Trained mesenchymal stromal cell-based therapy HXB-319 for treating diffuse alveolar hemorrhage in a pristane-induced murine model

Abstract

Introduction: Mesenchymal stromal cells (MSCs) can modulate immune responses and suppress inflammation in autoimmune diseases. Although their safety has been established in clinical trials, the efficacy of MSCs is inconsistent due to variability in potency among different preparations and limited specificity in targeting mechanisms driving autoimmune diseases.

Methods: We utilized high-dimensional design of experiments methodology to identify factor combinations that modulate gene expression by MSCs to mitigate inflammation. This led to a novel MSC-based cell therapy, HXB-319. Its anti-inflammatory properties were validated in vitro by flow cytometry, RT-PCR, and mass spectrophotometry. To evaluate in vivo efficacy, we treated a diffuse alveolar hemorrhage (DAH) mouse model (C57Bl/6). Seven days post-DAH induction with pristane, mice received either MSCs or HXB-319 (2X106 cells, IP). On day 14, peritoneal lavage fluid (PLF) and lung tissue were collected for flow cytometry, histopathological examination, and mRNA.

Results: HXB-319 increased gene expression levels of anti-inflammatory, angiogenic, and anti-fibrotic factors (eg, TSG-6, VEGF, and HGF). KEGG pathway analysis confirmed significant activation of relevant anti-inflammatory, angiogenic, and anti-fibrotic proteins, corroborating RT-PCR results. In the DAH model, HXB-319 significantly reduced lung inflammation and alveolar hemorrhage compared to MSC-treated and untreated DAH mice. HXB-319 treatment also significantly decreased neutrophils, plasmacytoid dendritic cells, and RORγT cells, increased FoxP3+ cells in PLF, and reversed alterations in mRNA encoding IL-6, IL-10, and TSG-6 in lung tissue compared to DAH mice.

Conclusion: HXB-319 effectively controls inflammation and prevents tissue damage in pristine-induced DAH, highlighting its therapeutic potential for autoimmune inflammatory diseases.

Keywords: autoimmune lung disease; cell therapy; interferon type I; lupus; mesenchymal stromal cells; pulmonary hemorrhage; vasculitis.

Graphical Abstract

Figure 1.

HXB-319’s characteristic anti-inflammatory, angiogenic, and anti-fibrotic gene expression. (A) The median IDO gene expression for fold difference between the MSCs and HXB-319, using RT-PCR (P < .001, Students t-test). (B) The IDO activity as measured by Kynurenine levels in the tissue culture media at 24 hours’ time point, demonstrated as fold difference as compared to the MSCs (P < .001, Student’s t-test). (C-F) RT-PCR bar graph charts showing fold differences (y-axis) between gene expressions from the same donor MSCs (n = 4) and HXB-319 (n = 4). Asterisk (*) shows statistical significance at P < .05 for all figures (Student’s t-test).

Figure 2.

Three major pathways were activated by HXB-319 (KEGG, Ipathway analysis). Anti-inflammatory and angiogenic signaling: IL-1RN (IL-1 receptor antagonist) activation that results in IL-1β, and control of IL-17 pathway by IK-βα, TSG-6, and potent angiogenic protein pathway upregulation.

Figure 3.

Anti-fibrotic signaling: MMP signaling activation, HGF/c-Met activation, INHBA (Inhibin A and Follistatin activation that suppress TGF-β). Red font/arrows are used for significantly upregulated, and blue font/arrows are used for significantly downregulated molecules.

Figure 4.

Images from mice representative of each experimental group. Grossly, the C57Bl/6 group had normal lungs that were pink and crepitant; microscopic examination of H&E revealed fully patent air spaces devoid of erythrocytes and delimited by thin walls (H&E) with no infiltration by leukocytes. The majority of mice in both the DAH and MSC groups had extensive hemorrhage: zones of deep red to black color and tactile induration grossly, large numbers of erythrocytes in alveolar walls alveoli (H&E), and infiltration of alveolar walls by prominent neutrophils (some indicated by red arrows) and less conspicuous monocytes/macrophages (some indicated by blue arrows). Most HXB-319 treated mice had no (grossly or microscopically) evident hemorrhage, but nearly all (88%) had neutrophils and sparse macrophages infiltrating alveolar walls (final microscopic magnification 50×).

Figure 5.

Proinflammatory and anti-inflammatory cytokine gene expression lung tissue. Lungs from mice treated with HXB-319 showed significantly reduced levels of mRNA encoding IL-6, IL-10, and significantly elevated levels of TSG-6 (*P < .05 between the arrows Students t-test) compared to untreated DAH mice lungs. No statistically significant differences were observed between mice treated with MSCs and untreated DAH mice.

Figure 6.

Survival of C57BL/6 mice after pristane induction and HXB319 and MSC treatment effects.