Topical cathelicidin (LL-37) an innate immune peptide induces acute olfactory epithelium inflammation in a mouse model

Abstract

Background: Cathelicidin (LL-37) is an endogenous innate immune peptide that is elevated in patients with chronic rhinosinusitis (CRS). The role of LL-37 in olfactory epithelium (OE) inflammation remains unknown. We hypothesized that: (1) LL-37 topically delivered would elicit profound OE inflammation; and (2) LL-37 induced inflammation is associated with increased infiltration of neutrophils and mast cells.

Methods: To test our hypothesis we challenged C57BL/6 mice intranasally with increasing concentrations of LL-37. At 24 hours tissues were examined histologically and scored for inflammatory cell infiltrate, edema, and secretory hyperplasia. In separate experiments, fluorescently conjugated LL-37 was instilled and tissues were examined at 0.5 and 24 hours. To test our last hypothesis, we performed tissue myeloperoxidase (MPO) assays for neutrophil activity and immunohistochemistry for tryptase to determine the mean number of mast cells per mm(2) .

Results: LL-37 caused increased inflammatory cell infiltrate, edema, and secretory cell hyperplasia of the sinonasal mucosa, with higher LL-37 concentrations yielding significantly more inflammatory changes (p < 0.01). Fluorescent LL-37 demonstrated global sinonasal epithelial binding and tissue distribution. Further, higher concentrations of LL-37 led to significantly greater MPO levels with dose-dependent increases in mast cell infiltration (p < 0.01).

Conclusion: LL-37 has dramatic inflammatory effects in the OE mucosa that is dose-dependent. The observed inflammatory changes in the olfactory mucosa were associated with the infiltration of both neutrophils and mast cells. Our biologic model represents a new model to further investigate the role of LL-37 in OE inflammation.

Keywords: cathelicidin; chronic disease; innate immunity; rhinosinusitis; sinusitis.

Figure 1. Gross images of sinonasal inflammation

Gross differences are observed in mouse hemi-sected heads demonstrating sinus mucosa (arrow) harvested 24 hours after (A) saline and (B) 320 μM LL-37 treatment. Animals treated with LL-37 (B) demonstrated increased inflammatory changes, as assessed by vascularity, erythema, hemorrhage, and edema compared to saline (A)-treated animals. Olfactory bulb (OB), posterior (Pos), anterior (Ant).

Figure 2. LL-37 causes inflammatory changes in the olfactory epithelium in a dose-dependent manner

H&E showing the gradual histologic changes in (A) saline, (B) 80 μM LL-37, (C) 160 μM LL-37, and (D) 320 μM LL-37. Increased inflammatory cell infiltrates (Boxes in D; magnified image), thickness of the lamina propria (LP), and mucus (arrow) are demonstrated in 320 μM LL-37-treated mice. Dotted circles are represented by the magnified images in the bottom panel.

Figure 3. Histological Scores

The mean score and standard deviations obtained from 6 animals of four treatment groups in each category: severity of inflammation, lamina propria thickness, and secretory hyperplasia was graphed. With the 0-4 score system, each score indicates the grade of inflammation: 0 (none), 1 (minimal), 2 (mild), 3 (moderate), and 4 (severe).

Figure 4

A dramatic significant increase in MPO was observed with higher concentration of LL-37 in sinonasal challenged tissue (p < 0.05).

Figure 5. Immunohistochemistry for mast cell tryptase

Representative immunofluorescence (IF) (A & B) and immunohistochemistry (IHC) (C & D) for mast cell tryptase from saline (A & C) and 320 μM LL-37-treated (B & D) mice. Tryptase is represented by red staining, and nuclei are stained green (A & B). Boxes and dark brown areas indicate mast cells, as detected by tryptase-DAB stain (C & D). LP (lamina propria), OE (olfactory epithelium).

Figure 6

Mast cells per mm² of tissue after LL-37 treatment.

Figure 7. Representative IF images following inoculation with fluorescently labeled LL-37

(A), (B), and (C) represent tissues harvested 30 minutes after inoculation with LL-37 at 10×, 20×, and 40× magnification, respectively. (D), (E), and (F) represent tissues harvested 24 hours after inoculation at 10×, 20×, and 40×, respectively. Nuclei are counterstained with DAPI (blue). Red represents fluorescent LL-37. At 30 minutes, there is coating of the epithelial layer, as well as bright staining within the lamina propria. At 24 hours, there is absence of fluorescent LL-37 coating the epithelium with minimal evidence of fluorescent LL-37 present in the lamina propria. OE (olfactory epithelium); LP (lamina propria).