Modeling the pasture-associated severe equine asthma bronchoalveolar lavage fluid proteome identifies molecular events mediating neutrophilic airway inflammation

Figures & data

Table 1 Definitions of neutrophil terms employed for biocuration

Activation	The change in morphology and behavior of a neutrophil resulting from exposure to a cytokine, chemokine, cellular ligand, or soluble factor.^Citation120
Adhesion	The process in which neutrophils attach to vascular endothelium as part of an event in recruitment into acutely inflammatory lesions, or to target tissues/cells following transmigration events.^Citation121,Citation122
Apoptosis	Regulated cell death.^Citation120
Cell spreading	The morphogenetic process that results in flattening of a cell as a consequence of its adhesion to a substrate.^Citation120
Chemotaxis	The directed movement of a neutrophil cell, the most common polymorphonuclear leukocyte found in the blood, in response to an external stimulus, usually an infection or wounding.^Citation120
Infiltration	The diffusion or accumulation of neutrophils in tissues or cells in response to a wide variety of substances released at the sites of inflammatory reactions.^Citation123
Migration	The movement of a neutrophil within or between different tissues and organs of the body.^Citation120
Phagocytosis	An endocytosis process that results in the engulfment of external particulate material.^Citation120
Respiratory burst	A phase of elevated metabolic activity, during which oxygen consumption increases; this leads to the production, by an NADH-dependent system, of H2O2, superoxide anions, and hydroxyl radicals.^Citation120
Transmigration	The migration of a neutrophil from the blood vessels into the surrounding tissue.^Citation120 Neutrophils pass through endothelium, interstitial tissues, and epithelium before arriving at the target location. No differentiation of stage was made for the purposes of our biocuration.

Figure 1 Cumulative effect of bronchoalveolar lavage fluid (BALF) proteins on neutrophil functions in asthmatic vs control horses. A total of 1,003 proteins were reviewed to identify their effects on ten neutrophil functions. Peer-reviewed manuscripts were read and actions involving ten neutrophil functions were scored for each protein as pro (+1), anti (−1), no effect (0), or no data. The contribution of each protein to each neutrophil function was calculated as the product of magnitude of expression multiplied by its effect (ie, −1, 0, +1), then these values were tabulated for category of neutrophil function. Bars represent the predicted net effect of BALF proteins on the respective neutrophil function.

Figure 2 Ingenuity® Pathway Analysis (IPA) predicted decreases in three and increases in four neutrophil functions. An interactive network was derived using IPA by screening 1,003 proteins in bronchoalveolar lavage fluid (BALF) of asthmatic vs control horses for their impact on neutrophil activity. Green shades indicate relative downregulation of proteins in asthmatic BALF (and relative upregulation in control BALF), while red shades indicate relative upregulation of proteins in asthmatic BALF (and relative downregulation of proteins in control BALF). Blue indicates predicted decreases in neutrophil functions, while orange shades indicate predicted increases. More intense (darker) colors indicate greater increases or decreases. Together, this network indicated net downregulation of neutrophil activation, chemotaxis, and phagocytosis, and upregulation of neutrophil migration, adhesion, infiltration, and respiratory burst. Broken lines indicate a relationship between the proteins and the neutrophil functions.

Table 2 Comparison of proteins curated by Ingenuity® Pathway Analysis (IPA) vs manual biocuration. Bolded proteins were common between the two methodologies

Neutrophil category	Proteins curated in IPA	Proteins manually curated
Migration	ALB – albumin ANXA1 – annexin A1 CCR7 – C-C chemokine receptor type 7 CSF2 – colony stimulating factor 2 FN1 – fibronectin 1 HCK – tyrosine-protein kinase HGF – hepatocyte growth factor IL17RA – interleukin 17A receptor INPP5D – inositol polyphosphate-5-phosphatase D ITGAM – integrin alpha M ITGB3 – integrin beta 3 NOS2 – nitric oxide synthase 2 S100A8 – S100 calcium binding protein A8 SERPINC1 – serpin family C, member 1 SLPI – secretory leukocyte peptidase inhibitor TNF – tumor necrosis factor TXN – thioredoxin	alpha-1-acid glycoprotein 2 alpha-1-acid glycoprotein-like alpha-1-antiproteinase 2-like isoform X1 annexin A1 apolipoprotein A-I-like apolipoprotein A-IV C-C chemokine receptor type 7 complement factor B dnaJ homolog subfamily C member 27 EGF-like module-containing mucin-like hormone receptor-like 2-like fibronectin isoform X1 gelsolin granulocyte-macrophage colony-stimulating factor precursor hemopexin integrin alpha-4 integrin alpha-M isoform X1 interleukin-17 receptor A isoform X1 nitric oxide synthase, inducible phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit alpha isoform X1 polypeptide N-acetylgalactosaminyltransferase 1 protein S100-A8 prothrombin pulmonary surfactant-associated protein D reactive oxygen species modulator 1-like thioredoxin toll-like receptor 8 precursor tumor necrosis factor tyrosine-protein kinase HCK
Activation	AHR – aryl hydrocarbon receptor ANXA1 – annexin A1 C3 – complement C3 CSF2 – colony stimulating factor 2 EPX – eosinophil peroxidase F2 – coagulation factor II, thrombin FN1 – fibronectin 1 IL18RAP – interleukin-18 receptor accessory protein ITGAM – integrin alpha M LCN2 – lipocalin 2 LTF – lactotransferrin SLPI – secretory leukocyte peptidase inhibitor TNF – tumor necrosis factor	alpha-1-acid glycoprotein 2 alpha-1-acid glycoprotein-like alpha-1-antiproteinase 2-like isoform X1 anionic trypsin annexin A1 annexin A5 isoform X2 apolipoprotein A-I-like apolipoprotein A-IV beta-2-glycoprotein 1 C-C chemokine receptor type 7 complement C2 complement C3 complement component C1q receptor complement factor B cytochrome c dnaJ homolog subfamily C member 27 dystrophin granulocyte-macrophage colony-stimulating factor precursor haptoglobin hexokinase-2 interleukin-18 receptor accessory protein isoform X1 lactotransferrin precursor leptin receptor-like mitogen-activated protein kinase kinase kinase 2 myoglobin nck-associated protein 1-like phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit alpha isoform X1 plasma protease C1 inhibitor polymeric immunoglobulin receptor prothrombin ral guanine nucleotide dissociation stimulator reactive oxygen species modulator 1-like serotransferrin precursor thioredoxin toll-like receptor 1 precursor toll-like receptor 8 precursor tropomyosin alpha-1 chain-like tumor necrosis factor
Chemotaxis	APOA1 – apolipoprotein A1 C3 – complement C3 CCR7 – C-C chemokine receptor type 7 CSF2 – colony stimulating factor 2 HCK – tyrosine-protein kinase INPP5D – inositol polyphosphate-5-phosphatase D ITGA4 – integrin alpha 4 ITGAM – integrin alpha M LCN2 – lipocalin 2 NCKAP1L – Nck-associated protein 1 like NOD2 – nucleotide-binding oligomerization domain-containing protein 2 PIGR – polymeric immunoglobulin receptor S100A8 - S100 calcium binding protein A8 SERPINA1 – serpin family A, member 1 SLPI – secretory leukocyte peptidase inhibitor TGFB2 – transforming growth factor-beta 2 THBS4 – thrombospondin-4 TNF – tumor necrosis factor	alpha-1-acid glycoprotein 2 alpha-1-acid glycoprotein-like alpha-1-antiproteinase 2-like isoform X1 amyloid beta A4 precursor protein-binding family A member 2 ankyrin repeat domain-containing protein 11 annexin A1 annexin A2 annexin A5 isoform X2 complement C2 complement C3 complement component C1q receptor fibronectin isoform X1 gamma-aminobutyric acid type B receptor subunit 2 gelsolin granulocyte-macrophage colony-stimulating factor precursor hemopexin hepatocyte growth factor isoformX1 integrin alpha-3 isoform 1 integrin alpha-4 leptin receptor-like microtubule-associated protein 2 isoform X8 nitric oxide synthase, inducible phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit alpha isoform X1 plasma protease C1 inhibitor polymeric immunoglobulin receptor protein S100-A8 prothrombin pulmonary surfactant-associated protein D thioredoxin tumor necrosis factor tyrosine-protein kinase HCK tyrosine-protein kinase JAK3 utrophin-like vitamin D-binding protein
Adhesion	ADGRE2 – adhesion G protein-coupled receptor E2 ANXA1 – annexin A1 APOA1 – apolipoprotein A1 C3 – complement C3 CSF2 – colony stimulating factor 2 F2 – coagulation factor II, thrombin FN1 – fibronectin 1 INPP5D – inositol polyphosphate-5-phosphatase D ITGAM – integrin alpha M KNG1 – kininogen 1 LCP1 – plastin 2 NOS2 – nitric oxide synthase 2 S100A8 – S100 calcium binding protein A8 SERPINA1 – serpin family A, member 1 TNF – tumor necrosis factor TXN – thioredoxin	A disintegrin and metalloproteinase with thrombospondin motifs 13 alkaline phosphatase, tissue-nonspecific isozyme isoform X1 alpha-1-antiproteinase 2-like isoform X1 annexin A1 apolipoprotein A-I-like apolipoprotein A-IV C-C chemokine receptor type 7 CD2-associated protein complement C3 dnaJ homolog subfamily C member 27 EGF-like module-containing mucin-like hormone receptor-like 2-like fibrinogen gamma chain fibronectin isoform X1 gelsolin granulocyte-macrophage colony-stimulating factor precursor hemopexin hepatocyte growth factor isoformX1 integrin alpha-4 integrin alpha-M isoform X1 kininogen-1 isoform X2 nitric oxide synthase, inducible plasma protease C1 inhibitor plastin-2 protein S100-A8 thioredoxin tumor necrosis factor tyrosine-protein kinase JAK2 isoform X1
Cell spreading	N/A	apolipoprotein A-I-like apolipoprotein A-IV serum albumin precursor tumor necrosis factor tyrosine-protein kinase HCK
Transmigration	N/A	annexin A1 annexin A2 antithrombin-III isoform X1 catenin alpha-3 isoform 1 C-C chemokine receptor type 7 hepatocyte growth factor isoformX1 integrin beta-3 precursor plasma protease C1 inhibitor polymeric immunoglobulin receptor tumor necrosis factor
Infiltration	ADAMTS13 – ADAM metallopeptidase with thrombospondin type 1 motif 13 ANXA1 – annexin A1 APOA1 – apolipoprotein A1 C2 – complement C2 C3 – complement C3 C4A/C4B – complement C4A/CFB CD36 – platelet glycoprotein 4 CFB – complement factor B F2 – coagulation factor II, thrombin HGF – hepatocyte growth factor IL17RA – interleukin 17A receptor ITGAM – integrin alpha M LCN2 – lipocalin 2 LRRK2 – leucine-rich repeat kinase 2 MAVS – mitochondrial antiviral-signaling protein NOS2 – nitric oxide synthase 2 PDK4 – pyruvate dehydrogenase kinase 4 PLG – plasminogen SERPINA1 – serpin family A, member 1 SERPINC1 – serpin family C, member 1 TNF – tumor necrosis factor	alpha-1-acid glycoprotein 2 alpha-1-antiproteinase 2-like isoform X1 annexin A1 annexin A2 antithrombin-III isoform X1 argininosuccinate synthase aryl hydrocarbon receptor BK potassium ion channel cadherin-1 complement C4-A dystrophin Fc receptor alpha-chain gelsolin granulocyte-macrophage colony-stimulating factor precursor group IIF secretory phospholipase A2 haptoglobin hepatocyte growth factor isoformX1 interleukin-17 receptor A isoform X1 leucine-rich repeat kinase 1 nitric oxide synthase, inducible pancreatic alpha-amylase peroxiredoxin-1 plasma protease C1 inhibitor plasminogen isoform X1 pulmonary surfactant-associated protein D thioredoxin tumor necrosis factor vitamin D-binding protein
Phagocytosis	C3 – complement C3 CD36 – platelet glycoprotein 4 CSF2 – colony stimulating factor 2 HGF – hepatocyte growth factor ITGAM – integrin alpha M LCN2 – lipocalin 2 NOD2 – nucleotide-binding oligomerization domain-containing protein 2 SFTPA1 – surfactant protein A1 SFTPD – surfactant protein D TNF – tumor necrosis factor	alpha-1-acid glycoprotein 2 alpha-1-acid glycoprotein-like alpha-fetoprotein precursor ankyrin repeat domain-containing protein 11 annexin A1 ceramide kinase ceruloplasmin isoform X2 complement C2 complement C3 complement component C1q receptor complement factor B complement factor I Fc receptor alpha-chain ferritin light chain fibronectin isoform X1 gelsolin granulocyte-macrophage colony-stimulating factor precursor hemopexin leptin receptor-like nucleotide-binding oligomerization domain-containing protein 2 plasma protease C1 inhibitor pulmonary surfactant-associated protein A precursor pulmonary surfactant-associated protein D toll-like receptor 1 precursor tumor necrosis factor
Respiratory burst	C3 – complement C3 FN1 – fibronectin 1 HCK – tyrosine-protein kinase ITGA4 – integrin alpha 4 ITGAM – integrin alpha M ITGB3 – integrin beta 3 TNF – tumor necrosis factor	alpha-1-acid glycoprotein 2 alpha-1-acid glycoprotein-like apolipoprotein A-II apolipoprotein A-IV C-C chemokine receptor type 7 complement C2 complement C3 complement component C1q receptor complement factor B dnaJ homolog subfamily C member 27 EF-hand calcium-binding domain-containing protein 3 granulocyte-macrophage colony-stimulating factor precursor group IIF secretory phospholipase A2 haptoglobin integrin alpha-M isoform lactotransferrin precursor leptin receptor-like mitogen-activated protein kinase kinase kinase 2 phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit alpha isoform X1 protein S100-A8 pulmonary surfactant-associated protein A precursor pulmonary surfactant-associated protein D thioredoxin toll-like receptor 1 precursor tumor necrosis factor tyrosine-protein kinase HCK
Apoptosis	N/A	alpha-1-acid glycoprotein-like alpha-1-antiproteinase 2-like isoform X1 amyloid beta A4 precursor protein-binding family A member 2 annexin A1 annexin A5 isoform X2 antithrombin-III isoform X1 argininosuccinate synthase bcl-2-related protein A1 cytochrome c dnaJ homolog subfamily C member 27 granulocyte-macrophage colony-stimulating factor precursor lactotransferrin precursor leptin receptor-like nitric oxide synthase, inducible phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit alpha isoform X1 protein phosphatase 2 (formerly 2A), regulatory subunit A (PR 65), beta isoform thioredoxin tumor necrosis factor

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