Impact of Hypothermia on Differentiation and Maturation of Neutrophils
Authors: Torikoshi Yusuke , Yokota Asumi , Kamio Naoka , Sato Atsushi , Shouji Tsukimi , Kashiwagi Takahiro , Miura Yasuo , Maekawa Taira , Hirai Hideyo 
Published: October 2019

Abstract
Accumulating evidence has suggested that low body temperature is associated with the risk of infection. Unintentional drops in the body temperature known as "accidental hypothermia" are occasionally accompanied with infections. Patients under therapeutic hypothermia for post-cardiac arrest care are also susceptible to infections. In addition, secondary hypothermia caused by severe sepsis is significantly associated with higher mortality. These observations suggest the negative impact of hypothermia on host defense. Neutrophils are continuously produced in the bone marrow (BM) and supplied to the peripheral blood (PB) or tissues, where they fight against microorganisms. In addition to the neutrophil functions, sufficient supply of neutrophils is a critical determinant of host defense. However, little is known about the impact of hypothermia on granulopoiesis, the process of neutrophil production in the BM. In this study, we investigated the changes in granulopoiesis under hypothermic conditions.
We first analyzed the neutrophils in the PB of mice exposed to low environmental temperature (4 °C). Under this condition, rectal temperature of the mice significantly declined from 36.7±0.4 °C to 35.5±0.4 °C. After 72-hour exposure to the low environmental temperature, PB neutrophil counts were significantly decreased. In order to understand the reason for the decrease, we analyzed their BMs by flow cytometry. Previously we developed a unique strategy to divide cells undergoing granulopoiesis into 5 subpopulations based on the expression of c-kit and Ly6G, which reflect successive differentiation/maturation from #1 (c-kithi Ly6G-) to #5 (c-kit- Ly6Ghi) (Satake S and Hirai H et al. J Immunol, 2012). In BM cells of the mice exposed to the low environmental temperature, a significant decrease in mature neutrophils (#5) and a significant increase in cellular intermediates (#3 and #4) were observed, while total BM cell numbers were unchanged. In order to clarify whether these changes were cell-intrinsic or -extrinsic, total BM cells were cultured in vitro at either 35 °C or 37 °C in the presence of G-CSF. Flow cytometric analysis of these cultured BM cells at 72 hours revealed the increase in the intermediates (#2 to #4) and a decrease in the mature subpopulation (#5), suggesting that these alterations were cell-intrinsic phenomena.
When neutrophil precursors (#1 or #2) were purified by cell sorter and subjected to in vitro culture at 35 °C for 48 hours, the number of resultant mature neutrophils (#5) were significantly less than those induced at 37 °C. These results clearly indicate that hypothermia delayed neutrophil differentiation/maturation.
Interestingly, mice with sepsis induced by cecal ligation and puncture (CLP) accompanied with lower body temperature revealed significantly fewer PB granulocytes and shorter survival when compared to those mice which maintained normal body temperature after CLP.
In order to understand the molecular mechanisms underlying the differentiation/maturation delay induced by hypothermia, we performed RNA sequencing of purified neutrophil precursors (#2) after 24-hour culture either at 35 °C or 37 °C. Interestingly, we found alterations in amino acid metabolic pathways and target genes of C/EBP, which is the transcription factor family required for granulopoiesis and cellular metabolism.
Collectively, these results indicate hypothermia causes neutropenia through delayed neutrophil differentiation/maturation. We are currently analyzing metabolic changes to understand more precise molecular mechanisms by which hypothermia regulates granulopoiesis. This study will facilitate the understanding of host defense at low body temperature, and shed novel insight into the management of hypothermia in patients.

Disclosures
Kashiwagi: Takara Bio Inc.: Employment. Hirai:Kyowa Hakko Kirin: Research Funding; Novartis Pharma: Research Funding.