Impact of ibrutinib on inflammation in a mouse model of Graves' orbitopathy

Abstract

Introduction: Bruton's tyrosine kinase (BTK) and interleukin (IL)-2 Inducible T-cell Kinase (ITK) inhibitors have anti-inflammatory properties. We investigated the therapeutic effect of ibrutinib, an orally bioavailable BTK/ITK inhibitor, in a mouse model of Graves' orbitopathy (GO).

Methods: Genetic immunization was performed through intramuscular administration of the recombinant plasmid, pCMV6-hTSHR cDNA, to 8-week-old female BALB/c mice. Serum levels of T3, T4, and thyroid-stimulating hormone receptor (TSHR) antibodies (TRAbs) were quantified using enzyme-linked immunosorbent assay. Histopathological changes in orbital tissues were examined using immunohistochemistry (IHC) staining for TSHR and various inflammatory markers. Following successful genetic immunization, ibrutinib was orally administered daily for 2 weeks in the GO model mice. After treatment, the mRNA and protein expression levels of BTK, ITK, IL-1β, and IL-6 in orbital tissues were evaluated using real-time PCR and Western blotting.

Results: In total, 20 mice were sacrificed to confirm successful genetic immunization. The GO mouse group exhibited significantly increased serum T3, T4, and TRAb levels. IHC revealed increased expression of TSHR, IL-1β, IL-6, transforming growth factor-β1, interferon-γ, CD40, CD4, BTK, and ITK in the GO mouse model. The orbital inflammation was significantly attenuated in ibrutinib-treated mice. The mRNA and protein expression levels of BTK, ITK, IL-1β, and IL-6 in orbital tissue were lower in ibrutinib-treated GO mouse group compared to the phosphate-buffered saline-treated GO mouse group.

Conclusion: The GO mouse model demonstrated enhanced BTK and ITK expression. Ibrutinib, a BTK/ITK inhibitor, suppressed the inflammatory cytokine production. These findings highlight the potential involvement of BTK/ITK in the inflammatory pathogenesis of GO, suggesting its role as a novel therapeutic target.

Keywords: Bruton’s tyrosine kinase; Graves’ orbitopathy; ibrutinib; inflammation; interleukin-2 inducible T-cell kinase; mouse.

Figure 1

Flowchart for the generation of pCMV6-hTSHR complementary deoxyribonucleic acid (cDNA)-induced Graves’ orbitopathy (GO) model mice (A–E). Ibrutinib administration and sacrifice of genetically immunized mice (F, G). (A) To induce an autoimmune condition and perform genetic immunization, cardiotoxin (CTX) was administered to 8-week-old female BALB/c mice. (B) The recombinant plasmid pCMV6-hTSHR cDNA was injected into the biceps femoris muscle. (C, E) Electroporation with an ECM 830 system and 100 mm electrode needle was performed at 200 V/cm immediately following CTX administration. (D) The vaccination process was repeated 3 times at 4-week intervals.

Figure 2

Serum messenger ribonucleic acid (mRNA) expression levels of (A) Bruton’s tyrosine kinase (BTK), (B) interleukin-2-inducible T-cell kinase (ITK), and (C, D) phospholipase C-γ (PLC-γ) 1 and 2 in Graves’ orbitopathy (GO) patients (n=15) and non-GO individuals (n=15). BTK, ITK, PLC-γ1, and PLC-γ2 transcription levels were assessed using reverse transcription-polymerase chain reaction. mRNA expression levels of BTK, ITK, PLC-γ1, and PLC-γ2 were higher in GO patients (2.21 ± 0.19, 1.35 ± 0.15, 1.97 ± 0.14, and 1.13 ± 0.16, respectively) than non-GO individuals (1.54 ± 0.20, 0.92 ± 0.05, 1.46 ± 0.12, and 0.76 ± 0.09, respectively). (p=0.022, 0.013, 0.01, and 0.029, respectively).

Figure 3

Phenotype, serum levels, and pathological analysis of Graves’ orbitopathy (GO) mice after successful immunization. (A, B) Mice immunized with the human thyrotropin hormone receptor (TSHR) plasmid exhibited significant proptosis. (C–E) Serum levels of T3, T4, and thyrotropin hormone receptor TSHR antibodies (TRAbs) were significantly higher in the GO mouse group (n=10) compared to the control group (n=10). (F, G) Hematoxylin–eosin (H&E) staining revealed retrobulbar inflammation of extraocular muscles and adipogenesis in human TSHR plasmid-immunized mice. Photos of H&E staining show 1 mice/group and are representative of 1 independent experiments with samples from 3 mice per group. (H–J) Immunohistochemistry (IHC) demonstrated increased hTSHR expression in the GO mouse model (22.1 ± 1.36% of area) compared to non-GO mouse model (1.9 ± 0.28% of area) (p=0.0016). (H, I) Photos of IHC show 1 mice/group and are representative of 1 independent experiments with samples from 3 mice per group. (J) Bar graphs use data from all 3 mice per group.

Figure 4

Inflammatory changes in the retrobulbar tissue of the Graves’ orbitopathy (GO) mouse model. (A, B) Immunohistochemistry (IHC) revealed higher expression levels of interleukin (IL)-1β (p=0.04), IL-6 (p<0.001), transforming growth factor-β1 (TGF-β1), (p<0.001), interferon-γ (IFN-γ) (p=0.001), cluster of differentiation (CD) 40 (p=0.002), and CD4 (p<0.001) in the GO mouse model. (A) Photos of IHC show 1 mice/group and are representative of 1 independent experiments with samples from 3 mice per group. (B) Bar graphs use data from all 3 mice per group. (C) Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated higher messenger ribonucleic acid expression levels of IL-1β (p=0.01), IL-6 (p=0.03), TGF-β1(p=0.065), IFN-γ (p=0.026), CD40 (p=0.132), and CD4 (p=0.009), in the GO mouse model (n=6) compared to the controls (n=6). Statistically significant differences were observed in all inflammatory cytokines, except TGF-β and CD40.

Figure 5

Comparison of Bruton’s tyrosine kinase (BTK) and interleukin-2-inducible T-cell kinase (ITK) signaling protein and mRNA expression levels between Graves’ orbitopathy (GO) and non-GO mouse models. (A, B) Immunohistochemistry (IHC) revealed elevated expression of BTK (p=0.002) and ITK (p<0.001) in the GO mouse model. (A) Photos of IHC show 1 mice/group and are representative of 1 independent experiments with samples from 3 mice per group. (B) Bar graphs use data from all 3 mice per group. (C) BTK (p=0.01) and ITK (p=0.065) expression levels were higher in human thyrotropin hormone receptor plasmid-immunized mice (n=6) compared to control plasmid-immunized mice (n=6).

Figure 6

Effects of the Bruton’s tyrosine kinase (BTK) and interleukin-2-inducible T-cell kinase (ITK) inhibitor on BTK and ITK expression in the Graves’ orbitopathy (GO) mouse model. (A, B) Following treatment with the BTK/ITK inhibitor ibrutinib, the BTK and ITK transcription levels were observed to be reduced. RT-PCR demonstrated that the BTK (p=0.029) and ITK (p=0.028) mRNA expression levels were lower in the ibrutinib-treated GO mouse model (0.50 ± 0.06 and 0.34 ± 0.06, respectively) (n=4) compared to PBS-treated GO mouse model (0.12 ± 0.05 and 0.09 ± 0.02, respectively) (n=4) (C–G) Western blotting demonstrated elevated expression levels of BTK, ITK, phospholipase C-γ (PLC-γ)1, and PLC-γ2 in the GO mouse model. Notably, the increased expression of BTK, ITK, PLC-γ1, and PLC-γ2 in the GO mouse model was suppressed by ibrutinib. WB show 2 mice/group and are representative of 2 independent experiments with samples from 4 control mice and 5 GO mouse and 5 ibrutinib-treated GO mouse group. (B) Bar graphs use data from all mice per group. ^* p<0.05.

Figure 7

Effects of the Bruton’s tyrosine kinase (BTK) and interleukin-2-inducible T-cell kinase (ITK) inhibitor on the transcription of proinflammatory cytokines in the Graves’ orbitopathy (GO) mouse model. Reverse transcription-polymerase chain reaction revealed a decrease in the messenger ribonucleic acid expression levels of proinflammatory cytokines, including interleukin (IL)-1β (p=0.197), IL-6 (p=0.538), transforming growth factor-β1 (p=0.256), interferon-γ (p=0.356), cluster of differentiation (CD)40 (p=0.119), and CD4 (p=0.395), in the GO mouse model following treatment with the BTK/ITK inhibitor ibrutinib. However, these changes did not reach statistical significance. Each group in this experiment consisted of 4 mice.

Figure 8

Effects of the Bruton’s tyrosine kinase (BTK) and interleukin-2-inducible T-cell kinase (ITK) inhibitor on inflammation in the Graves’ orbitopathy (GO) mouse model. (A) Western blotting (WB) analysis revealed elevated expression of interleukin (IL)-1β, IL-6, transforming growth factor-β1 (TGF-β1), and interferon-γ (IFN-γ) in the GO mouse model. (B–E) Treatment with the BTK/ITK inhibitor ibrutinib suppressed the increased expression of IL-1β, IL-6, TGF-β1, and IFN-γ in the GO mouse model. WB show 2 mice/group and are representative of 2 independent experiments with samples from 4 control mice and 5 GO mouse and 5 ibrutinib-treated GO mouse group. (B) Bar graphs use data from all mice per group. ^* p<0.05.