Increased M2 Macrophages and Higher Levels Of The Tribble Family Member Trib1 In Monocytes Are Associated With Progressive Disease In Multiple Myeloma (MM) Patients

Macrophages consist of two subgroups, M1 and M2. M1 macrophages are pro-inflammatory cells against bacterial and viral infections whereas M2 macrophages are anti-inflammatory and associated with tumor progression. The mammalian tribble (Trib) family of genes, Trib1, Trib2 and Trib3, encode pseudokinase proteins that have important roles in monocyte/macrophage proliferation, differentiation, and apoptosis. Trib1 is a critical factor that induces M2 macrophage differentiation in the bone marrow (BM).

First, we investigated the proportion of M1 and M2 macrophages in BM mononuclear cells (MCs) from multiple myeloma (MM) patients with progressive disease or in remission using flow cytometric analysis. The percentage of M2 (CD36⁺/CD86^-) macrophages in BM was significantly increased in MM patients with progressive disease (n=15) compared to those in remission (n=5; P<0.05) whereas there was no difference in the percentage of M1 (CD86⁺/CD14⁺) macrophages in BM derived from MM patients with progressive disease compared to those in remission. Using immunohistochemical (IHC) analysis, the proportion of M2 macrophages was also determined in MM BM biopsies from patients with progressive disease and remission. The samples were cut into five-micrometer sections and double stained with two antibodies following a standard IHC protocol. IHC demonstrated that the percentage of M2 macrophages (CD36⁺/CD14⁺) was markedly increased in BM sections from MM patients with progressive disease compared to those in remission. In contrast, the percentage of M1 (CD86⁺/CD14⁺) macrophages was not different among those patients with progressive disease compared to those in remission. Next, we analyzed Trib1, Trib2 and Trib3 gene expression in BMMCs obtained from MM patients with progressive disease or in remission. RT-PCR results showed Trib1 expression levels were much higher among patients with progressive disease compared to those in remission. In contrast, the expression Trib2 and Trib3 was not related to the MM patient's clinical status. To determine whether MM tumor cells affected monocyte/macrophage differentiation and Trib gene expression, we co-cultured fresh MM tumor cells with purified healthy human monocytes. BMMCs from MM patients were co-cultured with human monocytes from normal subjects using Transwell plates and the percentage of M1 and M2 macrophages was determined using flow cytometric analysis following 2, 5 and 7 days of culture. The percentage of M2 cells increased whereas the proportion of M1 cells decreased. Gene expression of Trib1, Trib2 and Trib3 was analyzed using RT-PCR following 2, 5, and 7 days of co-culture. The expression of Trib 1 increased during the 7 days of co-culture whereas the expression of Trib2 and Trib3 did not change. Moreover, when direct cell-to-cell contact occurred between the MM cells and the monocytes, the percentage of M2 macrophages (CD36⁺) markedly increased after 7 days of incubation.

We have shown that MM cells induce monocytes to increase Trib1 gene expression, which stimulates M2 differentiation in monocytes. M2 cells, in turn, induce tumor progression, providing a positive feedback loop on Trib1 expression, monocyte differentiation and tumor cell growth. Overall, we propose that Trib1 may be considered as a potential novel therapeutic target for the treatment of MM.