1. The Short-Chain Fatty Acid Sodium Butyrate Functions as a Regulator of the Skin Immune System 
Agatha Schwarz, Anika Bruhs, Thomas Schwarz 
Journal of Investigative Dermatology 
Volume 137, Issue 4, Pages (April 2017)
DOI: /j.jid
Copyright © 2016 The Authors Terms and Conditions

2. Figure 1
Sodium butyrate inhibits the elicitation phase of CHS via induction of skin Tregs. (a) SB was either injected s.c. or applied topically in/onto the left ears of mice, which were sensitized against TNCB 5 days before. After 24 hours, TNCB was applied on the same ear. Ear swelling was quantified 24 hours later. CHS was determined as the thickness of the hapten-challenged ear compared with the thickness of the vehicle-treated ear and expressed in cm × 10-3 (mean ± standard deviation). Positive control mice (PosCo) were sensitized and challenged; negative control mice (NegCo) were challenged only. Bar graph data show one from three independent experiments; n = 6 per group; PANOVA < 0.001, *PStudent SB vs. PosCo < (b) Paraffin sections of SB-treated and untreated ears were stained for the expression of Foxp3. Scale bar = 50 μm. (c) Single-cell suspensions were obtained from ears of mice treated as described in a. Cells were stained with an anti-Foxp3 antibody and subjected to FACS analysis. PANOVA < 0.001, ∗PStudent SB vs. control < Representative histogram plots are shown. (d) RNA was isolated from untreated and SB-treated ears. mRNA transcription of foxp3 and il-10 was measured by qRT-PCR. Relative transcription was normalized to the transcription of ß-actin and hprt1. Data show one representative of three independent experiments; n = 3 per group; PStudent SB vs. control < (e) RNA was isolated from SB-treated CD4+CD25– and CD4+CD25+ T cells and analyzed for il-10 transcripts by qRT-PCR. PANOVA < 0.01, ∗PStudent vs. CD4+CD25–-SB < (f) SB was applied topically on the left ears of TNCB-sensitized mice. Ear challenge was performed 24 hours later. One additional group was injected subcutaneously with a neutralizing anti-IL-10 antibody 3 hours before challenge. PANOVA < 0.001, ∗PStudent SB vs. PosCo < 0.001, ∗∗PStudent SB vs. SB + αIL-10 < CHS, contact hypersensitivity; FS, forward scatter; qRT-PCR, quantitative real-time reverse transcription-PCR; SB, sodium butyrate; s.c., subcutaneous; SS, side scatter; TNCB, 2,4,6-trinitro-1-chlorobenzene; Treg, regulatory T cell.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

3. Figure 2
Application of sodium butyrate to the skin attenuates an ongoing CHS response. (a) Mice were sensitized against TNCB on the shaved backs. One group was injected with SB s.c. in the left ears 5 days later (SB s.c.). After 24 hours, TNCB was applied on the same ear. In two groups SB was either injected s.c. or applied topically in/onto the left ears of mice 12 hours after the challenge (SB s.c., SB topically after challenge). Ear swelling was quantified 24 hours later. PANOVA < 0.001, ∗PStudent vs. PosCo < (b) Single-cell suspensions were obtained from ears of the PosCo and the SB-treated mice and double-stained with an anti-CD25 and anti-CD4 antibody or with an anti-Foxp3 antibody and subjected to FACS analysis. PANOVA <0.01, ∗PStudent vs. PosCo < Representative histogram plots are shown for both stainings. CHS, contact hypersensitivity; FS, forward scatter; NegCo, negative control; PosCo, positive control; SB, sodium butyrate; s.c., subcutaneous; SS, side scatter; TNCB, 2,4,6-trinitro-1-chlorobenzene.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

4. Figure 3
Sodium butyrate induces Foxp3-positive Tregs in vivo and switches nonregulatory to regulatory T cells in vitro. (a) DEREG mice were sensitized with TNCB, and 48 hours later DT was injected for 3 days (+DT) or left untreated (–DT). 24 hours after the last treatment, SB was either injected s.c. or applied topically on the left ear, and challenge of the same ear was performed 24 hours thereafter. n = 6 per group; PANOVA < 0.001; ∗PStudent PosCo vs. SB topical and vs. SB s.c. < 0.05; ∗∗PStudent SB topical vs. SB topical + DT < 0.001; ∗∗∗PStudent SB s.c. vs. SB s.c. + DT < (b) LN cells and splenocytes were obtained from naive mice and separated into CD4+CD25+ and CD4+CD25− T cells. One half of CD4+CD25– T cells were stimulated with 200 μmol/L SB. After 24 hours, cells were double-stained with anti-CD4 antibody and anti-CTLA-4, anti-GARP, or anti-Foxp3 antibodies. (c) SB-stimulated CD4+CD25– cells from the same suspensions used for FACS staining in b were injected s.c. into the ears of sensitized mice. As controls, the equal amount of Tregs or unstimulated CD4+CD25– T cells were injected. Recipients were challenged 24 hours later. n = 6 per group; PANOVA < 0.001; ∗PStudent CD4+CD25– +SB vs. CD4+CD25– < 0.05 and vs. PosCo < (d) CD4+CD25+ (Tregs), CD4+CD25− T cells, and SB-treated Tregs and CD4+CD25− T cells were co-cultured with anti-CD3 and anti-CD28 antibody–activated CD4+CD25− responder T cells. After 4 days, cell proliferation was measured using the MTT assay. Data are presented as percent suppression (mean ± standard error of difference from one of two independent experiments). ∗P < 0.05 CD4+CD25– vs. CD4+CD25– + SB; ∗∗P < 0.05 Treg vs. Treg + SB by two-tailed Mann-Whitney U test. DT, diphtheria toxin; LN, lymph node; SB, sodium butyrate; s.c., subcutaneous; TNCB, 2,4,6-trinitro-1-chlorobenzene; Treg, regulatory T cell.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

5. Figure 4
Skin T cells express GARP43. (a) Paraffin sections of SB- and untreated ears were stained for GPR43 and CD25. Scale bar = 50 μm. (b) Single-cell suspensions were prepared from SB- or untreated ears, simultaneously stained with an anti-CD25- and anti-GPR43 antibodies, and subjected to FACS analysis. PANOVA < 0.001; ∗PStudent SB vs. untreated < Representative histogram plots are shown. (c) T cells were isolated from murine skin, separated into CD4+CD25+ and CD4+CD25− T cells, and subjected to FACS analysis for the expression of GPR43. Representative histogram plots are shown. FS, forward scatter; SB, sodium butyrate; SS, side scatter; Treg, regulatory T cell.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

6. Figure 5
The immunomodulatory effect of sodium butyrate (SB) is histone acetylation dependent. (a) CD4+CD25– T cells were isolated from murine skin and treated with 200 μmol/L SB for 24 hours. Untreated cells served as a control. After 24 hours, cells were extensively washed and subjected to the histone acetylation assay. Bar graph data show mean ± standard deviation of one from three independent experiments; ∗PStudent < (b) Sensitized mice were injected intraperitoneally with anacardic acid (AA). After 30 minutes, SB was topically applied, and 24 hours later challenge was performed. Bar graph data show mean ± standard deviation of one from three independent experiments. n = 6 per group; PANOVA < 0.001; ∗PStudent SB vs. PosCo and vs. SB + AA < NegCo, negative control; PosCo, positive control.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

7. Figure 6
Sodium butyrate (SB) switches human nonregulatory T cells into regulatory T cells. (a) RNA was isolated from untreated and SB-treated human skin samples. mRNA transcription of foxp3, garp, il-10, il-6, and il-18 was measured by qRT-PCR. Relative transcription was normalized to the transcription of gapdh and hprt1. Data shown as mean ± standard deviation from three independent experiments; n = 4; ∗PStudent SB vs. control < (b) Paraffin sections of SB-treated and untreated human skin samples were stained for Foxp3 and GARP. Scale bar = 100 μm. (c) Human peripheral blood mononuclear cells were separated into CD4+CD25– and CD4+CD25+ T cells. Fractions were incubated with 200 μmol/L SB. CD4+CD25+ (Tregs), CD4+CD25− T cells, and SB-treated Treg and CD4+CD25− T cells were co-cultured with anti-CD2, anti-CD3 and anti-CD28 antibody–activated CD4+CD25− responder T cells. After 4 days, cell proliferation was measured using the MTT assay. Data are shown as mean ± standard deviation from one of three independent experiments. ∗P < 0.05 CD4+CD25– vs. CD4+CD25– + SB; ∗∗P < 0.05 Treg vs. Treg + SB by two-tailed Mann-Whitney U test. n.s., not significant; qRT-PCR, quantitative real-time reverse transcription-PCR; Treg, regulatory T cell.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions