THE GUT-LUNG AXIS IN VITRO: EXPLORING C. DIFFICILE'S INFLUENCE ON MACROPHAGES' M. TUBERCULOSIS UPTAKE

BARBERO, AM; MORICONI, ND; PALMA, S.; MENITE, N; ROMANO, L; VILLAFAÑE, G; MACHAIN, M; HERNANDEZ DEL PINO, RE; PASQUINELLI, V

San Luis

Congreso; LXXI REUNIÓN CIENTÍFICA ANUAL DE LA SOCIEDAD ARGENTINA DE INMUNOLOGÍA (SAI); 2023

SOCIEDAD ARGENTINA DE INMUNOLOGÍA (SAI)

Over the last years, interest has aroused in the gut-lung axis as a regulator of theimmune system homeostasis. Intestinal dysbiosis has been implicated innumerous respiratory infections or in the chronic exacerbation of certain lungdiseases such as Tuberculosis. Antibiotic-mediated dysbiosis can lead toClostridioides difficile infection (CDI), an opportunistic potentially lethal pathogenthat colonizes large intestine. Since the impact of CDI on immune cells functionsagainst M. tuberculosis (Mtb) infection remains unknown, here we evaluate theeffect of pre-exposing human macrophages to C. difficile (CD) on Mtb uptake.To this end, monocytes were obtained from healthy donors' blood after FicollHypaque gradient and CD14 positive magnetic selection. Monocytes werecultured in the absence of FBS for 2h and then in complete media ON.Afterwards, monocyte-derived macrophages (MΦs) were cultured in thepresence or absence of CD (NAP1/BI/027 strain) inactivated by heat treatment(CDH) for different times (24h, 48h, 5d and 7d). Then, MΦs were stimulated withFITC-coupled Mtb (BEI Resources/NR-14819, H37Rv) for 1h. Endocytic levelsfor Mtb were evaluated by flow cytometry and macrophages morphology bymicroscopy.Our results show that pre-exposure to CDH induce characteristic changes ofMΦs' differentiation. Increases in cytoplasmic volumes and granularity wereevidenced by enlarged forward and side scatter on flow cytometry. MΦs alsoexhibited typical endocytic structures (e.g. pseudopods). Moreover, cell viabilityseems not to be affected by CDH pre-exposure in none of the time pointsevaluated as analyzed by using eFluorTM 780 viability dye. When evaluating MtbFITC uptake, preliminary data indicate that MΦs differentiated for 5 and 7 daysenhanced their endocytic capacity compared to 24-48h-cultured MΦs (30 vs.60% of FITCpos MΦs). At day 5, MΦs also evidenced the highest median intensityof fluorescence, indicating that not only the percentage of endocytic cellsincreases, but also the amount of bacteria per MΦ. Interestingly, pre-exposure toCDH for 5d induced different Mtb-FITC uptake levels when CDH was washedbefore Mtb addition or not. While the presence of CDH in the cell culture inducedMtb-FITC uptake compared to MΦs without CDH pre-exposure (controls), theremoval of extracellular CDH led to reduced uptake capacity. Although we havenot studied it yet, these variations could be explained by the activation ofmacropinocytosis pathways by CDH, a mechanism that we have addressed in aprevious work.In conclusion, we observed a modulation of the endocytic range against M.tuberculosis elicited by C. difficile in human macrophages. These results are thefirst testing C. difficile-M. tuberculosis-macrophages crosstalk, exploring how anintestinal pathogen might affect innate immune responses against a respiratorybacterium.