Modulation of Neuro-Inflammatory Signals in Microglia by Plasma Prekallikrein and Neuronal Cell Debris

Abstract

Microglia, the resident phagocytes of the central nervous system and one of the key modulators of the innate immune system, have been shown to play a major role in brain insults. Upon activation in response to neuroinflammation, microglia promote the release of inflammatory mediators as well as promote phagocytosis. Plasma prekallikrein (PKall) has been recently implicated as a mediator of neuroinflammation; nevertheless, its role in mediating microglial activation has not been investigated yet. In the current study, we evaluate the mechanisms through which PKall contributes to microglial activation and release of inflammatory cytokines assessing PKall-related receptors and their dynamics. Murine N9-microglial cells were exposed to PKall (2.5 ng/ml), lipopolysaccharide (100 ng/ml), bradykinin (BK, 0.1 μM), and neuronal cell debris (16.5 μg protein/ml). Gene expression of bradykinin 2 receptor (B₂KR), protease-activated receptor 2 (PAR-2), along with cytokines and fibrotic mediators were studied. Bioinformatic analysis was conducted to correlate altered protein changes with microglial activation. To assess receptor dynamics, HOE-140 (1 μM) and GB-83 (2 μM) were used to antagonize the B₂KR and PAR-2 receptors, respectively. Also, the role of autophagy in modulating microglial response was evaluated. Data from our work indicate that PKall, LPS, BK, and neuronal cell debris resulted in the activation of microglia and enhanced expression/secretion of inflammatory mediators. Elevated increase in inflammatory mediators was attenuated in the presence of HOE-140 and GB-83, implicating the engagement of these receptors in the activation process coupled with an increase in the expression of B₂KR and PAR-2. Finally, the inhibition of autophagy significantly enhanced the release of the cytokine IL-6 which were validated via bioinformatics analysis demonstrating the role of PKall in systematic and brain inflammatory processes. Taken together, we demonstrated that PKall can modulate microglial activation via the engagement of PAR-2 and B₂KR where PKall acts as a neuromodulator of inflammatory processes.

Keywords: bradykinin 2 receptor; cytokines; interactome; neuroinflammation; plasma kallikrein-kinin system; protease-activated receptor 2.

FIGURE 1

Expression of components of the plasma kallikrein-kinin system (PKKS) in microglial cells. Microglial cells grown in 2% FBS were stimulated with LPS (100 ng/ml), BK (0.1 µM), PKall (2.5 ng/ml) and NCD (16.5 µg protein/ml) for 24 h. The boxplots demonstrate the mRNA levels of (A) PAR 2, (B) B2KR and (C) KNG expressed relative to GAPDH mRNA levels assessed concomitantly at the same time in the same samples (*p ≤ 0.05, ***p ≤ 0.005 vs. Control, n = 5–7). The boxplots (D) PAR 2 and (E) B₂KR, demonstrate the mRNA levels of these receptors expressed relative to GAPDH mRNA levels assessed concomitantly at the same time in the same samples in response to neuronal cell debris (*p ≤ 0.04, **p ≤ 0.033 vs. Control, n = 5). The bar graphs (F) and (G) represent the protein expression of PAR2 and B₂KR relative to GAPDH protein levels measured concomitantly at the same time in the same samples in response to LPS, BK, PKall, and NCD (*p ≤ 0.04 vs. C, n = 4).

FIGURE 2

Immunofluorescence and morphological changes of activated microglial cells. N9 microglial cells cultured on coverslips were stimulated with vehicle control (c), LPS (100 ng/ml), BK (0.1 µM), PKall (2.5 ng/ml), and NCD (16.5 µg protein/ml) for 24 h (A) Immunostaining was conducted using a specific anti-IBA-1 antibody (red), anti-Coronin antibody (green), DAPI to recognize the nucleus (blue), and the merged image. The yellow arrows indicate a change in the morphology of the microglial cells adopting a more rounded amoeboid shape indicative of phagocytosis. The immunofluorescence intensity of IBA-1 and coronin was not different between the groups. (B) Boxplot demonstrating the changes in microglial cell diameter (µm) in response to LPS, PKall, BK. and NCD stimulation measured by confocal microscopy. Data are representative of at least 50 cells from different representative images and expressed as mean ± SE (*p ≤ 0.03, ***p ≤ 0.001 vs C). (C) Cell viability of microglial cells. Microglial cell viability was determined by MTT assay in response to LPS, BK, PKall, and NCD. The bar graph represents the microglial cell viability expressed as a percentage of control (***p ≤ 0.001, LPS or PKall vs. C, respectively, n = 12).

FIGURE 3

Expression of inflammatory mediators in microglial cells. The gene expression levels of inflammatory cytokines measured in N9 microglial cells exposed to LPS (100 ng/ml), BK (0.1 µM), PKall (2.5 ng/ml) and NCD (16.5 µg protein/ml) for 24 h. Boxplot graphs depicts the changes in mRNA levels of (A) interleukin-6 (IL-6), (B) interleukin-1 β (IL-1β), (C) tumor necrosis factor-alpha (TNF-α), (D) cyclooxygenase-2 (COX-2), and (E) galectin-3 (Lgals3) expressed relative to control mRNA levels and to GAPDH mRNA levels were determined in the same samples at the same experiment (*p ≤ 0.05,**p ≤ 0.01, ***p ≤ 0.004 vs. Control, n = 5).

FIGURE 4

Engagement of PAR 2 and B2KR in cytokine production in the microglial cell. Microglial cells were stimulated for 24 h with PKall (2.5 ng/ml) or NCD (16.5 µg protein/ml) in the presence and absence of a B₂KR antagonist HOE 140 (1 µM) and a PAR 2 antagonist GB 83 (2 μM). Bar graphs depicts the levels of (A) interleukin-6 (IL-6, pg/ml), and (B) tumor necrosis factor-alpha (TNF-α, pg/ml) measured in the supernatant media by ELISA (**p ≤ 0.032, ***p ≤ 0.008 vs. C; $p ≤ 0.008, PKall vs. PKall + HOE 140, or PKall + GB 83; #p ≤ 0.025, NCD vs. NCD + GB 83, n = 5).

FIGURE 5

Autophagy and microglial inflammatory signals. To determine if autophagy plays a role in the microglial inflammatory response, microglial cells were exposed to LPS (100 ng), BK (10⁻⁷ M), PKall (2.5 ng/ml), or NCD (16.5 µg protein/ml) for 24 h, in the presence and absence of SAR 405 (1 µM), a phosphatidylinositol 3-kinase, catalytic subunit type 3 (PIK3C3) inhibitor, that inhibits autophagy. The bar graph shows the levels of (A) interleukin-6 (IL-6, pg/ml) and (B) tumor necrosis factor-alpha (TNF-α, pg/ml) released into the media and measured by ELISA (**p ≤ 0.02 or less vs. C, ***p ≤ 0.004 vs. C; $p ≤ 0.009, PKall vs. PKall + SAR405; +p ≤ 0.037, BK vs. BK + SAR405, n = 6).

FIGURE 6

Role of extra cellular regulated kinase1/2 (ERK1/2) in PKall and neuronal cell debris induced IL-6 production in microglial cells. (A) PKall and NCD stimulated ERK 1/2 phosphorylation (pERK 1/2) in microglial cells. N9 microglial cells were exposed to PKall (2.5 ng/ml) and/or NCD (16.5 μg protein/ml) for 10 min pERK and total ERK (TERK) levels were assessed by western blot. Bar graph represents the fold change in pERK relative to TERK protein levels (*p < 0.001 n = 6). (B) N9 microglial cells were stimulated with PKall (2.5 ng/ml) and/or NCD 16.5 μg protein/ml) for 24 h in the presence and absence of the MEK 1 inhibitor PD98059 (25 μM). Box plot represents the production and release of IL-6 levels into the media measured by ELISA in the different treatment groups. (*p ≤ 0.05, control vs. PD; ***p-value ≤ 0.001, PKall or NCD vs. Control; $p ≤ 0.004, PKall vs. PD + PKall; #p ≤ 0.002 NCD vs. PD + NCD, n = 6).

FIGURE 7

Pathway analysis of altered genes demonstrating direct functional interactions interactome networks in microglial cells. (A) Systemic related interactome, (B) Brain-related interactome. F2RL1 (Protease-activated receptor 2), IL-6 (Interleukin-6), KNG1 (Kininogen), KLKB1 (PKall), LGALS3 (Galectin-3), B₂KR (Bradykinin 2 receptor), IL-1B (Interleukin-1β), TNF-α (Tumor necrosis factor-α), COX2 (cyclooxygenase2), LPS (Lipopolysaccharide).