The manganese transporter SLC39A8 links alkaline ceramidase 1 to inflammatory bowel disease

Choi, Eun-Kyung; Rajendiran, Thekkelnaycke M.; Soni, Tanu; Park, Jin-Ho; Aring, Luisa; Muraleedharan, Chithra K.; Garcia-Hernandez, Vicky; Kamada, Nobuhiko; Samuelson, Linda C.; Nusrat, Asma; Iwase, Shigeki; Seo, Young Ah

doi:10.1038/s41467-024-49049-8

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Published: 05 June 2024

The manganese transporter SLC39A8 links alkaline ceramidase 1 to inflammatory bowel disease

Nature Communications volume 15, Article number: 4775 (2024) Cite this article

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Abstract

The metal ion transporter SLC39A8 is associated with physiological traits and diseases, including blood manganese (Mn) levels and inflammatory bowel diseases (IBD). The mechanisms by which SLC39A8 controls Mn homeostasis and epithelial integrity remain elusive. Here, we generate Slc39a8 intestinal epithelial cell-specific-knockout (Slc39a8-IEC KO) mice, which display markedly decreased Mn levels in blood and most organs. Radiotracer studies reveal impaired intestinal absorption of dietary Mn in Slc39a8-IEC KO mice. SLC39A8 is localized to the apical membrane and mediates ⁵⁴Mn uptake in intestinal organoid monolayer cultures. Unbiased transcriptomic analysis identifies alkaline ceramidase 1 (ACER1), a key enzyme in sphingolipid metabolism, as a potential therapeutic target for SLC39A8-associated IBDs. Importantly, treatment with an ACER1 inhibitor attenuates colitis in Slc39a8-IEC KO mice by remedying barrier dysfunction. Our results highlight the essential roles of SLC39A8 in intestinal Mn absorption and epithelial integrity and offer a therapeutic target for IBD associated with impaired Mn homeostasis.

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Introduction

Inflammatory bowel diseases (IBDs), which comprise Crohn’s disease and ulcerative colitis, are major chronic inflammatory disorders affecting the gastrointestinal tract in humans. The etiology and pathogenesis of IBD remain unknown; nevertheless, the prevailing notion is that a complex interplay among genetic, environmental, microbial, and immune factors contributes to the disorders¹. A significant pathogenic factor for IBD is the disruption of the epithelial barrier function, as this leads to intestinal hyperpermeability². The intestinal epithelial barrier consists of epithelial cells and intercellular junctions that seal the paracellular space and regulate the permeability of the mucosal barrier³. This barrier is selective and regulated, permitting the uptake of luminal nutrients while restricting the entry of pathogens and toxins into the intestine³. Consequently, a compromised barrier function leads to increased intestinal permeability of luminal antigens and bacteria into the lamina propria, resulting in pathologic immune responses and chronic inflammation^4,5. Thus, the treatment of intestinal barrier dysfunction may offer therapeutic options for IBDs.

Epidemiological studies have suggested an association between an increased risk of IBD and manganese (Mn) deficiency⁶. Mn is an essential nutrient required for fundamental physiological processes, such as bone formation, immune responses, and carbohydrate metabolism⁷. This multifunctionality likely reflects the action of this nutrient as a constituent of multiple enzymes and an activator of other enzymes⁸. Mn is acquired from the diet and is abundant in plant-based foods, such as whole grains, legumes, rice, nuts, and vegetables, whereas it is deficient in animal sources, including meat, fish, poultry, eggs, and dairy products⁹. Mn deficiency has long been considered rare in humans due to its abundance in dietary sources; therefore, the argument has been made that Mn deficiency might not contribute to IBD. However, increasing evidence has indicated that dietary Mn consumption has decreased by more than 40% in the past 15 years in developed countries, including the US^9,10,11, likely due to increasing consumption of a Western diet characterized by high intakes of meats, sugar, and refined grains^9,10,11. These studies suggest that dietary Mn deficiency is prevalent in the today’s animal-based diets, paralleling the increasing incidence of IBD¹². A causal relationship linking Mn deficiency to IBD was previously unknown; however, we have recently demonstrated that dietary Mn deficiency exacerbates the intestinal injury and inflammation associated with colitis in mice, whereas dietary Mn supplementation confers protection against colitis¹³. We also showed that dietary Mn deficiency increased intestinal permeability by impairing intestinal tight junctions¹³. This work provided evidence that Mn is necessary for proper maintenance of the intestinal barrier function and that it protects against colon injury, thereby highlighting a role for Mn in intestinal homeostasis and the occurrence of IBD¹³.

In addition to dietary Mn, several traits and diseases, including whole blood Mn level¹⁴ and Crohn’s disease^15,16, are significantly associated with a common genetic variant in the metal ion transporter SLC39A8 (p.Ala391Thr; A391T). Also known as ZIP8, SLC39A8 is a transmembrane transporter that can mediate the cellular uptake of zinc¹⁷, iron¹⁸, Mn^19,20, and cadmium²¹. Recent genetic studies on human subjects and animal models have revealed that SLC39A8 plays an essential role in regulating Mn levels. For example, SLC39A8 loss-of-function mutations have been identified in patients with severe Mn deficiency^22,23. These patients had inappropriately low blood levels of Mn, whereas the levels of other metals were normal^22,23. We used radiotracer studies to show that SLC39A8 is a cell-surface transporter that strongly stimulates ⁵⁴Mn incorporation into cells. By contrast, the disease-associated mutations completely abrogated the cellular uptake of ⁵⁴Mn²⁴, thereby providing a causal link between SLC39A8 deficiency and Mn deficiency. Further study of hepatocyte-specific Slc39a8 KO mice showed that SLC39A8 was localized to the apical membrane of hepatocytes, where it likely functions to reclaim Mn from the bile, suggesting that this transporter is essential for hepatic Mn homeostasis²⁵. These studies establish a role for SLC39A8 in the regulation of Mn levels, further supporting the hypothesis that the SLC39A8 A391T variant may increase the susceptibility to IBD by disturbing Mn homeostasis.

SLC39A8-related diseases have only been recently discovered, and the impact of the SLC39A8 A391T variant on human health and disease is just beginning to be appreciated; therefore, our understanding of the role of SLC39A8 in Mn homeostasis and its contribution to IBD remains limited. The important unresolved issues are: (1) the role of SLC39A8 in intestinal homeostasis; (2) the mechanisms by which SLC39A8 deficiency contributes to the pathophysiology of IBD; and (3) the therapeutic targets for IBD patients with SLC39A8 deficiency. In the present study, we hypothesized that epithelial SLC39A8 controls intestinal Mn homeostasis and epithelial integrity and that dysregulation of Mn homeostasis disrupts the epithelial barrier, thereby causing a predisposition to IBD development. We tested this hypothesis by generating Slc39a8 intestinal epithelial cell (IEC)-specific knockout (Slc39a8-IEC KO) mice and intestinal organoid monolayer cultures to test Mn transport and epithelial integrity. We first demonstrated that IEC-specific Slc39a8 deletion induced Mn deficiency in the blood and multiple organs, including the intestine. We also used radiotracer ⁵⁴Mn studies to show that Slc39a8 is essential for in vivo intestinal Mn absorption. We then confirmed that Slc39a8 mediates the uptake of ⁵⁴Mn at the apical membrane of intestinal organoid monolayer cultures. Our unbiased RNA-seq analysis in the Slc39a8-IEC KO intestine identified alkaline ceramidase 1 (ACER1), a lipid-generating enzyme that affects intestinal permeability, as a prominent target for maintaining intestinal epithelial integrity. We then demonstrated that treatment with an ACER1 inhibitor reduced intestinal permeability by enhancing tight junction proteins, thereby suppressing intestinal injury in Slc39a8-IEC KO mice and intestinal organoid monolayer cultures. Our study findings establish that intestinal epithelial SLC39A8 contributes to whole-body Mn homeostasis by controlling intestinal Mn absorption and that, through this mechanism, SLC39A8 maintains intestinal epithelial integrity and protects against epithelial injury.

Results

Loss of intestinal epithelial Slc39a8 leads to systemic Mn deficiency

The intestine plays a vital role in regulating Mn absorption and homeostasis in vivo²⁶. SLC39A8 is ubiquitously expressed, including in the intestine¹⁷. Quantitative PCR (qPCR) analysis of tissues from both female and male C57BL/6J mice revealed higher levels of Slc39a8 in the distal small intestine and colon (Fig. 1a), with no discernible sex-specific differences. Immunofluorescent staining further showed dominant localization of SLC39A8 in the apical membrane of enterocytes in both female and male C57BL/6J mice (Fig. 1b), with no apparent sex-specific differences. The SLC39A8 staining intensity increased, with the highest expression observed in the ileum and colon. In the colon, SLC39A8 immunostaining was localized to the enterocytes lining the epithelium in the crypts. To identify the function of intestinal SLC39A8, we generated Slc39a8 intestinal epithelial cell (IEC)-specific knockout mice (Slc39a8-IEC KO) by crossing floxed Slc39a8 (Slc39a8^fl/fl) mice with Villin-Cre mice (Supplementary Fig. 1a). The qPCR analysis confirmed that Slc39a8-IEC KO mice had significantly decreased Slc39a8 mRNA levels in the duodenum (‒90%, P < 0.001), jejunum (‒90%, P < 0.001), ileum (‒93%, P < 0.001), and colon (‒98%, P < 0.001), but normal Slc39a8 expression in other tissues, including liver, heart, lung, kidney, and brain (Fig. 1c). The Slc39a8-IEC KO mice were born at the expected Mendelian ratios and exhibited grossly normal appearance and body weight (Supplementary Fig. 1b), indicating that IEC-specific deletion of Slc39a8 did not lead to lethality.

**Fig. 1: Intestinal epithelial *Slc39a8* deletion leads to systemic Mn deficiency.**

We determined whether loss of Slc39a8 in IEC alters metal homeostasis by conducting inductively coupled plasma mass spectroscopy (ICP-MS) measurements of metal concentrations in a variety of tissues from Slc39a8-IEC KO and control mice at 20 weeks of age. Compared to the control mice, the Slc39a8-IEC KO mice showed a substantial reduction in Mn concentrations not only in the ileum and colon, but also in the liver, lung, heart, and brain (Fig. 1d). Notably, whole blood Mn was markedly decreased in Slc39a8-IEC KO mice (Fig. 1d). No sex-specific differences were observed in Mn concentrations (Fig. 1d). Although SLC39A8 can also mediate the cellular uptake of zinc¹⁷ and iron¹⁸, the tissue zinc and iron levels did not differ between Slc39a8-IEC KO mice and control mice (Supplementary Fig. 2a, b). Loss of Slc39a8 in the IECs also did not affect the concentrations of other metals, such as copper and selenium (Supplementary Fig. 2c, d). Thus, our Slc39a8-IEC KO mouse model induces a specific deletion of Slc39a8 in the intestine and leads to systemic Mn deficiency with little impact on other metal levels.

Slc39a8 is essential for intestinal absorption of ⁵⁴Mn

The lower Mn concentrations in whole blood and other tissues of the Slc39a8-IEC KO mice (Fig. 1d) suggested a possible role for SLC39A8 in Mn absorption. We tested this possibility by measuring radioactivity in whole blood of control and Slc39a8-IEC KO mice 15 min after administration of ⁵⁴Mn by oral-intragastric gavage. The 15 min time point was selected based on previous studies on intestinal absorption of Mn in mice after oral-intragastric gavage^27,28. We observed that the amount of ⁵⁴Mn in the whole blood was 33% lower (P < 0.05) in the Slc39a8-IEC KO mice than in the control mice (Fig. 2a). We also measured the amount of ⁵⁴Mn in the enterocytes, liver, and other tissues after administration of ⁵⁴Mn via oral-intragastric gavage. Notably, the amount of ⁵⁴Mn in the ileum was markedly reduced by 44% (P < 0.05) in Slc39a8-IEC KO mice compared to control mice (Fig. 2b). We also found that tissue ⁵⁴Mn levels were significantly reduced in the liver (‒61%, P < 0.05), lung (‒59%, P < 0.05), kidney (‒81%, P < 0.05), heart (‒69%, P < 0.05), and femur (‒41%, P < 0.05) in the Slc39a8-IEC KO mice (Fig. 2c).

**Fig. 2: Loss of *Slc39a8* in IECs results in impaired intestinal Mn absorption.**

The decreased ⁵⁴Mn level in the blood after oral-intragastric gavage could be due to increased clearance; therefore, we measured blood clearance 15 min after intravenous injection of ⁵⁴Mn. The ⁵⁴Mn levels in the whole blood did not differ between the control and Slc39a8-IEC KO mice (Fig. 2d). The amount of ⁵⁴Mn in the enterocytes, liver, and other tissues after administration of ⁵⁴Mn via intravenous injection also showed no differences between control and Slc39a8-IEC KO mice (Fig. 2e, f). One possibility is that the reduced ⁵⁴Mn levels in the tissues after a 4 h interval from gavage or intravenous injection may reflect enhanced Mn excretion. However, we found a significant reduction in transcript levels of the intestinal Mn excretion proteins, Slc39a14/ZIP14 and Slc30a10/ZnT10, in Slc39a8-IEC KO-derived enteroids (Supplementary Fig. 3a), thereby limiting the possibility of enhanced excretion of Mn at least in the intestine. Future studies are warranted to determine the contribution of absorption and excretion of Mn in each tissue. Overall, these data demonstrate that intestinal epithelial SLC39A8 is required for normal Mn uptake.

Slc39a8 mediates the uptake of ⁵⁴Mn at the apical membranes in intestinal organoid monolayer cultures

The major route of dietary Mn absorption is the intestine²⁶, where the IECs import Mn across the apical membrane into enterocytes. We examined the cellular mechanisms of Mn uptake by enterocytes by generating two-dimensional cultures of intestinal organoid monolayers directly from intestinal crypts derived from Slc39a8-IEC KO or control mice (Fig. 3a and Supplementary Fig. 3b)²⁹. The ileum and colon were chosen as the tissues for generating the 2D enteroids and colonoids over other intestinal regions, as we observed greater expression of Slc39a8 (Fig. 1a) and a larger reduction in Mn levels in those tissues (Fig. 1d). Immunofluorescence analysis confirmed the localization of SLC39A8 in control enteroid and colonoid monolayers, but its absence in Slc39a8-IEC KO monolayers, with an apical concentration suggested by expression next to the tight junction protein ZO-1 above the nuclear compartment (Fig. 3b). We then tested SLC39A8 Mn transport activity by adding ⁵⁴Mn to the apical chamber, and measuring radioactivity in the intestinal organoid monolayer cells (Fig. 3c). Importantly, apical ⁵⁴Mn accumulation was significantly impaired in the Slc39a8-IEC KO-derived enteroid (‒35%, P < 0.01) and colonoid (‒41%, P < 0.001) monolayer cells (Fig. 3d).

**Fig. 3: Slc39a8 mediates the uptake of ⁵⁴Mn at the apical membrane of intestinal organoid monolayer culture.**

The residual apical ⁵⁴Mn accumulation observed in Slc39a8-IEC KO-derived intestinal organoid monolayer cells suggests an alternative entry route for Mn at the apical membrane; therefore, we examined the expression of other metal ion transporters that could compensate Mn uptake. The Slc39a8-IEC KO-derived enteroids displayed significantly increased Slc11a2/DMT1 (divalent metal transporter 1) transcript levels and significantly decreased Slc39a14/ZIP14, Slc30a10/ZnT10, and Slc40a1/FPN (ferroportin-1) transcript levels (Supplementary Fig. 3a). These data suggest that DMT1 may compensate for the loss of SLC39A8 expression in Mn uptake. We then studied the consequences of the IEC-specific deletion of Slc39a8 on basolateral ⁵⁴Mn absorption by adding ⁵⁴Mn to the basolateral chamber and measuring radioactivity in the intestinal organoid monolayer cells (Fig. 3e). In contrast to the striking effect observed on apical ⁵⁴Mn absorption in the Slc39a8-deficient IECs, ⁵⁴Mn absorption from the basolateral chamber was similar in both monolayer cells (Fig. 3f). These data indicate that SLC39A8 mediates Mn uptake at the apical membrane of an intestinal organoid monolayer culture and that the loss of Slc39a8 in IEC does not substantially affect Mn uptake from the basolateral membrane of intestinal organoid monolayer cultures. Collectively, these observations strongly support a model in which SLC39A8 is localized to the apical surface of the enterocytes, where it mediates the uptake of Mn from the intestinal lumen into enterocytes, thereby facilitating the intestinal absorption of dietary Mn.

IEC-specific deletion of Slc39a8 exacerbates colitis following intestinal epithelial injury

Dietary Mn deficiency has been suggested to disrupt intestinal barrier function²⁰; therefore, our findings that the loss of Slc39a8 in IEC induces intestinal Mn deficiency led us to hypothesize that the IEC-specific deletion of Slc39a8 would alter susceptibility to colitis³⁰. Dextran sodium sulfate (DSS)-induced experimental colitis is a widely used mouse model to induce acute colonic epithelial injury³⁰. We treated Slc39a8-IEC KO and control mice with 3% DSS in the drinking water to induce colitis (inflammatory phase) and then placed on regular drinking water (recovery phase) (Fig. 4a). The DSS treatment induced colitis, characterized by rapid weight loss in control mice (Fig. 4b). Notably, Slc39a8-IEC KO mice displayed more pronounced body-weight loss (P < 0.05, Fig. 4b). Upon returning to regular water, control mice regained body weight, whereas Slc39a8-IEC KO mice did not show any recovery and continued to lose body weight until the experimental endpoint (Fig. 4b). As another indicator of tissue injury, the Slc39a8-IEC KO mice showed a dramatic decrease in colon length when compared with controls (24%, P < 0.001, Fig. 4c, d), indicating exacerbated DSS-induced colitis. The ratio of the spleen weight to body weight was also significantly higher in Slc39a8-IEC KO mice than in the control mice after DSS treatment (P < 0.01, Fig. 4e), indicating infiltration of splenic macrophages (Supplementary Fig. 3c)³⁰. Histological analysis of the colon tissue also revealed significant epithelial degeneration, crypt destruction, severe focal ulceration, and inflammation in Slc39a8-IEC KO mice following DSS treatment (Fig. 4f). The total pathological score was significantly higher in the colons of Slc39a8-IEC KO mice than in control mice (Fig. 4g). Consistent with previous studies^31,32, we observed lower sensitivity of female Slc39a8-IEC KO mice to the effects of DSS-induced colitis (Supplementary Fig. 3d). Since Slc39a8 expression and Mn levels did not differ between sexes (Figs. 1 and 2), the cause of the lower sensitivity of females to DSS is unknown. Based on these observations, we focused on our further study on male mice.

**Fig. 4: Loss of *Slc39a8* in IECs exacerbates DSS-induced acute model of colitis.**

We gained further insight into the change in intestinal permeability after DSS injury by examining barrier function using FITC-dextran (4 kDa molecular weight) as an indicator. Serum FITC-dextran levels on day 13 of DSS treatment were significantly higher in Slc39a8-IEC KO mice than in control mice (Fig. 4h). Increased permeability could be attributed to mucosal erosion and impaired epithelial barrier function³³. Therefore, we examined the expression of tight junction proteins, as these proteins can directly affect intestinal permeability. In Slc39a8-IEC KO mice, our qPCR analysis showed significantly greater reductions in the expression levels of tight junction genes Cldn2, Cldn3, Cldn5, and Cldn7 in the colonic mucosa after DSS treatment compared to control mice (Fig. 4i).

Immunofluorescence staining revealed that Zo1, Zo2, Cldn2, Cldn5, and Ocln were localized at the apical side of the colonic epithelium, while Cldn3 and Cldn7 were detected at the lateral membrane (Fig. 4j and Supplementary Fig. 3e). DSS treatment severely impaired the localization and expression of these tight junction proteins (Fig. 4j and Supplementary Fig. 3e). Notably, significantly reduced expression levels of Cldn3 and Cldn5 were observed in the Slc39a8-IEC KO mice after DSS treatment (Fig. 4j). Immunoblotting analysis confirmed diminished levels of Cldn3 and Cldn5 (Fig. 4k). While Cldn2 levels can increase in the inflammatory state in human tissues^34,35, our data and findings by others^36,37 have indicated decreases in Cldn2 levels (Fig. 4k and Supplementary Fig. 3f), implicating a complex context-dependent Cldn2 regulation. These data suggest a selective impact of Slc39a8 on the transcripts and proteins related to tight junctions. Compared to control mice, Slc39a8-IEC KO mice also showed significantly higher expression of the chemokine genes Ccl2 and Cxcl1 and the inflammatory cytokine gene Tnfa (Fig. 4l). No significant differences were observed between control and Slc39a8-IEC KO mice in the measured parameters without DSS treatment, (Fig. 4f–l).

Exacerbated colitis was also examined in Slc39a8-IEC KO mice using a second model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. Intrarectal administration of the haptenating agent TNBS induces a severe CD4⁺ T cell-dependent colitis in mice, resulting in increased permeability and inflammation³⁸. Our results were similar for both the TNBS-induced colitis model and the DSS-induced colitis model. Slc39a8-IEC KO mice exhibited exacerbated TNBS-induced body weight loss, clinical scores, and colonic injury compared to control mice (Supplementary Fig. 4a–e). Additionally, Slc39a8-IEC KO mice displayed increased gut permeability, reduced expression of tight junction protein transcripts of Zo1 and Cldn5, and increased expression of the chemokine genes Ccl2 and Cxcl1, and the inflammatory cytokine gene Tnfa (Supplementary Fig. 4f–h). These findings demonstrate that IEC-targeted Slc39a8 deficiency exacerbates mucosal damage by disrupting barrier function in both DSS and TNBS colitis models.

We further verified the colitis phenotype of Slc39a8-IEC KO mice in a third model of chronic DSS colitis that employs cyclical administration of DSS in drinking water followed by recovery with water alone. Mice received two cycles of 3% DSS treatment, each cycle consisting of 8 days of the inflammatory phase followed by 5 days of recovery phase. In the second DSS cycle, the clinical scores were assessed daily³⁹ (Fig. 5a). The Slc39a8-IEC KO mice developed clinical symptoms of colitis with increasing severity starting on day 2 of the second cycle and peaking on day 11, whereas the symptoms in control mice were delayed and significantly reduced in severity (Fig. 5b). The Slc39a8-IEC KO mice also showed an approximately 29% decrease in colon length compared with the control mice (Fig. 5c). Moreover, approximately 50% of the Slc39a8-IEC KO mice died by day 13 of the second cycle (Fig. 5d). Histological examination confirmed a marked increase in immune cell infiltration and ulceration in the Slc39a8-IEC KO colon (Fig. 5e), with significantly higher pathological scores on day 13 of the second DSS cycle (Fig. 5f). Without DSS treatment, we observed no significant differences between control and Slc39a8-IEC KO mice in the measured parameters (Fig. 5c–f). Taken together, the data from both the acute DSS colitis and TNBS colitis model, as well as the chronic DSS colitis model, indicated that the loss of epithelial Slc39a8 exacerbates mucosal barrier damage and promotes colon inflammation and colitis.

**Fig. 5: Loss of epithelial Slc39a8 aggravates the DSS-induced chronic model of colitis.**

Alkaline ceramidase 1 is upregulated in the intestines of Slc39a8-IEC KO mice

To identify the mechanisms by which IEC-specific Slc39a8 deficiency impairs intestinal integrity and exacerbates intestinal injury, we employed an unbiased RNA-seq approach using ileal and colonic mucosa from Slc39a8-IEC KO and control mice. Differential gene expression analyses identified four genes with consistently altered expression in the Slc39a8-IEC KO intestines compared to the control (Padj < 0.1, ileum and colon data were combined, Fig. 6a and Supplementary Table 1). Of the four genes, Slc39a8 expression was downregulated in the Slc39a8-IEC KO intestines, demonstrating the validity of the RNA-seq approach. Among the three upregulated genes, alkaline ceramidase 1 (Acer1) drew our attention (Fig. 6a, b). ACER1 is localized to the endoplasmic reticulum (ER)-Golgi network and catalyzes the hydrolysis of ceramides into sphingosine and free fatty acids at an alkaline pH 7.5–9.5⁴⁰. Therefore, ACER1 is a key enzyme in sphingolipid metabolism, a process that is involved in mouse models of colitis and human IBD^41,42,43. ACER1 is also critical for regulating ceramide content⁴⁴. Our qPCR analysis using an independent set of RNA samples confirmed the upregulation of Acer1 expression in the ileal and colonic mucosa from Slc39a8-IEC KO mice compared to control mice (Fig. 6c). The upregulation of Acer1 expression was also confirmed in the enteroid and colonoid monolayers derived from control and Slc39a8-IEC KO mice (Fig. 6d). Further analysis by immunoblotting confirmed an increase in Acer1 protein levels in the ileum (~1.4 fold, P < 0.05) and colon (~1.3 fold, P < 0.05) of Slc39a8-IEC KO mice and the enteroid monolayers (~4.5 fold) of Slc39a8-IEC KO mice (Fig. 6e, f). Although the increases in Acer1 protein levels were relatively smaller in the colonoid monolayers (1.3 fold) of Slc39a8-IEC KO mice, these smaller changes in the colon were consistent with the RNA-seq results. These results demonstrate that Acer1 is upregulated in the intestines of Slc39a8-IEC KO mice.

**Fig. 6: ACER1 is upregulated in the intestine of *Slc39a8*-IEC KO mice.**

Acer1 inhibitor enhances intestinal barrier function in Slc39a8-IEC KO mice-derived intestinal organoid monolayer culture

The finding of higher expression levels of Acer1 in the ileum and colon (Fig. 6) led us to test the contribution of ACER1 to the impaired intestinal barrier function and IBD susceptibility of Slc39a8-IEC KO mice. We selected the ACER1 inhibitor (1S,2R)-d-erythro−2-(N-myristoylamino)−1-phenyl-1-propanol (D-e-MAPP) as it potently and specifically inhibits alkaline ceramidase activity but not acid or neutral ceramidase activity⁴⁵. We first examined the effect of D-e-MAPP on epithelial barrier function in intestinal organoid monolayer cultures derived from Slc39a8-IEC KO and control mice under normal conditions. D-e-MAPP significantly upregulated the tight junction protein transcripts of Zo1, Cldn3, Cldn5, and Cldn7 in Slc39a8-IEC KO mice-derived enteroid and colonoid monolayers (Fig. 7a–d). Further analysis by immunoblotting confirmed that D-e-MAPP treatment enhanced levels of tight junction proteins Zo1, Cldn5, and Cldn7 in enteroid monolayers derived from Slc39a8-IEC KO mice (Fig. 7e). A similar trend was observed for the Cldn3 protein levels, although it did not reach statistical significance. We then performed FITC-dextran permeability assays and transepithelial electrical resistance (TEER) measurements. D-e-MAPP treatment significantly inhibited leakage of FITC-dextran into the bottom chambers in Slc39a8-IEC KO mice-derived enteroid and colonoid monolayers (Fig. 7f). Notably, at the highest D-e-MAPP dose tested, intestinal permeability, as determined by 4-kDa FITC-dextran, was increased to control-like levels, but no higher (Fig. 7f). Furthermore, the epithelial barrier function analysis by TEER measurements revealed that D-e-MAPP significantly increased TEER in the enteroid and colonoid monolayers derived from Slc39a8-IEC KO mice (Fig. 7g). The D-e-MAPP treatment restored the epithelial barrier function determined by TEER measurements back to control-like levels, but no lower (Fig. 7g). Treatment of intestinal organoid monolayer cultures with D-e-MAPP at the indicated doses also did not cause any observable toxicity (Supplementary Fig. 5a). Overall, these results suggest that ACER1 inhibition restores the gut barrier integrity in Slc39a8-deficient enterocytes by increasing the expression of tight junction proteins under normal conditions.

**Fig. 7: ACER1 inhibitor D-e-MAPP promotes upregulation of epithelial tight junction and epithelial barrier function in *Slc39a8-*deficient intestinal organoid monolayer cultures.**

Treatment with Acer1 inhibitor D-e-MAPP mitigates colitis in Slc39a8-IEC KO mice

Preventing increases in gut permeability by enhancing barrier functions could represent a potential therapeutic avenue for treating IBD patients with SLC39A8 deficiency. We hypothesized that targeting ACER1 may be critical for maintaining epithelial integrity and barrier function to protect against intestinal injury and inflammation in Slc39a8-IEC KO mice. We therefore examined the physiological relevance of ACER1 inhibition in the DSS-induced colitis model. The ACER1 inhibitor exhibited strong barrier protective activities by upregulating tight junction proteins in Slc39a8-deficient intestinal organoid monolayer culture under normal conditions (without DSS challenge) (Fig. 7). We therefore investigated whether regular exposure to this inhibitor would have sustained and beneficial colitis-preventing effects. Mice were pretreated with or without D-e-MAPP via oral gavage administration (10 nmol/g at 24 h intervals) for one week, followed by DSS treatment to induce colitis (inflammatory phase) (Fig. 8a). The mice were then placed on regular drinking water (recovery phase). The Slc39a8-IEC KO mice pretreated with D-e-MAPP were significantly protected from DSS-induced loss of body weight (Fig. 8b) and colon shortening (Fig. 8c, d). The pretreated Slc39a8-IEC KO mice also showed significantly reduced inflammation and pathological scores compared to untreated Slc39a8-IEC KO mice (Fig. 8e, f). Notably, we observed reduced intestinal permeability in the Slc39a8-IEC KO mice pretreated with D-e-MAPP (Fig. 8g). Further analysis of tight junction proteins in these mice also showed that pretreatment with D-e-MAPP protected against the downregulation of Zo1 and Cldn3 (Fig. 8h). Pretreatment with D-e-MAPP in Slc39a8-IEC KO mice also reduced the expression of inflammation markers, including Cxcl1 and Tnfa, which are hallmarks of ulcerative colitis (Fig. 8i). Pretreatment of Slc39a8-IEC KO or control mice with D-e-MAPP did not cause any signs of toxicity, as no changes were observed in mouse body weights or serum AST and ALT levels (Supplementary Fig. 5b–d).

**Fig. 8: Pretreatment with the ACER1 inhibitor D-e-MAPP confers protection against colitis in *Slc39a8*-IEC KO mice.**

The therapeutic applications of D-e-MAPP were also examined in the DSS-induced colitis model (Supplementary Fig. 6a). This treatment significantly protected the Slc39a8-IEC KO mice from DSS-induced body weight loss (Supplementary Fig. 6b) and colon shortening (Supplementary Fig. 6c, d). Histological analysis also showed significantly less tissue damage and inflammation scores in Slc39a8-IEC KO mice treated with D-e-MAPP (Supplementary Fig. 6e, f). The intestinal permeability was reduced in Slc39a8-IEC KO mice treated with D-e-MAPP compared to those without D-e-MAPP treatment, although this trend did not reach statistical significance (Supplementary Fig. 6g). Furthermore, D-e-MAPP treatment provided protection against DSS-induced downregulation of Cldn3 in the colon of Slc39a8-IEC KO mice (Supplementary Fig. 6h) and reduced the expression of inflammatory markers, such as Tnfa and I11b (Supplementary Fig. 6i). These results suggest that the ACER1 inhibitor has beneficial effects in preserving the barrier integrity and mitigating colitis in Slc39a8-IEC KO mice.

To determine the mechanisms underlying the upregulation of ACER1 and the rescue effect of D-e-MAPP, we performed global, unbiased lipidomics profiling of the intestine from WT and mutant mice, with or without the inhibitor, using triple time of flight liquid chromatography-mass spectrometry (Triple-TOF LC-MS). While ACER1 converts ceramide into sphingosine, sphingomyelin synthase (SMS1) converts ceramide into sphingomyelin; therefore, the two enzymes represent two major arms of ceramide metabolism (Fig. 8j). The lipidome profiling identified 40 differentially regulated lipids in Slc39a8-IEC KO intestines compared to control (Padj < 0.2, Supplementary Fig. 7a). Among the altered lipids, the heatmap (Fig. 8k) represents lipid species relevant to ceramide metabolism, including sphingomyelins, hexosylceramides, sphinganine, phosphatidylethanolamine ceramides, and ceramides. While we predicted a reduction in ceramides following ACER1 upregulation, the reduction was only observed for Cer 38:2;2O and Cer 43:1;2O, whereas four other ceramide species were upregulated in the Slc39a8-IEC KO intestines.

Notably, sphingomyelins were all significantly downregulated in the mutants, which is consistent with the fact that Mn is necessary for the activity of ceramide phosphoethanolamine synthase (Cpes)⁴⁶, the insect homolog of SMS1⁴⁷. Indeed, SMS activity was lower in Slc39a8-IEC KO intestines compared to control intestines (Fig. 8l). Furthermore, all three sphingomyelin species showed partial or complete restoration of their abundance upon D-e-MAPP treatment; the inhibitor alone did not affect the sphingomyelin levels (Fig. 8m). In contrast, we did not find any ceramide species whose changes completely agreed with the phenotypic outcomes, i.e., dysregulation in Slc39a8-IEC KO, restoration by D-e-MAPP, and no change by inhibitor alone (Supplementary Fig. 7b–g); the six KO-dysregulated ceramides largely satisfied the first two criteria, but D-e-MAPP alone also changed their levels in the control intestine, ruling them out as the mediator for gut phenotype rescue effects.

These results led us to propose the following mechanisms underlying Acer1 upregulation in Slc39a8-IEC KO intestines and phenotypic rescues by D-e-MAPP. First, the reduced SMS1 activity due to Mn deficiency led to reduced sphingomyelins and increased ceramide, triggering the compensatory increase of Acer1 expression to offset the ceramide increases. The increased Acer1 expression indirectly led to alterations in lipid composition, including the upregulation of some ceramide species and dysregulation of other detected lipids. The phenotypic rescues by D-e-MAPP (Figs. 7–9 and Supplementary Fig. 6) suggest that the upregulation of Acer1 and associated lipidome alterations contribute to the impaired gut function in Slc39a8-IEC KO mice. However, the rescue effect is unlikely to be mediated by ceramides; instead, the restoration of other lipid species, such as sphingomyelin, is the candidate mediator for the observed impact of D-e-MAPP on gut physiology.

**Fig. 9: Mn deficiency upregulates Acer1 expression and treatment with ACER1 inhibitor D-e-MAPP confers protection against colitis in Mn-deficient mice.**

Mn deficiency is sufficient to promote upregulation of Acer1 expression in the intestine, and treatment with an Acer1 inhibitor alleviates colitis in Mn-deficient mice

We also tested the protective effects of the Acer1 inhibitor on DSS-induced colitis in WT mice. Male C57BL/6J mice were fed either with a Mn-deficient (Mn-D) or Mn-adequate (Mn-A) diet for 14 days (Fig. 9a). The qPCR analysis revealed upregulation of Acer1 expression in the ileal and colonic mucosa from Mn-D mice compared to Mn-A mice (Fig. 9b), indicating that Mn deficiency is sufficient to promote upregulation of Acer1 expression in the intestine. Mice were then treated with D-e-MAPP via oral gavage (10 nmol/g at 24 h intervals) during DSS treatment, followed by placement on regular drinking water (Fig. 9a). The Mn-D mice displayed profound and sustained weight loss when compared to the Mn-A mice (Fig. 9c). Notably, D-e-MAPP treatment protected the Mn-D mice from this DSS-induced body weight loss (Fig. 9c), as well as against lower survival rate (Fig. 9d) and colon shortening (Fig. 9e, f), suggesting that the ACER1 inhibitor mitigated intestinal injury in Mn-D mice. Furthermore, the Mn-D mice treated with D-e-MAPP revealed marked amelioration of tissue inflammation and injury and significant reductions in their histological scores (Fig. 9g, h). Collectively, these results revealed Mn deficiency-induced ACER1 as a potential therapeutic target for IBDs with either genetic or environmental etiology.

Discussion

Our studies stem from the observational studies that common genetic variants at the SLC39A8 locus are significantly associated with various physiological traits and diseases, including whole-blood Mn levels¹⁴ and IBD^15,16. We used an IEC-specific Slc39a8 KO mouse model and intestinal organoid monolayer culture to gain insight into the roles of intestinal Slc39a8 in Mn homeostasis and how Slc39a8 contributes to the pathogenesis of IBD. Our studies revealed essential roles for intestinal Slc39a8 in controlling intestinal Mn absorption and epithelial integrity. Deletion of Slc39a8 in IEC impairs intestinal absorption of Mn, which in turn reduces the Mn content in other organs and tissues. Slc39a8 is localized to the apical surface of the ileum and colon and functions to import Mn into enterocytes. Moreover, Slc39a8 deficiency in IECs exacerbates colitis after intestinal epithelial injury. We identified increased levels of ACER1, a lipid-generating enzyme that affects intestinal permeability, in the intestines of Slc39a8-IEC KO mice. We further demonstrated that treatment with an ACER1 inhibitor significantly mitigated colitis in both preventive and therapeutic settings by enhancing the expression of tight junction proteins and inhibiting inflammation. Our results demonstrate that intestinal epithelial SLC39A8 controls intestinal Mn absorption and epithelial integrity, thereby providing potential insight into therapeutic options for IBD patients with SLC39A8 deficiency.

SLC39A8 was identified as a zinc transporter¹⁷ and thereafter as an iron transporter¹⁸; therefore, the early studies focused on the characterization of zinc and iron status using cell lines overexpressing SLC39A8. In 2016, SLC39A8 loss-of-function mutations were identified in human patients, and these patients were found to have abnormally low whole-blood Mn levels but normal levels of other metals^22,23. Furthermore, human carriers of the SLC39A8 A391T mutation, which is associated with reduced SLC39A8 activity, also showed reduced whole-blood Mn levels^48,49. Thus, human genetics studies have revealed an essential role for SLC39A8 in Mn homeostasis. The in vivo roles of SLC39A8 were largely unknown; however, a study of hepatocyte-specific Slc39a8 KO mice showed that SLC39A8 is essential for hepatic and whole-body Mn homeostasis without affecting other metal levels²⁵. Two research groups independently reported alterations in Mn levels, but not zinc and iron levels, in Slc39a8 A391T knock-in (KI) mice, although the tissue Mn levels reported in these studies were inconsistent^31,32. In the present study, expansion of the analysis of Mn, zinc, iron, copper, and selenium in multiple tissues (intestines, liver, lung, kidney, heart, brain, and whole blood; n = 10 per group in both sexes) revealed that a loss of Slc39a8 in the intestine reduced whole-body Mn levels without affecting zinc and iron concentrations in any of the tested tissues. Our findings in IEC-specific Slc39a8 KO mice are consistent with human data showing that patients with SLC39A8 loss-of-function mutations and human carriers of the SLC39A8 A391T mutation have reduced whole-body Mn levels. Collectively, these studies suggest that Mn is a major physiological substrate for SLC39A8 and that SLC39A8 is required for Mn homeostasis.

One of the key findings in our present study is that SLC39A8 plays an essential role in controlling intestinal Mn absorption. Whole-body Mn homeostasis is regulated by intestinal absorption and hepatobiliary excretion²⁶. The current model of intestinal Mn absorption is that enterocytes take up Mn from the diet at the apical membrane and export it into the bloodstream at the basolateral surface. The iron transporter SLC11A2, also known as divalent metal transporter 1 (DMT1), was considered to act as an intestinal Mn importer and to absorb Mn into duodenal enterocytes⁵⁰; however, IEC-specific DMT1 KO mice showed that DMT1 is dispensable for Mn uptake in the intestine²⁸. SLC39A14, a close family member of SLC39A8, localizes to the basolateral membrane of enterocytes, where it takes up Mn from the blood into the enterocytes to promote Mn elimination^51,52. SLC30A10, a Mn exporter, localizes to the duodenal enterocyte apical membrane, where it exports Mn into the intestinal lumen for subsequent Mn elimination via feces^53,54. These studies suggest that, in intestinal enterocytes, SLC39A14 at the basolateral membrane and SLC30A10 at the apical membrane limit Mn absorption and enhance intestinal Mn excretion^52,53,54. Our study shows that Slc39a8 localizes to the apical membrane of enterocytes, where it mediates intestinal Mn absorption.

Our data suggest that Slc39a8 mediates Mn uptake from the diet at the apical membrane, as markedly less ⁵⁴Mn was accumulated in the ileum from Slc39a8-IEC KO mice than from the intestines of controls after oral-intragastric gavage of ⁵⁴Mn. We also showed that Slc39a8-deficient intestinal organoid monolayer cultures exhibited reduced ⁵⁴Mn uptake at the apical membrane, but not at the basolateral membrane, thereby demonstrating that SLC39A8 mediates Mn uptake at the apical membrane of enterocytes. Consistent with our findings, single-cell transcriptome analysis in human intestines has revealed high expression of SLC39A8 in both the small and large intestines, implicating the importance of these segments for metal ion absorption⁵⁵. Thus, our data support a model in which SLC39A8 localizes to the apical surface of the enterocytes, where it mediates the uptake of Mn from the intestinal lumen into enterocytes for subsequent intestinal Mn absorption into the bloodstream.

Two recent studies have independently reported altered Mn homeostasis and exacerbated DSS-induced colitis in Slc39a8 A391T knockin (KI) mice^31,32. The study by Sunuwar et al. reported that Slc39a8 A391T KI mice showed reduced Mn levels in whole blood, liver, and kidney, but no alterations in the Mn levels in the distal small intestine, colon, lung, heart, and brain³¹. Nakata et al. reported reduced Mn levels in the whole blood, liver, and colon of Slc39a8 A391T KI mice, but they did not examine Mn levels in other tissues³². The colon Mn data reported in these two studies were inconsistent. Both studies measured the steady-state levels of metals by ICP-MS but did not directly measure intestinal Mn absorption using radiotracer studies. In addition, both studies investigated whole-body mutant mice, which developed a minimal phenotype, but neither study directly explored the cell-type-specific mechanism linking Slc39a8 deficiency to impaired intestinal barrier function. Our present study demonstrates the roles of intestinal SLC39A8 in the Mn supply for all peripheral tissues examined. Thus, our study provides insights into how Slc39a8 contributes to Mn absorption and protect animals from colitis.

Our RNA-seq analysis resulted in the identification of four differentially expressed genes in the Slc39a8-IEC KO intestines (Fig. 6a and Supplementary Table 1). Among these four genes, Slc39a8 was the only downregulated gene in the Slc39a8-IEC KO intestines (Fig. 6a and Supplementary Table 1), validating the RNA-seq approach. Acer1 encodes alkaline ceramidase 1 (ACER1), which catalyzes the hydrolysis of ceramides to generate sphingosine^56,57. Acer1 catalyzes the hydrolysis of ceramides with unsaturated long acyl chains (C18:1 and C20:1) or a very long saturated (C24:0) or unsaturated (C24:1) acyl chain⁵⁷. Considering the significant association between sphingolipid metabolism and IBD⁴¹, we further investigated ACER1 in our study. In addition to Acer1, two other members of the alkaline ceramidase family, Acer2 and Acer3, are present in both mice and humans^57,58. D-e-MAPP can inhibit ACER2 and ACER3^59,60; however, since we did not find any expression changes in Acer2 and Acer3 in Slc39a8-IEC KO mice (Supplementary Fig. 8a–e), the rescue effect observed with D-e-MAPP was likely due to ACER1 inhibition. Nevertheless, the lack of expression changes still does not rule out a potential involvement of Acer2 and Acer3. A previous study has demonstrated that ACER3 plays a crucial role in mediating the immune response in innate immune cells and colonic epithelial cells⁶¹. Acer3 deficiency has been shown to increase the local and systemic production of proinflammatory cytokines and exacerbated colitis in the DSS-induced murine colitis model⁶¹. These observations indicate that Acer activities are tightly regulated to ensure normal gut functions.

In addition to ACER1’s role in sphingosine production, sphingosine serves as the substrate for sphingosine kinases (SphKs), which have been extensively implicated in IBD^41,42. Thus, we tested their expression levels in our model. We found a significant upregulation of Sphk1 mRNA expression in the ileum and colon of Slc39a8-IEC KO mice, whereas Sphk2 expression levels remain unchanged (Supplementary Fig. 8f, g), suggesting the involvement of the SphK1/S1P pathway. However, our lipidomics data indicate no changes in sphingosine, likely due to compensatory mechanisms. Future studies are needed to explore the intricate regulation of sphingosine metabolism in response to Slc39a8 deficiency.

One important implication of our findings is that the use of an ACER1 inhibitor might be a better therapeutic avenue than dietary Mn supplementation because our data suggest that Slc39a8 deficiency impairs intestinal Mn absorption from diet. In addition to the findings for Slc39a8-IEC KO intestines, our data demonstrate that dietary Mn deficiency upregulates Acer1 expression in the intestine and that treatment with an Acer1 inhibitor mitigates colitis in Mn-deficient mice (Fig. 9). Epidemiological studies have shown a positive relationship between Mn deficiency and the risk of IBD. A recent epidemiological survey showed that pediatric patients with newly diagnosed Crohn’s disease and ulcerative colitis have significantly lower Mn levels in their hair samples⁶. Another human study reported significantly lower blood Mn levels in patients with active Crohn’s disease and ulcerative colitis compared to those in remission⁶². These data suggest that IBD patients have a risk of Mn deficiency, and may consequently upregulate Acer1 expression; this possibility can be tested in the future. Nevertheless, interventions aimed at restoring ACER1 levels may be beneficial in improving human IBD conditions. Our study has also revealed an involvement of sphingolipid metabolism and its therapeutic potential in IBD. Further studies are needed to examine whether aberrant sphingolipid metabolism is a general mechanism of IBD or whether it defines a subset of IBD conditions.

Methods

Animals

All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Michigan (PRO00008963). For IEC-specific disruption of Slc39a8, mice floxed for Slc39a8 (Slc39a8^fl/fl) on a C57BL/6J background (European Mouse Mutant Archive, EM: 05285) were crossed with Villin-Cre (Vil-Cre) mice on a C57BL/6J background (Jackson Lab, stock number 004586). The Slc39a8-flox allele was detected by PCR using the following primers: Forward: 5’-AAGGCGCATAACGATACCAC-3’ and Reverse: 5’-CCGCCTACTGCGACTATAGAGA-3’. The Villin-Cre transgenic allele was detected using the following primers: Forward: 5’-TTCTCCTCTAGGCTCGTCCA-3’ and Reverse: 5’-CATGTCCATCAGGTTCTTGC-3’. The Slc39a8-IEC KO (Slc39a8^fl/fl:Villin-Cre) and Slc39a8-IEC Het (Slc39a8^fl/+:Villin-Cre) mice were compared to controls, which included a mixture of three groups: Slc39a8-flox (Slc39a8^fl/fl and Slc39a8^fl/+), Slc39a8-WT (Slc39a8^+/+), and Vil-Cre (Slc39a8^+/+:Villin-Cre). We compared key experimental results, such as metal concentrations and experimentally induced colitis models, among the four genotypes of controls. We found no significant differences in Mn concentrations in the ileum and colon among the four genotypes of controls at 6–8 weeks of age (Supplementary Fig. 9a). We also observed that the four genotypes of controls did not display significant differences in DSS sensitivity at 6–8 weeks of age (Supplementary Fig. 9b). These data indicate that the flox sequence and Cre transgene did not impact Mn homeostasis or DSS sensitivity. The mice were housed in a pathogen-free animal facility at 22 °C with 40–60% humidity on a 12-h light/dark cycle, and provided the standard rodent diet of our institution (PicoLab Laboratory Rodent Diet 5L0D, LabDiet; 70 ppm Mn) and water ad libitum.

For the Mn-restriction diet experiment, WT C57BL/6 mice aged 4 weeks were purchased from Jackson Laboratory and maintained on a metal-basal diet containing 35 mg Mn/kg (TD120518, Harlan Teklad, Indianapolis, IN, USA)^13,27,63. The trace element levels in the diet were as recommended by the American Institute of Nutrition⁶⁴. For dietary Mn alterations, the mice were fed either Mn-deficient or Mn-adequate diets (<0.01 and 35 ppm Mn, respectively; Harlan Teklad)¹³. For colitis induction, mice were provided with water containing 3% (w/v) DSS for 6 days (inflammatory phase). The mice were then placed on regular drinking water for 8 days (recovery phase). All mice in the same experiment were from an age-matched, co-housed cohort. This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (Bethesda, MD, USA).

Materials

General laboratory chemicals and reagent solutions were purchased from Sigma-Aldrich. The ACER1 inhibitor D-erythro-MAPP was purchased from Enzo Life Sciences. Specific doses of D-erythro-MAPP were chosen based on a previous study⁶⁵. ELISA kits for AST and ALT were purchased from Sigma Aldrich.

Metal measurements

Tissue samples were analyzed for metals by inductively coupled plasma mass spectrometry (ICP-MS)^13,20,24. Briefly, tissue samples taken from mice were digested with 2 mL/g total wet weight nitric acid (BDH ARISTAR® ULTRA) for 24 h and then digested with 1 mL/g total wet weight hydrogen peroxide (BDH ARISTAR® ULTRA) for 24 h at room temperature. Specimens were preserved at 4 °C until quantification of metals. Ultrapure water was used for final sample dilution.

Absorption of Mn in vivo

To assess radiotracer Mn absorption, mice were dosed with 0.1 µCi ⁵⁴Mn (PerkinElmer) per gram body weight via oral-intragastric gavage or tail vein injection. Blood was collected at 15 min, and mice were killed at 4 h, after which tissue ⁵⁴Mn-associated radioactivity (counts per minute) was determined using a γ-counter^27,63.

Intestinal organoid monolayer cultures

Intestinal organoid monolayer cultures were created and maintained as previously described^29,66. Briefly, mouse ileum or colon was dissected and flushed with phosphate buffered saline (PBS) and transferred to chelation buffer (2 mM EDTA, PBS) for 30 min. The ileum or colon was shaken to remove crypt cells and then incubated in PBS with 43 mM sucrose and 55 mM sorbitol and filtered through a 70-μm filter. Isolated intestinal crypts from mice were seeded and maintained in L-WRN conditioned complete media supplemented with 50 ng/mL recombinant human EGF (R&D Systems) and an antibiotic–antimycotic (Corning).

⁵⁴Mn uptake study

Intestinal organoid monolayer cultures were grown on Transwell inserts, and ⁵⁴Mn uptake assay was determined as previously described^20,24. During the ⁵⁴Mn uptake experiment, the extracellular pH was maintained at pH 7.4 in the apical and basolateral compartments to mimic the physiological situation (i.e., the neutral intraluminal pH of the terminal ileum and colon and the normal blood pH)⁶⁷. Apical (basolateral) metal uptake experiments were initiated by adding 1 µM ⁵⁴Mn to the apical (basolateral) chamber, and the cells were incubated at 37 °C at the indicated time points. After uptake, cells were washed 3 times with PBS containing 1 mM EDTA to remove any unbound ⁵⁴Mn and directly harvested for intracellular radioactivity. Radioactivity was determined with a γ-counter and was normalized to the cell protein measured in lysates using the Bradford assay.

Immunofluorescence

Immunofluorescence staining was performed on frozen tissues or intestinal organoid monolayer cultures^20,24. Primary antibodies were incubated at room temperature for 1 h, followed by fluorescent secondary antibodies at room temperature for 20 min. Nuclei were detected with 4’,6-diamidino-2-phenylindole (DAPI). The following primary antibodies were used: SLC39A8 (Proteintech, 20459-1-AP, 1:50), ZO-1 (Santa Cruz, sc-33725, 1:50), ZO-2 (Santa Cruz; sc-515115, 1:50), Claudin-2 antibody (Thermo Fisher, # 51-6100, 1:100), Claudin-3 antibody (Thermo Fisher, # 34-1700, 1:100), Claudin-5 antibody (Thermo Fisher, # 35-2500, 1:50), Claudin-7 antibody (Thermo Fisher, # 34-9100, 1:50), and Occludin antibody (Santa Cruz, sc-133256, 1:50). The following secondary antibodies were used: Donkey anti-rat IgG H&L (Alexa Fluor 488) (Thermo Fisher, #A48269, 1:500), Donkey anti-mouse IgG H&L (Alexa Fluor 488) (Thermo Fisher, # A32766, 1:1000), Donkey anti-rabbit IgG H&L (Alexa Fluor 488) (Thermo Fisher, # A32790, 1:000), Goat anti-mouse IgG H&L (Alexa 488 Fisher A11001, 1:1500), and Goat anti-rabbit IgG H&L (Alexa 568 Fisher A11036, 1:1500), Confocal microscopy was performed using a Nikon Eclipse A-1 confocal microscope (Nikon Instruments). Image analysis was performed using ImageJ (NIH) software.

Chemically induced colitis models

For the acute DSS model, mice were treated with 3% DSS (Fisher Scientific) dissolved in the drinking water to induce colitis (inflammatory phase) and then placed on regular drinking water (recovery phase). The mice were sacrificed on Day 13 or 14 after DSS treatment. For the acute TNBS model, mice were intrarectally injected with 100 mg/kg TNBS (MilliporeSigma) in 50% ethanol, with 50% ethanol treatment used as a control. The mice were sacrificed 7 days after TNBS treatment. For the chronic DSS model, mice received two cycles of 3% DSS treatment, each cycle consisting of 8 days of inflammatory phase followed by 5 days of recovery phase. The mice were sacrificed on Day 26 after DSS treatment. Body weights, stool consistency, and GI bleeding were monitored daily. Clinical scores and colon damage scores were estimated as detailed previously³⁹.

Histology and immunohistochemical staining

Tissues were fixed in 4% paraformaldehyde at 4 °C overnight. Hematoxylin/eosin (H&E) analysis was performed in paraffin-embedded tissue sections (5 µm). The histological scoring was performed by a pathologist who blindly assessed the degree of surface epithelial loss, crypt destruction, and inflammatory cell infiltration into the mucosa, resulting in a score from 0 (no disease activity) to 12 (most severe disease activity).

Intestinal permeability assay

Intestinal permeability assay was performed as previously described¹³. Briefly, fluorescein isothiocyanate (FITC)-dextran (MW 4000; FD4, Sigma-Aldrich) was administered by oral gavage to examine the intestinal barrier function. Serum was collected 4 h later, and the fluorescence intensity of each sample (excitation, 485 nm; emission, 525 nm) was measured using a BioTek Synergy microplate reader (BioTek Instruments).

Transepithelial electrical resistance (TEER)

IECs were grown on permeable supports, and monolayers were monitored for electrical resistance using an epithelial volt-ohmmeter (EVOM/EndOhm; World Precision Instruments).

RNA isolation and sequencing

Total RNA was isolated from ileal or colonic mucosa from each mouse using the RNeasy mini kit (Qiagen, Valencia, CA). Three mice (n = 3 biological replicates) were used per experimental group. The sample size was determined based on previously described power calculations to optimize the detection of differentially expressed genes⁶⁸. RNA concentrations were measured with an Epoch Microplate Spectrophotometer (BioTek Instruments). RNA integrity was assessed with an Agilent Bioanalyzer 2100 using a Nano 6000 assay kit (Agilent Technologies). An RNA integrity number (RIN) > 7.2 was considered the minimum requirement for library preparation. RNA was reverse transcribed into cDNA using oligo-dT, and cDNA libraries were generated with a NEBNext Ultra II RNA Library Prep Kit (NEB #E7775). An insert size of 250–300 bp was used for cDNA library preparation. Libraries were sequenced on the Illumina NovaSeq 6000 platform with a 150-bp paired-end mode. Reference genome and annotation files were downloaded from Ensemble, and RNA-seq data were aligned to the reference genome using the Spliced Transcripts Alignment to a Reference (STAR) software⁶⁹. The DESeq2 package was used for differential expression analysis.

qPCR

Purified RNA was reverse-transcribed with SuperScript III First-Strand Synthesis System (Invitrogen, Thermo Fisher Scientific)⁷⁰. The qPCR was performed using the Power SYBRGreen PCR Master Mix (Applied Biosystems). The mRNA was normalized using 36B4. The primers used for qPCR are listed in Supplementary Table 2, and were all purchased from Integrated Genomics Technologies.

Immunoblot

Protein samples were loaded on SDS-polyacrylamide gels, separated by electrophoresis, and then transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA; Cat. No. 1620115). The membrane was immunoblotted with primary antibody ZO-1 (Santa Cruz, sc-33725, 1:500), ZO-2 (Santa Cruz; sc-515115, 1:500), Claudin-2 (Thermo Fisher, # 51-6100, 1:1000), Claudin-3 (Thermo Fisher, # 34-1700, 1:1000), Claudin-5 (Thermo Fisher, # 35-2500, 1:1000), Claudin-7 (Thermo Fisher, # 34-9100, 1:1000), Occludin (Santa Cruz, sc-133256, 1:1000), Cytokeratin 8/18 (Abcam, ab53280, 1:50000), ASAH3 antibody (Thermo Fisher, # PA5-75603, 1:500), and Actin (Proteintech, 66009-1-lg, 1:3000). The blots were visualized with infrared secondary antibodies, IRDye 680RD Goat anti-rat IgG Secondary antibody (Liborbio, #926-69076, 1:20000), IRDye 800CW Donkey anti-mouse IgG Secondary antibody (Liborbio, #925-32212, 1:20000), or IRDye 680LT Donkey anti-rabbit IgG Secondary antibody (Liborbio, #926-69021, 1:15000), using a LI-COR Odyssey fluorescent western blotting system (LI-COR Biosciences, Lincoln, NE, USA). Protein expression was quantified by densitometry (Image Studio Lite; LI-COR).

Lipidomics

Details of sample preparation and identification for untargeted lipidomic profiling have been previously reported⁷¹. Lipids were extracted using a modified Bligh-Dyer method⁷². Extraction was carried out using water:methanol:dichloromethane (2:2:2, v/v) at room temperature after spiking with internal standard lipids. The organic layer was collected and dried completely under nitrogen and the contained lipids were further analyzed by liquid chromatography-mass spectrometry (MS)-based lipidomics. The dried lipid extracts were injected onto a 1.8-μm particle 50 ×2.1 mm id Waters Corporation Acquity HSS T3 column, which was heated to 55 °C. A binary gradient system consisting of acetonitrile and water with 10 mM ammonium acetate (40:60, v:v) was used as eluent A. Eluent B consisted of water, acetonitrile, and isopropanol, both containing 10 mM ammonium acetate (5:10:85, v/v). The lipid extracts were reconstituted in buffer B and injected for MS. The MS analysis alternated between MS and data-dependent MS2 scans using dynamic exclusion in both positive and negative polarity. As quality controls (QC) to monitor the profiling process, pools of plasma and test plasma (a small aliquot from all test samples) were extracted and analyzed in tandem with experimental samples. These controls were incorporated multiple times into the randomization scheme to allow constant monitoring of the sample preparation and analytical variability.

Lipids were identified using the LIPIDBLAST library⁷³ by matching the product ion MS/MS data. To facilitate accurate lipid identification, a more stringent mass error rate of 0.001 m/z for the positive mode and 0.005 m/z for the negative mode was determined based on the mass accuracy of internal standards to reduce the rate of false positives and identify likely correct candidates. The sphingolipid species considered for identification were limited to features with (%RSD < 20) in pooled samples. More details regarding the spectral matching procedure used for compound identification against the LipidBlast library are outlined in the original report⁷⁴.

A manual review of spectral matches was performed in all potentially uncertain cases, including the sphingolipid species. Examples of spectral matches are included in Supplementary Fig. 10a, b. The product ion scan of [M+Na]+ species of sphingomyelin (SM 34:1, 2O) yields three fragments corresponding to neutral loss (NL) of C3H9N (m/z 666.477, C36H70NO6PNa+), NL of C5H14NO4P (m/z 542.486, C34H65NO2Na+), NL of C5H16NO5P (m/z 502.497, C34H64NO+), respectively. A product ion scan of m/z 300.2860, which corresponds to the [M + H]+ ion for sphingosine (SPB) 18:1;2O molecular species, was searched against the LipidBlast In-Silico library, revealing an exact match for the expected compound with high library scores (Supplementary Fig. 10a). MS/MS spectra of ceramides in the positive mode were dominated by fragments from the sphingoid base (Supplementary Fig. 10b). Collisional activation of ceramides in positive ion mode showed the characteristic product ions of m/z 264.2684 [C18H34N]+ and 282.279 that were attributed to the loss of N-linked fatty acid and one and two water molecules, respectively. The positive mode MSMS product ion spectrum of HexCer 18:0;3O/16:0;(2OH), which is illustrated in Supplementary Fig. 10b. This compound shows an NL of C6H10O5, the hexose ring, results m/z 572.5232 (Supplementary Fig. 10b), as well as other peaks that are m/z 554.5143 and m/z 264.2686 correspond to NL of C6H10O5 and H2O and SPB 18:0;O3 −3H2O respectively. Chromatographic retention time (RT) was utilized to validate identifications. The scatter plots (Supplementary Figs. 10c, d) demonstrate a linear relationship between sphingolipid RT, alkyl chain length, and desaturation, aligning with expected elution patterns. No significant deviations were observed, supporting the accuracy of our identifications.

MS data files were processed using MultiQuant 1.1.0.26 (Applied Biosystems/MDS Analytical Technologies). Identified lipids were quantified by normalizing against their respective internal standard. QC samples were used to monitor the overall quality of the lipid extraction and MS analyses and were mainly used to remove technical outliers and lipid species that were detected below the lipid class-based lower limit of quantification.

Statistics and reproducibility

Data are presented as individual values and represent the mean ± SEM. The exact sample size and the statistical tests are described in figure legends, exact p values are provided in the figure legends and Source Data. Test used include one-way ANOVA, two-way ANOVA, or two-tailed Student’s t test by GraphPad Prism software 8.0 (San Diego, CA, USA). Outliers were identified using the GraphPad ROUT (robust regression and outlier removal) method (Q = 1%).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All data generated or analyzed during this study are included in this published article (and its supplementary information files). All the datasets in this study are existing published and are available via the NCBI website, including Gene Expression Omnibus (GSE) accession number: GSE192695. Lipidomics data have been deposited at https://github.com/SeoResearchLab/IECKO2023. Source data are provided with this paper. Any additional information is available upon request to the corresponding author. Source data are provided with this paper.

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Acknowledgements

This work was supported by grants from the National Institutes of Health (R01DK123022 and R21NS112974) to Y.A.S.; (R01NS116008 and R01NS089896) to S.I. and from the University of Michigan Center for Gastrointestinal Research (DK034933) to Y.A.S. We acknowledge Theresa Keeley in the lab of Dr. Linda Samuelson for assistance with the immunofluorescence studies.

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Department of Nutritional Sciences, University of Michigan School of Public Health, Ann Arbor, MI, USA
Eun-Kyung Choi, Jin-Ho Park, Luisa Aring & Young Ah Seo
Department of Pathology, University of Michigan Medical School, Ann Arbor, MI, USA
Thekkelnaycke M. Rajendiran, Chithra K. Muraleedharan, Vicky Garcia-Hernandez, Nobuhiko Kamada & Asma Nusrat
Michigan Regional Comprehensive Metabolomics Resource Core, University of Michigan Medical School, Ann Arbor, MI, USA
Thekkelnaycke M. Rajendiran & Tanu Soni
Division of Gastroenterology and Hepatology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA
Nobuhiko Kamada & Linda C. Samuelson
Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, MI, USA
Linda C. Samuelson
Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI, USA
Shigeki Iwase

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Eun-Kyung Choi
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Thekkelnaycke M. Rajendiran
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Tanu Soni
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Jin-Ho Park
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Luisa Aring
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Chithra K. Muraleedharan
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Vicky Garcia-Hernandez
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Nobuhiko Kamada
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Linda C. Samuelson
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Asma Nusrat
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Shigeki Iwase
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Young Ah Seo
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Contributions

Y.A.S. conceived and designed the study. E.K.C., T.M.R., T.S., J.H.P., S.I., and Y.A.S. acquired the data. E.K.C., T.M.R., TS, J.H.P., C.K.M., V.G.H., N.K., T.S., T.M.R., L.C.S., A.N., S.I., and Y.A.S. developed the methodologies. E.K.C., T.M.R., T.S., J.H.P., T.M.R., S.I., and YAS performed experiments. E.K.C., T.M.R., T.S., J.H.P., L.A., N.K., L.C.S., A.N., S.I., and Y.A.S. analyzed and interpreted the data. YAS wrote the manuscript. Y.A.S. supervised the study. Y.A.S. was responsible for funding acquisition. Figures 3a, c, e, and 9i were created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

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Correspondence to Young Ah Seo.

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Choi, EK., Rajendiran, T.M., Soni, T. et al. The manganese transporter SLC39A8 links alkaline ceramidase 1 to inflammatory bowel disease. Nat Commun 15, 4775 (2024). https://doi.org/10.1038/s41467-024-49049-8

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Received: 25 February 2022
Accepted: 17 May 2024
Published: 05 June 2024
DOI: https://doi.org/10.1038/s41467-024-49049-8

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Abstract

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Mannose ameliorates experimental colitis by protecting intestinal barrier integrity

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Diosmetin has therapeutic efficacy in colitis regulating gut microbiota, inflammation, and oxidative stress via the circ-Sirt1/Sirt1 axis

Introduction

Results

Loss of intestinal epithelial Slc39a8 leads to systemic Mn deficiency

Slc39a8 is essential for intestinal absorption of 54Mn

Slc39a8 mediates the uptake of 54Mn at the apical membranes in intestinal organoid monolayer cultures

IEC-specific deletion of Slc39a8 exacerbates colitis following intestinal epithelial injury

Alkaline ceramidase 1 is upregulated in the intestines of Slc39a8-IEC KO mice

Acer1 inhibitor enhances intestinal barrier function in Slc39a8-IEC KO mice-derived intestinal organoid monolayer culture

Treatment with Acer1 inhibitor D-e-MAPP mitigates colitis in Slc39a8-IEC KO mice

Mn deficiency is sufficient to promote upregulation of Acer1 expression in the intestine, and treatment with an Acer1 inhibitor alleviates colitis in Mn-deficient mice

Discussion

Methods

Animals

Materials

Metal measurements

Absorption of Mn in vivo

Intestinal organoid monolayer cultures

54Mn uptake study

Immunofluorescence

Chemically induced colitis models

Histology and immunohistochemical staining

Intestinal permeability assay

Transepithelial electrical resistance (TEER)

RNA isolation and sequencing

qPCR

Immunoblot

Lipidomics

Statistics and reproducibility

Reporting summary

Data availability

References

Acknowledgements

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Slc39a8 is essential for intestinal absorption of ⁵⁴Mn

Slc39a8 mediates the uptake of ⁵⁴Mn at the apical membranes in intestinal organoid monolayer cultures

⁵⁴Mn uptake study