A mouse model characterizes the roles of ZIP8 in systemic iron recycling and lung inflammation and infection.

ZIP8 (SLC39A8) is a transmembrane divalent metal ion importer that is most highly expressed in the lung and is inducible by inflammatory stimuli. In addition to zinc and manganese, ZIP8 can transport iron, but its specific roles in iron regulation during homeostatic and pathologic processes remain poorly understood. Using a novel global inducible ZIP8 knockout (KO) mouse, we analyzed the role of ZIP8 in steady-state iron homeostasis and during inflammation and infection. We observed an unexpected phenotype of elevated spleen iron levels and decreased serum iron in ZIP8 KO mice, suggesting that ZIP8 plays a role in iron recycling. We also showed that ZIP8 is expressed on lung distal airspace epithelial cells and transports iron from the airway into lung tissue. LPS-induced inflammation induced ZIP8 expression in the lung, but ZIP8 deletion had no detrimental effect on the severity of LPS-induced acute lung injury or on the outcomes of Klebsiella pneumoniae lung infection. Thus, ZIP8 plays a role in systemic iron homeostasis but does not modulate the severity of inflammatory lung injury or the host defense against a common bacterial cause of pneumonia.

Bmpr2 mutant mice are an inadequate model for studying iron deficiency in pulmonary hypertension.

As bone morphogenetic protein receptor type II (Bmpr2) mutations are the most common genetic cause of pulmonary arterial hypertension (PAH), and iron deficiency (ID) is associated with worse clinical outcomes in PAH patients, we proposed to use Bmpr2 ± mice to induce a model of ID in pulmonary vascular disease. Our study shows that these transgenic mice are not a good model for this clinical phenomenon.

Expression of Iron-Regulatory Hormone Hepcidin and Iron Transporters Ferroportin and ZIP8 in Patients With and Without Chronic Rhinosinusitis.

Airway epithelia express intrinsic antimicrobial and nutrient-sequestering factors, which contribute to the host defense of the respiratory tract. Hepcidin is an endogenous peptide hormone that serves as a key regulator of iron metabolism, and ferroportin and ZIP8 are iron transporters. All exhibit innate antimicrobial activity. The purpose of this pilot study is to determine if molecules involved in iron regulation are expressed within sinus epithelia and to compare levels of expression between patients with and without chronic rhinosinusitis (CRS). Sinus mucosa was obtained from patients with (n = 19) and without (n = 14) CRS. Real-time polymerase chain reaction following RNA extraction was used to quantify expression of hepcidin, ferroportin, and ZIP8 mRNA. Hepcidin, ferroportin, and ZIP8 were all detected in the sinus epithelia of patients with and without CRS. However, only ZIP8 was significantly changed in CRS, with a 2.5-fold mean increase in mRNA expression relative to controls (P = .005). These findings suggest that ZIP8 may play a role in the innate epithelial defense of the paranasal sinuses.

Iron overload causes a mild and transient increase in acute lung injury.

Recent studies have demonstrated a strong link between acute respiratory distress syndrome (ARDS) and the levels of iron and iron-related proteins in the lungs. However, the role of iron overload in ARDS development has yet to be characterized. In this study, we compared the highly iron-overloaded hepcidin knockout mice (HKO) to their iron-sufficient wild-type (WT) littermates in a model of sterile acute lung injury (ALI) induced by treatment with oropharyngeal (OP) LPS. There were no major differences in systemic inflammatory response or airway neutrophil infiltration between the two groups at the time of maximal injury (days 2 and 3) or during the recovery phase (day 7). Hepcidin knockout mice had transiently increased bronchoalveolar lavage fluid (BALF) protein and MPO activity in the lung and BALF on day 3, indicating worse vascular leakage and increased neutrophil activity, respectively. The increased ALI severity in iron-overloaded mice may be a result of increased apoptosis of lung tissue, as evidenced by an increase in cleaved capsase-3 protein in lung homogenates from HKO mice versus WT mice on day 3. Altogether, our data suggest that even severe iron overload has a relatively minor and transient effect in LPS-induced ALI.

Iron in Lung Pathology.

The lung presents a unique challenge for iron homeostasis. The entire airway is in direct contact with the environment and its iron particulate matter and iron-utilizing microbes. However, the homeostatic and adaptive mechanisms of pulmonary iron regulation are poorly understood. This review provides an overview of systemic and local lung iron regulation, as well as the roles of iron in the development of lung infections, airway disease, and lung injury. These mechanisms provide an important foundation for the ongoing development of therapeutic applications.

In a Mouse Model of Sepsis, Hepcidin Ablation Ameliorates Anemia More Effectively than Iron and Erythropoietin Treatment.

Intensive care unit (ICU) anemia is an extreme version of anemia of inflammation that occurs commonly in critically ill patients and is associated with increased morbidity and mortality. Currently available therapies for ICU anemia have shown inconsistent efficacies in clinical trials. We conducted a systematic study of the effects of early versus delayed iron (Fe) and/or erythropoietin (EPO) therapy in our previously characterized mouse model of ICU anemia based on an injection of heat-killed Brucella abortus. To study the effects of ongoing inflammation on the response to therapy, inflamed wild-type (WT) and hepcidin knockout (HKO) mice were treated at either early (days 1 and 2) or delayed (days 7 and 8) time points after the inflammatory stimulus. In the early treatment group, Fe and/or EPO therapy did not increase hemoglobin (Hgb) levels or reticulocyte production in either the inflamed WT or HKO groups. In the delayed treatment group, combination Fe + EPO therapy did increase Hgb and reticulocyte production in WT mice (mean ΔHgb in WT saline group -9.2 g/dL vs. Fe/EPO -5.5 g/dL; P < 0.001). The HKO mice in the delayed treatment group did not improve their Hgb, but HKO mice in all treatment groups developed a milder anemia than the WT mice. Our findings indicate that combination Fe + EPO therapy is effective in partially reversing ICU anemia when administered after the phase of acute inflammation. Hepcidin ablation alone was more effective in attenuating ICU anemia than Fe + EPO therapy, which indicates the potential of antihepcidin therapeutics in treating ICU anemia.

Isocitrate treatment of acute anemia of inflammation in a mouse model.

Acute and severe anemia of inflammation (AI) is a common complication of various clinical syndromes, including fulminant infections, critical illness with multiorgan failure, and exacerbations of autoimmune diseases. Building on recent data showing beneficial results with isocitrate treatment for chronic low-grade AI in a rat model, we used a mouse model of acute and severe AI induced by intraperitoneal heat-killed Brucella abortus to determine if isocitrate would be effective in this more stringent application. Inflamed mice treated with isocitrate developed an early but transient improvement in hemoglobin compared to solvent-treated controls, with a robust improvement on day 7, and only a trend towards improvement by day 14. Reticulocyte counts were increased in treated mice transiently, with no significant difference by day 21. Serum erythropoietin (EPO) levels were similar in treated versus control mice, indicating that isocitrate increased sensitivity to EPO. Serum and tissue iron levels showed no significant differences between the treated and control mice, ruling out improved iron availability as the cause of the increased response to endogenous EPO. Compared to the milder rat model, much higher doses of isocitrate were required for a relatively modest benefit.

New insights into iron regulation and erythropoiesis.

PURPOSE OF REVIEW: Iron homeostasis and erythropoiesis regulate each other to ensure optimal delivery of oxygen and iron to cells and tissues. Defining the mechanisms of this crosstalk is important for understanding the pathogenesis of common conditions associated with disordered iron metabolism and erythropoiesis. RECENT FINDINGS: Stress erythropoiesis causes suppression of hepcidin to increase iron availability for hemoglobin synthesis. The erythroid hormone erythroferrone (ERFE) was identified as the mediator of this process. ERFE and additional candidates (TWSG1 and GDF15) may also mediate hepcidin suppression in ineffective erythropoiesis. Several mechanisms by which iron regulates erythropoiesis were also recently identified. Iron deficiency suppresses erythropoietin production via the IRP1-HIF2α axis to prevent excessive iron usage by erythropoiesis during systemic iron restriction. Iron restriction also directly impairs erythroid maturation by inhibiting aconitase, and this can be reversed by the administration of the aconitase product isocitrate. Another novel target is GDF11, which is thought to autoinhibit erythroid maturation. GDF11 traps show promising pharmacologic activity in models of both ineffective erythropoiesis and iron-restricted anemia. SUMMARY: This review summarizes exciting advances in understanding the mechanisms of iron and erythropoietic regulation, and development of novel therapeutic tools for disorders resulting from dysregulation of iron metabolism or erythropoiesis.

Mouse models of anemia of cancer.

Anemia of cancer (AC) may contribute to cancer-related fatigue and impair quality of life. Improved understanding of the pathogenesis of AC could facilitate better treatment, but animal models to study AC are lacking. We characterized four syngeneic C57BL/6 mouse cancers that cause AC. Mice with two different rapidly-growing metastatic lung cancers developed the characteristic findings of anemia of inflammation (AI), with dramatically different degrees of anemia. Mice with rapidly-growing metastatic melanoma also developed a severe anemia by 14 days, with hematologic and inflammatory parameters similar to AI. Mice with a slow-growing peritoneal ovarian cancer developed an iron-deficiency anemia, likely secondary to chronically impaired nutrition and bleeding into the peritoneal cavity. Of the four models, hepcidin mRNA levels were increased only in the milder lung cancer model. Unlike in our model of systemic inflammation induced by heat-killed Brucella abortus, ablation of hepcidin in the ovarian cancer and the milder lung cancer mouse models did not affect the severity of anemia. Hepcidin-independent mechanisms play an important role in these murine models of AC.

A mouse model of anemia of inflammation: complex pathogenesis with partial dependence on hepcidin.

Anemia is a common complication of infections and inflammatory diseases, but the few mouse models of this condition are not well characterized. We analyzed in detail the pathogenesis of anemia induced by an injection of heat-killed Brucella abortus and examined the contribution of hepcidin by comparing wild-type (WT) to iron-depleted hepcidin-1 knockout (Hamp-KO) mice. B abortus-treated WT mice developed severe anemia with a hemoglobin nadir at 14 days and partial recovery by 28 days. After an early increase in inflammatory markers and hepcidin, WT mice manifested hypoferremia, despite iron accumulation in the liver. Erythropoiesis was suppressed between days 1 and 7, and erythrocyte destruction was increased as evidenced by schistocytes on blood smears and shortened red blood cell lifespan. Erythropoietic recovery began after 14 days but was iron restricted. In B abortus-treated Hamp-KO compared with WT mice, anemia was milder, not iron restricted, and had a faster recovery. Similarly to severe human anemia of inflammation, the B abortus model shows multifactorial pathogenesis of inflammatory anemia including iron restriction from increased hepcidin, transient suppression of erythropoiesis, and shortened erythrocyte lifespan. Ablation of hepcidin relieves iron restriction and improves the anemia.

Investigation of the role of interleukin-6 and hepcidin antimicrobial peptide in the development of anemia with age.

Anemia is common in older adults and associated with adverse health outcomes in epidemiological studies. A thorough understanding of the complex pathophysiological mechanisms driving anemia in the elderly is lacking; but inflammation, iron restriction, and impaired erythroid maturation are thought to influence the phenotype. We hypothesized that interleukin-6 contributes to this anemia, given its pro-inflammatory activities, its ability to induce hepcidin antimicrobial peptide, and its negative impact on several tissues in older adults. We tested this hypothesis by comparing changes in indices of inflammation, iron metabolism and erythropoiesis in aged C57BL/6 mice to aged mice with targeted deletions of interleukin-6 or hepcidin antimicrobial peptide. Circulating neutrophil and monocyte numbers and inflammatory cytokines increased with age. Decline in hemoglobin concentration and red blood cell number indicated that C57BL/6, interleukin-6 knockout mice, and hepcidin antimicrobial peptide knockout mice all demonstrated impaired erythropoiesis by 24 months. However, the interleukin-6 knock out genotype and the hepcidin antimicrobial peptide knock out genotype resulted in improved erythropoiesis in aged mice. Increased erythropoietic activity in the spleen suggested that the erythroid compartment was stressed in aged C57BL/6 mice compared to aged interleukin-6 knockout mice. Our data suggest C57BL/6 mice are an appropriate mammalian model for the study of anemia with age. Furthermore, although interleukin-6 and hepcidin antimicrobial peptide are not required, they can participate in the development of anemia in aging mice, and could be targeted, pre-clinically, with existing interventions to determine the feasibility of such agents for the treatment of anemia in older adults.