Anti-inflammatory and immunomodulatory effects of Aquaphilus dolomiae extract on in vitro models

Figures & data

Table 1 PCR array analysis of the expression of 21 genes

Biological markers	Regulation of gene expression in 24-h-activated NHK	Effect of ES0 on gene expression of 24-h-activated NHK			Effect of DEX on gene expression of 24-h-activated NHK
		10 µg/mL	30 µg/mL	60 µg/mL	2 µM
Thymic stromal lymphopoietin (TSLP)	12.6±1.4 ***	0.44±0.04***	0.25±0.02***	0.08±0.02***	0.08±0.02***
Interleukin 1, alpha (IL1A)	35±1.4***	0.65±0.02**	0.54±0.04**	0.44±0.09**	0.42±0.08***
Interleukin 18 (IL18)	2±0.1***	0.73±0.06*	0.57±0.03**	0.37±0.09**	0.51±0.1**
Interferon, alpha 2 (IFNA2)	2.5±0.4***	1.1±0.05	1.16±0.31	1.1±0.3	0.57±0.21*
Interferon, beta 1 (IFNB1)	24.3±1.5***	0.35±0.08***	0.18±0.01***	0.1±0.02***	0.46±0.09**
Interleukin 8 (IL8)	732±155***	0.65±0.17*	0.47±0.08**	0.13±0.08***	0.24±0.08***
MIP-1α(CCL3)	5759±2977***	0.9±0.06	0.57±0.04**	0.27±0.06***	0.21±0.04***
RANTES (CCL5)	15591±1441***	0.85±0.03	0.57±0.04**	0.37±0.07**	0.8±0.15
MCP-3 (CCL7)	7.8±2.4***	0.61±0.18*	0.40±0.13**	0.14±0.07***	1.11±0.22
TARC (CCL17)	21.5±6.2***	0.44±0.08**	0.37±0.03**	0.67±0.17	1.11±0.21
MIP-3αCCL20	212±17***	0.6±0.04**	0.4±0.03***	0.21±0.04***	0.54±0.11*
MDC (CCL22)	33.1±4.2***	0.84±0.03	0.56±0.04**	0.45±0.09**	0.5±0.09**
Skinkine (CCL27)	57.8±0.7***	0.72±0.03*	0.68±0.05*	0.65±0.13	0.37±0.07***
Interleukin 4 receptor (IL4R)	6.3±0.6***	0.7±0.03*	0.56±0.04**	0.32±0.07**	0.23±0.05***
Cathelicidin antimicrobial peptide (CAMP)	5.7±0.8***	1.1±0.1	1.3±0.2	1.8±0.4*	0.42±0.08**
Ribonuclease, RNase A family, 7 (RNASE7)	7.7±0.1***	2.4±0.1***	3.5±0.2***	5.3±1.1***	0.27±0.05***
S100 calcium binding protein A11 (S100A11)	3±0.1***	0.88±0.03	0.88±0.06	0.84±0.18	0.6±0.11*
Psoriasin (S100A7)	16.6±0.2***	4.6±0.2***	11.4±1***	22.5±5.1***	0.42±0.08**
Ceramide synthase 6 (CERS6)	0.26±0.0***	0.94±0.04	0.95±0.16	0.93±0.23	1.3±0.2
Involucrin (IVL)	0.18±0.0***	1.6±0.1**	4.2±0.34***	11.8±2.4***	0.98±0.18
Filaggrin (FLG)	0.24±0.01***	1.02±0.04	0.83±0.05	0.74±0.15	1.47±0.27**

Notes: NHKs were incubated with 10 µg/mL poly I:C + 3 ng/mL of each cytokine (TNFα + IL-4 + IL-13). NHKs were exposed to three different doses of ES0 (10, 30, and 60 µg/mL) for 24 h compared with DEX of 2 µM. Relative quantity ±SE of mRNA expression compared to control cells (for 24 h-activated NHK) or to 2 h-activated NHKs (for the effect of ES0 or DEX). Data presented as mean ± standard deviation.

* P<0.05;

** P<0.01;

*** P<0.001.

Abbreviations: DEX, dexamethasone; ES0, extract from Aquaphilus dolomiae; IL, interleukin; NHK, normal human keratinocyte; PCR, polymerase chain reaction; SE, standard error; TNFα, tumor necrosis factor alpha.

Table 2 Effect of ES0 on normal human keratinocyte cell viability: MTT assay

	Treatment (24 h; µg/mL)	Cell viability (percentage of control ± SE)
Control		100±2
ES0	1.1	104±3
	3.3	105±4
	10	106±4
	30	107±4
	60	105±5
	100	105±4
	300	16±1
	900	2±0

Abbreviations: ES0, extract from Aquaphilus dolomiae; SE, standard error.

Figure 1 Stimulation of PAR-2 receptors by SCTE in HaCaT keratinocyte cells.

Notes: Dose-dependent inhibition by ES0 (30 and 100 µg/mL). ^*P<0.05; ^***P<0.001 versus SCTE; data are representative of three independent experiments.
Abbreviations: ES0, extract from A. dolomiae; PAR-2, protease-activated receptor-2; SCTE, stratum corneum tryptic enzyme.

Figure 2 Spectrophotometric measurement of TLR2, TLR4, and TLR5 activation in HEK 293 cell lines.

Notes: HEK were stimulated with ES0 (3–100 ng/mL) or positive controls: Pam2CSK4 = TLR2 ligand (A), LPS = TLR4 ligand (B), and flagellin = TLR5 ligand (C). ^***P<0.001 versus control; data are representative of three independent experiments.
Abbreviations: ES0, extract from A. dolomiae; HEK, human embryonic kidney; LPS, lipopolysaccharide; OD, optical density; TLR, toll-like receptor.

Figure 3 Relative quantity of hBD-2, CAMP, and psoriasin mRNA in NHK cells.

Notes: NHKs were stimulated by IL-1β (20 ng/mL, positive control), ES0 (6.5 µg/mL), flagellin (100 ng/mL), or alone (negative control). Pretreatment was performed for 1 h with anti-TLR2 (

), anti-TLR4 (

), anti-TLR5 (■) (1 µg/mL), or without antibody (□) to test whether stimulation was blocked in ES0- and flagellin-treated samples; data are the mean of three independent experiments. ^*P<0.05; ^**P<0.01; ^***P<0.001 versus control or comparator.
Abbreviations: CAMP, cathelicidin antimicrobial peptide; ES0, extract from A. dolomiae; hBD2, beta-defensin 2; NHK, normal human keratinocyte; TLR, toll-like receptor.

Figure 4 Lymphocyte stimulation by Staphylococcal enterotoxin B (SEB; 300 ng/mL).

Notes: Dose effect of ES0 (30, 60, and 100 µg/mL) on the production of Th1, Th2, and Th17 cytokines; data are representative of three independent experiments. ^***P<0.001 versus SEB-treated cells.
Abbreviations: ES0, extract from A. dolomiae; IL, interleukin; SEB, Staphylococcal enterotoxin B; Th, T helper cell type; TNFα, tumor necrosis factor alpha.

Table S1 Polymerase chain reaction primer sequences and amplicon length

Gene name	Gene symbol	Genebank accession number	Sense primer sequence	Antisense primer sequence	Amplicon Length (bp)
Thymic stromal lymphopoietin	TSLP	NM_033035, NM_138551	CAAAGAAATGTTCGCCATGA	TGGTGCTGTGAAATATGACCA	233
Interleukin 1, alpha	IL1A	NM_000575	ATCAGTACCTCACGGCTGCT	AACAAGTTTGGATGGGCAAC	283
Interleukin 18	IL18	NM_001562	CCAAGGAAATCGGCCTCTAT	TCAGGAGGATTCATTTCCTTAAA	201
Interferon, alpha 2	IFNA2	NM_000605	AGGAGGACTCCATTCTGGCTGTG	AGGTGAGCTGGCATACGAATCAATG	216
Interferon, beta 1, fibroblast	IFNB1	NM_002176	ATTGCTCTCCTGTTGTGCTTCTCC	TGCGGCGTCCTCCTTCTGG	207
Interleukin 8	IL8	NM_000584	CTCTTGGCAGCCTTCCTG	TTGGGGTCCAGACAGAGC	218
Chemokine (C-C motif) ligand 3	CCL3	NM_002983	CCTTTCTTGGCTCTGCTGACACTC	CTGGCTGCTCGTCTCAAAGTAGTC	222
Chemokine (C-C motif) ligand 5	CCL5	NM_002985	AGCTACTCGGGAGGCTAAGG	GAGGCATGCTGACTTCCTTC	263
Chemokine (C-C motif) ligand 7	CCL7	NM_006273	GGCTGAGACCAAACCAGAAA	GGACAGTGGCTACTGGTGGT	241
Chemokine (C-C motif) ligand 17	CCL17	NM_002987	TTCAAAACCAGGGTGTCTCC	CTGCCCTGCACAGTTACAAA	279
Chemokine (C-C motif) ligand 20	CCL20	NM_004591	TGAGCTAAAAACCATGTGCTGTA	AGCATTGATGTCACAGCCTTC	199
Chemokine (C-C motif) ligand 22	CCL22	NM_002990	TGTGCCAACTCTCTGCATTC	AGGCTGGAGACAGAGATGGA	258
Chemokine (C-C motif) ligand 27	CCL27	NM_006664	CAGCTCTACCGAAAGCCACT	GCATCCCAAAATTCAGCTTG	217
Interleukin 4 receptor	IL4R	NM_000418	CCTTGGGAAATCGATGAGAA	CAGGGTGGCTGAGCATATTT	268
Cathelicidin antimicrobial peptide	CAMP	NM_004345	CCCAGGTCCTCAGCTACAAG	GGTCACTGTCCCCATACACC	251
Ribonuclease, RNase A family, 7	RNASE7	NM_032572	CCTCCCCAGAAAAAGGACTC	GGAATTGAAGGTCAGCCAAA	106
S100 calcium binding protein A11	S100A11	NM_005620	CTTGCCATGACTCCTTCCTC	GCCTGCATGAGGTGGTTAGT	158
S100 calcium binding protein A7	S100A7	NM_002963	GCATGATCGACATGTTTCACAAATACAC	TGGTAGTCTGTGGCTATGTCTCCC	226
Ceramide synthase 6	CERS6	NM_203463	TTCGACAAAGACGCAATCAG	AAAACGACAGCTCCAGGATG	216
Involucrin	IVL	NM_005547	CTGGGAACAGCATGAGGAAT	GGAGCTCCAACAGTTGCTCT	194
Filaggrin	FLG	NM_002016	TGATGGTATTCAAGTTGGCTCA	TGTTTCTCTTGGGCTCTTGG	215
Glyceraldehyde-3-phosphate dehydrogenase	GAPDH	NM_002046	GGCTCTCCAGAACATCATCCCTGC	GGGTGTCGCTGTTGAAGTCAGAGG	269
Ribosomal protein L13a	RPL13A	NM_012423	TAAACAGGTACTGCTGGGCCGGAAGGTG	CACGTTCTTCTCGGCCTGTTTCCGTAGC	483