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The Veterinary Journal: M.E. Wilson, E.E. Mccandless, M.A. Olszewski, N.Edward Robinson
The Veterinary Journal: M.E. Wilson, E.E. Mccandless, M.A. Olszewski, N.Edward Robinson
The Veterinary Journal: M.E. Wilson, E.E. Mccandless, M.A. Olszewski, N.Edward Robinson
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.
http://dx.doi.org/10.1016/j.tvjl.2020.105436
1090-0233/© 2020 Elsevier Ltd. All rights reserved.
2 M.E. Wilson et al. / The Veterinary Journal 256 (2020) 105436
Data analysis
RNA extraction, reverse transcription and quantitative real-time PCR
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).
gene expression) were analyzed using an ANOVA with the fixed effect of group NC indicated by significantly elevated DPpl, and RL, and reduced
and time and the random effect of horse. Variables from stimulated AM were
Cdyn (Appendix: Supplementary material).
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 Gene expression in freshly harvested macrophages from horses with
comparisons using Bonferroni correction. Results were considered significant if SEA
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).
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
Results
horses with SEA. NC also induced a modest but significant
reduction in IL-12p40 expression in SEA-affected horses. There
Gene expression by polarized AM
were no significant differences in expression of other genes.
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.
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).
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 applied, the M2-like pre-polarization had a potent
subsequently with PAMPs, IL-4 significantly suppressed IL-10 immune-regulatory effect as demonstrated by profoundly reduced
(Fig. 4). Further, the IFNg + LPS-activated AM, despite being largely pro-inflammatory gene induction (IL-1β, IL-8, IL-12p40 and CD80)
pro-inflammatory, also robustly expressed IL-10. but, surprisingly also diminished IL-10. Furthermore, the M2 surface
The PAMPs were chosen based on their presence in HD (Pirie et al., receptor CD206 remained significantly elevated under these
2002). Our data on responses to PAMPs indicate that, in comparison conditions. Together, these observations demonstrate that equine
to non-polarized AM, M1-type polarization increased the expression AM pre-polarization significantly alters their abilities to respond to
of IL-1, IL-8, IL-6, IL-12p40, and CD-80 (Fig. 2). However, as also found PAMP stimulation.
by Karagianni et al. (2013), IL-10 expression did not increase (Fig. 4). When horses were rigorously protected from HD while at
This combination gives the AM the potential to be more pro- pasture, the AM from horses with SEA was characterized by an
inflammatory. In contrast, regardless of the stimulus we increased expression of IL-10 compared to control horses. However
6 M.E. Wilson et al. / The Veterinary Journal 256 (2020) 105436
Table 3 and the greater LPS- and HD-induced IL-8 expression in AM from
Effect of pathogen-associated molecular pattern (PAMP) incubation on expression
SEA than control horses (Laan et al., 2006).
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- The mannose receptor CD206 is the canonical receptor induced
incubated cells at pasture. by IL-4 (Stein et al., 1992). Although CD206 was elevated in both
horse groups during NC, the other characteristics of IL-4-activated
Pasture Natural challenge
equine macrophages (major suppression of pro-inflammatory
Control SEA Control SEA cytokines) were not concurrently observed in either horse group.
CD206 Therefore, it is unlikely that increasing CD206 expression under NC
LPS 2.1 0.2 3.0 1.2 3.8 0.2a 6.1 1.2a was driven by overwhelming IL-4 pulmonary production. The role
Peptoglycan 6.1 1.0 7.4 0.9 11.0 0.8a 11.6 4.8
of CD206 in airway inflammation is unknown but, mannose
Zymosan 5.2 0.4 4.9 0.7 7.8 1.2 8.1 1.1a
receptor ligation is mostly linked to anti-inflammatory effects
CD80 (Gazi and Martinez-Pomares, 2009).
LPS 1.9 0.1 1.8 0.1 2.5 0.8 2.2 0.2 Finally, one limitation of our study is that the purity of our
Peptoglycan 3.5 0.2 3.5 0.1 4.6 0.3 6.1 1.2
enriched macrophage population was not a 100%, thus lympho-
Zymosan 14.2 2.1 10.5 1.5 10.1 0.8 10.4 3.5
cytes may have contributed to the expression of genes studied.
LPS, Lipopolysaccharide. Studies with higher purity AMs may be needed to further refine our
a
Significant within group difference between pasture and natural challenge (P <
findings.
0.05). Please note that all treatments significantly increased gene expression over
values obtained when cells were incubated with medium alone.
Conclusions
during NC, the control AM gene expression became more like that The dynamic response of AM in control horses to environmental
of horses with SEA as a result of a large increase in IL-10 expression cues appears to be lacking in horses with SEA. Whether at pasture
only in control horses. The elevated IL-10 expression in unstimu- or during NC, SEA AM remain in a non-canonical phenotype that
lated AM (Table 2) suggests that even at pasture when the cellular appears to be programmed with an exaggerated response to
component of inflammation is well controlled (low BALF percent inhaled hay dust components that trigger SEA exacerbation. The
neutrophils, normal lung function), at the molecular level, horses cause of these persistent changes and determination of reversibil-
with SEA maintain an altered AM phenotype. A similar phenotype ity by longer-term protection from hay requires further inves-
with elevated IL-10 expression developed in control horses during tigations.
NC. Based on Table 1, increased IL-10 expression characterizes M1
polarization. However, the other feature of M1, increased CD80 Data statement
expression, was lacking. We conclude, therefore, that in horses
with SEA at pasture and in both groups during NC, the AM has a The research protocols used in this study were approved by
non-canonical M1/M2 polarization phenotype. Michigan State University's Institutional Animal Care and Use
Further evidence for non-canonical phenotypes, especially Committee. The research formed part of the first author's PhD
during NC, is provided by the response to PAMPs. Based on Protocol thesis entitled "Characterization of Equine Alveolar Macrophage
1, increased CD206 expression characterizes M2 polarization Phenotypes in Recurrent Airway Obstruction." The PhD was
(Fig. 1) but increased IL-10 lines up with equine M1 macrophage completed in 2014 in the Comparative Medicine and Integrative
response (Fig. 2). The co-existence of increased CD206 and IL-10 Biology Program of Michigan State University, East Lansing, Mi
expression suggests a non-canonical AM phenotype with features 48823, USA.
of M1 and M2 polarization in both control and horses with SEA
during NC. Additionally, increased IL-10 at pasture coupled with Conflict of interest
decreased IL-12p40 post-NC (Table 2) suggests a shift toward an
immune regulatory-type AM in horses with SEA. These findings None.
may not be surprising as AM have extensive plasticity (Davis et al.,
2013) and naturally occurring disease is complex, contrasting with Acknowledgements
the effect of a single polarizing agent.
A primary effect of IL-10 in macrophages is to inhibit the rate of The investigation was supported by the Matilda Wilson Equine
transcription of LPS-induced inflammatory genes (Murray, 2005). Research Endowment to the College of Veterinary Medicine at
However, the role of IL-10 in SEA may be two fold as IL-10 can also Michigan State University. Eilidh Wilson was supported by a Pfizer
potentiate pulmonary pathology by promoting mucous-cell Animal Health - Morris Animal Foundation Veterinary Fellowship
metaplasia, and airway remodeling (Lee et al., 2002), which are for Advanced Study. MAO was supported by VA RCS award
both features of airway inflammation in horses (Lugo et al., 2006; 1IK6BX003615-01. The authors are indebted to Ashley Bramman
Leclere et al., 2011). Also, given the inhibitory effects of IL-10 on LPS for her technical help with all aspects of the investigation.
response, it was surprising that SEA AM maintained responsive-
ness to LPS and IL-8 expression increased. This enhanced LPS Appendix A. Supplementary data
response was not mediated by differences in expression of TLR-4
(Table 2) but could be due to adenosine receptor activation, which Supplementary material related to this article can be found, in the
differentially enhances LPS-induced production of IL-10 and IL-8 in online version, at doi:https://doi.org/10.1016/j.tvjl.2020.105436.
equine monocytes (Sun et al., 2010). Regardless of underlying
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