ALX receptor ligands define a biochemical endotype for inflammation-based diseases
11519911 · 2022-12-06
Assignee
Inventors
Cpc classification
G01N2800/245
PHYSICS
A61P29/00
HUMAN NECESSITIES
G01N2800/56
PHYSICS
G01N33/566
PHYSICS
G01N2800/122
PHYSICS
International classification
Abstract
A method to determine the severity of a disease of chronic inflammation in a patient, comprising the steps of (1) collection or preparation of a bodily fluid, tissue or lavage and (2) measurement of ALX receptor ligands or ALX receptor expression in the fluid, tissue, or lavage, wherein the level of ALX receptor ligands predicts a clinical outcome or choice of treatment modality, is disclosed.
Claims
1. A method of mitigating or inhibiting asthma in a patient in need thereof comprising: (a) measuring ALX/FPR2 receptor expression and optionally ligands serum amyloid A (SAA) and lipoxin A4 (LXA4), and analogs or mimetics thereof in a fluid, tissue, or lavage; (b) comparing the expression of ALX/FPR2 to healthy control; and (c) administering LXA4 or analogs thereof in patients having elevated ALX/FPR2 and asthma.
2. The method of claim 1, wherein the fluid, tissue, or lavage is broncholalveolar lavage.
3. The method of claim 2 wherein the measurement is of at least one of ALX/FPR2, LXA.sub.4, 15-epi-LXA.sub.4 and SAA.
4. The method of claim 2 wherein the LXA.sub.4, analogs comprise 15-epi-LXA.sub.4, lipoxin A4 bioactive analogs and mimetics, 17-epi-resolvin D1, and resolvin D1 and resolvin D1 bioactive analogs and mimetics.
5. The method of claim 1 wherein the measurement is monitored over a course of treatment.
Description
DESCRIPTION OF FIGURES
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DETAILED DESCRIPTION OF THE INVENTION
(7) In General
(8) In the study of patient health, inflammation resolution is known as an active process governed by specialized pro-resolving mediators and receptors. ALX/FPR2 receptors (ALX) are targeted by both pro-resolving and pro-inflammatory ligands for opposing signaling events, which suggest to Applicants pivotal roles for ALX in the fate of inflammatory responses.
(9) In the Examples below, Applicants investigated whether ALX expression and ligands were linked to severe asthma (SA). ALX expression and levels of pro-resolving ligands (Lipoxin A.sub.4 (LXA.sub.4), 15-epi-LXA.sub.4 and Annexin A1 (ANXA1)), and a pro-inflammatory ligand (Serum Amyloid A (SAA)) were measured in bronchoscopy samples collected in Severe Asthma Research Program-3 (SA (n=69), non-severe asthma (NSA, n=51) or healthy donors (HD, n=47)).
(10) In brief, we found that bronchoalveolar lavage (BAL) fluid LXA.sub.4 and 15-epi-LXA.sub.4 were decreased and SAA was increased in SA relative to NSA (median SAA: 11.35 pg/μg protein in SA compared with 0 pg/μg protein in NSA). BAL macrophage ALX expression was increased in SA (BAL macrophage ALX index (MFI) ˜5 in SA compared with ˜3 in healthy donor). Subjects with LXA.sub.4low/SAAhigh levels had increased BAL neutrophils, more asthma symptoms, lower lung function, and increased relative risk for asthma exacerbation, sinusitis and gastroesophageal reflux disease and were assigned more frequently to SA clinical clusters. SAA and aliquots of LXA.sub.4low/SAAhigh BALF induced interleukin-8 production by lung epithelial cells expressing ALX receptors, which was inhibited by co-incubation with 15-epi-LXA.sub.4.
(11) Applicants conclude that these findings have established an association between select ALX receptor ligands and severity of chronic inflammatory diseases, such as asthma, that define a new biochemical endotype for inflammatory disease and support a pivotal functional role for ALX signaling in the fate of tissue inflammation.
(12) Present Invention
(13) a. Diagnostic Method
(14) In one embodiment, the present invention is a method of determining the severity of (and/or treatment for) a disease of chronic inflammation in a patient comprising: (1) collection or preparation of a bodily tissue, fluid, or lavage and (2) measurement of levels of ALX receptor ligands in the fluid, tissue, or lavage. The level of ALX receptor ligands or the ratio of the ligands predicts a clinical outcome or choice of treatment modality.
(15) By “ALX receptor ligands” we mean to specifically include Lipoxin A.sub.4 (LXA.sub.4) and 15-epi-LXA.sub.4 to levels of HD control or below to represent an inadequate counter-regulatory response to asthmatic inflammation and likely consistent with a diagnosis of SA relative to NSA.
(16) Preferably, one would also measure the ALX receptor ligand serum amyloid A (SAA). In some patients, the levels of SAA increase with airway inflammation in SA. We would correlate an increase in BALF SAA to 1 pg/μg protein or more to be associated with SA. Examined together, the levels of lipoxinA.sub.4 (low) and serum amyloid A (high) predict a severe disease clinical outcome, including more lung inflammation, more asthma symptomology, lower lung function and an increased risk for asthma co-morbidities.
(17) For example, severe immunological diseases, such as severe asthma, would have at least a 3-fold and preferably at least a 5-fold or greater increase in the ratio (SAA/LXA4) relative to non-severe asthma. For more information regarding the ratio, one is directed to FIG. 5 in JCI Insight, 2017:2[4] e93534, Ricklefs, et al, ALX receptor ligands define a biochemical endotype for severe asthma, incorporated by reference herein.
(18) Controls for the method of the present invention might be either milder/reversible forms of the same disease (as here with NSA) or potentially healthy subjects. Tissue would be a preferred standard for the measurements.
(19) In one embodiment of the invention, the disease of chronic inflammation disease is lung inflammation and may be severe asthma (SA) or cystic fibrosis. In another embodiment, the lung inflammation is part of COPD, bronchiectasis, bronchiolitis, interstitial lung disease, transplant rejection, pneumonitis, lung abscess, pulmonary vascular disease, pulmonary emboli, or ARDS (acute respiratory distress syndrome).
(20) In another embodiment of the invention, the chronic inflammation disease is selected from the group consisting of lung inflammation, arthritis, inflammatory bowel disease atherosclerosis and psoriasis.
(21) If one were to diagnose a non-SA inflammatory disease, the biomarkers and ranges the expected to be similar if not the same. One would most preferably use afflicted tissue, which would be specific to each indication.
(22) In the present invention, one must first examine patient tissue, fluid or lavage. In some embodiments of the invention, especially those involved with SA diagnosis, the fluid or lavage is bronchoalveolar lavage. In other embodiments, the tissue or fluid is selected from the group consisting of blood, serum, plasma, urine, nasal lavage, sputum, exhaled breath condensate, tears, breast milk, semen, pleural fluid, pericardial fluid, and joint fluid.
(23) In another embodiment of the invention, one would use the information obtained above to determine a treatment regimen. An evaluation of the markers listed above would allow one to determine that the patient has an inflammatory disease, such as SA, and a pro-resolving mediator would be indicated.
(24) Most important is that the pro-resolving mediator serve as a ligand for the ALX/FPR.sub.2 receptor. 15-epi-LXA.sub.4 is such a ligand. It serves as an allosteric inhibitor of the pro-inflammatory signaling serum amyloid A at the same receptor and also mediates pro-resolving actions via interactions with the same ALX/FPR.sub.2 receptor. In addition to 15-epi-LXA.sub.4, other pro-resolving ligands for ALX/FPR.sub.2 include lipoxin A.sub.4, lipoxin A.sub.4 bioactive analogs and mimetics, 17-epi-resolvin D1, and resolvin D1 and resolvin D1 bioactive analogs and mimetics. For example, one may examine U.S. Pat. Nos. 7,906,678 and 6,831,186 for exemplary Lipoxin A.sub.4 analogs. Other exemplary analogs are disclosed in U.S. Pat. No. 7,700,650, PCT/US02/06404, PCT/US02/35860, and PCT/US00/06582.
(25) In another embodiment, one would examine tissue, fluid or lavage for the expression of the ALX/FPR.sub.2 receptor by macrophages (in the tissue, fluid or lavage). An increase compared to control samples predicts a severe disease clinical outcome, including more lung inflammation, more asthma symptomology, lower lung function and an increased risk for asthma co-morbidities. The Example below discloses a preferred embodiment where the increase in receptor expression predicted SA.
(26) In this embodiment of the invention, one would be evaluating the increase in the following manner: An increase of preferably at least 5 would indicate an increased likelihood of a diagnosis of SA rather than NSA.
(27) a. Measurement of Biomarkers
(28) In another embodiment, the present invention is a method of examining a patient tissue, fluid, or lavage and measuring or examining the levels of specific ALX receptor ligands, such as Lipoxin A.sub.4 (LXA.sub.4), 15-epi-LXA.sub.4, and serum amyloid A (SAA). In one preferred embodiment, one would measure all three markers. In another embodiment, one would measure LXA.sub.4 and 15-epi-LXA.sub.4 or LXA.sub.4 along with SAA. In a preferred version of the invention, one would measure the markers and determine the ratio of LXA.sub.4 and SAA.
(29) In some embodiments of the invention, especially those involved with SA diagnosis, the fluid or lavage is bronchoalveolar lavage. In other embodiments, the tissue or fluid is selected from the group consisting of blood, serum, plasma, urine, nasal lavage, sputum, exhaled breath condensate, tears, breast milk, semen, pleural fluid, pericardial fluid, and joint fluid.
(30) The markers are typically measured by methods currently known in the art. For example, one might use the ELISA methods described below and in standard references. One could obtain useful antibodies at a custom research products organization, such as Cayman Chemical (Ann Arbor, Mich.). Other methods might include physical methods of detection such as lipidomics and proteomics.
EXAMPLES
(31) Subject Characteristics
(32) Subjects with SA and NSA, and non-asthmatic HD were recruited to participate in SARP-3 at seven research centers across the United States. Relative to NSA, subjects with SA had increased symptoms as manifested by lower Asthma Control Test (ACT) and higher Asthma Control Questionnaire (ACQ) scores. Spirometric measures of lung function were lower in SA than NSA and HD despite the SA cohort's use of more asthma-targeted medications (Table 1). A subset of subjects agreed to bronchoscopy with bronchoalveolar lavage (BAL) as part of their baseline phenotyping. SA subjects had more lung inflammation with increased BAL neutrophils (Table 1).
(33) Severe asthma subjects have decreased lipoxins and increased SAA and macrophage ALX receptor expression.
(34) The fate of innate inflammatory responses is dictated in part by ALX receptor signaling (16, 21), so the presence of BAL fluid (BALF) ALX ligands with pro-inflammatory (i.e., SAA) or pro-resolving properties (i.e., LXA.sub.4, 15-epi-LXA.sub.4 and ANXA1) and BAL cell surface ALX receptor expression were determined. SA subjects had significantly less BALF LXA.sub.4 (median: 0.23 pg/μg protein, mean: 0.28 pg/μg protein) and 15-epi-LXA.sub.4 (median: 1.02 pg/μg protein, mean: 1.24 pg/μg protein) than NSA subjects (LXA.sub.4: median 0.32 pg/μg protein, mean 0.40 pg/μg protein; 15-epi-LXA.sub.4: median 1.47 pg/μg protein, mean 1.88 pg/μg protein) (
(35) ALX ligands differentially correlate with asthma inflammation, symptoms, and lung function.
(36) To screen for relationships between the BALF ALX ligand levels and asthma clinical parameters, a Pearson correlation matrix was constructed using measures of disease activity from all asthma subjects (NSA and SA; n=120) (
(37) BAL neutrophilia in severe asthma is associated with decreased LXA.sub.4 and increased SAA levels.
(38) BAL leukocyte subsets were determined in NSA and SA subjects and HD. As expected, macrophages were the most numerous BAL cell type accounting for 88-92% of BAL leukocytes (
(39) ALX receptor ligands and expression are associated with asthma symptoms.
(40) To further investigate relationships between the ALX signaling pathway and measures of asthma symptoms, the continuous variables for BAL ALX ligands were converted to categorical high and low subgroups using the median value to define a cutoff between the subgroups. Using the median BALF SAA value (1.22 pg/μg protein;
(41) Because BALF LXA.sub.4 levels were inversely related to SAA levels and asthma symptoms by Pearson correlation (
(42) With opposing relationships for asthma symptoms for the ALX ligands SAA and LXA.sub.4, we next performed similar analyses for the BAL macrophage ALX index. Asthma subjects were categorized into ALX.sup.high and ALX.sup.low cohorts using the median ALX Index to define the cutoff between subgroups (median=4.61;
(43) SAA and LXA.sub.4 levels together represent a biochemical endotype that distinguishes SA from NSA.
(44) Individually, SAA and LXA.sub.4 levels were both associated with asthma severity. More SA subjects had SAA.sup.high and LXA.sub.4.sup.low levels and more NSA subjects had SAA.sup.low and LXA.sub.4.sup.high levels (
(45) Cluster analyses from SARP-1 used clinical parameters to identify five asthma subtypes (mild allergic asthma, mild-moderate allergic asthma, more severe older onset asthma, severe variable allergic asthma, and severe fixed airflow asthma) (5). Using this classification here with SAA and LXA.sub.4 as biochemical markers of ALX receptor signaling, it was notable that 71% of the LXA.sub.4.sup.highSAA.sup.low group were assigned to one of the NSA clusters and 64% of the LXA.sub.4.sup.lowSAA.sup.high subjects were assigned to one of the SA clusters (
(46) SAA is an acute phase protein and with the relationship for the LXA.sub.4.sup.lowSAA.sup.high endotype to asthma severity and neutrophilic lung inflammation, we next determined if there was a relationship for LXA.sub.4 and SAA to exacerbations and common asthma comorbidities. Both high SAA and low LXA.sub.4 levels were significantly related to sinusitis, gastroesophageal reflux disease (GERD) and obesity (BMI>30) and low LXA.sub.4 was also related to history of more frequent acute exacerbation over the prior year (Table 2). Together, the combination of low LXA.sub.4 levels and high SAA levels (i.e. the LXA.sub.4.sup.lowSAA.sup.high endotype) was even more closely associated with these asthma comorbidities (Table 2).
(47) SAA and 15-epi-LXA.sub.4 signaling via ALX receptors regulates production of the neutrophil chemoattractant IL-8.
(48) To determine the functional relationship for these ALX receptor ligands, we next turned to an ALX-dependent in vitro reporter assay that we have previously qualified in chronic obstructive pulmonary disease (18). A549 lung epithelial cells were stably transfected to express the human ALX/FPR2 receptor (hALX). BALF samples were selected from HD, NSA, and SA subjects with representative levels of ALX ligands (
DISCUSSION
(49) Here, in SARP-3, we measured the abundance of four ligands for ALX receptors, namely LXA.sub.4, 15-epi-LXA.sub.4, ANXA1 and SAA, with the potential for opposing effects on asthmatic airway responses. In SA, BAL macrophage ALX receptor expression was increased and BALF ligands for ALX were selectively regulated. BALF levels of pro-resolving lipoxins were decreased and levels of pro-inflammatory SAA were increased in SA. Levels of lipoxins inversely correlated to SAA, BAL neutrophils and asthma symptoms and lipoxins were positively correlated to measures of lung function. SAA.sup.high and ALX.sup.high subjects more commonly had SA with increased ACQ and decreased ACT scores. When LXA.sub.4 and SAA levels were considered together in a combined endotype, a stronger association with asthma symptoms, lung function, and airway neutrophilia was noted than when either mediator was considered individually. LXA.sub.4.sup.lowSAA.sup.high subjects had more lung inflammation and asthma symptoms and lower lung function relative to LXA.sub.4.sup.highSAA.sup.low subjects. LXA.sub.4.sup.lowSAA.sup.high and LXA.sub.4.sup.highSAA.sup.low subjects segregated to SA and NSA clinical clusters, respectively. Importantly, LXA.sub.4.sup.lowSAA.sup.high subjects had significantly increased likelihood for asthma exacerbation in the past year and for the asthma comorbidities of sinusitis, GERD and obesity. Exposure to SAA or BALF from SA subjects increased production of the neutrophil chemoattractant IL-8 by ALX-expressing human lung epithelial cells in vitro. This SAA-driven IL-8 production by epithelial cells was mitigated by exposure to 15-epi-LXA.sub.4 at pharmacological doses, supporting a functional interaction between the ALX ligands relevant to the neutrophilic inflammation in SA. Together, these findings support a mechanistic role for ALX receptor signaling by SAA and LXA.sub.4 in lung inflammation in SA that defines a new biochemical endotype for patient stratification in asthma.
(50) ALX receptors are intriguing targets for regulating the fate of inflammatory responses. LXA.sub.4 and SAA interact with ALX receptors to exert opposing effects on inflammation (18). ALX receptors are seven-membrane-spanning, G-protein coupled receptors that are present on several lung cell types relevant to asthma pathogenesis, including neutrophils (22), eosinophils (23), group 2 innate lymphoid cells (24), natural killer cells (24), lymphocytes (25), monocytes (26), dendritic cells (27, 28), macrophages (29) and airway structural cells (30). LXA.sub.4 is a high affinity ligand for ALX that signals for anti-inflammatory and pro-resolving cellular responses (16). SAA binds with lower affinity, yet this acute phase reactant is substantially more abundant than LXA.sub.4 during infection and the upstroke of acute inflammation (18). Also relevant in SA patients with comorbidities, corticosteroids can enhance SAA production, especially in conjunction with LPS (18). Distinct from LXA.sub.4's interaction with the seventh transmembrane domain and adjacent regions (31, 32), SAA interacts with the first and second extracellular loop domains (33), resulting in a marked shift in receptor conformation and dimerization that changes the receptor's pro-resolving signaling to pro-inflammatory signaling (17). Here in SA, ALX expression was increased on BAL macrophages and associated with increased asthma symptoms. Macrophage ALX expression correlated with surface CD206 expression, which marks “M2” macrophages that participate in endogenous pathways of inflammation resolution (34, 35). Together with the increased SAA and decreased lipoxins in SA BALF, the increase in ALX expression on BAL macrophages likely reflects SAA-driven outcomes, including the increased lung inflammation (i.e., BAL neutrophilia) despite higher doses of corticosteroids. With the presence of SAA and LXA.sub.4 in proximity to ALX receptors in the lung and their divergent influences on inflammatory responses, ALX receptors are poised to serve as a pivotal signaling nexus for acute inflammation or its resolution.
(51) Lipoxins are products of arachidonic acid metabolism that are structurally and functionally distinct from leukotrienes and prostaglandins. Lipoxin A.sub.4 was first detected in humans in BALF from patients with lung disease, including asthma (36). Lipoxins are the lead members of a new genus of endogenous chemical signals termed specialized pro-resolving mediators (SPMs), which are partially defined by their ability to inhibit granulocyte recruitment and activation in inflamed tissues as well as to promote macrophage-mediated clearance of dead cells, microbes and debris in catabasis (8). Distinct from increased leukotriene production by some asthmatic patients, lipoxin levels are decreased in uncontrolled and severe asthma (13). Current anti-leukotriene drugs would not be expected to increase lipoxins in asthma. There are likely multiple factors responsible for the defective lipoxin production in severe asthma; however, the increased oxidative stress in SA airways was recently determined to be a major cause for reduced lipoxin generation (11). In human studies, inhaled LXA.sub.4 dampens bronchoprovocation in asthma (10) and lipoxins regulate cytokine mediated increases in bronchial constriction to methacholine, histamine and thromboxane (11). Recently, a stable LXA.sub.4 analog was shown to markedly decrease allergic inflammation and symptoms in patients with juvenile eczema (37). Preclinical studies have established that LXA.sub.4 analogs that resist metabolic inactivation can prevent and potently reduce allergen-driven airway hyper-responsiveness to methacholine, airway mucus metaplasia and Type 2 inflammation (38, 39). Transgenic expression of hALX receptors also display decreased inflammatory responses to allergen, supporting a role for endogenous ALX ligands in anti-inflammation and pro-resolution (23). In addition to ALX, lipoxins can interact with additional receptors, including cysLT1—the pharmacological target of one of the classes of anti-leukotriene drugs (40). Together, these findings point to pivotal pro-resolving roles for lipoxins in health to control airway phlogistic responses and promote their resolution. Here, the diminished BALF levels of lipoxins in SA would be predicted from prior publications to disable a major endogenous regulatory pathway for airway inflammation, mucus and hyper-reactivity and our results show a strong and consistent correlation between low LXA.sub.4 and increased lung inflammation, asthma symptoms and comorbidities, and lower lung function in SA.
(52) In contrast to lipoxins, there are several peptide ligands for ALX receptors that engage these receptors to promote inflammatory responses. The acute phase reactant SAA is one of the ALX peptide ligands and can induce neutrophil chemotaxis and activation via ALX (41, 42). SAA is increased in severe allergic asthma (43) and can prevent dendritic cell apoptosis to induce glucocorticoid resistance for CD4.sup.+ T cells (44). Here, BALF SAA levels were increased in SA and strongly associated with BAL neutrophils. SAA.sup.high subjects had increased asthma symptoms and a higher relative risk of sinusitis, GERD and obesity. If subjects had both low LXA.sub.4 levels and high SAA levels (i.e., LXA.sub.4.sup.lowSAA.sup.high) then their relative risk for BAL neutrophils, asthma symptoms, and lower lung function were all increased. Recently, some subjects with SA with non-Type 2 inflammation were identified as IL-6.sup.high (45). IL-6 induces SAA expression (46) and may conspire with this acute phase protein to activate neutrophils and non-Type-2 lung inflammation in SA, in particular in those with systemic metabolic alterations associated with obesity. With A549.sup.hALX epithelial cells, BALF from LXA.sub.4.sup.lowSAA.sup.high subjects increased production of the neutrophil chemoattractant IL-8, which was inhibitable by 15-epi-LXA.sub.4. At ALX receptors, 15-epi-LXA.sub.4 inhibition of SAA is allosteric in nature (18), and when given at pharmacological doses, 15-epi-LXA.sub.4 can decrease SAA-driven IL-8 production by human airway epithelial cells in vitro and SAA-mediated acute inflammation in vivo in mice (18). Of interest for SA, SAA production is increased by corticosteroids and its expression is synergistically increased by the combination of steroids and LPS (18). Additional soluble mediators acting via distinct or synergistic pathways with SAA also are likely to contribute to epithelial cell IL-8 production and neutrophil chemoattraction in severe asthma. Subjects enrolled in SARP-3 were clinically characterized before and after intramuscular triamcinolone and adult subjects with SA continued to manifest lower FEV.sub.1 and worse asthma control as compared to NSA after the systemic corticosteroids (47). Of note, BALF levels of LXA.sub.4, 15-epi LXA.sub.4, and SAA were not significantly altered by a single dose of intramuscular triamcinolone when measured 3 to 6 weeks after steroid administration. Unlike 15-epi-LXA.sub.4, corticosteroids do not inhibit SAA-mediated lung inflammation (18), suggesting that for some subjects with SA their chronic neutrophilic lung inflammation could be perpetuated by corticosteroids and that SAA levels could inform more precise asthma management by helping to identify subjects at risk for this unintended consequence of corticosteroids.
(53) Biochemical analyses here have linked ALX receptor signaling to the pathophysiology of SA. Using clinical and statistical approaches, five phenotypes of adult asthma have been defined (5), but there remains a need to connect these phenotypes to distinct molecular mechanisms for SA pathogenesis (6). We chose a candidate pathway approach based on preclinical evidence that linked ALX signaling to SA and BAL LXA.sub.4 and SAA levels segregated subjects into discrete clinical clusters, suggesting that this biochemical pathway could convey additional value for patient stratification as an asthma endotype. Findings here suggest that these ALX ligands should be included in future studies designed to comprehensively model genetic, metabolic, and environmental influences and clinical characteristics for patient endotyping in SA.
(54) Here, we have identified significant associations for BALF LXA.sub.4.sup.lowSAA.sup.high levels with neutrophilic lung inflammation and poorly controlled asthma; however, there are several potential limitations to consider. While it was advantageous for biochemical analyses to have a relatively large number of BAL samples from this carefully phenotyped group of asthma subjects, it would not be practical to routinely perform bronchoscopy in a clinical (or clinical trial) setting. It will be important in future studies to obtain and analyze respiratory samples collected by less invasive means (i.e., sputum, exhaled breath condensate) to determine the influence of anatomic compartment on the relationships uncovered here with bronchoscopy specimens. The cross-sectional nature of the analyses here does not address the stability of this endotype, a question best addressed with samples obtained by less invasive means. Regarding additional ALX ligands, the ELISA used here for ANXA1 does not distinguish between intact and cleaved protein, so the absence of a relationship here does not preclude its potential existence when more specific experimental tools become available. There are also several additional peptide and lipid ligands for ALX receptors that might further enhance the discriminatory power of ALX signaling for identification of asthma endotypes.
(55) In summary, ALX receptor expression was increased in asthmatic BAL macrophages and we have identified a cassette of ALX receptor ligands that are selectively regulated in BALF in asthma. Levels of lipoxins and SAA correlated with lung inflammation and clinical parameters of asthma control. In particular, subjects with LXA.sub.4.sup.lowSAA.sup.high BALF were more likely to have SA with increased BAL neutrophils, asthma symptoms and asthma comorbidities and decreased lung function. At pharmacological levels, 15-epi-LXA.sub.4 functionally opposed SAA signaling at ALX receptors to inhibit production of the neutrophil chemoattractant IL-8. BALF LXA.sub.4.sup.lowSAA.sup.high subjects were assigned to discrete clinical clusters from LXA.sub.4.sup.highSAA.sup.low subjects, suggesting that these biochemical mediators could identify subgroups of asthma subjects and serve as a new asthma biochemical endotype for non-Type 2, steroid resistant inflammation in SA.
(56) Materials and Methods
(57) Study Design
(58) SARP-3 is an NHLBI-funded study designed to characterize molecular, cellular and physiological phenotypes in subjects with SA and NSA (NCT01606826). Asthmatic and healthy subjects were recruited and completed baseline characterization with some subjects agreeing to bronchoscopy. Details regarding SARP methods, subject enrollment, and study procedures can be found in reference (45).
(59) Participants and Sample Collection
(60) Subjects 13 years of age and older with asthma and healthy control subjects were recruited between November 2012 and February 2015 by seven geographically dispersed, research centers in the USA. European Respiratory Society/American Thoracic Society guidelines were used to categorize subjects as SA or NSA (48). Control subjects were individuals who reported general health and were non-smokers with no history of lung disease, atopic disease or allergic rhinitis. BAL was performed with three 50 mL aliquots of warm saline, and BALF were recovered by hand suction. Subjects received intramuscular triamcinolone (1 mg/kg up to a maximum dose of 40 mg) and some subjects agreed to undergo a second bronchoscopy 3 to 6 weeks later and BAL samples were collected in the same manner. BAL cells were enumerated and differential leukocyte counts determined. Cell-free BALF supernatant was divided into several aliquots. One aliquot of BALF (1 ml) was directly mixed with iced methanol (2 ml, for 1:2, vol/vol) before storing at −80° C. The other aliquots were directly stored at −80° C. The stored aliquots were later shipped to Brigham and Women's Hospital for analyses.
(61) Lipid Extraction
(62) Aliquots of BALF with methanol (1:2, vol:vol) were brought to dryness in vacuo using a BÜCHI Rotavapor R-200/205. The samples were resuspended with methanol (500 μl) and distilled/deionized water (10 ml) followed by extraction using C18 SepPak cartidges (Waters, Milford, Mass.) as in (13). The methyl formate fraction was brought to dryness under a gentle stream of nitrogen and each sample was resuspended in 1 ml of methanol and stored at −80° C. until LXA.sub.4 and 15-epi-LXA.sub.4 ELISAs were performed.
(63) Protein Assay
(64) Pierce BCA Protein Assay Kit (Thermo Fisher) was used for BALF protein determination. Samples with less than 25 μg of protein were excluded from further analysis (n=3; 1 NSA, 2 SA).
(65) ELISA
(66) LXA.sub.4 and 15-epi-LXA.sub.4 levels in the BALF were measured by ELISA (Neogen, Lexington, Ky.). Extracted BALF samples stored in methanol were brought to dryness under a gentle stream of nitrogen and resuspended in ELISA buffer. SAA and ANXA1 levels were measured by ELISA in aliquots of BALF stored in the absence of methanol (SAA: Abazyme, Needham, Mass.; ANXA1: Cloud-Clone, Houston, Tex.). LXA.sub.4, 15-epi-LXA.sub.4, SAA and ANXA1 levels were normalized to the total protein content of the BALF. Some subjects had an SAA level below the limit of detection and these samples were assigned a value of 0 pg/μg protein for analysis. The median value for LXA.sub.4 and SAA were used to segregate subjects into “high” and “low” subgroups.
(67) Flow Cytometry
(68) BAL cell pellets were available from a subgroup of subjects. For ex vivo staining, BALF cells were blocked with mouse serum (Sigma, St. Louis, Mo.) in PBS for 30 minutes at 4° C. The cells were then incubated with Viability Dye eFluorR660 (eBioscience, San Diego, Calif.) as per manufacturer's instructions followed by 30 minutes of incubation with the following antibodies to human proteins: anti-ALX (clone 304405, R&D Systems, Minneapolis, Minn.)-PerCP, anti-HLA-DR (major histocompatibility complex class II, L243)-allophycocyanin-Cy7 (APC-Cy7) (BD Biosciences, San Jose, Calif.) and anti-CD206 (macrophage mannose receptor, 19.2)-phycoerythrin (PE) (eBioscience) or with directly conjugated unrelated antibodies of the same isotype (BD Biosciences) at 4° C. Data were acquired on a Canto II flow cytometer (Becton Dickinson) and analyzed using FlowJo software version 10.1 (TreeStar, Ashland, Oreg.). Macrophages were identified as single cells (by doublet exclusion), viable (Viability Dye eFluorR660 negative), CD206.sup.+ cells. The MFI of ALX, MHC class II and CD206 was assessed on macrophages and normalized with the MFI of the isotype control antibody (MFI cell surface marker/MFI isotype control=MFI index).
(69) In Vitro A549 Cell Culture
(70) A549 transfected to stably express the human ALX receptor were used (as in (18)). A549.sup.hALX cells were seeded into a 48-well plate (5×10.sup.4 cells/well) and cultured in RPMI 1640 (Lonza) supplemented with 2 mM L-glutamine, 10% heat-inactivated fetal calf serum (Gibco), penicillin (100 IU/mL) and streptomycin (100 μg/mL) at 37° C. in 5% CO.sub.2 until confluent. When confluent, A549.sup.hALX cells were cultured with serum free media for 16 hours and then exposed to BALF (100 μl) and serum-free RPMI media (1001 μl, 1:1 vol:vol) for 24 hours (37° C., 5% CO.sub.2). In select experiments, recombinant human SAA (0-10 ng/ml, Peprotech), 15-epi-LXA.sub.4 (100 nM, Cayman Chemical) or vehicle control was added. At the end of the incubations, supernatants were collected on ice and stored at −80° C. IL-8 levels in the supernatants were measured by ELISA (R&D). If there was no increase in IL-8 production after exposure to BALF, the samples were assigned a value of zero (
(71) Statistical Analysis
(72) In figures, data are expressed either individually with indication of the median value or as mean±SEM; in tables, data are expressed as mean±SD. For violin plots in
(73) TABLE-US-00001 TABLE 1 Clinical characteristics and bronchoalveolar lavage leukocytes for subjects undergoing bronchoscopy.sup.# Healthy Non-Severe Severe Donors (HD) Asthma (NSA) Asthma (SA) NSA vs SA HD vs. NSA HD vs. SA No. of subjects 47 51 69 Clinical data Age 40.1 ± 12.9 36.9 ± 12.4 42.4 ± 13.6 ns ns ns (20-62) (18-61) (14-67) % Male 19 (40%) 17 (33%) 24 (35%) ns ns ns % African American 11 (23%) 14 (27%) 22 (32%) ns ns ns % White 31 (66%) 37 (74%) 45 (65%) ns ns ns BMI 27.6 ± 5.7 30.0 ± 9.2 31.4 ± 8.2 ns ns * (20-44) (18-61) (19-67) Symptom control % Uncontrolled.sup.§ n.a. 32 (63%) 68 (98%) **** ACQ n.a. 1.08 ± 0.9 1.93 ± 1.1 **** (0-3) (0-5) ACT n.a. 19.61 ± 34.0 14.48 ± 4.5 **** (7-25) (5-23) Lung function FEV1 % predicted 102.4 ± 12.5 89.8 ± 16.3 75.0 ± 19.1 **** *** **** (78.46-139.2) (42-124) (35-116) FVC % predicted 104.7 ± 14.2 102.6 ± 17.2 89.4 ± 17.6 **** ns **** (84.12-137.2) (64-145) (52-133) FEV1/FVC 97.7 ± 5.6 87.5 ± 9.0 82.8 ± 9.0 * **** **** (85.4-109.6) (61-109) (62-106) Medications Inhaled corticosteroids 0 (0%) 35 (69%) 67 (97%) **** High dose of inhaled 0 (0%) 4 (8%) 66 (96%) **** corticosteroids Oral steroids 0 (0%) 0 (0%) 16 (23%) **** Long acting beta agonists 0 (0%) 21 (37%) 64 (93%) **** Long acting anticholinergic 0 (0%) 0 (0%) 3 (4%) p = 0.13 medication Leukotriene receptor 0 (0%) 11 (22%) 24 (35%) p = 0.12 antagonists Omalizumab 0 (0%) 1 (2%) 8 (13%) * BAL leukocyte differentials Total cell count (millions) 4.9 ± 4.6 4.0 ± 2.7 6.4 ± 13.2 ns ns ns (0-25) (0-12) (0-107) Macrophages (%) 91.7 ± 5.8 92.2 ± 5.1 87.7 ± 10.1 ** ns * (73-99) (79-99) (53-99) Neutrophils (%) 1.6 ± 1.9 1.4 ± 1.4 3.3 ± 4.9 ** ns * (0-10) (0-7) (0-24) Eosinophils (%) 0.3 ± 0.4 1.1 ± 2.8 1.8 ± 5.0 ns ns ns (0-2) (0-17) (0-35) Lymphocytes (%) 6.5 ± 5.0 5.3 ± 4.5 7.3 ± 5.9 ns ns ns (0-22) (0-19) (0-34) .sup.#Values represent the mean ± SD (range). .sup.§Uncontrolled symptoms were defined as the occurrence of one of the following: ≥2 steroid bursts, hospitalization, intensive care unit admission, use of a ventilator, FEV1% predicted < 80%, ACT < 20, or self reported worsening with tapering steroids. Definition of abbreviations: BMI = body mass index, ACQ = asthma control questionnaire, ACT = asthma control test, FEV1 = forced expiratory volume in one second, FVC = forced vital capacity, n.a. = not applicable *P < 0.05, **P < 0.01, ****P < 0.001, ns = not significant. Comparison between three groups was performed by One-way-ANOVA followed by Tukey-test to adjust for multiple comparisons and Chi-squared test. Comparison between two groups was performed by Student's t-test.
(74) TABLE-US-00002 TABLE 2 Asthma exacerbations and comorbidities are associated with high SAA and low LXA.sub.4..sup.# p-value (Chi- Squared with SAA low SAA high p-value Bonferroni (n = 60) (n = 60) (Chi-Squared) correction) >1 AE 21 (35%) 24 (40%) n.s. n.s. Sinusitis 12 (20%) 24 (40%) 0.01 0.04 GERD 18 (30%) 28 (47%) 0.04 n.s. BMI > 30 19 (32%) 32 (53%) 0.01 0.04 LXA.sub.4 low LXA.sub.4 high (n = 60) (n = 60) >1 AE 29 (48%) 16 (27%) 0.01 0.04 Sinusitis 24 (40%) 12 (20%) 0.02 n.s. GERD 29 (48%) 17 (28%) 0.01 0.04 BMI > 30 34 (57%) 17 (28%) 0.002 0.008 SAA low SAA high LXA.sub.4 high LXA.sub.4 low (n = 40) (n = 39) >1 AE 10 (25%) 18 (46%) <0.05 n.s. Sinusitis 6 (15%) 18 (46%) 0.003 0.01 GERD 11 (28%) 22 (56%) 0.009 0.04 BMI > 30 11 (41%) 26 (74%) <0.001 <0.005 .sup.#Results are expressed as number of patients (percentage). Definition of abbreviations: AE = acute exacerbation, GERD = gastroesophageal reflux, BMI = body mass index; n.s. = not significant.
REFERENCES
(75) 1. National Current Asthma Prevalence (2014). https://www.cdc.gov/asthma/most recent data.htm. 2. Levy B D, Noel P J, Freemer M M, Cloutier M M, Georas S N, Jarjour N N, et al. Future Research Directions in Asthma. An NHLBI Working Group Report. Am J Respir Crit Care Med. 2015; 192(11):1366-72. 3. Ray A, Raundhal M, Oriss T B, Ray P, and Wenzel S E. Current concepts of severe asthma. The Journal of clinical investigation. 2016; 126(7):2394-403. 4. Modena B D, Bleecker E R, Busse W W, Erzurum S C, Gaston B M, Jarjour N N, et al. Gene Expression Correlated to Severe Asthma Characteristics Reveals Heterogeneous Mechanisms of Severe Disease. American journal of respiratory and critical care medicine. 2016. 5. Moore W C, Meyers D A, Wenzel S E, Teague W G, Li H, Li X, et al. Identification of asthma phenotypes using cluster analysis in the Severe Asthma Research Program. Am J Respir Crit Care Med. 2010; 181(4):315-23. 6. Wenzel S E. Asthma phenotypes: the evolution from clinical to molecular approaches. Nat Med. 2012; 18(5):716-25. 7. Fahy J V. Type 2 inflammation in asthma—present in most, absent in many. Nat Rev Immunol. 2015; 15(1):57-65. 8. Serhan C N. Pro-resolving lipid mediators are leads for resolution physiology. Nature. 2014; 510(7503):92-101. 9. Levy B D, and Serhan C N. Resolution of acute inflammation in the lung. Annual review of physiology. 2014; 76:467-92. 10. Christie P E, Spur B W, and Lee T H. The effects of lipoxin A4 on airway responses in asthmatic subjects. Am Rev Respir Dis. 1992; 145(6):1281-4. 11. Ono E, Dutile S, Kazani S, Wechsler M E, Yang J, Hammock B D, et al. Lipoxin generation is related to soluble epoxide hydrolase activity in severe asthma. American journal of respiratory and critical care medicine. 2014; 190(8):886-97. 12. Kazani S, Planaguma A, Ono E, Bonini M, Zahid M, Marigowda G, et al. Exhaled breath condensate eicosanoid levels associate with asthma and its severity. J Allergy Clin Immunol. 2013; 132(3):547-53. 13. Levy B D, Bonnans C, Silverman E S, Palmer L J, Marigowda G, Israel E, et al. Diminished lipoxin biosynthesis in severe asthma. Am J Respir Crit Care Med. 2005; 172(7):824-30. 14. Planaguma A, Kazani S, Marigowda G, Haworth O, Mariani T J, Israel E, et al. Airway lipoxin A4 generation and lipoxin A4 receptor expression are decreased in severe asthma. Am J Respir Crit Care Med. 2008; 178(6):574-82. 15. Vachier I, Bonnans C, Chavis C, Farce M, Godard P, Bousquet J, et al. Severe asthma is associated with a loss of LX4, an endogenous anti-inflammatory compound. The Journal of allergy and clinical immunology. 2005; 115(1):55-60. 16. Chiang N, Serhan C N, Dahlen S E, Drazen J M, Hay D W, Rovati G E, et al. The lipoxin receptor ALX: potent ligand-specific and stereoselective actions in vivo. Pharmacological reviews. 2006; 58(3):463-87. 17. Cooray S N, Gobbetti T, Montero-Melendez T, McArthur S, Thompson D, Clark A J, et al. Ligand-specific conformational change of the G-protein-coupled receptor ALX/FPR2 determines proresolving functional responses. Proc Natl Acad Sci USA. 2013; 110(45):18232-7. 18. Bozinovski S, Uddin M, Vlahos R, Thompson M, McQualter J L, Merritt A S, et al. Serum amyloid A opposes lipoxin A(4) to mediate glucocorticoid refractory lung inflammation in chronic obstructive pulmonary disease. Proceedings of the National Academy of Sciences of the United States of America. 2012; 109(3):935-40fff. 19. He R, Sang H, and Ye R D. Serum amyloid A induces IL-8 secretion through a G protein-coupled receptor, FPRL1/LXA4R. Blood. 2003; 101(4):1572-81. 20. Perretti M, Chiang N, La M, Fierro I M, Marullo S, Getting S J, et al. Endogenous lipid- and peptide-derived anti-inflammatory pathways generated with glucocorticoid and aspirin treatment activate the lipoxin A4 receptor. Nature medicine. 2002; 8(11):1296-302. 21. Morris T, Stables M, Colville-Nash P, Newson J, Bellingan G, de Souza P M, et al. Dichotomy in duration and severity of acute inflammatory responses in humans arising from differentially expressed proresolution pathways. Proceedings of the National Academy of Sciences of the United States of America. 2010; 107(19):8842-7. 22. Fiore S, Maddox J F, Perez H D, and Serhan C N. Identification of a human cDNA encoding a functional high affinity lipoxin A4 receptor. The Journal of experimental medicine. 1994; 180(1):253-60. 23. Levy B D, De Sanctis G T, Devchand P R, Kim E, Ackerman K, Schmidt B A, et al. Multi-pronged inhibition of airway hyper-responsiveness and inflammation by lipoxin A(4). Nat Med. 2002; 8(9):1018-23. 24. Barnig C, Cernadas M, Dutile S, Liu X, Perrella M A, Kazani S, et al. Lipoxin A4 regulates natural killer cell and type 2 innate lymphoid cell activation in asthma. Sci Transl Med. 2013; 5(174):174ra26. 25. Ariel A, Chiang N, Arita M, Petasis N A, and Serhan C N. Aspirin-triggered lipoxin A4 and B4 analogs block extracellular signal-regulated kinase-dependent TNF-alpha secretion from human T cells. J Immunol. 2003; 170(12):6266-72. 26. Romano M, Maddox J F, and Serhan C N. Activation of human monocytes and the acute monocytic leukemia cell line (THP-1) by lipoxins involves unique signaling pathways for lipoxin A4 versus lipoxin B4: evidence for differential Ca2+ mobilization. J Immunol. 1996; 157(5):2149-54. 27. Aliberti J, Hieny S, Reis e Sousa C, Serhan C N, and Sher A. Lipoxin-mediated inhibition of IL-12 production by DCs: a mechanism for regulation of microbial immunity. Nature immunology. 2002; 3(1):76-82. 28. Yang D, Chen Q, Le Y, Wang J M, and Oppenheim J J. Differential regulation of formyl peptide receptor-like 1 expression during the differentiation of monocytes to dendritic cells and macrophages. J Immunol. 2001; 166(6):4092-8. 29. Maderna P, Cottell D C, Toivonen T, Dufton N, Dalli J, Perretti M, et al. FPR2/ALX receptor expression and internalization are critical for lipoxin A4 and annexin-derived peptide-stimulated phagocytosis. FASEB journal: official publication of the Federation of American Societies for Experimental Biology. 2010; 24(11):4240-9. 30. Bonnans C, Fukunaga K, Levy M A, and Levy B D. Lipoxin A(4) regulates bronchial epithelial cell responses to acid injury. The American journal of pathology. 2006; 168(4):1064-72. 31. Chiang N, Fierro I M, Gronert K, and Serhan C N. Activation of lipoxin A(4) receptors by aspirin-triggered lipoxins and select peptides evokes ligand-specific responses in inflammation. The Journal of experimental medicine. 2000; 191(7):1197-208. 32. Chiang N, Gronert K, Clish C B, O'Brien J A, Freeman M W, and Serhan C N. Leukotriene B4 receptor transgenic mice reveal novel protective roles for lipoxins and aspirin-triggered lipoxins in reperfusion. The Journal of clinical investigation. 1999; 104(3):309-16. 33. Bena S, Brancaleone V, Wang J M, Perretti M, and Flower R J. Annexin A1 interaction with the FPR2/ALX receptor: identification of distinct domains and downstream associated signaling. The Journal of biological chemistry. 2012; 287(29):24690-7. 34. Chiang N, Dalli J, Colas R A, and Serhan C N. Identification of resolvin D2 receptor mediating resolution of infections and organ protection. The Journal of experimental medicine. 2015; 212(8):1203-17. 35. Dakin S G, Martinez F O, Yapp C, Wells G, Oppermann U, Dean B J, et al. Inflammation activation and resolution in human tendon disease. Science translational medicine. 2015; 7(311):311ra173. 36. Lee T H, Crea A E, Gant V, Spur B W, Marron B E, Nicolaou K C, et al. Identification of lipoxin A4 and its relationship to the sulfidopeptide leukotrienes C4, D4, and E4 in the bronchoalveolar lavage fluids obtained from patients with selected pulmonary diseases. The American review of respiratory disease. 1990; 141(6):1453-8. 37. Wu S H, Chen X Q, Liu B, Wu H J, and Dong L. Efficacy and safety of 15(R/S)-methyl-lipoxin A(4) in topical treatment of infantile eczema. Br J Dermatol. 2013; 168(1):172-8. 38. Haworth O, Cernadas M, Yang R, Serhan C N, and Levy B D. Resolvin E1 regulates interleukin 23, interferon-gamma and lipoxin A4 to promote the resolution of allergic airway inflammation. Nat Immunol. 2008; 9(8):873-9. 39. Levy B D, Lukacs N W, Berlin A A, Schmidt B, Guilford W J, Serhan C N, et al. Lipoxin A4 stable analogs reduce allergic airway responses via mechanisms distinct from CysLT1 receptor antagonism. FASEB journal: official publication of the Federation of American Societies for Experimental Biology. 2007; 21(14):3877-84. 40. Gronert K, Martinsson-Niskanen T, Ravasi S, Chiang N, and Serhan C N. Selectivity of recombinant human leukotriene D(4), leukotriene B(4), and lipoxin A(4) receptors with aspirin-triggered 15-epi-LXA(4) and regulation of vascular and inflammatory responses. The American journal of pathology. 2001; 158(1):3-9. 41. Su S B, Gong W, Gao J L, Shen W, Murphy P M, Oppenheim J J, et al. A seven-transmembrane, G protein-coupled receptor, FPRL1, mediates the chemotactic activity of serum amyloid A for human phagocytic cells. The Journal of experimental medicine. 1999; 189(2):395-402. 42. El Kebir D, Jozsef L, Khreiss T, Pan W, Petasis N A, Serhan C N, et al. Aspirin-triggered lipoxins override the apoptosis-delaying action of serum amyloid A in human neutrophils: a novel mechanism for resolution of inflammation. J Immunol. 2007; 179(1):616-22. 43. Buyukozturk S, Gelincik A A, Genc S, Kocak H, Oneriyidogan Y, Erden S, et al. Acute phase reactants in allergic airway disease. The Tohoku journal of experimental medicine. 2004; 204(3):209-13. 44. Ather J L, Fortner K A, Budd R C, Anathy V, and Poynter M E. Serum amyloid A inhibits dendritic cell apoptosis to induce glucocorticoid resistance in CD4(+) T cells. Cell death &disease. 2013; 4:e786. 45. Peters M C, McGrath K W, Hawkins G A, Hastie A T, Levy B D, Israel E, et al. Plasma interleukin-6 concentrations, metabolic dysfunction, and asthma severity: a cross-sectional analysis of two cohorts. Lancet Respir Med. 2016; 4(7):574-84. 46. Hagihara K, Nishikawa T, Sugamata Y, Song J, Isobe T, Taga T, et al. Essential role of STAT3 in cytokine-driven NF-kappaB-mediated serum amyloid A gene expression. Genes to cells: devoted to molecular & cellular mechanisms. 2005; 10(11):1051-63.
47. Phipatanakul W, Mauger D T, Sorkness R L, Gaffin J M, Holguin F, Woodruff P G, et al. Effects of Age and Disease Severity on Systemic Corticosteroid Responses in Asthma. Am J Respir Crit Care Med. 2016. 48. Chung K F, Wenzel S E, Brozek J L, Bush A, Castro M, Sterk P J, et al. International ERS/ATS guidelines on definition, evaluation and treatment of severe asthma. Eur Respir J. 2014; 43(2):343-73.