Naegleria fowleri Cathepsin B Induces a Pro-Inflammatory Immune Response in BV-2 Microglial Cells via NF-κB and AP-1 Dependent-MAPK Signaling Pathway

Abstract

Naegleria fowleri is a ubiquitous protozoa parasite that can cause primary amoebic meningoencephalitis (PAM), a fatal brain infection in humans. Cathepsin Bs of N. fowleri (NfCBs) are multifamily enzymes. Although their pathogenic mechanism in PAM is not clearly understood yet, NfCBs have been proposed as pathogenic factors involved in the pathogenicity of amoeba. In this study, the immune response of BV-2 microglial cells induced by NfCB was analyzed. Recombinant NfCB (rNfCB) evoked enhanced expressions of TLR-2, TLR-4, and MyD88 in BV-2 microglial cells. This enzyme also induced an elevated production of several pro-inflammatory cytokines such as TNF-α, IL-1α, IL-1β, and IL-6 and iNOS in cells. The inhibition of mitogen-activated protein kinases (MAPKs), including JNK, p38, and ERK, effectively reduced the production of these pro-inflammatory cytokines. The rNfCB-induced production of pro-inflammatory cytokines in BV-2 microglial cells was suppressed by inhibiting NF-kB and AP-1. Phosphorylation and nuclear translocation of p65 in cells were also enhanced by rNfCB. These results suggest that NfCB can induce a pro-inflammatory immune response in BV-2 microglial cells via the NF-κB- and AP-1-dependent MAPK signaling pathways. Such a NfCB-induced pro-inflammatory immune response in BV-2 microglial cells might contribute to the pathogenesis of PAM caused by amoeba, by exacerbating deleterious immune responses and tissue damages in N. fowleri-infected foci of the brain.

Keywords: MAPK; NF-κB; Naegleria fowleri; cathepsin B cysteine protease; microglial cells; pro-inflammatory response.

Figure 1

rNfCB activates BV-2 microglial cells via the MyD88-dependent TLR-2/TLR-4 pathway. BV-2 microglial cells were treated with different concentrations (20 µg/mL or 100 µg/mL) of active or heat-inactivated rNfCB. Cells were harvested at 6 h after treatment. Expression levels of TLR-2, TLR-3, and TLR-4, and the downstream adaptor MyD88 were analyzed via semi-quantitative RT-PCR. Bar graphs show the quantitative expression pattern of each gene, analyzed as the fold induction of each gene relative to GAPDH inthree independent experiments. One-way ANOVA with Dunnett’s post hoc test was performed as multiple comparisons with the negative control without treatment with either LPS or rNfCB. *** p < 0.0001, ** p < 0.001, * p < 0.01.

Figure 2

rNfCB induces the production of diverse cytokines/chemokines in BV-2 microglial cells. (a) Cytokine array assay. BV-2 microglial cells were stimulated with rNfCB (100 µg/mL). The culture supernatants were harvested and subjected to cytokine array analysis. The culture supernatant from rNfCB-untreated BV-2 microglial cells was used as a negative control (NC). Each cytokine or chemokine is represented as double dots. PC is a positive reference control to verify the reaction. (b) Quantitative analysis. The density of dots corresponding to each cytokine or chemokine was analyzed quantitatively, and the value was presented as mean ± SD. One-way ANOVA with Dunnett’s post hoc test was performed as multiple comparisons. *** p < 0.0001, ** p < 0.001, * p < 0.01.

Figure 3

rNfCB induces production of pro-inflammatory cytokines in BV-2 microglial cells. (a) mRNA expression. BV-2 microglial cells were treated with different concentrations (20 µg/mL or 100 µg/mL) of active and heat-inactivated rNfCB. Cells were harvested at 6 h after treatment. Semi-quantitative RT-PCR was performed to analyze expression patterns of cytokines (TNF-α, IL1-α, IL-1β, and IL-6) and iNOS. Bar graphs indicate the quantitative expression profile of each gene, represented as -fold induction of each gene relative to GAPDH in three independent experiments. (b) Quantitative ELISA. BV-2 microglial cells were treated with different concentrations (20 µg/mL and 100 µg/mL) of rNfCB for different time points (6 h, 9 h, or 12 h). Heat-inactivated rNfCB was administered to the cells for 12 h. At indicated time points, the supernatant was collected and protein levels of TNF-α and IL-6 were analyzed via ELISA. Values were presented as mean ± SD of three independent experiments. One-way ANOVA with Dunnett’s post hoc test was performed as multiple comparisons with the negative control without treatment, with either LPS or rNfCB. *** p < 0.0001, ** p < 0.001, * p < 0.01.

Figure 4

MAPK signaling pathways are involved in pro-inflammatory immune response of BV-2 microglial cells stimulated by rNfCB. (a) mRNA expression. BV-2 microglial cells were pre-treated with different concentrations (1 µM or 10 µM) of JNK inhibitor (SP600125), p38 inhibitor (SB239063), or ERK inhibitor (U0126) for 3 h. rNfCB (100 µg/mL) was then administered to the cells. The mRNA expressions of TNF-α, IL-1α, IL-1β, and IL-6 were analyzed via semi-quantitative RT-PCR. Bar graphs indicate the quantitative expression profile of each gene, represented as -fold induction of each gene relative to GAPDH in three independent experiments. (b) Quantitative ELISA. Production of TNF-α and IL-6 were measured using ELISA. Values are presented as mean ± SD of three independent experiments. One-way ANOVA with Dunnett’s post hoc test was performed as multiple comparisons with the control, treated with rNfCB. *** p < 0.0001, ** p < 0.001, * p < 0.01.

Figure 5

Phosphorylation levels of JNK, p38, and ERK in rNfCB-stimulated BV-2 microglial cells. To analyze phosphorylation levels of MAPKs, BV-2 microglial cells were pre-treated with JNK, p38, or ERK inhibitor, followed by treatment with rNfCB (100 µg/mL). Total proteins were extracted from the cells, and phosphorylation levels of JNK, p38, and ERK were analyzed via immunoblot using a specific antibody for each protein. The total JNK, p38, ERK, and β-actin were used as internal controls. Fold-change means relative density change compared to negative control without treatment with rNfCB and inhibitor.

Figure 6

Effects of NF-κB and AP-1 inhibitors on the pro-inflammatory immune response of BV-2 microglial cells stimulated by rNfCB. (a) mRNA expression. BV-2 microglial cells were pre-treated with different concentrations (1 µM or 10 µM) of NF-κB inhibitor (MG132) and AP-1 inhibitor (SR11302) for 3 h, followed by treatment with rNfCB (100 µg/mL). mRNA expression levels of TNF-α, IL-1α, IL-1β, and IL-6 were analyzed via semi-quantitative RT-PCR. Bar graphs indicate the quantitative expression profile of each gene, represented as -fold induction of each gene relative to GAPDH in three independent experiments. (b) Quantitative ELISA. Production of TNF-α and IL-6 were measured using ELISA. Values are presented as mean ± SD of three independent experiments. One-way ANOVA with Dunnett’s post hoc test was performed as multiple comparisons with the control treated with rNfCB. *** p < 0.0001, ** p < 0.001, * p < 0.01.

Figure 7

Phosphorylation and nuclear translocation of p65 in rNfCB-stimulated BV-2 microglial cells. To analyze phosphorylation level and translocation of p65, BV-2 microglial cells were treated with rNfCB (100 µg/mL) with or without pre-treatment with MG132. Cytoplasmic proteins and nuclear proteins were extracted from the cells separately. Phosphorylation of p65 was analyzed by immunoblot using a specific antibody for each protein. β-actin and Lamin A/C were used as internal controls. Fold-change means relative density change of P-p65 compared to negative control without treatment of rNfCB and inhibitor.

Figure 8

Summarized scheme of signaling pathways involved in rNfCB-induced pro-inflammatory immune responses of BV-2 microglial cells. NfCB induces a pro-inflammatory immune response of BV-2 microglial cells, which is initiated via MyD88-dependent TLR-2/TLR-4 pathway and mediated by NF-κB- and AP-1-dependent MAPK signaling pathways. The image was created with BioRender (https://biorender.com, accessed on 25 May 2022). NfESP, excretory and secretory products of N. fowleri.