Immunomodulatory action of synbiotic comprising of newly isolated lactic acid producing bacterial strains against allergic asthma in mice.

Given the reported role of gut-microbiota in asthma pathogenesis, the present work was carried to evaluate immunomodulatory action of newly isolated lactic acid producing bacterial strains Bifidobacterium breve Bif11 and Lactiplantibacillus plantarum LAB31 against asthma using ovalbumin (OVA) based mouse model. Our results show that both strains modulate Th2 immune response potentially through production of short chain fatty acids (SCFAs), resulting in suppression of OVA-induced airway inflammation. Furthermore, synbiotic comprising of both strains and prebiotic, Isomaltooligosaccharide exhibited superior potential in amelioration of OVA-induced airway inflammation through improved modulation of Th2 immune response. Further, synbiotic protects against OVA-induced mucus hyper-production and airway-hyperresponsiveness. Such protection was associated with normalization of gut microbiome and enhanced production of SCFAs in cecum which correlates closely with population of T-regulatory cells in spleen. Overall, our novel synbiotic possesses the ability to fine-tune the immune response for providing protection against allergic asthma.

Particulate matter in COPD pathogenesis: an overview.

Chronic obstructive pulmonary disease (COPD) is a progressive lung disorder with substantial patient burden and leading cause of death globally. Cigarette smoke remains to be the most recognised causative factor behind COPD pathogenesis. Given the alarming increase in prevalence of COPD amongst non-smokers in recent past, a potential role of air pollution particularly particulate matter (PM) in COPD development has gained much attention of the scientists. Indeed, several epidemiological studies indicate strong correlation between airborne PM and COPD incidence/exacerbations. PM-induced oxidative stress seems to be the major player in orchestrating COPD inflammatory cycle but the exact molecular mechanism(s) behind such a process are still poorly understood. This may be due to the complexity of multiple molecular pathways involved. Oxidative stress-linked mitochondrial dysfunction and autophagy have also gained importance and have been the focus of recent studies regarding COPD pathogenesis. Accordingly, the present review is aimed at understanding the key molecular players behind PM-mediated COPD pathogenesis through analysis of various experimental studies supported by epidemiological data to identify relevant preventive/therapeutic targets in the area.

Gallic acid ameliorates COPD-associated exacerbation in mice.

COPD is an inflammatory lung disease, which is often exacerbated with microbial infections resulting in worsening of respiratory symptoms. Gallic acid (GA), a naturally occurring phenolic compound is known to possess anti-oxidant/anti-inflammatory activity. We have recently reported that GA protects against the elastase (ET) induced lung inflammation and emphysema and the present work was designed to investigate the beneficial effects of Gallic acid against ET + Lipopolysachharide (LPS) induced COPD exacerbation like condition in mice model. Our data showed that i.t. administration of LPS at 21 days after ET instillation resulted in significant infiltration of inflammatory cells particularly neutrophils (p < 0.0001) into the lungs along with elevated levels of pro-inflammatory cytokines like TNF-α, IL-1ß and IL-6 (p < 0.0001). Interestingly, daily administration of GA (200 mg/Kg b. wt.) starting 7 days before ET instillation, significantly blunted the ET + LPS induced inflammation as indicated by reduced number of inflammatory cells particularly neutrophils (p < 0.0001) in BALF along with suppression of myeloperoxidase activity (p = 0.0009) and production of pro-inflammatory cytokines (p < 0.0001). Further, GA also restored the redox imbalance in the lungs towards normal. Additionally, phosphorylation of p65-NF-κB was found to be reduced (p = 0.015), which was associated with downregulation in the gene expression of IL-1ß (p = 0.022) and TNF-α (p = 0.04). Conversely, GA treatment resulted in increased protein levels of Nrf2 (p = 0.021) with concomitant increase in transcription of its downstream target genes HO-1 (p = 0.033) and Prdx-1 (p = 0.006). Overall, our data show that GA effectively modulates COPD exacerbation manifestations in mice potentially by restoring redox imbalance in lungs.

Cysteinyl leukotriene D4 (LTD4) promotes airway epithelial cell inflammation and remodelling.

OBJECTIVE: Cysteinyl leukotrienes (CysLTs), a group of inflammatory lipid mediators, are found elevated in obese-asthmatic patients. Leukotriene D4 (LTD4), a representative CysLT, is implicated in promoting lung inflammation and remodelling in allergic asthma, but its role in non-allergic asthma, especially in obese-asthmatic patients, is not known. Here, using primary human small airway epithelial cells (SAECs) we have investigated the mechanism of LTD4-induced inflammation and remodelling and assessed high proneness of obese mice to develop asthma upon challenge with allergen ovalbumin (OVA). METHODS: Primary human small airway epithelial cells (SAECs) were stimulated with different concentrations of LTD4 for different time intervals and various inflammatory markers were measured through cytokine array, membrane-based ELISA and Western blotting. An air-liquid interface (ALI) model of SAECs was used to study the effects of LTD4-induced remodelling in SAECs using Western blotting, H&E staining and PAS staining. Further, OVA-based murine model was used to examine the propensity of high-fat diet (HFD)-fed obese mice to develop asthma symptoms by studying the infiltration of inflammatory cells (assessed by bronchioalveolar lavage (BAL) cytology) and airway remodelling (assessed by histopathology) upon allergen exposure. RESULTS: The human primary small airway epithelial cells (SAECs) treated with LTD4 showed significant alterations in the levels of inflammatory markers such as GM-CSF, TNF-α, IL-1ß, EGF and eotaxin in dose- and time-dependent manner. Further, LTD4 enhanced the activation of inflammasomes as evidenced by increased levels of NALP3, cleaved caspase-1 and IL-1ß. LTD4 also enhanced inflammation by increasing the expression of COX-2 in SAECs. The airway remodelling markers Vimentin and Muc5AC were found elevated in ALI culture of SAECs when stimulated with LTD4, as it also increased TGF-ß levels and activation of Smad2/3 phosphorylation in SAECs. Last, sensitization and challenge of HFD-fed obese mice with OVA showed increased infiltration of inflammatory cells in BAL and enhanced levels of remodeling phenotypes like loss of cilia, mucus cell metaplasia and collagen deposition in mice lung tissues. CONCLUSION: The results suggest that LTD4 could induce inflammatory response in human airway epithelial cell by activating NALP3 inflammasome. LTD4 could further promote airway epithelial cells' remodelling through TGF-ß/smad2/3-mediated pathway. Our in vivo results suggested that obesity predisposed the OVA challenged mice to develop lung inflammation and remodelling akin to asthma-like phenotypes during obesity.

Gallic acid protects against the COPD-linked lung inflammation and emphysema in mice.

OBJECTIVE AND DESIGN: Gallic acid (GA) a naturally occurring phenolic compound, known to possess antioxidant/anti-inflammatory activities. The aim of the present work was to investigate the beneficial effects of GA against COPD-linked lung inflammation/emphysema by utilizing elastase (ET) and cigarette smoke (CS)-induced mice model. MATERIALS: Male BALB/c mice were treated with ET (1U/mouse) or exposed to CS (9 cigarettes/day for 4 days). GA administration was started 7 days (daily) prior to ET/CS exposure. Broncho-alveolar lavage was analyzed for inflammatory cells and pro-inflammatory cytokines. Lung homogenate was assessed for MPO activity/GSH/MDA/protein carbonyls. Further, Lung tissue was subjected to semi-quantitative RT-PCR, immunoblotting, and histological analysis. RESULTS: GA suppressed the ET-induced neutrophil infiltration, elevated MPO activity and production of pro-inflammatory cytokines (IL-6/TNF-α/IL-1ß) at 24 h. Reduced inflammation was accompanied with normalization of redox balance as reflected by ROS/GSH/MDA/protein carbonyl levels. Further, GA suppressed phosphorylation of p65NF-κB and IκBα along with down-regulation of IL-1ß/TNF-α/KC/MIP-2/GCSF genes. Furthermore, GA offered protection against ET-induced airspace enlargement and ameliorated MMP-2/MMP-9. Finally, GA suppressed the CS-induced influx of neutrophils and macrophages and blunted gene expression of TNF-α/MIP-2/KC. CONCLUSION: Overall, our data show that GA effectively modulates pulmonary inflammation and emphysema associated with COPD pathogenesis in mice.

Critical role of mitochondrial oxidative stress in acid aspiration induced ALI in mice.

Acute lung injury (ALI) is a pulmonary inflammatory disorder which causes significant mortality in critically ill patients. Intracellular oxidative stress has been considered to be the major component in the pathogenesis of ALI but exact source of intracellular ROS is not clearly known. The present study has been designed to elucidate the role of NADPH oxidase system and/or mitochondrial oxidative stress and its downstream pathway NLRP3 inflammasomes in mouse model of acid aspiration mediated ALI. Our data showed that acid aspiration induced lung inflammation was associated with enhanced oxidative stress as evident by data on MDA levels, nitrite levels and redox imbalance. Further acid aspiration resulted in elevation of expression of NADPH oxidase subunits (gp91 phox/p22 phox/p67 phox) as well as mitochondrial oxidative stress as reflected by aconitase activity, mitochondrial ROS levels. Interestingly, NADPH oxidase inhibitor, apocynin did not alter lung inflammation upon HCl instillation. Conversely, mitochondrial antioxidant mito-tempo resulted in significant amelioration of lung inflammation as indicated by suppression of pulmonary neutrophils and inflammatory cytokines namely IL-1ß, TNF-α, IL-6 in BALF. Analysis of mitochondrial enzymes aconitase/mitochondrial ROS/Mn-SOD confirmed that reduction in lung inflammation by mito-tempo was associated with normalization of oxidative stress in mitochondria. Further, mito-tempo administration blunted phosphorylation of p65- NF-κB at Ser 536. Finally, mito-tempo downregulated HCl-induced NF-κB-dependent pro-inflammatory cytokines (IL-1ß, TNF-α, IL-6) drastically at mRNA levels. Overall, our data support that mitochondrial oxidative stress is crucial in modulating the HCl induced lung inflammation and identifies mitochondrial-targeted antioxidant as a potential therapeutic agent.

PARP inhibition by olaparib alleviates chronic asthma-associated remodeling features via modulating inflammasome signaling in mice.

Despite the reported role of poly(ADP-ribose) polymerase (PARP) in asthma inflammation, its contribution during remodeling is not clearly known. The main aim of the current investigation was to examine the potential of olaparib, a pharmacological inhibitor of PARP against airway remodeling using an ovalbumin (OVA)-based murine model of chronic asthma. The results demonstrated that post-challenge olaparib treatment (5 mg/kg i.p., 30 min after OVA exposure) for six weeks (3 days/week) attenuates inflammation, mucus production, and collagen deposition in lungs. Additionally, olaparib blunted the protein expression of STAT-6 and GATA-3 considerably along with a modest reduction in p65-NF-κB phosphorylation. Furthermore, olaparib normalized the OVA-induced redox imbalance as reflected by data on reactive oxygen species, malondialdehyde, protein carbonyls, and reduced glutathione/oxidized glutathione ratio. Interestingly, the protection offered by olaparib was further linked with the altered level of NLRP3 inflammasome-mediated IL-1ß release and consequent expression of its downstream targets matrix metalloproteinase-9 and transforming growth factor beta. Suppressed collagen deposition in the lungs correlates well with the reduced expression of vimentin upon olaparib treatment. Finally, olaparib restored the expression of histone deacetylase 2, a steroid-responsive element in asthma. Overall, results suggest that olaparib prevents OVA-induced airway inflammation as well as remodeling via modulating inflammasome signaling in mice. © 2019 IUBMB Life, 1-11, 2019.

PARP-1 inhibition provides protection against elastase-induced emphysema by mitigating the expression of matrix metalloproteinases.

In our previous study, we have shown that PARP-1 inhibition (genetic or pharmacological) ameliorates elastase-induced inflammation and emphysema. Since matrix metalloproteinases (MMPs) particularly MMP-2 and MMP-9 are known to play a critical role in emphysema development, the present work was designed to evaluate the effects of PARP-1 inhibition on their expression utilizing elastase-induced mouse model of emphysema. Our data show that olaparib administration at a dose of 5 mg/kg b.wt. (daily) significantly prevented the elastase-induced inflammation as indicated by decreased inflammatory cells particularly macrophages in BALF at 1 week post-injury. In addition, the drug restored the altered redox balance in the lungs of elastase-treated mice toward normal. Further, PCR data show that olaparib administration ameliorates the elastase-induced expression of MMP-2 and MMP-9 without having much effect on the expressions of their inhibitors TIMP-1 and TIMP-2. Next, our data on immunoblot, gelatin zymography, and immunohistochemical analysis indeed confirm that olaparib reduced the elastase-induced expression of MMP-2 and MMP-9. Reduction in the expression of metalloproteinases correlate well with the PARP activity as olaparib treatment suppressed the elastase-induced expression of PAR modified proteins markedly. Overall, our data strongly suggest that PARP-1 inhibition blunts elastase-induced MMP-2 and MMP-9 expression, which may be partly responsible for prevention of emphysema.

Modulatory effect of 4-phenyl butyric acid on hyperoxaluria-induced renal injury and inflammation.

Hyperoxaluria-associated deposition of calcium oxalate crystals results from oxalate-induced renal injury and inflammation. The present study was designed to evaluate the effect of 4-Phenyl butyric acid (4-PBA), a chemical chaperone, in ethylene glycol-induced hyperoxaluria and compare its effect with antioxidant, N-acetyl cysteine (NAC). Male Sprague-Dawley rats were given ethylene glycol in drinking water for 28 days to induce hyperoxaluria. 4-PBA and NAC were given by oral gavage. Effect of 4-PBA was analyzed in both prophylactic and curative regimens. After every 7 days, 24-h urine samples were analyzed for kidney injury and inflammation markers. Increased amounts of kidney injury markers like Kidney injury molecule-1, Lactate dehydrogenase, and N-acetyl-ß-glucoseaminidase were found in the urine of hyperoxaluric rats which were significantly reduced by 4-PBA treatment in both prophylactic and curative regimens. Inflammatory markers IL-1ß, IL-6, and MCP-1 were also raised in the urine of hyperoxaluric rats which were significantly decreased by 4-PBA treatment. Hyperoxaluria was accompanied with renal oxidative stress as reflected by decreased glutathione redox status and increased reactive oxygen species which was significantly reduced by 4-PBA treatment. Histological study with H&E and Pizzolato staining showed numerous calcium oxalate crystal deposits in the renal tissues of hyperoxaluric rats. However, no significant crystal deposits were seen in the 4-PBA-treated hyperoxaluric rats. N-acetyl cysteine treatment effectively decreased renal oxidative stress but did not alter the production of inflammatory markers. Collectively, the present study suggested the potential protective effect of 4-PBA in hyperoxaluria-induced renal injury and inflammation.

Two hit induced acute lung injury impairs cognitive function in mice: A potential model to study cross talk between lung and brain.

Acute lung injury (ALI), a pulmonary inflammatory disorder, is associated with high morbidity and mortality rates. Interestingly, ALI survivors have been reported for some neurocognitive deterioration at/after discharge. However, the molecular factors behind such extra pulmonary manifestation are not clearly known. The present work was designed to investigate lung-brain cross talk in experimental mice for deciphering primary molecular factors that may be involved in ALI-mediated cognitive impairment. ALI was induced in Balb/c mice by intra-tracheal administration of either 0.1 N HCl (2 ml/kg) or LPS (1 mg/kg) as single hits or both agents were administered successively to mimic the 'two hit' model. Interestingly two hit-mediated ALI resulted in exaggerated inflammatory response as reflected by increased pulmonary neutrophils and inflammatory factors (TNF-α/IL-1ß/IL-6). Additionally, two hits resulted in delayed resolution of lung inflammation and was coupled with persistent decline in memory, as assessed by Morris water maze test. Further, two hits elevate serum levels of TNF-α/IL-1ß which was associated with compromised blood brain barrier (BBB), as evident by decreased expression of occludin/claudin-5 and consequent Evans-blue extravasation in hippocampus 1 week post injury. Finally, dexamethasone protects against the two hit mediated cognitive impairment by lowering the pro-inflammatory factors (TNF-α/IL-1ß) both in lungs and blood. Overall, we report for the first time that 'two hit' mediated ALI cause persistent cognitive impairment in mice partly via up-regulating systemic expression of TNF-α/IL-1ß that may disrupt BBB and hence the model may be a useful tool to examine the lung-brain cross-talk at the molecular level for exploring newer therapeutics.

Treatment with intranasal iloprost reduces disease manifestations in a murine model of previously established COPD.

Pulmonary endothelial prostacyclin appears to be involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). The effect of treatment with a prostacyclin analog in animal models of previously established COPD is unknown. We evaluated the short- and long-term effect of iloprost on inflammation and airway hyperresponsiveness (AHR) in a murine model of COPD. Nineteen mice were exposed to LPS/elastase, followed by either three doses of intranasal iloprost or saline. In the long-term treatment experiment, 18 mice were exposed to LPS/elastase and then received 6 wk of iloprost or were left untreated as controls. In the short-term experiment, iloprost did not change AHR but significantly reduced serum IL-5 and IFN-γ. Long-term treatment with iloprost for both 2 and 6 wk significantly improved AHR. After 6 wk of iloprost, there was a reduction in bronchoalveolar lavage (BALF) neutrophils, serum IL-1ß (30.0 ± 9.2 vs. 64.8 ± 7.4 pg/ml, P = 0.045), IL-2 (36.5 ± 10.6 vs. 83.8 ± 0.4 pg/ml, P = 0.01), IL-10 (75.7 ± 9.3 vs. 96.5 ± 3.5 pg/ml, P = 0.02), and nitrite (15.1 ± 5.4 vs. 30.5 ± 10.7 µmol, P = 0.01). Smooth muscle actin (SMA) in the lung homogenate was also significantly reduced after iloprost treatment (P = 0.02), and SMA thickness was reduced in the small and medium blood vessels after iloprost (P < 0.001). In summary, short- and long-term treatment with intranasal iloprost significantly reduced systemic inflammation in an LPS/elastase COPD model. Long-term iloprost treatment also reduced AHR, serum nitrite, SMA, and BALF neutrophilia. These data encourage future investigations of prostanoid therapy as a novel treatment for COPD patients.

DNA-dependent protein kinase inhibition blocks asthma in mice and modulates human endothelial and CD4⁺ T-cell function without causing severe combined immunodeficiency.

BACKGROUND: We reported that DNA-dependent protein kinase (DNA-PK) is critical for the expression of nuclear factor κB-dependent genes in TNF-α-treated glioblastoma cells, suggesting an involvement in inflammatory diseases. OBJECTIVE: We sought to investigate the role of DNA-PK in asthma. METHODS: Cell culture and ovalbumin (OVA)- or house dust mite-based murine asthma models were used in this study. RESULTS: DNA-PK was essential for monocyte adhesion to TNF-α-treated endothelial cells. Administration of the DNA-PK inhibitor NU7441 reduced airway eosinophilia, mucus hypersecretion, airway hyperresponsiveness, and OVA-specific IgE production in mice prechallenged with OVA. Such effects correlated with a marked reduction in lung vascular cell adhesion molecule 1 expression and production of several cytokines, including IL-4, IL-5, IL-13, eotaxin, IL-2, and IL-12 and the chemokines monocyte chemoattractant protein 1 and keratinocyte-derived chemokine, with a negligible effect on IL-10/IFN-γ production. DNA-PK inhibition by gene heterozygosity of the 450-kDa catalytic subunit of the kinase (DNA-PKcs(+/-)) also prevented manifestation of asthma-like traits. These results were confirmed in a chronic model of asthma by using house dust mite, a human allergen. Remarkably, such protection occurred without causing severe combined immunodeficiency. Adoptive transfer of TH2-skewed OT-II wild-type CD4(+) T cells reversed IgE and TH2 cytokine production but not airway hyperresponsiveness in OVA-challenged DNA-PKcs(+/-) mice. DNA-PK inhibition reduced IL-4, IL-5, IL-13, eotaxin, IL-8, and monocyte chemoattractant protein 1 production without affecting IL-2, IL-12, IFN-γ, and interferon-inducible protein 10 production in CD3/CD28-stimulated human CD4(+) T cells, potentially by blocking expression of Gata3. These effects occurred without significant reductions in T-cell proliferation. In mouse CD4(+) T cells in vitro DNA-PK inhibition severely blocked CD3/CD28-induced Gata3 and T-bet expression in CD4(+) T cells and prevented differentiation of TH1 and TH2 cells under respective TH1- and TH2-skewing conditions. CONCLUSION: Our results suggest DNA-PK as a novel determinant of asthma and a potential target for the treatment of the disease.

PARP inhibition by olaparib or gene knockout blocks asthma-like manifestation in mice by modulating CD4(+) T cell function.

BACKGROUND: An important portion of asthmatics do not respond to current therapies. Thus, the need for new therapeutic drugs is urgent. We have demonstrated a critical role for PARP in experimental asthma. Olaparib, a PARP inhibitor, was recently introduced in clinical trials against cancer. The objective of the present study was to examine the efficacy of olaparib in blocking established allergic airway inflammation and hyperresponsiveness similar to those observed in human asthma in animal models of the disease. METHODS: We used ovalbumin (OVA)-based mouse models of asthma and primary CD4(+) T cells. C57BL/6J WT or PARP-1(-/-) mice were subjected to OVA sensitization followed by a single or multiple challenges to aerosolized OVA or left unchallenged. WT mice were administered, i.p., 1 mg/kg, 5 or 10 mg/kg of olaparib or saline 30 min after each OVA challenge. RESULTS: Administration of olaparib in mice 30 min post-challenge promoted a robust reduction in airway eosinophilia, mucus production and hyperresponsiveness even after repeated challenges with ovalbumin. The protective effects of olaparib were linked to a suppression of Th2 cytokines eotaxin, IL-4, IL-5, IL-6, IL-13, and M-CSF, and ovalbumin-specific IgE with an increase in the Th1 cytokine IFN-γ. These traits were associated with a decrease in splenic CD4(+) T cells and concomitant increase in T-regulatory cells. The aforementioned traits conferred by olaparib administration were consistent with those observed in OVA-challenged PARP-1(-/-) mice. Adoptive transfer of Th2-skewed OT-II-WT CD4(+) T cells reversed the Th2 cytokines IL-4, IL-5, and IL-10, the chemokine GM-CSF, the Th1 cytokines IL-2 and IFN-γ, and ovalbumin-specific IgE production in ovalbumin-challenged PARP-1(-/-)mice suggesting a role for PARP-1 in CD4(+) T but not B cells. In ex vivo studies, PARP inhibition by olaparib or PARP-1 gene knockout markedly reduced CD3/CD28-stimulated gata-3 and il4 expression in Th2-skewed CD4(+) T cells while causing a moderate elevation in t-bet and ifn-γ expression in Th1-skewed CD4(+) T cells. CONCLUSIONS: Our findings show the potential of PARP inhibition as a viable therapeutic strategy and olaparib as a likely candidate to be tested in human asthma clinical trials.

PARP is activated in human asthma and its inhibition by olaparib blocks house dust mite-induced disease in mice.

Our laboratory established a role for poly(ADP-ribose)polymerase (PARP) in asthma. To increase the clinical significance of our studies, it is imperative to demonstrate that PARP is actually activated in human asthma, to examine whether a PARP inhibitor approved for human testing such as olaparib blocks already-established chronic asthma traits in response to house dust mite (HDM), a true human allergen, in mice and to examine whether the drug modulates human cluster of differentiation type 4 (CD4(+)) T-cell function. To conduct the study, human lung specimens and peripheral blood mononuclear cells (PBMCs) and a HDM-based mouse asthma model were used. Our results show that PARP is activated in PBMCs and lung tissues of asthmatics. PARP inhibition by olaparib or gene knockout blocked established asthma-like traits in mice chronically exposed to HDM including airway eosinophilia and hyper-responsiveness. These effects were linked to a marked reduction in T helper 2 (Th2) cytokine production without a prominent effect on interferon (IFN)-γ or interleukin (IL)-10. PARP inhibition prevented HDM-induced increase in overall cellularity, weight and CD4(+) T-cell population in spleens of treated mice whereas it increased the T-regulatory cell population. In CD3/CD28-stimulated human CD4 (+)T-cells, olaparib treatment reduced Th2 cytokine production potentially by modulating GATA binding protein-3 (gata-3)/IL-4 expression while moderately affecting T-cell proliferation. PARP inhibition inconsistently increased IL-17 in HDM-exposed mice and CD3/CD28-stimulated CD4(+) T cells without a concomitant increase in factors that can be influenced by IL-17. In the present study, we provide evidence for the first time that PARP-1 is activated in human asthma and that its inhibition is effective in blocking established asthma in mice.

PARP inhibitor, olaparib ameliorates acute lung and kidney injury upon intratracheal administration of LPS in mice.

We have previously shown that PARP-1 inhibition provides protection against lung inflammation in the context of asthma and acute lung injury. Olaparib is a potent new generation PARP inhibitor that has been approved for human testing. The present work was designed to evaluate its beneficial potential against LPS-induced acute lung injury and acute kidney injury upon intratracheal administration of the endotoxin in mice. Administration of olaparib at different doses, 30 min after LPS treatment showed that single intraperitoneal injection of the drug at 5 mg/kg b.wt. reduced the total number of inflammatory cells particularly neutrophils in the lungs. This was associated with reduced pulmonary edema as the total protein content in the bronchoalveolar fluid was found to be decreased substantially. Olaparib provided strong protection against LPS-mediated secondary kidney injury as reflected by restoration of serum levels of urea, creatinine, and uric acid toward normal. The drug restored the LPS-mediated redox imbalance toward normal in lung and kidney tissues as assessed by measuring malondialdehyde and GSH levels. Finally, RT-PCR data revealed that olaparib downregulates the LPS-induced expression of NF-κB-dependent genes namely TNF-α, IL-1ß, and VCAM-1 in the lungs without altering the expression of total p65NF-κB. Overall, the data suggest that olaparib has a strong potential to protect against LPS-induced lung injury and associated dysfunctioning of kidney in mice. Given the fact that olaparib is approved by FDA for human testing, our findings can pave the way for testing of the drug on humans inflicted with acute lung injury.

Apoptotic DNA fragmentation may be a cooperative activity between caspase-activated deoxyribonuclease and the poly(ADP-ribose) polymerase-regulated DNAS1L3, an endoplasmic reticulum-localized endonuclease that translocates to the nucleus during apoptosis.

Caspase-activated DNase (CAD) is the most favorable candidate for chromatin degradation during apoptosis. Ca(2+)-dependent endonucleases are equally important in internucleosomal DNA fragmentation (INDF), including the PARP-1-regulated DNAS1L3. Despite the elaborate work on these endonucleases, the question of whether these enzymes cooperate during INDF was not addressed. Here, we show a lack of correlation between INDF and CAD expression levels and inactivation by cleavage of its inhibitor (ICAD) during apoptosis. The cells that failed to induce INDF accumulated large amounts of 50-kb breaks, which is suggestive of incomplete chromatin processing. Similarly, INDF was blocked by Ca(2+) chelation without a block in ICAD cleavage or caspase-3 activation, which is consistent with the involvement of CAD in 50-kb DNA fragmentation and its Ca(2+) independence. However, DNAS1L3 expression in INDF-deficient cells promoted INDF during apoptosis and was blocked by Ca(2+) chelation. Interestingly, expression of DNAS1L3 in ICAD-deficient cells failed to promote tumor necrosis factor α-induced INDF but required the coexpression of ICAD. These results suggest a cooperative activity between CAD and DNAS1L3 to accomplish INDF. In HT-29 cells, endogenous DNAS1L3 localized to the endoplasmic reticulum (ER) and translocated to the nucleus upon apoptosis induction but prior to INDF manifestation, making it the first reported Ca(2+)-dependent endonuclease to migrate from the ER to the nucleus. The nuclear accumulation of DNAS1L3, but not its exit out of the ER, required the activity of cysteine and serine proteases. Interestingly, the endonuclease accumulated in the cytosol upon inhibition of serine, but not cysteine, proteases. These results exemplify the complexity of chromatin degradation during apoptosis.

Minocycline blocks asthma-associated inflammation in part by interfering with the T cell receptor-nuclear factor κB-GATA-3-IL-4 axis without a prominent effect on poly(ADP-ribose) polymerase.

Minocycline protects against asthma independently of its antibiotic function and was recently reported as a potent poly(ADP-ribose) polymerase (PARP) inhibitor. In an animal model of asthma, a single administration of minocycline conferred excellent protection against ovalbumin-induced airway eosinophilia, mucus hypersecretion, and Th2 cytokine production (IL-4/IL-5/IL-12(p70)/IL-13/GM-CSF) and a partial protection against airway hyperresponsiveness. These effects correlated with pronounced reduction in lung and sera allergen-specific IgE. A reduction in poly(ADP-ribose) immunoreactivity in the lungs of minocycline-treated/ovalbumin-challenged mice correlated with decreased oxidative DNA damage. The effect of minocycline on PARP may be indirect, as the drug failed to efficiently block direct PARP activation in lungs of N-methyl-N'-nitro-N-nitroso-guanidine-treated mice or H(2)O(2)-treated cells. Minocycline blocked allergen-specific IgE production in B cells potentially by modulating T cell receptor (TCR)-linked IL-4 production at the mRNA level but not through a modulation of the IL-4-JAK-STAT-6 axis, IL-2 production, or NFAT1 activation. Restoration of IL-4, ex vivo, rescued IgE production by minocycline-treated/ovalbumin-stimulated B cells. IL-4 blockade correlated with a preferential inhibition of the NF-κB activation arm of TCR but not GSK3, Src, p38 MAPK, or ERK1/2. Interestingly, the drug promoted a slightly higher Src and ERK1/2 phosphorylation. Inhibition of NF-κB was linked to a complete blockade of TCR-stimulated GATA-3 expression, a pivotal transcription factor for IL-4 expression. Minocycline also reduced TNF-α-mediated NF-κB activation and expression of dependent genes. These results show a potentially broad effect of minocycline but that it may block IgE production in part by modulating TCR function, particularly by inhibiting the signaling pathway, leading to NF-κB activation, GATA-3 expression, and subsequent IL-4 production.

Correlation between PDZK1, Cdc37, Akt and breast cancer malignancy: the role of PDZK1 in cell growth through Akt stabilization by increasing and interacting with Cdc37.

PDZ domain containing 1 (PDZK1) is a scaffold protein that plays a role in the fate of several proteins. Estrogen can induce PDZK1 gene expression; however, our recent report showed that PDZK1 expression in the breast cancer cell line MCF-7 is indirect and involves insulin-like growth factor (IGF)-1 receptor function. Such a relationship was established in cell culture systems and human breast cancer tissues. Here we show that overexpression of PDZK1 promoted an increase in cyclin D1 and enhanced anchorage-independent growth of MCF-7 cells in the absence of 17ß-estradiol, suggesting that PDZK1 harbors oncogenic activity. Indeed, PDKZ1 overexpression enhanced epidermal growth factor receptor (EGFR)-stimulated MEK/ERK1/2 signaling and IGF-induced Akt phosphorylation. PDZK1 appeared to play this role, in part, by stabilizing the integrity of the growth promoting factors Akt, human epidermal growth factor receptor 2 (Her2/Neu) and EGFR. Increased Akt levels occurred via a decrease in the ubiquitination of the kinase. PDZK1 overexpression was associated with resistance to paclitaxel/5-fluorouracil/etoposide only at low concentrations. Although the increased stability of Akt was sensitive to heat shock protein 90 (HSP90) inhibition, increased levels of the cochaperone cell division cycle 37 (Cdc37), as well as its ability to bind PDZK1, appear to play a larger role in kinase stability. Using human tissue microarrays, we show strong positive correlation between PDZK1, Akt and Cdc37 protein levels, and all correlated with human breast malignancy. There were no positive correlations between PDZK1 and Cdc37 at the mRNA levels, confirming our in vitro studies. These results demonstrate a relationship between PDZK1, Akt and Cdc37, and potentially Her2/Neu and EGFR, in breast cancer, representing a new axis that can be targeted therapeutically to reduce the burden of human breast cancer.

Guggulsterone protects against cigarette smoke-induced COPD linked lung inflammation.

Recently, we have shown that guggulsterone is the principal constituent responsible for protective effects of Commiphora wightii against elastase-induced chronic obstructive pulmonary disease (COPD)-linked inflammation/emphysema. Given that cigarette smoke (CS) exposure is a primary risk factor for COPD and beneficial effects of guggulsterone have not been investigated in CS-induced COPD-linked lung inflammation. The present work was designed to validate the potential of guggulsterone in amelioration of COPD-linked lung inflammation by using a CS-based mouse model of the condition. Male BALB/c mice were exposed to 9 cigarettes/day with 1 h interval for 4 days daily. Guggulsterone was administered daily at a dose of 10 mg/kg orally for 4 consecutive days, 1 h before initiation of CS exposure. Mice were subjected to measurement of lung function followed by procurement of bronchoalveolar lavage fluid (BALF)/lung tissue. BALF was analyzed for inflammatory cells and pro-inflammatory cytokines. Lung tissue was subjected to RT-PCR for gene expression analysis. Data showed that CS exposure resulted in a significant increase in total BALF cells, predominantly neutrophils, and macrophages. Interestingly, guggulsterone administration significantly blunted CS-induced inflammation as reflected by reduced neutrophil and macrophage count. Further, the compound inhibited CS-induced gene expression of pro-inflammatory mediators TNF-α/ IL-1ß/ G-CSF/and KC in lungs along with the production of pro-inflammatory mediators TNF-α/ IL-1ß/ IL-6/ G-CSF/ KC/and MCP-1 in BALF. Further, guggulsterone improved the lung function parameters upon CS exposure. Analysis of mRNA expression of matrix metalloproteinase (MMP)-9 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 suggests that guggulsterone may restore the fine balance between matrix-degrading proteases and its inhibitor in lung tissue upon CS exposure, which may contribute in the development of emphysema at later stages. Overall, our data show that guggulsterone protects against CS-induced COPD-linked lung inflammation by modulating relevant molecular players. Based on the potential effects of guggulsterone in the amelioration of CS-induced lung inflammation, we speculate that guggulsterone might alter chronic CS-induced emphysema.

PDZK1 is a novel factor in breast cancer that is indirectly regulated by estrogen through IGF-1R and promotes estrogen-mediated growth.

Although a relationship between PDZK1 expression and estrogen receptor (ER)-α stimulation has been suggested, the nature of such a connection and the function of PDZK1 in breast cancer remain unknown. Human tissue microarrays (cancer tissue: 262 cores; normal tissue: 87 cores) and breast cancer cell lines were used to conduct the study. We show that PDZK1 protein expression is tightly correlated with human breast malignancy, is negatively correlated with age and had no significant correlation with ER-α expression levels. PDZK1 exhibited an exclusive epithelial expression with mostly cytosolic subcellular localization. Additionally, 17ß-estradiol induced PDZK1 expression above its basal level more than 24 h after treatment in MCF-7 cells. PDZK1 expression was indirectly regulated by ER-α stimulation, requiring insulinlike growth factor 1 receptor (IGF-1R) expression and function. The molecular link between PDZK1 and IGF-1R was supported by a significant correlation between protein and mRNA levels (r = 0.591, p < 0.001, and r = 0.537, p < 0.001, respectively) of the two factors in two different cohorts of human breast cancer tissues. Interestingly, PDZK1 knockdown in MCF-7 cells blocked ER-dependent growth and reduced c-Myc expression, whereas ectopic expression of PDZK1 enhanced cell proliferation in the presence or absence of 17ß-estradiol potentially through an increase in c-Myc expression, suggesting that PDZK1 has oncogenic activity. PDKZ1 also appeared to interact with the Src/ER-α/epidermal growth factor receptor (EGFR) complex, but not with IGF-1R and enhanced EGFR-stimulated MEK/ERK1/2 signaling. Collectively, our results clarify the relationship between ER-α and PDZK1, propose a direct relationship between PDZK1 and IGF-1R, and identify a novel oncogenic activity for PDZK1 in breast cancer.