Immune profiles of heatstroke patients

Background: Heatstroke was the most life-threatening disease caused by hyperthermia, but lack of rapidly diagnostic biomarkers. Immune system disturbance was a common feature of the ‘Dual pathway model’ characterized by endotoxemia and hyperthermia, but the specific profiles of the immune system were unclear. Objectives: Our study aimed to explore the specific immune profiles that distinguish heatstroke from aseptic inflammation and sepsis patients, and find diagnostic biomarkers to differentiate heatstroke from sepsis. Methods: Patients in four groups were recruited: heatstroke patients (HS group), sepsis patients (Sepsis group), patients undergoing cardiopulmonary bypass (CPB group) and healthy controls (HC group). The HS patients were matched 1:1 with patients in HC, CPB and Sepsis groups based on age and sex using propensity score analysis. The primary outcome was all-cause mortality, and the secondary outcomes were the incidence of nervous dysfunction (including stroke and delirium), acute heart failure, acute kidney failure or acute lung injury within 30 days. Blood was collected within 24 hours after admission from healthy participants, HS and sepsis patients, and at the end of CPB in the case of CPB patients. This blood was used to perform spectral flow cytometry, measure the plasma level of inflammatory mediators and heat shock protein-70, as well as process single-cell RNA sequencing (sc-RNA seq) to assess the profiles of T cells, B cells, monocytes, and NK cells. Patients with HS and sepsis were followed up for 30 days after admission, while CPB patients were followed up for 30 days after surgery to record the incidence of primary and secondary outcomes. Results: The proportion of TLR4+ monocytes in HS higher than that in HC, CPB and Sepsis groups, and could differentiate heatstroke from sepsis. In HS, the upregulation of HLA genes (HLA-A, HLA-B, HLA-DRA, and HLA-DRB1), the activation of antigen presentation, as well as the inhibition of chemotaxis were found in monocytes highly expressed TLR4 based on sc-RNA seq analysis. In addition, the CCL1 and CXCL8 plasma levels were decreased in HS. Compared with HC and CPB, lymphopenia was found in HS and sepsis, which were attributed by the decreased of T cells counts and NK cells counts. T cells exhaustion was found in HS and sepsis, which was indicated by the upregulation of PD-1 in T cells and its PD-L1 in B cells, upregulation of CD69 and LAG3 but downregulation of TCF7 in T cells, and decreases of plasma levels of TNF-α and IFN-γ. In addition, the Treg/CD8+ T cells ratio was higher in HS patients who experienced death (0.58 vs. 0.35), nervous dysfunction (0.56 vs. 0.29), acute heart failure (0.67 vs. 0.35), and acute lung injury (0.58 vs. 0.34) within 30 days than those who did not. The NK cells cytotoxic activity was decreased in HS, which was indicated by downregulation of CD335, the inhibition of focal adhesion and cell adhesion, as well as the emergence of receptor-ligands pairs related to inhibit NK cells activation in HS, including HLA-A_ KIR3DL1, HLA-B_ KIR3DL2, HLA-C_ KIR2DL1 and HLA-E_ CD94:NKG2A expressed between various immune cells and NK cells. Conclusions: The immune system in HS was characterized by a significant increase in TLR4+ monocytes and immune suppression, the latter of which included lymphocytopenia, T cell exhaustion, and suppressed NK cell cytotoxic activity. TLR4+ monocytes may be regarded as a biomarker to distinguish heatstroke from sepsis, and the Treg/CD8+ T cell ratio may be associated with death and organ failure in heatstroke.