Abstract
ObjectiveTo investigate the activation of the IFNγ signaling pathway in monocytes of patients with secondary hemophagocytic lymphohistiocytosis (sHLH)/macrophage activation syndrome (MAS) and to evaluate whether levels of phosphorylated STAT1 represent a biomarker for the identification of patients at early stages of the disease.MethodsFresh whole blood samples from pediatric patients with active sHLH/MAS, not receiving (n=10) and receiving glucocorticoids (n=14) at time of sampling, were prospectively collected. As disease control groups, patients with active systemic juvenile idiopathic arthritis (sJIA) without MAS, patients with sHLH/MAS in remission and patients with other rheumatic diseases were also sampled. Whole blood cells were left unstimulated or stimulated with increasing concentrations of IFNγ for 10 minutes and the intracellular Tyrosine (701)-phosphorylated STAT1 (pSTAT1) levels were evaluated in monocytes by flow cytometry.ResultsMonocytes from untreated sHLH/MAS patients showed significantly higher basal levels of pSTAT1 compared to those observed in monocytes from glucocorticoid-treated sHLH/MAS patients and from all the other disease controls. In addition, a significant increase in responsiveness to IFNγ, as assessed by increased levels of pSTAT1 following ex vivo stimulation, was observed in monocytes from untreated sHLH/MAS patients. pSTAT1 levels in monocytes distinguished patients with sHLH/MAS not treated with glucocorticoids from patients with active sJIA or with other rheumatic diseases [AUC, 0.93; 95% confidence interval 0.85-1.00, p<0.001]. Statistically significant correlations between IFNG mRNA levels in whole blood cells, circulating IFNγ levels and pSTAT1 levels in sHLH/MAS monocytes were found.ConclusionOur data demonstrating higher basal levels of pSTAT1 as well as a hyperreactivity to IFNγ stimulation in monocytes from patients with sHLH/MAS point to perturbations in the activation of downstream IFNγ signaling pathway as a contributor to the hyperinflammation occurring in these patients. Finally, the observation that glucocorticoids affect pSTAT1 levels in vivo, makes it difficult to consider the measurement of pSTAT1 levels as a biomarker to identify patients at early stages of sHLH/MAS in clinical practice.
Highlights
Hemophagocytic lymphohistiocytosis (HLH) is a lifethreatening syndrome characterized by hyperinflammation caused by uncontrolled activation of immune cells, monocytes/macrophages and CD8+ T cells
Sixty-one patients were enrolled from 2017 to 2019 at Ospedale Pediatrico Bambino Gesù: patients with active sJIA (n=12; age 10.3 years, interquartile range (IQR) 5.7-14.2; 4/12 treated with glucocorticoids), patients with secondary HLH forms (sHLH)/macrophage activation syndrome (MAS) not treated with glucocorticoids (n=10; age 7.1 years, IQR 5.1-14.04/12; 0/10 treated with glucocorticoids), patients with sHLH/MAS treated with glucocorticoids (n=14; age 6.3 years, IQR 2.5-14.9; 14/14 treated with glucocorticoids), patients with sHLH/MAS in remission phase (n=12; age 11.8 years, IQR 5.7-17.8; 2/12 treated with glucocorticoids), patients with other rheumatic diseases (n=13; age 6.2 years, IQR 4.3-12.7; 0/13 treated with glucocorticoids)
These levels were compared with those of patients with active sJIA without MAS, with sHLH/MAS in remission phase, and with other inflammatory rheumatic diseases. sHLH/MAS patients showed significantly increased phosphorylated Tyrosine (701) STAT1 (pSTAT1) levels compared to sHLH/MAS patients in remission and a trend to higher levels of pSTAT1 compared to active sJIA patients (Figure 1A)
Summary
Hemophagocytic lymphohistiocytosis (HLH) is a lifethreatening syndrome characterized by hyperinflammation caused by uncontrolled activation of immune cells, monocytes/macrophages and CD8+ T cells. It is classified into primary and secondary forms, depending on whether they are inherited or acquired [1]. While primary HLH (pHLH) is caused by loss-of-function mutations in genes encoding proteins involved in the cytolytic secretory functions of natural killer (NK) cells and cytotoxic T lymphocytes, secondary HLH forms (sHLH)/macrophage activation syndrome (MAS) often occur as complication of malignancies, infections and rheumatic diseases [2]. A growing body of evidence demonstrated the involvement of IFNg in the pathogenesis of primary and secondary HLH as a major regulator of macrophage hyperactivation and hemophagocytosis [3,4,5,6]. Therapeutic neutralization of IFNg has been shown to be efficacious in the treatment of primary and secondary HLH forms [11,12,13]
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