Biomarkers and immunomodulatory effect of erythropoietin in postoperative ileus

Abstract

Objective

Postoperative ileus (POI) is a type of intestinal motility disorder (IMD) that can occur after abdominal surgery. At present, there is no accepted treatment for POI and no reliable biomarkers to assess the status of POI. Erythropoietin (EPO) can promote the recovery of the intestine from various injuries, including POI. This study aimed to investigate the immunomodulatory role of EPO and to identify reliable biomarkers of POI.

Results

The computational analysis suggested that immune signaling via EPO/EPOR pathways may vary in POI. Luminex multiplex analysis showed that EPO treatment considerably altered intestinal tissue pro-inflammatory (CRP, IL6, IL12p70, IL12p40, IL2, and IFNγ) and anti-inflammatory (IL4, IL10, and IL13) cytokines. EPO decreased pro-inflammatory intestinal chemokines (CCL12, CXCL2, and CCL4) and serum IL6, CRP, and CCL12 following POI. IL6, CRP, and CCL12 may serve as promising biomarkers for POI in both tissue and serum samples. The gene expression analysis of these markers confirmed their significance with increased levels in intestinal tissue of POI, and EPO treatment caused attenuation.

Conclusions

To our knowledge, this is the first study to investigate the immunomodulatory effect of EPO following IM, and suggests CCL12, CRP, and IL6 as reliable biomarkers of POI in both intestinal and serum samples.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13104-026-07659-z.

Introduction

Abdominal surgery is a cornerstone of treatment for several bowel disorders, such as tumors, obstructions, Crohn’s disease, ulcerative colitis, and pancreatitis [1, 2]. A common complication of surgery in these conditions is post-operative ileus (POI), which can last longer than five days after surgery and is associated with decreased intestinal motility, prolonged parenteral nutrition, abdominal discomfort, and increased morbidity [3, 4]. POI affects between 10% and 30% of patients after abdominal surgery, but unfortunately, there is a dearth of treatment options for POI [5, 6].

Erythropoietin (EPO) is a pleiotropic cytokine hormone used to treat anemia and has been shown to have beneficial effects against intestinal disorders [7–9]. Our recent study suggested novel beneficial effects of EPO against POI by reducing inflammation and oxidative stress using the POI mouse model [10]. Other studies have also shown that reactive cells such as neutrophils, macrophages, leucocytes, and T-cells play a vital role in intestine motility disorders (IMDs) [11, 12]. A few biomarker studies revealed increased expression of pro-inflammatory cytokines like CRP, IL1β, TNFα, and IL6 and chemokines like CCL2/3/4/5 and CXCL5/8/9/10/12 during POI [13, 14]. The complexities of POI are multifactorial and coupled with various biological mediators of intestine-responsive or reparative cells to control inflammation, intestinal transit, and homeostasis functions [15, 16].

Computational biology offers a variety of tools to reveal signal transduction networks, as well as more technical protein-protein interaction networks [17, 18]. Interaction network databases such as Reactome, String, BioGrid, or IntAct, can be used to compare and characterize functional molecular signaling sub-networks in complex systems [19]. By breaking down large networks into distinct components or sub-pathways, we can better understand how different treatments alter complex signal transduction networks [20]. This includes the elucidation of motifs, which are groups of nodes (intersections) in interaction maps that relay and modulate signals [21]. Treatments like EPO can therefore be analyzed for their direct effects on large subnetworks and downstream effector proteins. We focused on mapping the immune signaling agents downstream of EPO/EPOR pathways that are expected to be differentially regulated upon changes in POI to support possible clinical translation of EPO for IMDs.

The current study aimed to investigate the impact of EPO treatment on the expression of cytokines and chemokines that cause inflammation after IM and to identify potential biomarkers in both target intestinal tissue and circulation. We hypothesized that IM would worsen inflammation, and that EPO treatment would alleviate these effects in injured intestinal tissue, which are measurable in both tissue and serum. Furthermore, we hypothesized that some identified molecules could serve as confirmatory biomarkers for POI.

Main text

Materials and methods

Details of the methods used in the current study, including product and manufacturer information, are provided in Additional file 1.

Results

EPO is connected to multiple inflammatory cytokines and chemokines

The computational analysis revealed a complex network of immune signaling agents influenced by the EPO/EPOR pathways, which are expected to vary with POI (Additional file 2: Fig. S1). A more precise network was created by eliminating weaker nodes and concentrating on significant interacting hubs (Fig. S2). Further, we integrated the text-mined interactions enumerated by the Agilent Literature Search module in Cytoscape. We found that many of the key cytokines and chemokines related to EPO/EOPR proteins were highly significant. This strengthened our hypothesis that EPOR/EPO perturbations can modulate these cytokines. The shortlisted network was a smaller network (Fig. 1). The top 12 high-degree hub nodes (IL4, IL6, IL2, IL10, IL1β, CXCR3, CCL4, CCL2, CXCL2, CCL20, IL37, and CCL3) were cytokines and chemokines, which are a superfamily of secreted proteins involved in immune-regulatory and inflammatory processes.

Fig. 1.

PPi map of EPO and proinflammatory markers. Centrality degree and Betweenness were used to construct nodes and edges. Colored nodes (circles labeled with target protein) are the gene products of interest related to EPOR vs. proinflammatory signaling. Inset the node is the target protein’s 3D structure (known or predicted). Interactions are the edges (connecting lines): Turquoise colored connector represents database-curated interaction, and the purple connector represents experimentally demonstrated interaction. Predicted interactions by gene neighborhood (green), gene fusions (red), or gene co-occurrence (blue). Interactions from other sources are colored as black (co-expression), light blue (protein homology), and beryl green for text-mined interactions (Agilent Literature Search: Cytoscape)

We speculated that EPO may affect inflammatory cytokine expression during POI. Our network analysis suggests that EPO plays a crucial role in inflammatory conditions related to POI by modulating the expression of cytokines and chemokines, which can lead to pathological inflammatory responses.

EPO attenuated intestine inflammatory cytokines following POI

We aimed to investigate the effect of EPO on inflammatory cytokines and chemokines and identify POI tissue biomarkers. We used Luminex multiplex assay platforms to quantify proteins targeted at intestinal tissue following IM and EPO treatment (Fig. 2A). Our data showed that EPO, compared to saline, effectively reduced pro-inflammatory cytokines such as IL6, CRP, IL2, IL12p70, IL12p40, and IFNγ (Fig. 2B). Surprisingly, EPO also decreased anti-inflammatory cytokines such as IL-4, IL-10, and IL13 (Fig. 2C). This event indicates pro-inflammatory conditions in the saline-treated group, which are reduced in the EPO group, leading to increased anti-inflammatory cytokines after IM. Our data suggest that IL6, IL12p70, and CRP are potential intestinal tissue cytokine biomarkers of POI.

Fig. 2.

EPO treatment controlled intestine tissue inflammatory cytokines following 24-hour IM in mice. A The figure depicts the details of experimental plans and studies. Luminex multiplex analysis data showed EPO treatment (5000 IU/kg/IP, immediately after surgery) significantly controlled protein levels of pro-inflammatory (CRP, IL6, IL12p70, IL12p40, IL2, and IFNγ) B and anti-inflammatory (IL4, IL10, and IL13) C cytokines as compared to saline treatment (normal saline, 0.1 ml/IP/mouse). The results are represented as a bar graph. Data were analyzed using one-way ANOVA and Tukey’s multiple comparisons test and expressed as mean ± SEM, *P < 0.0332 and **P < 0.0021, n = 3 per group

EPO attenuated intestine inflammatory chemokines following POI

Chemokines are small (8–10 kDa) heparin-binding proteins, which attract white blood cells, neutrophils, and monocytes out of the blood and into tissues at the site of injury [22]. These cells contribute to local and systemic inflammation, exacerbating the pathological condition [23, 24]. Under the same experimental conditions depicted in Fig. 2A, we observed a significant or effective increase in the levels of inflammatory chemokines such as CCL12, CCL4, and CXCL2 in the saline-treated group following IM (Fig. 3A). However, this increase was attenuated in the EPO treatment. Our results confirmed CCL12 and CXCL2 as chemokine biomarkers of POI.

Fig. 3.

EPO treatment reduced intestine tissue and serum pro-inflammatory markers following 24-hour IM in mice. Luminex multiplex analysis data showed EPO treatment (5000 IU/kg/IP, immediately after surgery) significantly or effectively attenuated protein levels of intestine tissue pro-inflammatory chemokines (CCL12, CXCL2, and CCL4) A and serum markers (CRP, IL6, and CCL12) B as compared to saline treatment (normal saline, 0.1 ml/IP/mouse). C The figure depicts the details of gene expression experimental plans. D qRT-PCR gene expression analysis showed significant upregulations of pro-inflammatory cytokines (CRP and IL6) and chemokine (CCL12) in untreated (normal saline, 0.1 ml/IP/mouse) mice, where it attenuated in the EPO treatment (5000 IU/kg/IP, immediately after surgery). The results are represented as a bar graph. Data were analyzed using one-way ANOVA and Tukey’s multiple comparisons test and expressed as mean ± SEM, *P < 0.0332 and **P < 0.0021, n = 3 per group

Effect of EPO on Circulating serum inflammatory markers following POI

Serum biomarkers are useful for diagnosing, prognosis, and identification of potential targets for the development of novel molecules for various intestinal disorders [25–28]. In our study, EPO-treated mice had significantly attenuated circulatory proinflammatory cytokines of CRP, IL6, and CCL12, where these were elevated in the saline group as compared to healthy mice (Fig. 3B). Additionally, EPO reduced the serum levels of IL1β, which was upregulated in the saline-treated mice (Additional file 2: Fig. S3). These protein markers, except IL1 β, were also found in intestine tissue, confirming their relevance in POI recovery. Finally, we found reduced EPO serum levels in saline-treated mice with POI compared to uninjured mice (Additional file 2: Fig. S3).

Confirmation of novel biomarkers using gene expression analysis following POI

The experimental conditions are illustrated in Fig. 3C. Protein expression of CRP, IL6, and CCL12 in both intestinal tissue and serum samples showed a significant upregulation following IM. Therefore, we showed interest in confirming these markers’ expressions at mRNA levels, where gene expressions are translatable to protein synthesis. Results showed significant upregulation of the CRP, IL6, and CCL12 gene expression in the intestinal tissue of untreated mice following IM, and attenuation in EPO-treated mice (Fig. 3D). With confirmation of these biomarkers at both protein and gene expression levels, they may serve to assist in assessing recovery of POI. We believe EPO might be an advisable treatment for several IMDs, which we will address in future studies.

Discussion

The recovery of patients after abdominal surgery depends on the resolution of a pro-inflammatory ileus [29, 30]. POI is characterized by the recruitment of immunomodulatory cells such as leukocytes, neutrophils, and monocytes into the intestine-which aggravates inflammation and reduces bowel transit function [31–33]. Our previous study using wildtype POI mice has confirmed an excessive infiltration of neutrophils (MPO⁺ cells) and macrophages (F4/80⁺ cells) in the muscular layers and villi of the intestine after IM, where EPO ameliorated these changes [10]. Developing an effective strategy to treat POI is challenging. To date, several studies have investigated POI mechanisms in various surgical and inflammatory conditions associated with complex alterations in cellular cytokines and chemokines [16, 34–36]. Protein-protein interactions among GO-terms related to EPO showed that cytokines and chemokines downstream are important for a pro-inflammatory response confirming the immunome and anti-inflammatory effect of EPO/EPOR beneficial in the case of POI-like post-traumatic gut impairment. The heterodimer EPOR (EPOR + ß-common receptor {CD131}), also known as the innate repair receptor (IRR), blocks p65 (an NF-κB subunit) and thereby suppresses the expression of multiple cytokines and chemokines [37]. Nairz et al. reported an IRR-specific agonist-Cibinetide that blocked dextran sulfate sodium (DSS) induced colitis [38]. Protein-protein interactions among GO-terms related to EPO converged to many immune pathways through EPOR-CD131 (IRR) signaling. We hope to understand this complex signaling cascades for the recovery of POI.

In this study, we showed that clinically approved dosages of EPO protected against inflammation during POI by mitigating the expression of pro-inflammatory cytokines such as CRP, IL6, IL12p70, IL12p40, IL2, and IFNγ, and chemokines such as CCL4, CCL12, and CXCL12. We also confirmed CRP, IL6, and CCL12 may serve as a promising biomarker of POI in both intestinal tissue and serum using protein/gene expression analysis. EPO significantly reduced anti-inflammatory cytokines, in parallel to inflammatory cytokine reduction, like IL4, IL10, and IL13, which is a sign of early protection from inflammation following POI.

EPO-dosed animals, the circulatory levels of EPO increased compared with the saline group. Studies report that chronic inflammatory conditions increase pro-inflammatory (IL2, IL6, IFNγ, and TNFα) signaling, which suppresses EPO production by inhibiting the proliferation and differentiation of erythroid progenitor cells [39–41]. Our current data showed decreased levels of circulatory EPO in the saline-treated mice following POI (Fig. S3). EPO has known anti-inflammatory effect which results in augmented systemic EPO levels [37, 42, 43]. The timely recruitment and transition or polarization of immunomodulatory cells is critical in reducing inflammation and promoting recovery from POI [44–47]. Studies have shown that an excessive release of inflammatory mediators like CRP, IL6, IL1β, TNFα, and IL12 in POI conditions exerts stimulatory immunoreactive cellular signaling [11, 48, 49]. The present study in a POI mouse model confirmed significant upregulation of pro-inflammatory cytokines of CRP, IL6, and IL12 in the saline group, which were attenuated by EPO treatment. This validated the data mining findings of EPO-IRR-mediated attenuation of the inflammatory cascade. Also, these findings are consistent with other reports, we believe these cytokines can serve as potential tissue biomarkers of the recovery phase of POI [50, 51]. In addition to the above findings, we found CRP and IL6 can serve as serum biomarkers of POI. In our study, IL2 and IFNγ cytokines of the intestinal tissue were upregulated in the POI saline-treated control group, while the opposite was true in the EPO treatment group. Studies have shown that IFNγ can cause gut dysmotility by inducing inflammation and reducing contractility [52, 53]. Anti-inflammatory cytokines like IL4/10/13 are crucial for resolving inflammations following injuries or surgeries [54–56]. Surprisingly, our data revealed EPO significantly attenuated these cytokines in intestinal tissues following POI. Some studies showed that IL10 affects POI’s pathogenesis by triggering extravasation of leucocytes, neutrophils, and monocytes. Leucocyte-restricted IL10 deficiency appears protective against POI by reducing the infiltration of neutrophils in the muscularis externa of the intestine [31, 57].

Cytokine and chemokine interactions trigger signaling that regulates immune responses. Their dysregulation is crucial in bowel inflammation and immune-mediated disorders [14, 54, 58–60]. In this study, we found that pro-inflammatory chemokines CCL12, CCL4, and CXCL2 were significantly upregulated in POI, which was mitigated with EPO. We also confirmed that elevated serum CCL12 levels in POI were reduced in POI-EPO treatment. CCL12 may serve as a reliable biomarker for POI conditions.

In conclusions, to our knowledge, this is the first study to confirm that CCL12, CRP, and IL6 are reliable biomarkers of POI in both intestine and serum samples. Our findings also suggest that EPO has a beneficial effect against POI by regulating various inflammatory cytokines and chemokines.

Limitations

Our study has some limitations. The predictive value of inflammatory mediators, such as CCL12, CRP, and IL-6, is influenced by several factors, including the type of abdominal surgery, postoperative infections, and individual basal health conditions with pre-existing comorbidities. These mediators may not accurately indicate the POI condition, as elevated levels can persist even after bowel function resumes. EPO plays an effective role in mitigating inflammation during recovery, suggesting that its impact extends beyond just POI. Future studies using clinically relevant POI models and adjusted EPO dosages could provide valuable insights, allowing us to test variability in predictive markers across species. For now, the passage of flatulence and return of bowel movements are key indicators of POI recovery, although these markers are promising.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

EPO

Erythropoietin

IBD

Inflammatory bowel disease

IBS

Irritable bowel syndrome

IM

Intestinal manipulation

IMDs

Intestine motility disorders

POI

Postoperative ileus

PPI

Protein-protein interactions

HPPI2

Human PPI network 2

BC

Betweenness centrality

Author contributions

PKG (concept and design of the study, sample collections, data acquisition, analysis and interpretation, figure generation, manuscript drafting); JCE (concept and design of the study, data interpretation, manuscript drafting and finalization, and funding acquisition); YG (data analysis and bioinformatic analysis); GE (data analysis and figure generation); AAG (data organization and manuscript editing); MR (data analysis, bioinformatic analysis, and manuscript drafting); WE (concept and design of the study, animal surgery, and treatment, funding acquisition, data organization and interpretation, manuscript editing). All authors read and approved the final manuscript.

Funding

This study was supported by the National Institutes of Health (K08 AR060164-01 A), Department of Defense (W81XWH-16‐1‐0725), Children’s Miracle Network Grant, and institutional support from The Pennsylvania State University College of Medicine and The University of Arizona College of Medicine. The funding bodies played no role in the design of the study and collection, analysis, interpretation of data, and in writing the manuscript.

Data availability

The data supporting this study are stored on the institutional server and will be available from the corresponding author.

Declarations

Ethics approval and consent to participate

Our manuscript does not contain any human data. The experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the Pennsylvania State College of Medicine and the experiments were performed according to the guidelines of IACUC. The Committee’s composition and responsibilities regarding animal research are mandated by the Animal Welfare Act (Public Law 99–198).

Consent for publication

Not applicable.

Competing interests

John C. Elfar and Walaa Elfar are inventors on a patent (PCT/US2022/012854) submitted by the University of Rochester that covers “Erythropoietin for Gastrointestinal Dysfunction”. All other authors declare that they have no competing financial interests.