Pathogen-Associated Molecular Pattern-Induced TLR2 and TLR4 Activation Increases Keratinocyte Production of Inflammatory Mediators and is Inhibited by Phosphatidylglycerol

Abstract

Skin serves not only as a protective barrier to microbial entry into the body but also as an immune organ. The outer layer, the epidermis, is composed predominantly of keratinocytes, which can be stimulated to produce proinflammatory mediators. Although some inflammation is useful to defend against infection, excessive or persistent inflammation can lead to the development of inflammatory skin diseases, such as psoriasis, a common skin disorder affecting approximately 2% of the US population. We have previously found that phosphatidylglycerol (PG) derived from soy can inhibit inflammation in a contact irritant ear edema mouse model. Here, we investigated the ability of soy PG to inhibit inflammatory mediator expression in response to activators of the pattern recognition receptors, toll-like receptor-2 (TLR2) and -4 (TLR4). We found that in epidermal keratinocytes, soy PG inhibited TLR2 and TLR4 activation and inflammatory mediator expression in response to a synthetic triacylated lipopeptide and lipopolysaccharide, respectively, as well as an endogenous danger-associated molecular pattern. However, at higher concentrations, soy PG alone enhanced the expression of some proinflammatory cytokines, suggesting a narrow therapeutic window for this lipid. Dioleoylphosphatidylglycerol (DOPG), but not dioleoylphosphatidylcholine, exerted a similar inhibitory effect, completely blocking keratinocyte inflammatory mediator expression induced by TLR2 and TLR4 activators as well as NFκB activation in a macrophage cell line (RAW264.7); however, DOPG was not itself proinflammatory even at high concentrations. Furthermore, DOPG had no effect on NFκB activation in response to a TLR7/8 agonist. Our results suggest that DOPG could be used to inhibit excessive skin inflammation. SIGNIFICANCE STATEMENT: Although inflammation is beneficial for clearing an infection, in some cases, the infection can be excessive and/or become chronic, thereby resulting in considerable tissue damage and pathological conditions. We show here that the phospholipid phosphatidylglycerol can inhibit the activation of toll-like receptors 2 and 4 of the innate immune system as well as the downstream inflammatory mediator expression in response to microbial component-mimicking agents in epidermal keratinocytes that form the physical barrier of the skin.

Fig. 1.

The TLR1/2 agonist, Pam₃CSK₄, induced keratinocyte expression of inflammatory mediators in a dose-dependent manner. Keratinocytes were treated with various concentrations of the synthetic triacylated lipoprotein Pam₃CSK₄ (Pam) for 2 hours as indicated (in microgram per milliliter). RNA was then isolated, and the expression of the inflammatory mediators (A) IL1α, (B) IL1β, (C) IL6, and (D) TNFα was monitored by quantitative RT-PCR with GAPDH used as the housekeeping gene. Results represent the means ± S.D. of three separate experiments; *P < 0.05; **P < 0.01; ***P < 0.001 vs. the zero concentration (Control).

Fig. 2.

Pam₃CSK₄ induced keratinocyte expression of inflammatory mediators in a time-dependent manner. Keratinocytes were treated with vehicle or 2.5 µg/ml Pam₃CSK₄ (Pam) for 2, 4, and 6 hours. RNA was then isolated, and the expression of the inflammatory mediators (A) IL1α, (B) IL1β, (C) IL6, and (D) TNFα as well as (E) S100A8 and (F) S100A9 was monitored by quantitative RT-PCR with GAPDH used as the housekeeping gene. Results represent the means ± S.D. of three separate experiments; Pam₃CSK₄-treated values were analyzed at each time point using one-sample t tests vs. a hypothetical mean of 1.0 (to which the vehicle-treated control value was set), as performed by GraphPad Instat, with *P < 0.05; **P < 0.01; ***P<0.001 vs. the control at that time point.

Fig. 3.

Soy PG inhibited keratinocyte inflammatory mediator expression in response to Pam₃CSK₄. Keratinocytes were treated with 0, 1, and 2.5 µg/ml Pam₃CSK₄ (Pam) in the presence or absence of 10 or 50 µg/ml soy PG (s-PG) for 2 hours. RNA was then isolated, and the expression of the inflammatory mediators (A) IL1α, (B) IL1β, (C) IL6, and (D) TNFα was monitored by quantitative RT-PCR with GAPDH used as the housekeeping gene. Results represent the means ± S.D. of four separate experiments; *P < 0.05; **P < 0.01; ***P < 0.001 vs. the zero concentration control; ^ƒƒP < 0.01; ^ƒƒƒP < 0.001; ^ξξξP < 0.001 vs. the indicated groups.

Fig. 4.

Soy PG inhibited keratinocyte inflammatory mediator expression in response to LPS. Keratinocytes were treated with 0 or 1000 U/ml LPS in the presence or absence of 10 or 50 µg/ml soy PG (s-PG) for 2 hours. RNA was then isolated, and the expression of the inflammatory mediators (A) IL1α, (B) IL1β, (C) IL6, and (D) TNFα as well as (E) TLR1 and (F) TLR2 was monitored by quantitative RT-PCR with GAPDH used as the housekeeping gene. Results represent the means ± S.D. of three separate experiments; *P < 0.05; **P < 0.01; ***P < 0.001 vs. the zero concentration control; ^ƒP < 0.05; ^ƒƒP < 0.01; ^ƒƒƒP < 0.001; ^ξP < 0.05; ^ξξP < 0.01 vs. the indicated groups; ns, not significantly different.

Fig. 5.

Soy PG inhibited keratinocyte inflammatory mediator expression in response to S100A9. Keratinocytes were treated with 0 and 2.5 µg/ml recombinant S100A9 in the presence or absence of 10 or 50 µg/ml soy PG (s-PG) for 2 hours. RNA was then isolated, and the expression of the inflammatory mediators (A) IL1β, (B) IL6, and (C) TNFα as well as (D) TLR1 and (E) TLR2 was monitored by quantitative RT-PCR with GAPDH used as the housekeeping gene. Results represent the means ± S.D. of three separate experiments; *P < 0.05; **P < 0.01; ***P < 0.001 vs. the control; ^†††P < 0.001 vs. all other groups; ^ƒP < 0.05; ^ξP < 0.05; ^ξξP < 0.01 vs. the indicated groups.

Fig. 6.

DOPG inhibited keratinocyte inflammatory mediator expression in response to Pam₃CSK₄. Keratinocytes were treated with 0, 1, and 2.5 µg/ml Pam₃CSK₄ (Pam) in the presence or absence of 50 or 100 µg/ml DOPG for 2 hours. RNA was then isolated, and the expression of the inflammatory mediators (A) IL1α, (B) IL1β, (C) IL6, and (D) TNFα was monitored by quantitative RT-PCR with GAPDH used as the housekeeping gene. Results represent the means ± S.D. of four separate experiments; *P < 0.05; **P < 0.01; ***P < 0.001 vs. the zero concentration control; ^ƒƒƒP < 0.001; ^τP < 0.05; ^ξP < 0.05; ^ξξP < 0.01 vs. the indicated groups.

Fig. 7.

DOPG inhibited Pam₃CSK₄-induced inflammatory mediator expression in the macrophage cell line RAW264.7. RAW264.7 cells were treated with 0, 1, and 2.5 µM Pam₃CSK₄ (Pam) in the presence or absence of 50 or 100 µg/ml DOPG for 2 hours. RNA was then isolated, and the expression of the inflammatory mediators (A) IL1α, (B) IL1β, (C) IL6, and (D) TNFα was monitored by quantitative RT-PCR with GAPDH used as the housekeeping gene. Results represent the means ± S.D. of four separate experiments; *P < 0.05; **P < 0.01; ***P < 0.001 vs. the zero concentration control; ƒ P < 0.05; ^ƒƒP < 0.01; ^τP < 0.05; ^ξP < 0.05 vs. the indicated groups.

Fig. 8.

DOPG inhibited Pam₃CSK₄-induced NFκB activation in the macrophage cell line RAW264.7. (A) RAW264.7 cells were treated with or without 2.5 µg/ml Pam₃CSK₄ (Pam) in the presence and absence of 100 µg/ml DOPG for 10 minutes. Cells were harvested and the phosphorylation (activation) status of NFκB determined using an antibody recognizing phosphoserine⁵³⁶ in comparison with total NFκB levels. (A) shows a representative Western blot, whereas (B) presents the cumulative results from three separate experiments (means ± S.D.); ***P < 0.001 vs. the control; ξξξP < 0.001 as indicated. (C) RAW264.7 cells were treated with 0 and 2.5 µM Pam₃CSK₄ (Pam) in the presence or absence of 100 µg/ml DOPG or DOPC as indicated for 2 hours. NFκB was then monitored by immunocytochemistry and confocal microscopy (Scale bar, 5 µm). The final row shows the same micrographs illustrating grayscale NFκB staining as in the row above, with NFκB staining in green and nuclear staining (with DAPI) in blue in these merged color images. Results are representative of at least three separate experiments.

Fig. 9.

DOPG had no effect on R848-induced NFκB activation in the macrophage cell line RAW264.7. (A) RAW264.7 cells were treated with or without 1 µg/ml R848 (Resiquimod) in the presence and absence of 100 µg/ml DOPG for 10 minutes. Cells were harvested and the phosphorylation (activation) status of NFκB determined using an antibody recognizing phosphoserine⁵³⁶ in comparison with total NFκB levels. (A) shows a representative Western blot, whereas (B) presents the cumulative results from three separate experiments (means ± S.D.); *P < 0.05 vs. the control; ns, not significantly different.

Fig. 10.

DOPG, but not DOPC, inhibited Pam₃CSK₄- and LPS-induced TLR2 and TLR4 activation in reporter cell lines. (A) HEK-Blue-hTLR2 cells were incubated for 24 hours in the HEK-Blue detection medium with various concentrations of Pam₃CSK₄, as indicated. (B) HEK-Blue-hTLR4 cells were incubated for 24 hours in the HEK-Blue detection medium with various concentrations of LPS, as indicated. (C) HEK-Blue-hTLR2 cells were incubated in the HEK-Blue detection medium with or without 0.03 ng/ml Pam₃CSK₄ in the presence and absence of 100 µg/ml DOPG or DOPC for 24 hours. (D) HEK-Blue-hTLR4 cells were incubated in the HEK-Blue detection medium with or without 3 ng/ml LPS in the presence and absence of 100 µg/ml DOPG or DOPC for 24 hours. In all cases, SEAP activity was then measured as absorbance at 620 nm. Values represent the means ± S.D. from at least three separate experiments; ***P < 0.001 vs. the control; ^ƒƒƒP < 0.001; ^ξξξP < 0.001 as indicated.