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. 2024 Sep 1;8(9):652-667.
doi: 10.4049/immunohorizons.2400045.

Bruton Tyrosine Kinase Inhibition Decreases Inflammation and Differentially Impacts Phagocytosis and Cellular Metabolism in Mouse- and Human-derived Myeloid Cells

Rochelle Y Benoit  1 , Jennifer L Zagrodnik  1 , Samantha J Carew  1 , Craig S Moore  1
Affiliations

Bruton Tyrosine Kinase Inhibition Decreases Inflammation and Differentially Impacts Phagocytosis and Cellular Metabolism in Mouse- and Human-derived Myeloid Cells

Rochelle Y Benoit et al. Immunohorizons. .

Abstract

Bruton tyrosine kinase (BTK) is a kinase expressed by various immune cells and is often activated under proinflammatory states. Although the majority of BTK-related research has historically focused on B cells, understanding the role of BTK in non-B cell populations is critical given myeloid cells also express BTK at comparable levels. In this study, we investigated and compared how BTK inhibition in human and murine myeloid cells alters cell phenotype and function. All experiments were performed using two BTK inhibitors (evobrutinib and tolebrutinib) that are currently in late-stage clinical trials for the treatment of multiple sclerosis. Assays were performed to assess the impact of BTK inhibition on cytokine and microRNA expression, phagocytic capacity, and cellular metabolism. In all cells, both evobrutinib and tolebrutinib significantly decreased phosphorylated BTK and LPS-induced cytokine release. BTK inhibition also significantly decreased the oxygen consumption rate and extracellular acidification rate in myeloid cells, and significantly decreased phagocytosis in murine-derived cells, but not human macrophages. To further elucidate the mechanism, we also investigated the expression of microRNAs known to impact the function of myeloid cells. BTK inhibition resulted in an altered microRNA expression profile (i.e., decreased miR-155-5p and increased miR-223-3p), which is consistent with a decreased proinflammatory myeloid cell phenotype. In summary, these results provide further insights into the mechanism of action of BTK inhibitors in the context of immune-related diseases, while also highlighting important species-specific and cell-specific differences that should be considered when interpreting and comparing results between preclinical and human studies.

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Conflict of interest statement

The authors have no financial conflicts of interest.

Figures

FIGURE 1.
FIGURE 1.
BTKis tolebrutinib and evobrutinib significantly decrease phosphorylation of BTK in human and mouse myeloid cells. (A–C) ICC images of p-BTK (red) and DAPI (blue) in human MDMs, mouse BMDMs, and microglia (scale bars, 100 μm). (D–F) A significant decrease in p-BTK is shown in (D) MDMs, (E) BMDMs, and (F) microglia through quantification of ICC images in (A)–(C) (N = 3, n = 6 per treatment per cell type). DMSO: 0.1%, 10 μM. Statistical analysis was performed using a one-way ANOVA with the main comparator group being DMSO + LPS. **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bars represent mean ± SEM. AU, arbitrary units; NT, no treatment.
FIGURE 2.
FIGURE 2.
BTKis tolebrutinib and evobrutinib significantly decrease p-BTK protein expression in human and mouse myeloid cells. (A–C) Evobrutinib significantly decreased p-BTK levels in (A) human MDMs, (B) mouse BMDMs, and (C) microglia. (D–F) Tolebrutinib significantly decreased p-BTK levels in (D) MDMs, (E) BMDMs, and (F) microglia (n = 6 per treatment per cell type). DMSO: 0.1% at 10 μM. Statistical analysis was performed using a one-way ANOVA with the main comparator group being DMSO + LPS. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bars represent mean ± SEM. NT, no treatment; RFU, relative fluorescence units.
FIGURE 3.
FIGURE 3.
BTKis tolebrutinib and evobrutinib decrease cytokines in human and mouse myeloid cells. (A) Tolebrutinib and evobrutinib significantly decreased TNF and IL-6 in human whole blood. (B) Tolebrutinib and evobrutinib significantly decreased TNF and IL-10 in human MDMs. (C) Tolebrutinib and evobrutinib significantly decreased TNF and IL-6 from mouse BMDMs. (D) Tolebrutinib significantly decreased TNF and IL-10 in mouse microglia (n = 6 per treatment per cell type). DMSO: 0.1% at 10 μM. Statistical analysis was performed using a one-way ANOVA with the main comparator group being DMSO + LPS. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bars represent mean ± SEM. NT, no treatment.
FIGURE 4.
FIGURE 4.
BTKis tolebrutinib and evobrutinib decrease OCR and ECAR in human MDMs and mouse BMDMs, but only ECAR in mouse microglia. (A–L) Evobrutinib influences OCR and ECAR under mito-stress test conditions and baseline functions in (A–D) human MDMs, (E–H) mouse BMDMs, and (I–L) microglia. (M–X) Tolebrutinib influences OCR and ECAR under mito-stress test conditions and baseline functions in (M–P) MDMs, (Q–T) BMDMs, and (U–X) microglia (n = 6 per treatment per cell type). DMSO: 0.1% at 10 μM. Statistical analysis was performed using a one-way ANOVA with the main comparator group being DMSO + LPS. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bars represent mean ± SEM. NT, no treatment.
FIGURE 5.
FIGURE 5.
BTKis significantly decrease phagocytosis in mouse myeloid cells. (A–F) Evobrutinib effects on normalized pHrodo Zymosan Bead fluorescence intensity in (A) human MDMs, (C) mouse BMDMs, and (E) microglia and the effects on pHrodo Zymosan Bead (pHrodo) fluorescence intensity at the end of a 6-h kinetic interval showing phagocytosis trending upward in (B) MDMs, but significantly decreasing in (D) BMDMs and (F) microglia. (G–L) Tolebrutinib effects on normalized pHrodo Zymosan Bead fluorescence intensity in (G) MDMs, (I) BMDMs, and (K) microglia and the effects on pHrodo Zymosan Bead (pHrodo) fluorescence intensity at the end of the 6-h kinetic interval showing phagocytosis trending upward in (H) MDMs, but significantly decreasing in (J) BMDMs and (L) microglia (n = 6 per treatment per cell type). Statistical analysis was performed using a one-way ANOVA with the main comparator group being DMSO + LPS. ***p < 0.001, ****p < 0.0001. Error bars represent mean ± SEM. NT, no treatment; pHrodo, zymosan beads only.
FIGURE 6.
FIGURE 6.
BTKis significantly decrease miRNA-155-5p in mouse BMDMs and microglia, and miRNA-21-5p in mouse BMDMs. (A) Evobrutinib and tolebrutinib significantly decrease miR-155-5p and miR-21-5p in mouse BMDMs. (B) Evobrutinib and tolebrutinib significantly decrease miR-155-5p in mouse microglia. Mouse BMDMs: miR-223-3p and miR-21-5p; n = 4, miR-155-5p; n = 6; mouse microglia: miR-223-3p and miR-21-5p; n = 5, miR-155-5p; n = 6 (BTKi, 10 μM). Statistical analysis was performed using a one-way ANOVA with the main comparator group being LPS. **p < 0.01, ****p < 0.0001. Error bars represent mean ± SEM.
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