrophage Production and Increased Histologic and Biomechanical Tendon-Bone Healing Properties in a Rat Rotator Cuff Tear Model.

To explore the potential of growth hormone-releasing peptide 2 (GHRP-2) for tendon-bone healing in a rat rotator cuff tear (RCT) model. The impact of GHRP-2 on M1 macrophage polarization invitro was determined using real-time polymerase chain reaction, Western blot, and immunofluorescence staining. GHRP-2 was then applied in a rat RCT model, and the healing of the tendon-bone interface was systemically evaluated by histologic staining, radiologic assessments, gait analysis, and biomechanical tests. M1 macrophage polarization at the tendon-bone interface was assessed by immunofluorescence staining. GHRP-2 was found to reduce the expression of Cd86, Nos2, and tnfa (all P < .01), suggesting inhibited M1 macrophage polarization invitro. The invivo experiments showed that the proportion of M1 macrophages was reduced both 2 and 4 weeks after surgery (P < .01), and the number of M1 was reduced 4 weeks after surgery (P < .01) at the tendon-bone interface. The invivo experiments also showed that histologic scores and bone mineral density were increased by GHRP-2 at 8 weeks postsurgery (P < .01), suggesting improved healing of the tendon-bone interface. Furthermore, the GHRP-2 group showed a better biomechanical property at both 4 and 8 weeks postsurgery, including maximal failure load, stiffness, and tension (all P < .01), and better gait parameters at 8 weeks postsurgery, including mean area of the left front foot and mean intensity of the right front foot (all P < .05). GHRP-2 may be associated with decreased M1 macrophage production and increased histologic and biomechanical tendon-bone healing properties in a rat RCT model. The present study might be a transitional study to show the efficacy of GHRP-2 in enhancing bone-tendon healing and reduce retear rate after rotator cuff repair.

Hydroxyurea inhibits proliferation and stimulates apoptosis through inducible nitric oxide synthase in erythroid cells

Hydroxyurea (hydroxycarbamide, HU) arrests cells in the S-phase by inhibiting ribonucleotide reductase and DNA synthesis, significantly contributing to the release of nitric oxide (NO). We investigated the involvement of inducible NO synthase (NOS2) in the cytostatic effect of HU using in vitro shRNA-induced knockdown of the NOS2 transcript (NOS2kd) or a specific NOS2 inhibitor (1400W) in human erythroleukemic HEL92.1.7 cells, as well as murine erythroid progenitors (mERPs) from HU-treated wild-type (WT) and Nos2 knockout (Nos2–/–) mice. Over the long-term, HU increased NOS2 expression in HEL92.1.7 cells (via nuclear factor kappa B [NFκB] signaling) and in mERP. In the short-term, HU increased the activity of human recombinant and erythroleukemic cell-derived NOS2, as confirmed by NO metabolite nitrite/citrulline production. In silico molecular docking predicted that HU binds to the NOS2 active site and substrate L-arginine via hydrogen bonds. Molecular dynamics simulations showed reduced rigidity of the NOS2 active site upon interaction with HU, indicating stabilization of the enzyme-substrate complex. Both 1400W and NOS2kd prevented the in vitro reduction in proliferation and induction of apoptosis in HEL92.1.7 cells by HU. NOS2kd preferentially blocked early apoptosis and HU-induced S-phase arrest in HEL92.1.7 cells. The HU-induced decrease in proliferation and stimulation of early apoptosis in mERP were prevented in Nos2–/– mice and by 1400W in WT mice. This study demonstrated that HU induces NOS2 activity through direct interaction and increased protein expression via NFκB signaling. Moreover, NOS2 mediates the HU-induced inhibition of proliferation and stimulation of apoptosis in erythroid cells.

IMMU-47. HUMAN MICROBIOTA INFLUENCE THE GLIOMA-IMMUNE LANDSCAPE AS DETERMINED BY SINGLE CELL RNA-SEQUENCING IN A MURINE MODEL OF GLIOMA

Abstract We recently identified immunotherapy responder and nonresponder human microbiota compositions in the murine model of glioma. In this study, we sought to understand how human microbiota in mice influence the immune landscape in glioma. To test this, we utilized our novel humanized microbiome (HuM) mouse model (human microbiota transplanted into gnotobiotic mice) to determine the effect of immunotherapy in a mouse model of glioma utilizing single-cell RNA sequencing and 16S rRNA microbiome sequencing. We focused on two HuM mouse lines in this study: HuM1 (nonresponder) and HuM2 (responder). To confirm microbiota differences,16S rRNA sequencing revealed significant differences between alpha and beta diversity between HuM1 and HuM2 groups. HuM2 responder mice also displayed a significant increase in the relative abundances of Odoribacter, Muribaculaceae, and Ruminococcus genera compared to HuM1 mice. HuM1 and HuM2 mice were then injected with GL261 glioma cells into the brains, treated with one dose of anti-PD-1 or isotype control on day 13, and all mice were euthanized on day 14 for scRNA-sequencing of CD45+ cells in the tumors. We identified 21 clusters of immune cells in the tumors. Furthermore, we found significant differences between HuM1 and HuM2 mice in isotype control conditions including higher percentages of immature B cells, neutrophils, and microglia in HuM2 mice. In anti-PD-1 treated tumors, there was a robust increase in the percentage of innate immune cells in the tumor following, specifically the monocyte/macrophage (MM1; Cxcl10+) population in both HuM1 and HuM2 mice. However, the HuM2 MM1 cluster showed an increase in gene expression of Nos2 and decrease in Arg1, indicating that the MM1 population in HuM2 mice are more inflammatory (anti-tumor) compared to HuM1. Ultimately, our data demonstrate that the gut microbiome influences the tumor-immune landscape, and harnessing positive microbial compositions remains a viable translational avenue to pursue.

F-Box and WD repeat domain containing 7 induces infectious osteomyelitis by regulating MYB stability and ubiquitination.

Osteomyelitis is a bone inflammation initiated by invading pathogens. Macrophages and inflammation play essential roles in osteomyelitis. F-Box and WD repeat domain containing 7 (Fbxw7) is a tumour suppressor and E3 ubiquitin ligase. In the present study, the potential roles of Fbxw7 in osteomyelitis were explored. The mRNA level of Fbxw7 was measured in bone marrow cells from patients with osteomyelitis and Staphylococcus aureus (S. aureus)-infected macrophages. The conditional knockout mice with Fbxw7 deficiency in myeloid cells were generated. The expression of interleukin (IL)-6, IL-23a and nitric oxide synthase 2 (Nos2) was measured in S. aureus-infected Fbxw7-deficient bone marrow-derived macrophages (BMDMs). The body weight loss, bacterial burden, bone loss and formation and serum level of IL-6, IL-23 and TNF-α were measured in S. aureus-infected Fbxw7 conditional KO mice. The interacting partners of Fbxw7 were predicted using STRING and the interaction were tested. Elevated expression of Fbxw7 was observed in bone marrow cells from patients with osteomyelitis and in S. aureus-infected macrophages. The expression of IL-6, IL-23a and Nos2 was remarkably suppressed in S. aureus-infected Fbxw7-deficient BMDMs. Fbxw7 conditional knockout mice had less body weight loss, higher bacterial burden, less bone loss and formation and decreased serum level of cytokines. Fbxw7 interacted with MYB. S. aureus-infected Fbxw7-deficient BMDMs had higher level of MYB and less ubiquitination of MYB. Fbxw7 promotes osteomyelitis symptoms by regulating ubiquitination and stability of MYB.

Intestinal microbiome dysbiosis increases Mycobacteria pulmonary colonization in mice by regulating the Nos2-associated pathways

Increasing researches reveal gut microbiota was associated with the development of tuberculosis (TB). How to prevent or reduce Mycobacterium tuberculosis colonization in the lungs is a key measure to prevent TB. However, the data on gut microbiota preventing Mycobacterium colonization in the lungs were scarce. Here, we established the clindamycin-inducing intestinal microbiome dysbiosis and fecal microbial transplantation models in mice to identify gut microbiota’s effect on Mycobacterium’s colonization in the mouse lungs and explore its potential mechanisms. The results showed that clindamycin treatment altered the diversity and composition of the intestinal bacterial and fungal microbiome, weakened the trans-kingdom network interactions between bacteria and fungi, and induced gut microbiome dysbiosis in the mice. Gut microbiota dysbiosis increases intestinal permeability and enhances the susceptibility of Mycobacterium colonization in the lungs of mice. The potential mechanisms were gut microbiota dysbiosis altered the lung transcriptome and increased Nos2 expression through the ‘gut–lung axis’. Nos2 high expression disrupts the intracellular antimicrobial and anti-inflammatory environment by increasing the concentration of nitric oxide, decreasing the levels of reactive oxygen species and Defb1 in the cells, and promoting Mycobacteria colonization in the lungs of mice. The present study raises a potential strategy for reducing the risks of Mycobacteria infections and transmission by regulating the gut microbiome balance.

Targeting Krüppel-Like Factor 2 as a Novel Therapy for Glomerular Endothelial Cell Injury in Diabetic Kidney Disease.

DKD is a microvascular disease, and glomerular endothelial cell injury is a key pathological event in DKD development. Through unbiased screening of glomerular transcriptomes, we previously identified KLF2 as a highly regulated gene in diabetic kidneys. KLF2 exhibits protective effects in endothelial cells by inhibiting inflammation, thrombotic activation, and angiogenesis, all of which are protective for cardiovascular disease. We previously demonstrated that endothelial cell-specific ablation of Klf2 exacerbated diabetes-induced glomerular endothelial cell injury and DKD in mice. Therefore in this study, we sought to assess the therapeutic potential of KLF2 activation in murine models of DKD. We first examined the effects of endothelial cell-specific inducible overexpression of KLF2 (KLF2ov) in streptozotocin-induced diabetic mice. We developed small molecule KLF2 activators and tested whether increased KLF2 activity could impede DKD progression in type 2 diabetic db/db and BTBR ob/ob mice. Diabetic KLF2ov mice had attenuated albuminuria, glomerular endothelial cell injury, and diabetic glomerulopathy compared to control diabetic mice. Novel KLF2 activator, compound 6 (C-6) effectively induced downstream Nos3 expression and suppressed NF-kB activation in glomerular endothelial cells. The administration of C-6 improved albuminuria and glomerulopathy in db/db and BTBR ob/ob mice, which was associated with improved glomerular endothelial cell and podocyte injury. These results validate KLF2 as a potential drug target and KLF2 activators such as C-6 as a novel therapy for DKD.

Differential Regulation of L-Arginine Metabolism through NOS2 and Arginases during Infection with Trypanosoma cruzi.

L-arginine metabolism through arginases and inducible nitric oxide synthase (NOS2) constitutes a fundamental axis for the resolution or progression of Chagas disease. Infection with Trypanosoma cruzi can cause a wide spectrum of disease, ranging from acute forms contained by the host immune response to chronic ones, such as the chronic chagasic cardiomyopathy. Here, we analyzed, in an in vitro model, the ability of two T. cruzi isolates, with different degrees of virulence, to regulate the metabolism of L-arginine through arginase 1 (Arg-1) and NOS2 in macrophages and through arginase 2 (Arg-2) and NOS2 in cardiomyocytes. Stimulation of bone marrow-derived macrophages (BMMΦ), obtained from CD1 mice, with TNF-α + IFN-γ induced their polarization into classically activated macrophages (CAMΦ), which expressed functional NOS2, while stimulation with IL-4 induced their polarization into alternatively activated macrophages (AAMΦ), which expressed functional Arg-1. Interestingly, stimulation of cardiomyocytes, obtained from hearts of CD1 neonatal mice, with TNF-α + IFN-γ or IL-4 also resulted in functional NOS2 and arginase expression, as observed in CAMΦ and AAMΦ, but Arg-2 was the arginase isoform expressed instead of Arg-1. We observed that infection of BMMΦ with the more virulent T. cruzi isolate (QRO) importantly diminished NOS2 expression and nitric oxide (NO) production in CAMΦ, allowing parasite survival, while infection with the less virulent isolate (CI2) did not diminish NOS2 activity and NO production in CAMΦ to a great extent, which resulted in parasite killing. Regarding Arg-1, infection of BMMΦ with the QRO isolate significantly induced Arg-1 expression and activity in AAMΦ, which resulted in a higher parasite load than the one in the unstimulated BMMΦ. Even though infection with CI2 isolate did not increase Arg-1 expression and activity in AAMΦ, the parasite load was higher than the one in the unstimulated BMMΦ but at a lesser magnitude than that observed during infection with the QRO isolate. On the other hand, infection of cardiomyocytes with either QRO or CI2 isolates and further stimulation with TNF-α + IFN-γ inhibited NOS2 expression and NO production, leading to amelioration of infection. Surprisingly, infection of cardiomyocytes with either QRO or CI2 isolates and further stimulation with IL-4 strongly inhibited Arg-2 expression and function, which resulted in parasite loads similar to those observed in unstimulated cardiomyocytes. Our results suggest that T. cruzi isolates that exhibit variable virulence or pathogenicity degrees differentially regulate L-arginine metabolism through Arg-1/2 and NOS2 in macrophages and cardiomyocytes.

Cardiovascular effects of Roflumilast during sepsis: Risks or benefits?

BackgroundPhosphodiesterase-4 (PDE4) is responsible for terminating cyclic adenosine monophosphate (cAMP) signalling. PDE4 inhibitors, such as roflumilast (RFM), have anti-inflammatory activity and have been studied in inflammation-induced tissue damage in sepsis. However, the role of RFM on cardiovascular derangements induced by sepsis is still unknown. Thus, we aimed to evaluate the potential effects of RFM on cardiovascular collapse and multiorgan damage caused by sepsis. MethodsSepsis was induced by cecal ligation and puncture (CLP) in male rats. Six hours after the CLP or sham procedure, animals were randomly assigned to receive either RFM (0.3 mg/kg) or vehicle subcutaneously, and cardiovascular parameters were assessed 24 h after the surgery and organ/plasma samples were collected for further analyses. ResultsSepsis induced hypotension, tachycardia, reduced renal blood flow (RBF) and hyporeactivity to vasoconstrictors both in vivo and ex vivo. RFM treatment increased systemic cAMP levels and RBF. RFM also attenuated hypoperfusion and liver damage induced by CLP. Furthermore, RFM reduced systemic nitric oxide (NO) levels in septic rats, while there were no changes in hepatic NOS-2 expression. Nevertheless, RFM exacerbated sepsis-induced hypotension and tachycardia without ameliorating vascular hyporeactivity. ConclusionOur data show that PDE-4 inhibition protects septic rats from hepatic injury and improves renal perfusion. However, RFM worsened hemodynamic parameters and showed no protection against sepsis-induced cardiovascular dysfunction and mortality. Thus, despite the anti-inflammatory benefits of RFM, its application in sepsis should be approached cautiously.

Exercise-Induced Shear Stress Drives mRNA Translation In Vitro.

The vascular endothelium is the first line of defense to prevent cardiovascular disease. Its optimal functioning and health are maintained by the interaction of the proteins-endothelial nitric oxide synthase (eNOS), sirtuin 1 (SIRT1), and endothelin 1 (ET1)-and the genes that encode them-NOS3, SIRT1, and EDN1, respectively. Aerobic exercise improves endothelial function by allegedly increasing endothelial shear stress (ESS). However, there are no current data exploring the acute effects of specific exercise-induced ESS intensities on these regulatory proteins and genes that are associated with endothelial function. The purpose of this study was to assess the acute changes in endothelial proteins and gene expression after exposure to low-, moderate-, and high-intensity exercise-induced ESS. Human umbilical vein endothelial cells (HUVECs) were exposed to resting ESS (18 dynes/cm2, 60 pulses per minute (PPM)), low ESS (35 dynes/cm2, 100 PPM), moderate ESS (50 dynes/cm2, 120 PPM), and high ESS (70 dynes/cm2, 150 PPM). Protein and gene expression were quantified by fluorescent Western blot and RTqPCR, respectively. All exercise conditions showed an increase in eNOS and SIRT1 expression and a decrease in NOS3 and SIRT1 gene expression when compared to resting conditions. In addition, there was no expression of ET1 and an increase in EDN1 gene expression when compared to resting conditions. These results show that (1) exercise-induced ESS increases the expressions of vascular protective proteins and (2) there is an inverse relationship between the proteins and their encoding genes immediately after exercise-induced ESS, suggesting that exercise has a previously unexplored translational role catalyzing mRNA to proteins.

Comparison of efficacy and safety of different suction pressure for speeding non-ventilated lung collapse in uniport video-assisted thoracoscopic surgery: a randomized-controlled trial

BackgroundThe bronchial suction has been applied in speeding lung collapse. Low suction pressure may not speed lung collapse, but high pressure causes occult lung injury. The aim of the study was to explore efficacy and safety of different suction pressure for speeding lung collapse.MethodsEighty-four subjects undergoing uniport video-assisted thoracoscopic surgery (VATS) were randomly assigned for non-suction (Group 0), -10 cmH2O suction pressure (Group − 10), and − 30 cmH2O suction pressure (Group − 30). The primary outcome were the lung collapse scores (LCS) at 0 min (T0) after the visualization of the lung using a 10-point visual analogue scale and area under the curve (AUC) of LCS over time. The secondary outcomes included disconnection from the ventilator, the assessment of occult lung injury using NOS-3 expression, histologic scores of lung injury, and lung W/D weight ratio, intraoperative hypoxemia, the incidence of perioperative pulmonary complications.ResultsBoth the LCS at T0 and AUC analysis showed that compared with Group 0, Group − 10 and Group − 30 significantly achieved good lung collapse (P < 0.05), but no difference between Group − 10 and Group − 30. Four patients in Group 0 were treated with disconnection maneuver. The assessment of occult lung injury showed no differences.ConclusionsApplying − 10 cmH2O suction pressure for 1 min when pleural incision is a relatively safe method to promote lung collapse without the occurrence of occult lung injury.Trial registrationChinese Clinical Trial Registry number, ChiCTR2200062991. Registered on 26/08/2022.

Impact of apoptosis and oxidative stress on pancreatic beta cell pathophysiology in streptozotocin-induced Type 1 diabetes mellitus

AimsHyperglycemia plays a crucial role in the islet cells, especially pancreatic beta cell death in type 1 diabetes mellitus (T1DM). However, a few research have concentrated on the pathophysiology of apoptosis and oxidative stress in T1DM. The aim of this study was to determine the expression of Caspase 3, Caspase 9, 8-OHdG, Glutathione Reductase, endothelial and inducible nitric oxide synthase in the pancreatic tissue of streptozotocin (STZ)-induced T1DM patients and to compare the cellular mechanisms underlying this metabolic disorder. MethodsFor this purpose, a total of 20 Wistar albino rats were divided into two groups: Control (C) and Diabetes Mellitus (DM). In the DM group, T1DM was induced by STZ. Rats in the C group were injected intravenously with buffer solution. At the end of the day 20, rats were necropsied and immunohistochemical procedures were applied. ResultsThe immunohistochemical examination revealed, strong positive immunoreactions were observed in the islet cells of the DM groups, particularly when all antibody stains were considered. On the other hand, the C groups showed minimal changes. The difference between the C and DM groups in terms of all antibodies was statistically significant (p<0.01). ConclusionsIn the present study, it was concluded that apoptosis, oxidative stress and NOS expressions were involved in islet cell destruction in pancreatic tissue in STZ-induced T1DM.

A disturbed metabolite-GPCR axis is associated with microbial dysbiosis in IBD patients: Potential role of GPR109A in macrophages

Inflammatory Bowel Disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract characterized by disrupted immune function. Indeed, gut microbiota dysbiosis and metabolomic profile alterations, are hallmarks of IBD. In this scenario, metabolite-sensing G-protein coupled receptors (GPCRs), involved in several biological processes, have emerged as pivotal players in the pathophysiology of IBD. The aim of this study was to characterize the axis microbiota-metabolite-GPCR in intestinal surgical resections from IBD patients. Results showed that UC patients had a lower microbiota richness and bacterial load, with a higher proportion of the genus Cellulosimicrobium and a reduced proportion of Escherichia, whereas CD patients showed a decreased abundance of Enterococcus. Furthermore, metabolomic analysis revealed alterations in carboxylic acids, fatty acids, and amino acids in UC and CD samples. These patients also exhibited upregulated expression of most metabolite-sensing GPCRs analysed, which positively correlated with pro-inflammatory and pro-fibrotic markers. The role of GPR109A was studied in depth and increased expression of this receptor was detected in epithelial cells and cells from lamina propria, including CD68+ macrophages, in IBD patients. The treatment with β-hydroxybutyrate increased gene expression of GPR109A, CD86, IL1B and NOS2 in U937-derived macrophages. Besides, when GPR109A was transiently silenced, the mRNA expression and secretion of IL-1β, IL-6 and TNF-α were impaired in M1 macrophages. Finally, the secretome from siGPR109A M1 macrophages reduced the gene and protein expression of COL1A1 and COL3A1 in intestinal fibroblasts. A better understanding of metabolite-sensing GPCRs, such as GPR109A, could establish their potential as therapeutic targets for managing IBD.

Ephedrine attenuates LPS-induced M1 polarization of alveolar macrophages via the PKM2-mediated glycolysis.

Asthma is one of chronic inflammatory lung diseases in world. The important role of macrophage polarization and glycolysis in lung inflammation has attracted considerable attention. Ephedrine (EP) is a compound isolated from Ephedra and plays a regulatory role in inflammatory response, but its role in asthma and mechanism involved are not clear. Therefore, the purpose of this study was to investigate the molecular mechanism and effect of EP on lipopolysaccharide (LPS)-induced alveolar macrophage polarization and glycolysis. We investigated the expression of Tnf-a, Nos2, Il10, and Arg1 using RT-PCR, as well as PKM2 and LDHA protein expression with Western blot. A CCK-8 assay was performed to determine the viability of the cells. The extracellular acidification rate (ECAR), ATP and lactate level were detected using commercial kits. The results revealed that EP alleviated LPS-induced NR8383 cell glycolysis and M1 polarization. Further studies found that EP enhanced the effect of 2-DG on NR8383 cell glycolysis and M1 polarization. More importantly, PKM2 inhibitor alleviated LPS-induced NR8383 cell glycolysis and M1 polarization. In addition, EP alleviated LPS-induced NR8383 cell glycolysis and M1 polarization by targeting PKM2. It is suggested that EP alleviates LPS-induced glycolysis and M1 polarization in NR8383 cells by regulating PKM2, thereby alleviating lung injury, suggesting the involvment of alveolar macrophage polarization and glycolysis in the role of EP in asthma.

Anakinra Promotes M2 Microglia Activation during the Latent Phase of the Lithium-Pilocarpine Model of Temporal Lobe Epilepsy

Astrocytes and microglia and their polarization are thought to contribute to the progression of epilepsy. One of the processes affecting polarization is neuroinflammation, which plays an important role in epileptogenesis. However, the specific mechanisms of its involvement in shifting the pro- and anti-inflammatory reactivation of astro- and microglia have not been clarified. In this study, we examined the effect of 7-day interleukin-1 receptor antagonist (anakinra) administration on glial cell polarization during the latent phase of the lithium-pilocarpine model in 7-week-old male Wistar rats. In temporal cortex, dorsal and ventral hippocampus the mRNA expression levels of the following genes were analyzed: (i) markers of astroglia (S100b) and microglia (Aif1) activation, (ii) astrocytic proteins involved in glutamate transport and metabolism (Slc1a3, Glul, Gja1), (iii) pro-inflammatory pathway interleukin-1β (Nlrp3, Il1b, Il1rn) and transforming growth factor β1 (Tgfb1), (iv) markers of astroglia polarization (Lcn2, S100a10, Gbp2, Ptx3), and (v) microglia polarization (Nos2 and Arg1). The mRNA expression levels of S100b and Aif1 were significantly increased, and anakinra administration did not reduce their overexpression. This indicates reactivation of astroglia and microglia regardless of the anakinra administered. The expression of Slc1a3, Glul, and Gja1 genes increased in the hippocampus; anakinra administration did not affect their hyperexpression, but promoted increased expression of Gja1 in the temporal cortex. The mRNA production of Lcn2, S100a10, Gbp2, Ptx3, Nlrp3, Il1b, Il1rn and Tgfb1 increased in all structures. Administration of anakinra reduced the gene expression of Il1b. Among the markers of microglia polarization, downregulation of Arg1 expression in the dorsal hippocampus and Nos2 expression in the temporal cortex was detected. Anakinra administration enhanced the decrease in Nos2 expression and restored the level of Arg1 expression to control values. Thus, anakinra administration did not affect the intensity of glial cell reactivation, but improved M2 reactivation of microglia.

In vivo toxicity of upconversion nanoparticles (NaYF4:Yb, Er) in zebrafish during early life stages: Developmental toxicity, gut-microbiome disruption, and proinflammatory effects

Lanthanide-doped upconversion nanoparticles (Ln-UCNPs) have been considered promising materials for various fields, such as biomedical and industrial applications. However, data and reports regarding its toxicity and environmental risks are scarce. Under these circumstances, data must be obtained to fully understand potential toxicity and adverse outcome pathways. In the present study, the toxicity of uncoated Ln-UCNP cores (NaYF4:Yb, Er) was systematically assessed in zebrafish embryos during early developmental stages. Ln-UCNPs were found to have multiple toxic effects, such as effects on survival rates, delayed hatching times, shorter body lengths, altered heart rates and blood circulation (significantly reduced), and neurobehavioral impairments in response to photoperiod stimulation. Bioimaging showed that Ln-UCNPs were distributed on the chorion, eyes, and skin at 72 hpf. However, it accumulates in the pharynx, esophagus, and intestine after oral administration. Ln-UCNPs disrupt the diversity and abundance of host-associated microorganisms (gut microbiota) leading to an increase in the prevalence of harmful bacteria in zebrafish. Transcriptomic and Ingenuity Pathway Analysis (IPA) predicted Interleukin-8 (IL-8) signaling, neuroinflammation, cardiac hypertrophy signaling pathways, immune and inflammation-related response interferon-gamma (ifnγ), and miR-155 as key mediators in regulatory effects. Based on this, a causal network was built showing the strong links between the induced gene expression of differentially expressed genes (DEGs), such as nitric oxide synthase 2 (nos2) and tumor necrosis factor (tnf) upon Ln-UCNPs treatment, and with the downstream adverse outcomes, in particular, the promotion of apoptosis, liver damage, and inflammatory response. Finally, RT-qPCR analysis confirmed the up-regulated expression of nos2 and tnf in the exposed larvae, consistent with the observation of an increased number of fluorescence-labelled neutrophils and macrophages in lyz: DsRed transgenic zebrafish until 120 hpf exposure, which together demonstrated the proinflammatory effects of Ln-UCNPs on organisms. In conclusion, we illustrated the developmental toxicity, disruption of gut-microbiome, and proinflammatory effects of Ln-UCNP cores on zebrafish, and the causal network from IPA analysis may help further elucidate the adverse outcome pathway of Ln-UCNPs.

The role of PI3K-Akt-mTOR axis in Warburg effect and its modification by specific protein kinase inhibitors in human and rat inflammatory macrophages

The Warburg effect occurs both in cancer cells and in inflammatory macrophages. The aim of our work was to demonstrate the role of PI3K-Akt-mTOR axis in the Warburg effect in HL-60 derived, rat peritoneal and human blood macrophages and to investigate the potential of selected inhibitors of this pathway to antagonize it. M1 polarization in HL-60-derived and human blood monocyte-derived macrophages was supported by the increased expression of NOS2 and inflammatory cytokines. All M1 polarized and inflammatory macrophages investigated expressed higher levels of HIF-1α and NOS2, which were reduced by selected kinase inhibitors, supporting the role of PI3K-Akt-mTOR axis. Using Seahorse XF plates, we found that in HL-60-derived and human blood-derived macrophages, glucose loading reduced oxygen consumption (OCR) and increased glycolysis (ECAR) in M1 polarization, which was antagonized by selected kinase inhibitors and by dichloroacetate. In rat peritoneal macrophages, the changes in oxidative and glycolytic metabolism were less marked and the NOS2 inhibitor decreased OCR and increased ECAR. Non-mitochondrial oxygen consumption and ROS production were likely due to NADPH oxidase, expressed in each macrophage type, independently of PI3K-Akt-mTOR axis. Our results suggest that inflammation changed the metabolism in each macrophage model, but a clear relationship between polarization and Warburg effect was confirmed only after glucose loading in HL-60 and human blood derived macrophages. The effect of kinase inhibitors on Warburg effect was variable in different cell types, whereas dichloroacetate caused a shift toward oxidative metabolism. Our findings suggest that these originally anti-cancer inhibitors may also be candidates for anti-inflammatory therapy.

Atriplex hortensis var. 'rubra' extracts and purified amaranthin-type pigments reduce oxidative stress and inflammatory response in LPS-stimulated RAW264.7 cells

The use of direct injection ion mobility mass spectrometry (DI-IM-MS) to detect and identify betacyanin pigments in A. hortensis ‘rubra’ extracts was explored for the first time, with results compared to conventional LC-MS/MS analysis. The anti-inflammatory activities of leaf and seed extracts, alongside purified amaranthin and celosianin pigments, were investigated using a model of lipopolysaccharide (LPS)-activated murine macrophages. Extracts and purified pigments significantly inhibited the production of prostaglandin E2 and NO by up to 90% and 70%, respectively, and reduced the expression of Il6, Il1b, Nos2, and Cox2. Leaf and seed extracts also decreased secretion of Il6 and Il1b cytokines and reduced protein levels of Nos2 and Cox2. Furthermore, extracts and purified pigments demonstrated potent dose-dependent radical scavenging activity in a cellular antioxidant activity assay (CAA) without any cytotoxic effects. Our research highlights the promising biological potential of edible, climate-resilient A. hortensis ‘rubra’ as a valuable source of bioactive compounds.

Identification and multimodal characterization of a specialized epithelial cell type associated with Crohn’s disease

Crohn’s disease (CD) is a complex chronic inflammatory disorder with both gastrointestinal and extra-intestinal manifestations associated immune dysregulation. Analyzing 202,359 cells from 170 specimens across 83 patients, we identify a distinct epithelial cell type in both terminal ileum and ascending colon (hereon as ‘LND’) with high expression of LCN2, NOS2, and DUOX2 and genes related to antimicrobial response and immunoregulation. LND cells, confirmed by in-situ RNA and protein imaging, are rare in non-IBD controls but expand in active CD, and actively interact with immune cells and specifically express IBD/CD susceptibility genes, suggesting a possible function in CD immunopathogenesis. Furthermore, we discover early and late LND subpopulations with different origins and developmental potential. A higher ratio of late-to-early LND cells correlates with better response to anti-TNF treatment. Our findings thus suggest a potential pathogenic role for LND cells in both Crohn’s ileitis and colitis.