Brucella infects and induces a mild inflammatory response in human intestinal epithelial cells

FERRERO MC,; RUMBO M,; FOSSATI CA; BALDI PC,

Buenos Aires

Congreso; Brucellosis 2011 International Research Conference; 2011

In humans, one of the most common forms of transmission of brucellosis is consumption of food contaminated with Brucella spp. We investigated the ability of Brucella smooth strains (B. abortus 2308, B. suis) and rough strains (B. canis and B. abortus RB51) to infect, replicate and induce the secretion of cytokines in the human intestinal epithelial cell lines Caco-2 and HT-29. Cells were infected at 200 bacteria/cell for 16 hours. At 2, 24 and 48 hours postinfection (pi) intracellular bacteria were quantified by gentamicin protection assays. All the tested strains adhered and invaded Caco-2 and HT29 cells, and in all cases the adherence and invasion of rough strains was higher than that of smooth strains. In HT-29 cells adherence was 6.5 ± 0.7 x107 for B. canis and 5.7 ± 0.2 x107 for RB51 versus 5.6 ± 0.9 x106 and 2.43 ± 0.13 x106 CFU/ml for B. abortus 2308 and B. suis, respectively, whereas invasion was 7.9 ± 2.6 x106 and 9.0 ± 1.0 x106 versus 2.4 ± 0.4 x103 and 9.8 ± 0.7x102 CFU/ml, respectively. Invasion was dependent on microfilaments and microtubules since it was inhibited by 90 ± 0.8% by cytochalasin D and by 52 ± 1.3% by colchicine. Only smooth strains exhibited intracellular replication (CFU increased ~ 2 log at 48 hours pi in HT-29). However, as in other cell types, a virB10 mutant (type IV secretion system-T4SS-) failed to replicate intracellularly in both cell lines. As assessed with fluorescent probes, B. abortus 2308 and B. abortus RB51 infection did not affect the viability of Caco-2 and HT-29 cells, but increased the percentage of damaged cells in HT-29 cultures. LDH levels were increased only in supernatants of HT-29 cells infected with B. abortus RB51. In HT-29 cells the infection induced a moderate production of IL-8 and a low secretion of GM-CSF, but did not induce MCP-1, IL-1β or TNF-α. Cytokine induction was dependent on bacterial viability (heat-killed B. abortus did not induce cytokines) but did not depend on T4SS function or intracellular replication, as cytokine levels induced by the virB10 mutant were similar to those induced by the wild type strain. Infection did not induce cytokine secretion in Caco-2 cells. These results indicate that Brucella can invade and replicate in human intestinal epithelium without affecting cell viability or inducing a significant inflammatory response.