Identification and functional analysis of type 2 innate lymphoid cells in the skin and in lesional skin biopsies of patients with atopic dermatitis

Over the past four years, a previously unrecognised family of innate effector cells has been identified. Their comprehensive functional capabilities range from lymphoid organogenesis, tissue remodelling, wound healing, immune protection and homeostasis to contribution to inflammation and allergic responses. Here we investigate the presence and function of type 2 innate lymphoid cells (ILC2) in the skin. We show that human ILC2 are resident in human skin and express RORA and GATA3, and skin homing receptors. ILC2 further infiltrate the skin after allergen challenge, where they produce the type 2 cytokines IL-5 and IL-13. Skin-derived ILC2 express the IL-33 receptor ST2, which is up-regulated during activation. Signalling via IL-33 induces type 2 cytokine and amphiregulin expression, and increases ILC2 migration.
Atopic dermatitis (AD) is a chronic inflammatory skin disorder. Current evidence suggests that both skin barrier dysfunction and immune system abnormalities, particularly those of a type 2 phenotype, contribute to disease pathogenesis. We demonstrated that ILC2 are enriched in lesional skin biopsies from atopic patients and show higher expression of cytokine receptors, reflecting an activated phenotype. Down-regulation of E-cadherin is characteristic of filaggrin insufficiency, a cardinal feature of AD. Interestingly, E-cadherin binding to KLRG1 on human ILC2 dramatically inhibits IL-5 and IL-13 production. ILC2 may contribute to increases in type 2 cytokine production in the absence of the inhibitory E-cadherin ligation through this novel mechanism of barrier sensing.
CRTH2, a receptor for prostaglandin D2 (PGD2), is expressed by human ILC2. However, the function of CRTH2 in these cells is unclear. We sought to determine the role of PGD2 and CRTH2 in human ILC2 and compare it with that of the established ILC2 activators IL-25 and IL-33. PGD2 binding to CRTH2 induced ILC2 migration and production of type 2 cytokines IL-4, IL-5, IL-13 and release of other pro-inflammatory cytokines IL-3, IL-8, IL-9, IL-21, GM-CSF, and CSF-1 in a dose-dependent manner. ILC2 activation through CRTH2 also upregulated the expression of IL-33 and IL-25 receptor subunits (ST2 and IL-17RA) suggesting a synergistic role. The effects of PGD2 on ILC2 could be mimicked by the supernatant from activated human mast cells and inhibited by a CRTH2 antagonist. Therefore, PGD2 can be considered as an important and potent activator of ILC2 through CRTH2 mediating strong inflammatory responses.
Cell surface interaction mechanisms that activate ILC2 function are unknown. We observed the expression of NKp30 on ILC2 ex vivo and after culture. Using quantitative PCR we confirmed that ILCs express NKp30c splice variant, an immune-modulatory isoform. Incubation of ILC2 with the NKp30 ligand B7H6 and tumour cell lines expressing this protein induced production of type 2 cytokines. This interaction can be inhibited by NKp30 blocking antibodies. We further established that activation of NKp30 induces the canonical pathway of NFƙB signalling.
Overall the work in thesis shows for the first time that ILC2 are resident in human skin and infiltrate rapidly after allergen challenge and in AD lesional skin. We have defined cytokine and lipid mediators that contribute to migration and activation of ILC2 and shown that KLRG1 and NKp30 act as inhibitory and activatory receptors respectively. The work defines novel pathways for barrier sensing and cutaneous inflammation, and identifies potential new targets for therapeutic intervention.

Keywords: Innate lymphoid cells
Skin
Atopic dermatitis
Advisor: Ogg, G