Publications & Presentations
International Colloquium on Lung and Airway Fibrosis
Sept. 2014, Mont-Tremblant, Quebec, Canada
Stem cell factor (SCF) and its receptor c-Kit have been implicated in tissue remodeling and fibrosis. Using Ingenuity Software to analyze public IPF patient microarray databases it was demonstrated that SCF is highly upregulated in IPF patients compared to normal and further upregulated in exacerbated patients compared to stable IPF. The Ingenuity analysis also demonstrated the linkage of SCF to multiple pro-fibrotic pathways that have previously been shown to be important in IPF progression. Our studies have previously demonstrated that by blocking SCF the fibrotic responses in bleomycin and chronic allergic asthma models, the pathophysiologic aspects of disease are mitigated. We have recently made a mouse anti-human SCF monoclonal antibody that cross-reacts to mouse SCF and utilized it in three independent pulmonary fibrotic disease models to determine its suitability as a human therapeutic. In the first model, fibrosis was induced by intratracheal instillation of bleomycin (BLM), which causes increased SCF levels in plasma, bronchoalveolar lavage fluid (BALF) and lung tissue. The intra-tracheal administration of the monoclonal anti SCF antibody (OpSCF) in a therapeutic modality on days 8 and 12 after bleomycin treatment significantly reduced the histologic pathology and collagen staining. In addition, the treatment of animals with OpSCF significantly reduced the pulmonary mRNA expression of collagen genes (1 & 3) and Th2 cytokines. In a second model of chronic lung fibrosis using fibroblasts from IPF patients injected into SCID mice, OpSCF was administered intranasally using a therapeutic regimen beginning on day 35 of the pulmonary fibrotic model once every 3 days until termination of the experiment on day 65. The results demonstrated a reduction in the developing fibrotic response by both histologic examination and by hydroxyproline analysis. Finally, using a model of chronic cockroach allergen-induced asthma that results in remodeling of the airways, the admini-stration of OpSCF into the airways during the terminal phases of the disease model alleviated the airway disease, including airway hyperreactivity, peribronchial inflammation and eosinophilia, mucus, and airway thickening/remodeling as well as key pathologic cytokines. Together, these data demonstrate that by blocking SCF in a therapeutic manner during the development and within established disease a significant reduction in lung remodeling disease can be achieved. Thus, OpSCF provides an avenue to alter the development and progression of chronic remodeling pulmonary diseases and provides important pre-clinical data to demonstrate efficacy of targeting this novel target.
Anti-KITLG Therapy in Renal Disease
May 2019, O'Brien retreat
RENAL FIBROSIS AND KITLG ACTIVITY:
Renal fibrosis is the final common consequence of a multitude of chronic kidney diseases (CKD) and is the culmination of multifactorial processes which ultimately result in renal dysfunction and failure.
• KIT/KITLG signaling regulates inflammation and fibrosis in humans and animal models and cross-talks with TGFB signaling (1-6); however, its molecular mechanism in mediating kidney fibrosis remains unclear.
• Anti-fibrotic therapies exist for other organs; however, they are ineffective, non-specific and have numerous side effects in CKD patients (7).
• Aim: Evaluate the effect of inhibiting isoform-specific KITL on morphometric and transcriptomic parameters in TGFB1 transgenic mice.
CONCLUSIONS AND FUTURE DIRECTIONS:
• Gene expression in human kidneys revealed that declining kidney function (eGFR) in IgAN patients had significantly higher levels of tissue Kitlg expression. Moreover, patients with RPGN had significantly higher Kitlg expression than living donors. This data demonstrates that inhibition of KITLG may be beneficial for treating CKD progression in different kidney diseases.
• In the four week aKITL treatment study, aKITL did not significantly, but trended towards, improved TGFB1-TG mouse survival suggesting increasing the sample size and/or a longer treatment could improve survival. Moreover, aKITL reduced the accumulation of interstitial fibrosis. For these reasons, we proceeded with a 2 week treatment to determine how aKITL protects the kidney from fibrosis during disease initiation.
• Because TGFB1-TG mice have a heterogeneous phenotype, separation of mice by a PCA of RNA-seq data demonstrated a viable approach to determine mild and severe phenotypes. This approach revealed previously published gene expression differentials defining progressive and non-progressive groups (Fig. 3B) were maintained and further supports the use of these genes in group separation for future studies.
• Body measurements and morphometry from the two week treatment revealed severe IgG mice had significantly higher mesangial index, mesangial volume, plasma KITL and lower podocyte density compared to mild IgG mice, while treatment with aKITL ameliorated these differences in mild versus severe mice.
• Matrisome genes Fndc1, Col15a1, Col6a3 and Fbln1 were significantly reduced in kidney cortex of aKITL vs IgG mice providing early transcriptomic evidence in support of aKITL reduction of interstitial fibrosis from study 1.
• Myofibroblasts marker gene, Acta2, was the only immune cell marker gene to be significantly elevated in severe IgG-treated mice. Because the antibody does not inhibit TGFB1 activation of myofibroblasts, but there is an amelioration of myofibroblasts via aKITL (aKITL severe is not different from either mild group or severe IgG), this suggests aKITL may have an affect on myofibroblast abundance and provides evidence for a potential mechanism for the reduction in fibrosis and related fibrosis gene expression.
• In summary, our findings findings suggest that pKITL increased with disease severity and aKITL antibody ameliorated kidney damage in TGFB1-TG mice. These data support aKITL therapy as a candidate intervention for fibrotic kidney disease. Studies examining human biofluids and aKITL therapy in other mouse models of kidney injury are ongoing.
ILC2 Regulated by Stem Cell Factor during Asthmatic Disease
Stem cell factor (SCF) binds to the receptor c-Kit that is expressed on a number of myeloid and lymphoid cell populations, including Type 2 innate lymphoid cells (ILC2). However the importance of the SCF/c-Kit interaction in ILC2 has not been studied. Here we investigate the role of a specific SCF isoform, SCF248, in the allergic asthmatic response and SCF/c-Kit in ILC2 activation during chronic allergy. We observed that mice treated with a monoclonal antibody specific for SCF248 attenuated the development of chronic asthmatic disease by decreasing the number of mast cells, ILC2 and eosinophils, as well as reducing the accompanying pathogenic cytokine responses. These data were supported using SCFfl/fl-Col1-Cre-ERT mice and W/Wv mice that demonstrated the importance of the stem cell factor/c-Kit activation during chronic allergy and the accumulation of c-kit+ cells. Finally, these data demonstrate for the first time that SCF could activate ILC2 cells in vitro for the production of key allergic cytokines. Together these findings indicate that SCF is a critical cytokine involved in the activation of ILC2 that lead to more severe outcomes during chronic allergy and that the SCF248 isoform could be an important therapeutic target to control the disease progression.
Inhibition of the Stem Cell Factor 248 Isoform Attenuates the Development of
Pulmonary Remodeling Disease
Am J Physiol Lung Cell Mol Physiol 318 (2020)
Stem cell factor (SCF) and its receptor c-kit have been implicated in inflammation, tissue remodeling, and fibrosis. Ingenuity Integrated Pathway Analysis of gene expression array data sets showed an upregulation of SCF transcripts in idiopathic pulmonary fibrosis (IPF) lung biopsies compared with tissue from nonfibrotic lungs that are further increased in rapid progressive disease.
SCF248, a cleavable isoform of SCF, was abundantly and preferentially expressed in human lung fibroblasts and fibrotic mouse lungs relative to the SCF220 isoform. In fibroblast-mast cell coculture
studies, blockade of SCF248 using a novel isoform-specific anti-SCF248 monoclonal antibody (anti-SCF248), attenuated the expression of COL1A1, COL3A1, and FN1 transcripts in cocultured IPF but not normal lung fibroblasts. Administration of anti-SCF248 on days 8 and 12 after bleomycin instillation in mice significantly reduced fibrotic lung remodeling and col1al, fn1, acta2, tgfb, and ccl2 transcript expression. In addition, bleomycin increased numbers of c-kit-positive mast cells, eosinophils, and ILC2 in lungs of mice, whereas they were not significantly increased in anti-SCF248-treated animals. Finally, mesenchymal cell-specific deletion of SCF significantly attenuated
bleomycin-mediated lung fibrosis and associated fibrotic gene expression. Collectively, these data demonstrate that SCF is upregulated in diseased IPF lungs and blocking SCF248 isoform significantly ameliorates fibrotic lung remodeling in vivo suggesting that it may be a therapeutic target for fibrotic lung diseases.