Credit: Figure from Ramakrishnan, L. Nat. Rev. Immunol. 12, 352–366 (2012), Nature Publishing Group.

Granulomas — immune cell aggregates formed in response to chronic inflammatory stimuli such as Mycobacterium spp. infection — contain a range of macrophage morphologies, including polyploid, multinuclear giant cells. Formation of these giant cells was thought to occur by cell-to-cell fusion, in a similar manner to osteoclast differentiation. New evidence now shows instead that macrophage mitotic defects downstream of Toll-like receptor 2 (TLR2) signalling promote genome duplications that are stabilized by activation of the DNA damage response.

In vitro stimulation of common macrophage and dendritic cell progenitors and macrophage precursors with M-CSF (macrophage colony-stimulating factor) and bacterial lipoprotein (BLP; a ligand for TLR2–TLR6) resulted in the formation of large multinuclear macrophages, in a manner dependent on cell-autonomous MYD88 signalling. TLR3 and TLR4 ligands were markedly less efficient at inducing multinuclear macrophages.

Using genome-wide transcriptome analysis and single-cell RNA sequencing, Herrtwich et al. showed that BLP stimulation through TLR2 drives a non-canonical macrophage differentiation programme, including the downregulation of macrophage cell fate-determining factors such as Mafb. BLP-stimulated macrophages had altered expression of genes related to cholesterol transport and metabolism, leading to lipid body accumulation, as well as increased expression of genes associated with remodelling of the extracellular matrix. Similar gene signatures were confirmed for macrophages from mycobacterial granulomas in vivo. Furthermore, in mixed bone marrow-chimeric mice infected with Mycobacterium bovis, the authors were not able to detect multinuclear macrophages containing mixed surface markers from the two bone marrow donors. Thus, it seems that multinuclear granuloma macrophages form via a distinct differentiation pathway rather than by the fusion of 'canonical' macrophages.

Analysing the DNA content of BLP-stimulated macrophages showed the presence of polyploid nuclei mainly in binuclear cells, which is indicative of recurrent cytokinesis failure. An increased rate of cytokinesis failure due to cleavage furrow regression was observed in mononuclear macrophages stimulated with BLP. Whereas BLP initially suppressed the proliferation of macrophage precursors, at later time points it reprogrammed binuclear macrophages to express cell cycle genes and re-enter mitosis. Therefore, BLP stimulation seems to allow macrophages to overcome the p53-dependent barriers to proliferation of polyploid cells. Re-proliferation of polyploid cells required chronic TLR2−MYC signalling. Such signalling was shown to activate the DNA damage response and the expression of DNA repair genes in BLP-stimulated macrophages, thus securing the genomic stability of polyploid macrophages. A role for the DNA damage response pathway in promoting the differentiation of polyploid macrophages was also shown for human granulomas caused by Mycobacterium tuberculosis infection, as well as by non-infectious mechanisms.

This study sheds new light onto the molecular mechanisms that control macrophage differentiation during chronic inflammation. The macrophage composition of granulomas can determine disease outcome for M. tuberculosis infections, so understanding the macrophage cell fate decisions that occur in granulomas could offer new opportunities for therapeutic targeting of this and other granuloma-associated diseases.