Protective and Anti-Inflammatory Effects of Protegrin-1 on Citrobacter rodentium Intestinal Infection in Mice

Abstract

Infectious intestinal colitis, manifesting as intestinal inflammation, diarrhea, and epithelial barrier disruption, affects millions of humans worldwide and, without effective treatment, can result in death. In addition to this, the significant rise in antibiotic-resistant bacteria poses an urgent need for alternative anti-infection therapies for the treatment of intestinal disorders. Antimicrobial peptides (AMPs) are potential therapies that have broad-spectrum antimicrobial activity due to their (1) unique mode of action, (2) broad-spectrum antimicrobial activity, and (3) protective role in GI tract maintenance. Protegrin-1 (PG-1) is an AMP of pig origin that was previously shown to reduce the pathological effects of chemically induced digestive tract inflammation (colitis) and to modulate immune responses and tissue repair. This study aimed to extend these findings by investigating the protective effects of PG-1 on pathogen-induced colitis in an infection study over a 10-day experimental period. The oral administration of PG-1 reduced Citrobacter rodentium intestinal infection in mice as evidenced by reduced histopathologic change in the colon, prevention of body weight loss, milder clinical signs of disease, and more effective clearance of bacterial infection relative to challenged phosphate-buffered saline (PBS)-treated mice. Additionally, PG-1 treatment altered the expression of various inflammatory mediators during infection, which may act to resolve inflammation and re-establish intestinal homeostasis. PG-1 administered in its mature form was more effective relative to the pro-form (ProPG-1). To our knowledge, this is the first study demonstrating the protective effects of PG-1 on infectious colitis.

Keywords: Citrobacter rodentium infection; antimicrobial peptide; immunomodulatory effect; infectious colitis; protegrin-1.

Figure 1

Animal Study Design. (A) The trial timeline for induction of colitis. (B) Treatment groups. Mice were challenged with C. rodentium (2 × 10⁹ CFU/mL) or left unchallenged (PBS) and were treated daily with ProPG-1, mPG-1, or PBS. Two mice within the ProPG-1 group died due to gavage complications; six mice in this group completed the study.

Figure 2

Effects of PG-1 on body weight, Disease Activity Index (DAI), and bacterial load. Protegrin treatments reduced body weight loss (A), DAI (B), and C. rodentium infection (C) compared to challenged mice receiving PBS. A higher DAI score represents more severe disease signs. ** p < 0.01 indicates DAI on day 10 is significantly different than in challenged mice receiving PBS. Body weight and DAI data represent the mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001 calculated by a one-way repeated measures ANOVA post hoc Tukey test over the course of the trial. Bacterial colony counts are expressed as Log₁₀ CFU/mL.

Figure 3

Effects of PG-1 on histologic lesions (A) and colonic histomorphology (B–E). Distal and proximal colonic sections on Day 10 showed altered mucosal morphology in C. rodentium-challenged mice (upper center and upper right images [enlarged]) compared to unchallenged mice (upper left image). The challenged mice had crypt hyperplasia with loss of goblet cells (yellow arrow–lesional area; green arrow–normal area), necrosis of individual enterocytes (arrowhead), and infiltration of neutrophils (arrows). Both ProPG-1- and mPG-1-treated challenged mice (lower left and right images, respectively) had milder lesions than PBS-treated mice, but mPG-1-treated challenged mice had less immune cell infiltration. Representative hematoxylin-and-eosin-stained sections of colon (magnification: 400×). Compared to untreated mice challenged with C. rodentium, both ProPG-1- and mPG-1-treated challenged mice had a decrease in (B) average total number of neutrophils. No significant differences were observed among treatments for (C) average crypt depth or (D) goblet cell counts, but both ProPG-1- and mPG-1-treated challenged mice showed a decrease in overall histologic score (E). Data represent the mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001 compared to controls; one-way ANOVA post hoc Tukey test.

Figure 4

Effects of PG-1 on antimicrobial peptide (AMP) gene expression (A–C), self-protecting gene expression (D–H), and apoptotic, innate immune response, and cytokine gene expression (H–M) within the colon. Protegrin treatments differentially affect gene expression of the AMPs Reg3β (A), Reg3γ (B), and sPLA2 (C), Muc1 (D), Muc2 (E), cell adhesion molecule VCAM-1 (F), cell proliferation gene EGR1 (G), stress response factor HIF1α (H), apoptotic markers BAX (I) and BCL3 (J), innate immune response genes TLR2 (L) and TLR6 (L), and the cytokine regulator SOCS3 (M). Colonic gene expression levels were determined by RT-qPCR, with values normalized against GAPDH and β-actin. Data represent the mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001 compared to controls calculated by a one-way ANOVA post hoc Tukey test. All values in gene expression analysis were compared to those of challenged mice set to a relative expression of 1.