Mycoplasmalipoproteins are major determinants of neutrophil extracellular trap formation

Abstract

Neutrophil granulocytes are paramount to innate responses as major effectors of acute inflammation. Among the various strategies enacted by neutrophils to eliminate microbes NETosis is a novel distinct antimicrobial activity in which an interlacement of chromatin fibres rich in granule-derived antimicrobial peptides and enzymes is extruded (NETs, neutrophils extracellular traps ). NETs contribute to the pathogenesis of acute and chronic inflammatory disorders. The interactions of mycoplasmas and innate immune cells, in particular neutrophil granulocytes, are poorly defined. Here, we describe NET formation in vivo in the mammary gland and milk of sheep naturally infected by Mycoplasma agalactiae. Also, we assess the contribution of liposoluble proteins, the most abundant component of the Mycoplasma membrane, in inducing NETosis. We demonstrate that Mycoplasma liposoluble proteins induce NET release at levels comparable to what observed with other stimuli, such as lipopolysaccharides and phorbol 12-myristate 13-acetate. Stimulation of neutrophils with synthetic diacylated lipopeptides based on the M. agalactiae P48, P80, and MAG_1000 proteins, combined in a mix or used individually, suggests that NETosis might not be dependent on a specific lipopeptide sequence. Also, NETosis is partially abolished when TLR2 is blocked with specific antibodies. The results presented in this work provide evidences for the mechanisms underlying NET activation in mycoplasma infections, and on their contribution to pathogenesis of mycoplasmosis.

In vitro induction of NETosis: Comprehensive live imaging comparison and systematic review

“…For example, in LPS-activated neutrophils multiple papers state to have observed NETosis after 30 min with 100 ng/ml [76, 83, 158, 161, 187], whereas other authors did not observe NETosis using a concentration of 10 μg/ml [18]. …”

Comparison of Macrophage Responses to African Swine Fever Viruses Reveals that the NH/P68 Strain is Associated with Enhanced Sensitivity to Type I IFN and Cytokine Responses from Classically Activated Macrophages

“…21 h pi, media was removed and replaced with cRPMI supplemented with 1 µg/mL of a S-[2-bis(palmitoyl)-propyl]cysteine (Pam2Cys) lipopeptide (kindly provided by Prof. Bernardo Chessa and Dr. Carla Cacciotto, University of Sassari, Italy), and after 24 h supernatants were removed, cleared by centrifugation (2000× g for 3 min) and stored at −80 • C until determination of cytokine levels. Pam2Cys lipopeptide was synthesized based on the 14 amino acids following the cysteine immediately downstream the signal peptide of a Mycoplasma agalactiae lipoprotein (P48: CGDKYFKETEVDGV) [31].…”

Comprehensive RNA-Seq Profiling to Evaluate the Sheep Mammary Gland Transcriptome in Response to Experimental Mycoplasma agalactiae Infection

“…Another study has described neutrophil extracellular trap (NET) formation in vivo in the mammary gland and milk of sheep naturally infected with M . agalactiae [16]. Although mycoplasma liposoluble proteins are demonstrated to induce NET release, the exact role of the recently identified phase variable β-(1→6)-Glucan is yet to be elucidated in its disease progression [17].…”