No abstract

Multidrug-resistant bacterial strains are a rapidly emerging healthcare threat; therefore it is critical to develop new therapies to combat these organisms. Prior antibacterial strategies directly target pathogen growth or viability. Host-directed strategies to increase antimicrobial defenses may be an effective alternative to antibiotics and reduce development of resistant strains. In this study, we demonstrated the efficacy of a pyrimidine synthesis inhibitor, N-phosphonacetyl-l-aspartate (PALA), to enhance clearance of methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Acinetobacter baumannii strains by primary human dermal fibroblasts in vitro. PALA did not have a direct bactericidal effect, but enhanced cellular secretion of the antimicrobial peptides human β-defensin 2 (HBD2) and HBD3 from fibroblasts. When tested in porcine and human skin explant models, a topical PALA formulation was efficacious to enhance MRSA, P. aeruginosa, and A. baumannii clearance. Topical PALA treatment of human skin explants also resulted in increased HBD2 and cathelicidin (LL-37) production. The antimicrobial actions of PALA required expression of nucleotide-binding, oligomerization domain 2 (NOD2), receptor-interacting serine/threonine-protein kinase 2 (RIP2), and carbamoyl phosphatase synthase II/aspartate transcarbamylase/dihydroorotase (CAD). Our results indicate that PALA may be a new option to combat multidrug-resistant bacterial infections of the skin through enhancement of an integral pathway of the cutaneous innate immune defense system.

Food additives are common components of processed foods consumed in a Western diet. In inflammatory bowel disease patients, some diets that exclude food additives improved clinical disease parameters, suggesting a link between food additives and disease pathogenesis. Food additives also enhanced disease severity in mouse colitis models through incompletely described mechanisms. This study examined the mechanisms by which the food additive maltodextrin (MDX) alters the development of colitis in a murine model. Interleukin-10 knockout (IL10KO) mice were fed diets supplemented with MDX or carboxymethyl cellulose (CMC) to determine their impact on colitis onset and severity; microbiome composition, function, and location; colonic immune cell infiltrates; and mucus layer integrity. Primary IL10KO colonic epithelial monolayers were used to dissect the impact of MDX directly on epithelial differentiation and mucus production. MDX or CMC consumption increased the incidence and severity of colitis, as well as decreased microbiome diversity, altered microbial composition, and decreased fecal acetic acid levels. The number of mucus producing cells were decreased in food additive fed mice and resulted in increased microbial proximity to the intestinal epithelium. Additionally, MDX supplementation resulted in crypt hyperplasia and expansion of the HopX+ injury renewal stem cell niche. In primary intestinal epithelial-derived monolayers devoid of microbes and immune cells, MDX exposure decreased goblet cell number and mucus production in association with downregulated expression of the transcription factor Klf4, a marker of terminally differentiated goblet cells. These results suggest MDX disrupts the balance of epithelial cell differentiation and proliferation to contribute to disease pathogenesis through direct and indirect actions on the intestinal epithelial barrier.

Pyoderma gangrenosum (PG) is a debilitating skin condition often accompanied by inflammatory bowel disease (IBD). Strikingly, ~40% of patients that present with PG have underlying IBD, suggesting shared but unknown pathogenesis mechanisms. Impeding the development of effective treatments for PG is the absence of an animal model that exhibits features of both skin and gut manifestations. This study describes the development of the first experimental drug-induced mouse model of PG with concurrent intestinal inflammation. Topical application of pyrimidine synthesis inhibitors on wounded mouse skin generates skin ulcers enriched in neutrophil extracellular traps (NETs) and pro-inflammatory cellular as well as soluble mediators mimicking human PG. The mice also develop spontaneous intestinal inflammation demonstrated by histologic damage. Further investigations revealed increased circulating immature low-density IL-1β primed granulocytes that undergo enhanced NETosis at inflamed tissue sites supported by increase in circulatory citrullinated histone 3, a marker of aberrant NET formation. Granulocyte depletion dampens the intestinal inflammation in this model, further supporting the notion that granulocytes contribute to the skin-gut crosstalk in PG mice. We anticipate that this novel murine PG model will enable researchers to probe common disease mechanisms and identify more effective targets for treatment for PG patients with IBD.

Severe burns are traumatic and physically debilitating injuries with a high rate of mortality. Bacterial infections often complicate burn injuries, which presents unique challenges for wound management and improved patient outcomes. Currently, pigs are used as the gold standard of pre-clinical models to study infected skin wounds due to the similarity between porcine and human skin in terms of structure and immunological response. However, utilizing this large animal model for wound infection studies can be technically challenging and create issues with data reproducibility. We present a detailed protocol for a porcine model of infected burn wounds based on our experience in creating and evaluating partial thickness burn wounds infected with Staphylococcus aureus on six pigs. Wound healing kinetics and bacterial clearance were measured over a period of 27 days in this model. Enumerated are steps to achieve standardized wound creation, bacterial inoculation, and dressing techniques. Systematic evaluation of wound healing and bacterial colonization of the wound bed is also described. Finally, advice on animal housing considerations, efficient bacterial plating procedures, and overcoming common technical challenges is provided. This protocol aims to provide investigators with a step-by-step guide to execute a technically challenging porcine wound infection model in a reproducible manner. Accordingly, this would allow for the design and evaluation of more effective burn infection therapies leading to better strategies for patient care.