The interrelationship between hepcidin, vitamin d, and anemia in children with acute infectious disease

ABSTRACT BACKGROUND Hepcidin is a master regulator of iron metabolism. Recently, it has been shown that vitamin D suppresses hepcidin expression. Our hypothesis was that hepcidin levels

inversely correlate with vitamin D levels in anemic children during acute infection. METHODS A prospective study was performed on 90 patients (45 females, 45 males, mean age 7.3 ± 5 years)

who were admitted to the pediatric ward. Sixty-two patients had infectious disease (32 with coexisting anemia, 30 without anemia), and 28 patients were hospitalized for noninfectious causes.

Blood samples for IL-6, hepcidin, iron status parameters, and 25-hydroxyvitamin D (25-OHD) were obtained within 72 h after admission. RESULTS Serum concentrations of IL-6 and hepcidin were

significantly higher and 25-OHD, iron, and transferrin were significantly lower in anemic children with infectious disease compared with controls. Children with a serum 25-OHD level < 20 

ng/ml had significantly increased odds of having anemia than those with a level > 20 ng/ml (OR: 6.1, CI: 1.15–32.76). Correlation analyses found positive associations between hepcidin

levels and ferritin (_R_2 = 0.47, _P_ < 0.001) and negative associations between hepcidin and transferrin (_R_2 = 0.57, _P_ < 0.001). CONCLUSION Higher IL-6 and lower 25-OHD levels may

lead to higher hepcidin levels and subsequently to hypoferremia and anemia in children with acute infection. You have full access to this article via your institution. Download PDF SIMILAR

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2023 INTRODUCTION Anemia is known to occur in the setting of chronic infection as well as in inflammatory disease.1,2 Hepcidin, a master regulator of iron metabolism, is an important factor

in the development of anemia associated with inflammation.3,4,5,6 Hepcidin functions as a regulator of cellular iron export by decreasing the amount of ferroportin, a membrane protein that

is the major exporter of iron from cells, including macrophages that recycle iron, duodenal enterocytes that absorb dietary iron, and hepatocytes that store iron.5,7 During inflammatory

states or infection, hepcidin expression is upregulated by several proinflammatory cytokines, including interleukin-6 (IL-6) and IL-18,9 via both the BMP/SMAD and the JAK STAT3 inflammatory

signaling pathways.5,10 This upregulation of hepcidin limits the pool of extracellular iron by preventing iron release from the cells and its availability for erythropoiesis, causing

iron-restrictive anemia.5,7,8 Hepcidin was initially described as an antimicrobial protein because hepcidin-induced hypoferremia is thought to be a defense against bacterial infection by

withholding iron from invading pathogens.5 It has recently been demonstrated that vitamin D is a potent regulator of hepcidin in humans.11,12 Treatment of cultured hepatocytes or monocytes

with 25-OHD3 or 1,25(OH)2D3 decreased the expression of hepcidin mRNA by 0.5-fold. Promotor–receptor and chromatin immunoprecipitation analysis indicated that direct transcriptional

expression of hepcidin gene by 1,25(OH)2D3 caused a decrease in hepcidin mRNA levels. Furthermore, two pilot studies on healthy volunteers reported that supplementation with a single oral

high dose of vitamin D significantly reduced the circulating levels of hepcidin.11,13 Indeed, the association between vitamin D deficiency and anemia has been described in a number of

observational studies in both healthy and diseased populations.14,15,16,17 In the present study, we aimed to investigate the association between anemia associated with infection, hepcidin,

and vitamin D status in hospitalized pediatric patients, and to examine the role of hepcidin and vitamin D in the setting of anemia during acute inflammation in children. METHODS SUBJECTS We

prospectively recruited male and female children aged 1–16 years who were admitted to the ward at Dana-Dwek Children’s Hospital of the Tel Aviv Medical Center between 1 February, 2016, and

3 December, 2016. The study population consisted of three groups: (1) children with acute bacterial infections (i.e., osteomyelitis/septic arthritis, pyelonephritis, pneumonia, mastoiditis,

etc.) and coexisting anemia (defined as hemoglobin levels < 11 mg/dl upon admission),13,18 (2) children with acute infection without anemia, and (3) children hospitalized for elective

surgery who were free of infection and served as controls. All patients in the infectious groups had high fever and all needed hospitalization for treatment with IV antibiotics. Children who

were receiving medications known to alter vitamin D metabolism or who had a malabsorption, hepatic, or renal disease, as well as those known to have nutritional deficiencies, such as B12 or

folic acid, were excluded from the study. The study protocol was approved by the institutional review board of the medical center. A written informed consent was obtained from the parents

of all the participants. MEASUREMENTS Blood samples for the measurement of IL-6, hepcidin, iron, ferritin, transferrin, complete blood count (CBC), C-reactive protein (CRP), and 25-OHD were

obtained from all children within 72 h after admission. Sera were separated and frozen at –80°C until analysis. The 25-OHD concentration was measured by a radioimmunoassay commercial kit

(25-hydroxyvitamin D RIH kit, 68100E, DiaSorin, Via Crescento, Italy) which has a sensitivity of 1.5 ng/ml and a coefficient of variance of 8.1%. Vitamin D deficiency was defined as a 25-OHD

level < 20 ng/ml. IL-6 was measured using Human IL-6 ELISA Ready-SET-Go!® eBioscience, Thermo Fisher Scientific (Paisley, UK) that has a sensitivity of 2 pg/ml. Hepcidin was measured by

DRG Hepcidin 25 (bioactive) HS ELISA (Springfield, NJ) with an analytical sensitivity of 0.143 ng/ml, an intra-assay variability of 5.3%, and an inter-assay variability of 9.5%. Blood

chemistry and CBC were measured by standard laboratory methods. DATA ANALYSIS The statistical analyses were performed using Minitab version 16 (Minitab Inc, State College, PA). Descriptive

statistics were examined for all variables. Continuous variables were expressed as median with range when they were not normally distributed, and as mean ± standard deviation (SD) for

normally distributed variables. Continuous variables that did not follow a normal distribution were logarithmically transformed (serum ferritin and IL-6) for modeling. Categorical variables

were presented as number and percentage. Differences in demographic and biochemical variables between the three studied groups were examined using one-way ANOVA for continuous variables and

the _χ_2 or Fisher’s exact test for categorical variables. The Pearson correlation and simple linear-regression analysis were performed to examine bivariate associations of vitamin D and

hepcidin with biomarkers related to anemia and inflammation. A _P_ value ≤ 0.05 was considered significant. RESULTS Ninety patients (45 females and 45 males, mean age 7.3 ± 5 years) were

enrolled after their admission to the pediatric ward. Sixty-two patients had infectious disease, 32 with coexisting anemia and 30 without anemia. Twenty-eight patients were hospitalized for

noninfectious causes (elective surgery) and served as controls. Table 1 depicts the distribution of the acute infectious diseases between the study groups. There was no correlation between

specific diseases and the development of anemia, although a more severe disease was observed in children with both infection and anemia, as reflected by higher mean CRP and WBC levels (_P_ 

_<_ 0.01 and _P_ < 0.001, respectively, Fig. 1). Table 2 displays the laboratory data of the patients with infection and anemia, those with infection without anemia, and controls. The

hepcidin, ferritin, IL-6 levels, and platelet count were significantly higher, and serum iron parameters (serum iron and transferrin) were significantly lower in patients with infections and

anemia compared with the other two groups of patients. Six patients with infection and anemia, two patients with infection without anemia, and none in the control group had vitamin D

deficiency. The mean 25-OHD levels were significantly lower among the  infectious  and anemic patients, and the patients with a 25-OHD level < 20 ng/ml had significantly increased odds of

having anemia than those with a 25-OHD level > 20 ng/ml (odds ratio (OR): 6.1, confidence interval (CI): 1.15–32.76). However, there was no significant correlation between serum 25-OHD

levels and circulating levels of hepcidin. There was significant positive correlation between serum hepcidin and ferritin levels (_R_2=0.47, _P_ < 0.001), and a significant negative

correlation between hepcidin and transferrin levels (_R_2 = 0.57, _P_ < 0.001, Fig. 2). DISCUSSION The findings of the present study demonstrate that the IL-6 and hepcidin levels were

significantly higher, and that the iron and transferrin levels were significantly lower in children with acute infectious disease and coexisting anemia than in children with infectious

disease and no anemia as well as in the noninfection controls. These results suggest that hepcidin-induced hypoferremia might be involved in the pathogenesis of anemia of acute infection in

children. It is now widely accepted that hepcidin has a pivotal role in the pathogenesis of iron-deficiency anemia of chronic infection and inflammatory disease.3,4,5 Anemia is also a

recognized feature of acute, moderate, and severe infection in previously healthy children who were hospitalized for a variety of acute infections,18 and in children with acute infection

seen in pediatric outpatient clinics.17 Children with moderately severe acute infections reportedly experienced a mean hemoglobin drop of 1.8 mg/dl within less than 1 week of illness onset,

regardless of the specific cause of infection.18 Anemia was reversible after resolution of the infection without the need for iron supplementation in the majority of children.17 The rapid

effect of acute bacterial infection on the IL-6-hepcidin axis and the development of hypoferremia have been studied in both humans and in mice models. Kemna et al.19 examined the temporal

association and responses of plasma IL-6 levels, hepcidin levels, and serum iron parameters in 10 healthy volunteers following injections of lipopolysaccharides. IL-6 was dramatically

reduced within 6 h, followed by a significant decrease in serum iron concentration. Darton et al.20 also demonstrated a strong early hepcidin upregulation and hypoferremia following the

injection of _Salmonella typhi_ to 50 healthy volunteers. Studies with models of wild-type and hepcidin knockout mice challenged with sublethal doses of either lipopolysaccharides21 or

heat-killed _Brucella abortus_22 demonstrated that hepcidin and ferroportin are important but not the sole mediators of acute hypoferremia. Those authors showed that the development of acute

hypoferremia during infection relied on both hepcidin-dependent and hepcidin-independent mechanisms. Furthermore, in addition to hepcidin-induced restrictive hypoferremia, those mouse

models showed a multifactorial pathogenesis of inflammatory anemia, including suppression of erythropoiesis and shortened erythrocyte lifespan.22,23 The association between vitamin D

deficiency and anemia—particularly anemia of inflammation—has been previously described by observational and epidemiologic studies.13,14,15,16 There are several possible mechanisms that

could explain this association. One is that vitamin D has both direct and indirect suppressive effects on hepcidin expression.11,12 Another is that 1,25(OH)2D3 interacts directly with

vitamin D response elements on the promotor of the hepcidin gene in monocytes and hepatocytes and suppresses hepcidin mRNA transcription.11,12 Vitamin D also has an indirect effect on

hepcidin expression by the suppression of proinflammatory cytokines which stimulate hepcidin production during inflammation.12 In addition, 1,25(OH)2D3 has been shown to directly support

erythropoiesis by increasing burst-forming unit erythroid proliferation and to have a synergistic effect with erythropoietin to further enhance erythroid progenitor cell proliferation.23

Serum 25-OHD levels were significantly lower and hepcidin levels were significantly higher in children with an infectious disease and coexisting anemia than in children with an infectious

disease without anemia and in patients without any infection. A serum 25-OHD level < 20 ng/ml held significantly increased odds of anemia than a serum 25-OHD level > 20 ng/ml. However,

there was no significant correlation between serum 25-OHD levels and circulating levels of hepcidin. One possible explanation for this is that although the intracellular production of

1,25(OH)2D3 in monocytes and macrophages appears to be sensitive to the availability of substrate 25-OHD in the serum, the hepcidin–ferroportin axis is suppressed by the 1,25(OH)2D3 produced

by intracellular 25-OHD3-1 alpha hydroxylase, and is actually regulated by several different cytokines and Toll-like receptors.24 Each of these factors may influence intracellular

1,25(OH)2D3 production and, subsequently, hepcidin expression. Moreover, hepcidin synthesis and release from hepatocytes and macrophages into the circulation are controlled by at least eight

different proteins.25 Interestingly, Adams et al.26 who studied the direct effect of vitamin D on the expression of cathelicidin, another antibacterial protein, in monocytes found that

although 1,25(OH)2D3 directly stimulates cathelicidin expression, there was a lack of any correlation between serum 25-OHD levels and circulating levels of cathelicidin. Those authors

emphasized the importance of a local intracrine mechanism regulating 1,25(OH)2D3 and cathelicidin production. In summary, the findings of the present study indicate that the

hepcidin–ferroportin axis is involved in the pathogenesis of hypoferremia and iron-restrictive anemia in children with acute infectious diseases, and suggests that a low vitamin D status may

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AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Pediatrics, Dana Dwek Children Hospital and The Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Hadar

Moran-Lev, Yosef Weisman & Ronit Lubetzky * Department of Pediatric Gastroenterology, Dana Dwek Children Hospital and The Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv,

Israel Hadar Moran-Lev & Shlomi Cohen * Department of Hematology Laboratories, Tel Aviv Sourasky Medical Center and The Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

Varda Deutsch & Michal Cipok * Preclinical Research Pulmonary Laboratory, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel Ekaterina Bondar * Department of Neonatology, Dana Dwek

Children Hospital and The Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Dror Mandel Authors * Hadar Moran-Lev View author publications You can also search for this

author inPubMed Google Scholar * Yosef Weisman View author publications You can also search for this author inPubMed Google Scholar * Shlomi Cohen View author publications You can also

search for this author inPubMed Google Scholar * Varda Deutsch View author publications You can also search for this author inPubMed Google Scholar * Michal Cipok View author publications

You can also search for this author inPubMed Google Scholar * Ekaterina Bondar View author publications You can also search for this author inPubMed Google Scholar * Ronit Lubetzky View

author publications You can also search for this author inPubMed Google Scholar * Dror Mandel View author publications You can also search for this author inPubMed Google Scholar

CORRESPONDING AUTHOR Correspondence to Hadar Moran-Lev. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER'S NOTE:

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THIS ARTICLE Moran-Lev, H., Weisman, Y., Cohen, S. _et al._ The interrelationship between hepcidin, vitamin D, and anemia in children with acute infectious disease. _Pediatr Res_ 84, 62–65

(2018). https://doi.org/10.1038/s41390-018-0005-0 Download citation * Received: 27 September 2017 * Revised: 17 January 2018 * Accepted: 21 January 2018 * Published: 23 May 2018 * Issue

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