Imaging approaches to understand the effects of bisphosphonate drugs on macrophages outside the skeleton — ASN Events
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  2. Workshop 11: Innate Immunity
  3. Imaging approaches to understand the effects of bisphosphonate drugs on macrophages outside the skeleton

Imaging approaches to understand the effects of bisphosphonate drugs on macrophages outside the skeleton (#127)

Marcia A Munoz 1 , Kristen Perry 1 , Niall Byrne 1 , Julie Jurczyluk 1 , Tri G Phan 1 , Simon Junankar 1 , Hristo Zlatev 2 , Seppo Auriola 2 , Michael Rogers 1
  1. Garvan Institute of Medical Research, Darlinghurst, NSW, Australia
  2. School of Pharmacy, University of Eastern Findland, Kuopio, Findland

Bisphosphonates (BPs) are a class of calcium-seeking drugs used clinically to inhibit bone resorption. BPs such as zoledronic acid (ZOL) act by inhibiting FPP synthase, (an enzyme of the mevalonate pathway) in osteoclasts, causing accumulation of the upstream isoprenoid lipid IPP and preventing the prenylation and regulation of small GTPase proteins that govern cytoskeletal dynamics and bone resorption. A small proportion of BP users, however, experience adverse inflammatory side effects that remain poorly understood.

Accumulating evidence suggests that BPs have pleiotropic effects outside the skeleton. Our findings reveal that BPs can target soft tissue macrophages. We have shown that intravenous ZOL administration in mice inhibits the prenylation of Rab GTPases in peritoneal macrophages. Using 2-photon intravital imaging of fluorescently-tagged BPs, we discovered that BPs accumulate in the draining lymph nodes after subcutaneous injection. We have also demonstrated that BPs diffuse from leaky vasculature and accumulate in 4T1 mammary tumour tissue by binding to microcalcifications that are then engulfed by tumour-associated macrophages. Four weekly doses of intravenous ZOL resulted in clear accumulation of IPP in tumour tissue and quantified by LC-MS/MS analysis.

Live cell imaging and immunofluorescence staining of IC-21 immortalised mouse macrophages revealed that treatment with ZOL (10uM for 48h) impaired cell adhesion (~50%), motility (<60% speed and displacement), and loss of morphological polarisation -characterised by the absence of clear leading (lamellipodia) and lagging (uropod) ends, without compromising cell viability. Importantly, these defects in morphology and migration were proportional to the accumulation of unprenylated proteins, and restoration of prenylation by supplementation of the lipid metabolite completely rescued cell morphology and cell migration. We are now translating these findings to determine the effects of BPs on macrophage behaviour in vivo.

Our data support the notion that BPs can target macrophages outside bone, potentially altering innate immune function.

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