Substantial heterogeneity of inflammatory cytokine production and its inhibition by a triple cocktail of toll-like receptor blockers in early sepsis

Introduction: Early sepsis is a life-threatening immune dysregulation believed to feature a cytokine storm due to activation of pattern recognition receptors by pathogen and danger associated molecular patterns. However, treatments with single toll-like receptor (TLR) blockers have shown no clinical benefit. We speculated that sepsis patients at the time of diagnosis are heterogeneous in relation to their cytokine production and its potential inhibition by a triple cocktail of TLR blockers. Accordingly, we analyzed inflammatory cytokine production in whole blood assays from early sepsis patients and determined the effects of triple TLR-blockade. Methods: Whole blood of 51 intensive care patients sampled within 24h of meeting Sepsis-3 criteria was incubated for 6h without or with specific TLR2, 4, and 7/8 stimuli or suspensions of heat-killed S. aureus or E. coli bacteria as pan-TLR challenges, and also with a combination of monoclonal antibodies against TLR2 and 4 and chloroquine (endosomal TLR inhibition), subsequent to dose optimization. Concentrations of tumor necrosis factor (TNF), Interleukin(IL)-6, IL-8, IL-10, IL-1 alpha and IL-1 beta were measured (multiplex ELISA) before and after incubation. Samples from 11 sex and age-matched healthy volunteers served as controls and for dose-finding studies. Results: Only a fraction of sepsis patient samples revealed ongoing cytokine production ex vivo despite sampling within 24 h of first meeting Sepsis-3 criteria. In dose finding studies, inhibition of TLR2, 4 and endosomal TLRs reliably suppressed cytokine production to specific TLR agonists and added bacteria. However, inflammatory cytokine production ex vivo was only suppressed in the high cytokine producing samples but not in the majority. The suppressive response to TLR-blockade correlated both with intraassay inflammatory cytokine production (r=0.29-0.68; p<0.0001-0.04) and cytokine baseline concentrations (r=0.55; p<0.0001). Discussion: Upon meeting Sepsis-3 criteria for less than 24 h, a mere quarter of patient samples exhibits a strong inflammatory phenotype, as characterized by increased baseline inflammatory cytokine concentrations and a stark TLR-dependent increase upon further ex vivo incubation. Thus, early sepsis patient cohorts as defined by Sepsis-3 criteria are very heterogeneous in regard to inflammation. Accordingly, proper ex vivo assays may be useful in septic individuals before embarking on immunomodulatory treatments.

Automated summary

For early sepsis patients who meet the Sepsis-3 criteria, only a fraction of the patients exhibit ongoing cytokine production ex vivo, and the suppressive response to TLR-blockade is only seen in high cytokine producing samples. Therefore, early sepsis patients are heterogeneous in terms of inflammation, and proper ex vivo assays may be useful in guiding treatment.