1. Differential Activation of Human Keratinocytes by Leishmania Species Causing Localized or Disseminated Disease 
Breanna M. Scorza, Mark A. Wacker, Kelly Messingham, Peter Kim, Aloysius Klingelhutz, Janet Fairley, Mary E. Wilson 
Journal of Investigative Dermatology 
Volume 137, Issue 10, Pages (October 2017)
DOI: /j.jid
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2. Figure 1
Low level of Leishmania species infection in human keratinocytes in vitro. (a) Primary human keratinocytes harboring intracellular Leishmania infantum were visualized by Wright-Giemsa stain (left, scale bar = 10 μm) or confocal microscopy using DAPI and Alexa Flour 488-phalloidin (right, scale bar = 20 μm). White arrows indicate parasite nucleus and kinetoplastid staining. (b) NHSK1 cells (solid lines) or primary human monocytes (dashed lines) were incubated with L. infantum (black circles) or Leishmania major (white squares). Percentage infection at indicated time points were quantified in 250 cells/coverslip in triplicate by light microscopy; mean ± standard deviation, n = 2–3 independent experiments. Hrs, hours; Li, Leishmania infantum; Lm, Leishmania major; p.i., post infection.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

3. Figure 2
Differential gene expression by human keratinocytes exposed to cutaneous or visceralizing Leishmania parasites. (a–b) Keratinocytes exposed to Li or Lm. Log2(fold change) expression over control. Mean ± standard deviation, t test. (a) The 24-hour primary human keratinocytes, β-actin normalized, n = 3 separate experiments from one donor. (b) The 4-hour NHSK1s, glyceraldehyde-3-phosphate dehydrogenase normalized, n = 5–7 independent experiments. (c–d) NHSK1 24-hour supernatant protein, exposure to indicated cell-to-promastigote ratio or 10 ng/ml TNF by ELISA. Mean ± standard error of the mean, analysis of variance. (c) IL-8: Tukey posttest, n = 3 independent experiments. (d) IL-6: NHSK1s exposed to 1:100 Li. Dunnett posttest, n = 2–3 independent experiments. (e–h) The 4-hour log2(fold change) NHSK1 gene expression, glyceraldehyde-3-phosphate dehydrogenase normalized. Mean ± standard deviation, analysis of variance, n = 3 independent experiments. (e) NHSK1s treated with 10 ug/ml Li, Lm, or NHSK1 control exosomes. Tukey posttest. (f–h) NHSK1s exposed to different strains of (f) Li or (g) Lm plus 10 ng/ml TNF or (h) indicated Leishmania species. Dunnett post-test. Ctrl, control; Lb, Leishmania braziliensis; Ld, Leishmania donovani; Li, Leishmania infantum; Lm, Leishmania major; NHSK1, normal human skin keratinocyte immortalized cell line; TNF, tumor necrosis factor.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

4. Figure 3
Factors contributing to keratinocyte inflammatory responses to L. infantum or L. major. (a, b) Pooled densitometry of phospho- compared with total NF-κBp65 in NHSK1 lysates. Analysis of variance, Dunnett posttest, n = 3 independent experiments, mean ± standard deviation. (a) NHSK1s exposed to 1:100 Li or Lm for indicated time. Normalized to β-tubulin. (b) NHSK1s exposed to media or indicated ratio of Lm promastigotes ± 10 ng/ml TNF for 30 minutes. Normalized to α-tubulin. (c–e) The 4-hour NHSK1s log2(fold change) gene expression compared with control, normalized to glyceraldehyde-3-phosphate dehydrogenase. IL-6 expression: NHSK1s exposed to (c) live or paraformaldehyde-fixed Li or (d) Lm promastigotes mean ± standard deviation. t test, n = 3–4 independent experiments. (e) IL-8 expression: NHSK1s exposed to media, Li, or TNF. Within groups, NHSK1s exposed to media (-), vehicle (V), or metalloprotease inhibitor (I) (o-phenathroline) for initial 2 hours. Mean ± standard deviation. Two-way analysis of variance, Tukey posttest, n = 2–5 independent experiments. Li, Leishmania infantum; Lm, Leishmania major; m, minute; NHSK1, normal human skin keratinocyte immortalized cell line; ns, not significant; p-, phosphorylated; TNF, tumor necrosis factor.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

5. Figure 4
Co-culture with human keratinocytes alters monocyte infection rate and gene expression. NHSK1s and primary human monocytes were exposed to parasites separately for 2 hours, washed, and co-cultured across a Transwell. Fold change in percentage (a, c) infected monocytes and (b, d) parasite burden infected with (a, b) Li or (c, d) Lm from the 2-hour time point. Monocytes alone (white circles). Monocytes with unexposed keratinocytes (+K, white squares) Monocytes with parasite-exposed keratinocytes (+Li-K or +Lm-K, black squares). Analysis of variance, Tukey posttest, mean ± standard error of the mean, n = 3 separate donors. Average 2-hour monocyte infection: Li = %, Lm = %. Transwell monocytes using Li were analyzed by quantitative real-time reverse transcriptase–PCR. Log2(fold change) of selected transcripts (e) 8 hours or (f) 24 hours compared with uninfected monocytes cultured alone. Hatched bars: uninfected monocytes. Filled bars: Li-infected monocytes. Analysis of variance with a Dunnett posttest, mean ± standard error of the mean, n = 3 separate donors. h, hour; hrs, hours; Li, Leishmania infantum; MO, monocyte; p.i., post infection.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions