Impact of Chronic Inflammation and Cytotoxic Therapy on Bone Marrow Microenvironment of Patients with Autoimmune Rheumatic Diseases and Myeloid Neoplasms
Authors: Kutyna Monika , Thomas Daniel , Hiwase Devendra 
Published: December 2024

BACKGROUND
Patients with autoimmune rheumatic diseases (AIRD) are at greater risk of developing myeloid neoplasms (MN) due to both the use of immunosuppressive agents and underlying immune dysregulation. The higher frequency of AIRD in MN, especially in myelodysplastic syndrome (MDS), suggests a common link, such as a chronic inflammatory microenvironment. Although AIRD patients are treated with long-term cytotoxic and disease-modifying antirheumatic drugs (DMARD), the effect on the bone marrow (BM) microenvironment is not well understood. Our previous research showed that senescent BM mesenchymal stromal cells (BMSC) has a highly active proinflammatory microenvironment and exhibits defective proliferative and differentiation capacities (Kutyna et al., Leukemia 2022).

METHODS
We conducted a functional study of BMSC obtained from MN-AIRD patients and carefully selected control populations to evaluate the damage induced by cytotoxic and DMARD, including non-cytotoxic biological and cytotoxic DMARD such as methotrexate, cyclophosphamide, and azathioprine in three cohorts: (1) MN-AIRD, patients who developed myeloid malignancy after long-term use of cytotoxic drugs and DMARD (n=33); (2) de novo MN without AIRD (de novo MN without AIRD; n=16); and (3) age-matched controls (n=17). We assessed morphology, proliferation by maximum cumulative population doublings (CPD), clonogenic potential by colony forming unit fibroblast (CFU-F), cellular senescence by the percentage of β-galactosidase-stained cells at passage 3, adipo- and osteogenic differentiation potential of BMSC, and cytokine levels (38-plex magnetic bead panel, Millipore).

RESULTS
BMSC isolated from MN-AIRD patients were significantly different from both age-matched healthy controls and MN without AIRD, with a decreased number of CFU-F (0.68±0.71 vs. 4.5±2.79 vs. 1.81±0.79 per 104 cells, respectively, P &lt; 0.001) and maximum CPD (0.71±1.48 vs. 11.46±6.74 vs. 4.43±2.56 doublings respectively, P &lt; 0.001).
Morphological indicators of senescence, as measured by β-galactosidase staining, were distinctly higher in MN-AIRD compared to de novo MN without AIRD and healthy controls (54±16% vs. 27±9% vs. 6±4%, respectively; P &lt; 0.001). Remarkably, several inflammatory cytokines in the marrow interstitial fluid, including fractalkine, IFNα2, IL-1β, IFNγ and GM-CSF, were higher in MN-AIRD compared to de novo MN without AIRD.
The BMSC osteogenic differentiation potential, assessed by mineralization matrix formation, was significantly higher in MN-AIRD (134.4±65.76 Ca2+/µg DNA) compared to healthy controls (52.76±34.29 Ca2+/µg DNA) and de novo MN without AIRD (53.78±34.29 Ca2+/µg DNA; P = 0.0088). This was further confirmed by Alizarin red-stained mineralized deposits. In contrast, the quantification of lipid-laden Nile Red- marked adipocytes showed a significant reduction in MN-AIRD compared to de novo MN without AIRD and healthy controls (0.07±0.06 vs. 0.33±0.17 vs. 0.67±0.06; P&lt;0.0001).
We further analyzed the association between the type of cytotoxic therapy or DMARD and differences in the BM stroma. Surprisingly, we did not observe a significant differences in clonogenic capacity (1.13±0.98, 0.41±0.33, 0.35±0.40, 0.83±0.85 per 104 cells; P=0.28), proliferative capacity (0.81±0.8, 0.46±0.76, 0±0, 0.67±1.6 doublings; P=0.83) or degrees of senescence (53±28.28%, 56±15.56%, 53±0%; P=0.99) in AIRD cases treated with azathioprine vs. methotrexate vs. other cytotoxics vs. biological DMARD.
To test the hypothesis that prior cytotoxic and DMARD therapy leads to long-term damage to the BM microenvironment, we assessed the proliferation, cellular senescence, and differentiation potential of BMSC samples before MN diagnosis in AIRD patients. Our preliminary results indicate a reduction in the proliferation capacity of BMSC years before MN diagnosis but subsequent to DMARD treatment.

CONCLUSION
Our study demonstrates that the marrow microenvironment in patients with MN-AIRD exhibits significant functional and morphological differences compared to healthy controls and patients with de novo MN without AIRD, indicating that long-term cytotoxic or DMARD therapy adversely affects BMSC. These findings suggest the need for more tailored and effective therapeutic strategies for MN-AIRD patients.