Characterization of innate lymphoid cells in human skin and blood demonstrates increase of NKp44+ ILC3 in psoriasis

Abstract

Innate lymphoid cells (ILCs) are increasingly appreciated as key regulators of tissue immunity. However, their role in human tissue homeostasis and disease remains to be fully elucidated. Here we characterize the ILCs in human skin from healthy individuals and from the inflammatory skin disease psoriasis. We show that a substantial proportion of IL-17A and IL-22 producing cells in the skin and blood of normal individuals and psoriasis patients are CD3-negative innate lymphocytes. Deep immunophenotyping of human ILC subsets showed a statistically significant increase in the frequency of circulating NKp44+ ILC3 in the blood of psoriasis patients compared with healthy individuals or atopic dermatitis patients. More than 50% of circulating NKp44+ ILC3 expressed cutaneous lymphocyte-associated antigen, indicating their potential for skin homing. Analysis of skin tissue revealed a significantly increased frequency of total ILCs in the skin compared with blood. Moreover, the frequency of NKp44+ ILC3 was significantly increased in non-lesional psoriatic skin compared with normal skin. A detailed time course of a psoriasis patient treated with anti-tumor necrosis factor showed a close association between therapeutic response, decrease in inflammatory skin lesions, and decrease of circulating NKp44+ ILC3. Overall, data from this initial observational study suggest a potential role for NKp44+ ILC3 in psoriasis pathogenesis.

Figure 1. Identification of CD3 negative lymphocytes in blood and skin which produce IL-17 and IL-22

PBMC or skin cells from psoriasis patients (P), healthy individuals (N) or AD patients were stimulated with PMA/ionomycin and stained for surface markers and cytokines for flow cytometric analysis. a) Representative plots showing IL-17A and IL-22 production in PBMC from N (n=16) and P (n=16). b) Frequencies of CD3 negative cells within IL-17A+ or IL-22+ cells in PBMC c) Representative dot plots for CD3− cells producing IL-17 and IL-22 from epidermis and dermis of normal (NN, n=4 [epidermis], 5[dermis]), psoriasis non-lesional (PN, n=4 [epidermis], 3 [dermis]), psoriasis lesional (PP, n=5 [epidermis and dermis]), AD non-lesional (AN, n=5 [epidermis], 2 [dermis]), and AD lesional (AA, n=8 [epidermis], 7 [dermis) skin. d) Representative plots of dermal skin explants from NN (n=2), PN (n=2) and PP (n=5) skin.

Figure 2. Deep immuno-phenotyping of PBMC shows increased frequency of NKp44+ ILC3 in blood of psoriasis patients

a) Mean frequencies ± SEM of myeloid (top) and lymphocytic (bottom) cell populations within PBMC of Psoriasis (P) (black, n=9), healthy individuals N (white, n=7) and AD patients (A) (grey, n=10). b) Analysis of Total ILC defined as Lin-CD127+ within live CD45+ lymphocytes in P (n=11), N (n=9) and A (n=10) (mean % ±SEM). ILC sub-populations were defined as shown in representative plots: ILC1 (pink), ILC2 (green), NKp44+ ILC3 (orange) and NKp44− ILC3 (purple). c) Pie charts showing average frequencies of ILC sub-populations in P, N and A. d) Median frequencies of NKp44+ ILC3; each filled (P), empty (N) or shaded triangle (A) represents an individual donor. Kruskal-Wallis, followed by Dunn’s Multiple Comparison test was performed, *P <0.05.

Figure 3. Reduction in NKp44+ ILC3 population during successful treatment of psoriasis with anti-TNF therapy

Case study of a psoriasis patient before and at indicated intervals after commencing therapy with the anti-TNF monoclonal antibody adalimumab. Time-course analysis showing a) the frequency of myeloid cell populations within CD45+/CD3−/CD19−/CD20− cells (top left); the frequency of B and T cell populations within CD45+ lymphocytes (top right); b) the frequency of ILC1, ILC2, NKp44− ILC3 and NKp44+ ILC3 cells within total ILCs; c) the absolute count of NKp44+ ILC3 within 10⁶ PBMC, (left y axis) plotted against disease severity expressed as “PASI” (right y axis).

Figure 4. Circulating NKp44+ ILC3 express skin homing CLA and are enriched in skin of psoriasis patients

PBMC (a-b) and collagenase-digested skin cells (c-f) were immuno-phenotyped. a) Frequency (mean ± SEM) of CLA+ cells within CD4s, CD8s and ILC from psoriasis (P, n=9), healthy individuals (N, n=6) and AD patients (A, n=7). b) Frequency (median ± IQ range) of CLA+ cells within ILC subsets. c) Plots representative of NKp44+ ILC3, show gating of ILC populations in normal (NN, n=6), psoriasis non-lesional (PN, n=5) and psoriasis lesional (PP, n=5) skin. d-f) Total ILC frequency (d) ILC subset distribution (e) and frequency of NKp44⁺ ILC3 (f) in NN, PN and PP skin. Each open (NN), shaded (PN) and filled (PP) triangle represents an individual donor (d&f). One-way ANOVA, with Bonferroni post test (a) or Kruskal-Wallis, with Dunn’s Multiple Comparison test (b,d&f), *P<0.05, **P<0.01, ***P<0.001.