Putative link between Polo-like kinases (PLKs) and Toll-like receptor (TLR) signaling in transformed and primary human immune cells

Abstract

Toll-like receptors (TLRs) are important sentinels of bacterial and viral infection and thus fulfil a critical sensory role in innate immunity. Polo-like kinases (PLKs), a five membered family of Ser/Thr protein kinases, have long been studied for their role in mitosis and thus represent attractive therapeutic targets in cancer therapy. Recently, PLKs were implicated in TLR signaling in mice but the role of PLKs in TLR signaling in untransformed primary immune cells has not been addressed, even though PLK inhibitors are in clinical trials. We here identified several phospho-serine and phospho-threonine residues in the known TLR pathway kinases, Interleukin-1 receptor-associated kinase (IRAK) 2 and IRAK4. These sites lie in canonical polo-box motifs (PBM), sequence motifs known to direct recruitment of PLKs to client proteins. Interestingly, PLK1 was phosphorylated and PLK 2 and 3 mRNA induced upon TLR stimulation in primary immune cells, respectively. In whole blood, PLK inhibition disparately affected TLR mediated cytokine responses in a donor- and inhibitor-dependent fashion. Collectively, PLKs may thus potentially interface with TLR signaling in humans. We propose that temporary PLK inhibitor-mediated blockade of TLR-signaling in certain patients receiving such inhibitors during cancer treatment may cause adverse effects such as an increased risk of infections due to a then compromised ability of the TLR recognition system to sense and initiate cytokine responses to invading microbes.

Figure 1

IRAK2 and 4 contain novel phospho-sites that map to Polo-box motifs. (A) Overview of human IRAK2 primary sequence (yellow) with DD and KD indicated in dark grey. Putative PBM are in blue with red boxes, and the newly identified phospho-S144 highlighted. (B) MS-MS fragment spectrum for the IRAK2 135–147 tryptic fragment containing the S144 phosphorylation (red). (C) as in A but shown for human IRAK4. Already published sites are shown in blue, sites newly identified in this study in red. S152 is highlighted.

Figure 2

TLR stimulation induces PLK2 and 3 mRNA expression. Whole blood was treated with R848 or LPS at the indicated concentrations and for 2 or 6 h. Subsequently, total RNA was isolated, genomic DNA digested, reverse-transcribed and used in quantitative real-time PCR for the indicated PLKs relative to the TBP housekeeping gene. Four donors were assayed in triplicates. Biological means +/− SEM are shown.

Figure 3

PLK1 is phosphorylated in primary immune cells upon TLR stimulation. Freshly drawn whole blood was treated with R848 or left untreated for 10 min before erythrocytes were lysed, cells fixed, permeabilized and stained using anti-phospho-PLK1, -p38, -ERK1/2 or –p65. Gates were set to distinguish monocytes, peripheral blood lymphocytes (PBL) and PMN as shown in Fig. S1. Event # are given as indicated. In (a) one representative donor is shown, in (b) data from 2–3 donors are summarized. Each dot represents one donor. Differences were not statistically significant by one-way ANOVA with Sidak correction for multiple testing due to donor-to-donor variations and the low number of donors.

Figure 4

PLK inhibitors BI2536 and BI6272 donor-independently modulate TLR- but not NLRP3 inflammasome-dependent cytokine responses. Freshly drawn whole blood was treated with 5 µg/ml R848 in the presence or absence of the indicated concentrations of BI2536 (a), BI6272 (b) or GSK461364 (c), respectively, for 3 h or left unstimulated. Subsequently, total RNA was isolated, genomic DNA digested, reverse-transcribed and used in quantitative real-time PCR for IL6 and CXCL10 mRNA relative to TBP. Each sample was measured in triplicates and individual symbols represent the mean for each donor coded by symbol type and color. The effects of inhibition were tested with reference to R848 + DMSO control using an unpaired two-tailed t-test. Technical triplicates that were statistically significantly (p < 0.05) higher or lower than the donor’s R848 + DMSO are marked by *. A combined statistical analysis did not yield significant differences due to high inter-individual variation. (d) ELISA analysis of IL-1ß in supernatants from PMA-differentiated THP-1 cells treated for 1 h with the indicated PLK1 inhibitors and then stimulated with the NLRP3 agonist, nigericin. (e) Cell viability using CCK8 reagent normalized to DMSO control. In D and E data are representative of two independent experiments.