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Department of Dermatology, Cutaneous Biology Research Institute, Yonsei University College of Medicine, Seoul, South KoreaBrain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea
Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, South KoreaSeverance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, South Korea
Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, South KoreaSeverance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, South Korea
Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, South KoreaDepartment of Environmental Medical Biology, Institute of Tropical Medicine, Yonsei University College of Medicine, Seoul, South Korea
Correspondence: Chae Gyu Park, Severance Biomedical Science Institute, Yonsei University College of Medicine, 50-1, Yonsei-ro, Seodaemun-gu, Seoul 03722, Korea.
Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, South KoreaSeverance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, South Korea
Min-Geol Lee, Department of Dermatology, Severance Hospital, Cutaneous Biology Research Institute, Yonsei University College of Medicine, 50-1, Yonsei-ro, Seodaemun-gu, Seoul 03722, Korea.
Department of Dermatology, Cutaneous Biology Research Institute, Yonsei University College of Medicine, Seoul, South KoreaBrain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea
Conventional dendritic cells (cDCs) are composed of heterogeneous subsets commonly arising from dendritic cell (DC)−committed progenitors. A population of CD301b-expressing DCs has recently been identified in non-lymphoid barrier tissues such as skin. However, whether CD301b+ DCs in the skin represent an ontogenetically unique subpopulation of migratory cDCs has not been fully addressed. Here, we demonstrated that CD301b+ dermal DCs were distinct subpopulation of FMS-like tyrosine kinase 3 ligand (FLT3L)−dependent CD11b+ cDC2 lineage, which required an additional GM-CSF cue for the adequate development. Although the majority of lymphoid-resident cDC2 lacked CD301b expression, dermal migratory cDC2 contained a substantial fraction of CD301b+ subset. Similar to CD301b− population, CD301b+ dermal DC development was closely regulated by FLT3 signaling, suggesting their common origin from FLT3L-responsive cDC progenitors. However, FLT3L-driven cDC progenitor culture was not sufficient, but additional GM-CSF treatment was required to produce CD301b+ cDC2. In vivo development of CD301b+ cDC2 was significantly augmented by exogenous GM-CSF, while the repopulation of CD301b+ dermal cDC2 was abrogated by GM-CSF neutralization. Functionally, CD301b+ cDC2 was capable of producing a high level of IL-23, and the depletion of CD301b+ cDC2 effectively prevented IL-17−mediated psoriasiform dermatitis. Therefore, our findings highlight the differentiation program of a distinct CD301b+ dermal cDC2 subset in the skin and its involvement in psoriatic inflammation.
Dendritic cell (DC) lineage is composed of a large group of heterogeneous subsets arising from the defined cascades of DC-committed progenitors in bone marrow (BM) (
). Recent ontogenetic and functional studies have demonstrated two distinct conventional DC (cDC) subsets in both lymphoid and nonlymphoid tissues; these cDC subsets are characterized as IRF8-dependent CD24+CD172α−CD11b− cDC1 and IRF4-dependent CD24−CD172α+CD11b+ cDC2 (
). DCs found in nonlymphoid peripheral tissues, such as skin, lung, and intestine, are called migratory DCs, which continuously migrate to the draining lymph nodes to present peripheral antigens to T cells; there also exist lymphoid tissue–resident DC populations which sample blood-borne antigens (
). Currently, at least three different DC subsets in the skin have been well-described: (i) epidermal Langerhans cells (LCs), (ii) dermal cDC1, and (iii) dermal cDC2 (
). CD301b+ DCs were characterized by lack of expressing Langerin, EpCAM, and CD103 markers and its developmental requirement of IRF4 transcription factor, indicating that they might belong to the dermal Langerin−/cDC2 lineage of the skin (
). Nevertheless, the precise ontogeny and the immunologic role for CD301b+ DCs in the skin have not yet been fully understood.
Here we showed that CD301b+ dermal DCs are distinct subpopulation of FLT3 signaling-dependent migratory cDC2 originating from pre-cDC progenitors. Interestingly, GM-CSF is additionally required for the optimal development of CD301b+ cDC2 both in vitro and in vivo. Functional studies demonstrated that CD301b+ dermal cDC2 plays a pathogenic role by driving IL-17−mediated psoriasis-like inflammation via producing IL-23. Thus, our report reveals the unique features of GM-CSF−dependent differentiation pathway for CD301b+ cDC2 in the skin, which is co-opted in the pathogenesis of psoriasis.
Results
CD301b+ DCs are distinct FLT3 signaling-dependent cDC2 subpopulation of migratory dermal DCs
Although CD301b+ dermal DCs have been described as Langerin− subset, whether CD301b+ dermal DCs depict overall or certain population of migratory dermal cDC2 has not been examined carefully. We first characterized CD301b expression by heterogeneous DC populations from the epidermis, dermis, skin-draining lymph nodes, and spleen using multicolor flow cytometry (Supplementary Figure S1 online). Epidermal LCs and all examined CD24+ cDC1 lineage were devoid of CD301b expression (Figure 1a, 1b ). However, CD11b+ dermal migratory cDC2 lineage was clearly subdivided into two populations by CD301b expression (Figure 1b). Interestingly, in sharp contrast, resident CD11b+ cDC2 in skin-draining lymph node revealed a much lesser CD301b+ population and splenic cDC2 did rarely express CD301b marker (Figure 1b). Next, we examined whether CD301b+ dermal cDC2 was specifically depleted in Mgl2−diphtheria toxin receptor (DTR) mice by diphtheria toxin (DT) treatment. Mgl2-DTR mice have been utilized to demonstrate the role of dermal cDC2 (
). Notably, DT-treated Mgl2-DTR mice showed a significant but incomplete reduction of CD11b+CD24− dermal cDC2 population with a particular depletion of CD301b+ cDC2 (Figure 1c, 1d), which was also observed in migratory and resident cDC2 in skin-draining lymph node (Supplementary Figure S2 online). Because our data suggested that CD301b+ dermal DCs were a subset of migratory dermal cDC2, we reasoned that CD301b+ dermal DC development is regulated by FLT3L-FLT3 signaling. In vivo overexpression of FLT3L by daily subcutaneous injection of FLT3L led to a significant expansion of both CD301b+ and CD301b− dermal DC subsets in conjunction with cDC1 and double-negative cDCs (Figure 1e). Next, we analyzed dermal DC populations from wild-type and Flt3−/− mouse skin. The number of CD301b+ dermal cDC2 and other types of cDCs was commonly decreased in Flt3−/− mice (Figure 1f), suggesting that FLT3 signaling was critical for the development of overall cDCs in the dermis. Taken together, these results indicate that CD301b+ DCs are unequivocal subgroup of FLT3 signaling-dependent migratory cDC2 lineage in the dermis.
Figure 1CD301b defines a subpopulation of FLT3 signaling-responsive dermal migratory CD11b+ cDC2. (a, b) Representative flow cytometric plots of CD301b expression by EpCAM+ LCs (a) and cDCs (CD24+ cDC1 and CD11b+ cDC2) (b) isolated from the indicated organs. (c, d) Representative flow cytometric plots (c) and summarized bar graph (d) of CD301b+ cDC2 population from the dermal cell suspensions of wild-type (WT), Langerin-DTR, and Mgl2-DTR mice 24 hours after DT treatment. (e, f) The number of cDC population from the isolated dermis of FLT3L-treated WT (e) and in Flt3−/− mice (f) was analyzed by flow cytometry. Results are from at least two independent experiments with four to five mice per group. cDC, conventional dendritic cell; DC, dendritic cell; DNDC, double-negative conventional dendritic cell; DTR, diphtheria toxin receptor; L-DTR, Langerin diphtheria toxin receptor; M-DTR, Mgl2 diphtheria toxin receptor; migDC, migratory dendritic cell; PBS, phosphate buffered saline; SDLN, skin-draining lymph node; WT, wild-type. Error bars show mean ± standard error of the mean. ∗∗P < 0.01, ∗∗∗P < 0.001.
GM-CSF is required for FLT3L-driven CD301b+ cDC2 development in vitro
We next examined whether FLT3L was sufficient to generate CD301b+ cDCs from BM progenitors. FLT3L-driven BM cultures convincingly produced both CD172α− cDC1 and CD172α+ cDC2 subsets mimicking splenic cDC populations (FLT3L-cDCs) (
). However, interestingly, we found the scarce number of CD301b+ cDC2 among FLT3L-cDCs, suggesting that FLT3L alone was not sufficient to produce CD301b+ cDC2 in this system (Figure 2a). GM-CSF−supplemented BM culture has been used extensively to generate DCs in vitro (GM-DCs) (
), it is unclear whether GM-DCs suitably reflect dermal circumstances because dermal cDC development showed FLT3L dependency. We found that, in contrast to the FLT3L-cDCs, GM-DCs comprised exclusively CD172α+ subset with or without CD301b expression, suggesting that GM-DCs could not mirror the homeostatic dermal cDC1 and cDC2 population (Figure 2a). Therefore, we reasoned that FLT3L-based BM culture conditioned with additional GM-CSF might be a feasible approach to produce each dermal cDC subset. GM-CSF supplemented at day 0 led to the predominant cDC2 but highly limited cDC1 differentiation, possibly due to the suppressive effect of GM-CSF downstream STAT5 on FLT3L-activated Irf8 (Figure 2b) (
). However, when GM-CSF was introduced at the late stage of FLT3L-based culture, we found apparent subset division of CD172α− cDC1 and CD172α+ cDC2, and there was further CD301b+ cDC2 differentiation, which adequately imitated dermal cDCs (Figure 2c). In vitro−generated cDC1 revealed the highest expression level of Batf3 and Irf8, whereas Irf4 was profoundly expressed by CD301b− and CD301b+ cDC2 subsets (Figure 2d). As GM-CSF preferentially activates STAT5 for inducing DC development, we explored the potential STAT5-binding sites of Irf4 promoter. Mulan software predicted possible cross-species conserved STAT5-binding sites upstream of the Irf4 gene (Figure 2e). Taken together, these results suggest that GM-CSF is additionally required for the FLT3L-dependent CD301b+ cDC2 development.
Figure 2GM-CSF augments CD301b+ cDC2 development in vitro. (a) Representative flow cytometric plots of CD301b expression from FLT3L-derived (upper) and GM-CSF−derived (lower) bone marrow dendritic cells (BMDCs). (b) Representative flow cytometric plots of cDC1 (CD11b−CD172α−) and cDC2 (CD11b+CD172+) differentiation from GM-CSF−supplemented FLT3L-based BMDC culture at the indicated time points. (c) Representative flow cytometric plots and summarized bar graph of CD301b expression by cDCs derived from FLT3L and D6 GM-CSF−supplemented FLT3L BM culture. (d) Gene expression analysis of Batf3, Irf8, and Irf4 transcripts by the sorted cDC subsets derived from D6 GM-CSF−supplemented FLT3L BM culture. (e) Promoter analysis of murine Irf4 gene using Mulan software. Results are from three independent experiments with four mice per group. cDC, conventional dendritic cell; ns, not significant. Error bars show mean ± standard error of mean. ∗∗∗P < 0.001.
). We first examined whether pre-cDCs possess a CD301b-expressing subpopulation. Pre-cDCs in BM or spleen did not show CD301b expression, suggesting that CD301b+ cDC2 development might not originate from the CD301b+ pre-cDCs (Figure 3a). We next explored whether pre-cDCs could directly differentiate into CD301b+ cDC2 subset. We sorted CD45.2+ BM pre-cDCs and cultured them with CD45.1+ supporting BM under the FLT3L and additional GM-CSF−supplemented condition. Importantly, although FLT3L alone culture showed a limited CD301b+ cDC2 differentiation, GM-CSF supplement highly facilitated CD301b+ cDC2 development from pre-cDCs (Figure 3b). These data indicate that pre-cDCs are direct precursors for CD301b+ cDC2.
Figure 3CD301b+ cDC2 arise from pre-cDCs. (a) Representative flow cytometric plots of CD301b expression by pre-cDCs in BM and spleen. (b) The experimental scheme of the mixed pre-cDC culture (left) and representative flow cytometric plots of CD301b expression by cDCs derived from the cultured CD45.2+ pre-cDCs (right). Results are from three independent experiments with three mice. BM, bone marrow; cDC, conventional dendritic cell; MHC, major histocompatibility complex.
GM-CSF is required for CD301b+ cDC2 development in vivo
We next tested whether GM-CSF is required for CD301b+ cDC2 development in vivo. A greater level of Csf2 expression was detected in the skin compared to that of other lymphoid organs, implying that GM-CSF expressed in the skin may locally facilitate CD301b+ cDC2 differentiation (Figure 4a). To test whether exogenously administered GM-CSF could boost CD301b+ cDC2 subset differentiation, we subcutaneously implanted a B16 melanoma cell line engineered to produce GM-CSF (B16-GM-CSF) and analyzed DC subsets (
). Although the frequency of CD301b+ population among migratory dermal cDC2 was not affected, GM-CSF significantly increased CD301b+ resident cDC2 differentiation in the lymphoid organs, which further supported that CD301b+ cDC2 development might be limited in the lymphoid tissues due to low expression of GM-CSF (Figure 4b, 4c). We next examined repopulation kinetics of CD301b+ dermal cDC2 subset from DT-treated Mgl2-DTR mice under the treatment of anti−GM-CSF neutralizing antibody. CD301b+ cDC2 reappeared in the dermis at 14 days after a single DT injection in the isotype antibody-treated group, but in contrast, GM-CSF neutralization significantly abrogated their repopulation (Figure 4d, 4e). These results establish that GM-CSF actively supports CD301b+ dermal cDC2 subset development in vivo.
Figure 4In vivo CD301b+ cDC2 development shows GM-CSF dependency. (a) Gene expression analysis of Csf2 transcript by the indicated organs. (b, c) Representative flow cytometric plots (b) and summarized bar graphs (c) of CD301b expression by cDC2 subset from the different organs 12 to 14 days after subcutaneous B16 or B16−GM-CSF tumor injection. (d, e) Representative flow cytometric plots (d) and summarized line graphs (e) of repopulating CD301b+ dermal cDC2 from the dermal cell suspensions of diphtheria toxin−treated Mgl2 diphtheria toxin receptor mice at the indicated time points during isotype or GM-CSF neutralizing antibody injection. Results are from three independent experiments with four to five mice per group. cDC, conventional dendritic cell; DC, dendritic cell; migDC, migratory dendritic cell; ns, not significant; resDC, resident dendritic cell; SDLN, skin-draining lymph node. Error bars show mean ± standard error of mean. ∗∗P < 0.01.
CD301b+ dermal cDC2 are required for imiquimod-induced psoriasis-like inflammation
Psoriasis is one of chronic inflammatory skin diseases driven by IL-23/IL-17 pathway, and lesional DCs play a pivotal role for stimulating IL-17 production from T cells via IL-23 secretion (
). Langerin− dermal DCs have been shown to produce IL-23 in imiquimod (IMQ)-induced psoriasiform dermatitis model and thought to be a possible pathogenic DC subset (
). We investigated whether CD301b+ dermal DCs are specifically able to mediate psoriatic inflammation. DT-treated Mgl2-DTR mice developed significantly less severe inflammation compared with wild-type mice (Figure 5a). Gene expression analysis of the lesional skin showed that the expression level of DC-derived cytokines, including IL-23p19 and T-cell−derived IL-17A, was generally decreased in the absence of CD301b+ dermal DCs (Figure 5b). Because pathogenic IL-17A found in the IMQ-treated skin is mainly produced by skin-penetrating dermal γδ T cells and RORγt+ innate lymphocytes, we subsequently examined IL-17A production (
). The psoriatic skin of DT-treated Mgl2-DTR mice had a decrease in the frequency of skin-infiltrating γδTCRint dermal γδT cells and αβT cells (Supplementary Figure S3a online). Importantly, the frequency of IL-17A−producing dermal γδT cells and γδTCR− lymphocytes was significantly diminished upon CD301b+ dermal DC depletion both in the skin (Figure 5c) and skin-draining lymph node (Supplementary Figure S3b). For unknown reasons, DT-treated Mgl2-DTR mice showed a partial depletion of epidermal LCs (Supplementary Figure S4a online) (
IL-23 from Langerhans cells is required for the development of imiquimod-induced psoriasis-like dermatitis by induction of IL-17A-producing gammadelta T cells.
). CD301b was not expressed by CD86+-activated LCs in the steady-state and IMQ-applied inflamed skin, which excluded the possible depletion of mature LCs in Mgl2-DTR mice upon DT treatment (Supplementary Figure S4b). To selectively deplete CD301b+ dermal DCs, we generated BM chimeras reconstituted with Mgl2-DTR BM (Supplementary Figure S5 online) (
). Notably, CD301b+ dermal DC-depleted chimeras were significantly protected from IMQ-induced psoriatic inflammation (Figure 5d, 5e) and demonstrated lesser degree of epidermal thickness (Figure 5f). Furthermore, specific depletion of CD301b+ dermal cDC2 led to a significant decrease in IL-23p19 and IL-17A lesional expression (Figure 5g). These data implicate that CD301b+ cDC2 critically drive psoriatic inflammation in mice.
Figure 5CD301b+ dermal cDC2 drives psoriasiform dermatitis in mice. (a) Changes of ear thickness in IMQ-treated WT and Mgl2-DTR mice. (b) Gene expression of indicated cytokines in skin was analyzed. (c) Representative flow cytometric plots and summarized bar graphs of the frequencies of IL-17A−producing cells from the CD45+Thy1.2+ lymphocytes of whole skin cell suspensions. (d) Changes of ear thickness in IMQ-applied bone marrow chimeras. (e) Representative histologic sections of IMQ-treated ears. Scale bar = 100 μm. (f) Summarized bar graphs of epidermal thickness. (g) Gene expression of indicated cytokines in IMQ-treated skins of BM chimeras. Results are from at least two independent experiments with five to six mice per group. DTR, diphtheria toxin receptor; IMQ, imiquimod; WT, wild-type. Error bars show mean ± standard error of mean. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.
IL-23 production by CD301b+ cDC2 in response to IMQ treatment
Next, we tested the direct expression of IL-23 by CD301b+ cDC2. Although LCs and CD301b− cDC2 contained a small population for generating IL-23 in the IMQ-treated skins, CD301b+ dermal cDC2 was a principal subset of IL-23 production (Figure 6a). Finally, we cultured and sorted three cDC progenies from the combined FLT3L and GM-CSF BM culture, and treated them with soluble IMQ (
). Remarkably, CD301b+ cDC2 generated higher levels of IL-23 mRNA and protein compared to other DCs (Figure 6b, 6c). Collectively, we concluded that CD301b+ dermal DCs are crucial DC subset for driving psoriasis-like inflammation through producing IL-23.
Figure 6IL-23 production from CD301b+ cDC2 subset. (a) Representative flow cytometric plots of the frequencies of IL23-producing cells among each DC subset from the dermal cell suspensions of IMQ-treated skin. (b) Gene expression kinetics from each BMDC subset after soluble IMQ treatment. (c) The level of IL-23 and IL-12 protein produced by each BMDC subset with or without 18-hour IMQ treatment was measured by ELISA. Results are from at least two independent experiments with five mice per group. cDC, conventional dendritic cell; IMQ, imiquimod; LC, Langerhans cell. Error bars show mean ± standard error of mean.
). Compared to lymphoid-resident cDCs, migratory cDCs in nonlymphoid tissues exhibit much greater heterogeneity, which might be imprinted by local tissue microenvironments (
). In the current study, we first investigated ontogenetic properties of CD301b+ DCs in the skin. Our results showed that CD301b+ dermal DCs were specifically restricted to the FLT3L-dependent CD11b+ cDC2 subset. Moreover, we also found that CD301b+ cDC2 could arise directly from pre-cDCs, which strongly suggest that CD301b+ DCs are an authentic member of cDC lineage.
FLT3L-driven BMDC culture has been widely used to induce cDC1 and cDC2 analogues in vitro (
). Surprisingly, however, our results showed that FLT3L culture of DC progenitors did not adequately produce CD301b+ cDC2 subset. Population of CD301b+ DCs has been described in GM-CSF−driven BM culture suggesting that GM-CSF might be involved in CD301b+ cDC2 differentiation (
). In line with this, we found that additional GM-CSF highly promoted an efficient emergence of Irf4-expressing CD301b+ subset from FLT3L-induced cDC2, which convincingly reflected CD301b+ dermal cDC population. The role of GM-CSF has been revisited recently because studies showed that GM-CSF was required for the homeostatic maintenance of cDC1 and cDC2 in the nonlymphoid organs (
). In addition, the lack of GM-CSF signaling led to a prominent decrease in CD301b+ DCs in the lung, implying that GM-CSF is critical for the nonlymphoid CD301b+ DC development and/or homeostasis (
). Accordingly, a reduced repopulation of CD301b+ dermal cDC2 by GM-CSF neutralization as described in our work further highlights the idea that GM-CSF is additionally required for the CD301b+ subset development in the skin. Intriguingly, the gene expression patterns of GM-CSF−driven BMDCs most closely resembled those of migratory cDCs from dermis (
). Thus, our results suggest that there might be a conserved CD301b+ cDC2 differentiation pathway within the skin mediated by locally expressed GM-CSF (Supplementary Figure S6 online). However, we did not observe further induction of CD301b expression in cDC1 and cDC2 from the IMQ-treated inflamed skin where GM-CSF and other inflammatory molecules are commonly increased (Supplementary Figure S4c). Additional studies will be needed to elucidate how GM-CSF expression is maintained in the skin, especially focusing on the cellular crosstalk as described in the gut immune system (
IL-23 from Langerhans cells is required for the development of imiquimod-induced psoriasis-like dermatitis by induction of IL-17A-producing gammadelta T cells.
). In the chronic human psoriatic lesions, CD11c+CD1c− dermal DCs rather than LCs and CD11c+CD1c+ cDC2 are characterized as inflammatory type of DCs producing multiple pro-inflammatory factors (
). However, it remains to be determined whether CD11c+CD1c− inflammatory DCs are an ontogenetically and functionally different subset from CD11c+CD1c+ resident dermal cDC2, because the global gene expression profile of psoriatic CD1c− inflammatory DCs was most closely related to that of CD1c+ dermal cDC2, indicating that CD1c− inflammatory DCs might be derived from IRF4-dependent CD1c+ resident cDC2 under the chronic inflammatory settings (
Identification of TNF-related apoptosis-inducing ligand and other molecules that distinguish inflammatory from resident dendritic cells in patients with psoriasis.
). Indeed, the IMQ-induced psoriatic mouse model generally represents an acute phase of psoriasis, which is sharply different from the chronic nature of human psoriasis. Thus, we speculate the possibility that IRF4-dependent dermal cDC2 may be equipped for IL-23 production during the initiation phase of psoriatic inflammation and undergo changes in surface marker expression depending on the inflammatory milieu (
). In line with this, our data clearly showed that CD301b+ dermal cDC2 were critically involved in the development of psoriasis-like dermatitis by actively producing IL-23. In cutaneous Candida albicans infection, nociceptive sensory nerves stimulate CD301b+ dermal DCs to produce IL-23, which principally drives a protective IL-17 secretion from dermal γδT cells (
). Therefore, CD301b+ cDC2 subset would be a major pathogenic cellular player, which initiates IL-17−mediated psoriatic inflammation of the skin (Supplementary Figure S6). Further studies will definitely be needed to elucidate the underlying nature of psoriatic inflammatory DCs. Elucidating and modulating human analogue of CD301b+ cDC2 would be a potential therapeutic approach for psoriasis and other types of IL-17−mediated diseases, which commonly affect the barrier areas in our body (
WT C57BL/6 (CD45.2) and B6.SJL (CD45.1) mice were purchased from Orient Bio (Seongnam, Gyeonggi-do, Korea) and The Jackson Laboratory (Bar Harbor, ME), respectively. Langerin-DTR mice (
) were a gift from Heung Kyu Lee at Korea Advanced Institute of Science and Technology. Mgl2-DTR mice were kindly provided by Akiko Iwasaki at Yale University (
). Age- and sex-matched 2- to 5-month-old mice were used in this study. Mice were bred and housed under a specific pathogen-free condition in the animal facilities of Yonsei University College of Medicine and Hanyang University approved by the Institutional Animal Care and Use Committees.
IMQ-induced psoriasis-like dermatitis model
Mice were treated with daily 20 mg of 5% IMQ cream (Aldara; 3M Pharmaceuticals, Leicestershire, UK) on ears for 5−6 consecutive days. Ear thickness was measured using a dial thickness gauge (PEACOCK, Ozaki MFG Co. Ltd., Tokyo, Japan). Twenty-four hours after the last treatment, mice were euthanized and tissues were harvested for further analyses. Epidermal thickness was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD).
DT-induced cell depletion and repopulation assay
Mice were treated intraperitoneally with 1 μg DT (Sigma, St. Louis, MO) dissolved in phosphate buffered saline at day −3 and −1 before starting experiments. For GM-CSF blockade, 300 μg anti−GM-CSF neutralizing antibody (clone MP1-22E9; BioXCell, West Lebanon, NH) was administered intraperitoneally three times per week starting from 1 week before DT treatment and throughout the repopulation period. An appropriate isotype antibody was used as control (clone 2A3; BioXCell).
Cytokine-induced DC expansion in vivo
Mice were subcutaneously injected with either daily 10 μg mouse FLT3L or phosphate buffered saline nine times and tissues were analyzed 24 hours after the last injection, as described in our previous studies (
). B16 mouse melanoma cells and B16-GM-CSF cells (kindly provided by Sang Jun Ha at Yonsei University) were cultured in complete DMEM containing 10% fetal bovine serum (Gibco, Carlsbad, CA) (
). At the day of tumor transplantation, cells were washed with phosphate buffered saline and mice were intradermally injected with 2−3 × 106 tumor cells. The DC subsets were analyzed 12−14 days later.
BM chimera
BM chimeras were generated as described previously (
Isolation and flow cytometric characterization of DC subsets was performed according to our previous work and other studies as indicated in the Supplementary Materials (
) were produced in-house. For inducing FLT3L-DCs, 2 × 106 BM cells were cultured in complete RPMI medium (Gibco) containing 10% FLT3L-conditioned supernatant and half of culture medium was replenished at 3-day intervals. In some experiments, 1% GM-CSF−conditioned supernatant was added to the culture at the indicated time points. At day 10, BMDCs were underwent flow cytometric analyses or sorted using FACSAria sorter (BD Biosciences, San Jose, CA). Each 5 × 105 sorted DCs were stimulated with 1 μg/ml imiquimod (R837; Invivogen, San Diego, CA) for the indicated times.
Pre-cDC sorting and mixed culture
Pre-cDCs from CD45.2+ BM were determined and sorted as described in our previous study (
). Total 2 × 106 CD45.1+ supporting BM cells and 1 × 104 sorted CD45.2+ pre-cDCs were gently mixed and cultured in the complete RPMI medium containing FLT3L-conditioned supernatant with or without additional GM-CSF supplement. CD45.2+ progeny were subsequently examined 3 to 4 days later by flow cytometry (
To measure IL-23 and IL-12p70 from the cultured supernatants of BMDCs, the appropriate ELISA kits for mouse IL-23 (R&D Systems, Minneapolis, MN) and IL-12p70 (BioLegend, San Diego, CA) were used according to the manufacturer’s recommendations.
Real-time quantitative polymerase chain reaction
Total RNA isolation, cDNA synthesis, and quantitative real-time PCR were performed as described in our previous study (
). Results were normalized to Hprt mRNA. Primer sequences are listed in Supplementary Table S1 (online).
Statistics
Data were analyzed with unpaired Student two-tailed t tests, unless otherwise stated, using Prism 5 software (GraphPad Software Inc., San Diego, CA). Analysis of variance with Bonferroni correction was used for multiple comparisons. All P values <0.05 were considered statistically significant.
This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute, funded by the Ministry of Health and Welfare, Korea (HI17C1659 to MGL) and by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2017R1D1A1B03035571 to TGK; NRF-2014R1A4A1008625 to CGP).
IL-23 from Langerhans cells is required for the development of imiquimod-induced psoriasis-like dermatitis by induction of IL-17A-producing gammadelta T cells.
Identification of TNF-related apoptosis-inducing ligand and other molecules that distinguish inflammatory from resident dendritic cells in patients with psoriasis.
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