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\begin{document}

\title{NK cells and Lipoxin A 4 promote resolution of eosinophilic inflammation
after nasal allergen challenge}



\author[1]{Anh Poirot}%
\author[1]{Guillaume Wacht}%
\author[2]{Lehalle Christine}%
\author[3]{Philippe Saas}%
\author[2]{Nelly Frossard}%
\author[1]{Bernard Geny}%
\author[1]{Frédéric De Blay}%
\author[3]{Cindy Barnig}%
\affil[1]{Universite de Strasbourg Institut de Finance Strasbourg}%
\affil[2]{Universite de Strasbourg Faculte de pharmacie}%
\affil[3]{Interaction hote-greffon-tumeur et ingenierie cellulaire et genique}%


\vspace{-1em}



  
  \date{May 18, 2024}


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\textbf{NK cells and Lipoxin A\textsubscript{4} promote resolution of
eosinophilic inflammation after nasal allergen challenge}

\emph{To the Editor:}

Allergic airway diseases, such as rhinitis and asthma, are one of the
most common chronic inflammatory respiratory diseases in the world (1).
Although the mechanisms underlying the pathology and treatment of
allergic airway inflammation have been widely studied, many aspects
remain unclear, including how allergic eosinophilic inflammation
effectively resolves in the airways (2). Recent studies implicated
natural killer (NK) cells in the regulation of eosinophilic airway
inflammation, notably by inducing apoptosis of autologous
eosinophils\emph{in vitro} (3). Moreover, lipoxin
A\textsubscript{4}(LXA\textsubscript{4}), a lead member of a larger
family of specialized pro-resolving lipid mediators, can enhance the
ability of NK cells to cause eosinophil-apoptosis i\emph{n vitro} (3)
and decrease allergic eosinophilic inflammation in animal models (4).

In this study, twenty subjects \textbf{(} mean age 28.07 \selectlanguage{ngerman}± 7.1, ten men
and ten women) with confirmed grass pollen allergic rhinitis were
included. All the subjects underwent two standardized nasal allergen
challenges with either a single pre-titrated threshold dose of a grass
pollen allergen extract or a diluent in a randomized order and at least
4 weeks apart (for details see Supp. Material and Methods). Nasal lavage
fluid and cells were collected at baseline and at different time points
after challenge. The study was approved by the local ethics committee
and all subjects gave informed written consent.

The nasal allergen challenge induced typical allergic symptoms and a
local inflammatory response in all patients that resolved spontaneously
within 72 hrs (Figure S1). No significant response was observed after
the diluent challenge. Leukocyte sub-populations were further identified
in nasal lavage samples by FACS (Figure 1A). Neutrophils were the most
abundant population recruited after allergen challenge, peaking 1 hr
after. As expected, eosinophils were also rapidly recruited.
Interestingly, NK cells were recruited as early as 1 h after the
challenge, persisted for 6 hrs before falling to their baseline levels.
Monocyte and lymphoid cell recruitment was also observed (Figure 1B).
There was a positive relationship between the total number of nasal
lavage NK cells and that of eosinophils 1, 6 and 24 hrs after allergen
challenge (Figure 1C).

As LXA\textsubscript{4} can decrease allergic eosinophilic inflammation
and regulate NK cell-eosinophil interaction (3, 4), we next quantified
LXA\textsubscript{4} levels in nasal lavage samples after the allergen
challenge. We observed that LXA\textsubscript{4} was produced in the
nasal mucosa at baseline and significantly increased in the nasal lavage
fluid 1 hr after allergen challenge (Figure 2A). LXA\textsubscript{4} is
produced by multistep enzymatic processes in different cell types,
including neutrophils (6). Interestingly, increased
LXA\textsubscript{4}levels 1 hr after allergen challenge correlated with
the peak of nasal neutrophil infiltration, suggesting a potential role
of neutrophils in LXA\textsubscript{4} biosynthesis during the early
phase of the allergic inflammatory response (Figure 2B).
LXA\textsubscript{4} levels also significantly increased at 48 and 72
hrs post-allergen challenge when compared to baseline levels and
correlated at these time points to monocyte recruitment.

As previously shown, we observed that peripheral blood NK cells isolated
from healthy donors induce apoptosis of autologous blood
eosinophils\emph{in vitro} (Figure S2)\emph{.} Moreover, to induce
eosinophil apoptosis a direct contact and a combined action of
CD56\textsuperscript{bright} and CD56\textsuperscript{dim} NK cells were
needed (Figure S2). Besides a potential pro-resolving effect of NK cells
on eosinophils, recent data has shown that NK cells can also trigger
superoxide release by eosinophils, that can worsen inflammation (5). In
our study, eosinophils isolated from healthy donors were by far the most
important producers of superoxide anion among different leukocyte
populations (Figure 2C). In the presence of NK cells, superoxide release
from eosinophils was significantly reduced after 1 hr of co-incubation.
In contrast, this inhibitor effect was no longer present after 4 hrs of
co-incubation (Figure 2D). When eosinophils from healthy donors were
exposed to LXA\textsubscript{4}, they significantly reduced their
superoxide release in a dose-dependent manner (Figure 2E). Superoxide
release by eosinophils co-incubated with NK cells in the presence of
LXA\textsubscript{4} was still inhibited in a dose-dependent manner 4
hrs later (Figure 2F) in contrast without LXA\textsubscript{4} (Figure
2D).

Our study underlines the complex network between cellular and molecular
actors during resolution of allergic airway inflammation. Here we report
for the first time that NK cells are recruited to the nasal mucosa of
subjects with allergy in response to nasal allergen challenge and
correlate with eosinophilic inflammation. The accumulation of
neutrophils along with monocytes during the allergic inflammatory
response may furthermore be an important regulatory feedback to initiate
and promote resolution of allergic inflammation as our data suggest
involvement of these cells in LXA\textsubscript{4} biosynthesis.
Moreover, we identified a combined role for NK cells and
LXA\textsubscript{4} in mediating resolution of eosinophilic
inflammation \emph{in vitro} .

Anh Poirot, BSc\textsuperscript{1};

Guillaume Wacht, MD, MSc\textsuperscript{2};

Christine Lehalle, MSc\textsuperscript{3};

Philippe Saas, PhD\textsuperscript{4};

Nelly Frossard, PhD\textsuperscript{3};

Bernard Geny, MD, PhD\textsuperscript{2};

Fréderic de Blay, MD\textsuperscript{1};

Cindy Barnig, MD, PhD\textsuperscript{4,5}

\textsuperscript{1} Department of Chest Disease, University Hospital of
Strasbourg, Strasbourg, France

\textsuperscript{2} EA 3072, University of Strasbourg, France

\textsuperscript{3} UMR 7200 CNRS / Université de~Strasbourg,
Laboratoire d'Innovation Thérapeutique and LabEx MEDALIS, Faculté de
Pharmacie, Strasbourg, France

\textsuperscript{4} Univ. Bourgogne Franche-Comté, INSERM, EFS BFC,
UMR1098, Interactions Hôte-Greffon-Tumeur/Ingénierie Cellulaire et
Génique, LabEx LipSTIC, F-25000 Besançon, France

\textsuperscript{5} Department of Chest Disease, University Hospital of
Besançon, Besançon, France

\emph{Corresponding author:}

Cindy Barnig

Univ. Bourgogne Franche-Comté, INSERM, EFS BFC, UMR1098, Interactions
Hôte-Greffon-Tumeur/Ingénierie Cellulaire et Génique, LabEx LipSTIC,
F-25000 Besançon, France

Tel: +33 3 81 66 88 02

e-mail~: cindy.barnig@univ-fcomte.fr

\textbf{Acknowledgments}

We thank Pr Seiamak Bahram and Dr Beatrice Uring-Lambert for access to
flow cytometer.

\textbf{Funding Statement~}

This study was supported by «~Appel à Projets Jeunes Chercheurs 2013
GIRCI Est~» and «~Subvention 2015 de la Société Française
d'Allergologie~».

\textbf{Conflict of interest~}

The authors declare that there is no conflict of interest.

\textbf{References}

1. Bousquet J, Anto JM, Bachert C, Baiardini I, Bosnic-Anticevich S,
Walter Canonica G, et al. Allergic rhinitis. Nat Rev Dis Primers.
2020;6(1):95.

2. Barnig C, Frossard N, Levy BD. Towards targeting resolution pathways
of airway inflammation in asthma. Pharmacol Ther. 2018;186:98-113.

3. Barnig C, Cernadas M, Dutile S, Liu X, Perrella MA, 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.

4. Levy BD, De Sanctis GT, Devchand PR, Kim E, Ackerman K, Schmidt BA,
et al. Multi-pronged inhibition of airway hyper-responsiveness and
inflammation by lipoxin A(4). Nat Med. 2002;8(9):1018-23.

5. Awad A, Yassine H, Barrier M, Vorng H, Marquillies P, Tsicopoulos A,
et al. Natural killer cells induce eosinophil activation and apoptosis.
PLoS One. 2014;9(4):e94492.

6. Chavis C, Vachier I, Chanez P, Bousquet J, Godard P.
5(S),15(S)-dihydroxyeicosatetraenoic acid and lipoxin generation in
human polymorphonuclear cells: dual specificity of 5-lipoxygenase
towards endogenous and exogenous precursors. J Exp Med.
1996;183(4):1633-43.

\textbf{Figure 1. NK cells appear early during the allergic inflammatory
response and correlate with eosinophils.} Subjects (n=20) suffering from
confirmed seasonal grass pollen rhinitis were challenged intranasally
with a single threshold dose of a grass pollen extract or
diluent.\textbf{(A)} Representative dot plots for flow cytometric
sequential gating of leukocyte populations in nasal lavage. \textbf{(B)}
Time course of neutrophils, eosinophils, NK cells, lymphoid cells, T
lymphocytes and monocytes following allergen nasal challenge or diluent.
Data are displayed as mean ± SEM; *P \textless{} 0.05; **P \textless{}
0.01; ***P \textless{} 0.001; 2-way ANOVA with Sidaks's multiple
comparison test was used to compare allergen vs diluent at various time
points. \textbf{(C)} Correlation between total NK cells and eosinophils
1, 6 and 24h after the allergen challenge (Pearson correlation
\emph{r}value and significance are noted).

\textbf{Figure 2. LXA\textsubscript{4} is produced in the nasal mucosa
after nasal allergen and is essential to inhibit NK cells triggered
eosinophil superoxide release \emph{in vitro}.}
\textbf{(A)}LXA\textsubscript{4} concentrations in nasal lavage samples;
*\emph{P}\textless{} 0.05 compared to diluent,
\textsuperscript{\$}P\textless{} 0.05 compared to baseline levels;
allergen \emph{vs} diluent: 2-way ANOVA with Sidaks's multiple
comparison test, levels \emph{vs} baseline levels: 2-way ANOVA with
Tukey's multiple comparison test. \textbf{(B)}Correlation of
LXA\textsubscript{4} levels and neutrophil and monocyte counts after
allergen challenge; Pearson correlation \emph{r} value and significance
are noted). \textbf{(C)} Superoxide release by different leukocyte
population at rest (vehicle) and when activated by phorbol myristate
acetate (PMA), \emph{n} = 5 healthy donors, *P \textless{} 0.05, at rest
(vehicle) when cells are compared to each other; **P \textless{} 0.05,
after PMA when cells are compared to each other;\textsuperscript{£}P
\textless{} 0.05, when cells are compared between rest (vehicle) and
PMA; multiple \emph{t} -tests with Bonferroni correction. \textbf{(D)}
Superoxide release from eosinophils co-incubated with NK cells, \emph{n}
= 7 healthy donors, *p\textless{}0.05, paired Student's \emph{t} test.
\textbf{(E)}Superoxide release from eosinophils pre-treated with
increasing doses of LXA\textsubscript{4} and stimulated with PMA;
\emph{n} = 6 healthy donors; *p\textless{}0.05 compared to
vehicle;\textsuperscript{£}p\textless{}0.05 compared to 1 nM; one-way
ANOVA with Tukey's multiple comparison test. \textbf{(F)} , Superoxide
release from eosinophils pre-treated with LXA\textsubscript{4} and
co-incubated with NK cells, \emph{n} = 3 healthy donors; *P \textless{}
0.05; multiple\emph{t} -tests with Bonferroni correction. Results are
expressed as means ± SEM.



\textbf{Hosted file}

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\textbf{Hosted file}

\verb`image2.emf`  available at \url{https://authorea.com/users/414290/articles/529258-nk-cells-and-lipoxin-a-4-promote-resolution-of-eosinophilic-inflammation-after-nasal-allergen-challenge}

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