Impaired clearance of apoptotic cells leads to HMGB1 release in the bone marrow of patients with myelodysplastic syndromes and induces TLR4-mediated cytokine production

Abstract

Excessive pro-inflammatory cytokine production in the bone marrow has been associated with the pathogenesis of myelodysplastic syndromes. We herein investigated the involvement of toll-like receptors and their endogenous ligands in the induction/maintenance of the inflammatory process in the marrow of patients with myelodysplastic syndromes. We evaluated the expression of toll-like receptors in marrow monocytes of patients (n=27) and healthy controls (n=25) by flow-cytometry and also assessed the activation of the respective signaling using a realtime polymerase chain reaction-based array. We measured the high mobility group box-1 protein, a toll-like receptor-4 ligand, in marrow plasma and long-term bone marrow culture supernatants by an enzyme-linked immunosorbent assay and we performed cross-over experiments using marrow plasma from patients and controls in the presence/absence of a toll-like receptor-4 inhibitor to evaluate the pro-inflammatory cytokine production by chemiluminescence. We assessed the apoptotic cell clearance capacity of patients' macrophages using a fluorescence microscopy-based assay. We found over-expression of toll-like receptor-4 in patients' marrow monocytes compared to that in controls; this over-expression was associated with up-modulation of 53 genes related to the respective signaling. Incubation of patients' monocytes with autologous, but not with normal, marrow plasma resulted in over-production of pro-inflammatory cytokines, an effect that was abrogated by the toll-like receptor-4 inhibitor suggesting that the pro-inflammatory cytokine production in myelodysplastic syndromes is largely mediated through toll-like receptor-4. The levels of high mobility group box-1 protein were increased in patients' marrow plasma and culture supernatants compared to the levels in controls. Patients' macrophages displayed an impaired capacity to engulf apoptotic cells and this defect was associated with excessive release of high mobility group box-1 protein by dying cells. A primary apoptotic cell clearance defect of marrow macrophages in myelodysplastic syndromes may contribute to the induction/maintenance of the inflammatory process through aberrant release of molecules inducing toll-like receptor-4 such as high mobility group box-1 protein.Impaired clearance of apoptotic cells leads to HMGB1 release in the bone marrow of patients with myelodysplastic syndromes and induces TLR4-mediated cytokine production

Extracellular HMGB1 promotes differentiation of nurse-like cells in chronic lymphocytic leukemia

“…39,48 TLR4 is the most widely studied receptor for HMGB1-mediated inflammation. 17,20 However, our results showed that most CLL samples have absent or weak TLR4 expression, and TLR4 blockade by the neutralizing antibody failed to prevent NLC differentiation. RAGE and TLR9 are ubiquitously expressed in CLL cells and are highly expressed in stromal-like cells in the CLL microenvironment.…”

“…44,45 Increased circulating HMGB1 is found in some cancers, including malignant peritoneal mesothelioma 46 and myelodysplastic syndromes. 20 Higher circulating HMGB1 is associated with worse clinical outcomes, and plasma samples with higher concentrations of HMGB1 (.150 ng/mL) in our study were from relapsed or progressive CLL patients and are correlated with Ten untreated and 6 relapsed CLL peripheral mononuclear cells were stored at 280°C and lysed directly from the pellets. Fifty micrograms of proteins was loaded onto each lane.…”

“…6,7,19 DAMPs are associated with acute inflammatory responses, chronic inflammation, and wound healing, but are also important components of the disordered tumor microenvironment. 8,20 HMGB1 is a DNA-binding protein, and increased serum concentrations of the HMGB1-DNA complex can activate the immune system and cause systemic autoimmune disease via the receptor for advanced glycation end products (RAGE) and toll-like receptor-9 (TLR9). 21 Interactions of HMGB1-RAGE-TLR9 constitute a tripod that triggers nuclear factor kB (NF-kB) activation 22 and promotes dendritic cell maturation.…”

Aberrant overexpression of CD14 on granulocytes sensitizes the innate immune response in mDia1 heterozygous del(5q) MDS

“…by guest www.bloodjournal.org From myeloid dysplasia. 32 Together with previously reported evidence of dyserythropoiesis attributable to loss of RPS14, 7,8 and dysmegakaryopoiesis attributable to loss of miR-145/146a, 12,13 the combined loss of RPS14, miR-145/146a, and mDia1 may recapitulate the pathogenesis of trilineage dysplasia in MDS with del(5q).…”

The NLRP3 inflammasome functions as a driver of the myelodysplastic syndrome phenotype

“…4,7 In addition, the BM plasma concentration of HMGB1, a nuclear DAMP and TLR4 ligand, was significantly increased in MDSs (n 5 55) compared with controls (n 5 11) (P 5 2.6 3 10 23 ) ( Figure 2C), consistent with intracellular DAMP release upon cytolysis. 26,27 Moreover, S100A9 and HMGB1 transcripts were upregulated 104.5-fold and 1.5-fold in MDSs, respectively, compared with normal donors (Figure 2D-E). Further, flow cytometry analyses of phenotypically distinct hematopoietic lineages confirmed a corresponding increase in intracellular S100A9 in MDS stem cells and progeny ( Figure 2F-G).…”