Anti-IFNG monoclonal antibody, clone YNH2.3 [Alexa Fluor® 488]

Mycobacterium bovis is the causative agent of bovine tuberculosis (TB), a cattle disease of global importance. M. bovis infects bovine macrophages (Mo) and subverts the host cell response to generate a suitable niche for survival and replication. We investigated the role of the anti-inflammatory cytokine interleukin (IL) 10 during in vitro infection of bovine monocyte-derived Mo (bMDM) with two divergent UK strains of M. bovis, which differentially modulate expression of IL10. The use of IL10-targeting siRNA revealed that IL10 inhibited the production of IL1B, IL6, tumour necrosis factor (TNF) and interferon gamma (IFNG) during infection of bMDM with the M. bovis strain G18. In contrast, IL10 only regulated a subset of these genes; TNF and IFNG, during infection with the M. bovis reference strain AF2122/97. Furthermore, nitric oxide (NO) production was modulated by IL10 during AF2122/97 infection, but not at the nitric oxide synthase 2 (NOS2) mRNA level, as observed during G18 infection. However, IL10 was found to promote survival of both M. bovis strains during early bMDM infection, but this effect disappeared after 24 h. The role of IL10-induced modulation of TNF, IFNG and NO production in M. bovis survival was investigated using siRNA targeting TNF, IFNG receptor 1 (IFNGR1) and NOS2. Knock-down of these genes individually did not promote survival of either M. bovis strain and therefore modulation of these genes does not account for the effect of IL10 on M. bovis survival. However, TNF knock-down was found to be detrimental to the survival of the M. bovis strain G18 during early infection. The results provide further evidence for the importance of IL10 during M. bovis infection of Mo. Furthermore, they highlight M. bovis strain specific differences in the interaction with the infected bMDM, which may influence the course of infection and progression of bovine TB.

During the last two decades, mesenchymal stem cells (MSCs) gained a place of privilege in the field of regenerative medicine. Recently, extracellular vesicles (EVs) have been identified as major mediators of MSCs immunosuppressive as well as pro-regenerative activities in many disease models, including inflammatory/degenerative conditions as joint diseases and osteoarthritis. In order to shed light on EVs potential, a rigorous profiling of embedded proteins, lipids and nucleic acids (mRNA/miRNA) is mandatory. Nevertheless, reliable strategies to efficiently score miRNA cargo and modulation under diverse experimental conditions or treatments are missing. The aim of this work was to identify reliable reference genes (RGs) to analyze miRNA content in EVs secreted by adipose-derived MSCs (ASCs) and verify their consistency under inflammatory conditions that were proposed to enhance ASC-EVs immunomodulatory and regenerative potential. RefFinder algorithm, that integrates the currently available major computational programs (geNorm, NormFinder, BestKeeper, and Delta Ct method), allowed to identify miR-22-5p and miR-29a-5p as the most stable RGs. Notably, both miRNAs maintained the highest stability when EVs isolated from IFNg-treated ASCs were included in the analysis. In addition, considerable effects of suboptimal RGs choice on the reliable quantification of miRNAs involved at different levels (tissue homeostasis or macrophage polarization) in the osteoarthritis phenotype, and thus considered as promising therapeutic molecule, have clearly been demonstrated. In conclusion, a proper normalization method is not only needed for research purposes but also mandatory to characterize clinical products and predict their therapeutic potential, especially in the emerging field of MSCs derived-EVs as new tools for regenerative medicine.