Ibrutinib modifies the function of monocyte/macrophage population in chronic lymphocytic leukemia

Abstract

In lymphoid organs, nurse-like cells (NLCs) show properties of tumor-associated macrophages, playing a crucial role in chronic lymphocytic leukemia (CLL) cell survival. Ibrutinib, a potent inhibitor of Bruton's tyrosine kinase (BTK), is able to counteract pro-survival signals in CLL cells. Since the effects on CLL cells have been studied in the last years, less is known about the influence of ibrutinib on NLCs properties. We sought to determine how ibrutinib modifies NLCs functions focusing on the balance between immunosuppressive and inflammatory features. Our data show that ibrutinib targets BTK expressed by NLCs modifying their phenotype and function. Treatment with ibrutinib reduces the phagocytic ability and increases the immunosuppressive profile of NLCs exacerbating the expression of M2 markers. Accordingly, ibrutinib hampers LPS-mediated signaling, decreasing STAT1 phosphorylation, while allows IL-4-mediated STAT6 phosphorylation. In addition, NLCs treated with ibrutinib are able to protect CLL cells from drug-induced apoptosis partially through the secretion of IL-10. Results from patient samples obtained prior and after 1 month of treatment with ibrutinib show an accentuation of CD206, CD11b and Tie2 in the monocytic population in the peripheral blood. Our study provides new insights into the immunomodulatory action of ibrutinib on monocyte/macrophage population in CLL.

Keywords: CLL; ibrutinib; immune modulation; microenvironment; nurse-like cells.

Figure 1. Ibrutinib specifically targets BTK in NLCs

NLCs (n=5) were treated or not with ibrutinib 1μM for 1h. Then, cell lysates were analyzed by immunoblotting using anti-phosphoBTK Tyr⁵⁵¹, Tyr²²³, total BTK and anti-actin antibodies. Three representative samples are depicted in panel A. In panel B bar diagram represents densitometric quantification of bands relative to phospho-BTK Tyr⁵⁵¹ and phospho-BTK Tyr²²³ normalized on β-actin. Data are presented as mean ± SEM of 5 different NLCs samples (**P<0.01). NLCs were treated with ibrutinib for 1h before assessing expression of phospho-BTK Tyr⁵⁵¹ and Tyr²²³ by flow cytometry C. and immunofluorescence microscopy D. (n=5, *P<0.05).

Figure 2. Ibrutinib impairs the phaghocytic activity of NLCs

A. Bar diagram represents the formazan release by metabolically active NLCs treated with ibrutinib 1 μM for 24 hours compared to untreated control (n=6, ns). In the bottom panels, representative phase-contrast micrographs demonstrate NLCs activation (as insoluble formazan precipitate) after treatment with ibrutinib. B. Box-plots summarize FITC-Dextran uptake by NLCs treated or not with ibrutinib 1 μM relative to 6 independent experiments (**P<0.01). In the bottom panels, two representative NLCs samples show confocal staining of NLCs with FITC-dextran, phalloidin and DAPI. C. Bar diagram shows the percentage of positive CD14+ NLCs stained for MAC-1 Ab or isotype control (n=6, P<0.05). Contour plots show a representative sample.

Figure 3. Exposure of NLCs to ibrutinib intensifies the expression of genes involved in M2 polarization

A. NLCs from 8 CLL patients were exposed to ibrutinib or vehicle (DMSO) for 24 hours. Transcriptional levels of CD163, IL-10, MRC1, NAMPT, CCL18 and PD-L1 (M2 polarization) and IL-1, TNFα, IL-2 (M1 polarization) were measured by quantitative reverse-transcription PCR (n=8, *P<0.05, **P<0.01). B. Diagrams show CD163 fluorescence intensity of NLCs treated with ibrutinib for 24 hours. Values of untreated and treated samples (n=7) are connected by lines (P<0.01). C. Bar diagram shows cumulative analysis of CD206 pixel intensity scoring at least 10 different cells for 3 different samples (P<0.01). On the right one representative sample stained for CD206, DAPI and phalloidin. D. NAMPT expression on NLCs, treated with ibrutinib 1 μM for the indicated time, was evaluated by confocal microscopy (n=6) using anti-NAMPT and secondary Alexa-488-anti-rabbit antibodies. Phalloidin and DAPI were used to counterstain. Graph shows cumulative data of green fluorescence pixel intensity (n=6, P<0.05).

Figure 4. Ibrutinib supports M2 signaling pathways by interfering with M1 polarization

NLCs were treated for 1 hour with ibrutinib 1 μM and then stimulated with LPS (100 ng/ml) or IL-4 (10 ng/ml) for 3 hours or 30 minutes respectively. Lysates were probed for pSTAT1, tSTAT1 A. pERK, tERK, pIĸB, tIĸB, pAKT, tAKT and actin B. following LPS stimulation investigating M1 polarization. C. PSTAT6, tSTAT6 and actin were determined after IL-4 stimulation for M2 polarization. D. NLCs were treated with ibrutinib and SHIP1 induction was monitored by western blot and by immunofluorescence. Bar diagram depicts densitometric quantification of bands relative to pSTAT1, pSTAT6 and SHIP1, either in presence or absence of the corresponding stimulus, normalized on β-actin. Data are presented as mean ± SEM of 5 different NLCs samples (*P<0.05).

Figure 5. IL-10 mediates pro-survival signals in CLL cells during treatment with ibrutinib

A. Viability of CLL cells were investigated treating both CLL cells and NLCs with ibrutinib 1 μM. CLL were harvested and divided into two fractions, one was placed back onto the autologous NLCs and the second was placed into wells without NLCs. Bar diagrams represent the mean relative CLL cell viabilities after 24, 48, 72h (n=9, *P<0.05, **P<0.01). B. Box plots show the percentage of IL-10 secreting cells in CD14+ NLCs population treated or not with ibrutinib for 1 hour relative to five independent experiments. C. Bar diagrams show the relative viability of CD19+ CLL cells treated with ibrutinib for 1 hour and then stimulated for 48 hours with IL-10 compared to untreated control (n=7, *P<0.05, **P<0.01). D. Western blot represents the activation of pro-survival signals induced by IL-10 either in presence or not of ibrutinib in one representative CD19+ CLL sample. Blots display pSTAT3, tSTAT3, pERK 1/2, tERK and actin.

Figure 6. Ibrutinib alters the circulating monocytes in CLL patients

A. Bar diagrams show cumulative analysis of CD206 (up) and CD11b (down) MFI for 5 different CLL patients in pre-treatment and after treatment samples. On the right, histograms show MFI of CD206 (up) and CD11b (down) for 3 representative CLL samples calculated using the corresponding isotype in pre-treated and after treatment samples. The blue histogram represents the post-treatment condition and the red shows the pre-treatment condition. B. Contour plots show the percentage of CD14+ Tie2+ monocytes before and after treatment with ibrutinib. On the right, diagram represents the percentage of positive CD14+ cells stained for Tie2 either before or after treatment with ibrutinib (n=5). C. Phase contrast photomicrographs and May-Grunwald Giemsa staining document the morphology of NLCs before and after treatment with ibrutinib.