Yildirim/Conlon Lab

RATIONALE Lung adenocarcinoma (LUAD) frequently harbors KRAS mutations and is highly aggressive and resistant to conventional therapies. KRAS mutations drive tumorigenesis by altering pathways like NF-κB, suggesting that CCL2, VCAN and IL-1ß signaling within the tumor microenvironment is important to LUAD's etiology. Using Isunakinra, a potent IL-1 receptor antagonist, we tested whether these signaling pathways and immune cell recruitment contribute to suppressing the pro-tumorigenic environment fostered by KRAS mutations. METHODS and RESULTS Here we developed a bioluminescence-based reporting system (NF-κB.GFP.Luc; pNGL) to monitor NF-κB activity in four KRAS-mutant lines (KM: LLC, MC38, AE17, FULA1) and two KRAS wild-type lines (KW: B16F10, PANO2). When treated with Isunakinra, NF-κB activity was significantly inhibited in KM vs KW cell lines (p<0.001). FVB mice were injected subcutaneously with KM or KW and treated with Isunakinra (n= 5-6/cell line) for 7 days Tumor growth, pulmonary metastasis and malignant pulmonary effusion were all significantly decreased by Isunakinra (KM vs KW, p<0.001). Next, mice were treated with urethane (1g/kg/mouse, n=10/group), a reliable system for generating KRAS-mutant tumors. Isunakinra (20mg/kg; twice-weekly) was administered for 30 days either early during the first month post-induction or later during the fifth month post-induction. Tumor number and burden were assessed, and KRAS mutations (Q61 and G12/G13) were identified using ddPCR. Immunofluorescent labeling of lung sections was used to assess the presence of macrophages and CCL2/VCAN protein expression. Early treatment reduced both tumor number and burden by approximately 25% (vs non-treated, p < 0.05), while late treatment achieved a 40% reduction (p < 0.05), with no differences between early and late timing. KRAS mutations were confirmed in our model, and subtypes remained unchanged across groups (p > 0.05). CD68+ macrophages increased within the tumor area during late treatment (p= 0.053), while peritumor CD68+ macrophages decreased significantly (p < 0.05). CCR2 expression in tumor regions was reduced by 40% in the late-treated group (vs non-treated, p = 0.051). Interestingly, late-treated mice increased CCL2 expression, correlating with reduced tumor number and burden in early-treated mice (p<0.05) and suggesting compensatory changes in chemokine-receptor dynamics. VCAN levels were not significantly altered. CONCLUSIONS Overall, our data suggest that Isunakinra effectively influences the KRAS tumorgenicity via CCL2 signaling loop, reducing tumor growth by modulating the immune microenvironment. These results support its further investigation as a modulator of immune signaling and immune cell recruitment, and as a therapeutic approach for KRAS-mutated LUAD.

Plants and animals/humans have evolved sophisticated innate immune systems to cope with microbial attack. Innate immunity implies the presence of membrane-located and intracellular receptors to recognize compounds released by damage or by invading pathogens. After detection the receptor molecules initiate intracellular defense signaling, resulting in cell death and/or production of defense molecules. Interestingly, the defense response includes also memory mechanisms, which allow the organisms to better cope with future microbial attacks. Redox mechanisms play an important role in defense signaling. In this review article, we compare the innate immune memory of animals/humans and plants and describe how reversible nitric oxide- and reactive oxygen species-dependent protein modifications enable the activation of defense signaling proteins and transcription factors and regulate the activity of chromatin modifying enzymes to establish innate immune memory. We hope to encourage efforts to characterize further molecular redox mechanisms of the innate immune memory, which might enable the development of new immunotherapies.

RATIONALE: Human precision-cut lung slices (hPCLS) are a unique platform for functional, mechanistic, and drug discovery studies in respiratory research. Their relevance lies in their ability to maintain all resident cellular compartments (epithelial, mesenchymal, and immune cells) as well as the extracellular matrix (ECM) in their native three-dimensional structure. However, tissue availability, transportation, generation, and cultivation time represent important challenges for their usage. To address this, the present study aimed to evaluate the efficacy of a specifically designed tissue preservation solution (TiProtec) in the absence (-) or presence (+) of iron chelators as an alternative for long-term cold storage of hPCLS. METHODS: 500 µm hPCLS were generated and stored either in DMEM/F-12 medium or TiProtec (-/+) for up to 28 days. Viability, metabolic activity, and tissue structure were longitudinally determined. Bulk-RNA sequencing was used to study transcriptional changes, regulated signaling pathways, and changes in cellular composition after cold storage. Moreover, the induction of cold storage-associated cellular senescence was determined by transcriptomics and immunofluorescence (IF). To evaluate their potential for mechanistic studies in lung research, we evaluated the response to a previously described fibrotic cocktail after 7 and 14 days of cold storage in TiProtec (-/+) by IF and RT-qPCR. RESULTS: We demonstrated that TiProtec (+) preserves the viability, metabolic activity, transcriptional profile, and cellular composition of hPCLS for up to 14 days when compared to freshly sliced hPCLS. Moreover, cold storage did not significantly induce cellular senescence in hPCLS. Notably, TiProtec (+) downregulated pathways associated with cell death and inflammation while activating pathways protective against oxidative stress. Finally, cold-stored hPCLS remained responsive for up to 14 days to a fibrotic cocktail upregulating the expression of fibrosis-associated proteins such as fibronectin, alpha-smooth muscle actin, and alpha-1 type I collagen. CONCLUSION: This study provides for the first time insights into the transcriptional and functional changes associated with cold storage preservation of hPCLS. Moreover, it contributes to an optimized use of hPCLS, enabling banking, sharing, and on-demand processing and usage of hPCLS for translational lung research.

There is an urgent need for innovative therapies targeting defective epithelial repair in chronic diseases like COPD. The mesenchymal niche is a critical regulator in epithelial stem cell activation, suggesting that their secreted factors are possible potent drug targets. Utilizing a proteomics-guided drug discovery strategy, we explored the lung fibroblast secretome to uncover impactful drug targets. Our lung organoid assays identified several regenerative ligands, with osteoglycin (OGN) showing the most profound effects. Transcriptomic analyses revealed that OGN enhances alveolar progenitor differentiation, detoxifies reactive oxygen species, and strengthens fibroblast-epithelial crosstalk. OGN expression was diminished in COPD patients and smoke-exposed mice. An active fragment of OGN (leucine-rich repeat regions 4-7) replicated full-length OGN's regenerative effects, significantly ameliorating elastase-induced lung injury in lung slices and improving lung function in vivo. These findings highlight OGN as a pivotal secreted protein for alveolar epithelial repair, positioning its active fragment as a promising therapeutic for COPD.

BACKGROUND: Human precision-cut lung slices (hPCLS) are a unique platform for functional, mechanistic, and drug discovery studies in the field of respiratory research. However, tissue availability, generation, and cultivation time represent important challenges for their usage. Therefore, the present study evaluated the efficacy of a specifically designed tissue preservation solution, TiProtec, complete or in absence (-) of iron chelators, for long-term cold storage of hPCLS. METHODS: hPCLS were generated from peritumor control tissues and stored in DMEM/F-12, TiProtec, or TiProtec (-) for up to 28 days. Viability, metabolic activity, and tissue structure were determined. Moreover, bulk-RNA sequencing was used to study transcriptional changes, regulated signaling pathways, and cellular composition after cold storage. Induction of cold storage-associated senescence was determined by transcriptomics and immunofluorescence (IF). Finally, cold-stored hPCLS were exposed to a fibrotic cocktail and early fibrotic changes were assessed by RT-qPCR and IF. RESULTS: Here, we found that TiProtec preserves the viability, metabolic activity, transcriptional profile, as well as cellular composition of hPCLS for up to 14 days. Cold storage did not significantly induce cellular senescence in hPCLS. Moreover, TiProtec downregulated pathways associated with cell death, inflammation, and hypoxia while activating pathways protective against oxidative stress. Cold-stored hPCLS remained responsive to fibrotic stimuli and upregulated extracellular matrix-related genes such as fibronectin and collagen 1 as well as alpha-smooth muscle actin, a marker for myofibroblasts. CONCLUSIONS: Optimized long-term cold storage of hPCLS preserves their viability, metabolic activity, transcriptional profile, and cellular composition for up to 14 days, specifically in TiProtec. Finally, our study demonstrated that cold-stored hPCLS can be used for on-demand mechanistic studies relevant for respiratory research.

Transplanted organs are inevitably exposed to ischemia-reperfusion (IR) injury, which is known to cause graft dysfunction. Functional and structural changes that follow IR tissue injury are mediated by neutrophils through the production of oxygen-derived free radicals, as well as from degranulation which entails the release of proteases and other pro-inflammatory mediators. Neutrophil serine proteases (NSPs) are believed to be the principal triggers of post-ischemic reperfusion damage. Extended preservation times for the transplanted donor organ correlate with heightened occurrences of vascular damage and graft dysfunction. Preservation with α1-antitrypsin, an endogenous inhibitor of NSPs, improves primary graft function after lung or heart transplantation. Furthermore, pre-operative pharmacological targeting of NSP activation in the recipient using chemical inhibitors suppresses neutrophilic inflammation in transplanted organs. Hence, effective control of NSPs in the graft and recipient is a promising strategy to prevent IR injury. In this review, we describe the pathological functions of NSPs in IR injury and discuss their pharmacological inhibition to prevent primary graft dysfunction in lung or heart transplantation.

SARS-CoV-2 infection is associated with long-lasting neurological symptoms, although the underlying mechanisms remain unclear. Using optical clearing and imaging, we observed the accumulation of SARS-CoV-2 spike protein in the skull-meninges-brain axis of human COVID-19 patients, persisting long after viral clearance. Further, biomarkers of neurodegeneration were elevated in the cerebrospinal fluid from long COVID patients, and proteomic analysis of human skull, meninges, and brain samples revealed dysregulated inflammatory pathways and neurodegeneration-associated changes. Similar distribution patterns of the spike protein were observed in SARS-CoV-2-infected mice. Injection of spike protein alone was sufficient to induce neuroinflammation, proteome changes in the skull-meninges-brain axis, anxiety-like behavior, and exacerbated outcomes in mouse models of stroke and traumatic brain injury. Vaccination reduced but did not eliminate spike protein accumulation after infection in mice. Our findings suggest persistent spike protein at the brain borders may contribute to lasting neurological sequelae of COVID-19.

Targeted (nano-)drug delivery is essential for treating respiratory diseases, which are often confined to distinct lung regions. However, spatio-temporal profiling of drugs or nanoparticles (NPs) and their interactions with lung macrophages remains unresolved. Here, we present LungVis 1.0, an AI-powered imaging ecosystem that integrates light sheet fluorescence microscopy with deep learning-based image analysis pipelines to map NP deposition and dosage holistically and quantitatively across bronchial and alveolar (acinar) regions in murine lungs for widely-used bulk-liquid and aerosol-based delivery methods. We demonstrate that bulk-liquid delivery results in patchy NP distribution with elevated bronchial doses, whereas aerosols achieve uniform deposition reaching distal alveoli. Furthermore, we reveal that lung tissue-resident macrophages (TRMs) are dynamic, actively patrolling and redistributing NPs within alveoli, contesting the conventional paradigm of TRMs as static entities. LungVis 1.0 provides an advanced framework for exploring pulmonary delivery dynamics and deepening insights into TRM-mediated lung immunity.

Initially, identified as a Hodgkin lymphoma marker, CD30 was subsequently detected on a subset of human B cells within and around germinal centers (GCs). While CD30 expression is typically restricted to a few B cells, expansion of CD30-expressing B cells occurs in certain immune disorders and during viral infections. The role of CD30 in B cells remains largely unclear. To address this gap in knowledge, we established a conditional CD30-knockin mouse strain. In these mice, B-cell-specific CD30 expression led to a normal B-cell phenotype in young mice, but most aged mice exhibited significant expansion of B cells, T cells and myeloid cells and increased percentages of GC B cells and IgG1-switched cells. This may be driven by the expansion of CD4+ senescence-associated T cells and T follicular helper cells, which partially express CD30-L (CD153) and may stimulate CD30-expressing B cells. Inducing CD30 expression in antigen-activated B cells accelerates the GC reaction and augments plasma cell differentiation, possibly through the posttranscriptional upregulation of CXCR4. Furthermore, CD30 expression in GC B cells promoted the expansion of IgG1-switched cells, which displayed either a GC or memory-like B-cell phenotype, with abnormally high IgG1 levels compared with those in controls. These findings shed light on the role of CD30 signaling in GC B cells and suggest that elevated CD30+ B-cell numbers lead to pathological lymphocyte activation and proliferation.