Lactoferrin Protects Rabbits fromShigella flexneri-Induced Inflammatory Enteritis

Abstract

Shigella species cause bacillary dysentery in humans by invasion, intracellular multiplication, spread to adjacent cells, and induction of brisk inflammatory responses in the intestinal epithelium. In vitro data suggest that lactoferrin, a glycoprotein present in human mucosal secretions, has a role in protection from bacterial enteric infections. We sought to determine the activity of lactoferrin in vivo, using the concentration present in human colostrum, to investigate its effect on the development of clinical and pathological evidence of inflammation in a rabbit model of enteritis. Lactoferrin protected rabbits infected with Shigella flexneri from developing inflammatory intestinal disease. Typical histological changes in ill animals included villous blunting with sloughing of epithelial cells, submucosal edema, infiltration of leukocytes, venous congestion, and hemorrhage. Lactoferrin at a concentration normally found in human colostrum blocks development of S. flexneri-induced inflammatory enteritis.Dysentery due to Shigella spp. is among the most communicable and severe forms of bacterial gastroenteritis in humans (43). The virulence of Shigella is due to its ability to invade, multiply, spread intracellularly, and induce inflammation within the intestinal epithelium (33). Most of the genes responsible for invasion of eukaryotic cells are located on a 230-kb virulence plasmid (1). Entry into mammalian cells is regulated by a plasmid-encoded type III secretory system located on a 31-kb locus that codes for the invasion plasmid antigens IpaA, IpaB, IpaC, and IpaD. This secretory mechanism is activated upon contact with epithelial cells (49). Upon entry into host cells, bacteria gain access to the cytoplasm by lysing the phagocytic vacuole and rapidly multiplying and spreading (26,32,41) Epidemiologic studies have shown that breast-feeding decreases the severity of Shigella sp. infection in infants who become colonized early in life (2,12,13,20,22,25). Immune and nonimmune components of milk may be relevant to this protection. Among the nonantibody factors is lactoferrin, an iron-binding glycoprotein of 78 kDa that is resistant to proteolytic enzymes (10). However, epidemiologic data cannot separate the effect of lactoferrin from those of other potentially protective human milk factors. Lactoferrin, antilipopolysaccharide (anti-LPS) and anti-invasion plasmid antigen secretory immunoglobulin A (IgA), lysozyme, and oligosaccharides may each play a role in protection of infants from shigellosis. Although multiple effects of lactoferrin have been demonstrated in vitro, there are no animal model studies demonstrating a role in the gut in vivo. The objective of these studies was to determine whether lactoferrin at concentrations found in human colostrum (0.125 mM) could prevent the development of clinical and pathological changes in an in vivo rabbit model of inflammatory enteritis. MATERIALS AND METHODSLactoferrin was obtained from Agennix Inc. Lactoferrin is expressed in Aspergillus awamori as a glucoamylase fus...

New Animal Model of Shigellosis in the Guinea Pig: Its Usefulness for Protective Efficacy Studies

“…Previous reports have also shown that IL-8 expression correlates with the severity of ulcerative colitis, the inflammatory signs of which resemble those observed in human shigellosis (29,30). The main function of IL-8, which can be produced by epithelial cells, is to recruit and activate neutrophils, which in turn activate NO and trigger the release of granular contents, i.e., elastase (31), lactoferrin (18), and myeloperoxidase (32). In addition, IL-8 appears to be a major mediator of the recruitment of neutrophils in the human (22) and the rabbit-ligated ileal loop model (33).…”

“…To overcome this hurdle, considerable effort over the years has been devoted to establishing a reliable animal model of bacillary dysentery. Several established Shigella infection models have proven to be useful; for instance, the pneumonia model by intranasal challenge in mice, keratoconjunctivitis by eye infection in guinea pig, intestinal inflammation by ileal ligated loop assay in rabbit, and typical bacillary dysentery following intragastric inoculation in macaques monkeys (15)(16)(17)(18)(19). However, these models are not sufficiently reliable, clinically relevant, or cost effective to be used in the screening for Shigella vaccine candidates.…”

Effect of lactoferrin on enteric pathogens

“…This complex machine secretes virulence proteins in response to sensing the presence of mammalian target cells. Lactoferrin binds to Shigella flexneri [28] and decreases invasiveness of S. flexneri in HeLa cells [29] associated with release and degradation of invasion plasmid antigens B and C, proteins that are responsible for entry of Shigella into mammalian cells [30,31]. Recombinant human lactoferrin also protects rabbits from Shigella flexneri-induced inflammatory enteritis [30].…”

The Comparison of Effect of Human Milk and Powdered Milk on the Shigella dysenteriae Invasion in Cell Culture

“…Since the recombinant Lf does not have the same structure, the ability of Shigella in the inhibition of adhesion is missed. 17 Gomez et al in 2002 and 2003 showed that high concentrations of recombinant Lf in Saccharomyces cerevisiae protect rabbits against inflammatory bowel diseases caused by S. flexneri 21 and interfere with the invasion of S. flexneri M90T but do not attach to Hela cells through inducing the separation and decomposition of ApaB. Similarly, recent studies have found that fSC prevents E. coli from binding to the gut and inhibits the adhesion of ETEC.…”

“…19 In addition, 2 glycol proteins, Lf and fSC, are involved in the inhibition of adhesion of enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), and enterotoxigenic E. coli (ETEC) . 20,21 These glycol proteins also prevent the adhesion of Shigella dysentery in lower concentrations, found in human milk.…”