DNA methyltransferase inhibitors increase NOD-like receptor activity and expression in a monocytic cell line.

Background: The intracellular NOD-like receptor (NLR) family of pathogen recognition receptors (PRRa) is involved in initiating the innate immune response of which NOD1 and NOD2 are the best-characterized members. Aberrant expression of NOD1 and NOD2 has been uncovered in a number of chronic inflammatory diseases, such as inflammatory bowel disease and rheumatoid arthritis. However, the mechanism underlying NOD1/NOD2 gene expression regulation is still in its infancy. Epigenetic modifications such as DNA methylation and histone acetylation regulate the expression of genes and alterations in their patterns have been linked to many inflammatory diseases. This study investigated whether epigenetic modifying drugs affect the regulation of NOD1/NOD2 activity and expression. DNA methyltransferase inhibitors have recently been used in the treatment of myelodysplastic syndrome and as combination therapy in cancer but the full extent of their effects has not been quantified.Methods: Pharmacological inhibition of epigenetic enzymes in a human monocytic THP-1 cell line was carried out and NOD1/NOD2 expression and pro-inflammatory responses were quantified.Results: Cells primed with a DNA methyltransferase inhibitor (but not a histone deacetylase [HDAC] inhibitor) were found to be consistently more responsive to NOD1/NOD2 stimulation and had increased basal expression.Conclusion: The novel experimentation carried out here suggests for the first time that NOD1/NOD2 receptor activity and expression in monocytes are possibly regulated directly by DNA methylation.

Cyclophosphamide alters the tumor cell secretome to potentiate the anti-myeloma activity of daratumumab through augmentation of macrophage-mediated antibody dependent cellular phagocytosis.

Multiple Myeloma (MM) is a malignant disorder of plasma cells which, despite significant advances in treatment, remains incurable. Daratumumab, the first CD38 directed monoclonal antibody, has shown promising activity alone and in combination with other agents for MM treatment. Daratumumab is thought to have pleiotropic mechanisms of activity including natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC). With the knowledge that CD38-expressing NK cells are depleted by daratumumab, we sought to investigate a potential mechanism of enhancing macrophage-mediated antibody-dependent cellular phagocytosis (ADCP) by combining daratumumab with cyclophosphamide (CTX). Cyclophosphamide's immunomodulatory function was investigated by conditioning macrophages with tumor cell secretome collected from cyclophosphamide treated MM cell lines (CTX-TCS). Flow cytometry analysis revealed that CTX-TCS conditioning augmented the migratory capacity of macrophages and increased CD32 and CD64 Fcγ receptor expression on their cell surface. Daratumumab-specific tumor clearance was increased by conditioning macrophages with CTX-TCS in a dose-dependent manner. This effect was impeded by pre-incubating macrophages with Cytochalasin D (CytoD), an inhibitor of actin polymerization, indicating macrophage-mediated ADCP as the mechanism of clearance. CD64 expression on macrophages directly correlated with MM cell clearance and was essential to the observed synergy between cyclophosphamide and daratumumab, as tumor clearance was attenuated in the presence of a FcγRI/CD64 blocking agent. Cyclophosphamide independently enhances daratumumab-mediated killing of MM cells by altering the tumor microenvironment to promote macrophage recruitment, polarization to a pro-inflammatory phenotype, and directing ADCP. These findings support the addition of cyclophosphamide to existing or novel monoclonal antibody-containing MM regimens.

Understanding the regulation of pattern recognition receptors in inflammatory diseases - a 'Nod' in the right direction.

Nucleotide-binding oligomerization domain (NOD) -like receptors (NLRs) are a family of 23 receptors known as pattern recognition receptors; they are expressed in many cell types and play a key role in the innate immune response. The NLRs are activated by pathogen-associated molecular patterns, which include structurally conserved molecules present on the surfaces of bacteria. The activation of these NLRs by pathogens results in the downstream activation of signalling kinases and transcription factors, culminating in the transcription of genes coding for pro-inflammatory factors. Expression of NLR is altered in many cellular, physiological and disease states. There is a lack of understanding of the mechanisms by which NLR expression is regulated, particularly in chronic inflammatory states. Genetic polymorphisms and protein interactions are included in such mechanisms. This review seeks to examine the current knowledge regarding the regulation of this family of receptors and their signalling pathways as well as how their expression changes in disease states with particular focus on NOD1 and NOD2 in inflammatory bowel diseases among others.