Therapeutic doses of isoniazid (INH) interfere with NETosis by targeting Myeloperoxidase. A possible mechanism for INH-induced autoimmune reactions.

PABLO SCHIERLOH,; LUIS CASTILLO,; NAHUEL RODRIGUEZ-RODRIGUES; GARCIA MARINA; FERNÁNDEZ, GC; LANDONI V.I

Buenos Aires

Congreso; The 18th International Congress on Infectious Diseases (ICID), Buenos Aires, Argentina,; 2018

Background: Drug induced lupus-like reactions are characterizedby the presence of anti-nuclear antibodies (ANA) withouthypersensitivity to the drug itself. One of such drugs is the isoniazid(INH), an effective antimicrobial worldwide used to treattuberculosis (TB). Recent a study has reported increased prevalenceof anti-cytoplasmic Neutrophil antibodies (ANCA) in TB patientsafter INH-combined anti-TB therapy. INH is a prodrug that needsto be activated by the mycobacterial peroxidase/catalase KatG(Rv1908c) to give the INH? toxic radical. Considering that NeutrophilExtracellular Traps (NETs) formation requires enzymaticallyactive myeloperoxidase (MPO), we asked if the extensive neutrophil(PMN) activation typically observed during active TB maylead to undesirable autoimmune reactions when it is combinedwith an INH-based therapy.Methods & Materials: Signed informed consent was obteinedfrom all blood donors. PMN were isolated from normal humanblood by Ficoll gradient followed by dextran sedimentation. Primarygranules were isolated after mechanical rupture of PMNfollowed by high speed centrifugation. Inmunofluorescence studieswere were carried out by confocal-mycroscopy and flow cytomtry.ELISA and enzimatic activities were achieved by UV/visible spectrophotometry.Results: Primary granules containing MPO were incubated withdiferent doses of INH. We found that INH acts as both substrateand inhibitor for human MPO at therapeutic concentrations(Km = 30 ± 5 M; Cmax = 40 ± 15 M) giving an active productwith oxidant properties (p < 0.01). Moreover, in the presence ofINH, in vitro induced NETs adopted a differential conformationwith several unique features including reduced DNA spreading(p < 0.01), reduced DNA-MPO colocalization (p < 0.01), reducedDNA-Neutrophil Elastase colocalization (p < 0.05), diminished actindegradation (p < 0.05), reduced DNAse 1 sensitivity (p < 0.01)and diminished phagocytic removal by autologous macrophages(p < 0.05). As a clinical correlate, we observe that prevalence ofANCA + sera were higher in a subset of TB patients under prolongedINH-combined chemotherapy (n = 11/168; p < 0.05) compared withdrug-resistant TB patients treated with second line antimicrobials(n = 0/61).Conclusion: Our results suggest a possible mechanism for adrug-induced autoimmune reaction in which MPO-oxidation ofINH alters the NET structure and its later processing, leading topathogenic accumulation of potentially immunogenic self-antigens(i.e: DNA + Core Histones + primary granules enzymes).Ongoingexperiments with a mice model of INH treatment will shed morelight on this regard