Ming Hui Fan

An Essential Role for Lipopolysaccharide-binding Protein in Pulmonary Innate Immune Responses

Lipopolysaccharide (LPS)-binding protein (LBP) greatly facilitates LPS activation of monocytic cells through the CD14 receptor, triggering activation of innate immune responses. An acute phase protein, LBP is produced predominantly by the liver; however, we and others have shown that LBP is produced extrahepatically in multiple locations, including the lung. The importance of LBP in the lung has remained unclear. LBP may make the host more acutely sensitive to LPS and development of septic complications; alternatively, it may be protective, aiding in detection, opsonization, and killing of bacteria. Our objective was to determine the role LBP plays in local pulmonary immune defenses to bacterial challenge. LBP knockout mice and age-matched C57BL/6 wild-type controls were challenged with direct intratracheal inoculation of Klebsiella pneumoniae. We observed a significant increase in mortality, earlier onset of bacteremia, and greater pulmonary bacterial loads in LBP knockout mice compared with controls. Total lung myeloperoxidase (MPO) activity, neutrophil recruitment to the alveolar space, and levels of KC—a chemokine involved in neutrophil recruitment—in bronchoalveolar lavage (BAL) fluid and lung homogenates were found to be significantly diminished in knockout mice compared with controls. Together, our findings suggest that LBP is essential in local pulmonary innate immune responses against bacteria.

Lipopolysaccharide-binding protein modulates acetaminophen-induced liver injury in mice†

Acetaminophen toxicity is the most common cause of acute liver failure in the United States and Europe. Although much is known about the metabolism of acetaminophen, many questions remain regarding the pathogenesis of liver injury. In this study, we examined the role of lipopolysaccharide‐binding protein (LBP), a protein important in mediating cellular response to lipopolysaccharides, by using LBP wild‐type and knockout (KO) mice. We found that LBP KO mice were protected from acetaminophen‐induced hepatotoxicity. At 350 mg/kg of acetaminophen, LBP KO mice had significantly less liver injury and necrosis than wild‐type mice. Repletion studies in LBP KO mice using an LBP–adenoviral construct resulted in significantly more hepatic injury and necrosis after acetaminophen exposure compared with mice receiving the control adenoviral construct. In conclusion, LBP KO mice are protected from toxicity with a decrease in hepatic necrosis following acetaminophen challenge. This suggests a novel role for LBP in modulating acetaminophen‐induced liver injury. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2005;41:187–195.)

Protegrin-1 enhances bacterial killing in thermally injured skin.

ObjectiveSeptic complications and the emergence of drug-resistant microbes represent serious risks to patients. Recently, naturally occurring peptides have been discovered that possess potent and broad-spectrum antimicrobial activity. Protegrin-1 is particularly attractive for clinical use in human wounds because, unlike defensins, protegrin-1 retains broad antimicrobial and antifungal activity at physiologic salt concentration and in the presence of serum. The objective of this study was to examine the efficacy of protegrin-1 in killing multiple drug-resistant microbes isolated from human burn patients. DesignFor the in vitro experiment, bilayer radial diffusion was performed comparing standard antibiotics with protegrin-1 on multiple-drug-resistant microbial organisms isolated from infected burn wounds. In vivo, rats received a 20% total body surface area partial-thickness burn by immersion in 60°C water for 20 secs followed by wound seeding with 106 colony forming units of Silvadene-resistant Pseudomonas aeruginosa. SettingUniversity of Michigan research laboratory. SubjectsAdult, male Sprague-Dawley rats. InterventionsRats were randomized into three groups: those receiving synthetic protegrin-1, acetic acid (carrier), or gentamicin (positive control). Protegrin-1 was administered by topical application or intradermal injection. Wound tissues were harvested aseptically at different time points for quantitative bacterial counts. Measurements and Main Results In vivo and in vitro experiments revealed rapid and significant decreases in bacterial counts for protegrin-1-treated groups compared with controls. ConclusionsThis study shows that protegrin-1 potentially may be used as an alternative or adjunct therapy to standard agents used to treat wound infections.

Protegrin-1 increases bacterial clearance in sepsis but decreases survival.

Despite recent progress in antibiotic and critical care therapy, sepsis remains a major clinical problem, especially after surgery or trauma. Sepsis ranks as the leading cause of death in intensive care units, and its incidence is increasing steadily (1). In the United States alone, it is estimated that the incidence of sepsis encompasses 300,000 to 500,000 cases each year, with mortality rates ranging from 20% to 40% (2). The therapy for septic patients usually consists of empirical treatment with broad-spectrum antibiotics before definitive bacterial culture and sensitivity results are available. In recent years, no substantial increase in survival has been accomplished; however, resistance to commonly used antibiotics in bacterial populations has increased dramatically with over-usage of antimicrobial drugs (3–5). Furthermore, antifungals are generally not a part of empirical antibiotic coverage and patients often develop candidal sepsis (6). Recently, two studies demonstrated that inadequate empirical antimicrobial treatment in critically ill patients is associated with greater hospital mortality, especially in individuals infected with antibioticresistant bacteria and candida species (7, 8). Facing growing bacterial resistance to conventional antibiotics, difficulty with fungal coverage, and persistence of high morbidity and mortality from infection, research has been undertaken to identify potential therapeutic alternatives. Among the potential candidates are antimicrobial peptides such as protegrin (PG)-1. In mammals, such peptides have an important role in the innate immune system’s primary defenses against invading microbes. The protegrin family consists of five antimicrobial peptides (PG-1 to PG5), which were initially identified in pigs (9–11). Protegrins are small (~2 kDa) antimicrobial peptides found in porcine neutrophils, where they are stored as cathelin-containing precursors (12). The mechanisms by which protegrins kill bacteria are not fully understood. Preliminary evidence suggests that they create voltage-dependent ion channels into the bacterial membrane (13–15). Protegrins have moderately high affinity for integral components of the bacterial membrane such as lipopolysaccharide found in Gram negatives and lipoteichoic acid found in Gram positives. When a threshold concentration of bound protegrin is reached, voltage-dependent channels are formed, which rapidly kill the microbe (16–19). At lower concentrations, no effect is seen and the bound protegrins are eventually digested by extracellular proteases. In vitro and in vivo studies have shown that PG-1 acts in an extremely rapid manner to kill logand stationary-phase Grampositive and Gram-negative bacteria and fungus, including methicillin-resistant Staphylococcus aureus (ATCC 19636), vancomycin-resistant Enterococcus faecalis (ATCC 29212), and E. faecium (clinical isolates VREF 032 and 033) (19) as well as Candida albicans (20). PG-1 may hold advantages for clinical application because, unlike human defensins, PG-1 retains broad antimicrobial and antifungal activity at physiologic salt concentrations and in the presence of serum, both of which are found in sites of inflammation where capillary leak is taking place. The broad spectrum, rapid microbicidal and antifungal activity of PG-1 may allow for single-agent therapy systemically. This would facilitate drug monitoring and minimize the considerable risk of adverse drug reactions. Although antimicrobial peptides have been shown to be quite effective against microorganisms in vitro, little work has been done in vivo. It remains unclear if, and under what conditions, antimicrobial peptides retain antimicrobial properties. It is also unknown what effect exogenous peptides have on the native innate immune response to infection. This latter issue is of particular interest because much of the morbidity and mortality of sepsis is thought to be caused by an inappropriately prolonged and overly exuberant host immune response. Several attempts have been undertaken to shed light on the pathophysiology of sepsis and to improve survival using experimental animal models. Although animal studies revealed significant improvement in survival using a strategy whereby specific cytokines were inhibited either by using neutralizing antibodies or by receptor antagonists (21, 22), large-scale clinical trials have failed to confirm these findings in humans (23, 24). A possible explanation for these failures is that virtually all of the preclinical trials were done using injection of either a single strain of live bacteria or lipopolysaccharide only as a model for sepsis. Although appropriate for the acute study of immune modulation of cytokines, these models do not adequately reflect the complex immunology and microbiology of sepsis seen in the clinical setting. A clinically more relFrom the Departments of Surgery (LS, MHF, SCW), Medicine (GLS), and Pathology (JN, AM, DIR), University of Michigan, Ann Arbor, MI; and the Department for Plastic Surgery (LS, OB, HUS), BG University Hospital Bergmannsheil, Bochum, Germany.

Thermal injury induces expression of CD14 in human skin

BACKGROUND Skin is equipped with an array of immune mediators aimed at fighting invading microbes. CD14 has been shown to play a key role in modulating the activation of cells by LPS. Since LPS levels within burn wounds are often found to be elevated, we sought to examine the expression of CD14 within human skin following thermal injury. METHODS Patients who sustained partial thickness burns, were recruited into the study (n=57). Total RNA was isolated from both burn and normal (control) skin. Northern blot analysis and TaqMan RT-PCR were used to determine skin CD14 mRNA levels. Immunohistochemistry was used to localize CD14 expression in burned and normal skin. RESULTS Quantitative PCR showed significantly increased CD14 expression levels in the immediate post-burn period (P<0.05 burn versus non-burn). Immunohistochemistry revealed more pronounced CD14 staining 24 h after the injury, reaching normal levels approximately 5-7 days post-burn. CONCLUSION CD14 expression peaks within the first week post-burn before declining, reaching normal levels after 14 days. This loss of supranormal CD14 expression locally within the wound may contribute to a weakened host defense response 5-6 days after injury, when patients become especially vulnerable to infection.

Zytokin-Expressionsprofile in infizierten Verbrennungswunden

Inspite of dramatic improvements in the management of burns, infection still remains a serious risk for the burn patient. Bacterial infection and thermal injury have been shown to trigger the inflammatory response. The aim of this study was to shed light on the impact of acute and subacute burn injury with or without infection on cytokine profiles. Sprague-Dawley rats (n =20) were randomized into three groups: 1) burn only 2) burn and bacteria (106 colony forming units of multi-drug resistant pseudomonas aeruginosa) and 3) sham burn. Animals in the first two groups received a 30% TBSA partial thickness burn. Weight changes were monitored daily. Blood was collected after 12,24,48,72 h and 6 and 12 days, and was used for cytokine ELISAs, LPS quantification, and peripheral blood analysis. Animals were sacrificed either after 6 or 12 days. Skin was harvested for bacterial counts, histological analysis and RNA isolation for microarray analysis. Infected animals showed substantial weight loss until day 6 postburn. We also found higher plasma levels (p <0.05) of IL-1β, TNF-α and IL-10 in the infected group compared to the uninfected burn group throughout the observation periods. The m-RNA expression levels showed no difference in cytokine expression, except for IL-1. Endotoxin levels were elevated significantly in the infected burn group during the first 2 days postinjury. Our results suggest that burn wound infection elicits a systemic and local release of cytokines with corresponding upregulation of m-RNA levels. High TNF-α levels were seen in the infected group during the initial period of weight loss, with a later significant upregulation of the anti-inflammatory cytokine IL-10 seen when weight gain resumed.