The TLR4-Active Morphine Metabolite Morphine-3-Glucuronide Does Not Elicit Macrophage Classical Activation In Vitro

Abstract

Macrophages are abundant in the tumor microenvironment where they adopt a pro-tumor phenotype following alternative polarization induced by paracrine factors from cancer and stromal cells. In contrast, classically activated macrophages have tumoricidal activities, such that the polarization of tumor-associated macrophages has become a novel therapeutic target. Toll-like receptor 4 engagement promotes classical activation of macrophages, and recent literature suggests TLR4 agonism to prevent metastasis and promote survival in experimental metastasis models. A growing number of studies indicate that TLR4 can respond to opioids, including the opioid receptor-inactive morphine metabolite morphine-3-glucuronide (M3G). We measured the activation of TLR4 in a reporter cell line exogenously expressing TLR4 and TLR4 co-receptors, and confirmed that M3G weakly but significantly activates TLR4. We hypothesized that M3G would promote the expression of classical activation signature genes in macrophages in vitro. We exposed mouse and human macrophage cell lines to M3G or the TLR4 activator lipopolysaccharide (LPS), alone or in combination with interferon gamma (IFN-γ). The classical macrophage activation markers tested were iNOS, CD86, IL-6, or TNF-α in RAW 264.7 cells and IL-6, IL-12, IL-23, TNF-α, CXCL10, and CXCL11 in THP1 cells. Our results show that despite exhibiting TLR4-activation ability, M3G does not elicit the expression of classical activation markers in LPS-responsive macrophages.

Morphine-induced hyperalgesia involves mu opioid receptors and the metabolite morphine-3-glucuronide

“…TLR4 activation has been reported as a key event in morphine analgesic tolerance and OIH, although conflicting results have been obtained 4 , 24 . Thus, to investigate if the lack of OIH in MOR KO animals may be caused by a difference in TLR4 expression in KO mice, we compared TLR4 transcripts in WT and KO mice.…”

“…Besides, M3G was shown to display low affinity binding to TLR4 and to induce cellular effects distinct from the classical TLR4 activator LPS. In the HEK-Blue-TLR4 Secreted alkaline phosphatase (SEAP) signalling assay, M3G weakly activates TLR4 but also partly inhibits LPS-induced TLR4 activation 24 . Furthermore M3G does not elicit classical LPS-induced activation markers in human and mouse macrophages 24 , while morphine effects on macrophages were reported to be MOR-dependent and independent 53 , 54 .…”

“…In the HEK-Blue-TLR4 Secreted alkaline phosphatase (SEAP) signalling assay, M3G weakly activates TLR4 but also partly inhibits LPS-induced TLR4 activation 24 . Furthermore M3G does not elicit classical LPS-induced activation markers in human and mouse macrophages 24 , while morphine effects on macrophages were reported to be MOR-dependent and independent 53 , 54 . In addition, the investigation of TLR4 involvement in OIH by using TLR4 knockout mice led to conflicting results 4 , 9 , 11 , 13 , 14 .…”

Influence of opioids on immune function in patients with cancer pain: from bench to bedside

“…TLR4 is involved in innate immune system activation. In vitro and in silico techniques have shown that many commonly used opioids can have direct effects on innate immunity via activation of the TLR4 (Hutchinson et al, ; Franchi et al, ; Grace et al, ; Khabbazi et al, ,b).…”

“…However, morphine has been shown to be protective in a murine Lewis lung carcinoma model by reducing immune cell infiltration and angiogenesis (Koodie et al, ). Morphine can also elicit anti‐tumour effects that are mediated via modulation of paracrine communication between cancer cells and non‐malignant cells in the tumour micro‐environment (Afsharimani et al, ) and can prevent the pro‐angiogenic interaction between macrophages and breast cancer cells (Khabbazi et al, ,b). Furthermore, morphine modulates cells in the tumour micro‐environment via many mechanisms, some of which increase and others decrease tumour aggressiveness (Khabbazi et al, ).…”