Equine asthma is an immune-mediated disease

Airway inflammation is a component of an asthma-affected horse's response to aeroallergens and is considered one of the primary characteristics of this disease. Equine asthma has been extensively studied, but the precise sequence of disease events is still not well-understood (Leguillette, Reference Leguillette2003). Generally, airway inflammation involves the activation of pathogen-specific inflammatory cells, the modulation of gene transcription factors, and the release of inflammatory mediators (Bureau et al., Reference Bureau, Bonizzi, Kirschvink, Delhalle, Desmecht, Merville, Bours and Lekeux2000a, Reference Bureau, Delhalle, Bonizzi, Fievez, Dogne, Kirschvink, Vanderplasschen, Merville, Bours and Lekeuxb). The immunological background of severe equine asthma remains not fully elucidated, despite many studies on its pathogenesis. Type I hypersensitivity, which is IgE-mediated (Curik et al., Reference Curik, Fraser, Eder, Achmann, Swinburne, Binns, Crameri, Brem, Sölkner and Marti2003; Künzle et al., Reference Künzle, Gerber, van der Haegen, Wampfler, Straub and Marti2007; Tahon et al., Reference Tahon, Baselgia, Gerber, Doherr, Straub, Robinson and Marti2009; Morán et al., Reference Morán, Burgos, Araya and Folch2010a, Reference Morán, Folch, Burgos, Araya and Barriab; Morán et al., Reference Moran, Folch, Henriquez, Ortloff and Barria2012), and type III hypersensitivity reactions have been suggested to play a role in airway inflammation (Lavoie et al., Reference Lavoie, Maghni, Desnoyers, Taha, Martin and Hamid2001; Robinson, Reference Robinson2001). IgE plays an important role in the induction of type I immediate hypersensitivity reactions in asthma-affected horses (Morán et al., Reference Morán, Burgos, Araya and Folch2010a, Reference Morán, Folch, Burgos, Araya and Barriab; Morán et al., Reference Moran, Folch, Henriquez, Ortloff and Barria2012). The inflammatory response associated with equine asthma is also characterized by neutrophilic bronchiolitis, which is considered evidence of a type III hypersensitivity response resulting from antigen–antibody complex formation and the subsequent activation of the complement cascade, with the release of anaphylatoxin and C3a and C5a peptides (Lavoie et al., Reference Lavoie, Maghni, Desnoyers, Taha, Martin and Hamid2000). Additional reports suggest that the inflammatory influx of neutrophils to the airways of chronically affected horses may be maintained by chemokines released from the same marginated granulocytes (Bureau et al., Reference Bureau, Bonizzi, Kirschvink, Delhalle, Desmecht, Merville, Bours and Lekeux2000a, Reference Bureau, Delhalle, Bonizzi, Fievez, Dogne, Kirschvink, Vanderplasschen, Merville, Bours and Lekeuxb; Ainsworth et al., Reference Ainsworth, Wagner, Erb, Young and Retallick2007).

T cells also play an important role in the modulation of the immune response in asthma equine pathogenesis. Many results suggest that pulmonary helper T lymphocytes may be implicated in heaves through the secretion of Th1-type or Th2-type cytokines (Lavoie et al., Reference Lavoie, Maghni, Desnoyers, Taha, Martin and Hamid2001; Giguere et al., Reference Giguere, Viel, Lee, MacKay, Hernandez and Franchini2002; Ainsworth et al., Reference Ainsworth, Grünig, Matychak, Young, Wagner, Erb and Antczak2003; Cordeau et al., Reference Cordeau, Joubert, Dewachi, Hamid and Lavoie2004; Ainsworth et al., Reference Ainsworth, Wagner, Erb, Young and Retallick2007; Riihimäki et al., Reference Riihimäki, Raine, Art, Lekeux, Couëtil and Pringle2008). Asthma-affected horses produce both type 1 and 2 cytokines, depending on the stage of their disease and the timing of sample collection. Cytokine expression in airway lymphocytes is also influenced by the length of time that an asthma-affected horse has experienced clinical disease (Pietra et al., Reference Pietra, Peli, Bonato, Ducci and Cinotti2007). Furthermore, lymphocytes retrieved from asthma horses after prolonged exposure to allergens (months) demonstrate an increase in the production of interleukin (IL)-8 and interferon-γ (Horohov et al., Reference Horohov, Beadle, Mouch and Pourciau2005). Moreover, IL-17 is known to induce the expression of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, IL-1B, and IL-6 as well as chemokines CXCL1, 2, and 8, all of which are hallmarks of acute inflammatory processes (Schmidt-Weber et al., Reference Schmidt-Weber, Akdis and Akdis2007). Finally, regulatory T cells (Treg) appear to play a role in the immune response in asthma-affected horses (Henriquez et al., Reference Henriquez, Perez, Morales, Sarmiento, Carrasco, Morán and Folch2014).

Neutrophil migration and activation in the lungs of horses with asthma

The neutrophil recruitment cascade is mediated by the sequential interaction of receptors present on neutrophils with ligands induced on the surface of the activated endothelium (Mayadas et al., Reference Mayadas, Cullere and Lowell2014). Neutrophils are observed in higher concentrations in the pulmonary capillaries compared with systemic blood even in the absence of inflammatory stimuli. This phenomenon allows neutrophils to readily migrate into the lungs in response to inflammatory stimuli (Cheng and Palaniyar, Reference Cheng and Palaniyar2013). During inflammation, neutrophils become activated upon stimulation and may produce ROS and NETs, undergo degranulation, or exhibit other functions. The activation of neutrophils is required before migration into the lungs (Ley et al., Reference Ley, Laudanna, Cybulsky and Nourshargh2007). In asthma-affected horses, the neutrophils migrate within hours into the airway lumen followed by the development of airway obstruction and a late phase of migration (Fairbairn et al., Reference Fairbairn, Page, Lees and Cunningham1993; Franchini et al., Reference Franchini, Gilli, Akens, Fellenberg and Bracher1998; Brazil et al., Reference Brazil, Dagleish, McGorum, Dixon, Haslett and Chilvers2005).

The principal lesion in asthma-affected horses is bronchiolitis. The peribronchiolar accumulation of lymphocytes is accompanied by the intraluminal accumulation of neutrophils (Leguillette, Reference Leguillette2003) and occurs within 7 h after environmental challenge (Fairbairn et al., Reference Fairbairn, Page, Lees and Cunningham1993). A type III hypersensitivity reaction explains, in part, the neutrophilic inflammation in the airways of asthma-affected horses, but the factors that initiate neutrophilia in the airways of affected horses have not been completely elucidated. As previously mentioned, bronchoalveolar cells retrieved from asthma-affected horses after antigenic challenge demonstrate the increased expression of the neutrophil chemokine, IL-8 (Giguere et al., Reference Giguere, Viel, Lee, MacKay, Hernandez and Franchini2002; Ainsworth et al., Reference Ainsworth, Grünig, Matychak, Young, Wagner, Erb and Antczak2003). An increase in the concentration of IL-8 in broncheoalveolar lavage fluid (BALF) has also been demonstrated (Franchini et al., Reference Franchini, Gill, Von Fellenberg and Bracher2000; Ainsworth et al., Reference Ainsworth, Grünig, Matychak, Young, Wagner, Erb and Antczak2003), and Riihimaki et al. (Reference Riihimäki, Raine, Art, Lekeux, Couëtil and Pringle2008) reported that IL-8 mRNA expression was upregulated in BALF cells and in endobronchial biopsies from asthma-affected horses in acute crisis. Several authors also suggest that alveolar macrophages can contribute to the airway inflammation by the release of IL-8, macrophage inflammatory protein-2 and TNF-α (Giguere et al., Reference Giguere, Viel, Lee, MacKay, Hernandez and Franchini2002; Ainsworth et al., Reference Ainsworth, Appleton, Antczak, Santiago and Aviza2002; Joubert et al., Reference Joubert, Cordeau and Lavoie2011). In addition, bronchial nuclear factor-κB (NF-κB) activity strongly correlates to the percentage of neutrophils present in the bronchi; this result suggests that the sustained NF-κB activity in the airways of asthma-affected horses is driven mainly by the granulocytic and non-granulocytic cells that remain or appear in the bronchi after antigen challenge (Bureau et al., Reference Bureau, Bonizzi, Kirschvink, Delhalle, Desmecht, Merville, Bours and Lekeux2000a, Reference Bureau, Delhalle, Bonizzi, Fievez, Dogne, Kirschvink, Vanderplasschen, Merville, Bours and Lekeuxb). BALF granulocytes from asthma-affected horses demonstrate a significant delay in apoptosis compared with blood granulocytes from the same horses or blood and BALF granulocytes from healthy horses (Bureau et al., Reference Bureau, Delhalle, Bonizzi, Fievez, Dogne, Kirschvink, Vanderplasschen, Merville, Bours and Lekeux2000b; Turlej et al., Reference Turlej, Fievez, Sandersen, Dogné, Kirschvink, Lekeux and Bureau2001). Furthermore, since airway neutrophilia is a well-recognized characteristic of clinical equine asthma, several researchers have attempted to establish a relationship between IL-17 and the immediate influx of neutrophils into the airways of asthma-affected horses (Murcia et al., Reference Murcia, Vargas and Lavoie2016). IL-17 is produced by CD4 + T helper 17 cell and other types of cells such as γ/δ T cells, natural killer cells, lymphoid tissue inducer cells, macrophages, eosinophils, and neutrophils (Gaffen Reference Gaffen2009; Ramirez-Velazquez et al., Reference Ramirez-Velazquez, Castillo, Guido-Bayardo and Ortiz-Navarrete2013). IL-17 can indirectly promote the activation and recruitment of neutrophils into the airways by inducing the production of such chemokines as IL-8, CXCL1, and granulocyte colony-stimulating factor (G-CSF) in endothelial and epithelial cells (Ouyang et al., Reference Ouyang, Kolls and Zheng2008). Neutrophil influx into the airways and surrounding pulmonary tissues coincides with a significant increase in IL-17 mRNA expression in the airway cells obtained from endobronchial biopsies and BALF compared with controls during provocation studies (Ainsworth et al., Reference Ainsworth, Wagner, Franchini, Grunig, Erb and Tan2006; Riihimaki et al., Reference Riihimäki, Raine, Art, Lekeux, Couëtil and Pringle2008). Additionally, Debrue et al. (Reference Debrue, Hamilton, Joubert, Lajoie-Kadoch and Lavoie2005) suggested that IL-17 may induce neutrophil chemotaxis and activation, mucus hypersecretion and alterations in airway function. Korn et al. (Reference Korn, Miller, Dong, Buckles, Wagner and Ainsworth2015) also found an increased IL-17 response focused on NF-κB and a downregulation of the IL-4 gene in asthma-affected horses through immunohistochemistry and global gene expression profiles in mediastinal lymph nodes. This finding provides additional evidence of the involvement of IL-17 in the chronic stages of equine asthma.

Conversely, the innate immune response plays an important role in neutrophil activation during allergic airway diseases in both humans and horses (Feleszko et al., Reference Feleszko, Jaworska and Hamelmann2006; Berndt et al., Reference Berndt, Derksen, Venta, Ewart, Yuzbasiyan-Gurkan and Robinson2007). Among the innate mechanisms described is the formation of NETs, which serve as possible promotors of disease in asthma-affected horses. The pathogenic role of NETs has been described for many infectious and non-infectious human diseases, including respiratory cases with a massive influx of neutrophils into the airways (Porto and Stain, Reference Porto and Stein2016). Excessive NET release is particularly deleterious in lung diseases because NETs can expand easily in the pulmonary alveolar space and cause lung injury. NETs and their associated molecules can also directly induce epithelial and endothelial cell death (Xu et al., Reference Xu, Zhang, Pelayo, Monestier, Ammollo, Semeraro, Taylor, Esmon, Lupu and Esmon2009; Saffarzadeh et al., Reference Saffarzadeh, Juenemann, Queisser, Lochnit, Barreto, Galuska, Lohmeyer and Preissner2012). NETs have been identified in lungs with CF, ALI, neutrophilic asthma, and bacterial, viral, or fungal infections. The primary role of NETs is to prevent microbial dissemination because of their stringy structure and to kill pathogens due to the high local concentrations of antimicrobial molecules (Manzenreiter et al., Reference Manzenreiter, Kienberger, Marcos, Schilcher, Krautgartner, Obermayer, Huml, Stoiber, Hector, Griese, Hannig, Studnicka, Vitkov and Hartl2012). However, studies reveal that NETs can exert adverse effects in a number of diseases, including diseases of the lung (Cheng and Palaniyar, Reference Cheng and Palaniyar2013). NETs are composed of a backbone of nuclear DNA combined with a multitude of nuclear proteins, as well as the contents of neutrophil granules, including Myeloperoxidase (MPO) and elastase and peptidylarginine deiminase type IV (Martinelli et al., Reference Martinelli, Urosevic, Daryadel, Oberholzer, Baumann, Fey, Dummer, Simon and Yousefi2004; Urban et al., Reference Urban, Ermert, Schmid, Abu-Abed, Goosmann, Nacken, Brinkmann, Jungblut and Zychlinsky2009; Papayannopoulos et al., Reference Papayannopoulos, Metzler, Hakkim and Zychlinsky2010). These DNA–protein complexes are then released extracellularly as NETs. The potent neutrophil chemoattractant, IL-8, has also been shown to induce NETosis (Brinkmann et al., Reference Brinkmann, Reichard, Goosmann, Fauler, Uhlemann, Weiss, Weinrauch and Zychlinsky2004; Gupta et al., Reference Gupta, Hasler, Holzgreve, Gebhardt and Hahn2005). However, their contribution to disease severity is not clearly understood. In asthma-affected horses, NETs were present in BALF in exacerbated cases but not in fluid from horses in remission periods or in healthy challenged horses (Côté et al., Reference Côté, Clark, Viel, Labbé, Seah, Khan, Douda, Palaniyar and Bienzle2014).

Pathogen-associated molecules are recognized by pattern recognition receptors, such as Toll-like receptors (TLRs). Thus, TLRs are important for the activation of antigen-presenting cells during innate and adaptive immune responses (Casale and Stoke, Reference Casale and Stoke2008). TLRs are expressed by different cells involved in asthma-related airway inflammation, such as epithelial cells, macrophages, dendritic cells, and mast cells (Takeda et al., Reference Takeda, Kaisho and Akira2003) and airway smooth muscle (Sukkar et al., Reference Sukkar, Xie, Khorasani, Kon, Stanbridge, Issa and Chung2006). In asthma-affected horses, TLR4 mRNA expression is increased in the BALF of horses that have been exposed to stable dust compared with unaffected horses (Ainsworth et al., Reference Ainsworth, Wagner, Franchini, Grunig, Erb and Tan2006). This finding suggests that exposure to stable dust leads to increased TLR4 mRNA expression in bronchial epithelial cells from asthmatic horses. In addition, the upregulation of epithelial TLR4 mRNA correlates with IL-8 mRNA expression (Berndt et al., Reference Berndt, Derksen, Venta, Ewart, Yuzbasiyan-Gurkan and Robinson2007). These results could explain the exacerbated neutrophilic airway inflammation of asthma-affected horses in response to airborne endotoxin (Pirie et al., Reference Pirie, Collie, Dixon and McGorum2002, Reference Pirie, Collie, Dixon and McGorum2003; Berndt et al., Reference Berndt, Derksen, Venta, Ewart, Yuzbasiyan-Gurkan and Robinson2007). Interestingly, other reports also state that microbial-derived products, such as endotoxins, play an important role in allergy-induced human lung disease (Feleszko et al., Reference Feleszko, Jaworska and Hamelmann2006).

Role of ROS in asthma-affected horses

ROS are produced during oxygen reduction and are characterized by high reactivity. ROS participate in many important physiological processes, but if they are produced in high concentrations, they may lead to oxidative stress development and disturb the pro-oxidative/antioxidative balance toward an oxidation reaction, thereby leading to the damage of lipids, proteins, carbohydrates, or nucleic acids (Kleniewska and Pawliczak, Reference Kleniewska and Pawliczak2017). Oxidative stress has been shown to occur in many human respiratory conditions, including COPD and human asthma (Kirkham and Barnes, Reference Kirkham and Barnes2013; Zuo et al., Reference Zuo, Otenbaker, Rose and Salisbury2013). ROS derived from inflammatory cells (neutrophils, macrophages), which migrate in large numbers to the lungs, are crucial in the oxidant–antioxidant imbalance observed during the course of the above-mentioned diseases (Niedzwiedz and Jaworski, Reference Niedzwiedz and Jaworski2014).

ROS formation is a multi-step process involving the translocation of cytosolic components of NADPH (p47phox, p67phox, and Rac) to the NADPH components found on the cytoplasmic or phagosome membrane (gp91phox and gp22phox). When this occurs, NADPH transports electrons to molecular oxygen, generating a superoxide anion. This phenomenon, in turn, undergoes a rapid and spontaneous dismutation to hydrogen peroxide, which serves as a substrate for myeloperoxidase for the subsequent generation of hypohalous acids, the most important of which is hypochlorite acid (Tintinger et al., Reference Tintinger, Anderson and Feldman2013).

Horses that suffer from asthma have a decreased pulmonary antioxidant capacity, which may render them more susceptible to oxidative challenge. Research on oxidative stress in horses with asthma has been conducted, and some authors demonstrated that neutrophilia induced by exposure to organic dust is associated with increases in elastase and decreases in ascorbic acid concentrations in BALF retrieved from horses with asthma (Deaton et al., Reference Deaton, Marlin, Smith, Roberts, Harris, Schroter and Kelly2005a, Reference Deaton, Marlin, Smith, Harris, Dagleish, Schroter and Kellyb). Concurrently, affected horses experience significant antioxidant depletion in the trachea, which may be related to inflammation, and oxidative processes in peripheral airways (Deaton et al., Reference Deaton, Marlin, Smith, Harris, Schroter and Kelly2006). Acute exacerbations are associated with a significant increase in the levels of markers of oxidative stress (oxidized glutathione and glutathione redox ratio) in pulmonary epithelial lining fluid (Robinson, Reference Robinson2001). These markers correlate significantly with the number of neutrophils in BALF (Art et al., Reference Art, Kirschvink, Smith and Lekeux1999).

Asthmatic patients and mouse models typically exhibit varying degrees of airway inflammation; oxidizing agents interfere with the structure of epithelial cells, resulting in the increased production of mucus. This phenomenon eventually leads to structural changes and bronchial remodeling (Weiss and Bellino, Reference Weiss and Bellino1986; Adler et al., Reference Adler, Holden-Stauffer and Repine1990; Doelman et al., Reference Doelman, Leurs, Oosterom and Bast1990; Katsumata et al., Reference Katsumata, Miura, Ichinose, Kimura, Takahashi, Inoue and Takishima1990), although the precise role of ROS in modulating equine airway smooth muscle tone and the airway wall is unclear and may depend on the presence of other inflammatory mediators (Deaton et al., Reference Deaton, Marlin, Smith, Roberts, Harris, Schroter and Kelly2005a). The destructive nature of ROS may contribute to increased inflammation, apoptosis, or necrosis by modifying nucleotide chains and disrupting DNA stability. This property may also lead to the proliferation of smooth muscle cells in the airways, or an increase in the amount of mucus in the lungs (Cooke et al., Reference Cooke, Evans, Dizdaroglu and Lunec2003; Kamiya, Reference Kamiya2003; Reddy et al., Reference Reddy, Beyaz, Perry, Cooke, Sayre and Smith2005; Höhn et al., Reference Höhn, König and Grune2013); these changes have been described in horses with asthma (Herszberg et al., Reference Herszberg, Ramos-Barbon, Tamaoka, Martin and Lavoie2006; Bullone and Lavoie Reference Bullone and Lavoie2015). ROS overproduction may also activate transcription factors, such as NF-κB or AP-1 (activator protein 1) proteins (Csiszar et al., Reference Csiszar, Wang, Lakatta and Ungvari2008; Noutsios and Floros., Reference Noutsios and Floros2014; Schuliga, Reference Schuliga2015), which in turn may lead to the expression of many pro-inflammatory cytokines, including TNF-α, IL-4, IL-5, IL-6, and IL-13, and aggravate the disease (Frossi et al., Reference Frossi, Carli, Daniel, Rivera and Pucillo2003). Moreover, studies reveal that NETosis is dependent on the generation of ROS by NADPH oxidase (Porto and Stein, Reference Porto and Stein2016). Furthermore, several authors suggest that dietary antioxidant cocktails may improve the lung function of asthma-affected horses by modulating oxidant–antioxidant balance and airway inflammation (Kirschvink et al., Reference Kirschvink, Smith, Fievez, Marlin and Gustin2002). Finally, all of the described data support the hypothesis that defects in the intracellular antioxidant defense system may be critical contributors to the development of equine asthma under increased ROS production. Further studies on oxidative stress markers and the efficacy of selected antioxidants in equine asthma treatment are needed to determine the optimal control of this disease.

Neutrophils and the resolution of inflammation

Airway epithelial cells are the first line of defense against inhaled pathogens and antigens in the airways. Once the triggering antigen reaches the airways, epithelial cells launch signals that activate tissue-resident cells of the innate immune system, initiating the inflammatory response and recruiting circulating neutrophils (Hallstrand et al., Reference Hallstrand, Hackett, Altemeier, Matute-Bello, Hansbro and Knight2014). To leave their intravascular location, neutrophils must interact with the endothelial cells of the local vessels, which increase adhesion molecule expression after being activated and allow the migration of neutrophils to the underlying tissue. This effect, in turn, promotes the recruitment of inflammatory monocytes and potentiates the pro-inflammatory environment, allowing control of the insulting agent that triggered the initial inflammation (Mantovani et al., Reference Mantovani, Cassatella, Costantini and Jaillon2011) and permitting the resolution of the inflammatory event. However, in some cases, such as neutrophilic asthma, an exacerbated inflammatory response occurs. The resolution of this acute process relies on many soluble molecules; such molecules as Annexin A1 (AnxA1) (Perretti and D'Acquisto, Reference Perretti and D'Acquisto2009), ChemerinC15 (Cash et al., Reference Cash, Hart, Russ, Dixon, Colledge, Doran, Hendrick, Carlton and Greaves2008), lipoxins (Serhan et al., Reference Serhan, Chiang and Van Dyke2008), and resolvins (Ariel and Serhan, Reference Ariel and Serhan2007) play an important role in stopping the recruitment of neutrophils. These signals act in conjunction with the apoptosis of neutrophils, which is central in the resolution process; dying neutrophils are known to stimulate their own efferocytosis, inducing macrophagic transition from a pro-inflammatory (M1) to an anti-inflammatory (M2) profile (Ortega-Gomez et al., Reference Ortega-Gomez, Perretti and Soehnlein2013). AnxA1 (37 kDa) is an abundant protein in the cytosol of resting neutrophils. AnxA1 translocates to the plasma membrane when the cell is activated and interacts with the formyl peptide receptor 2 to moderate leukocyte adhesion and migration (Perretti et al., Reference Perretti, Chiang, La, Fierro, Marullo, Getting, Solito and Serhan2002; Dalli et al., Reference Dalli, Norling, Renshaw, Cooper, Leung and Perretti2008). AnxA1 also promotes neutrophil apoptosis and clearance by macrophages (Perretti and Solito, Reference Perretti and Solito2004; Scannell et al., Reference Scannell, Flanagan, deStefani, Wynne, Cagney, Godson and Maderna2007). Another neutrophil-derived protein with similar activities is lactoferrin. This protein is contained within the secondary granules of neutrophils, and when released, lactoferrin binds to specific receptors that trigger Mitogen-Activated Protein Kinases (MAPK)-mediated intracellular signaling, which is crucial in the regulation of cytoskeletal remodeling and cell adhesion (Bournazou et al., Reference Bournazou, Pound, Duffin, Bournazos, Melville, Brown, Rossi and Gregory2009). Additionally, in a model of ALI, lactoferrin application prevented neutrophil tissue infiltration and edema formation and improved lung function (Li et al., Reference Li, Liu, Chen, Pan, Kong and Pang2012). Neutrophils that have started their apoptotic process are cleared by macrophages via efferocytosis. Apoptotic neutrophils promote their own clearance by expressing ‘find me’ and ‘eat me’ signals. ‘Find me’ signals are secreted factors that attract scavengers. To date, four major ‘find me’ signals have been described (Lauber et al., Reference Lauber, Bohn, Kröber, Xiao, Blumenthal, Lindemann, Marini, Wiedig, Zobywalski, Baksh, Xu, Autenrieth, Schulze-Osthoff, Belka, Stuhler and Wesselborg2003; Gude et al., Reference Gude, Alvarez, Paugh, Mitra, Yu, Griffiths, Barbour, Milstien and Spiegel2008; Truman et al., Reference Truman, Ford, Pasikowska, Pound, Wilkinson, Dumitriu, Melville, Melrose, Ogden, Nibbs, Graham, Combadiere and Gregory2008; Elliott et al., Reference Elliott, Chekeni, Trampont, Lazarowski, Kadl, Walk, Park, Woodson, Ostankovich, Sharma, Lysiak, Harden, Leitinger and Ravichandran2009). ‘Eat me’ signals are surface markers that permit the identification of a dying cell. These signals can either be molecules exposed de novo at the cell membrane or existing ones that undergo modifications during apoptosis; the best-known such molecule is phosphatidylserine, which is also the best-studied marker of early apoptosis (Ortega-Gomez et al., Reference Ortega-Gomez, Perretti and Soehnlein2013). Furthermore, neutrophils induce a change in phagocytic macrophages from a pro-inflammatory to an anti-inflammatory mode. Upon apoptotic cell efferocytosis, macrophages turn off the production of pro-inflammatory cytokines and lipid mediators and launch an anti-inflammatory transcriptional program characterized by the release of IL-10 and Transforming growth factor (TGF)-β (Fadok et al., Reference Fadok, Bratton, Konowal, Freed, Westcott and Henson1998) and the secretion of lipid mediators that play a key role in the orchestration of inflammation and its resolution (Serhan et al., Reference Serhan, Chiang and Van Dyke2008). Moreover, neutrophils may also stimulate regulatory-suppressive cells. Apoptotic neutrophils or efferocytes induce the recruitment of myeloid-derived suppressor cells (MDSC) after phagocytosis (Bronte et al., Reference Bronte, Apolloni, Cabrelle, Ronca, Serafini, Zamboni, Restifo and Zanovello2000; Ribechini et al., Reference Ribechini, Greifenberg, Sandwick and Lutz2010) that secrete IL-10 and TGF-β. Lymphoid regulatory cells, such as B regulatory cells and Treg, are also attracted to the inflammatory site, where IL-10 and TGF-β secreted by efferocytes and MDSC induce their expansion and potentiate their suppressor activity, increasing the expression of FoxP3 (Savage et al., Reference Savage, de Boer, Walburg, Joosten, van Meijgaarden, Geluk and Ottenhoff2008). Treg cells are important players in the pro-resolution mechanism that occurs after injury, because their absence delays the resolution of lung inflammation (D'Alessio et al., Reference D'Alessio, Tsushima, Aggarwal, West, Willett, Britos, Pipeling, Brower, Tuder, McDyer and King2009). In this sense, several authors demonstrated that more Tregs are present in the airways of asthma-affected horses, probably due to allergic inflammation, and that these cells are possibly a heterogeneous population with different physiologic attributes and roles in the regulation and final resolution of airway allergic inflammation (Henriquez et al., Reference Henriquez, Perez, Morales, Sarmiento, Carrasco, Morán and Folch2014).

On the other hand, in human asthma patients with more severe disease, the asthmatic-repairing epithelium can generate pro-neutrophilic factors that can have profound chemotactic and apoptosis-delaying actions (Uddin et al., Reference Uddin, Lau, Seumois, Vijayanand, Staples, Bagmane, Cornelius, Dorinsky, Davies and Djukanovi´c2013). There is a persistence of apoptosis-resistant neutrophils in the airways of patients with severe asthma that may impede timely neutrophil clearance and thereby delay the resolution of airway inflammation (Louis and Djukanovic, Reference Louis and Djukanovic2006). Moreover, neutrophilic asthma is relatively resistant to glucocorticoids (GCs) (Bruijnzeel et al., Reference Bruijnzeel, Uddin and Koenderman2015). A similar phenomenon occurs in asthma-affected horses. Murcia et al. (Reference Murcia, Vargas and Lavoie2016) showed that IL-17 directly activates equine neutrophils at 24 h and that the expression of IL-8 is not attenuated by GCs. Additionally, IL-17 increases neutrophil viability and decreases apoptosis. Therefore, treatments that target neutrophilic inflammation could be useful to modify the course of the disease and improve clinical outcomes in both humans and horses. Several alternative treatments with proposed resolution effects on inflammation have been evaluated. These include the optimization of GC treatment protocols (Cesarini et al., Reference Cesarini, Hamilton, Picandet and Lavoie2006; DeLuca et al., Reference DeLuca, Erb, Young, Perkins and Ainsworth2008; Robinson et al., Reference Robinson, Berney, Behan and Derksen2009; Leclere et al., Reference Leclere, Lefebvre-Lavoie, Beauchamp and Lavoie2010; Franke and Abraham, Reference Franke and Abraham2014; Barton et al., Reference Barton, Shety, Bondzio, Einspanier and Gehlen2016), autologous bone marrow-derived mononuclear cell therapy (Barussi et al., Reference Barussi, Bastos, Leite, Fragoso, Senegaglia, Brofman, Nishiyama, Pimpão and Michelotto2016), nanoparticulate immunotherapy (Klier et al., Reference Klier, Lehmann, Fuchs, Reese, Hirschmann, Coester, Winter and Gehlen2015), and tamoxifen treatment (Sarmiento et al., Reference Sarmiento, Perez, Morales, Henriquez, Vidal, Folch, Galecio and Morán2013; Perez et al., Reference Perez, Henriquez, Sarmiento, Morales, Folch, Galesio, Uberti and Morán2016; Borlone et al., Reference Borlone, Morales, Henriquez, Folch, Olave, Sarmiento, Uberti and Moran2017). Tamoxifen promotes early neutrophil apoptosis and dampens the chemotactic index and respiratory burst production in vitro (Borlone et al., Reference Borlone, Morales, Henriquez, Folch, Olave, Sarmiento, Uberti and Moran2017). Overall, extensive research is still required to identify effective therapeutic targets and interventions to achieve the resolution of inflammation in diseased patients’ lungs.