Department of Technology and Health, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, ItalyENEA, Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Department of Radiobiology and Human Health, Vianguillarese 301, 00123 Rome, ItalyGiellepi S.p.A., Via G. Verdi, 41/Q, 20831 Seregno (MB), ItalyDepartment of Pediatrics, Sapienza University of Rome, Viale Regina Elena, 324, 00161 Rome, Italy
r t i c l e i n f o
rticle history:eceived 29 November 2013ccepted 9 February 2014vailable online xxx
Background: Crohn’s disease is a multifactorial disease in which an aberrant immune response to com-mensal intestinal microbiota leads to chronic inflammation. The small intestine of patients with Crohn’sdisease is colonized by a group of adherent-invasive Escherichia coli strongly able to adhere and invadeintestinal epithelial cells lactoferrin is an iron-binding glycoprotein known to have anti-bacterial andanti-inflammatory activities.Aims: We explore the ability of bovine lactoferrin to modulate the interactions between the adherent-invasive E. coli strain LF82 and intestinal epithelial cells as well as the inflammatory response.Methods: Bacterial adhesion and invasion assays were used to assess the antimicrobial activity of lacto-ferrin. Electron microscopy was used to characterize bacteria–cell interactions. The mRNA expression ofpro-inflammatory cytokines was measured both in cultured cells and in biopsies taken from intestine ofpatients affected by Crohn’s disease.Results: Lactoferrin inhibited bacterial invasion through minimally affecting adhesion. This divergencewas due to a mannose-dependent lactoferrin binding to the bacterial type 1 pili and consequent bacterial
aggregation on the intestinal epithelial cell surface. Expression of pro-inflammatory cytokines, such asTNF-alpha, IL-8, and IL-6, was markedly inhibited by lactoferrin both in cultured and Crohn-derivedintestinal cells.Conclusions: Bovine lactoferrin might function via an antibacterial and/or anti-inflammatory mechanismin the treatment of Crohn’s disease.
Crohn’s disease (CD) is an inflammatory bowel disease (IBD)haracterized by a chronic transmural, segmental, and typicallyranulomatous inflammation of the intestine in humans withnknown aetiology. Deregulation of the innate and adaptive
mmune responses directed against luminal bacteria or their prod-cts, which are normally under tight immunological tolerance, are
mmune factors characteristic of CD and other IBDs [1].
Please cite this article in press as: Bertuccini L, et al. Lactoferrin prevLF82 infection in experimental model of Crohn’s disease. Dig Liver Dis
Among the bacteria that may play a role as target of de-regulatedmmunity in the pathogenesis of CD, a pathovar of Escherichia colialled AIEC (adherent-invasive E. coli) has been strongly implicated
roenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.
[2]. AIEC encompass a subgroup of E. coli spp. named from theircharacteristic capability to adhere to intestinal cells, to invadethe infected eukaryotic cells, and to replicate in epithelial cellsand macrophages, ultimately causing intestinal diseases in humans[3–7]. LF82, an E. coli strain (serotype O83:H1) originally isolatedfrom a patient with CD [3], represents the prototype AIEC strain.
Lactoferrin (Lf), a member of the transferrin family [8] presentsin milk and other exocrine secretions, constitutes one of the majorantimicrobial components of the innate immune system [9]. Biolog-ical properties reported for Lf include antimicrobial activity againsta wide range of pathogenic bacteria, fungi, protozoa and virus,as well as anti-inflammatory, antitumor and immunomodulatory
ents invasion and inflammatory response following E. coli strain (2014), http://dx.doi.org/10.1016/j.dld.2014.02.009
activities [10,11]. Antimicrobial functions attributed to this proteinor its peptides include iron sequestration [12], destabilization ofGram-negative bacteria outer membrane through binding of bacte-rial lipopolysaccharides (LPS) [13–16], selective penetration of ions
17], and modulation of bacterial entry into host cells [11,18,19].he immunomodulatory activity of Lf affects the production ofytokines such as IL-1, IL-6, and TNF-alpha [20–22].
Despite Lf broad spectrum of antimicrobial and immunomodu-atory activities mentioned above, relatively little is known aboutf capacity to modulate the complex interactions between bacte-ia and intestinal epithelial cells, and the resultant inflammatoryesponse by these latter. We addressed this issue by testing bovineactoferrin (bLf) for its efficacy as potential control agent of E. coliF82-intestinal epithelial cell interactions as well as modulator ofhe inflammatory bowel response.
. Materials and methods
.1. LF82
The AIEC strain utilized in this study was LF82 (ileal Crohn’strain, kindly provided by Arlette Darfeuille-Michaud, Clermontniversité, Clermont-Ferrand, France).
LF82 was cultured in MacConkey agar plates for 24 h at 37 ◦C andhen subcultured in Luria Bertani Broth (LB, Oxoid) with overnightncubation in air (120 rpm) at 37 ◦C. Before infection of cells, bacte-ia were washed and re-suspended in cell culture medium at theuitable concentrations.
.2. Cell line culture
Caco-2 (human colorectal adenocarcinoma) cells were obtainedrom the American Type Culture Collection (ATCC, Manassas, VA)ere grown in DMEM with high glucose (Euroclone) supplementedith 10% FCS (growth medium) as previously described [23].
.3. Organ culture
The study was approved by the ethical committee of the Univer-ity Hospital Umberto I, Rome, Italy, where patients were admitted.or each patient informed consent from parents was obtained. Afterritten consent, CD subject biopsies were obtained from intestinal
issue not involved in chronic inflammation processes. For organulture, specimens were immediately placed in Trowell T8 mediumnd treated as previously described [23].
.4. Bovine lactoferrin
BLf (Morinaga Milk Industry Co., Ltd., Japan) was deprived ofndotoxin as previously described [24], filtered, and dissolved inBS (80 mg/ml).
.5. Cytotoxicity assay
To establish the maximal non-cytotoxic dose of protein, two-old serial dilutions of bLf were incubated at 37 ◦C with Caco-2ells grown in 96-well tissue culture microplates (Flow Laborato-ies) in a humidified 5% CO2 incubator as previously described [25].fter 24, 48, and 72 h, the following parameters were evaluated:ell morphology and viability (determined by neutral red staining)ere examined by light microscopy, cell proliferation was eval-ated quantitatively by microscopic counts after dispersion into
ndividual cells with trypsin. Protein dilutions that did not affectny of these parameters were considered as non-cytotoxic concen-rations and utilized for adhesion and invasion assays.
Please cite this article in press as: Bertuccini L, et al. Lactoferrin prevLF82 infection in experimental model of Crohn’s disease. Dig Liver Dis
.6. Antibacterial activity
The minimal inhibitory concentration (MIC) of bLf was deter-ined by broth microdilution methods. LF82 cells were grown up
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at a final concentration of 5 × 105 CFU/ml. Bacteria (100 �l/well)were added to a 96-well plate with 100 �l of different bLf concen-trations (serial two-fold dilutions ranging from 0.08 to 40 mg/ml).The MIC was defined as the lowest protein concentration causinga complete inhibition of bacterial growth after 24 h incubation at37 ◦C.
2.7. Influence of bLf on LF82 adhesion and entry into Caco-2 cells
Cell monolayers, prepared in 24-well tissue culture plates (Fal-con), were incubated for 2 h at 37 ◦C in growth medium with 2-foldserial dilutions of bLf, starting from the highest non-cytotoxic con-centration. Similarly incubated cell monolayers without bLf servedas controls. Then cells were infected with LF82 at a multiplicity ofinfection (MOI) of 1 or 10 bacteria/cell and, after 3 h, were washed3 times and lysed with 0.1% TritonX-100 in H2O. The number ofadherent bacteria was determined by CFU counts on LB agar plates.Bacterial adhesion was defined as adhesion index, calculated as thepercentage of adherent bacteria compared with the initial inocu-lum, taken as 100%. The LF82 adhesion index to lactoferrin treatedcells was always normalized to that one of control monolayers.
To determine bacterial invasion, cell monolayers were infectedwith bacteria at MOI 1 or 10. After 3 h at 37 ◦C, cells were washed3 times and incubated for additional 1 h in medium containing0.1 mg/ml gentamicin to kill extracellular bacteria. The inhibitionactivity of bLf was assessed by incubating cells 2 h before and duringthe infection with different protein concentrations. Similarly incu-bated cell monolayers without bLf served as controls. CFU counts onLB agar plates were performed as described above. Bacterial inva-sion was defined as invasion index, calculated as the percentageof internalized bacteria compared with the initial inoculum, takenas 100%. The LF82 invasion index of bLf treated monolayers wasalways normalized to that one of control cells.
All assays were performed in triplicate and repeated at leastthree times.
2.8. Cytokine analysis in Caco-2 infected cells
Cells were incubated with bLf (1 mg/ml) before (2 h) and dur-ing (3 h) infection with LF82 (MOIs 1 or 10). Similarly incubatedcell monolayers without bLf served as controls. After infection,culture supernatants were collected, cleared from any cells andpathogens by centrifugation and kept frozen at −80 ◦C until use.Cell extracts were processed for tumour necrosis factor-alpha (TNF-alpha), interleukin 8 (IL-8), and interleukin 6 (IL-6) gene expressionby quantitative real time-PCR as described below. All assays wereperformed in triplicate and repeated at least three times.
2.9. Cytokine analysis in infected organ cultures
Organ cultures were incubated with bLf before (2 h at 37 ◦C)and during infection with LF82 (108 CFU/mL). Similarly incubatedcell monolayers without bLf served as controls. Bioptic material-containing dishes were placed in a modular incubator chamber (MPBiomedicals, Aurora, OH) at high oxygen saturation (95%) and incu-bated at 37 ◦C. After 6 and 24 h, biopsies were collected and totalRNA was extracted for reverse-transcription PCR (RT-PCR) analysis.Each experiment was independently repeated three times.
2.10. Cytokine induction in Caco-2 cells
ents invasion and inflammatory response following E. coli strain (2014), http://dx.doi.org/10.1016/j.dld.2014.02.009
mRNA of proinflammatory cytokines was detected by treatingcells, in the absence or presence of 1 mg/ml bLf, with 5 ng/mL ofinterferon gamma for 6 h. Cell extracts were processed for cytokinemRNA (IL-6, IL-8, and TNF-alpha) by quantitative RealTime-PCR.
Fig. 1. Effect of lactoferrin on the invasion of E. coli strain LF82 into epithelial cells.Caco-2 cells were infected with 1 or 10 CFU/cell. The invasion index was calculatedas the percentage of internalized bacteria compared with the initial inoculum, takenas 100%. The LF82 invasion index of lactoferrin-treated monolayers was always
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L. Bertuccini et al. / Digestive a
ll assays were performed in triplicate and repeated at least threeimes.
.11. Real-time PCR
Expression of TNF-alpha, IL-8, and IL-6 genes was detected byeal-time PCR. Primers were designed to non-redundant sequencessing Primer Express V3.0 (Applied Biosystems, Foster City,alifornia, United States).
Total RNA (1 �g) was reverse-transcribed to cDNA by a Highapacity cDNA Reverse Transcription Kit (Applied Biosystems).eal-time PCR amplification was done with an ABI®PRISM 7300equence Detection System, using the SYBR®Green kit (Appliediosystems). Relative transcript levels were determined usingAPDH as the endogenous control genes: primers used: 5′-CATCAATGGAAATCCCATCA-3 and 5′-GCCAGCATCGCCCCACTT-3′.
.12. Scanning electron microscopy (SEM)
BLf-treated (2 h at 37 ◦C) or untreated Caco-2 cell monolayersere infected with LF82 (MOI 10) in the presence or absence of
mg/ml bLf (4 h at 37 ◦C), processed as already reported [26] andxamined by SEM QUANTA (PHILIPS) scanning electron micro-cope.
.13. Transmission electron microscopy (TEM)
Caco-2 cells were infected with LF82 (MOI 10) in the presencer absence of 1 mg/ml bLf. At 4 h post-infection, cells were fixed,rocessed as already reported [25], and examined by a Philips 208Slectron microscope.
.14. Light microscopy
LF82 strain, cultured overnight in LB broth at 37 ◦C, was dilutedn DMEM with or without 1 mg/ml of bLf (107 CFU/ml), in the pres-nce or absence of 2% (w/v) d-mannose, and incubated at 37 ◦Cor 4 h. Bacterial suspensions were observed in bright light by anlympus BX53 optical microscope.
.15. Statistical analysis
Statistical analysis was performed using the Student’s t-test fornpaired data. Data were expressed as the mean ± S.D. and P valuesf <0.05 were considered significant.
. Results
.1. Effect of bLf on LF82 adhesion and invasion of Caco-2 cells
A preliminary set of experiments was carried out in order tossess the highest non-cytotoxic and non-bactericidal concentra-ion of bLf. To this purpose, 2-fold serial dilutions of protein from
mg/ml in MEM were incubated with Caco-2 cells for 24 h at7 ◦C. Under these conditions, protein did not affect any cyto-oxic end-points (data not shown). The antibacterial activity of
Please cite this article in press as: Bertuccini L, et al. Lactoferrin prevLF82 infection in experimental model of Crohn’s disease. Dig Liver Dis
Lf was then investigated and shown to be absent up to a con-entration of 40 mg/ml. Hence, the effect of bLf on LF82 adhesionnd internalization into Caco-2 cells was investigated within theange of non-cytotoxic, non-antibacterial bLf concentrations. It was
normalized to that one of control cells. **p < 0.05; *p < 0.01.
found that LF82 attachment to Caco-2 cell membranes was not sig-nificantly affected by bLf, whereas bacterial internalization wasinhibited by bLf in a dose-dependent manner, reaching degreesof inhibition as high as about 75 and 60% at a MOI of 1 and 10,respectively (Fig. 1).
3.2. Scanning electron microscope examination of LF82 adhesionto Caco-2 cells
Scanning electron microscope (SEM) examination of LF82-infected Caco-2 cells revealed that, as already reported in literature[5,26], adherent bacteria induced the elongation of microvilli at thesite of adherence (Fig. 2A). Moreover, the elongated microvilli wereseen to enwrap the adherent bacteria (Fig. 2B).
When Caco-2 cells were infected in the presence of bLf, thebacteria were seen to adhere to the epithelial cell membranes with-out causing, however, significant morphological modifications ofthe host cell membranes (Fig. 2C and D). High magnification scan-ning electron microscopy showed bacteria in tight aggregates onthe cellular surface, with tiny interconnecting digitations betweenindividual bacteria in the aggregates (Fig. 2D, arrows).
3.3. Light microscopy
When samples of untreated E. coli LF82 were examined by lightmicroscopy, bacteria were found to be quite evenly distributed andtypically occurring as single cells or cell pairs (Fig. 3A). Samplesof bLf-treated E. coli LF82 had a very different appearance in thatthe majority of them were in clusters (Fig. 3B). To assess the roleof bacterial type 1 pili in the formation of above clusters, somesamples were incubated with 2% d-mannose. In the presence of thisinhibitor, (Fig. 3C and D), bLf was unable to aggregate the bacterialcells (Fig. 3D).
3.4. Transmission electron microscope examination ofLF82-infected Caco-2 cells
Transmission electron microscope (TEM) examination ofuntreated and bLf-treated Caco-2 infected cells confirmed theresults obtained with both invasion tests and SEM observations.As shown in Fig. 4, adherent bacteria markedly induced the
ents invasion and inflammatory response following E. coli strain (2014), http://dx.doi.org/10.1016/j.dld.2014.02.009
elongation of microvilli from the surface of Caco-2 untreated cells(A, C), with some bacteria also being detected intracellularly, eitherinside endocytic vacuoles (A, C) or free in the cytoplasm (C). Otherbacteria were also observed within gaps between cells (B).
4 L. Bertuccini et al. / Digestive and Liver Disease xxx (2014) xxx–xxx
Fig. 2. Scanning electron microscopy of Caco-2 cells infected with E. coli LF82 in the absence or presence of lactoferrin. E. coli LF82 interaction with Caco-2 cells in the absence( nningb . B: hil C). Th
tco
3i
uioaoaeibbstw
o
A and B) or in presence (C and D) of 1 mg/ml of lactoferrin was visualized by scaacteria with the eukaryotic cell membranes were observed in untreated cells (A)
actoferrin treated cell bacteria adhere to the cell membranes in aggregated forms (
In the presence of bLf, the bacteria were seen to interact withhe epithelial cells but neither appreciable morphological modifi-ations of the host cell membranes nor intracellular bacteria werebserved (Fig. 4D).
.5. Effect of bLf on pro-inflammatory cytokine mRNA expressionn Caco-2 cells and cultured mucosal explants infected with LF82
In order to understand whether bLf inhibition of bacteriaptake by the IECs was associated to modulation of some pro-
nflammatory cytokine production by these cells. mRNA expressionf tumour necrosis factor-alpha (TNF-alpha), interleukin 8 (IL-8),nd interleukin 6 (IL-6) was first analyzed in Caco-2 cells infectedr not with LF82 (MOI 1 and 10), and treated or not with bLf (Fig. 5And B). Of note, the absence of antibacterial activity of bLf in ourxperimental conditions was relevant to exclude that variationsn cytokine mRNA expression could be related to a different num-er of viable adherent bacteria. We observed that the addition ofLf did not influence pro-inflammatory cytokines mRNA expres-ion in mock-infected Caco-2 cells whereas significantly decreased
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he TNF-alpha, IL-8, and IL-6 messenger quantity in cells infectedith LF82 either at MOI 1 (Fig. 5A) or at MOI 10 (Fig. 5B).
In an attempt to simulate the in vivo conditions, the effect of bLfn pro-inflammatory cytokine mRNA expression was also analyzed
electron microscopy. Elongation of membrane microvilli in the site of contact ofgher magnification: the elongated microvilli were seen to enwrap the bacteria. Inin projections are visible between individual bacteria in the aggregates (D, arrows).
in the biopsies from CD subjects infected with LF82 and culturedfor 6 and 24 h (Fig. 5C and D). Similarly to in vitro experiments,the addition of bLf decreased the TNF-alpha, IL-8, and IL-6 mRNAexpression in cultured mucosal explants infected with LF82, thisinhibition being more evident after 24 h (Fig. 5D).
3.6. Effect of bLf on pro-inflammatory cytokine mRNA expressionin Caco-2 cells treated with IFN-gamma
Lactoferrin expression is upregulated in response to inflam-matory stimuli. The anti-inflammatory activity occurs throughinhibition of binding of lipopolysaccharide endotoxin to inflamma-tory cells, as well as through interaction with epithelial cells at localsites of inflammation to inhibit inflammatory cytokine production[28].
To evaluate a possible direct effect of bLf on pro-inflammatorycytokine synthesis, Caco-2 cells were treated with IFN-gamma for6 h in the presence or absence of bLf. As shown in Fig. 6, the additionof bLf decreased the pro-inflammatory cytokine mRNA expres-
ents invasion and inflammatory response following E. coli strain (2014), http://dx.doi.org/10.1016/j.dld.2014.02.009
sion, in particular TNF-alpha, in cells treated with IFN-gamma,demonstrating that the immunoregulatory activity exerted by bLf isnot limited to a particular kind of bacterial stimulation of epithelialcells.
Please cite this article in press as: Bertuccini L, et al. Lactoferrin prevents invasion and inflammatory response following E. coli strainLF82 infection in experimental model of Crohn’s disease. Dig Liver Dis (2014), http://dx.doi.org/10.1016/j.dld.2014.02.009
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Fig. 3. Effect of d-mannose on bacterial aggregation induced by lactoferrin: light micrographs. Bacterial aggregation was assessed under the light microscope after incubationof E. coli LF82 in DMEM alone (A), DMEM containing 1 mg/ml lactoferrin (B), DMEM containing d-mannose (C), or DMEM containing d-mannose plus 1 mg/ml lactoferrin (D)and viewed with the light microscope.
Fig. 4. Transmission electron micrographs of untreated and lactoferrin-treated Caco-2 cells infected with E. coli strain LF82. In untreated cells (A–C) the invading bacteria causethe elongation of membrane extensions upon contact with the eukaryotic cell membranes (A and C). Bacteria are engulfed by the elongated microvilli and are internalizedwithin endocytic vacuoles (A and C). Bacteria were also observed within gaps between cells (B). In lactoferrin-treated cells LF82 adheres to the cell membranes withoutinducing any morphological alteration in the host cells and bacterial internalization was not observed (D).
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Fig. 5. Effect of lactoferrin on pro-inflammatory cytokines mRNA expression in Caco-2 cells and cultured mucosal explants infected with E. coli LF82. mRNA expressionof TNF-alpha, IL-8, and IL-6 was analyzed in Caco-2 cells infected or not with E. coli strain LF82 (MOI 1, A; MOI 10, B), and treated or not with 1 mg/ml lactoferrin. mRNApro-inflammatory cytokine expression was analyzed in Crohn’s disease subject biopsies, obtained from intestinal tissue macroscopically not involved in chronic inflammationprocesses. Biopsies were cultured and infected with E. coli strain LF82 in the presence or absence of 1 mg/ml lactoferrin for 6 (C) or 24 (D) h. Relative transcript levels weredetermined using GAPDH as the endogenous control genes. Fold of activation was calculated as the ratio between expression level in mock-infected and infected cells. Barsrepresent the mean of three experiments ± SD. **p < 0.05; *p < 0.01.
L. Bertuccini et al. / Digestive and Liver Disease xxx (2014) xxx–xxx 7
Fig. 6. Effect of lactoferrin on pro-inflammatory cytokines mRNA expression in Caco-2 cells treated with IFN-gamma. Caco-2 cells were treated with IFN-gamma for 6 h in thep mentc
4
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resence or absence of 1 mg/ml lactoferrin. Bars represent the mean of three experiontrol genes. **p < 0.05; *p < 0.01.
. Discussion
Crohn’s disease (CD) is a chronic inflammatory disorder result-ng from a deregulated response of the intestinal immune systemo commensal microbiota in genetically predisposed individuals.
E. coli are commonly found in the lumen of human gut andre mostly considered non-harmful. Nevertheless, some strains ofhem, while not containing virulence factors, have a capacity todhere to and invade epithelial cells in vivo and in vitro and there-ore should be considered a separate category of E. coli which mayause intestinal diseases in humans. These strains, called AIEC, haveeen put forward as causative factors in CD. AIEC are true invasiveacteria, as their uptake by intestinal epithelial cells is dependentpon actin microfilaments and microtubules and they survive andeplicate in the host cell cytoplasm after lysis of endocytic vac-oles [5]. Pathogenicity of AIEC is mediated by a number of factors
nvolved in adhesion to and invasion of intestinal epithelial cells. Inarticular, type 1 pili-mediated adherence plays an essential part inhe invasive ability of these strains by inducing membrane exten-ions, which surround the bacteria at the sites of contact betweenhe entering bacteria and epithelial cells [27].
Immune features identified in CD are: deregulation of the innatend adaptive immune system directed against luminal bacteria orheir products found in the intestinal lumen as well as inappropri-te immune responses to organisms in the intestine that normallyo not elicit a response, possibly because of intrinsic alterations inucosal barrier functions.The presence of these close connections between infection and
nflammation in CD strongly justifies an in depth investigation intohe potential benefit of multifunctional drugs, such as lactoferrin.
Lactoferrin, a multifunctional milk protein responsible fornnate-immunity, is considered to be an important factor in theefence against microbial infections [11]. It exerts many func-ions such as anti-microbial activity related and unrelated to itsron-withholding ability, modulation of bacterial aggregation andiofilm formation through its iron-withholding ability, inhibitionf microbial adhesion and invasion through its binding to bothicrobial and host cell surfaces [11]. In addition, lactoferrin exerts
potent anti-inflammatory and immunomodulating activity, con-ributing to protect mucosa from infections and inflammation.
In the present study we analyzed the ability of bovine lactoferrin
Please cite this article in press as: Bertuccini L, et al. Lactoferrin prevLF82 infection in experimental model of Crohn’s disease. Dig Liver Dis
bLf) to modulate the interactions between LF82, the prototype ofIEC strains, and intestinal target cells, including those retrieved
rom human biotic material, with special emphasis on pro-nflammatory cytokine production. We found that bLf treatment
s ± SD. Relative transcript levels were determined using GAPDH as the endogenous
had no effect on bacterial adhesion, whereas, it was able to pre-vent LF82 invasion in a dose-dependent manner, at non-cytotoxicand non-bactericidal concentration, as shown in gentamicin killingassay. These findings are in agreement with our previous datademonstrating that addition of lactoferrin to cells had a little influ-ence on adhesiveness of Burkholderia cenocepacia and Pseudomonasaeruginosa, whereas it strongly inhibited bacterial invasion[29].
Results from scanning electron microscopy analysis showedthat, in the presence of bLf, LF82 was unable to trigger the forma-tion of cell membrane extensions that enwrapped bacteria. Similarobservations have already been reported with an LF82 type 1 pili-negative mutant by Boudeau and co-workers [27] demonstratingthat type 1 pilus-mediated adherence is involved in LF82-inducedalterations in epithelial cells and that this active phenomenon isa key step in the establishment of its invasive process. Effectively,our transmission electron microscopy results clearly demonstratedthat LF82-cell interaction in the presence of bLf does not resultin either host membrane morphological modifications or bacterialinternalization.
It is well known that adhesion of AIEC bacteria to cells is medi-ated by the recognition of FimH adhesin, located at the tip of thetype 1 pilus, by the mannose residues on cellular receptors [30].As bLf is known to contain highly mannosylated glycans [31,32],it is likely that a direct interaction between bLf and FimH adhesinresults in inhibition of type 1 pilus-mediated adherence and, conse-quently, prevention of bacterial internalization. Indeed it has beenreported that human lactoferrin is able to bind to enterotoxigenicE. coli fimbrial CFA I adhesin [33], and that the oligosaccharide moi-eties of lactoferrin are involved in its interaction with E. coli type 1pili and Porphyromonas gingivalis fimbriae [34,35].
Our scanning electron microscopy studies also demonstratedthat, in bLf treated cells, adherent bacteria form aggregates on thecellular surfaces. The presence of projections between individualbacteria in the aggregates suggests that bLf could act as a bridgeamong bacterial cells by binding pili. Actually, we have previouslydemonstrated that bLf is able to specifically bind to Burkholde-ria cenocepacia cable pili, leading to an increased aggregation ofbacterial cells [36]. It was also reported that lactoferrin promoteaggregation of other bacteria, such as Streptococcus mutans [37],E. coli [34], and Clostridium spp. [38]. On the basis of this evidence,
ents invasion and inflammatory response following E. coli strain (2014), http://dx.doi.org/10.1016/j.dld.2014.02.009
it is conceivable that bLf binding to pili may favour bacterial aggre-gation by preventing diffuse cell-to-cell interactions.
This hypothesis has been confirmed by light microscopy resultsdemonstrating that bLf-treatment of E. coli LF82 produces a
-mannose-sensitive bacterial aggregation. These results also sug-est that bLf might favour the clearance of bacteria through highggregation efficiency.
As a general rule, the interaction between adherent and/ornvasive E. coli and host cells induces inflammatory reactions. Con-erning the inflammatory response of infected IECs, we observedhat the treatment with bLf of Caco-2 cells or cultured mucosalxplants results in the reduction of mRNA expression of thero-inflammatory cytokines TNF-alpha, IL-8, and IL-6. Since theevelopment of inflammation is dependent on AIEC bacteria inter-ction with the gut mucosa via type 1 pili [39], the observedlock of inflammation by bLf could be due to the inhibition ofype 1 pili functions. It has been observed with uropathogenic. coli (UPEC) in the context of urinary infections [40] that thelock of the interaction between type 1 pili and receptor moleculesllows disrupting colonization and the subsequent inflammatorymplification loop. Indeed, the entrance of AIEC strains in IECspregulate the TNF-alpha production creating a loop of coloniza-ion and inflammation leading to chronic disease with the secretionf other pro-inflammatory cytokines. In this contest, of particularelevance may be the down-modulation of IL-8 production by bLfeing this cytokine the one most able to call for local recruitmentf neutrophils, a landmark of CD [41].
However, we cannot exclude that the observed anti-nflammatory effect of bLf could be due also to a direct effectf this protein on the cytokine transcription. To verify this hypoth-sis, we have tested whether the cytokine down-modulatingffects of bLf could take place also when cytokine gene expressionas not induced by a bacterial infection. Thus, Caco-2 cells were
reated with IFN-gamma for 6 h in the presence or absence of bLfnd pro-inflammatory cytokine mRNA synthesis was evaluated.ur results, showing that bLf treatment brings about a reduction ofro-inflammatory cytokine transcription, indicates that bLf exertsn anti-inflammatory effect on intestinal cells, regardless whetherhe stimulant is the bacterial cell or a pro-inflammatory cytokine.n this context, there is a possible additional or even synergisticffect of bLf on inflammation due to inhibition by bLf of bacterialnvasion that is a potent stimulant of inflammation itself.
In conclusion, this study shows that lactoferrin strongly inhibitsIEC invasion of IECs and reduces inflammation, including aytokine-generated amplification loop, suggesting that in princi-le it might be beneficial in the treatment of chronic inflammatoryonditions. Of course this requires future investigations focussedn pharmacokinetics of bLf, inclusive of its bioavailability at thentestinal level. It will also require a further insight into the cel-ular and molecular mechanism of bLf activity. This established,he known safety of bLf as natural component of host immu-ity and the absence of adverse events suggests that blf couldxert a benefit in the maintenance of the remission state of CDatients.
unding
This work was supported by grant from the “Bovine lactoferrinnd inflammatory bowel diseases” ISS/Giellepi S.p.A. agreement.
FT is employee of Giellepi S.p.A.
onflict of interest statementhe funder had no role in study design, data collection and analysis,ecision to publish, or preparation of the manuscript.
Please cite this article in press as: Bertuccini L, et al. Lactoferrin prevLF82 infection in experimental model of Crohn’s disease. Dig Liver Dis
cknowledgement
We thank Fabio Minelli (ISS, Department of Food Safety andeterinary Public Health) for microbiological analysis.
[
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