Luis Ramirez

Reduced Neutrophil Chemotaxis and Infiltration Contributes to Delayed Resolution of Cutaneous Wound Infection with Advanced Age

Advanced age is associated with alterations in innate and adaptive immune responses, which contribute to an increased risk of infection in elderly patients. Coupled with this immune dysfunction, elderly patients demonstrate impaired wound healing with elevated rates of wound dehiscence and chronic wounds. To evaluate how advanced age alters the host immune response to cutaneous wound infection, we developed a murine model of cutaneous Staphylococcus aureus wound infection in young (3–4 mo) and aged (18–20 mo) BALB/c mice. Aged mice exhibit increased bacterial colonization and delayed wound closure over time compared with young mice. These differences were not attributed to alterations in wound neutrophil or macrophage TLR2 or FcγRIII expression, or age-related changes in phagocytic potential and bactericidal activity. To evaluate the role of chemotaxis in our model, we first examined in vivo chemotaxis in the absence of wound injury to KC, a neutrophil chemokine. In response to a s.c. injection of KC, aged mice recruited fewer neutrophils at increasing doses of KC compared with young mice. This paralleled our model of wound infection, where diminished neutrophil and macrophage recruitment was observed in aged mice relative to young mice despite equivalent levels of KC, MIP-2, and MCP-1 chemokine levels at the wound site. This reduced leukocyte accumulation was also associated with lower levels of ICAM-1 in wounds from aged mice at early time points. These age-mediated defects in early neutrophil recruitment may alter the dynamics of the inflammatory phase of wound healing, impacting macrophage recruitment, bacterial clearance, and wound closure.

Aberrant acute-phase response in aged interleukin-6 knockout mice

ABSTRACT This study was designed to determine whether the acute-phase response in aged mice is altered by interleukin (IL) 6 deficiency. Young and aged wild-type (WT) and IL-6 knockout (KO) BALB/C female mice were injected with lipopolysaccharide (LPS; 1.5 &mgr;g/g body weight). After 24 h, aged IL-6 KO mice had an improved survival when compared with aged WT mice. Serum levels of IL-6 in aged WT animals given LPS were determined and, as expected, were significantly higher when compared with young LPS-treated WT animals (P < 0.05). Serum levels of the acute-phase protein, serum amyloid A, were 50% lower in aged LPS-treated IL-6 KO mice relative to aged WT mice given LPS (P < 0.001). In contrast, the induction of LPS-binding protein was not affected by age or IL-6 deficiency in LPS-treated animals. Circulating levels of corticosterone were markedly reduced in aged LPS-treated IL-6 KO mice relative to aged WT mice given LPS. These data indicate that IL-6 is an important contributor to the outcome of the acute-phase response of aged individuals challenged with endotoxin. We conclude that the absence of IL-6, a cytokine that contributes to the elevated basal proinflammatory state observed in aging, can improve the ability of aged mice to withstand an otherwise lethal challenge of bacterial endotoxin.

The acute pulmonary inflammatory response to the graded severity of smoke inhalation injury.

Objectives:To determine whether the graded severity of smoke inhalation is reflected by the acute pulmonary inflammatory response to injury. Design:In a prospective observational study, we assessed the bronchoalveolar lavage fluid for both leukocyte differential and concentration of 28 cytokines, chemokines, and growth factors. Results were then compared to the graded severity of inhalation injury as determined by Abbreviated Injury Score criteria (0, none; 1, mild; 2, moderate; 3, severe; 4, massive). Setting:All patients were enrolled at a single tertiary burn center. Patients:The bronchoalveolar lavage fluid was obtained from 60 patients within 14 hrs of burn injury who underwent bronchoscopy for suspected smoke inhalation. Interventions:None. Measurements and Main Results:Those who presented with worse grades of inhalation injury had higher plasma levels of carboxyhemoglobin and enhanced airway neutrophilia. Patients with the most severe inhalation injuries also had a greater requirement for tracheostomy, longer time on the ventilator, and a prolonged stay in the intensive care unit. Of the 28 inflammatory mediators assessed in the bronchoalveolar lavage fluid, 21 were at their highest in those with the worst inhalation injury scores (grades 3 and 4), the greatest of which was interleukin-8 (92,940 pg/mL, grade 4). When compared in terms of low inhalation injury (grades 1–2) vs. high inhalation injury (grades 3–4), we found significant differences between groups for interleukin-4, interleukin-6, interleukin-9, interleukin-15, interferon-&ggr;, granulocyte-macrophage colony-stimulating factor, and monocyte chemotactic protein-1 (p < .05 for all). Conclusions:These data reveal that the degree of inhalation injury has basic and profound effects on burn patient morbidity, evokes complex changes of multiple alveolar inflammatory proteins, and is a determinant of the pulmonary inflammatory response to smoke inhalation. Accordingly, future investigations should consider inhalation injury to be a graded phenomenon. (Crit Care Med 2012; 40:–1121)

The protective role of laparoscopic antireflux surgery against aspiration of pepsin after lung transplantation.

BACKGROUND The goal of this study was to determine, in lung transplant patients, if laparoscopic antireflux surgery (LARS) is an effective means to prevent aspiration as defined by the presence of pepsin in the bronchoalveolar lavage fluid (BALF). METHODS Between September 2009 and November 2010, we collected BALF from 64 lung transplant patients at multiple routine surveillance assessments for acute cellular rejection, or when clinically indicated for diagnostic purposes. The BALF was tested for pepsin by enzyme-linked immunosorbent assay (ELISA). We then compared pepsin concentrations in the BALF of healthy controls (n = 11) and lung transplant patients with and without gastroesophageal reflux disease (GERD) on pH-monitoring (n = 8 and n = 12, respectively), and after treatment of GERD by LARS (n = 19). Time to the development of bronchiolitis obliterans syndrome was contrasted between groups based on GERD status or the presence of pepsin in the BALF. RESULTS We found that lung transplant patients with GERD had more pepsin in their BALF than lung transplant patients who underwent LARS (P = .029), and that pepsin was undetectable in the BALF of controls. Moreover, those with more pepsin had quicker progression to BOS and more acute rejection episodes. CONCLUSION This study compared pepsin in the BALF from lung transplant patients with and without LARS. Our data show that: (1) the detection of pepsin in the BALF proves aspiration because it is not present in healthy volunteers, and (2) LARS appears effective as a measure to prevent the aspiration of gastroesophageal refluxate in the lung transplant population. We believe that these findings provide a mechanism for those studies suggesting that LARS may prevent nonallogenic injury to the transplanted lungs from aspiration of gastroesophageal contents.

Early Pulmonary Immune Hyporesponsiveness Is Associated With Mortality After Burn and Smoke Inhalation Injury

This prospective study aims to address mortality in the context of the early pulmonary immune response to burn and inhalation injury. The authors collected bronchoalveolar lavage fluid from 60 burn patients within 14 hours of their injury when smoke inhalation was suspected. Clinical and laboratory parameters and immune mediator profiles were compared with patient outcomes. Patients who succumbed to their injuries were older (P = .005), had a larger % TBSA burn (P < .001), and required greater 24-hour resuscitative fluids (P = .002). Nonsurvivors had lower bronchoalveolar lavage fluid concentrations of numerous immunomodulators, including C5a, interleukin (IL)-1&bgr;, IL-1RA, IL-8, IL-10, and IL-13 (P < .05 for all). Comparing only those with the highest Baux scores to account for the effects of age and % TBSA burn on mortality, nonsurvivors also had reduced levels of IL-2, IL-4, granulocyte colony-stimulating factor, interferon-&ggr;, macrophage inflammatory protein-1&bgr;, and tumor necrosis factor-&agr; (P < .05 for all). The apparent pulmonary immune hyporesponsiveness in those who died was confirmed by in vitro culture, which revealed that pulmonary leukocytes from nonsurvivors had a blunted production of numerous immune mediators. This study demonstrates that the early pulmonary immune response to burn and smoke inhalation may be attenuated in patients who succumb to their injuries.

Inhibition of long myosin light-chain kinase activation alleviates intestinal damage after binge ethanol exposure and burn injury

Laboratory evidence suggests that intestinal permeability is elevated following either binge ethanol exposure or burn injury alone, and this barrier dysfunction is further perturbed when these insults are combined. We and others have previously reported a rise in both systemic and local proinflammatory cytokine production in mice after the combined insult. Knowing that long myosin light-chain kinase (MLCK) is important for epithelial barrier maintenance and can be activated by proinflammatory cytokines, we examined whether inhibition of MLCK alleviated detrimental intestinal responses seen after ethanol exposure and burn injury. To accomplish this, mice were given vehicle or a single binge ethanol exposure followed by a sham or dorsal scald burn injury. Following injury, one group of mice received membrane permeant inhibitor of MLCK (PIK). At 6 and 24 h postinjury, bacterial translocation and intestinal levels of proinflammatory cytokines were measured, and changes in tight junction protein localization and total intestinal morphology were analyzed. Elevated morphological damage, ileal IL-1β and IL-6 levels, and bacterial translocation were seen in mice exposed to ethanol and burn injury relative to either insult alone. This increase was not seen in mice receiving PIK after injury. Ethanol-exposed and burn-injured mice had reduced zonula occludens protein-1 and occludin localization to the tight junction relative to sham-injured mice. However, the observed changes in junctional complexes were not seen in our PIK-treated mice following the combined insult. These data suggest that MLCK activity may promote morphological and inflammatory responses in the ileum following ethanol exposure and burn injury.

Inhalation injury severity and systemic immune perturbations in burned adults.

Objective:We aimed to determine whether the severity of inhalation injury evokes an immune response measurable at the systemic level and to further characterize the balance of systemic pro- and anti-inflammation early after burn and inhalation injury. Background:Previously, we reported that the pulmonary inflammatory response is enhanced with worse grades of inhalation injury and that those who die of injuries have a blunted pulmonary immune profile compared with survivors. Methods:From August 2007 to June 2011, bronchoscopy was performed on 80 patients admitted to the burn intensive care unit when smoke inhalation was suspected. Of these, inhalation injury was graded into 1 of 5 categories (0, 1, 2, 3, and 4), with grade 0 being the absence of visible injury and grade 4 corresponding to massive injury. Plasma was collected at the time of bronchoscopy and analyzed for 28 immunomodulating proteins via multiplex bead array or enzyme-linked immunosorbent assay. Results:The concentrations of several plasma immune mediators were increased with worse inhalation injury severity, even after adjusting for age and % total body surface area (TBSA) burn. These included interleukin (IL)-1RA (P = 0.002), IL-6 (P = 0.002), IL-8 (P = 0.026), granulocyte colony-stimulating factor (P = 0.002), and monocyte chemotactic protein 1 (P = 0.007). Differences in plasma immune mediator concentrations in surviving and deceased patients were also identified. Briefly, plasma concentrations of IL-1RA, IL-6, IL-8, IL-15, eotaxin, and monocyte chemotactic protein 1 were higher in deceased patients than in survivors (P < 0.05 for all), whereas IL-4 and IL-7 were lower (P < 0.05). After adjusting for the effects of age, % TBSA burn, and inhalation injury grade, plasma IL-1RA remained significantly associated with mortality (odds ratio, 3.12; 95% confidence interval, 1.03–9.44). Plasma IL-1RA also correlated with % TBSA burn, inhalation injury grade, fluid resuscitation, Baux score, revised Baux score, Denver score, and the Sequential Organ Failure Assessment score. Conclusions:The severity of smoke inhalation injury has systemically reaching effects, which argue in favor of treating inhalation injury in a graded manner. In addition, several plasma immune mediators measured early after injury were associated with mortality. Of these, IL-1RA seemed to have the strongest correlation with injury severity and outcomes measures, which may explain the blunted pulmonary immune response we previously found in nonsurvivors.

Decreased Pulmonary Inflammation Following Ethanol and Burn Injury in Mice Deficient in TLR4 but not TLR2 Signaling

BACKGROUND Clinical and laboratory evidence suggests that alcohol consumption prior to burn injury leads to dysregulated immune function and subsequent higher rates of morbidity and mortality. Our laboratory previously observed higher levels of pro-inflammatory cytokines and leukocyte infiltration in the lungs of mice following ethanol and burn injury. To understand the mechanism of the increased inflammatory response, we looked at different signaling initiators of inflammation including toll-like receptors 2 and 4 (TLR2 and 4) pathways. METHODS Wild-type, TLR2, and TLR4 knockout mice were treated with vehicle or a single binge dose of ethanol (1.11 g/kg) and subsequently given a sham or burn injury. Twenty-four hours postinjury, systemic and pulmonary levels of pro-inflammatory cytokines were quantified, and differences in neutrophil infiltration were determined by histological examination. RESULTS Higher numbers of neutrophils were observed in the lungs of wild-type mice following the combined insult of ethanol and burn injury relative to either injury alone. This increase in leukocyte accumulation was absent in the TLR4 knockout mice. Circulating levels of IL-6 and tumor necrosis factor-α were also elevated in wild-type mice but not in TLR4 knockout mice. Consistent with these findings, pulmonary levels of KC and IL-6 were increased in wild-type mice following burn and ethanol compared to burn injury alone as well as to their TLR4 knockout counterparts. In contrast, TLR2 knockout mice displayed similar levels, to wild-type mice, of neutrophil infiltration as well as IL-6 and KC in the lung. CONCLUSIONS These data suggest that TLR4 signaling is a crucial contributory component in the exuberant inflammation after ethanol and burn injury. However, TLR2 does not appear to play a vital role in the aberrant pulmonary inflammation.

Acute Ethanol Exposure Attenuates Pattern Recognition Receptor Activated Macrophage Functions

Both clinical and experimental data have linked acute ethanol exposure to increased susceptibility to infection as well as increased morbidity and mortality after injury. Macrophages play an integral role in the innate immune system and are important in priming the adaptive immune system. In this study, we investigated the effect of a single in vivo exposure of macrophages to physiologically relevant levels of ethanol (1.2 and 2.9 g/kg) followed by ex vivo stimulation with lipopolysaccharide (LPS) or bacteria. Our study confirms the work of others showing that a single administration of ethanol suppresses the production of tumor necrosis factor-alpha(TNF-alpha), interleukin-6 (IL-6), and IL-12 in response to LPS. There was no effect of ethanol on LPS induction of cytokine production at 30 min after treatment. In contrast, at 3 h, both doses of ethanol exposure decreased ex vivo TNF-alpha production by splenic and alveolar macrophages. Interestingly, the higher dose of ethanol resulted in sustained suppression of LPS-induced TNF-alpha production at 3 and 6 h after ethanol administration, as well as decreased IL-6 and IL-12 production after 6 h, as compared to control (saline-treated groups). Alveolar macrophages behaved similarly at 3 h after ethanol treatment. LPS-stimulated production of TNF-alpha and IL-6 was reduced at 3 h after ethanol administration, when compared with the saline-treated animals. Alveolar macrophages stimulated for 3 h with bacteria also showed decreased TNF-alpha and IL-6 production after harvested from mice given 2.9 g/kg ethanol for 3 h. This time point and high dose of ethanol also resulted in decreased Pseudomonas aeruginosa phagocytosis by alveolar macrophages. Taken together, we conclude that the effects of physiological levels of ethanol are dose dependent, have effects that last after ethanol is cleared from the circulation, and can affect multiple macrophage functions.

Interleukin-6 Contributes to Age-Related Alteration of Cytokine Production by Macrophages

Here, we studied in vitro cytokine production by splenic macrophages obtained from young and aged BALB/c wild type (WT) and IL-6 knockout (IL-6 KO) mice. Relative to macrophages obtained from young WT mice given lipopolysaccharide (LPS), those from aged WT mice had decreased production of proinflammatory cytokines. In contrast, when compared to macrophages from young IL-6 KO mice, LPS stimulation yielded higher levels of these cytokines by cells from aged IL-6 KO mice. Aging or IL-6 deficiency did not affected the percentage of F4/80+ macrophages, or the surface expression of Toll-like receptor 4 (TLR4) and components of the IL-6 receptor. Overall, our results indicate that IL-6 plays a role in regulating the age-related defects in macrophages through alteration of proinflammatory cytokines, adding to the complexity of IL-6-mediated impairment of immune cell function with increasing age.