The TIGIT+ T regulatory cells subset associates with nosocomial infection and fatal outcome in COVID-19 patients under mechanical ventilation

Abstract

The TIGIT⁺FOXP3⁺Treg subset (TIGIT⁺Tregs) exerts robust suppressive activity on cellular immunity and predisposes septic individuals to opportunistic infection. We hypothesized that TIGIT⁺Tregs could play an important role in intensifying the COVID-19 severity and hampering the defense against nosocomial infections during hospitalization. Herein we aimed to verify the association between the levels of the TIGIT⁺Tregs with the mechanical ventilation requirement, fatal outcome, and bacteremia during hospitalization. TIGIT⁺Tregs were immunophenotyped by flow cytometry from the peripheral blood of 72 unvaccinated hospitalized COVID-19 patients at admission from May 29th to August 6th, 2020. The patients were stratified during hospitalization according to their mechanical ventilation requirement and fatal outcome. COVID-19 resulted in a high prevalence of the TIGIT⁺Tregs at admission, which progressively increased in patients with mechanical ventilation needs and fatal outcomes. The prevalence of TIGIT⁺Tregs positively correlated with poor pulmonary function and higher plasma levels of LDH, HMGB1, FGL2, and TNF. The non-survivors presented higher plasma levels of IL-33, HMGB1, FGL2, IL-10, IL-6, and 5.54 times more bacteremia than survivors. Conclusions: The expansion of the TIGIT⁺Tregs in COVID-19 patients was associated with inflammation, lung dysfunction, bacteremia, and fatal outcome.

Figure 1

COVID-19 was marked by decreased levels of the FOXP3 + Treg repertoire. (A) Blood samples of 42 COVID-19 patients collected at admission and 18 healthy volunteers were evaluated according to the FOXP3⁺Treg repertoire by flow cytometry. The data was expressed in frequency relative to CD4⁺cells (%), and absolute numbers per μL of blood (#). (B) Healthy Controls are represented by gray bars (n = 18), patients that did not require mechanical ventilation are represented by the yellow bars (n = 18), and the intubated patients are represented by the brown bars (n = 26). (C–D) The intubated patients were stratified in survivors (blue bars, n = 13) and non-survivors (red bars, n = 13). Statistical significance was determined by either the one-way ANOVA followed by Tukey’s post hoc test, or the unpaired Student t test for data that reached normal distribution, and the Mann–Whitney test for not normally distributed data. *p < 0.05.

Figure 2

The TIGIT⁺Treg subset was increased in COVID-19 patients with worst prognosis. (A) Blood samples of 42 COVID-19 patients collected at admission and 18 healthy volunteers were evaluated according to the TIGIT⁺ and TIGIT^- FOXP3⁺Treg subsets by flow cytometry. The data was expressed in frequency relative to FOXP3⁺Treg repertoire (%), and absolute numbers per μL of blood (#). (B) Healthy Controls are represented by gray bars (n = 18), patients that did not require mechanical ventilation are represented by the yellow bars (n = 18), and the intubated patients are represented by the brown bars (n = 26). The intubated patients were stratified in survivors (blue bars, n = 13) and non-survivors (red bars, n = 13). (C) Frequency of TIGIT^-FOXP3⁺ Tregs, and Frequency of TIGIT⁺FOXP3⁺Tregs. (D) Absolute number of TIGIT^-FOXP3⁺Tregs and TIGIT⁺FOXP3⁺Tregs subsets. Statistical significance was determined by either the one-way ANOVA followed by Tukey’s post hoc test, or the unpaired Student t test for data that reached normal distribution, and the Mann–Whitney test for not normally distributed data. *p < 0.05.

Figure 3

Cytokine measurement of intubated COVID-19 patients stratified in survivors and non-survivors. The cohort of the intubated COVID-19 patients (n = 26) were evaluated according to their cytokine levels in the plasma at admission. The intubated patients were stratified in survivors (blue bars, n = 13) and non-survivors (red bars, n = 13). (A) Interleukin-33, IL-33; High Mobility Group Box 1, HMGB1; (B) Fibrinogen-like 2, FGL2; Interleukin-10, IL-10; (C) Interferon-γ, IFN-γ; Tumor necrosis fator, TNF; (D) Interleukin-6, IL-6. Statistical significance was determined by either the unpaired Student t test for data that reached normal distribution, and the Mann–Whitney test for not normally distributed data. *p < 0.05.

Figure 4

The prevalence of the TIGIT⁺Tregs correlates with lung dysfunction and increased inflammation. Correlation between the frequency of TIGIT⁺Treg subset among the FOXP3⁺Treg repertoire and the (A) PaO₂/FiO₂, and the plasmatic levels of (B) CRP, LDH, HMGB1, FGL2, and TNF; (C) IL-33; (D) IL-10 and IL-6. All parameters were analyzed during admission of 26 COVID-19 patients that did require mechanical ventilation during hospitalization. The blue-dots represent the survivors and red-dots is related to non-survivors. Spearman’s rank-order correlation (r) was calculated to describe correlations.

Figure 5

The COVID-19 fatal outcome was marked by increased bacteremia during hospitalization. The cohort of 26 COVID-19 patients that did require mechanical ventilation during hospitalization was evaluated in terms of (A) the prevalence of bacteremia among survivors (n = 13, blue bar) and non-survivors (n = 13, red bar). (B) Isolated microorganisms in blood culture among survivors (n = 13, left panel) and non-survivors (n = 13, right panel).