Immune cells play diverse roles in cancer development. Myeloid cells are key drivers of tumor-escape mechanisms as they suppress immune responses, facilitate metastasis, and contribute to therapy resistance. In particular, macrophages can be polarized into an inflammatory M1 (anti-tumor) or anti-inflammatory M2 (pro-tumor) phenotype. M2 macrophages are associated with tumor progression, as they secrete factors that promote tumor angiogenesis, suppress T-cell activity, and correlate with poor clinical outcomes in squamous cell carcinoma (SCC). Given this context, this study aims to demonstrate the biological effects of monocytes and both M1 and M2 macrophages in squamous cell carcinoma. Our data indicate higher CD163 immunoreactivity in biopsies from SCC patients. Furthermore, we found that a conditioned medium (CM) containing bioactive compound generated by M2 macrophages enhances the proliferation and invasion of the SCC-25 cell line in vitro. Surprisingly, CM derived from blood CD14+ monocytes increased SCC-25 proliferation at the same rate of M2 macrophages-CM. M1 macrophages conditioned medium significantly enhanced the motility and decreased proliferation in Detroit 562 cells. The analysis of tumor-associated transcripts showed that both M1 and M2 conditioned medium induced high levels of EPCAM mRNA and significantly decreased the expression of MYC, an epithelial-to-mesenchymal transition marker, in SCC cell lines. Detroit cells exposed to conditioned medium from monocytes and macrophage also showed elevated SOX2 mRNA levels. The findings suggest that monocytes and macrophage mediators exert distinct biological effects on SCC cell lines.

Idiopathic pulmonary fibrosis (IPF) is a progressive, fatal lung disease marked by alveolar type 2 (AT2) stem cell dysfunction and excessive matrix deposition, with no effective treatments. Recent advances have recognized that AT2 cells act as stem cells, in addition to their role in the production of pulmonary surfactants in the distal alveolar space. We and others have reported a failure of AT2 regeneration and a loss of AT2 cells in IPF. We recently further reported that there is a defect in lipid metabolism in IPF AT2 cells and we discovered a selective loss of lysophosphatidylcholine acyltransferase 1 (LPCAT1) in AT2 cells from IPF, as well as in AT2 cells from bleomycin-injured mice. Pharmacological and genetic experiments confirm that LPCAT1 is required for AT2 cell renewal in 3D organoid assays. AT2 cell-specific Lpcat1 deletion resulted in reduced AT2 renewal, spontaneous lung fibrosis, and heightened susceptibility to bleomycin-induced fibrosis in mice in vivo. Expression-based high-content drug screening with an LPCAT1 knock-in cell line identified several drug families that upregulated LPCAT1 expression. We further confirmed that anti-malarial artesunate and PLA2 inhibitor ONO-RS-082 increased LPCAT1 mRNA expression, promoted AT2 renewal, and attenuated bleomycin-induced lung fibrosis in mice in vivo. Our findings establish LPCAT1 as a critical regulator of AT2 renewal and lipid metabolism in IPF, suggesting that reactivation of LPCAT1 could offer a novel therapeutic strategy for restoring alveolar progenitor function and mitigating lung fibrosis.

RATIONALE: Alveolar type 2 (AT2) cells play critical roles as lung stem cells in initiating lung repair and alveologenesis following injury via self-renewal and differentiation into alveolar type 1 (AT1) cells. Hence, effective AT2-to-AT1 differentiation is essential for lung repair. The over-expression of syndecan-1 in AT2 cells in lung fibrosis impacts their proliferative and reparative roles by promoting AT2 senescence. We proposed to examine the pathological roles of excess syndecan-1 in AT2 differentiation and its roles in unresolved fibrosis. METHODS: We analyzed human and mouse scRNAseq of Idiopathic pulmonary fibrosis (IPF) and bleomycin-injured WT and Sdc1-/-mice. IPF and control lungs were immunostained for transitional (KRT5, KRT8, and KRT17), AT2 (proSP-C), and AT1 (Pdpn) cell markers. Aged-WT and Sdc1-/- mice were given a single-dose bleomycin intratracheally. At day 21, the lungs were immunostained with all epithelial cell markers. The bleomycin-injured WT and Sdc1-/-AT2 3D organoid cell cultures were assessed for colony-forming efficiency and AT2 differentiation by serial imaging and immunostaining for AT1, AT2, and transitional cell markers. RESULTS: Human scRNAseq data showed that syndecan-1 was over-expressed in IPF AT2 and transitional epithelial cells compared to controls. The transcriptomic composite SenMayo Senescence score was higher in IPF AT2 cells. Spatial transcriptomic data of IPF indicated that syndecan-1 was co-expressed with KRT17 and CDKN1A [p21]. Immunostaining corroborated that syndecan-1 was co-expressed with KRT8+ KRT17+ cells in IPF. In aged mice, syndecan-1 was profibrotic, and excess syndecan-1 in the aged – WT mice was associated with the accumulation of Krt8+ cells. These findings were validated in AT2 3D organoids, where we found a significant Krt8+ and Krt17+ cell expansion in bleomycin-injured WT. Additionally, Krt17+ cells were significantly less in Sdc1-/- AT2 organoids. Further, we found normal morphology and AT1 differentiation in Sdc1-/- organoids, whereas WT AT2 organoids developed atypical morphology and maldistribution of AT1 cells (Figure 1). CONCLUSIONS: We concluded that syndecan-1 was upregulated in aberrant transitional epithelial cells of human and mouse lung fibrosis. An excess of syndecan-1 promoted an accumulation of aberrant transitional epithelial cells, thereby preventing normal AT2 differentiation and leading to ineffective lung repair in lung fibrosis. We proposed that syndecan-1 mediated an abnormal accumulation of aberrant transitional cells by maintaining the senescence state of these cells. Our future work includes the identification of regulatory molecules that syndecan-1 regulates to promote the persistent senescence state of aberrant transitional epithelial cells.

Rationale: Idiopathic pulmonary fibrosis is a progressive disease characterized by unrelenting lung remodeling ultimately leading to lung failure and death. Single cell RNAseq studies have revealed that there is marked decrease in the abundance of alveolar type 2 cells (AT2) in lung samples from IPF patients. Reduction of AT2 viability impairs alveolar regeneration since these cells are a source of alveolar type 1 cells (AT1). In this study, we optimized a 3D alveolosphere model generated with isolated and cultured alveolar type 2 cells from IPF and donor, non-IPF control patients and evaluated the effect of CSD peptides LTI-03 and LTI-2355.Methods: Cryo-preserved IPF and normal donor lung explants were processed using enzymatic and mechanical digestion to obtain lung epithelial cell preparations. Primary human AT2 cells (EPCAM+ HTII-280+) were isolated using fluorescence-activated cell sorting (FACS). FACS-sorted AT2 cells were subsequently resuspended in Matrigel (1:1 Matrigel to media). A 30 μL drop containing 5 x104 AT2 cells was applied to the center of each well in a 24 well plate. Either vehicle control, CSD peptide (LTI-03 or LTI-2355 at 0.5, 3, or 10 μM) or Nintedanib (80 nM) were added into each drop, and to the overlying tissue culture media. Treatments were changed every other day for 28 days. Results: Both LTI-03 and LTI-2355 CSD peptides at the 10 μM dose increased the number of IPF alveolosperes compared with the control treatment of IPF alveolospheres at day 28 in culture. CSD peptide treatment did not alter the average size of IPF alveolospheres. Quantitative PCR analysis of SFTPC levels in IPF and normal donor alveolospheres revealed that CSD peptides increase SFTPC transcripts in both groups of alvelospheres. Finally, Nintedanib treatment of normal donor alveolospheres significantly reduced the number and size of alveolospheres at day 28 of culture compared with vehicle or CSD treated normal donor alveolospheres.Conclusion: LTI-03 and LTI-2355 increased the outgrowth of IPF alveolospheres in 3D tissue culture indicating that LTI CSD peptides exert a putative pro-regenerative effect on IPF AT2 cells. In contrast, nintedanib exhibited inhibitory effects on the outgrowth of normal donor alveolospheres. Ethical approval: Institutional Review Boards both at Cedars-Sinai Medical Center and Aileron Therapeutics approved all experiments with human tissue.

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.

"The Role of Inflammation and Fibrosis in ILD Treatment Decisions." American Journal of Respiratory and Critical Care Medicine, 0(ja), pp.

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.

Telomere shortening is a prominent hallmark of aging and is emerging as a characteristic feature of Myelodysplastic Syndromes (MDS) and Idiopathic Pulmonary Fibrosis (IPF). Optimal telomerase activity prevents progressive shortening of telomeres that triggers DNA damage responses. However, the upstream regulation of telomerase holoenzyme components remains poorly defined. Here, we identify RIOK2, a master regulator of human blood cell development, as a critical transcription factor for telomere maintenance. Mechanistically, loss of RIOK2 or its DNA-binding/transactivation properties downregulates mRNA expression of both TRiC and dyskerin complex subunits that impairs telomerase activity, thereby causing telomere shortening. We further show that RIOK2 expression is diminished in aged individuals and IPF patients, and it strongly correlates with shortened telomeres in MDS patient-derived bone marrow cells. Importantly, ectopic expression of RIOK2 alleviates telomere shortening in IPF patient-derived primary lung fibroblasts. Hence, increasing RIOK2 levels prevents telomere shortening, thus offering therapeutic strategies for telomere biology disorders.

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.

A percentage of COVID-19 affected patients develop post-acute sequalae of SARS-CoV-2, with evidence that SARS-CoV-2 causes ARDS leading to lung fibrosis (PASC-Fibrosis) that shares similarities with idiopathic pulmonary fibrosis (IPF). Herein, we addressed the hypothesis that fatal COVID-19, PASC, and IPF share key biomarkers of interest in lung fibrosis. Autopsy and explanted lung samples were subjected to histopathological and immunohistochemical (IHC) analysis for key biomarkers of interest in fibrosis. PASC-fibrosis-derived 2D and 3D cell cultures were treated with BRD4 inhibitors to evaluate proliferation and differentiation of fibroblasts, airway epithelial cells and lung bronchospheres. Single-cell RNA sequencing determined altered immune subpopulations. PASC-Fibrosis survivors had diffuse interstitial fibrosis, with organization into a non-specific interstitial fibrosis (NSIP) pattern. Transcriptomic analysis from fatal COVID-19 lungs showed BRD4, GDF15, IFNα2 and IL11 pathways markedly active. These findings were confirmed by IHC on PASC-fibrosis, with increased expression in myeloid cells. Altered frequencies in lung PASC-fibrosis subpopulations included loss of FABP4+ and increase of MERTK+SSP1+ macrophages and monocytes. Pharmacological inhibition of PASC-Fibrosis lung cultures and bronchospheres with BRD4 inhibitors limited outgrowth and differentiation of fibroblasts, airway epithelial cells and the formation of lung bronchospheres. In conclusion, shared profibrotic pathways among COVID-19 and chronic fibrotic ILDs provides the impetus to further explore treatment strategies to chronically ventilated COVID-19 patients.

Rationale Contribution of central lung tissues to pathogenesis of idiopathic pulmonary fibrosis (IPF) remains unknown. Objective To ascertain the relationship between cell types of IPF-central and IPF-peripheral lung explants using RNA sequencing (RNA-seq) transcriptome. Methods Biopsies of paired IPF-central and IPF-peripheral along with non-IPF lungs were selected by reviewing H&E data. Criteria for differentially expressed genes (DEG) were set at false discovery rate <5% and fold change >2. Computational cell composition deconvolution was performed. Signature scores were computed for each cell type. Findings Comparison of central IPF versus non-IPF identified 1723 DEG (1522 upregulated and 201 downregulated). Sixty-two per cent (938/1522) of the mutually upregulated genes in central IPF genes were also upregulated in peripheral IPF versus non-IPF. Moreover, 85 IPF central-associated genes (CAG) were upregulated in central IPF versus both peripheral IPF and central non-IPF. IPF single-cell RNA-seq analysis revealed the highest CAG signature score in myofibroblasts and significantly correlated with a previously published activated fibroblasts signature (r=0.88, p=1.6×10−4). CAG signature scores were significantly higher in IPF than in non-IPF myofibroblasts (p=0.013). Network analysis of central-IPF genes identified a module significantly correlated with the deconvoluted proportion of myofibroblasts in central IPF and anti-correlated with inflammation foci trait in peripheral IPF. The module genes were over-represented in idiopathic pulmonary fibrosis signalling pathways. Interpretation Gene expression in central IPF lung regions demonstrates active myofibroblast features that contributes to disease progression. Further elucidation of pathological transcriptomic state of cells in the central regions of the IPF lung that are relatively spared from morphological rearrangements may provide insights into molecular changes in the IPF progression.

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.### Competing Interest StatementBreAnne MacKenzie is an employee of Lung Therapeutics, Inc. Cory Hogaboam is a consulting CSO for Lung Therapeutics, Inc.* AM : Alveolar macrophage. CAV : Caveolin CD : Cluster of differentiation CH3IL1 : Chitinase 3-like-1 COPD : Chronic obstructive pulmonary disease CSD : Caveolin scaffolding domain DMEM : Dulbecco’s Modified Eagle Medium ECM : Extracellular matrix FABP : Fatty Acid Binding Protein IL : Interleukin IPF : Idiopathic Pulmonary Fibrosis MERTK MER : Proto-Oncogene, Tyrosine Kinase MMR/MRC : Mannose receptor MMP : Matrix metalloprotease Mo-MA : Monocyte derived macrophage PASC-F : Post-acute sequelae of COVID fibrosis RCU : Red Calibrated Unit SA : Staphylococcus aureus sCD : soluble CD SPP/OPN : Osteopontin TLR : Toll like receptor TNF : Tumor necrosis factor TR-AM : Tissue-resident alveolar macrophage

Rationale Cellular senescence is crucial in the progression of idiopathic pulmonary fibrosis (IPF), but it is yet unclear whether the standard-of-care (SOC) drugs nintedanib and pirfenidone have senolytic properties. Objectives We attempted to illuminate the effects of SOC drugs on senescent normal and IPF lung fibroblasts in vitro . Methods Colorimetric/fluorimetric assays, qRT-PCR, and western blotting were used to evaluate the effect of SOC drugs on senescent normal and IPF lung fibroblasts. Results SOC drugs did not induce apoptosis in the absence of death ligands in either normal or IPF senescent cells. Nintedanib increased caspase-3 activity in the presence of Fas Ligand (FasL) in normal but not in IPF senescent fibroblasts. Conversely, nintedanib enhanced B cell lymphoma (Bcl)-2 expression in senescent IPF lung fibroblasts. Moreover, in senescent IPF cells, pirfenidone alone induced mixed lineage kinase domain-like pseudokinase (MLKL) phosphorylation, provoking necroptosis. However, fragmented gasdermin D, indicating pyroptosis, was not detected under any condition. In addition, SOC drugs increased transcript levels of fibrotic and senescence markers in senescent IPF fibroblasts, whereas D+Q inhibited all these markers. Finally, D+Q enhanced growth differentiation factor 15 (GDF15) transcript and protein levels in both normal and IPF senescent fibroblasts. Conclusions In the presence and absence of the extrinsic pro-apoptotic ligands, SOC drugs failed to trigger apoptosis in senescent fibroblasts, possibly due to enhanced Bcl-2 levels and the activation of the necroptosis pathway. SOC drugs elevated fibrotic and senescence markers in IPF lung fibroblasts. Together, these data demonstrated the inefficacy of SOC in targeting senescent cells. Further investigation is required to fully elucidate the therapeutic implications of SOC drugs on other senescent cell types in IPF.