A POSSIBLE ROLE OF HEPCIDIN IN INTESTINAL INFLAMMATION

PIXIOLINE, Michelle; CHRISTOFOLETTI, Camila Rubia; CONSTANTINO, Julia Aun; MACEDO, Juliana Alves; GAMBERO, Alessandra

doi:10.1590/S0004-2803.24612024-045

ABSTRACT

Background: Hepcidin’s main function is to control iron availability to hematopoiesis. However, it has been shown that hepcidin may have an additional role in intestinal inflammation, as intestinal cells and leukocytes increase the production in experimental colitis and Crohn’s disease.

Objective: Using an HT-29 cell as a model, we investigated the role of hepcidin in intestinal inflammation.

Methods: The ability of HT-29 cells to produce hepcidin was evaluated after stimulus with IL-6, TNF-α, and lipopolysaccharide (LPS) for 24 h. Experiments were performed in the presence of stat-3 or IκBα phosphorylation inhibitor. The release of IL-8 by HT-29 cells was evaluated after hepcidin stimulus in the presence or absence of ferroportin (FPN) antibody. Nuclear factor (NF) κB translocation and reactive oxidative species (ROS) production in response to hepcidin were also studied.

Results: HT-29 cells can produce hepcidin under IL-6, TNF-α, and LPS stimulation. The Stat-3 inhibitor reduces hepcidin production induced by IL-6, and the IκBα inhibitor reduces hepcidin production by all stimuli tested. IL-8 is produced by HT-29 cells in response to hepcidin, and the FPN antibody did not modify IL-8 release. Il-8 production induced by hepcidin was NFκB dependent, but when cells were co-stimulated with LPS, IL-8 release, and NFκB translocation were inhibited, and HPN antibody reduced it. Hepcidin increases ROS production by HT-29 cells.

Conclusion: We used HT-29 cells to demonstrate that hepcidin is produced at low levels in response to inflammatory stimuli. The hepcidin action is dual in HT-29 cells, performing a proinflammatory action by producing ROS and IL-8 under physiological conditions but an anti-inflammatory action by reducing IL-8 release and NFκ-B activation when LPS is present, suggesting that hepcidin has a modulatory role in intestinal inflammation.

Keywords: Hepcidin; interleukin-8; intestinal inflammation; nuclear factor κB

RESUMO

Contexto: A principal função da hepcidina é controlar a disponibilidade de ferro para a hematopoiese. No entanto, foi demonstrado que a hepcidina pode ter um papel adicional na inflamação intestinal, uma vez que a produção é aumentada pelas células intestinais e leucócitos na colite experimental e na doença de Crohn.

Objetivo: Utilizando células da linhagem HT-29 como modelo, investigamos o papel da hepcidina na inflamação intestinal.

Métodos: A capacidade das células HT-29 em produzir hepcidina foi avaliada após estimulação com IL-6, TNF-α e lipopolissacarídeo (LPS) por 24 horas. Os experimentos foram realizados na presença do inibidor de fosforilação Stat-3 ou de IκBα. A liberação de IL-8 foi avaliada após estímulo das células HT-29 com hepcidina, na presença ou ausência de anticorpo anti-ferroportina (FPN). A translocação do fator nuclear (NF) κB e a produção de espécies reativas de oxigênio (ERO) em resposta à hepcidina também foram estudadas.

Resultados: As células HT-29 são capazes de produzir hepcidina sob estimulação com IL-6, TNF-α e LPS. O inibidor Stat-3 reduz a produção de hepcidina induzida por IL-6, e o inibidor IκBα reduz a produção de hepcidina por todos os estímulos testados. A IL-8 é produzida pelas células HT-29 em resposta à hepcidina e o anticorpo anti-FPN não modificou a liberação de IL-8. A produção de IL-8 induzida pela hepcidina foi dependente de NFκB, mas quando as células foram co-estimuladas com LPS, a liberação de IL-8 e a translocação de NFκB foram inibidas e o anticorpo HPN a reduziu. A hepcidina aumenta a produção de EROs pelas células HT-29.

Conclusão: Utilizando células HT-29, demonstramos que a hepcidina é produzida em baixos níveis em resposta a estímulos inflamatórios. A ação da hepcidina é dupla nas células HT-29, realizando uma ação pró-inflamatória ao produzir ERO e IL-8 sob condições fisiológicas, mas uma ação anti-inflamatória ao reduzir a liberação de IL-8 e a ativação de NFk-B quando LPS está presente, sugerindo que a hepcidina tem um papel modulador na inflamação intestinal.

Palavras-chave: Hepcidina; interleucina-8; inflamação intestinal; fator nuclear κB

HIGHLIGHTS

•Increased hepcidin expression has previously been detected in the gastrointestinal tract, H. pylori-infected gastric mucosa and experimental models of colitis.

•Understanding the gastrointestinal functions of hepcidin was our goal.

•In this in vitro approach, we showed that hepcidin has a dual role. It is capable of isolating ROS and IL-8 production but is protective when cells are stimulated with LPS, reducing IL-8 production and NF-KB translocation.

•Hepcidin may play a beneficial local role in modulating the gastrointestinal inflammatory response.

INTRODUCTION

Hepcidin is a protein encoded by the HAMP gene in humans that has two functions. The main function is to regulate iron metabolism by inhibiting post-translational ferroportin (FPN), thus reducing iron absorption by enterocytes and releasing iron by hepatocytes and macrophages into circulation. The other function is innate antimicrobial activity, which limits iron availability in plasma and extracellular fluids. Hepcidin synthesis is significantly induced by iron overload, infection, or inflammation and inhibited in iron deficiency and hypoxia¹^,².

Hepcidin is mainly produced in the liver, but studies have shown that hepcidin is also made in other tissues and cells, like the kidney³, heart⁴, brain⁵, pulmonary vasculature⁶, pancreatic beta cells⁷, adipose tissue⁸, macrophages, lymphocytes, neutrophils, dendritic cells⁹ and mononuclear cells¹⁰^-¹³, being in sites where hepcidin can participate in the local regulation of iron and inflammation.

As cited, hepcidin synthesis can be induced by inflammation mediated by the cytokines produced during immune system activation, mainly by interleukin (IL)-6 that activates the JAK-STAT3 signaling pathway (Janus kinase-signal transducer and activator of transcription)¹⁴. Studies have shown the existence of a highly responsive lipopolysaccharide (LPS)-iron-IL-6-hepcidin axis linking innate immunity and iron metabolism in the liver¹⁵, while results for an important inflammatory cytokine, tumor necrosis factor (TNF)- a demonstrated that blood hepcidin was found to be decreased in patients with Crohn’s disease after successful therapy with anti-TNF-α mAb therapy¹⁶.

In a rodent model of non-infectious colitis, we demonstrated previously an increased expression of hepcidin-25 in colon tissue associated with the IL-6/Stat-3 signaling pathway and local iron sequestration¹⁷. Biopsies from inflamed mucosa of Crohn’s disease patients expressed greater amounts of mRNA of HAMP compared with non-inflamed areas (internal control)¹⁸ or with health subjects⁹. Initial clinical studies have found elevated systemic levels of hepcidin in patients with Helicobacter pylori infection-associated gastritis¹⁹^,²⁰. After, analysis of stomach biopsies demonstrated that hepcidin expression was increased in lymphocytes of the gastric mucosal lymphoid follicles²¹ and parietal cells from patients with Helicobacter pylori infection²², strengthening the idea that hepcidin can play a local, in addition to systemic, roles in gastrointestinal inflammation, either through modular responses to infection or by directly altering the inflammatory response.

Although there is consensus that hepcidin-induced hypoferremia demonstrates clear antimicrobial effects that favor inflammation associated with infectious processes²³ and that iron-fortified diets or luminal iron concentrations negatively impact intestinal inflammatory responses²⁴, the role of hepcidin in inflammation is just beginning to be understood. Luminal iron content, microbiota, and cell-immune cell interactions could interfere with the response of intestinal epithelium to hepcidin, and our question was, what is the function of hepcidin during epithelial injury?

In this in vitro work, we investigated the role of hepcidin in intestinal inflammation using HT-29 cells. HT-29 cells can be used in in vitro models to study intestinal inflammation and the pathogenesis of inflammatory bowel diseases, being able to respond to microorganisms²⁵, bacterial products such as LPS and inflammatory cytokines²⁶, altering tight junction expression²⁷, producing mucus²⁸ and proinflammatory factors²⁶. HT-29 cell models have been extensively used to screen new anti-inflammatory drugs or foods. Using an in vitro cell system, we obtained responses not altered by systemic or luminal factors, seeking to understand the direct role of hepcidin on intestinal epithelial cells during inflammation.

METHODS

Cell line

The human HT-29 intestinal cell line was purchased from the Rio de Janeiro Cell Bank (BCRJ 0111) (OS.C.16.28.12; Lote:000412; Passage:151). The cell line was stored in cryogenic conditions in our laboratory. Cells were cultured in McCoy’s 5A (Sigma, Saint Louis, MO, USA) supplemented with 2mM Glutamine (Gibco, Carlsbad, CA USA), 10% fetal bovine serum (Gibco), penicillin, and streptomycin (Life Technologies, Carlsbad, CA USA) in a humidified incubator at 37°C under an atmosphere of 5% CO₂.

Experimental protocols

HT-29 cells were seeded onto 96-well plates at a density of 3 x 10⁴ cells/well. They were incubated with 10-100 ng/mL of IL-6 (Peprotech, Rocky Hill, NJ, USA), 10-200 ng/mL of TNF-α (Peprotech), or 0.01-10 μg/mL of the TLR-4 ligand lipopolysaccharide from E. coli (LPS; Sigma) for 24 h. The supernatant was collected and stored for hepcidin measurements (R&D Systems, Minneapolis, MN, USA) and cells were assessed for cellular viability by performing an MTT assay. Additional experiments were performed with stimulus in the presence of stat-3 inhibitor (Stattic; Sigma) or an irreversible inhibitor of IκBα phosphorylation (BAY 11-7085; Calbiochem, CA, USA). Hepcidin (20-200 pg/mL, R&D Systems) was employed as a stimulus for HT-29, and IL-8 was measured in the supernatant (R&D Systems). Antibody anti-ferroportin (FPN) was also added to additional experiments to evaluate IL-8 release. All experiments were performed in triplicate.

MTT assay

After removing the culture medium, the MTT solution (Sigma, 5 mg.mL⁻¹ in phosphate-buffered saline; PBS) was added to each well for 3 hours at 37°C with 5% CO₂. After that, isopropanol was added to solubilized formazan crystals formed. Absorbance was measured at 540 nm using a microplate reader (Glomax; Promega, USA). The assay was performed in triplicate.

NF-kB assay

After the culture medium was removed, nuclear proteins were extracted using Cayman’s Nuclear Extraction Kit (Cayman, Ann Arbor, MI, USA), and the detection of NF-kB (p65) DNA binding activity was determined by using NF-κB (p65) Transcription Factor Assay Kit (Cayman) following manufacturer instructions.

Reactive oxygen species (ROS) detection

After the culture medium was removed, cells were loaded with the cell-permeant 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA, 10 μmol/L in Hanks’ balanced salt solution HBSS, Invitrogen, Eugene, OR, USA) and incubated for 30 min at 37°C with 5% CO₂. The fluorescence at excitation and emission wavelengths of 490 nm and 520 nm were monitored every half hour for 5 h (NOVOstar, BMG LABTECH, Germany). The results were expressed as the percentage increase in fluorescence from the respective baseline value.

Statistical analysis

All data are expressed as mean ± SEM. Data comparisons were performed using ANOVA followed by Bonferroni’s Multiple Comparison Test. An associated probability (P value) of less than 0.05 was considered significant.

RESULTS

Hepcidin production by HT-29 cells

HT-29 cells produced a detectable amount of hepcidin after 24 h of IL-6, TNF-a, and LPS stimulus. We did not observe a synergistic effect when LPS was combined with TNF-a or IL-6 regarding hepcidin production (Figure 1). No alterations in OD values in the MTT assay were detected in this assay (^{S1 in Supplementary material} S1 HT-29 cell viability after 24 h incubation with different IL-6, TNF-α, and LPS concentrations. No reductions in cellular viability were observed. ). When HT-29 cells were incubated with a stat-3 inhibitor (Static) or an IκBα inhibitor (BAY 11-7085) before the IL-6 stimulus, hepcidin production was reduced significantly. Only the IκBα inhibitor inhibited hepcidin production stimulated by TNF-a and LPS (Figure 2). No alterations in OD values of the MTT assay were detected in this assay (^{S2 in Supplementary file} S2 HT-29 cell viability after 24 h of incubation with IL-6, TNF-α, and LP and different concentrations of Static and BAY 11-7085 isolated or IL-6, TNF-α, and LPS previously incubated with Static and BAY 11-7085. No reductions in cellular viability were observed. ).

FIGURE 1
Hepcidin released by HT-29 cells after TNF-α (10-200 ng/mL), IL-6 (10-100 ng/mL), and LPS (0.01-10 µg/mL) or combinations of TNF-α (20 ng/mL) and LPS or IL-6 (50 ng/mL) and LPS (0.1 µg/mL) stimulus. *P<0.05 when compared with non-stimulated (NS) cells.

FIGURE 2
Hepcidin released by HT-29 cell after TNF-α (20 ng/mL), IL-6 (50 ng/mL), and LPS (0.1 µg/mL) pretreated with Stattic (1 and 10 µM) or BAY 11-7085 (1 and 5 µM) *P<0.05 when compared with non-pretreated (NT) cells.

HT-29 cells stimulated by hepcidin release IL-8

As a model of intestinal inflammation, we assessed the HT-29 cell’s ability to produce IL-8 when stimulated by hepcidin, LPS alone, or LPS and hepcidin combined. Hepcidin induces IL-8 release by HT-29 cells when used as an isolated stimulus. LPS alone also promoted a great IL-8 release by HT-29 cells. However, when hepcidin was co-incubated with LPS, a significant inhibition in IL-8 release was assessed (Figure 3A). As we could register, the concentration of 200 pg/mL of hepcidin reduced cell viability in HT-29 after 24 h (Figure 3B). Another experiment using hepcidin (20 pg/mL) and LPS was performed to evaluate IL-8 release, but in this experimental set, HT-29 cells were previously incubated with an antibody anti-ferroportin for 30 min. No reduction of viability was observed (data not shown), but the FPN blockade was able to reverse the ability of hepcidin to inhibit IL-8 release induced by LPS (Figure 4A). However, no inhibition was observed in IL-8 release induced by hepcidin alone when antibody anti-FPN was employed (Figure 4B).

FIGURE 3
IL-8 released induced by hepcidin (20 and 200 pg/mL) alone or in combination with LPS (0.1 µg/mL) by HT-29 cell (A) and cellular viability after hepcidin (20 and 200 pg/mL) alone or in combination with LPS (0.1 µg/mL) in HT-29 cell. *P<0.05 when compared with non-stimulated (NS) cells. # P<0.05 when compared with LPS stimulus alone (LPS).

FIGURE 4
IL-8 released induced by hepcidin (20 pg/mL;) in HT-29 cells without or with pre-treatment with antibody anti-ferroportin (Ab anti-FPN) (A). IL-8 release induced by LPS alone (0.1 µg/mL) or combined with hepcidin (20 pg/mL) in HT-29 cells without or with pre-treatment with antibody anti-FPN (Ab hf). *P<0.05 when compared with non-stimulated (NS) cells. # P<0.05 when compared with LPS stimulus alone (LPS).

Hepcidin blockade NF-kB translocation induced by LPS

An additional experiment with hepcidin at a non-toxic dose (20 pg/mL) in combination with LPS was carried out. The nuclear protein extract was evaluated for the amount of p65 NF-kB. Hepcidin significantly decreases the presence of p65 NF-kB at the HT-29 nucleus after LPS stimulation (Figure 5).

FIGURE 5
NFkb activity induced by LPS alone (0.1 µg/mL), hepcidin (20 pg/mL) combined with LPS in HT-29 cells. *P<0.05 when compared with non-stimulated (NS) cells. # P<0.05 when compared with LPS stimulus alone (LPS).

HT-29 cells were stimulated by hepcidin

Hepcidin showed the ability to increase ROS in HT-29 cells (Figure 6).

FIGURE 6
ROS production induced by hepcidin (20 pg/mL) by HT-29 cell. *P<0.05 when compared to non-stimulated (NS) cells.

DISCUSSION

The main stimulus for hepcidin production is iron overload signaling via the bone morphogenetic protein-SMAD (BMP-SMAD) pathways²⁹. The second most studied stimulator of hepcidin production is inflammation. In this situation, the interleukin-6/janus kinase 2/signal transducer and activator of transcription 3 (IL-6/JAK2/STAT3) pathway are considered the most important as well, studies support the concept that hepcidin production by iron and inflammation are interconnected, suggesting crosstalk between IL-6-STAT3 and BMP-SMAD pathways³⁰^-³¹. We demonstrated that HT-29 can produce hepcidin when stimulated by IL-6, TNF-a, and LPS. We previously showed Hamp expression in these cells after IL-6 and LPS stimulus¹⁷. The amount of hepcidin produced by HT-29 cells is low, and the combined inflammatory stimulus did not act synergistically. Ghadimi et al. (2020) registered in a triple cell co-culture model with HT-29/B6/HEPG-2 cells after 24 h of LPS stimulus amounts of hepcidin about 1.25 ng/mL³². For example, hepcidin production in brain cells is highest in glial cells and lowest in neurons after IL-6 and LPS stimulation³³, suggesting that different cells can produce hepcidin in various quantities.

We observed that inhibition of STAT-3 signaling by STATTIC was able to inhibit hepcidin production in HT-29 after the IL-6 stimulus but not after LPS or TNF-a stimulus, suggesting that IL-6/JAK2/STAT3 is present in HT-29 cells. Therefore, the IκBα inhibitor (BAY 11-7085) inhibited hepcidin production induced by IL-6, LPS, and TNF-a, suggesting that NFk-B signaling could also participate in hepcidin production in intestinal cells. In human peripheral blood leukocytes, NFk-B was pointed as a mediator of LPS-induced mRNA expression of hepcidin¹¹.

Our next step was to evaluate if epithelial intestinal cells respond to hepcidin stimulation, altering cytokine release upon the basal or inflammatory state by measuring IL-8 released induced by LPS from E. coli as a model. We demonstrated that HT-29 cells produce IL-8 in response to hepcidin stimulation. We used a higher hepcidin concentration than we can register as produced by HT-29 cells isolated but lower than reported in co-culture assays or in vivo systemic measurements. The concentration of hepcidin 20 pg/mL was more effective in inducing IL-8 release than 200 pg/mL, but cellular viability assay demonstrated a significant reduction of it at 200 pg/mL treated cells. In gastric cells, in patients and an experimental model with chronic atrophic gastritis, hepcidin seems to induce ferroptosis throughout IL-6/STAT3 pathways, contributing to gastric injury³⁴. We cannot fail to consider the hypothesis that in vitro, high concentrations of hepcidin show different results than those observed with the lowest concentration that we used in this study, added to the fact that we do not know what concentrations are produced locally in the gastrointestinal tract in a physiological situation or during inflammatory and infectious processes.

Interestingly, when cells were pretreated with 20 pg/mL hepcidin and stimulated with LPS as an inflammatory stimulus, the amount of IL-8 produced by HT-29 cells was reduced without reductions in cellular viability, indicating that in the inflammatory context, the response is contrary. In inflammatory diseases, like ulcerative colitis and Cron’s disease, the release of IL-8 has an important role in inducing neutrophil infiltration in human intestinal mucosa³⁵. In macrophages, as previously demonstrated by De Domenico et al., binding hepcidin to ferroportin results in the altered transcription of several genes, including Socs3. Hepcidin reduces IL-6 and TNF-a production induced by LPS in macrophages by inducing SOCS3 (suppressors of cytokine signaling-3) dampened the cytokine release induced by TLR stimulation³⁶. It is assumed that the same is occurring in our in vitro model, as in the presence of anti-FNP antibody, the IL-8 released induced by LPS was modified. In vivo, hepcidin protected mice from a lethal dose of LPS, and Hamp^-/- mice were more susceptible to death induced by LPS than wild-type mice³⁶. After hepcidin treatment, we registered a direct inhibition of NFk-B activation induced by LPS in HT-29 cells, a classical signaling pathway in intestinal inflammation³⁷^-³⁹.

Hepcidin induces an accumulation of ROS in HT-29 cells, which could be related to IL-8 production in these cells under basal conditions. We searched for iron accumulation in HT-29 cells stimulated by hepcidin; however, the Prussian blue stain analysis did not detect any iron accumulation (data not shown) in cells stimulated or unstimulated. In vivo, in colitis induced by TNBS, where we detected a local production of hepcidin, we also did not detect iron accumulation in the epithelium¹⁷. Using another intestinal line cell, Phoaubon et al. demonstrated that Caco-2 cells uptake iron when cells were exposed to ascorbic acid together with ferric ammonium citrate, and uptake was suppressed by hepcidin. Still, calcium uptake under physiological conditions is increased by hepcidin. Calcium uptake could explain ROS increases and IL-8 release in HT-29 after hepcidin exposure⁴⁰.

Our research question in this work was about the direct effect of hepcidin in intestinal epithelial cells during the inflammatory process, without interference by microbiome or systemic/luminal iron levels. Chronic inflammation of the epithelial barrier in the gastrointestinal lumen is dangerous and associated with cancer development, for example⁴¹. However, alterations in the microbial composition of the gastrointestinal tract (dysbiosis) are believed to contribute to the establishment of a chronic inflammatory process, and it is not present in our experimental model, limiting the broader understanding of what the role of hepcidin may be in this context.

In conclusion, HT-29 cells, as a model of the intestinal epithelium, can produce hepcidin at low levels in response to an inflammatory stimulus. Hepcidin presents a dual action in HT-29 cells, playing a proinflammatory action by producing ROS and IL-8 at physiological conditions but an anti-inflammatory action by reducing IL-8 release and NFk-B activation induced by LPS at the concentration used in this in vitro study. The role of hepcidin on intestinal inflammation could represent a modulatory mechanism to limit the inflammatory response to bacterial infections that, through TLRs, increase the local production of cytokines and hepcidin itself.

REFERENCES

¹ Xu Y, Alfaro-Magallanes VM, Babitt JL. Physiological and pathophysiological mechanisms of hepcidin regulation: clinical implications for iron disorders. Br J Haematol. 2021;193:882-93.
² Aksoyalp ZS, Temel A, Erdogan BR. Iron in infectious diseases friend or foe?: The role of gut microbiota. J Trace Elem Med Biol. 2023;75:127093.
³ Kulaksiz H, Theilig F, Bachmann S, Gehrke SG, Rost D, Janetzko A, et al. The iron-regulatory peptide hormone hepcidin: expression and cellular localization in the mammalian kidney. J Endocrinol. 2005;184:361-70.
⁴ Lakhal-Littleton S, Wolna M, Chung YJ, Christian HC, Heather LC, Brescia M, et al. An essential cell-autonomous role for hepcidin in cardiac iron homeostasis. Elife. 2016;5.
⁵ Vela D. The Dual Role of Hepcidin in Brain Iron Load and Inflammation. Front Neurosci. 2018;12:740.
⁶ Lakhal-Littleton S, Crosby A, Frise MC, Mohammad G, Carr CA, Loick PAM, et al. Intracellular iron deficiency in pulmonary arterial smooth muscle cells induces pulmonary arterial hypertension in mice. Proc Natl Acad Sci U S A. 2019;116:13122-30.
⁷ Kulaksiz H, Fein E, Redecker P, Stremmel W, Adler G, Cetin Y. Pancreatic beta-cells express hepcidin, an iron-uptake regulatory peptide. J Endocrinol. 2008;197:241-9.
⁸ Gotardo EM, dos Santos AN, Miyashiro RA, Gambero S, Rocha T, Ribeiro ML, et al. Mice that are fed a high-fat diet display increased hepcidin expression in adipose tissue. J Nutr Sci Vitaminol (Tokyo). 2013;59:454-61.
⁹ Bessman NJ, Mathieu JRR, Renassia C, Zhou L, Fung TC, Fernandez KC, et al. Dendritic cell-derived hepcidin sequesters iron from the microbiota to promote mucosal healing. Science. 2020;368:186-9.
¹⁰ Barton JC, Acton RT. Hepcidin, iron, and bacterial infection. Vitam Horm. 2019;110:223-42.
¹¹ Wu S, Zhang K, Lv C, Wang H, Cheng B, Jin Y, et al. Nuclear factor-kappaB mediated lipopolysaccharide-induced mRNA expression of hepcidin in human peripheral blood leukocytes. Innate Immun. 2012;18:318-24.
¹² Pinto JP, Dias V, Zoller H, Porto G, Carmo H, Carvalho F, et al. Hepcidin messenger RNA expression in human lymphocytes. Immunology. 2010;130:217-30.
¹³ Peyssonnaux C, Zinkernagel AS, Datta V, Lauth X, Johnson RS, Nizet V. TLR4-dependent hepcidin expression by myeloid cells in response to bacterial pathogens. Blood. 2006;107:3727-32.
¹⁴ Conde Diez S, de Las Cuevas Allende R, Conde Garcia E. Anemia of inflammation and iron metabolism in chronic diseases. Rev Clin Esp (Barc). 2024;224:598-608.
¹⁵ Kemna E, Pickkers P, Nemeth E, van der Hoeven H, Swinkels D. Time-course analysis of hepcidin, serum iron, and plasma cytokine levels in humans injected with LPS. Blood. 2005;106:1864-6.
¹⁶ Atkinson MA, Leonard MB, Herskovitz R, Baldassano RN, Denburg MR. Changes in Hepcidin and Hemoglobin After Anti-TNF-alpha Therapy in Children and Adolescents With Crohn Disease. J Pediatr Gastroenterol Nutr. 2018;66:90-4.
¹⁷ Gotardo EM, Ribeiro Gde A, Clemente TR, Moscato CH, Tome RB, Rocha T, et al. Hepcidin expression in colon during trinitrobenzene sulfonic acid-induced colitis in rats. World J Gastroenterol. 2014;20:4345-52.
¹⁸ Giudici F, Lombardelli L, Russo E, Cavalli T, Zambonin D, Logiodice F, et al. Multiplex gene expression profile in inflamed mucosa of patients with Crohn’s disease ileal localization: A pilot study. World J Clin Cases. 2019;7(17):2463-76.
¹⁹ Sato Y, Yoneyama O, Azumaya M, Takeuchi M, Sasaki SY, Yokoyama J, et al. The relationship between iron deficiency in patients with Helicobacter pylori-infected nodular gastritis and the serum prohepcidin level. Helicobacter. 2015;20:11-8.
²⁰ Yokota S, Konno M, Mino E, Sato K, Takahashi M, Fujii N. Enhanced Fe ion-uptake activity in Helicobacter pylori strains isolated from patients with iron-deficiency anemia. Clin Infect Dis. 2008;46:e31-3.
²¹ Nishigaki Y, Sato Y, Sato H, Iwafuchi M, Terai S. Influence of Helicobacter pylori Infection on Hepcidin Expression in the Gastric Mucosa. Kurume Med J. 2023;68:107-13.
²² Schwarz P, Kubler JA, Strnad P, Muller K, Barth TF, Gerloff A, et al. Hepcidin is localised in gastric parietal cells, regulates acid secretion and is induced by Helicobacter pylori infection. Gut. 2012;61:193-201.
²³ Arezes J, Jung G, Gabayan V, Valore E, Ruchala P, Gulig PA, et al. Hepcidin-induced hypoferremia is a critical host defense mechanism against the siderophilic bacterium Vibrio vulnificus. Cell Host Microbe. 2015;17:47-57.
²⁴ Mahalhal A, Burkitt MD, Duckworth CA, Hold GL, Campbell BJ, Pritchard DM, et al. Long-Term Iron Deficiency and Dietary Iron Excess Exacerbate Acute Dextran Sodium Sulphate-Induced Colitis and Are Associated with Significant Dysbiosis. Int J Mol Sci. 2021;22:3646.
²⁵ Tandon A, Baral B, Saini V, Kandpal M, Dixit AK, Parmar HS, et al. The role of Helicobacter pylori in augmenting the severity of SARS-CoV-2 related gastrointestinal symptoms: An insight from molecular mechanism of co-infection. Heliyon. 2024;10:e37585.
²⁶ Haddad MJ, Zuluaga-Arango J, Mathieu H, Barbezier N, Anton PM. Intestinal Epithelial Co-Culture Sensitivity to Proinflammatory Stimuli and Polyphenols Is Medium-Independent. Int J Mol Sci. 2024;25:7360.
²⁷ Wang A, Guan C, Wang T, Mu G, Tuo Y. Indole-3-Lactic Acid, a Tryptophan Metabolite of Lactiplantibacillus plantarum DPUL-S164, Improved Intestinal Barrier Damage by Activating AhR and Nrf2 Signaling Pathways. J Agric Food Chem. 2023;71:18792-801.
²⁸ Zhao J, Zhao R, Cheng L, Yang J, Zhu L. Peroxisome proliferator-activated receptor gamma activation promotes intestinal barrier function by improving mucus and tight junctions in a mouse colitis model. Dig Liver Dis. 2018;50:1195-204.
²⁹ Pettinato M, Aghajan M, Guo S, Bavuso Volpe L, Carleo R, Nai A, et al. A functional interplay between the two BMP-SMAD pathway inhibitors TMPRSS6 and FKBP12 regulates hepcidin expression in vivo. Am J Physiol Gastrointest Liver Physiol. 2024;326:G310-G7.
³⁰ Silvestri L, Nai A, Dulja A, Pagani A. Hepcidin and the BMP-SMAD pathway: An unexpected liaison. Vitam Horm. 2019;110:71-99.
³¹ Lanser L, Fuchs D, Kurz K, Weiss G. Physiology and Inflammation Driven Pathophysiology of Iron Homeostasis-Mechanistic Insights into Anemia of Inflammation and Its Treatment. Nutrients. 2021;13:3732.
³² Ghadimi D, Yoness Hassan MF, Folster-Holst R, Rocken C, Ebsen M, de Vrese M, et al. Regulation of hepcidin/iron-signalling pathway interactions by commensal bifidobateria plays an important role for the inhibition of metaflammation-related biomarkers. Immunobiology. 2020;225:151874.
³³ Urrutia P, Aguirre P, Esparza A, Tapia V, Mena NP, Arredondo M, et al. Inflammation alters the expression of DMT1, FPN1 and hepcidin, and it causes iron accumulation in central nervous system cells. J Neurochem. 2013;126:541-9.
³⁴ Zhao Y, Zhao J, Ma H, Han Y, Xu W, Wang J, et al. High Hepcidin Levels Promote Abnormal Iron Metabolism and Ferroptosis in Chronic Atrophic Gastritis. Biomedicines. 2023;11:2338.
³⁵ Mitsuyama K, Toyonaga A, Sasaki E, Watanabe K, Tateishi H, Nishiyama T, et al. IL-8 as an important chemoattractant for neutrophils in ulcerative colitis and Crohn’s disease. Clin Exp Immunol. 1994;96:432-6.
³⁶ De Domenico I, Zhang TY, Koening CL, Branch RW, London N, Lo E, et al. Hepcidin mediates transcriptional changes that modulate acute cytokine-induced inflammatory responses in mice. J Clin Invest. 2010;120:2395-405.
³⁷ Zhang D, Mi M, Jiang F, Sun Y, Li Y, Yang L, et al. Apple polysaccharide reduces NF-Kb mediated colitis-associated colon carcinogenesis. Nutr Cancer. 2015;67:177-90.
³⁸ Engevik MA, Herrmann B, Ruan W, Engevik AC, Engevik KA, Ihekweazu F, et al. Bifidobacterium dentium-derived y-glutamylcysteine suppresses ER-mediated goblet cell stress and reduces TNBS-driven colonic inflammation. Gut Microbes. 2021;13:1-21.
³⁹ Wang K, Qiu L, Zhu J, Sun Q, Qu W, Yu Y, et al. Environmental contaminant BPA causes intestinal damage by disrupting cellular repair and injury homeostasis in vivo and in vitro. Biomed Pharmacother. 2021;137:111270.
⁴⁰ Sun W, Wu X, Gao H, Yu J, Zhao W, Lu JJ, et al. Cytosolic calcium mediates RIP1/RIP3 complex-dependent necroptosis through JNK activation and mitochondrial ROS production in human colon cancer cells. Free Radic Biol Med. 2017;108:433-44.
⁴¹ Kania B, Sotelo A, Ty D, Wisco JJ. The Prevention of Inflammation and the Maintenance of Iron and Hepcidin Homeostasis in the Gut, Liver, and Brain Pathologies. J Alzheimers Dis. 2023;92:769-89.

Disclosure of funding:
This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq 303625/2019-8)
Declaration of use of artificial intelligence:
The author wrote the manuscript in English and used Grammarly® to revise spelling and grammar

Supplementary material

A possible role of hepcidin in intestinal inflammation. Prione et al.

S1
HT-29 cell viability after 24 h incubation with different IL-6, TNF-α, and LPS concentrations. No reductions in cellular viability were observed.

S2
HT-29 cell viability after 24 h of incubation with IL-6, TNF-α, and LP and different concentrations of Static and BAY 11-7085 isolated or IL-6, TNF-α, and LPS previously incubated with Static and BAY 11-7085. No reductions in cellular viability were observed.

Edited by

Associate Editor:
Wellington Andraus

Publication Dates

Publication in this collection
20 Dec 2024
Date of issue
2024

History

Received
22 Apr 2024
Accepted
01 Nov 2024

This is an open-access article distributed under the terms of the Creative Commons Attribution License

[1] ¹ Xu Y, Alfaro-Magallanes VM, Babitt JL. Physiological and pathophysiological mechanisms of hepcidin regulation: clinical implications for iron disorders. Br J Haematol. 2021;193:882-93.

[2] ² Aksoyalp ZS, Temel A, Erdogan BR. Iron in infectious diseases friend or foe?: The role of gut microbiota. J Trace Elem Med Biol. 2023;75:127093.

[3] ³ Kulaksiz H, Theilig F, Bachmann S, Gehrke SG, Rost D, Janetzko A, et al. The iron-regulatory peptide hormone hepcidin: expression and cellular localization in the mammalian kidney. J Endocrinol. 2005;184:361-70.

[4] ⁴ Lakhal-Littleton S, Wolna M, Chung YJ, Christian HC, Heather LC, Brescia M, et al. An essential cell-autonomous role for hepcidin in cardiac iron homeostasis. Elife. 2016;5.

[5] ⁵ Vela D. The Dual Role of Hepcidin in Brain Iron Load and Inflammation. Front Neurosci. 2018;12:740.

[6] ⁶ Lakhal-Littleton S, Crosby A, Frise MC, Mohammad G, Carr CA, Loick PAM, et al. Intracellular iron deficiency in pulmonary arterial smooth muscle cells induces pulmonary arterial hypertension in mice. Proc Natl Acad Sci U S A. 2019;116:13122-30.

[7] ⁷ Kulaksiz H, Fein E, Redecker P, Stremmel W, Adler G, Cetin Y. Pancreatic beta-cells express hepcidin, an iron-uptake regulatory peptide. J Endocrinol. 2008;197:241-9.

[8] ⁸ Gotardo EM, dos Santos AN, Miyashiro RA, Gambero S, Rocha T, Ribeiro ML, et al. Mice that are fed a high-fat diet display increased hepcidin expression in adipose tissue. J Nutr Sci Vitaminol (Tokyo). 2013;59:454-61.

[9] ⁹ Bessman NJ, Mathieu JRR, Renassia C, Zhou L, Fung TC, Fernandez KC, et al. Dendritic cell-derived hepcidin sequesters iron from the microbiota to promote mucosal healing. Science. 2020;368:186-9.

[10] ¹⁰ Barton JC, Acton RT. Hepcidin, iron, and bacterial infection. Vitam Horm. 2019;110:223-42.

[11] ¹¹ Wu S, Zhang K, Lv C, Wang H, Cheng B, Jin Y, et al. Nuclear factor-kappaB mediated lipopolysaccharide-induced mRNA expression of hepcidin in human peripheral blood leukocytes. Innate Immun. 2012;18:318-24.

[12] ¹² Pinto JP, Dias V, Zoller H, Porto G, Carmo H, Carvalho F, et al. Hepcidin messenger RNA expression in human lymphocytes. Immunology. 2010;130:217-30.

[13] ¹³ Peyssonnaux C, Zinkernagel AS, Datta V, Lauth X, Johnson RS, Nizet V. TLR4-dependent hepcidin expression by myeloid cells in response to bacterial pathogens. Blood. 2006;107:3727-32.

[14] ¹⁴ Conde Diez S, de Las Cuevas Allende R, Conde Garcia E. Anemia of inflammation and iron metabolism in chronic diseases. Rev Clin Esp (Barc). 2024;224:598-608.

[15] ¹⁵ Kemna E, Pickkers P, Nemeth E, van der Hoeven H, Swinkels D. Time-course analysis of hepcidin, serum iron, and plasma cytokine levels in humans injected with LPS. Blood. 2005;106:1864-6.

[16] ¹⁶ Atkinson MA, Leonard MB, Herskovitz R, Baldassano RN, Denburg MR. Changes in Hepcidin and Hemoglobin After Anti-TNF-alpha Therapy in Children and Adolescents With Crohn Disease. J Pediatr Gastroenterol Nutr. 2018;66:90-4.

[17] ¹⁷ Gotardo EM, Ribeiro Gde A, Clemente TR, Moscato CH, Tome RB, Rocha T, et al. Hepcidin expression in colon during trinitrobenzene sulfonic acid-induced colitis in rats. World J Gastroenterol. 2014;20:4345-52.

[18] ¹⁸ Giudici F, Lombardelli L, Russo E, Cavalli T, Zambonin D, Logiodice F, et al. Multiplex gene expression profile in inflamed mucosa of patients with Crohn’s disease ileal localization: A pilot study. World J Clin Cases. 2019;7(17):2463-76.

[19] ¹⁹ Sato Y, Yoneyama O, Azumaya M, Takeuchi M, Sasaki SY, Yokoyama J, et al. The relationship between iron deficiency in patients with Helicobacter pylori-infected nodular gastritis and the serum prohepcidin level. Helicobacter. 2015;20:11-8.

[20] ²⁰ Yokota S, Konno M, Mino E, Sato K, Takahashi M, Fujii N. Enhanced Fe ion-uptake activity in Helicobacter pylori strains isolated from patients with iron-deficiency anemia. Clin Infect Dis. 2008;46:e31-3.

[21] ²¹ Nishigaki Y, Sato Y, Sato H, Iwafuchi M, Terai S. Influence of Helicobacter pylori Infection on Hepcidin Expression in the Gastric Mucosa. Kurume Med J. 2023;68:107-13.

[22] ²² Schwarz P, Kubler JA, Strnad P, Muller K, Barth TF, Gerloff A, et al. Hepcidin is localised in gastric parietal cells, regulates acid secretion and is induced by Helicobacter pylori infection. Gut. 2012;61:193-201.

[23] ²³ Arezes J, Jung G, Gabayan V, Valore E, Ruchala P, Gulig PA, et al. Hepcidin-induced hypoferremia is a critical host defense mechanism against the siderophilic bacterium Vibrio vulnificus. Cell Host Microbe. 2015;17:47-57.

[24] ²⁴ Mahalhal A, Burkitt MD, Duckworth CA, Hold GL, Campbell BJ, Pritchard DM, et al. Long-Term Iron Deficiency and Dietary Iron Excess Exacerbate Acute Dextran Sodium Sulphate-Induced Colitis and Are Associated with Significant Dysbiosis. Int J Mol Sci. 2021;22:3646.

[25] ²⁵ Tandon A, Baral B, Saini V, Kandpal M, Dixit AK, Parmar HS, et al. The role of Helicobacter pylori in augmenting the severity of SARS-CoV-2 related gastrointestinal symptoms: An insight from molecular mechanism of co-infection. Heliyon. 2024;10:e37585.

[26] ²⁶ Haddad MJ, Zuluaga-Arango J, Mathieu H, Barbezier N, Anton PM. Intestinal Epithelial Co-Culture Sensitivity to Proinflammatory Stimuli and Polyphenols Is Medium-Independent. Int J Mol Sci. 2024;25:7360.

[27] ²⁷ Wang A, Guan C, Wang T, Mu G, Tuo Y. Indole-3-Lactic Acid, a Tryptophan Metabolite of Lactiplantibacillus plantarum DPUL-S164, Improved Intestinal Barrier Damage by Activating AhR and Nrf2 Signaling Pathways. J Agric Food Chem. 2023;71:18792-801.

[28] ²⁸ Zhao J, Zhao R, Cheng L, Yang J, Zhu L. Peroxisome proliferator-activated receptor gamma activation promotes intestinal barrier function by improving mucus and tight junctions in a mouse colitis model. Dig Liver Dis. 2018;50:1195-204.

[29] ²⁹ Pettinato M, Aghajan M, Guo S, Bavuso Volpe L, Carleo R, Nai A, et al. A functional interplay between the two BMP-SMAD pathway inhibitors TMPRSS6 and FKBP12 regulates hepcidin expression in vivo. Am J Physiol Gastrointest Liver Physiol. 2024;326:G310-G7.

[30] ³⁰ Silvestri L, Nai A, Dulja A, Pagani A. Hepcidin and the BMP-SMAD pathway: An unexpected liaison. Vitam Horm. 2019;110:71-99.

[31] ³¹ Lanser L, Fuchs D, Kurz K, Weiss G. Physiology and Inflammation Driven Pathophysiology of Iron Homeostasis-Mechanistic Insights into Anemia of Inflammation and Its Treatment. Nutrients. 2021;13:3732.

[32] ³² Ghadimi D, Yoness Hassan MF, Folster-Holst R, Rocken C, Ebsen M, de Vrese M, et al. Regulation of hepcidin/iron-signalling pathway interactions by commensal bifidobateria plays an important role for the inhibition of metaflammation-related biomarkers. Immunobiology. 2020;225:151874.

[33] ³³ Urrutia P, Aguirre P, Esparza A, Tapia V, Mena NP, Arredondo M, et al. Inflammation alters the expression of DMT1, FPN1 and hepcidin, and it causes iron accumulation in central nervous system cells. J Neurochem. 2013;126:541-9.

[34] ³⁴ Zhao Y, Zhao J, Ma H, Han Y, Xu W, Wang J, et al. High Hepcidin Levels Promote Abnormal Iron Metabolism and Ferroptosis in Chronic Atrophic Gastritis. Biomedicines. 2023;11:2338.

[35] ³⁵ Mitsuyama K, Toyonaga A, Sasaki E, Watanabe K, Tateishi H, Nishiyama T, et al. IL-8 as an important chemoattractant for neutrophils in ulcerative colitis and Crohn’s disease. Clin Exp Immunol. 1994;96:432-6.

[36] ³⁶ De Domenico I, Zhang TY, Koening CL, Branch RW, London N, Lo E, et al. Hepcidin mediates transcriptional changes that modulate acute cytokine-induced inflammatory responses in mice. J Clin Invest. 2010;120:2395-405.

[37] ³⁷ Zhang D, Mi M, Jiang F, Sun Y, Li Y, Yang L, et al. Apple polysaccharide reduces NF-Kb mediated colitis-associated colon carcinogenesis. Nutr Cancer. 2015;67:177-90.

[38] ³⁸ Engevik MA, Herrmann B, Ruan W, Engevik AC, Engevik KA, Ihekweazu F, et al. Bifidobacterium dentium-derived y-glutamylcysteine suppresses ER-mediated goblet cell stress and reduces TNBS-driven colonic inflammation. Gut Microbes. 2021;13:1-21.

[39] ³⁹ Wang K, Qiu L, Zhu J, Sun Q, Qu W, Yu Y, et al. Environmental contaminant BPA causes intestinal damage by disrupting cellular repair and injury homeostasis in vivo and in vitro. Biomed Pharmacother. 2021;137:111270.

[40] ⁴⁰ Sun W, Wu X, Gao H, Yu J, Zhao W, Lu JJ, et al. Cytosolic calcium mediates RIP1/RIP3 complex-dependent necroptosis through JNK activation and mitochondrial ROS production in human colon cancer cells. Free Radic Biol Med. 2017;108:433-44.

[41] ⁴¹ Kania B, Sotelo A, Ty D, Wisco JJ. The Prevention of Inflammation and the Maintenance of Iron and Hepcidin Homeostasis in the Gut, Liver, and Brain Pathologies. J Alzheimers Dis. 2023;92:769-89.