Title:

Emerging roles of innate lymphoid cells in inflammatory diseases: clinical implications

Short title:

Role of innate lymphoid cells in inflammatory diseases

Authors:

Inge Kortekaas Krohn 1, Medya Mara Shikhagaie 2, Korneliusz Golebski 2,3, Jochem HJ

Bernink 2, Christine Breynaert 1,4, Brecht Creyns 1, Zuzana Diamant 5,6 Wytske J Fokkens 3,

Philippe Gevaert 7, Peter Hellings 8,1,3, Rudi W Hendriks 9, Ludger Klimek 10, Jenny

Mjösberg 11, Hideaki Morita 12,13 Graham Ogg 14, Liam O’Mahony 13, Jürgen Schwarze

15,16 Sven F Seys 1, Mohamed H Shamji 17,18, Suzanne M Bal 2

Affiliations:

1 Laboratory of Clinical Immunology, Dpt. Microbiology & Immunology, KU Leuven, Leuven,

Belgium

2 Department of Experimental Immunology, Academic Medical Center, Amsterdam, The

Netherlands

3 Department of Otorhinolaryngology, Academic Medical Center, Amsterdam, The

Netherlands

4 Department of General Internal Medicine, Allergy and Clinical Immunology, University

Hospitals of Leuven, Leuven, Belgium

5 Department of Respiratory Medicine and Allergology, Skåne University Hospital, Lund,

Sweden

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6 Department of General Practice and Department of Clinical Pharmacy & Pharmacology,

University Medical Centre Groningen, University of Groningen, Groningen, The Netherlands

7 Upper Airways Research Laboratory, Ghent University, Ghent, Belgium

8 Clinical Division of Otorhinolaryngology, Head and Neck Surgery, University Hospitals

Leuven, Leuven, Belgium

9 Department of Pulmonary Medicine, Erasmus MC, Rotterdam, the Netherlands

10 Center for Rhinology and Allergology, Wiesbaden, Germany

11 Center for Infectious Medicine, Department of Medicine Huddinge, Karolinska Institutet,

Stockholm, Sweden

12 Department of Allergy and Clinical Immunology, National Research Institute for Child

Health and Development, Tokyo, Japan

13 Swiss Institute of Allergy and Asthma Research (SIAF), University of Zurich, Davos,

Switzerland

14 MRC Human Immunology Unit and Oxford University Hospitals NHS Trust, Weatherall

Institute of Molecular Medicine, Oxford, UK

15 MRC Centre for Inflammation Research, The University of Edinburgh, Edinburgh, UK

16 Child Life & Health, The University of Edinburgh, Edinburgh, UK

17 Immunomodulation and Tolerance group, Allergy and Clinical Immunology, Inflammation,

Repair and Development, Imperial College London, London, United Kingdom

18 MRC & Asthma UK Centre in Allergic Mechanisms of Asthma, London, United Kingdom

Conflicts of interest:

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Address for correspondence:

Inge Kortekaas Krohn

Laboratory of Clinical Immunology

Dpt. Microbiology & Immunology

KU Leuven

Herestraat 49, bus 811

3000 Leuven, Belgium

Telephone: +3216346165

Ingekortekaas@gmail.com

Key words:

airways, gut, inflammatory diseases, innate lymphoid cells, lungs, mucosa, skin

Acknowledgements:

We would like to thank the European Academy of Allergy and Clinical Immunology for the

financial support of the Task Force.

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Abstract

Innate lymphoid cells (ILC) represent a group of lymphocytes that lack specific antigen

receptors and are relatively rare as compared to adaptive lymphocytes. ILCs play important

roles in allergic and non-allergic inflammatory diseases due to their location at barrier

surfaces within the airways, gut and skin and they respond to cytokines produced by

activated cells in their local environment. ILCs contribute to the immune response by the

release of cytokines and other mediators, forming a link between innate and adaptive

immunity. In recent years, these cells have been extensively characterized and their role in

animal models of disease has been investigated. Data to translate the relevance of ILCs in

human pathology, and the potential role of ILCs in diagnosis, as biomarkers and/or as future

treatment targets are also emerging.

This review, produced by a task force of the Immunology Section of the European Academy

of Allergy and Clinical Immunology (EAACI) encompassing clinicians and researchers,

highlights the role of ILCs in human allergic and non-allergic diseases in the airways,

gastrointestinal tract and skin, with a focus on new insights into clinical implications,

therapeutic options and future research opportunities.

Word count: 187

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1. Introduction

Innate lymphoid cells (ILCs) are a recently identified family of effector cells, which are

important early regulators of immune responses at barrier surfaces. ILCs are instructed by

cytokines and lipid mediators produced by epithelial, stromal and myeloid cells and so

contribute in shaping the type and intensity of the immune response by producing cytokines

and other mediators, and via cell-cell interactions.

Three subsets of ILCs have been identified: group 1 (ILC1s), group 2 (ILC2s), and group 3 ILCs

(ILC3s), which can be viewed as innate equivalents of T helper (Th) cell subsets Th1, Th2 and

Th17, respectively. Extensive information on the ILC surface markers and cytokine

production can be found in recent reviews (1, 2). Beyond this canonical classification, ILCs

display plasticity and heterogeneity (3).

ILC1s

ILC1s include various populations of non-cytotoxic IFN-γ-producing ILC1s and the

developmentally distinct cytotoxic natural killer (NK) cells. Here we will focus on the non-NK

ILC1s, which can be activated by macrophages, dendritic cell (DC)-derived interleukin (IL)-12

and IL-18. To date, four distinct ILC1 subsets have been described based on their anatomical

location and function (4). In steady state, ILC1s are present at low numbers in the dermis,

lung, liver, and the intestine, but increase in number upon exposure to intracellular

pathogens to protect against viruses and bacteria. In non-resolving chronic inflammatory

Th1-like diseases including Crohn’s disease (5) and chronic pulmonary obstructive disease

(COPD), the number of ILC1s remains elevated (6).

ILC2s

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ILC2s were originally characterized as a critical early source of type 2 cytokines for the

expulsion of parasitic worms and can be distinguished from other ILC subtypes by the CRTH2

receptor (7). When stimulated by epithelial cell-derived innate mediators i.e. IL-25, IL-33,

thymic stromal lymphopoietin (TSLP), or prostaglandin D2 (PGD2), these cells rapidly expand

and secrete large amounts of IL-5, IL-13, and under certain conditions, also IL-4 (2).

Presently, the pivotal role of ILC2s in allergic airway inflammation is well established in

various murine models. In humans, ILC2s were found in healthy lung parenchyma and

bronchoalveolar lavage (BAL) fluid from patients who underwent lung transplantation, in

nasal polyps, and at low numbers in peripheral blood (7-9). By production of cytokines and

via direct interaction with myeloid and Th2 cells, they promote mucus production,

eosinophilia and accumulation of mast cells in allergic diseases (10).

ILC3s

ILC3s represent a heterogeneous group of cells comprising of lymphoid tissue inducer cells

(LTi) and two subsets of non-LTi ILC3s that can be distinguished by the expression of the

natural cytotoxicity receptor NKp44. NKp44- ILC3s can be found in the blood of healthy

individuals, whereas all ILC3s subsets reside in tissues (1). ILC3s produce IL-17A, IL-17F, IL-22,

granulocyte-macrophage colony-stimulating factor (GM-CSF), and/or tumor necrosis factor

(TNF)-α in response to IL-1β, IL-23, and aryl hydrocarbon receptor (AhR) ligands (11).

Depending on specific tissue-derived signals, ILC3s can promote immunity against

pathogens, and/or enhance an immunologically tolerogenic state (2). Evidence is

accumulating that ILC3s play a critical role in (chronic) non-allergic inflammatory conditions

including psoriasis (12) or inflammatory bowel disease (13).

2. ILCs in human disease

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ILCs in the airways

Under homeostatic conditions, all three ILC subsets are present in the human lung with ILC2s

and ILC3s being the most prevalent (6, 14, 15). In healthy mucosal tissue of the nose also all

ILC subsets are present, but ILC3s are the predominant population (6). Airway epithelial cells

can actively contribute to local tissue inflammatory responses by detecting and responding

to environmental pathogens and potential threats by producing cytokines and chemokines

that can activate ILCs (16). Based on murine data, where in the lungs almost all ILCs are of

ILC2 origin, studies in human have mostly focused on ILC2s. However, in pulmonary

homeostasis and in many Th2-low inflammatory diseases the role of other ILC subsets

remains to be elucidated. Upper airway inflammation can be studied in mucosal biopsies or

by the removal of nasal polyps. Useful tools to study lower airway inflammation are sputum

and BAL samples, although these airway sampling methods may not accurately reflect the

tissue state, especially as ILCs are thought to be tissue resident cells (17).

Allergic rhinitis (AR)

AR is characterized by an IgE-mediated response at the nasal mucosa upon exposure to

airborne allergens in sensitized individuals (18). In AR, the barrier function is disrupted,

which is likely to facilitate allergen passage through the epithelium. Moreover, respiratory

viruses can further interfere with the barrier integrity and induce the secretion of cytokines,

such as IL-33 and TSLP, that may skew the differentiation of immune cells towards the

allergic type 2 response involving ILC2s (19) (Figure 1). Additionally, antigen binding to IgE on

mast cells results in rapid degranulation and release of mediators such as cysteinyl

leukotrienes (CysLTs) and PGD2, which are activators of ILC2s (20, 21).

Increased (IL-13-producing) ILC2s were found in the blood of patients with house dust mite

allergy (22). Moreover, elevated ILC2 numbers are found in the blood after intranasal

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allergen challenge of cat-sensitized adults (23) and during the grass pollen season in grass

allergic patients and in the nasal fluid upon grass pollen challenge (24). However, allergen-

dependent differences have been reported (22, 25).

Chronic rhinosinusitis (CRS) with and without nasal polyps

CRS is a chronic inflammation of the nose and paranasal sinuses which can be categorized

into CRS without nasal polyps (CRSsNP) or with NP (CRSwNP) (26). In western countries,

CRSwNP is usually characterized by a type 2 inflammation and often associated with asthma

(27). It has been demonstrated that ILC2s represent the dominant ILC subset in CRSwNP,

their numbers are increased compared to nasal tissue of healthy donors (9, 28), and are

enriched in CRSwNP compared to CRSsNP (29, 30). Allergy is an additional factor positively

influencing the ILC2 numbers in CRS (31) and a positive correlation between eosinophil and

ILC2 numbers in NP tissue of patients with CRSwNP has been reported (32). Also in nasal

scrapings from patients with aspirin-exacerbated respiratory disease, elevated ILC2 numbers

were found upon COX-1 inhibitor-induced reactions, which coincided with decreased blood

ILC2 levels, indicating migration into the nasal mucosa (33). At the same time, elevated PGD2

metabolites were found in the urine of these patients. The increased numbers of ILC2s in NP

tissue can be explained by tissue enrichement with ILC2-activating cytokines, i.e.: IL-25, IL-33

and TSLP. Under homeostatic conditions, IL-33 is retained in the nucleus of basal epithelial

cells and its release is associated with tissue injury or cell death. Therefore, elevated IL-33

levels may be related to tissue damage in treatment-recalcitrant NP (34), which may drive

the perpetuating eosinophilic inflammation via ILC2-derived IL-5. This vicious circle is

maintained by eosinophil-derived IL-4, a key cytokine required for ILC2 maintenance (6).

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Th2-high asthma

Asthma is characterized by a variable and often reversible airway obstruction due to chronic

inflammation of the respiratory tract. It is a heterogeneous disease that can be associated

with allergy. Like in CRS, asthma phenotypes are often based on the distinction between

Th2-high and Th2-low disease asthma (35).

IL-5 and IL-13-containing CD34+ non-T cells, likely representing ILC2s, were found in the

sputum of patients with allergic asthma following allergen inhalation (36). The involvement

of ILC2s in asthma pathogenesis is also supported by (i) genome-wide association study data

showing association of asthma with loci encoding IL-33, IL33 receptor, IL-13 and the

transcription factor ROR, which are all essential for ILC2s (37), and by (ii) the finding of

increased expression of IL-25, IL-33 and TSLP in lungs and sputum of asthma patients (38-

41). Higher numbers of total and IL-5/IL-13-producing ILC2s are detected in sputum of

patients with severe eosinophilic asthma versus mild asthma (42) and after allergen

challenge (43). In another study, ILC2 and IL-33 levels were elevated in BAL of patients with

asthma and IL-33 correlated with disease severity (44). The proportions of ILC2s were also

found to be higher in BAL from children with severe therapy-resistant asthma (45). Increased

ILC2s are found in blood of asthma patients and these cells produced more IL-5 and IL-13 in

response to IL-25 or IL-33 compared to healthy controls or AR patients (46). Whereas similar

peripheral blood ILC2 proportions were found in asthma patients and controls in an earlier

study (47), the finding of increased ILC2 numbers in patients with asthma was confirmed by

several subsequent reports (42, 48, 49). Taken together, these studies suggest the

involvement of ILC2s in specific subgroups of asthma (Figure 1). Recently, it has been shown

that ILC2-derived IL-13 disrupts the bronchial integrity of airway epithelial cells in asthma,

thereby sustaining the type 2 inflammation in the airways (50).

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Th2-low asthma

Different non-allergic factors, such as pathogens, air pollutants, and obesity can exacerbate

asthma and contribute to the heterogeneous pattern of asthma (35). Human data are

scarce, but in mice IL-17A producing ILC3s were implicated in obesity-induced asthma (51,

52), indicating a possible role for IL-1β (51) as this cytokine can stimulate both ILC2s and

ILC3s (6, 53, 54). In a pilot study , IL-17A-producing-lineage negative cells in BAL were

demonstrated to be enriched in patients with severe asthma (51). So far, no other studies

have been performed analysing ILC subsets in patients with Th2-low asthma.

Chronic obstructive pulmonary disease (COPD)

Even though asthma and COPD share some clinical features, the inflammatory pattern differs

(55). COPD is usually characterized by a more pronounced neutrophilic and/or type 1

inflammation. In patients with GOLD stage I and II the ILC profile tended to shift towards IL-

17-producing NKp44- ILC3 (14). In a different study, GOLD stage IV patients had significantly

more ILC3s and ILC1s, due to the differentiation of ILC2s into ILC1s (6). Elevated ILC1

numbers were also found in peripheral blood of COPD patients, correlating with disease

severity and susceptibility to exacerbations (56). Recently, ILC3s were detected in

inflammatory aggregates and near blood vessels in lungs of smokers and COPD patients

providing insight into the initiation of smoke-induced ectopic pulmonary lymphoid

aggregates (Figure 1) (15).

Viral infections and exacerbations

Upper respiratory viral infections are the main cause of asthma exacerbations (57). In mouse

models, ILC2 activation upon influenza infection, can induce bronchial hyperreactivity as well

as restore barrier integrity by amphiregulin production (8, 58). In respiratory syncytial virus

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(RSV) infections, ILC2s can confer a type 2-cytokine-associated disease (59). In humans, ILC2s

have not been described directly in these pathologies. However, IL-33 and IL-25 have been

detected in nasal fluids of infants hospitalised with RSV bronchiolitis (59) and in asthmatic

subjects following experimental rhinovirus (RV)16-infection (60, 61). The supernatant of RV-

infected primary bronchial epithelial cells activates ILC2s in an IL-33-dependent manner (60).

In conclusion, circumstantial evidence suggests that ILC2s may be involved in the immune

pathophysiology of viral infections and asthma exacerbations.

Pulmonary fibrosis

Both ILC2s and ILC3s are implicated in fibrosis (62). ILC2s produce IL-5 and IL-13, which

contribute to the pathogenesis of fibrosis, but at the same time can produce amphiregulin.

Elevated IL-33 and IL-25 levels were found in the lungs of patients with cystic fibrosis (CF)

and interstitial pulmonary fibrosis (IPF) (63-65). IL-25 positively correlated with periostin,

and elevated numbers of ILC2s were found in the BAL fluid of fibrosis patients (63). IL-13

contributes to the differentiation of macrophages towards a profibrotic phenotype inducing

collagen deposition by fibroblasts (63). Also in patients with systemic sclerosis increased ILC2

numbers were reported, which correlated with the presence of pulmonary fibrosis (66).

ILC3s may play a role in the generation and progression of IL-17-mediated fibrosis as IL-17A

and IL-1β levels were elevated in sputum, BAL and bronchial epithelium of patients with CF

and IPF (67-69). This implies that the IL-1β – ILC3 – IL-17 axis can be an important target in

fibrotic diseases (Figure 1).

Other pulmonary inflammatory diseases

Elevated ILC2 numbers have been described in other eosinophilic lung diseases, such as in

the pleural fluid of patients with primary spontaneous pneumothorax (70) and in blood of

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patients during helminth infections (71, 72). An expansion of CD117+ ILCs (ILC3s and subset

of ILC2s) was found in blood of filarial-infected subjects, compared to filarial-uninfected

controls (71). ILC2s are diminished in young children infected with Schistosoma

haematobium (bilharzia), whereby anti-helminthic treatment resulted in increased TSLP

levels and normalization of ILC2 numbers (72).

ILCs in the skin

ILCs are present in healthy dermis at a relatively high abundance compared to other barrier

tissues, which might be partly explained by the expression of the skin homing marker

cutaneous leukocyte-associated antigen on circulating ILCs (12). The most prevalent ILC

population in human skin is the ILC2 (3). As skin ILC2s express high levels of amphiregulin,

they are implicated in tissue homeostasis and repair (73). This is supported by increased ILC2

numbers in healing cutaneous wounds (74). IL-17-producing ILC3s were also elevated in the

dermis upon wounding (75) and in mice these cells were shown to be crucial for restoring

the epithelium (Figure 2).

Atopic dermatitis (AD)

AD is a common chronic inflammatory skin disorder characterized by eczematous lesions

with lichenification of the skin (76). In lesions of AD, increased IL-4 and IL-13 levels as well as

accumulation of skin-resident ILC2s were observed as compared to normal skin (73, 77).

There are some differences between the studies regarding whether these skin ILC2s

preferentially respond to IL-33 or TSLP (12, 73). Several ILC-modulating factors are

dysregulated in AD skin. Elevated levels of IL-25, IL-33, TSLP, and PGD2 were reported, but

also cell-cell interactions affect the ILC2 response (Figure 2). Keratinocytes express E-

cadherin and B7-H6, thereby interacting with the killer-cell lectin like receptor G1 or with the

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activating receptor NKp30, respectively, on ILC2s (73, 78). These molecules have opposing

functions, resulting in suppression or activation of ILC2s. In AD patients, filaggrin mutations

cause defects in the skin barrier function and lead to lower E-cadherin expression (73),

resulting in elevated IL-1β levels and an accumulation of ILC2s (79). This is further amplified

by widespread expression of B7-H6 in AD skin, promoting type 2 cytokine production (78).

These two mechanisms lead to persistent activation of ILC2s. Moreover, ILC2s in AD skin

reside in close proximity with basophils (80) and mast cells. IL-4 and PGD2 derived from

these cells are potent ILC2-recruiting factors and activators (21).

Psoriasis

Psoriasis is a chronic inflammatory skin disorder characterized by scaly patches resulting

from epidermal thickening and infiltration of inflammatory cells (81). The numbers of IL-22-

producing ILC3s were increased in both lesional and non-lesional skin (Figure 2) and blood of

patients with psoriasis, compared to healthy subjects (12, 82, 83). Additionally, the increased

ILC3 numbers in skin lesions correlated with psoriasis severity (12) and ILC3sproduced IL-22

in response to stimulation with IL-1β and IL-23. IL-22 may limit the ongoing inflammation,

paralleling its role in epithelial maintenance in the gut. Its overproduction in diseased skin

inhibits terminal differentiation of keratinocytes and promotes the epidermal thickening

characteristic for psoriasis (12).

ILCs in the intestines

In steady state (Figure 3A), ILC3s represent the predominant ILC subset within the intestinal

lamina propria (5). Furthermore, a distinct intra-epithelial ILC1 subset resides (5, 84). ILC3s,

including lymphoid tissue inducer cells and postnatal ILC3s, are essential for the

development of lymphoid structures, including Peyer’s patches and isolated lymphoid

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follicles that emerge from cryptopatches (85). Moreover, ILC3s contribute to the control of

the intestinal homeostasis by rapidly responding to environmental cues and interacting with

other immune and non-immune cells. For example, ILC3s directly sense diet-derived retinoic

acid and AhR-ligands, which in turn instruct these cells to produce IL-22, contributing to the

integrity of the epithelial barrier by inducing mucus production, antimicrobial production,

and fucosylation. Furthermore, sensing of intestinal commensals by macrophages induces IL-

1β which triggers GM-CSF production by ILC3s, which instructs intestinal phagocytes to

produce regulatory molecules, such as retinoic acid and IL-10, to expand regulatory T cells,

thereby inducing oral tolerance (86, 87).

Inflammatory bowel diseases (IBD)

IBD, such as Crohn’s disease (CD) and ulcerative colitis (UC), are chronic inflammatory

disorders of the intestine with a relapsing and remitting disease course (88). Whereas in

homeostasis, the intestinal lamina propria is predominantly populated by an IL-22-producing

ILC3 subset, the ILC composition and cytokine profile within the colon and ileum in IBD is

markedly altered. CD is thought to involve both type 1 (IFN-γ) and type 3 (IL-17) cytokines,

whereas in UC, both type 2 cytokines and IL-17 are predominantly involved (88). In CD,

elevated numbers of IL-17-producing ILC3s were observed (89) and the frequency of IFN-γ-

producing ILC1s was dramatically enhanced, which inversely correlated with a decrease of

IL-22-producing ILC3s compared to controls (5, 13, 54, 84, 90, 91) and potentially interfering

with the maintenance of the epithelial barrier, induction of antimicrobial peptides and

wound healing (13). The enhanced frequency of IFN-γ-producing ILC1s directly affects the

epithelial barrier, amplifying its disruption. Although low in number at steady state, ILC2s

acquired an ILC1-like phenotype by the production of IFN-γ next to IL-13 in the gut of

patients with CD (92). Overall, ILCs may contribute to intestinal inflammation (Figure 3B)

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through cytokine production, lymphocyte recruitment, and organization of the inflammatory

tissue and may represent a novel tissue-specific target for subtypes of IBD.

Celiac disease and non-celiac gluten hypersensitivity

Celiac disease is a T cell-mediated enteropathy characterized by the loss of tolerance to

dietary gluten resulting in villous atrophy. The pathogenesis is based on induction of gluten-

specific inflammatory Th1 cells and targeted killing of intestinal epithelial cells.

Patient data on ILC involvement are scarce, but in refractory celiac disease type II, an

expansion of aberrant non-T cell intraepithelial lymphocytes was observed. These cells are

heterogeneous and include ILC-like precursor cells that respond to either IL-15 or IL-21,

indicating differential cytokine responsiveness in subsets of patients with refractory celiac

disease (93). Another study showed that ILCs producing TNF-α and IFN-γ are significantly

increased in the inflamed mucosa of patients with celiac disease (94). The rapid production

of TNF-α upon ILC activation, suggest a possible role for ILCs in early phases of tissue damage

in celiac disease (94).

In rectal biopsies from wheat-challenged non-celiac wheat sensitivity (NCWS) patients,

increased mucosal IFN-γ-producing ILC1 infiltration was found as compared to IBD controls.

Approximately 30% of the IFN-γ-producing CD45+ cells consisted of ILC1s. Two weeks after

resuming a wheat-free diet, IFN-γ-producing ILC1 cells significantly decreased (95),

suggesting their potential role in NCWS pathogenesis.

ILC2s might well be implicated in eosinophilic esophagitis, but the available data so far is still

very limited (96, 97).

3. Clinical implications

ILCs as a diagnostic tool

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Currently, ILCs cannot be used for diagnosis because a well-defined relationship with clinical

manifestations and/or therapeutic is missing, blood ILC numbers are low, there is no

advantage over measuring eosinophils and the detection of ILC2s is complex and non-

standardized. However, the following observations have been reported (summarized in

Figure 4A and Table 1):

o Peripheral blood ILC2 numbers correlated with clinical symptoms in patients

with seasonal AR (25).

o ILC2s in mucosal biopsies from patients with CRS correlated with tissue and

blood eosinophilia and the presence of nasal polyps (28) and negatively

correlated with the number of previous sinus surgeries (31).

o In aspirin-exacerbated respiratory disease nasal ILC2 numbers correlate

positively and blood ILC2 numbers negatively with maximum symptom scores

(33).

o Peripheral blood IL-13-producing ILC2 numbers correlated with asthma control

status (49).

o In peripheral blood of patients with COPD, elevated ILC1 numbers negatively

correlated with lung function (56).

o Increased numbers of ILC2s were found in BAL fluid from fibrosis patients (63).

o Increased ILC2 numbers were shown in pleural fluid of patients with primary

spontaneous pneumothorax, and in blood from patients with helminth

infections (70-72).

o In esophagal biopsies of patients with active EoE, ILC2s numbers were increased

(97).

o Increased numbers of ILC2 were found in lesions from patients with AD (73, 77,

79).

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o Increased ILC3 numbers were detected in psoriatic skin lesions correlating with

disease severity (12).

o In CD, the numbers of ILC3s and ILC1s were increased and the frequency of ILC1s

inversely correlated with disease severity (5, 13, 54, 84, 90).

o Increased infiltration of ILC1s were found in rectal biopsies from wheat-

challenged NCWS patients (95).

Consequences of ILC plasticity in disease

In response to microenvironmental changes, ILCs display plasticity, i.e. one subset is able to

transdifferentiate into another, allowing a quick adaptation to environmental changes. A

conversion of ILC3s into ILC1s, induced by IL-12, was found within the intestines of CD

patients (5, 92). In a similar IL-12-dependent manner, ILC2s are converted into ILC1s within

the lungs of patients with severe COPD (6). It is thought that IFN-γ production from ILC1s

worsens the disease outcome. ILC1s can be reconverted into ILC3s by activation with IL-1β,

IL-23 and retinoic acid, while IL-4 can convert ILC1s into ILC2s. Targeting these processes

may interfere with disease progression and severity.

ILCs as therapeutic targets

Although the therapeutic benefit of selectively targeting ILCs has not been systematically

investigated, many ILC-related pathways have been shown to affect pathologies.

Therapeutic strategies can include prevention of ILC activation, interference with

intracellular signalling pathways, neutralization of effector cytokines or limiting cell

trafficking (Figure 4B). To translate in vitro findings of potential drug candidates in in vivo

models, humanized mice (mice with a humanized immune system) are an excellent tool to

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study ILCs (6, 98). In this manner several ILC2-targeting options have already been evaluated

(99, 100).

Potential future targets:

Cytokines that activate ILCs such as IL-1β (all ILC subtypes), IL-33 (ILC2), IL-23 (ILC3s),

or IL-12p35 (ILC1s).

Cytokines produced by ILCs, such as IL-13, IL-9 and IL-22.

ILC modulators that promote protective ILC functions, such as the vitamin A

metabolite retinoic acid and vitamin D3 (101).

Cytokines and lipid mediators that limit pathologic ILC functions, such as IL-27, type

I interferons and lipoxin A4 (47, 102).

Molecules critical for migration and recruitment of ILCs, such as α4β7 and MAdCAM-

1.

Cytokines that induce ILC trans-differentiation.

Targeting ILCs in respiratory diseases

Although targeting ILCs in rhinitis or asthma has not been systematically investigated,

(existing) therapeutic options are able to modulate the inflammatory effects of ILCs.

Currently, corticosteroids represent the first line medication in allergic diseases, although

patients with more severe disease often are refractory to corticosteroids. Reduced ILC2

numbers were found in NP from CRS patients treated with systemic corticosteroids (32).

Even though this suggests corticosteroid responsiveness, in another study it was shown that

ILC2s are less susceptible to corticosteroid inhibition than Th2 cells (49). TSLP was reported

to be a key regulator of corticosteroid resistance (103).

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Another treatment option is immunotherapy. Immune tolerance induced following grass

pollen immunotherapy was associated with reduction in blood ILC2 numbers and IL-13

producing ILC2s. Reduction in ILC2s was associated with decreased severity of self-reported

symptoms (25).

More specific ILC2-modulating therapies can be realized via targeting of the leukotriene

and/or eicosanoid pathways. ILC2s express both the CysLT1 (20) and the CRTH2 receptor

(21). A leukotriene receptor antagonist (montelukast) was shown to inhibit ILC2 activation

(104) and showed clinical effectiveness in (corticosteroid-refractory) obese asthmatics (105),

although ILC2s were not analyzed. Similarly, a CRTH2 antagonist (OC000459) improved lung

function in moderate and severe eosinophilic asthma (106) and reduced symptoms in grass

pollen-sensitized patients (107). A combination of CRTH2 and CysLT1-receptor antagonist

may provide synergistic blockade of both Th2- and ILC2- inflammatory pathways, and hence

may have therapeutic potential in both allergic and non-allergic eosinophilic airway diseases.

Clinical studies in appropriate endotypes should provide evidence for this hypothesis.

Another eicosanoid, lipoxin A4 (LXA4) functions as an inhibitor of ILC2s (47) and severe

asthma patients have a LXA4 deficiency (108), indicating LXA4 supplementation as a

treatment option in these patients.

In addition, approaches with monoclonal antibodies (mAb) against IL-5 or its receptor and

anti-IL-4 α receptor led to improved disease in patients with severe nasal polyps (CRSwNP)

(109, 110) and in severe refractory eosinophilic asthma (111). Treatment with anti-IL-4 α

receptor may also target ILC2s in these airway disorders (109, 112). Upstream blocking of IL-

25, IL-33 and TSLP could potentially inhibit both the Th2- and ILC2-inflammatory pathways.

Therapeutic approaches targeting these cytokines (‘alarmins’) are presently in early stage

development. So far, an anti-TSLP showed protection against allergen-induced airway

responses in allergic asthma (113).

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The effects of ILC3s in non-allergic asthma may be modified by targeting IL-17. However, an

IL-17 receptor antibody did not improve asthma control in poorly controlled moderate to

severe asthma, although in a small subgroup, with high bronchodilator reversibility some

effect was seen (114).

Targeting ILCs in skin diseases

Many components of the pathways relevant to ILC2 activation are being explored to target

ILC2 in AD, for example CRTH2 antagonism and anti-IL-13 treatment. Anti-IL4Rα seems

promising for the management of AD (115). It may be that combination approaches will be

required to obtain optimal efficacy, as described above for leukotriene and prostaglandin co-

inhibition (104), whereby interventions typically affect pathways beyond ILC2s.

In psoriasis, ILC3s have emerged as important sources of IL-22 and IL-17. The IL-23/IL-17

cytokine axis can be targeted with anti-IL-17A and anti-IL-23p40 which are licensed for

therapeutic use, and clinical trials of anti-IL23p19 show significant and prolonged efficacy

(116). Further therapeutic aspects of blocking this pathway are being actively investigated.

Targeting ILCs in intestinal diseases

The shift from IL-22 to IL-17-producing cytokines in CD patients led to targeting IL-17 (using

secukinumab) in a therapeutic setting. However, clinical trials did not reach their endpoint

due to exacerbated disease activity (117). ILC plasticity may be utilized to induce a shift from

pathological ILC1s into beneficial ILC3s in IBD patients. Remarkably, previous clinical trials

targeting IFN-γ (using fontolizumab) (118) or the p40 subunit of IL-12 (as main IFN-γ inducing

cytokine) did not reach clear therapeutic efficacy (119). Furthermore, p40 directed therapy

does not specifically target IL-12, as IL-23 shares the same domain. A p35 antibody specific

to the IL-12 axis should be further examined. In addition, targeting IL-17 and IL-22 is difficult,

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as for both cytokines tissue-protective and pro-inflammatory effects have been described.

Nevertheless, ILC3s play an important role in maintenance of homeostatic conditions and in

disease pathogenesis. Additionally, therapeutic approaches supporting the induction of ILC2

and the IL-33-ILC2-amphiregulin pathway could be beneficial in the treatment of IBD

patients (120).

4. Research needs

There remains a need for further investigation of the role of ILCs in human allergic and non-

allergic diseases for better understanding of the pathophysiology and to promote the

development of new targeted therapeutic strategies. In many diseases, the numbers or

activity of ILCs are altered, but it still needs to be established whether these changes

correlate with symptoms and disease severity.

Although, in general, there is consensus on how to classify the different ILC populations,

further efforts should be made to uniform protocols regarding the markers to be used to

selectively identify each ILC subset and to exclude contaminating cells. A recent report

elegantly showed that there is both inter-individual heterogeneity and variation across

tissues with respect to the surface marker expression (3). Better instruments/methods to

quantify ILC subsets are crucial, because currently available methods can be used in a

research setting, but are not suitable in routine clinical practice.

Current word count: 4466

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ILC1 ILC2 ILC3AirwaysAllergic rhinitis (22-25)CRS (6, 9, 28, 29, 31-33)Allergic asthma (36-50, 103)Non-allergic asthma (51)COPD (6, 56) (14, 15)Viral infections (59-61)CF (64) (68, 69)IPF (63, 65) (67)Primary spontaneous pneumothorax

(70)

Helminth infections (71, 72) (71)SkinHomeostasis (74) (75)AD (73, 77-80)Psoriasis (12, 82, 83)GutHomeostasis (5, 54, 84) (54, 85) EoE (96, 97)IBD (5, 13, 54, 84, 90, 92) (5, 13, 54, 84, 89-91)Celiac disease (93, 94)NCWS (95)

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Figures and Table legends

Figure 1: Innate lymphoid cells in airway diseases

In homeostasis, all innate lymphoid cell (ILC) subsets are present in the human lungs. In non-

allergic inflammation, IL-1β secreted from (alveolar) macrophages can activate both ILC2s

and ILC3s. This leads to the secretion of type 2 cytokines by ILC2s, including IL-13 which is

implicated in fibrosis, and IL-17A from ILC3s which recruits monocytes and neutrophils. In

response to inhaled allergens, the epithelium produces cytokines, such as IL-33 and TSLP,

inducing a type 2 response. Cysteinyl leukotrienes (CysLTs) and prostaglandin D2 (PGD2),

secreted by activated mast cells, are activators of ILC2s. Upon activation, ILC2s rapidly

expand and secrete large amounts of IL-5, IL-13. ILC2s contribute to the allergic airway

inflammation by direct interaction with myeloid and Th2 cells, promoting cytokine and

mucus production, eosinophilia and accumulation of mast cells.

Figure 2: Innate lymphoid cells in skin diseases

ILCs are abundant in the dermis and they are implicated in tissue homeostasis and wound

repair due to the secretion of amphiregulin and IL-22 by ILC2s and ILC3s, respectively. In

psoriasis, the numbers of IL-22 producing ILC3s are elevated. IL-17A, secreted from ILC3s,

recruits monocytes and neutrophils. In patients with atopic dermatitis, elevated levels of IL-4

and IL-13 are found in parallel with increased levels of IL-33, TSLP and PGD2. Keratinocytes

interact with ILC2s via the B7-H6 - NKp30 axis and the E-cadherin – killer-cell lectin like

receptor G1 (KLRG1) interaction, leading to activation or suppression of ILC2s, respectively.

Figure 3: Innate lymphoid cells in intestinal homeostasis and diseases

A: ILC3s are important regulators of the homeostasis as they rapidly respond to the local

environment by the secretion of IL-22 and granulocyte-macrophage colony-stimulating

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factor (GM-CSF). IL-22 contributes to the epithelial barrier function and mucus production,

while GM-CSF stimulates intestinal macrophages to secrete IL-10 and retinoic acid regulating

the expansion of regulatory T cells (Tregs) and the induction of tolerance. ILC3s are also

important for the development of lymphoid structures, such as Peyer’s patches and isolated

lymphoid follicles.

B: The altered cytokine profile in the intestines of patients with Crohn’s disease (CD) or

ulcerative colitis (UC) leads to elevated IL-17-producing ILC3s and IFN-y producing ILC1s in

the intestines of CD patients, while IL-17 and type 2 cytokines are elevated in UC patients. In

food allergy, the allergic inflammation is characterized by a type 2 response with enhanced

levels of IgE, IL-4 and IL-13 and increased numbers of basophils and mast cells.

Figure 4: Clinical and therapeutic implications of innate lymphoid cells in allergic and

inflammatory diseases

A: Summary of the clinical implications of the subtypes of ILCs in allergic and inflammatory

diseases of the airways, gut and skin.

B: Overview of potential therapeutic implications for targeting ILCs via effector cytokines or

mediators that implicate ILC function.

Supplementary Table 1: Innate lymphoid cells being implicated in disease

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