Sarah A. Gardner

Hypothermia and Surgery: Immunologic Mechanisms for Current Practice

Objective:To examine cellular and immunologic mechanisms by which intraoperative hypothermia affects surgical patients. Summary Background Data:Avoidance of perioperative hypothermia has recently become a focus of attention as an important quality performance measure, aimed at optimizing the care of surgical patients. Anesthetized surgical patients are particularly at risk for hypothermia, which has been directly linked to the development of sequelae, such as coagulopathy, infection, morbid myocardial events, and death after surgery. However, many of the underlying immunologic mechanisms remain unclear. Methods:Venous blood samples from healthy volunteers were exposed for up to 4 hours to various temperatures following the addition of a 1 ng/mL lipopolysaccharide challenge. Innate immune function, assessed by the ability of monocytes to present antigen and coordinate cytokine release, was determined by qualitative and quantitative measurements of HLA-DR surface expression 2 hours following incubation, and proinflammatory tumor necrosis factor-α (TNF-α) and anti-inflammatory (IL-10) cytokine release in the first 4 hours. Results:Monocyte incubation at hypothermic temperatures (34°C) reduced HLA-DR surface expression, delayed TNF-α clearance, and increased IL-10 release. Conversely, hyperthermia (40°C) increased monocyte antigen presentation and resulted in rapid decay of TNF-α. However, IL-10 release was also increased. Normothermia (37°C) attenuated IL-10 release following the initial proinflammatory surge. Conclusion:Hypothermia exerts multiple effects at the cellular level, which impair innate immune function, and are associated with increased septic complications and mortality. These findings provide a physiological basis for perioperative temperature monitoring, which is a valid surgical performance measure that can be used to reduce surgical complications associated with avoidable hypothermia.

Oxygen and surgical site infection: a study of underlying immunologic mechanisms.

Background:Recent clinical trials investigating the role of hyperoxia in decreasing surgical site infection have reported conflicting results. Immunologic mechanisms through which supplemental oxygen could act have not been elucidated fully. The authors sought to investigate the effects of hyperoxia on previously tested and prognostically significant innate immune parameters to uncover the potential effects of hyperoxia at the cellular level. Methods:After formal approval and informed consent, venous blood samples were collected from young healthy volunteers. Corresponding samples were incubated at 21 or 80% O2 following a 1 ng/ml lipopolysaccharide challenge and analyzed to determine human leukocyte antigen-DR surface receptor expression, cytokine release, phagocytic capacity, and formation of reactive oxygen species. Data are presented as mean ± SD. Results:After the 2 h of incubation at 21% O2 (room air) and in 80% O2 chambers, the change in human leukocyte antigen-DR mean channel fluorescence in lipopolysaccharide-stimulated monocytes was 2,177 ± 383 and 2,179 ± 338 (P = 0.96), respectively. Tumor necrosis factor-&agr; concentrations were significantly lower for samples incubated at 80% O2 when compared with 21% O2 (P < 0.05). The phagocytic capacity of the innate immune system was not significantly enhanced by supplemental oxygen. However, the formation of reactive oxygen species increased by 87% (P < 0.05). Conclusion:Hyperoxia exerts significant effects on multiple cellular and immunologic parameters, providing a potential mechanism for benefits from the use of supplemental oxygen. However, the ability to translate positive basic scientific findings to the operating suite or bedside require the existence of similar innate immune processes in vivo and the efficient transfer of oxygen to the sites where it may be used.

Quantification of Phagocytosis in Human Neutrophils by Flow Cytometry

Phagocytosis represents a central element of the host response to microbial invasion. We describe a flow cytometric method for measuring the kinetics of phagocytosis of two bacteria by human polymorphonuclear leukocytes (PMNs). Over a 60‐min period, isolated human PMNs were exposed to Staphylococcus aureus (rapidly phagocytosed) and Klebsiella pneumoniae (slowly phagocytosed). This method distinguished adherent from ingested bacteria by quenching fluorescein isothiocyanate‐labeled extracellular bacteria with ethidium bromide. This further allowed the exclusion of dead, highly permeable, and subsequently bright‐red fluorescent PMNs. Our experiments with two different bacteria, various PMN‐to‐bacteria ratios (1:1, 1:10, 1:100), and different individuals proved that 1) flow cytometric analysis is accurate and useful for characterizing phagocytosis, 2) adherent bacteria can be distinguished from ingested bacteria after quenching with ethidium bromide, and that 3) phagocytosis kinetics of two bacteria with different onsets of phagocytosis can be determined by flow cytometry and the assessment of a score that quantifies phagocytosis.

MicroRNA-155 potentiates the inflammatory response in hypothermia by suppressing IL-10 production

Therapeutic hypothermia is commonly used to improve neurological outcomes in patients after cardiac arrest. However, therapeutic hypothermia increases sepsis risk and unintentional hypothermia in surgical patients increases infectious complications. Nonetheless, the molecular mechanisms by which hypothermia dysregulates innate immunity are incompletely understood. We found that exposure of human monocytes to cold (32°C) potentiated LPS‐induced production of TNF and IL‐6, while blunting IL‐10 production. This dysregulation was associated with increased expression of microRNA‐155 (miR‐155), which potentiates Toll‐like receptor (TLR) signaling by negatively regulating Ship1 and Socs1. Indeed, Ship1 and Socs1 were suppressed at 32°C and miR‐155 antagomirs increased Ship1 and Socs1 and reversed the alterations in cytokine production in cold‐exposed monocytes. In contrast, miR‐155 mimics phenocopied the effects of cold exposure, reducing Ship1 and Socs1 and altering TNF and IL‐10 production. In a murine model of LPS‐induced peritonitis, cold exposure potentiated hypothermia and decreased survival (10 vs. 50%; P < 0.05), effects that were associated with increased miR‐155, suppression of Ship1 and Socs1, and alterations in TNF and IL‐10. Importantly, miR‐155‐deficiency reduced hypothermia and improved survival (78 vs. 32%, P < 0.05), which was associated with increased Ship1, Socs1, and IL‐10. These results establish a causal role of miR‐155 in the dysregulation of the inflammatory response to hypothermia.— Billeter, A. T., Hellmann, J., Roberts, H., Druen, D., Gardner, S. A., Sarojini, H., Galandiuk, S., Chien, S., Bhatnagar, A., Spite, M., Polk, H. C., Jr. MicroRNA‐155 potentiates the inflammatory response in hypothermia by suppressing IL‐10 production. FASEB J. 28, 5322–5336 (2014). www.fasebj.org

Glucose and Surgical Sepsis: A Study of Underlying Immunologic Mechanisms

BACKGROUND Early clinical trials investigating the role of tightly controlled glucose levels showed marked benefit in survival of critically ill patients. However, a recent meta-analysis and large randomized controlled trial have failed to reproduce the benefit, showing instead substantially increased risk of dangerous hypoglycemia. We sought to investigate the effects of varying glucose concentrations on previously tested, prognostically significant, innate immune parameters, to define any potential effects of glucose at the cellular level. STUDY DESIGN After formal approval and informed consent, venous blood samples were collected from young healthy volunteers. Up to 11 corresponding (same-subject) samples were incubated at 100, 350, or 600 mg/dL glucose concentrations and analyzed to determine human leukocyte antigen-DR surface receptor expression, cytokine release, phagocytic capacity, and formation of reactive oxygen species. Data are presented as mean +/- SEM. RESULTS After incubation, the change in human leukocyte antigen-DR mean channel fluorescence from resting baseline values in lipopolysaccharide-stimulated monocytes was not significantly different between 100, 350, and 600 mg/dL (1,749 +/- 110; 1,748 +/- 120; and 1,725 +/- 96, respectively; p = 0.89). Tumor necrosis factor-alpha concentrations were significantly lower for samples incubated at higher glucose concentrations (179 +/- 50 pg/mL, 125 +/- 30 pg/mL, and 107 +/- 29 pg/mL; p < 0.05). The phagocytic capacity of the innate immune system was marginally enhanced by glucose. However, the formation of reactive oxygen species was markedly impaired by rising glucose (55% to 66% impairment; p < 0.05). CONCLUSIONS Increasing glucose concentrations exert considerable opposing effects on several well-established innate immunologic processes. The opposing findings might contribute to recent clinical controversies. Physician judgment and experience are essential to imminent treatment of critically ill and perioperative surgical patients.

Differential microRNA (miRNA) expression could explain microbial tolerance in a novel chronic peritonitis model.

We observed persistent peritoneal bacteria despite a transient early innate immune response to intraperitoneal (IP) Klebsiella pneumoniae. Pretreatment with LPS prior to peritonitis induced a tolerant pattern of pro-inflammatory cytokine protein production over 72 h, but not at the mRNA level. MicroRNAs (miRNAs) regulate inflammatory cytokines and may explain this paradox. After pretreatment with IP LPS or saline, C57BL/6 mice were given 103 CFU of K. pneumoniae IP. Total RNA was isolated from peritoneal exudate cells (4 h, 24 h and 48 h following infection). mRNA and miRNA expression levels were detected and bioinformatics pathway analysis was performed, followed by measuring TNF-α, IL-1β, IL-6 and High-mobility Group Box 1 (HMBG1) protein levels. Of 88 miRNAs studied, 30 were significantly dysregulated at all time points in the LPS-pretreated group, including MiR-155, -146a, -142-3p, -299, and -200c -132 and -21. TNF-α, regulated by miR-155 and miR-146a, was decreased in the LPS-pretreated group at all time points (P < 0.05), as were HMGB1, a key alarmin regulated by miR-146, -142-3p, -299 and -200c (P < 0.05), and IL-1β and IL-6, both regulated by miR-132and miR-21 respectively (P < 0.05). Specific dysregulation of miR-155, -146a, -142-3p, -299, and -200c -132 and -21 with their corresponding effects on the TLR and NF-κB signaling pathways during inflammation, suggests a plausible mechanism for tolerance in this novel chronic model with persistent peritoneal infection.

Impact of microbial tolerance in persistent secondary Klebsiella pneumoniae peritonitis

PURPOSE AND METHODS Microbial tolerance represents a diminished pro-inflammatory response following repeated stimulation by a host of pathogen-associated molecular patterns (PAMPs) of varying origins. Toll-like receptors (TLRs) have been centrally implicated in the development of tolerance. The purpose of this study was to investigate the impact of tolerance in a previously described murine model. C57BL/6 mice were pretreated intraperitoneally with phosphate buffered saline (PBS), heat-killed Klebsiella 2×10(8) CFU (hkKlebsiella), LPS 10mg/kg (LPS 10), or BLP 10mg/kg (BLP 10). Following pretreatment, peritonitis was induced 24h later using 10(3) intraperitoneal Klebsiella CFU. Peritoneal concentrations of TNF-α, IL-10 and nitric oxide (NO), as well as characteristic cell patterns, were determined. Long-term consequences of microbial tolerance were assessed by measuring survival and weight-loss. RESULTS Following in vitro stimulation with Klebsiella 10(5) and 10(3) CFU, TNF-α and IL-10 secretion were diminished in macrophages harvested from mice pretreated with hkKlebsiella, LPS 10 and BLP 10. Pretreated animals had significantly lower bacterial counts. Conversely, local NO levels were elevated. Survival was not different between the groups. CONCLUSION Pretreatment with TLR ligands induced microbial tolerance, with reduced peritoneal cytokine concentrations and enhanced early bacterial clearance. However, this did not translate into improved survival.

Microbial tolerance in secondary peritonitis is dose dependent.

Local microbial tolerance was investigated in a murine model of peritonitis. Peritoneal bacterial burden and inflammatory cytokine concentrations were determined at different times, within 48h after infection. Peritoneal macrophages were harvested from naïve mice or from mice 48h after infection and underwent ex vivo stimulation with different concentrations of Klebsiella. Cytokine secretion was determined in the supernatants. Peritoneal bacteria concentrations, remained relatively steady between 24h (median: 5.04 log CFU) and 48h (median: 5.19 log CFU) after infection. Peritoneal cytokine concentrations peaked early but were already diminished at 48h after infection, despite persistent high bacteria levels. Macrophages, harvested from naïve mice responded vigorously to ex vivo stimulation with 10(5) CFU and 2 x 10(8) CFU Klebsiella. Cells harvested from animals 48h after infection, were unresponsive to an ex vivo stimulation with 10(5) CFU Klebsiella, but fully responded to 10(8) CFU. Persistent intraabdominal bacterial infection induced dose dependent microbial tolerance in peritoneal macrophages.

Warming to 39°C but Not to 37°C Ameliorates the Effects on the Monocyte Response by Hypothermia.

Objective:To investigate whether warming to normal body temperature or to febrile range temperature (39°C) is able to reverse the detrimental effects of hypothermia. Background:Unintentional intraoperative hypothermia is a well-described risk factor for surgical site infections but also sepsis. We have previously shown that hypothermia prolongs the proinflammatory response whereas normothermia and especially febrile range temperature enhance the anti-inflammatory response. Methods:Primary human monocytes were isolated from healthy volunteers. After stimulation with LPS (Lipopolysaccharide), the monocytes were exposed to 32°C for 3 hours or 6 hours and then warmed at either 37°C or 39°C for the remaining 33 hours or 36 hours, respectively. Tumor necrosis factor &agr;, interleukin 10, and the expression of miR-155 and miR-101 were assessed at 24 hours and 36 hours. Results:Warming to 37°C does not normalize monocyte cytokine secretion within 36 hours, whereas warming to 39°C partially reverses the effects of hypothermia on monocyte function. Both miR-155 and miR-101 were suppressed after the warming episode. However, 39°C had a stronger suppressive effect than 37°C. The duration of hypothermia and the warming temperature seem to be critical for a full reversibility of the effects of hypothermia. Conclusion:Warming to normal body temperature (37°C) does not restore normal monocyte function in vitro. These data suggest that hypothermic patients should be warmed to febrile range temperatures. Furthermore, febrile range temperatures should be investigated as a means to modulate the inflammatory response in patients with systemic infections.

Rapid tissue regeneration induced by intracellular ATP delivery—A preliminary mechanistic study

We have reported a new phenomenon in acute wound healing following the use of intracellular ATP delivery—extremely rapid tissue regeneration, which starts less than 24 h after surgery, and is accompanied by massive macrophage trafficking, in situ proliferation, and direct collagen production. This unusual process bypasses the formation of the traditional provisional extracellular matrix and significantly shortens the wound healing process. Although macrophages/monocytes are known to play a critical role in the initiation and progression of wound healing, their in situ proliferation and direct collagen production in wound healing have never been reported previously. We have explored these two very specific pathways during wound healing, while excluding confounding factors in the in vivo environment by analyzing wound samples and performing in vitro studies. The use of immunohistochemical studies enabled the detection of in situ macrophage proliferation in ATP-vesicle treated wounds. Primary human macrophages and Raw 264.7 cells were used for an in vitro study involving treatment with ATP vesicles, free Mg-ATP alone, lipid vesicles alone, Regranex, or culture medium. Collagen type 1α 1, MCP-1, IL-6, and IL-10 levels were determined by ELISA of the culture supernatant. The intracellular collagen type 1α1 localization was determined with immunocytochemistry. ATP-vesicle treated wounds showed high immunoreactivity towards BrdU and PCNA antigens, indicating in situ proliferation. Most of the cultured macrophages treated with ATP-vesicles maintained their classic phenotype and expressed high levels of collagen type 1α1 for a longer duration than was observed with cells treated with Regranex. These studies provide the first clear evidence of in situ macrophage proliferation and direct collagen production during wound healing. These findings provide part of the explanation for the extremely rapid tissue regeneration, and this treatment may hold promise for acute and chronic wound care.