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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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PDGFD-induced pericyte contractility drives pathological neoangiogenesis

Neovascular tufts (NVTs) are a defining feature of proliferative retinopathies such as diabetic retinopathy and retinopathy of prematurity, in which they compromise retinal integrity and vision. Excessive endothelial cell (EC) sprouting underlies their formation, yet the cellular and mechanical mechanisms that shape NVTs, and the contribution of pericytes (PCs) to this process, remain unclear. In this work, we investigated the role of actomyosin contractility in ECs and PCs during pathological angiogenesis and NVT formation. We identified non-muscle myosin IIA (NMIIA) and phosphorylated myosin light chains (MLCs) as a key signature of PCs and ECs in retinopathy and NVTs. Using inducible deletion of Myh9 in the oxygen-induced retinopathy mouse model, we uncover distinct and opposing roles for NMIIA in the two cell types. Endothelial NMIIA promotes neovascularisation and prevents enlargement of NVTs, whilst PC NMIIA drives neovascular tuft formation. Mechanistically, we identify platelet-derived growth factor D (PDGFD), but not PDGFB, as a driver of pathological PC actomyosin contractility through Rho-associated coiled-coil kinase (ROCK). Genetic deletion of Pdgfd partially phenocopies the loss of NMIIA in PCs, reducing NVT formation and lowering NMIIA and phosphorylated MLC levels within NVTs. Our findings establish cell-type-specific actomyosin contractility as a determinant of NVT pathogenesis and identify PDGFD-ROCK-NMIIA signalling in PCs as a therapeutic target in proliferative retinopathies.

cell biology↗

QuPath: Open source software for digital pathology image analysis

QuPath is new bioimage analysis software designed to meet the growing need for a user-friendly, extensible, open-source solution for digital pathology and whole slide image analysis. In addition to offering a comprehensive panel of tumor identification and high-throughput biomarker evaluation tools, QuPath provides researchers with powerful batch-processing and scripting functionality, and an extensible platform with which to develop and share new algorithms to analyze complex tissue images. Furthermore, QuPaths flexible design make it suitable for a wide range of additional image analysis applications across biomedical research.

pathology↗

FalseColor-Python: a rapid intensity-leveling and digital-staining package for fluorescence-based slide-free digital pathology

Slide-free digital pathology techniques, including nondestructive 3D microscopy, are gaining interest as alternatives to traditional slide-based histology. In order to facilitate clinical adoption of these fluorescence-based techniques, software methods have been developed to convert grayscale fluorescence images into color images that mimic the appearance of standard absorptive chromogens such as hematoxylin and eosin (H&E). However, these false-coloring algorithms often require manual and iterative adjustment of parameters, with results that can be inconsistent in the presence of intensity nonuniformities within an image and/or between specimens (intra- and inter-specimen variability). Here, we present an open-source (Python-based) rapid intensity-leveling and digital-staining package that is specifically designed to render two-channel fluorescence images (i.e. a fluorescent analog of H&E) to the traditional H&E color space for 2D and 3D microscopy datasets. However, this method can be easily tailored for other false-coloring needs. Our package offers (1) automated and uniform false coloring in spite of uneven staining within a large thick specimen, (2) consistent color-space representations that are robust to variations in staining and imaging conditions between different specimens, and (3) GPU-accelerated data processing to allow these methods to scale to large datasets. We demonstrate this platform by generating H&E-like images from cleared tissues that are fluorescently imaged in 3D with open-top light-sheet (OTLS) microscopy, and quantitatively characterizing the results in comparison to traditional slide-based H&E histology.Competing Interest StatementA.K.G. and J.T.C.L. are co-founders and shareholders of Lightspeed Microscopy Inc.View Full Text

pathology↗

Expression of ACE2, TMPRSS2, and CTSL in human airway epithelial cells under physiological and pathological conditions: Implications for SARS-CoV2 infection

SARS-CoV-2 enters into human airway epithelial cells via membrane fusion or endocytosis, and this process is dependent on ACE2, TMPRSS2, and cathepsin L. In this study, we examined the expression profiles of the three SARS-CoV-2 entry-related genes in primary human airway epithelial cells isolated from donors with different physiological and pathological backgrounds such as smoking, COPD, asthma, lung cancer, allergic rhinitis, cystic fibrosis, or viral infections. By reanalyzing 54 GEO datasets comprising transcriptomic data of 3428 samples, this study revealed that i) smoking is associated with an increased expression of ACE2 and TMPRSS2 and a decreased expression of cathepsin L; ii) infection of rhinovirus as well as poly(I:C) stimulation leads to high expression of all three SARS-CoV-2 entry-related genes; iii) expression of ACE2 and cathepsin L in nasal epithelial cells are decreased in patients with asthma and allergic rhinitis. In conclusion, this study implicates that infection of respiratory viruses, cigarette smoking and allergic respiratory diseases might affect the susceptibility to and the development of COVID-19.

pathology↗

Immunological and pathological outcomes of SARS-CoV-2 challenge after formalin-inactivated vaccine immunisation of ferrets and rhesus macaques

There is an urgent requirement for safe and effective vaccines to prevent novel coronavirus disease (COVID-19) caused by SARS-CoV-2. A concern for the development of new viral vaccines is the potential to induce vaccine-enhanced disease (VED). This was reported in several preclinical studies with both SARS-CoV-1 and MERS vaccines but has not been reported with SARS-CoV-2 vaccines. We have used ferret and rhesus macaques challenged with SARS-CoV-2 to assess the potential for VED in animals vaccinated with formaldehyde-inactivated SARS-CoV-2 (FIV) formulated with Alhydrogel, compared to a negative control vaccine in ferrets or unvaccinated macaques. We showed no evidence of enhanced disease in ferrets or rhesus macaques given FIV except for mild transient enhanced disease seen at seven days post infection in ferrets. This increased lung pathology was observed early in the infection (day 7) but was resolved by day 15. We also demonstrate that formaldehyde treatment of SARS-CoV-2 reduces exposure of the spike receptor binding domain providing a mechanistic explanation for suboptimal immunity.

pathology↗

Speckle illumination microscopy enables slide-free and non-destructive pathology of human lung adenocarcinoma

Histopathology based on formalin-fixed and paraffin-embedded tissues remains the gold standard for surgical margin assessment (SMA). However, routine pathological practice is lengthy and laborious, failing to provide immediate feedback to surgeons and pathologists for intraoperative decision-making. In this report, we propose a cost-effective and easy-to-use histological imaging method with speckle illumination microscopy (i.e., HiLo). HiLo can achieve rapid and non-destructive imaging of large and fluorescently-labelled resection tissues at an acquisition speed of 5 cm2/min with 1.3-m lateral resolution and 5.8-m axial resolution, demonstrating a great potential as an intraoperative SMA tool that can be used by surgeons and pathologists to detect residual tumors at surgical margins. It is experimentally validated that HiLo enables rapid diagnosis of different subtypes of human lung adenocarcinoma and hepatocellular carcinoma, producing images with remarkably recognizable cellular features comparable to the gold-standard histology. This work will facilitate the clinical translations of HiLo microscopy to improve the current standard-of-care.

pathology↗

Tracing the origin of pathologic pulmonary fibroblasts

Fibroblasts substantially remodel extracellular matrix (ECM) in response to tissue injury and generate fibrotic scars in chronic diseases. Recent studies have identified diverse fibroblast subsets in healthy and injured tissues. However, the origin(s) and functional importance of injury-induced fibroblast lineages remain unclear. Here we show that alveolar fibroblasts, which provide a niche for maintaining alveolar type 2 cells in uninjured lungs, are the dominant source of multiple emergent fibroblast subsets that sequentially arise to facilitate fibrosis after lung injury. We demonstrate that Cthrc1+ fibroblasts, which express the highest levels of ECM proteins at injured sites, are effector cells for fibrogenesis using a novel mouse tool, Cthrc1-CreER. We use another novel mouse tool, Scube2-CreER, that uniquely targets alveolar fibroblasts, to reveal that alveolar fibroblasts are the dominant origin for multiple fibroblast subsets that emerge after lung injury. Pseudotime and in vitro analysis suggest that inflammatory cytokines initially induce chemokine-producing inflammatory fibroblasts from alveolar fibroblasts, which can differentiate into Cthrc1+ fibrotic fibroblasts in response to TGF-{beta}. We identify similar fibroblast lineages from scRNA-seq in human pulmonary fibrosis. These results elucidate the pathologic fibroblast lineage development in response to lung injury and suggest that targeting key steps in transitions among these subsets could provide novel strategies for the treatment of fibrotic diseases.

pathology↗

Label-free and non-destructive pathology of human lung adenocarcinomas with ultraviolet single-plane illumination microscopy

Lung cancer is one of the leading causes of cancer death worldwide. The diagnosis of lung cancer based on the analysis of formalin-fixed and paraffin-embedded (FFPE) tissues is laborious and time-consuming, failing to guide surgeons intraoperatively. Here we proposed a rapid histological imaging method, termed microscopy with ultraviolet single-plane illumination (MUSI), to enable rapid ex-or in-vivo imaging of fresh and unprocessed tissues in a label-free and non-destructive manner. The MUSI system allows surgical specimens with large irregular surfaces to be screened at a speed of 0.5 mm2/s with a subcellular resolution, which is sufficient to provide immediate feedback to surgeons and pathologists for intraoperative decision-making. We demonstrate that MUSI can differentiate between different subtypes of human lung adenocarcinomas, revealing diagnostically important features that are comparable to the gold standard FFPE histology. As an assistive imaging platform, MUSI could facilitate the development of precise image-guided surgery and revolutionize the current practice in surgical pathology.

pathology↗

Benchmarking robust spatial transcriptomics approaches to capture the molecular landscape and pathological architecture of archived cancer tissues

AbtractsApplying spatial transcriptomics (ST) to explore a vast amount of formalin-fixed paraffin-embedded (FFPE) archival cancer tissues has been highly challenging due to several critical technical issues. In this work, we optimised ST protocols to generate unprecedented spatial gene expression data for FFPE skin cancer. Skin is among the most challenging tissue types for ST due to its fibrous structure and a high risk of RNAse contamination. We evaluated tissues collected from ten years to two years ago, spanning a range of tissue qualities and complexity. Technical replicates and multiple patient samples were assessed. Further, we integrated gene expression profiles with pathological information, revealing a new layer of molecular information. Such integration is powerful in cancer research and clinical applications. The data allowed us to detect the spatial expression of non-coding RNAs. Together, this work provides important technical perspectives to enable the applications of ST on archived cancer tissues.

pathology↗

Drosophila model to clarify the pathological significance of OPA1 in autosomal dominant optic atrophy

Autosomal dominant optic atrophy (DOA) is a progressive form of blindness caused by degeneration of retinal ganglion cells and their axons, mainly caused by mutations in the OPA1 mitochondrial dynamin like GTPase (OPA1) gene. OPA1 encodes a dynamin-like GTPase present in the mitochondrial inner membrane. When associated with OPA1 mutations, DOA can present not only ocular symptoms but also multi-organ symptoms (DOA plus). DOA plus often results from point mutations in the GTPase domain, which are assumed to have dominant negative effects. However, the presence of mutations in the GTPase domain does not always result in DOA plus. Therefore, an experimental system to distinguish between DOA and DOA plus is needed. In this study, we found that loss-of-function mutations of the dOPA1 gene in Drosophila can imitate the pathology of optic nerve degeneration observed in DOA. We successfully rescued this degeneration by expressing the human OPA1 (hOPA1) gene, indicating that hOPA1 is functionally interchangeable with dOPA1 in the fly system. However, mutations previously identified did not ameliorate the dOPA1 deficiency phenotype. By expressing both WT and DOA plus mutant hOPA1 forms in the optic nerve of dOPA1 mutants, we observed that DOA plus mutations suppressed the rescue, facilitating the distinction between loss-of-function and dominant negative mutations in hOPA1. This fly model aids in distinguishing DOA from DOA plus and guides initial hOPA1 mutation treatment strategies.

pathology↗

Low circulating choline, a modifiable dietary factor, is associated with the pathological progression and metabolome dysfunction in Alzheimers disease.

Most Americans ([~]90%) are deficient in dietary choline, an essential nutrient. Associations between circulating choline and pathological progression in Alzheimers disease (AD) remain unknown. Here, we examined these associations and performed a metabolomic analysis in blood serum from severe AD, moderate AD, and healthy controls. Additionally, to gain mechanistic insight, we assessed the effects of dietary choline deficiency (Ch-) in 3xTg-AD mice and choline supplementation (Ch+) in APP/PS1 mice. In humans, we found AD-associated reductions in choline, its derivative acetylcholine (ACh), and elevated pro-inflammatory cytokine TNF. Choline and ACh were negatively correlated with Plaque density, Braak stage, and TNF, but positively correlated with MMSE and brain weight. Metabolites L-Valine, 4-Hydroxyphenylpyruvic, Methylmalonic, and Ferulic acids were associated with choline levels. In mice, Ch-paralleled AD severe, but Ch+ was protective. In conclusion, low circulating choline is associated with AD-neuropathological progression, illustrating the importance of dietary choline consumption to offset disease.

pathology↗

A transcriptomics-based drug repositioning approach to identify drugs with similar activities for the treatment of muscle pathologies in spinal muscular atrophy (SMA) models

Spinal muscular atrophy (SMA) is a genetic neuromuscular disorder caused by the reduction of survival of motor neuron (SMN) protein levels. Although three SMN-augmentation therapies are clinically approved that significantly slow down disease progression, they are unfortunately not cures. Thus, complementary SMN-independent therapies that can target key SMA pathologies and that can support the clinically approved SMN-dependent drugs are the forefront of therapeutic development. We have previously demonstrated that prednisolone, a synthetic glucocorticoid (GC) improved muscle health and survival in severe Smn-/-;SMN2 and intermediate Smn2B/- SMA mice. However, long-term administration of prednisolone can promote myopathy. We thus wanted to identify genes and pathways targeted by prednisolone in skeletal muscle to discover clinically approved drugs that are predicted to emulate prednisolones activities. Using an RNA-sequencing, bioinformatics and drug repositioning pipeline on skeletal muscle from symptomatic prednisolone- treated and untreated Smn-/-;SMN2 SMA and Smn+/-;SMN2 healthy mice, we identified molecular targets linked to prednisolones ameliorative effects and a list of 580 drug candidates with similar predicted activities. Two of these candidates, metformin and oxandrolone, were further investigated in SMA cellular and animal models, which highlighted that these compounds do not have the same ameliorative effects on SMA phenotypes as prednisolone; however, a number of other important drug targets remain. Overall, our work further supports the usefulness of prednisolones potential as a second-generation therapy for SMA, identifies a list of potential SMA drug treatments and highlights improvements for future transcriptomic-based drug repositioning studies in SMA.

pathology↗

Spatial Omics Driven Crossmodal Pretraining Applied to Graph-based Deep Learning for Cancer Pathology Analysis

Graph-based deep learning has shown great promise in cancer histopathology image analysis by contextualizing complex morphology and structure across whole slide images to make high quality downstream outcome predictions (ex: prognostication). These methods rely on informative representations (i.e., embeddings) of image patches comprising larger slides, which are used as node attributes in slide graphs. Spatial omics data, including spatial transcriptomics, is a novel paradigm offering a wealth of detailed information. Pairing this data with corresponding histological imaging localized at 50-micron resolution, may facilitate the development of algorithms which better appreciate the morphological and molecular underpinnings of carcinogenesis. Here, we explore the utility of leveraging spatial transcriptomics data with a contrastive crossmodal pretraining mechanism to generate deep learning models that can extract molecular and histological information for graph-based learning tasks. Performance on cancer staging, lymph node metastasis prediction, survival prediction, and tissue clustering analyses indicate that the proposed methods bring improvement to graph based deep learning models for histopathological slides compared to leveraging histological information from existing schemes, demonstrating the promise of mining spatial omics data to enhance deep learning for pathology workflows.

pathology↗

A weighted and cumulative point system for accurate scoring of intestinal pathology in a piglet model of necrotizing enterocolitis

BackgroundNecrotizing enterocolitis (NEC) is a serious condition, primarily affecting premature infants, in which a portion of the gut undergoes inflammation and necrosis. Symptoms of NEC are unspecific, and together with a rapid progression, the disease remains a significant concern. The preterm pig develops NEC spontaneously, making it a suitable model for exploring novel treatments. During piglet necropsy, NEC-lesions closely resemble the pathologies found during surgery or autopsy of preterm infants. As such, the systematic gross inspection enables direct evaluation of gut lesions, which is not possible in the medical preterm patient. Here, we introduce a revised intestinal scoring system with an expanded score range and more detailed descriptive features to accurately describe the diversity of NEC-lesions in the preterm piglet model. MethodsWe included 333 preterm piglets from four separate experiments, each delivered via cesarian section at 90% gestation. The pigs were fed either a gently processed (GP) or harshly processed (HP) milk formula for 96 hours and were subsequently euthanized. At necropsy, the gastrointestinal tract was assessed with 1) an established 6-grade scoring system and 2) a systematic and descriptive approach focusing on the distribution and severity of hyperemia, hemorrhage, pneumatosis intestinalis (intramural gas), and necrosis. Lesion biopsies were sampled for cytokine measurement and a subset (n = 62) was sampled for histopathological assessment. ResultsThe systematic and descriptive registrations were evaluated and converted into a weighted and cumulative point (WCP) score. Compared to the 6-grade score, the WCP score enabled a higher discrepancy in severity levels, especially among organs with more prominent NEC lesions. IL-1{beta} in small intestinal lesions and both IL-8 and IL-1{beta} in colon lesions correlated positively with the WCP scale. A histopathological grade system (0-8) was established and revealed mucosal lesions not recognized macroscopically. Finally, the WCP score showed a higher NEC-promoting effect of the HP formula compared to the GP formula. ConclusionThe validation of the weighted and cumulative scoring system demonstrated an expanded score range, enhancing the accuracy in describing NEC-lesions in gastrointestinal segments of preterm pigs. This approach may increase the efficiency of preclinical NEC experiments.

pathology↗

Neuroinflammation and Spinal Cord Pathology Associated with Hypertension in Spontaneously Hypertensive Stroke-Prone Rats

Hypertension is a major contributor to cardiovascular and cerebrovascular disease, leading to significant central nervous system (CNS) damage, including in the spinal cord. This study examines the spinal cord in uncontrolled hypertension of stroke-prone spontaneously hypertensive rats (SHRsp) compared to normotensive Wistar-Kyoto rats (WKY). Histological analyses, including Sudan Black B staining, NADPH-diaphorase histochemistry, immunohistochemistry, and Western blotting, were used to assess inflammation, gliosis, oxidative stress, and axonal injury. SHRsp rats displayed extensive spinal cord pathology, including macrophage infiltration, astrocyte hypertrophy, and demyelination. ED1 immunostaining revealed significant microglial activation in SHTsp, particularly in the ventrolateral white matter, while Sudan Black B staining showed edema and lipid-laden macrophages in dilated perivascular spaces. Increased NADPH-diaphorase activity in SHRsp indicated heightened nitric oxide synthase activity. Glial fibrillary acidic protein (GFAP) immunostaining confirmed significant astrogliosis, with hypertrophic astrocytes more prominent in SHRsp. Western blot analysis showed elevated amyloid precursor protein (APP) levels in SHRsp, associated with axonal injury and oxidative stress. APP-positive phagocytes in white matter further suggested active axonal degeneration. These findings demonstrate significant neuroinflammation, gliosis, and spinal cord tissue damage in hypertensive rats, highlighting the spinal cords vulnerability to hypertension. This underscores the importance of addressing hypertensive damage in the CNS, particularly in patients at risk of neurodegenerative or motor impairments.

pathology↗

Molecular and Pathological Characterization of Cervicovaginal Specimens Evince Significant Protection Against HPV Infections, Bacterial Vaginosis, and Epithelial Cell Abnormalities by Lactobacillus species

BackgroundBacterial vaginosis (BV) results in dysbiosis in the vaginal microbiome and facilitates secondary human papilloma virus (HPV) infections, which can cause cervical abnormalities. The effects of Lactobacilli on BV-HPV infections and cervicovaginal pathology were investigated. MethodElectronic molecular and cytology laboratory results, and demographic records of 19,105 females screened for BV, HPV, and cervical cancer between August 2021 and April 2023 were collected. After data cleaning and reorganization, 15, 607 sample results were used. The data was subjected to epidemiological, statistical, and machine learning analyses to identify patterns, correlations, associations, and interactive effects of age, State, Lactobacilli, BV-associated pathogens, high-risk HPV (hrHPV) genotypes, BV, and cervical cytology. ResultsThe final dataset comprised of data from anonymized females (n = 15541, 99.57%), males (n = 13, 0.08%) and unknown gender (n = 53, 0.34%) between 14 and 95 years from 32 States and Washington D.C. Most samples were BV positive (n = 8282, 53.07%) and/or NILM (n = 11749) followed by 14.29% ASCUS (n = 2231), 7.14% LSIL (n = 1115), 1.04% RCC (n = 163), 0.53% HSIL (n = 82), 0.39% ASC-H (n = 61), and 0.16% AGC (n = 25). HPV-positive samples had higher BV pathogens and BV-positive diagnosis (between 51.90% and 68.60%) than BV-negative diagnosis. Lactobacillus gasseri (79.75%), Lactobacillus jensenii (82.68%), Lactobacillus crispatus (90.91%) were mostly found in BV-negative and NILM specimens. BV and ECA outcomes were significantly associated with age and State. Statistical, correlation, and machine learning models predictively confirmed these findings. ConclusionL. crispatus, followed by L. gasseri and L. jensenii, significantly maintains a healthy cervicovaginal microbiome by inhibiting harmful bacterial pathogens and high-risk HPV subtypes; L. iners have an opposing or neutral effect. Lay summary/importance/highlightsBacterial vaginosis (BV) and human papillomavirus (HPV) infections mostly occur in sexually active and reproductive-age women, adversely affecting their quality of life and predisposing them to sequelae of reproductive disorders. In this study, the protective effects of Lactobacilli against BV and HPV infections to maintain a healthy cervicovaginal microbiome were investigated. Lactobacillus crispatus, L. gasseri, and L. jensenii were common in clinical specimens that had no or relatively lower concentrations of hrHPV, BV, and cervico-vaginal cell abnormalities and vice versa. This shows how important these Lactobacilli are in maintaining a healthy cervix and vagina. Age and BV were found to be strongly associated with the incidence of HPV infections and epithelial cell abnormalities (ECAs). Some States had higher prevalence of BV and ECAs than others. Co-infection with multiple hrHPV and/or hrHPV-BV pathogens segued into negative health effects; some interactions were beneficial for the microbiome, albeit those were few. This makes multiple HPV genotype infections dangerous.

pathology↗

Pathology of Influenza A (H5N1) infection in pinnipeds reveals novel tissue tropism and vertical transmission.

In 2023, an unprecedented outbreak of highly pathogenic avian influenza (HPAI) H5N1 resulted in the death of thousands of pinnipeds along the Argentinean coast, raising concerns about its ecological and epidemiological impact. Here, we present clinical, pathological, and molecular findings associated with HPAI H5N1 infection in pinnipeds from Chubut, Argentina. Necropsies were conducted on three South American Sea Lions (SASLs) (Otaria flavescens) and one Southern Elephant Seal (SES) (Mirounga leonina), followed by histopathological, immunohistochemical and RT-sqPCR analyses. Neurological clinical signs were observed in two SASLs, with one also exhibiting respiratory distress. Neuropathological findings included lymphoneutrophilic meningoencephalomyelitis and choroiditis, neuronal necrosis, gliosis, hemorrhages, and perivascular cuffing. Viral antigen was localized in neurons, glial cells, choroid plexus epithelial cells, ependymal cells, and the neuropil. Systemic manifestations included HPAI-related necrotizing myocarditis in the elephant seal and placental necrosis in a sea lion, with fetal tissues testing positive for HPAIV. Pulmonary lesions were minimal, limited to bronchial glands in one individual. RT-sqPCR confirmed HPAI H5 in all tested animals. Our findings highlight the neurotropism of HPAI H5N1 in pinnipeds, and expand the known systemic effects of the virus, revealing new tissue tropism and vertical transmission.

pathology↗

Mouse pulmonary pathological characteristics induced by Asian-mineral dust transported with Coniothyrium fuckelii at a high altitude of 2,000 meters

Asian sand dust (ASD) events are known to transport not only mineral particles but also bioaerosols such as fungi across East Asia, posing complex respiratory health risks. Previously, we isolated Coniothyrium fuckelii (Strain Con-15H316)--a dematiaceous phytopathogenic fungus--from the ASD samples collected at an altitude of 2000 m over the Noto Peninsula, Japan. While Con-15H316 elicited potent immunological responses in our earlier cytokine-focused studies, its histopathological impact on mammalian lungs remained uncharacterized. Here, we re-evaluated formalin-fixed murine lung tissues from mice intratracheally administered heat-treated ASD (H-ASD: only mineral particles), Con-15H316, and Con-15H316 in combination with H-ASD. H-ASD alone induced macrophage-dominant alveolar granulomas, and Con-15H316 alone triggered peribronchiolitis with eosinophilic infiltration. Strikingly, co-exposure resulted in a qualitatively distinct and synergistically worsened phenotype, featuring neutrophil-rich suppurative alveolar pneumonia, granulomas, and peribronchiolitis. Bronchoalveolar lavage fluid (BALF) analysis revealed marked co-elevation of lactate dehydrogenase (LDH) and CCL2 (C-C motif chemokine 2, also known as monocyte chemoattractant protein-1, MCP-1), suggesting concurrent tissue injury and monocyte-driven immune activation. To elucidate the cellular origin of the CCL2 protein, we reanalyzed two publicly available single-cell RNA-seq datasets, identifying monocytes and interstitial macrophages as the dominant Ccl2-expressing populations in microbially injured lungs. Although C. fuckelii has been recognized generally as a plant pathogen, this study exhibited the first histological evidence of the potential of this fungus to induce or exacerbate mammalian lung injury, especially in combination with mineral dust. Our findings uncover a novel, non-allergic alveolar injury phenotype associated with microbial-particulate co-exposure, expanding current toxicological paradigms beyond pathologies associated with excessive immune reactions to inhaled particulates and bioaerosols.

pathology↗