Analysis of subsets and function of monocytes by flow cytometry in septic patients and correlation with clinical outcome

Sepsis is a life-threatening dysregulated host response to infection responsible of multiple organs dysfunction (Sepsis-3 International Consensus Definition) where the clinical outcome is a balance between inflammation and immune suppression. Monocytes are critical immune effectors, cross-linking innate and adaptative immunity, responsible of antigen presentation and release of pro-inflammatory cytokines. Different subsets of monocytes perform different functions. We aimed to evaluate monocytes polarization and reprogramming from initial inflammation-phase to that of immune-suppression and monocytes anergy in association with increased risk of worst outcome and/or secondary infections. We analyzed 93 patients with procalcitonin level >0.5 ng/mL (hPCT) and suspected/confirmed sepsis (after microbial culture assay) and 84 controls by analysis of CD14, CD16 and HLA-DR expression on blood monocytes using fluorescent labeled monoclonal antibodies and BD FACS CANTO II. Complete blood cell count, procalcitonin and other biochemical markers were evaluated. Intermediate monocytes CD14++CD16+ increased in hPCT patients (with both positive and negative culture) compared to controls (13.6% ± 0.8 vs 6.2% ± 0.3, p<0.001), while classical monocytes CD14++CD16- were significantly reduced (72.5% ± 1.6 vs 82.6% ± 0.7, p<0.001). Among hPCT patients having positive microbial culture, the intermediate monocytes percentage was significantly higher in septic compared with non-septic/localized-infection patients (17.4% vs 11.5%; p<0.05) whilst classical monocytes percentage was lower (68.0% vs 74.5%, p=0.087). Three-four days following the diagnosis of sepsis, HLA-DR expression on monocyte (mHLA-DR) was lower (94.3%) compared to controls (99.4%) (p<0.05). Septic patients with the worst clinical conditions showed higher incidence of secondary infections, long-time hospitalization and lower HLA-DR+ monocytes compared to septic patients with better clinical outcome (88.4% vs 98.6%, p=0.05). Worst conditions patients didn’t show restoration of normal values of white blood cells, platelets, monocytes, neutrophils and lymphocytes counts compared to better outcome patients The dynamic nature of sepsis correlates with monocytes functional polarization and reprogramming from a lonely CD14++CD16+-pro-inflammatory-phenotype, in infected-not-septic hPCT patients to a decrease of HLA-DR surface expression in hPCT patients with confirmed sepsis, making HLA-DR reduction a marker of immune-paralysis and sepsis outcome. Moreover, worst clinical outcome showed a significant reduction of mHLA-DR percentage quantified as MFI (mode of florescence intensity), indicator of a reduced number of HLA-DR molecule per monocytes. Time course evaluation of hemato-chemical markers showed degeneration of the clinical conditions of worst-outcome-patients. Analysis of monocytes plasticity opens to new mechanisms responsible for pro/anti-inflammatory responses during sepsis, and new immunotherapies.