ORIGINAL ARTICLE

The Major Orphan Forms of Ichthyosis Are

Characterized by Systemic T-Cell Activation
and Th-17/Tc-17/Th-22/Tc-22 Polarization
in Blood
Tali Czarnowicki1,2, Helen He1, Alexandra Leonard1, Kunal Malik1,3, Shai Magidi4, Stephanie Rangel5,
Krishna Patel5, Kara Ramsey5, Morgan Murphrey5, Teresa Song1, Yeriel Estrada1, Hue-Chi Wen1,
James G. Krueger2, Emma Guttman-Yassky1,2,6 and Amy S. Paller5,6

The ichthyoses are rare skin disorders with immune and barrier aberrations. Identifying blood phenotypes may
advance targeted therapeutics. We aimed to compare frequencies of skin homing/cutaneous lymphocyte antigen (þ)
versus systemic/cutaneous lymphocyte antigen (e) “polar” CD4þ/CD8þ and activated T-cell subsets in ichthyosis
versus atopic dermatitis, psoriasis, and control blood, with appropriate clinical correlations. Flow cytometry was used
to measure IFN-g, IL-13, IL-9, IL-17, and IL-22 cytokines in CD4þ/CD8þ T cells, with inducible co-stimulator molecule
and HLA-DR defining mid- and long-term T-cell activation, respectively. We compared peripheral blood from 47
patients with ichthyosis (congenital ichthyosiform erythroderma, lamellar ichthyosis, epidermolytic ichthyosis, and
Netherton syndrome) with 43 patients with atopic dermatitis and 24 patients with psoriasis and 59 age-matched
controls. Clinical measures included the ichthyosis severity score, with subsets for erythema and scaling, trans-
epidermal water loss, and pruritus. All ichthyoses had excessive inducible co-stimulator molecule activation
(P < 0.001), particularly epidermolytic ichthyosis. Significantly elevated IL-17- (P < 0.05) and IL-22-producing (P < 0.01)
T cells characterized ichthyoses, mainly Netherton syndrome and congenital ichthyosiform erythroderma (P < 0.05).
Increased T helper 2/cytotoxic T cell 2/T helper 9 (P < 0.05) and similar IFN-g frequencies (P > 0.1) versus controls were
also noted. IL-17/IL-22-producing cells clustered with clinical measures, whereas IFN-g clustered with age. Our data
show peripheral blood IL-17/IL-22 activation across the ichthyoses, correlating with clinical measures. Targeted
therapies should dissect the relative contribution of polar cytokines to disease pathogenesis.
Journal of Investigative Dermatology (2018) 138, 2157e2167; doi:10.1016/j.jid.2018.03.1523

INTRODUCTION defective epidermal barrier (Kawashima et al., 2005) with

The ichthyoses encompass a spectrum of syndromic and
nonsyndromic dermatoses with varying underlying genetic
bases (Oji et al., 2010). Their shared features include a
1
Department of Dermatology and the Immunology Institute, Icahn School
of Medicine at Mount Sinai, New York, New York, USA; 2Laboratory for
Investigative Dermatology, The Rockefeller University, New York, New
York, USA; 3College of Medicine, SUNY Downstate, Brooklyn, New York,
USA; 4Department of Pharmacological Sciences, Mount Sinai Center for
Bioinformatics, BD2K-LINCS Data Coordination and Integration Center,
Icahn School of Medicine at Mount Sinai School, New York, New York,
USA; and 5Departments of Dermatology and Pediatrics, Northwestern
University Feinberg School of Medicine, Illinois, USA
6
These authors contributed equally to this work.
Correspondence: Amy S. Paller, Department of Dermatology, Northwestern
University Feinberg School of Medicine, 676 N. St. Clair, Suite 1600,
Chicago, Illinois 60611-2997, USA. E-mail: apaller@northwestern.edu
Abbreviations: AD, atopic dermatitis; ARCI, autosomal recessive congenital
ichthyosis; CIE, congenital ichthyosiform erythroderma; CLA, cutaneous
lymphocyte antigen; EI, epidermolytic ichthyosis; IASI, ichthyosis area
severity index; IASI-E, ichthyosis area severity index-erythema; IASI-S, ich-
thyosis area severity index-scaling; ICOS, inducible co-stimulator molecule;
LI, lamellar ichthyosis; NS, Netherton syndrome; Tc, cytotoxic T cell; Tcm,
central memory T cell; Tem, effector memory T cell; TEWL, transepidermal
water loss; Th, T helper; Treg, T-regulatory cell
Received 5 February 2018; revised 15 March 2018; accepted 23 March 2018;
accepted manuscript published online 14 April 2018; corrected proof
published online 7 June 2018
scaling and variable cutaneous erythema. Patients suffer from
reduced quality of life and recurrent infections, posing an
economic burden on health care (Dreyfus et al., 2015).
The best-characterized and most common phenotype is
ichthyosis vulgaris, owing to FLG (encoding filaggrin) gene
mutations (Smith et al., 2006). The more prevalent rare forms
of ichthyosis are lamellar ichthyosis (LI; usually TGM1,
encoding transglutaminase 1) and congenital ichthyosiform
erythroderma (CIE; multiple causative genes) (Takeichi and
Akiyama, 2016), collectively known as autosomal recessive
congenital ichthyosis (ARCI; Oji et al., 2010), keratinopathic
ichthyosis, epidermolytic ichthyosis (EI), involving mutations
in KRT1 and KRT10 (Vahlquist et al., 2017), and Netherton
syndrome (NS), with its atopic manifestations and underlying
deficiency of a serine peptidase inhibitor, Kazal type 5
(SPINK5 mutations) (Sarri et al., 2017).
Recently, the cutaneous ichthyosis phenotype of these
orphan ichthyoses was linked to robust IL-17/tumor necrosis
factor-a cytokine activation, which is highly associated with
disease severity and transepidermal water loss (TEWL), the
functional barrier measure (Paller et al., 2017b). These data
are supported by studies on a limited number of patients
with restricted panels, mostly in NS (Briot et al., 2009; Fontao
et al., 2011), showing a pro-Th2/TARC/CCL17/TSLP signature

ª 2018 The Authors. Published by Elsevier, Inc. on behalf of the Society for Investigative Dermatology. www.jidonline.org 2157
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Th17/Tc17/Th22/Tc22-Skewing in Ichthyosis Blood

(Akagi et al., 2013; Briot et al., 2009) with innate (IL-1b,
tumor necrosis factor-a) and T-cell activation (IL-2) cytokine
increases (Renner et al., 2009). A previous study in nine
children with NS using flow cytometry found normal
numbers of T, B, and T-regulatory cells (Tregs), with small
increases in natural killer cells in affected individuals (Renner
et al., 2009). Another study on 10 children and one adult
with NS showed altered B-cell expression and decreased
natural killer cytotoxic activity (Hannula-Jouppi et al., 2016).
Comprehensive blood phenotyping of a larger number of
these pediatric and adult patients with ichthyosis is not
available.
There remains a huge unmet need for safer, more effective
treatments for ichthyoses. Current treatments are empirical,
often ineffective, and largely directed at reducing scale.
These include topical moisturizers, keratolytics, retinoids,
vitamin D analogs, corticosteroids, and calcineurin in-
hibitors, all off-label. Systemic retinoids have variable effi-
cacy and multiple potential side effects (DiGiovanna and
Robinson-Bostom, 2003). Experimental gene therapies are
early in preclinical development (Gorell et al., 2014).
Although cutaneous and systemic immune inflammation has
been successfully targeted in atopic dermatitis (AD) and
psoriasis (Czarnowicki et al., 2015c; Guttman-Yassky et al.,
2017), little attention has been paid to targeting inflammation
in ichthyosis. Indeed, specific immune-targeting therapeutics
have shown efficacy for NS (anti-tumor necrosis factor-a)
(Fontao et al., 2011; Paller et al., 2017a) and ichthyotic dis-
order resulting from mutations in DSP, encoding desmopla-
kin (ustekinumab) (Paller et al., 2017a).
Cutaneous lymphocyte antigen (CLAþ) memory T cells are
peripheral biomarkers in inflammatory skin disorders
(Czarnowicki et al., 2017b). We evaluated activated CD4þ/
CD8þ T cells and their differentiation to IFN-g-, IL-13-, IL-22-,
IL-17A-, and IL-9-producing T cells within skin homing/CLAþ
and systemic/CLAe compartments in blood of patients with
ichthyosis, compared to patients with AD and psoriasis
and healthy controls. Blood of patients with ichthyosis had
increases in T-cell activation, and in IL-22-, IL-17A-, IL-13-,
and IL-9-producing T cells. IL-17/IL-22 axes were linked with
disease severity, whereas the IFN-g pathway was associated
with disease chronicity.
RESULTS
Flow cytometry was used to measure frequencies of IFN-g-,
IL-9-, IL-13-, IL-17-, and IL-22-producing T cells, defining
T helper (Th) 1/cytotoxic T cell (Tc)1, Th9/Tc9, Th2/Tc2,
Th17/Tc17, and Th22/Tc22 subsets in CD4þ/CD8þ T cells,
respectively. Cell surface staining was used to measure
expression of mid (inducible co-stimulator molecule [ICOS])
and late (HLA-DR) activation markers in central (Tcm/
CCR7þCD45ROþ) and effector (Tem/CCR7eCD45ROþ)
memory T cells in skin homing/CLAþ and systemic/CLAe
subsets. To assess immune polarity, samples were analyzed in
parallel with blood from healthy controls and previously
published cohorts of moderate-to-severe AD and psoriasis
(Czarnowicki et al., 2015b, 2015c), representing different
immune polarizations (Th2/Th22 and Th1/Th17, respec-
tively). To understand the common immune skewing in ich-
thyosis, we first compared frequencies of T-cell populations
between ichthyosis overall and healthy controls, patients with
psoriasis and AD, and subsequently evaluated differences
among the various ichthyosis subtypes (ARCI-LI, ARCI-CIE,
EI, and NS).
To enhance representation of the distribution of values on
the significance of results, comparison plots incorporate both
means (black) and medians (red)  standard error, with
respective P-values. Although results discuss mean values,
both are detailed in Supplementary Tables S1 and S2 online.
Ichthyosis is characterized by increased mid/ICOS and late/
HLA-DR T-cell activation
Tcm and Tem cells, key components of adaptive immunity,
are characterized by diverse homing capacities (Sallusto
et al., 2004). Both express the skin homing marker CLA,
but Tcm are also CCR7þ, allowing them to migrate into
lymph nodes, creating an immunologic reserve (Sallusto
et al., 1999). Ichthyosis showed the highest skin-homing
CD4þTem frequencies (normal: 20%, psoriasis: 21%, AD:
23%, ichthyosis: 28%; P < 0.01; Figure 1a and b) and higher
CLAþCD8þ Tem (12% vs. 9%, P ¼ 0.05; Figure 1c) and Tcm
(13.5% vs. 9.8%, P ¼ 0.03; Figure 1d) cells than controls
(Supplementary Table S1).
T-cell activation via the T-cell receptor leads to sequential
expression of T-cell activation markers. ICOS, a mid-
activation marker, is upregulated within 24 hours (Tafuri
et al., 2001), whereas HLA-DR indicates chronic T-cell acti-
vation (Ferenczi et al., 2000). Ichthyosis showed increased
pan activation of ICOS and HLA-DR in both the skin-homing/
CLAþ and systemic/CLAe compartments, particularly among
CD4þ populations (Figure 1eel). Most striking was ICOS
activation, in both CD4þ and CD8þ subsets (CD4þ Tem:
CLAþ: ichthyosis 43.5% vs. normal 20.5%, P < 0.0001;
CLAe: ichthyosis 18.5% vs. normal 11.7%, P < 0.001
[Figure 1eef]; and CD8þ Tcm: CLAþ: ichthyosis 29.8% vs.
normal 15.6%, P < 0.0001; CLAe: ichthyosis 6.3% vs.
normal 4.2%, P ¼ 0.001 [Supplementary Figure S1a, b, e,
and f online]). These frequencies were even higher than in
AD, a highly inflammatory skin disease with excessive T-cell
activation (Czarnowicki et al., 2015c). Conversely, HLA-DR
demonstrated differential expression, with increases
restricted to CD4þ subsets (Tem: CLAþ: ichthyosis 10% vs.
normal 5.5%, P ¼ 0.004; CLAe: ichthyosis 4.2% vs. normal
2.2%, P ¼ 0.05; Tcm: CLAþ: ichthyosis 8.8% vs. normal
4.6%, P ¼ 0.004; CLAe: ichthyosis 4% vs. normal 4%,
P ¼ 0.03 [Figure 1g, h, k, and l]), but significantly lower
frequencies in CD8þ Tem/Tcm/CLAþ/CLAe cells (P < 0.02;
Supplementary Figure S1c, d, g and h, Supplementary
Table S1).
Prominent Th17/Th22 signatures characterize ichthyoses
T-cell activation is followed by T-cell subset differentiation.
Thus, we next evaluated the frequencies of polar T-cell sub-
sets, including IFN-g-, IL-9-, IL-13-, IL-17-, and IL-22-
producing CD4þ/CD8þ T cells, in both the cutaneous/CLAþ
and systemic/CLAe compartments.
Congruent with our recent skin findings (Paller et al.,
2017b), IL-17 was increased in ichthyosis versus control
blood, particularly among skin-homing CD4þ T cells (CLAþ:
4.5% vs. 2.4%, P ¼ 0.004; Figure 2a), demonstrating levels
either similar to or higher than in psoriasis (CD8þ/CLAþ/CLAe:

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a + +
CLA CD4 Tem cells b CLA+CD4+ Tcm cells c + +
CLA CD8 Tem cells d CLA+CD8+ Tcm cells
* + 6 *
100 * *
+ +
* +
*** **
*** 30 40 **

75
4
Frequency (%)

30
20
50
20
2
25 10
10

0 0 0

Normal Psoriasis AD Ichthyosis Normal Psoriasis AD Ichthyosis Normal Psoriasis AD Ichthyosis Normal Psoriasis AD Ichthyosis

e ICOS+CLA+CD4+ Tem cells

f ICOS+CLA–CD4+ Tem cells
g HLA-DR+CLA+CD4+ Tem cells
h HLA-DR+CLA–CD4+ Tem cells
*** *** * **
*** *** *
* ** *** 40 *
*** * *** * ** +
*** *** 60 *
100 *** 60 *** **
*** *** ***
*** ** ***
*** ***
30
Frequency (%)

75
40
40
20
50

20
20 10
25

0 0 0
0
Normal Psoriasis AD Ichthyosis Normal Psoriasis AD Ichthyosis Normal Psoriasis AD Ichthyosis Normal Psoriasis AD Ichthyosis

i ICOS+CLA+CD4+ Tcm cells

j k HLA-DR+CLA+CD4+ Tcm cells
l
ICOS+CLA–CD4+ Tcm cells + – +
HLA-DR CLA CD4 Tcm cells
*** *** *** ***
*** + *** *** ***
*** * 40 * ***
*** *** ** *** 30 ***
*** *** 60 *** **
90 *** * **
* * *
* *** ** ***
** *** *** ***
** **
** 30
Frequency (%)

20
60 40
20

30 20 10
10

0 0 0 0

Normal Psoriasis AD Ichthyosis Normal Psoriasis AD Ichthyosis Normal Psoriasis AD Ichthyosis Normal Psoriasis AD Ichthyosis

Figure 1. T-cell memory subset activation. (aed) CLA CD4 /CD8 Tcm (CD45RO CCR7 ), Tem (CD45RO CCR7 ) subset frequencies, and (eel) ICOSþ and
þ þ þ þ þ þ e

HLA-DRþ activation in CLAþ/e CD4þ Tcm/Tem cells in control (red), psoriasis (green), AD (purple), and ichthyosis (blue). Bar plots represent means (black)/
medians (red)  SEMs. P-values are designated as ***<0.001, **<0.01, *<0.05, þ<0.1. AD, atopic dermatitis; CLA, cutaneous lymphocyte antigen; ICOS,
inducible co-stimulator molecule; SEM, standard error of the mean/median; Tcm, central memory T cell; Tem, effector memory T cell.

P < 0.01; Figure 2bed), a prototypical Th17/IL-17 disease
(Raychaudhuri, 2013). Even more prominent were the
increased frequencies of IL-22-producing CD4þ (CLAþ: 7% vs.
4%, P ¼ 0.006; CLAe: 3.4% vs. 1.65%, P ¼ 0.001; Figure 2e
and f) and CD8þ (CLAþ: 6.6% vs. 2%, P ¼ 0.004; CLAe: 1.5%
vs. 0.5%, P ¼ 0.001; Figure 2g and h) T cells in ichthyosis
versus controls, seen consistently across the cutaneous and
systemic compartments. IL-22 levels in ichthyosis were similar
or higher (CD8þ/CLAe: 1.5% vs. 0.6%, P ¼ 0.004; Figure 2h)
than in AD, an inflammatory disease with characteristic IL-22
skewing in blood and skin (Czarnowicki et al., 2015b; Gittler
et al., 2012). Intriguingly, similar to AD (Czarnowicki et al.,
2015b), IL-13-producing CD4þ/CD8þ cells (besides CD4þ/
CLAþ cells, P > 0.1) were increased in ichthyosis to levels
much higher than in psoriasis across all subsets (P < 0.01;
Figure 3aed). IL-9-producing cells, recently associated with
psoriasis (Ruiz-Romeu et al., 2017; Singh et al., 2013) and
AD (Ma et al., 2014), were also upregulated in ichthyosis,
specifically in CD4þ (CLAþ: 6.9% vs. 3.7%, P ¼ 0.01; CLAe:
0.4% vs. 0.2%, P ¼ 0.02; Figure 3e and f), but not CD8þ T cells
(P > 0.1; Figure 3g and h). Finally, IFN-g was largely similar
between ichthyoses and control (P > 0.1; Figure 3iel).
Epidermolytic ichthyosis/EI shows the highest ICOS and
HLA-DR expression
To detect whether inflammatory profiles of ichthyosis sub-
types vary, we next compared disease subcategories and
controls. Although all subtypes had increased Tem ICOS
activation, EI showed highest expressions in both CLAþ
(55.3% vs. 20.5%, P ¼ 0.0001; Figure 4a) and CLAe (29%

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CD4+ T cells CD8+ T cells

a IL-17+CLA+ b IL-17+CLA– c IL-17+CLA+ d +
IL-17 CLA
–

30 + + 40 **
** 10.0 *** +
* ** **
** * +
**
* 6
* 30
7.5
20
Frequency (%)

Frequency (%)
4
20
5.0

10
10 2
2.5

0 0.0 0 0

Normal Psoriasis AD Ichthyosis Normal Psoriasis AD Ichthyosis Normal Psoriasis AD Ichthyosis Normal Psoriasis AD Ichthyosis

e IL-22+CLA+
f IL-22+CLA–
g +
IL-22 CLA
+ h +
IL-22 CLA
–

**
**
*
** + ***
60 + +
***
*** * ** **
*** ***
*** *** *
*** 15 **
75 ** *
*** *** *** 10
*** ***
Frequency (%)

Frequency (%)
40
10 50

5
20
5 25

0 0 0 0

Normal Psoriasis AD Ichthyosis Normal Psoriasis AD Ichthyosis Normal Psoriasis AD Ichthyosis Normal Psoriasis AD Ichthyosis
D D þ þ þ/e þ þ
Figure 2. IL-17 /IL-22 cytokine frequencies. (aed) IL-17 and (eeh) IL-22 frequencies in CLA , CD4 /CD8 T cells in control (red), psoriasis (green), AD
(purple), and ichthyosis (blue). Bar plots represent means (black)/medians (red)  SEMs. P-values are designated as ***<0.001, **<0.01, *<0.05, þ<0.1. AD,
atopic dermatitis; CLA, cutaneous lymphocyte antigen; SEM, standard error of the mean/median.

vs. 11.7%, P ¼ 0.01; Figure 4b) CD4þ, but not CD8þ Tem Th17/Tc17 and Th22/Tc22 cells cluster with ichthyosis
populations (Supplementary Table S2; Supplementary clinical measures
Figure S2a and b online). HLA-DR Tem activation was We also assessed how clinical characteristics, including
largely similar among ichthyoses (Figure 4c and d; ichthyosis severity (total ichthyosis area severity index [IASI],
Supplementary Figure S2c and d). EI also showed the with subsets for erythema [IASI-E] and scaling [IASI-S]),
highest Tcm ICOS (CLAþ: 37% vs. 14.7%, P ¼ 0.008; CLAe: pruritus, and TEWL, a functional barrier measure, relate to
15.6% vs. 7.2%, P ¼ 0.02) and HLA-DR activation, polar T-cell subsets. Unsupervised hierarchical clustering of
restricted to CD4þ subsets (Figure 4eeh; Supplementary all parameters was performed using Pearson correlations, as
Figure S2eeh). shown in the correlation heatmap and dendrogram in
Figure 6 (red: positive; blue: negative correlations, stars and
plus signs: significant correlations). Selected individual cor-
NS and CIE demonstrate the highest IL-17/L-22 frequencies relation scatter plots are presented in Supplementary
NS showed elevated IL-17 and IL-22 frequencies in CLAþ or Figures S4 and S5 online. As shown in Figure 6, a tight
CLAe, CD4þ or CD8þ cells (P < 0.05 for all), not just versus cluster gathering of TEWL, IASI-S, IASI, IASI-E, and IL-17-
controls but also compared with other ichthyosis subtypes producing cells (purple square) was adjacent to a Th22/
except CIE (P > 0.1) (Figure 5, Supplementary Table S2). Tc22 cluster (green square). Clinical severity and TEWL
Contrary to the Th2 fingerprint reported in past NS serum measures (red ellipses) branched around IL-17/IL-22 axes,
studies (Akagi et al., 2013; Briot et al., 2009), our results whereas pruritus clustered with IL-13-producing cells (yellow
showed comparable IL-13 frequencies in NS and controls square). IFN-g clustered with age (blue square). Correlations
(Supplementary Figure S3aed online). In contrast, CIE and LI are listed in Supplementary Table S3 online.
had increased IL-13 frequencies, particularly among the IASI-E/inflammation and IASI-S/scaling components highly
CLAe CD4þ (CIE 1.8% vs. control 0.7%, P ¼ 0.007; correlated with total IASI (P < 0.0001; Supplementary
Supplementary Figure S3b) and CD8þ (CIE 1.3% vs. control Figure S4a and b). Th17 (r ¼ 0.4, P ¼ 0.004;
0.3%, P ¼ 0.01; LI 1.4% vs. control 0.3%, P ¼ 0.005; Supplementary Figure S4c)/Tc17 (r ¼ 0.34, P ¼ 0.016;
Supplementary Figure S3d) subsets. Th9/Tc9 occurrence was Supplementary Figure S4d) and Th22 (r ¼ 0.43, P ¼ 0.001;
upregulated in NS and LI (Supplementary Figure S3eeh). Supplementary Figure S4e)/Tc22 (r ¼ 0.32, P ¼ 0.02;
Only EI showed significantly lower IFN-g levels than controls Supplementary Figure S4f) correlated significantly with total
(P < 0.05; Supplementary Figure S3iel). IASI. Analogous correlations were observed between Th17/

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CD4+ T cells CD8+ T cells

a IL-13+CLA+ b IL-13+CLA–
c IL-13+CLA+ d IL-13+CLA–
*** *** *** 10.0 *
*** *** 50 *** **
*** 8 *** *** +
*** * *** *** *
*** * *** *
*** * *** ***
*** ** 40 ** ***
*** *** *** 7.5 ***
40 *** 6 ** *** **
** ***
Frequency (%)

Frequency (%)
30
5.0
4
20
20

2 2.5
10

0 0 0 0.0

Normal Psoriasis AD Ichthyosis Normal Psoriasis AD Ichthyosis Normal Psoriasis AD Ichthyosis Normal Psoriasis AD Ichthyosis

e IL-9+CLA+ f IL-9+CLA– g IL-9+CLA+ h IL-9+CLA–

60 *** + 30 +
** 1.5 +
***
*** +
**
* * **
* +
4
40
Frequency (%)

20

Frequency (%)
1.0

20 2
10 0.5

0 0 0 0.0

Normal Psoriasis AD Ichthyosis Normal Psoriasis AD Ichthyosis Normal Psoriasis AD Ichthyosis Normal Psoriasis AD Ichthyosis

i IFN-γ+CLA+ j +
IFN-γ CLA
– k IFN-γ+CLA+ l IFN-γ+CLA–
+
* +
* 120
* *
* *
60
60 60
Frequency (%)

Frequency (%)

80
40
40 40

40
20 20 20

0 0 0 0

Normal Psoriasis AD Ichthyosis Normal Psoriasis AD Ichthyosis Normal Psoriasis AD Ichthyosis Normal Psoriasis AD Ichthyosis

Figure 3. IL-13D/IL-9D/IFN-gD cytokine frequencies. (aed) IL-13þ, (eeh) IL-9þ, and (iel) IFN-gþ frequencies in CLAþ/e, CD4þ/CD8þ T cells in control (red),
psoriasis (green), AD (purple), and ichthyosis (blue). Bar plots represent means (black)/medians (red)  SEMs. P-values are designated as ***<0.001, **<0.01,
*<0.05, þ<0.1. AD, atopic dermatitis; CLA, cutaneous lymphocyte antigen; SEM, standard error of the mean/median.

Tc17/Th22/Tc22 and IASI-E (Supplementary Figure S5aed).
IASI-S correlated with both CD8þ/CLAþ cells (r ¼ 0.31, P ¼
0.02; Supplementary Figure S5e) and Th9/Tc9 cells (P < 0.05;
Supplementary Figure S5f and g). Although Th22 cells
correlated positively with TEWL (r ¼ 0.28, P ¼ 0.06;
Supplementary Figure S4g), negative correlations were
observed between Th9/Tc9 (r ¼ e0.35, P ¼ 0.02 for both;
Supplementary Figure S4h and i) and TEWL. Tc2 (r ¼ 0.44,
P ¼ 0.003; Supplementary Figure S4j) significantly correlated
with the 5D-pruritus scale, with lesser correlations found
with Th17 (r ¼ 0.32, P ¼ 0.04) and Th22 (r ¼ 0.28, P ¼ 0.07;
Supplementary Figure S4k and l). To evaluate for the effect of
disease chronicity, we also correlated parameters with age.
IFN-g, previously associated with disease chronicity
(Czarnowicki et al., 2015a, 2017a; Gittler et al., 2012), was
significantly correlated with age (P < 0.04; Supplementary
Figure S4m and n). Ichthyosis severity showed a trend of
amelioration with age (Supplementary Figure S4o). Lastly,
IL-22-producing cells highly correlated with both IL-17 and
IL-13 CD4þ/CD8þ T-cell subsets (P < 0.01 for all;
Supplementary Figure S5hek).
To assess how skin-homing T cells in blood correlate with
cutaneous T cells and disease severity in ichthyosis, we per-
formed immunostaining for CD3þ T cells in skin from avail-
able ichthyosis samples (n ¼ 27). CD3þ cell counts
correlated significantly with ICOS-activated systemic Tcm
cells (r ¼ 0.45, P ¼ 0.02; Supplementary Figure S5l), with a
trend toward significance for IASI-E (r ¼ 0.34, P ¼ 0.08;
Supplementary Figure S5m), but not IASI-S (P ¼ 0.9;
Supplementary Figure S5n, Supplementary Table S3).

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a ICOS+CLA+CD4+ Tem cells b + – +

ICOS CLA CD4 Tem cells c HLA-DR+CLA+CD4+ Tem cells d HLA-DR+CLA-CD4+ Tem cells
* ** +
* * 30
60 *
* ** +
* *
75 *
*** *
***
100 *** *** 20
***
Frequency (%)

*** +
* 40
*** +
***
** 50

50 10
20
25

0
0 0 0

Normal NS LI EI CIE Normal NS LI EI CIE Normal NS LI EI CIE Normal NS LI EI CIE

e ICOS+CLA+CD4+ Tcm cells f ICOS+CLA–CD4+ Tcm cells g HLA-DR+CLA+CD4+ Tcm cells h HLA-DR+CLA-CD4+ Tcm cells
+
** *** +
*
*** 60 *
** + + 30
**
90 ** *
*
+ *
*
* 40
* **
Frequency (%)

*
40 20
60

20 10
20
30

0
0 0 0

Normal NS LI EI CIE Normal NS LI EI CIE Normal NS LI EI CIE Normal NS LI EI CIE

D þ þ þ/e þ
Figure 4. Ichthyosis subtypes CD4 T-cell activation. (aeh) ICOS and HLA-DR activation in CLA CD4 Tcm/Tem cells in control (red), NS, LI, EI, and CIE
(shades of blue). Bar plots represent means (black)/medians (red)  SEMs. P-values are designated as ***<0.001, **<0.01, *<0.05, þ<0.1. CIE, congenital
ichthyosiform erythroderma; CLA, cutaneous lymphocyte antigen; EI, epidermolytic ichthyosis; ICOS, inducible co-stimulator molecule; LI, lamellar ichthyosis;
NS, Netherton syndrome; SEM, standard error of the mean/median; Tcm, central memory T cell; Tem, effector memory T cell.

Total Tregs are increased in ichthyosis and correlate with biomarkers to monitor treatment responses and advance
T-cell activation. targeted therapeutic development.
Ichthyosis showed significantly increased total (defined Similar to our recent findings of Th17-dominant inflam-
as CD25þCD127eCCR4þ) (Gorski et al., 2010) (P < 0.01), mation in skin (Paller et al., 2017b), the current data
but not skin-homing (CCR4þCLAþ) Treg frequencies versus establish shared elevation of IL-17 frequencies in ichthyoses
control, psoriasis, and AD, with largely similar blood across skin homing/CLAþ and systemic/CLAe CD4þ
distribution among ichthyosis subtypes (Supplementary and CD8þ populations, supporting their systemic immune
Figure S6aed online). Similar to AD report (Czarnowicki dysregulation and hinting at possible associated comorbid-
et al., 2015c), Tregs significantly correlated with skin- ities. IL-22 was profoundly upregulated in ichthyosis to
homing Tcm/Tem, HLA-DR activated T-cell subsets levels even higher than in AD, a disease with a prominent
(P < 0.013 for all; Supplementary Figure S6eeh, IL-22 signature (Czarnowicki et al., 2015b; Gittler et al.,
Supplementary Table S3). 2012). Furthermore, Th17/Tc17/Th22/Tc22 subsets corre-
late with total IASI and IASI-E, and cluster close to TEWL,
DISCUSSION 5D pruritus score and IASI-S, suggesting their possible role
This study details all recognized effector T-cell subsets within in ichthyosis skin manifestations. The close link of barrier
CLAþ and CLAe compartments in the circulation of the four measures to dysregulated cytokines indicates ongoing
more common subtypes among orphan ichthyoses (ARCI-LI, crosstalk between these components in ichthyosis, similar to
ARCI-CIE, EI, and NS) compared with healthy controls. AD and psoriasis (Czarnowicki et al., 2014; Guttman-Yassky
Because of the suggested phenotypical similarities to psori- et al., 2011). NS and CIE show the highest IL-17/IL-22
asis and AD in skin, ichthyosis blood samples were analyzed expression, reflecting their more erythrodermic phenotype
in parallel with previously published AD (Th2/Th22-skewed) (Richard, 1993) and higher IASI-E and TEWL values
and psoriasis (Th1/Th17-polarized) samples (Czarnowicki (Supplementary Table S4 online), further highlighting the
et al., 2015c). We not only found a common blood pheno- association between inflammation and barrier impairment.
type in the largest group of patients with ichthyosis, but also IL-22 and IL-17 are extensively involved in protective anti-
characterized the unique blood profiles of different subtypes. bacterial immune responses (Cho et al., 2010; Jin and
Expanding the ichthyosis phenotyping to blood, including Dong, 2013), antimicrobial peptide secretion, regulation
identification of activation markers and polarized cytokines, of chronic inflammation, and maintenance of epithelial
carries the potential for the development of noninvasive integrity (Sonnenberg et al., 2011; Wolk et al., 2004). The

2162 Journal of Investigative Dermatology (2018), Volume 138

 T Czarnowicki et al.
Th17/Tc17/Th22/Tc22-Skewing in Ichthyosis Blood

CD4+ T cells CD8+ T cells

a IL-17+CLA+ b IL-17+CLA– c IL-17+CLA+ d IL-17+CLA–

+ + +
** * 50
+ 15
*
+
+
* 40 7.5 *
20 +
Frequency (%)

Frequency (%)
*
10 + *
30 *
5.0

10 20
5
2.5
10

0 0 0 0.0

Normal NS LI EI CIE Normal NS LI EI CIE Normal NS LI EI CIE Normal NS LI EI CIE

e IL-22+CLA+
f IL-22+CLA–
g +
IL-22 CLA
+ h IL-22+CLA–

***
+ +
25 +
*
+
+ +
+
** * 15
** * 100
** 20 + *
60 *** **
** * *
** **
+
Frequency (%)

Frequency (%)
***
** 75 *
15 *
+ **
+ **
10
40
10 50

5
20
5 25

0 0 0 0

Normal NS LI EI CIE Normal NS LI EI CIE Normal NS LI EI CIE Normal NS LI EI CIE

D D þ þ þ/ þ þ
Figure 5. Ichthyosis subtypes IL-17 /IL-22 cytokine frequencies. (aed) IL-17 and (e-h) IL-22 frequencies in CLA , CD4 /CD8 T-cells control (red), NS,
LI, EI, and CIE (shades of blue). Bar plots represent means (black)/medians (red)  SEMs. P-values are designated as ***<0.001, **<0.01, *<0.05, þ<0.1. CIE,
congenital ichthyosiform erythroderma; CLA, cutaneous lymphocyte antigen; EI, epidermolytic ichthyosis; LI, lamellar ichthyosis; NS, Netherton syndrome;
SEM, standard error of the mean/median.

increased IL-17/IL-22 blood subsets are consistent with the et al., 2009). IL-9 is increased in ichthyosis blood, perhaps
elevated IL-17/IL-22-related markers in skin, including perpetuating the Th17/Th22 deviation. Although positively
antimicrobial peptides (Paller et al., 2017b). Although pos- correlated with IASI-S, IL-9 correlates negatively with TEWL,
itive correlations between IL-13 and IL-22 observed here are suggesting a complex interaction between Th9/Tc9 subsets
similar to AD findings (Czarnowicki et al., 2015b), IL-17 and barrier homeostasis. Similar to alopecia areata
and IL-22 associations might partially derive from the abil- (Czarnowicki et al., 2017a) and AD (Czarnowicki et al.,
ity of Th17 cells to produce both cytokines (Liang et al., 2015a; Gittler et al., 2012), in which IFN-g marks disease
2006). chronicity, Th1/Tc1 subsets correlate with age. Ichthyosis has
Despite the cutaneous resemblance to psoriasis (Paller similar or even higher Tem/Tcm cell frequencies than AD,
et al., 2017b), the ichthyosis blood profile blends character- suggesting excessive circulatory activation with concurrent
istics of both AD and psoriasis, with increased ICOS/HLA-DR immune reserve accumulation in lymph nodes, as indicated
and IL-22 activation, features of both AD and psoriasis, but by increased Tcm cells, potentially explaining disease chro-
also elevated IL-17 level characteristic of psoriasis and nicity, recurrent exacerbations, and treatment failures.
increased IL-13 frequencies typifying AD. Clustering (and ICOS, the mid-activation marker, is necessary for intact
positive correlations) of Th2/Tc2 cells and the 5D pruritus immune circuits (Tafuri et al., 2001) and shapes adaptive
scale not only support the IL-13 role as an itch mediator immunity (Coyle et al., 2000). Although ICOS augments Th2
(Mollanazar et al., 2016) but may also suggest a tool for responses (Tesciuba et al., 2008), it is also critical for Th17
patient targeted therapeutic stratification. development (Nurieva, 2005; Paulos et al., 2010). ICOS
Surprisingly, despite the accepted association between NS activation is profoundly increased across all Tcm/Tem/CD4þ/
and atopy (Samuelov and Sprecher, 2014), IL-13 is not CD8þ/CLAþ/systemic subsets in ichthyosis to levels even
increased in NS blood or skin (Paller et al., 2017b). Lack of higher than the exceedingly activated AD (Czarnowicki et al.,
Th2 skewing in NS may result from the reciprocal regulation 2015c), probably augmenting IL-17 responses in ichthyosis
by Th17 (Lynch et al., 2016), which is highest in NS. IL-9 has blood and skin (Paller et al., 2017b). The fact that the chronic
been implicated in allergic/atopic disorders (Wisniewski and activation marker HLA-DR (Reddy et al., 2004) is increased
Borish, 2011), including AD (Ma et al., 2014) and more in CD4þ Tem/Tcm cells, but significantly low among CD8þ
recently psoriasis (Ruiz-Romeu et al., 2017; Singh et al., subsets, suggests a specific T-cell activation sequence in
2013). IL-9 induces Th17 differentiation and promotes ichthyosis with persistent ICOS/HLA-DR CD4þ activation,
Th17-driven inflammation (Elyaman et al., 2009; Nowak and a minor contribution of CD8þ cells in chronic disease.

www.jidonline.org 2163
 T Czarnowicki et al.
Th17/Tc17/Th22/Tc22-Skewing in Ichthyosis Blood

TEWL

5-D-Pruritus Scale
Age

IASI-S
+

CD4+IL13+CLA+
CD8+IL13+CLA+
CD4 IFNγ CLA

CD4+IL9+CLA+
CD4+CLA+
CD8+CLA+
+

CD8+IFNγ+CLA+

CD8 IL9+CLA+

+
CD4 IL17 CLA
+

CD8+IL17+CLA+
+

CD4+IL22+CLA+
IASI-E
IASI
CD4 IL9+
CD4+IL9+CLA–
+

CD8+IL9+
CD8+IL9+CLA–

CD8+IL22+CLA+
Correlation

+
+

CD4+IL13+
CD4+IL13+CLA–
1.0

CD8+IL13+
CD8+IL13+CLA–
CD4+IL22+
CD4 IL22+CLA–
CD4 IL17+
CD4+IL17+CLA–

CD8+IL17+
CD8 IL17+CLA–

CD8+IL22+
CD8+IL22+CLA–
CD4+IFNγ+CLA–
CD8+IFNγ+
CD8+IFNγ+CLA–

CD4+IFNγ+

0.5

+
0.0

+
+
-0.5

-1.0

Age ** * * *** ** * + * * **
+ + + + + +
CD4 IFNγ CLA *** ** ** *** *** * *
CD8+IFNγ+CLA+ *** *** *** *** **
CD8+IFNγ+ *** *** *** * * + + *
CD8+IFNγ+CLA– *** *** * * + + *
CD4+IFNγ+ *** + +

CD4+IFNγ+CLA– +

CD4+CLA+ *** ** * * ** * + +

CD8+CLA+ ** ** *** *** ** * * * * * * *

CD8+IL9+CLA+ *** *** *** *** * ** +

CD8+IL9+ *** *** *** ** * ** *

CD8+IL9+CLA– + * ** + * +

CD4+IL9+CLA+ *** ** + * + + + + +

CD4+IL9+ *** *
CD4+IL9+CLA–
TEWL + + + + +
IASI-S *** +
IASI *** + * ** ** * * ** ** * * *
IASI-E + * ** ** * * *** *** * ** ** ** *
+ + + + +
CD4 IL17 CLA *** *** *** *** *** *** *** * *
CD8+IL17+CLA+ *** *** *** *** *** *** ** ** **
CD4+IL17+ *** *** *** *** *** ** ** *** *** *
CD4+IL17+CLA– *** *** *** *** ** ** *** *** *
CD8+IL17+ *** *** *** * ** *** *** +

CD8+IL17+CLA– *** *** * ** *** *** +

CD8+IL13+ *** ** ** *** *** *** *** * ** *** ***

CD8+IL13+CLA– ** ** *** *** *** *** + ** *** **
CD4+IL22+ *** *** *** *** *** + * **
CD4+IL22+CLA– *** *** *** *** + * **
CD4+IL22+CLA+ *** *** *** * *
CD8+IL22+CLA+ *** *** + *
CD8+IL22+ *** + *
CD8+IL22+CLA– + *
5-D-Pruritus Scale **
CD4+IL13+ *** *** ***
CD4+IL13+CLA– * **
CD4+IL13+CLA+ ***
CD8+IL13+CLA+
Age
CD4+IFNγ+CLA+
CD8+IFNγ+CLA+
CD8+IFNγ+
CD8+IFNγ+CLA–
+

CD4+IFNγ+CLA–
CD4+CLA+
+

CD8+IL9+
CD8+IL9+CLA–
+

CD4+IL9+CLA–
TEWL
IASI-S
IASI
IASI-E
+

CD4+IL17+
CD4+IL17+CLA–
CD8+IL17+
–

CD8+IL13+
CD8+IL13+CLA–
CD4+IL22+
–

CD4+IL22+CLA+
+

5-D-Pruritus Scale

CD4+IL13+
CD4+IL13+CLA–
+

CD8+IL13+CLA+
CD8 IL17 CLA

CD4 IL22 CLA

CD8 IL22 CLA

CD4 IFNγ

CD8 CLA
CD8 IL9 CLA

CD4 IL9 CLA

CD4 IL9

CD4 IL17 CLA

CD8 IL17 CLA

CD8 IL22 CLA

CD8 IL22

CD4 IL13 CLA

+

+
+

+
+

+
+

***p<0.001 **p<0.01 *p<0.05 +p<0.1

Figure 6. Unsupervised hierarchical clustering of polarized cytokine subsets with ichthyosis clinical measures, using Pearson correlation as a similarity
metric. CLAþ/e Th17/Tc17 clustered with or adjacent to total IASI, IASI-E, IASI-S and TEWL (blue box), and close to CLAþ/e Th22/Tc22 (orange box). Th2/Th2
clustered with the 5-D Pruritus Scale (teal box). All clinical measures clustered close to each other (red ellipses). CLAþ/e Th1/Tc1 cells clustered with age
(red box). The heatmap shows the positive (red) or negative (blue) correlations of all parameters with color intensity reflecting the strength of the correlation
(e1 to þ1). Dendrograms are shown as a tree, representing the distance between variables. P-values are designated as ***<0.001, **<0.01, *<0.05, þ<0.1.
CLA, cutaneous lymphocyte antigen; IASI, ichthyosis area severity index; IASI-E, ichthyosis area severity index- erythema; IASI-S, ichthyosis area severity
index-scaling; Tc, cytotoxic T cell; TEWL, transepidermal water loss; Th, T helper.

2164 Journal of Investigative Dermatology (2018), Volume 138


Tregs are crucial in maintaining immune tolerance (Long
and Buckner, 2011), and their levels may influence activa-
tion of effector cells (McHugh and Shevach, 2002). Our data
show significantly increased systemic Treg frequencies in
ichthyosis, even higher than in AD. Conversely, CLAþ Tregs
were lower than AD and psoriasis, possibly resulting from the
complex counterequilibrium between Th17 cells and Tregs
(Diller et al., 2016; Eisenstein and Williams, 2009). The
positive correlations between activated skin-homing memory
T cells and Treg may be secondary to the possible perpetu-
ation of inflammation via Tregs, in addition to their sup-
pressive effects.
Whether expanded Th17/Th22 cytokines are primary
pathogenic factors in ichthyosis or secondary to chronic
antigenic stimulation and barrier impairment still needs
investigation. Our preliminary skin and blood data showing
Th1/Th17/IL-23 elevation in a patient with an ichthyotic
syndrome resulting from DSP mutation led to initiation of
targeted Th1/Th17/IL-23 blockade with ustekinumab in two
patients. Ustekinumab treatment reduced skin erythema,
scaling, and TEWL, supporting a possible pathogenic role for
IL-17 (Paller et al., 2017a). Furthermore, the significant Th17
skewing in our recent ichthyosis skin phenotyping in 21 pa-
tients with the four orphan forms studied herein (Paller et al.,
2017b) engendered an ongoing clinical trial of secukinumab
(anti-IL17-antibody) in patients with ichthyosis
(NCT03041038).
Ichthyoses are characterized by overt systemic inflamma-
tion, as reflected by increased T-cell activation and immune
dysregulation of multiple polar cytokines among CLAe sub-
sets. These data further highlight ichthyosis as a systemic,
rather than skin-limited disease, similar to psoriasis and AD,
both currently conceptualized as systemic disorders
(Guttman-Yassky and Krueger, 2017; Guttman-Yassky et al.,
2017). Both were linked to systemic comorbidities, including
the increased risk of cardiovascular disease (Brunner et al.,
2017; Puig, 2017; Silverberg and Greenland, 2015). In pso-
riasis, IL-17 was suggested to drive cutaneous and systemic
inflammation, as well as the comorbidities (Krueger and
Brunner, 2017). Although there is no consensus on comor-
bid associations in ichthyoses, our results suggest the need for
early monitoring for systemic extracutaneous involvement,
particularly for metabolic and cardiovascular disease. It is
possible that excessive T-cell activation and IL-17 polariza-
tion in ichthyosis promote systemic manifestations that,
similar to psoriasis (Conrad and Gilliet, 2018), might be
addressed or even prevented through targeted therapeutics.
Our study had limitations, including the fact that a small
subset of ichthyosis patients was younger than 18 years,
limiting our ability to compare absolute numbers between
age groups. However, their immune polarity was comparable
to that of adults (data not shown). Also, although our study
presents the largest dataset of ichthyoses blood profiles, there
were patient number variations across different subtypes due
to their rarity.
In sum, ichthyosis blood is accompanied by increased
systemic and skin-homing T-cell activation and multicytokine
polarization, with IL-17/IL-22 predominance, similar to the
skin compartment. Although the pathogenic contribution of
each cytokine pathway will only be determined through
T Czarnowicki et al.
Th17/Tc17/Th22/Tc22-Skewing in Ichthyosis Blood
targeted treatment investigation, positive correlations be-
tween CLAþ/CLAe IL-17/IL-22-producing cells and clinical
measures support their possible roles in ichthyosis. Further-
more, our findings suggest that “nonsyndromic/skin-limited”
ichthyoses may have systemic manifestations and support
systemic therapeutic approaches for severely affected
patients.
MATERIALS AND METHODS
Patient characteristics and samples
Blood was obtained from 47 patients with ichthyosis (30 females, 17
males; ages 1e57 years, mean 22 years; 13 ARCI-LI, 18 ARCI-CIE, 8
NS, 8 EI) (Paller et al., 2017b), 43 patients with moderate-to-severe
AD (19 females, 24 males; ages 18e81 years; mean 43 years), 24
patients with moderate-to-severe psoriasis (19 females, 24 males;
ages 18e81 years; mean 43 years), and 59 controls (30 females, 29
males; ages 6e66 years; mean 25 years). No age (P ¼ 0.32, one-way
analysis of variance), ethnicity (P ¼ 0.57, chi-square test), or sex
(P ¼ 0.18, chi-square test) disparities were observed between
ichthyosis patients and controls. Eighteen patients with ichthyosis
were younger than 18 years. All patients were classified clinically,
but genotyping was performed on 96% of patients and was consis-
tent with the clinical diagnosis. All NS resulted from SPINK5
mutations; seven of the eight EI had a KRT10 and one a KRT1
mutation; clinically patients with LI had TGM1 mutations, but one
had a NIPAL4 mutation; and the patients with CIE included three
with harlequin ichthyosis (all ABCA12 mutations), but others had
one of various genes mutated as have been described with CIE
(ABCA12, ALOXE3, ALOX12B, CerS3, NIPAL4, and PNPLA1). Of
the 47 patients, 2 (4%) took a retinoid orally (one acetretin, one
isotretinoin), 3 (6%) used topical tazarotene to localized areas, 1 had
used budesonide ointment, and 1 tacrolimus ointment for pruritus
in the previous few days. No patient was treated with an oral
immunosuppressant medication.
Although cytokine polarization was similar between children and
adults by sensitivity analysis, adults showed higher T-cell activation
than children, probably secondary to disease chronicity and ongoing
immune stimulation. Written institutional review board-approved
informed consents were obtained from subjects (12 years) and
parents (<18 years). Ichthyosis severity was calculated using the IASI
score, as previously described (Paller et al., 2017b), a nonvalidated
scale that combines the IASI-E/inflammation and IASI-S into a total
IASI score. Additional clinical measures, including TEWL on the
upper arm and 5D pruritus score, were captured as well.
Demographic data are summarized in Supplementary Tables S4 and
S5 online.
Isolation of peripheral blood mononuclear cells
Peripheral blood mononuclear cells were isolated from whole blood
by Ficoll-Paque Plus (GE Healthcare, Uppsala, Sweden), as previ-
ously described (Czarnowicki et al., 2017a) (see Supplementary
Materials and Methods online).
Stimulation of blood cell populations for cytokine responses
Ex vivo cell activation is required to detect cytokine production, as
less than 1% of nonstimulated cells produce cytokines. Whole blood
was incubated with phorbol 12-myristate 13-acetate (25 ng/ml) plus
ionomycin (2 mg/ml) in the presence of brefeldin A (10 mg/ml) for 4
hours at 37 C to induce cytokine responses. After stimulation, red
blood cells were lysed with FACS lysing solution to obtain leuko-
cytes (see Supplementary Materials and Methods).
www.jidonline.org 2165
 T Czarnowicki et al.
Th17/Tc17/Th22/Tc22-Skewing in Ichthyosis Blood
Cell surface staining and intracellular staining on peripheral
blood mononuclear cells and stimulated and nonstimulated
CD4/CD8 T cells
Peripheral blood mononuclear cells were stained with
fluorochrome-labeled antibodies to cell surface markers (CD3, CD8,
CD4, CD45RO, CCR7, ICOS, HLA-DR, and CLA). Stimulated and
nonstimulated blood cells were stained for cell surface markers
(CD3, CD4, CD69, and CLA) and permeabilized with FACS/perm to
stain for cytokines, including IL-13, IL-22, IL-9, IFN-g, and IL-17A
(see Supplementary Materials and Methods).
Immunohistochemistry
Immunohistochemistry staining for CD3 was performed on frozen
OCT-embedded cryostat sections using purified mouse anti-human
mAb (Clone SK7, BD Biosciences), as described (Esaki et al.,
2015; Suarez-Farinas et al., 2015). Immunohistochemistry staining
was performed on a subset of 27 available patient samples. Cell
counts were quantified with ImageJ V1.42 (National Institute of
Health, Bethesda, MD). See Supplementary Materials and Methods
for details.
Statistical analysis
Welch’s t-test and the Wilcoxon Mann-Whitney test were used to
compare means and medians of variables, respectively. Unsuper-
vised hierarchical clustering of variables was performed using the R
package hclust with Pearson correlation as a similarity metric with
average agglomeration algorithm and presented as a heatmap with a
dendrogram (using the R package ape). Individual scatter plots
display coefficients, 95% confidence intervals, and P-values.
P-values were designated as ***<0.001, **<0.01, *<0.05, þ<0.1.
CONFLICT OF INTEREST
JGK: Institutional research grants and funding from Novartis, Pfizer, Boeh-
ringer, Janssen, Abbvie, Amgen, Innovaderm, BMS, EMD Serono, Paraxial,
Leo Pharma, Vitae, Kinta, Regeneron, Novan, AkrosNovartis, Sienna, UCB;
consulting fees from Novartis, Pfizer, Boehringer, Amgen, Lilly, BiogenIdec,
Janssen, Abbvie,Leo Pharma, Escalier, Acros, Roche, Valiant, Allergen, UCB,
Aurigene. EGY: Institutional grants from Celgene, Dermira, Janssen, Leo,
Merck Novartis, Regeneron BMS; consulting fees from Abbie, Allergen,
Amgen Anacor, Asana,BMS, Celgene, Celsus,Curadim, Dermira, Drais,
Escalier, Galderma, Genentech, GlaxoSmithKline, Glenmark, Kymab Limited,
Kyowa Kirin, LEAD, Leo, Novartis, Pfizer, Regeneron, Sanofi, Sienna, Ther-
avance. The remaining authors state no conflict of interest.
ACKNOWLEDGMENTS
This research was supported by the Foglia Family Foundation Endowment. We
acknowledge Core resources provided by the Northwestern University Skin
Disease Research Center (NIAMS P30AR057216); genetic testing of most
patients was performed in the laboratory of Dr Keith Choate, Yale University.
We acknowledge the assistance of the Foundation for Ichthyosis and Related
Skin Types, which facilitated enrollment of many of these subjects at the 2016
FIRST meeting.
SUPPLEMENTARY MATERIAL
Supplementary material is linked to the online version of the paper at www.
jidonline.org, and at https://doi.org/10.1016/j.jid.2018.03.1523.
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