Phosphorylated toll-like receptor 4 defines a high-risk sepsis endotype

Background: Sepsis is a life-threatening condition characterized by a dysregulated immune response to infection. Toll-like receptor 4 plays a central role in pathogen recognition and inflammatory signalling and has been considered a key driver of sepsis pathophysiology. Pharmacological inhibition of this receptor showed beneficial effects in experimental models but failed in clinical trials. We therefore aimed to quantify in vivo activation of Toll-like receptor 4 in patients with sepsis and to determine its association with 30-day survival. Methods: Peripheral blood mononuclear cells were obtained from 100 patients with sepsis enrolled in the SepsisDataNet.NRW cohort. Samples were collected on day 1 (within 36 h after diagnosis) and day 4. Activation of TLR4 was quantified by measuring receptor phosphorylation using a validated proximity ligation assay. Survival analyses were performed using Kaplan-Meier curves and Cox proportional hazards regression models to assess the association between receptor activation and 30-day mortality. Results: Overall activation of TLR4 was low, with median values below one signal per cell at both day 1 and day 4. Despite the generally low levels, a subgroup of patients showed increased receptor activation. Higher activation was associated with significantly reduced 30-day survival. Patients with elevated activation had a higher risk of death both at day 1 (HR 2.03, 95% CI 1.01-4.07, p = 0.048) and day 4 (HR 2.77, 95% CI 1.14-6.73, p = 0.025). This association remained significant after adjustment for SOFA score at admission, age, infection focus and sex in multivariable Cox regression analysis (p = 0.006). Conclusions: In vivo activation of TLR4 is not uniformly present in patients with sepsis but occurs only in a subset of individuals. In those patients, increased activation is strongly associated with mortality. These findings suggest the presence of a distinct high-risk sepsis endotype characterized by enhanced receptor activation. This may help explain the failure of previous clinical trials of TLR4 inhibitors and supports the concept of biomarker-guided precision medicine approaches in sepsis. Trial registration: German Clinical Trials Register (DRKS), DRKS00018871, retrospectively registered on 14 November 2019.