Commensal Peptoniphilus harei induce activation of monocytes via TLR2/CD14 signalling in whole blood

Myeloid cells, such as monocytes, are central in the immune response to infections. However, limited knowledge exists regarding the inflammatory response to Gram-positive anaerobic cocci (GPAC).To explore the ability of GPACs to induce monocyte activation, we compared two GPAC species, Parvimonas micra and Peptoniphilus harei, with E. coli, a major cause of bloodstream infections. We stimulated whole blood with heat-killed bacteria or conditioned medium (CM) and analysed monocytes for signs of activation by flow cytometry. The mechanisms were explored using anti-CD14- or anti-TLR2 antibodies. Finally, to investigate potential pathogen-associated molecular patterns in CM, we performed mass spectrometry (MS).Our findings revealed that P. harei significantly induced activation of monocytes, as evidenced by increased expression of activation markers (PDL1, HLA-DR and CD11b), phosphorylation of members of signalling pathways (NFκBp65, p38 and Akt) and production of cytokines (IL-1β, TNF, IL-6). Interestingly, P. micra had minimal impact on monocyte activation, and we thus focused on P. harei. Pre-treatment with anti-CD14 attenuated the activation of monocytes by heat-killed P. harei. Moreover, P. harei CM also induced clear monocyte activation, though, in contrast to heat-killed P. harei, this activation was dependent on TLR2 signalling. MS analysis of CM primarily identified four proteins possibly associated with monocyte activation. Finally, we also explored neutrophil activation and found a similar activation pattern to that of monocytes, suggesting that our observations are not exclusive to monocytes.Our study demonstrates that P. harei induce prominent activation of monocytes in blood, highlighting a pathogenic potential of these otherwise harmless perceived bacteria.