Oral Presentation ANZBMS-MEPSA-ANZORS 2022

Bisphosphonates may boost immune responses to pulmonary infection by acting on tissue-resident macrophages in the lung (#5)

Marcia A Munoz 1 , Emma K Fletcher 1 , Oliver P Skinner 1 , Esther Kristianto 2 , Mark P Hodson 3 , Michael J Rogers 1
  1. Skeletal Diseases Program, Garvan Institute of Medical Research, Sydney, NSW, Australia
  2. VCCRI Innovation Centre, Victor Chang Cardiac Research Institute, Sydney, NSW, Australia
  3. School of Pharmacy, University of Queensland, Woolloongabba, Queensland, Australia

Bisphosphonate drugs inhibit bone resorption but also appear to have actions outside the skeleton by mechanisms that are poorly understood. Clinical and epidemiological studies suggest that nitrogen-containing bisphosphonate (N-BP) therapy is associated with reduced risk of infection and mortality from pneumonia. To better understand how N-BPs may have beneficial effects in the lung we employed various approaches including multicolour flow cytometry, mass spectrometry and single-cell RNA sequencing, and identified alveolar macrophages (aMac) as targets of N-BPs.

Flow cytometric analysis of bronchoalveolar lavage cells, isolated from mice after a single intravenous injection of a fluorescently‑tagged analogue of the N-BP zoledronate (AF647-ZOL), revealed drug uptake by >97% of aMac (CD11blo/-CD11chiCD64+) and 34% of interstitial (CX3CR1+CD11bhi) macrophages. AF647-ZOL was still detectable in aMac one week after administration. Intracellular accumulation of unprenylated proteins and build-up of the metabolite isopentenyl diphosphate (IPP) are hallmarks of the pharmacological actions of N-BPs. Consistent with intracellular uptake of labelled ZOL, using mass spectrometry and a customised biochemical assay we detected a clear build-up of IPP and unprenylated Rab GTPases in aMac 48 hours after one intravenous dose of ZOL. Importantly, single-cell RNA sequencing of aMac, isolated from mice challenged intranasally with LPS endotoxin, revealed enhanced gene expression of cytokines and chemokines (including Il1b, Cxcl2, Cxcl3, Ccl3 and Ccl4) in a subpopulation of aMac in ZOL‑treated mice compared to controls. This was accompanied by higher levels of these cytokines and chemokines in bronchoalveolar lavage fluid from ZOL‑treated mice after LPS-challenge, as measured by multiplex cytokine assays.

These studies dispel the dogma that N-BPs act only in bone and clearly demonstrate that they also affect lung-resident macrophages. In addition to preventing bone loss, we propose that N-BP treatment may boost the initial immune response of aMac to pulmonary infections, therefore conferring protection from pneumonia.