Prdx6 Regulates Nlrp3 Inflammasome Activation-Driven Inflammatory Response in Lens Epithelial Cells.

The continuum of antioxidant response dysregulation in aging/oxidative stress-driven Nlrp3 inflammasome activation-mediated inflammatory response is associated with age-related diseases. Peroxiredoxin (Prdx) 6 is a key antioxidant that provides cytoprotection by regulating redox homeostasis. Herein, using lens epithelial cells (LECs) derived from the targeted inactivation of Prdx6 gene and aging lenses, we present molecular evidence that Prdx6-deficiency causes oxidative-driven Nlrp3 inflammasome activation, resulting in pyroptosis in aging/redox active cells wherein Prdx6 availability offsets the inflammatory process. We observed that Prdx6-/- and aging LECs harboring accumulated reactive oxygen species (ROS) showed augmented activation of Nlrp3 and bioactive inflammatory components, like Caspase-1, IL-1ß, ASC and Gasdermin-D. Similar to lipopolysaccharide treatment, oxidative exposure led to further ROS amplification with increased activation of the Nlrp3 inflammasome pathway. Mechanistically, we found that oxidative stress enhanced Kruppel-like factor 9 (Klf9) expression in aging/Prdx6-/- mLECs, leading to a Klf9-dependent increase in Nlrp3 transcription, while the elimination of ROS by the delivery of Prdx6 or by silencing Klf9 prevented the inflammatory response. Altogether, our data identify the biological significance of Prdx6 as an intrinsic checkpoint for regulating the cellular health of aging or redox active LECs and provide opportunities to develop antioxidant-based therapeutic(s) to prevent oxidative/aging-related diseases linked to aberrant Nlrp3 inflammasome activation.

Hydralazine Revives Cellular and Ocular Lens Health-Span by Ameliorating the Aging and Oxidative-Dependent Loss of the Nrf2-Activated Cellular Stress Response.

A major hallmark of aging-associated diseases is the inability to evoke cellular defense responses. Transcriptional protein Nrf2 (nuclear factor erythroid-derived 2-related factor) plays a pivotal role in the oxidative stress response, cellular homeostasis, and health span. Nrf2's activation has been identified as a therapeutic target to restore antioxidant defense in aging. Here, we demonstrated that FDA-approved drug, hydralazine (Hyd), was a reactivator of the Nrf2/ARE (antioxidant response element) pathway in various ages and types of mouse (m) or human (h) lens epithelial cells (LECs) and mice lenses in-vitro/in-vivo. This led to Hyd-driven abatement of carbonyls, reduced reactive oxygen species (ROS), and reduced 4-HNE/MDA-adducts with cytoprotection, and extended lens healthspan by delaying/preventing lens opacity against aging/oxidative stress. We elucidated that Hyd activated the protective signaling by inducing Nrf2 to traverse from the cytoplasm to the nucleus and potentiated the ARE response by direct interaction of Nrf2 and ARE sequences of the promoter. Loss-of-function study and cotreatment of Hyd and antioxidant, N-acetyl cysteine (NAC) or Peroxiredoxin (Prdx)6, specified that Nrf2/ARE-driven increase in the promoter activity was Hyd-dependent. Our study provides proof-of concept evidence and, thereby, paves the way to repurposing Hyd as a therapeutic agent to delay/prevent aging and oxidative-related disorders.

Occupational lead exposure is an independent modulator of hypertension and poor pulmonary function: A cross-sectional comparative study in lead-acid battery recycling workers.

Blood lead level (BLL) is the primary biomarker for lead-exposure monitoring in occupationally exposed workers. We evaluated occupational lead-exposure (OE) impact on cardiopulmonary functions in lead-acid battery recycling unit workers. Seventy-six OE cases and 30 control subjects were enrolled for questionnaire-based socio-demographic, dietary, tobacco usage, and medical history data. Anthropometric measurements, systolic and diastolic blood pressure (SBP and DBP), and pulmonary function tests were performed. Venous blood was collected for BLL, hematological analysis, and biochemical analysis. OE caused a significant increase in BLL, SBP, DBP, and small airways obstruction in lung function tests. It also impaired platelet indices, affected renal and liver biochemical measurements, and promoted oxidative stress and DNA damage. Multilinear regression analysis suggested that BLL affected SBP (ß = 0.314, p = .034) and increased small airways obstruction (FEV1/FVC, ß = -0.37, p = .05; FEV25-75%, ß = -0.351, p = .016). Higher BLL appears to be an independent modulator of hypertension and poor pulmonary function upon occupational lead exposure in lead-acid battery recyclers.

Role of Decorin in the Lens and Ocular Diseases.

Decorin is an archetypal member of the small leucine-rich proteoglycan gene family and is involved in various biological functions and many signaling networks, interacting with extra-cellular matrix (ECM) components, growth factors, and receptor tyrosine kinases. Decorin also modulates the growth factors, cell proliferation, migration, and angiogenesis. It has been reported to be involved in many ischemic and fibrotic eye diseases, such as congenital stromal dystrophy of the cornea, anterior subcapsular fibrosis of the lens, proliferative vitreoretinopathy, et al. Furthermore, recent evidence supports its role in secondary posterior capsule opacification (PCO) after cataract surgery. The expression of decorin mRNA in lens epithelial cells in vitro was found to decrease upon transforming growth factor (TGF)-ß-2 addition and increase upon fibroblast growth factor (FGF)-2 addition. Wound healing of the injured lens in mice transgenic for lens-specific human decorin was promoted by inhibiting myofibroblastic changes. Decorin may be associated with epithelial-mesenchymal transition and PCO development in the lens. Gene therapy and decorin administration have the potential to serve as excellent therapeutic approaches for modifying impaired wound healing, PCO, and other eye diseases related to fibrosis and angiogenesis. In this review, we present findings regarding the roles of decorin in the lens and ocular diseases.

Role of Decorin in Posterior Capsule Opacification and Eye Lens Development.

Decorin (DCN) is involved in a variety of physiological and pathological processes. Epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) has been proposed as a major cause for the development of posterior capsule opacification (PCO) after cataract surgery. We investigated the plausible target gene(s) that suppress PCO. The expression of Dcn was significantly upregulated in rat PCO tissues compared to that observed in the control using a microarray-based approach. LECs treated with fibroblast growth factor (FGF) 2 displayed an enhanced level of DCN expression, while LECs treated with transforming growth factor (TGF)ß-2 showed a decrease in DCN expression. The expression of tropomyosin 1 (Tpm1), a marker of lens EMT increased after the addition of TGFß-2 in human LEC; however, upregulation of Tpm1 mRNA or protein expression was reduced in human LECs overexpressing human DCN (hDCN). No phenotypic changes were observed in the lenses of 8- and 48-week-old transgenic mice for lens-specific hDCN (hDCN-Tg). Injury-induced EMT of the mouse lens, and the expression patterns of α smooth muscle actin, were attenuated in hDCN-Tg mice lenses. Overexpression of DCN inhibited the TGFß-2-induced upregulation of Tpm1 and EMT observed during wound healing of the lens, but it did not affect mouse lens morphology until 48 weeks of age. Our findings demonstrate that DCN plays a significant role in regulating EMT formation of LECs and PCO, and suggest that for therapeutic intervention, maintenance of physiological expression of DCN is essential to attenuate EMT progression and PCO formation.

Identification of Differential Gene Expression Pattern in Lens Epithelial Cells Derived from Cataractous and Noncataractous Lenses of Shumiya Cataract Rat.

The Shumiya cataract rat (SCR) is a model for hereditary cataract. Two-thirds of these rats develop lens opacity within 10-11 weeks. Onset of cataract is attributed to the synergetic effect of lanosterol synthase (Lss) and farnesyl-diphosphate farnesyltransferase 1 (Fdft1) mutant alleles that lead to cholesterol deficiency in the lenses, which in turn adversely affects lens biology including the growth and differentiation of lens epithelial cells (LECs). Nevertheless, the molecular events and changes in gene expression associated with the onset of lens opacity in SCR are poorly understood. In the present study, a microarray-based approach was employed to analyze comparative gene expression changes in LECs isolated from the precataractous and cataractous stages of lenses of 5-week-old SCRs. The changes in gene expression observed in microarray results in the LECs were further validated using real-time reverse transcribed quantitative PCR (RT-qPCR) in 5-, 8-, and 10-week-old SCRs. A mild posterior and cortical opacity was observed in 5-week-old rats. Expressions of approximately 100 genes, including the major intrinsic protein of the lens fiber (Mip and Aquaporin 0), deoxyribonuclease II beta (Dnase2B), heat shock protein B1 (HspB1), and crystallin γ (γCry) B, C, and F, were found to be significantly downregulated (0.07-0.5-fold) in rat LECs derived from cataract lenses compared to that in noncataractous lenses (control). Thus, our study was aimed at identifying the gene expression patterns during cataract formation in SCRs, which may be responsible for cataractogenesis in SCR. We proposed that cataracts in SCR are associated with reduced expression of these lens genes that have been reported to be related with lens fiber differentiation. Our findings may have wider implications in understanding the effect of cholesterol deficiency and the role of cholesterol-lowering therapeutics on cataractogenesis.

Clock Protein Bmal1 and Nrf2 Cooperatively Control Aging or Oxidative Response and Redox Homeostasis by Regulating Rhythmic Expression of Prdx6.

Many disorders of aging, including blinding-diseases, are associated with deficiency of brain and muscle arnt-like protein 1 (Bmal1) and, thereby, dysregulation of antioxidant-defense pathway. However, knowledge is limited regarding the role of Bmal1 regulation of antioxidant-pathway in the eye lens/lens epithelial cells (LECs) at the molecular level. We found that, in aging human (h)LECs, a progressive decline of nuclear factor erythroid 2-related factor 2 (Nrf2)/ARE (antioxidant response element)-mediated antioxidant genes was connected to Bmal1-deficiency, leading to accumulation of reactive oxygen species (ROS) and cell-death. Bmal1-depletion disrupted Nrf2 and expression of its target antioxidant genes, like Peroxiredoxin 6 (Prdx6). DNA binding and transcription assays showed that Bmal1 controlled expression by direct binding to E-Box in Prdx6 promoter to regulate its transcription. Mutation at E-Box or ARE reduced promoter activity, while disruption of both sites diminished the activity, suggesting that both sites were required for peak Prdx6-transcription. As in aging hLECs, ROS accumulation was increased in Bmal1-deficient cells and the cells were vulnerable to death. Intriguingly, Bmal1/Nrf2/Prdx6 and PhaseII antioxidants showed rhythmic expression in mouse lenses in vivo and were reciprocally linked to ROS levels. We propose that Bmal1 is pivotal for regulating oxidative responses. Findings also reveal a circadian control of antioxidant-pathway, which is important in combating lens/LECs damage induced by aging or oxidative stress.

Relative gene expression analysis of human pterygium tissues and UV radiation-evoked gene expression patterns in corneal and conjunctival cells.

A sight threatening, pterygium is a common ocular surface disorders identified by fibrovascular growth of the cornea and induced by variety of stress factors, like ultraviolet (UV) exposure. However, the genes involved in the etiopathogenesis of this disease is not well studied. Herein, we identified the gene expression pattern of pterygium and examined the expression of pterygium-related genes in UV-B-induced human primary cultured corneal epithelial cells (HCEpCs), telomerase immortalized human corneal epithelial (hTCEpi), primary conjunctival fibroblast (HConFs) and primary pterygium fibroblast cells (HPFCs). A careful analysis revealed that the expression of 10 genes was significantly modulated (by > 10-fold). Keratin 24 (KRT24) and matrix metalloproteinase 9 (MMP-9) were dramatically upregulated by 49.446- and 24.214-fold, respectively. Intriguingly, UV-B exposure (50 J/m2) induced the upregulation of the expressions of MMP-9 in corneal epithelial cells such as HCEpCs and hTCEpi. Furthermore, UV-B exposure (100 and/or 200 J/m2) induced the upregulation of the expressions of MMP-9 in fibroblast such as HConFs and HPFCs. The exposure of HCEpCs to 100 and 200 J/m2 UV-B induced significant expressions of KRT24 mRNA. Nevertheless, no expression of KRT24 mRNA was detected in HConFs and HPFCs. The findings provide evidence that the progression of pterygium may involve the modulation of extracellular matrix-related genes and vasculature development and the up-regulation of KRT24 and MMP-9 by UV stress. UV radiation may promote the modulation of these pterygium-related genes and induce the initiation and progression of human pterygium.

Sulforaphane-Induced Klf9/Prdx6 Axis Acts as a Molecular Switch to Control Redox Signaling and Determines Fate of Cells.

Sulforaphane (SFN), an activator of transcription factor Nrf2 (NFE2-related factor), modulates antioxidant defense by Nrf2-mediated regulation of antioxidant genes like Peroxiredoxin 6 (Prdx6) and affects cellular homeostasis. We previously observed that dose levels of SFN are crucial in determining life or death of lens epithelial cells (LECs). Herein, we demonstrated that higher doses of SFN (>6 µM) activated death signaling by overstimulation of Nrf2/ARE (antioxidant response element)-mediated Kruppel-like factor (Klf9) repression of Prdx6 expression, which increased reactive oxygen species (ROS) load and cell death. Mechanistically, Klf9 bound to its repressive Klf9 binding elements (RKBE; 5-CA/GCCC-3) in the Prdx6 promoter, and repressed Prdx6 transcription. Under the condition of higher dose of SFN, excessive Nrf2 abundance caused death signaling by enforcing Klf9 activation through ARE (5-RTGAYnnnGC-3) in Klf9 promoter that suppress antioxidant genes such as Prdx6 via a Klf9-dependent fashion. Klf9-depletion showed that Klf9 independently caused ROS reduction and subsequent cell survival, demonstrating that Klf9 upregulation caused cell death. Our work revealed the molecular mechanism of dose-dependent altered activity of SFN in LECs, and demonstrated that SFN activity was linked to levels of Nrf2/Klf9/Prdx6 axis. We proposed that in the development of therapeutic interventions for aging/oxidative disorders, combinations of Klf9-ShRNA and Nrf2 inducers may prove to be a promising strategy.

Ginkgolic Acid Rescues Lens Epithelial Cells from Injury Caused by Redox Regulated-Aberrant Sumoylation Signaling by Reviving Prdx6 and Sp1 Expression and Activities.

Sumoylation is a downstream effector of aging/oxidative stress; excess oxidative stress leads to dysregulation of a specificity protein1 (Sp1) and its target genes, such as Peroxiredoxin 6 (Prdx6), resulting in cellular damage. To cope with oxidative stress, cells rely on a signaling pathway involving redox-sensitive genes. Herein, we examined the therapeutic efficacy of the small molecule Ginkgolic acid (GA), a Sumoylation antagonist, to disrupt aberrant Sumoylation signaling in human and mouse lens epithelial cells (LECs) facing oxidative stress or aberrantly expressing Sumo1 (small ubiquitin-like modifier). We found that GA globally reduced aberrant Sumoylation of proteins. In contrast, Betulinic acid (BA), a Sumoylation agonist, augmented the process. GA increased Sp1 and Prdx6 expression by disrupting the Sumoylation signaling, while BA repressed the expression of both molecules. In vitro DNA binding, transactivation, Sumoylation and expression assays revealed that GA enhanced Sp1 binding to GC-boxes in the Prdx6 promoter and upregulated its transcription. Cell viability and intracellular redox status assays showed that LECs pretreated with GA gained resistance against oxidative stress-driven aberrant Sumoylation signaling. Overall, our study revealed an unprecedented role for GA in LECs and provided new mechanistic insights into the use of GA in rescuing LECs from aging/oxidative stress-evoked dysregulation of Sp1/Prdx6 protective molecules.

Roles of TGF ß and FGF Signals in the Lens: Tropomyosin Regulation for Posterior Capsule Opacity.

Transforming growth factor (TGF) ß and fibroblast growth factor (FGF) 2 are related to the development of posterior capsule opacification (PCO) after lens extraction surgery and other processes of epithelial⁻mesenchymal transition (EMT). Oxidative stress seems to activate TGF ß1 largely through reactive oxygen species (ROS) production, which in turn alters the transcription of several survival genes, including lens epithelium-cell derived growth factor (LEDGF). Higher ROS levels attenuate LEDGF function, leading to down-regulation of peroxiredoxin 6 (Prdx6). TGF ß is regulated by ROS in Prdx6 knock-out lens epithelial cells (LECs) and induces the up-regulation of tropomyosins (Tpms) 1/2, and EMT of LECs. Mouse and rat PCO are accompanied by elevated expression of Tpm2. Further, the expression of Tpm1/2 is induced by TGF ß2 in LECs. Importantly, we previously showed that TGF ß2 and FGF2 play regulatory roles in LECs in a contrasting manner. An injury-induced EMT of a mouse lens as a PCO model was attenuated in the absence of Tpm2. In this review, we present findings regarding the roles of TGF ß and FGF2 in the differential regulation of EMT in the lens. Tpms may be associated with TGF ß2- and FGF2-related EMT and PCO development.

Sumoylation-deficient Prdx6 repairs aberrant Sumoylation-mediated Sp1 dysregulation-dependent Prdx6 repression and cell injury in aging and oxidative stress.

Progressive deterioration of antioxidant response in aging is a major culprit in the initiation of age-related pathobiology induced by oxidative stress. We previously reported that oxidative stress leads to a marked reduction in transcription factor Sp1 and its mediated Prdx6 expression in lens epithelial cells (LECs) leading to cell death. Herein, we examined how Sp1 activity goes awry during oxidative stress/aging, and whether it is remediable. We found that Sp1 is hyper-Sumoylated at lysine (K) 16 residue in aging LECs. DNA binding and promoter assays revealed, in aging and oxidative stress, a significant reduction in Sp1 overall binding, and specifically to Prdx6 promoter. Expression/overexpression assay revealed that the observed reduction in Sp1-DNA binding activity was connected to its hyper-Sumoylation due to increased reactive oxygen species (ROS) and Sumo1 levels, and reduced levels of Senp1, Prdx6 and Sp1. Mutagenesis of Sp1 at K16R (arginine) residue restored steady-state, and improved Sp1-DNA binding activity and transactivation potential. Extrinsic expression of Sp1K16R increased cell survival and reduced ROS levels by upregulating Prdx6 expression in LECs under aging/oxidative stress, demonstrating that Sp1K16R escapes the aberrant Sumoylation processes. Intriguingly, the deleterious processes are reversible by the delivery of Sumoylation-deficient Prdx6, an antioxidant, which would be a candidate molecule to restrict aging pathobiology.

Membrane microdomains regulate NLRP10- and NLRP12-dependent signalling in A549 cells challenged with cigarette smoke extract.

Chronic obstructive pulmonary disease (COPD) is predicted to become the third leading cause of death and disability worldwide by 2030; with cigarette smoking (active or passive) being one of the chief cause of its occurrence. Cigarette smoke exposure has been found to result in excessive inflammation and tissue injury, which might lead to COPD, although the exact pathophysiology of the disease remains elusive. While previous studies have demonstrated the role of membrane-bound Toll-like receptors (TLRs) in cigarette smoke (CS)-induced inflammation, scant information is available about the role of cytosolic NOD-like receptors (NLRs) in regulating CS-mediated inflammatory responses. Thus, we investigated the role of NLRP10 and NLRP12 in regulating inflammatory responses in human alveolar type II epithelial cells (A549) and human monocytic cells (THP-1) in response to a challenge with cigarette smoke extract (CSE). We observed CSE-mediated increase in caspase-1 activity; production of IL-1ß and IL-18; and expression of NLRP10 and NLRP12 in A549 and THP-1 cells. Interestingly, immunofluorescence imaging results demonstrated an increase in the membrane recruitment of NLRP10 and NLRP12 proteins in CSE-challenged A549 cells. We also observed an increase in the expression of lipid raft proteins (caveolin-1, caveolin-2, and flotillin-1) and an induction of lipid raft assembly following CSE-exposure in A549 cells. Lipid rafts are cholesterol-rich membrane microdomains well known to act as harbours for signalling molecules. Here we demonstrate  the recruitment of NLRP10 and NLRP12 in lipid raft entities as well as the interaction of NLRP12 with the lipid raft protein caveolin-1 in CSE-challenged A549 cells. Furthermore, enrichment of lipid raft entities with poly-unsaturated fatty acids (PUFA) rescued A549 cells from CSE-mediated membrane recruitment of NLRP10 and NLRP12, and also from inflammatory responses and inflammasome activation. Enrichment of membrane microdomains with PUFA was able to reverse filipin (chemical agent used for disrupting lipid rafts)-mediated enhanced inflammation in CSE-challenged A549 cells. Overall, our findings unveil a novel mechanism by identifying an important role of membrane microdomains (lipid rafts) in regulating CSE-induced inflammation and NLRP10/NLRP12-dependent signalling in A549 cells.

Tropomyosin 2 heterozygous knockout in mice using CRISPR-Cas9 system displays the inhibition of injury-induced epithelial-mesenchymal transition, and lens opacity.

The process of epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) after cataract surgery contributes to tissue fibrosis, wound healing and lens regeneration via a mechanism not yet fully understood. Here, we show that tropomyosin 2 (Tpm2) plays a critical role in wound healing and lens aging. Posterior capsular opacification (PCO) after lens extraction surgery was accompanied by elevated expression of Tpm2. Tpm2 heterozygous knockout mice, generated via the clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) system showed promoted progression of cataract with age. Further, injury-induced EMT of the mouse lens epithelium, as evaluated histologically and by the expression patterns of Tpm1 and Tpm2, was attenuated in the absence of Tpm2. In conclusion, Tpm2 may be important in maintaining lens physiology and morphology. However, Tpm2 is involved in the progression of EMT during the wound healing process of mouse LECs, suggesting that inhibition of Tpm2 may suppress PCO.

Cigarette smoke-induced inflammation: NLRP10-mediated mechanisms.

Chronic obstructive pulmonary disease (COPD) is a progressive, life-threatening disease that causes irreversible lung damage. Cigarette smoking is the chief etiologic factor for the commencement of this condition. Despite constant efforts to develop therapeutic interventions and to ascertain the molecular mechanism leading to the pathophysiology of this disease, much remains unknown. However, pattern recognition receptors (PRRs), i.e., Toll-like-receptors (TLRs) and NOD-like receptors (NLRs) are believed to play important roles in COPD and could serve as effective therapeutic targets. Although the role of TLRs in COPD has been well studied, the importance of NLRs has not yet been explored in detail. The NLR family member NLRP10 (aka NOD8, PAN5, PYNOD) is the only member of this family of proteins that lacks the leucine rich repeat (LRR) domain responsible for detection of pathogen and danger-associated molecular patterns (PAMPs/DAMPs). Therefore, instead of functioning as a PRR, NLRP10 may have a broader regulatory role. To elucidate the role of NLRP10 in secondhand smoke (SHS)-induced inflammation, we exposed C57Bl/6 (WT) and Nlrp10-deficient mice (Nlrp10-/-) on the C57Bl/6 background to filtered air- or SHS- for 6 weeks (acute exposure) and assessed the resulting molecular events. Leukocyte recruitment in SHS-exposed Nlrp10-/- mice was found to be significantly lower compared to SHS-exposed WT mice. In addition, we observed an important role for NLRP10 in SHS-mediated caspase-1 activation, cytokine/chemokine production (IL-1ß, IL-18, MCP-1 and IL-17A), and induction of NF-κB and MAPKs in the lungs of C57Bl/6 mice. The reduced influx of CD4+IL-17A+ and CD8+IL-17A+ cells into the lungs of SHS-exposed Nlrp10-/- mice and impaired differentiation of Nlrp10-/- Th0 cells into Th17 cells (ex vivo) provide insight into the mechanistic details underlying NLRP10-dependent IL-17 production. We further substantiated our in vivo findings by challenging human alveolar type II epithelial cells (A549) transfected with scrambled- or Nlrp10-siRNA with cigarette smoke extract (CSE). We observed an important role of NLRP10 in cytokine and chemokine production as well as expression of NF-κB and MAPKs in CSE-exposed A549 cells. Furthermore, replenishment of A549 cell culture with recombinant IL-17A (rIL-17A) during NLRP10 knockdown rescued CSE-induced inflammatory responses. To identify upstream mediators of NLRP10 regulation we investigated epigenetic markers within the Nlrp10 promoter following cigarette smoke exposure and observed significant changes in active as well as repressive gene markers on histone 3 and histone 4 using both in vivo and in vitro study models. Further, alterations in the respective histone acetyl- and methyltransferases (PCAF, SET1, ESET, SUV20H1) correlated well with the observed histone modifications. Overall, our findings suggest a novel role of epigenetically regulated NLRP10 in Th17/IL-17 signaling during CS exposure.

Immune-related gene polymorphisms in pulmonary diseases.

Between the DNA sequences of two randomly-selected human genomes, which consist of over 3 billion base pairs and twenty five thousand genes, there exists only 0.1% variation and 99.9% sequence identity. During the last couple of decades, extensive genome-wide studies have investigated the association between single-nucleotide polymorphisms (SNPs), the most common DNA variations, and susceptibility to various diseases. Because the immune system's primary function is to defend against myriad infectious agents and diseases, the large number of people who escape serious infectious diseases underscores the tremendous success of this system at this task. In fact, out of the third of the global human population infected with Mycobacterium tuberculosis during their lifetime, only a few people develop active disease, and a heavy chain smoker may inexplicably escape all symptoms of chronic obstructive pulmonary disease (COPD), lung cancer, and other smoke-associated lung diseases. This may be attributable to the genetic makeup of the individual(s), including their SNPs, which provide some resistance to the disease. Pattern recognition receptors (PRRs), transcription factors, cytokines and chemokines all play critical roles in orchestrating immune responses and their expression/activation is directly linked to human disease tolerance. Moreover, genetic variations present in the immune-response genes of various ethnicities may explain the huge differences in individual outcomes to various diseases and following exposure to infectious agents. The current review focuses on recent advances in our understanding of pulmonary diseases and the relationship of genetic variations in immune response genes to these conditions.

Kodo millet whole grain and bran supplementation prevents high-fat diet induced derangements in a lipid profile, inflammatory status and gut bacteria in mice.

The protective role of kodo millet whole grain and bran supplementation in diet induced obesity has not been investigated. Here we have studied the role of kodo millet supplementation in age matched Swiss albino mice that were randomly divided into groups and fed their respective diets for 16 weeks. A high fat diet increased weight gain, reduced glucose tolerance, increased serum lipids, altered hepatic and adipocyte gene expression and caused dysbiosis in the intestinal beneficial bacteria. Kodo millet supplementation did not affect weight gain but it improved glucose tolerance and prevented an increase in the serum cholesterol and lipid parameters (P ≤ 0.05), modulated adipogenesis related gene expression, decreased serum IL-6 and LPS levels (P ≤ 0.05), promoted selected beneficial gut bacterial abundances (Lactobacillus sp., Bifidobacteria, Akkermansia and Roseburia spp.) and improved the total short chain fatty acid production (P ≤ 0.05) and acetate levels (P ≤ 0.05) in cecal contents. This study provides evidence that kodo millet supplementation alleviates high-fat diet induced changes and hence can be incorporated as a functional ingredient for the management of obesity.

Sumoylation-deficient Prdx6 gains protective function by amplifying enzymatic activity and stability and escapes oxidative stress-induced aberrant Sumoylation.

Aberrant Sumoylation of protein(s) in response to oxidative stress or during aging is known to be involved in etiopathogenesis of many diseases. Upon oxidative stress, Peroxiredoxin (Prdx) 6 is aberrantly Sumoylated by Sumo1, resulting in loss of functions and cell death. We identified lysines (K) 122 and 142 as the major Sumo1 conjugation sites in Prdx6. Intriguingly, the mutant Prdx6 K122/142 R (arginine) gained protective efficacy, increasing in abundance and promoting glutathione (GSH) peroxidase and acidic calcium-independent phospholipase A2 (aiPLA2) activities. Using lens epithelial cells derived from targeted inactivation of Prdx6-/- gene and relative enzymatic and stability assays, we discovered dramatic increases in GSH-peroxidase (30%) and aiPLA2 (37%) activities and stability in the K122/142 R mutant, suggesting Sumo1 destabilized Prdx6 integrity. Prdx6-/-LECs with EGFP-Sumo1 transduced or co-expressed with mutant TAT-HA-Prdx6K122/142 R or pGFP-Prdx6K122/142 R were highly resistant to oxidative stress, demonstrating mutant protein escaped and interrupted the Prdx6 aberrant Sumoylation-mediated cell death pathway. Mutational analysis of functional sites showed that both peroxidase and PLA2 active sites were necessary for mutant Prdx6 function, and that Prdx6 phosphorylation (at T177 residue) was essential for optimum PLA2 activity. Our work reveals the involvement of oxidative stress-induced aberrant Sumoylation in dysregulation of Prdx6 function. Mutant Prdx6 at its Sumo1 sites escapes and abates this adverse process by maintaining its integrity and gaining function. We propose that the K122/142R mutant of Prdx6 in the form of a TAT-fusion protein may be an easily applicable intervention for pathobiology of cells related to aberrant Sumoylation signaling in aging or oxidative stress.

FGF2 antagonizes aberrant TGFß regulation of tropomyosin: role for posterior capsule opacity.

Transforming growth factor (TGF) ß2 and fibroblast growth factor (FGF) 2 are involved in regulation of posterior capsule opacification (PCO) and other processes of epithelial-mesenchymal transition (EMT) such as cancer progression, wound healing and tissue fibrosis as well as normal embryonic development. We previously used an in vivo rodent PCO model to show the expression of tropomyosin (Tpm) 1/2 was aberrantly up-regulated in remodelling the actin cytoskeleton during EMT. In this in vitro study, we show the Tpms family of cytoskeleton proteins are involved in regulating and stabilizing actin microfilaments (F-actin) and are induced by TGFß2 during EMT in lens epithelial cells (LECs). Importantly, we found TGFß2 and FGF2 played contrasting roles. Stress fibre formation and up-regulation of α-smooth muscle actin (αSMA) induced by TGFß2 could be reversed by Tpm1/2 knock-down by siRNA. Expression of Tpm1/2 and stress fibre formation induced by TGFß2 could be reversed by FGF2. Furthermore, FGF2 delivery to TGFß-treated LECs perturbed EMT by reactivating the mitogen-activated protein kinase (MAPK)/ extracellular signal-regulated kinase (ERK) pathway and subsequently enhanced EMT. Conversely, MEK inhibitor (PD98059) abated the FGF2-mediated Tpm1/2 and αSMA suppression. However, we found that normal LECs which underwent EMT showed enhanced migration in response to combined TGFß and FGF2 stimulation. These findings may help clarify the mechanism reprogramming the actin cytoskeleton during morphogenetic EMT cell proliferation and fibre regeneration in PCO. We propose that understanding the physiological link between levels of FGF2, Tpm1/2 expression and TGFßs-driven EMT orchestration may provide clue(s) to develop therapeutic strategies to treat PCO based on Tpm1/2.

Aberrant sumoylation signaling evoked by reactive oxygen species impairs protective function of Prdx6 by destabilization and repression of its transcription.

Loss of the cytoprotective protein peroxiredoxin 6 (Prdx6) in cells that are aging or under oxidative stress is known to be linked to the pathobiology of many age-related diseases. However, the mechanism by which Prdx6 activity goes awry is largely unknown. Using Prdx6-deficient (Prdx6(-/-) ) cells as a model for aging or redox active cells, human/mouse lens epithelial cells (LECs) facing oxidative stress and aging lenses, we found a significant increase in the levels of small ubiquitin-like modifier (Sumo)1 conjugates. These cells displayed increased levels of Sumo1 and reduced the expression of Prdx6. Specifically, we observed that Prdx6 is a target for aberrant sumoylation signaling, and that Sumo1 modification reduces its cellular abundance. LECs overexpressing Sumo1 showed reduced expression and activity of Prdx6 and its transactivator specificity protein 1 (Sp1), mRNA and protein with increased levels of reactive oxygen species; those cells were vulnerable to oxidative stress-induced cell death. A significant reduction in Prdx6, Sp1 protein and mRNA expression was observed in redox active Prdx6(-/-) cells and in aging lenses/LECs. The reduction was correlated with increased expression of Sumo1 and enrichment of the inactive form (dimeric) of Sumo-specific protease (Senp)1. Experiments with Sumo1-fused Prdx6 and Prdx6 promoter-linked to chloramphenicol acetyltransferase reporter gene constructs indicated that Sumo1 dysregulated Prdx6 activity by reducing its abundance and attenuating its transcription; in contrast, the delivery of Senp1 or Prdx6 reversed the process. The data show that reactive oxygen species-evoked aberrant sumoylation signaling affects Prdx6 activity by reducing Prdx6 abundance, as well as transcription. The findings of the present study may provide a foundation for a strategy to repair deleterious oxidative signaling generated by a reduced activity of Prdx6.