N. J. Galbraith1, A. T. Billeter1, C. Lawson1, J. D. Rice1, H. C. Polk1 1University Of Louisville,Hiram C. Polk Jr MD Department Of Surgery,Louisville, KY, USA
Introduction:
Unintentional hypothermia is a known risk factor for peri-operative septic and cardiovascular complications. We have previously shown that hypothermia increases the pro-inflammatory response, while the anti-inflammatory response is suppressed via Interleukin 10 (IL-10) and microRNA-155 (miR -155). However, the exact molecular mechanisms by which hypothermia affects the monocyte immune response are unknown. Transient receptor potential ion channels (TRP) are a large group of cell membrane bound receptors, implicated in pain, cancer and sepsis. These channels are activated by a variety of chemical substances or physical stress such as mechanical forces or temperature. Three TRP-channels (TRPA1, TRPM8, and TRPV1) are temperature sensitive of which TRPA1 and TRPM8 sense cold whereas TRPV1 is heat activated. The purpose of this study was to investigate whether these channels mediate the effects of hypothermia on the monocyte inflammatory response.
Methods:
Primary human monocytes were freshly isolated from healthy donors, treated for 24h then stimulated with LPS (100ng/ml) at 32°C or 37°C. Cell culture supernatant and total RNA were isolated. Expression of TRPA1, TRPM8, and TRPV1 mRNA as well as miR-155 was determined using qRT-PCR. Cytokines in the supernatant were measured using ELISAs. Specific inhibitors of TRPA1 (HC- 030031) and a specific activator of TRPV1 (capsaicin) were used to block or activate the specific channels. Statistical analysis was performed using the Wilcoxon signed-rank test.
Results:
TRPM8 mRNA was not expressed in monocytes whereas TRPA1 and TRPV1 were highly expressed. TRPV1 mRNA-expression was significantly suppressed at 32°C but not at 37°C; TRPA1 was strongly induced at 32°C and 37°C. Immunofluorescence microscopy confirmed that monocytes express TRPA1 and TRPV1 on their surface. Blocking TRPA1 at 32°C tended to decrease TNF secretion after 24h. Similarly, TRPV1 activation at 32°C tended to suppress TNF secretion. By comparison, TRPA1 blockade (93.7 ± 29.8pg/ml vs. 43.6 ± 9pg/ml, p<0.05) and TRPV1 (5.0 ± 6.1pg/ml vs. 43.6 ± 9pg/m, p<0.05) activation at 32°C significantly increased IL-10. In addition, TRPA1 blockade and TRPV1 activation at 32°C both suppressed miR-155 expression p<0.05).
Conclusion:
These findings suggest that hypothermia mediates its effects on the monocyte inflammatory response through TRP channels. Both the blockade of the cold activated TRPA1 channels, and the activation of the heat activated TRPV1 channels can restore normal cytokine production, thus alleviating the detrimental effects of hypothermia. This modulation of the monocyte inflammatory response may provide critical new treatment options in patients suffering hypothermic associated complications.