Narasaiah Kolliputi

NLRP3 deletion protects from hyperoxia-induced acute lung injury

Inspiration of a high concentration of oxygen, a therapy for acute lung injury (ALI), could unexpectedly lead to reactive oxygen species (ROS) production and hyperoxia-induced acute lung injury (HALI). Nucleotide-binding domain and leucine-rich repeat PYD-containing protein 3 (NLRP3) senses the ROS, triggering inflammasome activation and interleukin-1β (IL-1β) production and secretion. However, the role of NLRP3 inflammasome in HALI is unclear. The main aim of this study is to determine the effect of NLRP3 gene deletion on inflammatory response and lung epithelial cell death. Wild-type (WT) and NLRP3(-/-) mice were exposed to 100% O2 for 48-72 h. Bronchoalveolar lavage fluid and lung tissues were examined for proinflammatory cytokine production and lung inflammation. Hyperoxia-induced lung pathological score was suppressed in NLRP3(-/-) mice compared with WT mice. Hyperoxia-induced recruitment of inflammatory cells and elevation of IL-1β, TNFα, macrophage inflammatory protein-2, and monocyte chemoattractant protein-1 were attenuated in NLRP3(-/-) mice. NLRP3 deletion decreased lung epithelial cell death and caspase-3 levels and a suppressed NF-κB levels compared with WT controls. Taken together, this research demonstrates for the first time that NLRP3-deficient mice have suppressed inflammatory response and blunted lung epithelial cell apoptosis to HALI.

Mir-206 Regulates Pulmonary Artery Smooth Muscle Cell Proliferation and Differentiation

Pulmonary Arterial Hypertension (PAH) is a progressive devastating disease characterized by excessive proliferation of the Pulmonary Arterial Smooth Muscle Cells (PASMCs). Studies suggest that PAH and cancers share an apoptosis-resistant state featuring excessive cell proliferation. MicroRNA-206 (miR-206) is known to regulate proliferation and is implicated in various types of cancers. However, the role of miR-206 in PAH has not been studied. In this study, it is hypothesized that miR-206 could play a role in the proliferation of PASMCs. In the present study, the expression patterns of miR-206 were investigated in normal and hypertensive mouse PASMCs. The effects of miR-206 in modulating cell proliferation, apoptosis and smooth muscle cell markers in human pulmonary artery smooth muscle cells (hPASMCs) were investigated in vitro. miR-206 expression in mouse PASMCs was correlated with an increase in right ventricular systolic pressure. Reduction of miR-206 levels in hPASMCs causes increased proliferation and reduced apoptosis and these effects were reversed by the overexpression of miR-206. miR-206 over expression also increased the levels of smooth muscle cell differentiation markers α-smooth muscle actin and calponin implicating its importance in the differentiation of SMCs. miR-206 overexpression down regulated Notch-3 expression, which is key a factor in PAH development. These results suggest that miR-206 is a potential regulator of proliferation, apoptosis and differentiation of PASMCs, and that it could be used as a novel treatment strategy in PAH.

IL-6 Protects against Hyperoxia-Induced Mitochondrial Damage via Bcl-2–Induced Bak Interactions with Mitofusions

Overexpression of IL-6 markedly diminishes hyperoxic lung injury, hyperoxia-induced cell death, and DNA fragmentation, and enhances Bcl-2 expression. We hypothesized that changes in the interactions between Bcl-2 family members play an important role in the IL-6-mediated protective response to oxidative stress. Consistent with this hypothesis, we found that IL-6 induced Bcl-2 expression, both in vivo and in vitro, disrupted interactions between proapoptotic and antiapoptotic factors, and suppressed H(2)O(2)-induced loss of mitochondrial membrane potential in vitro. In addition, IL-6 overexpression in mice protects against hyperoxia-induced lung mitochondrial damage. The overexpression of Bcl-2 in vivo prolonged the survival of mice exposed to hyperoxia and inhibited alveolar capillary protein leakage. In addition, apoptosis-associated DNA fragmentation was substantially reduced in these animals. This IL-6-mediated protection was lost when Bcl-2 was silenced, demonstrating that Bcl-2 is an essential mediator of IL-6 cytoprotection. Finally, Bcl-2 blocked the dissociation of Bak from mitofusin protein (Mfn) 2, and inhibited the interaction between Bak and Mfn1. Taken together, our results suggest that IL-6 induces Bcl-2 expression to perform cytoprotective functions in response to oxygen toxicity, and that this effect is mediated by alterations in the interactions between Bak and Mfns.

IL-6 cytoprotection in hyperoxic acute lung injury occurs via PI3K/Akt-mediated Bax phosphorylation

IL-6 overexpression protects mice from hyperoxic acute lung injury in vivo, and treatment with IL-6 protects cells from oxidant-mediated death in vitro. The mechanisms of protection, however, are not clear. We characterized the expression, localization, and regulation of Bax, a proapoptotic member of the Bcl-2 family, in wild-type (WT) and IL-6 lung-specific transgenic (Tg(+)) mice exposed to 100% O(2) and in human umbilical vein endothelial cells (HUVEC) treated with H(2)O(2) and IL-6. In control HUVEC treated with H(2)O(2) or in WT mice exposed to 100% O(2), a marked induction of Bax translocation and dimerization was associated with increased JNK and p38 kinase activity. In contrast, specific JNK or p38 kinase inhibitors or treatment with IL-6 inhibited Bax mitochondrial translocation and apoptosis of HUVEC. IL-6 Tg(+) mice exposed to 100% O(2) exhibited enhanced phosphatidylinositol 3-kinase (PI3K)/Akt kinase and increased serine phosphorylation of Bax at Ser(184) compared with WT mice. The PI3K-specific inhibitor LY-2940002 blocked this IL-6-induced Bax phosphorylation and promoted cell death. Furthermore, IL-6 potently blocked hyperoxia- or oxidant-induced Bax insertion into mitochondrial membranes. Thus IL-6 functions in a cytoprotective manner, in part, by suppressing Bax translocation and dimerization through PI3K/Akt-mediated Bax phosphorylation.

The Inflammasome Mediates Hyperoxia-Induced Alveolar Cell Permeability

A hallmark of hyperoxic acute lung injury is the influx of inflammatory cells to lung tissue and the production of proinflammatory cytokines, such as IL-1β; however, the mechanisms connecting hyperoxia and the inflammatory response to lung damage is not clear. The inflammasome protein complex activates caspase-1 to promote the processing and secretion of proinflammatory cytokines. We hypothesized that hyperoxia-induced K+ efflux activates the inflammasome via the purinergic P2X7 receptor to cause inflammation and hyperoxic acute lung injury. To test this hypothesis, we characterized the expression and activation of inflammasome components in primary murine alveolar macrophages exposed to hyperoxia (95% oxygen and 5% CO2) in vitro, and in alveolar macrophages isolated from mice exposed to hyperoxia (100% oxygen). Our results showed that hyperoxia increased K+ efflux, inflammasome formation, release of proinflammatory cytokines, and induction of caspase-1 and IL-1β cleavage both in vitro and in vivo. The P2X7 agonist ATP enhanced hyperoxia-induced inflammasome activation, whereas the P2X7 antagonist, oxidized ATP, inhibited hyperoxia induced inflammasome activation. In addition, when ATP was scavenged with apyrase, hyperoxia-induced inflammasome activation was significantly decreased. Furthermore, short hairpin RNA silencing of inflammasome components abrogated hyperoxia-induced secretion of proinflammatory cytokines in vitro. These results suggest that hyperoxia induces K+ efflux through the P2X7 receptor, leading to inflammasome activation and secretion of proinflammatory cytokines. These events would affect the permeability of the alveolar epithelium and ultimately lead to epithelial barrier dysfunction and cell death.

TXNIP shuttling: missing link between oxidative stress and inflammasome activation

Thioredoxin-interacting protein (TXNIP) has been linked to cell apoptosis and inflammation in a number of diseases, including type 2 diabetes (Shah et al., 2013), atherosclerosis (Berk, 2007), and myocardial ischemia (Yoshioka et al., 2012). TXNIP has been identified as a tumor suppressor gene and in cancer, a loss of TXNIP can lead to cell proliferation (Zhou and Chng, 2013). The primary role of TXNIP is inhibition of thioredoxin (TRX), an important redox protein and promoter of cell growth. TRX is a ubiquitous protein that reduces thiols, especially insulin disulfides, and controls levels of reactive oxygen species (ROS) in cells, limiting damage from oxidative stress. Inhibition of TRX by TXNIP carries lethal consequences for cells and can promote destructive inflammation (Junn et al., 2000; Spindel et al., 2012). In human aortic endothelial cells with TXNIP knockdown, cultured under high glucose conditions to promote oxidative stress, there was a decrease in the amount of ROS generated as compared with control cells. This suggests that high levels of TXNIP inhibit the redox activity of cytoplasmic thioredoxin (TRX1) correlating with increased levels of ROS (Li et al., 2009). Mouse mesangial cells from wild type (C3H) and TXNIP-deficient mice (Hcb-19) were exposed to high glucose. After 3 h, ROS generation was 2.5–3 times greater in the cells from C3H compared to Hcb-19 (Shah et al., 2013). Mitochondrial thioredoxin, TRX2, like its cytoplasmic counterpart, TRX1, was found to regulate ROS and manage oxidative stress within mitochondria. HeLa cells were transiently transfected with a TRX2 expression vector and when treated with tumor necrosis factor alpha (TNF-a), a pro-inflammatory cytokine, generated approximately 50% less ROS than TNF-a treated control HeLa cells (Hansen et al., 2006). In this opinion piece, we interpret the facts on mitochondrial danger signaling: TXNIPs role in the mitochondria, interactions with mitochondrial TRX2 and activation of the NOD-like receptor protein 3 (NLRP3) inflammasome [an oligomeric complex activated by cellular infections or stress (Schroder and Tschopp, 2010)] signaling pathway not previously described. While the role of TXNIP is defined, details of its localization within the cell remain unsolved. Based on previous research, which implicated TXNIP in mitochondrial death signaling, Saxena et al. explored the intracellular localization of TXNIP. They found that it is shuttled into mitochondria under oxidative stress while it is found in the nucleus under normal conditions (Saxena et al., 2010). TXNIP and TRX2 interactions were examined based on the parallel roles of cytoplasmic TRX1 and mitochondrial TRX2. Indeed, TXNIP and TRX2 are bound within the mitochondria, inhibiting the reductive power of TRX2, thus leaving ROS levels unchecked. The redox-regulated apoptosis-signal kinase (ASK1), a member of the mitogen-activated protein kinase family, has been deemed an important link between cellular stress and innate immunity (Barton and Medzhitov, 2003; Kolliputi and Waxman, 2009). ASK1 is usually bound to TRX2 under basal conditions, however, during stress and following TXNIP translocation to the mitochondria, ASK1-TRX2 binding is disrupted, triggering an apoptotic signal cascade. Unbound ASK1 is phosphorylated, signaling cytochrome C release and caspase-3 cleavage, eventually causing apoptosis (Bhattacharyya et al., 2003). Zhou et al. showed that ROS causes TXNIP to associate with NLRP3 leading to inflammasome activation (Zhou et al., 2011). This study suggests that in the unstressed cell, TXNIP is bound to TRX1 and is inactive. In response to oxidative stress, ROS generation facilitates TRX1-TXNIP dissociation, thus increasing NLRP3-TXNIP association. Also, TXNIP translocates to the mitochondria, where it binds to TRX2 leading to mitochondrial dysfunction. However, the function of TRX2 in regulating the inflammasome has not been studied to determine whether it is an essential component of NLRP3 activation. The NLRP3 inflammasome, consists of NLRP3 oligomers and apoptosis-associated speck-like (ASC) adapter protein. The assembly of the NLRP3 inflammasome triggers caspase-1 activation leading to processing of interleukin-1s (IL-1s) (Schroder and Tschopp, 2010). Studies by Shimada et al. and Saxena et al. elucidate the mystery of inflammasome activation that could hold a key to a therapeutic approach to limit unchecked cell apoptosis and inflammation with limited side effects. Shimada et al. reported that oxidized mitochondrial DNA (mtDNA) directly activates the NLRP3 inflammasome (Shimada et al., 2012). Their work showed that direct binding of oxidized mtDNA with NLRP3 serves as a trigger for inflammasome activation, indicating that apoptosis signaling leads to inflammasome activation and IL-1s secretion. A key signaling point occurs when mtDNA reacts with ROS to become oxidized mtDNA, threatening imminent cell death (Shimada et al., 2012). Earlier, Nakahira et al. showed that mtDNA interacts with NLRP3 (Nakahira et al., 2011) but the exact mechanism is unknown. Significant cytosolic levels of free oxidized mtDNA could not be found in the absence of NLRP3, suggesting that it may stabilize mtDNA. As mentioned earlier, Saxena et al. described a novel mitochondrial apoptosis signaling cascade showing that TXNIP, under oxidative stress, translocates from the nucleus to mitochondria (Saxena et al., 2010). It is therefore tempting to speculate that mitochondrial shuttling of TXNIP may affect mitochondrial dysfunction and oxidation of mtDNA leading to NLRP3 inflammasome activation. Exploration of TXNIPs role in the mitochondria, interactions with mitochondrial TRX2 and mitochondrial danger signaling has shed light on a new NLRP3 signaling pathway not previously described. ROS within mitochondria must initiate the mitochondrial cascade upstream, causing inflammasome activation. Several studies show that mitochondrial ROS are present prior to NLRP3 activation (Zhou et al., 2011). A possible source of ROS could stem from early TXNIP translocation following upstream cellular oxidative stress signals (Figure ​(Figure1).1). Mitochondrial shuttling of TXNIP, followed by displacement of ASK1 by TXNIP-TRX2 binding, initiates downstream apoptotic signaling. Also, TXNIP inhibits TRX2 protection of mitochondria against ROS, allowing free oxidation of mtDNA, signaling distress, binding NLRP3, and triggering inflammasome activation. Downstream cleavage of caspase-3 by ASK1 leads to apoptosis serving as a secondary signal for NLRP3 activation. Figure 1 Oxidative stress mediated TXNIP shuttling: TXNIP localization under basal conditions (Left), TXNIP rests in the nucleus. TRX1-ASK1 binding in the cytosol and TRX2-ASK1 binding in the mitochondria maintains low ROS. Under oxidative stress (Right), TXNIP ... Based on the mechanisms described, early apoptotic signals play a crucial role in inflammation. The earliest signal found is the shuttling of TXNIP from the nucleus to the mitochondria. A key therapeutic approach to limit inflammasome activation could be to inhibit mitochondrial shuttling of TXNIP. It was previously discussed that TXNIP inhibits reductive properties of TRX. In the mitochondria, this inhibition allows ROS to accumulate until TXNIP is shuttled to the cytoplasm where it interacts with NLRP3 (Davis and Ting, 2010). However, this mechanism needs to be defined. The increase in ROS causes the release of oxidized mtDNA, cytochrome C, and caspase-3 cleavage. Combined, these signals induce cell apoptosis and inflammasome activation, leading to IL-1s secretion and localized inflammation. When TXNIP is in the mitochondria, it allows the accumulation of ROS. Therefore, finding and blocking the shuttling mechanism for TXNIP would allow TRX2 to maintain its reductive role and prevent downstream activation of NLRP3. The role of TXNIP in inflammasome activation has proven invaluable in efforts to limit damage to cells and surrounding tissue. Once TXNIP reaches the mitochondria, it triggers a rapid cascade of apoptotic signals that end with TXNIP-NLRP3 binding and inflammasome activation. Early warning signals may be present prior to TXNIP translocation to mitochondria; however, such signals need to be identified. Further investigations into TXNIP localization and mitochondrial shuttling should be undertaken to completely map the signaling pathway, find factors that block TXNIP, and ultimately limit undesirable inflammasome activation. In recent years, there is continued evidence of the central role of inflammasomes in sensing danger signals and orchestrating a subsequent inflammatory program (Kolliputi et al., 2010). However, due to the multitude of danger signals sensed by the NLRP3 inflammasome, it has remained a mystery how a single molecule can achieve this almost impossible task. A plausible explanation is that instead of detecting each danger signal individually, the NLRP3 inflammasome monitors the activity of the mitochondrion, which acts as an integrator of danger signals, including those of metabolic origin. Via a mechanism that remains elusive, excessive ROS production by mitochondria leads to activation of the inflammasome. Therefore, a clear understanding of this mechanism and regulation, will allow development of new therapeutic approaches to diseases involving the NLRP3 inflammasome.

NALP-3 inflammasome silencing attenuates ceramide-induced transepithelial permeability

The hallmark of acute lung injury (ALI) is the influx of proinflammatory cytokines into lung tissue and alveolar permeability that ultimately leads to pulmonary edema. However, the mechanisms involved in inflammatory cytokine production and alveolar permeability are unclear. Recent studies suggest that excessive production of ceramide has clinical relevance as a mediator of pulmonary edema and ALI. Our earlier studies indicate that the activation of inflammasome promotes the processing and secretion of proinflammatory cytokines and causes alveolar permeability in ALI. However, the role of ceramide in inflammasome activation and the underlying mechanism in relation to alveolar permeability is not known. We hypothesized that ceramide activates the inflammasome and causes inflammatory cytokine production and alveolar epithelial permeability. To test this hypothesis, we analyzed the lung ceramide levels during hyperoxic ALI in mice. The effect of ceramide on activation of inflammasome and production of inflammatory cytokine was assessed in primary mouse alveolar macrophages and THP‐1 cells. Alveolar transepithelial permeability was determined in alveolar epithelial type‐II cells (AT‐II) and THP‐1 co‐cultures. Our results reveal that ceramide causes inflammasome activation, induction of caspase‐1, IL‐1β cleavage, and release of proinflammatory cytokines. In addition, ceramide further induces alveolar epithelial permeability. Short‐hairpin RNA silencing of inflammasome components abrogated ceramide‐induced secretion of proinflammatory cytokines in vitro. Inflammasome silencing abolishes ceramide‐induced alveolar epithelial permeability in AT‐II. Collectively, our results demonstrate for the first time that ceramide‐induced secretion of proinflammatory cytokines and alveolar epithelial permeability occurs though inflammasome activation. J. Cell. Physiol. 227: 3310–3316, 2012.

IL-6 Cytoprotection in Hyperoxic Acute Lung Injury Occurs via Suppressor of Cytokine Signaling-1-Induced Apoptosis Signal-Regulating Kinase-1 Degradation

Hyperoxic acute lung injury (HALI) is characterized by a cell death response that is inhibited by IL-6. Suppressor of cytokine signaling-1 (SOCS-1) is an antiapoptotic negative regulator of the IL-6-mediated Janus kinase-signal transducer and activator of transcription signaling pathway. We hypothesized that SOCS-1 is a critical regulator and key mediator of IL-6-induced cytoprotection in HALI. To test this hypothesis, we characterized the expression of SOCS-1 and downstream apoptosis signal-regulating kinase (ASK)-1-Jun N-terminal kinase signaling molecules in small airway epithelial cells in the presence of H(2)O(2), which induces oxidative stress. We also examined these molecules in wild-type and lung-specific IL-6 transgenic (Tg(+)) mice exposed to 100% oxygen for 72 hours. In control small airway epithelial cells exposed to H(2)O(2) or in wild-type mice exposed to 100% oxygen, a marked induction of ASK-1 and pJun N-terminal kinase was observed. Both IL-6-stimulated endogenous SOCS-1 and SOCS-1 overexpression abolished H(2)O(2)-induced ASK-1 activation. In addition, IL-6 Tg(+) mice exposed to 100% oxygen exhibited reduced ASK-1 levels and enhanced SOCS-1 expression compared with wild-type mice. Interestingly, no significant changes in activation of the key ASK-1 activator, tumor necrosis factor receptor-1/tumor necrosis factor receptor-associated factor-2 were observed between wild-type and IL-6 Tg(+) mice. Furthermore, the interaction between SOCS-1 and ASK-1 promotes ubiquitin-mediated degradation both in vivo and in vitro. These studies demonstrate that SOCS-1 is an important regulator in IL-6-induced cytoprotection against HALI.

MicroRNA-133a-1 regulates inflammasome activation through uncoupling protein-2

Inflammasomes are multimeric protein complexes involved in the processing of IL-1β through Caspase-1 cleavage. NLRP3 is the most widely studied inflammasome, which has been shown to respond to a large number of both endogenous and exogenous stimuli. Although studies have begun to define basic pathways for the activation of inflammasome and have been instrumental in identifying therapeutics for inflammasome related disorders; understanding the inflammasome activation at the molecular level is still incomplete. Recent functional studies indicate that microRNAs (miRs) regulate molecular pathways and can lead to diseased states when hampered or overexpressed. Mechanisms involving the miRNA regulatory network in the activation of inflammasome and IL-1β processing is yet unknown. This report investigates the involvement of miR-133a-1 in the activation of inflammasome (NLRP3) and IL-1β production. miR-133a-1 is known to target the mitochondrial uncoupling protein 2 (UCP2). The role of UCP2 in inflammasome activation has remained elusive. To understand the role of miR-133a-1 in regulating inflammasome activation, we either overexpressed or suppressed miR-133a-1 in differentiated THP1 cells that express the NLRP3 inflammasome. Levels of Caspase-1 and IL-1β were analyzed by Western blot analysis. For the first time, we showed that overexpression of miR-133a-1 increases Caspase-1 p10 and IL-1β p17 cleavage, concurrently suppressing mitochondrial uncoupling protein 2 (UCP2). Surprisingly, our results demonstrated that miR-133A-1 controls inflammasome activation without affecting the basal expression of the individual inflammasome components NLRP3 and ASC or its immediate downstream targets proIL-1β and pro-Caspase-1. To confirm the involvement of UCP2 in the regulation of inflammasome activation, Caspase-1 p10 and IL-1β p17 cleavage in UCP2 of overexpressed and silenced THP1 cells were studied. Suppression of UCP2 by siRNA enhanced the inflammasome activity stimulated by H2O2 and, conversely, overexpression of UCP2 decreased the inflammasome activation. Collectively, these studies suggest that miR-133a-1 suppresses inflammasome activation via the suppression of UCP2.

MicroRNA 16 Modulates Epithelial Sodium Channel in Human Alveolar Epithelial Cells

Acute lung injury (ALI) is a devastating disease characterized by pulmonary edema. Removal of edema from the air spaces of lung is a critical function of the epithelial sodium channel (ENaC) in ALI. The molecular mechanisms behind resolution of pulmonary edema are incompletely understood. MicroRNAs (miRNA) are crucial gene regulators and are dysregulated in various diseases including ALI. Recent studies suggest that microRNA-16 (miR-16) targets serotonin transporter (SERT) involved in the serotonin (5-HT) transmitter system. Alterations in serotonin levels have been reported in various pulmonary diseases. However, the role of miR-16 on its target SERT, and ENaC, a key ion channel involved in the resolution of pulmonary edema, have not been studied. In the present study, the expression patterns of miR-16, SERT, ENaC and serotonin were investigated in mice exposed to room air and hyperoxia. The effects of miR-16 overexpression on ENaC, SERT, TGF-β and Nedd4 in human alveolar epithelial cells were analyzed. miR-16 and ENaC were downregulated in mice exposed to hyperoxia. miR-16 downregulation in mouse lung was correlated with an increase in SERT expression and pulmonary edema. Overexpression of miR-16 in human alveolar epithelial cells (A549) suppressed SERT and increased ENaCβ levels when compared to control-vector transfected cells. In addition, miR-16 over expression suppressed TGFβ release, a critical inhibitor of ENaC. Interestingly Nedd4, a negative regulator of ENaC remained unaltered in miR-16 over expressed A549 cells when compared to controls. Taken together, our data suggests that miR-16 upregulates ENaC, a major sodium channel involved in resolution of pulmonary edema in ALI.