Fig 2: Spic Regulates Ferroportin-Mediated Iron Export in Macrophages(A) Spic+/− and Spic−/− mice (three mice per group) were treated with LPS (30 μg/mouse). Patrolling monocytes (CD45+CD11B+CD115+LY6C-) were purified 72 h after treatment and subjected to microarray-based (affymetrix, mouse gene 2.0_ST) gene expression profiling. Shown are the GSEA plots for the hallmark of heme metabolism (left) and corresponding heatmap based on differentially expressed genes (nominal p < 0.01) between the two genotypes (right).(B) Expression (y axis, linear scale) of Fpn from the microarray data.(C) WT and Spic−/− BMDMs (two mice per group) were treated with LPS (100 ng/ml), RNA was extracted 24 h later, and microarray-based (affymetrix, mouse gene 2.0_ST) gene expression profiling was performed. Shown are the GSEA plots for the hallmark of heme metabolism (left) and the corresponding heatmap based on differentially expressed genes (nominal p = 0.457) between the two genotypes (right).(D) Mo-MACs from WT and Spic−/− mice were treated with LPS (1 μg/ml), RNA was extracted 16 h ater, and the expression (normalized to 18S rRNA)ofFpn was measured (y axis relative to a non-treated group) by qRT-PCR.(E) WT or Spic−/− mice were treated (i.p.) with LPS (150 μg/mouse) twice 48 h apart. Lungs were harvested 48 h after the final LPS treatment, and levels of FPN protein measured by flow cytometry (left). The bar graph (right) shows quantification of mean fluorescent intensity (MFI) of FPN staining.(F) PreRPM (CD11BhiF4/80lo) from WT and Spic−/− spleen were purified by FACS, RNA was extracted, and the levels (normalized to 18S rRNA)ofSpic and Fpn were measured (y axis, relative a WT PreRPM) by qRT-PCR.(G) BMDMs from WT mice were treated with LPS (100 ng/ml), RNA was extracted at indicated time points (x axis), and the levels (normalized to 18S rRNA) of Fpn were measured (y axis, relative to non-treated group) by qRT-PCR.(H) BMDMs of indicated genotypes were cultured for 7 days, after which the medium was removed, and fresh medium containing 100 mM of FeSO4 was added, followed by treatment with LPS (1 mg/mL) or PBS (control). Intracellular iron was measured 24 h later. Graph is representative of five independent experiments.The replicates for each individual experiment are technical replicates for the assay.qRT-PCR, data representative of ≥3 independent experiments. Plots show a single experiment with n ≥2 per group. Results are expressed as mean ± SEM. p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), and p ≤ 0.0001 (****). See also Figures S2 and S3.

Fig 3: Monocytes in the skin support CHIKV infection(A–D) Cohorts of 4-week-old male C57BL/6J mice were inoculated with 103 FFU of CHIKV-LR and sacrificed at 0, 8, or 18 hpi. Ipsilateral foot skin was harvested and processed. Cell suspensions were incubated with antibodies against CHIKV E1 (Ag), CD45, B220, CD3, NK1.1, Ly6G, Ly6C, CD115, and CD11b, and flow cytometry was performed to assess the percentages (A and C) and numbers (B and D) of Ly6Chi (monocytes, top panels) and Ly6G+ (neutrophils, bottom panels) of cells expressing CHIKV antigen (n = 6, two experiments).(E and F) Four-week-old C57BL/6J mice were inoculated in the footpad with 105 FFU of CHIKV AF15561-Venus reporter virus. At 0, 18, or 36 hpi, ipsilateral foot skin and DLN were harvested, and cell staining and flow cytometry were performed. The percentages (E) and numbers (F) of Ly6Chi monocytes expressing YFP are shown (n = 6, two experiments). Cells from naive mice were used to set staining gates. Gating strategies for this figure are shown in Figure S5. See also Figures S6 and S7. Bars indicate median values.

Fig 4: Humoral and cellular immune responses to C. albicans and Kazachstania sp. (A) Flow cytometric analysis of myeloid blood cell compartment of mice 4 wk colonized with C. albicans or K. weizmannii. Neutrophils, classical, and non-classical monocytes are defined as Ly6G+ CD115−, Ly6C+ CD115+, and Ly6C− CD115+ cells, respectively. N = 8–21 mice, pooled from three independent experiments. Gating strategy, left; results, right. The corresponding statistical analysis shows mean; significance is calculated with two-tailed Student’s t test. (B) Flow cytometric analysis of humoral anti-fungal reactivity. Gating strategy for the determination of serum immunoglobulin binding to cultured C. albicans or K. weizmannii. (C) Anti-C. albicans and K. weizmannii IgA and IgG serum reactivity of single-colonized animals, measured as shown in B. Note that most but not all animals harbor cross-reactive sera. Data shown as percentage of Ig-positive gate, two pooled independent experiments, N = 11–13 per group. (D) Representative gating strategy of RORγt+ T cells (left); percentage of RORγt+ cells among TCRβ CD4+ T cells 1 mo following C. albicans or K. weizmannii colonization, n = 3–5 mice per group, three independent experiments (right). The corresponding statistical analysis shows mean; significance is calculated with two-tailed Student’s t test. (E) Representative gating strategy of IL17 producing Th17 cells (left); percentage of IL17+ cells among TCRβ CD4+ T cells 1 mo following C. albicans or K. weizmannii colonization, n = 3–5 mice per group, three independent experiments (right). The corresponding statistical analysis shows mean; significance is calculated with two-tailed Student’s t test. (F) Quantitative PCR analysis of genomic DNA of C. albicans SC5314 and K. weizmannii recovered from feces of Rag2−/− animals colonized with C. albicans SC5314 (C) or a mixed culture (C/K) compared to Abx-treated SPF animals (−) 1 wk after administration; n = 3–4 mice per group, pooled two independent experiments; data shown as mean ± SEM; statistical significance calculated using ANOVA, P < 0.05. (G) EC coculture assay of C. albicans, S. cerevisiae, and K. weizmannii. EC damage was assessed using the LDH assay as described in Allert et al. (2018). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.