Erdafitinib diminishes LPS-mediated neuroinflammatory responses through NLRP3 in wild-type mice

Abstract

Introduction: Erdafitinib is an FDA-approved inhibitor of fibroblast growth factor receptor (FGFR) that is used clinically to treat metastatic urothelial cancer. FGFR activation is involved in proinflammatory responses, but the potential effects of FGFR inhibitors like erdafitinib on neuroinflammatory responses in the brain have not been fully established.

Methods: The effects of pretreatment with 1 μM or 5 μM erdafitinib on proinflammatory responses induced by 1 μg/mL or 200 ng/mL LPS in vitro were evaluated in BV2 microglial cells. For in vivo experiments, 3-month-old C57BL6/N mice were injected (i.p.) daily for 7 days with vehicle (5% DMSO +40% PEG +5% Tween80 + 50% saline) or 10 mg/kg erdafitinib. On the final day, the mice were injected (i.p.) with 10 mg/kg LPS or PBS after erdafitinib administration and sacrificed after 8 h. The mRNA and protein expression of neuroinflammatory-associated molecules were assessed in cells or mouse brain tissue by real-time PCR, immunofluorescence staining, and/or Western blotting.

Results and discussion: In BV2 microglial cells, erdafitinib pretreatment significantly reduced the increases in proinflammatory cytokines, NLRP3 inflammasome activation and JNK/PLCγ signaling induced by LPS. In C57BL6/N mice, erdafitinib pretreatment significantly suppressed LPS-stimulated microglial/astroglial activation and proinflammatory cytokine expression. Importantly, erdafitinib pretreatment significantly downregulated LPS-induced NLRP3 inflammasome activation and astroglial neuroinflammation-associated molecules in C57BL6/N mice. Collectively, our experiments demonstrate that erdafitinib pretreatment diminishes LPS-induced neuroinflammation by suppressing NLRP3 inflammasome activation in vitro and in vivo and suggest that erdafitinib is a potential therapeutic agent for neuroinflammation-related diseases.

Keywords: FGFR; JNK; NLRP3; erdafitinib; neuroinflammation.

FIGURE 1

Erdafitinib treatment significantly diminishes LPS-stimulated proinflammatory cytokine levels by downregulating NLRP3 and JNK/PLCγ1/c-JUN signaling in BV2 microglial cells. (A) MTT assays of BV2 microglial cells treated with vehicle or erdafitinib (n = 6/group). (B) Real-time PCR analysis of proinflammatory cytokine mRNA levels in BV2 microglial cells treated with 1 μM or 5 μM erdafitinib or vehicle (1% DMSO) for 30 min and then treated with 1 μg/mL LPS or PBS for 5.5 h (n = 9/group). (C) Real-time PCR analysis of neuroinflammation-associated molecular target expression in BV2 microglial cells treated with 5 μM erdafitinib or vehicle and then treated with 200 ng/mL LPS or PBS (n = 7–8/group). (D) Real-time PCR of nlrp3, cox-2, il-1β, il-6 and tnf-α mRNA expression in BV2 microglial cells transfected with nlrp3 siRNA (40 nM) or scramble siRNA for 41 h and subsequently treated as described above (n = 8/group). (E–F) Western blotting with anti-p-JNK, anti-JNK, anti-p-PLCγ1, anti-PLCγ1, and anti-β-actin antibodies of BV2 microglial cells treated with 5 μM erdafitinib or vehicle for 45 min and then treated with 1 μg/mL LPS or PBS for 45 min (n = 9/group). (G–H) Western blotting with anti-p-c-JUN, anti-NF-κB and anti-PCNA antibodies of the nuclear fraction of BV2 microglial cells treated with 5 μM erdafitinib or vehicle and then treated with 1 μg/mL LPS or PBS as described above (n = 9/group). *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 2

Erdafitinib treatment ameliorates LPS-induced microgliosis in C57BL6/N mice. (A, B) Immunofluorescence staining of Iba-1 expression in C57BL6/N mice injected (i.p.) with vehicle (5% DMSO +40% PEG +5% Tween80 + 50% saline) or 10 mg/kg erdafitinib daily for 7 days and subsequently injected (i.p.) with 10 mg/kg LPS or PBS for 8 h on day 7. (C–E) Quantification of data from A and B (n = 19–20 slices from 5 mice/group). *p < 0.05, **p < 0.01, and ***p < 0.001. Scale bar = 100 μm.

FIGURE 3

Erdafitinib treatment reduces LPS-mediated astrogliosis in C57BL6/N mice. (A, B) Immunofluorescence staining of GFAP expression in C57BL6/N mice treated as described above (C–E) Quantification of data from A and B (n = 19–20 slices from 5 mice/group). *p < 0.05, **p < 0.01, and ***p < 0.001. Scale bar = 100 μm.

FIGURE 4

Erdafitinib administration suppresses LPS-stimulated proinflammatory cytokine il-6 mRNA and IL-6 protein expression in C57BL6/N mice. (A–D) Real-time PCR analysis of cox-2 and il-6 mRNA expression in C57BL6/N mice treated as described above (n = 7–8/group). (E) Immunofluorescence staining of IL-6 expression in C57BL6/N mice treated as described above. (F) Quantification of data from E (n = 20 slices from 5 mice/group). *p < 0.05, **p < 0.01, and ***p < 0.001. Scale bar = 100 μm.

FIGURE 5

Erdafitinib treatment downregulates LPS-evoked proinflammatory cytokine IL-1β expression and NLRP3 inflammasome activation in C57BL6/N mice. (A–B) Real-time PCR analysis of il-1β mRNA expression in C57BL6/N mice (n = 7–8/group). (C–D) Immunofluorescence staining of IL-1β protein expression in C57BL6/N mice treated as described above (E) Quantification of data from C and D (n = 19–20 slices from 5 mice/group). (F–G) Real-time PCR analysis of nlrp3 and pro-il-1β mRNA expression in C57BL6/N mice (n = 6–8/group). *p < 0.05, **p < 0.01, and ***p < 0.001. Scale bar = 100 μm.

FIGURE 6

Erdafitinib administration diminishes LPS-mediated reactive astrocyte-related neuroinflammatory dynamics in C57BL6/N mice. (A–C) Real-time PCR analysis of cxcl10, chi3l1 and serpina3n mRNA expression in C57BL6/N mice treated as described above (n = 6–8/group). (D) Model of the effects of erdafitinib on astrocyte-associated neuroinflammatory dynamics induced by LPS in C57BL6/N mice. *p < 0.05, **p < 0.01, and ***p < 0.001.