ORMDL3 regulates NLRP3 inflammasome activation by maintaining ER-mitochondria contacts in human macrophages and dictates ulcerative colitis patient outcome.

Genome-wide association studies in inflammatory bowel disease have identified risk loci in the orosomucoid-like protein 3/ORMDL sphingolipid biosynthesis regulator 3 (ORMDL3) gene to confer susceptibility to ulcerative colitis (UC), but the underlying functional relevance remains unexplored. Here, we found that a subpopulation of the UC patients who had higher disease activity shows enhanced expression of ORMDL3 compared to the patients with lower disease activity and the non-UC controls. We also found that the patients showing high ORMDL3 mRNA expression have elevated interleukin-1ß cytokine levels indicating positive correlation. Further, knockdown of ORMDL3 in the human monocyte-derived macrophages resulted in significantly reduced interleukin-1ß release. Mechanistically, we report for the first time that ORMDL3 contributes to a mounting inflammatory response via modulating mitochondrial morphology and activation of the NLRP3 inflammasome. Specifically, we observed an increased fragmentation of mitochondria and enhanced contacts with the endoplasmic reticulum (ER) during ORMDL3 over-expression, enabling efficient NLRP3 inflammasome activation. We show that ORMDL3 that was previously known to be localized in the ER also becomes localized to mitochondria-associated membranes and mitochondria during inflammatory conditions. Additionally, ORMDL3 interacts with mitochondrial dynamic regulating protein Fis-1 present in the mitochondria-associated membrane. Accordingly, knockdown of ORMDL3 in a dextran sodium sulfate -induced colitis mouse model showed reduced colitis severity. Taken together, we have uncovered a functional role for ORMDL3 in mounting inflammation during UC pathogenesis by modulating ER-mitochondrial contact and dynamics.

Comprehensive Study on Solomonamides: Total Synthesis, Stereochemical Revision, and SAR Studies toward Identification of Simplified Lead.

Solomonamides, a pair of macrocyclic peptide natural products originating from marine sources, have garnered significant attention within the synthetic community owing to their marked anti-inflammatory efficacy and intricate molecular architectures. In this paper, we present a very detailed investigation into solomonamides, including the challenges associated with the total synthesis, the evolution of our synthetic strategies, structural reassignment, synthesis of all possible stereoisomeric macrocycles, biological assessment, structure-activity relationship (SAR) studies, etc. Within the ambit of this total synthesis, diverse strategies for macrocyclization were rigorously explored, encompassing the Friedel-Crafts acylation, cyclization involving the aniline NH2 moiety, macrolactamization utilizing Gly-NH2, and Heck macrocyclization methodologies. In addition, an array of intriguing chemical transformations were devised, including but not limited to photo-Fries rearrangement, Wacker oxidation, ligand-free Heck macrocyclization, oxidative cleavage of indole, synthesis of contiguous stereocenters via substrate/reagent-controlled protocols, and simultaneous making and breaking of olefinic moieties. The findings of this investigation revealed a structurally simplified lead compound. Remarkably, the lead compound, while possessing structural simplification in comparison to the intricate solomonamide counterparts, demonstrates equipotent in vivo anti-inflammatory efficacy.

CLUH functions as a negative regulator of inflammation in human macrophages and determines ulcerative colitis pathogenesis.

Altered mitochondrial function without a well-defined cause has been documented in patients with ulcerative colitis (UC). In our efforts to understand UC pathogenesis, we observed reduced expression of clustered mitochondrial homolog (CLUH) only in the active UC tissues compared with the unaffected areas from the same patient and healthy controls. Stimulation with bacterial Toll-like receptor (TLR) ligands similarly reduced CLUH expression in human primary macrophages. Further, CLUH negatively regulated secretion of proinflammatory cytokines IL-6 and TNF-α and rendered a proinflammatory niche in TLR ligand-stimulated macrophages. CLUH was further found to bind to mitochondrial fission protein dynamin related protein 1 (DRP1) and regulated DRP1 transcription in human macrophages. In the TLR ligand-stimulated macrophages, absence of CLUH led to enhanced DRP1 availability for mitochondrial fission, and a smaller dysfunctional mitochondrial pool was observed. Mechanistically, this fissioned mitochondrial pool in turn enhanced mitochondrial ROS production and reduced mitophagy and lysosomal function in CLUH-knockout macrophages. Remarkably, our studies in the mouse model of colitis with CLUH knockdown displayed exacerbated disease pathology. Taken together, this is the first report to our knowledge explaining the role of CLUH in UC pathogenesis, by means of regulating inflammation via maintaining mitochondrial-lysosomal functions in the human macrophages and intestinal mucosa.

Design, synthesis and evaluation of novel pyrrole-hydroxybutenolide hybrids as promising antiplasmodial and anti-inflammatory agents.

In the pursuance of novel scaffolds with promising antiplasmodial and anti-inflammatory activity, a series of twenty-one compounds embraced with most promising penta-substituted pyrrole and biodynamic hydroxybutenolide in single skeleton was designed and synthesized. These pyrrole-hydroxybutenolide hybrids were evaluated against Plasmodium falciparum parasite. Four hybrids 5b, 5d, 5t and 5u exhibited good activity with IC50 of 0.60, 0.88, 0.97 and 0.96 µM for chloroquine sensitive (Pf3D7) strain and 3.92, 4.31, 4.21 and 1.67 µM for chloroquine resistant (PfK1) strain, respectively. In vivo efficacy of 5b, 5d, 5t and 5u was studied against the P. yoelii nigeriensis N67 (a chloroquine-resistant) parasite in Swiss mice at a dose of 100 mg/kg/day for 4 days via oral route. 5u was found to show maximum 100% parasite inhibition with considerably increased mean survival time. Simultaneously, the series of compounds was screened for anti-inflammatory potential. In preliminary assays, nine compounds showed more than 85% inhibition in hu-TNFα cytokine levels in LPS stimulated THP-1 monocytes and seven compounds showed more than 40% decrease in fold induction in reporter gene activity analyzed via Luciferase assay. 5p and 5t were found to be most promising amongst the series, thus were taken up for further in vivo studies. Wherein, mice pre-treated with them showed a dose dependent inhibition in carrageenan induced paw swelling. Moreover, the results of in vitro and in vivo pharmacokinetic parameters indicated that the synthesized pyrrole-hydroxybutenolide conjugates abide by the required criteria for the development of orally active drug and thus this scaffold can be used as pharmacologically active framework that should be considered for the development of potential antiplasmodial and anti-inflammatory agents.

Standardized fraction of Xylocarpus moluccensis inhibits inflammation by modulating MAPK-NFκB and ROS-HIF1α-PKM2 activation.

OBJECTIVE: Present study investigates the effect of Xylocarpus moluccensis (Lamk.) M. Roem fruit fraction (CDR) on endotoxemia and explores the underlying mechanisms. MATERIALS AND METHODS: The effect of CDR (1-100 µg/ml) was assessed on cytokines, MAPKs, ROS, and metabolic reprogramming in LPS-induced cells (J774.2 and THP-1) by the conventional methodology of ELISA, PCR, and Western blotting. The effect of CDR (1-50 mg/kg, p.o.) was also evaluated in the mice model of endotoxemia and sepsis. RESULTS: CDR prevents LPS-induced cytokine production from murine and human whole blood and cell lines. CDR suppressed total cellular and mitochondrial superoxide generation and preserved mitochondrial function in LPS-stimulated phagocytes. Additionally, CDR abrogated LPS-induced MAPK's phosphorylation and IκBα degradation in J774.2 cells. Moreover, CDR suppressed LPS-induced glycolytic flux as indicated from PKM2, HK-2, PDK-2, and HIF-1α expression in J774.2 cells. In vivo, CDR pre-treatment inhibited pro-inflammatory cytokines release, metabolic reprogramming from oxidative phosphorylation to glycolysis in both LPS-induced endotoxemia and cecal slurry-induced sepsis mice model. CONCLUSION: Present study demonstrates the protective effect of CDR on LPS-induced inflammation and sepsis and identifies MAPK-NFκB and ROS-HIF1α-PKM2 as the putative target axis.

Galectin-3 S-glutathionylation regulates its effect on adipocyte insulin signaling.

Protein-S-glutathionylation promotes redox signaling in physiological and oxidative distress conditions. Galectin-3 (Gal-3) promotes insulin resistance by down-regulating adipocyte insulin signaling, however, its S-glutathionylation and significance is not known. In this context, we report reversible S-glutathionylation of Gal-3. Site-directed mutagenesis established Gal-3 Cys187 as the putative S-glutathionylation site. Glutathionylated Gal-3 prevents Gal-3(WT)-Insulin Receptor interaction and facilitates insulin-induced murine adipocyte p-IRS1(tyr895) and p-AKT(ser473) signaling and glucose uptake in a Gal-3 Cys187 glutathionylation dependent manner in murine adipocytes, as assessed by Western blotting and 2-NBDG uptake assay respectively. Pre-glutathionylated Gal-3 at Cys187 resisted irreversible oxidation by H2O2. M2 macrophages showed enhanced Gal-3 S-glutathionylation when compared to M1 phenotype. Serum and stromal vascular fraction (SVF) isolated from control mice showed increased Gal-3 S-glutathionylation as compared to db/db mice. A significant increase in Gal-3 S-glutathionylation was observed in metformin-treated db/db mice when compared to db/db mice alone. Similar to murine, enhanced Gal-3 S-glutathionylation is observed in primary human monocyte derived M2 macrophages when compared to the M1 macrophage phenotype and Gal-3 regulates primary human adipocyte insulin signaling in a glutathionylation dependent manner. Collectively, we identified Gal-3 S-glutathionylation as a protective phenomenon, which relieves its inhibitory effect on adipocyte insulin signaling.

FOXO3a acetylation regulates PINK1, mitophagy, inflammasome activation in murine palmitate-conditioned and diabetic macrophages.

Nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain containing 3 inflammasome and mitophagy play an important role in cytokine release and diabetes progression; however, the role of saturated fatty acid that is induced under such conditions remains little explored. Therefore, the present study evaluates mechanisms regulating mitophagy and inflammasome activation in primary murine diabetic and palmitate-conditioned wild-type (WT) peritoneal macrophages. Peritoneal macrophage, from the diabetic mice and WT mice, challenged with LPS/ATP and palmitate/LPS/ATP, respectively, showed dysfunctional mitochondria as assessed by their membrane potential, mitochondrial reactive oxygen species (mtROS) production, and mitochondrial DNA (mtDNA) release. A defective mitophagy was observed in the diabetic and palmitate-conditioned macrophages stimulated with LPS/ATP as assessed by translocation of PTEN-induced kinase 1 (PINK1)/Parkin or p62 in the mitochondrial fraction. Consequently, increased apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) oligomerization, caspase-1 activation, and IL1ß secretion were observed in LPS/ATP stimulated diabetic and palmitate-conditioned macrophages. LPS/ATP induced Forkhead box O3a (FOXO3a) binding to PINK1 promoter and increased PINK1 mRNA expression in WT macrophages. However, PINK1 mRNA and protein expression were significantly decreased in diabetic and palmitate-conditioned macrophages in response to LPS/ATP. Palmitate-induced acetyl CoA promoted FOXO3a acetylation, which prevented LPS/ATP-induced FOXO3a binding to the PINK1 promoter. C646 (P300 inhibitor) and SRT1720 (SIRT1 activator) prevented FOXO3a acetylation and restored FOXO3a binding to the PINK1 promoter, PINK1 mRNA expression, and mitophagy in palmitate-conditioned macrophages treated with LPS/ATP. Also, a significant decrease in the LPS/ATP-induced mtROS production, mtDNA release, ASC oligomerization, caspase-1 activation, and IL-1ß release was observed in the palmitate-conditioned macrophages. Similarly, modulation of FOXO3a acetylation also prevented LPS/ATP-induced mtDNA release and inflammasome activation in diabetic macrophages. Therefore, FOXO3a acetylation regulates PINK1-dependent mitophagy and inflammasome activation in the palmitate-conditioned and diabetic macrophages.

nNOS induction and NOSIP interaction impact granulopoiesis and neutrophil differentiation by modulating nitric oxide generation.

Nitric oxide (NO), a versatile free radical and a signalling molecule, plays an important role in the haematopoiesis, inflammation and infection. Impaired proliferation and differentiation of myeloid cells lead to malignancies and Hematopoietic deficiencies. This study was aimed to define the role of nNOS derived NO in neutrophil differentiation (in-vitro) and granulopoiesis (in-vivo) using multipronged approaches. The results obtained from nNOS over-expressing K562 cells revealed induction in C/EBPα derived neutrophil differentiation as evident by an increase in the expression of neutrophil specific cell surface markers, genes, transcription factors and functionality. nNOS mediated response also involved G-CSFR-STAT-3 axis during differentiation. Consistent increase in NO generation was observed during neutrophil differentiation of mice and human CD34+ HSPCs. Furthermore, granulopoiesis was abrogated in the nNOS inhibitor treated mice, depicting a decrease in the numbers of BM mature and progenitor neutrophils. Likewise, in vitro inhibition of nNOS in human CD34+ HSPCs indicated an indispensable role of nNOS in neutrophil differentiation. Expression of nNOS inhibitory protein, NOSIP was significantly and consistently decreased during the final stage of differentiation and was linked with the augmentation in NO release. Moreover, neutrophils from CML patients had more NOSIP and less NO generation as compared to the PMNs from healthy individuals. The present study thus indicates a critical role of nNOS, and its interaction with NOSIP during neutrophil differentiation. The study also highlights the importance of nNOS in the neutrophil progenitor proliferation and differentiation warranting investigations to assess its role in the haematopoiesis-related disorders.

Macrophage p47phox regulates pressure overload-induced left ventricular remodeling by modulating IL-4/STAT6/PPARγ signaling.

NADPH oxidase (Nox) mediates ROS production and contributes to cardiac remodeling. However, macrophage p47phox, a Nox subunit regulating cardiac remodeling, is unclear. We aimed to investigate the role of macrophage p47phox in hypertensive cardiac remodeling. Pressure-overload induced by Angiotensin II (AngII) for two weeks in young adult male p47phox deficient (KO) mice showed aggravated cardiac dysfunction and hypertrophy as indicated from echocardiographic and histological studies in comparison with wild-type littermates (WT). Additionally, LV of AngII-infused KO mice showed augmented interstitial fibrosis, collagen deposition and, myofibroblasts compared to AngII-infused WT mice. Moreover, these changes in AngII-infused KO mice correlated well with the gene analysis of hypertrophic and fibrotic markers. Similar results were also found in the transverse aortic constriction model. Further, AngII-infused KO mice showed elevated circulating immunokines and increased LV leukocytes infiltration and CD206+ macrophages compared to AngII-infused WT mice. Likewise, LV of AngII-infused KO mice showed upregulated mRNA expression of anti-inflammatory/pro-fibrotic M2 macrophage markers (Ym1, Arg-1) compared to AngII-infused WT mice. AngII and IL-4 treated bone marrow-derived macrophages (BMDMs) from KO mice showed upregulated M2 macrophage markers and STAT6 phosphorylation (Y641) compared to AngII and IL-4 treated WT BMDMs. These alterations were at least partly mediated by macrophage as bone marrow transplantation from KO mice into WT mice aggravated cardiac remodeling. Mechanistically, AngII-infused KO mice showed hyperactivated IL-4/STAT6/PPARγ signaling and downregulated SOCS3 expression compared to AngII-infused WT mice. Our studies show that macrophage p47phox limits anti-inflammatory signaling and extracellular matrix remodeling in response to pressure-overload.

Expression of inducible NOS is indispensable for the antiproliferative and proapoptotic effect of imatinib in BCR-ABL positive cells.

Chronic myeloid leukemia (CML) is characterized by constitutive BCR-ABL kinase activity, an aggressive proliferation of immature cells, and reduced differentiation. Targeting tyrosine kinase activity of BCR-ABL with imatinib is an effective therapy for the newly diagnosed CML patients; however, 20%-30% of the patients initially treated with imatinib eventually experience treatment failure. Therefore, early identification of these patients is of high clinical relevance. In the present study, we by undertaking a direct comparison of inducible NOS (iNOS) status in neutrophils from healthy volunteers, newly diagnosed, imatinib responder, and resistant CML patients as well as by conducting in vitro studies in K562 cells demonstrated that inhibition of BCR-ABL by imatinib or siRNA significantly enhanced NO generation and iNOS expression. Indeed, patients exhibiting treatment failure or imatinib resistance were less likely to induce NO generation/iNOS expression. Our findings further demonstrated that imatinib mediated antiproliferative and proapoptotic effect in BCR-ABL+ cells associated with enhanced iNOS expression, and it was significantly prevented in the presence of L-NAME, 1400W, or iNOS siRNA. Overexpression of iNOS in K562 cells expectedly enhanced imatinib sensitivity on cytostasis and apoptosis, even at lower concentration (0.1 µM) of imatinib. Mechanistically, imatinib or BCR-ABL siRNA following deglutathionylation of NF-κB, enhanced its binding to iNOS promoter and induced iNOS transcription. Deglutathionylation of procaspase-3 however associated with increased caspase-3 activity and cell apoptosis. Taken together, results obtained suggest that monitoring NO/iNOS level could be useful to identify patients likely to be responsive or resistant to imatinib and can be used to personalized alternative therapy.

Lin28B Regulates Angiotensin II-Mediated Let-7c/miR-99a MicroRNA Formation Consequently Affecting Macrophage Polarization and Allergic Inflammation.

Angiotensin-II (Ang-II) receptor plays a role in allergic airway inflammation; however, the underlying mechanism and role of macrophages need better understanding. In the present study, angiotensin-II infusion (1 µg/kg/min) in ovalbumin-induced airway inflammation mice model significantly decreased immune cell infiltration, goblet cell hyperplasia, and eosinophil numbers in lungs. Ang-II infusion increased M1 and decreased M2 macrophage population in bronchoalveolar lavage fluid and respective macrophage markers in lung macrophages. Similarly, in vitro Ang-II treatment in murine bone marrow-derived macrophages (BMDMs) induced M1 and reduced M2 macrophage phenotype with enhanced bactericidal activity. Mechanistically, Ang-II inhibits Let-7c and miR-99a expression in BMDMs and in vivo as well. Lentiviral overexpression of Let-7c and miR-99a miRNAs in BMDMs abrogated Ang-II-induced M1 phenotype activation and promoted M2 phenotype, which is governed by targeting TNFα by miR-99a. In lung macrophages, ovalbumin-induced TNFα inhibition was rescued after Ang-II treatment. In BMDMs, knockdown of TNFα abrogated Ang-II-induced M2 to M1 macrophage phenotype switch and associated bactericidal activity. Ang-II affects mature miRNA formation by enhancing Lin28B levels in macrophages in vivo and in vitro. Furthermore, Lin28B knockdown prevented Ang-II-mediated inhibition of mature Let-7c/miR-99a miRNA formation, M2 to M1 macrophage phenotype switch, and increased bactericidal activity. Therefore, present study suggests a role of Lin28B in Ang-II-induced Let-7c/miR-99a miRNA formation that consequently affects TNFα production, M1 phenotype activation, and allergic airway inflammation. Graphical Abstract Ovalbumin inhibits LIN28B expression thereby fails to inhibit premature to mature Let-7c/miR-99a miRNA formation. Mature miR-99a miRNA that inhibits TNFα consequently promotes M2 polarization and allergic airway inflammation. While Ang-II induces Lin28B, which inhibits Let-7c/miR-99a miRNA processing and mature miRNA formation, this results in increased TNFα levels that lead to M1 polarization and allergic airway inflammation inhibition.

Non-coding RNAs: Regulators of valvular calcification.

There is currently a growing global burden of valvular heart diseases due to aging populations and changing lifestyles. Valvular heart diseases mainly include the malfunctioning of aortic and mitral valves and are characterized by extensive tissue remodeling, which includes calcification, endothelial dysfunction, and endothelial-mesenchymal transition. These valvular remodeling processes are known to be regulated by protein-coding genes as well as non-coding genes. Here, we have summarized studies highlighting the non-coding RNA mediated regulation of valvular tissue remodeling and their potential therapeutic benefits. Additionally, studies investigating the diagnostic capability of circulating non-coding RNA molecules in valvular diseases are also summarized. Overall, of the various candidates, several studies have highlighted miR-214 and miR-204 as central regulators of valvular calcification.

Standardized Xylocarpus moluccensis fruit fraction mitigates collagen-induced arthritis in mice by regulating immune response.

OBJECTIVE: This study was undertaken to evaluate the effect of Xylocarpus moluccensis fruit fraction (F018) on the pathogenesis of collagen-induced arthritis in mice. METHODS: Arthritis was induced by intradermal injection of collagen (2 mg/ml) with complete Freund's adjuvant in DBA/1J mice. F018 was administered orally at 1, 3 and 10 mg/kg for 20 days. Disease progression and mechanism were assessed by micro-CT analysis, RT-PCR, flow cytometry assay, myeloperoxidase (MPO) and MTT assay. RESULTS: F018 at 3 and 10 mg/kg significantly reduced paw thickness, clinical score, mononuclear cell infiltration and collagen layer depletion in the knee section of collagen-induced arthritis (CIA) mice when compared with collagen-induced arthritis mice alone. Furthermore, F018 treatment in collagen-induced arthritis mice significantly recovered bone volume and trabecular number and decreased the trabecular space by modulating RANKL and OPG mRNA expression in the synovial tissue. F018 treatment in collagen-induced arthritis mice significantly attenuated spleen index, lymphocyte proliferation and paw myeloperoxidase (MPO) activity, pro-inflammatory cytokine TNFα, IL1ß, and IL6 mRNA expression and enhanced IL10 mRNA expression in paw tissue. Furthermore, F018 treatment in collagen-induced arthritis mice significantly reduced splenic dendritic cell maturation and Th17 cells. In culture, F018 significantly decreased collagen-induced arthritis-FLS proliferation and promoted apoptosis. CONCLUSION: F018 may serve as a potential curative agent for arthritis.

Role of pyruvate kinase M2 in oxidized LDL-induced macrophage foam cell formation and inflammation.

Pyruvate kinase M2 (PKM2) links metabolic and inflammatory dysfunction in atherosclerotic coronary artery disease; however, its role in oxidized LDL (Ox-LDL)-induced macrophage foam cell formation and inflammation is unknown and therefore was studied. In recombinant mouse granulocyte-macrophage colony-stimulating factor-differentiated murine bone marrow-derived macrophages, early (1-6 h) Ox-LDL treatment induced PKM2 tyrosine 105 phosphorylation and promotes its nuclear localization. PKM2 regulates aerobic glycolysis and inflammation because PKM2 shRNA or Shikonin abrogated Ox-LDL-induced hypoxia-inducible factor-1α target genes lactate dehydrogenase, glucose transporter member 1, interleukin 1ß (IL-1ß) mRNA expression, lactate, and secretory IL-1ß production. PKM2 inhibition significantly increased Ox-LDL-induced ABCA1 and ABCG1 protein expression and NBD-cholesterol efflux to apoA1 and HDL. PKM2 shRNA significantly inhibited Ox-LDL-induced CD36, FASN protein expression, DiI-Ox-LDL binding and uptake, and cellular total cholesterol, free cholesterol, and cholesteryl ester content. Therefore, PKM2 regulates lipid uptake and efflux. DASA-58, a PKM2 activator, downregulated LXR-α, ABCA1, and ABCG1, and augmented FASN and CD36 protein expression. Peritoneal macrophages showed similar results. Ox-LDL induced PKM2- SREBP-1 interaction and FASN expression in a PKM2-dependent manner. Therefore, this study suggests a role for PKM2 in Ox-LDL-induced aerobic glycolysis, inflammation, and macrophage foam cell formation.

Pyroptotic and apoptotic cell death in iNOS and nNOS overexpressing K562 cells: A mechanistic insight.

Previous studies from this lab and others have demonstrated that nitric oxide (NO) in a concentration dependent manner, modulated neutrophil and leukemic cell survival. Subsequent studies delineated importance of iNOS in neutrophil differentiation and leukemic cell death. On the contrary, role of nNOS in survival of these cells remains least understood. Present study was therefore undertaken to assess and compare the role of iNOS and nNOS in the survival of NOS overexpressing myelocytic K562 cells. Cells with almost similar iNOS and nNOS activities displayed comparable cell cycle perturbation, Annexin V positivity, mitochondrial dysfunction, augmented DCF fluorescence, and also attenuated expression of antioxidants. Moreover, induction in cell death was also accompanied by the activation of pJNK/p38MAPK/Erk1/2 and reduction in PI3K/Akt/mTOR signaling. Treatment of NOS isoform overexpressing K562 cells with NAC, a potent free radical scavenger prevented cell death and also the modulations in the signaling proteins. In addition, enhanced expression of CASP1 and CASP4 genes, along with increased Caspase-1 cleavage and increased IL-1ß release were significantly more in K562iNOS cells, which indicate priming of these cells for pyroptotic cell death. On the other hand, K562nNOS cells, displayed much enhanced CASP3 gene expression, Caspase-3 cleavage and Caspase-3 activity. Results obtained indicate that similar level of iNOS or nNOS activation in K562 cells, preferred pyroptotic and apoptotic cell death respectively.

Augmentation of iNOS expression in myeloid progenitor cells expedites neutrophil differentiation.

Neutrophils play important role in immunity and inflammation through diverse mechanisms. Reports from this lab and others have demonstrated involvement of NO in neutrophil adhesion, chemotaxis, bacterial killing, reactive oxygen species generation, neutrophil extracellular traps' formation, and apoptosis. Constitutive expression of iNOS in human neutrophils has also been documented. The role of NO-iNOS in neutrophil differentiation however remains ill-defined. The present study was undertaken to understand the role of NO generated from iNOS in the neutrophil differentiation by using iNOS-overexpressing K562 cells (K562iNOS ) and iNOS-deficient murine progenitor cells (lineage negative cells; lin-ve ). We observed that iNOS overexpression led to increased neutrophilic differentiation in K562 cells; more specifically an early and accelerated neutrophilic differentiation was spotted in K562iNOS . These observations were further validated using iNOS knockout lin-ve cells or hematopoietic progenitor cells that exhibited delayed neutrophil differentiation in comparison to its wild-type counterpart. In addition, a significant increase in the gene expression of iNOS during neutrophilic differentiation of CD34+ hematopoietic stem and progenitor cells derived from human bone marrow further substantiates importance of iNOS in neutrophil differentiation. Moreover, a significant increase in NO generation during neutrophil differentiation was observed and enhanced neutrophil differentiation with NO donor was also observed, implying the importance of NO in neutrophil differentiation. Collectively, using alternative approaches, we demonstrated that neutrophil differentiation is significantly influenced by iNOS or NO, suggesting the possibility of exploiting this novel link for therapeutic aspects of NO generated from iNOS and neutrophil differentiation in hematopoiesis-related disorders.

MicroRNA-99a mimics inhibit M1 macrophage phenotype and adipose tissue inflammation by targeting TNFα.

In human adipose tissue and obesity, miR-99a expression is negatively correlated with inflammation. Therefore, the present study investigated the role of miR-99a in macrophage phenotype activation and adipose tissue inflammation. M2 BMDMs showed a significant increase in miR-99a expression when compared to the M0 and M1 phenotypes. Phenotype-switching experiments established an association between upregulated miR-99a expression and the M2 phenotype. Overexpression of miR-99a prevented M1 phenotype activation and attenuated bactericidal activity. Likewise, knockdown of miR-99a abolished M2 phenotype activation. By means of in silico target prediction tools and a luciferase reporter assay, TNFα was identified as a direct target of miR-99a. Knockdown of TNFα recapitulated the effect of miR-99a overexpression in M1 BMDMs. In a db/db mice model, miR-99a expression was reduced in eWAT and F4/80+ ATMs. Systemic overexpression of miR-99a in db/db mice attenuated adipocyte hypertrophy with increased CD301 and reduced CD86 immunostaining. Flow cytometry analysis also showed an increased M2 and a reduced M1 macrophage population. Mimics of miR-99a also improved the diabetic dyslipidemia and insulin signaling in eWAT and liver, with an attenuated expression of gluconeogenesis and cholesterol metabolism genes in the liver. Furthermore, adoptive transfer of miR-99a-overexpressing macrophages in the db/db mice recapitulated in vivo miR-99a mimic effects with increased M2 and reduced M1 macrophage populations and improved systemic glucose, insulin sensitivity, and insulin signaling in the eWAT and liver. The present study demonstrates that miR-99a mimics can regulate macrophage M1 phenotype activation by targeting TNFα. miR-99a therapeutics in diabetic mice reduces the adipose tissue inflammation and improves insulin sensitivity.

Total synthesis of the potent anti-inflammatory natural product solomonamide A along with structural revision and biological activity evaluation.

Herein, we report the total synthesis of solomonamide A along with its structural revision for the first time. The natural product possesses very potent anti-inflammatory activity, and it contains a macrocyclic peptide having four consecutive stereocenters on an unnatural amino acid component. The key features in the present synthesis include the application of an Evans aldol reaction, ligand-free Heck macrocyclization and chemoselective oxidations. The challenging task of fixing the stereochemistry of OH at the C5-position was accomplished with the help of DFT calculations, applying a quantum-mechanical (QM)/NMR combined approach. Biological evaluation in a mouse paw edema model revealed that a low dose (0.3 mg kg-1) of the synthesized solomonamide A showed 74% reduction at 6 h, which was comparable to a high dose (10 mg kg-1) standard drug dexamethasone effect (75% at 6 h). Thus, we further confirmed the revised structure of solomonamide A.