Elevated levels of damage-associated molecular patterns HMGB1 and S100A8/A9 coupled with toll-like receptor-triggered monocyte activation are associated with inflammation in patients with myelofibrosis

Abstract

Inflammation plays a pivotal role in the pathogenesis of primary and post-essential thrombocythemia or post-polycythemia vera myelofibrosis (MF) in close cooperation with the underlying molecular drivers. This inflammatory state is induced by a dynamic spectrum of inflammatory cytokines, although recent evidence points to the participation of additional soluble inflammatory mediators. Damage-associated molecular patterns (DAMPs) represent endogenous signals released upon cell death or damage which trigger a potent innate immune response. We assessed the contribution of two prototypical DAMPs, HMGB1 and S100A8/A9, to MF inflammation. Circulating HMGB1 and S100A8/A9 were elevated in MF patients in parallel to the degree of systemic inflammation and levels increased progressively during advanced disease stages. Patients with elevated DAMPs had higher frequency of adverse clinical features, such as anemia, and inferior survival, suggesting their contribution to disease progression. Monocytes, which are key players in MF inflammation, were identified as a source of S100A8/A9 but not HMGB1 release, while both DAMPs correlated with cell death parameters, such as serum LDH and cell-free DNA, indicating that passive release is an additional mechanism leading to increased DAMPs. HMGB1 and S100A8/A9 promote inflammation through binding to Toll-like receptor (TLR) 4, whereas the former also binds TLR2. Monocytes from MF patients were shown to be hyperactivated at baseline, as reflected by higher CD11b and tissue factor exposure and increased expression levels of proinflammatory cytokines IL-1β and IL-6. Patient monocytes showed preserved TLR4 and TLR2 expression and were able to mount normal or even exacerbated functional responses and cytokine upregulation following stimulation of TLR4 and TLR2. Elevated levels of endogenous TLR ligands HMGB1 and S100A8/A9 coupled to the finding of preserved or hyperreactive TLR-triggered responses indicate that DAMPs may promote monocyte activation and cytokine production in MF, fueling inflammation. Plasma IL-1β and IL-6 were elevated in MF and correlated with DAMPs levels, raising the possibility that DAMPs could contribute to cytokine generation in vivo. In conclusion, this study highlights that, in cooperation with classic proinflammatory cytokines, DAMPs represent additional inflammatory mediators that may participate in the generation of MF inflammatory state, potentially providing novel biomarkers of disease progression and new therapeutic targets.

Keywords: HMGB1; S100A8/A9; Toll-like receptors; inflammation; monocyte; myelofibrosis.

Figure 1

Levels of HMGB1 in patients with myelofibrosis. HMGB1 was measured in plasma by ELISA in (A) myelofibrosis (MF) (n=50), essential thrombocythemia (ET) (n=15) and polycythemia vera (PV) (n=15) patients and controls (n=20), *P<0.05, **P<0.01, ****P<0.0001, Kruskal-Wallis test. (B) MF patients grouped according to driver mutations, CALR (n=14), including CALR type 1 (n=13) and CALR type 2 (n=1), MPL (n=4), triple-negative (TN) (n=4) and JAK2V617F-positive (n=28) patients, P=NS, Kruskal-Wallis test. (C) MF patients positive (n=19) or negative (n=31) for high-molecular risk (HMR) mutations, P=NS, Mann-Whitney test. (D) MF patients stratified in low (n=10), intermediate (Int.)-1 (n=15), intermediate-2 (n=16) and high (n=9) risk groups according to the DIPSS score, ***P<0.001, Mann-Whitney test and (E) low (n=5), intermediate (n=28), and high (n=17) risk groups according to the MIPSS70 score, ***P<0.001, Mann-Whitney test. (F) Correlation between HMGB1 and C-reactive protein (CRP), P<0.0001, Spearman correlation. Dashed lines in (A–C) indicate reference values. Median with interquartile range values are shown.

Figure 2

Levels of S100A8/A9 in patients with myelofibrosis. S100A8/A9 was measured in plasma by ELISA in (A) myelofibrosis (MF), essential thrombocythemia (ET) (n=15) and polycythemia vera (PV) (n=15) patients and controls (n=20), *P<0.05, Kruskal-Wallis test. (B) MF patients grouped according to driver mutations, CALR (n=14), including CALR type 1 (n=13) and CALR type 2 (n=1), MPL (n=4), triple-negative (TN) (n=4) and JAK2V617F-positive (n=28) patients, P=NS, Kruskal-Wallis test. (C) MF patients positive (n=19) or negative (n=31) for high-molecular risk (HMR) mutations, P=NS, Mann-Whitney test. (D) MF patients stratified in low (n=10), intermediate (Int.)-1 (n=15), intermediate-2 (n=16) and high (n=9) risk groups according to the DIPSS score, ***P<0.001, Mann-Whitney test and (E) low (n=5), intermediate (n=28), and high (n=17) risk groups according to the MIPSS70 score, P=0.06, Mann-Whitney test. (F) Correlation between S100A8/A9 and C-reactive protein (CRP), P<0.01, Spearman correlation. Dashed lines in (A–C) indicate reference values. Median with interquartile range values are shown.

Figure 3

Survival analysis according to levels of circulating alarmins. (A) Overall survival in patients with myelofibrosis (MF) stratified according to high (upper two quartiles of the patient population) and low (lower two quartiles) HMGB1 levels, P<0.01, log rank test. (B) Overall survival in MF patients stratified according to high (upper two quartiles of the patient population) and low (lower two quartiles) S100A8/A9 levels, P<0.01, log rank test. (C) Combined survival analysis in MF patients grouped according to the presence of high or low levels of one or both alarmins, P<0.001, log rank test.

Figure 4

Monocyte release of damage-associated molecular patterns HMGB1 and S100A8/A9. Monocytes were purified from peripheral blood, cultured during 20 hours in basal conditions or stimulated with LPS 100 ng/mL and the release of HMGB1 and S100A8/A9 to the culture supernatant was measured by ELISA. (A) HMGB1 release in patients with myelofibrosis (MF) and controls (C) (n=10). (B) S100A8/A9 release in patients and controls (n=21), *P<0.05, **P<0.01, *** P<0.001 paired student t test. (C) Absolute monocyte count in patients and controls. **P<0.01, Mann-Whitney test (D) Correlation between monocyte counts and S100A8/A9 plasma levels in patients (n=48), P<0.001, Spearman correlation.

Figure 5

Correlation between levels of circulating HMGB1 and S100A8/A9 and cell death parameters in patients with myelofibrosis. Correlation between serum LDH and (A) HMGB1 and (B) S100A8/A9 plasma levels. Correlation between cell-free DNA and (C) HMGB1 and (D) S100A8/A9 plasma levels (n=50), P<0.05, P<0.001, P<0.0001, Spearman correlation.

Figure 6

Expression of Toll-like receptors (TLR) 4 and TLR2 and TLR-triggered functional responses in monocytes. (A) Surface expression of TLR4 and TLR2 in monocytes from myelofibrosis (MF) patients (n=25) and controls (n=25) by flow cytometry. RFI, relative fluorescence intensity, P=NS, Wilcoxon test. (B) Monocytes from MF patients (n=25) and controls (n=25) were incubated with increasing concentrations of LPS or Pam3CSK4 (PAM) and surface expression of adhesion molecule CD11b was measured by flow cytometry. MFI, mean fluorescence intensity *P<0.05, ***P<0.001, Wilcoxon test for comparison between patients and controls. (C) A representative example of CD11b expression in MF and control monocytes is shown. (D) Monocytes from MF patients (n=25) and controls (n=25) were incubated with increasing concentrations of LPS or Pam3CSK4 and surface exposure of tissue factor was assessed by flow cytometry. *P<0.05, Wilcoxon test for comparison between patients and controls. (E) A representative example of tissue factor exposure in MF and control monocytes is shown.

Figure 7

Monocyte gene expression and plasma levels of proinflammatory cytokines IL-1β and IL-6. Purified monocytes from patients with myelofibrosis (MF) (n=25) and controls (n=25) were incubated in the absence or presence of increasing concentrations of LPS or Pam3CSK4 (PAM) during 4 hs and gene expression of (A) IL-1β and (B) IL-6 was measured by qPCR. *P<0.05, **P<0.01 ***P<0.001 Wilcoxon test for comparison between patients and controls. Plasma levels of (C) IL-1β and (F) IL-6 in patients with MF (n=50) and controls (N=10) measured by ELISA, ****P<0.0001, Mann-Whitney test. Correlation between plasmatic IL-1β and circulating levels of (D) HMGB1 and (E) S100A8/A9. Correlation between plasmatic IL-6 and circulating levels of (G) HMGB1 and (H) S100A8/A9, P<0.01 P<0.001, P<0.0001, Spearman correlation.