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. 2014 Jul;21(1):208-19.
doi: 10.1016/j.intimp.2014.05.001. Epub 2014 May 14.

A novel ligand-independent peptide inhibitor of TREM-1 suppresses tumor growth in human lung cancer xenografts and prolongs survival of mice with lipopolysaccharide-induced septic shock

Alexander B Sigalov  1
Affiliations

A novel ligand-independent peptide inhibitor of TREM-1 suppresses tumor growth in human lung cancer xenografts and prolongs survival of mice with lipopolysaccharide-induced septic shock

Alexander B Sigalov. Int Immunopharmacol. 2014 Jul.

Abstract

Triggering receptor expressed on myeloid cells-1 (TREM-1) amplifies the inflammatory response and plays a role in cancer and sepsis. Inhibition of TREM-1 by short hairpin RNA (shRNA) in macrophages suppresses cancer cell invasion in vitro. In the clinical setting, high levels of TREM-1 expression on tumor-associated macrophages are associated with cancer recurrence and poor survival of patients with non-small cell lung cancer (NSCLC). TREM-1 upregulation on peritoneal neutrophils has been found in human sepsis patients and in mice with experimental lipopolysaccharide (LPS)-induced septic shock. However, the precise function of TREM-1 and the nature of its ligand are not yet known. In this study, we used the signaling chain homooligomerization (SCHOOL) model of immune signaling to design a novel, ligand-independent peptide-based TREM-1 inhibitor and demonstrated that this peptide specifically silences TREM-1 signaling in vitro and in vivo. Utilizing two human lung tumor xenograft nude mouse models (H292 and A549) and mice with LPS-induced sepsis, we show for the first time that blockade of TREM-1 function using non-toxic and non-immunogenic SCHOOL peptide inhibitors: 1) delays tumor growth in xenograft models of human NSCLC, 2) prolongs survival of mice with LPS-induced septic shock, and 3) substantially decreases cytokine production in vitro and in vivo. In addition, targeted delivery of SCHOOL peptides to macrophages utilizing lipoprotein-mimicking nanoparticles significantly increased peptide half-life and dosage efficacy. Together, the results suggest that ligand-independent modulation of TREM-1 function using small synthetic peptides might be a suitable treatment for sepsis and NSCLC and possibly other types of inflammation-associated disorders.

Keywords: HDL nanoparticles; Non-small cell lung cancer; Sepsis; TREM-1 receptor; Targeted delivery; Therapeutic SCHOOL peptides.

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Figures

Figure 1
Figure 1
Schematic representation of the proposed concepts. (A) Inhibiting TREM-1 by synthetic peptides designed using a novel model of immune signaling, the Signaling Chain HOmoOLigomerization (SCHOOL) model. In contrast to current approaches, these inhibitors employ ligand-independent mechanisms of action and block transmembrane interactions between TREM-1 and its signaling partner, DAP-12. (B) Targeting high density lipoproteins (HDL) to macrophages by using a specific and naturally occurring modification (sulfoxidation) of the HDL major protein, apolipoprotein (apo) A-I. In the human body, native unmodified HDL (depicted by blue) function to deliver excess cholesterol from peripheral tissues to the liver and are not normally uptaken by macrophages. In contrast, synthetic HDL containing oxidized apo A-I or its peptides (depicted by red) are uptaken by macrophages, thus delivering the incorporated payload(s) directly to the cells of interest. Abbreviations: apo, apolipoprotein; DAP-12, DNAX activation protein of 12 kDa; IL, interleukin; ITAM, immunoreceptor tyrosine-based activation motif; M-CSF, macrophage colony-stimulating factor; TNF, tumor necrosis factor; TREM-1, triggering receptor expressed on myeloid cells-1.
Figure 2
Figure 2
Representative electron microscopy images of HDL of discoidal (A) and spherical (B) morphology that contain the incorporated TREM-1 SCHOOL peptide GF9. Characteristic rouleaux (depicted by circles and zoomed in on the right panels) are found in disc-shaped HDL particles (A). Particles with unmodified apo A-I are shown for illustrative purposes. Similar shape, size and size distribution are observed for HDL with oxidized apo A-I or its unmodified and oxidized peptides. Abbreviations: apo, apolipoprotein; high density lipoproteins, HDL; SCHOOL, signaling chain homooligomerization; TREM-1, triggering receptor expressed on myeloid cells-1.
Figure 3
Figure 3
Oxidation of apo A-I or synthetic apo A-I peptides enhances in vitro macrophage uptake of HDL that contain the incorporated TREM-1 SCHOOL peptide GF9. (A) Mean fluorescence intensities of cell lysates normalized to cell protein content (mean ± SD, n = 3): J774 macrophages were incubated at 37°C for 4 h with medium only (white bars) or with 4 µM Rho-B fluorescent GF9-HDL(A-I) or GF9-HDL(H4+H6) of discoidal or spherical morphology. Unmodified and oxidized protein/peptide species are depicted by light and dark gray bars, respectively. ***, P = 0.0001 to 0.001 as compared with unmodified apo A-I protein/peptides. (B) Mean fluorescence intensities of cell lysates normalized to cell protein content (mean ± SD, n = 3): J774 macrophages were incubated at 37°C for 4, 12 and 24 h (white, light gray and dark gray bars, respectively) with 4 µM Rho-B fluorescent GF9-HDL(A-I) or GF9-HDL(H4+H6) of discoidal or spherical morphology that contain oxidized apo A-I or its peptides, H4 and H6. ***, P = 0.0001 to 0.001 as compared with 4 h time point. Abbreviations: apo, apolipoprotein; high density lipoproteins, HDL; HDL(A-I), HDL with apo A-I; HDL(H4+H6), HDL with a 1:1 mixture of synthetic peptides H4 and H6 that correspond to apo A-I helixes 4 and 6, respectively; dHDL and sHDL, discoidal and spherical HDL, respectively; Rho-B, rhodamine B; SCHOOL, signaling chain homooligomerization; TREM-1, triggering receptor expressed on myeloid cells-1.
Figure 4
Figure 4
The TREM-1 SCHOOL peptide GF9 in free form inhibits tumor growth in the H292 (A) and A549 (B) xenograft mouse models of NSCLC. H292 or A549 cells were injected subcutaneously into nude mice with randomization (n = 10) once tumors reached an average of 200 mm3. Vehicle, paclitaxel (PTX) as a positive control, and GF9 peptide were all intraperitoneally administered twice a week. Bars, SEM. For statistical analysis, each treatment was compared with the vehicle control using repeated measures ANOVA followed by Bonferroni test. ****, P < 0.0001. Abbreviations: NSCLC, non-small cell lung cancer; SCHOOL, signaling chain homooligomerization; TREM-1, triggering receptor expressed on myeloid cells-1.
Figure 5
Figure 5
The TREM-1 SCHOOL peptide GF9 incorporated into macrophage-targeted HDL that contain oxidized apo A-I inhibits tumor growth in the H292 (A) and A549 (B) xenograft mouse models of NSCLC. H292 or A549 cells were injected subcutaneously into nude mice with randomization (n = 10) once tumors reached an average of 200 mm3. Vehicle (HDL containing no GF9) and paclitaxel (PTX) as a positive control were intraperitoneally administered twice a week, while GF9-dHDL and GF9-sHDL were all intraperitoneally administered twice or once (indicated as "1/wk") a week. Bars, SEM. For statistical analysis, each treatment was compared with the vehicle control using repeated measures ANOVA followed by Bonferroni test. **, P = 0.01 to 0.001; ****, P < 0.0001. Abbreviations: apo, apolipoprotein; HDL, high density lipoproteins; dHDL and sHDL, discoidal and spherical HDL, respectively; NSCLC, non-small cell lung cancer; SCHOOL, signaling chain homooligomerization; TREM-1, triggering receptor expressed on myeloid cells-1.
Figure 6
Figure 6
The TREM-1 SCHOOL peptide GF9 incorporated into macrophage-targeted HDL that contain an equimolar mixture of oxidized apo A-I peptides H4 and H6 inhibits tumor growth in the A549 xenograft mouse model of NSCLC. (A) A549 cells were injected subcutaneously into nude mice with randomization (n = 10) once tumors reached an average of 200 mm3. Vehicle (HDL containing no GF9), paclitaxel (PTX) as a positive control, GF9-dHDL(H4+H6), and GF9- sHDL(H4+H6) were all intraperitoneally administered twice a week. Bars, SEM. For statistical analysis, each treatment was compared with the vehicle control using repeated measures ANOVA followed by Bonferroni test. ****, P < 0.0001. (B) Average weights of excised tumors postnecroscopy from mice in each group treated with vehicle, PTX and GF9-HDL that contain either oxidized apo A-I or its oxidized peptides H4 and H6. Once a week administration is indicated as "1/wk". Bars, SEM. For statistical analysis, each treatment was compared with the vehicle control using Student’s t test. **, P = 0.001 to 0.01. Abbreviations: apo, apolipoprotein; HDL, high density lipoproteins; HDL(H4+H6), HDL with a 1:1 mixture of synthetic peptides H4 and H6 that correspond to apo A-I helixes 4 and 6, respectively; dHDL and sHDL, discoidal and spherical HDL, respectively; NSCLC, non-small cell lung cancer; SCHOOL, signaling chain homooligomerization; TREM-1, triggering receptor expressed on myeloid cells-1.
Figure 7
Figure 7
The TREM-1 SCHOOL peptide GF9 in free form prolongs survival of mice with LPS-induced septic shock at a dose of 25 mg/kg but contributes to death at a dose of 150 mg/kg, while being non-toxic in healthy mice up to a dose of 300 mg/kg. (A) C57BL/6 mice (n = 10) were intraperitoneally administered with vehicle or the indicated doses of GF9 peptide 1 h before LPS administration (30 mg/kg). The Kaplan-Meier survival curves were analyzed using the log-rank test. **, P = 0.001 to 0.01 as compared with vehicle-treated animals. (B) The average weight of healthy C57BL/6 mice (n=5) intraperitoneally administered with the indicated doses of GF9 peptide in free form. Bars, SEM. Abbreviations: LPS, lipopolysaccharide; SCHOOL, signaling chain homooligomerization; TREM-1, triggering receptor expressed on myeloid cells-1.
Figure 8
Figure 8
The TREM-1 SCHOOL peptide GF9 incorporated into macrophage-targeted HDL that contain oxidized apo A-I prolongs survival of mice with LPS-induced septic shock. (A) C57BL/6 mice (n=10) were intraperitoneally administered with vehicle (HDL containing no GF9) or the indicated doses of GF9, GF9-dHDL, and GF9-sHDL 1 h before LPS administration (30 mg/kg). Survival curves during the 40-h (A) and 7-d (B) periods after LPS injection are illustrated. Expected apparent half-lifes of agents in circulation are shown in the inset (B). The Kaplan-Meier survival curves were analyzed using the log-rank test. **, P = 0.001 to 0.01 as compared with vehicle-treated animals. Abbreviations: apo, apolipoprotein; HDL, high density lipoproteins; dHDL and sHDL, discoidal and spherical HDL, respectively; LPS, lipopolysaccharide; SCHOOL, signaling chain homooligomerization; TREM-1, triggering receptor expressed on myeloid cells-1.
Figure 9
Figure 9
The TREM-1 SCHOOL peptide GF9 in free and HDL-bound form inhibits cytokine production in vitro and in vivo. (A) Release of cytokines from J774 macrophages. Cells were cultured for 24 h at 37°C in the presence of LPS (1 µg/ml) in combination with 50 ng/ml control peptide or GF9 peptide in free and HDL-bound form. After incubation, culture medium samples were subjected to TNF-α, IL-6, and IL-1β determination by the appropriate ELISA kits. Results represent the mean ± S.D. of three independent experiments. ***, P = 0.0001 to 0.001 as compared with medium-treated LPS-challenged cells. (B) Analysis of serum cytokine levels in mice during LPS-induced endotoxemia. Mice (n = 10) were treated with PBS or the indicated doses of dexamethasone (DEX), control peptide or GF9 peptide in free and HDL-bound form 1 h before LPS administration (30 mg/kg). Blood samples were obtained at 90 min post LPS challenge, and serum was screened for levels of TNF-α, IL-6, and IL-1β using the appropriate ELISA kits. *, P = 0.01 to 0.05 as compared with animals treated with 25 mg/kg GF9; ***, P = 0.0001 to 0.001 as compared with PBS-treated animals. Abbreviations: ELISA, enzyme-linked immunosorbent assay; HDL, high density lipoproteins; dHDL and sHDL, discoidal and spherical HDL, respectively; IL, interleukin; LPS, lipopolysaccharide; SCHOOL, signaling chain homooligomerization; PBS, phosphate-buffered saline; TNF, tumor necrosis factor; TREM-1, triggering receptor expressed on myeloid cells-1.
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