Phosphatidylglycerol Inhibits Toll-Like Receptor-Mediated Inflammation by Danger-Associated Molecular Patterns

Abstract

Psoriasis is a common skin disorder characterized by hyperproliferation and aberrant differentiation of epidermal keratinocytes and inflammation. We previously showed that phosphatidylglycerol (PG) can regulate keratinocyte function and suppress skin inflammation. Based on data suggesting that PG can inhibit toll-like receptor (TLR) activation induced by microorganisms and their components, we determined whether PG can inhibit TLR activation in response to antimicrobial peptides. These peptides, which are up-regulated in psoriasis, are known to function as danger-associated molecular patterns (i.e., DAMPs) to activate TLRs and the innate immune system. Because S100A9 is elevated in psoriatic skin and in animal models of psoriasis, we selected S100A9 as a representative antimicrobial peptide DAMP. We showed that in primary keratinocytes and a macrophage cell line, PG suppressed inflammatory mediator production induced by recombinant S100A9 functioning through both TLR2 and TLR4. In addition, PG, but not phosphatidylcholine, inhibited downstream S100A9-elicited TLR2 and NF-κB activation. These results, to our knowledge previously unreported, show PG's ability to inhibit DAMP-induced TLR activation, thereby reducing inflammatory signals. In addition, topical PG ameliorated skin lesions and inflammation in a mouse model of psoriasis. Together, these results suggest the possibility of developing PG as a therapy for psoriasis.

Figure 1.. DOPG inhibits expression of inflammatory mediators induced by the DAMP, recombinant S100A9, in keratinocytes and a macrophage cell line.

(a-c) Keratinocytes or (d-g) RAW264.7 cells were treated with 2.5μg/mL S100A9 in the presence and absence of 100μg/mL DOPG or DOPC for 2 hours. RNA was then isolated and the expression of the inflammatory mediators, (a and d) IL1β, (b and e) IL6 and (c and f) TNFα monitored by quantitative RT-PCR with GAPDH used as the housekeeping gene. Results represent the means ± SEM of 3 separate experiments; ##p<0.01, ###p<0.001 versus all other conditions; *p<0.05, ***p<0.001 versus control; †††p<0.001 as indicated. (g) Media collected from RAW264.7 cells treated with or without 2μg/mL recombinant S100A9 (or S100A9 that had been boiled) in the presence and absence of 100μg/mL DOPG were analyzed by ELISA for TNFα levels. Results represent the means ± SEM of 3 separate experiments; ***p<0.001 versus the control; †††p<0.001 versus boiled S100A9.

Figure 2.. A TLR1/2 and/or TLR4 antagonist inhibits S100A9-induced inflammatory mediator expression in keratinocytes and the macrophage cell line.

(a-c) Keratinocytes or (d-f) RAW264.7 cells were treated with or without 2μg/mL recombinant S100A9 in the presence and absence of 25μM CU-CPT22, a TLR1/2 antagonist, or 10μM TAK242, a TLR4 antagonist, for 2 hours. RNA was then isolated and the expression of the inflammatory mediators, (a and d) IL-1β, (b and e) IL6 and (c and f) TNFα was monitored by quantitative RT-PCR with GAPDH used as the housekeeping gene. Results represent the means ± SEM of 4–5 separate experiments; **p<0.01, ***p<0.001 versus the control; ^† p<0.05, ^†† p<0.01, and ^†††p<0.001 as indicated.

Figure 3.. DOPG, but not DOPC, inhibits p65-NFκB activation by recombinant S100A9 protein.

RAW264.7 cells were treated with or without 2μg/mL recombinant S100A9 (A9) in the presence and absence of 100μg/mL DOPG or 100μg/mL DOPC for 30 minutes. Cells were harvested and the phosphorylation (activation) status of p65-NFκB determined as described in Methods. (a) A representative Western blot is shown. (b) The cumulative results from 3 separate experiments are presented as means ± SEM. (c) RAW264.7 cells plated on coverslips were treated with or without 2μg/mL recombinant S100A9 (A9) in the presence and absence of 100μg/mL DOPG or DOPC, as indicated, for 60 minutes. Immunocytochemistry for p65-NFκB was performed as described in Methods and shown in grayscale (scale bar = 5μm). The figures are representative of 3 separate experiments, all showing similar results. (d) Quantification of nuclear staining by two observers blinded to the experiment and counted in at least two random micrographs; results from each observer were averaged and expressed as the percent maximal value. Values represent the means ± SEM of 3 independent experiments and were analyzed by ANOVA with Student-Newman-Keuls post-hoc tests; *p<0.05, ***p<0.001 versus the control; ^†p<0.05, ^††††p<0.001 as indicated.

Figure 4.. DOPG inhibits anti-microbial peptide DAMP-induced TLR activation in a TLR2 reporter cell line.

(a) HEK-Blue-hTLR2 cells were incubated with or without S100A9 (10μg/mL) in the presence and absence of 100μg/mL DOPG or DOPC for 24 h or (b) with and without human β-defensin-2 (hBD2, 25μg/mL) in the presence and absence of 100μg/mL DOPG in the HEK-Blue detection medium for 18 h. SEAP activity was measured as absorbance at 620 nm. Values represent the means ± SEM from 3 separate experiments; ***p<0.001 versus the control; ^††p<0.01, ^†††p<0.001 as indicated.

Figure 5.

DOPG improves psoriasiform lesions in the imiquimod-induced mouse model of psoriasis. Mice (n=4–5) were treated with vehicle (vaseline) or imiquimod daily for 5 days in the morning. In the afternoon water or DOPG in water (prepared by sonicating a DOPG lipid film in deionized water) was applied to the treated skin. (a) Ear thickness and (b) weight were measured and are shown as means ± SEM. (c) Psoriasis area severity index (PASI) scores were quantified in a blinded fashion (means ± SEM). Sections of formalin-fixed, paraffin-embedded back skin were stained with H&E. (d) Representative images are shown and (e) epidermal thickness quantified in multiple sections from each mouse as described in Methods. Additional sections were incubated with an antibody recognizing TNFα and visualized with a Cy3-conjugated secondary antibody. (f) Representative images and (g) TNFα immunoreactivity quantified in multiple sections from each mouse as described in Methods. One-way analysis of variance with Student-Newman-Keuls multiple comparison post-hoc testing was used to determine significant differences; **p<0.01, ***p<0.001 versus control; †p<0.05, †††p<0.001 versus IMQ alone.