a-delayed-inoculation-model-chronic-pseudomonas-aeruginosa-wound

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

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of ...

Stanford University

We recently reported that abundant deposits of the extracellular matrix polysaccharide hyaluronan (HA) are characteristic of autoimmune insulitis in patients with type 1 diabetes (T1D), but the relevance of these deposits to disease was unclear. Here, we have demonstrated that HA is critical for the pathogenesis of autoimmune diabetes. Using the DO11.10xRIPmOVA mouse model of T1D, we determined that HA deposits are temporally and anatomically associated with the development of insulitis. Moreover, treatment with an inhibitor of HA synthesis, 4-methylumbelliferone (4-MU), halted progression to diabetes even after the onset of insulitis. Similar effects were seen in the NOD mouse model, and in these mice, 1 week of treatment was sufficient to prevent subsequent diabetes. 4-MU reduced HA accumulation, constrained effector T cells to nondestructive insulitis, and increased numbers of intraislet FOXP3+ Tregs. Consistent with the observed effects of 4-MU treatment, Treg differentiation was inhibited by HA and anti-CD44 antibodies and rescued by 4-MU in an ERK1/2-dependent manner. These data may explain how peripheral immune tolerance is impaired in tissues under autoimmune attack, including islets in T1D. We propose that 4-MU, already an approved drug used to treat biliary spasm, could be repurposed to prevent, and possibly treat, T1D in at-risk individuals.

Inhibition of hyaluronan synthesis restores immune tolerance during autoimmune insulitis.

Hyaluronan (HA), an extracellular matrix glycosaminoglycan, is implicated in the pathogenesis of both type 1 diabetes (T1D) as well as type 2 diabetes (T2D) and has been postulated to be increased in these diseases due to hyperglycemia. We have examined the serum and tissue distribution of HA in human subjects with T1D and T2D and in mouse models of these diseases and evaluated the relationship between HA levels and glycemic control. We found that serum HA levels are increased in T2D but not T1D independently of hemoglobin-A1c, C-peptide, body mass index, or time since diabetes diagnosis. HA is likewise increased in skeletal muscle in T2D subjects relative to non-diabetic controls. Analogous increases in serum and muscle HA are seen in diabetic db/db mice (T2D), but not in diabetic DORmO mice (T1D). Diabetes induced by the β-cell toxin streptozotozin (STZ) lead to an increase in blood glucose but not to an increase in serum HA. These data indicate that HA levels are increased in multiple tissue compartments in T2D but not T1D independently of glycemic control. Given that T2D but not T1D is associated with systemic inflammation, these patterns are consistent with inflammatory factors and not hyperglycemia driving increased HA. Serum HA may have value as a biomarker of systemic inflammation in T2D.

Hyaluronan levels are increased systemically in human type 2 but not type 1 diabetes independently of glycemic control.

Bacteriophage are abundant at sites of bacterial infection, but their effects on mammalian hosts are unclear. We have identified pathogenic roles for filamentous Pf bacteriophage produced by  () in suppression of immunity against bacterial infection. Pf promote  wound infection in mice and are associated with chronic human  wound infections. Murine and human leukocytes endocytose Pf, and internalization of this single-stranded DNA virus results in phage RNA production. This triggers Toll-like receptor 3 (TLR3)- and TIR domain-containing adapter-inducing interferon-β (TRIF)-dependent type I interferon production, inhibition of tumor necrosis factor (TNF), and the suppression of phagocytosis. Conversely, immunization of mice against Pf prevents  wound infection. Thus, Pf triggers maladaptive innate viral pattern-recognition responses, which impair bacterial clearance. Vaccination against phage virions represents a potential strategy to prevent bacterial infection.

Bacteriophage trigger antiviral immunity and prevent clearance of bacterial infection.

Therapeutic increase of brown adipose tissue (BAT) thermogenesis is of great interest as BAT activation counteracts obesity and insulin resistance. Hyaluronan (HA) is a glycosaminoglycan, found in the extracellular matrix, which is synthesized by HA synthases (Has1/Has2/Has3) from sugar precursors and accumulates in diabetic conditions. Its synthesis can be inhibited by the small molecule 4-methylumbelliferone (4-MU). Here, we show that the inhibition of HA-synthesis by 4-MU or genetic deletion of  improves BAT`s thermogenic capacity, reduces body weight gain, and improves glucose homeostasis independently from adrenergic stimulation in mice on diabetogenic diet, as shown by a magnetic resonance T2 mapping approach. Inhibition of HA synthesis increases glycolysis, BAT respiration and uncoupling protein 1 expression. In addition, we show that 4-MU increases BAT capacity without inducing chronic stimulation and propose that 4-MU, a clinically approved prescription-free drug, could be repurposed to treat obesity and diabetes.

4-Methylumbelliferone improves the thermogenic capacity of brown adipose tissue.

 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  infection.  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  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) .  Using this delayed inoculation model and 7.5 ± 2.5 × 10 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  biofilms were not more virulent than infections with planktonic , whereas treatment with HK  only induces a short-term inflammatory state.  We describe a versatile wound model of chronic  infection that lasts 10 days without causing sepsis or other excessive morbidity.  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  infection.

Vivekananda Sunkari

Division of Infectious Diseases,
School of Medicine,
Division of Infectious Diseases, School of Medicine

Stanford University

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

Vivekananda Sunkari

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Stanford University

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

Division of Infectious Diseases,
School of Medicine,
Division of Infectious Diseases, School of Medicine