Cell Type-Specific Targeting Dissociates the Therapeutic from the Adverse

Cell type-speciﬁc targeting dissociates the therapeutic from the adverse effects of protein kinase inhibition in allergic skin disease

Patcharee Ritprajaka,b, Morisada Hayakawaa, Yasuyo Sanoa, Kinya Otsuc,d, and Jin Mo Parka,1

aCutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129; bDepartment of Microbiology and Immunology and Dental Research Unit of Oral Microbiology, Faculty of Dentistry, Chulalongkorn University, Patumwan, Bangkok 10330, Thailand; cDepartment of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan; and dCardiovascular Division, King’s College London, London SE5 9NU, United Kingdom

Edited by Melanie H. Cobb, University of Texas Southwestern Medical Center, Dallas, TX, and approved April 25, 2012 (received for review February 19, 2012) The kinase p38α, originally identified because of its endotoxin- loss on allergic skin inflammation and propose that cell-selective and cytokine-inducible activity and affinity for antiinflammatory targeting may help increase the therapeutic index of p38α compounds, has been posited as a promising therapeutic target inhibition. for various immune-mediated disorders. In clinical trials, however, p38α inhibitors produced adverse skin reactions and other toxic Results and Discussion effects that often outweighed their benefits. Such toxicity may Keratinocytes, the epithelial cells of the skin, play an active role arise from a perturbation of physiological functions unrelated to in inflammatory responses, not only serving the epidermal bar- or even protective against the disease being treated. Here, we rier function, but also producing various inflammatory mediators show that the effect of interfering with p38α signaling can be (8). Best characterized as sentinels and effectors of innate im- therapeutic or adverse depending on the targeted cell type. Using munity, myeloid cells such as macrophages and neutrophils are a panel of mutant mice devoid of p38α in distinct cell types and an central to the triggering and regulation of inflammation (9, 10). experimental model of allergic skin disease, we find that dendritic Our previous study showed that p38α protein kinase signaling cell (DC)-intrinsic p38α function is crucial for both antigen-specific was pivotal in eliciting inflammatory responses to acute tissue T-cell priming and T-cell–mediated skin inflammation, two inde- injury, yet the contribution of epithelial and myeloid p38α dif- pendent processes essential for the immunopathogenesis. By fered depending on the mode of injury (11). Meanwhile, the contrast, p38α in other cell types serves to prevent excessive in- precise roles of epithelial and myeloid cells, let alone those of flammation or maintain naïve T-cell pools in the peripheral lym- p38α in these cell types, in antigen-specific T-cell–mediated in- phoid tissues. These findings highlight a dilemma in the clinical use flammation remain ill defined. To address this knowledge gap, of p38α inhibitors, yet also suggest cell-selective targeting as a po- we sought to determine the effects of cell type-specific p38α tential solution for improving their therapeutic index. deficiency on contact hypersensitivity (CH). In this skin immune reaction, topical contact with a small-molecule allergen (hapten) allergic contact dermatitis | contact hypersensitivity | hapten leads to a priming of specific T cells (sensitization phase); the resulting effector T cells, particularly CD8+ effectors (12–15), he kinase p38α, the most abundant and ubiquitously ex- are recruited to and activated in the skin upon reencounter with Tpressed p38 MAP kinase isoform in mammals, was dis- the hapten, a process associated with actual skin disease covered based on its binding affinity for antiinflammatory (challenge phase). fi α compounds (1). The nature of the stimuli that elicited p38α ac- Keratinocyte- and myeloid cell-speci c p38 KO mice (11), tivation and enabled its identification—proinflammatory cyto- designated K-KO and M-KO, respectively, were examined for — the severity of CH reaction to the hapten 2,4-dinitroflu- kines, microbial products, and injurious environmental insults α fi also hinted at a role for p38α in the immune and stress response orobenzene (DNFB). Expression of p38 was ef ciently ablated (2–4). Pharmacological inhibition of p38α has since held promise and activation of its downstream kinases (16), such as MAP ki- for the treatment of allergic, autoimmune, and other diseases of nase-activated protein kinase 2 (MK2) and mitogen- and stress- activated kinase 1 (MSK1), was attenuated in keratinocytes and inflammatory etiology. A series of recent clinical studies, how- macrophages from K-KO and M-KO mice, respectively (Fig. 1 A ever, revealed the frequent occurrence of adverse events, ranging and B). Both mutant mice exhibited more severe DNFB-induced from skin rashes to liver damage, after the use of p38α inhibitors edema and rash compared with wild-type (WT) mice, as in- (5–7). These toxicities limited the dose and frequency of p38α dicated by greater swelling of ear skin as early as 24 h after inhibitor treatment and have become a liability to fulfilling its hapten challenge (Fig. 1 C–F). We performed adoptive T-cell promise as an effective therapeutic strategy. α fi transfer experiments to assess the role of p38 during the sen- The therapeutic index is a relative measure of the ef cacy sitization and the challenge phase. Hapten-sensitized K-KO and versus toxicity of a treatment regimen. Toxic side effects have often been the cause of the failure of an otherwise effective therapeutic agent such as p38α inhibitors. We questioned α Author contributions: P.R., M.H., and J.M.P. designed research; P.R., M.H., Y.S., and J.M.P. whether the adverse effects of p38 inhibitors arose from in- performed research; K.O. contributed new reagents/analytic tools; P.R., M.H., Y.S., and

terference with a physiological function of the protein kinase J.M.P. analyzed data; and P.R. and J.M.P. wrote the paper. MEDICAL SCIENCES and, if so, whether the therapeutic and the adverse effects of The authors declare no conflict of interest. p38α inhibition were based on distinct cell type-specific mecha- This article is a PNAS Direct Submission. nisms. In this study, we used a mouse model of allergic contact Freely available online through the PNAS open access option. α dermatitis to examine the disease responses of conditional p38 Data deposition: The data reported in this paper have been deposited in the Gene Ex- knockout (KO) mice. In these mice, ablation of p38α expression pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE35318). was targeted to keratinocytes, myeloid cells, dendritic cells, or T 1To whom correspondence should be addressed. E-mail: [email protected]. α cells, which represented a simulation of p38 inhibition in one This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cell type at a time. We observe cell type-specific effects of p38α 1073/pnas.1202984109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1202984109 PNAS | June 5, 2012 | vol. 109 | no. 23 | 9089–9094 Downloaded by guest on September 30, 2021 ABWT K-KO WT M-KO TPA (min) : 0 30 60 120 0 30 60 120 LPS (min) : 0 15 30 60 120 0 15 30 60 120 p38 p38

p-MK2 p-MK2

p-MSK1 p-MSK1

Actin Actin

WT K-KO C WT K-KO D 30 ** ** 25 ** 20 15 10 5 0 Ear swelling (0.01 mm) 0244872 Time (h)

WT M-KO E WT M-KO F 30 * * 25 20 15 10 5 0 Ear swelling (0.01 mm) 0244872 Time (h) GH * 20 20 20 20 * 15 15 15 15

10 10 10 10

5 5 5 5

0 0 0 0 Ear swelling (0.01 mm) Ear swelling (0.01 mm) Ear swelling (0.01 mm) Ear swelling (0.01 mm) D : WT K-KO D : WT WT D : WT M-KO D : WT WT R : WT WT R : WT K-KO R : WT WT R : WT M-KO

Fig. 1. Keratinocytes and myeloid cell-specific p38α ablation exacerbates skin inflammatory responses during the challenge phase of CH. (A and B) Kera- tinocytes and bone marrow-derived macrophages from the indicated mice were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA; 100 nM) and li- popolysaccharide (LPS; 100 ng/mL). Whole-cell lysates were prepared after the indicated durations of stimulation and analyzed by immunoblotting with antibodies against the proteins indicated on the left. p-, phosphorylated. (C–F) WT, K-KO, and M-KO mice were sensitized on two consecutive days by topical application of DNFB on the shaved abdomen and chest; 4 d later, the left ear was challenged with DNFB and the right ear with vehicle. Skin tissue sections from the auricles of the indicated mice were prepared 48 h after hapten treatment and analyzed by hematoxylin and eosin staining (C and E). (Scale bars: 100 μm.) Hapten-specific ear swelling was determined at the indicated time points (D and F). Data represent mean ± SE (n =5–7). (G and H) LN T cells were prepared from donor (D) mice 4 d after hapten sensitization and transferred to naïve recipient (R) mice as indicated. One day later, the left and right ears of the R mice were challenged as in C–F, and ear swelling was determined 24 h after hapten challenge. **P < 0.01; *P < 0.05.

M-KO mice could generate lymph node (LN) T-cell populations hapten-induced inflammatory responses. We generated mice in that transfer hapten sensitivity to naïve WT recipient animals. which Mapk14, the p38α gene, was specifically deleted in DCs, The severities of inflammatory reactions in the recipients that the most proficient antigen-presenting cells and a proximal reg- received T cells from WT, K-KO, and M-KO donors were ulator of antigen-induced T-cell responses. Expression of p38α in comparable (Fig. 1 G and H). However, naïve K-KO and M-KO LN DCs from these mutant mice, D-KO, was almost abolished mice developed more severe inflammation compared with WT (Fig. 2A) and, consequently, MK2 and MSK1 activation after after passive acquisition of hapten sensitivity from donor-derived treatment with CD40 ligand (CD40L), a potent DC stimulant, T cells (Fig. 1 G and H). These results show that p38α in kera- was markedly reduced (Fig. 2B). D-KO mice did not display any tinocytes and myeloid cells is dispensable for hapten-specific apparent phenotypic changes or spontaneously develop immune- T-cell priming; rather, the role of p38α in these cells appears to mediated disorders in specific pathogen-free conditions. The be antiinflammatory and confined to moderating inflammatory numbers (Fig. 2C) and relative fractions (Fig. S1) of specific responses during the challenge phase. Pharmacological in- immune cell subpopulations in the lymphoid tissues of naïve D- terference with antiinflammatory mechanisms in epithelial and KO mice were normal, indicating no major baseline abnormali- myeloid cells may account for the adverse effects of p38α ties in the immune system. In marked contrast to the CH re- inhibitors seen in clinical settings (5–7). sponse in K-KO and M-KO mice, DNFB-induced skin swelling We extended our investigation of p38α function to other cell and rash in D-KO mice were greatly diminished compared with types that participate in hapten-specific T-cell priming and WT (Fig. 2 D and E). T cells from hapten-sensitized D-KO mice

9090 | www.pnas.org/cgi/doi/10.1073/pnas.1202984109 Ritprajak et al. Downloaded by guest on September 30, 2021 A WT D-KO C #1 #2 #3 #1 #2 #3 ) 6 p38 60 40 ERK WT 20 JNK D-KO 0

Cell number (x10 LN Sp LN Sp LN Sp Actin CD3+CD4+ CD3+CD8+ B220+ ) 6 B WT D-KO 6 CD40L (min) : 0 15 30 60 0 15 30 60 4

p-MK2 2 0 p-MSK1

Cell number (x10 LN Sp LN Sp LN Sp LN Sp + + + + Actin CD11c PDCA-1 F4/80 CD49b

WT D-KO D WT D-KO E 20 ** 15 **

10 *

Ear swelling (0.01 mm) 0 0244872 Time (h) F H 20 15 WT D-KO WT D-KO 15 10 6000 600 ** ** * 10 * 5000 500 5 5 4000 400 ** ** 0 0 Ear swelling (0.01 mm) Ear swelling (0.01 mm) (pg/ml) 3000 300 D : WT D-KO D : WT WT 2000 200 IL-13 (pg/ml) R : WT WT R : WT D-KO IFN- G 1000 100 15

+ 0 0 10 * 1:100 1:30 1:10 1:100 1:30 1:10 WT + APC : CD8+ T 5 D-KO APC : CD8 T % CD69

0 CD3+CD4+ CD3+CD8+

Fig. 2. DC-specific p38α ablation diminishes CH reactions, attenuating both the sensitization and the challenge phase. (A) Whole-cell lysates from WT and D- KO LN DCs were analyzed by immunoblotting with antibodies against the proteins indicated on the left. The numbers indicate individual animals. (B)WTand D-KO LN DCs were treated with CD40 ligand (CD40L; 1 μg/ml). Whole-cell lysates were prepared after the indicated durations of stimulation and analyzed by immunoblotting with antibodies against the proteins indicated on the left. p-, phosphorylated. (C) The number of cells expressing the indicated markers in the lymph nodes (LN) and spleen (Sp) of the indicated mice was determined by flow cytometry. (D and E) WT and D-KO mice (n = 5) were sensitized and challenged with DNFB, and hapten-specific ear swelling was determined as in Fig. 1. (F) Recipient (R) mice received LN T cells from donor (D) mice and were challenged with hapten and analyzed as in Fig. 1. (G) CD69+ cells among the indicated T-cell subpopulations in the LNs of WT and D-KO mice were analyzed by flow cytometry 1 d after hapten sensitization. (H) LN CD8+ T cells were prepared from WT or D-KO mice 4 d after hapten sensitization, mixed with hapten- pulsed LN DCs from C57BL/6 mice as antigen-presenting cells (APC) at the indicated ratios. Seventy-two hours later, amounts of IFN-γ and IL-13 in the culture supernatant were determined by ELISA (n = 3). **P < 0.01; *P < 0.05.

could not transfer hapten sensitivity to naïve recipient mice as showed that D-KO mice failed to generate a population of ac- efﬁciently as WT donor T cells, nor were D-KO recipient mice tivated hapten-responsive T cells. Nevertheless, the ability of competent to develop hapten-induced skin reactions after re- p38α-KO DCs to uptake antigens and present them to T cells in ceiving T cells from sensitized WT mice (Fig. 2F). Therefore, DC general seemed intact, given the undiminished capacity of p38α-

p38α was critically required not only for hapten sensitization, but KO DCs to mediate in vitro T-cell responses to DNFB (Fig. MEDICAL SCIENCES also for hapten-induced skin inﬂammation. S3A). Also, in vitro migration of WT and p38α-KO DCs in DNFB-sensitized D-KO mice showed very weak T-cell ex- gradients of the chemokines CCL19 and CCL21 was comparable pansion in the LNs draining the sensitization site, mainly owing (Fig. S3B). Hence, ablation of p38α in DCs likely preserved most to the lack of increase in CD8+ T-cell number (Fig. S2). Fur- of the fundamental cell-autonomous properties required for thermore, LN CD8+ T cells isolated from sensitized D-KO mice antigen-presenting cell function. contained much lower fractions of activated CD69+ cells (Fig. To further substantiate the functional defects of p38α-KO 2G) and produced substantially reduced amounts of cytokines DCs and identify the associated gene expression changes, we upon hapten restimulation in vitro (Fig. 2H). These results compared gene expression in WT and p38α-KO DCs by DNA

Ritprajak et al. PNAS | June 5, 2012 | vol. 109 | no. 23 | 9091 Downloaded by guest on September 30, 2021 microarray analysis. As an in vitro model for inducing p38α ac- failure of D-KO mice to support hapten-specific CD8+ T-cell + tivity and p38α-dependent gene expression, we used Pam3CSK4, priming and CD8 T-cell-mediated skin inflammation. an agonist of Toll-like receptor 2 (TLR2), as a stimulant because Having identified the suppressive effect of DC-specific p38α it strongly induced p38α phosphorylation in DCs, and TLR2 ablation on T-cell–mediated allergic skin disease, we turned our signaling has been shown to be relevant to and crucial for CH investigation to the T cell itself and generated T-cell–specific (17, 18). TLR2-activated p38α-KO DCs underwent functional p38α-KO mice. These mice, T-KO, showed highly efficient p38α maturation, gaining the ability to present a peptide antigen to ablation in T cells from the lymphoid tissues (Fig. 4A). Naïve and induce the proliferation of CD4+ and CD8+ T cells (Fig. adult T-KO mice presented no signs of spontaneous disease, but 3A). Correspondingly, the WT and p38α-KO DCs exhibited gene examination of their lymphoid tissues revealed moderate atrophy expression profiles indicative of maturation, inducing cytokine with the size and cellularity of the thymus, LNs, and spleen de- and other immune regulatory gene expression at similar in- creased to varying extents ranging between 60% and 80% of − tensities and with similar kinetics (Fig. 3B). There was, however, those of WT organs (Fig. S6A). The CD4+CD8+, CD4+CD8 , − a small group of genes whose expression was substantially de- and CD4 CD8+ T-cell subsets developed normally in the thymus creased in p38α-KO compared with WT DCs. Among the p38α- of T-KO mice (Fig. S6B). In contrast, the fractions of CD8+ T dependent genes identified was Ccl17, which encodes the che- cells, but not CD4+ T cells, among total LN cells and splenocytes mokine CCL17, also known as thymus- and activation-regulated were markedly reduced in T-KO mice (Fig. S6C). Consequently, chemokine (TARC). The induction of CCL17 mRNA and pro- T-KO mice had significantly lower absolute numbers of T cells, + tein was much lower in Pam3CSK4- and CD40L-stimulated p38α- particularly CD8 T cells, in the peripheral lymphoid tissues KO DCs (Fig. 3 C and D), yet the production of cytokines such (Fig. 4B). as tumor necrosis factor, interleukin (IL)-6, and IL-12 (p70) was CH reactions in T-KO mice were weaker than in WT mice not impaired in the same stimulation conditions (Fig. S4). It has (Fig. 4 C and D). This result could either indicate a role for T- been demonstrated that CCL17 expression is restricted to DCs cell p38α in the immune response to hapten or simply reflect the and that CCL17 deficiency attenuates CH reactions (19). DC- decreased pool of available CD8+ T cells in T-KO mice. When derived CCL17 was found to promote the interaction of DCs equal numbers of T cells from hapten-sensitized WT and T-KO with CD8+ T cells and, hence, the activation of CD8+ T cells mice were transferred to naïve WT mice, the two recipient (20, 21) and the migration of skin-resident dendritic cells (22). groups developed hapten-induced skin reactions at similar Hapten-induced emigration of epidermal dendritic cells was strengths (Fig. 4E), suggesting that p38α-KO T cells were primed impaired in D-KO mice (Fig. S5). Of note, however, CCL17-KO in the donor and gained full capacity to induce skin disease. DCs (22), but not p38α-KO DCs (Fig. S3B), displayed cell-au- Indeed, CD8+ T cells from sensitized WT and T-KO mice tonomous defects in CCL19- and CCL21-directed migration contained comparable fractions of activated CD69+ cells (Fig. in vitro. This discrepancy is presumably due to incomplete loss of 4F) and produced similar amounts of cytokines upon hapten CCL17 expression in p38α-KO DCs. Overall, p38α-dependent restimulation (Fig. 4G). T-KO mice on the recipient side de- CCL17 expression in DCs accounts, at least in part, for the veloped skin reactions similar in severity to those in WT

WT D-KO A 100 100 B WT D-KO 89.9 2.31 89.1 3.99 75 75 Time (h): 0 2 4 0 2 4 Tnf OT-I 50 50 Jag1 Nfkbiz 25 25 Cell number Nfkbiz 0 0 Tnfsf9 100 101 102 103 104 100 101 102 103 104 Dusp14 Cbx4 100 100 83.9 13.1 83.4 13.9 Jag1 75 75 Dll4 Dll4 OT-II 50 50 Mki67 Ampd3 25 25 Cell number Slc22a19 0 0 Il23a 100 101 102 103 104 100 101 102 103 104 Gpr85 Phlda2 ﬁ CFSE Fig. 3. Transcriptional induction of speci c genes in DCs Edil3 requires p38α.(A) CFSE-labeled naïve CD8+ T cells from OT-I Il1a mice and CD4+ T cells from OT-II mice were mixed with ov- C Cd70 WT D-KO WT D-KO albumin peptide-pulsed WT and D-KO LN DCs as indicated, 100 100 Ebi3 Ccl6 cultured for 3 d, and analyzed by ﬂow cytometry. Cell pro- 75 75 Ptgs2 liferation was determined by CFSE dilution. (B) WT and D-KO 50 50 Gng4 LN DCs were treated with Pam CSK (1 μg/mL). Total RNA was Cxcl9 3 4 ( Ccl17 ) 25 ( Ccl17 ) 25 Ifng isolated at the indicated time points and subjected to DNA 0 0 Il12a microarray analysis. Relative RNA amounts of the genes in- Relative expression 01234 Relative expression 01234 Fam107b dicated on the left are presented in color-coded arbitrary Ccl17 Pam CSK (h) CD40L (h) units. Arrows on the right indicate genes showing lower 3 4 Tnnt2 expression in D-KO relative to WT. Genes with maximum Gsto1 Serpinb1b expression in WT cells at 2 h and 4 h posttreatment are WT D-KO D 1500 Il2ra arranged in upper (Tnf to Edil3) and lower (Il1a to Serpinb9b) Serpinb9b rows, respectively. (C and D) WT and D-KO LN DCs were 1000 μ Relative expression treated with Pam3CSK4 (1 g/mL) and CD40 ligand (CD40L; 1 500 Low High μg/mL). Total RNA was isolated at the indicated time points and analyzed by quantitative PCR (C). Culture supernatants CCL17 (pg/ml) 0 were collected 24 h after stimulation, and CCL17 amounts None Pam CSK CD40L 3 4 were determined by ELISA (D).

9092 | www.pnas.org/cgi/doi/10.1073/pnas.1202984109 Ritprajak et al. Downloaded by guest on September 30, 2021 Th LN Sp A Lv B CD3+ CD3+ CD3+ Sp WT WT WT WT LN WT T-KO WT T-KO ) 6 ) 20

T-KO T-KO T-KO T-KO 6 6 5 p38 * 15 4 JNK 3 10 ** ** 2 5 ERK 1 ** Cell number (x10 0 Cell number (x10 0 Actin CD3+CD4+ CD3+CD8+ CD3+CD4+ CD3+CD8+ CD WT T-KO WT T-KO 25 ** * 20 * 15 10 5 Fig. 4. T-cell-speciﬁc p38α ablation results in

Ear swelling (0.01 mm) 0 + 0 244872 smaller lymphoid tissues and decreased CD8 Time (h) T-cell pools. (A) Whole-cell lysates were pre- + E G pared from CD3 cells in the thymus (Th), LNs WT T-KO WT T-KO (LN), and spleen (Sp), and from liver (Lv) tis- 15 15 sues of WT and T-KO mice, and analyzed by 8000 2000 immunoblotting with antibodies against the 10 10 proteins indicated on the left. (B) The num- 6000 1500 bers of cells expressing the indicated markers in the LNs (Left) and spleen (Right) were 5 5 ﬂ 4000 1000 determined by ow cytometry. (C and D)WT (pg/ml) and T-KO mice (n = 5) were sensitized and 0 challenged with DNFB, and hapten-speciﬁc

Ear swelling (0.01 mm) 0 Ear swelling (0.01 mm) IL-13 (pg/ml) IFN- 2000 500 < D : WT T-KO D : WT WT ear swelling determined as in Fig. 1. **P 0.01; *P < 0.05. (E) Recipient (R) mice re- R : WT WT R : WT T-KO 0 0 ceived LN T cells from donor (D) mice and 1:100 1:30 1:10 1:3 1:100 1:30 1:10 1:3 were challenged with hapten and analyzed F + 15 APC : CD8+ T APC : CD8+ T as in Fig. 1. (F) CD69 cells among the in-

+ dicated T-cell subpopulations in the LNs of 10 WT and D-KO mice were analyzed as in Fig. 2. WT (G)LNCD8+ T cells were prepared from hap- 5 T-KO ten-sensitized WT or D-KO mice, restimulated % CD69 in vitro, and amounts of IFN-γ and IL-13 in the 0 culture supernatant were determined by ELISA + + + + CD3 CD4 CD3 CD8 (n = 3)asinFig.2.

recipients after transfer of WT donor T cells (Fig. 4E). There- background. All animal studies were conducted under Institutional Animal fore, T-KO mice appeared to retain the ability to generate Care and Use Committee-approved protocols. hapten-primed T cells, and p38α-KO T cells remained fully functional throughout the course of CH. The main reason for the Cell Isolation and Culture. Primary keratinocytes and macrophages were pre- impaired immune response in T-KO is likely that they had fewer pared and cultured as described (11). DCs were isolated from cervical, axillary, and inguinal LNs by collagenase digestion and enriched by positive selection T cells before and after hapten exposure (Fig. S7), a condition using CD11c-specific magnetic microbeads (Miltenyi Biotec). CD3+ T cells were that may be difficult to achieve by a short-term pharmacolo- isolated from the thymus, LNs, and spleen by negative selection using the Pan T gical approach. + + fi Cell Isolation Kit II (Miltenyi Biotec). CD4 and CD8 T cells were isolated from Our ndings indicate cell-selective targeting as a solution for the LNs and spleen similarly by using biotinylated anti-CD4 (GK 1.5) and anti- fi bypassing the toxicity while preserving the ef cacy of protein CD8 (53-6.70) antibodies (eBioscience). DCs were stimulated with 1 μg/mL fi kinase inhibitors. In our speci c model of allergic skin disease, Pam3CSK4 (InvivoGen) and 1 μg/mL CD40L (R&D Systems). DC-intrinsic p38α function was specifically linked to the immunopathogenesis. Cell type-specific delivery of p38 inhibitors or Allergic Skin Disease Model. CH to DNFB (Sigma-Aldrich) was induced as targeting of p38α-dependent disease mechanisms uniquely op- described (26, 27). For hapten sensitization, 0.5% DNFB in 20 μL of acetone: erating in the specific cell type will help improve the clinical olive oil (4:1) was applied to the shaved abdominal skin on day 0 and 1. For outcome, an idea widely applicable to other therapeutics with hapten challenge, 0.35% DNFB in 20 μL of acetone:olive oil (4:1) was applied MEDICAL SCIENCES a low therapeutic index. to the left auricle and 20 μL of acetone:olive oil (4:1) was applied onto the right auricle on day 5. Hapten-specific skin swelling was measured by sub- Materials and Methods tracting the increase in right ear thickness from that in left ear thickness. For fl fl fl fl + Animals. M-KO (Mapk14 / -LysMCre) and K-KO (Mapk14 / -K14Cre) mice adoptive T-cell transfer, CD3 T cells were isolated from the draining LNs of 7 were described (11). D-KO and T-KO mice were generated by crossing donor mice 4 d after DNFB sensitization. Donor T cells (3 × 10 ) were injected fl fl Mapk14 / mice (23) with CD11cCre (24) and LckCre (25) mice, respectively. i.v. to recipient mice. Recipient mice were challenge with DNFB 1 d after These two Cre mice, and OT-I and OT-II T-cell receptor transgenic mice, were T-cell transfer. Mouse ear skin samples were embedded in paraffin, and obtained from the Jackson Laboratory. All animals were on a C57BL/6J sections were stained with hematoxylin and eosin.

Ritprajak et al. PNAS | June 5, 2012 | vol. 109 | no. 23 | 9093 Downloaded by guest on September 30, 2021 T-Cell Activation in Vitro. Hapten-induced T-cell activation was performed as Protein and RNA Analysis. Whole-cell lysates were prepared and analyzed by described (27). LN DCs were incubated with 20 mM dinitrobenzene sulfonate immunoblot as described (28). Antibodies against the following proteins (Sigma-Aldrich), a water-soluble analog of DNFB, in serum-free medium for 30 were used in immunoblotting: p38α (sc-535; Santa Cruz Biotechnology), ERK min, and mixed with 3 × 105 LN CD8+ T cells isolated from mice 4 d after DNFB (9102; Cell Signaling Technology), JNK (554285; BD Pharmingen), phos- sensitization. Culture supernatants were analyzed after 72 h of coculture. For phorylated MK2 (3007; Cell Signaling Technology), phosphorylated MSK1 ovalbumin peptide-induced T-cell activation, LN DCs were treated with (04-384; Millipore), and actin (A4700; Sigma-Aldrich). The following proteins in culture supernatants were measured by ELISA: CCL17 (R&D Systems); IFN- Pam3CSK4 (1 μg/mL) for 24 h and then incubated with ovalbumin peptides, + + γ, and IL-13 (eBioscience). Total RNA was extracted by using TRIzol (Invi- OVA – and OVA – (GeneScript), for 4 h. Splenic CD8 and CD4 T cells 257 264 323 339 trogen). DNA microarray analysis was performed with GeneChip Mouse were isolated from OT-I and OT-II mice, respectively, and incubated with car- Genome 430 2.0 Array (Affymetrix) at the Partners HealthCare Cetner for boxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen). CFSE-labeled Personalized Genetic Medicine. RNA analysis by quantitative PCR was per- + × 6 + × 6 CD8 (0.5 10 ) and CD4 (1 10 ) T cells were mixed with OVA peptide-pulsed formed as described (28) using Ccl17 primers (foward, 5′-CAGGAAGTTGGT- + + DCs at the ratio of 1:3 (DC:T cell). Proliferation of CD8 and CD4 T cells was GAGCTGGT-3′; reverse, 5′-CATCCCTGGAACACTCCACT-3′). analyzed by flow cytometry after 48 h and 72 h of coculture, respectively. Statistical Analysis. Data values are expressed as mean ± SD unless indicated Flow Cytometry. Fluorescent-conjugated antibodies against markers were otherwise. P values were obtained with the unpaired, two-tailed Student used as follows: CD3 (2C11), CD4 (RM4-5), CD8 (53-6.7), CD11c (N418), CD45R t test. (RA3-6B2), CD49b (DX5), CD69 (H1.2F3), F4/80 (BM8), and PDCA-1 (eBio927; all from eBioscience). Stained cells were analyzed by flow cytometry using ACKNOWLEDGMENTS. This study was supported by National Institutes of FACSCanto (BD) and the FlowJo software (Tree Star). Health Grant AI074957 (to J.M.P.).

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