Supporting Information

Plasmodium berghei infection ameliorates atopic dermatitis-like skin lesions in NC/Nga mice C. Kishi 1, H. Amano 1, K. Suzue 2 & O. Ishikawa1 1 Department of Dermatology, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan; 2 Department of Parasitology, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan

Data S1. Materials and methods Mice and parasites All experiments that involved mice were reviewed and approved by the Committee for Ethics on Animal Experiments in the Graduate School of Gunma University (approval number 8-50). Blood-stage Pb XAT parasites are spontaneously cleared in immuno-competent mice after 2 peaks of parasitemia, whereas parasitemia in mice infected with blood-stage Pb NK65 progressively increases, and all of these mice die within 2–3 weeks. Blood-stage Pb XAT parasites were used in all experiments. These were obtained after fresh passage through a donor mouse 2–3 days after inoculation with frozen stock. The mice were then intraperitoneally infected with 1 × 105 Pb-infected erythrocytes obtained from mice freshly inoculated with a frozen stock of the parasites.

Determination of parasitemia Thin blood films were prepared and fixed with methanol before Giemsa staining. At least 1,000 erythrocytes were examined in each film every other day under light microscopy, and the percentage of parasitized erythrocytes (i.e., parasitemia) out of the total number of erythrocytes was calculated. Inter-observer error did not vary by more than 5%.

Dermatitis evaluation The clinical skin severity score of dermatitis was assessed for 4 symptoms: erythema/hemorrhage, scaring/dryness, edema, and excoriation/erosion. Each symptom was graded from 0 to 3 (none, 0; mild, 1; moderate, 2; severe, 3) and the final dermatitis

1 score was defined as the sum of each symptom score (range, 0–12). Two investigators who were blinded to the study made the score determinations. Inter-observer error did not vary by more than 3%.

Immunohistochemistry The skin biopsy specimens were cut into 2 pieces. One piece was fixed in 10% neutral buffered formalin overnight and embedded in paraffin. These pieces were then deparaffinized, cut into sections (3–5 μm thick), and stained with hematoxylin and eosin. The other piece of the original biopsy specimen was embedded in an optimal cutting temperature compound (Sakura Finetechnical Co. Ltd., Tokyo, Japan) and frozen in liquid nitrogen. Thick cryosections, 6 μm, were prepared using a CM1850 cryostat (Leica) and mounted on MAS-coated glass slides. After blocking in phosphate-buffered saline (PBS) with 5% normal goat serum and 2% bovine serum albumin (BSA), the sections were incubated with several primary antibodies at 4°C overnight. The primary antibodies used were monoclonal anti-mouse CD3 antibodies (MAB4841, R&D Systems, Minneapolis, MN, USA), purified anti-mouse NKp46 (bs-10027R, Bioss, Woburn, MA, USA), and LEAFTM purified anti-mouse CD25 (102013 BioLegend, San Diego, CA, USA). The following day, anti-rat Ig HRP detection kit (551013, BD Biosciences-Pharmingen, San Diego, CA, USA) was used to visualize the signal. The cell density was expressed as the number of positive cells per 5 high-power fields (×400) in each section. The CD3 molecule is one of the TCR complexes, and used to detect T cells. NKp46/NCR1 are termed natural cytotoxicity receptors (NCR). NCR1 has been shown to represent a novel NK cell-specific molecule involved in human NK cell activation. CD25 is the alpha chain of the IL-2 receptor and is used as a marker to identify CD4+FOXP3+ regulatory T cells in mice.

Cytokine assay To determine cytokine production in Pb XAT infected mice, the culture supernatant was harvested three days after stimulation and the cytokines were measured by ELISA using the DuoSet® ELISA Development Systems (R&D).

Real-time quantitative PCR Total RNA was extracted from the spleen and skin, respectively. The spleen and skin samples were homogenized using a Multi-Beads Shocker (Yasui-kikai, Osaka, Japan) and then cooled with liquid nitrogen. The total RNA was extracted using the Isogen (Nippon

2 Gene, Toyama, Japan) according to the manufacturer’s protocol. RNA, 1 μg, from each skin or spleen sample, respectively was subjected to reverse transcription using SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Using an ABI PRISM 7700 instrument (Applied Biosystems, Foster, CA, USA), real-time PCR was performed by TaqMan Gene Expression Assays (Applied Biosystems, Foster, CA, USA). For each duplicate sample, the gene expression was corrected against the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The primers used for PCR reactions were as follows: (1) FOXP3 (Mm00475164_m1); (2) GAPDH (Mm99999915_gl); (3) KLRB1C (Mm00824341_m1); (4) IFNG (Mm0116814_m1); (5) NFIL3 (Mm00600292_s1); (6) IL10 (Mm00439614_m1); (7) IL4 (Mm00445259_m1); and (8) IL13 (Mm00434204_m1). The relative quantitation of gene expression was performed using the comparative CT method.

Measurement of total IgE IgE levels were measured by a sandwich ELISA method using the mouse IgE ELISA Quantitation Set (Bethyl Laboratories, Inc., Montgomery, TX, USA) according to the manufacturer’s instructions. Blood was collected from the tail vein, and serum was separated by centrifugation at 500 g for 30 min. The sera were stored at −20°C until use. The sensitivity of this system was >3.9 ng/mL.

Detection of parasite-specific IgE( antibodies in sera from Pb XAT-infected mice) For detection of parasite-specific IgE antibodies, 96-well microtiter plates were coated fwith Pb XAT freeze-thaw crude lysate in pH 9.6 carbonate buffer for 2 hours at 37°C. After twice washing with 0.05% Tween 20-containing PBS, the plates were blocked with 1% bovine serum albumin. Sera were assayed and incubated for 2 hours at 37°C. The specifically binding IgE was detected with HRP-conjugated goat anti-mouse IgE (Bethyl Laboratories, Inc., Montgomery, TX, USA).

Adoptive transfer of NK cells NK cells were enriched form spleen PBMCs using EasySep, NK cells negative selection isolation kit (Stemcell Technologies, Inc, Vancouver, British Columbia, Canada). Negative selection was chosen in order to avoid altering detection of CD49b expression by flow cytometry. NK cells purity were evaluated using flow cytometry. A minimum of 10,000

3 events was acquired using a BDTM Likes flow cytomery, and analyzed with BD FACSCalibur and FlowJo software. NK cells purity was over 80%. Also, NK cells viability was assessed using trypan blue exclusion. The viability was over 98%. A total of 1.0 x 106 NK cells from Pb XAT infected mice were transferred intravenously per mouse. In the control group, PBS or adoptive transfer of the cells except NK cells were injected intravenously.

Figure S1. Time course of malaria parasitemia in SPF NC/Nga and conventional NC/Nga mice. Parasitemia of SPF NC/Nga mice (●) and conventional NC/Nga mice (○) infected with 10,000 Pb XAT-parasitized RBCs. Under both SPF and conventional conditions, the NC/Nga mice that were infected with Pb XAT developed parasitemia within 2 weeks and maintained high levels of parasitemia at 4 weeks. Most mice were spontaneously cured from parasitemia at 4–6 weeks. No difference was observed between the infection period and the parasitemia level in the both groups. The bars represent mean ± SEM for 5–7 mice in each group. A significant difference in the time course of malaria parasitemia was not observed between the SPF condition and the conventional condition mice.

Figure S2. Relationship between the clinical skin severity score and parasitemia in the conventional condition NC/Nga mice. Clinical skin severity score (■) decreased in direct proportion to the number of days after Pb XAT infection. Parasitemia (○) reached a plateau at approximately 3 weeks. The clinical skin severity score and parasitemia were inversely related.

Figure S3. (A) Serum levels of IgE Serum levels of IgE in the NC/Nga mice kept under SPF and conventional conditions with/without Pb XAT infection. IgE serum levels in the conventional condition NC/Nga mice were comparably elevated with or without infections. SPF condition mice did not have elevated levels of serum IgE. Data are mean ± SD of 5 different mice. (B) Serum levels of parasite-specific IgE

4 Serum levels of IgE in the NC/Nga mice kept under SPF and conventional conditions with Pb XAT infection. The levels of parasite-specific IgE slightly increased upon infection with Pb XAT in conventional NC/Nga mice, but not SPF NC/Nga mice. Data are mean ± SD of 5 different mice.

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