Toll-like Receptors (TLR) are major sensors of the innate immune system and are involved in the recognition of Pathogen Associated Molecular Patterns (PAMPs) and Damage Associated Molecular Patterns (DAMPs). When activated, TLR signaling pathways trigger an intracellular response that leads to the production of pro-inflammatory cytokines, chemotactic factors, antimicrobial peptides, and interferons. Overexpression of certain TLRs on neutrophils has been associated with increased mortality in patients with infection-induced shock, although the role of TLR4 overexpression is still debated. While TLRs play a role in recognizing in bacterial structures, they can also detect viral patterns, particularly TLR2, TLR3, and TLR7. The activation of the TLR signaling pathway is an early step recognizing viral genomic patterns of SARS-CoV-2 and its spike protein activates TLR4.
In this prospective single-center observational cohort study, we conducted a comprehensive analysis of the protein expression of the ten known human TLR in white blood cells from patients with either bacterial and viral infections using flow cytometry, together with CD180, a 95-105 kDa TLR-like glycoprotein expressed on peripheral blood monocytes and B-lymphocytes. 73 patients were included, divided into 4 groups based on infection severity (sepsis, septic shock, moderate and severe COVID-19), along with 7 control subjects. Septic shock was assessed according to the third international consensus definition. COVID-19 patients with confirmed SARS-CoV-2 infection using reverse transcriptase-polymerase chain reaction (RT-PCR), admitted either to the emergency department (ED) or in the ICU were included. This study protocol was reviewed and approved by the local ethic committee, and written informed consent was obtained from participants of their relatives. For endosomal TLRs (TLR3, TLR7, TLR8, TLR9), plasma cell permeabilization was performed using the Perm2 Solution® (BD Biosciences, USA) prior to incubation of monoclonal antibodies staining. Cell surface expression was studied for the other TLRs.
In neutrophils, antibody staining for TLR1-10 and CD180 revealed the expression of TLR4, with a large variation in positivity between groups, TLR8, TLR9 were also expressed, while TLR2, TLR3, and TLR7 were weakly expressed. In monocytes, only TLR2, TLR4, and CD180 were detected
Notably, we observed a significant reduction in the expression of TLR4 and its co-receptor CD14 in neutrophils of patients with shock compared to controls (MFI=19 273 vs. 111 691, p<0.001 and MFI 2 028 vs. 3 025, p <0.02 respectively). Furthermore, severe COVID-19 patients exhibited a significant increase in TLR3 and TLR7 levels in neutrophils (p=0.003 and p=0.02 respectively) compared to the control group. Patients with shock also showed a similar increase in TLR7 expression in neutrophils, along with elevated intermediate monocytes compared to controls.
TLR expression remained unchanged in lymphocytes. We also observed that CD16 expression in neutrophils was significantly lower in septic shock patients compared to healthy donors (p<0.0001), and to a lesser extent in sepsis patients (p<0.01), consistent with previous studies
This study provided further insights into the mechanisms of TLR activation in various severe forms of infection. Additional analysis is required to assess their correlation with patient outcomes and evaluate the potential impact of TLR-related therapy on neutrophils activation.
Disclosures
No relevant conflicts of interest to declare.
