The innate immune system is comprised of many components that function coordinately to prevent bacterial sepsis. However, thermal injury suppresses many of these factors, and the opportunistic pathogen Pseudomonas aeruginosa takes advantage of this condition, making it one of the leading causes of morbidity and mortality in the setting of thermal injury.
The pathogenicity of P. Syndecan 1 is a major heparan sulfate proteoglycan present on many host cells involved in thermal injury. Syndecan 1 anchored to the cell surface can be cleaved in a process termed ectodomain shedding. Syndecan 1 shedding results in the release of intact, soluble proteoglycan ectodomains that have diverse roles in innate immunity.
Here we show for the first time that thermal injury results in shedding of syndecan 1 from host tissue.
Our data show that syndecan 1 null mice are significantly less susceptible to P. These results suggest that shed syndecan 1 plays an important role in the pathogenesis of P. The gram-negative opportunistic pathogen Pseudomonas aeruginosa is particularly prevalent in burn wound infections. The extensive damage induced during P. However, the increased ability of P. Beyond the initial barrier of the skin, several components of the host's innate immune system are designed to act as sentinels that guard against systemic bacterial attacks.
Toll-like receptors TLRs are transmembrane receptors found on host cells that recognize pathogen-associated molecular patterns Upon recognition of a pathogen-associated molecular pattern, TLRs activate downstream signaling events, which induce an immediate inflammatory response Acute inflammation, as seen in thermal injury, is characterized by vasodilatation and neutrophil infiltration.
Consequences of inflammation include the production of cytokines and chemokines. Chemokines, such as interleukin-8 IL-8 , recruit neutrophils into the damaged area, which normally engulf the infecting bacteria before high numbers are reached. Among their many roles, cytokines regulate neutrophil functions and perpetuate the inflammatory response. Although these first lines of defense are normally effective, the highly devascularized environment of a burn wound and overall dysregulation of immunity in burned individuals provide an ideal setting for the proliferation and spread of bacteria within the injured tissue and subsequent dissemination throughout the immunocompromised host.
In this environment, inflammation can shift from a protective process to a destructive one. The massive neutrophil infiltration, characteristic of thermal injury, may actually have a negative biological effect.
Activated neutrophils release oxidants and hydrolytic enzymes that, when combined with bacterial degradative enzymes, can result in increased proteolysis within the wound and may actually aid spread and eventual dissemination of bacteria 14 , This neutrophil paradox has been implicated in the pathology of many chronic inflammatory conditions Dozens of inflammatory mediators play important roles in balancing the protective and destructive effects of inflammation.
One important group of inflammatory mediators are heparan sulfate proteoglycans HSPGs 7. HSPGs are ubiquitous molecules expressed by all adherent cells and are composed of a core protein and one or more covalently attached heparan sulfate HS chains 7.
The HS chains are made up of repeating units of hexuronic acid and N-substituted glucosamine disaccharide units. One family of HSPGs implicated in inflammation is the syndecans.
Four syndecan family members syndecan 1 to 4 have been cloned in mammals and are implicated in mediating a wide range of effects, including leukocyte rolling on endothelial cells, wound repair, angiogenesis, vascular permeability, and modulation of chemokine activity 7.
Syndecans attached to the cell surface can be cleaved in a process termed shedding. Syndecan shedding results in the release of intact, soluble proteoglycan ectodomains into the host cell environment that can mediate cellular physiology at distant sites 7. Although syndecan ectodomains are shed at low levels as part of normal cell turnover, tissue injury can markedly activate syndecan shedding Thus, shed syndecan 1 Sdc-1 is not found at detectable levels in the biological fluids of healthy individuals, but high levels of shed Sdc-1 and Sdc-4 have been documented in human dermal wound fluids 37 and in tracheal aspirates of ventilated preterm infants However, it has also been shown that the LasA protease produced by P.
Shed Sdc-1 contributed to P.
MATERIALS AND METHODS
Furthermore, treating P. Considering the demonstrated importance of shed Sdc-1 to P. These data provide support for the prospect of using Sdc-1 neutralizing agents in the treatment of burn wounds. Mice used in experiments were 6 to 8 weeks old and weighed 17 to 20 g. The exact inoculum of each strain was determined by plating serial dilutions of the inoculum on LB agar plates. The virulence of PAO1 was examined by using the modified burned-mouse model of Stieritz and Holder In this modified model, a scald burn is induced.
Mice were anesthetized by intraperitoneal injection of 0. The mice were securely placed into a template with an opening 4.
What is Sepsis? (Life-threatening Infection)
Such an injury is nonlethal but causes a third-degree full-thickness burn. Fluid replacement therapy consisting of a subcutaneous injection of 0. In some experiments, porcine mucosal heparan sulfate Sigma, St. Louis, Mo. During recovery, the mice were continuously observed under warming lights. Skin sections of approximately 5 by 5 mm were obtained from the burned skin of both the control and challenged mice.
Simultaneously the entire liver of each animal was obtained. Skin sections from control nonburned mice, mice that had been administered a third-degree scald burn, and mice that were burned and infected with P.
The tissue was placed in phosphate-buffered saline PBS and gently vortexed for 10 s. The same blot was stripped and then reprobed with an antibody directed against the cytoplasmic domain of Sdc-1 21 to confirm that Sdc-1 detected by the antibody was shed ectodomain and not intact Sdc-1 released from cells damaged by vortexing. The tissue was then processed through a graded series of ethanol, cleared in xylene, and infiltrated with paraffin.
The tissue was embedded on edge in paraffin, and blocks were sectioned with a Cut Olympus microtome.
MRSA (Staph) Infection
Mouse skin that had been burned and infected with P. Mouse skin that had been burned but not infected was treated in a similar manner to use as a control.
Tissue was then frozen in liquid nitrogen and fractured with a frozen blade to expose more surface. The tissue was critically point dried with a Tousimis critical point dryer Rockville, MD.
The sections were mounted on edge on stubs coated with carbon tape. Some of the infected skin tissue samples were carved using a razor blade under a dissecting scope to reveal more of the inner structure. All the tissues were gold coated using a Polaron sputter coater. The sections were then scanned for bacteria, and pictures were taken using a Hitachi S scanning electron microscope Hitachi America, Ltd. Gels were dried, and protected bands representing different cytokines were detected after exposure of the gels to a phosphorimager screen.
The signal of the band representing a cytokine of interest was normalized to the signal of the L32 ribosomal gene band from the same sample. Tissue injury activates syndecan shedding 12 , and shed Sdc-1 is found at high levels in human dermal wound fluids 37 and in tracheal aspirates of ventilated preterm infants However, Sdc-1 has not been demonstrated in burn wounds.
To determine if thermal injury results in Sdc-1 shedding, Sdc-1 ectodomain levels in burn fluid were measured by dot immunoblotting.
Samples were then dot blotted onto a cationic nylon membrane and quantified by immunoblotting with an Sdc-1 ectodomain antibody. Sdc-1 was detected in the fluid extract of burned, but not nonburned, skin; however, increased levels of Sdc-1 were detected in the wound fluid extracts of mice that were burned and infected with PAO1 Fig.
Sdc-1 ectodomains were not detected when the membrane was reprobed with an antibody against the cytoplasmic domain of Sdc-1, confirming that Sdc-1 was shed and not released intact from cells data not shown. Immunoblotting with Sdc-1 antibody. Skin sections from two control nonburned mice, two mice that had been administered a third-degree scald burn, and two mice that were burned and infected with PAO1 were placed into PBS and gently vortexed.
Samples in triplicate from these wound fluid extracts were transferred to cationic nylon membranes and quantified by dot immunoblotting with a monoclonal antibody to Sdc-1 ectodomain. Cont, control. To determine if the increased levels of shed Sdc-1 that are associated with thermal injury correlated with the severity of P. The thermally injured mouse model of P.
We have previously determined that the P.
Three groups of mice 5 to 8 mice per group were thermally injured, inoculated with PAO1, allowed to recover, and monitored over 5 days h. Mortality was assessed at h time points. Data shown represent three independent experiments. Shed Sdc-1, via its HS chains, can function as a soluble effector to regulate various cellular processes, including proliferation, adhesion, and differentiation 3.
Taken together, these results indicate that the high levels of Sdc-1 shed due to thermal injury exacerbate the virulence of P. Data shown represent two independent experiments. The first phase involves colonization of the burned tissue by P. The devascularized state of the burn eschar burn wound area provides an environment ideally suited for bacterial growth, and P.
To determine if Sdc-1 influenced either stage of P.
Bacteremia and septicemia pdf merge
We examined the local colonization of bacteria within the eschar by determining the number of PAO1 CFU in epithelial sections taken at two specific sites within the area of thermal injury, the inoculation site and a site 15 mm distal to the inoculation site. Using this approach, we have previously shown that PAO1 spreads efficiently from the inoculation site to the distal site by 24 h postburn As in the mortality experiments, mice were thermally injured and inoculated with PAO1.
These results indicate that within 24 h, P. The livers and sections of thermally injured tissue from two sites the bacterial inoculation site and a site 15 mm distal were taken from each mouse and homogenized. To examine the systemic spread of P.
On average, 3. Taken together, these results indicate that P.
However, Sdc-1 appears to be needed for dissemination of PAO1 into the bloodstream and secondary colonization of internal organs. Two distinctive clinical features of P.