DL-2-hydroxyisocaproic acid attenuates inflammatory responses in a murine Candida albicans biofilm model

Abstract

Chronic biofilm infections are often accompanied by a chronic inflammatory response, leading to impaired healing and increased, irreversible damage to host tissues. Biofilm formation is a major virulence factor for Candida albicans and a challenge for treatment. Most current antifungals have proved ineffective in eradicating infections attributed to biofilms. The biofilm structure protects Candida species against antifungals and provides a way for them to evade host immune systems. This leads to a very distinct inflammatory response compared to that seen in planktonic infections. Previously, we showed the superior efficacy of dl-2-hydroxyisocaproic acid (HICA) against various bacteria and fungi. However, the immunomodulatory properties of HICA have not been studied. Our aim was to investigate the potential anti-inflammatory response to HICA in vivo. We hypothesized that HICA reduces the levels of immune mediators and attenuates the inflammatory response. In a murine model, a robust biofilm was formed for 5 days in a diffusion chamber implanted underneath mouse skin. The biofilm was treated for 12 h with HICA, while caspofungin and phosphate-buffered saline (PBS) were used as controls. The pathophysiology and immunoexpression in the tissues surrounding the chamber were determined by immunohistochemistry. Histopathological examination showed an attenuated inflammatory response together with reduced expression of matrix metalloproteinase 9 (MMP-9) and myeloperoxidase (MPO) compared to those of chambers containing caspofungin and PBS. Interestingly, the expression of developmental endothelial locus 1 (Del-1), an antagonist of neutrophil extravasation, increased after treatment with HICA. Considering its anti-inflammatory and antimicrobial activity, HICA may have enormous therapeutic potential in the treatment of chronic biofilm infections and inflammation, such as those seen with chronic wounds.

FIG 1

Summary of distribution of staining intensities representing protein expression within biofilm (A) or nonbiofilm (B) groups after 12 h of treatment with 5% (wt/vol) HICA or 10 mg/liter caspofungin or PBS. Sections of subcutaneous tissue surrounding the diffusion chamber were stained using polyclonal antibodies against MMP-8, MMP-9, MPO, NE, TNF-α, IL-1β, and Del-1. Intensities of staining were analyzed semiquantitatively by two evaluators and graded as no staining or mild, moderate, or strong staining.

FIG 2

Representative images of the tissue sections immunostained with polyclonal antibodies for matrix metalloproteinase (MMP-9) (A), myeloperoxidase (MPO) (B), and developmental endothelial locus 1 (Del-1) (C). The serrated lines show the location of the semipermeable membrane of the chamber. Framed panels on the right are magnifications of the areas marked with black rectangles in the left-hand panels. The expression of proinflammatory proteases MMP-9 and MPO localized predominantly in the inflammatory cells. The staining intensities for MMP-9 and MPO were lower in the HICA-treated biofilm group than in controls (treated with caspofungin or PBS), and histopathology was similar to that of nonbiofilm controls but with a thinner and less dense inflammatory cell infiltrate (A and B). Expression of neutrophil extravasation antagonist Del-1 was localized in the endothelium and was stronger in the HICA-treated group (C).