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; MACHAÍN M,; HERNÁNDEZ DEL PINO RE; PASQUINELLI V

San Luis

Congreso; LXXI Reunión Anual de la Sociedad Argentina de Inmunología (SAI); 2023

SAI

Over the last years, interest has aroused in the gut-lung axis as a regulator of the immune system homeostasis. Intestinal dysbiosis has been implicated in numerous respiratory infections or in the chronic exacerbation of certain lung diseases such as Tuberculosis. Antibiotic-mediated dysbiosis can lead to Clostridioides difficile infection (CDI), an opportunistic potentially lethal pathogenthat colonizes large intestine. Since the impact of CDI on immune cells functions against M. tuberculosis (Mtb) infection remains unknown, here we evaluate the effect of pre-exposing human macrophages to C. difficile (CD) on Mtb uptake. To this end, monocytes were obtained from healthy donors' blood after Ficoll- Hypaque gradient and CD14 positive magnetic selection. Monocytes were cultured in the absence of FBS for 2h and then in complete media ON. Afterwards, monocyte-derived macrophages (MΦs) were cultured in the presence 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 with FITC-coupled Mtb (BEI Resources/NR-14819, H37Rv) for 1h. Endocytic levels for Mtb were evaluated by flow cytometry and macrophages morphology by microscopy.Our results show that pre-exposure to CDH induce characteristic changes of MΦs' differentiation. Increases in cytoplasmic volumes and granularity were evidenced by enlarged forward and side scatter on flow cytometry. MΦs also exhibited typical endocytic structures (e.g. pseudopods). Moreover, cell viability seems not to be affected by CDH pre-exposure in none of the time points evaluated as analyzed by using eFluorTM 780 viability dye. When evaluating Mtb- FITC uptake, preliminary data indicate that MΦs differentiated for 5 and 7 days enhanced their endocytic capacity compared to 24-48h-cultured MΦs (30 vs. 60% of FITCpos MΦs). At day 5, MΦs also evidenced the highest median intensity of fluorescence, indicating that not only the percentage of endocytic cells increases, but also the amount of bacteria per MΦ. Interestingly, pre-exposure to CDH 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 induced Mtb-FITC uptake compared to MΦs without CDH pre-exposure (controls), the removal of extracellular CDH led to reduced uptake capacity. Although we have not studied it yet, these variations could be explained by the activation of macropinocytosis pathways by CDH, a mechanism that we have addressed in a previous work.In conclusion, we observed a modulation of the endocytic range against M. tuberculosis elicited by C. difficile in human macrophages. These results are the first testing C. difficile-M. tuberculosis-macrophages crosstalk, exploring how an intestinal pathogen might affect innate immune responses against a respiratory bacterium.