Anti-infectious and anti-inflammatory activities of lactoferrin and fragments thereof

Abstract: Lactoferrin (LF), a major protein present in milk, mucosal secretions and secondary granules of neutrophils, has been suggested to participate in host defense at mucosal surfaces and to mediate anti-inflammatory activities. A pepsin-derived fragment of LF has been shown to contain the antibacterial domain of the protein. Despite this proposed important dual capacity of LF at the mucosal membranes there are few in vivo studies to support these effects. Our aim was to investigate if anti-infectious or anti-inflammatory activities on mucosal surfaces could be mediated by perorally given LF or synthetic fragments of the antibacterial region of LF in experimental animal models, to gain insight into how LF mediates the anti-inflammatory activities in vitro, and to define the sequence in the antibacterial region of the LF molecule responsible for the antimicrobial activity. Experimental mouse models of urinary tract infection (UTI) induced by E.coli O6K5 and dextran sulphate (DX)-induced acute colitis were used. The number of E.coli present in the urinary tract and the urinary and systemic inflammatory response (urinary leukocytes, urinary and systemic IL-6 levels) in mice with UTI were reduced by the LF treatment compared to the control group. Mice treated with LF peptide fragments (HLD1 and 2) also showed reduced numbers of bacteria in the kidneys. The perorally given LF was found to pass over to the circulation and urine. HLD2 mediated significant bactericidal activity against E.coli when tested in vitro in mouse urine. The damaging effects induced by DX exposure (presence of blood in the faeces, colon shortening, IL-1b serum levels, crypt score) were delayed or reduced in mice treated with LF or the peptides. The number of inflammatory cells present in the colon after 7 days of DX exposure (F4/80 macrophages, CD4- and TNF-a- positive cells) was lower in the LF treated group compared to the control. LF also reduced shortening of the colon when given orally to animals with an already established inflammatory response as induced by two days of DX exposure. LF was shown to down-regulate the secretion of TNF-a, IL-1b, IL-6, IL-8 and IL-10 in monocytic cell lines (THP-1, Mono Mac 6) stimulated with LPS. The down regulation of the cytokines was reflected at the transcriptional level. Thus LPS-induced TNF-a-, IL-1b-, IL-6-, and IL-8 mRNA as shown by reversed transcription PCR were reduced as well. The known binding of LF to LPS could not explain the reduced cytokine mRNA expression and protein secretion since the effects were observed also when LF was added 30 min after the LPS to the cell assay. In addition, also IL-1b induced IL-6 secretion was down-regulated in the presence of LF. Moreover, LF was detected by immunocytochemistry in the cell nucleoli already after 30 min of incubation with the THP-1 cells and found by electromobility shift assay to decrease the binding of nuclear factor (NF)-kB to the TNF-a promoter. The antimicrobial activity against E.coli, S.aureus and C.albicans of synthetic peptides homologous to the surface exposed a-helix and b-sheet region from the N-terminal end of human LF showed that a short region comprising 12-15 a.a. corresponding to the major part of the helix region were optimal for the killing activity against all three microorganisms. In addition certain amino acids such as cystein, and hydrophobic and positively charged amino acids in the 12-amino acid long peptide were found to be important for the expression of antimicrobial activity. In summary, orally given LF can reduce infection and inflammation in a remote site such as the urinary tract, and mediates anti-inflammatory activities in the colon. This dual effect may partly reside in the antimicrobial region of the LF molecule, since synthetic fragments also provide similar activities. One possibly important mechanism of its anti-inflammatory effects is through the ability to down-regulate cytokine production via interference with the transcription factor NF-kB. The anti-infectious and anti-inflammatory activities of LF on mucosal surfaces is being utilized by the suckling child which obtains large amounts of LF via the maternal milk. However, therapeutic use of LF or its fragments may be possible in other fields of application.