Deficiency of the BMP Type I receptor ALK3 partly protects mice from anemia of inflammation

Background: Inflammatory stimuli induce the hepatic iron regulatory hormone hepcidin, which contributes to anaemia of inflammation (AI). Hepcidin expression is regulated by the bone morphogenetic protein (BMP) and the interleukin-6 (IL-6) signalling pathways. Prior results indicate that the BMP type I receptor ALK3 is mainly involved in the acute inflammatory hepcidin induction four and 72 h after IL-6 administration. In this study, the role of ALK3 in a chronic model of inflammation was investigated. The intact, heat-killed bacterium Brucella abortus (BA) was used to analyse its effect on the development of inflammation and hypoferremia in mice with hepatocyte-specific Alk3-deficiency (Alk3fl/fl; Alb-Cre) compared to control (Alk3fl/fl) mice. Results: An iron restricted diet prevented development of the iron overload phenotype in mice with hepatocyte-specific Alk3 deficiency. Regular diet leads to iron overload and increased haemoglobin levels in these mice, which protects from the development of AI per se. Fourteen days after BA injection Alk3fl/fl; Alb-Cre mice presented milder anaemia (Hb 16.7 g/dl to 11.6 g/dl) compared to Alk3fl/fl control mice (Hb 14.9 g/dl to 8.6 g/dl). BA injection led to an intact inflammatory response in all groups of mice. In Alk3fl/fl; Alb-Cre mice, SMAD1/5/8 phosphorylation was reduced after BA as well as after infection with Staphylococcus aureus. The reduction of the SMAD1/5/8 signalling pathway due to hepatocyte-specific Alk3 deficiency partly suppressed the induction of STAT3 signalling. Conclusion: The results reveal in vivo, that 1) hepatocyte-specific Alk3 deficiency partly protects from AI, 2) the development of hypoferremia is partly dependent on ALK3, and 3) the ALK3/BMP/hepcidin axis may serve as a possible therapeutic target to attenuate AI

Intravenous Iron Carboxymaltose as a Potential Therapeutic in Anemia of Inflammation

<div><p>Intravenous iron supplementation is an effective therapy in iron deficiency anemia (IDA), but controversial in anemia of inflammation (AI). Unbound iron can be used by bacteria and viruses for their replication and enhance the inflammatory response. Nowadays available high molecular weight iron complexes for intravenous iron substitution, such as ferric carboxymaltose, might be useful in AI, as these pharmaceuticals deliver low doses of free iron over a prolonged period of time. We tested the effects of intravenous iron carboxymaltose in murine AI: Wild-type mice were exposed to the heat-killed <i>Brucella abortus</i> (BA) model and treated with or without high molecular weight intravenous iron. 4h after BA injection followed by 2h after intravenous iron treatment, inflammatory cytokines were upregulated by BA, but not enhanced by iron treatment. In long term experiments, mice were fed a regular or an iron deficient diet and then treated with intravenous iron or saline 14 days after BA injection. Iron treatment in mice with BA-induced AI was effective 24h after iron administration. In contrast, mice with IDA (on iron deficiency diet) prior to BA-IA required 7d to recover from AI. In these experiments, inflammatory markers were not further induced in iron-treated compared to vehicle-treated BA-injected mice. These results demonstrate that intravenous iron supplementation effectively treated the murine BA-induced AI without further enhancement of the inflammatory response. Studies in humans have to reveal treatment options for AI in patients.</p></div

Hepatic mRNA levels of pSTAT3, MCP-1, SOD2 and activin B in the liver of WT mice after BA and intravenous iron injection.

<p><b>(A)</b> Phosphorylated STAT3 levels compared to α-tubulin in the liver of C57BL/6 mice 4h after intraperitoneal BA or PBS administration followed by intravenous iron or PBS treatment for an additional 2h. <b>(B)</b> Hepatic MCP-1 mRNA levels were determined in WT mice after intraperitoneal BA or PBS administration followed by intravenous iron or PBS injection for an additional 2h (n = 3, 2-way ANOVA P<0,0001; **P = 0.007: PBS/PBS vs BA/PBS; *P = 0.02: PBS/PBS vs BA/iron; **P = 0.007: PBS/iron vs BA/PBS); (*P = 0.02: PBS/iron vs BA/iron; **P = 0.008: BA/PBS vs BA/iron; not shown in the graph). <b>(C)</b> Hepatic SOD2 mRNA levels in WT mice 4h after intraperitoneal BA or PBS administration followed by intravenous iron or PBS treatment for an additional 2h (n = 3, 2-way ANOVA P = 0.0002; **P = 0.004: PBS/PBS vs BA/PBS; **P = 0.004: PBS/PBS vs BA/iron); (**P = 0.006: PBS/iron vs BA/PBS; **P = 0.007: PBS/iron vs BA/iron, not shown in graph). <b>(D)</b> Hepatic activin B mRNA levels in WT mice 4h after intraperitoneal BA or PBS administration followed by intravenous iron or PBS injection for an additional 2h (n = 3, 2-way ANOVA P<0,0001; **P = 0.009: PBS/PBS vs BA/PBS; ***P = 0.0002: PBS/PBS vs BA/iron); (**P = 0.009: PBS/iron vs BA/PBS; ***P = 0.0003: PBS/iron vs BA/iron, not shown in graph).</p

Serum cytokine levels measured after BA and intravenous iron administration.

<p>(<b>A</b>) Serum IL-6 protein levels were determined in C57BL/6 mice 4h after intraperitoneal BA or PBS administration followed by intravenous iron or PBS injection for an additional 2h (n = 3, 2-way ANOVA P = 0.003; *P = 0.04: PBS/PBS vs BA/PBS; *P = 0.04: PBS/iron vs BA/PBS). <b>(B)</b> Serum TNF-α levels (n = 3, 2-way ANOVA P = 0.03; **P = 0.009: PBS/PBS vs BA/PBS; *P = 0.03: PBS/PBS vs BA/iron; **P = 0.009: PBS/iron vs BA/PBS; *P = 0.03: PBS/iron vs BA/iron).</p

Philippe Lachaud, architecte et urbaniste (1935-2012)

Liver and spleen iron content from Alk3fl/fl and Alk3fl/fl; Alb-Cre mice 14 days after BA challenge. (a) Liver iron content in Alk3fl/fl and Alk3fl/fl; Alb-Cre mice 14 days after heat-killed Brucella abortus (BA) injection (*P = 0.04: Alk3fl/fl; Alb-Cre injected with saline [n = 4] vs Alk3fl/fl; Alb-Cre injected with BA [n = 6]). (b) Spleen iron content from Alk3fl/fl and Alk3fl/fl; Alb-Cre mice 14 days after heat-killed Brucella abortus (BA) injection. (TIFF 82 kb

Intravenous iron treated BA-induced anemia in mice.

<p>WT mice were challenged with intraperitoneal BA injection and 14d later treated with intravenous iron. <b>(A)</b> Hemoglobin levels 24h after the iron treatment in mice fed a regular (black bars) or iron deficient (white bars) diet (regular diet: n = 3, 2-way ANOVA P < 0,0001; ***P = 0.0006: PBS/PBS vs BA/PBS; *P = 0.02: BA/PBS vs BA/iron). <b>(B)</b> Hemoglobin levels 7d after the intravenous iron treatment as in (A) (iron deficient diet: n = 4, 2-way ANOVA P = 0.002; ***P = 0.0007: PBS/PBS vs BA/PBS; **P = 0.003: BA/PBS vs BA/iron). <b>(C)</b> Serum iron levels 24h after intravenous iron treatment in mice on a regular or iron deficient diet (iron deficient diet: n = 4, 2-way ANOVA P = 0.007; **P = 0.006: PBS/PBS vs BA/PBS; **P = 0.006: BA/PBS vs BA/iron). <b>(D)</b> Serum iron levels 7d after intravenous iron treatment as in (C).</p

Hepcidin response to iron treatment depends on the diets.

<p>WT mice were challenged with or without intraperitoneal BA injection and 14d later treated with or without intravenous iron. <b>(A)</b> Hepatic hepcidin mRNA levels 24h after iron treatment in mice fed a regular iron diet (black bars) or an iron deficient diet (white bars) (iron deficient diet: n = 4, 2-way ANOVA P = 0.006; *P = 0.01: PBS/PBS vs BA/iron; ***P = 0.0002: BA/PBS vs BA/iron). <b>(B)</b> Hepcidin mRNA levels 7d after the iron treatment in mice as in (A) (iron deficient diet: n = 4, 2-way ANOVA P = 0.0007; *p = 0.02: PBS/PBS vs BA/PBS; **P = 0.001: BA/PBS vs BA/iron). <b>(C)</b> Liver iron content (LIC) was determined in C57BL/6 mice fed a regular or iron deficient diet. LIC 14d after BA and 24h after intravenous iron administration are shown (regular diet: n = 3–4, 2-way ANOVA P = 0.002; **P = 0.008: PBS/PBS vs BA/PBS, iron deficient diet: n = 4, 2-way ANOVA, P < 0.0001; **P = 0.005: PBS/PBS vs BA/iron; **P = 0.004: BA/PBS vs BA/iron). <b>(D)</b> LIC 14d after BA and 7d after iron treatment in mice fed a regular or an iron deficient diet (iron deficient diet: n = 4, 2-way ANOVA P = 0.0002; *P = 0.01: PBS/PBS vs BA/iron; *P = 0.01: BA/PBS vs BA/iron).</p

Reticulocyte production index (RPI) in BA-challenged mice fed a regular or an iron deficient diet and treated with intravenous iron or PBS.

<p>WT mice were fed a regular or iron deficient diet and injected with BA intraperitoneally followed 14d later by intravenous iron or PBS treatment. <b>(A)</b> Reticulocyte production Index (RPI = Retic%xHb/14.46, with 14.46g/dL as the mean baseline hemoglobin (Hb) level of healthy WT mice) 24h after iron or PBS treatment (2-way ANOVA P = 0.01, regular diet: n = 3, *P = 0.03: PBS/PBS vs BA/PBS; iron deficient diet: n = 3; *P = 0.02: PBS/PBS vs BA/iron). <b>(B)</b> Reticulocyte production 7d after iron or PBS treatment (iron deficient diet: n = 3–4, 2-way ANOVA P = 0.008; *P = 0.04: PBS/PBS vs BA/iron).</p

Additional file 1: of Deficiency of the BMP Type I receptor ALK3 partly protects mice from anemia of inflammation

Experimental design. (a) Mice were fed an iron deficient diet since weaning and throughout the experiment. At the age of 12 weeks, female Alk3fl/fl; Alb-Cre and Alk3fl/fl mice were intraperitoneally injected with 5 × 108 particles/mouse of heat-killed Brucella abortus (BA) or saline. Two weeks later blood and organs were collected. (b) 12 week old Alk3fl/fl; Alb-Cre and Alk3fl/fl female mice fed a regular diet were intravenously inoculated with 1 × 106 colony forming units (CFUs) of Staphylococcus aureus. Twenty-four hours later blood and organs were collected. (TIFF 164 kb

Induction of hepatic hepcidin mRNA levels and serum iron levels after intraperitoneal injection of BA and/or intravenous iron carboxymaltose administration.

<p><b>(A)</b> Hepatic hepcidin mRNA levels were determined by quantitative RT-PCR in C57BL/6 mice 4h after intraperitoneal BA or PBS administration followed by intravenous iron or PBS treatment for an additional 2h (values represent mean±SD, n = 3, 2-way ANOVA, P = 0.002; **P = 0.004: PBS/PBS vs BA/PBS, and **P = 0.002: PBS/PBS vs BA/iron). <b>(B)</b> Serum iron levels were measured (values represent mean±SD, n = 3, 2-way ANOVA, P = 0.0008; *P = 0.02: PBS/PBS vs PBS/iron, *P = 0.01: PBS/PBS vs BA/PBS, *P = 0.04: PBS/PBS vs BA/iron, and *P = 0.03: BA/PBS vs BA/iron).</p