DUGi

Supervivència i replicació intracel·lular de soques AIEC i no-AIEC en dues línies cel·lulars de macròfags

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Escherichia coli (E. coli) is a common microorganism of the human intestinal microbiota. A new pathotype called AIEC (Adherent Invasive Escherichia coli) associated with Crohn’s disease (CD) has been described. It has the ability to adhere to and invade intestinal epithelial cells, as well as to survive and replicate inside macrophages. Most studies have been done using a murine cell line (J774), but recent studies demonstrate its ability to replicate in human cell lines such as THP1. No study comparing the bacterial replication in the two types of cell lines with a broad set of AIEC strains exist. Additionally, there is no sufficient knowledge of the survival and replication abilities in macrophages of non-adherent (non-AIEC) strains that inhabit in the intestine. For these reasons, we wanted to study and compare the survival and replication of E. coli strains, including AIEC and non-AIEC, in human and murine macrophages. A total of 50 strains, 21 AIEC and 29 non-AIEC (isolated from patients with CD and controls, the same from which the AIEC strains have been isolated) have been tested. An AIEC reference LF82 and commensal laboratory strain K12 (non-AIEC) have been used as control strains. The murine macrophages J774 and the human monocyte THP1 cell lines have been tested. The survival and replication index (I_REPL) is measured based on the bacterial infection of these cell cultures using gentamicin to eliminate the extracellular bacteria. It is calculated as the percentage of intracellular bacteria after 24h of the infection with respect to the internalized ones (1h post-infection). To analyze the replication capacity in J774 and THP1 according to the pathotype, a t-test has been performed. To compare frequency data, Pearson’s χ2 has been used, and the possible relationship between cell lines has been determined by Pearson or Spearman correlation study. In this study, it has been possible to verify that AIEC strains presented high replication capacity in J774 macrophages (with an average of I_REPL = 1146%), but it replicated less in THP1 (mean of I_REPL = 89%). In contrast, non-AIEC strains did not show large differences in replication between both cell lines (mean of I_REPL = 116% in THP1 and mean of I_REPL = 281% in J774). Finally, it has been possible to establish a possible categorization threshold (I_REPL = 500%) that allows the classification of E. coli strains according to pathotype. In conclusion, THP1 is not a good option for conducting these studies with E. coli and, therefore, the difference between cell lines determines that the ability of survival and replication of these bacteria is not independent of the host but depends on the infected cell