Congenital Deficiency of Conventional Dendritic Cells Promotes the Development of Atopic Dermatitis-Like Inflammation

Atopic dermatitis (AD) is a common pruritic inflammatory skin disease characterized by impaired epidermal barrier function and dysregulation of Thelper-2 (TH2)-biased immune responses. While the lineage of conventional dendritic cells (cDCs) are implicated to play decisive roles in T-cell immune responses, their requirement for the development of AD remains elusive. Here, we describe the impact of the constitutive loss of cDCs on the progression of AD-like inflammation by using binary transgenic (Tg) mice that constitutively lacked CD11chi cDCs. Unexpectedly, the congenital deficiency of cDCs not only exacerbates the pathogenesis of AD-like inflammation but also elicits immune abnormalities with the increased composition and function of granulocytes and group 2 innate lymphoid cells (ILC2) as well as B cells possibly mediated through the breakdown of the Fms-related tyrosine kinase 3 ligand (Flt3L)-mediated homeostatic feedback loop. Furthermore, the constitutive loss of cDCs accelerates skin colonization of Staphylococcus aureus (S. aureus), that associated with disease flare. Thus, cDCs maintains immune homeostasis to prevent the occurrence of immune abnormalities to maintain the functional skin barrier for mitigating AD flare.


Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR)
Total RNA from cells was extracted by using ISOGEN II (Nippon Gene) and the first-strand complementary DNA (cDNA) was synthesized from 100 ng of total RNA with oligo(dT)20 primer using the PrimeScript RT Master Mix (Takara) according to the manufacturer's instructions. Transcriptional expression levels were analyzed as described previously (9, 36) by using SYBR ® Premix Ex Taq II on Thermal Cycler Dice (Takara) with specific primer pairs listed in TABLE S1 after normalization for bactin expression.

AD-Like Inflammation
To induce AD-like skin lesions (13,15,(38)(39)(40), mice received topical application of 2 nmol of MC903 (calcipotriol, Sigma-Aldrich) in 20 µl of ethanol (vehicle) on the left ear skin for five consecutive days a week during 2 weeks followed by two consecutive days a week during last week for 16 days. As vehicle control, the same amount of ethanol was applied. The development of ADlike inflammation was determined by ear thickness at every day after topical application with MC903 for 16 days using digital calipers (PK-1012CPX; Mitsutoyo) (9, 37). In some experiments, Spl, EDLNs, and ear were obtained from the mice before or after the first topical application of MC903 at 16 days. For the improvement of the compromised skin barrier function, petrolatum (Wako Pure Chemicals) was topically applied twice daily at the days of MC903 application to the ventral and dorsal portions of the ear for the duration of the treatment with MC903. For antibiotic treatment, mice were treated topically with 1% Acuatim ointment (Nadifloxacin, Otsuka) on the ventral and dorsal portions of the ear twice daily at the days of MC903 application for the duration of the treatment with MC903.

Histopathologic Assessment
Tissues from the ear were fixed with 4% paraformaldehyde (PFA) in PBS and embedded in paraffin. The tissue sections (5 µm thickness) were stained with H&E and toluidine blue to detect MCs. The stained slides were examined with a bright-field microscopy (BX53; Olympus). The areas of the epidermis and dermis of ear were quantified by thickness using ImageJ (National Institutes of Health) by a blinded observer as described previously (9, 37).

Immunohistochemical Analysis
Spl and ear skin were embedded in OCT compound (Sakura Fineteck) and frozen in liquid N2.

Bacterial Culture
To measure CFU of S. aureus (16), the homogenates of ear skin were placed into 1 ml of PBS, and serial dilutions were prepared. 100 µl of each dilution was transferred on Mannitol salt agar with egg yolk plates (BD), and colonies were counted after 2-day incubation. CFUs were calculated by dividing the number of colonies per plate by the dilution factor.  (E) Frozen sections obtained from epidermis obtained from WT mice and ΔCD11c hi cDC mice (n = 3 per group) were stained for CD207 (green) and DAPI (blue). Bars indicate 50 µm. Data are obtained from two to four individual samples in a single experiment. *P < .05 compared with WT mice. All data are representative of at least 3 independent experiments. FIGURE S3. Inflammatory status of ΔCD11c hi cDC mice in the homeostatic conditions. (A) Serum production of cytokines in WT mice and ΔCD11c hi cDC mice (n =4 per group). n.d.; not determined. (B) Transcriptional expression of inflammation-and epithelium-related molecules in ear skin obtained from WT mice and ΔCD11c hi cDC mice (n = 6 per group). Data are obtained from three to six individual samples in a single experiment. *P < .05, **P < .01 compared with WT mice. All data are representative of at least 3 independent experiments.   EDLNs (C,E) obtained from WT mice and ΔCD11c hi cDC mice (n = 8 per group). (F) The frequency of the subsets of IgM + plasma cells, IgG 1 + plasma cells, and IgE + plasma cells of BM obtained from WT mice and ΔCD11c hi cDC mice (n = 8 per group). Data are obtained from three to eight individual samples in a single experiment. *P < .05, **P < .01 compared with WT mice. All data are representative of at least 3 independent experiments.