Signaling through the IL-6-STAT3 Pathway Promotes Proteolytically-Active Macrophage Accumulation Necessary for Development of Small AAA

Abstract

Introduction: Elevated interleukin-6 (IL-6) plasma levels have been associated with abdominal aortic aneurysm (AAA), but whether this cytokine plays a causative role in the degenerative remodeling or represents an effect from the inflammatory cascades initiated by infiltrating leukocytes remained unclear. This project aims to demonstrate that within the aortic wall, signaling from IL-6 through the STAT3 transcription factor is necessary for infiltration of proteolytically-active macrophages and development of small AAA.

Methods: Following measurement of baseline infrarenal aortic diameter (AoD, digital microscopy), C57Bl/6 and IL-6 knockout (IL-6KO) mice underwent AAA induction by application of peri-adventitial CaCl₂ (0.5 M) +/- implantation of an osmotic mini-pump delivering IL-6 (4.36 µg/kg/day over 21 days). At the terminal procedure, AoDs were measured by digital microscopy and aortas harvested for immunoblot (pSTAT3/STAT3), matrix metalloproteinase (MMP) quantification, or flow cytometric analysis of macrophage content. Plasma was collected for cytokine analysis.

Results: IL-6 infusion significantly increased the plasma IL-6 levels in C57Bl/6 and IL-6KO animals. The C57Bl/6 + CaCl₂ group developed AAA (AoD >50% above baseline) but IL-6KO + CaCl₂ did not. In the IL-6KO + IL-6+CaCl2 group, AAA developed to match that of C57Bl/6 + CaCl₂ mice. STAT3 activity was significantly increased in animals with advanced stages of dilation (>40% from baseline), compared to those with ectasia (≤25%). Although cytokine profiles did not support T-cells or neutrophils as being active contributors in this stage of aortic remodeling, changes in the profile of elaborated MMPs suggested macrophage activity with a trend toward alternatively activated pathways. Flow cytometry confirmed significantly increased macrophage abundance specifically in animals with upregulated STAT3 activity and advanced aortic dilation.

Conclusion: In this murine model of AAA, progressive dilation to development of true AAA was only accomplished when IL-6 signaling upregulated STAT3 activity to effect accumulation of proteolytically-active macrophages. This pathway warrants further investigation to identify potential therapeutic avenues to abrogate growth of small AAA.

Keywords: STAT3; abdominal aortic aneurysm; interleukin-6.

Figure 1.

Plasma concentration of IL-6 in treatment groups (n = 3–5). (a): P = .001 vs C57Bl/6; (b): P < .001 vs C57Bl/6; (c): P = .020 vs C57Bl/6; (d): P = .031 vs IL-6KO; (e): P = .011 vs C57Bl/6; (f): P = .024 vs IL-6KO.

Figure 2.

(A) The percent change in aortic diameter (AoD) on day 21 after IL-6 infusion, CaCl₂ application, or both as compared to the internal baseline (AoD on day 0; n = 5–10). (a): P < .001 vs C57Bl/6; (b): P < .001 vs C57Bl/6 +IL-6; (c): P = .002 vs IL-6KO; (d): P < .001 vs IL-6KO; (e): P < .001 vs IL-6KO + IL-6, IL-6KO + CaCl₂. (B) Representative in vivo digital images utilized to measure AoD (outer wall to outer wall).

Figure 3.

(A) The STAT3 activity (pSTAT3/STAT3 ratio) in homogenized aorta and normalized to the C57Bl/6 control mice. (a): P = .007 vs C57Bl/6; (b): P = .015 vs C57Bl/6; (c): P = .039 vs C57Bl/6+IL-6; (d): P < .001 vs C57Bl/6; (e): P < .001 vs C57Bl/6 + IL-6; (f): P < .027 vs C57Bl/6 + CaCl₂; (g): P < .001 vs IL-6KO, IL-6KO + IL-6, IL-6KO + CaCl₂ (B) Representative Western Blot is displayed. (n = 4–6).

Figure 4.

Plasma concentration of (A) IL-2, (B) IL-5 a: P < .001 vs C57Bl/6; (b): P = .044 vs C57Bl/6 +IL-6; (c): P = .027 vs C57Bl/6 +IL-6, (C) IL-10, (D) IL-13 a: P = .009 vs C57Bl/6; b: P = .038 vs C57Bl/6 +IL-6; c: P = .001 vs IL-6KO; d: P = .006 vs IL-6+IL-6+CaCl₂, and (E) IL-17a (n = 3–5).

Figure 5.

Plasma concentration of neutrophil recruiting chemokines (A) CXCL1, (B) CXCL2, (C) CXCL5 (n = 3–5).

Figure 6.

Aortic homogenate concentration of (A) MMP-2 a: P < .001 vs C57Bl/6; (b): P < .001 vs C57BL/6+IL-6; c: P = .05 vs C57Bl/6; d: P = .002 vs C57Bl/6 +IL-6; e: P = .013 vs IL-6KO; f: P = .008 vs IL-6KO + IL-6; g: P = .002 vs C57Bl/6 +IL-6; h: P < .001 vs IL-6KO; i: P < .001 vs IL-6KO + IL-6, (B) MMP-3 a: P < .001 vs C57Bl/6 and C57BL/6+IL-6; b: P = .021 vs C57Bl/6; c: P = .009 vs IL-6KO; d: P = .036 vs IL-6KO, (C) MMP-8 a: P = .045 vs C57Bl/6 +IL-6 b: P = .018 vs C57Bl/6 + CaCl₂, (D) pro-MMP-9 a: P = .043 vs C57Bl/6; b: P = .007 vs C57Bl/6 + IL-6; c: P = .001 vs C57Bl/6 + CaCl₂; d: P = .008 vs C57Bl/6 + CaCl₂; e: P = .004 vs IL-6KO + IL-6; f: P = .034 vs IL-6KO+ CaCl₂, and (E) MMP-12 a: P = .035 vs C57Bl/6; b: P < .001 vs C57Bl/6 and C57Bl/6 +IL-6; c: P < .001 vs C57Bl/6; d: P < .001 vs IL-6KO and IL-6KO + IL-6; e: P < .001 vs IL-6KO, IL-6KO + IL-6, IL-6KO + CaCl₂ (n = 3–5).

Figure 7.

Macrophage accumulation in the infrarenal aorta as quantified by flow cytometric analysis for cells positive for CD11b (myeloid lineage marker) and F480 (mature murine macrophage marker) (a): P = .032 vs C57Bl/6; (b): P < .001 vs C57Bl/6; c: P = .001 vs C57Bl/6 +IL-6; d: P < .001 vs C57Bl/6 +IL-6; e: P = .002 vs C57Bl/6 + CaCl₂; f: P = .027 vs IL-6KO; g: P < .001 vs IL-6KO; h: P < .001 vs IL-6KO + IL-6 and IL-6KO + CaCl₂ (n = 4–8).