Study of toll -like receptor expression on haematopoietic and stromal bone marrow cells of patients with myelodysplastic syndromes and hypoplastic neutropenias

Background: Excessive pro-inflammatory cytokine production in the bone marrow (BM) has been recognized as a prominent pathogenetic mechanism for the disturbed hematopoiesis in patients with Myelodysplastic Syndromes (MDS) and Chronic Idiopathic Neutropenia (CIN). However, the upstream pathways, the exact cellular source and the triggering events related to cytokine excess in MDS BM remain unknown. Furthermore, in patients with MDS, infection vulnerability does not always reflect the severity of neutropenia. This observation suggests that additional factors may predispose MDS patients to infections. Aim: The first objective of this study was to investigate the possible involvement of toll-like receptors (TLRs) and their endogenous ligands in the induction/maintenance of the inflammatory process in patients’ BM. Besides, based on available evidence suggesting that the rs4986790 and rs4986791 Single Nucleotide Polymorphisms (SNP) of human TLR4 may be associated with increased susceptibility to bacterial infections, we sought to examine the possible association between TLR4 SNPs and infection susceptibility in MDS patients from a homogeneous Greek population. Methods: TLR expression was evaluated in the BM of patients and healthy controls using flow-cytometry. Quantitative PCR analysis of 84 genes related to TLR-mediated signal transduction was performed, using a commercially available PCR array, in immunomagnetically sorted CD14+ BM cells of patients and controls. The results were confirmed by testing separately three significantly over-expressed genes. The levels of various cytokines produced by patient monocytes treated with autologous plasma in the presence/absence of a TLR4 inhibitor were measured with chemiluminescence and the levels of the high mobility group box 1 (HMGB1) protein, a TLR4 endogenous ligand, in long-term 16 Abstract BM culture supernatants were assayed with ELISA. To further examine if the excess of HMGB1 in the BM of MDS patients is due to macrophage dysfunction, a fluorescent microscopy-based assay was developed to estimate macrophage capacity to phagocytose apoptotic cells, and the time-course of HMGB1 release in a co-culture system of patient macrophages with different numbers of apoptotic cells was monitored. Genotyping for TLR4 rs4986790 and rs4986791 SNPs was performed by PCR – Restriction Fragment Length Polymorphisms (RFLPs) as described previously, using the NcoI and HinfI restriction enzymes, which digest the G allele-containing and T allele-containing PCR products of the rs4986790 and rs4986791 SNPs, respectively. To examine the possible correlation of the rs4986790 and rs4986791 polymorphic genotypes with MDS patient susceptibility to infections, we recorded the number of infections per patient tested, since the day of diagnosis. Results: A statistically significant increase in the proportion of TLR4+ cells within the CD14+ BM cells was observed in both patient groups compared to controls along with an up-regulation of TLR4 expression as was indicated by the TLR4 Μean Ratio of relative fluorescence intensity (MRFI) in patients. The TLR signaling gene array study in purified BM CD14+ cells showed that many of the TLR-related genes displayed at least two-fold increase in patients compared to controls. When examined separately, the relative mRNA expression of MyD88, TRIF/TICAM1 and TRAM/TICAM2, three adaptor molecules necessary for MyD88-dependent and MyD88-independent TLR4 signaling, was also found significantly increased in patients compared to controls confirming the results obtained from the PCR-arrays. Incubation of patient monocytes with autologous BM plasma resulted in the TLR4 dependent production of the cytokines measured, as been indicated by the significant decrease of IL-1, IL-6 and TNFα levels in the presence of the TLR4 inhibitor compared to cultures treated with the BM plasma alone. HMGB1 levels were significantly increased in 17 Abstract long-term BM cultures of patients compared to controls suggesting that HMGB1 might constitute an endogenous TLR4-activating ligand in MDS and CIN BM. MDS-derived macrophages displayed impaired capacity to engulf apoptotic cells in comparison to healthy individuals. HMGB1 release by MDS BM macrophages loaded with increasing numbers of apoptotic cells for different time periods was dependent on the apoptotic cell load and incubation time. Regarding the genetic study, there was no statistically significant difference in the frequency of infectious episodes between MDS patients carrying the risk allele and the MDS patients that did not carry the risk allele for either SNP studied. However, it is interesting, that the frequency of the minor allele G of the rs4986790 SNP was found significantly increased in MDS patients compared to healthy controls. Furthermore, the heterozygous polymorphic genotype A/G seemed to be more frequent among MDS patients compared to controls. The frequency of the minor allele T and the polymorphic genotype C/T of rs4986791 SNP was also higher in MDS patients, although not at a statistically significant level. Conclusions: These data demonstrate a significant role of BM monocytes in the pathophysiology of CIN and MDS through the production of pro-inflammatory cytokines in a TLR4-mediated mechanism under the influence of endogenous ligands such as HMGB1. A primary apoptotic cell clearance defect of BM macrophages in MDS might contribute to the inflammatory process through aberrant release of HMGB1 from the late apoptotic/necrotic cells. The presence of the polymorphic genotype of rs4986790 does not seem to correlate with MDS patients’ vulnerability to infections. However, the frequency of the mutant allele G and the A/G genotype is significant higher in MDS patients compared to healthy controls, representing probably a genetically defined risk factor for MDS development