Imbalance of Circulating Innate Lymphoid Cell Subpopulations in Patients With Septic Shock

Abstract

Background: Septic shock, a major cause of death in critical care, is the clinical translation of a cytokine storm in response to infection. It can be complicated by sepsis-induced immunosuppression, exemplified by blood lymphopenia, an excess of circulating Treg lymphocytes, and decreased HLA-DR expression on circulating monocytes. Such immunosuppression is associated with secondary infections, and higher mortality. The effect of these biological modifications on circulating innate lymphoid cells (ILCs) has been little studied. Methods: We prospectively enrolled patients with septic shock (Sepsis-3 definition) in the intensive care unit (ICU) of Timone CHU Hospital. ICU controls (trauma, cardiac arrest, neurological dysfunction) were recruited at the same time (NCT03297203). We performed immunophenotyping of adaptive lymphocytes (CD3+ T cells, CD19+ B cells, CD4+CD25+FoxP3+ Treg lymphocytes), ILCs (CD3-CD56+ NK cells and helper ILCs - ILC1, ILC2, and ILC3), and monocytes by flow cytometry on fresh blood samples collected between 24 and 72 h after admission. Results: We investigated adaptive and innate circulating lymphoid cells in the peripheral blood of 18 patients in septic shock, 15 ICU controls, and 30 healthy subjects. As expected, the peripheral blood lymphocytes of all ICU patients showed lymphopenia, which was not specific to sepsis, whereas those of the healthy volunteers did not. Circulating CD3+ T cells and CD3-CD56+ NK cells were mainly concerned. There was a tendency toward fewer Treg lymphocytes and lower HLA-DR expression on monocytes in ICU patients with sepsis. Although the ILC1 count was higher in septic patients than healthy subjects, ILC2, and ILC3 counts were lower in both ICU groups. However, ILC3s within the total ILCs were overrepresented in patients with septic shock. The depression of immune responses has been correlated with the occurrence of secondary infections. We did not find any differences in ILC distribution according to this criterion. Conclusion: All ICU patients exhibit lymphopenia, regardless of the nature (septic or sterile) of the initial medical condition. Specific distribution of circulating ILCs, with an excess of ILC1, and a lack of ILC3, may characterize septic shock during the first 3 days of the disease.

Keywords: NK cells; immunosuppression; innate lymphoid cells (ILC); lymphopenia; sepsis; sepsis-induced immunosuppression; septic shock.

Copyright © 2019 Carvelli, Piperoglou, Bourenne, Farnarier, Banzet, Demerlé, Gainnier and Vély.

Figures

Figure 1

Phenotypic analysis of circulating lymphocyte subsets in ICU patients. (A) Comparison of helper T-cell, cytotoxic T-cell, B-cell, and NK-cell counts (cells/μl) of septic patients (red circles) with those of healthy controls (dark circles), and ICU patients without sepsis (blue circles). (B) Comparison of helper T-cell, cytotoxic T-cell, B-cell, and NK cell counts (cells/μl) of patients with severe tissue injuries (green circles) with those of healthy controls (dark circles), and patients with less severe lesions (orange circles). The bars show the median. Statistical analyses were performed using the Mann Whitney U-test. Differences were considered significant when P < 0.05: *P < 0.05, ***P < 0.001, ****P < 0.0001. ns: not significant.

Figure 2

Phenotypic analysis of circulating Tregs and monocytes in ICU patients. (A, upper) Comparison of Treg cell counts (cells/μl) of septic patients (red circles) with those of ICU patients without sepsis (blue circles). (A, lower) Comparison of Treg cell counts (cells/μl) of patients with severe tissue injuries (green circles) with those of patients with less severe lesions (orange circles). (B, upper) Comparison of the MFI of HLA-DR expression and the number of HLA-DR molecules on monocytes of septic patients (red circles) with those of ICU patients without sepsis (blue circles). (B, lower) Comparison of the MFI of HLA-DR expression and the number of HLA-DR molecules on monocytes of patients with severe tissue injuries (green circles) with those of patients with less severe lesions (orange circles). The bars show the median. Statistical analyses were performed using the Mann Whitney U-test. Differences were considered significant when P < 0.05: *P < 0.05. ns: not significant.

Figure 3

Flow cytometry gating strategy for the identification of human peripheral blood ILCs. ILCs are defined as Lin−CD127+ cells with a lineage cocktail containing antibodies directed against CD3, CD19, CD14, TCRαβ, TCRγδ, CD94, CD16, FcεRI, CD34, CD123, and CD303 (Left). Within the ILC gate, ILC2s are CD294+ cells, whereas ILC1s are CD294−CD117− cells and ILC3s are CD294−CD117+ cells (Right).

Figure 4

Phenotypic analysis of circulating ILCs in ICU patients. (A, upper) Comparison of total ILC, ILC1, ILC2, and ILC3 counts (cells/ml) of septic patients (red circles) with those of healthy controls (dark circles) and ICU patients without sepsis (blue circles). (A, lower) Distribution of each ILC subset among total ILCs. (B, upper) Comparison of total ILC, ILC1, ILC2, and ILC3 counts (cells/ml) of patients with severe tissue injuries (green circles) with those of healthy controls (dark circles) and patients with less severe lesions (orange circles). (B, lower) Distribution of each ILC subset among total ILCs. The bars show the median. Statistical analyses were performed using the Mann Whitney U-test. Differences were considered significant when P < 0.05: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns: not significant.