Personalized Severe Asthma Management

• Project 1

  Severe asthma is asthma that remains problematic despite maximal intervention with conventional asthma therapies, and it accounts for the majority of the mortality and cost of asthma. Dysfunction of airway β2-adrenergic receptor (β2AR) signaling contributes to severe asthma pathogenesis, but the precise signaling mechanisms responsible are unclear. While activation of β2AR using β-agonist drugs is important in acute asthma treatment, overactivation of β2AR is detrimental and can be fatal. Airway β2ARs signal both through G proteins and through G protein-coupled receptor kinase (GRK)/β-arrestin pathways. Our group has found that airways are regulated by nitric oxide (NO) through S-nitrosylation of thiols to form S-nitrosothiols, including on cysteine residues in proteins, a post-translational modification that alters protein functions. In addition, the endogenous NO-modified low mass thiol, S-nitrosoglutathione (GSNO) lung protein-SNO and is protective in asthma. GSNO reductase (GSNOR) that inactivates GSNO, and mice lacking GSNOR are protected from developing asthma. Since the β2AR can activate NO synthase in the airways to generate NO, there is a need to discover how this promotes endogenous SNO-mediated bronchoprotection.  Our recent work has shown that β2AR is S-nitrosylated after activation, and preventing SNO-β2AR with a point mutation augments β2AR signaling. Importantly, mice bearing β2AR with a knock-in of this mutation are protected from developing asthma. We have previously shown that β2AR regulators GRK2 and β-arrestin2 are S-nitrosylated to inhibit their activity to desensitize the β2AR, and mice bearing GRK2-C340S and β-arrestin2-C253S knock-in exhibit heightened β2AR activity and worsened injury in cardiac models.

  We have begun to test the efficacy of inhaled GSNO to affect bronchorelaxation and improved lung function in patients, and these data and clinical samples uniquely position us to examine the role of S-nitrosylation in regulating the β2AR pathway from bench to bedside.   Additionally, we have shown that GSNO degradation is an important determinant of the clinical biomarker, fraction of exhaled NO (FENO).  These data suggest that FENO reports airway GSNO concentration.

  The Central Hypothesis of Project 1 is that the β2AR signaling system is a key target and mediator of SNO-GSNOGSNOR protective effects in the airways in severe asthma. Our studies will define the role of S-nitrosylation of specific β2AR signaling pathway components in a murine model of asthma, delineate the roles of inhaled GSNO and of GSNO dinitrosylases on β2AR signaling in mice and in humans.

• Project 2

  The airway epithelium, like the renal tubular epithelium, expresses proteins that regulate luminal pH. For example, glutaminase regulates ammonia production; it is inhibited by Th1 cytokines, acidifying the airway. Cystic fibrosis transmembrane regulatory protein regulates outward HCO - transit, and its absence results in luminal acidification. Decreased airway lining fluid pH can promote airway inflammation as well as viral and bacterial replication. We hypothesize that the distal airway epithelial surface is acidic, both because of high CO2 levels and low- pH lamellar bodies; that pH increases as airway lining fluid is swept proximally; and that impaired airway luminal buffering can contribute to the pathophysiology of asthma. In particular, there is evidence that acute asthma exacerbations are associated with a fall in airway luminal pH; an effect reversed by glucocorticoids. Further, some asthma patients have a low luminal pH at baseline. Here, we will study the cellular determinants of airway pH regulation. We will make use of the effect of airway pH to alter airway NO metabolism and bioactivities (Project 1) to map the location and effects of low pH in the asthmatic airway in vivo. We will study the recently discovered effects of androgens (Project 3) to promote the expression of beneficial pH regulatory enzymes in asthma. Finally, we will study the potential benefit of inhaled buffer in asthma patients with exacerbations. Specifically, we will carry out three aims.

  In Aim 1, we will characterize the cellular determinants of airway pH. In Aim 2, we will map the epithelial pH of the normal and asthmatic airway. In Aim 3, we will measure the effect of inhaled buffer on inflammation and response to β2 agonists in asthma. At the end of the project, we anticipate having mapped both the cellular regulation of airway epithelial pH and the anatomic variations in airway pH in health and in asthma; and we will have developed a new, personalized approach to target pH abnormalities in patients with asthma exacerbations.

• Project 3

  Asthma is a sexually dimorphic disease, with women exhibiting higher asthma prevalence and more severe manifestations than men. Projects 3 builds upon the rationale that androgens such as testosterone and dehydroepiandrosterone (DHEA) have been linked to better outcomes in asthma. However, mechanisms of androgen-mediated regulation of airway biology are only incompletely understood. In addition, it is unclear if androgen therapy is beneficial in severe asthma and if specific patient populations exist that could be targeted via a precision medicine approach. Project 3 aims to fill this knowledge gap and identify novel, therapeutically targetable mechanisms of androgen-mediated improvement in lung function. Ultimately, this will lead to novel, targeted and corticosteroid-sparing therapies for patients with severe asthma of either sex. We have generated intriguing new data suggesting a causal relationship between androgens and relief of human asthmatic airflow obstruction. We have found that in women with mild asthma and low DHEA-sulfate plasma levels, DHEA treatment was associated with a significant improvement in FEV1. We’ve also discovered in a cohort of severe asthma patients that a variant in position 1245 of the gene HSD3B1 (encoding an enzyme that increases tissue androgen levels) is associated with a lower FEV1 if DHEA-S plasma levels are low. Lastly, we found that increased androgen receptor mRNA abundance in airway epithelial cells (AECs) is associated with improved lung function and fewer asthma symptoms. These data led us to hypothesize that androgens exert beneficial effects on S- nitrosylation, pH regulation and inflammatory signaling that lead to improved lung function in patients with severe asthma, and that genetic variations in androgen signaling are clinically relevant modifiers of the therapeutic response in these patients. We propose the following: 1) To determine whether androgens favorably impact S-nitrosylation and pH regulation in brushed AECs from patients with severe asthma; 2) To study if androgens regulate pro-inflammatory mediator gene and protein expression in AECs from patients with severe asthma; and 3) To investigate if DHEA improves FEV1 and decreases FeNO in asthma patients with low DHEA-S levels and a favorable HSD3B1 genotype. These studies will provide a better understanding of the yet unknown mechanisms and targets of androgen signaling in severe asthma.

Publications

• A multienzyme S-nitrosylation cascade regulates cholesterol homeostasis.
  Stomberski CT, Venetos NM, Zhou HL, Qian Z, Collison BR, Field SJ, Premont RT, Stamler JS.
  Cell Rep. 2022 Oct 25;41(4):111538. doi: 10.1016/j.celrep.2022.111538.
  PMID: 36288700
• S-Nitroso-l-cysteine and ventilatory drive: A pediatric perspective.
  Hubbard D, Tutrow K, Gaston B.
  Pediatr Pulmonol. 2022 Oct;57(10):2291-2297. doi: 10.1002/ppul.26036. Epub 2022 Jul 24.
  PMID: 35785452
• S-nitrosylation is required for β2AR desensitization and experimental asthma.
  Fonseca FV, Raffay TM, Xiao K, McLaughlin PJ, Qian Z, Grimmett ZW, Adachi N, Wang B, Hausladen A, Cobb BA, Zhang R, Hess DT, Gaston B, Lambert NA, Reynolds JD, Premont RT, Stamler JS.
  Mol Cell. 2022 Aug 18;82(16):3089-3102.e7. doi: 10.1016/j.molcel.2022.06.033. Epub 2022 Aug 4.
  PMID: 35931084
• The global prevalence and ethnic heterogeneity of primary ciliary dyskinesia gene variants: a genetic database analysis.
  Hannah WB, Seifert BA, Truty R, Zariwala MA, Ameel K, Zhao Y, Nykamp K, Gaston B.
  Lancet Respir Med. 2022 May;10(5):459-468. doi: 10.1016/S2213-2600(21)00453-7. Epub 2022 Jan 17.
  PMID: 35051411
• The enzymatic function of the honorary enzyme: S-nitrosylation of hemoglobin in physiology and medicine.
  Premont RT, Singel DJ, Stamler JS.
  Mol Aspects Med. 2022 Apr;84:101056. doi: 10.1016/j.mam.2021.101056. Epub 2021 Nov 28.
  PMID: 34852941
• B-value and empirical equivalence bound: A new procedure of hypothesis testing.
  Zhao Y, Caffo BS, Ewen JB.
  Stat Med. 2022 Mar 15;41(6):964-980. doi: 10.1002/sim.9298. Epub 2022 Jan 10.
  PMID: 35014082
• Photolytic Measurement of Tissue S-Nitrosothiols in Rats and Humans In Vivo.
  Neidigh N, Alexander A, van Emmerik P, Higgs A, Plack L, Clem C, Cater D, Marozkina N, Gaston B.
  Molecules. 2022 Feb 15;27(4):1294. doi: 10.3390/molecules27041294.
  PMID: 35209089
• Hypoxic vasodilatory defect and pulmonary hypertension in mice lacking hemoglobin β-cysteine93 S-nitrosylation.
  Zhang R, Hausladen A, Qian Z, Liao X, Premont RT, Stamler JS.
  JCI Insight. 2022 Feb 8;7(3):e155234. doi: 10.1172/jci.insight.155234.
  PMID: 34914637
• The manifold roles of protein S-nitrosylation in the life of insulin.
  Zhou HL, Premont RT, Stamler JS.
  Nat Rev Endocrinol. 2022 Feb;18(2):111-128. doi: 10.1038/s41574-021-00583-1. Epub 2021 Nov 17.
  PMID: 34789923 
• Use of Fractional Exhaled Nitric Oxide to Guide the Treatment of Asthma: An Official American Thoracic Society Clinical Practice Guideline.
  Khatri SB, Iaccarino JM, Barochia A, Soghier I, Akuthota P, Brady A, Covar RA, Debley JS, Diamant Z, Fitzpatrick AM, Kaminsky DA, Kenyon NJ, Khurana S, Lipworth BJ, McCarthy K, Peters M, Que LG, Ross KR, Schneider-Futschik EK, Sorkness CA, Hallstrand TS; American Thoracic Society Assembly on Allergy, Immunology, and Inflammation.
  Am J Respir Crit Care Med. 2021 Nov 15;204(10):e97-e109. doi: 10.1164/rccm.202109-2093ST.
  PMID: 34779751