Peptidomimetics, method of synthesis and uses thereof
11472844 · 2022-10-18
Assignee
- Centre National De La Recherche Scientifique (Paris, FR)
- INSERM (Institut National de la Santé et de la Recherche Médicale) (Paris, FR)
- Institut Pasteur (Paris, FR)
Inventors
Cpc classification
G01N1/30
PHYSICS
International classification
C07K14/00
CHEMISTRY; METALLURGY
Abstract
The invention relates to a peptidomimetic comprising or consisting of a D amino-acid sequence having at least 75% identity with SEQ ID NO: 1 or SEQ ID NO: 2, or variants or fragments thereof, in particular a peptidomimetic having the capability to interact at least with: neutrophils and/or neutrophil granules, and/or lactoferrin, and/or globet-cells and/or Muc2 proteins, and/or mucus and/or airway sputum. The peptidomimetic may have the capacity to adopt a multimeric, especially a trimeric, organization, and can be labelled, or associated with a reporter or a carrier entity, or associated with an active molecule. The invention also relates to a Solid-Phase Synthesis method for synthesizing a peptidomimetic of the invention, compositions comprising the same and use of the peptidomimetics as a medicamentor an inflammation marker or a neutrophilic inflammation marker. The invention also relates to the use of a peptidomimetic as a probe or marker for staining purposes, or to detect mucus production, or neutrophils, or to detect or monitor diseases or conditions, especially neutrophilic inflammation. The invention also relates to the use of a polypeptide comprising or consisting of SEQ ID NO: 3, or variants or fragments thereof, as probe or marker for staining lactoferrin, in particular neutrophil lactoferrin, or a probe or marker for investigating neutrophilic inflammation, especially in an imaging method.
Claims
1. A method of detecting lactoferrin, comprising: a) contacting lactoferrin with a D amino-acid peptidomimetic, wherein the D amino-acid peptidomimetic comprises the amino acid sequence of SEQ ID NO: 1; and b) detecting the D amino-acid peptidomimetic bound to the lactoferrin.
2. The method of claim 1, wherein the D amino-acid peptidomimetic comprises the amino acid sequence of SEQ ID NO: 2.
3. The method of claim 2, wherein the D amino-acid peptidomimetic consists of the amino acid sequence of SEQ ID NO: 2.
4. The method of claim 1, wherein the D amino-acid peptidomimetic further comprises a label and/or a reporter or a carrier entity, and/or is associated with an active molecule.
5. The method of claim 4, wherein the D amino-acid peptidomimetic is labelled by coupling with a fluorophore or a biotin.
6. The method of claim 1, wherein the D amino-acid peptidomimetic has the capacity to adopt a multimeric organization and/or is a trimer.
7. The method of claim 1, wherein the D amino-acid peptidomimetic has a D-Cysteine residue at its extremity-terminus.
8. The method of claim 1, wherein the D amino-acid peptidomimetic has an amide group at its C-terminus and/or an acetyl group at its N-terminus.
9. The method of claim 1, wherein the lactoferrin is present in a patient sample or in a patient (in vivo).
10. The method of claim 9, wherein lactoferrin associated with neutrophils is detected.
11. The method of claim 9, wherein lactoferrin associated with neutrophil granules is detected.
12. The method of claim 9, wherein lactoferrin associated with mucus is detected.
13. The method of claim 9, wherein lactoferrin secreted by neutrophils that is detected.
14. The method of claim 9, wherein the presence of detected lactoferrin indicates neutrophilic inflammation.
15. The method of claim 9, wherein the method comprises administering the D amino-acid peptidomimetic to the patient.
16. The method of claim 9, wherein the patient has or is suspected to have a pathogen infection.
17. The method of claim 16, wherein the pathogen infection is a bacterial infection.
18. The method of claim 9, wherein the patient has or is suspected to have a neoplastic disease.
19. The method of claim 18, wherein the neoplastic disease is selected from mucinous carcinoma, gastric cancer and colorectal cancer.
20. The method of claim 9, wherein the patient has or is suspected to have cystic fibrosis.
21. The method of claim 9, wherein the patient has or is suspected to have an intestine inflammatory disease.
22. The method of claim 9, wherein the sample comprises living cells.
23. The method of claim 1, wherein the lactoferrin is glycosylated.
24. The method of claim 1, wherein the lactoferrin is one or more of glycosylated lactoferrin, neutrophil lactoferrin stored in neutrophil specific granules (β1), neutrophil lactoferrin stored in tertiary granules (β2), and lactoferrin secreted by neutrophils.
Description
LEGEND OF THE FIGURES
(1)
(2) (A) Schematic representation of the four 40-amino acid peptides named MUB.sub.40#1 (SEQ ID NO: 3), MUB.sub.40#2 (SEQ ID NO: 4), MUB.sub.40#3 (SEQ ID NO: 5), MUB.sub.40#4 (SEQ ID NO: 6), overlapping and covering the MUB.sub.70 sequence (SEQ ID NO: 11) (Coïc et al., 2012) (from C-terminal to N-terminal, see
(3)
(4) (a) Operational sequences for the syntheses of MUB.sub.40#1 (SEQ ID NO: 3), MUB.sub.40#2 (SEQ ID NO: 4), MUB.sub.40#3 (SEQ ID NO: 5), MUB.sub.40#4 (SEQ ID NO: 6), where the where secondary amino acid surrogates are underlined (pseudoproline dipeptides) or in bold (Dmb dipeptides). Prolines are in italic bold. (b) As described in
(5)
(6) Muc2 was stained with an anti-Mucin2 antibody (green). Tissue was additionally stained with MUB.sub.40-Cy5 (1 μg/mL, magenta). DNA was stained with Dapi (blue).
(7)
(8) Muc2 was stained with an anti-Mucin2 antibody (green). Muc5ac was stained with an anti-Mucin5ac antibody (red). Tissue was additionally stained with MUB.sub.40-Cy5 (1 μg/mL, magenta). DNA was stained with Dapi (blue).
(9)
(10) (A) Human polymorphonuclear neutrophils were similarly labelled with MUB.sub.40 (SEQ ID NO: 3) conjugated with Cy5 (magenta) or Alexa405 (blue) and with a retro-inverso (RI) MUB.sub.40 peptide, designed with non-natural D-amino acids (SEQ ID NO: 2), conjugated with Cy5 (magenta). Nuclei were stained with Dapi (blue or white). Bars, 20 μm. (B) Human hematopoietic stem cells (CD34+) were not labelled by MUB.sub.40 (SEQ ID NO: 3)-Cy5 during their proliferation, when a positive staining (magenta) was obtained upon their differentiation in polymorphonuclear neutrophils in the presence of G-CSF, IL-3, and IL-6 (2 weeks). Nuclei were stained with Dapi (blue). Bars, 50 μm. (C-D) RI-MUB.sub.40 peptide (SEQ ID NO: 2) was not degraded by trypsin. (C) MUB.sub.40 (SEQ ID NO: 3) and RI-MUB.sub.40 (SEQ ID NO: 2) final peptide concentration was 0.25 mg/mL and trypsin to protein ratio was 1:20 (w/w). HPLC profiles of purified MUB.sub.40 (SEQ ID NO: 3) and RI-MUB.sub.40 (SEQ ID NO: 2) peptides incubated with Trypsin during the 0, 1, 3, and 24 h at 37° C. The percentage of peptide stability over the time are shown in (D). Results are expressed with Mean±S.D. (n=3).
(11)
(12) PBMC were purified from human blood samples and fixed in PFA 3%. CD3+ (T lymphocyte), CD14+ (monocytes, macrophage), and CD19+ (B lymphocyte) cells (red) were not stained with MUB.sub.40-Cy5 (magenta), using the same protocol as in
(13)
(14) (A) Guinea pig and mouse neutrophils were fixed and labelled with MUB.sub.40 (SEQ ID NO: 3)-Cy5 (magenta). Nuclei were stained with Dapi (blue). Bars, 10 μm. (B) Upon Shigella flexneri 5a pGFP (green) infection of the guinea pig colonic mucosa, infiltrated neutrophils were labelled with MUB40 (SEQ ID NO: 3)-Cy5 (magenta) or MUB40 (SEQ ID NO: 3)-Alexa405 (blue). Bars, 100 μm. (C) Upon Shigella sonnei pMW211 (pDsRed) (red) oral challenge of mice, a local colonization of the colonic mucosa was observed, associated with a recruitment of neutrophils labelled with MUB.sub.40 (SEQ ID NO: 3)-Cy5 (magenta). Actin was stained with Phalloidin-FITC (green), bar, 100 μm.
(15)
(16) Immunofluorescence detection of neutrophils (MMP-9, green) in a rabbit ileum section. Actin was stained with RRX-phalloidin (red), and neutrophils were labeled with MUB.sub.40 (SEQ ID NO: 3)-Dylight405 (blue) at a final concentration of 1 μg/mL and a anti-gelatinase (MMP-9) antibody (green). Bar is 30 μm.
(17)
(18) HPLC analysis was performed on a symmetry 300C18 column (Waters) by applying a 15%-40% B gradient in 20 min at a 0.35 mL/min flow rate, where A=ammonium acetate 50 mM pH6.5 and B═CH.sub.3CN. Arrow (1): target peptide, Arrow (2): Asp-Gly deletion
(19)
(20) The experimental mass of Cy5-RI-MUB40 (SEQ ID NO: 2) was checked by negative electrospray mass spectroscopy on a quadrupole-TOF Micro mass spectrometer (Waters) equipped with a Z-spray API source, giving 5551.265 Da after MaxEnt1 (Masslynx, Waters) deconvolution (expected 5550,115)
(21)
(22) The experimental mass of Biot-RI-MUB40 was checked by negative electrospray mass spectroscopy on a quadrupole-TOF Micro mass spectrometer (Waters) equipped with a Z-spray API source, giving 5296.994 Da after MaxEnt1 (Masslynx, Waters) deconvolution (expected 5296.792)
(23)
(24) HPLC analysis of purified biot-RI-MUB40 was performed on an Aeris C18 column (Phenomenex) by applying a 25%-50% B gradient in 20 min at a 0.35 mL/min flow rate, where A=0.08% aqueous TFA and B═CH.sub.3CN
(25)
(26) HPLC analysis of purified Cy5-RI-MUB40 (SEQ ID NO: 2) was performed on a symmetry 300C18 column (Waters) by applying a 15%-40% B gradient in 20 min at a 0.35 mL/min flow rate, where A=ammonium acetate 50 mM pH6.5 and B═CH.sub.3CN
(27)
(28) (A) Human neutrophil granules were purified and fractionated on a 3-layers Percoll gradient (Kjeldsen et al., 1999). Total granule, α (azurophil granules), β1 (specific granules), β2 (tertiary granules), and γ (secretory vesicles) fractions were separated on a 10% SDS Page gel and stained with Coomassie. Most abundant proteins in each fraction was identified by mass spectrometry (right panel) (raw data presented in
(29)
(30) Neutrophil granules were purified as described in Methods and fractionated on a three-layer Percoll gradient, as described previously.sup.1. =10 μg of each sample (α, β1, β2 and γ granule fractions) were separated on a 12% SDS-PAGE gel and stained with InstantBlue Protein Stain (Sigma-Aldrich). Indicated bands (a-g) were cut and further analysed by Mass Spectrometry for protein identification.
(31)
(32) (A) The kinetics of the lactoferrin degranulation (cell surface exposure, white arrows) was assessed by live microscopy on neutrophils infected by Shigella flexneri pGFP (green) (at MOI 20) in the presence of RI-MUB.sub.40 (SEQ ID NO: 2)-Cy5 (1 μg/mL, magenta) (RPMI 1640 medium supplemented with 10 mM Hepes at 37° C.). Images were acquired every 60 s for 9 min (see Movie S1), bar 10 μm. RI-MUB.sub.40 (SEQ ID NO: 2)-Cy5 did not cross the plasma membrane of living cells (data not shown), similarly to MUB.sub.70 (SEQ ID NO: 11) (Coic et al., 2012). (B) Neutrophil lactoferrin degranulation is detected in Shigella flexneri 5a pGFP (green) foci of infection in the guinea pig mucosa with MUB.sub.40 (SEQ ID NO: 3)-Cy5 (1 μg/mL, magenta) (white arrow) DNA was stained with Dapi (blue). Bar, 20 μm. (C-D) RI-MUB.sub.40 (SEQ ID NO: 2)-Cy5 accumulates at sites of inflammation in vivo. C5761/6J mice were injected i.v. with serum from K/B×N mice (K/B×N, n=4) or saline (saline, n=5) (day 0). (C) Photon emission corresponding to luminol degradation by myeloperoxidase activity present in the joints was measured 6 days post arthritis induction (top panels). Bioluminescence in regions of interest (red circles) is expressed as average radiance (p/s/cm.sup.2/sr; left scatter plot). Accumulation of RI-MUB.sub.40-Cy5 fluorescent signal (640 nm/700 nm) in the joints of arthritic mice but not in control mice (middle panels and lower panels as merged picture with CT). Epifluorescence in regions of interest (red circles) is expressed as average radiant efficiency (p/s/cm.sup.2/sr/[uW/cm.sup.2]; right scatter plot). (D) Correlation of bioluminescent signal and RI-MUB.sub.40-Cy5 fluorescent signal in indicated regions of interest (hind ankle joints). Data in (C) are representative of two independent experiments. Error bars correspond to the SEM, **** p≤0.0001 (unpaired t-test).
(33)
(34) Tissue inflammation is characterized by neutrophil recruitment and potentially associated with lactoferrin degranulation. The validation of MUB.sub.40 peptides (here MUB.sub.40 (SEQ ID NO: 3)-Cy5) as markers of inflammation was confirmed on human biopsies, here (A) a malignant fibrous hystiocytoma (sterile inflammation) and (B) a streptococcal skin abscess (infectious inflammation). Lactoferrin was stained with an anti-lactoferrin antibody (green) and MUB.sub.40 (SEQ ID NO: 3)-Cy5 (1 μg/mL, magenta), DNA was stained with Dapi (blue). Bars, 150 μm. Surface plots and correlation of fluorescent signals were obtained with the Fiji software.
(35)
(36) K/B×N mice were injected intravenously with RI-MUB40-Cy5 (SEQ ID NO: 2), MUB40-Cy5 (SEQ ID NO: 3) or Cy5 only. The inventors demonstrated that 2 days post-injection, the fluorescent signal associated with RI-MUB40-Cy5 (SEQ ID NO: 2) accumulation at hind and front inflamed ankles was significantly higher compared to MUB40-Cy5 (SEQ ID NO: 3) (p<0.01) or Cy5 only (p<0.001). This result confirms the increased stability of RI-MUB40-Cy5 (SEQ ID NO: 2) compared to MUB40-Cy5 (SEQ ID NO: 3) in vivo.
(37) (A) Ankles of K×B/N mice were imaged 2 Days after intravenous injection of 0.9 nmoles of RI-MUB40-Cy5 (SEQ ID NO: 2), MUB40-Cy5 (SEQ ID NO: 3) or Cy5 only. Fluorescence was quantified at the level of front or hind ankles (in ROI, as indicated).
(38) (B) Quantification of the fluorescent signal associated with RI-MUB40 (SEQ ID NO: 2)-Cy5, MUB40 (SEQ ID NO: 3)-Cy5 or Cy5 accumulation at hind and front ankles (top) or hind ankles only (bottom). Results are average from at least three animals. ** indicates p<0.01 and *** indicates p<0.001 (Student's T-test).
EXAMPLES
(39) A. MATERIALS AND METHODS
(40) MUB.sub.40 Peptides Series Synthesis
(41) The synthesis were carried out on a 100 μmoles scale on an ABI 433 synthesizer (Applied Biosystems, Foster City, Calif.) from a polystyrene AM-RAM resin and using conventional FMOC chemistry. N-terminal acetylation was achieved by treating the peptide resins at the end of the synthesis with acetic anhydride for 30 minutes. As a result, all peptides were N-terminal amide and C-terminal acetylated. For the purpose of structural analysis (CD and gel filtration), MUB.sub.40 peptides were submitted to a N-ethyl maleimide treatment in order to prevent covalent dimer formation. Fluorophore labeling and biotin derivatization were performed through the conjugation of their maleimide derivatives to the free sulfhydryle peptides. All purification steps and HPLC analysis were done by C18 Reverse Phase columns. Final characterization by electro-spray mass analysis were consistent with the expected masses (between brackets): Cy5-labeled MUB.sub.40#1 (SEQ ID NO: 3): 5549.740 (5550.115); Cy5-labeled MUB.sub.40#2 (SEQ ID NO: 4): 5447.472 (5447.933); Cy5-labeled MUB.sub.40#3 (SEQ ID NO: 5): 5588.273 (5589.170); Cy5-labeled MUB.sub.40#4 (SEQ ID NO: 6): 5501.778 (5502.093); Cy5-labeled RI-MUB.sub.40#1 (SEQ ID NO: 2) (RI-MUB40): 5551.265 (5550.115); Dylight 405-labeled MUB.sub.40#1 (SEQ ID NO: 3) (MUB.sub.40-D405): 5519,849 (5518, 9 . . . —restricted proprietary information); RI-MUB.sub.40#1 (SEQ ID NO: 3)-Biotin (RI-MUB.sub.40-Biotin): 5296.994 (5296.792). Detailed synthesis and derivatization steps and the biophysical analysis of MUB.sub.40 peptides are described below.
(42) Synthesis and cleavage. The synthesis were carried out on a 100 μmoles scale on an ABI 433 synthesizer (Applied Biosystems, Foster City, Calif.) equipped with a conductivity flow cell to monitor Fmoc deprotection from a polystyrene AM-RAM resin (capacity 0.41 mmol/g for MUB.sub.40 peptides and 0.62 mmol/g for retro-inverso RI-MUB.sub.40#1, Rapp Polymere GmbH, Tuebingen, Germany). Standard Fmoc amino acids, Dmb, and pseudoproline dipeptides were activated with HCTU (2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate) DIPEA (N,N-diisopropylethylamine). Fmoc-D-amino acids and Hmb dipeptide were activated with HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo-[4,5-13]pyridinium hexafluorophosphate 3-oxide)/DIPEA). All Fmoc-AAs and surrogates were single-coupled with eight-fold molar excess regarding the resin. Both coupling reagents, N-methyl pyrrolidone (NMP) and standard Fmoc amino acids were obtained from Applied Biosystems. Fmoc D-amino acids were obtained from Eurogentec (Eurogentec, Seraing, Belgium). Fmoc L and D-amino acids were side-protected as follows: tBu for aspartic acid, glutamic acid, serine, threonine, and tyrosine; Trt for cysteine and histidine: Boc for lysine; and Pbf (2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl) for arginine. Fmoc-Asp(OtBu)-(Dmb)Gly-OH dipeptides and pseudoproline (oxazolidine) dipeptides were purchased from Merck-Novabiochem. Fmoc-D-Asp(OtBu)-(Hmb)Gly-OH was purchased from Bachem (Bubendorf, Switzerland). Piperidine was purchased from Sigma-Aldrich (St Louis, Mo., USA).
(43) N-terminal acetylation was achieved by treating the peptide resin at the end of the synthesis with acetic anhydride for 30 minutes. As a result, all peptides were N-terminal amide and C-terminal acetylated. Cleavage from the solid support and deprotection of the amino acid side chains were accomplished in one step by 3 h treatment at room temperature with a mixture of TFA/ethanedithiol/triisopropylsilane/water (92.5/2.5/2.5/2.5) for MUB.sub.40 peptides or TFA/ethanedithiol/triisopropylsilane/water/phenol (92/2.5/2/2.5/1) for 4 h for RI-MUB.sub.40. After filtration of the resin, the cleavage mixtures were poured into ice-cold diethyl ether. The precipitates were recovered by centrifugation, washed three times with cold diethyl ether, dried, resuspended in a mixture of water and acetonitrile, and freeze dried.
(44) HPLC analysis of synthesized peptides. Analysis of crude mixtures and purity control of the final peptides were performed by RP-HPLC on an Agilent (Santa Clara, Calif., USA) 1100 Series liquid chromatograph and monitored with a photodiode array detector by absorbance at 230 nm, according to the following methods. A linear gradient from 15% to 40% of acetonitrile in aqueous solvent A (50 mM ammonium acetate, pH 6.5) over 20 min was applied at a 0.35 ml/min flow rate on a Symmetry 300 C18 3.5 μm 2.1×100 mm column (Waters, Manchester, UK). To check the purity of RI-MUB.sub.40 derivatives, supplementary analyses were done in 0.08% aqueous TFA by applying a 25%-50% acetonitrile gradient on an Aeris Peptide 3.6μ XB-C18 column.
(45) Peptides purification. The free sulfhydryl crude peptides MUB.sub.40#1, #2, & #3 (SEQ ID NOS: 3-5) were solubilized at a final concentration of 20 mg/ml in a mixture of solvent A and acetonitrile, 8:2 v/v. Crude MUB.sub.40#4 was solubilized at the same concentration in water with aqueous ammonia (pH8) and 10 equivalent of DTT (1,4-Dithio-DL-threitol). Crude RI-MUB.sub.40 was solubilized at a final concentration of 3.5 mg/ml in solvent A. Those materials were purified by RP-MPLC (AP-100/200 flash, Armen Instrument, Saint Ave, France) on a preparative column (26×313 mm) packed with 100 Å 20 μm C18 Nucleoprep packing (Macherey & Nagel GmbH & Co, Düren, Germany), by applying a linear gradient of 15-70% (MUB.sub.40 peptides) or 15-50% (RI-MUB.sub.40) solvent B (mixture of acetonitrile and solvent A, 8/2 v/v) in solvent A over 60 min at a 20 ml/min flow rate. The purification was monitored at 214 nm (UV detector K2501, Knauer, Berlin, Germany). The suitable fractions were pooled and freeze dried. The overall isolated yields (from 20% to 30%) were in concordance with the observed synthesis yields deducted from the crude's HPLC analysis.
(46) MUB.sub.40 peptides conjugation. Cy5 and Dylight 405 (Thermofisher Scientific) conjugations were operated in a 0.1 M Phosphate buffer pH=6 (MUB.sub.40 peptides) or pH=7.2 (RI-MUB.sub.40), using 1.2 equivalent of the correspondent maleimide derivative (InvitroGen) in the presence of 1.5 equivalent of TCEP (Tris (2-carboxyethyl)phosphine) per mole of cysteine residue. Repeating one time, this 30 minute coupling protocol was necessary to achieve completion of RI-MUB.sub.40 labeling. The labeled peptides were purified by RP-HPLC using a linear gradient of 15-40% acetonitrile over 20 min at a 6 ml/min flow rate, either on a Nucleosil 5 μm C18 300 Å semi-preparative column equilibrated in solvent A (MUB.sub.40 peptides) or on a Kromasil 5 μm C18 300 Å semi-preparative column (AIT, Houilles) equilibrated in 50 mM Triethyl ammonium acetate (RI-MUB.sub.40). The purity was checked according to the formerly-described HPLC analytical method. The exact concentration was determined by quantitative amino acid analysis (Hitachi, L-8800 analyzer), giving a 50% to 60% conjugation isolated yield.
(47) Biotinylated RI-MUB.sub.40 was obtained by adding the free sulfhydrile peptide to 5 equivalents of the maleimide derivative (EZ-linked maleimide-PEG2-biotin, Thermoscience) in 0.1M phosphate buffer (pH6). The biotinylated peptide was purified by HPLC in 50 mM ammonium acetate on a Kromasil 5 μm C18 300 Å semi-preparative column, using a linear gradient of 15-40% acetonitrile over 20 min at a 6 ml/min flow rate. A double peak was observed in the analytical HPLC profile, which was attributed to the resolution of the two isomers resulting from the addition of the sulfhydryl peptide on the maleimide double bond.
(48) Electrospray ionisation mass spectrometry. Mass spectrometry was carried out on a quadrupole-TOF Micro mass spectrometer (Waters) equipped with a Z-spray API source. Capillary, sample cone, and extraction cone voltages were set at 3 kV, 40V, and 10V, respectively. Source and desolvation temperatures were set at 80 and 250° C., respectively. Data were acquired by scanning over the m/z range 150-2000 at a scan rate of 1 s and an interscan delay of 0.1 s. Peptides were dissolved in a mixture of water/methanol/acetic acid (49.5/49.5/1, v/v/v) at a concentration of 1 μg/μl and analyzed in positive-ion mode by infusion at a flaw rate of 5 μl/min. Around fifty spectra were combined and the resultant raw multi-charged spectra were processed using the MaxEnt1 deconvolution algorithm embedded in the Masslynx software. Given the deconvolution process of MaxEnt1, applied to the charged molecules (the Cy5 moiety is positively charged), final characterization was consistent with the expected masses (between brackets): Cy5-labeled MUB.sub.40#1 (SEQ ID NO: 3): 5549.740 (5550.115); Cy5-labeled MUB.sub.40#2 (SEQ ID NO: 4): 5447.472 (5447.933); Cy5-labeled MUB.sub.40#3 (SEQ ID NO: 5): 5588.273 (5589.170); Cy5-labeled MUB.sub.40#4 (SEQ ID NO: 6): 5501.778 (5502.093); Cy5-labeled RI-MUB.sub.40#1 (SEQ ID NO: 3) (RI-MUB.sub.40): 5551.265 (5550.115); Dylight 405-labeled MUB.sub.40#1 (SEQ ID NO: 3) (MUB.sub.40-D405): 5519,849 (5518.9.); RI-MUB.sub.40#1 (SEQ ID NO: 3)-Biotin (RI-MUB.sub.40-Biotin): 5296.994 (5296.792).
(49) Neutrophil Granules Purification and Fractionation.
(50) Neutrophil granules were collected from purified polymorphonuclear neutrophils, following the procedure described previously (Kjeldsen et al., 1999). Neutrophils were resuspended in PBS (2.7. 10.sup.6 cell/mL) with 0.5 μL/mL DFP (Sigma-Aldrich) and incubated on ice for 15 min prior centrifugation (1300 rpm; 10 min). Cells were resuspended (20.10.sup.6 cell/mL) in a relaxation buffer (KCl 100 mM, NaCl 3 mM, MgCl.sub.2 3.5 mM, PIPES 10 mM, adjusted at pH 6.8) with a cocktail of protease inhibitors (400 mM leupeptin, 400 mM pepstatin, 3 mM PMSF, 1 mM orthovanadate) and supplemented with 1 mM ATP, 1 mM EDTA, and 1.25 mM EGTA. Cells were lysed by nitrogen cavitation (350 psi, 20 min). Cell lysates were centrifuged at 3000 rpm for 15 min to remove remaining cells and nuclei. For total granule recovery, lysates were centrifuged at 16000 rpm for 45 min; granules were resuspended in protease inhibitor-containing relaxation buffer (described above) and stored at −80° C. For granule fractionation, lysates were centrifuged onto a 3-layers Percoll gradient (densities 1.120 g/mL-1.090 g/mL-1.050 g/mL) at 37.000×g for 30 min at 4° C., as described in (Kjeldsen et al. 1999). From the top to the bottom, γ (secretory vesicles), β2 (tertiary granules), β1 (specific granules), and α (azurophil granules) fractions were collected. Remaining Percoll solution was removed by ultracentrifugation (100.000×g for 90 min at 4° C.); purified granules were collected in inhibitor-containing relaxation buffer and stored at −80° C.
(51) Human and Mouse Models of Inflammation
(52) Colon explant surgical collection. In summary, human colon segments (ascending, descending, and sigmoid colon) were obtained from fully informed patients undergoing surgery for colon carcinoma and were analyzed anonymously. Patient written consent was obtained, according to the French bioethics law. None of the patients had undergone radiotherapy or chemotherapy. According to the pathologist's examination rules for the longitudinally bisected colon, a healthy segment of tissue, which was distant from the tumor region and devoid of metastatic cells, was removed. Tissues were processed according to the French Government guidelines for research on human tissues and the French Bioethics Act with the authorization no RBM 2009-50.
(53) Human inflammatory tissues. Human biopsies from patients diagnosed for malignant fibrous histiocytoma and for a streptococcal skin abscess were collected and processed at the Kremlin Bicêtre Hospital, Anatomy and Pathological Histology Department. Tissue samples were fixed in formaldehyde and further embedded in paraffin. 10 μm sections were obtained using a microtome (Leica Biosystem). Tissues were labeled with a mouse anti-lactoferrin primary monoclonal antibody (Hycult biotech, clone 265-1K1, 1:50 dilution), MUB40-Cy5 (SEQ ID NO: 3) (1 μg/mL), and Dapi (1:1000 dilution) as described below.
(54) Ex vivo infection of human colonic tissue. Human colon explants were infected with S. flexneri 5a (M90T) pGFP, as described in (Nothelfer et al. 2014) and adapted from (Coron et al. 2009). Briefly, colonic tissues were cut into ˜5 cm.sup.2 segments and pinned flat, with the submucosa facing down, onto a 4% agarose layer in tissue culture Petri dishes containing DMEM/F12 culture medium (Invitrogen) supplemented with 10% FBS, glutamine, and 2.1 g/L NaHCO.sub.3(Sigma-Aldrich). S. flexneri (M90T) 5a pGFP was added at ˜2×10.sup.8 bacteria per cm.sup.2 of tissue. Bacteria were allowed to settle for 15 min at room temperature before incubation at 37° C., 5% CO.sub.2 for 6 h on a slowly rocking tray. Tissue was fixed by overnight incubation with 4% PFA (Euromedex) and 0.1 M Hepes (Gibco) in PBS. For whole-mount staining, tissues were fixed on a 40×11-mm tissue culture dish (TPP) with Histoacryl tissue glue (Braun). To obtain 150-μm-thick sections, the tissue was embedded in low-melting agarose according to (Snippert et al. 2011) and cut with a vibratome (VT1000E; Leica).
(55) Mouse arthritis model. 6-7 weeks old female C57BL/6J mice were purchased from Charles River France, housed under specific pathogen-free (SPF) conditions and handled in accordance with French and European directives. Mouse protocols were approved by the Animal Ethics committee CETEA number 89 (Institut Pasteur, Paris, France) and registered under #2013-0103, and by the French Ministry of Research under agreement #00513.02. Arthritis was induced by i.v. injection of 120 μL K/B×N serum and arthritis scored as described previously (Bruhns et al. 2003). Mice injected with physiological saline were used as controls. On day 6 after serum transfer, mice were anesthetized, shaved, depilated and injected i.p. with luminol (10 mg/mouse) and i.v. with 5 μg/mouse MUB-40 Cy5. Epifluorescence, bioluminescence and CT images were acquired 10-90 min after injection using an IVIS SpectrumCT (Perkin Elmer).
(56) Flow Cytometry
(57) Human. Neutrophils and PBMCs were separated as described below. Naïve cells were resuspended in PBS+EDTA 2 mM. Fixed and permealized were were obtained by incubation in PFA 3.2% for 30 min and resuspention in PBS+Triton 0.1% for 30 min. Naïve and fixed/permeabilized cells were incubated with CD45-FITC and MUB.sub.40-Alexa405 peptide (1 μL/mL) for 15 min at room temperature. Cells were analyzed with a FACSCANTO II (BD) and data were analyzed using Flowjo software.
(58) Mouse. Blood leucocytes were purified by dextran, washed in PBS EDTA and kept on ice. For hematopoietic populations staining, 2 to 3 million cells were resuspended in PBS EDTA, blocked with 16/32 for 15 minutes and stained with antibodies for 30 minutes at 4° C. (CD45 PE-CF594, CD3e FITC, B220 PE-CY7, NKP46 FITC, CD11b PE from BD biosciences, and Ly6G APC-H7, Ly6C APC from Biolegend). Cells were washed with PBS EDTA and fixed in PFA 3.3% at room temperature during 15 minutes. Cells were washed and resuspended in PBS-0.1% Triton for 5 minutes. Cells were then incubated with MUB40-Alexa405 peptide (1 μL/mL) for 15 minutes at RT and washed. Not fixed cells incubated with peptide or fixed cells not incubated with peptide were used as negative control. Cell fluorescence was determined using a Fortessa (BD biosciences) and analyzed with Kaluza Software (Beckman Coulter). The fluorescence intensity was quantified.
(59) Fluorescent Markers and Cell Labeling
(60) Fixation and staining procedures. For microscopy studies, purified or cultured cells and polymerized lactoferrin were resuspended onto 24-well plates containing 12 mm coverslips in RMPI 1640+10 mM Hepes (when cultured in the autologous plasma) and centrifuged at 300×g for 10 min. Culture media were removed and cells were fixed in 4% Paraformaldehyde (PFA) for two hours. Fixed cells were washed three times in PBS+0.1% saponin (Sigma-Aldrich), prior immunolabeling with primary antibodies in the same buffer for 1 hour (Triton 0.1% can alternatively be used for cell permeabilization). After three additional washes in PBS+0.1% saponin, secondary antibodies and fluorescent markers were incubated for one hour. Coverslips were washed three time in PBS+0.1% saponin, three times in PBS, and three times in deionized H.sub.2O and mounted with prolong gold.sup.tm mounting media.
(61) Antibodies, fluorescent markers. For immunofluorescence assay, lactoferrin was detected with a mouse anti-lactoferrin primary monoclonal antibody (Hycult biotech, clone 265-1K1, 1:50 dilution) and an anti-mouse-FITC conjugated secondary antibody (1:1000). Nuclei were detected with DAPI (Life Technologies), Lyophilized MUB.sub.40 peptides (MUB.sub.40#1 (SEQ ID NO: 3)-Cy5, MUB.sub.40#2 (SEQ ID NO: 4)-Cy5, MUB.sub.40#3 (SEQ ID NO: 5)-Cy5, MUB.sub.40#4 (SEQ ID NO: 6)-Cy5, MUB.sub.40#1 (SEQ ID NO: 3)-Dylight, RI-MUB.sub.40 (SEQ ID NO: 2)-Cy5, and RI-MUB.sub.40 (SEQ ID NO: 2)-Biotin) were solubilized in a phosphate buffer pH8 at a 1 mg/mL concentration and used at a 1:1000 dilution. For PBMC staining CD19-APC (B lymphocytes), CD3-APC (T lymphocytes), and CD14-PercP (monocytes, macrophages) (BD Bioscience) were used at a 1:1000 dilution together with Dapi (1:1000) and MUB.sub.40-Cy5 (1 mg/mL). Actin was stained with Phalloidin-Rhodamine red X (RRX) (Jackson Immunoresearch Antibodies) used at a 1:1000 final dilution.
(62) Biochemistry
(63) Electrophororesis. Proteins were separated by either SDS Page (4-12% gradient, at 150 volts for 90 min) or Ag-Page (0.7% (w/v) agarose gels in 1×TAE (40 mM Tris-acetate, 1 mM EDTA pH 8), containing 0.1% (w/v) SDS at 65 volts for 3 hours).
(64) Transfer. Protein samples separated by SDS Page were transferred to nitrocellulose membrane performing electrophoretic protein transfer. Protein samples separated by Ag-Page were transferred to nitrocellulose membrane using a vacuum blotter (GE HealthCare); 40 mbar pressure in 4×SSC (0.6 M sodium chloride, 60 mM tri-sodium citrate).
(65) RI-MUB.sub.40-Biotin immunoblotting. Transferred nitrocellulose membranes were blotted in PBS with RI-MUB.sub.40-Biotin (SEQ ID NO: 2) (1 μg/mL) for 3 hours, washed three times (15 min) in PBS, incubated with HRP-conjugated Streptavidin (Thermo Scientific ref. N100, 1:1000 dilution) for one hour, and washed three times (15 min) in PBS. RI-MUB.sub.40 (SEQ ID NO: 2)-Biotin binding was detected with chemiluminescence (ECL kit, GE Healthcare) using a an imaging system (B:Box, Syngene).
(66) RI-MUB.sub.40-Biotin Pulldown assay. Lyophilized, biotinylated retro-inverso MUB.sub.40 (RI-MUB.sub.40-Biotin) was solubilized in 1 mL (185 μg/ml final concentration) of binding buffer (20 mM NaH.sub.2PO.sub.4, 0.15 M NaCl, pH 7.5). 150 μL (277 ng) of solubilized RI-MUB40-Biotin was loaded onto 200 μL of washed/packed Streptavidin Sepharose High Performance beads (GE HealthCare). The loaded beads were incubated with gentle rocking for 1 hour at room temperature with 1 mg of purified neutrophil granule fractions. Pulldown fractions were transferred to columns and washed with 10 mL fresh binding buffer. Bound RI-MUB.sub.40-Biotin and α-purified proteins were eluted with 500 μL 8M Guanidine-HCl, pH 1.5. Eluted proteins were mixed 1:1 with 2× Laemmli buffer (4% SDS, 20% Glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue, and 0.125 M Tris HCl, pH 6.8). Samples were run on 4-20% SDS-Page gels and stained with InstantBlue™ (Expedeon, ref. ISB1L) for 1 hour before destaining in deionized H.sub.2O. Stained gels were imaged and =80 kDa α-purified band was cut out and sent for mass spectrometry identification.
(67) Lactoferrin polymerization. Human purified lactoferrin (ref: L1294, Sigma-Aldrich) was resuspended in deionized H.sub.2O (35 μg/mL). Lactoferrin was incubated in RPMI+10 mM Hepes supplemented with 10 mM FeCl.sub.3, at indicated concentrations (0.35 or 3.5 μg/mL) onto 24-well plates containing 12 mm coverslips overnight at 37° C., adapted from similar lactoferrin polymerization experiments performed in iron saturated phosphate buffer (Mantel et al. 1994).
(68) Lactoferrin deglycolysation. About 10 μg of human purified lactoferrin (ref: L1294, Sigma-Aldrich) was treated by Peptide-N-Glycosidase F (PNGaseF, New England Biolabs, Ipswich Mass., USA) for N-deglycosylation. Protein was treated exactly as described in manufacturer protocol. Twelve unit of PNGase F were added for 4 h at 37° C. for the removal of N-glycosylation.
(69) Lactoferrin polymers interaction with MUB.sub.40-Cy5. Lactoferrin polymers were centrifuged at 2000 rpm for 10 min and fixed in Paraformaldehyde (PFA) 4% for one hour. Lactoferrin polymers were immunolabeled in PBS+0.1% saponin (Sigma-Aldrich) with a mouse anti-lactoferrin primary monoclonal antibody (Hycult biotech, clone 265-1K1, 1:50 dilution) and an anti-mouse-FITC conjugated secondary antibody (1:1000), together with MUB.sub.40-Cy5 (SEQ ID NO: 3) (1 μg/mL). Slides were washed three times in PBS and three times in deionized H.sub.2O and mounted with prolong gold.sup.tm mounting media.
(70) Lactoferrin polymers interaction with RI-MUB.sub.40-Biotin. 5 μg lactoferrin polymers pre-formed in RPMI 1640+10 mM Hepes (see above) were separated by Ag-Page, transferred to nitrocellulose membrane and blotted with RI-MUB.sub.40 (SEQ ID NO: 2)-Biotin (1 μg/mL) in PBS, as described above.
(71) MUB.sub.40 Peptides Biochemical and Biophysical Characterization
(72) Size Exclusion Chromatography. Gel filtration was performed using an Agilent 1100 system (Agilent Technologies; Palo Alto, Calif., USA) and monitored by absorbance at 280 nm. MUB.sub.40 peptides were solubilized in the gel filtration buffer (20 mM phosphate buffer, 150 mM NaCl, pH7.4) at a concentration of 10 μg/ml, 100 μg/ml, or 1 mg/ml), separately injected on a Yarra™ 3μ SEC-2000 300×7.8 mm column (Phenomenex, Le Pecq, France), and eluted at a 0.5 ml/min flow rate. The column was calibrated with a mixture of standards proteins (ribonuclease A, 13.7 kDa; carbonic anhydrase 29 kDa; ovalbumin 44 kDa; GE Healthcare) complemented with a custom synthetic peptide of our own library (peptide x, 5.1 kDa; Institut Pasteur). The logarithm of the molecular weights were plotted versus the corresponding partition coefficients (Kay=(Ve−Vo)/(Vc−Vo); Ve, elution volume; Vo, void volume; Vc, geometric column volume), giving log(Mr)=2,3108−2,2361Kav as a calibration curve equation.
(73) Circular dichroism. Far-UV Circular Dichroism (CD) spectra were recorded on an Aviv215 spectropolarimeter (Aviv Biomedical) between 190 and 260 nm using a cylindrical cell with a 0.02 cm path length and an averaging time of 1 s per step. Prior analysis, MUB.sub.40 peptides were solubilized at a 60 μM final concentration in 20 mM sodium phosphate buffer (pH 7.4) in the presence of 50 mM NaCl. Scans were repeated consecutively three times and merged to produce an averaged spectrum. Results were corrected using buffer baselines measured under the same conditions and normalized to the molar peptide bond concentration and path length as mean molar differential coefficient per residue. MUB40 Peptides were solubilized at a 60 μM final concentration in 20 mM sodium phosphate buffer (pH 7.4) in the presence of 50 mM NaCl.
(74) Trypsin proteolysis. MUB.sub.40 (SEQ ID NO: 3)-Cy5 and RI-MUB.sub.40 (SEQ ID NO: 2)-Cy5 were dissolved in 50 mM ammonium bicarbonate, pH 8. Prior to digestion and owing to the propensity of maleimide derivatives to undergo ring-opening in a basic environment (Fontaine et al., 2015), we incubated both peptides at 37° C. overnight. Ring-opening completion was checked before trypsin addition. Doing so, we avoided overlapping of N-terminal digest fragments resulting from α-existing succinimidyl thioether and succinamic acid thioether peptides. Along this treatment, minor foot-peaks appeared besides the major peak, which was linked with the well-known side reaction of deamidation and concomitant isomerization, leading to aspartyl and isoaspartyl forms of the peptides (Yang et al., 2010). Lyophilized trypsin (Thermo scientific) was reconstituted using 50 mM acetic acid, diluted with 50 mM ammonium bicarbonate and added to the peptide solution so as to obtain a final peptide concentration of 0.25 mg/mL and a protease to protein ratio of 1:20 (w/w). Samples were directly incubated on the injection sampler thermostated at 37° c. HPLC and LC-MS analyses were performed as previously described above, applying a 15% to 40% linear gradient of acetonitrile in 10 mM ammonium acetate over 20 minutes. An Aeris Peptide 3.6μ XB-C18 column was employed for LC-MS analysis of the L-peptide digest fragments, which were identified in positive electrospray ionization mode (data not shown).
(75) Cell Biology and Neutrophil Fractionation
(76) Ethics. All participants gave written informed consent in accordance with the Declaration of Helsinki principles. Peripheral Human blood was collected from healthy patients at the ICAReB service of the Pasteur Institut (authorization DC No.2008-68). Hematopoietic Stem Cells were purified from cytapheresis products collected from healthy patients stimulated 5 days with G-CSF at the Gustave Roussy Cancer Campus (Villejuif, France). Human blood was collected from the antecubital vein into tubes containing sodium citrate (3.8% final) as anticoagulant molecules.
(77) Polymorphonuclear neutrophils purification. Human polymorphonuclear neutrophils were purified as described previously (Monceaux et al. 2016). Briefly, plasma was removed by centrifugation (450×g, 15 min); blood cells were resuspended in 0.9% NaCl solution supplemented with 0.72% Dextran. After red blood cells sedimentation, white blood cells were pelleted and further separated on a two layer Percoll (GE Healthcare) (51%−42%) by centrifugation (at 240×g, 20 min). PBMC (top layer) were isolated from polymorphonuclear neutrophils (bottom layer). Red blood cells were removed from the latter fraction using CD235a (glycophorin) microbeads (negative selection) (Miltenyi Biotec). PBMCs and polymorphonuclear neutrophils were resuspended in the autologous plasma. Guinea pig and mouse polymorphonuclear neutrophils were purified with the same procedure.
(78) Hematopoietic Stem cells (CD34+) purification, proliferation and differentiation. HSC were purified from cytapheresis products with a CD34 Microbead Kit Ultrapure, according to the manufacturer procedure (Miltenyi). Lin.sup.−/CD34+ HSC purity yield was >98%. CD34+ HSC were cultured in StemSpan SFEM II supplemented with SCF (100 ng/ml), IL-3 (10 ng/ml), and IL-6 (100 ng/ml) at 37° C. with 5% CO.sub.2. Neutrophil differentiation was induced in StemSpan SFEM II containing G-CSF (10 ng/ml), SCF (100 ng/ml), and IL-3 (10 ng/ml) for 13 days at 37° C. with 5% CO.sub.2.
(79) Bacterial strains and growth conditions. Shigella flexneri 5a pGFP (M90T) strain was grown in GCTS broth or on TCS agar plates supplemented with 0.01% Congo Red (Sigma-Aldrich) and Ampicillin (100 lag/ml). Shigella sonnei was acquired from the Institut Pasteur strain collection (CIP 106347) and is a clinical isolate from a 1999 Paris infection. The strain was grown in GCTS supplemented with Ampicillin (100 μg/ml) to maintain the pMW211 plasmid.
(80) Neutrophil infection. Human purified neutrophils were cultured in glass-bottom dishes (Mattek) for live fluorescent microscopy in a RMPI 1640 (Life Technologies) culture medium supplemented with 10% of heat inactivated Human Serum (Sigma-Aldrich). Neutrophils (5.10.sup.5 cell/mL in 2 mL) were infected with exponentially-grown Shigella flexneri 5a pGFP at a Multiplicity Of Infection (MOI) 20 in RMPI 1640 (Life Technologies) culture medium supplemented with 10 mM Hepes (Life Technologies) at 37° C. Infected neutrophils were centrifuged at 2000 rpm for 10 min prior imaging.
(81) Animal Models of Shigellosis
(82) Guinea pig. The experimental protocol was approved by the french Ethic Committee Paris (n° 20140069, 2014). Young guinea pigs (Hartley, <150 g, Charles River) were anaesthetized and infected intrarectally with 10.sup.9 CFU exponentially grown Shigella flexneri 5a (M90T) pGFP as previously described (Shim et al. 2007) (Monceaux et al. 2016). Infection occurred during 8 hours before animals were sacrificed and infected colons collected and fixed in 4% Paraformaldehyde (PFA) for two hours. For immunohistochemical staining, infected guinea pig colon samples were washed in PBS and incubated at 4° C. in PBS containing 16% sucrose for 4 hours, followed by incubation in PBS with 30% sucrose overnight. Samples were frozen in OCT (VWR 361603E) on dry ice. Frozen blocks were stored at −80 until sectioning. 10 to 30 μm sections were obtained using a cryostat CM-3050S (Leica). Tissue slices were labeled in PBS+0.1% saponin (Sigma-Aldrich) with MUB.sub.40-Cy5 or MUB40-Dylight (1 μg/ml) to localize recruited neutrophils. Slides were washed three time in PBS+0.1% saponin, three times in PBS, and three times in deionized H.sub.2O and mounted with prolong gold.sup.tm mounting media.
(83) Mouse. The experimental protocol was approved by the french Ethic Committee Paris (number 20150042). Female 6 week-old BALB/cJRi mice from Charles River were orally gavaged with streptomycin (100 μL of 200 mg/ml solution) 24 hours prior to Shigella sonnei infection. Mice were orally gavaged with 10.sup.10 CFUs S. sonnei carrying pMW211 expressing DsRed and monitored for 24 hours. At the end of the experiment, animals were sacrificed and tissue sections from the colon were extracted. Colon sections were placed in 4% paraformaldehyde (PFA) solution for 2 hours. PFA fixed tissue sections were passaged for 16 hours in 16% sucrose followed by 4 hours in 30% sucrose solutions. Fixed colon slices were embedded in OCT (VWR 361603E) and flash frozen in dry ice chilled 2-methylbutane. Frozen blocks were stored at −80 until sectioning. A Leica CM3050S cryostat was used to cut 30 μM thick colon slices, which were absorbed to glass microscope slides. Tissue slices were prepared for fluorescence microscopy by incubation in 0.1% saponin for 1 hour followed by incubation with fluorescent markers specific for actin (phalloidin-FITC, Life Technology) and MUB.sub.40-Cy5 at a final 1 μg/ml concentration. Slides were washed in deionized H.sub.2O and mounted with prolong gold.sup.tm mounting media.
(84) Rabbit. The experimental protocol was approved by the french Ethic Committee Paris 1 (number 20070004, December 9th 2007). New Zealand White rabbits weighting 2.5-3 kg (Charles River) were used for experimental infections. For each animal, up to 12 intestinal ligated loops, each 5 cm in length, were prepared as described previously (Jones et al. 2007; Martinez et al. 1988; West et al. 2005) and infected with 10.sup.5 CFU S. flexneri pGFP per loop. After 16 h, animals were sacrificed and collected tissue were fixed in 4% Paraformaldehyde (PFA) for two hours. For immunohistochemical staining, infected rabbit ileum samples were washed in PBS, incubated at 4° C. PBS containing 12% sucrose for 90 min, then in PBS with 18% sucrose overnight, and frozen in OCT (Sakura) on dry ice. 7 μm sections were obtained using a cryostat CM-3050 (Leica). Fluorescent staining was performed in PBS+0.1% saponin using Phalloidin-RRX (1:1000 dilution) to stain Actin, MUB.sub.40-Cy5 (1 μg/mL) and a mouse anti-lactoferrin primary monoclonal antibody (Hycult biotech, clone 265-1K1, 1:50 dilution) and an anti-mouse-FITC conjugated secondary antibody (1:1000) to stain infiltrated neutrophils. Slides were washed three time in PBS+0.1% saponin, three times in PBS, and three times in deionized H.sub.2O and mounted with prolong gold.sup.tm mounting media.
(85) Mass Spectrometry Analyses
(86) Digestion of proteins. Coomassie-stained bands detected on gel were cut and rinsed three times in a 50/50 mix of water/acetonitrile (ACN). Proteins were reduced (10 mM Dithiothreitol (DTT)) and further alkylated (50 mM Iodoacetamide) in-gel. In-gel tryptic digestion was performed by adding 400 ng sequencing grade modified trypsin (Promega France, Charbonnières, France) in 50 mM NH.sub.4HCO.sub.3 for 18 h at 37° C. Tryptic peptides were recovered by washing the gel pieces twice in 0.5% FA-50% ACN and once in 100% acetonitrile, and all supernatants were collected in the same tube and evaporated to almost dryness.
(87) LC-MS/MS of tryptic digest. Digested peptides were analyzed by nano LC-MS/MS using an EASY-nLC 1000 (Thermo Fisher Scientific) coupled to an Orbitrap Q Exactive HF mass spectrometer (Thermo Fisher Scientific, Bremen). Half of each sample was loaded and separated at 250 nl.Math.min.sup.−1 on a home-made C.sub.18 30 cm capillary column picotip silica emitter tip (75 μm diameter filled with 1.9 μm Reprosil-Pur Basic C.sub.18-HD resin, (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany)) equilibrated in solvent A (0.1% FA). Peptides were eluted using a gradient of solvent B (ACN, 0.1% FA) from 2% to 5% in 5 min, 5% to 35% in 30 min, 30% to 50% in 5 min at 250 nL/min flow rate (total length of the chromatographic run was 50 min including high ACN level steps and column regeneration). Mass spectra were acquired in data-dependent acquisition mode with the XCalibur 2.2 software (Thermo Fisher Scientific, Bremen) with automatic switching between MS and MS/MS scans using a top-15 method. MS spectra were acquired at a resolution of 60000 with a target value of 3×10.sup.6 ions. The scan range was limited from 300 to 1700 m/z. Peptide fragmentation was performed via higher-energy collision dissociation (HCD) with the energy set at 28 NCE. Intensity threshold for ions selection was set at 1×10.sup.5 ions with charge exclusion of z=1 and z>6. The MS/MS spectra were acquired at a resolution of 17500 (at m/z 400). Isolation window was set at 2 Th. Dynamic exclusion was employed within 30 s.
(88) Data were searched using MaxQuant.sup.1 (version 1.5.3.8) (with the Andromeda search engine) against a human database (20202 entries, downloaded from Uniprot the 2016.05.26).
(89) The following search parameters were applied: carbamidomethylation of cysteines was set as a fixed modification, and oxidation of methionine and protein N-terminal acetylation were set as variable modifications. The mass tolerances in MS and MS/MS were set to 5 ppm and 20 ppm, respectively. Maximum peptide charge was set to 7, and 5 amino acids were required as minimum peptide length. Results were filtered by a 0.01 false discovery rate at both protein and peptide levels.
(90) Microscopy and Image Analysis
(91) Confocal microscopy. Fixed cells (polymorphonuclear neutrophils, PBMC, haematopoietic stem cells), guinea pig colon and rabbit ileum infected with S. flexneri pGFP were imaged on a laser-scanning TCS SP5 confocal microscope (Leica). Z-stack images were taken with 1 μM step-size increments. Obtained Z-stack images were processed with Fiji software
(92) Spinning disk microscope. Mouse colons infected with S. sonnei pDsRed were imaged on a Bruker Opterra fluorescence microscope using a Zeiss Plan-Apochromat 63×/1.40 oil immersion lense and Prairie View software version 5.3. Z-stack images were taken with 0.5 μM step-size increments. Obtained Z-stack images were stitched with Fiji software version 2.0.0-rc-30/1.49u and visualized using IMARIS software version 8.3.1.
(93) Live epifluorescence microscopy. Viable neutrophils infected with S. flexneri pGFP were imaged on a Definite focus live microscope (Zeiss) equipped with a temperature control chamber (37° C.) and a 63× oil immersion objective. Images were acquired every 60 s. Z-stack images were taken with 2 μM step-size increments. Obtained Z-stack images were processed with Fiji software to generate a Movie and the corresponding extracted images are shown in
(94) Two-photons microscopy. Human colonic tissue segments infected with S. flexneri pGFP were imaged using a commercial laser-scanning microscope (LSM710, Meta, Zeiss, Germany) as described previously (Tyanova et al. 2016). MUB.sub.40-Cy5 was detected using multiphoton excitation (MPE, magenta), Acquisitions were performed with Zen 2008 SP 1.1 software acquisition package developed by ZEISS. Imaris software (http://www.bitplane.com) was used to prepare final images.
(95) B. Results
(96) MUB.sub.40-Peptides Chemical Synthesis, Structure, and Binding Properties
(97) MUB.sub.40 is a 40-amino acid peptide, derived from the MUB.sub.70 marker, originally characterized for its ability to bind the human colonic mucus (Coïc et al., 2012). Four overlapping 40-amino acid peptides (named MUB.sub.40#1 (SEQ ID NO: 3), MUB.sub.40#2 (SEQ ID NO: 4), MUB.sub.40#3 (SEQ ID NO: 5), and MUB.sub.40#4) (SEQ ID NO: 6) covering the MUB.sub.70 sequence were designed (
(98) MUB.sub.40#1 (SEQ ID NO: 3) (corresponding to MUB.sub.70 C-terminal part), conjugated to Cy5 (MUB.sub.40#1-Cy5) was the only peptide, which conserved the ability to bind to and fluorescently label the human colonic mucus (
(99) MUB70 (SEQ ID NO: 11) was also previously found to assemble as a trimer, as evidenced by analytical gel filtration (Coïc et al., 2012). Further, among the four 40-AAs MUB40 polypeptides disclosed in WO 2013/034749 A1 (SEQ ID NOS: 3-6), the inventors validated that one kept the mucus-binding property, i.e. probe MUB40 #1 (SEQ ID NO: 3)-Cy5. In fact, the inventors have previously shown that the C-terminal part of MUB70, i.e. the part covered by MUB40 #1 probe (SEQ ID NO: 3) in WO 2013/034749 A1, adopt a trimeric organization as compared to its theoretical molecular weight. On the contrary, MUB40 #2 (SEQ ID NO: 4), MUB40 #3 (SEQ ID NO: 5) and MUB40 #4 (SEQ ID NO: 6) probes have presently been shown to adopt a different multimeric status in phosphate buffer as shown in
(100) MUB.sub.40 peptides were almost similarly shaped and could be assigned to unstructured peptide chains or polyproline II-like scaffolds (by Circular Dichroism, see Methods) (
(101) Also, as reported before, inflammatory tissues can be associated with mucus pathological accumulation. The specific labelling of these inflammatory samples was assessed with MUB40 #1 (SEQ ID NO: 3). As an example, colonic mucinous carcinoma, which was demonstrated to be specifically labelled with the original MUB70 peptide (Coïc et al., 2012), was similarly labelled with MUB40 #1, together with an anti-Muc2 antibody. The accumulation of Muc2 in this pathology was additionally confirmed (
(102) The inventors also demonstrated in vitro that cystic fibrosis (CF) patients sputum was labeled with MUB40 #1 (SEQ ID NO: 3)-Cy5, together with anti-Muc2 and anti-Muc5ac monoclonal antibodies (
(103) MUB.sub.40 Labels Human and Other Mammalian Granulocytes
(104) In addition to its colonic mucus binding property, confirmed on fixed human colon explants infected with Shigella flexneri (S. flexneri) (
(105) The inventors further confirmed that regardless of the fluorophore conjugated to MUB.sub.40 (MUB.sub.40 (SEQ ID NO: 3)-Cy5 or MUB.sub.40 (SEQ ID NO: 3)-Dylight405, see Methods), human neutrophil granules were specifically labeled (
(106) The inventors have here demonstrated that all mammalian neutrophils tested were efficiently labeled with MUB.sub.40-(SEQ ID NO: 3) Cy5, as illustrated with mouse and guinea pig circulating neutrophils (
(107) These results confirm the potential use of MUB.sub.40 peptide as a specific neutrophil marker in physiological and pathophysiological conditions, in humans and in various animal models. The inventors next aimed at synthesizing the so-called RI-MUB40 polypeptide and identifying the specific MUB.sub.40 (and RI-MUB40) target(s) in neutrophil granules.
(108) In fact, in order to anticipate its use in the presence of living cells, a non-cleavable retro-inverso version of MUB.sub.40, named RI-MUB.sub.40, was synthesized with non-natural D-amino acids. RI-MUB.sub.40 was resistant to trypsin proteolysis, whereas MUB.sub.40 was rapidly degraded (
(109) Synthesis of RI-MUB40
(110) Considering Solid Phase Peptide Synthesis (SPPS), most of the deprotection and coupling difficulties are related to inter or intra-molecular hydrogen bonds occurring over the synthesis. N-alkylated amino-acids such as Dmb/Hmb (Johnson et al., 1995) or pseudoproline (Mutter et al., 1995) (Coïc et al., 2010) have been developed to overcome the resulting aggregation propensity of the protected peptide chain anchored to the resin. Moreover, aspartimide (Asp) formation (Mergler et al., 2003) and subsequent base-catalyzed ring-opening during Fmoc-SPPS have been described to be one of the most prevalent side reaction occurring in Asp-containing peptides, Asp-Gly sequences being particularly prone to aspartimide formation. Therefore MUB70 was obtained by using a rational incorporation of both pseudoproline and Asp(OtBu)-(Dmb)Gly-OH dipeptides (Coïc et al, 2012).
(111) The synthesis strategy of RI-MUB40 was designed by taking into account the commercial availability of dipeptides surrogates in the D-AAs series. Along this line, Fmoc-D-Asp(OtBu)-(Hmb)Gly-OH was incorporated instead of Asp31 and Gly32 (as referred to the Retro-Inverso sequence of SEQ ID NO: 2, D-amino-acid residues in bold and underlined), in order to minimize aspartimide formation.
(112) TABLE-US-00001 MUB40 retro-inverso sequence (SEQ ID NO: 1) FIVTYFQDNTDDNDFKEGAPFNDKFLEYGDGEFKKIGEAT Synthesized RI-MUB40 sequence (SEQ ID NO: 2) acetyl-CFIVTYFQDNTDDNDFKEGAPFNDKFLEYGDGE FKKIGEAT-amide
RI-MUB40 was finally obtained with an overall isolated yield of 9%, mainly due to a major side-product corresponding to the Asp-Gly deletion peptide, suggesting a poor Hmb dipeptide coupling yield. Careful LC-MS analysis of the RI-MUB40 crude mixture allowed the inventors to clearly identify a poor Hmb-Asp-Gly dipeptide coupling step (
First Experimental Validation of RI-MUB40
(113) RI-MUB40 synthesis allowed the inventors to validate conservation of mucus and neutrophil granules binding properties of retro-inverso MUB-type polypeptides.
(114) RI-MUB40 mucus-binding properties were validated on human colonic mucus samples. The inventors confirmed that MUB40 #1-Cy5 labeled neutrophil granules, as anticipated, and demonstrated that RI-MUB40-Cy5 has similar properties (
(115) Resistance of MUB-40 and RI-MUB40 to Enzymatic Degradation
(116) The resistance improvement of the MUB40 retro-inverso analogue to protease degradation was evaluated through a trypsin digestion performed on the Cy5 labeled peptides (0.25 mg/mL) with a final protease to peptide ratio of 1:20 (w/w). Hydrolysis experiments were done in solution at 37° c., pH8 and monitored by HPLC by measuring the peak area of the remaining intact peptides. Unlike the L-peptide, which was almost completely degraded within 3 h, no degradation was observed for the D-peptide until 24 h, demonstrating the enhanced stability of the retro-inverso peptide (
(117) TABLE-US-00002 TABLE 1 Cy5-MUB40#1 Cy5-RI-MUB40 Time % (SEQ ID NO: 3) of remaining % of (SEQ ID NO: 2) remaining (hours) peptide peptide 0.033 90 100 1.25 13 100 3 3 100 5 0.5 100 24 0 100
MUB.sub.40 Binds to Lactoferrin Stored in Specific and Tertiary Granules
(118) The four classes of neutrophil granules (α, β1, β2, and γ) were fractionated on a three-layer Percoll gradient (see Methods), as previously described (Kjeldsen et al., 1999). To confirm the appropriateness of the approach the inventors subjected the different fractions to mass spectrometry and could identify the most abundant proteins stored in each granule population (see Methods): cathepsin G, neutrophil elastase, and myeloblastin in azurophil granules; lactoferrin, NGAL, cathelicidin C, and lysozyme C in specific granules; and lactoferrin, MMP-9, NGAL, cathelicidin C, and protein S100-A9 in tertiary granules, as previously reported (
(119) The MUB.sub.40 target was mainly stored in specific (β1) and tertiary (β2) granules, as revealed by western blot using a biotinylated version of RI-MUB.sub.40 (RI-MUB.sub.40-biotin) (see Methods) when granule contents were analyzed on SDS Page gel (
(120) Lactoferrin was identified as a target of MUB.sub.40 in neutrophil granules, by a pulldown assay with RI-MUB.sub.40-Biotin (
(121) As a conclusion, MUB.sub.40 (SEQ ID NO: 3) is a marker of lactoferrin, which is the most abundant protein stored in neutrophil specific and tertiary granules. The potential use of MUB.sub.40 (SEQ ID NO: 3)-Cy5 as a marker of lactoferrin secretion was further assessed during Shigella flexneri infection in vitro and in in vivo models of inflammation.
(122) Detection of Lactoferrin Degranulation with MUB.sub.40-Cy5 In Vitro and In Vivo
(123) Neutrophil granule inducible exocytosis (or degranulation) occurs in the presence of an inflammatory stimulus, such as bacterial infection. Here, for the first time, the degranulation process could be assessed in vitro on living neutrophils infected with Shigella flexneri by live fluorescence microscopy in the presence of RI-MUB.sub.40-Cy5: transient, dot-shaped fluorescent signals were detected on the cells' surface (
(124) Further validation of RI-MUB.sub.40 in living animals suffering of shigellosis was not possible due to the localization of the infected organ, the colon, deep into the abdomen, making the assessment of fluorescent signals difficult given the current state of technology or possibilities offered to the inventors. Alternatively, the capacity of RI-MUB.sub.40 to specifically label inflammatory tissues was evaluated in an arthritis mouse model (sterile inflammation), using the K/B×N serum transfer model (Bruhns et al., 2003). Indeed, following systemic administration (i.v. injection) of RI-MUB40-Cy5 into arthritic mice, but not their naïve controls, a fluorescent signal accumulated at inflammatory sites, localized in joints, which are known to contain a large population of activated neutrophils (
(125) In conclusion, we demonstrated here that MUB.sub.40 and RI-MUB.sub.40 peptides allowed the assessment of neutrophil lactoferrin detection in vitro or in vivo. Therefore, the potential use of RI-MUB.sub.40 as an inflammatory events investigation tool or marker, associated with neutrophil recruitment and activation, was further evaluated on human inflamed tissues.
(126) Neutrophil Labeling with MUB.sub.40 on Human Inflamed Tissues
(127) Results reported herein indicated that neutrophil lactoferrin could be labeled with MUB.sub.40 or RI-MUB.sub.40 peptides intracellularly on fixed samples (cells and tissues) or extracellularly upon its secretion by living neutrophils upon activation. Neutrophil recruitment and activation was further successfully assessed on various human biopsies from patients diagnosed with sterile or infectious inflammatory diseases with MUB.sub.40 and illustrated here with a malignant fibrous histiocytoma (
(128) In conclusion the inventors demonstrated that so-called MUB.sub.40 peptides, including retro-inverso ones, allow detection of human lactoferrin and therefore offers a new tool for the assessment of neutrophil recruitment and activation in human inflammatory tissues.
(129) C. Discussion
(130) Using maleimide-biotin and maleimide-Cy5 derivatives, the inventors showed that RI-MUB40 has similar binding properties as MUB40 for colonic mucus (human), mucus produced by human cell lines (L174T) or neutrophil granules. Therefore, RI-MUB40 promises to be a more valuable tool as it is designed to resist to protease degradation, especially as a novel inflammatory marker in particular within mucinous inflammatory tissues, or in the context of bio-conjugates targeting overproducing mucus pathologies (i.e. Cystic fibrosis) for drug delivery, in particular as a vehicle when conjugated with mucolytic molecules.
(131) In addition, the peptidomimetics described herein promise to be interesting tools for innovative therapeutical applications, combining for example such peptidomimetics with mucolytic molecules (acetylcysteine, bromalaine, human DNase I or bacterial mucinases) in order to improve mucus clearance in various pathologies such as Cystic fibrosis, arthritis or inflammatory cancers in order to either improve patients state or existing treatments efficiently promoting the access of therapeutical molecules to inflammatory sites.
(132) As disclosed herein, the inventors designed, synthesized and further validated MUB.sub.40 and RI-MUB.sub.40 peptides as novel markers of neutrophil lactoferrin, which may be considered as universal markers of mammalian neutrophils and will facilitate neutrophil detection and study in animal models of inflammation, including mouse, rabbit, or guinea pig (
(133) As a novel lactoferrin marker, MUB.sub.40 type probes and/or RI-MUB.sub.40 type probes will contribute to a better understanding of lactoferrin modulatory and antimicrobial functions in vitro and in vivo. To date, most studies have focused on the importance of mucus lactoferrin in the protection of the respiratory tract from inflammation or infection in vivo (Valenti et al., 2011), (Sagel et al., 2009), (Dubin et al., 2004) or in lung epithelial cell culture models (Calu3 (Babu et al., 2004)). Lactoferrin abundance and function in the intestinal tract has been less investigated, although confirmed in humans (Peen et al., 1996); its protective role was confirmed in a mouse model of colitis (Ye et al., 2014). The resistance of lactoferrin to bacteria secreted serine proteases (SPATE) proteolysis strongly support its importance in preserving the epithelial lineage from bacterial aggression (Gutierrez-Jimenez et al., 2008). The use of MUB.sub.40 type probes and/or RI-MUB.sub.40 type probes will promote lactoferrin studies in intestinal inflammation and infection.
(134) The inventors previously characterized MUB.sub.70 colonic mucus-binding property mediated by its ability to interact with a Mucin 2 glycosylation moiety (Coïc et al., 2012). Here the inventors have confirmed that its shorter derivative, MUB.sub.40-Cy5, similarly labeled colonic mucus and goblet cells' granules (
(135) Using RI-MUB.sub.40-Cy5, the inventors could reveal neutrophil degranulation for the first time in inflammatory tissues in vivo with a non-invasive method (
(136) The inventors demonstrated that MUB.sub.40 is a specific neutrophil marker, which may be used, in a broad range of in vitro assays. Further investigations will be required to validate RI-MUB.sub.40 as a potentially new inflammation marker in vivo, including a pre-clinical study for evaluating toxicity, bioavailability, specificity and sensitivity of its lactoferrin-binding property. Labeling RI-MUB.sub.40 with radioactive elements can be envisaged for inflammation site localization with non-invasive inflammation-imaging methods such as scintigraphy, PET or SPECT (Wu et al., 2013) (Zhang et al., 2010) (Zhang et al., 2007), (Locke et al., 2009).
(137) Significance
(138) Neutrophils are major players of the innate immune response during inflammation and infection, although their detection remains difficult in animal models and humans. Here the inventors have described MUB.sub.40 type probes and/or RI-MUB.sub.40 type probes as specific markers of neutrophils, binding to lactoferrin, stored in specific and tertiary granules or released upon neutrophil activation. MUB.sub.40 and its retro-inverso derivative RI-MUB.sub.40 allow the imaging of neutrophils in vitro and in vivo in inflammation animal models; these markers will open new doors in neutrophil study, non-invasive live imaging of inflammation and diagnostic of inflammatory diseases.
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