The enzymatic oxidation of arachidonic acid is proposed to yield trihydroxytetraene species (termed lipoxins) that resolve inflammation via ligand activation of the formyl peptide receptor, FPR2. While cell and murine models activate signaling responses to synthetic lipoxins, primarily 5S,6R,15S-trihydroxy-7E,9E,11Z,13E-eicosatetraenoic acid (lipoxin A4, LXA4), there are expanding concerns about the biological formation, detection and signaling mechanisms ascribed to LXA4 and related di- and tri-hydroxy ω-6 and ω-3 fatty acids. Herein, the generation and actions of LXA4 and its primary 15-oxo metabolite were assessed in control, LPS-activated and arachidonic acid supplemented RAW 264.7 macrophages. Despite protein expression of all enzymes required for LXA4 synthesis, both LXA4 and its 15-oxo-LXA4 metabolite were undetectable. Moreover, synthetic LXA4 and the membrane permeable 15-oxo-LXA4 methyl ester that is rapidly de-esterified to 15-oxo-LXA4, displayed no ligand activity for the putative LXA4 receptor FPR2, as opposed to the FPR2 ligand WKYMVm. Alternatively, 15-oxo-LXA4, an electrophilic α,β-unsaturated ketone, alkylates nucleophilic amino acids such as cysteine to modulate redox-sensitive transcriptional regulatory protein and enzyme function. 15-oxo-LXA4 activated nuclear factor (erythroid related factor 2)-like 2 (Nrf2)-regulated gene expression of anti-inflammatory and repair genes and inhibited nuclear factor (NF)-κB-regulated pro-inflammatory mediator expression. LXA4 did not impact these macrophage anti-inflammatory and repair responses. In summary, these data show an absence of macrophage LXA4 formation and receptor-mediated signaling actions. Rather, if LXA4 were present in sufficient concentrations, this, and other more abundant mono- and poly-hydroxylated unsaturated fatty acids can be readily oxidized to electrophilic α,β-unsaturated ketone products that modulate the redox-sensitive cysteine proteome via G-protein coupled receptor-independent mechanisms.

Obesity and associated changes to the gut microbiome worsen airway inflammation and hyperresponsiveness in asthma. Obesogenic host-microbial metabolomes have altered production of metabolites that may influence lung function and inflammatory responses in asthma. To understand the interplay of the gut microbiome, metabolism, and host inflammation in obesity-associated asthma, we used a multi-omics approach to profile the gut-lung axis in the setting of allergic airway disease and diet-induced obesity. We evaluated an immunomodulator, nitro-oleic acid (NO2-OA), as a host- and microbial-targeted treatment intervention for obesity-associated allergic asthma. Allergic airway disease was induced using house dust mite and cholera toxin adjuvant in C57BL6/J mice with diet-induced obesity to model obesity-associated asthma. Lung function was measured by flexiVent following a week of NO2-OA treatment and allergen challenge. 16S rRNA gene (from DNA, taxa presence) and 16S rRNA (from RNA, taxa activity) sequencing, metabolomics, and host gene expression were paired with a Treatment-Measured-Response model as a data integration framework for identifying latent/hidden relationships with linear regression among variables identified from high-dimensional meta-omics datasets. Targeting both the host and gut microbiota, NO2-OA attenuated airway inflammation, improved lung elastance, and modified the gut microbiome. Meta-omics data integration and modeling determined that gut-associated inflammation, metabolites, and functionally active gut microbiota were linked to lung function outcomes. Using Treatment-Measured-Response modeling and meta-omics profiling of the gut-lung axis, we uncovered a previously hidden network of interactions between gut levels of amino acid metabolites involved in elastin and collagen synthesis, gut microbiota, NO2-OA, and lung elastance. Further targeted metabolomics analyses revealed that obese mice with allergic airway disease had higher levels of proline and hydroxyproline in the lungs. NO2-OA treatment reduced proline biosynthesis by downregulation of pyrroline-5-carboxylate reductase 1 (PYCR1) expression. These findings are relevant to human disease: adults with mild-moderate asthma and BMI ≥ 25 had higher plasma hydroxyproline levels. Our results suggest that changes to structural proteins in the lung airways and parenchyma may contribute to heightened lung elastance and serve as a potential therapeutic target for obese allergic asthma.

Out of all breast cancers (BC), ~20% are triple negative breast cancers (TNBC) devoid of the three receptors that define breast cancer treatment strategies such as estrogen receptor (ER), progesterone receptor (PR) and ERBB2 (also known as HER2). Unlike other BC, TNBC disproportionally affects younger women and those of African origins and recurs early within 5 years after diagnosis in ~ 40% of the patients. Standard care for TNBC patients includes surgery, ionizing radiation (IR) treatment and chemotherapy. Successful targeting of the poly (ADP-ribose) polymerase (PARP) has revolutionized therapy for cancer patients carrying gmBRCA1/2. However, in TNBC patients optimal clinical usage of PARP inhibitors (PARPi) is stifled by a) only 15-20% of TNBC patients being positive for gmBRCA1/2 and currently receiving PARPi therapy, b) most PARPi-treated gmBRCA1/2 patients developing resistance due to restoration of HR-mediated DNA DSB repair and, c) PARPi treatment-associated toxicities that require dose adaptations or drug discontinuation. Our novel therapeutic approach addresses this clinical unmet need by further advancing a small molecule inhibitor of HR that includes the 80% TNBC patients wild type for BRCA1/2 genes, re-sensitizes patients resistant to PARPi due to restoration of HR-mediated DNA DSB repair and lowers PARPi doses by conferring greater sensitivity to PARPi actions through synthetic lethality approaches.We first defined OA-NO2 ((E)-9/10-nitro-octadec-9-enoic acid), an endogenous electrophilic fatty acid nitroalkene as an RAD51 inhibitor in TNBC. RAD51 is essential in HR-mediated DNA DSB repair, is a highly coveted drug target in cancer and is a revealing functional biomarker for PARPi sensitivity. OA-NO2 reacts with protein thiols via reverse Michael addition reaction and has broad spectrum anticancer activities. OA-NO2 inhibits RAD51 function by selectively alkylating RAD51 on Cys319, a residue essential in RAD51- guided HR. In combination with the PARPi talazoparib, OA-NO2 synergizes the killing of TNBC cells in vitro and in vivo. Two reasons motivated the further development of OA-NO2 as RAD51 inhibitor: 1) X-ray crystal structure modeling predicted superior fitting with the RAD51 C-terminus if the nitroalkene substituent is closer to the carboxylate terminus of the nitro-fatty acid, and 2) OA-NO2 exhibits off target effects by inhibiting c-GAS-STING signaling and activating Nrf2 activity, thus, possibly offsetting anti-cancer activities. To optimize nitroalkene-mediated RAD51 inhibition, a library of >50 compounds was built that expands upon 5 chemical classes. The library was evaluated for structure activity relationships (SAR) and a new lead nitroalkene emerged, CP-23. This electrophilic small molecule kills as a monotherapy TNBC and not benign breast epithelial cells and unlike OA-NO2, has no impact on STING and Nrf2 signaling, or the cell cycle. CP-23 inhibits RAD51 DNA binding, shows potent inhibition of RAD51 nuclear foci formation after ionizing radiation (IR) treatment and HR in a GFP-reporter cell line, and demonstrated drug synergism with PARP inhibition and IR. At present, in vivo tumor growth inhibition, expansion of PARPi-CP-23 synthetic lethality studies and pilot PK/toxicology evaluation are underway. Citation Format: Carola Anke Neumann, John J Skoko, Bruce A Freeman, Francisco Schopfer, Steven Robert Woodcock, Fei Chang, Lisa Hong, Dennis Carl Braden, Crystal Uvalle. Targeting homologous recombination-proficient triple negative cancer cells with a novel RAD51 inhibitor [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-16-17.

Abstract Obesity-associated asthma is phenotypically characterized by low grade inflammation and resistance to standard therapies. Like many other asthma endotypes, the molecular mechanisms contributing to obesity-associated asthma are diverse and ambiguous. A panoply of metabolites that act as signaling mediators are altered by obesity and may also promote lung dysfunction and a pro-asthma phenotype. Recent metabolomics studies indicate that bile acid profiles are altered in individuals with asthma, thus these metabolites were investigated in a murine model of obesity-associated asthma and clinically. In obese mice with allergic airway disease (AAD), the levels of β-muricholic acid (βMCA) and tauro-β-muricholic (tβMCA) were significantly increased in serum and positively correlated with impaired lung function. Two conjugated bile acids, glycocholic acid (GCA) and glycoursodeoxycholic acid (GUDCA), were elevated in the plasma of high body mass index (BMI) individuals and correlated with both BMI and airway hyperreactivity as measured by FEV1, forced expiratory volume in one second. In vitro differentiated T helper (Th) cells were treated with tβMCA and βMCA, which resulted in changes in T cell metabolism that were specific to Th17 cells. In addition, tβMCA treatment increased IL-17 production from Th17 cells upon stimulation. Finally, the anti-inflammatory and metabolic regulatory activities of nitro-oleic acid (NO2-OA) were evaluated in the murine model of obesity-associated asthma. NO2-OA reduced levels of βMCA and tβMCA, with a concomitant reduction in tissue resistance and elastance, providing a potential approach to improving the current standard of care for obese individuals with asthma. Supported by R01HL146445 (MLM), S10OD023402 (SGW), R61HL157069 (SGW, BAF, FH), R01HL132550 (BAF)

Bile acid profiles are altered in obese individuals with asthma. Thus, we sought to better understand how obesity-related systemic changes contribute to lung pathophysiology. We also test the therapeutic potential of nitro-oleic acid (NO 2 -OA), a regulator of metabolic and inflammatory signaling pathways, to mitigate allergen and obesity-induced lung function decline in a murine model of asthma. Bile acids were measured in the plasma of healthy subjects and individuals with asthma and serum and lung tissue of mice with and without allergic airway disease (AAD). Lung function, indices of inflammation and hepatic bile acid enzyme expression were measured in obese mice with house dust mite-induced AAD treated with vehicle or NO 2 -OA. Serum levels of glycocholic acid and glycoursodeoxycholic acid clinically correlate with body mass index and airway hyperreactivity whereas murine levels of β-muricholic acid and tauro-β-muricholic acid were significantly increased and positively correlated with impaired lung function in obese mice with AAD. NO 2 -OA reduced murine bile acid levels by modulating hepatic expression of bile acid synthesis enzymes, with a concomitant reduction in small airway resistance and tissue elastance. Bile acids correlate to body mass index and lung function decline and the signaling actions of nitroalkenes can limit AAD by modulating bile acid metabolism, revealing a potential pharmacologic approach to improving the current standard of care.

Asthma biomarkers from a variety of sources have variable clinical utility, performance characteristics, and applicability. Sputum mast cells (MC) have been identified in stable, asymptomatic asthmatics, as well as more severe asthmatics. [Foresi, JACI, 1997; Fajt, AAAAI, 2011] Little is known about the treatment response to steroids in sputum MCs from different subtypes of asthma. Induced sputum, lung function, exhaled nitric oxide and questionnaire data was collected at baseline and two weeks after triamcinolone injection (40mg) from 10 asthmatics (6 non-severe, 4 severe) from the University of Pittsburgh site of the Severe Asthma Research Program. Subjects were classified as Th2 high or low based on peripheral blood eosinophilia (≥300/mL). Sputum tryptase was measured by qPCR. Data was analyzed nonparametrically. Th2 high asthmatics (n=4) had significantly greater sputum tryptase mRNA at baseline than Th2 low [median(IQR) 27.5(14.2-696.4) vs. 2.2(0-9.20), p=0.01]. After triamcinolone, Th2 high asthmatics had decreases in sputum tryptase mRNA vs. mixed responses in Th2 low asthma (p=0.03). FEV1%predicted tended to increase following triamcinolone in Th2 high compared to Th2 low asthma [median increase= 6.5% vs. 0.5%, p=0.13]. Th2 high asthma (based on blood eosinophils) associated with higher basal levels of MC biomarkers. These higher levels predicted both a reduction in MC marker mRNA and improvement in lung function. Changes in MC mRNA (and FEV1) in response to triamcinolone in Th2 low asthmatics were inconsistent. While asthmatics with persistent Th2-like inflammation may respond clinically to additional steroid therapy, other therapies targeting alternate pathways may be needed in Th2 low asthma.

BACKGROUND:Severe asthma remains poorly characterized, although it likely consists of at least 1 phenotype with features of TH2-like inflammation. IL1RL1, encoding both the IL-33 receptor, ST2L, and decoy receptor, sST2, has been genetically associated with asthma, though the mechanism for susceptibility remains unknown.OBJECTIVE:Given previous data supporting a role for IL1RL1 in TH2 inflammation, we hypothesized that ST2L expression might be increased in TH2-like asthma and that expression levels would be associated with single nucleotide polymorphisms in IL1RL1, possibly explaining its genetic relationship with asthma. We also sought to evaluate the regulation of ST2L and sST2 in vitro.METHODS:Endobronchial brushings and biopsies were obtained and expression of ST2L compared by severity levels, as well as by TH2-like biomarkers. Subjects were genotyped and the relationship of dichotomous expression of ST2L and sST2 to single nucleotide polymorphisms in IL1RL1 were determined. Epithelial cells were grown in air-liquid interface culture, and ST2L and sST2 responses to IFN-γ and IL-13 were evaluated.RESULTS:ST2L expression was increased in severe asthma (P = .02) and associated with multiple indicators of TH2-like inflammation, including blood eosinophils (P = .001), exhaled nitric oxide (P = .003), and epithelial CLCA1 (P < .0001) and eotaxin-3 (P = .001) mRNA expression. Multiple single nucleotide polymorphisms in IL1RL1 were found in relation to dichotomous expression of both ST2L and sST2. sST2 expression was associated with IFN-γ expression in bronchoalveolar lavage, while inducing its expression in vitro in primary human epithelial cells.CONCLUSION:Both pathologic and genetic approaches support a role for IL1RL1 in severe asthma, as well as TH2-lke asthma, suggesting that targeting this pathway may have therapeutic benefits.

Background: Bronchoalveolar lavage (BAL) fluid prostaglandin D-2 (PGD(2)) levels are increased in patients with severe, poorly controlled asthma in association with epithelial mast cells (MCs). PGD(2), which is generated by hematopoietic prostaglandin D synthase (HPGDS), acts on 3 G protein-coupled receptors, including chemoattractant receptor-homologous molecule expressed on T(H)2 lymphocytes (CRTH2) and PGD(2) receptor 1 (DP1). However, much remains to be understood regarding the presence and activation of these pathway elements in asthmatic patients.Objective: We sought to compare the expression and activation of PGD(2) pathway elements in bronchoscopically obtained samples from healthy control subjects and asthmatic patients across a range of disease severity and control, as well as in relation to T(H)2 pathway elements.Methods: Epithelial cells and BAL fluid were evaluated for HPGDS (quantitative real-time PCR/immunohistochemistry [IHC]) and PGD(2) (ELISA/liquid chromatography mass spectrometry) in relation to levels of MC proteases. Expression of the 2 inflammatory cell receptors DP1 and CRTH2 was evaluated on luminal cells. These PGD(2) pathway markers were then compared with asthma severity, level of control, and markers of T(H)2 inflammation (blood eosinophils and fraction of exhaled nitric oxide).Results: Confirming previous results, BAL fluid PGD(2) levels were highest in patients with severe asthma (overall P = .0001). Epithelial cell compartment HPGDS mRNA and IHC values differed among groups (P = .008 and P < .0001, respectively) and correlated with MC protease mRNA. CRTH2 mRNA and IHC values were highest in patients with severe asthma (P = .001 and P = .0001, respectively). Asthma exacerbations, poor asthma control, and T(H)2 inflammatory markers were associated with higher PGD(2), HPGDS, and CRTH2 levels.Conclusion: The current study identifies coordinated upregulation of the PGD(2) pathway in patients with severe, poorly controlled, T(H)2-high asthma despite corticosteroid use.