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. 2014 Mar 5;9(3):e90399.
doi: 10.1371/journal.pone.0090399. eCollection 2014.

TWEAK promotes peritoneal inflammation

Ana Belen Sanz  1 , Luiz Stark Aroeira  2 , Teresa Bellon  3 , Gloria del Peso  4 , Jose Jimenez-Heffernan  5 , Beatriz Santamaria  6 , Maria Dolores Sanchez-Niño  4 , Luis Miguel Blanco-Colio  6 , Manuel Lopez-Cabrera  7 , Marta Ruiz-Ortega  8 , Jesus Egido  9 , Rafael Selgas  10 , Alberto Ortiz  11
Affiliations

TWEAK promotes peritoneal inflammation

Ana Belen Sanz et al. PLoS One. .

Abstract

Peritoneal dialysis (PD) is complicated by peritonitis episodes that cause loss of mesothelium and eventually sclerosing peritonitis. An improved understanding of the molecular contributors to peritoneal injury and defense may increase the therapeutic armamentarium to optimize peritoneal defenses while minimizing peritoneal injury. There is no information on the expression and function of the cytokine TWEAK and its receptor Fn14 during peritoneal injury. Fn14 expression and soluble TWEAK levels were measured in human PD peritoneal effluent cells or fluids with or without peritonitis. Fn14 expression was also analyzed in peritoneal biopsies from PD patients. Actions of intraperitoneal TWEAK were studied in mice in vivo. sTWEAK levels were increased in peritoneal effluent in PD peritonitis. Effluent sTWEAK levels correlated with the number of peritoneal macrophages (r=0.491, p=0.002). Potential TWEAK targets that express the receptor Fn14 include mesothelial cells and macrophages, as demonstrated by flow cytometry of peritoneal effluents and by analysis of peritoneal biopsies. Peritoneal biopsy Fn14 correlated with mesothelial injury, fibrosis and inflammation, suggesting a potential deleterious effect of TWEAK/Fn14. In this regard, intraperitoneal TWEAK administration to mice promoted peritoneal inflammation characterized by increased peritoneal effluent MCP-1, Fn14 and Gr1+ macrophages, increased mesothelial Fn14, MCP-1 and CCL21 expression and submesothelial tissue macrophage recruitment. Taken together these data suggest that the TWEAK/Fn14 system may promote inflammation and tissue injury during peritonitis and PD.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Increased peritoneal effluent sTWEAK in PD peritonitis.
A) sTWEAK levels were measured in peritoneal effluents from PD patients with peritonitis (days 1 and 4±1) or without peritonitis. Clinical data in tables 1 and 2. *p<0.002 vs non-peritonitis. B) Evolution of sTWEAK levels through the peritonitis episode in 6 individual patients with at least three samples that cover the whole peritonitis episode. sTWEAK levels decrease when peritonitis resolves. Peritonitis episodes correspond to those presented in table 1. C) Scatter plot showing the significant positive correlation between sTWEAK levels and the number of peritoneal macrophages in peritoneal effluents during 17 episodes of peritonitis in PD patients.
Figure 2
Figure 2. Fn14 is expressed by cultured human mesothelial cells and by leukocytes in peritoneal effluents from PD patients.
A) Cultured human mesothelial cells (HMC) express Fn14 and TWEAK as assessed by western blot. Representative images of three different experiments. B) Fn14 expression was analyzed by flow cytometry in peripheral blood and peritoneal effluent leucocytes. Cells were stained with Fn14-PE and monocyte/macrophages, T cells and NK cells were identified with anti-CD14, anti-CD3 and anti-CD56 antibodies respectively within the appropriate gates according to FSC and SSC parameters. Controls for the technique were stained with isotype immunoglubulin. Peripheral blood mononuclear cells (PBMCs) were used as positive controls for leukocyte population markers. Numbers within histograms indicate mean fluorescence intensity (MFI) for Fn14 staining. Note increased Fn14 expression mainly in peritoneal macrophages from patients with peritonitis.
Figure 3
Figure 3. Colocalization of Fn14 and mesothelial cell markers in human preserved peritoneum.
Human non-infected peritoneum from stable chronic PD patient with preserved mesothelium was stained with both anti-Fn14 antibody (green) and anti-cytokeratin 8 antibody (red). Mesothelial cells (red) express Fn14 (green). Confocal microscopy. Original magnification x200, detail x400 and x1000.
Figure 4
Figure 4. CD68 positive macrophages express Fn14 in human injured peritoneum.
Human peritoneum biopsies from non-infected, stable, chronic PD patients were stained with both anti-Fn14 antibody (green) and anti-CD68 antibody (red). CD68+ macrophages (red) are among the submesothelial cells that express Fn14 (green)(white arrows). Additional submesothelial cells express Fn14 (arrowhead). Confocal microscopy. Original magnification x200, detail x400 and x1000.
Figure 5
Figure 5. Increased Fn14 protein expression in peritoneal biopsies from non-infected patients.
A) Fn14 immunohistochemistry in peritoneal tissue from non-infected, stable, chronic PD patients with different degrees of injury. Representative images from each group. Controls for the technique are stained with nonspecific immunoglobulin (Ig (-)). B) Quantification of Fn14 protein expression in peritoneal biopsies from non-infected patients. Fn14 staining was higher in patients with a more severe peritoneal histological injury score that evaluated mesothelial integrity, peritoneal fibrosis and peritoneal inflammation ( table 3 )32. Mean ± SEM *p<0.04, n = 4–5 patients per group.
Figure 6
Figure 6. Fn14 protein expression in peritoneal tissue correlates with peritoneal injury, fibrosis and inflammation in humans.
Scatter plot showing the significant positive correlation between Fn14 expression in peritoneal tissue from non-infected patients (n = 9) and histological peritoneal injury, peritoneal fibrosis and peritoneal inflammation scores.
Figure 7
Figure 7. TWEAK increases inflammatory mediators in murine peritoneal effluents.
A) MCP-1 protein levels were quantified by ELISA in peritoneal lavage of mice that had received intraperitoneal TWEAK or vehicle (control). Mean ± SEM of 5 animals per group. *p<0.001 vs control. B) MCP-1 and Fn14 mRNA levels measured in peritoneal lavage cells. Median (25th to 75th percentile) of 5 animals per group. *p<0.03. C) Quantification by flow cytometry of inflammatory cells present in murine peritoneal lavage. Mean ± SEM of 5 animals per group. *p<0.05. Macrophages were defined as CD11b+F4/80+ cells and Gr1+ macrophages as CD11b+F4/80+Gr1+ cells.
Figure 8
Figure 8. TWEAK increases Fn14 expression in murine peritoneal tissue in vivo.
A) Intraperitoneal TWEAK administration increased Fn14 mRNA in peritoneal tissue. Mean ± SEM of 5 animals per group. *p<0.02. B) Fn14 immunohistochemistry. TWEAK administration increased Fn14 expression in mesothelial cells and submesothelial infiltrating cells (arrows). Controls for the technique are stained with nonspecific Ig (-). Images representative of 5 animals per group.
Figure 9
Figure 9. TWEAK promotes murine peritoneal tissue chemokine mRNA expression in vivo.
MCP-1 and CCL21 mRNA levels measured by RT-PCR in murine peritoneal tissue 4 and 24 hours following TWEAK administration. Median (25th to75th percentile) of 5 animals per group. *p<0.05.
Figure 10
Figure 10. TWEAK induces chemokine protein expression and peritoneal inflammation in mice in vivo.
A and B) Increased MCP-1 (A) and CCL21 (B) protein expression in mesothelium from mice 24 h after TWEAK injection. C) F4/80 antigen immunohistochemistry. An increased number of submesothelial macrophages stained with F4/80 were noted 24 h after TWEAK injection (arrows). Controls for the technique are stained with nonspecific Ig. Images representative images of 5 animals per group.
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This work was supported by FIS PS09/00447, PI08/1564, PI10/00234, MS12/03262, FEDER funds ISCIII-RETIC REDinREN/RD06/0016, RD12/0021, Comunidad de Madrid (Fibroteam S2010/BMD-2321, S2010/BMD-2378). Programa Intensificación Actividad Investigadora (ISCIII/Agencia Laín-Entralgo/CM) to AO, Programa Estabilización Investigadores to LB-C, Miguel Servet to ABS, Sara Borrell to BS, MDSN. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.