The relation between BTK expression and iron accumulation of myeloid cells in multiple sclerosis

Abstract

Activation of Bruton's tyrosine kinase (BTK) has been shown to play a crucial role in the proinflammatory response of B cells and myeloid cells upon engagement with B cell, Fc, Toll-like receptor, and distinct chemokine receptors. Previous reports suggest BTK actively contributes to the pathogenesis of multiple sclerosis (MS). The BTK inhibitor Evobrutinib has been shown to reduce the numbers of gadolinium-enhancing lesions and relapses in relapsing-remitting MS patients. In vitro, BTK inhibition resulted in reduced phagocytic activity and modulated BTK-dependent inflammatory signaling of microglia and macrophages. Here, we investigated the protein expression of BTK and CD68 as well as iron accumulation in postmortem control (n = 10) and MS (n = 23) brain tissue, focusing on microglia and macrophages. MS cases encompassed active, chronic active, and inactive lesions. BTK⁺ and iron⁺ cells positively correlated across all regions of interests and, along with CD68, revealed highest numbers in the center of active and at the rim of chronic active lesions. We then studied the effect of BTK inhibition in the human immortalized microglia-like HMC3 cell line in vitro. In particular, we loaded HMC3 cells with iron-dextran and subsequently administered the BTK inhibitor Evobrutinib. Iron treatment alone induced a proinflammatory phenotype and increased the expression of iron importers as well as the intracellular iron storage protein ferritin light chain (FTL). BTK inhibition of iron-laden cells dampened the expression of microglia-related inflammatory genes as well as iron-importers, whereas the iron-exporter ferroportin was upregulated. Our data suggest that BTK inhibition not only dampens the proinflammatory response but also reduces iron import and storage in activated microglia and macrophages with possible implications on microglial iron accumulation in chronic active lesions in MS.

Keywords: Bruton's tyrosine kinase; Evobrutinib; microglia; multiple sclerosis; myeloid cells; neuroinflammation.

FIGURE 1

Immunohistochemistry (microglia/macrophage phagocytosis marker CD68, BTK) and iron histochemistry of a representative control white matter (NWM, line A), MS NAWM (line B), a chronic active MS lesion (C) and an active MS lesion (D). Scale bar = 50 μm; Inset scale bar = 20 μm. NAWM, normal‐appearing white matter; NWM, normal white matter.

FIGURE 2

Representative triple immunofluorescent stainings of chronic active lesion rim from MS patient using DAPI (blue) (A), CD68 (green) (B), FTL (yellow) (C), and BTK (pink) (D). BTK, Bruton's Tyrosine Kinase; CD68, cluster of differentiation of 68; DAPI, 4′,6‐diamidino‐2 phenylindole; FTL, ferritin light chain. Scale bar = 50 μm.

FIGURE 3

Quantification of BTK (A), TBB (B) and CD68 (C) positive cells in ROIs of controls (n = 10), inactive (n = 10), chronic active (n = 10), and active lesions (n = 7). Dot plot displays the correlation between BTK and TBB, including cell counts of control WM as well as inactive, chronic active, and active MS lesions (D). Each dot represents one patient sample. Colors indicate distinct MS lesion types. NWM, normal white matter; PPWM, periplaque white matter. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 4

Fold changes of expression of inflammation‐related (A, C) and iron‐associated genes (B, D) in HMC3 cells. (A, B) Expression in HMC3 cells upon 20 h of iron‐dextran treatment, compared with untreated cells cultured in EMEM medium. (C, D) Each bar depicts fold changes of gene expression in iron‐loaded HMC3 cells with Evobrutinib application, compared with iron‐loaded cells without BTK inhibition. mRNA levels were detected with quantitative real‐time PCR. Error bars represent standard error of the mean (SEM).

FIGURE 5

Expression of inflammation‐related and iron‐related proteins in HMC3 cells. Representative immunocytochemistry of iron‐loaded only (A) and iron‐loaded Evobrutinib‐treated HMC3 cells (B) stained for Iba‐1, ferritin, and ferroportin (A, B). (C) Differential protein expression of selected markers in iron‐loaded HMC3 cells with BTK inhibition compared with iron‐loaded cells. Samples were normalized to beta‐actin. Each bar depicts the fold changes in protein expression between the two treatment groups, with error bars representing the standard error of the mean (SEM). Statistical significance was determined using two‐tailed paired t‐test on densitometry values and the associated p‐values are indicated by asterisks (*). Asterisks denote the level of significance as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

FIGURE 6

Immunohistochemistry of chronic active MS lesion with microglial/macrophage marker CD68 (A), low‐density lipoprotein receptor–related protein 1 (Lrp‐1) (B), the ferritin receptor Tim‐1 (C) and the inflammasome NLRP3. Cells with microglia/macrophage (arrowheads) and astrocyte morphology (arrow) (D). Scale bar = 125 μm, inset scale bar = 25 μm.