Chitinase 3-like 1 regulates cellular and tissue responses via IL-13 receptor α2

Abstract

Members of the 18 glycosyl hydrolase (GH 18) gene family have been conserved over species and time and are dysregulated in inflammatory, infectious, remodeling, and neoplastic disorders. This is particularly striking for the prototypic chitinase-like protein chitinase 3-like 1 (Chi3l1), which plays a critical role in antipathogen responses where it augments bacterial killing while stimulating disease tolerance by controlling cell death, inflammation, and remodeling. However, receptors that mediate the effects of GH 18 moieties have not been defined. Here, we demonstrate that Chi3l1 binds to interleukin-13 receptor α2 (IL-13Rα2) and that Chi3l1, IL-13Rα2, and IL-13 are in a multimeric complex. We also demonstrate that Chi3l1 activates macrophage mitogen-activated protein kinase, protein kinase B/AKT, and Wnt/β-catenin signaling and regulates oxidant injury, apoptosis, pyroptosis, inflammasome activation, antibacterial responses, melanoma metastasis, and TGF-β1 production via IL-13Rα2-dependent mechanisms. Thus, IL-13Rα2 is a GH 18 receptor that plays a critical role in Chi3l1 effector responses.

Figure 1. Binding and Localization of Chi3l1/YKL-40 and IL-13Rα2

(A) A549 cells were transfected with Chi3l1/YKL-40 (Chi3l1) and/or human IL-13Rα2 (IL-13Rα2), lysates were prepared and immunoprecipitated (IP) with either anti-Chi3l1 or anti-iL-13Rα2, and the precipitates were evaluated using immunoblot (IB) analysis as noted. (B) Triple-label IHC to detect the colocalization of IL-13Rα2 and BRP-39 in the macrophages (upper panels) and type 2 alveolar epithelial cells (lower panels) in lungs from IL-13 Tg mice using antibodies to BRP-39, IL-13Rα2, and cell-specific markers of macrophages (anti-F4/80) and type 2 epithelial cells (anti-SP-C). Arrows highlight some of the colocalized cells. (C) Cell surface colocalization of Chi3l1/YKL-40 (Chi3l1) and IL-13Rα2 (IL-13Rα2). THP-1 cells were incubated in the presence or absence of anti-YKL-40-biotin antibody and anti-iL-13Rα2 immunoglobulin G (IgG) antibody without permeabilization. They were then washed and stained with streptavidin (SA)-PE and anti-igG-APC and subjected to flow cytometric analysis. (D) Measurement of the affinity and kinetics of IL-13Rα2 binding to Chi3l1/YKL-40 by surface plasmon resonance (SPR). Chil3l1/YKL-40 was immobilized and IL-13Rα2 was in the mobile phase. (E) Yeast two-hybrid characterization of the structures in hChi31 that bind to IL-13Rα2. The 18 GH catalytic domain (CD), chitin binding motif (CBM), signal peptide (SP), and C-terminal fragment are illustrated. (F) Yeast two-hybrid characterization of the structures in IL-13Rα2 that bind to hChi31. The extracellular domain (ECD), transmembrane domain (TD), intracellular domain (ICD), signal peptide (SP), and sites of N-glycosylation (N) are illustrated. Each panel is representative of a minimum of three evaluations. Scale bars in (B) represent 10 μm. See also Figure S1.

Figure 2. Chi3l1 and IL-13Rα2 Regulation of Macrophage Signaling

(A and B) THP-1 cells were incubated with recombinant Chi3l1/YKL-40 (rChi3l1) for the noted periods of time at the noted doses. Western blot evaluations were used to evaluate ERK1/2 phosphorylation (ERK-P), total ERK 1/2 (ERK-T), AKT phosphorylation (AKT-P), total AKT (AKT-T), β-catenin nuclear translocation, and nuclear c-fos accumulation. (C and D) The role(s) of IL-13Rα2 in these responses was assessed by comparing these signaling events in THP-1 cells treated with IL-13Rα2 siRNA (siRNA⁺) or scrambled controls (siRNA⁻) (C) and peritoneal macrophages from wild-type (+/+) and IL-13Rα2 null (−/−) mice (D). Each panel is representative of a minimum of three evaluations. (E) The effects of recombinant soluble IL-13Rα2 (rsIL-13Rα2) on Chi3l1-stimulated signaling were also assessed. In these experiments, peritoneal macrophages from WT and IL-13Rα2 null mice were treated with PBS or rsIL-13Rα2 and rChi3l1 as indicated and ERK and AKT activation was evaluated by western blot analysis. (F) Peritoneal macrophages from IL-13Rα2 null (−/−) mice was transfected with full-length (FL) IL-13Rα2, a truncated construct (TC) of IL-13Rα2 that lacked its intracellular domain, or an empty vector. After incubation of the cells with rChi3l1 (500 ng/ml) as indicated, cell lysates were prepared and ERK and AKT activation was evaluated by western blot analysis. See also Figure S2.

Figure 3. Interactions of IL-13, IL-13Rα2, and Chi3l1/YKL-40/BRP-39

(A) Triple-label IHC was used to detect the colocalization of IL-13, IL-13Rα2, and Chi3l1/BRP-39 in lungs from IL-13 Tg mice using antibodies to Chi3l1/BRP-39, IL-13Rα2, and IL-13. Arrows highlight some of the colocalized cells. (B) Lung lysates were prepared from WT and IL-13 transgenic mice and immunoprecipitated (IP) with anti-Chi3l1, anti-iL-13Rα2, or anti-iL-13, and the precipitates were evaluated using immunoblot (IB) analysis as noted. (C) THP-1 cells were incubated with recombinant (r) IL-13 or Chi3l1/YKL-40 (rChi3l1) alone and in combination as noted. Western blot evaluations were used to evaluate ERK1/2 phosphorylation, total ERK1/2, AKT phosphorylation, and total AKT. (D) Peritoneal macrophages from Chi3l1^−/− mice were incubated overnight with rIL-13 (10 ng/ml), cell lysates were prepared, rChi3l1 was added at the noted doses, and immunoprecipitation and immunoblot analysis was undertaken with anti-iL-13 or anti-iL-13Rα2 antibodies as noted. (E) THP-1 cells were incubated with rIL-13 and rChi3l1 as indicated and western blot evaluations were used to evaluate STAT6 phosphorylation (STAT6-P) and total STAT 6 (STAT6-T). (F) Peritoneal macrophages were isolated from IL-13Rα2^−/− mice and transfected with empty vector, full-length IL-13Rα2 (IL-13Rα2-myc), or deletion mutants lacking IL-13 binding site 2 (IL-13Rα2^dS2-myc) or site 3 (IL-13Rα2^dS3-myc). Cell lysates were prepared, incubated with rChi3l1 (500 ng) and rIL-13 (10 ng) overnight, and evaluated using IP and IB with anti-myc, anti-iL-13, or anti-Chi3l1 as noted.

Figure 4. Chi3l1 and IL-13Rα2 Regulation of Cell Death and Acute Oxidant Injury

WT (+/+), Chi3l1/BRP-39 null (−/−), IL-13Rα2 null (−/−), Chi3l1/YKL-40 Tg (+), Chi3l1/YKL-40 Tg(+)/BRP-39(−/−), and Chi3l1/YKL-40 Tg(+)/IL-13Rα2(−/−) mice were exposed to 100% O₂ or room air (100% O₂ −) for 48 hr. (A) TUNEL staining of lung sections from those mice. (B) BAL fluid total protein. (C) Airway epithelial cells (1HAEo) were exposed for 6 hr to H₂O₂ (800 μM). Cells were used that had been treated with IL-13Rα2-specific siRNA or scrambled controls and incubated in the presence and absence of recombinant(r) Chi3l1. Annexin V and propidium iodide staining was evaluated by FACS analysis. (D and E) Peritoneal macrophages from WT (+/+), Chi3l1 null (−/−), and IL-13Rα2 null (−/−) mice were incubated with H₂O₂ (H₂O₂ +) or its vehicle control (H₂O₂ −) in the presence and absence of rChi3l1/BRP-39, and TUNEL staining (D) and LDH release (E) were evaluated. Values in (A), (B), (D), and (E) represent the means ± SEM of evaluations of a minimum of four mice. (C) is representative of at least three similar evaluations. *p < 0.05, **p < 0.01. NS, not significant. See also Figure S4.

Figure 5. Chi3l1 and IL-13Rα2 Regulation of SP-Induced Responses

(A and B) Peritoneal macrophages from WT (+/+), Chi3l1 null (−/−), and IL-13Rα2 null (−/−) mice were incubated with SP (SP+) or its vehicle control (SP−) in the presence and absence of rChi3l1/BRP-39, and TUNEL staining (A) and LDH release (B) were assessed. (C) Western blotting was also used to evaluate cell lysate caspase-1 activation. (D) Peritoneal macrophages from WT (+/+), Chi3l1 null (−/−), and IL-13Rα2 null (−/−) mice were also incubated with SP (SP+) or SP preincubated for 60 min without antibiotics with rChi3l1/BRP-39 at a 10:1 SP-to-cell ratio (SP/rChi3l1). They were then incubated with gentamicin to kill extracellular bacteria and the viable bacteria in the cell lysates were assessed 6 hr later. Values in (A), (B), and (D) are the means ± SEM of triplicate measurements and are representative of a minimum of three similar evaluations; (C) is representative of three similar evaluations. *p < 0.05, **p < 0.01. NS, not significant.

Figure 6. Chi3l1 and IL-13Rα2 Regulation of SP-Induced Inflammasome Activation

Peritoneal macrophages were obtained from WT mice, Chi3l1/BRP-39 null (−/−) mice, and IL-13Rα2 null (−/−) mice and incubated with SP. (A) The kinetics of IL-1β production were evaluated by ELISA. (B) IL-1β maturation and release into the cell supernatant (SN) was evaluated by western blotting. (C–F) The ability of rmChi3l1/BRP-39 to modulate IL-1β production (C) and maturation (D) caspase-1 activity measured by ELISA (E) and bacterial load were also evaluated. Values in (A), (C), (E), and (F) are the means ± SEM of triplicate measurements and are representative of a minimum of three similar evaluations. (B) and (D) are representative of three evaluations. *p < 0.05. NS, not significant.

Figure 7. Chi3l1 and IL-13Rα2 in Melanoma Metastasis and TGF-β1 Production In Vivo

WT mice, IL-13Rα2 null (−/−) mice, Chi3l1/YKL-40 Tg mice (Tg+), and Chi3l1/YKL-40 Tg/IL-13Rα2 null mice received B16-F10 melanoma cells by tail-vein injection. (A) Fourteen days later, melanoma metastasis was visually assessed. (B) Quantification of pleural melanoma colonies. (C) ELISA evaluation of total BAL TGF-β1. (D) ELISA evaluation of activated TGF-β1. (A) is representative of three similar evaluations. Values in (B)–(D) are the means ± SEM of triplicate measurements and are representative of a minimum of three similar evaluations. *p < 0.05.