Human primary monocytes are heterogeneous in terms of phenotype and function, but are sub-divided only based on CD16 and CD14 expression. CD16 expression distinguishes a subset of monocytes with highly pro-inflammatory properties from non-CD16 expressing “classical” monocytes. CD14 expression further subdivides the CD16 + monocytes into non-classical CD14 low and intermediate CD14 high subsets. This long-standing CD16–CD14 classification system, however, has limitations as CD14 is expressed in a continuum, leading to subjectivity in delineating the non-classical and intermediate subsets; in addition, CD16 expression is unstable, making identification of the subsets impossible after in vitro culture or during inflammatory conditions in vivo . Hence, we aimed to identify the three monocyte subsets using an alternative combination of markers. Additionally, we wanted to address whether the monocyte subset perturbations observed during infection is real or an artifact of differential CD16 and/or CD14 regulation. Using cytometry by time-of-flight (CyTOF), we studied the simultaneous expression of 34 monocyte markers on total monocytes, and derived a combination of five markers (CD33, CD86, CD64, HLA-DR, and CCR2), that could objectively delineate the three subsets. Using these markers, we could also distinguish CD16 + monocytes from CD16 − monocytes after in vitro stimulation. Finally, we found that the observed expansion of intermediate (CD14 high ) monocytes in dengue virus-infected patients was due to up-regulated CD16 expression on classical monocytes. With our new combination of markers, we can now identify monocyte subsets without CD16 and CD14, and accurately re-examine monocyte subset perturbations in diseases.

Plasma leakage is a major pathogenic mechanism of severe dengue, but the etiology remains unclear. The association between endothelial glycocalyx integrity and vascular permeability in older adults with dengue has not been evaluated. A prospective cohort study of adults with undifferentiated fever screened for dengue by RT-PCR or NS1 antigen testing was performed. Patients were assessed daily while symptomatic and at convalescence. Serum hyaluronic acid (HA), heparan sulfate (HS) and selected cytokines (TNF-α, IL-6, IL-10) were measured on enrollment and convalescence. Patients were diagnosed as dengue fever (DF, n = 30), dengue hemorrhagic fever (DHF, n = 20) and non-dengue (ND) febrile illness (n = 11). Acute HA and HS levels were significantly higher in all dengue patients compared to ND (p = 0.0033 and p = 0.0441 respectively), but not different between DF and DHF (p = 0.3426 and p = 0.9180 respectively). Enrolment HA inversely correlated with serum albumin, protein and platelets in all dengue and DHF (p < 0.05). HA and HS in all dengue patients decreased significantly at convalescence. Serum IL-10 was significantly associated with HA in all dengue patients (p = 0.002). Serum HA and HS levels were increased in adult dengue and HA was associated with markers of disease severity. Endothelial glycocalyx damage may have a role in vascular leakage in dengue.