Triggering of Toll-Like Receptor 4 Expression in Human Multiple Myeloma Promotes Proliferation and Protects the Cells From Immune and Chemotherapy Drug Attack.
Authors: Cai Zhen , Bao Hanying , Wang Lijuan , Yang Yang , Zhao Yi , He Donghua , Lu Peilin , Wang Michael 
Published: October 2019

Abstract 2843

Poster Board II-819

Multiple myeloma (MM) is an incurable B-cell malignancy characterized by accumulation of malignant plasma cells in bone marrow (BM) and recurrent or persistent infections. Toll like receptors (TLRs) are essential in the host defense against infections. In MM, recurrent infections could promote tumor growth and favor escape from standard therapies. TLRs initiated responses in B cells include proliferation, antiapoptosis and immune escape.

In this study, we first screened four MM cell lines, MM1-r, MM1-s, RPMI8226, and U266, for the expression of major TLRs (including TLR 1–2,4-7and 9) by RT-PCR. Surprisingly, all the MM cell lines expressed multiple TLRs. We focused on TLR4, which had the most strongly expressed mRNA. Consistent with the RT-PCR result, fluorescence-activated cell sorting (FACS) analysis revealed that TLR4 protein was also present on the surface of MM cell lines. By FACS analysis, we found that MM1-s and MM1-r cells had increased TLR4 co-receptor CD14 expression when induced by lipopolysaccharide (LPS).

We next asked if TLR4 was functional in MM cells. To activate TLR4 in MM cells (MM1-r, MM1-s, RPMI8226, and U266), we incubated the cells with LPS, the natural ligand for TLR4 and measured cell proliferation by [3H]thymidine incorporation. Proliferation of MM1-s and MM1-r cells increased significantly, but that of U266 and RPMI8226 cells did not. As mechanisms involved in the resistance to apoptosis play a major role in MM escape to therapies, we sought to determine the capacity of TLR4 ligand to promote the survival of MM cells. We pretreated MM cells with lipopolysaccharide followed by induction by adriamycin. Our results showed that LPS could save MM1-s cells from adriamycin induced apoptosis.

In addition to proliferation and apoptosis, we would like to learn whether TLR4 ligand could change cell cycle, We found that MM1-s and MM1-r MM cells showed decreased number in G0/G1 phase but increased number in S phase when induced by LPS. To explore whether MAPK pathways were implicated in MM1-s and MM1-r cells to LPS stimulation, we investigated the role of ERK1/2, p38, and JNK MAPKs in LPS-stimulated cells by using specific inhibitors of MAPK pathways, Firstly, time-dependent MAPKs phosphorylation was measured to assess the activation of these kinases upon treatment with LPS. ERK1/2, p38, and JNK phosphorylation and NF-κB were significantly up-regulated following LPS treatment, with the maximum phosphorylation occurring at 20min after stimulation. It is interesting to note that JNK inhibitors inhibited phosphorylation of JNK and inhibited phosphorylation of p-38 simultaneously. Moreover, inhibitors of ERK1/2 inhibited phosphorylation of ERK1/2, increased phosphorylation of p-38 simultaneously, suggesting that ERK, JNK and p38 pathways were associated. Our findings demonstrated that LPS-induced cell proliferation dependent on JNK, ERK and p38 signaling, suggesting that ERK and p38 pathways were involved in MM1-s and MM1-r cells proliferation.

Since TLRs activate innate and adaptive immune responses, we further study immunoregulatory factor IL-6, IL-12 and IL-18 secretion in the culture supernatants induced by LPS. LPS increased the production of IL-6 in RPMI8226 and U266 cells, and increased the production of IL-18 in MM1-r and MM1-s cells. To further examine the effect of LPS on immune escape, MM1-s cells were co-cultured with T cells from donors and 3H incorporation was examined for T cells proliferation. Our results showed that T cells proliferation was decreased when the cells were treated by LPS, suggesting that TLR4 ligand LPS facilitates MM1-s cells' evasion of immune surveillance.

Disclosures:

No relevant conflicts of interest to declare.