Devin Druen

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

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.

MicroRNAs: New helpers for surgeons?

MICRORNAS ARE VERY SMALL RNA molecules that are approximately 22 nucleotides in length. These microRNAs do not encode proteins; therefore, they are part of the family of noncoding RNA molecules. Because of their short length, they have been ignored for some time, despite being first described in 1993. MicroRNAs have started to gain interest after the discovery by Fire and Mello that these short, double-stranded RNAs, known as silencing RNA (siRNA), would inhibit quite strongly the protein translation after injection into cells of Caenorhabditis elegans. For this contribution, they received The Nobel Prize in Physiology or Medicine 2006. All of a sudden, these earlier described (but so far not understood) small RNA molecules seemed to have a profound impact on cell function by inhibiting protein translation. Indeed, microRNAs exist in all animals and plants, and their effect on the inhibition of protein translation has been demonstrated in all cells investigated to date. In addition to the classic regulation of protein production via upregulation ofmessenger RNA (mRNA) transcription, there exists an additional system regulating protein production by microRNAs. Subsequent research revealed the profound impact of microRNAs on cell development and cell function in health and disease. This short review provides a concise overview of the function ofmicroRNAs with an emphasis on clinical application for diagnosis, prognosis determination and, hopefully soon, therapeutic applications. Because of the huge amount of newly

MicroRNA as a New Factor in Lung and Esophageal Cancer

Lung cancer is the most lethal cancer due to late detection in advanced stages; early diagnosis of lung cancer allows surgical treatment and improves the outcome. The prevalence of gastroesophageal reflux-related adenocarcinomas of the esophagus is increasing; repetitive surveillance endoscopies are necessary to detect development of cancer. A blood-based biomarker would simplify the diagnosis and treatment of both diseases. MicroRNAs (miRNAs) are short RNA strands that interfere with protein production. miRNAs play pivotal roles in cell homeostasis, and dysregulation of miRNAs can lead to the development of cancer. miRNAs can be found in all body fluids and have been proposed to serve as messengers between closely localized cells but also distant organs. Cancer cells actively secrete miRNAs, and these miRNA profiles can be found in blood. We outline, here, how these miRNAs may aid in diagnosis and treatment of lung and esophageal cancers, as well as their apparent limitations.

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.

Does Clinically Relevant Temperature Change miRNA and Cytokine Expression in Whole Blood

Unintentional hypothermia is a well-described risk factor for death and complications after elective and emergency surgery. The molecular mechanisms by which hypothermia exerts its detrimental effects are not well understood. Differences in cytokine production and the overall cell function have been reported under hypothermic conditions. We investigated the effect of a range of clinically relevant temperatures on cytokine production and microRNA (miRNA) expression in a whole-blood model. We found that there was a wide variation in tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-10 production among different subjects, ranging from low to high TNF-α producers. The intersubject variation can also be found on the transcriptional level: high producers had higher upregulation of TNF-α messenger RNA than intermediate and low producers. This variation in TNF-α was reproducible in each individual. Temperature seems to modulate TNF-α production among these different groups. miRNA expression was modulated by temperature. miRNA-181a might control, or be a part of the mechanism which controls, TNF-α production. However, an analysis of whole-leukocyte RNA does not allow the investigation of mechanisms in a specific leukocyte subpopulation such as monocytes, because these changes may be concealed by miRNA expression changes in the other leukocyte subsets. In conclusion, TNF-α, IL-6, and IL-10 production is highly variable among different persons, but temperature affects the expression of miRNAs, which may consequently alter the production of TNF-α.

Development of a human cadaver model for training in laparoscopic donor nephrectomy

The organ procurement network recommends a surgeon record 15 cases as surgeon or assistant for laparoscopic donor nephrectomies (LDN) prior to independent practice. The literature suggests that the learning curve for improved perioperative and patient outcomes is closer to 35 cases. In this article, we describe our development of a model utilizing fresh tissue and objective, quantifiable endpoints to document surgical progress, and efficiency in each of the major steps involved in LDN.