Iron is a trace element important for the proper folding and function of various proteins. Physiological regulation of iron stores is of critical importance for RBC production and antimicrobial defense. Hepcidin is a key regulator of iron levels within the body. Under conditions of iron deficiency, hepcidin expression is reduced to promote increased iron uptake from the diet and release from cells, whereas during conditions of iron excess, induction of hepcidin restricts iron uptake and movement within the body. The cytokine IL-6 is well established as an important inducer of hepcidin. The presence of this cytokine during inflammatory states can induce hepcidin production, iron deficiency, and anemia. In this study, we show that IL-22 also influences hepcidin production in vivo. Injection of mice with exogenous mouse IgG1 Fc fused to the N terminus of mouse IL-22 (Fc–IL-22), an IL-22R agonist with prolonged and enhanced functional potency, induced hepcidin production, with a subsequent decrease in circulating serum iron and hemoglobin levels and a concomitant increase in iron accumulation within the spleen. This response was independent of IL-6 and was attenuated in the absence of the IL-22R–associated signaling kinase, Tyk2. Ab-mediated blockade of hepcidin partially reversed the effects on iron biology caused by IL-22R stimulation. Taken together, these data suggest that exogenous IL-22 regulates hepcidin production to physiologically influence iron usage.

Iron is an essential biological element; however, as a result of the potential toxicity associated with iron excess, iron uptake from the diet and movement within the body are tightly regulated. The acute-phase protein hepcidin is produced primarily by the liver and is the central regulator of iron homeostasis (13). Hepcidin binds to the iron-transport protein, ferroportin, causing its internalization and degradation (4). Loss of ferroportin decreases the absorption of dietary iron by enterocytes and the release of iron from intracellular stores. Thus, elevated hepcidin causes reduced iron uptake from the diet and decreased iron release from tissues, resulting in decreased serum iron availability and, thus, relative iron deficiency. Because iron is an essential nutrient for cellular and microbial proliferation, hepcidin-mediated iron sequestration in response to inflammatory cytokines may contribute to the reduction in microbial proliferation in vivo. IL-6 was reported to be the primary inducer of hepcidin in inflammation (57), although IL-1 (8), oncostatin M (9), and bone morphogenic proteins may also induce hepcidin expression (10, 11).

Recently, IL-22 was shown to induce hepcidin in a human hepatocyte cell line (12). IL-22 is a member of the IL-10–related family of cytokines and is predominantly produced by T cells and NK cells (13, 14). The IL-22R complex is a heterodimer consisting of IL-22R1 and IL-10R2 (15-17). Although IL-10R2 is ubiquitously expressed, IL-22R1 expression is largely restricted to nonhematopoietic cells, including epithelial cells, keratinocytes, and hepatocytes (18, 19). IL-22R signaling activates Jak1 and Tyk2 and induces phosphorylation of STAT1, STAT3, and STAT5, as well as several members of the MAPK and Akt pathways (14, 20).

IL-22 expression is altered in multiple chronic inflammatory diseases, including inflammatory bowel disease (21, 22) and psoriasis (23). IL-22 is a multifunctional cytokine that has both protective and inflammatory properties, depending upon the disease context. For example, IL-22 is protective in mouse models of inflammatory bowel disease (24, 25) and liver necrosis (26, 27); however, it contributes to the pathogenesis of skin inflammation (28, 29). IL-22 was also identified as a strong regulator of the innate immune response. In this capacity, IL-22 induces the production of antimicrobial proteins, including β-defensins and several members of the S100 protein family, and promotes barrier function and wound healing by enhancing epithelial homeostasis and keratinocyte proliferation and mobility (18, 30). IL-22 stimulates the production of acute-phase proteins by the liver, including serum amyloid A (SAA) and haptoglobin (15). Additionally, adenoviral delivery of IL-22 was shown to modulate both RBC count and hematocrit in mice (31).

In the current study, significant decreases in circulating iron were observed while assessing the in vivo consequences of treatment with exogenous rIL-22 or mouse (m)IgG1 Fc fused to the N terminus of mIL-22 (Fc–IL-22). These effects were associated with the induction of hepcidin by IL-22. Collectively, these data identify IL-22 as a cytokine involved in regulating hepcidin expression and iron homeostasis.

Female B10.Q/Ai (B10) and Tyk2 naturally deficient (B10.D1-2 < q > -Tyk2 < E775 > /J) mice were obtained from Amgen breeding colonies housed at Taconic Laboratories (Germantown, NY). The Tyk2-deficient mice can be obtained from The Jackson Laboratory (Bar Harbor, ME) as B10.D1-H2q/SgJ. Female FcRγ-chain–deficient (B6.129P2-Fcer1gtm1Rav N12) mice were purchased from Taconic Laboratories. Female IL-6–deficient (B6.129S6-Il6tm1Kopf) mice were purchased from The Jackson Laboratory. Female C57BL/6 mice were purchased from either Taconic Laboratories or The Jackson Laboratory. Mice were fed standard rodent chow and water ad libitum and maintained under specific pathogen–free conditions, according to standard animal husbandry procedures. All experimental protocols were approved by the Amgen Institutional Animal Care and Use Committee. Mice were used between 8 and 12 wk of age.

Recombinant murine (rm)IL-22, recombinant human (rh)IL-22, and rhIL-6 were expressed in Escherichia coli and purified using standard laboratory techniques at Amgen. An isotype-control mIgG1 Ab, clone 4D2, was produced and purified at Amgen. Mouse IgG1 Fc fused to the C terminus of m4-1BB (4-1BB-Fc) was produced and purified at Amgen.

Two separate IL-22–Fc fusion constructs were generated. Fc–IL-22 and mouse IgG1 Fc fused to the C terminus of mIL-22 (IL-22–Fc) were constructed by PCR overlap extension using a DNA template encoding the mFc sequence (gb: U65534.1) and mIL-22 (gb: NM016971.2). A 5-aa linker (GGGGS) was inserted between mFc and mIL-22. The following primers (Integrated DNA Technologies, Coralville, IA) were used for the PCR overlap extension: 5′- GGCGCGCCTGTAAGCCTTGCATATG-3′, 5′-GGCTTCCACCGCCTCCTTTACCAGGAGAGTGGG-3′, 5′-GGAGGCGGTGGAAGCCAGGAGGCAAATGCGCTGCCCG-3′, and 5′-GCGGCCGCTCAGACGCAAGCATTTCTC-3′.

Fc–IL-22 and IL-22–Fc cassettes were cloned into an Amgen proprietary vector downstream of a modified signal sequence for human Ig VH5-a. The proteins were expressed in HEK-293 cells and purified using a MabSelect SuRe protein A affinity column (GE Healthcare Biosciences, Piscataway, NJ). The molecular mass of the IL-22–Fc dimer proteins is 84.99 kDa.

A cohort of 10 Hep2 mice [a mouse strain in which mouse Hamp1 and Hamp2 genes are replaced with human Hamp (32)] was immunized with a conjugate of mouse hepcidin 1 (DTNFPICIFCCKCCNNSQCGICCKT), human Fc, and a Th cell–stimulation peptide following a conventional immunization schedule, as described below. Day 1: Subcutaneous injection of 25 μg the conjugate in CFA (Pierce/Thermo Scientific, Rockford, IL). Day 14: Subcutaneous and i.p. injection of 15 μg the conjugate in Ribi Adjuvant (Sigma-Aldrich, St. Louis, MO). Day 28: Subcutaneous and i.p. injection of 7.5 μg the conjugate in Ribi Adjuvant. Blood samples were collected on day 38, and mice producing anti-mouse hepcidin Abs were identified. Candidate mice were boosted i.p. with 50 μg the conjugate; 4 d later, the spleens were removed, hybridomas were generated, and Abs were purified from hybridoma supernatants using standard laboratory techniques. Ab 2C10, an mIgG1 that binds to mouse hepcidin 1 with ∼0.5 nM affinity (ELISA, data not shown) was used for all studies. Strategies to determine the mouse hepcidin 1 epitope recognized by Ab 2C10 were unsuccessful, because 2C10 does not recognize truncated versions of mouse hepcidin 1. Ab 2C10 reacts very weakly with human hepcidin; however, this binding was not quantified. The interaction between Ab 2C10 and mouse hepcidin 2 binding was not evaluated, because it was not possible to generate sufficient correctly folded synthetic mouse hepcidin 2 peptide. The hepcidin-neutralizing ability of 2C10 was established, as described previously (32) (Supplemental Fig. 3).

Induction of SAA.

Mice treated with rmIL-22 or the Fc–IL-22 constructs were injected i.p. with 25 μg the indicated reagent and harvested at the indicated time point.

Induction of hepcidin and mRNA analysis.

Mice treated with mouse serum albumin (MSA) control protein (Sigma-Aldrich), rhIL-6, or rmIL-22 were injected i.p. with 50 μg the indicated reagent and harvested at the indicated time point.

Iron-deficiency studies.

Mice treated with MSA, rhIL-6, or rmIL-22 were injected i.p. twice daily with 25 μg the indicated reagent at a 7-h interval, for a total dose of 50 μg MSA or cytokine/d, up to the final day of injection. Mice were injected with a single dose of 50 μg the indicated reagent on the final day of injection. Mice treated with mIgG1 control Ab 4D2 or Fc–IL-22 were injected i.p. with 150–200 μg the indicated reagent for the indicated number of days, as described in the figure legends.

Anti-hepcidin inhibitor studies.

Mice treated with mIgG1 control Ab 4D2 or anti-hepcidin Ab, 2C10 (Amgen), were injected i.p. with 2.5 mg on the indicated days, in addition to injections of rIL-22 or Fc–IL-22, as described above. All reagents were tested for endotoxin activity (≤0.5 EU/mg) and resuspended in endotoxin-free PBS prior to use.

Harvest dates are indicated for individual experiments. Unless otherwise indicated, mice were euthanized 4–5 h after the final injection. Whole blood was obtained by intracardiac puncture, with the collected volume split between EDTA tubes for blood cell parameter analysis and serum-collection tubes (both from BD Biosciences, San Jose, CA) for serum analysis. Spleens or livers were removed and prepared for histopathology analysis or RNA isolation from select studies.

Spleens were fixed for 18–24 h in 10% neutral-buffered formalin and transferred to 70% ethanol. Tissues were then processed, embedded, sectioned at 4–6 μm, and stained for ferric iron with Prussian Blue Stain (Perls’ iron). To calculate the percentage of iron content, a digital image of Perls’ iron–stained spleens was generated using a Hamamatsu NanoZoomer 2.0 HT Slide Scanner (Hamamatsu Photonics K.K, Hamamatsu City, Japan). Spleen images were analyzed, and iron content as a percentage of total splenic area was calculated using the Visiomorph Image Analysis Software system (Visiopharm, Hoersholm, Denmark).

Blood cell parameters were determined using a Bayer Advia 120 hematology analyzer (Bayer Instruments, Tarrytown, NY). Serum iron concentrations were determined using an Olympus AU400e clinical chemistry analyzer with Olympus Iron Reagent (Olympus Diagnostics, Melville, NY).

Serum hepcidin concentrations were determined, as previously described (33), using solid-phase extraction, followed by liquid chromatography–tandem mass spectrometry analysis interfaced with a Sciex API 4000 mass spectrometer for detection. Data represent a total of full-length mature hepcidin M25 and hepcidin M24, a partial degradation product of hepcidin, and are reported as hepcidin M25 + M24. Values below limit of quantitation are reported as 0.

Serum concentrations of SAA were determined using a commercially available SAA ELISA kit (Invitrogen, Carlsbad CA), according to the manufacturer’s instructions. Absorbance was measured at 450 nm on a Thermo Max plate reader (Molecular Devices, Sunnyvale, CA). Concentrations were determined by interpolation of the standard curve using Delta Soft Analysis Software, version 2.22 (E. Becktold and BioMetallics, Princeton, NJ).

Human hepatoma HepG2/C3a cells (American Type Culture Collection, Manassas, VA) were maintained in DMEM/10% FBS supplemented with 1× l-glutamine and 1× nonessential amino acids. Cells were serum starved overnight in DMEM/0.5% FBS prior to analysis. The following day, cells were stimulated with DMEM/0.5% FBS containing 1 μg/ml the indicated reagent for 30 min at 37°C. Cells were fixed in 2% paraformaldehyde/PBS and then permeabilized in 90% methanol. Cells were stained for 1 h with anti–phospho-STAT3 (Y705) mAb and analyzed using standard flow cytometry techniques on a FACSCalibur cytometer using Cell Quest Pro software version 5.2.1 (all from BD Biosciences).

Total RNA was isolated from snap-frozen liver tissue using the QIAGEN RNeasy Mini isolation kit (QIAGEN Sciences, Germantown, MD), according to the manufacturer’s protocol. RNA quantity and quality were assessed using the Agilent RNA 6000 Nano Chip kit (Agilent Technologies, Santa Clara, CA) and/or NanoDrop (Thermo Scientific, Rockford IL). All RNA samples were then normalized to 110 ng/μl in nuclease-free water. cDNA was synthesized using the High-Capacity cDNA Reverse Transcription kit (Life Technologies/Applied Biosystems, Carlsbad, CA), according to the manufacturer’s protocol. After synthesis, TaqMan reactions were run on a 7900HT Real-time PCR system in a 384-well optical reaction plate. All reactions were run in triplicate using default cycling parameters.

Cryopreserved hepatocytes were thawed at 37°C, spun at 100 × g in Cryopreserved Hepatocyte Recovery Medium, and resuspended in plating media (all from Invitrogen). A total of 56,000 cells/well was plated onto 96-well collagen I–coated plates. Hepatocytes were allowed to attach for 4–6 h, and plating media were removed and replaced with maintenance media (Invitrogen) containing 0.25 mg/ml Matrigel (BD Biosciences). Cells were treated with the indicated cytokines or Fc–IL-22 on the following day. Maintenance medium plus appropriate concentrations of cytokines were replaced daily.

Total RNA was isolated using the MagMax96 RNA isolation kit (Applied Biosystems/Ambion, Austin, TX), according to the manufacturer’s protocol. RNA quantity and quality were assessed using the Agilent RNA 6000 Nano Chip kit (Agilent Technologies) and/or NanoDrop (Thermo Scientific). All RNA samples were then normalized to 10 ng/μl in nuclease-free water. cDNA was synthesized using the High-Capacity cDNA Reverse Transcription kit (Life Technologies/Applied Biosystems), according to the manufacturer’s protocol. After synthesis, TaqMan reactions were run on a 7900HT real-time PCR system in a 384-well optical-reaction plate. All reactions were run in triplicate using default cycling parameters.

Statistics are reported as the mean ± SEM for each treatment group and were calculated using GraphPad Prism v. 5.04 software (GraphPad, La Jolla, CA). Significant differences among treatment groups were determined using an unpaired t test.

To enable studies of the long-term effects of signaling induced by IL-22 in vivo, two IL-22–Fc reagents were produced to generate a more potent and longer-lasting IL-22R agonist compared with rIL-22. The activity of the IL-22–Fc constructs was first demonstrated in vitro by stimulating HepG2/C3a cells with rmIL-22, Fc–IL-22, or IL-22–Fc, followed by detection of STAT3 tyrosine phosphorylation (pSTAT3) by flow cytometry. As shown in Fig. 1A, rhIL-22 and Fc–IL-22 induced comparable levels of pSTAT3, whereas IL-22–Fc induced minimal pSTAT3.

FIGURE 1.

Characterization of the activity of IL-22–Fc fusion proteins relative to rIL-22. (A) STAT3 tyrosine phosphorylation was induced in HepG2/C3a cells with rmIL-22 or Fc–IL-22 but not IL-22–Fc. Cells were stimulated with 1 μg/ml of the indicated reagent for 30 min, fixed, permeabilized, stained with an anti–phospho-STAT3 (Y705) mAb, and analyzed by flow cytometry. (B) rmIL-22 and Fc–IL-22 induce SAA in female C57BL/6 mice. Mice were injected i.p. with 25 μg of the indicated reagent. Serum was collected at the indicated time points and analyzed for SAA by ELISA. The data shown are the mean concentration of SAA ± SEM for each group (n = 5 mice/group). Data are representative of results from three separate experiments. *Data below the level of detection.

FIGURE 1.

Characterization of the activity of IL-22–Fc fusion proteins relative to rIL-22. (A) STAT3 tyrosine phosphorylation was induced in HepG2/C3a cells with rmIL-22 or Fc–IL-22 but not IL-22–Fc. Cells were stimulated with 1 μg/ml of the indicated reagent for 30 min, fixed, permeabilized, stained with an anti–phospho-STAT3 (Y705) mAb, and analyzed by flow cytometry. (B) rmIL-22 and Fc–IL-22 induce SAA in female C57BL/6 mice. Mice were injected i.p. with 25 μg of the indicated reagent. Serum was collected at the indicated time points and analyzed for SAA by ELISA. The data shown are the mean concentration of SAA ± SEM for each group (n = 5 mice/group). Data are representative of results from three separate experiments. *Data below the level of detection.

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To compare the in vivo potency of the two IL-22–Fc reagents relative to rmIL-22, the kinetics of SAA induction in the serum of naive mice were measured following a single 25-μg injection of rmIL-22, Fc–IL-22, or IL-22–Fc. As shown in Fig. 1B, circulating SAA was elevated within 1 d after injection of rmIL-22, but it became undetectable by day 2. Injection of Fc–IL-22 resulted in elevated serum SAA concentrations for ≥7 d, with the highest SAA concentration detected at day 2. In addition to having a longer-lasting effect, Fc–IL-22 appeared to be more potent than rmIL-22. On day 1, increased concentrations of SAA were observed following injection of Fc–IL-22 relative to rmIL-22, despite rmIL-22 being dosed ∼8-fold higher on a molar basis. Based on the kinetics of SAA induction, it was concluded that Fc–IL-22 was active in vivo and maintained an increased functional effect relative to rmIL-22. In contrast, circulating SAA was not elevated in mice injected with the IL-22–Fc construct. Thus, based on both in vitro and in vivo results, Fc–IL-22 was determined to be fully active, and IL-22–Fc was determined to be a less-active construct.

To evaluate the long-term effects of Fc–IL-22 administration in a nondisease setting, C57BL/6 wild-type (WT) mice were injected with Fc–IL-22 three times/wk for 28 d. Mice injected with Fc–IL-22 had significant decreases in serum iron, hemoglobin (HGB), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration, mean corpuscular volume, and hematocrit relative to control Ab treatment (Table I). Overall, the changes observed in serum iron and red cell parameters were consistent with a microcytic, hypochromic anemia and were suggestive of relative iron deficiency.

Table I.
Long-term administration of Fc–IL-22 induces iron deficiency in C57BL/6 mice
MeasurementNaiveControl AbFc–IL-22p Value (t Test)a
Serum iron (μg/dl) 255.4 ± 18.1 211.3 ± 13.4 69.3 ± 1.7 <0.0001 
HGB (g/dl) 14.6 ± 0.17 14.7 ± 0.15 10.7 ± 0.07 <0.0001 
MCH (pg) 15.0 ± 0.06 15.3 ± 0.11 11.2 ± 0.15 <0.0001 
MCHC (g/dl) 29.7 ± 0.18 30.0 ± 0.19 26.0 ± 0.14 <0.0001 
MCV (fL) 50.6 ± 0.18 51.0 ± 0.17 43.3 ± 0.42 <0.0001 
MPV (fL) 6.4 ± 0.12 5.9 ± 0.14 6.1 ± 0.12 0.278 
HCT (%) 49.3 ± 0.69 49.1 ± 0.64 41.4 ± 0.27 <0.0001 
RBC (×106/μl) 9.8 ± 0.12 9.6 ± 0.14 9.6 ± 0.09 0.769 
RDW (%) 12.3 ± 0.20 12.0 ± 0.08 19.0 ± 0.25 <0.0001 
MeasurementNaiveControl AbFc–IL-22p Value (t Test)a
Serum iron (μg/dl) 255.4 ± 18.1 211.3 ± 13.4 69.3 ± 1.7 <0.0001 
HGB (g/dl) 14.6 ± 0.17 14.7 ± 0.15 10.7 ± 0.07 <0.0001 
MCH (pg) 15.0 ± 0.06 15.3 ± 0.11 11.2 ± 0.15 <0.0001 
MCHC (g/dl) 29.7 ± 0.18 30.0 ± 0.19 26.0 ± 0.14 <0.0001 
MCV (fL) 50.6 ± 0.18 51.0 ± 0.17 43.3 ± 0.42 <0.0001 
MPV (fL) 6.4 ± 0.12 5.9 ± 0.14 6.1 ± 0.12 0.278 
HCT (%) 49.3 ± 0.69 49.1 ± 0.64 41.4 ± 0.27 <0.0001 
RBC (×106/μl) 9.8 ± 0.12 9.6 ± 0.14 9.6 ± 0.09 0.769 
RDW (%) 12.3 ± 0.20 12.0 ± 0.08 19.0 ± 0.25 <0.0001 

Hematologic results (mean ± SEM) of female C57BL/6 mice (n = 5 mice/group) injected i.p. with 150 μg of control Ab or Fc–IL-22 three times/wk and harvested on day 28 (48 h after the final injection).

a

Control Ab versus Fc–IL-22.

HCT, Hematocrit; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; MPV, mean platelet volume; RDW, red cell distribution width.

Given hepcidin’s role in regulating iron availability, the changes in iron parameters observed following long-term administration of Fc–IL-22 suggested that hepcidin expression was increased. Indeed, when serum hepcidin concentrations were measured, they were significantly elevated in mice injected with Fc–IL-22 relative to control Ab on day 2 (213.9 ± 39.7 ng/ml for control mice, 391.3 ± 39.4 ng/ml for Fc–IL-22–treated mice); however, the significance of the elevated hepcidin was variable at later time points (Fig. 2A). Thus, serum hepcidin concentrations were measured on day 2 for subsequent long-term studies.

FIGURE 2.

Administration of Fc–IL-22 increases serum hepcidin expression and splenic iron accumulation in C57BL/6 mice. (A and B) Female C57BL/6 mice were injected i.p. with 150 μg of control Ab or Fc–IL-22 three times/wk on days 0, 2, 5, 7, 9, 12, 14, 17, 19, 21, 23, and 26 and harvested on days 2, 7, 14, 21, and 28 (48 h after the final injection). (A) Serum hepcidin concentration (M25 + M24), as measured by mass spectrometry. Data are mean ± SEM (days 0 and 28 time points: n = 10 mice/group; days 2, 7, 14, and 21 time points: n = 5 mice/group). (B) Representative analyses of ferric iron in the spleen of a naive (left), control Ab–treated (middle), or Fc–IL-22–treated (right) mouse on day 28. Iron was measured by Perls’ stain (blue pigment) (original magnification ×100). (CE) Female C57BL/6 mice were injected i.p. with a single dose of 150 μg of control Ab or Fc–IL-22 and harvested at the indicated time points. (C) Serum hepcidin (M25 + M24) concentration, as measured by mass spectrometry (n = 5 mice/group). (D) Serum iron concentration, as measured by an Olympus clinical chemistry analyzer (n = 5 mice/group). (E) HGB concentration, as measured by an Advia 120 hematology analyzer (n = 5 mice/group). Data are representative of two separate experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired t test.

FIGURE 2.

Administration of Fc–IL-22 increases serum hepcidin expression and splenic iron accumulation in C57BL/6 mice. (A and B) Female C57BL/6 mice were injected i.p. with 150 μg of control Ab or Fc–IL-22 three times/wk on days 0, 2, 5, 7, 9, 12, 14, 17, 19, 21, 23, and 26 and harvested on days 2, 7, 14, 21, and 28 (48 h after the final injection). (A) Serum hepcidin concentration (M25 + M24), as measured by mass spectrometry. Data are mean ± SEM (days 0 and 28 time points: n = 10 mice/group; days 2, 7, 14, and 21 time points: n = 5 mice/group). (B) Representative analyses of ferric iron in the spleen of a naive (left), control Ab–treated (middle), or Fc–IL-22–treated (right) mouse on day 28. Iron was measured by Perls’ stain (blue pigment) (original magnification ×100). (CE) Female C57BL/6 mice were injected i.p. with a single dose of 150 μg of control Ab or Fc–IL-22 and harvested at the indicated time points. (C) Serum hepcidin (M25 + M24) concentration, as measured by mass spectrometry (n = 5 mice/group). (D) Serum iron concentration, as measured by an Olympus clinical chemistry analyzer (n = 5 mice/group). (E) HGB concentration, as measured by an Advia 120 hematology analyzer (n = 5 mice/group). Data are representative of two separate experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired t test.

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With an upregulation of hepcidin, there should also be an increase in macrophage iron accumulation resulting from downregulation of ferroportin activity. Indeed, long-term administration of Fc–IL-22 resulted in an increase in iron sequestration in the spleen, as measured by Perls’ staining (Fig. 2B). An increase in iron accumulation in liver was not observed (data not shown).

To further characterize the relationship between IL-22R stimulation and hepcidin induction, changes in hepcidin, serum iron, and HGB were evaluated over time following Fc–IL-22 injection. Mice had significantly elevated serum hepcidin concentrations within 2 h after a single injection of Fc–IL-22, and this elevation was maintained for ≥48 h (Fig. 2C), with the highest concentration of serum hepcidin detected at 6 h postinjection (145.9 ± 14.6 ng/ml for control Ab–treated mice, 299.4 ± 21.1 ng/ml for Fc–IL-22–treated mice). Serum iron concentrations began decreasing 2 h after Fc–IL-22 injection (Fig. 2D) and were significantly reduced at the 6-h time point (203.4 ± 2.3 g/dl for control Ab–treated mice, 149.3 ± 7.2 g/dl for Fc–IL-22–treated mice). HGB concentrations began to decrease 6 h after Fc–IL-22 injection (Fig. 2E) and were significantly reduced at the 48-h time point (14.9 ± 0.07 g/dl for control Ab–treated mice, 13.4 ± 0.14 g/dl for Fc–IL-22–treated mice).

Iron deficiency was also observed following injection of Fc–IL-22 in two other mouse strains: C57BL/6 SCID mice and FcRγ−/− mice (Supplemental Figs. 1, 2). Thus, the observed iron deficiency was not mediated by an adaptive immune response or FcR-mediated response to Fc–IL-22. To further confirm the lack of a role for the Fc portion of Fc–IL-22 in these effects, iron and red cell analyses were done with various irrelevant Fc-containing proteins, as well as the Fc–IL-22 and IL-22–Fc proteins (Fig. 3). Fc–IL-22 induced a significant increase in splenic iron accumulation (Fig. 3A) and a decrease in serum iron (Fig. 3B), HGB (Fig. 3C), and RBC counts (Fig. 3D). IL-22–Fc, the less-active fusion protein, induced no change in splenic iron accumulation and modest decreases in serum iron, HGB, and RBC counts, inducing comparable responses to IL-22 cytokine injection. Control IgG1 Ab, as well as a 4-1BB–Fc fusion protein, induced no effect on the measured parameters.

FIGURE 3.

Iron deficiency is not induced by the presence of mIgG1 Fc. Indicated female C57BL/6 mice were injected i.p. daily with 50 μg of MSA control or rIL-22 reagent from day 0-4 as described in 2Materials and Methods. Indicated mice were injected i.p. with 150 μg of control Ab, 4-1BB-Fc, IL-22–Fc, or Fc–IL-22 on days 0, 2, and 4. Mice were harvested 4–5 h after the final injection on day 4. (A) The percentage of splenic iron content was calculated using Perls’ stain and Visiomorph Software (n = 5 mice/group). (B) Serum iron concentration, as measured by an Olympus clinical chemistry analyzer (n = 2–5 mice/group). HGB (C) and RBC count (D), as measured by an Advia 120 hematology analyzer (n = 5 mice/group). Data shown are representative of a single experiment. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired t test.

FIGURE 3.

Iron deficiency is not induced by the presence of mIgG1 Fc. Indicated female C57BL/6 mice were injected i.p. daily with 50 μg of MSA control or rIL-22 reagent from day 0-4 as described in 2Materials and Methods. Indicated mice were injected i.p. with 150 μg of control Ab, 4-1BB-Fc, IL-22–Fc, or Fc–IL-22 on days 0, 2, and 4. Mice were harvested 4–5 h after the final injection on day 4. (A) The percentage of splenic iron content was calculated using Perls’ stain and Visiomorph Software (n = 5 mice/group). (B) Serum iron concentration, as measured by an Olympus clinical chemistry analyzer (n = 2–5 mice/group). HGB (C) and RBC count (D), as measured by an Advia 120 hematology analyzer (n = 5 mice/group). Data shown are representative of a single experiment. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired t test.

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To determine whether IL-22R signaling was required for Fc–IL-22–mediated induction of hepcidin expression, B10 WT mice and B10 mice containing a natural deficiency in Tyk2 (34, 35) were treated with Fc–IL-22, and both serum hepcidin concentrations and associated iron deficiency were evaluated. Within the first 48 h after Fc–IL-22 injection, serum hepcidin was increased in B10 WT mice; this increase was similar to that seen in C57BL/6 WT mice (compare Fig. 4A with Fig. 2C). In contrast, there was no detectable increase in serum hepcidin in the Tyk2-deficient mice treated with Fc–IL-22 (Fig. 4B). Seven-day treatment of B10 WT mice with Fc–IL-22 induced an increase in splenic iron accumulation and a reduction in HGB, MCH, and RBC count similar to those observed in C57BL/6 WT mice (compare Fig. 4C–F with Table I). In comparison, Fc–IL-22 treatment of Tyk2-deficient mice induced no significant increase in splenic iron accumulation (Fig. 4C) and no significant reduction in MCH (Fig. 4E). A reduction in HGB and RBC counts was observed in Tyk2-deficient mice treated with Fc–IL-22; however, it was less significant than that observed in B10 WT mice (Fig. 4D, 4F). Importantly, there were no significant differences in any of the measured parameters between B10 WT and Tyk2-deficient mice treated with control Ab. Similar results were observed in samples harvested following either 14 or 28 d of Fc–IL-22 treatment in these mice (data not shown).

FIGURE 4.

Fc–IL-22–induced hepcidin expression and iron deficiency are dependent on Tyk2. (A and B) Administration of Fc–IL-22 does not induce hepcidin expression in Tyk2-deficient mice. Mice were injected i.p. with 150 μg of control Ab or Fc–IL-22 and harvested at the indicated time points. Serum hepcidin (M25 + M24) concentration, as measured by mass spectrometry in female B10 WT (A) and Tyk2-deficient (B) mice (n = 4 mice/group). (CF) Fc–IL-22–induced tissue iron accumulation and iron deficiency were less evident in Tyk2-deficient mice than in B10 WT mice. B10 WT and Tyk2-deficient mice were injected i.p. with 150 μg of control Ab or Fc–IL-22 on days 0, 2, 5, and 7. Mice were harvested 4–5 h after the final injection on day 7. (C) The percentage of iron content in the spleen was calculated using Perls’ stain and Visiomorph Software. HGB (D), MCH (E), and RBC count (F), as measured by an Advia 120 hematology analyzer. The data shown are the mean concentration ± SEM for each group (n = 4–7 mice/group) and are representative of three separate experiments harvested on days 7, 14, or 28. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired t test.

FIGURE 4.

Fc–IL-22–induced hepcidin expression and iron deficiency are dependent on Tyk2. (A and B) Administration of Fc–IL-22 does not induce hepcidin expression in Tyk2-deficient mice. Mice were injected i.p. with 150 μg of control Ab or Fc–IL-22 and harvested at the indicated time points. Serum hepcidin (M25 + M24) concentration, as measured by mass spectrometry in female B10 WT (A) and Tyk2-deficient (B) mice (n = 4 mice/group). (CF) Fc–IL-22–induced tissue iron accumulation and iron deficiency were less evident in Tyk2-deficient mice than in B10 WT mice. B10 WT and Tyk2-deficient mice were injected i.p. with 150 μg of control Ab or Fc–IL-22 on days 0, 2, 5, and 7. Mice were harvested 4–5 h after the final injection on day 7. (C) The percentage of iron content in the spleen was calculated using Perls’ stain and Visiomorph Software. HGB (D), MCH (E), and RBC count (F), as measured by an Advia 120 hematology analyzer. The data shown are the mean concentration ± SEM for each group (n = 4–7 mice/group) and are representative of three separate experiments harvested on days 7, 14, or 28. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired t test.

Close modal

It is well-established that IL-6 is a major inducer of hepcidin expression. To determine whether IL-22–induced iron deficiency required IL-6, C57BL/6 WT and C57BL/6 IL-6−/− (IL-6−/−) mice were treated with Fc–IL-22 three times/wk for 28 d. During this time, changes in various parameters relevant to iron deficiency were monitored. Serum hepcidin concentrations were significantly elevated on day 2 in Fc–IL-22–treated C57BL/6 WT and IL-6−/− mice (416.6 ± 19.6 and 387.7 ± 27.6 ng/ml, respectively) relative to mice treated with control Ab (266.6 ± 32.2 and 174.9 ± 27.6 ng/ml, respectively) (Fig. 5A). Splenic iron content was significantly increased in both Fc–IL-22–treated C57BL/6 WT mice and IL-6−/− mice relative to control Ab–treated mice on day 2, and it remained elevated through day 28 (Fig. 5B). Serum iron and HGB were significantly decreased in both strains within 2 d after Fc–IL-22 injection, and they continued to decline through day 28 (Fig. 5C, 5D). A rapid decrease in RBC count was also observed in both strains by day 4 after Fc–IL-22 injection. Although RBC counts gradually increased at each measured time point after day 4, RBC counts in Fc–IL-22–injected C57BL/6 WT or IL-6−/− mice remained significantly reduced relative to control Ab–injected mice through day 28 (Fig. 5E).

FIGURE 5.

Fc–IL-22 induces hepcidin and splenic iron accumulation independently of IL-6. Female C57BL/6 WT mice or C57BL/6 IL-6−/− mice were injected i.p. with 150 μg of control Ab or Fc–IL-22 three times/wk and harvested 4–5 h after the final injections on days 2, 4, 7, 14, 21, or 28. (A) Day-2 serum hepcidin (M25 + M24) concentration, as measured by mass spectrometry (n = 5 mice/group). (B) The percentage of iron content in the spleen was calculated using Perls’ stain and Visiomorph Software. (C) Serum iron concentration, as measured by an Olympus clinical chemistry analyzer. HGB (D) and RBC count (E), as measured by an Advia 120 hematology analyzer. Data are mean ± SEM (n = 5 mice/group) and are representative of two separate experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (for WT mice), control Ab versus Fc–IL-22, unpaired t test. +p < 0.05, ++p < 0.01, +++p < 0.001, ++++p < 0.0001 (for IL-6−/− mice), control Ab versus Fc–IL-22, unpaired t test.

FIGURE 5.

Fc–IL-22 induces hepcidin and splenic iron accumulation independently of IL-6. Female C57BL/6 WT mice or C57BL/6 IL-6−/− mice were injected i.p. with 150 μg of control Ab or Fc–IL-22 three times/wk and harvested 4–5 h after the final injections on days 2, 4, 7, 14, 21, or 28. (A) Day-2 serum hepcidin (M25 + M24) concentration, as measured by mass spectrometry (n = 5 mice/group). (B) The percentage of iron content in the spleen was calculated using Perls’ stain and Visiomorph Software. (C) Serum iron concentration, as measured by an Olympus clinical chemistry analyzer. HGB (D) and RBC count (E), as measured by an Advia 120 hematology analyzer. Data are mean ± SEM (n = 5 mice/group) and are representative of two separate experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (for WT mice), control Ab versus Fc–IL-22, unpaired t test. +p < 0.05, ++p < 0.01, +++p < 0.001, ++++p < 0.0001 (for IL-6−/− mice), control Ab versus Fc–IL-22, unpaired t test.

Close modal

rhIL-6 was shown to be active in mice (36, 37), and it was used to directly compare the in vivo effects of IL-6 on hepcidin expression and iron deficiency with those induced by rmIL-22. The effect of IL-22 on liver hepcidin mRNA expression was compared with the effect induced by IL-6 following a single injection of cytokine. As shown in Fig. 6A, rmIL-22 induced a significant elevation in Hamp1 mRNA expression relative to the MSA control mice at 2 h, but not 4 h, postinjection (∼1.4- and 1.1-fold increases, respectively). In contrast, injection of rhIL-6 did not induce a significant elevation in Hamp1 mRNA expression relative to MSA control mice at 2 h postinjection (∼1.1-fold increase), but it significantly elevated Hamp1 expression at the 4-h time point (∼1.6-fold increase). Despite the increase in hepcidin expression detected in the liver, serum hepcidin concentrations were not significantly elevated (data not shown).

FIGURE 6.

rIL-22 and rIL-6 equivalently induce iron deficiency and hepcidin in C57BL/6 mice. (A) Female C57BL/6 mice were injected i.p. with 50 μg of the indicated reagent. Liver was collected at the indicated time point. RNA was isolated, and Hamp1 mRNA expression was determined by TaqMan analysis. Data are mean concentration of Hamp1 gene expression relative to β-actin gene expression ± SEM (n = 5 mice/group). (BE) Female C57BL/6 mice were injected i.p. daily with 50 μg of the indicated reagent from day 0 to 4, as described in 2Materials and Methods. Mice were harvested 4–5 h after the final injection on day 4. (B) The percentage of splenic iron content was calculated using Perls’ stain and Visiomorph Software (n = 5 mice/group). (C) Serum iron concentration, as measured by an Olympus clinical chemistry analyzer (n = 5 mice/group). HGB (D) and RBC count (E), as measured by an Advia 120 hematology analyzer (n = 5 mice/group). Data shown are representative of two separate experiments. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired t test.

FIGURE 6.

rIL-22 and rIL-6 equivalently induce iron deficiency and hepcidin in C57BL/6 mice. (A) Female C57BL/6 mice were injected i.p. with 50 μg of the indicated reagent. Liver was collected at the indicated time point. RNA was isolated, and Hamp1 mRNA expression was determined by TaqMan analysis. Data are mean concentration of Hamp1 gene expression relative to β-actin gene expression ± SEM (n = 5 mice/group). (BE) Female C57BL/6 mice were injected i.p. daily with 50 μg of the indicated reagent from day 0 to 4, as described in 2Materials and Methods. Mice were harvested 4–5 h after the final injection on day 4. (B) The percentage of splenic iron content was calculated using Perls’ stain and Visiomorph Software (n = 5 mice/group). (C) Serum iron concentration, as measured by an Olympus clinical chemistry analyzer (n = 5 mice/group). HGB (D) and RBC count (E), as measured by an Advia 120 hematology analyzer (n = 5 mice/group). Data shown are representative of two separate experiments. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired t test.

Close modal

The effects of rIL-22 and rIL-6 on iron deficiency were evaluated following multiple injections of cytokine over 4 d. Splenic iron accumulation was increased in both rhIL-6–and rmIL-22–injected mice, although the increase induced by rhIL-6 was not significant relative to the MSA controls (Fig. 6B). Serum iron (Fig. 6C), HGB (Fig. 6D), and RBC counts (Fig. 6E) were significantly decreased on day 4 in both rhIL-6– and rmIL-22–treated mice relative to MSA control–injected mice.

To demonstrate a direct effect of IL-22 on the induction of human hepcidin in vitro, primary human hepatocytes were stimulated with a titration of rhIL-22 or Fc–IL-22, and hepcidin mRNA was measured. The effect of IL-22 on hepcidin was compared with the effect induced by rhIL-6. As shown in Fig. 7A and 7B, increased expression of hepcidin mRNA was detected following both IL-22 and IL-6 stimulation. Treatment with 10 nM of rIL-22 induced an ∼2- and ∼3.5-fold increase in hepcidin expression at 24 and 48 h, respectively. Although not as potent as the recombinant human cytokine, treatment with 10 nM of Fc–IL-22 induced an ∼2-fold increase in hepcidin at 48 h. Treatment with the same concentration of IL-6 resulted in ∼7- and ∼11-fold increases in hepcidin at 24 and 48 h, respectively. These results are not necessarily reflective of an increased potency of IL-6 relative to IL-22, because the mRNA expression of the receptors for IL-6 and IL-22 were not comparable in these cells (Fig. 7C).

FIGURE 7.

rIL-22 and rIL-6 induce hepcidin mRNA expression in primary human hepatocytes. (A and B) Human primary hepatocytes were incubated with a titration of rhIL-6, Fc–IL-22, or rhIL-22. RNA was isolated at 24 h (A) or 48 h (B), and hepcidin mRNA expression was determined by TaqMan analysis. Data are representative of two separate donors. (C) RNA was isolated from unstimulated human primary hepatocytes cultured for 72 h, and IL-6Rα, gp130, IL-10R2, and IL-22R1 mRNA expression was determined by TaqMan analysis. IL-6Rα was set as the calibrator with a relative quantitation (RQ) equal to one. Error bars represent the average minimum and maximum RQ values obtained for three individual donors. The RQ values for gp130, IL-10R2, and IL-22R1 represent the average and SD of three individual donors compared with IL-6Rα levels.

FIGURE 7.

rIL-22 and rIL-6 induce hepcidin mRNA expression in primary human hepatocytes. (A and B) Human primary hepatocytes were incubated with a titration of rhIL-6, Fc–IL-22, or rhIL-22. RNA was isolated at 24 h (A) or 48 h (B), and hepcidin mRNA expression was determined by TaqMan analysis. Data are representative of two separate donors. (C) RNA was isolated from unstimulated human primary hepatocytes cultured for 72 h, and IL-6Rα, gp130, IL-10R2, and IL-22R1 mRNA expression was determined by TaqMan analysis. IL-6Rα was set as the calibrator with a relative quantitation (RQ) equal to one. Error bars represent the average minimum and maximum RQ values obtained for three individual donors. The RQ values for gp130, IL-10R2, and IL-22R1 represent the average and SD of three individual donors compared with IL-6Rα levels.

Close modal

A monoclonal anti-mouse hepcidin Ab (2C10; Supplemental Fig. 3) was used to determine whether neutralization of hepcidin would reverse IL-22–induced iron deficiency in vivo. To evaluate the efficacy of anti-hepcidin treatment on different severities of iron deficiency, mice were treated with either Fc–IL-22 (Fig. 8) or rmIL-22 (Fig. 9) to induce severe or mild iron deficiency, respectively. Treatment of mice with anti-hepcidin Ab alone resulted in decreased splenic iron accumulation (Fig. 8A) and increased serum iron concentrations (Figs. 8B, 9A) relative to naive mice or mice injected with control protein. These results were expected, because hepcidin neutralization was shown to increase serum iron concentrations (32), and decreased hepcidin levels were shown to lead to a decrease in splenic iron accumulation (38). Treatment with anti-hepcidin Ab alone had no effect on HGB concentrations (Fig. 8C, 9B), but it significantly elevated MCH measurements in one study (Fig. 8D). Mice injected with either Fc–IL-22 or rmIL-22 had decreased serum iron (Figs. 8B, 9A) and HGB (Figs. 8C, 9B) and an increase in splenic iron (Fig. 8A). Treatment with the anti-hepcidin Ab significantly, but not completely, inhibited the Fc–IL-22–induced decreases in serum iron, HGB, and MCH (Fig. 8B–D) and significantly reduced the amount of splenic iron accumulation (Fig. 8A). Similar results were seen in mice treated with rmIL-22 in combination with anti-hepcidin Ab. Specifically, treatment with anti-hepcidin Ab partially inhibited the rmIL-22–induced iron deficiency, as measured by serum iron concentration (Fig. 9A) and MCH (Fig. 9C). Also of note, mice injected with either Fc–IL-22 or rmIL-22 had a significant reduction in RBC count that was not affected by treatment with the anti-hepcidin Ab (Figs. 8E, 9D).

FIGURE 8.

Anti-hepcidin Ab ameliorates Fc–IL-22–induced iron deficiency. (AE) Indicated female C57BL/6 mice were injected i.p. with 2.5 mg of anti-hepcidin Ab (2C10) or control Ab on days −1, 1, 4, and 6. Indicated mice were injected i.p. with 150 μg of Fc–IL-22 or control Ab on days 0, 2, 5, and 7. Mice were harvested 4–5 h after the final injection on day 7. (A) The percentage of splenic iron content was calculated using Perls’ stain and Visiomorph Software, as described in 2Materials and Methods (n = 5 mice/group). (B) Serum iron concentration, as measured by an Olympus clinical chemistry analyzer (n = 5 mice/group). HGB (C), MCH (D), and RBC count (E), as measured by an Advia 120 hematology analyzer (n = 5 mice/group). *p < 0.05, **p < 0.01, ***p < 0.001, unpaired t test.

FIGURE 8.

Anti-hepcidin Ab ameliorates Fc–IL-22–induced iron deficiency. (AE) Indicated female C57BL/6 mice were injected i.p. with 2.5 mg of anti-hepcidin Ab (2C10) or control Ab on days −1, 1, 4, and 6. Indicated mice were injected i.p. with 150 μg of Fc–IL-22 or control Ab on days 0, 2, 5, and 7. Mice were harvested 4–5 h after the final injection on day 7. (A) The percentage of splenic iron content was calculated using Perls’ stain and Visiomorph Software, as described in 2Materials and Methods (n = 5 mice/group). (B) Serum iron concentration, as measured by an Olympus clinical chemistry analyzer (n = 5 mice/group). HGB (C), MCH (D), and RBC count (E), as measured by an Advia 120 hematology analyzer (n = 5 mice/group). *p < 0.05, **p < 0.01, ***p < 0.001, unpaired t test.

Close modal
FIGURE 9.

Anti-hepcidin Ab ameliorates IL-22–induced iron deficiency. (AD) Indicated female C57BL/6 mice were injected i.p. with 2.5 mg of anti-hepcidin Ab (2C10) on days −1, 1, 4, and 6. Indicated mice were injected i.p. with 50 μg of MSA control or rmIL-22 on days 0–7, as described in 2Materials and Methods. Mice were harvested 4–5 h after the final injection on day 7. (A) Serum iron concentration, as measured by an Olympus clinical chemistry analyzer (n = 5 mice/group). HGB (B), MCH (C), and RBC count (D), as measured by an Advia 120 hematology analyzer (n = 5 mice/group). *p < 0.05, **p < 0.01, ***p < 0.001, unpaired t test.

FIGURE 9.

Anti-hepcidin Ab ameliorates IL-22–induced iron deficiency. (AD) Indicated female C57BL/6 mice were injected i.p. with 2.5 mg of anti-hepcidin Ab (2C10) on days −1, 1, 4, and 6. Indicated mice were injected i.p. with 50 μg of MSA control or rmIL-22 on days 0–7, as described in 2Materials and Methods. Mice were harvested 4–5 h after the final injection on day 7. (A) Serum iron concentration, as measured by an Olympus clinical chemistry analyzer (n = 5 mice/group). HGB (B), MCH (C), and RBC count (D), as measured by an Advia 120 hematology analyzer (n = 5 mice/group). *p < 0.05, **p < 0.01, ***p < 0.001, unpaired t test.

Close modal

The studies described in this article demonstrate that IL-22R signaling induces hepcidin expression in vitro and in vivo and that this effect is independent of IL-6. Hepcidin limits iron uptake and mobility, and increased expression would be expected to reduce serum iron availability and increase iron accumulation in macrophage-rich tissues. Because iron is withheld in the tissues, HGB production for nascent RBCs would also decrease, resulting in smaller RBCs (microcytic) containing less HGB (hypochromic). Indeed, treatment of mice with exogenous rmIL-22 induced a significant decrease in serum iron and HGB; however, large doses of rmIL-22 and frequent administration were required. To better facilitate in vivo analysis of IL-22R stimulation, Fc–IL-22 was generated and shown to be active and potentially more potent than rmIL-22. Treatment of mice with exogenous Fc–IL-22 induced a significant increase in serum hepcidin expression, concomitant reductions in serum iron and HGB concentrations, and increased tissue iron accumulation (Fig. 2). Neutralization of hepcidin with a mAb incompletely restored the HGB and iron levels to baseline, demonstrating that the effects of IL-22 on iron metabolism can be partially reversed by neutralizing hepcidin. This incomplete restoration of baseline serum iron levels could be due to additional hepcidin-independent mechanisms induced by IL-22 or Fc–IL-22 or due to the incomplete neutralization of hepcidin by the injected Ab. The increased potency of the Fc–IL-22 molecule may make it harder to fully neutralize the induced hepcidin; indeed, the anti-hepcidin Ab is much more effective at restoring serum iron levels to baseline in mice treated with rIL-22 compared with Fc–IL-22.

The in vivo detection of hepcidin presented some challenges in this study. Although most of our studies cumulatively detected the M25 and M24 peptides at ∼200 ng/ml in the serum, some studies detected lower amounts or saw the levels decrease over time. The reason for this is unclear. Hepcidin can be cleaved into various shorter forms, and peptides smaller than 24 aa may be generated that are not being measured. Augmented cleavage of hepcidin could account for some of the variation in our results, and this could come from variation in the quality of the blood draws or time in between sample preparation and analysis. Additionally, it is quite possible that some type of feedback mechanism is being activated. SOCS3 is a candidate protein that could be mediating such a response (39). Regulation of such a response would make sense, and it is a potentially fruitful area for future study. Alternatively, the Fc–IL-22 protein may be immunogenic, and neutralization of this reagent could dampen the hepcidin induction response over time.

In vitro treatment of primary human hepatocytes with rhIL-22 alone induced hepcidin mRNA expression (Fig. 7). As a control, primary human hepatocytes were treated with rhIL-6. Although treatment with rhIL-6 seemed to be more potent, this difference could be due to the greater expression of IL-6R relative to IL-22R on these cells. Nevertheless, more careful biochemical studies are warranted to understand the relative potencies more thoroughly. It is worth noting that the Fc–IL-22 protein is less potent than IL-22 at inducing hepcidin in vitro (Fig. 7). The reason for this is unclear, but it suggests that dimerizing the cytokine does not inherently improve potency and that the in vivo potency increase is more likely to be due to the improved half-life afforded by the Fc.

Recent work by Armitage et al. (12) also showed that IL-22 can induce hepcidin expression in hepatocyte cell lines and that this effect was independent of IL-6. Additionally, Liang et al. (31) observed decreases in RBCs after sustained IL-22 production driven by an adenoviral vector. Our work extends these observations by showing that Fc–IL-22 treatment of both C57BL/6 WT and IL-6−/− mice induced similar increases in serum hepcidin and tissue iron accumulation, as well as similar decreases in HGB (Fig. 5). Together, these results indicate that IL-22–mediated induction of hepcidin is independent of IL-6 both in vitro and in vivo.

Previous reports showed that the IL-6–mediated induction of hepcidin was dependent on STAT3 signaling (4042). Because IL-22 primarily signals through STAT3 (18, 23), it is presumed that, similar to IL-6, induction of hepcidin by IL-22 also requires the STAT3 pathway. Fc–IL-22 induced STAT3 activation in vivo (data not shown), but the specific STAT3 dependence of the downstream responses assessed in this study was not investigated. However, related to this, the dependence on a less-understood aspect of IL-22R signaling was investigated using mice spontaneously deficient in Tyk2 (Fig. 4). Prior studies showed that IL-22 induces signaling via Tyk2 (20), but the absolute dependence of IL-22R signaling on this kinase has not been clarified. A requirement for Tyk2 signaling in Fc–IL-22–induced hepcidin production was demonstrated by comparing the effect of Fc–IL-22 in B10 WT and Tyk2-deficient mice. B10 WT mice treated with Fc–IL-22 had robust hepcidin accumulation in serum after 48 h, whereas Fc–IL-22 had no effect on serum hepcidin levels in Tyk2-deficient mice. Chronic dosing of Fc–IL-22 in B10 WT mice resulted in a microcytic, hypochromic anemia, whereas chronic dosing of Fc–IL-22 in Tyk2-deficient mice resulted in a mild anemia associated with subtle microcytosis and hypochromasia. In this strain, the deficiency in Tyk2 is due to a natural mutation that produces an unstable protein and not because of a targeted knockout (35); thus, it is possible that some Tyk2 is available to signal and induce the mild responses observed in the Tyk2-deficient mice. Altogether, these results demonstrate that the iron-modulating effects of IL-22 are dependent on IL-22R–associated signaling pathways. This ability to attribute the effects of Fc–IL-22 to Tyk2 is significant because it provides further support, along with the FcγR−/− data (Supplemental Fig. 2) and the use of various mIgG1 Fc containing proteins (Fig. 3), that the iron-modulating effects observed in this study are not simply due to the Fc portion of the protein.

In addition to the effects of rmIL-22 and Fc–IL-22 on components of iron metabolism, injection of C57BL/6 WT mice with either rmIL-22 or Fc–IL-22 induced an acute reduction in RBCs within 96 or 48 h, respectively (Figs. 6E and 5E, respectively). Such a rapid effect on RBC count is unlikely to be due to iron restriction and suggests that IL-22 may influence RBC count by an additional mechanism, such as inducing a hemolytic anemia or altering the homeostatic erythrophagocytosis process. In mice injected with Fc–IL-22, time-course analysis of RBC counts showed that they were maximally reduced on day 4 but gradually recovered until day 28, presumably as the result of an increase in the production and maturation of reticulocytes. This reduction in RBCs after Fc–IL-22 treatment was observed in most, but not all, studies. Interestingly, short-term studies, done within 4–7 d, found marked decreases in RBC counts, yet longer-term studies, going out to 28 d, often found minimal reductions. Indeed, Fig. 5 shows that RBC counts recover over 28 d, despite the sustained decrease in serum iron and HGB. The reason for this disconnect between the iron and HGB levels and RBC counts is unclear. Treatment with an anti-hepcidin Ab ameliorated the IL-22–induced iron deficiency in a short-term study; however, it did not result in a recovery of RBC count, supporting the hypothesis that the acute IL-22–mediated effect on RBCs is not hepcidin related.

We attempted various studies to determine the cause of the IL-22–induced loss of RBCs but have not found an explanation. Based on preliminary results evaluating the effects of IL-22 on serum bilirubin levels, there is no conclusive evidence to suggest that the observed reduction in RBC counts is due to intravascular hemolytic anemia (data not shown). Additionally, studies performed in FcRγ−/− mice (Supplemental Fig. 2A) or with irrelevant Fc fusions (Fig. 3) suggest that Fc–IL-22 is not artificially inducing an FcR-mediated extravascular removal of RBCs. The process of erythrophagocytosis is performed primarily by splenic macrophages, although it was reported that neutrophils can phagocytose RBCs as well (43). Ab depletion of macrophages and neutrophils in vivo using Abs to CD11b or Gr-1 had no effect on Fc–IL-22–induced RBC loss (data not shown). Thus, additional studies will be needed to further identify the effect of IL-22R agonism on RBC loss and erythrophagocytosis.

IL-22 is well established as an important cytokine involved in the innate immune response, including barrier function maintenance and repair, as well as pathogen defense, particularly against extracellular bacteria. It was reported that IL-22−/− mice have an increased rate of mortality in mouse models of infection (44). IL-22 induces the production of a number of antimicrobial proteins, as well inflammatory cytokines that promote neutrophil production or migration (31). The induction of hepcidin and subsequent restriction of essential iron by IL-22 may be an additional mechanism of the innate immune response with regard to pathogen defense. IL-22 was also reported to be elevated in a number of chronic diseases, and it is tempting to speculate that IL-22 may also participate in anemia of chronic disease (ACD) through hepcidin-induced alterations in iron metabolism. ACD is characterized by low serum iron concentration and reduced serum transferrin saturation, with increased intracellular iron storage (45). Although the pathogenesis of ACD is not fully elucidated, the induction of hepcidin by inflammatory cytokines is likely involved (45), and IL-22 may be worth investigating in this condition. The results reported in this article demonstrate potent effects on iron regulation resulting from exogenously administered IL-22, and it remains to be determined what role endogenous levels of cytokine play in iron restriction and ACD. Nevertheless, numerous publications have demonstrated a beneficial role for IL-22 in inflammatory disease, and therapies that may try to capitalize on its protective side will have to consider the effects that such treatments may have on iron levels. In all, the dual nature of IL-22 continues to be unraveled; although IL-22 benefits the innate immune response by triggering antimicrobial and other acute-phase responses, it may have detrimental effects by limiting iron availability through hepcidin upregulation.

We thank numerous people in the Departments of Pathology and Protein Sciences and the Center for Comparative Animal Research for assistance.

The online version of this article contains supplemental material.

Abbreviations used in this article:

ACD

anemia of chronic disease

B10

B10.Q/Ai

4-1BB-Fc

mouse IgG1 Fc fused to the C terminus of mouse 4-1BB

Fc–IL-22

mouse IgG1 Fc fused to the N terminus of mouse IL-22

HGB

hemoglobin

IL-22–Fc

mouse IgG1 Fc fused to the C terminus of mouse IL-22

m

mouse

MCH

mean corpuscular hemoglobin

MSA

mouse serum albumin

rh

recombinant human

rm

recombinant murine

SAA

serum amyloid A

WT

wild-type.

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All authors were employees of Amgen, Inc. when this study was conducted.