Pre-clinical proof-of-concept of anti-fibrotic activity of caveolin-1 scaffolding domain peptide LTI-03 in ex vivo precision cut lung slices from patients with Idiopathic Pulmonary Fibrosis.

Rationale: While rodent lung fibrosis models are routinely used to evaluate novel antifibrotics, these models have largely failed to predict clinical efficacy of novel drug candidates for Idiopathic Pulmonary Fibrosis (IPF). Moreover, single target therapeutic strategies for IPF have failed and current multi-target standard of care drugs are not curative. Caveolin-1 (CAV-1) is an integral membrane protein, which, via its caveolin scaffolding domain (CSD), interacts with caveolin binding domains (CBD). CAV-1 regulates homeostasis, and its expression is decreased in IPF lungs. LTI-03 is a seven amino acid peptide derived from the CSD and formulated for dry powder inhalation; it was well tolerated in normal volunteers ( NCT04233814 ) and a safety trial is underway in IPF patients ( NCT05954988 ). Objectives: Anti-fibrotic efficacy of LTI-03 and other CSD peptides has been observed in IPF lung monocultures, and rodent pulmonary, dermal, and heart fibrosis models. This study aimed to characterize progressive fibrotic activity in IPF PCLS explants and to evaluate the antifibrotic effects of LTI-03 and nintedanib in this model. Methods: First, CBD regions were identified in IPF signaling proteins using in silico analysis. Then, IPF PCLS (n=8) were characterized by COL1A1 immunostaining, multiplex immunoassays, and bulk RNA sequencing following treatment every 12hrs with LTI-03 at 0.5, 3.0, or 10 µM; nintedanib at 0.1 µM or 1 µM; or control peptide (CP) at 10 µM. Measurements and Main Results: CBDs were present in proteins implicated in IPF, including VEGFR, FGFR and PDGFR. Increased expression of profibrotic mediators indicated active fibrotic activity in IPF PCLS over five days. LTI-03 dose dependently decreased COL1A1 staining, and like nintedanib, decreased profibrotic proteins and transcripts. Unlike nintedanib, LTI-03 did not induce cellular necrosis signals. Conclusion: IPF PCLS explants demonstrate molecular activity indicative of fibrosis during 5 days in culture and LTI-03 broadly attenuated pro-fibrotic proteins and pathways, further supporting the potential therapeutic effectiveness of LTI-03 for IPF.

Caveolin scaffolding domain (CSD) peptide LTI-2355 modulates the phagocytic and synthetic activity of lung derived myeloid cells in Idiopathic Pulmonary Fibrosis (IPF) and Post-acute sequelae of COVID-fibrosis (PASC-F).

Rationale: The role of the innate immune system in Idiopathic Pulmonary Fibrosis (IPF) remains poorly understood. However, a functional myeloid compartment is required to remove dying cells and cellular debris, and to mediate innate immune responses against pathogens. Aberrant macrophage activity has been described in patients with Post-acute sequelae of COVID fibrosis (PASC-F). Therefore, we examined the functional and synthetic properties of myeloid cells isolated from normal donor lung and lung explant tissue from both IPF and PASC-F patients and explored the effect of LTI-2355, a Caveolin Scaffolding Domain (CSD) peptide, on these cells. Methods & Results: CD45 + myeloid cells isolated from lung explant tissue from IPF and PASC-F patients exhibited an impaired capacity to clear autologous dead cells and cellular debris. Uptake of pathogen-coated bioparticles was impaired in myeloid cells from both fibrotic patient groups independent of type of pathogen highlighting a cell intrinsic functional impairment. LTI-2355 improved the phagocytic activity of both IPF and PASC-F myeloid cells, and this improvement was paired with decreased pro-inflammatory and pro-fibrotic synthetic activity. LTI-2355 was also shown to primarily target CD206-expressing IPF and PASC-F myeloid cells. Conclusions: Primary myeloid cells from IPF and PASC-F patients exhibit dysfunctional phagocytic and synthetic properties that are reversed by LTI-2355. Thus, these studies highlight an additional mechanism of action of a CSD peptide in the treatment of IPF and progressive fibrotic lung disease.

Interleukin 33 supports squamous cell carcinoma growth via a dual effect on tumour proliferation, migration and invasion, and T cell activation.

Interleukin (IL)-33 is an important cytokine in the tumour microenvironment; it is known to promote the growth and metastasis of solid cancers, such as gastric, colorectal, ovarian and breast cancer. Our group demonstrated that the IL-33/ST2 pathway enhances the development of squamous cell carcinoma (SCC). Conversely, other researchers have reported that IL-33 inhibits tumour progression. In addition, the crosstalk between IL-33, cancer cells and immune cells in SCC remains unknown. The aim of this study was to investigate the effect of IL-33 on the biology of head and neck SCC lines and to evaluate the impact of IL-33 neutralisation on the T cell response in a preclinical model of SCC. First, we identified epithelial and peritumoural cells as a major local source of IL-33 in human SCC samples. Next, in vitro experiments demonstrated that the addition of IL-33 significantly increased the proliferative index, motility and invasiveness of SCC-25 cells, and downregulated MYC gene expression in SCC cell lines. Finally, IL-33 blockade significantly delayed SCC growth and led to a marked decrease in the severity of skin lesions. Importantly, anti-IL-33 monoclonal antibody therapy increase the percentage of CD4+IFNγ+ T cells and decreased CD4+ and CD8+ T cells secreting IL-4 in tumour-draining lymph nodes. Together, these data suggest that the IL-33/ST2 pathway may be involved in the crosstalk between the tumour and immune cells by modulating the phenotype of head and neck SCC and T cell activity. IL-33 neutralisation may offer a novel therapeutic strategy for SCC.

Standard of care drugs do not modulate activity of senescent primary human lung fibroblasts.

Cellular senescence is crucial in the progression of idiopathic pulmonary fibrosis (IPF), but it is not evident whether the standard-of-care (SOC) drugs, nintedanib and pirfenidone, have senolytic properties. To address this question, we performed colorimetric and fluorimetric assays, qRT-PCR, and western blotting to evaluate the effect of SOC drugs and D + Q on senescent normal and IPF lung fibroblasts. In this study, we found that SOC drugs did not provoke apoptosis in the absence of death ligand in normal or IPF senescent lung fibroblasts. Nintedanib increased caspase-3 activity in the presence of Fas Ligand in normal but not in IPF senescent fibroblasts. Conversely, nintedanib enhanced B cell lymphoma 2 expression in senescent IPF lung fibroblasts. Moreover, in senescent IPF cells, pirfenidone induced mixed lineage kinase domain-like pseudokinase phosphorylation, provoking necroptosis. Furthermore, pirfenidone increased transcript levels of FN1 and COL1A1 in senescent IPF fibroblasts. Lastly, D + Q augmented growth differentiation factor 15 (GDF15) transcript and protein levels in both normal and IPF senescent fibroblasts. Taken together, these results establish that SOC drugs failed to trigger apoptosis in senescent primary human lung fibroblasts, possibly due to enhanced Bcl-2 levels by nintedanib and the activation of the necroptosis pathway by pirfenidone. Together, these data revealed the inefficacy of SOC drugs to target senescent cells in IPF.

HER2 drives lung fibrosis by activating a metastatic cancer signature in invasive lung fibroblasts.

Progressive tissue fibrosis, including idiopathic pulmonary fibrosis (IPF), is characterized by excessive recruitment of fibroblasts to sites of tissue injury and unremitting extracellular matrix deposition associated with severe morbidity and mortality. However, the molecular mechanisms that control progressive IPF have yet to be fully determined. Previous studies suggested that invasive fibroblasts drive disease progression in IPF. Here, we report profiling of invasive and noninvasive fibroblasts from IPF patients and healthy donors. Pathway analysis revealed that the activated signatures of the invasive fibroblasts, the top of which was ERBB2 (HER2), showed great similarities to those of metastatic lung adenocarcinoma cancer cells. Activation of HER2 in normal lung fibroblasts led to a more invasive genetic program and worsened fibroblast invasion and lung fibrosis, while antagonizing HER2 signaling blunted fibroblast invasion and ameliorated lung fibrosis. These findings suggest that HER2 signaling may be a key driver of fibroblast invasion and serve as an attractive target for therapeutic intervention in IPF.

Antibody-mediated depletion of CCR10+EphA3+ cells ameliorates fibrosis in IPF.

Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant repair that diminishes lung function via mechanisms that remain poorly understood. CC chemokine receptor (CCR10) and its ligand CCL28 were both elevated in IPF compared with normal donors. CCR10 was highly expressed by various cells from IPF lungs, most notably stage-specific embryonic antigen-4-positive mesenchymal progenitor cells (MPCs). In vitro, CCL28 promoted the proliferation of CCR10+ MPCs while CRISPR/Cas9-mediated targeting of CCR10 resulted in the death of MPCs. Following the intravenous injection of various cells from IPF lungs into immunodeficient (NOD/SCID-γ, NSG) mice, human CCR10+ cells initiated and maintained fibrosis in NSG mice. Eph receptor A3 (EphA3) was among the highest expressed receptor tyrosine kinases detected on IPF CCR10+ cells. Ifabotuzumab-targeted killing of EphA3+ cells significantly reduced the numbers of CCR10+ cells and ameliorated pulmonary fibrosis in humanized NSG mice. Thus, human CCR10+ cells promote pulmonary fibrosis, and EphA3 mAb-directed elimination of these cells inhibits lung fibrosis.

Differential Responses to Targeting Matrix Metalloproteinase 9 in Idiopathic Pulmonary Fibrosis.

Rationale: Aberrant lung remodeling in idiopathic pulmonary fibrosis (IPF) is characterized by elevated MMP9 (matrix metalloproteinase 9) expression, but the precise role of this matrix metalloproteinase in this disease has yet to be fully elucidated.Objectives: To evaluate antifibrotic effects of MMP9 inhibition on IPF.Methods: Quantitative genomic, proteomic, and functional analyses both in vitro and in vivo were used to determine MMP9 expression in IPF cells and the effects of MMP9 inhibition on profibrotic mechanisms.Measurements and Main Results: In the present study, we demonstrate that MMP9 expression was increased in airway basal cell (ABC)-like cells from IPF lungs compared with ABC cells from normal lungs. The inhibition of MMP9 activity with an anti-MMP9 antibody, andecaliximab, blocked TGF-ß1 (transforming growth factor ß1)-induced Smad2 phosphorylation. However, in a subset of cells from patients with IPF, TGF-ß1 activation in their ABC-like cells was unaffected or enhanced by MMP9 blockade (i.e., nonresponders). Further analysis of nonresponder ABC-like cells treated with andecaliximab revealed an association with type 1 IFN expression, and the addition of IFNα to these cells modulated both MMP9 expression and TGF-ß1 activation. Finally, the inhibition of MMP9 ameliorated pulmonary fibrosis induced by responder lung cells but not a nonresponder in a humanized immunodeficient mouse model of IPF.Conclusions: Together, these data demonstrate that MMP9 regulates the activation of ABC-like cells in IPF and that targeting this MMP might be beneficial to a subset of patients with IPF who show sufficient expression of type 1 IFNs.

DNA-PKcs modulates progenitor cell proliferation and fibroblast senescence in idiopathic pulmonary fibrosis.

BACKGROUND: Recent studies have highlighted the contribution of senescent mesenchymal and epithelial cells in Idiopathic Pulmonary Fibrosis (IPF), but little is known regarding the molecular mechanisms that regulate the accumulation of senescent cells in this disease. Therefore, we addressed the hypothesis that the loss of DNA repair mechanisms mediated by DNA protein kinase catalytic subunit (DNA-PKcs) in IPF, promoted the accumulation of mesenchymal progenitors and progeny, and the expression of senescent markers by these cell types. METHODS: Surgical lung biopsy samples and lung fibroblasts were obtained from patients exhibiting slowly, rapidly or unknown progressing IPF and lung samples lacking any evidence of fibrotic disease (i.e. normal; NL). The expression of DNA-Pkcs in lung tissue was assessed by quantitative immunohistochemical analysis. Chronic inhibition of DNA-PKcs kinase activity was mimicked using a highly specific small molecule inhibitor, Nu7441. Proteins involved in DNA repair (stage-specific embryonic antigen (SSEA)-4+ cells) were determined by quantitative Ingenuity Pathway Analysis of transcriptomic datasets (GSE103488). Lastly, the loss of DNA-PKc was modeled in a humanized model of pulmonary fibrosis in NSG SCID mice genetically deficient in PRKDC (the transcript for DNA-PKcs) and treated with Nu7441. RESULTS: DNA-PKcs expression was significantly reduced in IPF lung tissues. Chronic inhibition of DNA-PKcs by Nu7441 promoted the proliferation of SSEA4+ mesenchymal progenitor cells and a significant increase in the expression of senescence-associated markers in cultured lung fibroblasts. Importantly, mesenchymal progenitor cells and their fibroblast progeny derived from IPF patients showed a loss of transcripts encoding for DNA damage response and DNA repair components. Further, there was a significant reduction in transcripts encoding for PRKDC (the transcript for DNA-PKcs) in SSEA4+ mesenchymal progenitor cells from IPF patients compared with normal lung donors. In SCID mice lacking DNA-PKcs activity receiving IPF lung explant cells, treatment with Nu7441 promoted the expansion of progenitor cells, which was observed as a mass of SSEA4+ CgA+ expressing cells. CONCLUSIONS: Together, our results show that the loss of DNA-PKcs promotes the expansion of SSEA4+ mesenchymal progenitors, and the senescence of their mesenchymal progeny.

PD-L1 on invasive fibroblasts drives fibrosis in a humanized model of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with unremitting extracellular matrix deposition, leading to a distortion of pulmonary architecture and impaired gas exchange. Fibroblasts from IPF patients acquire an invasive phenotype that is essential for progressive fibrosis. Here, we performed RNA sequencing analysis on invasive and noninvasive fibroblasts and found that the immune checkpoint ligand CD274 (also known as PD-L1) was upregulated on invasive lung fibroblasts and was required for the invasive phenotype of lung fibroblasts, is regulated by p53 and FAK, and drives lung fibrosis in a humanized IPF model in mice. Activating CD274 in IPF fibroblasts promoted invasion in vitro and pulmonary fibrosis in vivo. CD274 knockout in IPF fibroblasts and targeting CD274 by FAK inhibition or CD274-neutralizing antibodies blunted invasion and attenuated fibrosis, suggesting that CD274 may be a novel therapeutic target in IPF.

Characterization of CD28null T cells in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fibrotic lung disease, with unknown etiopathogenesis and suboptimal therapeutic options. Previous reports have shown that increased T-cell numbers and CD28null phenotype is predictive of prognosis in IPF, suggesting that these cells might have a role in this disease. Flow cytometric analysis of explanted lung cellular suspensions showed a significant increase in CD8+ CD28null T cells in IPF relative to normal lung explants. Transcriptomic analysis of CD3+ T cells isolated from IPF lung explants revealed a loss of CD28-transcript expression and elevation of pro-inflammatory cytokine expression in IPF relative to normal T cells. IPF lung explant-derived T cells (enriched with CD28null T cells), but not normal donor lung CD28+ T cells induced dexamethasone-resistant lung remodeling in humanized NSG mice. Finally, CD28null T cells expressed similar CTLA4 and significantly higher levels of PD-1 proteins relative to CD28+ T cells and blockade of either proteins in humanized NSG mice, using anti-CTLA4, or anti-PD1, mAb treatment-accelerated lung fibrosis. Together, these results demonstrate that IPF CD28null T cells may promote lung fibrosis but the immune checkpoint proteins, CTLA-4 and PD-1, appears to limit this effect.

Expansion of commensal fungus Wallemia mellicola in the gastrointestinal mycobiota enhances the severity of allergic airway disease in mice.

The gastrointestinal microbiota influences immune function throughout the body. The gut-lung axis refers to the concept that alterations of gut commensal microorganisms can have a distant effect on immune function in the lung. Overgrowth of intestinal Candida albicans has been previously observed to exacerbate allergic airways disease in mice, but whether subtler changes in intestinal fungal microbiota can affect allergic airways disease is less clear. In this study we have investigated the effects of the population expansion of commensal fungus Wallemia mellicola without overgrowth of the total fungal community. Wallemia spp. are commonly found as a minor component of the commensal gastrointestinal mycobiota in both humans and mice. Mice with an unaltered gut microbiota community resist population expansion when gavaged with W. mellicola; however, transient antibiotic depletion of gut microbiota creates a window of opportunity for expansion of W. mellicola following delivery of live spores to the gastrointestinal tract. This phenomenon is not universal as other commensal fungi (Aspergillus amstelodami, Epicoccum nigrum) do not expand when delivered to mice with antibiotic-depleted microbiota. Mice with Wallemia-expanded gut mycobiota experienced altered pulmonary immune responses to inhaled aeroallergens. Specifically, after induction of allergic airways disease with intratracheal house dust mite (HDM) antigen, mice demonstrated enhanced eosinophilic airway infiltration, airway hyperresponsiveness (AHR) to methacholine challenge, goblet cell hyperplasia, elevated bronchoalveolar lavage IL-5, and enhanced serum HDM IgG1. This phenomenon occurred with no detectable Wallemia in the lung. Targeted amplicon sequencing analysis of the gastrointestinal mycobiota revealed that expansion of W. mellicola in the gut was associated with additional alterations of bacterial and fungal commensal communities. We therefore colonized fungus-free Altered Schaedler Flora (ASF) mice with W. mellicola. ASF mice colonized with W. mellicola experienced enhanced severity of allergic airways disease compared to fungus-free control ASF mice without changes in bacterial community composition.

CCR10+ epithelial cells from idiopathic pulmonary fibrosis lungs drive remodeling.

Idiopathic pulmonary fibrosis (IPF) is a devastating fibrotic lung disease of unknown etiology and limited therapeutic options. In this report, we characterize what we believe is a novel CCR10+ epithelial cell population in IPF lungs. There was a significant increase in the percentage of CCR10+ epithelial cells in IPF relative to normal lung explants and their numbers significantly correlated to lung remodeling in humanized NSG mice. Cultured CCR10-enriched IPF epithelial cells promoted IPF lung fibroblast invasion and collagen 1 secretion. Single-cell RNA sequencing analysis showed distinct CCR10+ epithelial cell populations enriched for inflammatory and profibrotic transcripts. Consistently, cultured IPF but not normal epithelial cells induced lung remodeling in humanized NSG mice, where the number of CCR10+ IPF, but not normal, epithelial cells correlated with hydroxyproline concentration in the remodeled NSG lungs. A subset of IPF CCR10hi epithelial cells coexpress EphA3 and ephrin A signaling induces the expression of CCR10 by these cells. Finally, EphA3+CCR10hi epithelial cells induce more consistent lung remodeling in NSG mice relative to EphA3-CCR10lo epithelial cells. Our results suggest that targeting epithelial cells, highly expressing CCR10, may be beneficial in IPF.

TRAIL-Dependent Resolution of Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is the most common form of interstitial lung disease characterized by the persistence of activated myofibroblasts resulting in excessive deposition of extracellular matrix proteins and profound tissue remodeling. In the present study, the expression of tumor necrosis factor- (TNF-) related apoptosis-inducing ligand (TRAIL) was key to the resolution of bleomycin-induced pulmonary fibrosis. Both in vivo and in vitro studies demonstrated that Gr-1+TRAIL+ bone marrow-derived myeloid cells blocked the activation of lung myofibroblasts. Although soluble TRAIL was increased in plasma from IPF patients, the presence of TRAIL+ myeloid cells was markedly reduced in IPF lung biopsies, and primary lung fibroblasts from this patient group expressed little of the TRAIL receptor-2 (DR5) when compared with appropriate normal samples. IL-13 was a potent inhibitor of DR5 expression in normal fibroblasts. Together, these results identified TRAIL+ myeloid cells as a critical mechanism in the resolution of pulmonary fibrosis, and strategies directed at promoting its function might have therapeutic potential in IPF.

Recognition of Candida albicans by gingival fibroblasts: The role of TLR2, TLR4/CD14, and MyD88.

Recent evidence indicates that nonprofessional immune cells such as epithelial cells, endothelial cells, and fibroblasts also contribute to innate immunity via secretion of cytokines. Fibroblasts are the principal type of cell found in the periodontal connective tissues and they are involved in the immune response during periodontal disease. The role of fibroblasts in the recognition of pathogens via Toll-like receptors (TLRs) has been established; however, few studies have been conducted concerning the involvement of innate immune receptors in the recognition of Candida albicans by gingival fibroblast. In the current study, we investigate the functional activity of TLR2, cluster of differentiation 14 (CD14), and myeloid differentiation primary response gene 88 (MyD88) molecules in the recognition of C. albicans by gingival fibroblast. First, we identified that gingival fibroblasts expressed TLR2, TLR3, and TLR4. Our results showed that TLR agonists had no effect on these receptors' expression by TLR2, MyD88, and CD14-deficient cells. Notably, C. albicans and a synthetic triacylated lipoprotein (Pam3CSK4) induced a remarkable increase of TLR3 expression on MyD88-deficient gingival fibroblasts. TLR4 expression levels were lower than TLR2 and TLR3 levels and remained unchanged after TLR agonist stimulation. Gingival fibroblasts presented morphological similarities; however, TLR2 deficiency on these cells leads to a lower proliferative response, whereas the deficiency on CD14 expression resulted in lower levels of type I collagen by these cells. In addition, the recognition of C. albicans by gingival fibroblasts had an effect on the secretion of cytokines and it was dependent on a specific recognition molecule. Specifically, tumor necrosis factor-α (TNF-α) production after the recognition of C. albicans was dependent on MyD88, CD14, and TLR2 molecules, whereas the production of interleukin-1ß (IL-1ß) and IL-13 was dependent on TLR2. These findings are the first to describe a role of gingival fibroblast in the recognition of C. albicans and the pathways involved in this process. An understanding of these pathways may lead to alternative treatments for patients with periodontal disease.

Divergent roles for Clusterin in Lung Injury and Repair.

Lung fibrosis is an unabated wound healing response characterized by the loss and aberrant function of lung epithelial cells. Herein, we report that extracellular Clusterin promoted epithelial cell apoptosis whereas intracellular Clusterin maintained epithelium viability during lung repair. Unlike normal and COPD lungs, IPF lungs were characterized by significantly increased extracellular Clusterin whereas the inverse was evident for intracellular Clusterin. In vitro and in vivo studies demonstrated that extracellular Clusterin promoted epithelial cell apoptosis while intercellular Clusterin modulated the expression of the DNA repair proteins, MSH2, MSH6, OGG1 and BRCA1. The fibrotic response in Clusterin deficient (CLU-/-) mice persisted after bleomycin and it was associated with increased DNA damage, reduced DNA repair responses, and elevated cellular senescence. Remarkably, this pattern mirrored that observed in IPF lung tissues. Together, our results show that cellular localization of Clusterin leads to divergent effects on epithelial cell regeneration and lung repair during fibrosis.

Antifibrotic role of vascular endothelial growth factor in pulmonary fibrosis.

The chronic progressive decline in lung function observed in idiopathic pulmonary fibrosis (IPF) appears to result from persistent nonresolving injury to the epithelium, impaired restitution of the epithelial barrier in the lung, and enhanced fibroblast activation. Thus, understanding these key mechanisms and pathways modulating both is essential to greater understanding of IPF pathogenesis. We examined the association of VEGF with the IPF disease state and preclinical models in vivo and in vitro. Tissue and circulating levels of VEGF were significantly reduced in patients with IPF, particularly in those with a rapidly progressive phenotype, compared with healthy controls. Lung-specific overexpression of VEGF significantly protected mice following intratracheal bleomycin challenge, with a decrease in fibrosis and bleomycin-induced cell death observed in the VEGF transgenic mice. In vitro, apoptotic endothelial cell-derived mediators enhanced epithelial cell injury and reduced epithelial wound closure. This process was rescued by VEGF pretreatment of the endothelial cells via a mechanism involving thrombospondin-1 (TSP1). Taken together, these data indicate beneficial roles for VEGF during lung fibrosis via modulating epithelial homeostasis through a previously unrecognized mechanism involving the endothelium.

Senescent stromal cell-induced divergence and therapeutic resistance in T cell acute lymphoblastic leukemia/lymphoma.

T cell Acute Lymphoblastic Leukemia/Lymphoma (T-ALL/LBL) is a precursor T cell leukemia/lymphoma that represents approximately 15% of all childhood and 25% of adult acute lymphoblastic leukemia. Although a high cure rate is observed in children, therapy resistance is often observed in adults and mechanisms leading to this resistance remain elusive. Utilizing public gene expression datasets, a fibrotic signature was detected in T-LBL but not T-ALL biopsies. Further, using a T-ALL cell line, CCRF-CEM (CEM) cells, we show that CEM cells induce pulmonary remodeling in immunocompromised mice, suggesting potential interaction between these cells and lung fibroblasts. Co-culture studies suggested that fibroblasts-induced phenotypic and genotypic divergence in co-cultured CEM cells leading to diminished therapeutic responses in vitro. Senescent rather than proliferating stromal cells induced these effects in CEM cells, due, in part, to the enhanced production of oxidative radicals and exosomes containing miRNAs targeting BRCA1 and components of the Mismatch Repair pathway (MMR). Collectively, our studies demonstrate that there may be bidirectional interaction between leukemic cells and stroma, where leukemic cells induce stromal development in vivo and senescent stromal cells generates genomic alterations in the leukemic cells rendering them therapeutic resistant. Thus, targeting senescent stroma might prove beneficial in T-ALL/LBL patients.

Axl receptor blockade protects from invasive pulmonary aspergillosis in mice.

Aspergillus fumigatus is a sporulating fungus found ubiquitously in the environment, which is quickly contained in the immunocompetent host but can cause lethal invasive aspergillosis in the immunocompromised host. We have recently demonstrated that Axl (one member of the Tyro3, Axl, Mertk receptor family) is a key regulator of antiviral immune responses in the lung. In this study, we investigated the role of Axl in antifungal immunity in a model of invasive pulmonary aspergillosis (IPA). In this model, Aspergillus fumigatus conidia were administered into the lungs of neutrophil-depleted mice, and the mice were monitored for survival, lung inflammatory response, and fungal clearance. The lethal effect of IPA was significantly reduced in anti-Axl mAb-treated mice compared with IgG control-treated mice. Targeting Axl significantly inhibited pulmonary inflammation, including the expression of IL-1ß, IL-6, TNF-α, and chitinase-like proteins in whole lung. Further, anti-Axl mAb treatment significantly increased M1 macrophages that highly expressed inducible NO synthase and decreased M2 macrophages that expressed Arginase 1 and were found in inflammatory zone protein (Fizz1). More importantly, anti-Axl mAb treatment significantly increased the number of IFN-γ-producing T cells and NK cells compared with the IgG control group during IPA. Together, our results demonstrate that the Axl mAb treatment is protective during invasive aspergillosis in neutropenic mice. Collectively, these data suggest a potential deleterious role for Axl during primary immune responses directed against A. fumigatus and novel therapeutic strategy for IPA.

Role of growth arrest-specific gene 6 in the development of fungal allergic airway disease in mice.

Growth arrest-specific gene (Gas)6 is a secreted vitamin K-dependent protein with pleiotropic effects via activation of receptor tyrosine kinase Tyro3, Axl, and Mertk receptors, but little is known about its role in allergic airway disease. We investigated the role of Gas6 in the development of fungal allergic airway disease in mice. The immune response was evaluated in Gas6-deficient (Gas6-/-) and wild-type (WT) mice and in recombinant Gas6-treated WT mice during Aspergillus fumigatus-induced allergic airway disease. Gas6 plasma levels were significantly elevated in adult clinical asthma of all severities compared with subjects without asthma. In a murine model of fungal allergic airway disease, increased protein expression of Axl and Mertk were observed in the lung. Airway hyperresponsiveness (AHR), whole lung Th2 cytokine levels, goblet cell metaplasia, and peribronchial fibrosis were ameliorated in Gas6-/- mice compared with WT mice with fungal allergic airway disease. Intranasal Gas6 administration into WT mice had a divergent effect on airway inflammation and AHR. Specifically, a total dose of 2 µg of exogenous Gas6 (i.e., low dose) significantly increased whole lung Th2 cytokine levels and subsequent AHR, whereas a total dose of 7 µg of exogenous Gas6 (i.e., high dose) significantly suppressed Th1 and Th2 cytokines and AHR compared with appropriate control groups. Mechanistically, Gas6 promoted Th2 activation via its highest affinity receptor Axl expressed by myeloid DCs. Intranasal administration of Gas6 consistently exacerbated airway remodeling compared with control WT groups. These results demonstrate that Gas6 enhances several features of fungal allergic airway disease.

Targeting interleukin-13 with tralokinumab attenuates lung fibrosis and epithelial damage in a humanized SCID idiopathic pulmonary fibrosis model.

The aberrant fibrotic and repair responses in the lung are major hallmarks of idiopathic pulmonary fibrosis (IPF). Numerous antifibrotic strategies have been used in the clinic with limited success, raising the possibility that an effective therapeutic strategy in this disease must inhibit fibrosis and promote appropriate lung repair mechanisms. IL-13 represents an attractive target in IPF, but its disease association and mechanism of action remains unknown. In the present study, an overexpression of IL-13 and IL-13 pathway markers was associated with IPF, particularly a rapidly progressive form of this disease. Targeting IL-13 in a humanized experimental model of pulmonary fibrosis using tralokinumab (CAT354) was found to therapeutically block aberrant lung remodeling in this model. However, targeting IL-13 was also found to promote lung repair and to restore epithelial integrity. Thus, targeting IL-13 inhibits fibrotic processes and enhances repair processes in the lung.