Chemerin Produced By Mesenchymal Stromal Cells (MSC) Is an Important Factor for In Vivo macrophage Migration

Mesenchymal Stromal Cells (MSC) are multipotent cells currently used for treating several inflammatory disorders thanks to their ability to modulate the immune response. However, the mechanisms by which MSC are able to suppress the immune response have not been fully understood. Chemerin has been recently identified as a chemotactic protein, secreted as a precursor, named Prochemerin, and converted into its active form through the proteolitic cleavage of the last six-seven amino acids at the C-terminal domain by different serine and cysteine proteases derived from the fibrinolitic, coagulation and inflammatory cascade.

In particular, we observed that both human and mouse bone marrow-derived MSC were able to produce Chemerin under basal conditions and its production was strongly increased after stimulation with inflammatory cytokines.

The aim of this study was to understand whether Chemerin produced by MSC is involved in their potent immune-modulatory activity.

Chemerin was immune-purified from supernatant of human MSC (MSC-Chem) and utilized for measuring in vitro migration index (MI) of pre-B cells expressing the human ChemR23 receptor (L1.2-ChemR23). MSC-Chem was able to induce the migration of ChemR23-expressing cells in a dose-depend manner (MI 1nM=85, MI 5nM=480, MI 10nM=1131). However, recombinant human (rh)-chemerin induced higher migration of L1.2-ChemR23 cells compared to MSC-Chem (MI 5nM=1938), suggesting that only a fraction of MSC-Chem was converted into its active form by MSC themselves. In accordance, LC/MS mass spectrometry analysis on purified MSC-Chem did not identify the active form of the protein. Interestingly, pre-incubation of MSC-Chem with Neutrophil Elastase and Cathepsin L induced a strong migration of L1.2-ChemR23 cells compared to MSC-Chem alone (MI MSC-Chem alone 1 nM=23.33; MI MSC-Chem 1 nM + Neutrophil Elastase=328; MI MSC-Chem 1 nM + Cathepsin L=4950; p=0.002), suggesting that MSC-Chem were converted in its active form, after cleavage by proteases.

Starting from these data, we established an in vivo migration assay by injecting under the abdominal skin of C57BL6 mice a mix of matrigel and murine (m)MSC (secreting or not Chemerin). After 5 days, the matrigel plug was excided, digested and infiltrating immune cells were analyzed by FACS analysis. Chemerin production by mMSC was totally abrogated by using RNA interference approach (sChem-MSC). Interestingly, mMSC features, such as phenotype and differentiation ability, were not affected by the gene-silencing process.

Preliminary results showed that 5 days after injection, scramble Chem-MSC were able to recruit macrophages (CD45⁺CD11b⁺F4/80⁺ cells) into the matrigel plug. On the other hand, sChem-MSC drastically decreased their ability to induce macrophages migration, (sChem-MSC mean=2.38%, range=0.8%-6.4%; scramble MSC mean=8.2%; range=4%-11.5%; p=0.01; n=3).

These findings identify a new mechanism by which MSC, through Chemerin production, attract macrophages in vivo. Further studies are needed to understand whether recruited macrophages are also affected by the immunomodulatory activity of MSC