The Veterinary Journal 256 (2020) 105436

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The Veterinary Journal

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Alveolar macrophage phenotypes in severe equine asthma

M.E. Wilsona,1, E.E. McCandlessb , M.A. Olszewskic , N.Edward Robinsona,*
a
Department of Large Animal Clinical Sciences, Michigan State University, East Lansing, MI 48824, USA
b
Global Therapeutics Research, Zoetis, 333 Portage St, Kalamazoo, MI, 49007, USA
c
Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical School, VA Ann Arbor Healthcare
System, Research Service (151), Ann Arbor, MI, 48105, USA

A R T I C L E I N F O A B S T R A C T

Because the alveolar macrophage (AM) phenotype of horses with severe equine asthma (SEA) is
Keywords: unknown, the cytokines expressed by M1- and M2-polarized AM were determined and the hypothesis
Horse
that natural hay/straw challenge (NC) induces divergent AM phenotypes in control horses and horses
Immunology
Pulmonary inflammation
with SEA was tested. Macrophages from control horses were activated either with eIFNg +
SEA lipolysaccharide (LPS) or eIL-4 to characterize M1- or M2-polarized AM gene expression, respectively
and determine the response of polarized cells to pathogen-associated molecular patterns (PAMPS): LPS,
zymosan, peptidoglycan and hay dust. Subsequently, gene expression was explored in AM of control
horses and horses with SEA at pasture and after NC.
M1 polarization increased expression of pro-inflammatory cytokines (TNFα, IL-8, IL-12p40), IL-10, and
CD80. M2 polarization increased CD206 and down-regulated arginase-II and IL-10. Expression of pro-
inflammatory cytokines and CD80 in response to PAMPS was further increased by M1 pre-polarization
whereas M2 pre-polarization down-regulated expression of pro-inflammatory cytokines and IL-10 but
increased CD206. In horses with SEA, AMs had elevated expression of IL-10 both at pasture and after NC,
but only after NC in control horses. CD206 expression increased in both groups during NC. At pasture,
stimulation by PAMPS augmented expression of IL-8 and IL-10 in horses with SEA compared to control
horses. NC eliminated this difference by selectively increasing expression of IL-10 in control horses. A
fundamental shift in the macrophage phenotype in SEA is supported by consistently elevated production
of IL-10. A similar non-canonical phenotype develops temporarily in control horses upon NC suggesting
that AMs in horses with SEA have lost the ability to respond dynamically to environmental cues.
© 2020 Elsevier Ltd. All rights reserved.

Introduction and allergic responses (Mosser and Edwards, 2008). Importantly,

Alveolar macrophages (AM) provide one of the first lines of
defense when pro-inflammatory stimuli enter the lung. Depending
on the challenge and the local microenvironment AM can assume
various phenotypes that display differential roles in host-defense
and inflammation (Mantovani et al., 2013). Pro-inflammatory
stimuli such as pathogen-associated molecular patterns (PAMPS)
and gamma-interferon (IFNg) induce the M1 phenotype (Man-
tovani et al., 2013) while a variety of stimuli can induce
alternatively-activated phenotypes, generally referred to as M2
(Gordon and Martinez, 2010). The M2 phenotype tends to be anti-
inflammatory and participates in parasite defense, wound healing
* Corresponding author.
E-mail address: robohand@comcast.net (N.E. Robinson).
1
Current address: VPG Exeter, Building 1B Exeter Science Park, Babbage Way,
Exeter, EX5 2FN, UK.
interleukins (IL)-4 and IL13, key cytokines in many atopic diseases
including allergic asthma, polarize AM into the M2 phenotype
(Arora et al., 2018).
Severe equine asthma (SEA) is an inflammatory obstructive
airway disease in which inhalation of organic dust, particularly hay
dust (HD), worsens inflammation and obstruction (Pirie, 2014;
Bond et al., 2018). The immuno-pathogenesis of SEA is the subject
of debate with evidence for involvement of Th1, Th2, and Th17
pathways (Bullone and Lavoie, 2015). Although several inves-
tigators have measured cytokines released by AM in control horses
and horses with SEA (Laan et al., 2006, 2005; Joubert et al., 2011;
Karagianni et al., 2013, 2017), there have been no reports of AM
phenotypes in these populations in the presence and absence of
HD challenge.
The first goal of our investigation was to define the gene
expression profile of MI- and M2-polarized equine AM and to
examine the effect of pre-polarization on their responses to HD and
its relevant PAMP components. The second aim was to test the

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2 M.E. Wilson et al. / The Veterinary Journal 256 (2020) 105436

hypothesis that the exaggerated inflammatory response of horses Table 1

The effect of IFNg + LPS and IL-4 stimulation on the gene expression of M1 and M2
with SEA to HD would be reflected by either the promotion of a
candidate genes in equine alveolar macrophages. Gene expression is presented as
pro-inflammatory or absence of an anti-inflammatory AM pheno- fold change (mean  standard deviation) compared to untreated non-polarized
type in horses with SEA. We did this by comparing the AM cells.
expression of M1- and M2-associated genes (with and without HD
Gene Agonists
and PAMP incubation) in SEA and control horses at pasture and
following chronic exposure to hay in the stable (natural challenge, IFNg + LPS IL-4

NC). Anticipated M1 genes

TNFα 48.0  23.0a,b 1.4  1.9
IL-8 17.2  14.3a,b 2.7  0.9
Materials and methods
IL-12p40 305.0  55.8a,c ND
IL-6 38.7  28.5a 12.8  12.0
The study was approved by Michigan State University's Institutional Animal CD80 50.0  65.0a,b 6.0  6.4
Care and Use Committee (Application number 01/12-005-00; Date of approval 8 iNOS ND ND
February, 2012). TLR2 1.00  0.7 1.1  1.5
TLR4 1.0  3.4 2.1  0.7
Animals
Anticipated M2 genes
Horses were classified as SEA if NC induced a maximal change in pleural Arginase-II 1.4  3.0 11.5  7.3a
pressure during tidal breathing (DPplmax) of >20 cm H2O and this was CD206 1.6  1.1 13.7  2.9a,b
ameliorated by anti-cholinergic treatment and reversed by removal from NC β-glucan R 25.1  45.9 28.0  48.0
(Robinson, 2001). Control horses had no history of SEA and did not develop IL-10 46.2  45.2a 9.4  7.2a,b
airway obstruction under NC. All horses were maintained at pasture for at least 1 TGFβ 5.8  7.2 1.4  2.7
month before cell collection and were fed a supplemental complete pelleted feed. Chitotriosidase 1.3  1.9 1.3  0.2
Protocol 1 enrolled eight control horses (mean age, 13.4 +/ 5.2 years); protocol 2
enrolled five control horses (mean age, 14 +/ 5 years) and six horses with SEA TNF, tumour necrosis factor; IL, Interleukin; iNOS, Inducible nitric oxide synthase;
(mean age, 20 +/ 2 years). There were no significant differences in horse age. TLR, Toll-like receptor; TGF, Transforming growth factor.
a
Except during NC, all horses were maintained on pasture and supplemented with P  0 .05, compared to non-polarized alveolar macrophages.
complete pelleted feed. Horses with SEA were in remission (DPplmax <10 cm
b
P  0.05 comparing IFNg +LPS- and IL-4-stimulated alveolar macrophages.
c
H20) at the start of the protocol. Data are fold change calculated using empirical CT value in non-polarized
control cells. ND, not detectable.

Pulmonary function and bronchoalveolar lavage

In a separate experiment, AM were stimulated as described above and then
incubated for 16 h with either medium alone, medium plus LPS (100 ng/mL), HD
Pulmonary function was assessed by measurement of DPplmax, pulmonary
(0.02 mg/mL), peptidoglycan (Pep, 1 mg/mL) or zymosan (Zym, 10 mg/mL).
resistance (RL) and dynamic compliance (Cdyn; Behan et al., 2013). Bronchoalveolar
lavage (BAL) was performed on sedated horses (detomidine hydrochloride 0.01
Protocol 2: Effects of natural challenge on alveolar macrophages
mg/kg IV, butorphanol tartrate 0.02 mg/kg, IV) by use of a 3 m endoscope passed
intra-nasally and wedged in a peripheral bronchus. Five hundred mL of sterile saline
Control and horses with SEA came from pasture to the laboratory and were
was infused in 250 mL aliquots and lavage fluid was retrieved by gentle aspiration
bedded on straw and fed hay (NC) (Fig. 1). For horses with SEA, NC continued until
with a 60 mL syringe. The BAL fluid (BALF) was placed on ice and processed within
labored breathing occurred and DPplmax was 15 cm H2O. Because of the
30 min.
complexity of the investigation, it was not possible to harvest macrophages and
process them from two horses in one day. For this reason control horses overlapped
Isolation of alveolar macrophages
in the stable with horses with SEA and received a similar duration of hay/straw
exposure. Lung function measurements were made and BALF harvested immedi-
Macrophages were isolated from BALF by magnetically-activated cell sorting
ately after horses came from pasture and from the contralateral lung at the end of
using negative selection against antibody labelled lymphocytes and neutrophils
NC.
(Appendix: Supplementary material). The enriched macrophage population was re-
An aliquot of enriched AM was taken to quantify gene expression in ‘freshly
suspended (6.25  105 cells/mL) in sterile medium (minus EDTA). A small aliquot
harvested AM.’ The remaining cells were plated into sterile tissue culture dishes
was collected for cell counting and cytology by hemocytometer and light
(12-well at 5  105 cells/well) and incubated (5% CO2, 37  C) for 2 h to allow
microscopy, respectively, and to assess viability by trypan blue exclusion.
adherence. The medium was changed, and adherent cells were incubated for 16 h
Macrophages constituted 87  8% (mean  standard deviation) of cells: the
with medium alone (control) or, medium plus PAMPs as described above. These
remainder were lymphocytes and there no differences between groups or
cultured AM were then harvested and RNA was extracted and stored ( 80  C).
treatments.

Data analysis
RNA extraction, reverse transcription and quantitative real-time PCR

Normality of errors was assessed by visual inspection of the error histogram,

Isolated AM were homogenized and RNA was extracted and 100 ng was reverse
probability plots, and normality testing using SAS-Proc Univariate procedure.
transcribed to create cDNA. Thirty ng of cDNA were pre-amplified and stored at 80
 Normally distributed data (cytology, pulmonary function, freshly harvested AM
C. Quantitative PCR was performed using TaqMan Gene Expression Assays and the
average of two endogenous genes, hyoxanthine ribosyltransferase (HPRT) and
elongation factor-1α (ELF1), was used to normalize each sample (see Appendix:
Supplementary material for details and Table 1 for a list of candidate genes).

Collection of hay dust

A single batch of dust collected and size-fractionated from hay with proven
ability to induce pulmonary inflammation in horses with SEA was used for all parts
of the investigation (Appendix: Supplementary material).

Protocol 1: Polarization of cells

Protocols are summarized in Fig. 1. Enriched AM from pastured control horses

were plated into sterile 12-well cell culture dishes (5  105 cells/well) and
incubated (2 h, 5% CO2, 37  C) to allow adherence. After a medium change, adherent
cells were incubated for 20 h. with either medium (non-polarized control), medium
plus recombinant equine IFNg (20 ng/mL) plus lipopolysaccharide (LPS, 1 ng/mL) or
recombinant equine IL-4 (20 ng/mL). Cells were harvested and RNA extracted and
stored ( 80  C) until gene expression was evaluated. Fig. 1. Schematic representation of Protocols 1 and 2. SEA, severe equine asthma.
 M.E. Wilson et al. / The Veterinary Journal 256 (2020) 105436
gene expression) were analyzed using an ANOVA with the fixed effect of group
and time and the random effect of horse. Variables from stimulated AM were
analyzed with the fixed effect of group, time and treatment and the random
effect of horse (SAS Proc Mixed). Errors that were not normally distributed were
log transformed. Data from cultured AM were corrected for multiple treatment
comparisons using Bonferroni correction. Results were considered significant if
the difference in cycle threshold (delta CT) between comparisons was 1 (2-fold
difference) and P  0.05. All statistical analyses were run on SAS 9.4 (SAS
Institute).
Results
Gene expression by polarized AM
In comparison to incubation with medium alone, incubation of
AM with IFNg + LPS significantly up-regulated TNFα, IL-8, IL-12p40,
IL-6, CD80, and IL-10. TLR2 and TLR4 expression were unaffected.
IL-4 incubation significantly up-regulated CD206 and down-
regulated both IL-10 and Arginase II (Table 1).
PAMP stimulation of polarized AM phenotypes
Interferon and LPS polarization increased expression of the
canonical M1 receptor CD80, while IL-4 increased expression of the
canonical M2 receptor CD206 (Fig. 2). Compared to PAMP-induced
responses of non-polarized cells, IFNg + LPS polarization enhanced
while IL-4 polarization down-regulated the expression of pro-
inflammatory cytokines IL-1β, IL-8, IL-6 and IL-12p40 (Fig. 3).
Significant changes occurred in LPS and HD responses but not
those of Pep or Zym where large variability was observed. IL-10
expression was significantly suppressed by IL-4 polarization but no
significant effects of polarization were observed on TGFβ expres-
sion (Fig. 4).
BALF cytology and pulmonary function
The median duration of natural challenge was 9 days (range
7–21). At pasture, the only significant difference between groups
was a greater BALF lymphocyte percentage in SEA than control
horses (Appendix: Supplementary material). Natural challenge
significantly increased neutrophil percentage in both groups but
the magnitude was significantly greater in horses with SEA.
Lymphocyte percentage decreased in SEA only. In keeping with SEA
phenotype (Robinson, 2001) horses with SEA but not control
horses developed impairments in pulmonary function following
Fig. 2. The effect of polarization on gene expression of surface markers CD80 and CD206 by alveolar macrophages in response to pathogen-associated molecular pattern
(PAMP) stimulation. Responses of non-polarized, IFNg/LPS- and IL-4-polarized cells are shown in white, black, and grey bars, respectively. Macrophages were incubated with
peptidoglycan (Pep), zymosan (Zym) lipopolysaccharide (LPS) or hay dust (HD) for 16 h. Data are expressed as fold change compared to non-polarized-non-stimulated control
horses (mean  standard error). *P  0.05 compared to non-polarized control; †P  0.05 compared to IFNg/LPS treatment.
3
NC indicated by significantly elevated DPpl, and RL, and reduced
Cdyn (Appendix: Supplementary material).
Gene expression in freshly harvested macrophages from horses with
SEA
At pasture, SEA-affected horses had greater expression of IL-10
than control horses (Table 2). This difference was eliminated after
NC because IL-10 expression increased in the control horses but not
horses with SEA. NC also induced a modest but significant
reduction in IL-12p40 expression in SEA-affected horses. There
were no significant differences in expression of other genes.
Effect of PAMPs on gene expression of AM from horses with SEA
In response to LPS, expression of TNFα, IL-1β, IL-12p40, IL-8,
IL-10 significantly increased in comparison to incubation with
medium alone. However AM from pastured horses with SEA
demonstrated significantly greater expression of both IL-8 and
IL-10 than control horses (P < 0.05; Fig. 5). Natural challenge
eliminated these differences because of selectively increased IL-8
and IL-10 expression in AM from control horses but not horses with
SEA (Fig. 5). Although IL-1β expression never differed between
groups its expression in control horses was significantly increased
by NC. In response to Pep and Zym, directional changes in IL-1β and
IL-10 expression were similar to LPS stimulation but not significant
(Appendix: Supplementary material). After incubation with
PAMPS, expression of CD206 never differed between horse groups
but increased significantly in both groups during NC (Table 3).
There were no significant changes in CD80 expression between
groups or in response to NC (Table 3).
Discussion
We hypothesized that the exaggerated inflammatory response of
horses with SEA to HD would be reflected by either the promotion of a
pro-inflammatory or absence of an anti-inflammatory AM pheno-
type in horses with SEA. The results did not support the hypothesis:
the SEA AM phenotype was characterized by elevated expression of
IL-10 both at pasture and during NC. Control horses only develop this
phenotype during NC.
Macrophage polarization in control horses (Protocol 1) resulted
in distinct transcriptional signatures and significantly
impacted the response to PAMPs. The gene expression pattern
4 M.E. Wilson et al. / The Veterinary Journal 256 (2020) 105436

Fig. 3. The effect of polarization on gene expression of inflammatory cytokines by alveolar macrophages in response to pathogen-associated molecular pattern (PAMP)
stimulation. Responses of non-polarized, IFNg/LPS- and IL-4-polarized cells are shown in white, black, and grey bars, respectively. Macrophages were incubated with
peptidoglycan (Pep), zymosan (Zym) lipopolysaccharide (LPS) or hay dust (HD) for 16 h. Data are expressed as fold change compared to non-polarized-non-stimulated control
horses (mean  standard error). *P  0.05 compared to non-polarized control; †P  0.05 compared to IFNg/LPS treatment.

Fig. 4. The effect of polarization on gene expression of regulatory cytokines IL-10 and TGFβ by alveolar macrophages in response to pathogen-associated molecular pattern
(PAMP) stimulation. Responses of non-polarized, IFNg/LPS- and IL-4-polarized cells are shown in white, black, and grey bars, respectively. Macrophages were incubated with
peptidoglycan (Pep), zymosan (Zym) lipopolysaccharide (LPS) or hay dust (HD) for 16 h. Data are expressed as fold change compared to non-polarized-non-stimulated control
horses (mean  standard error). *P  0.05 compared to non-polarized control.

after IFNg + LPS activation of equine AM was typical of the pro- showed a more anti-inflammatory expression pattern typical of
inflammatory M1 AM, i.e., increased TNFα, IL-8, IL-12p40, and the M2/alternative macrophage phenotype with low induction of
CD80 (Table 1) but, no upregulation of iNOS, similar to the findings pro-inflammatory genes accompanied by increased CD206 ex-
of Karagianni et al. (2013, 2017). In contrast IL-4-activated AM pression. Unlike other species, IL-4-treated equine AM lacked
 M.E. Wilson et al. / The Veterinary Journal 256 (2020) 105436 5

Table 2
Comparison of gene expression in freshly-harvested alveolar macrophages in severe equine asthma-affected (SEA) and control horses at pasture and following natural
challenge. Expression of genes is presented as a ratio of fold change between horses with SEA and control horses (left columns) and between natural challenge and pasture
(right columns).

Gene SEA/Control Natural challenge/Pasture

Pasture Natural challenge Control SEA

TNFα 1.26  0.94 1.06  0.37 0.98  0.25 1.27  0.74
IL-1β 1.34  0.40 1.79  2.16 2.89  3.04 2.21  1.20
IL-12 p40 1.38  0.83 0.35  0.18b 1.79  1.24 0.59  0.47b
CD80 1.03  0.33 0.98  0.37 0.70  0.16 0.70  0.65
IL-8 1.07  0.83 0.84  0.47 1.00 0.20 1.00  0.47
IL-6 1.70  1.75 1.40  1.69 1.71  0.90 1.47  1.10
Arginase-II 0.76  0.17 0.89  0.16 1.42  0.39 0.97  0.28
CD206 1.00  0.34 1.1  0.51 1.02  0.16 1.15  0. 34
β-Glucan Receptor 0.76  0.17 0.89  0.16 0.81  0.14 0.99  0.07
TLR4 0.87  0.35 0.91  0.11 0.74  0.24 0.99  0. 07
TLR2 1.27  0.35 0.86  0.45 1.00  0.09 0.99  0.35
TGFβ 1.13  0.38 0.92  0.22 1.04  0.29 0.89  0.40
IL-10 4.35  2.32a 0.80  0.70 15.59  13.24a 1.76  0.77

TNF, tumour necrosis factor; IL, Interleukin; TLR, Toll-like receptor; TGF, Transforming growth factor.
a
P < 0.006.
b
P < 0.027.

Fig. 5. Gene expression in response to LPS in alveolar macrophages of control (black bars, n = 5) and horses with severe equine asthma (SEA; grey bars, n = 5) at pasture and
after natural challenge. Data are expressed as fold change (mean  standard error) relative to control cells harvested at pasture. *Significant difference between SEA and
control (P  0.05) within challenge conditions. †Significant within group difference (P  0.05) between natural challenge and pasture. Please note that all treatments
significantly increased gene expression over that values obtained when cells were incubated with medium alone.

increased IL-10 expression (Table 1) and, even when stimulated
subsequently with PAMPs, IL-4 significantly suppressed IL-10
(Fig. 4). Further, the IFNg + LPS-activated AM, despite being largely
pro-inflammatory, also robustly expressed IL-10.
The PAMPs were chosen based on their presence in HD (Pirie et al.,
2002). Our data on responses to PAMPs indicate that, in comparison
to non-polarized AM, M1-type polarization increased the expression
of IL-1, IL-8, IL-6, IL-12p40, and CD-80 (Fig. 2). However, as also found
by Karagianni et al. (2013), IL-10 expression did not increase (Fig. 4).
This combination gives the AM the potential to be more pro-
inflammatory. In contrast, regardless of the stimulus we
subsequently applied, the M2-like pre-polarization had a potent
immune-regulatory effect as demonstrated by profoundly reduced
pro-inflammatory gene induction (IL-1β, IL-8, IL-12p40 and CD80)
but, surprisingly also diminished IL-10. Furthermore, the M2 surface
receptor CD206 remained significantly elevated under these
conditions. Together, these observations demonstrate that equine
AM pre-polarization significantly alters their abilities to respond to
PAMP stimulation.
When horses were rigorously protected from HD while at
pasture, the AM from horses with SEA was characterized by an
increased expression of IL-10 compared to control horses. However
6 M.E. Wilson et al. / The Veterinary Journal 256 (2020) 105436
Table 3
Effect of pathogen-associated molecular pattern (PAMP) incubation on expression
of CD206 and CD80 in control and horses with severe equine asthma (SEA) at
pasture and during natural challenge. Data are fold change relative to media-
incubated cells at pasture.
Pasture Natural challenge
Control SEA Control SEA
CD206
LPS 2.1  0.2 3.0  1.2 3.8  0.2a 6.1  1.2a
Peptoglycan 6.1  1.0 7.4  0.9 11.0  0.8a 11.6  4.8
Zymosan 5.2  0.4 4.9  0.7 7.8  1.2 8.1  1.1a
CD80
LPS 1.9  0.1 1.8  0.1 2.5  0.8 2.2  0.2
Peptoglycan 3.5  0.2 3.5  0.1 4.6  0.3 6.1  1.2
Zymosan 14.2  2.1 10.5  1.5 10.1  0.8 10.4  3.5
LPS, Lipopolysaccharide.
a
Significant within group difference between pasture and natural challenge (P <
0.05). Please note that all treatments significantly increased gene expression over
values obtained when cells were incubated with medium alone.
during NC, the control AM gene expression became more like that
of horses with SEA as a result of a large increase in IL-10 expression
only in control horses. The elevated IL-10 expression in unstimu-
lated AM (Table 2) suggests that even at pasture when the cellular
component of inflammation is well controlled (low BALF percent
neutrophils, normal lung function), at the molecular level, horses
with SEA maintain an altered AM phenotype. A similar phenotype
with elevated IL-10 expression developed in control horses during
NC. Based on Table 1, increased IL-10 expression characterizes M1
polarization. However, the other feature of M1, increased CD80
expression, was lacking. We conclude, therefore, that in horses
with SEA at pasture and in both groups during NC, the AM has a
non-canonical M1/M2 polarization phenotype.
Further evidence for non-canonical phenotypes, especially
during NC, is provided by the response to PAMPs. Based on Protocol
1, increased CD206 expression characterizes M2 polarization
(Fig. 1) but increased IL-10 lines up with equine M1 macrophage
response (Fig. 2). The co-existence of increased CD206 and IL-10
expression suggests a non-canonical AM phenotype with features
of M1 and M2 polarization in both control and horses with SEA
during NC. Additionally, increased IL-10 at pasture coupled with
decreased IL-12p40 post-NC (Table 2) suggests a shift toward an
immune regulatory-type AM in horses with SEA. These findings
may not be surprising as AM have extensive plasticity (Davis et al.,
2013) and naturally occurring disease is complex, contrasting with
the effect of a single polarizing agent.
A primary effect of IL-10 in macrophages is to inhibit the rate of
transcription of LPS-induced inflammatory genes (Murray, 2005).
However, the role of IL-10 in SEA may be two fold as IL-10 can also
potentiate pulmonary pathology by promoting mucous-cell
metaplasia, and airway remodeling (Lee et al., 2002), which are
both features of airway inflammation in horses (Lugo et al., 2006;
Leclere et al., 2011). Also, given the inhibitory effects of IL-10 on LPS
response, it was surprising that SEA AM maintained responsive-
ness to LPS and IL-8 expression increased. This enhanced LPS
response was not mediated by differences in expression of TLR-4
(Table 2) but could be due to adenosine receptor activation, which
differentially enhances LPS-induced production of IL-10 and IL-8 in
equine monocytes (Sun et al., 2010). Regardless of underlying
causes, IL-10 regulatory function is profoundly altered in SEA AM
with respect to both its natural expression kinetics in response to
presence and absence of hay dust and its potency as an
immunosuppressive mediator. Such disruption of IL-10 regulatory
function could be a contributing factor in SEA pathogenesis,
especially the element of exaggerated inflammatory response to
the inhaled hay dust components such as LPS (Pirie et al., 2001)
and the greater LPS- and HD-induced IL-8 expression in AM from
SEA than control horses (Laan et al., 2006).
The mannose receptor CD206 is the canonical receptor induced
by IL-4 (Stein et al., 1992). Although CD206 was elevated in both
horse groups during NC, the other characteristics of IL-4-activated
equine macrophages (major suppression of pro-inflammatory
cytokines) were not concurrently observed in either horse group.
Therefore, it is unlikely that increasing CD206 expression under NC
was driven by overwhelming IL-4 pulmonary production. The role
of CD206 in airway inflammation is unknown but, mannose
receptor ligation is mostly linked to anti-inflammatory effects
(Gazi and Martinez-Pomares, 2009).
Finally, one limitation of our study is that the purity of our
enriched macrophage population was not a 100%, thus lympho-
cytes may have contributed to the expression of genes studied.
Studies with higher purity AMs may be needed to further refine our
findings.
Conclusions
The dynamic response of AM in control horses to environmental
cues appears to be lacking in horses with SEA. Whether at pasture
or during NC, SEA AM remain in a non-canonical phenotype that
appears to be programmed with an exaggerated response to
inhaled hay dust components that trigger SEA exacerbation. The
cause of these persistent changes and determination of reversibil-
ity by longer-term protection from hay requires further inves-
tigations.
Data statement
The research protocols used in this study were approved by
Michigan State University's Institutional Animal Care and Use
Committee. The research formed part of the first author's PhD
thesis entitled "Characterization of Equine Alveolar Macrophage
Phenotypes in Recurrent Airway Obstruction." The PhD was
completed in 2014 in the Comparative Medicine and Integrative
Biology Program of Michigan State University, East Lansing, Mi
48823, USA.
Conflict of interest
None.
Acknowledgements
The investigation was supported by the Matilda Wilson Equine
Research Endowment to the College of Veterinary Medicine at
Michigan State University. Eilidh Wilson was supported by a Pfizer
Animal Health - Morris Animal Foundation Veterinary Fellowship
for Advanced Study. MAO was supported by VA RCS award
1IK6BX003615-01. The authors are indebted to Ashley Bramman
for her technical help with all aspects of the investigation.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.tvjl.2020.105436.
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