Ventilator-induced inflammatory lung injury (VILI) is mechanistically linked to increased NAMPT transcription and circulating levels of nicotinamide phosphoribosyl-transferase (NAMPT/PBEF). Although VILI severity is attenuated by reduced NAMPT/PBEF bioavailability, the precise contribution of NAMPT/PBEF and excessive mechanical stress to VILI pathobiology is unknown. We now report that NAMPT/PBEF induces lung NFκB transcriptional activities and inflammatory injury via direct ligation of Toll–like receptor 4 (TLR4). Computational analysis demonstrated that NAMPT/PBEF and MD-2, a TLR4-binding protein essential for LPS-induced TLR4 activation, share ~30% sequence identity and exhibit striking structural similarity in loop regions critical for MD-2-TLR4 binding. Unlike MD-2, whose TLR4 binding alone is insufficient to initiate TLR4 signaling, NAMPT/PBEF alone produces robust TLR4 activation, likely via a protruding region of NAMPT/PBEF (S402-N412) with structural similarity to LPS. The identification of this unique mode of TLR4 activation by NAMPT/PBEF advances the understanding of innate immunity responses as well as the untoward events associated with mechanical stress-induced lung inflammation.

Purpose Cisplatin is one of the most commonly used chemotherapy drugs worldwide and one of the most ototoxic. We sought to identify genetic variants that modulate cisplatin-associated ototoxicity (CAO). Experimental Design We performed a genome-wide association study (GWAS) of CAO using quantitative audiometry (4–12 kHz) in 511 testicular cancer survivors of European genetic ancestry. We performed polygenic modeling and functional analyses using a variety of publicly available databases. We used an electronic health record cohort to replicate our top mechanistic finding. Results One SNP, rs62283056, in the first intron of Mendelian deafness gene WFS1 (wolframin ER transmembrane glycoprotein) and an expression quantitative trait locus (eQTL) for WFS1 met genome-wide significance for association with CAO (P=1.4×10−8). A significant interaction between cumulative cisplatin dose and rs62283056 genotype was evident, indicating that higher cisplatin doses exacerbate hearing loss in patients with the minor allele (P=0.035). The association between decreased WFS1 expression and hearing loss was replicated in an independent BioVU cohort (n=18,620 patients, Bonferroni adjusted P<0.05). Beyond this top signal, we show CAO is a polygenic trait and that SNPs in and near 84 known Mendelian deafness genes are significantly enriched for low P-values in the GWAS (P=0.048). Conclusions We show for the first time the role of WFS1 in CAO and document a statistically significant interaction between increasing cumulative cisplatin dose and rs62283056 genotype. Our clinical translational results demonstrate that pre-therapy patient genotyping to minimize ototoxicity could be useful when deciding between cisplatin-based chemotherapy regimens of comparable efficacy with different cumulative doses.

Increased nicotinamide phosphoribosyltransferase (NAMPT) transcription is mechanistically linked to ventilator-induced inflammatory lung injury (VILI), with VILI severity attenuated by reduced NAMPT bioavailability. The molecular mechanisms of NAMPT promoter regulation in response to excessive mechanical stress remain poorly understood. The objective of this study was to define the contribution of specific transcription factors, acute respiratory distress syndrome (ARDS)-associated single nucleotide polymorphisms (SNPs), and promoter demethylation to NAMPT transcriptional regulation in response to mechanical stress. In vivo NAMPT protein expression levels were examined in mice exposed to high tidal volume mechanical ventilation. In vitro NAMPT expression levels were examined in human pulmonary artery endothelial cells exposed to 5 or 18% cyclic stretch (CS), with NAMPT promoter activity assessed using NAMPT promoter luciferase reporter constructs with a series of nested deletions. In vitro NAMPT transcriptional regulation was further characterized by measuring luciferase activity, DNA demethylation, and chromatin immunoprecipitation. VILI-challenged mice exhibited significantly increased NAMPT expression in bronchoalveolar lavage leukocytes and in lung endothelium. A mechanical stress-inducible region (MSIR) was identified in the NAMPT promoter from 22,428 to 22,128 bp. This MSIR regulates NAMPT promoter activity, mRNA expression, and signal transducer and activator of transcription 5 (STAT5) binding, which is significantly increased by 18% CS. In addition, NAMPT promoter activity was increased by pharmacologic promoter demethylation and inhibited by STAT5 silencing. ARDS-associated NAMPT promoter SNPs rs59744560 (2948G/T) and rs7789066 (22,422A/G) each significantly elevated NAMPT promoter activity in response to 18% CS in a STAT5-dependent manner. Our results show that NAMPT is a key novel ARDS therapeutic target and candidate gene with genetic/epigenetic transcriptional regulation in response to excessive mechanical stress.Keywords: acute respiratory distress syndrome; cyclic stretch; nicotinamide phosphoribosyltransferase; B cell colony-enhancing factor; signal transducer and activator of transcription 5 Clinical RelevanceNicotinamide phosphoribosyltransferase (NAMPT)/pre-B cell colony-enhancing factor is a key novel molecular marker and therapeutic target for acute lung injury. This study promotes the interpretation of the genetic and epigenetic regulation of NAMPT in response to excessive mechanical stress.Acute respiratory distress syndrome (ARDS) is characterized by severe hypoxemia and a persistently high mortality rate (z 30%) (1, 2). Mechanical ventilation is a life-saving intervention in critically ill patients with respiratory failure due to ARDS; however, excessive mechanical ventilation contributes directly to inflammatory lung injury, a process known

Increased lung vascular permeability and alveolar edema are cardinal features of inflammatory conditions such as acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI). We previously demonstrated that pre-B-cell colony-enhancing factor (PBEF)/NAMPT, the proinflammatory cytokine encoded by NAMPT, participates in ARDS and VILI inflammatory syndromes. The present study evaluated posttranscriptional regulation of PBEF/NAMPT gene expression in human lung endothelium via 39-untranslated region (UTR) microRNA (miRNA) binding. In silico analysis identified hsa-miR-374a and hsa-miR-568 as potential miRNA candidates. Increased PBEF/NAMPT transcription (by RT-PCR) and expression (by Western blotting) induced by 18% cyclic stretch (CS) (2 h: 3.4 6 0.06 mRNA fold increase (FI); 10 h: 1.5 6 0.06 protein FI) and by LPS (4 h: 3.8 6 0.2 mRNA FI; 48 h: 2.6 6 0.2 protein FI) were significantly attenuated by transfection with mimics of hsa-miR-374a or hsa-miR-568 (40-60% reductions each). LPS and 18% CS increased the activity of a PBEF/NAMPT 39-UTR luciferase reporter (2.4-3.25 FI) with induction reduced by mimics of each miRNA (44-60% reduction). Specific miRNA inhibitors (antagomirs) for each PBEF/ NAMPT miRNA significantly increased the endogenous PBEF/ NAMPT mRNA (1.4-3.4 6 0.1 FI) and protein levels (1.2-1.4 6 0.1 FI) and 39-UTR luciferase activity (1.4-1.7 6 0.1 FI) compared with negative antagomir controls. Collectively, these data demonstrate that increased PBEF/NAMPT expression induced by bioactive agonists (i.e., excessive mechanical stress, LPS) involves epigenetic regulation with hsa-miR-374a and hsa-miR-568, representing novel therapeutic strategies to reduce inflammatory lung injury.

Purpose: Cisplatin, a commonly used chemotherapeutic, results in tinnitus, the phantom perception of sound. Our purpose was to identify the clinical and genetic determinants of tinnitus among testicular cancer survivors (TCS) following cisplatin-based chemotherapy. Experimental Design: TCS (n ¼ 762) were dichotomized to cases (moderate/severe tinnitus; n ¼ 154) and controls (none; n ¼ 608). Logistic regression was used to evaluate associations with comorbidities and SNP dosages in genome-wide association study (GWAS) following quality control and imputation (covariates: age, noise exposure, cisplatin dose, genetic principal components). Pathway over-representation tests and functional studies in mouse auditory cells were performed. Results: Cisplatin-induced tinnitus (CisIT) significantly associated with age at diagnosis (P ¼ 0.007) and cumulative cisplatin dose (P ¼ 0.007). CisIT prevalence was not significantly greater in 400 mg/m 2-treated TCS compared with 300 (P ¼ 0.41), but doses >400 mg/m 2 (median 580, range 402-828) increased risk by 2.61-fold (P < 0.0001). CisIT cases had worse hearing at each frequency (0.25-12 kHz, P < 0.0001), and reported more vertigo (OR ¼ 6.47; P < 0.0001) and problems hearing in a crowd (OR ¼ 8.22; P < 0.0001) than controls. Cases reported poorer health (P < 0.0001) and greater psychotropic medication use (OR ¼ 2.4; P ¼ 0.003). GWAS suggested a variant near OTOS (rs7606353, P ¼ 2 Â 10 À6) and OTOS eQTLs were significantly enriched independently of that SNP (P ¼ 0.018). OTOS overexpression in HEI-OC1, a mouse auditory cell line, resulted in resistance to cisplatin-induced cytotoxicity. Pathway analysis implicated potassium ion transport (q ¼ 0.007). Conclusions: CisIT associated with several neurootological symptoms, increased use of psychotropic medication, and poorer health. OTOS, expressed in the cochlear lateral wall, was implicated as protective. Future studies should investigate otoprotective targets in supporting cochlear cells.