# The IL-15 / sIL-15Rα complex modulates immunity without effect on asthma features in mouse

Antoine Moui, Martin Klein, Dorian Hassoun, Eléonore Dijoux, Marie Aude Cheminant, Antoine Magnan, Grégory Bouchaud

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Antoine Moui, Martin Klein, Dorian Hassoun, Eléonore Dijoux, Marie Aude Cheminant, et al.. The IL-15 / sIL-15Rα complex modulates immunity without effect on asthma features in mouse. Respiratory Research, 2020, 21 (1), pp.1-10. 10.1186/s12931-020-1301-x. hal-02619754

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# The IL-15 / sIL-15Rα complex modulates immunity without effect on asthma features in mouse

Antoine Moui<sup>1,2</sup>, Martin Klein<sup>1</sup>, Dorian Hassoun<sup>1,2</sup>, Eléonore Dijoux<sup>1</sup>, Marie-Aude Cheminant<sup>1</sup>, Antoine Magnan<sup>1,2</sup> and Grégory Bouchaud<sup>3* ID</sup>

## Abstract

**Background:** Interleukin 15 (IL-15) is a growth and modulating factor for B, T lymphocytes and natural killer cells (NK). Its action on innate and adaptive immunity is modulated by its alpha chain receptor (IL-15Rα). The IL-15/sIL-15Rα complex (IL-15Cx) increases the bioavailability and activity of the cytokine in vivo. IL-15Cx has been used in diseases to dampen IL-15 inflammation by the use of soluble IL-15Ralpha specificity. Here, we aim to evaluate the interest of IL-15Cx in a mouse model of asthma.

**Methods:** Using a mouse model of asthma consisting in percutaneous sensitization and intranasal challenge with total house dust mite extract, we evaluated the effect of IL-15Cx injected intraperitoneally four times after a first nasal challenge. Respiratory function was assessed by the technique of forced oscillations (Flexivent®). The effect on bronchial remodeling was evaluated by lung histology. The inflammatory status was analyzed by flow cytometry.

**Results:** We observed that the IL-15Cx modulates lung and systemic inflammation by increasing NK cells, CD8+ memory T cells and regulatory cells. However, IL-15Cx displays no effect on bronchial hyperreactivity, bronchial remodeling nor cellular bronchial infiltrate, but limits the secretion of bronchial mucus and modulates only inflammatory response in a HDM-allergic asthma murine model.

**Conclusions:** IL-15Cx has a limited effect on immune response in asthma and has no effect on lung function in mice. Thus, it limits its therapeutic potential but might suggest a combinatory potential with other therapeutics.

**Keywords:** Interleukin-15, Interleukin-15 complex, Asthma, Allergy

## Background

Interleukin 15 is a pleiotropic cytokine of the family of cytokines with 4 α-helices also including members such as IL-2, IL-3, IL-4, IL-6 and IL-21. It was discovered in 1994 as a cytokine inducing the proliferation of T cells independently of IL-2 but thanks to the β receptor of IL-2 (IL-2Rβ) [1]. It has similar functionalities to IL-2, with which it shares common receptor chains, IL-2 / 15Rβ (CD122) and γc (CD132) [2]. Cytokine specificity is conferred by the presence of their respective α-receptor chains, IL-15Rα (CD215) and IL-2Rα (CD25). IL-15Rα has a high affinity for IL-15 (kD ~ 10⁻¹¹ M) independently of the β and γc chain [3], and its mRNA is found

in many tissues. Kennedy et al. determined the cellular response to IL-15 through the use of IL-15 and IL-15Rα deficient mice. Indeed, these mice had a deficiency in Natural Killer (NK) cells, Natural Killer T (NKT) cells, γδ T lymphocytes, naive and memory CD8+ T lymphocytes [4], demonstrating a role in innate and adaptive immunity. Furthermore, the main targets of IL-15 in vivo seem to be NK cells and CD8+ T lymphocytes since the administration of recombinant IL-15 to mice causes a rapid increase of these cells [5]. Its role in the proliferation and survival of memory CD8+ T cells is also well established [6]. IL-15 is synthesized in small amounts mainly by antigen presenting cells such as dendritic cells, monocytes and macrophages. When the IL-15 / IL-15Rα complex (IL-15Cx) is routed to the surface of these cells, it can stimulate NK cells and CD8+ T cells

<footnote>* Correspondence: gregory.bouchaud@inra.fr</footnote>

<footnote n="3">³INRA, UR1268 BIA, rue de la Géraudière, F-44316 Nantes, France
Full list of author information is available at the end of the article</footnote>
via their IL-2Rβ and γc receptor, thus allowing a func-
tional response to IL-15. This mechanism of action of
IL-15 in vivo is called trans-presentation [7, 8]. In 2004,
it was shown that a soluble form of human IL-15Rα
could also be released naturally from cells bearing this
receptor, through an excretion process involving matrix
metalloproteinases [9]. This soluble IL-15Rα receptor
was able to bind to IL-15 with high affinity and effect-
ively blocked IL-15 dependent proliferation of cells
through the IL-15Rα/β/γ high affinity heterotrimeric sig-
naling complex. In addition, the complex formed by IL-
15 and the soluble IL-15Rα (IL-15Cx) is able to improve
the binding as well as the bioactivity of IL-15 via the IL-
15Rβ/γ intermediate affinity heterodimeric receptor [10].
These divergent results led to study the IL-15Cx in vivo
in mouse models. Biological activity of IL-15 is improved
after interaction with recombinant soluble IL-15Rα [11]
and injection of the complex rapidly induced prolifera-
tion of memory CD8+ T cells and NK cells. These re-
sults could be explained by a conformational change of
IL-15 after interaction with soluble IL-15Rα during IL-
15Cx formation which potentiates the recognition of IL-
15 by the βγc receptor on T lymphocytes, via a 13-
amino acid (aa) peptide present on the encoded ectodo-
main by exon 3 [12]. The administration of an IL-15Cx
will have a modulatory effect on the activity of the cyto-
kine by allowing fixation only to cells expressing the di-
meric βγc receptor. Another advantage of this complex
is that it increases the biological activity of IL-15 by 50-
fold and its half-life by 20-fold [13], regardless of the
route of administration. The therapeutic utility was also
tested by the same team by demonstrating that com-
plexed IL-15 compared to IL-15 alone, significantly re-
duced tumor burden in a B16 melanoma model. The
pulmonary inflammatory modulation of the IL-15Cx is
not well documented and could be interesting for in-
flammatory diseases. Asthma is a common pathology
with a significant inflammatory component, conducive
to the study of the complex. IL-15 seems to have a role
in the pathophysiology of allergic asthma. Recently, Ven-
kateshaiah et al. showed that IL-15 deficiency increased
airway resistance and decreased compliance in a mouse
model of asthma [14]. Furthermore, administration of
recombinant IL-15 decreased allergic airway obstruction.
Overexpression of IL-15 increased interferon gamma
and decreased pro-inflammatory Th2 cytokine levels as
well as endothelial hyperplasia. Despite these interesting
results, some points are still to be clarified. In fact, the
model used was more a model of pulmonary inflamma-
tion than a model of allergy. The use of an allergic
asthma model with a TH2 / TH17 inflammatory compo-
nent approximating asthma in humans would better re-
flect the effect of IL-15 in asthma. In addition, using the
IL-15Cx would prolong half-life and bioavailability of

the cytokine, still in a human therapy projection. Here
we investigated inflammatory modulation of IL-15Cx in
steady state and its therapeutic potential in a mouse
model of asthma.

Methods

Mouse model of acute allergic asthma to house dust
mites

Female BALB/c mice 6 weeks old (JAX Mice BALB /
cByJ, Charles River) were used for animal experimenta-
tion. As described previously [15], the model was con-
structed with two phases, a sensitization phase and a
challenge phase (Fig. 4a). Before each allergenic expos-
ure, mice were anesthetized by intraperitoneal injection
(IP) of 100 µl of a mixture of ketamine (80 mg/kg) and
xylazine (15 mg/kg). The allergen used was house dust
mites (HDM) (Stallergenes, Antony, France) containing
*Dermatophagoides pteronyssinus*. Five hundred micro-
grams of pure HDM diluted in dimethylsulfoxide
(DMSO, Sigma-Aldrich, L'Isle d'Abeau Chesnes, France)
were applied percutaneously to both sides of each ear.
Four weekly applications over 3 weeks were performed
(D0, D7, D14 and D21). For control mice, DMSO 70%
diluted in isotonic saline (NaCl) was applied percutan-
eously. The challenge phase included two challenges 1
week apart (D27 and D34). Two hundred and fifty µg of
HDM diluted in phosphate buffered saline (PBS) were
inoculated intranasally using a micropipette. The control
mice received only PBS. This protocol received a favor-
able opinion from the Pays de la Loire ethics committee
under number 9456. The analyzes were performed the
day after the second challenge (J35), after a sacrifice by
injecting a lethal dose of EUTHASOL (150 mg/kg
intraperitoneally).

IL-15Cx

Human recombinant IL-15 (12.9 k Dalton, Peprotech
France) and human IL-15 Rα-Fc (42.6 k Dalton R & D
Systems) were reconstituted in PBS at concentrations of
10 µg/mL and 60 µg/mL respectively. The complex was
prepared with a molar ratio of 1 to 1. Both components
were mixed together for 20 min at room temperature be-
fore injection. Between the two challenge phases, control
and asthmatic treated mice received four injections of
100 µL of the complex composed by 0.5 µg of rhIL-15
and 3 µg of rhIL-15Rα-Fc. The mice not treated with the
complex received 100 µL of PBS intraperitoneally.

Airway hyperresponsiveness measurements

Tracheotomy was performed surgically on anesthetized
mice and a cannula was inserted. The mice were placed
in the Finepoint RC system room (Data Sciences Inter-
national, St Paul, Minnesota) and connected to a ventila-
tor. Mechanical ventilation was started with a
respiratory rate of 150/min and a tidal volume of 0.15 mL as previously described [16]. The mice were nebulized with NaCL (baseline reference data) then with methacholine at increasing doses (5, 10, 15 and 20 mg/ L). Aerosols of 20 µL were generated by an ultrasonic nebulizer and aspirated through the chamber for 30 s. Resistance (cmH2O.sec/mL) was measured with an average of the values generated within 3 min after each nebulization. At the end of the protocol, the mice were sacrificed.

## Histology

For each condition, lungs were perfused with 4% paraformaldehyde, then removed and deposited in the same solution. The right and left lungs were separated and cut perpendicular to the insertion of the bronchus and then embedded in paraffin using an inclusion automaton. Histological sections of 7 µm thick were made using a microtome on the paraffin blocks. These sections were then stained with Hematoxylin & Eosin staining for morphological studies and calculation of a histological score. Micrographs of the stained sections were then taken under the microscope to allow analysis on the ImageJ software (Cambridge, UK). The histological score was calculated on 12 points, of which 8 points to evaluate the bronchial inflammation and 4 points for the bronchial remodeling. Schiff periodic acid staining (PAS) was also applied for each condition on different sections to evaluate bronchial mucus secretion score. This score was calculated by counting 5 bronchial sections per group, the total number of PAS positive cells (goblet cells) relative to the total number of epithelial cells of this bronchus. Scores were evaluated by blinded observers.

## Flow cytometry

After mice dissection, lungs, mediastinal lymph nodes and spleens were collected in 15 mL of RPMI, milled and filtered through 40 µm cell sieves. We also collected the bronchoalveolar lavages by intra-tracheal injection then aspiration of 1 ml of PBS. After centrifugation for 5 min at 260 relative centrifugal force (RCF), the lung pellet and spleen were suspended in Red Blood Cell lysis (RBC, Sigma) to destroy the red blood cells followed by an addition of 9 mL of FACS Buffer (1% 0.5 M EDTA pH 8, 5% FCS, 1X DPBS without Ca2+ without Mg2+, Gibco) to stop the action of RBC. The sample cells were then counted on a KOVA slide (California, USA). In parallel, each sample was deposited on a 96-well conical bottom plate. The cells were then centrifuged for 2 min at 310 RCF and then incubated for 20 min at 4 °C with the various antibodies in the presence of Fc blocker CD16/32 (BD Bioscience). For extracellular labeling each antibody was diluted in FACS Buffer: CCR3-AF647,

CD3-PerCP Cy5.5, Ly6C-PE, CD4-APC-Cy7, CD25-PE-Cy7, CD8-FITC, CD44-BV421, CD122-PE, CD11c-APC and MHC II-BV510 (Sony, UK), NKp46-AF700, DX5-PeCy7 and CXCR3-BV510 (Biosciences, Laix Bridge) and F4/80-FITC (Biolegend, San Diego, USA). After extracellular labeling, the samples were centrifuged and then resuspended in 200 µL of FACS Buffer. The cells were centrifuged for 2 min at 310 RCF at 4 °C and then permeabilized with a fixing/ permeabilization solution (BD Phosflow™, BD Biosciences) for 15 min at 4 °C in the dark. They were then incubated for 40 min at 4 °C with the FoxP3-AF647 antibody (Sony) in the permeabilization solution. Finally, we resuspended them in 200 µL of FACS Buffer. The cells were analyzed by flow cytometry on the Fortessa TM LSRII (BD Biosciences) and the data acquired on the FACS Diva 8 software (BD Biosciences). Data were analyzed on the Flowjo software (TreeStar, version 10.4).

## Statistical analysis

Statistical analysis to compare the different groups of mice were performed using the GraphPad Prism 7 software (version 7.0a; GraphPad Software, San Diego, California). The D'Agostino Pearson and Bartlett's tests were performed to test the normality of the values and the difference of the variances respectively. Subsequently, the nonparametric Mann Whitney test was used to compare for non-normal-law variables and the parametric t-test for normal-law variables, as indicated in figure legend. Values are represented on figures with mean ± SEM (Standard Error of the Mean). A value of *p* < 0.05 was considered statistically significant.

## Results

### IL-15 complex modulates lung inflammation

In order to explore the potential of IL-15Cx on the lungs, we analyzed innate and adaptive responses in control mice (CTL) and to mice which received four injections of the IL-15 complex (IL-15Cx group) over 1 week. We first looked at NK cells (NKP46+, DX5+, CD3-), a population increased by injection of the complex in spleen but not in lungs and mediastinal lymph nodes (Fig. 1). In the lungs, there was no statistically significant difference but a tendency to an increase of NK cells in IL-15Cx compared to CTL (4267 ± 1100 cells versus 1983 ± 685 respectively, *p* = 0,09) (Fig. 1a). There was no evidence of a change in the number of NK cells in the mediastinal lymph nodes after injection of the IL-15Cx (Fig. 1b). However, based on findings from past studies on IL-15Cx, we observed an increase in NK cells in IL-15Cx treated group compared to CTL in the spleen (23, 630 ± 2120 cells versus 8060 ± 1080 respectively, *p* = 0, 002) (Fig. 1c). Given an almost significant difference on NK cells in the lung, we wanted to further explore the
<figure><img src="image_3.png" /></figure>

pulmonary and systemic inflammatory effects of the complex on other cell populations. To this end, we analyzed CD8 and CD4 lymphocytes subpopulations (Fig. 2). We observed an increase in CD8⁺ lymphocytes (CD8⁺, CD44⁺, CD122⁺) in lung after administration of the complex (9,3% versus 17,4% respectively, *p* = 0,009) (Fig.

2a), but not in the spleen (Fig. 2b). On the contrary, CD8⁺ lymphocytes expressing the CXCR3 receptor (CD8⁺, CXCR3⁺) were increased in the spleen (6,6% versus 13,22% respectively, *p* = 0,001) (Fig. 2d) but not in the lung (Fig. 2c). Moreover, we analyzed CD4⁺ T-helper cells in the lungs (Fig. 2e) and in the spleen (Fig.

<figure><img src="image_4.png" /></figure>
2f) and no effect was observed. On the other hand, when we explored the effect of IL-15Cx on T regulatory lymphocytes (Treg) (CD4+, CD25+, FOXP3+), and found an increase of their percentage in the lungs (2,62% in CTL versus 5,88% in IL15Cx, p = 0,02) (Fig. 2g) but not in the spleen (2,81% versus 1,13% respectively, p = 0,15) (Fig. 2h). Altogether, IL-15Cx modulates adaptive immunity and may have an effect on innate cells. Among them, dendritic cells and macrophages are known to be partially dependent of IL-15 for their homeostasis. These cells secrete IL-15 and activate other cells by transpresentation through the IL-15Rα. Administration of the IL-15 complex containing the IL-15Rα should theoretically not activate these cells but we wanted to clarify this hypothesis in vivo. We performed flow cytometry on lungs (Fig. 3a and d), mediastinal lymph nodes (Fig. 3b and e) and spleen (Fig. 3c and f) in CTL and IL-15Cx group to analyze macrophages and dendritic cells respectively. We did not observe significant difference after administration of the IL-15Cx (Fig. 3) on both cell populations. These results are consistent with the mechanism of action of the complex and in particular the absence of activation of the cells expressing the soluble receptor alpha of IL-15.

## The IL-15 complex does not improve asthma features

Having shown that IL-15Cx has interesting properties and plays a role in modulation of NK cells, CD8+ lymphocytes and T regulatory lymphocytes, we aimed to evaluate effect of IL-15Cx in airway inflammation induced by house dust mite (HDM). Asthma results in chronic inflammation of the lower airways, bronchoconstriction and bronchial hypersecretion, associated with bronchial remodeling (smooth muscle hypertrophy, epithelial cell hyperplasia, basement membrane thickening). We decided to analyze these different pillars of pathophysiology with a murine model of allergic asthma to HDM (Fig. 4a) characterized by a mixed inflammation Th2 and Th17 [15]. We verified the levels of endogenous human and mouse IL-15 after injection of the complex in wild type mice and in asthma challenged mice. As expected, endogenous mouse IL-15 levels were not modified after IL-15Cx injection, but human IL-15 levels were increased (data not shown). An essential component for studying asthma is bronchial hyperreactivity. This can be evaluated by an invasive method that uses forced oscillometry, Flexivent*. We performed it on control group (CTL), asthmatic mice group (HDM group) and on asthmatic mice treated by IL-15Cx group (HDM IL-15Cx group) (Fig. 4b). As expected, asthmatic mice

<figure><img src="image_5.png" /></figure>
<figure><img src="image_6.png" /></figure>

showed an increase in lung resistances in response to increasing doses of methacholine compared to control mice. However, there was no difference in pulmonary resistance between the asthmatic and asthmatic IL-15Cx treated mice. To better understand the lack of effect of the IL-15Cx on bronchial hyperreactivity, we performed histology on lung tissue to measure anatomical observation (Fig. 4c-d). In HDM mice, we observed a strong peri-bronchial and perivascular cell infiltrate, as well as hyperplasia of the epithelium with the appearance of numerous mucus cells (Fig. 4c). The evaluation of this histological score confirms a significant increase in the score between CTL and HDM groups (Fig. 4d). As for lung resistances, we did not show any reduction in the histological score between the HDM and HDM IL-15Cx groups. Mucus production is increased in asthma and contributes to increase pulmonary resistance. We evaluated mucus production on our model by calculating a score of goblet cells colored by periodic acid of Schiff (Fig. 4c). We found an increase in this score between CTL and HDM groups (Fig. 4d). We also observed a

decrease in the score between the HDM group and the HDM IL-15Cx group. This decrease, however, seems insufficient to improve the respiratory function of our asthmatic murine model. To better assess lung inflammation in our model, we performed broncho-alveolar lavages. We found an increase in eosinophil count in our asthma model but IL-15Cx injection did not decrease eosinophilia (Fig. 4e). Then the inflammatory action of the IL-15 complex, was analyzed in lungs, mediastinal lymph nodes and spleen in HDM and HDM IL-15Cx mice. We first evaluated NK cells (NKP46⁺, DX5⁺, CD3⁻) (Fig. 5). In lungs and in mediastinal lymph nodes we found no significant difference after the injection of the IL-15Cx (Fig. 5a-b). However, we found an increase in NK cells in the spleen between HDM and HDM IL-15Cx groups. In the allergic asthmatic murine model, IL-15 complex injected intraperitoneally has an effect on NK cells in spleen but not in local sites of inflammation. Then, we explored adaptive immunity to investigate whether the complex modulates inflammation (Fig. 6). Regarding memory CD8⁺ lymphocytes (CD8⁺, CD44⁺,
<figure><img src="image_7.png" /></figure>

CD122⁺), there was no statistical difference in the lungs and in the spleen (Fig. 6a-b) between groups. The percentage of CD8⁺ CXCR3⁺ lymphocytes was not increased in the lungs after the injection of the complex (Fig. 6c) but it tended to increase in the spleen (8,66% versus 12,54% respectively, *p* = 0,07) (Fig. 6d). Moreover,

the percentage of Th1 lymphocytes (CD4⁺, CD25⁺, CXCR3⁺) was not modified in the lungs (Fig. 6e) nor in the spleen (Fig. 6f). T regulatory lymphocytes (CD4⁺, CD25⁺, FOXP3⁺) were not modified in the spleen (Fig. 6h) but tended to increase in the lungs (6,24% versus 3,67% respectively, *p* = 0,07) (Fig. 6g). In conclusion, we

<figure><img src="image_8.png" /></figure>
did not find any impact on the adaptative immunity with
the IL-15 complex in our model of allergic asthma.

Discussion

In this study, we have evaluated the therapeutic effect of
an IL-15 / sIL-15Rα complex on pulmonary inflamma-
tion and in particular through a mouse model of acute
HDM allergic asthma. We found that the IL-15Cx can
modulate lung inflammation but has no effect on asthma
features in mouse. Indeed, focusing on the three main
physiopathological pillars of asthma, our results show
that the IL-15Cx did not decrease bronchial hyperreac-
tivity, did not reduce bronchial remodeling or cellular
bronchial infiltrate, but reduced bronchial mucus secre-
tion and modulated systemic inflammatory response.
Using ovalbumin sensitized transgenic mice with an in-
creased number of memory-like CD8⁺ T cells in periph-
eral lymphoid tissues, Th2-type cytokine production was
found to be severely attenuated after inhalation of ov-
albumin. Transgenic mice overexpressing IL-15 prefer-
entially developed CD8⁺ T cell-mediated Th1 responses
after ovalbumin sensitization [17]. Mathias et al. have
made it possible to better characterize the role of this
cytokine in the inflammatory response during the devel-
opment of an allergic airway disease by using
interleukin-15 deficient mice [18]. They used wild-type
and IL-15 deficient mice that were sensitized and then
exposed to ovalbumin. In the absence of IL-15,
ovalbumin-sensitive mice exhibited increased bronchial
eosinophil inflammation, high IL-13 production, and se-
vere pulmonary histopathology damages compared to
wild type mice. These results clearly demonstrate that
mice with endogenous IL-15 deficiency are likely to de-
velop a strong, potent and Th2-mediated allergic airway
disease, which could be regulated by CD8⁺ T cells. Re-
cently it has been shown that IL-15 deficiency increases
airway resistance and decreases compliance in a mouse
model of asthma [14]. These discrepancies with our ob-
servation might be linked to several points. First, we
used a lower dose of IL-15 than others. Therefore, an in-
sufficient dose injected could explain the absence of an
effect on bronchial hyperreactivity. It is of note that the
dose we used demonstrated a biological effect stronger
than IL-15 alone [19]. Secondly, we used a mouse model
of asthma induced by HDM mimicking the human situ-
ation in a more complex manner than the airway inflam-
mation induced by ovalbumin. In fact, our model
displays a mix Th2/Th17 inflammation associated with a
mix eosinophil/neutrophil infiltrate. Therefore, it is likely
that in our model, mechanisms implicated might be
more complicated, yet closer to the human situation,
and several compensatory effects may explain our obser-
vation. Despite the potential effect of IL-15Cx on Th2
inflammation, we did not use it on an ovalbumin-

induced asthma model, a pure Th2 model. Indeed, if the
development of new therapies in asthma remains essen-
tial, the last years have seen the emergence of biothera-
pies efficiently targeting the Th2 pathway (anti-IL5, anti-
RIL-5) in severe asthma [20, 21]. Research must there-
fore now focus on patients with a part of non-Th2 in-
flammation in which oral corticosteroids is often the
only option, with unacceptable side effects. We therefore
went directly on a mouse model with a mixed Th2 and
non-Th2 inflammation, which has already proven itself
[15]. After the study of bronchial hyperreactivity we
were interested in the action of the complex on bron-
chial remodeling and cellular infiltrate. The complex
does not seem to interfere with bronchial remodeling.
Nevertheless, the analysis of the secretion of mucus by
bronchial goblet cells through the use of a score after
PAS staining, found a significant decrease thereof. The
mechanism of action explaining this effect remains to be
elucidated by studying the components of the IL-15 re-
ceptor present on the goblet cells of asthmatic subjects.
With respect to inflammatory modulation, we observed
that in control mice, injection of the IL-15 / sIL-15Rα
complex had systemic and a local effect on NK cells. In
addition, we observed an increase in CD8+ memory and
T regulatory lymphocytes. NK cells have an immunoreg-
ulatory role in many inflammatory pathologies and their
involvement in asthma remains highly controversial in
the literature. The development of house dust mite aller-
gic asthma in mice has been shown to increase NK, par-
ticularly in the mediastinal lymph nodes, but NK
blockade by antibodies or their genetic deficiency does
not alter the characteristics of asthma, namely Th2 in-
flammation, bronchial hyperreactivity, specific IgE titers,
and mucus production [22]. The study of the prostaglan-
din 12 receptor deficiency in a HDM allergic murine
model shows an increase in IFN-γ producing NK cells in
the lungs, inversely correlated with the number of type 2
innate lymphoid cells (ILC2). In addition, anti-NK-1.1
monoclonal antibody treatment restored inflammation
of the allergic airways and increased the level of IL-5
producing, attracting eosinophils. This study thus reveals
that NK cells prevent allergic pulmonary inflammation
by limiting the number of ILC2 [23]. Furthermore, the
mode of administration of the complex can influence
the biological activity and explain our results showing a
difference in the spleen (systemic) and not at the tissue
level. In this context, the administration of the complex
by aerosol would be an interesting track. Finally, viral
exacerbations are the leading cause of exacerbation of
asthma in winter, and an increase in memory T8 lym-
phocytes would perhaps reduce these exacerbations in
targeted populations. Indeed, Laza-Stanca et al demon-
strated that IL-15 deficiency in humans could be a part
of the virus-induced asthma exacerbations pathogenesis
[24]. In asthma, Th1 and Treg populations are not dir-
ectly involved in the allergic reaction but are rather in-
volved as antagonists and regulators [25]. Th1
population, secreting interferon gamma (IFNγ), will
modulate the allergic reaction by limiting the expansion
of the Th2 population and inducing cell-mediated im-
munity [26]. The other regulatory T cell population, via
the secretion of IL-10 and tumor growth factor beta
(TGF-β), will inhibit the Th1, Th2 and Th17 responses
to restore the pro-inflammatory/anti-inflammatory bal-
ance [27]. Tregs inactivate the inflammatory response in
order to stop bronchial inflammation but are quantita-
tively and qualitatively deficient in this pathology [28,
29]. In our model, the increase in Th1 CXCR3+ in the
Th1 pathway may also counteract the Th2 inflammation
but these modulations do not seem to be sufficient. In
the same way, a tendency to increase regulatory T cells
in the lungs after injection of the complex is observed.
Though we did not demonstrate a therapeutic effect of
the IL-15Cx in our mouse model of house dust mite-
allergic asthma, other therapies implicating IL-15 in
asthma should not be completely abandoned. Indeed, it
is clearly established that asthma syndrome recovers sev-
eral different phenotypes with different endotypes, so
that targeting one molecule could be relevant for one
endotype but not for another. For instance, targeting IL-
5 with mepolizumab on benralizumab is relevant for the
eosinophilic phenotype whereas omalizumab concerns
allergic asthma. IL-15 could be involved in another type
of asthma for which our model, mainly Th2 and Th17-
driven, is not relevant. Nevertheless, the modulation of
inflammation induced by the IL-15 / sIL-15Rα complex
is of major interest in inflammatory diseases such as
psoriasis, transplantation or in oncology where the role
of IL-15 is well established. Indeed, we demonstrated be-
fore that endogenous soluble IL-15Rα derived from epi-
dermal stroma, protects against dendritic cell/IL-15-
mediated, T cell-driven skin inflammation in vivo, and is
relevant to human psoriasis [30]. Regarding tolerance in
lung transplantation, Jungraithmayr et al. showed that
expansion of recipient NK cells through IL-15/IL-15Rα
complex treatment resulted in decreased T-cell infiltra-
tion and alloreactive T-cell priming as well as improved
function of the allogeneic lung transplant in a mouse
model [31]. Other forms of the complex have been
tested in oncology. A fusion protein called RLI, com-
posed of the sushi domain of IL-15 receptor α coupled
via a linker to IL-15, seems to have a high antitumor ac-
tivity in metastatic melanoma and colorectal cancer in
mice [32]. ALT-803, is a new IL-15 superagonist com-
posed of a mutant of human IL-15 and the sushi domain
of human IL-15 Rα fused with the Fc domain IgG1. Re-
cently, the use of ALT-803 in combination with NIVO-
LUMAB (anti-PD1) in non-small-cell metastatic lung

cancers has been the subject of an encouraging Phase 1
study that has conducted to a Phase 2 study still in pro-
gress [33]. A novel fusion protein encompassing anti-
PD-L1 and the IL-15 superagonist fusion complex
(ALT-803) called N-809 has been created and has very
promising antitumor functionalities [34]. The association
of the complex with other existing targeted therapies
could therefore also be an interesting research avenue.

Conclusions

In conclusion, we did not demonstrate an effect of the
IL-15 / sIL-15Rα complex in our mouse model of
HDM-allergic asthma on bronchial hyperreactivity,
bronchial remodeling and inflammatory response. A de-
crease of mucus secretion has been observed but needs
further explanation. On the other hand, we showed a
modulation of lung and systemic inflammation with an
increase of NK cells, CD8+ memory and CD8+ CXCR3+
lymphocytes in control mice. This could be of interest
for other inflammatory disease associated or not with
other biologics. Indeed, the addition of therapies could
potentiate the inflammatory modulation or act on other
inflammatory pathways.

Abbreviations

HDM: House dust mite; IL-15 Ra: Alpha chain receptor of IL-15; IL-15Cx: IL-15/
sIL-15R α complex; NK: Natural killer cells; Treg: T regulatory lymphocytes

Acknowledgments

First the author would like to thanks the Cytocell platform for assistance with
the flow cytometry analysis, and we would like to thank the Therassay
platform for the use of respiratory function equipment (Flexivent system).
We are grateful to the members of the UTE IRS-UN animal facility for the
support they provided to us with the mice. We would also like to thank the
MicroPicell platform for performing the histological analysis. Finally, we
would like to thank Stallergenes Greer for kindly providing the HDM total
extract.

Authors' contributions

AMo performed the experiments, analyzed the data and wrote the
manuscript. MK participated in the cell experiments and analysis. M-AC, DH
and ED provided support for the experiments in mice. AMa and GB designed
the study and supervised manuscript writing. All authors read and approved
the final manuscript.

Funding

This work benefited from state aid managed by the "Agence Nationale de la
Recherche" under the "Programme d'Investissement d'Avenir" (bearing the
reference ANR-16-IDEX-0007) and financial support from the Region Pays de
la Loire and the Université de Nantes.

Availability of data and materials

The datasets used and/or analyzed during the current study are available
from the corresponding author on reasonable request.

Ethics approval and consent to participate

This protocol including mice received a favorable opinion from the Pays de
la Loire ethics committee under number 9456.

Competing interests

The authors declare that they have no competing interests.
Author details

¹L'institut du thorax, Inserm, CNRS, Université de Nantes, Nantes, France.
²L'institut du thorax, CHU de Nantes, service de pneumologie, Nantes, France.
³INRA, UR1268 BIA, rue de la Géraudière, F-44316 Nantes, France.

Received: 14 October 2019 Accepted: 20 January 2020
Published online: 29 January 2020

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