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C-FGF23 peptide alleviates hypoferremia
ABC
Figure 4. Inhibition of fibroblast growth factor 23 (FGF23) signaling decreases Fgf23 expression and circulating levels induced by lipopolysaccharide (LPS). C57BL/6J wild-type mice were injected intraperitoneally (i.p.) with C-tail FGF23 (1 mg/kg, indicated as FGF23 BL) or vehicle (HEPES buffer) for 8 hours (h). Mice were then challenged with LPS (i.p. 50 mg/kg) or vehicle (0.9% NaCl) for 4 h. (A) Quantitative real-time polymerase chain reaction (qRT-PCR) for Fgf23 expression in bone. Data are expressed as fold change (2-DDCt) relative to housekeeping gene Gapdh. (B and C) Serum concentration of (B) C-terminal FGF23 (cFGF23) and (C) intact FGF23 measured by ELISA. Samples were measured in duplicates (n=5-7 per group). Data are represented as mean+standard deviation. All data were analyzed for normality with Shapiro-Wilk test and equivalence of variance using Levene’s test. Because the samples did not show normal distribution, data were aligned in RANK transformation, and confirmed for normality. As the samples showed normal distribution, two-way ANOVA was performed with Bonferroni’s multiple comparison test. Ctl: control (vehicle), ns: not significant, *P<0.05, **P<0.01, ***P<0.001.
confirmed this effect in mice injected with LPS or saline (Figure 7D) using a 10 times lower concentration of the FGF23 C-tail blocking peptide than the one we used in our previous work.29 However, we found that 4 h of LPS treat- ment at the concentration of 50 mg/kg were not sufficient to alter circulating Epo levels (Figure 7D). In addition to its effect on renal Epo and EpoR, LPS significantly decreased the expression of splenic and hepatic Epo and EpoR (Figure 7E-H), which was rescued by treatment with the FGF23 C-tail blocking peptide (Figure 7E-H). These data clearly demonstrate the efficiency of the C-tail FGF23 in increas- ing renal and extra-renal Epo and EpoR expression in the presence of inflammation, leading to increased serum Epo levels. Our findings also indicate that in the absence of inflammation, the C-tail FGF23 increases circulating Epo levels by upregulating splenic Epo expression without affecting renal or hepatic Epo expression.
Discussion
Iron is an essential micronutrient for host-pathogen interaction. During infection, pathogens develop multiple strategies to acquire iron for proliferation and virulence, such as secretion of ferric iron-binding molecules known as siderophores.41 A host defense mechanism to inhibit pathogen growth is to deprive invading pathogens of iron, a process known as nutritional immunity by sequestering iron in tissues, thus limiting availability of circulating iron and resulting in hypoferremia.42 Iron homeostasis is con- trolled by two proteins, the hepatic hormone hepcidin which regulates iron absorption, and the iron exporter fer- roportin which exports iron into the circulation from duo-
denal enterocytes, hepatocytes, and splenic reticulo- endothelial macrophages that recycle the iron of senescent erythrocytes.43 Toll-like receptors (TLR) are key compo- nents of the innate immune system that recognize pathogen-associated molecular patterns (PAMP). Activation of TLR signaling plays a pivotal role in the development of the hypoferremic host response.2,3,44 Several toll like receptors such as TLR3, 4, 7, 7/8 and 9 have been described to induce hepcidin expression in vitro and in vivo.44
Several studies have demonstrated that iron deficiency induces FGF23 production.12,13,45 We recently reported that inhibition of FGF23 signaling using the C-tail FGF23 res- cues anemia and iron deficiency in a CKD mouse model characterized by chronic inflammation, by increasing EPO, serum iron and ferritin levels, and attenuating inflammation.29 Our goals in the present study were: (i) to decipher the kinetics of Fgf23 in response to LPS in com- parison to the kinetics of inflammatory, iron metabolism, and erythropoiesis parameters; and (ii) to investigate the impact of FGF23 signaling inhibition during LPS-induced acute inflammation.
Our study showed that FGF23 is induced as early as pro- inflammatory cytokines in response to LPS (Figure 1), fol- lowed by upregulation of hepcidin and decrease in serum iron and transferrin saturation (Figure 3A-C), suggesting that the early increase in FGF23 by LPS may contribute to hypoferremia. Previous studies have shown that FGF23 is produced by macrophages and acts locally to stimulate TNF-α expression via activation of FGFR1/α-Klotho sig- naling.46 Moreover, acute inflammation induced by inter- leukin-1β (IL-1β) or heat-killed Brucella abortus in mice sig- nificantly increased Fgf23 production.12 Thus, since FGF23
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