Inhibiting triggering receptor expressed on myeloid cells 1 signaling to ameliorate skin fibrosis

Abstract

Systemic sclerosis (SSc) is characterized by immune system failure, vascular insult, autoimmunity, and tissue fibrosis. TGF-β is a crucial mediator of persistent myofibroblast activation and aberrant extracellular matrix production in SSc. The factors responsible for this are unknown. By amplifying pattern recognition receptor signaling, triggering receptor expressed on myeloid cells 1 (TREM-1) is implicated in multiple inflammatory conditions. In this study, we used potentially novel ligand-independent TREM-1 inhibitors in preclinical models of fibrosis and explanted SSc skin fibroblasts in order to investigate the pathogenic role of TREM-1 in SSc. Selective pharmacological TREM-1 blockade prevented and reversed skin fibrosis induced by bleomycin in mice and mitigated constitutive collagen synthesis and myofibroblast features in SSc fibroblasts in vitro. Our results implicate aberrantly activated TREM-1 signaling in SSc pathogenesis, identify a unique approach to TREM-1 blockade, and suggest a potential therapeutic benefit for TREM-1 inhibition.

Keywords: Autoimmune diseases; Autoimmunity; Infectious disease; Rheumatology.

Figure 1. Pharmacological inhibition of TREM-1 prevents early loss of dermal adipose tissue.

C57BL/6J mice received daily subcutaneous injections of PBS or bleomycin alone or together with GF9 and GA31-LPC or vehicle for 5 days. Mice were sacrificed on day 8, and skin was harvested for analysis. (A) Representative images of Trichrome staining. Scale bar: 100 μm. (B) Assessment of dermal thickness from A (8 determinations/hpf) and real-time quantitative PCR. Results were normalized with GAPDH and are shown as mean ± SD of triplicate determinations from 6 mice per group. One-way ANOVA followed by Šidák’s multiple comparison test. P values are as shown. (C) Immunolabeling using antibodies against perilipin (green), procollagen I (red), and DAPI (blue). Representative images and P values are shown. Scale bar: 100 μm. (D) Immunolabeling using antibodies against ASMA (green) and DAPI (blue). Quantification of ASMA-positive cells is shown (an average of 4 randomly selected regions from 4 mice/group). One-way ANOVA followed by Šidák’s multiple comparisons test. Scale bar: 100 μm. P values are shown.

Figure 2. Inhibition of TREM-1 signaling by GA31-LPC treatment prevents skin fibrosis.

C57BL/6J mice were treated for 2 weeks. They were sacrificed on day 22, and skin was harvested for analysis. (A) Representative images of Trichrome staining and assessment of dermal thickness. Scale bar: 100 μm. Results are shown as the mean ± SD of 8 determinations/hpf. One-way ANOVA followed by Šidák’s multiple comparisons test. P values are shown. (B) Skin hydroxyproline assays. Results are shown as mean ± SEM. P values are shown. (C) Immunolabeling using antibodies against ASMA (green) and DAPI (blue). Representative images are shown. Scale bar: 100 μm. ASMA-positive cells (an average of 4 randomly selected regions per group). One-way ANOVA followed by Šidák’s multiple comparisons test. P values are shown.

Figure 3. Inhibition of TREM-1 signaling by GF9 and GA31-LPC treatment mitigates established skin fibrosis.

C57BL/6J mice were randomized to 4 treatment groups (n = 5–8 mice/group). They were euthanized on day 28, and skin was harvested. (A) Representative images of Trichrome staining and assessment of dermal thickness. Scale bar: 100 μm. Results are shown as the mean ± SD of 5–8 determinations/hpf. One-way ANOVA was followed by Šidák’s multiple comparisons test. P values are shown. (B) Hydroxyproline assays. One-way ANOVA followed by Šidák’s multiple comparisons test. P values are shown.

Figure 4. TREM-1 signaling is activated in SSc skin.

(A) Skin biopsies from patients with SSc (n = 8) and individuals acting as healthy controls (n = 4) were immunolabeled with antibodies against pSyk or ASMA, and immunofluorescence was visualized by Nikon A1R laser scanning confocal microscope. The percentage of immuno-positive cells (mean percentages from 4 randomly selected regions) was quantified. Mann-Whitney U test. P values are shown. Original magnification, ×40. (B) Confluent SSc skin fibroblasts (n = 8, top; n = 3, bottom) were incubated with GF9 for 24 hours, and mRNA levels were quantitated by real-time quantitative PCR. Results were normalized with GAPDH and are shown as the mean ± SD of triplicate determinations from individual patients. Paired t test. P values are shown. (C) SSc fibroblasts (n = 6) were immunolabeled using antibodies against collagen I, ASMA, or pSyk. Scale bar: 100 μm. Representative images and relative fluorescence intensities (mean ± SEM from 3 randomly selected regions) are shown.