Monocytes enhance the inflammatory response to TLR2 stimulation in aortic valve interstitial cells through paracrine up-regulation of TLR2 level

Abstract

Background and Objectives: Chronic valvular inflammation associated with monocyte infiltration promotes calcific aortic valve disease (CAVD) progression. Further, innate immunity in aortic valve interstitial cells (AVICs), mediated by Toll-like receptors (TLRs), up-regulates cellular inflammatory, fibrogenic and osteogenic activities. Currently, the pro-inflammatory communication between monocytes and AVICs and the underlying mechanism are unclear. We hypothesized that monocytes up-regulate AVIC inflammatory activity. This study sought to characterize the interaction between monocytes and AVICs and to elucidate the mechanism underlying cell-to-cell communication. Methods and Results: AVICs, monocytes and co-cultures were exposed to a low concentration of TLR2 activator Pam3CSK4 (0.03 µg/ml). The TLR2 activator at this dose induced a marked increase in AVIC production of ICAM-1 and VCAM-1 only when co-cultured with monocytes. Adding conditioned medium from Pam3CSK4-treated monocytes (Pam3 CM, containing 0.1 µg/ml of Pam3CSK4) to AVIC culture (30% vol/vol; diluting Pam3CSK4 to 0.03 µg/ml) greatly increased the expression of adhesion molecules while adding conditioned medium from untreated monocytes (control CM) had no effect. Inhibition or knockdown of TLR2 in AVICs markedly reduced ICAM-1 and VCAM-1 expression induced by Pam3 CM. Further, Pam3 CM increased TLR2 levels in AVICs. Multiplex-ELISA analysis of Pam3 CM identified greater levels of TNF-α. Neutralization of TNF-α abolished the effect of Pam3 CM on AVIC TLR2 levels, resulting in marked attenuation of its potency in the induction of adhesion molecule expression. Conclusions: This study demonstrates that activated monocytes use paracrine signaling to sensitize AVICs for inflammatory responses to a low level of TLR2 activator. The mechanism of sensitization involves up-regulation of AVIC TLR2 levels by TNF-α from monocytes. Infiltrated monocytes in aortic valve tissue may exacerbate valvular inflammation by rendering AVICs hypersensitive to TLR2 activators.

Keywords: Aortic valves; Toll-like receptor; adhesion molecules; inflammation; monocytes.

Figure 1

Pam3CSK4 induces inflammatory responses in AVIC and monocyte co-culture. A. AVICs and monocytes were cultured separately and treated with a low dose of Pam3CSK4 (0.03 µg/ml) for 24 h. Representative immunoblots and ELISA data of 4 separate experiments show that Pam3CSK4 elevates ICAM-1 level moderately and causes a slight increase in MCP-1 production in monocytes while Pam3CSK4 in this low dose has no effect on the levels of ICAM-1, VCAM-1 and MCP-1 in AVICs. B. AVIC and monocyte co-cultures were treated with Pam3CSK4 (0.03 µg/ml) for 24 hours or left untreated. Representative immunoblots and ELISA data show that low levels of ICAM-1, VCAM-1 and MCP-1 are present in untreated control. However, stimulation of TLR2 with a low dose of Pam3CSK4 markedly up-regulates the production of ICAM-1, VCAM-1 and MCP-1 in co-cultures. Data are presented as mean ± SEM. n=4 cell isolates from distinct donor valves, *P<0.05 vs. control. C. Representative images of ICAM-1 immunofluorescence staining from 3 experiments show that both AVICs and monocytes express ICAM-1 (red) following the exposure to a low dose of Pam3CSK4 (0.03 µg/ml). Immunofluorescence staining of vimentin (green) and DAPI counterstaining (blue) were applied to identify AVICs and to outline all nuclei in the co-culture. Original magnification 40× objective.

Figure 2

Conditioned medium from Pam3CSK4-treated monocytes induces inflammatory responses in human AVICs. Conditioned medium was collected from monocyte cultures treated with Pam3CSK4 (0.10 µg/ml, Pam3 CM) or untreated controls (control CM). Pam3 CM and control CM, 0.15 ml each, were added to AVIC culture in 0.35 ml growth medium [achieving a 30% (vol/vol) for CM in AVIC culture], and AVICs were treated with control CM or Pam3 CM for 24 hours. A. Representative immunoblots and densitometric data show that Pam3 CM markedly up-regulates ICAM-1 and VCAM-1 expression in AVICs while neither control CM nor 0.03 µg/ml of Pam3CSK4 (equivalent to the concentration in Pam3 CM-treated AVIC culture) demonstrated this effect. *P<0.05 vs. untreated control. B. ELISA data shows that Pam3 CM stimulates AVICs to produce and release MCP-1. *P<0.05 vs. untreated control. C. AVICs were treated with DMSO or CU CPT 22 for 1 hour before Pam3 CM treatment for 24 hours. Inhibition of TLR2 in AVICs markedly attenuated ICAM-1 and VCAM-1 expression induced by Pam3 CM. *P<0.05 vs. untreated control, #P<0.05 vs. Pam3 CM+DMSO. All data are presented as mean ± SEM. n=4 cell isolates from distinct donor valves.

Figure 3

Pam3 CM up-regulates TLR2 expression in AVICs to elicit cellular inflammatory responses. A. AVICs were exposed to Pam3CSK4 (0.03 μg/ml), control CM (30%) or Pam3 CM (30%) for 24 hours. Representative immunoblots and densitometric data show that Pam3 CM elevates TLR2 levels, but not MyD88 levels in AVICs. *P<0.05 vs. untreated control, Pam3CKS4-treated and control CM-treated. B. AVICs were treated with control CM or Pam3 CM for 4 to 24 hours. A representative immunoblot and densitometric data show that cellular TLR2 levels increase at 8, 12 and 24 hours following the exposure to Pam3 CM. *P<0.05 vs. control CM treatment for the same time period. Representative images of immunofluorescence staining confirmed the up-regulation of TLR2 (red) at 8-24 hours of treatment with Pam3 CM. The nuclei were visualized with DAPI (blue) counterstaining. Original magnification 40× objective. C and D. AVICs were transduced with lentivirus expressing control (mock) shRNA or TLR2 shRNA. After expression for 5 days, cells were treated with Pam3 CM for 24 hours. Knockdown of TLR2 abolished TLR2 up-regulation induced by Pam3 CM, and reduced the production of adhesion molecules and MCP-1. *P<0.05 vs. mock transduction, #P<0.05 vs. Pam3 CM + mock transduction. All quantitative data are presented as mean ± SEM. n=4 cell isolates from distinct donor valves in each group.

Figure 4

Denatured Pam3 CM fails to induce AVIC inflammatory responses. AVICs were treated with control CM, Pam3 CM or heat-denatured Pam3 CM for 24 hours. A representative immunoblot of 3 experiments shows that denatured Pam3 CM lost its effect on the expression of ICAM-1 and VCAM-1 in AVICs.

Figure 5

TNF-α up-regulates TLR2 and contributes to the mechanism underlying the inflammatory responses in AVICs, but recombinant TNF-α is inadequate to induce the inflammatory responses. A. ELISA assay confirmed that Pam3 CM has greater levels of TNF-α in comparison to control CM. Data are presented as mean ± SEM. n=5, *P<0.05 vs. control CM. B. AVICs were treated for 24 hours with Pam3 CM pre-incubated with TNF-α neutralizing antibody (10 µg/ml) or non-immune mouse IgG (10 µg/ml). Neutralization of TNF-α abolished the effect of Pam3 CM on TLR2 up-regulation and attenuated the capacity of Pam3 CM to induce ICAM-1 and VCAM-1 expression in AVICs. *P<0.05 vs. untreated control, #P<0.05 vs. Pam3 CM and Pam3 CM + IgG. C. AVICs were treated with recombinant TNF-α (1.0 or 2.0 ng/ml) or Pam3 CM for 24 hours. TNF-α in the tested doses increased TLR2 levels in AVICs, but had no effect on the expression of ICAM-1 and VCAM-1. Data are presented as mean ± SEM. n=4 cell isolates from distinct donor valves in each group. *P<0.05 vs. untreated control.

Figure 6

Schematic diagram depicting the mechanism underlying the interaction between monocytes and AVICs. Monocytes activated by TLR2 agonist release pro-inflammatory cytokines. Among these cytokines, TNF-α is responsible for up-regulating AVIC expression of TLR2. AVICs with greater levels of TLR2 become hypersensitive to TLR2 stimulation by a low level of activator.