MSMD Patients with IFN-g-STAT1 Signaling Defect Present Enhanced Osteoclastogenesis and Bone Resorption

Mendelian susceptibility to mycobacterial diseases (MSMD) is a rare congenital phagocytic disorder characterized by susceptibility to clinical diseases caused by weakly virulent mycobacteria, such as Mycobacterium bovis Bacille Calmette-Guérin (BCG) and non-tuberculous mycobacteria. In individuals with MSMD, mutations have been found in several genes that all encode proteins in the type 1 cytokine pathway that is essential for killing or controlling mycobacteria.  The various gene mutations, such as IL12B, IL12RB1, IFNGR1 IFNGR2, and STAT1, have been identified for the responsible genes in patients with MSMD.  STAT1 mediates most of the biological functions of both type I IFN (IFN-α and β) and type II IFN (IFN-γ ) and is important in immune response to viruses and other intracellular pathogens. The loss-of-function formed IFN-γ receptor 1 and STAT1 deficiency presents MSMD. Osteomyelitis is a bone infection by bacteria and other microorganism, and is one of clinical features presented in more than 80% of patients with MSMD. The histopathology of biopsied specimen shows noncaseating granuloma with abundant number of osteoclasts. Osteoclasts, which play a critical role in pathological bone loss, are multinucleated cells of hematopoietic origin that are formed from monocyte/macrophage lineage precursor cells. Osteoclastogenesis is an intricate process that involves many stages, such as differentiation to tartrate-resistant acid phosphatase (TRAP)-positive cells, fusion to form multinucleated cells, activation to resorb bone, and spontaneous apoptosis. IFN-γ is known to strongly suppress osteoclast formation in mice. However, the mechanisms underlying the effects of IFN-γ osteoclast formation and function still remain unclear in human. In this study, we examined the effect of IFN-γ on the formation and function of osteoclasts derived from patients with AD IFNGR1 or STAT1 deficiency. Osteoclast precursor cells derived from the bone marrow mononuclear cells were enriched by incubation with SCF, IL-3 and GM-CSF followed by the culture of M-CSF and RANKL-containing media with or without IFN-γ.  In the absence of IFN-γ, no significant difference in in vitro osteoclastogenesis observed by TRAP staining was noted between patients and healthy subjects. The addition of IFN-γ in the culture dose-dependently suppressed osteoclastogenesis. Ten IU/ml of IFN-γ sufficiently inhibited osteoclast formation in normal subjects, while more than 50 IU/ml of IFN-γ was required to suppress osteoclastogenesis in patients. Osteoclast formation of patients with IFN-γ-STAT1 signaling defect was less susceptible to IFN-γ than those of healthy subjects, suggesting the association with the abundant osteoclast involvement in patients’ bony inflammation.  Next, bone resorption assay, pit-formation was microscopically measured to evaluate the osteoclast activity. Bone resorption by osteoclasts was inhibited by the addition of IFN-γ. However, the suppressive activity of IFN-γ for pit formation was significantly lower in patients with IFN-γ-STAT1 signaling defect than that in healthy subjects, resulting in the enhanced activity of patients’ osteoclasts.  The down-regulation of nuclear factor of activated T cells (NFATc1) mRNA expression induced by IFN-γ is essential for the osteoclast formation and its function. Osteoclasts derived from patients showed the significant impairment of down-regulation of NFATc1 mRNA expression in the stimulation with IFN-γ. These results suggest that the qualitative and quantitative augmentations of osteoclasts may be associated with the development of chronic and multifocal osteomyelitis in patients with MSMD through the impairment of IFN-γ-STAT1 signaling.