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. 2024 May 15;27(6):109993.
doi: 10.1016/j.isci.2024.109993. eCollection 2024 Jun 21.

Host defense peptides human β defensin 2 and LL-37 ameliorate murine necrotizing enterocolitis

Shiloh R Lueschow-Guijosa  1 , Amy H Stanford  1 , Jennifer N Berger  2 , Huiyu Gong  1 , Timothy J Boly  1 , Benjamin A H Jensen  3 , Peter Nordkild  4 , Alexandra J Leegwater  5 , Jan Wehkamp  6 , Mark A Underwood  5 , Steven J McElroy  5
Affiliations

Host defense peptides human β defensin 2 and LL-37 ameliorate murine necrotizing enterocolitis

Shiloh R Lueschow-Guijosa et al. iScience. .

Abstract

Necrotizing enterocolitis (NEC) is a leading cause of preterm infant morbidity and mortality. Treatment for NEC is limited and non-targeted, which makes new treatment and prevention strategies critical. Host defense peptides (HDPs) are essential components of the innate immune system and have multifactorial mechanisms in host defense. LL-37 and hBD2 are two HDPs that have been shown in prior literature to protect from neonatal sepsis-induced mortality or adult inflammatory bowel disease, respectively. Therefore, this article sought to understand if these two HDPs could influence NEC severity in murine preclinical models. NEC was induced in P14-16 C57Bl/6 mice and HDPs were provided as a pretreatment or treatment. Both LL-37 and hBD2 resulted in decreased NEC injury scores as a treatment and hBD2 as a pretreatment. Our data suggest LL-37 functions through antimicrobial properties, while hBD2 functions through decreases in inflammation and improvement of intestinal barrier integrity.

Keywords: Biological sciences; Immunology; Molecular biology.

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Conflict of interest statement

P.N. and J.W. (co-authors) are shareholders of Defensin Therapeutics ApS (DTA), Denmark. P.N. is CEO of DTA and B.J. (co-author) is next to his fulltime position as Ass. Prof., Head of Nutritional Immunology, University of Copenhagen, CSO of DTA. D.T. owns a patent to pursue the therapeutic use of hBD2 in graft versus host disease and the ethical pharmaceutical rights to pursue prophylactic or therapeutic use of hBD2 in NEC. DTA has supported this work by providing relevant amounts of hBD2. The company had no share in the data analysis, integration, and presentation. The company solely delivered the defensins used in the current study. The views expressed in this manuscript are those of the authors and not necessarily those of DTA or other funding bodies that could be construed as a potential conflict of interest.

Figures

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Graphical abstract
Figure 1
Figure 1
Oral hBD2 treatment at increasing doses does not induce inflammation or small intestinal injury in neonatal mice No mortality was seen in either P7 or P14 (n = 3–5 in all groups at both ages) C57Bl/6J mice following oral gavage of hBD2 at increasing doses as compared to sham (A). P7 and P14 C57Bl/6J mice were given hBD2 at increasing doses and their small intestine was evaluated for generalized intestinal injury. Injury was scored as either no injury (score of 0), mild intestinal injury (score of 1 representing mild separation of the lamina propria and/or mild villus vacuolization), or severe intestinal injury (score of 2 representing significant villous vacuolization, mucosal ulceration, lamina propria damage and/or presence of hemorrhage within villi) and compared to sham. No significant differences were seen in mice treated with hBD2 compared to controls. The percentage of mild intestinal injury for each dose is denoted by yellow bars. No animal sustained severe injury (B). Example histology of intestinal injury is shown. No significant differences were seen in combined villus height (C), combined villus area (D), or combined crypt depth (E) (n = 6–10 mice per group for all histologic measurement with 300 villi or 100 crypts measured per animal). Mice treated with hBD2 had no significant change in Paneth cell numbers per crypt compared to shams (2.4 vs. 2.3) (F). Serum was collected from P7 (G) and P14 (H) mice with increasing doses of hBD2 and quantified for TNF, KCGRO, IL6, and IL10 compared to both sham and non-lethal LPS treatments (0.01 mg/kg bw) as a positive control. (n = 3–5 per group, ∗p < 0.001). Only LPS treatments were significantly different from sham controls. Error bars represent SEM.
Figure 2
Figure 2
LL-37 and hBD2 significantly reduce experimental NEC induced by Paneth cell disruption with bacterial dysbiosis P14-P16 C57Bl/6J mice were treated with dithizone and K. pneumoniae to induce NEC. Following euthanasia, ileal samples were harvested and scored for NEC-like injury. NEC injury scores of sham (gray), Dithizone (Dith) (orange), K. pneumoniae (Kleb) (green), NEC (red), hBD2 pre (light blue), hBD2 post (dark blue), hBD2 subQ (aqua), LL-37 pre (purple), and LL-37 post (plum) are shown with the dotted horizontal line indicating a NEC like injury (score of 2 or greater). Each circle represents a single animal. Mice with NEC had increased injury compared to sham, hBD2 pre, hBD2 post, hBD2 subQ, and LL-37 post (n = sham: 37, dithizone: 60, Klebsiella: 9, NEC: 99, hBD2 pre: 26, hBD2 post: 24, hBD2 subQ: 17, LL-37 pre: 25, LL-37 post: 20; p < 0.004 for all) (A). Total percentage of animals developing significant disease (score of ≥ 2) (B). On necropsy, mice with NEC (second panel) had dark discolored intestines with adhesions, while animals treated with hBD2 or LL-37(third and fourth panels) had healthier appearing intestines and less adhesions that were more similar to sham conditions (first panel). Yellow arrows indicate small intestine (C). Serum levels of IL-17a, IL-22, and TNF were quantified at time of tissue harvest (D–F). Serum levels of IL17A, IL-22, and TNF were significantly elevated in NEC compared to sham (p = 0.0016, 0.0006, and 0.0044 respectively), but hBD2 and LL-37 treatment/prevention did not significantly alter any cytokine levels compared to NEC. P14-P16 C57Bl/6J mice were euthanized 30 min following SQ injection or oral gavage with hBD2 or oral gavage or intraperitoneal (IP) injection with LL-37. Serum and homogenized intestinal samples were obtained and quantified for hBD2 (G) and serum, homogenized intestinal samples, and intraperitoneal wash (IP Wash) samples were obtained and quantified for LL-37 (H). Intestinal homogenates from SQ treated mice had hBD2 levels >1000 pg/mL, while all other hBD2 treated samples had hBD2 levels that exceeded the detectable limit by the ELISA plate (n = 3 animals per group). Elevation of LL-37 was detected in the IP wash of IP injected animals, but elevation was not detected in the serum or intestine in LL-37 IP injection or gavage (n = 3 animals per group). Error bars in all figures represent SEM.
Figure 3
Figure 3
LL-37 and hBD2 prevention and treatment approaches do not cause detectable alterations in the cecal microbiome Cecal samples were analyzed for microbial composition using 16S sequencing. Phylum and family level analysis showed that induction of NEC altered the microbial composition by increasing the relative amount of Proteobacteria (p < 0.0001), and in particular Enterobacteriaceae (p < 0.0001), while decreasing the relative amount of Firmicutes. hBD2 and LL-37 treatment (pre- or post-induction of NEC) had no impact (n = sham: 6, NEC: 9, hBD2 pre: 11, hBD2 post: 10, hBD2 subQ 5, LL-37 pre 6, LL-37 post 5, p < 0.05) [(A): Phyla, (B): Enterobacteriaceae only)]. Alpha diversity analysis via chao1 demonstrated decreases in diversity in NEC and HDP pre-treated animals (C). Principle coordinate analysis of the 16S cecal microbiome indicates animals exposed to K. pneumoniae gavage (Klebsiella, NEC, pre hBD2, post hBD2, subQ hBD2, pre LL-37, and post LL-37) clustered together independent of HDP treatment and separate from non-Klebsiella animals (sham, dithizone) (D). Error bars in all figures represent SEM.
Figure 4
Figure 4
LL-37 can directly kill K pneumoniae 10031, while hBD2 has more limited antimicrobial capacity K. pneumoniae abundance was measured by PCR (A). NEC treatment significantly increased detection of K. pneumoniae (p = 0.0002, n = 9). Pre-treatment with HDPs LL-37 and hBD2 had no effect on NEC-induced increase in K. pneumoniae (p > 0.9999, n = 3), while post-treatment with HDPs LL-37 and hBD2 reduced K. pneumoniae to sham-levels (sham vs. Post LL-37 p = 0.1648; sham vs. Post hBD2 p = 0.8622 n = 3). Similar to oral therapeutic treatment with HDPs, SQ hBD2 decreased NEC-induced increases in K. pneumoniae toward sham levels (p = 0.1501). K. pneumoniae (6.3 × 106 - 1.1 × 107 CFU/mL) and E. coli (2.25 × 106 - 1.585 × 107 CFU/mL) were exposed to increasing concentrations of hBD2. hBD2 decreased growth for E. coli at 80 μg/mL, and achieved a MIC at 160 μg/mL, but no concentration of hBD2 significantly inhibited growth of K. pneumoniae (n = 2.5 μg/mL: 3, 160 μg/mL: 6, and all other doses 9) (B). K. pneumoniae grown in Nutrient Broth or Tryptic Soy Broth were exposed to increasing concentrations of hBD2. hBD2 induced a sharp decline in survival at concentrations of 25 μg/mL in both media types, but complete killing of K. pneumoniae was not observed in either media or at any concentration of hBD2 tested (n = 10 replicates for all strains and all doses) (C). In a turbidity assay, hBD2 caused significantly decreased bacterial growth for Bifidobacterium breve and Bifidobacterium adolescentis, but not in S. thermophilus (D). K. pneumoniae (5.15 × 106 - 1.18 × 107 CFU/mL) was exposed to increasing concentrations of LL-37 (E). LL-37 inhibited growth of K. pneumoniae at 25 μg/mL and completely inhibited growth at 32 μg/mL. Error bars in all figures represent SEM.
Figure 5
Figure 5
Impact of hBD2 on NEC injury scores and cytokine levels is NEC model dependent Experimental NEC independent of K. pneumoniae was induced in P14-P16 C57Bl/6J mice using RMS formula feeds and dithizone injection. Exposure to RMS formula feeds and Paneth cell disruption induced significant NEC-like injury compared to controls (p = 0.0003); however, hBD2 treatment did not prevent RMS NEC-induced injury (n = 10–15 per group) (A). Representative histology from groups (B). Paneth cell disruption with bacterial dysbiosis NEC induction of injury is associated with significantly greater relative abundance of Proteobacteria and decreased relative abundance of Firmicutes and Bacteroidetes species compared to Paneth cell disruption with RMS NEC induced injury (n = 7 Paneth NEC, 5 RMS NEC, p = 0.01 Bacteroidetes, 0.007 Firmicutes, and <0.0001 Proteobacteria) (C). Microbial composition of the cecum showed no significant differences between RMS NEC and RMS NEC treated with SQ hBD2 at either the phylum (D), or the family (E) levels, and had no significant clustering in Beta diversity (F) (n = 9 for all groups). Ileal samples were harvested, and gene expression was quantified. While the Paneth cell disruption with bacterial dysbiosis NEC model induced an upregulation of IL-1b and TNF genes, the Paneth cell disruption with RMS NEC model induced IL-6 and KCGRO genes (n = 3–4 per group, p values as shown) (G). Serum cytokine levels of IL-1b, IL-6, KCGRO, and TNF were all significantly increased after induction of NEC through the RMS NEC model. Treatment with hBD2 significantly blocked the RMS NEC-induced upregulation (n = 4–10 per group, p values as shown) (H) Error bars in all figures represent SEM.
Figure 6
Figure 6
hBD2 improves epithelial restitution and tight junction expression Intestinal samples were harvested from P14-P16 mice following NEC induction with or without treatment with subcutaneous hBD2 and compared to sham animals for ZO-1 expression by immunohistochemistry. NEC induced visual loss of intestinal architecture and ZO-1 expression compared to sham and was partially recovered by hBD2 treatment. Representative samples shown at 20× with ZO-1 stain (brown) shown, n = 5 animals per treatment (A). Intestinal samples were homogenized and quantified for Claudin 3 by Western blot analysis. NEC significantly decreased Claudin 3 expression compared to sham controls (p = 0.007, n = 4), while hBD2 treated NEC animals were not statistically different from sham controls (B). IEC-18 monolayers were wounded with a rotating silicone disk and treated with 12.5 μg/mL or 25 μg/mL of hBD2 and compared to treatment with 10 ng/mg EGF and sham control. Both hBD2 doses and EGF showed significantly increased wound closure at 6 (48%, 54%, and 50% respectively) and 12 h (76%, 80%, and 80%) compared to sham controls (25% and 52%) (p = 0.02, n = 9 wounds per condition) Left panel shows closure curves, Right panel shows individual points with statistics and SEM. For all p values, ∗ ≤0.05, ∗∗ ∗ ≤0.01, ∗∗∗ ≤0.001, and ∗∗∗∗ ≤0.0001 (C). Representative micrographs taken at 40 × 6 h after wounding. The initial wound size can be seen as the solid line, while the leading edge of the wound was marked using NIS elements software and is noted by the dotted line (D).
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References

    1. Blencowe H., Cousens S., Oestergaard M.Z., Chou D., Moller A.-B., Narwal R., Adler A., Vera Garcia C., Rohde S., Say L., Lawn J.E. National, regional, and worldwide estimates of preterm birth rates in the year 2010 with time trends since 1990 for selected countries: a systematic analysis and implications. Lancet. 2012;379:2162–2172. - PubMed
    1. Romero R., Dey S.K., Fisher S.J. Preterm labor: one syndrome, many causes. Science. 2014;345:760–765. - PMC - PubMed
    1. Liu L., Johnson H.L., Cousens S., Perin J., Scott S., Lawn J.E., Rudan I., Campbell H., Cibulskis R., Li M., et al. Global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since 2000. Lancet. 2012;379:2151–2161. - PubMed
    1. Howson C.P., Kinney M.V., McDougall L., Lawn J.E., Born Too Soon Preterm Birth Action Group Born Too Soon: Preterm birth matters. Reprod. Health. 2013;10 doi: 10.1186/1742-4755-10-S1-S1. - DOI - PMC - PubMed
    1. March of Dimes P.M.N.C.H., Save the Children W. 2012. Born Too Soon: The Global Action Report on Preterm Birth.