Hepcidin antagonists for use in the treatment of inflammation

Abstract

The present invention relates to a hepcidin antagonist for use in the treatment of inflammatory diseases.

Claims

1. A method for treating psoriasis, said method comprising a step of administering a hepcidin antagonist to a patient in need thereof, wherein said hepcidin antagonist is administered topically to the skin, and wherein said hepcidin antagonist is selected from the group consisting of anti-hepcidin antibodies, anti-hepcidin aptamers, lexaptepid pegol, the PRS-080 compound and an antisense oligonucleotide targeting the hepcidin gene, wherein the anti-hepcidin aptamer is a peptide aptamer having an anti-hepcidin antibody variable region and wherein the antisense oligonucleotide comprises a sequence that is complementary to a region of hepcidin mRNA.

2. The method according to claim 1, wherein the hepcidin antagonist is administered in an amount sufficient to inhibit and/or reduce neutrophil migration.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1. LPS induced intestinal hepcidin expression

(2) a, Expression of hepcidin in intestine (duodenum (D), Jejunum (J), Ileum (I), colon (C)) 7 h after PBS or LPS injection (Q-PCR). (n=4 per group; bars represent mean+/−standard error of the mean (s.e.m.); *p<0.05). b, Scheme representing the generation of the Hepc.sup.Δint mice (top). Hepcidin expression in intestine and liver of WT (black bars) and Hepc.sup.Δint (open bars) mice (n=5 per group; bars represent mean+/−s.e.m.); **p<0.01; *p<0.05). c, Hepcidin expression in intestine of WT (black bars) and Hepc.sup.Δint (open bars) mice after PBS or LPS injection (n=5) (mean+/−s.e.m; *p<0.05).

(3) FIG. 2. Hepcidin is a chemoattractant and proinflammatory molecule

(4) a, Flow cytometry analysis of neutrophils in lamina propria of WT and KO mice. Left: Representative experiment with Ly6G.sup.+ Ly6C.sup.low cells shown after gating on CD45.sup.+CD11b.sup.+ cells; Right: pool of four experiments (mean+/−s.e.m; *p<0.05). b, Migration of WT bone marrow isolated neutrophils in response to hepcidin-25. (3 experiments performed in triplicate; mean+/−s.e.m; *p<0.05). c, CXCL1, CXCL2, IL-1beta, iNOS mRNA levels in macrophages incubated for 1 or 3 hours with 1 μg/ml, 10 μg/ml Hepcidin-25 or PBS (control). d, Immunohistochemistry using anti-mouse iNOS antibody on intestine of WT and Hepc.sup.Δint mice upon PBS or LPS injection.

(5) FIG. 3. Intestinal hepcidin is required for systematic inflammation

(6) Cytokines levels measured in the plasma of WT (black square) and Hepc.sup.Δint (open square) mice (n=10) (mean+/−s.e.m.).

(7) FIG. 4. Iron parameters in WT and Hepc.sup.Δint mice

(8) a, Plasma iron and transferrin saturation in WT (black bars) and Hepc.sup.Δint (open bars) mice after PBS or LPS injection (n=10) (mean+/−s.e.m.). ns: not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. b, Expression of hepcidin in liver 7 h after PBS or LPS injection (Q-PCR). (n=10 per group; bars represent mean+/−standard error of the mean (s.e.m.); ***p<0.001). c, Kaplan-Meier survival curve following i.p. LPS (30 mg/kg) injection in WT (n=10) and Hepc.sup.Δint (n=12). Statistical analysis by a Log-Rank (Mantel-Cox) test. *p<0.05.

(9) FIG. 5. Hepcidin is expressed in the differentiated compartment of the intestinal epithelium.

(10) a, Scheme of the fractionation of an intestinal villus. b, Tissue fractionation procedure was validated by quantitative PCR analysis of either villus (Fabp2, and KLF4) or crypt intestinal markers (Sox9). Expression of hepcidin in the duodenum 7 h after PBS or LPS injection (Q-PCR). n=4 per group; bars represent mean+/−s.e.m.; *p<0.05; **p<0.01; ***p<0.001.

(11) FIG. 6. Immune cell population in lamina propria.

(12) Flow cytometry analysis on lamina propria of the small intestine of WT (black square) and HepcΔint (open square) mice 7 h after PBS or LPS injection. n=4 mice per group; Bars represent mean+/−s.e.m.

(13) FIG. 7. A proposed model for the mechanisms linking intestinal hepcidin to systemic inflammation.

(14) FIG. 8. TLRs agonists induce hepcidin expression in the intestine. Q-PCR on duodenal extracts of WT and HampΔint mice injected for 7 hours with Zymosan, Flagellin, Poly:IC or PBS. n=3 per group; bars represent mean+/−s.e.m.; *p<0.05; ***p<0.001.

(15) FIG. 9: Serum starvation enhances the proinflammatory action of Hepc-25 in macrophages. Expression by Q-PCR of CXCL2 and TNF-α in Raw 264.7 (ATCC® TIB-71™) murine macrophage cell line serum starved or not for 1 hour before the addition of 10 μg/ml of Hepc-25 (Peptide International) for an additional hour. N=3 per group; mean+/−s.e.m. Statistical analysis by a two-way analysis of variance (ANOVA) followed by a Bonferroni posttest. **p<0.01; ***p<0.001.

(16) FIG. 10: Hepc-25 induces a robust expression of Cxcl2 at 1 μg/ml in conditions of serum starvation. Expression by Q-PCR of CXCL2 in Raw 264.7 (ATCC® TIB-71™) murine macrophage cell line serum starved for 1 hour or not before the addition of 1 or 10 μg/ml of Hepc-25 (Peptide International) for an additional hour. N=3 per group; mean+/−s.e.m. Statistical analysis by a two-way analysis of variance (ANOVA) followed by a Bonferroni posttest. **p<0.01; ****p<0.0001.

(17) FIG. 11: Intestinal deletion of hepcidin in the intestine prevents neutrophil migration in the intestine. Ly6G expression by Q-PCR in intestine of WT and HepcΔint mice after 7 hours PBS or 2 mg/kg LPS injection. n=5 per group; mean+/−s.e.m.

(18) FIG. 12: Inhibition of Cxcr2 prevents neutrophil migration towards hepcidin. Migration of WT bone marrow isolated neutrophils in response to hepcidin (1 μg/ml) in presence or not of the Cxcr2 inhibitor (sb225002, Sigma-Aldrich). n=3. mean+/−s.e.m. It indicates that Cxcr2 may be the receptor of hepcidin in neutrophils

EXAMPLES

(19) Hepcidin is a 25 amino acid peptide demonstrated to be the key iron regulatory hormone, produced by the liver, capable of blocking iron absorption from the duodenum and iron release from macrophages.sup.1. Here, is disclosed a completely new role of hepcidin in the intestine. The gut is the motor of the systemic inflammatory response in critical illness but the mechanisms by which it acts is unclear.sup.2. While, during acute inflammation, hepcidin has been shown to be poorly induced in the liver.sup.3,4, it has been found here that it was highly produced by the intestinal epithelium. Our results showed that gut hepcidin acts both as a neutrophil chemoattractant protein and a proinflammatory molecule. Generation of intestinal specific hepcidin KO mice demonstrated that gut hepcidin was required for the recruitment of intestinal neutrophils and the induction of chemokines (CXCL1, CXCL2) and proinflammatory molecules by the macrophages. Importantly, hepcidin is critical to the systemic production of key inflammatory cytokines (IL-6, KC/GRO, TNF, IL-1beta . . . ) and the setting of the hypoferremia of inflammation. Therefore, this study unravels intestinal hepcidin as a critical component to systemic inflammation initiation and a potential new target in systemic inflammatory diseases, which currently lack effective therapeutics.

(20) If hepatic hepcidin is now recognized as the key iron regulatory hormone.sup.5, it was originally identified as a cationic antimicrobial peptide (AMP).sup.6. However, the potential expression and role of hepcidin in intestinal epithelia, a major source of AMPs, has never been investigated. In this study, basal hepcidin mRNA level was detected in murine intestine, with an increasing expression gradient from the duodenum to the colon of WT mice (FIG. 1a). As the gut is considered as the motor of systemic inflammation.sup.2, a major cause of morbidity and mortality across many diseases, it has herein been investigated whether intestinal hepcidin expression was modified under these conditions. WT Mice were challenged intraperitonealy (ip) with the immune activator lipopolysaccharide (LPS), which derives from the Gram-negative bacterial cell wall. Seven hours after LPS induction, we found a marked increase of hepcidin gene expression in all the segments of the intestine (FIG. 1a) in contrast to the modest induction in the liver, as previously reported.sup.3,4 (FIG. 4b). Using a sequential isolation of intestinal epithelial cells along the crypt villus axis (CVA) (FIG. 5a), we determined that hepcidin expression was restricted to mature differentiated intestinal cells, its expression being undetectable in the proliferative crypt compartment (FIG. 5b). After LPS stimulation, hepcidin was maximally expressed in villus cells with an increasing gradient along the CVA. LPS is a microbial activator of TLR4, a pattern recognition molecule critical for initiating innate immune signaling cascades and proinflammatory responses. TLR4 expression by intestinal epithelial cells is low at basal level, but increases during inflammation (Abreu et al., Nat Rev Immunol 10, 131-144, 2010). The inventors have further demonstrated that the loss of TLR4 clearly reduced the LPS induced-level of hepcidin in the gastrointestinal tract (Data not shown) demonstrating that the hepcidin response to LPS was TLR4-dependent.

(21) To determine the role of gut-derived hepcidin, mice with specific invalidation of hepcidin in the intestinal epithelium (Hepc.sup.lox/lox-VillinCre: Hepc.sup.Δint) were generated. Those were obtained by breeding recently generated Hamp1.sup.lox/lox mice.sup.8 with a transgenic strain expressing the Cre recombinase under the control of the murine villin promoter.sup.9 (FIG. 1b). The deletion efficiency of hepcidin in isolated epithelial cells of the small and large intestines was approximately 97% as determined by quantitative PCR on mouse genomic DNA (data not shown). It confirmed that hepcidin mRNA levels were totally abolished in the intestine (duodenum, jejunum, ileum, colon) of the Hepc.sup.Δint mice and not affected in the liver of these mice compared to WT mice (FIG. 1b). Hepcidin was not induced in the purified intestinal cells enriched in enterocytes of Hepc.sup.Δint mice upon LPS injection, confirming that the intestinal epithelial cells are the source of hepcidin production (FIG. 1c).

(22) It is now well established that in addition to their barrier and absorptive functions, intestinal epithelial cells can be activated to produce mediators that recruit, activate and condition cells of the immune system.sup.10-12 In particular, during intestinal inflammation, cytokines and chemokines have been reported as critical regulators for recruitment and infiltration of the major effectors of acute inflammation, i.e. the neutrophils, the first leukocytes to be recruited to an inflammatory site.sup.13. As gut-derived hepcidin expression is induced upon acute systemic inflammation, flow cytometric analyses were performed to determine whether locally it could contribute to the recruitment of immune cells in the lamina propria. Among all the tested immune populations (FIG. 6), only neutrophils were recruited upon LPS injection (FIG. 2a). Importantly, the deletion of intestinal hepcidin decreased neutrophil population by almost two fold (FIG. 2a) suggesting that intestinal hepcidin contributed to the recruitment of neutrophils in the intestine after LPS injection.

(23) Following LPS stimulation, the intestine of WT mice was severely inflamed as demonstrated by a high expression level of inducible nitric oxide synthase (NOS2), an essential provider for NO-mediated signaling during the initiation of systemic inflammation.sup.22 (Data not shown). In sharp contrast, the Hepc.sup.Δint mice intestine had a dramatic reduction of NOS2. Addition of synthetic mature hepcidin on the basolateral or apical side of the Caco-2 intestinal cell line did not induce iNOS expression (data not shown) nor increased the expression of inflammatory cytokines, such as TNF-α or the neutrophil chemokine IL-8.

(24) Hepcidin was originally identified as a cationic AMP by its close structural similarity to the beta defensins.sup.6. Hepcidin contains disulfide bridges, highly conserved among vertebrates, like typical chemokine proteins (C, CC, CXC, CX.sub.3C), which contain intramolecular disulfide bonds critical for their functions. Through a close analysis of 1-D sequences and 3-D structural alignments of hepcidin with chemokines, the inventors highlighted the striking structural similarity of hepcidin with known chemokines (CCL5, CCL11, CCL20, and XCL1; Data not shown), and therefore hypothesized that hepcidin could display a direct chemokine function. They thus tested the ability of synthetic hepcidin to attract purified neutrophils using a classical migration assay. As shown in FIG. 2b, hepcidin induced a dose-dependent migration of neutrophils in the typical bimodal manner.sup.14-16 demonstrating for the first time that hepcidin displays chemoattractant properties. The peak response was observed at 0.1 μg/ml, a dose at which prototypical AMPs exert maximum chemotaxis.sup.14-16. As disclosed in FIG. 11, intestinal deletion of hepcidin in the intestine prevents neutrophil migration in the intestine.

(25) By studying the direct chemoattractant effect the inventors have observed that inhibition of Cxcr2 was also able to prevent neutrophil migration (see FIG. 12). These results clearly suggest that Cxcr2 may be the receptor of hepcidin in neutrophils.

(26) The gastrointestinal tract contains the largest reservoir of macrophages in the organism.sup.17. Besides its proper chemoattractant property, the inventors hypothesized that hepcidin may influence the production of chemokines by the macrophages. Macrophages were incubated with hepcidin-25 at different time points (30 min, 1 h, 3 h, 24 h, 48 h). At 1 hour, the expression of the specific neutrophil chemoattractants CXCL-1 and CXCL2, was slightly increased with 1 μg/ml hepcidin and maximally induced with 10 μg/ml hepcidin, suggesting that hepcidin may also indirectly promote neutrophil migration through macrophage-induced specific neutrophil chemoattractants such as CXCL-1 and CXCL2, thus contributing to neutrophil recruitment cascade (FIG. 2c). Strikingly, the only set of genes statistically induced by hepcidin at 1 hour (with a fold change >2) was involved in the inflammatory response: TNF-α, the neutrophil-specific chemoattractants CXCL1 and CXCL2, NF-kappa-B inhibitor zeta, an atypical IκB family member and transcriptional coactivator required for the selective expression of a subset of NF-κB target genes (Hildebrand et al., J Immunol 190, 4812-4820, 2013) and Mir-146, recently demonstrated to have a critical role in limiting an excessive acute inflammatory reaction (Brudecki et al., Immunol Cell Biol 91, 532-540, 2013) (Data not shown).

(27) Kinetic response of macrophage-derived CXCL1 and TNF-α following hepcidin treatment was further determined. Transient mRNA induction peaked at 1 hour, suggesting a direct proinflammatory effect of hepcidin. Massive protein accumulation of CXCL1 and TNF-α reached respectively 300 and 150 pg/ml at 7 hours (Data not shown). Hepcidin stimulated IL-1beta, IL-6 and iNOS expression with a maximal induction at 3 hours with 10 μg/ml hepcidin (FIG. 2c) while the peak of TNF-alpha and Cox-2 expression occurred after 1 hour of hepcidin treatment. AMPs, such as LL37, typically function in this high concentration range.sup.18,19 to induce proinflammatory molecules.sup.20,21. This allows the production of inflammatory mediators in pathological conditions where AMPs concentrations can reach high values.sup.18,19 and not in healthy tissues where inflammatory damage might otherwise harm host cells. Altogether, these data reveal for the first time that hepcidin functions as both a chemoattractant for neutrophils and a regulator of immune response through induction of proinflammatory mediators by macrophages. Importantly, this new proinflammatory role of hepcidin was independent of its known role in iron homeostasis as the expression of the iron-responsive gene TfR1, was unchanged by the addition of hepcidin (Data not shown) and supplementation of iron or iron chelators did not modify either the expression of inflammatory genes (data not shown).

(28) The inventors have further demonstrated that inhibition of the MAPK, PI3K and AKT pathways had no effect on the hepcidin-induced expression of CXCL1 and TNF-α. In contrast, inhibitors of the NF-kB pathway (BAY 7082 and BAY 7085) strongly blunted the hepcidin-induced expression of these genes (Data not shown). Moreover, MyD88, a canonical adaptor for inflammatory signaling pathway, but not TLR4, was required for the hepcidin-triggered induction of CXCL1 and TNF-α. Indeed, macrophages isolated from TLR4−/− but not MyD88−/− mice were still able to mount an inflammatory response after addition of hepcidin.

(29) As disclosed in FIGS. 9 and 10, the stimulatory effect of hepcidin on macrophage-mediated cytokine release in clearly enhanced in serum-free conditions.

(30) Upon LPS stimulation, the intestine of the WT mice was largely inflamed with a high expression of iNOS (FIG. 2d), iNOS-derived NO acting as a signalling element essential for the initiation of systemic inflammation.sup.22. In sharp contrast to this LPS-induced inflammation of the WT mice intestinal mucosa, we observed a dramatic reduction of the inflammatory status of the Hepc.sup.Δint mice intestine with almost no immunostaining of iNOS.

(31) The effect of intestinal hepcidin deletion on systemic inflammation has been further evaluated by measuring blood cytokinic expression profile in WT and mutant mice. As expected, acute inflammation induced by ip LPS injection highly increased the production of proinflammatory cytokines in the plasma of WT mice, with the highest expression of IL-6 and CXCL-1 (KC/GRO). Strikingly, deletion of intestinal hepcidin largely blunted the production of IL-6, CXCL-1 (KC/GRO), IL-12p70, TNF, IFN-gamma, IL-1beta, IL-2, IL-5 and IL-4 (FIG. 3) and also largely prevented the production of the anti-inflammatory cytokine, IL-10. This dramatic result shows that intestinal hepcidin is critically required for triggering systemic inflammation upon LPS stimulation, its deficiency mimicking the phenotype of mice made deficient for the LPS receptor, TLR4.sup.23.

(32) Under physiological conditions, hepatic hepcidin is the predominant reservoir for systemic hepcidin.sup.8 and controls serum iron levels by regulating intestinal iron absorption and macrophage iron recycling. Hepatic hepcidin is induced by many inflammatory cytokines, such as IL-6.sup.25, TLR-agonists and pathogens.sup.26 and has therefore been proposed to be responsible for the hypoferremia of inflammation.sup.27. To determine to what extent the high production of hepcidin produced by the intestine after acute inflammation could contribute to the hypoferremia of inflammation, iron levels in both WT and mutant mice were measured. At basal level, Hepc.sup.Δint mice presented similar iron levels than WT littermates. However, upon inflammation, the decrease in plasma iron and transferrin saturation observed in WT mice was blunted in the Hepc.sup.Δint mice (FIG. 4a), suggesting that intestinal hepcidin is participating to the mechanisms leading to hypoferremia. The increase in hepatic hepcidin in WT mice prevented in the mutant mice was correlated to the level of inflammation (FIG. 4b). It has been proposed that hepcidin evolved from an antimicrobial peptide to an iron-regulatory hormone. However, the data presented herein suggest that hypoferremia of inflammation is triggered by the evolutionary conserved pleiotropic effects of hepcidin, as an AMP.

(33) Inflammatory cytokines, released in massive amounts in response to bacterial toxins such as LPS, are important biological mediators of sepsis, a major cause of mortality and morbidity. Importantly, we found that whereas challenge of WT mice with lethal doses of LPS provoked only 10% survival by 96 h, Hepc.sup.Δint mice experienced 58% survival (FIG. 4c).

(34) Although the results presented here were performed with classical TLR4 agonist LPS, hepcidin expression in the intestinal epithelium was likewise induced by zymosan, flagellin, and poly:IC, agonists of TLR2, TLR5, and TLR3 respectively, suggesting that hepcidin may be a convergence point in inflammatory response to a wide array of microbial triggers (FIG. 8).

(35) Clinical relevance of the inflammatory role of hepcidin in intestine was further assessed using the DSS-induced colitis model, mimicking some characteristics of human inflammatory bowel diseases. The expression of NOS2, IL-17A and TNF-α, key proinflammatory cytokines in the setting of human IBDs (Duma et al., 2011; Leppkes et al., Gastroenterology 136, 257-267, 2009; Neurath et al., Eur J Immunol 27, 1743-1750 1997), are stronly induced in the colon of DSS-induced WT mice, but was significantly dampened in Hepc.sup.Δint littermates compared to WT mice (FIG. 5C). Intestinal hepcidin may therefore have a protective role in IBDs by regulating cytokines, important for epithelial repair.

(36) Finally, the inventors have analyzed the hepcidin expression in the colonic mucosa of healthy subjects CD patients and UC patients. In healthy subjects, hepcidin immunoreactivity was concentrated apically in colonic epithelial cells. Its expression was increased and relocated to the basolateral surface and into the cytoplasm of the intestinal epithelium as well as in the lamina propria in CD and UC patients.

(37) Herein is disclosed a new model where hepcidin acts as an intestinal chemokine to trigger neutrophil infiltration and macrophage activation in the lamina propria and provoking a systemic inflammatory response (FIG. 7).

(38) The systemic inflammatory response is biologically complex and had led to the failure of hundreds clinical trial strategies to modify the systemic inflammatory response by targeting endogenous mediator molecules.sup.28. The gut is considered for many years as a cytokine-generating organ in systemic inflammation.sup.29. However, there is currently no unifying hypothesis that encompasses the diverse ways in which the gut influences outcome in critical illness.sup.30. Importantly, the results presented herein bring a new paradigm and show that the trigger of systemic inflammation depends on the induction of a single gut-derived molecule: hepcidin. It therefore opens new therapeutics avenues in the treatment of inflammatory diseases.

(39) Methods

(40) Mice

(41) Hamp1.sup.lox/lox mice.sup.8 were bred with villin-Cre transgenic mice.sup.9, in which the Cre recombinase is under the control of the murine villin promoter. Studies were performed in a mixed C57BL6/129SV genetic background, using male littermates.

(42) The animal studies described here were reviewed and approved (Agreement no CEEA34.CP.003.13) by the “Président du Comité d'Ethique pour l'Expérimentation Animale Paris Descartes” and are in accordance with the principles and guidelines established by the European Convention for the Protection of Laboratory Animals (Council of Europe, ETS 123, 1991).

(43) Mice were ip injected with LPS (from E. coli O111:B4; 2 mg/kg), Zymosan A (from Saccharomyces cerevisiae, Sigma-Aldrich Z4250; 250 mg/kg), Poly:IC (Polyinosinic-polycytidylic acid sodium salt, Sigma-Aldrich P0913; 12.5 mg/kg), Flagellin (FLA-ST from Salmonella Typhimirium, InvivoGen, #12B06-MM; 1.5 mg/kg) or with a sterile saline solution (PBS) and sacrificed 7 hours later.

(44) Fractionation of Murine Epithelial Cells

(45) The sequential isolation of mouse small intestinal epithelial cells along the crypt villus axis has been previously described and validated.sup.31-33.

(46) Briefly, mice were sacrificed by cervical dislocation. The duodenum was removed separately, tied off at one end, everted and filled to distension with phosphate buffered saline (PBS) prior to closing the remaining open end. It was then incubated with shaking at 37° C. for 10 min in 20 ml of citrate buffer (96 mMNaCl, 1.5 mMKCl, 27 mMNa-citrate, 8 mM KH2PO4, 5.6 mM Na2HPO4, and 1 mM dithiothreitol (DTT), pH 7.3). Duodenum was then transferred in an EDTA buffer (PBS containing 1.5 mM EDTA, and 0.5 mM DTT) to a 37° C. shaking incubator and dissociated epithelial cells were collected after each of 4 consecutive incubation steps lasting 10 min (fraction A), 10 min (fraction B), 30 min (fraction C), and 20 min (fraction D). Cells isolated in the resulting fractions were harvested by centrifugation at 1500 rpm at 4° C. for 5 min, snap frozen in liquid nitrogen, and stored at −80° C.

(47) Lamina Propria Cell Isolation

(48) Seven hours after PBS or LPS (2 μg/g) injection, mice were sacrificed and the small and large intestine were harvested. After Peyer's patches removal, the tissue was opened longitudinally and washed in Ca.sup.2+/Mg.sup.2+-free PBS containing 100 U/ml Penicillin, 100 μg/ml Streptomycin (GIBCO), 100 U/ml polymixin B (Sigma-Aldrich). Next, the tissue was cut into 1 cm pieces and incubated twice in Ca.sup.2+/Mg.sup.2+-PBS containing 2 mM EDTA, 2% FCS, and antibiotics for 20 min at 37° C., vigorously shaking. Tissues were washed for 10 min in Ca.sup.2+/Mg.sup.2+-PBS containing 0.01 M HEPES (GIBCO) and antibiotics, and then minced and incubated in RPMI 1640 containing 0.05 mg/ml Collagenase D (Roche), 0.05 mg/ml DNAse (Roche), and 0.025 mg/ml Hyaluronidase (Roche) for 20 min at 37° C., vigorously shaking. Tissues were then filtered through a 70 μm cell strainer, cells were counted and resuspended in cold Ca.sup.2+/Mg.sup.2+-PBS containing 2% FCS.

(49) Flow Cytometry

(50) The lamina propria cell suspension (4×10.sup.6 cells/well) was washed in Ca.sup.2+/Mg.sup.2+-PBS and incubated with LiveDead reagent (Blue fluorescent reactive dye, Invitrogen) for 20 min at room temperature. Prior to staining, Fc receptors were blocked with FcBlock™ (anti-CD16 and anti-CD32, 5 μg/ml, BD Pharmingen) and cells were further incubated 15 min at 4° C. with antibodies in 96-wells round bottom plates with agitation. Cells were either stained with biotinylated anti-Ly6C (myeloid analysis) or biotinylated anti-TCRβ (lymphoid analysis). After 2 washes in PBS 2% FCS, cells were stained with antibodies specific for CD45.2/CD45.1-PerCP-Cy5.5, CD11b-APC, CD11c-PE-Cy7, NK1.1-PE, streptavidin-Pac Blue and Ly6G-FITC (for myeloid analysis) or with antibodies anti-CD45.2-APC, CD4-Pacific Blue, CD8-APC-H7, streptavidin-PE-Cy7, CD19-FITC, NK1-PerCP-Cy5.5 and anti-TCRβ-PE (for lymphoid analysis). All antibodies were purchased from BD Pharmingen, except for the CD11b-APC from eBiosciences. Cells were washed, fixed in 1% paraformaldehyde and stored at 4° C. until further analysis. Cells were acquired using the multicolour flow cytometer Fortessa (BD) and analysed with FACSDIVA (BD).

(51) Reverse Transcription and Real-Time Quantitative PCR

(52) RNA extraction, reverse transcription, quantitative PCR and sequences of the primers used have been previously described.sup.8. All samples were normalized to the threshold cycle value for cyclophilin-A.

(53) Cytoplex Assay

(54) Cytokines in the plasma of mice were measured with the V-PLEX Proinflammatory Panel1 (mouse) Kit (Meso Scale Discovery), according to the manufacturer's instruction.

(55) Macrophages

(56) Primary Mouse Bone Marrow-Derived Neutrophils

(57) Bone marrows from WT mice were flushed from femurs and tibias with PBS using a 25 G needle. Tissues were homogenized through a 18 G needle and the suspension was passed through 70 μm and 40 μm cell strainers. Cells were centrifuged at 400 g for 10 min and resuspended in cold MACS buffer (PBS, BSA 0.5%, EDTA 2 mM, pH 7.4). After counting, cells were incubated on ice with FcBlock™ for 10 min, anti-Ly6C for 15 min and anti-biotin antibody microbeads for 15 min, following the manufacturer's instructions. Cells were washed with MACS buffer and centrifuged at 400 g for 10 min. Neutrophils were immuno-magnetically separated following filtration of the cell suspension through an LS column (Miltenyi) according to manufacturer's instructions.

(58) Migration Assay

(59) Neutrophil migration was assessed using transwell (3 μm; BD-Falcon) coated with 10 μg/ml fibronectin (# F1141, Sigma-Aldrich). The cells (5×10.sup.5/well) were diluted in 700 μL DMEM+10% SVF and migration towards hepcidin (Peptide International) in the bottom well was allowed for 2 hours at 37° C. The resulting migrated cells recovered from the bottom well were counted using hemocytometer.

(60) Immunostaining

(61) Tissues were fixed in 4% formaldehyde and embedded in paraffin. Sections were subjected to antigen retrieval in a pressure cooker for 15 min at 95° C., blocking endogenous peroxidases and immunohistochemistry with mouse anti-iNOS primary antibody (BD Biosciences ref. 610328) 1 h30 at room temperature. Bound primary antibody was detected by sequential incubation of the samples with goat anti-mouse biotinylated IgG's (Vector BA-9200) and streptavidin/horseradish peroxidase (VECTOR, Vectastain ABC Kit, PK-6100) for 30 min at room temperature. iNOS was revealed with DAB method (VECTOR, ImmPACT NovaRED SK4805). Images were captured with a light microscope using a 10× resolution objective (Leica DMI 3000 B).

(62) Iron Measurements

(63) Plasma iron was quantified colorimetrically by a previously described method.sup.34.

(64) 1-D Sequence Alignment Method

(65) Clustal W/X version 2.0.sup.35 has been used to carry out the 1-D sequence alignment. Clustal programs incorporate a position-specific scoring scheme and a weighting scheme for down weighting over-represented sequence groups. All pairs of sequences to be aligned are compared by pair-wise alignment and a score matrix of similarity has been produced, indicating the divergence or similarities of each pair. The “quality score” represents the main method of alignment and has been performed with default parameters.sup.36.

(66) 3-D Structural Alignment Method

(67) TM-align algorithm.sup.37 has been used to perform 3-D structural alignment between protein pairs. This method combines the TM-score rotation matrix.sup.38 and dynamic programming.sup.39. TM-align only employs the backbone C-alpha coordinates of the given protein structures. TM-align process combines two methods that are initial alignments and heuristic iterative algorithm. Initial alignments are exploited using dynamic programming and then heuristic iteration using TM-score rotation.

(68) Statistical Analysis

(69) Analysis was performed using GraphPad Prism 5.0 and the significance of experimental differences was evaluated by a t-test or one-way ANOVA analysis followed by a Bonferroni posttest.

REFERENCES

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