Rat hepatocyte invasion by Listeria monocytogenes and analysis of TNF-alpha role in apoptosis

Abstract

Listeria monocytogenes, etiological agent of severe human foodborne infection, uses sophisticated mechanisms of entry into host cytoplasm and manipulation of the cellular cytoskeleton, resulting in cell death. The host cells and bacteria interaction may result in cytokine production as Tumor Necrosis Factor (TNF) alpha. Hepatocytes have potential to produce pro-inflammatory cytokines as TNF-alpha when invaded by bacteria. In the present work we showed the behavior of hepatocytes invaded by L. monocytogenes by microscopic analysis, determination of TNF-alpha production by bioassay and analysis of the apoptosis through TUNEL technique. The presence of bacterium, in ratios that ranged from 5 to 50,000 bacteria per cell, induced the rupture of cellular monolayers. We observed the presence of internalized bacteria in the first hour of incubation by electronic microscopy. The levels of TNF-alpha increased from first hour of incubation to sixth hour, ranging from 0 to 3749 pg/mL. After seven and eight hours of incubation non-significant TNF-alpha levels decrease occurred, indicating possible saturation of cellular receptors. Thus, the quantity of TNF-alpha produced by hepatocytes was dependent of the incubation time, as well as of the proportion between bacteria and cells. The apoptosis rate increased in direct form with the incubation time (1 h to 8 + 24 h), ranging from 0 to 43%, as well as with the bacteria : cells ratio. These results show the ability of hepatocyte invasion by non-hemolytic L. monocytogenes, and the main consequences of this phenomenon were the release of TNF-alpha by hepatocytes and the induction of apoptosis. We speculate that hepatocytes use apoptosis induced by TNF-alpha for release bacteria to extracellular medium. This phenomenon may facilitate the bacteria destruction by the immune system.

Brucella-infected hepatocytes mediate potentially tissue-damaging immune responses

“…Hepatocytes have been shown to respond to bacterial and viral infections with the secretion of proinflammatory cytokines and chemokines [5,6,22]. Therefore, hepatocytes may not only constitute a site for Brucella replication in the liver, but may also mediate the recruitment of inflammatory cells to the infectious focus.…”

“…Kupffer cells are critical for the rapid clearance of microorganisms from the systemic circulation, and can facilitate the generation of a local inflammatory response leading to recruitment of inflammatory cells such as neutrophils, monocytes, and lymphocytes. On the other hand, hepatocytes can also produce proinflammatory cytokines and chemokines in response to bacterial invasion and/or cytokine activation [5][6][7]. The inflammatory microenvironment can then activate recruited neutrophils to release toxic oxygen radicals, cytokines, and matrix metalloproteases, all of which have been shown to mediate hepatocyte injury in vitro [8,9].…”

Quantification of the Expression of HIF-1alpha by Real-Time PCR in Rat Hepatocytes Cultures Invaded by <i>Shigella flexneri</i> under Normoxic and Hypoxic Conditions

“…Hepatocytes monolayers grown on glass coverslips and were stained by hematoxylin-eosin as described elsewhere [27]. Images were analyzed in an optical microscope with digital image capture system (Leica).…”

Análise comparativa do processo de invasão de hepatócitos de rato por Listeria monocytogenes e Salmonella Typhimurium: caracterização morfológica, quantificação da liberação de TNF-alfa e da morte celular por apoptose

“…al., 1989;Conlan e North, 1991;Havell e Sehgal, 1991, Gregory, et al, 1992 (Santos et al, 2005). Assim, é provável que as cepas de L. monocytogenes utilizadas em nossos experimentos não produzam listeriolisina O (LLO), e por isso tenham dificuldades na etapa de invasão célula-a-célula, quando se forma o vacúolo de dupla membrana.…”

“…Foram utilizadas concentrações bacterianas proporcionais ao número de células, denominadas "multiplicidade de infecção" (MOI), à saber: MOI 0,5 (5x10 5 bactérias : 1x10 6 células), MOI 5 (5x10 6 bactérias : 1x10 6 células), MOI 50 (5x10 7 bactérias : 1x10 6 células), MOI 500 (5x10 8 bactérias : 1x10 6 células), MOI 5000 (5x10 9 bactérias : 1x10 6 células), e MOI 50000 (5x10 10 bactérias : 1x10 6 células) (Santos et al, 2005).…”