EGFR transactivation contributes to neuroinflammation in Streptococcus suis meningitis

Abstract

Background: Streptococcus suis serotype 2 (SS2) is an important zoonotic bacterial pathogen in both humans and animals, which can cause high morbidity and mortality. Meningitis is one of the major clinical manifestations of SS2 infection. However, the specific process of SS2 meningitis and its molecular mechanisms remain unclear. Epidermal growth factor receptor (EGFR) has been reported to initiate transduction of intracellular signals and regulate host inflammatory responses. Whether and how EGFR contributes to the development of S. suis meningitis are currently unknown.

Methods: The tyrosine phosphorylation of cellular proteins, the transactivation of EGFR, as well as its dimerization, and the associated signal transduction pathways were investigated by immunoprecipitation and western blotting. Real-time quantitative PCR was used to investigate the transcriptional level of the ErbB family members, EGFR-related ligands, cytokines, and chemokines. The secretion of cytokines and chemokines in the serum and brain were detected by Q-Plex™ Chemiluminescent ELISA.

Results: We found an important role of EGFR in SS2 strain SC19-induced meningitis. SC19 increasingly adhered to human brain microvascular endothelial cells (hBMEC) and caused inflammatory lesions in the brain tissues, with significant induction and secretion of proinflammatory cytokines and chemokines in the serum and brains. SC19 infection of hBMEC induced tyrosine phosphorylation of cellular EGFR in a ligand-dependent manner involving the EGF-like ligand HB-EGF, amphiregulin (AREG), and epiregulin (EREG) and led to heterodimerization of EGFR/ErbB3. The EGFR transactivation did not participate in SS2 strain SC19 adhesion of hBMEC, as well as in bacterial colonization in vivo. However, its transactivation contributed to the bacterial-induced neuroinflammation, via triggering the MAPK-ERK1/2 and NF-κB signaling pathways in hBMEC that promote the production of proinflammatory cytokines and chemokines.

Conclusions: We investigated for the first time the tyrosine phosphorylation of cellular proteins in response to SS2 strain SC19 infection of hBMEC and demonstrated the contribution of EGFR to SS2-induced neuroinflammation. These observations propose a novel mechanism involving EGFR in SS2-mediated inflammatory responses in the brain, and therefore, EGFR might be an important host target for further investigation and prevention of neuroinflammation caused by SS2 strains.

Keywords: Brain microvascular endothelial cell; EGFR; Neuroinflammation; Streptococcus suis.

Fig. 1

SS2 strain SC19 infection induced a strong neuroinflammation. a Increasing adherence of SC19 to hBMEC along with time. b 5-week-old CD1 mice were injected intravenously with 2 × 10⁸ CFU SC19 strain. The histopathological changes of the brain in infected mice were investigated. (a, d) Normal cerebral meninges and brain tissue, (b) meningeal thickening, (c) subdural hemorrhage, (e) perivascular inflammatory infiltrates, (f) slight neuronophagia, and (g) partial magnification of panel (f). Scale bar = 50 μm. c Brain lysates and serum were harvested at various time points post infection and cytokines production were determined by Q-Plex™ Chemiluminescent ELISA. Results were expressed as the mean ± SD from three infected mice at each time point. Statistical analysis was carried out between the infected group at each time point and the uninfected group (0 h). d Total RNAs of infected and uninfected hBMEC were extracted and reversely transcribed. The mRNA levels of cytokines and chemokines were markedly increased in response to SC19 stimulation

Fig. 2

SS2 strain SC19 induces tyrosine phosphorylation of several cellular proteins. a Tyrosine phosphorylation of the hBMEC extracts in response to SS2 infection were analyzed by immunoblotting with anti-phosphotyrosine antibody (PY). The tyrosine-phosphorylation of several proteins could be markedly inhibited by AG1478. β-actin was analyzed as a normalization control. b The hBMEC was lysed and subjected to immunoprecipitation with an anti-EGFR antibody and immunoblotting with the anti-phosphotyrosine mAb 4G10. c Bacteria were cultured in TSB containing 10 % newborn bovine serum, and treatment with 5 M AG1478 or vehicle (DMSO) had no influence on bacterial growth at 37 °C. d Treatment with 5 μM AG1478 did not influence the hBMEC proliferation, as demonstrated by MTT assay

Fig. 3

SS2 infection of hBMEC induces the ligand-dependent transactivation as well as the dimerization of EGFR. a Total RNAs extracted from hBMEC were analyzed by real-time PCR for the transcription of ErbB family genes. GAPDH was used as an internal control for normalization. b Immunoblot analysis of ErbB family members in whole cell extracts after infection of hBMEC with SC19 at an MOI of 10 for the indicated times. β-actin in cell lysates was analyzed as normalization control. c, d EGFR or ErbB3 was immunoprecipitated and immunoblotted with an anti-phosphotyrosine antibody (4G10) and then with the anti-EGFR or anti-ErbB3 antibody (top and middle). After stripping, the blots were reprobed with anti-ErbB3 or anti-EGFR antibodies to determine the possible interaction of EGFR with ErbB3. e Determination of ErbB3 expression in hBMEC transfected with either vehicle control or ErbB3 shRNA. f Knocking down of ErbB3 via shRNA attenuated SS2-induced EGFR activation. g AG1478 treatment significantly inhibited SS2-stimulated ErbB3 activation in hBMEC. h Heat-inactivated SC19 was unable to induce transactivation of EGFR, compared with the time-dependent activation of EGFR in response to viable SS2. i The induction of EGFR-related ligands in hBMEC in response to viable or heat-inactivated SS2 were compared by real-time PCR. GAPDH was used as the internal control. j Pretreatment with batimastat decreased SS2-induced transactivation of EGFR

Fig. 4

Effects of EGFR inhibition on SS2 adherence and colonization. a AG1478 pretreatment (5 μM) did not significantly affect SC19 adhesion to hBMEC in vitro. b–d Mice (n = 5) were administrated with AG1478, or DMSO as vehicle control, and then challenged with 2 × 10⁸ CFU SC19 strain. At the indicated time points, bacterial colonization in the blood, brain, and lung from both groups of mice were compared

Fig. 5

EGFR selective inhibitor AG1478 could significantly attenuate the SS2-induced neuroinflammation. a SC19 infection of hBMEC induced a time-dependent increase of the proinflammatory cytokines and chemokines, while this could be significantly blocked by the treatment of AG1478. b Serum and brains lysates were collected from challenged mice with or without AG1478 pretreatment. The levels of representative cytokine and chemokine, IL-6 and MCP-1, were determined and compared. Results were expressed as mean ± SD of three infected mice in each group

Fig. 6

SS2-induced transactivation of EGFR contributes to the neuroinflammation via MAPK-ERK1/2 and NF-κB signaling pathways. a, b NF-κB inhibitors BAY11-7082 or CAY10657 and the ERK1/2 inhibitor U0126 dose-dependently inhibited SS2-induced upregulation of IL-6 and MCP-1. c SC19 infection of hBMEC induced an obvious phosphorylation of p65 subunit, as well as the degradation of IκBα. d SC19 infection of hBMEC induced obvious activation of ERK1/2. e, f EGFR-selective inhibitor AG1478 could significantly decrease the SC19-induced NF-κB signaling (including p65 phosphorylation and IκBα degradation), as well as ERK1/2 phosphorylation. g Blockage of ERK1/2 signaling by U0126 did not affect SS2-induced p65 phosphorylation and IκBα degradation. h Fluorescence microscopy of SS2-induced p65 subunit translocation to the nucleus in hBMEC with pretreatment of AG1478 (5 μM), U0126 (5 μM), or BAY-11-7082 (5 μM). Cells were incubated with anti-p65 antibody and visualized with a FITC-labeled goat anti-mouse IgG antibody. Scale bar = 100 μm