Hepcidin is a key mediator of anemia of inflammation in Crohn's disease

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1 Journal of Crohn's and Colitis (203) 7, e286 e29 Available online at Hepcidin is a key mediator of anemia of inflammation in Crohn's disease Robert J. Basseri a, Elizabeta Nemeth b, Maria E. Vassilaki c, Benjamin Basseri a, Pedram Enayati a, Omid Shaye a, Leonidas A. Bourikas d, Tom Ganz b, Konstantinos A. Papadakis d, a Department of Gastroenterology, Cedars-Sinai Medical Center, Los Angeles, CA, USA b Department of Pathology and Laboratory Medicine, UCLA School of Medicine, Los Angeles, CA, USA c Department of Social Medicine, University of Crete, Faculty of Medicine, Heraklion, Crete, Greece d Department of Gastroenterology, University of Crete, Faculty of Medicine, Heraklion, Crete, Greece Received 23 December 20; received in revised form 9 October 202; accepted 25 October 202 KEYWORDS Anemia; Crohn's disease; Anemia of chronic disease; Hepcidin Abstract Anemia often complicates the course of Inflammatory Bowel Disease (IBD). Hepcidin, a liver-produced peptide hormone, is a key mediator of anemia of chronic disease (ACD). We hypothesized that hepcidin is significantly elevated in anemic CD patients and that hepcidin may cause iron restriction and, therefore, mediate ACD. Methods: We enrolled 7 patients with CD and ACD recruited from the Cedars-Sinai IBD Center. Routine blood tests included hemoglobin (Hgb), hematocrit, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP). Anemia was defined as hemoglobin b2 g/dl and b3.5 g/dl, in men and women, respectively. ACD was diagnosed on the basis of a combination of the following: a) normal or elevated ferritin b) lowered serum iron and total iron binding capacity and c) normal percent iron saturation. Serum and urine hepcidin, as well as IL-6 levels were also measured. Patients with documented iron-deficiency anemia were excluded. Results: There was an excellent correlation between urine (expressed as ng/mg of creatinine) and serum hepcidin levels expressed as ng/ml (r=0.853, pb0.00). We also found a strong positive correlation between serum hepcidin and ferritin levels (r=0.723, p=0.005). There was a positive correlation between serum hepcidin and IL-6 levels (r =0.546, p =0.023). We found a strong negative correlation between serum hepcidin concentrations and Hgb levels (r= 0.528, p= 0.029). Corresponding author at: Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA. address: Papadakis.konstantinos@mayo.edu (K.A. Papadakis) /$ - see front matter 202 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.

2 Hepcidin is a key mediator of anemia of inflammation in Crohn's disease e287 Conclusion: We demonstrate that ACD in CD is characterized by high serum IL-6 and hepcidin levels, which negatively correlate with Hgb levels. Our data support the hypothesis that IL-6-driven hepcidin production mediates ACD in patients with CD. 202 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.. Introduction Crohn's disease (CD) and ulcerative colitis (UC) are chronic gastrointestinal diseases characterized by relapsing and remitting inflammation of the intestines. Interestingly, anemia may often complicate the course of IBD. 2,3 A recent systematic review showed that anemia is common and may affect roughly 7% of patients with IBD. 4 Nevertheless, the awareness of anemia in IBD has received little attention and is often overlooked by treating gastroenterologists. 5 Anemia in IBD, the pathogenesis of which is multi-factorial, is frequently the result of chronic intestinal blood loss from inflamed gastrointestinal mucosa and iron-deficiency, but inflammation may also contribute to the development of anemia. 6 This is especially true for CD, where impaired intestinal absorption of folate and/or vitamin B2 occur secondary to inflamed mucosa and/or bowel resection. Moreover, anemia may result from IBD therapies, such as sulfasalazine, which may confound the problem. 7 Anemia of inflammation or anemia of chronic disease (ACD) is characterized by normochromic, normocytic or mildly microcytic erythrocytes, low or normal serum iron and total iron-binding capacity, normal or increased iron stores reflected by elevated ferritin levels, low transferrin levels, and an inappropriately low reticulocyte response relative to the degree of anemia Table ). ACD is unresponsive to treatment with iron, vitamin B2 or folic acid. Anemia in IBD could be the effect of cytokines on cells of the reticuloendothelial system inducing changes in iron homeostasis, the proliferation of erythroid progenitor cells, as well as the production of erythropoietin, and reducing the longevity of red blood cells. 8 Several in vitro and in vivo studies demonstrate that inflammatory cytokines, such as interleukin (IL)-β, TNF-α and IFN-γ, may suppress erythropoiesis, impair the availability of iron from reticuloendothelial stores, and impair erythropoietin production in response to anemia. 9 A recent study indicated that children with active CD have impaired oral iron absorption and elevated IL-6 levels compared with subjects with inactive disease suggesting that oral iron may be of limited benefit to these patients. Hepcidin, a key regulator of iron metabolism, is expressed in the liver, distributed in blood, and excreted in urine. Hepcidin is a key mediator of ACD. 2 The synthesis of hepcidin is greatly stimulated by inflammation or by iron overload. Hepcidin appears to be the main inhibitor of iron absorption from the small bowel (SB) and iron release from macrophages. 2,3 We hypothesized that hepcidin is significantly elevated in anemic adult CD patients not suffering from iron deficiency, and that hepcidin may cause iron restriction and, therefore, mediate ACD. We aimed to assess serum and urine hepcidin concentrations in patients with CD and ACD and to correlate hepcidin levels with hematologic parameters and markers of inflammation. 2. Methods 2.. Study patients Subjects were recruited from the inpatient and outpatient services of the Cedars-Sinai IBD Center. Patients were eligible to participate in the study if they were between 8 and 65 years of age, had a diagnosis of CD and concomitant ACD. Subjects were excluded if there was a known history of iron deficiency anemia (IDA), 4 UC, or macrocytic anemia. Furthermore, patients who used iron supplementation, B2 or folate within 3 months of study enrollment were also excluded. The study was approved by the Cedars-Sinai Institutional Review Board Study design Subjects who met inclusion criteria underwent laboratory evaluation of ACD, 4 and subsequently underwent routine blood tests including complete blood cell count, iron studies, ferritin, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP). Serum and urine were also obtained for measurement of hepcidin levels as described below. Table Characteristics of the CD patient population. Number 7 (%) Median age (y), (range) 35, (20 67) M:F 8:9 Disease duration (y), median, (range) 5, ( 30) Disease phenotype a Inflammatory 5 (88) FS 3 (7) IP 7 (4) Disease extent Small bowel 9 (53) Ileocolonic 4 (23.5) Colonic 4 (23.5) Prior surgeries 8 (47) Medical therapy 5-ASA 8 (47) Corticosteroids 5 (29) Thiopurines 7 (4) Anti-TNF 5 (29) a FS = fibrostenosing, IP = internal penetrating; 2 patients had both inflammatory and FS phenotype, 5 patients had both inflammatory and IP phenotype and patient had both FS and IP phenotypes.

3 e288 R.J. Basseri et al Data analysis Serum IL-6 measurement Serum IL-6 levels in the CD study population were measured using a human IL-6 ELISA assay (Bio Source International, Camarillo, California, USA) according to the manufacturer's recommendations Urinary hepcidin assay Urinary creatinine concentrations were measured by the UCLA clinical laboratories as previously described. 5 Briefly, cationic peptides were extracted from urine using CM Macro-prep (Bio-Rad Laboratories Inc.), eluted with 5% acetic acid, lyophilized, and suspended in 0.0% acetic acid. Hepcidin urinary concentrations were determined by immunodot assay. Urine extracts equivalent to mg of creatinine were dotted on Immobilon-P membrane (Millipore Corp., Bedford, Massachusetts, USA), along with a range of synthetic hepcidin standards (0 40 ng). Hepcidin was detected on the blots using rabbit anti-human hepcidin Abs' with goat anti-rabbit HRP as a secondary Ab. Dot blots were developed by the chemiluminescent detection method (SuperSignal West Pico Chemiluminescent Substrate; Pierce Chemical Co., Rockford, Illinois, USA) and quantified with the Chemidoc cooled camera running Quantity One software (Bio-Rad Laboratories Inc.). Hepcidin quantity in each sample was normalized using urinary creatinine concentrations measured in UCLA Clinical Laboratories, and urinary hepcidin levels were expressed as ng hepcidin/mg creatinine. history of prior surgery. The disease characteristics of the CD patient population are shown in Table Correlation of urinary and serum hepcidin levels We collected serum and urine concomitantly from our patient cohort and measured hepcidin levels in both the serum and the urine as described in materials and methods. We initially tested whether the two assays used to measure hepcidin (urine and serum) could correlate. As demonstrated in Fig. A, there was an excellent correlation between urine (expressed as ng/mg of creatinine) and serum hepcidin levels expressed as ng/ml (r 2 =0.853, pb0.00). We also illustrate a strong positive correlation between serum hepcidin and ferritin levels (r 2 =0.723, p=0.005) (Fig. B) Correlation of serum hepcidin levels with markers of inflammation and hemoglobin IL-6, but not TNF-α, induces hepcidin production by hepatocytes in vitro and in vivo. 5 It has been previously reported that hepcidin levels correlate with inflammatory markers such as IL-6 and CRP in a cohort of pediatric patients with CD. We, therefore, tested whether hepcidin serum levels correlated A Serum hepcidin assay Serum hepcidin levels were measured using a commercially available ELISA (Intrinsic Life Sciences LLC, La Jolla, CA) according to the manufacturer's recommendations. The development and validation of this first serum ELISA for hepcidin, which previously posed great difficulty, was recently reported in detail. 6 0 r=0.853 p< Statistical analysis Correlations between serum cytokine levels of IL-6 and urine/serum hepcidin, as well as hematologic parameters, were performed. Statistical analysis was performed using a linear regression and Pearson Product Moment Correlation on normal or log-transformed data. Data were reported as mean±standard error with statistical significance defined as pb Results 3.. Patient demographics Seventeen CD subjects (9 females and 8 males) with ACD were included in the study. The median age of the CD subjects was 35 years (range, 20 to 67 years). The median disease duration was 5 years (range, to 30 years). Most patients (88%) had an inflammatory disease phenotype, 53% of the patients had small bowel involvement and 47% had a B urinary hepcidin (ng/mg creat) 0 r=0.723 p= ferritin (ng/ml) Figure Correlation between serum and urine hepcidin levels (A) and serum hepcidin with ferritin levels (B) in the CD patient cohort.

4 Hepcidin is a key mediator of anemia of inflammation in Crohn's disease e289 with serum IL-6 levels in our adult patient cohort with CD, which we used as a surrogate marker for disease activity and CRP levels. 7,8 As shown in Fig. 2A, there was a positive correlation between serum hepcidin and IL-6 levels (r=0.546, p=0.023). Since hepcidin has been associated with ACD, we further tested whether hepcidin correlated with hemoglobin levels in patients with CD. We found a strong negative correlation between serum hepcidin concentrations and hemoglobin as shown in Fig. 2B (r=0.528, p=0.029). Furthermore, our data clearly indicate that serum hepcidin levels correlate positively with markers of inflammation (such as IL-6) and, negatively with hemoglobin levels. 4. Discussion In this study, we show that serum and urine hepcidin levels show an excellent positive correlation in adult CD patients with ACD. Serum hepcidin levels positively correlated with serum ferritin and IL-6 and negatively correlated with hemoglobin levels. These data suggest that hepcidin is an important mediator of ACD in adult CD. Our data are in agreement with the study by Semrin et. al. who showed similar findings in a pediatric cohort of CD patients. A A B r=0.546 p= IL-6 (pg/ml) r = p= Hgb (mg/dl) Figure 2 Correlation between serum hepcidin and IL-6 levels (A) and serum hepcidin with hemoglobin (B) in the CD patient cohort. recent study by Oustamanolakis et al. has also reported higher serum levels of hepcidin in both patients with ulcerative colitis (UC) and CD and a good correlation with ferritin levels and disease activity for UC. 9 The study by Nagy et al. found no significant differences in prohepcidin levels between UC and CD patients compared to healthy controls. 20 The study by Arnold et al. showed low serum hepcidin levels in patients with IBD with or without irondeficiency anemia but a significant positive correlation was present between hepcidin and IL-6 levels. 2 The discrepancies between serum hepcidin levels in patients with IBD reported in these studies may be partly related to the method used to determine hepcidin serum levels. The serum assay we used in the current study has been previously developed and validated as the first serum enzyme-linked immunosorbent assay for hepcidin, that has been very difficult to measure in the past. 6 In addition, we provide excellent correlation of the serum and urine hepcidin levels in our CD patient cohort as previously shown by Ganz et al. 6 The strongest negative and positive correlations that we observed in our study with hepcidin serum levels and hemoglobin and ferritin levels, respectively, compared to the ones reported in the study by Oustamanolakis et al., 9 could be related to the fact that we excluded patients with IDA and UC and studied hepcidin levels in CD patients in the context of anemia of inflammation. Indeed, serum hepcidin concentrations were shown to be undetectable or low in patients with IDA (ferritin b ng/ml) and high in patients with inflammation or chronic kidney disease. 6 Hepcidin was independently discovered by Park et al. 22 and Krause et al. 23 from the urine and blood, respectively. The predominant form of hepcidin in human urine contains 25 amino acids (hepcidin-25), two peptides shorter at the amino terminus were also found, hepcidin-22 and hepcidin-20. Subsequent studies have demonstrated that hepcidin is produced in the liver in response to infection or inflammation, and plays a significant role in iron metabolism. 2,5,24 Thus, overproduction of hepcidin during infection and inflammation may be responsible for ACD in chronic infectious or inflammatory disorders. Recent literature indicates that IL-6 is the necessary and sufficient cytokine that induces hepcidin production by hepatocytes. 5 Indeed,wefoundapositivecorrelationbetween hepcidin and IL-6 levels in our CD patient cohort with ACD (Fig. 2A). Hepcidin, in turn, acts as a negative regulator of intestinal iron absorption and macrophage iron release. 24 A schematic presentation of the pathophysiology of ACD is shown in Fig. 3. Evidence from transgenic mouse models indicates that hepcidin is the predominant negative regulator of iron absorption in the small intestine, iron transport across the placenta, and iron release from macrophages. The key role of hepcidin is confirmed by the presence of non-sense mutations in the hepcidin gene, homozygous in the affected members, in 2 families with severe juvenile hemochromatosis. 25 Moreover, hepcidin production is increased up to 0-fold in ACD, and this may account for the defining feature of this condition the sequestrationofironinmacrophages. 2 A general correlation exists between IBD activity and the degree of the anemia. 26 Previous studies have indicated that anemia in chronic diseases (i.e. renal failure) can improve with recombinant erythropoietin, 27 meanwhile several studies have reported encouraging results regarding the efficacy

5 e290 R.J. Basseri et al. Spleen Liver hepcidi n hepcidin Fpn hepcidi n Fpn Plasma Fe-Tf RBC Fpn Duodenum Bone marrow Figure 3 Proposed mechanisms of hepcidin-mediated anemia of inflammation in CD. IL-6 produced during inflammation induces hepcidin production from the liver. The hepcidin-ferroportin (Fpn) axis is the principal regulator of extracellular iron homeostasis. of erythropoietin in treating anemia in IBD patients. 28,29 However, treatment with iron supplementation and control of the underlying inflammation appears to be the cornerstone therapeutic approach for IBD patients with anemia. More specifically, treating Gastroenterologists should not overlook anemia in their IBD population and should further dissect the etiology of anemia as ACD and iron deficiency merit different therapeutic approaches. While iron replacement may be beneficial in chronic blood loss and iron deficiency, stringent treatment of the underlying CD should, theoretically, treat anemia of chronic disease. In conclusion, in this prospective study we suggest that ACD in CD is mediated by hepcidin, as exemplified by the elevated serum and urine levels of hepcidin, its positive correlation with IL-6 levels and its negative correlation with Hgb levels. This finding of elevated serum and urine hepcidin may be an important addition in defining the etiology of anemia in IBD. Moreover, serum or urine hepcidin may be used as a simple method in monitoring response to treatment as it relates to anemia in patients with ACD. Conflict of interest The authors have no conflict of interest related to the present manuscript. References. Podolsky DK. Inflammatory bowel disease. N Engl J Med 2002;347(6): Gasche C. Anemia in IBD: the overlooked villain. Inflamm Bowel Dis 2000;6(2):42 50 [discussion 5]. 3. Kulnigg S, Gasche C. Systematic review: managing anaemia in Crohn's disease. Aliment Pharmacol Ther 2006;24( 2): de la Morena F, Gisbert JP. Anemia and inflammatory bowel disease. Rev Esp Enferm Dig 2008;0(5): Gasche C, Lomer MC, Cavill I, Weiss G. Iron, anaemia, and inflammatory bowel diseases. Gut 2004;53(8): de Silva AD, Mylonaki M, Rampton DS. Oral iron therapy in inflammatory bowel disease: usage, tolerance, and efficacy. Inflamm Bowel Dis 2003;9(5): Gisbert JP, Gomollon F. Common misconceptions in the diagnosis and management of anemia in inflammatory bowel disease. Am J Gastroenterol 2008;3(5): Gasche C, Reinisch W, Lochs H, Parsaei B, Bakos S, Wyatt J, et al. Anemia in Crohn's disease. Importance of inadequate erythropoietin production and iron deficiency. Dig Dis Sci 994;39(9): Morceau F, Dicato M, Diederich M. Pro-inflammatory cytokine-mediated anemia: regarding molecular mechanisms of erythropoiesis. Mediators Inflamm 2009;2009: Semrin G, Fishman DS, Bousvaros A, Zholudev A, Saunders AC, Correia CE, et al. Impaired intestinal iron absorption in Crohn's disease correlates with disease activity and markers of inflammation. Inflamm Bowel Dis 2006;2(2): 6.. Tomosugi N, Kawabata H, Wakatabe R, Higuchi M, Yamaya H, Umehara H, et al. Detection of serum hepcidin in renal failure and inflammation by using ProteinChip System. Blood 2006;8(4): Ganz T. Hepcidin, a key regulator of iron metabolism and mediator of anemia of inflammation. Blood 2003;2(3): Ganz T, Nemeth E. Hepcidin and disorders of iron metabolism. Annu Rev Med 20;62: Weiss G, Goodnough LT. Anemia of chronic disease. N Engl J Med 2005;352(): Nemeth E, Rivera S, Gabayan V, Keller C, Taudorf S, Pedersen BK, et al. IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin. J Clin Invest 2004;3(9): Ganz T, Olbina G, Girelli D, Nemeth E, Westerman M. Immunoassay for human serum hepcidin. Blood 2008;2(): af Bjorkesten CG, Nieminen U, Turunen U, Arkkila P, Sipponen T, Farkkila M. Surrogate markers and clinical indices, alone or combined, as indicators for endoscopic remission in anti- TNF-treated luminal Crohn's disease. Scand J Gastroenterol 202;47(5):

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