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.

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