The Immune Response to Chronic Pseudomonas aeruginosa Wound Infection in Immunocompetent Mice

Abstract

Objective: Our goal was to develop a chronic wound model in mice that avoids implantation of foreign material or impaired immunity and to use this to characterize the local and systemic immune response associated with Pseudomonas aeruginosa infection. Approach: We generated bilateral full-thickness dermal wounds in healthy 10-12-week-old C57Bl6 mice. We waited 24 h to inoculate the developing wound eschar at these sites. We performed careful titration experiments with luminescent strains of P. aeruginosa to identify bacterial inoculation concentrations that consistently established stable infections in these animals. We performed flow cytometry-based immunophenotyping of immune cell infiltrates at the wound site, spleen, and draining lymph nodes over time. Finally, we compared inflammatory responses seen in wound inoculation with planktonic bacteria, preformed biofilm, and heat-killed (HK) P. aeruginosa. Results: Using this delayed inoculation model and 7.5-2.5 • 10 2 CFU/mL of PAO1 we consistently established stable infections that lasted at 10 days in duration. During early infection, we detected a strong upregulation of inflammatory cytokines and neutrophil infiltration at the wound site, while natural killer (NK) cells and dendritic cells (DCs) were reduced. At the systemic level, only plasmacytoid DCs were increased early in infection. During later stages, there was systemic upregulation of B cells, T cells, and macrophages, whereas NK cells and interferon killer DCs were reduced. Infections with P. aeruginosa biofilms were not more virulent than infections with planktonic P. aeruginosa, whereas treatment with HK P. aeruginosa only induces a short-term inflammatory state. Innovation: We describe a versatile wound model of chronic P. aeruginosa infection that lasts 10 days without causing sepsis or other excessive morbidity. Conclusion: This model may facilitate the study of chronic wound infections in immunocompetent mice. Our findings also highlight the induction of early innate immune cell populations during P. aeruginosa infection.

A Delayed Inoculation Model of Chronic <em>Pseudomonas aeruginosa</em> Wound Infection

“…Doses higher than 10 4 CFU/mL resulted in 100% infection rate ( Figure 3F). These results were adapted from previously published data 13 .…”

Insights into Host–Pathogen Interactions in Biofilm-Infected Wounds Reveal Possibilities for New Treatment Strategies

“…Furthermore, it is extremely challenging to avoid the unintended colonization of the pigs’ wounds, and there exist only sparse reagents for the evaluation of host responses [ 34 ]. Still, rodents reveal several similar healing mechanisms to humans and are the most used species in wound research, albeit with different approaches and set ups [ 35 , 36 , 37 ]. Wounds are often incisional full thickness or burn wounds, although several additional strategies to generate the necessary initial skin defect have been described [ 38 , 39 , 40 ].…”

Filamentous Bacteriophage Delay Healing of Pseudomonas-Infected Wounds

“…We first examined the impact of a Pf phage-positive (Pf+) strain of Pa (PAO1) versus a Pf phagenegative (Pf-) isogenic strain (PAO1∆Pf4) on morbidity and mortality in a delayed inoculation murine wound infection model in which a provisional wound matrix is allowed to form for 24 hours after wounding before introduction of bacteria. We previously pioneered this model to study the initial establishment of Pa wound infections in otherwise healthy C57BL/6 mice in the absence of foreign material, diabetes, or other forms of immune suppression (de Vries et al;Sweere et al, 2019a;Sweere et al, 2019b). Here, we have adapted this model to study wound healing in the setting of infection over a two-week time period.…”

“…This was done as recently described (Sweere et al, 2019a;Sweere et al, 2019b). In brief, ten-to-twelveweek old male mice were anesthetized using 3% isoflurane, and their backs were shaved using a hair clipper and further depilated using hair removal cream (Nair).…”

“…To test this, we first examined the effects of Pf phage in a well-established in vitro wound healing model (a scratch assay). We then examined the effects of Pf phage on wound healing in an in vivo mouse model in the absence or presence of live Pa (Sweere et al, 2019a;Sweere et al, 2019b).…”