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Accelerated T-cell activation and differentiation of polar subsets characterizes early atopic dermatitis development

2016; Elsevier BV; Volume: 138; Issue: 5 Linguagem: Inglês

10.1016/j.jaci.2016.04.052

1097-6825

Hitokazu Esaki, Tali Czarnowicki, Juana Gonzalez, Margeaux Oliva, Sreya Talasila, Isabel Haugh, Giselle Rodriguez, Lauren Becker, James G. Krueger, Emma Guttman‐Yassky, Amy S. Paller,

Allergic Rhinitis and Sensitization

Infancy is a crucial period for immune development. Cellular immune responses are immature at birth, with decreased natural killer cell activity and regulatory responses.1Gasparoni A. Ciardelli L. Avanzini A. Castellazzi A.M. Carini R. Rondini G. et al.Age-related changes in intracellular TH1/TH2 cytokine production, immunoproliferative T lymphocyte response and natural killer cell activity in newborns, children and adults.Biol Neonate. 2003; 84: 297-303Crossref PubMed Scopus (121) Google Scholar Little is known about T-cell activation and subset differentiation in patients with atopic dermatitis (AD) during disease initiation in the first years of life. To study early AD development, blood was obtained from 29 infants and toddlers aged 0 to 3 years (mean, 14.5 months; SCORAD score: range, 21-84; mean, 54) with moderate-to-severe AD, 13 children aged 3 to 6 years with infancy-onset and persistent disease (mean, 57.1 months; SCORAD score: range, 36-73; mean, 53), and their respective age-matched control subjects (demographic data are shown in Table E1 in this article's Online Repository at www.jacionline.org). T-cell surface markers and intracellular flow cytometric panels were generated (see the Methods section in this article's Online Repository at www.jacionline.org) to measure polar differentiation and activated CD4+/CD8+ T-cell frequencies in central memory T (TCM/CCR7+CD45RO+) cell and effector memory T (TEM/CCR7−CD45RO+) cell subsets in skin-homing/cutaneous lymphocyte antigen (CLA)+ and CLA− subsets. Circulating CLA+ T cells constitute blood biomarkers for skin inflammation.2Ferran M. Romeu E.R. Rincon C. Sagrista M. Gimenez Arnau A.M. Celada A. et al.Circulating CLA+ T lymphocytes as peripheral cell biomarkers in T-cell-mediated skin diseases.Exp Dermatol. 2013; 22: 439-442Crossref PubMed Scopus (28) Google Scholar TCM and TEM cell populations were further characterized by mid (inducible costimulator [ICOS]) and late (HLA-DR) activation markers. Data were analyzed with the Student t test and Pearson correlation coefficient to correlate variables. P values of less than .05 were considered significant. At birth, most T cells are naive. Antigen exposure induces T cells to differentiate into TEM, TCM, and terminally differentiated effector memory (TEMRA/CCR7−CD45RO−) cells.3Cossarizza A. Ortolani C. Paganelli R. Barbieri D. Monti D. Sansoni P. et al.CD45 isoforms expression on CD4+ and CD8+ T cells throughout life, from newborns to centenarians: implications for T cell memory.Mech Ageing Dev. 1996; 86: 173-195Crossref PubMed Scopus (213) Google Scholar In both control subjects and patients with AD, CD4+ naive cell counts progressively decreased (control subjects: r = −0.62, P = .002; patients with AD: r = −0.56, P < .001; Fig 1, A and B) parallel to increases in TCM/TEM cell subsets (P < .05; Fig 1, C-F). Uniquely in patients with AD, numbers of TEM (but not TCM) cells, which have rapid effector function, were higher in 3- to 6-year-old patients with AD versus those in 0- to 3-year-old patients with AD (14.1% vs 4.9%, P = .02; Fig 1, G and H). More CLA+ TCM cells were present in 3- to 6-year-old patients with AD (but not 0- to 3-year-old patients with AD) versus control subjects (28.0% vs 13.9%, P = .004; Fig 1, I). In contrast, the CLA+/TEM cell subset was higher in both the 0- to 3-year-old (40.7% vs 28.5%, P = .02; Fig 1, J) and 3- to 6-year-old (42.9% vs 25.1%, P = .003; Fig 1, J) groups. Opposite trends were observed in the CLA− populations (Fig 1, K and L). Differences were not observed between the 2 AD cohorts (0-3/3-6 year olds; P > .16; Fig 1, I-L). Numbers of TEMRA cells, the most differentiated effector memory subset, positively correlated with age only in control subjects (r = 0.5, P = .01; Fig 1, M and N), likely based on their earlier differentiation in patients with AD (0-3 years old: 5.5% vs 3.6%, P = .02; Fig 1, O). These data demonstrate increased effector T-cell differentiation at AD initiation exclusively in the skin-homing compartment. Past studies showed fewer CD8+ than CD4+ T cells in infants.1Gasparoni A. Ciardelli L. Avanzini A. Castellazzi A.M. Carini R. Rondini G. et al.Age-related changes in intracellular TH1/TH2 cytokine production, immunoproliferative T lymphocyte response and natural killer cell activity in newborns, children and adults.Biol Neonate. 2003; 84: 297-303Crossref PubMed Scopus (121) Google Scholar Consistently, neither patients with AD nor control subjects showed significant increases in CD8+ TCM/TEM cell counts (P > .14; see Fig E1 in this article's Online Repository at www.jacionline.org), supporting our recent demonstration that the TH1/TH2 imbalance in early AD is CD4+ selective, with CD8+ T cells playing a lesser role in initial AD development.4Czarnowicki T. Esaki H. Gonzalez J. Malajian D. Shemer A. Noda S. et al.Early pediatric atopic dermatitis shows only a cutaneous lymphocyte antigen (CLA) 1 T(H)2/T(H)1 cell imbalance, whereas adults acquire CLA1(+) T(H)22/T(C)22 cell subsets.J Allergy Clin Immunol. 2015; 136: 941-951Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar We recently reported that TCM/TEM ICOS activation, which also stimulates TH2 expansion and IgE switching, is greater in children with AD.4Czarnowicki T. Esaki H. Gonzalez J. Malajian D. Shemer A. Noda S. et al.Early pediatric atopic dermatitis shows only a cutaneous lymphocyte antigen (CLA) 1 T(H)2/T(H)1 cell imbalance, whereas adults acquire CLA1(+) T(H)22/T(C)22 cell subsets.J Allergy Clin Immunol. 2015; 136: 941-951Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar Indeed, ICOS activation was greater in younger (0- to 3 years old) children with AD, particularly in CLA− cells (TCM cells: 7.3% vs 4.1%, P = .02; TEM cells: 18.6% vs 9.6%, P = .02; see Fig E2, A and B, in this article's Online Repository at www.jacionline.org), with CLA+ differences more obvious in the 3- to 6-year-old group (see Fig E2, C and D). Chronological decreases of memory subset activation were observed in both groups but reached significance only in control subjects (P < .05; see Fig E2, E-L). Ongoing TCM ICOS activation in patients with early AD indicates maintenance of a large lymph node T-cell reservoir, whereas early high numbers of TEM cells are probably crucial for disease initiation. No differences in HLA-DR chronic activation were seen between control subjects and patients with AD (P > .2, data not shown). However, the age-related increases exclusively in patients with AD (TCM cells: CLA+ r = 0.4, P = .02 and CLA− r = 0.47, P = .004; see Fig E2, M-P) suggest a later contribution of HLA-DR to AD. Skin residence could explain why chronological HLA-DR increases in blood were not significant for TEM cells (P > .13, data not shown). Because activation of memory cells precedes T-cell differentiation, we next studied polar subsets. The smaller skin-homing TH1 subset in patients with AD versus control subjects5Czarnowicki T. Gonzalez J. Shemer A. Malajian D. Xu H. Zheng X.Z. et al.Severe atopic dermatitis is characterized by selective expansion of circulating T(H)2/T(C)2 and T(H)22/T(C)22, but not T(H)17/T(C)17, cells within the skin-homing T-cell population.J Allergy Clin Immunol. 2015; 136: 104-115Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar was particularly prominent in patients with AD aged 0 to 3 years (6.8% vs 11.6%, P = .009; Fig 2, A). As previously reported,6Kawamoto N. Kaneko H. Takemura M. Seishima M. Sakurai S. Fukao T. et al.Age-related changes in intracellular cytokine profiles and Th2 dominance in allergic children.Pediatr Allergy Immunol. 2006; 17: 125-133Crossref PubMed Scopus (35) Google Scholar significant increases in IFN-γ+CLA+ cells were observed in older versus younger control subjects (20.1% vs 11.6%, P = .04; Fig 2, A) but not in patients with AD (P = 0.08; Fig 2, A). These changes were not seen in the CLA− subset (Fig 2, B), emphasizing the role of inappropriate TH1 developmental delay exclusively among the skin-homing compartment in early AD. Conversely, IL-13+ levels were significantly higher in the younger AD group in both CLA+ (10.0% vs 1.9%, P < .001; Fig 2, C) and CLA− (1.1% vs 0.4%, P = .008; Fig 2, D) subsets, without further increases in patients with AD aged 3 to 6 years (P > .29; Fig 2, C and D), supporting the frequent concurrence of noncutaneous atopic manifestations beginning early in AD. These results underscore the role of primary CLA+ TH1/TH2 cytokine dysregulation in AD initiation, which is further demonstrated by a low TH1/TH2 ratio in patients with AD aged 0 to 3 years versus control subjects (CLA+: 1.7% vs 8%, P = .001 and CLA−: 7.5% vs 18.5%, P = .003; Fig 2, E and F). Children with AD demonstrated "normalization" of the TH1/TH2 ratio with age only in the CLA− subset (r = 0.58, P < .001; Fig 2, G and H), potentially correlating temporally with resolution of associated atopic disorders (eg, food allergies).7Sicherer S.H. Sampson H.A. Food allergy: epidemiology, pathogenesis, diagnosis, and treatment.J Allergy Clin Immunol. 2014; 133: 291-308Abstract Full Text Full Text PDF PubMed Scopus (969) Google Scholar Early TH2 activation might be a driver of the subsequent atopic march because the 0- to 3-year-old AD cohort had low rates of associated atopic conditions unlike the 3- to 6-year-old group, in which most patients had atopic associations (38% vs 92%, P = .002; see Table E1). This discrepancy in atopic disorder occurrence supports the notion that early cutaneous sensitization predisposes to other atopic diseases. IL-9 was linked to peanut allergy in older children.8Brough H.A. Cousins D.J. Munteanu A. Wong Y.F. Sudra A. Makinson K. et al.IL-9 is a key component of memory TH cell peanut-specific responses from children with peanut allergy.J Allergy Clin Immunol. 2014; 134: 1329-1338Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar However, CLA+/CLA− IL-9 levels in our young children with AD, 10 of whom had IgE sensitization to peanuts (vs 1 control subject), were similar to those seen in control subjects, without a difference between age groups (P > .16; Fig 2, I and J, and see Fig E3, A-D, in this article's Online Repository at www.jacionline.org). The possibility that TH9 cells reside in the skin of patients with early AD but later migrate to blood to mediate systemic allergic responses deserves further study. IL-22 levels positively correlated with age only in the CLA− compartment of children with AD (r = 0.34, P = .04; Fig 2, K and L, and see Fig E3, E and F), whereas IL-17+ exclusively correlated with age in control subjects (CLA+: r = 0.5, P = .03; CLA−: r = 0.52, P = .02; Fig 2, M and N, and see Fig E3, G and H). TH22 is involved in host defenses, and TH17 is involved in antimicrobial production and neutrophil chemotaxis.9Korn T. Bettelli E. Oukka M. Kuchroo V.K. IL-17 and Th17 Cells.Annu Rev Immunol. 2009; 27: 485-517Crossref PubMed Scopus (3803) Google Scholar Failure to increase CLA+ TH17 and TH22 subsets in early AD might contribute to cutaneous immune compromise. Absence of increased IL-22+ and IL-17+ blood levels result from high TH2 signals or, as hypothesized for TH9, reflect migration of these subsets into lesional skin. Finally, early TH17, TH9, and TH22 cell frequencies were much higher in CLA+ than CLA− subsets in both patients with AD and control subjects (Fig 2, I and J, and see Fig E3, I-L), suggesting that cutaneous antigenic exposure drives early subset development. In conclusion, pediatric AD development is characterized by early excessive T-cell activation and imbalanced subset differentiation. Effector memory subsets and polarized T-cell frequencies are most distinct from control subjects in the very young AD cohort (0-3 years old), suggesting that future studies elucidating immune subset fluctuations and evaluating AD developmental mechanisms should focus on this population. PBMCs were obtained by means of gradient centrifugation with Ficoll-Paque Plus (Amersham Biosciences, Pittsburgh, Pa). Blood was placed under the Ficoll gradient. After spinning, PBMCs were collected at the interface between the plasma and the Ficoll gradient and washed with PBS before staining for flow cytometric analysis. For T-cell surface staining, PBMCs were washed and incubated for 30 minutes on ice with fluorochrome-conjugated mAbs to cell-surface molecules (CD3-phycoerythrin [PE] Alex Fluor 610, CD8-V-500, CD4-Qdot800, CD45RO-PECy7, CLA–fluorescein isothiocyanate, ICOS-PerCpCy5.5, CCR7 Alexa Fluor 700, HLA-DR–allophycocyanin-H7, and Live/dead-Blue Dye). Then the cells were fixed in ice with 4% paraformaldehyde (BD Biosciences, San Jose, Calif) for 20 minutes. For intracellular cytokine staining, whole blood was activated with 25 ng/mL phorbol 12-myristate 13-acetate and 2 μg/mL ionomycin in the presence of 10 μg/mL brefeldin A (protein transporter inhibitor) for 4 hours at 37°C. All reagents were purchased from Sigma-Aldrich (St Louis, Mo). Nonactivated control subjects were only treated with brefeldin A. After this incubation, EDTA (2 mmol/L; Invitrogen, Grand Island, NY) was added for 15 minutes at room temperature (RT) to stop activation. The blood was then incubated with FACS lysing solution for 10 minutes at RT and washed with PBS twice. Then the cells were incubated for 20 minutes with fluorochrome-conjugated mAbs to cell-surface molecules (CD3-PE Alex Fluor 610, CD4-Qdot800, and CLA–fluorescein isothiocyanate). Subsequently, the cells were permeabilized (Perm/Wash, BD Biosciences) and incubated for 20 minutes with fluorochrome-conjugated mAbs (IL-13–PerCpCy5.5, IL-22–PECy7, IL-9–PE, and IFN-γ–Alexa Fluor 700) in 5% mouse serum (eBioscience, San Diego, Calif). All incubations for cell-surface and intracellular staining were done at RT. Appropriate FMO-isotype controls were used. After incubation, cells were washed and suspended in PBS. This was followed by acquisition with a BD LSR II flow cytometer (BD Biosciences) and analysis with FlowJo software (TreeStar, Ashland, Ore).Fig E2CLA+/CLA− ICOS-activated TCM/TEM cell frequencies and correlations versus age in patients with AD and control subjects. A-D, The youngest (0-3 years) AD group had higher TCM (Fig E2, A) and TEM (Fig E2, B) ICOS+CLA− activation compared with control subjects, whereas CLA+ differences were more prominent between the 3- to 6-year-old groups (Fig E2, C and D). E-P, ICOS activation decreased over time exclusively in control subjects (Fig E2, E-L), whereas HLA-DR chronic activation increased selectively in patients with AD (Fig E2, M-P; CI values appear in parentheses). Bar plots represent means ± SEMs.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3CLA+/CLA− TH9, TH22, and TH17 cell subset frequencies and correlations versus age in patients with AD and control subjects. A-D, Nonsignificant correlations were found between TH9 cell frequencies and age in either control subjects or patients with AD (CI values appear in parentheses). E-H, Nonsignificant correlations were found between TH22 (Fig E3, E and F) and TH17 (Fig E3, G and H) cell counts with age in control subjects and pediatric patients with AD, respectively. I-L, CLA+/CLA− TH22/TH17 cell frequencies among the different groups. Bar plots represent means ± SEMs.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Epidemiologic, laboratory, and clinical data for children with AD and control subjectsPatients with AD aged 0-3 yControl subjects aged 0-3 yP valuePatients with AD aged 3-6 yControl subjects aged 3-6 yP valueAge (mo)14.5 ± 1.8 (4-35)16.8 ± 1.9 (6-33).557.1 ± 3.1 (37-70)53.4 ± 3.3 (36-67).4Sex (n).3.7 Female11863 Male18676Ethnicity (n).1.9 Hispanic3011 Asian5041 African American5132 White161355SCORAD score54.1 ± 2.6 (21-84)NA—53.0 ± 3.6 (36-73)NA—Duration of AD (mo)11.3 ± 1.5 (3-29)NA—48.9 ± 3.5 (18-62)NA—IgE∗Reference range: 0 to 100 kU/L. (kU/L)984.4 ± 509.1 (5.3-5,000)16.8 ± 4.4 (1-43.3).01825.4 ± 382.1 (95-2,248)83.7 ± 31.8 (3.2-159).05Other atopy110<.0001120<.0001Data are presented as means ± SEMs unless otherwise specified.NA, Not applicable.∗ Reference range: 0 to 100 kU/L. Open table in a new tab Data are presented as means ± SEMs unless otherwise specified. NA, Not applicable.