Peptidoglycan Recognition Peptide 2 Aggravates Weight Loss in a Murine Model of Chemotherapy-Induced Gastrointestinal Toxicity

Abstract

Introduction: Chemotherapy-induced gastrointestinal toxicity (CIGT) is a frequent, severe and dose-limiting side effect. Few treatments have proven effective for CIGT. CIGT is characterized by activation of the nuclear factor kappa B pathway which, leads to upregulation of proinflammatory cytokines. The innate immune protein peptidoglycan recognition peptide 2 (PGLYRP2) binds to and hydrolyzes microbial peptidoglycan. Expression of PGLYRP2 is upregulated in the intestine of chemotherapy-treated piglets. In this experimental study, we investigated the role of Pglyrp2 in the development and severity of murine CIGT. Methods: Pglyrp2 wildtype and Pglyrp2 knockout mice received intraperitoneal injections of chemotherapy (Doxorubicin 20 mg/kg) to induce CIGT. Weight was monitored daily, and animals were euthanized after 2 or 7 days. Expression of proinflammatory cytokines in the jejunum was measured by quantitative real-time polymerase-chain reaction and enzyme-linked immunosorbent assay. Villus height, crypt depth, and histologic inflammation were evaluated on haematoxylin and eosin stained tissue specimens. Results: Chemotherapeutic treatment induced weight loss (p < 0.05), shortening of the small intestine (p < 0.05), elongation of villus height (p < 0.05), increased crypt depth (p < 0.05), and led to elevated mRNA levels of II1β (p < 0.05), II6 (p < 0.05), and Tnf (p < 0.001) at day 2. Protein levels of IL1β, IL6, and TNFα did not change after exposure to chemotherapy. Doxorubicin treated wildtype mice had a more pronounced weight loss compared to knockout mice from day 3 to day 7 (D3-D6: p < 0.05 and D7: p < 0.01). No other phenotypic differences were detected. Conclusion: Pglyrp2 aggravates chemotherapy-induced weight loss but does not induce a specific pattern of inflammation and morphological changes in the small intestine.

Keywords: chemotherapy; gastrointestinal mucositis; inflammation; mice; peptidoglycan recognition peptide 2.

Figure 1

Weight loss. Change of weight in % each day as compared to baseline weight (Day−1) in wildtype (WT) and knockout (KO) mice. WT-NaCl, n = 5. KO-NaCl, n = 5. WT-Doxo 2, n = 10. KO-Doxo 2, n = 10. WT-Doxo 7, n = 16. KO-Doxo 7, n = 11. Data is presented as means ± standard error of the mean. Two-way ANOVA followed by Tukey multiple comparison test was used to analyze results. *p < 0.05 and **p < 0.01.

Figure 2

Small intestinal length in cm in wildtype (WT) and knockout (KO) mice 7 days after IP injection with saline (NaCl) or 2 or 7 days after IP injection with doxorubicin (Doxo). WT-NaCl, n = 5. KO-NaCl, n = 5. WT-Doxo 2, n = 10. KO-Doxo 2, n = 10. WT-Doxo 7, n = 16. KO-Doxo 7, n = 11. Data is presented as means ± standard error of the mean. Two-way ANOVA followed by Holm-Sidak multiple comparison test was used to analyze results. *p < 0.05, ***p < 0.001, and ns, no significance.

Figure 3

Histopathology. Villus height (μm) and crypt depth (μm) were measured at haematoxylin and eosin stained tissue from duodenum, jejunum and ileum in wildtype (WT) and knockout (KO) mice after IP injection with saline (NaCl) or 2 or 7 days after IP injection with doxorubicin (Doxo). This figure shows examples of tissue slides from KO-NaCl, jejunum (A), KO-Doxo 7, ileum (B), WT-NaCl, ileum (C), and WT-Doxo 7, ileum (D).

Figure 4

Villus height (μm), crypt depth (μm) and villus crypt ratio measured at haematoxylin and eosin stained tissue from duodenum, jejunum and ileum 7 days after IP injection with saline (NaCl) or 2 or 7 days after IP injection with doxorubicin (Doxo) in wildtype (WT) and knockout (KO) mice. Note the differences in y-axis. WT-NaCl, n = 5. KO-NaCl, n = 5. WT-Doxo 2, n = 10. KO-Doxo 2, n = 10. WT-Doxo 7, n = 16. KO-Doxo 7, n = 11. Data is presented as mean ± standard error of mean. Two-way ANOVA followed by Tukey multiple comparison test (within genotypes) and Holm-Sidak comparison test (between genotypes) were used to analyze data following a Gaussian distribution (data in D–H). Kruskal–Wallis test with Dunn's multiple comparison test was used to analyze data not following a Gaussian distribution (data in A–C,I). *p < 0.05 and **p < 0.01.

Figure 5

Histological score 2 days after exposure to doxorubicin (Doxo) and 7 days after exposure to saline (NaCl) or Doxo in wildtype (WT) and knockout (KO) mice. WT-NaCl, n = 5. KO-NaCl, n = 5. WT-Doxo 2, n = 10. KO-Doxo 2, n = 10. WT-Doxo 7, n = 16. KO-Doxo 7, n = 11. Data is presented as means ± standard error of the mean. Kruskal–Wallis test with Dunn's multiple comparison test was used to analyze results.

Figure 6

Quantitative real-time polymerase chain reaction analyses showing gene expression of Pglyrp2, Il1b, Il6, and Tnf in jejunal tissue obtained from wildtype (WT) and knockout (KO) mice either 7 days after IP injection with saline (NaCl) or 2 or 7 days after IP injection with doxorubicin (Doxo). Data are shown relative to the housekeeping gene Hprt. Please notice scale differences in y-axis. WT-NaCl, n = 5. KO-NaCl, n = 5. WT-Doxo 2, n = 10. KO-Doxo 2, n = 10. WT-Doxo 7, n = 16. KO-Doxo 7, n = 11. Data is presented as means ± standard error of the mean. Two-way ANOVA followed by Holm–Sidak multiple comparison test was used to analyze Gaussian distributed data (data in B). Non-Gaussian distributed data were analyzed by Kruskal–Wallis test followed by Dunn's multiple comparison test (data in A, C, D). *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 7

Enzyme-linked immunosorbent assay analyses showing protein expression of IL1β, IL6 and TNFα in jejunal tissue obtained from wildtype (WT) and knockout (KO) mice either 7 days after IP injection with saline (NaCl) or 2 or 7 days after IP injection with Doxorubicin (Doxo). Data are shown in pg/g tissue. Please notice scale differences in y-axis. WT-NaCl, n = 4. KO-NaCl, n = 4. WT-Doxo 2, n = 10. KO-Doxo 2, n = 9. WT-Doxo 7, n = 16. KO-Doxo 7, n = 11. Data is presented as means ± standard error of the mean. All data was Gaussian distributed and two-way ANOVA followed by Holm–Sidak multiple comparison test was used to analyze data.