Butyrate Prevents the Pathogenic Anemia-Inflammation Circuit by Facilitating Macrophage Iron Export

Abstract

Most patients with inflammatory bowel disease (IBD) develop anemia, which is attributed to the dysregulation of iron metabolism. Reciprocally, impaired iron homeostasis also aggravates inflammation. How this iron-mediated, pathogenic anemia-inflammation crosstalk is regulated in the gut remains elusive. Herein, it is for the first time revealed that anemic IBD patients exhibit impaired production of short-chain fatty acids (SCFAs), particularly butyrate. Butyrate supplementation restores iron metabolism in multiple anemia models. Mechanistically, butyrate upregulates ferroportin (FPN) expression in macrophages by reducing the enrichment of histone deacetylase (HDAC) at the Slc40a1 promoter, thereby facilitating iron export. By preventing iron sequestration, butyrate not only mitigates colitis-induced anemia but also reduces TNF-α production in macrophages. Consistently, macrophage-conditional FPN knockout mice exhibit more severe anemia and inflammation. Finally, it is revealed that macrophage iron overload impairs the therapeutic effectiveness of anti-TNF-α antibodies in colitis, which can be reversed by butyrate supplementation. Hence, this study uncovers the pivotal role of butyrate in preventing the pathogenic circuit between anemia and inflammation.

Keywords: anemia; butyrate; ferroportin; inflammatory bowel disease; macrophages.

Figure 1

Insufficient SCFA production is correlated with inflammation‐associated anemia in IBD patients. A) The levels of indicated SCFAs in the serum from A‐IBD patients (n = 20), NA‐IBD patients (n = 20), and healthy controls (n = 10) were measured. ^* p < 0.05; ^** p < 0.01; ^*** p < 0.001, unpaired, two‐tailed Student's t‐test. B) The proportions of A‐IBD and NA‐IBD patients were analyzed in SCFAlow patients (n = 20). C) ROC curve analysis indicated the predictive roles of serum SCFAs in distinguishing between A‐IBD and NA‐IBD patients. D) The correlation between hemoglobin and serum iron was analyzed using Spearman's correlation test. D) The correlations between serum iron and SCFA levels in IBD patients were analyzed using Spearman's correlation test, ^* p < 0.05; ^*** p < 0.001.

Figure 2

Butyrate prevents anemia in colitis by maintaining iron homeostasis. A,B) Mice were fed 2.5% DSS with or without 150 mm butyrate in drinking water. Body weight change (A) and colon length (B) were measured (n = 4 for H₂O groups, n = 7 for DSS groups). C) The levels of hemoglobin, serum iron, and transferrin saturation were evaluated. D) Iron levels in colon tissues were evaluated. E) Mice were pretreated with 150 mm sodium butyrate in drinking water for two days, followed by intraperitoneal (i.p.) injection of 5 mg k⁻¹g LPS for 6 h. The levels of serum iron were measured (n = 3 to 5 per group). F) Control or butyrate‐administered mice were intraperitoneally injected with 40 mg k⁻¹g PHZ, the levels of hemoglobin and serum iron were evaluated 2 days after injection (n = 5 per group). G) Mice were fed on a low iron diet (8 ppm, n = 7 per group) or control diet (45 ppm, n = 3 per group) for four weeks, with or without 100 mm butyrate in drinking water. The levels of serum iron were measured. H–J) Mice were intraperitoneally injected with clodronate liposomes to deplete macrophages, followed by 2.5% DSS challenge with or without butyrate administration (n = 4 to 5 per group). Body weight change (H) and colon length (I) were measured. J) The levels of hemoglobin, serum iron, and colonic iron were evaluated. ^* p < 0.05; ^** p < 0.01; ^*** p < 0.001, unpaired, two‐tailed Student's t‐test.

Figure 3

Butyrate prevents iron overload in macrophages by promoting FPN expression. A) Peritoneal macrophages (PMs) were treated with 2 mm butyrate for 12 h, RNA sequencing was performed. The levels of iron metabolism‐related genes were analyzed. B) PMs were treated with 1 mm butyrate for 12 h, the protein levels of FPN were evaluated by flow cytometry. C) Human PBMC‐derived macrophages were treated with 2 mm butyrate for 12 h, FPN expression was evaluated by QPCR. D) PMs were pretreated with 1 mm butyrate followed by 1 µg mL⁻¹ LPS stimulation for 12 h, FPN expression was evaluated by QPCR. E) Mice were fed 2.5% DSS with or without 150 mm butyrate in drinking water, the expression of FPN in colonic macrophages was evaluated by QPCR. (n = 4 to 7 per group). F) FPN expression in human colon immune cells was explored using Single Cell Portal (https://singlecell.broadinstitute.org/single_cell, accession number SCP259). G) The quantification of monocyte and macrophage FPN expression in (F). H) Mice were fed 2.5% DSS for 8 days, the protein levels of FPN on CD45⁺CD11b⁺F4/80⁺ colonic macrophages were evaluated by flow cytometry. I,J) PMs were pretreated with 1 mm butyrate for 4 h, followed by 1 µ g mL⁻¹ LPS stimulation overnight. The intracellular iron contents were measured (I), the fluorescence intensity of calcein‐AM was evaluated by flow cytometry (J). K) The expression levels of FPN in the colonic mucosa from A‐IBD and NA‐IBD patients were evaluated by QPCR (n = 20 per group). ^* p < 0.05; ^** p < 0.01; ^*** p < 0.001, unpaired, two‐tailed Student's t‐test. L) The correlations between mucosal FPN expression and serum butyrate, serum iron, and hemoglobin concentrations in IBD patients (n = 40) were analyzed by Spearman's correlation test, ^*** p < 0.001.

Figure 4

FPN deficiency in macrophages aggravates colitis‐associated iron disequilibrium. A) Construction strategy of the FPN ^MKO mice. B) FPN ^MWT and FPN ^MKO mice were fed 2.5% DSS with or without 150 mm butyrate in drinking water (n = 5 to 6 per group). Body weight changes were measured. C) The levels of serum iron were evaluated. D) The levels of colonic iron were evaluated. E) PMs from FPN ^MWT and FPN ^MKO mice were pretreated with 1 mm butyrate for 4 h, followed by 1 µ g mL⁻¹ LPS stimulation overnight. The intracellular iron contents were measured. ^* p < 0.05; ^** p < 0.01; ^*** p < 0.001, unpaired, two‐tailed Student's t‐test.

Figure 5

Butyrate inhibits TNF‐α production in macrophages by facilitating iron export. A) PMs were pre‐treated with butyrate for 6 h, followed by 1 µ g mL⁻¹ LPS stimulation for 12 h, the culture supernatants were harvested and were subjected to a cytokine array. Orange boxes indicate TNF‐α (left). Fold changes (LPS + BR vs LPS) were quantified (right). B) The correlations between serum TNF‐α and serum iron, mucosal FPN expression, or serum butyrate concentrations in IBD patients (n = 40) were analyzed using Spearman's correlation test. ^** p < 0.01; ^*** p < 0.001. C,D) The protein levels of TNF‐α in colon homogenates (C) and culture supernatants of colonic macrophages (D) from DSS‐fed mice were evaluated by ELISA (n = 7 per group). E) The protein levels of TNF‐α in colon homogenates from FPN ^MWT and FPN ^MKO mice were evaluated by ELISA (n = 5 to 6 per group, left). The mean inhibition rates of butyrate in FPN ^MWT and FPN ^MKO mice were calculated (right). F) PMs from FPN ^MWT and FPN ^MKO mice were pretreated with PBS or 1 mm butyrate for 6 h, followed by 1 µg mL⁻¹ LPS stimulation for 6 h. The levels of TNF‐α in culture supernatants were evaluated by ELISA (left). The mean inhibition rates of butyrate in FPN ^MWT and FPN ^MKO macrophages were calculated (right). G) PMs were pretreated with PBS or 1 mm butyrate for 6 h, followed by 1 µg mL⁻¹ LPS stimulation for 6 h in the presence of FAC (100 µm) or DFO (40 µm). The levels of TNF‐α in culture supernatants were evaluated by ELISA (left). The mean inhibition rates of butyrate in control or DFO‐treated groups were calculated (right). ^* p < 0.05; ^** p < 0.01; ^*** p < 0.001, unpaired, two‐tailed Student's t‐test.

Figure 6

FPN‐mediated macrophage iron export is required for the optimal response to αTNF‐α therapy. A) The serum iron levels in IFX^NR (n = 10) and IFX^R IBD patients (n = 20) were evaluated. B) The expression levels of mucosal FPN in IFX^NR and IFX^R IBD patients were evaluated by QPCR. C,D) ROC curve analysis indicated the predictive roles of mucosal FPN expression (C) or serum iron (D) in distinguishing between IFX^NR and IFX^R IBD patients. E,F) FPN ^MWT and FPN ^MKO mice were fed 2.5% DSS and were treated with IgG or αTNF‐α. Body weight change was evaluated (E), colon length was measured on day 8 (F). G,H) Mice fed with 2.5% DSS were treated with IgG or αTNF‐α, with or without 150 mm butyrate in drinking water. Body weight change was evaluated (G), colon length was measured on day 8 (H). ^* p < 0.05; ^** p < 0.01; ^*** p < 0.001, unpaired, two‐tailed Student's t‐test.

Figure 7

Butyrate increases FPN expression via inhibiting HDAC1‐Sp1 interaction. A) PMs were treated with 2 mm butyrate for 12 h after a 2‐h pertussis toxin (PT) pretreatment, or treated with 1 mm niacin for 10 h, FPN expression was evaluated by QPCR. B) PMs were treated with TSA for 12 h, FPN expression was evaluated by QPCR. C) PMs were treated with HDAC subtype inhibitors (all at 5 µm) for 12 h, FPN expression was evaluated by QPCR. D) PMs were treated with 1 mm butyrate for indicated times, the levels of H3K18Ac were evaluated by immunoblotting. E) PMs were treated with PBS or 1 mm butyrate for 4 h. ChIP assay was performed to assess the levels of H3K18Ac at Slc40a1 promoter. F) Venn diagram showing proteins that can potentially interact with both human Slc40a1 promoter and HDAC1. G) Sp1 binding sites within the Slc40a1 promoter were predicted using the JASPAR database. H) PMs were treated with PBS or 1 mm butyrate for 4 h. ChIP assay was performed to assess the enrichment of Sp1 at Slc40a1 promoter. I) Peritoneal macrophages were treated with PBS or 1 mm butyrate for 4 h, the interaction between Sp1 and HDAC1 was evaluated by Co‐IP. ^** p < 0.01; ^*** p < 0.001, unpaired, two‐tailed Student's t‐test.

Figure 8

Model illustrating the mechanism that butyrate prevents the vicious cycle between anemia and inflammation in the gut.