A synthetic glycosaminoglycan reduces sinonasal inflammation in a murine model of chronic rhinosinusitis

Abstract

Chronic rhinosinusitis (CRS) is characterized by sustained mucosal inflammation, impaired mucociliary clearance, loss of cilia and epithelial barrier breakdown, and tissue remodeling. Certain glycosaminoglycans inhibit various inflammatory mediators, suppress bacterial growth, and provide important functions in mucosal tissue repair and mucociliary clearance. Herein, we evaluated the effects of a synthetic glycosaminoglycan, GM-1111, on the clinical signs and inflammatory tissue changes associated with CRS in mice. CRS was generated by repeated intranasal applications of Aspergillus fumigatus (A. fumigatus) extracts over 4 weeks. Mice were then intranasally administered GM-1111 (600 μg per dose, 5 times a week) or vehicle (phosphate buffered saline, PBS) for an additional 4 weeks while still being given A. fumigatus extracts to maintain a chronic inflammatory environment with acute exacerbations. Clinical signs indicative of sinonasal inflammation were recorded throughout the study. After 9 weeks, whole blood and sinonasal tissues were harvested for hematological, histological, and biochemical examination. The clinical signs, white blood cell counts, tissue markers of sinonasal inflammation, and histological changes caused by A. fumigatus extract administration were compared to the healthy (PBS vehicle) and GM-1111-treated groups (n = 12 per treatment group). Compared to vehicle-treated animals, animals treated with GM-1111 demonstrated significant reductions in clinical signs (p<0.05), degenerative tissue changes, goblet cell hyperplasia, inflammatory cell infiltration (p<0.01), innate immunity- (tlr2, tlr4, myd88, il1b, tnfa, il6, and il12) and adaptive immunity-associated (ccl11, ccl24, ccl5, il4, il5, and il13) cytokine gene expression (p<0.05 to p<0.0001) in sinonasal tissues, and serum IgE levels (p<0.01). Our data suggest that GM-1111 significantly reduces local and systemic effects of CRS-associated sinonasal inflammation.

Fig 1. The chemical structure of synthetic glycosaminoglycan, GM-1111.

Fig 2. Study design to examine the anti-inflammatory properties of GM-1111 in a murine model of CRS.

At week 0, control animals (PBS group) were sensitized with an intraperitoneal (i.p.) injection of PBS vehicle in alum adjuvant, whereas the A. fumigatus + PBS (red arrow) and A. fumigatus + GM-1111 (blue arrow) groups received A. fumigatus extracts in alum adjuvant. After 1 week, the animals were subjected to an 8-week regiment (3 times weekly) of intranasal PBS or A. fumigatus extracts. At week 5, intranasal treatment of GM-1111 or PBS was initiated (5 times weekly) and continued for 4 weeks with continued A. fumigatus extract administration (upper right inset). At week 9, whole blood was collected, and the animals were sacrificed and examined for histologic changes and inflammatory tissue biomarkers associated with CRS.

Fig 3. GM-1111 treatment significantly reduces clinical signs of CRS in mice.

Clinical signs include nasal erythema and edema, itchy nose, sneezing, and difficulty breathing (n = 12 per group). The data are represented as the number of recorded clinical signs measured after beginning treatment with GM-1111, reported for each animal per treatment group with the median. *p<0.05; ***p<0.001; NS: Not Significant.

Fig 4. GM-1111 reduces CRS-associated sinonasal inflammation in mice.

Microscopic images of sinonasal tissues stained with hematoxylin and eosin show coronal sections and respective higher magnification images of the indicated region of the nasal septum (box) and ethmoturbinate tissue (circle). A. fumigatus administration resulted in marked degenerative changes and loss of cilia in all epithelial layers (arrows), increased inflammatory cell infiltration, and thickening of the nasal septum (star). These changes were much less pronounced in animals treated with GM-1111. Histopathological interpretations were generated using images from all animals (n = 12 per treatment group) with representative images demonstrated.

Fig 5. GM-1111 reduces CRS-associated changes in mucus-secreting cells in the sinuses of mice.

The ethmoturbinates from A. fumigatus-treated mice stained with Alcian Blue (turquoise = acid mucopolysaccharides) show the loss of luminal surface mucus lining and ciliated cells (arrows) and decreased alcian blue staining, indicating a reduction in glandular contents with concomitant goblet cell hyperplasia in the epithelium (dotted box). Anti-PCNA immunolabeling (brown = dividing cells) demonstrates increased cellular activity to regenerate in tissues from A. fumigatus-treated mice. These changes were much less pronounced in animals treated with GM-1111. Histopathological interpretations were generated using images from all animals (n = 12 per treatment group) with representative images demonstrated.

Fig 6. GM-1111 reduces TLR2 and TLR4 protein and NFκB-regulated gene expression of downstream inflammatory cytokines associated with innate immunity.

The sinonasal tissues from A. fumigatus-treated animals demonstrate increased (A) protein (n = 10, PBS; n = 12, A. fumigatus + PBS; n = 12, A. fumigatus + GM-1111) and (B) gene expression of TLR2 and TLR4 compared to controls and animals treated with GM-1111. The gene expression of adaptor molecule myd88 and cytokines il1b, tnfa, il6, and il12 associated with TLR2 and TLR4 activation were also elevated in the sinus tissues of animals treated with A. fumigatus (n = 12, PBS; n = 8, A. fumigatus + PBS; n = 12, A. fumigatus + GM-1111). Treatment with GM-1111 showed significant reductions in these innate immune biomarkers compared to disease controls. The genes from each group were normalized to cltc, gapdh, gusb, hprt, pgk1, and tubb5 and plotted as the mean ± SEM of gene transcript copy in each treatment group. The images demonstrating TLR2 are representative for each treatment group. *p<0.05; **p<0.01; p<0.001; NS: Not Significant.

Fig 7. GM-1111 reduces blood and tissue biomarkers associated with allergy and Th2-mediated adaptive immunity.

Compared to controls, animals treated with A. fumigatus exhibited significant increases in (A) CD4+ T cells found in the sinonasal tissues (p<0.0001) (n = 10, PBS; n = 12, A. fumigatus + PBS; n = 12, A. fumigatus + GM-1111), expressed as an infiltration severity index based on numbers of CD4+ cells counted per high power field, (B) blood eosinophils (% WBC) determined by CBC count (p<0.01) (n = 9, PBS; n = 10, A. fumigatus + PBS; n = 8, A. fumigatus + GM-1111), (C) eosinophils found in the sinonasal tissues (p<0.0001) (n = 10, PBS; n = 12, A. fumigatus + PBS; n = 12, A. fumigatus + GM-1111), (D) serum IgE levels (kIU/mL) (n = 10, PBS; n = 5, A. fumigatus + PBS; n = 10, A. fumigatus + GM-1111), and (E) eosinophil-associated cytokine and chemokine gene expression (ccl11, ccl24, ccl5, il4, il5, and il13) (n = 12, PBS; n = 8, A. fumigatus + PBS; n = 12, A. fumigatus + GM-1111). Treatment with GM-1111 showed significant reductions in the influx of CD4+ T cells (p<0.01) and eosinophils (p<0.05), as well as IgE levels (p<0.0001) and gene expression levels (p<0.01 or p<0.001) compared to disease controls. The genes from each group were normalized to cltc, gapdh, gusb, hprt, pgk1, and tubb5 and plotted as the mean ± SEM of gene transcript copy in each treatment group. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; NS: Not Significant.

Fig 8. Schematic illustrating the proposed mechanism by which GM-1111 reduces allergic CRS-associated inflammation.

GM-1111 blocks the innate immune response through TLR activation, thereby suppressing the expression of proinflammatory cytokines, chemokines, and allergy signaling molecules and inhibiting the chemotaxis of CD4+ T cells and eosinophils, which amplify adaptive immune signaling.