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Comparison between human (a) and mouse (b) precision‐cut lung slices (PCLS) structure (Blue: DAPI staining of nuclei). (c) Section of an hPCLS infected with Legionella pneumophila (Red: expressing mScarlet‐I) 24 hr post infection (White: CellMask staining of cytoplasmic membranes). * mark alveolar spaces in (a) and (c). (a, c) were acquired by FV and (b) was acquired by FV and John Stegmayr
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Respiratory infections are leading causes of mortality worldwide. Most of the research on the underlying disease mechanisms is based on cell culture, organoid or surrogate animal models. Although these provide important insights, they have limitations. Cell culture models fail to recapitulate cellular interactions in the lung and animal models ofte...
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Small animal models that accurately model pathogenesis of SARS-CoV-2 variants are required for ongoing research efforts. We modified our human immune system mouse model to support replication of SARS-CoV-2 by implantation of human lung tissue into the mice to create TKO-BLT-Lung (L) mice and compared infection with two different variants in a human...
Citations
... However, there is currently no suitable culture model available to simulate the natural lung environment. PCLSs, an ex vivo model, have been effectively used to study pathogenic lung infections, including those caused by paramyxoviruses, rhinoviruses, adenoviruses and influenza viruses (Viana et al. 2021). In the study, we successfully established a porcine PCLSs ex-vivo model. ...
Respiratory coronaviruses (RCoVs) significantly threaten human health, necessitating the development of an ex vivo respiratory culture system for investigating RCoVs infection. Here, we successfully generated a porcine precision-cut lung slices (PCLSs) culture system, containing all resident lung cell types in their natural arrangement. Next, this culture system was inoculated with a porcine respiratory coronavirus (PRCV), exhibiting clinical features akin to humans who were infected by SARS-CoV-2. The results demonstrated that PRCV efficiently infected and replicated within PCLSs, targeting ciliated cells in the bronchioles, terminal bronchioles, respiratory bronchioles, and pulmonary alveoli. Additionally, through RNA-Seq analysis of the innate immune response in PCLSs following PRCV infection, expression levels of interferons, inflammatory cytokines and IFN stimulated genes were significantly upregulated. This ex vivo model may not only offer new insights into PRCV infection in the porcine respiratory tract but also serve as a valuable tool for studying human respiratory CoVs infection.
Supplementary Information
The online version contains supplementary material available at 10.1186/s13568-024-01717-0.
... Due to these characteristics, PCLS are bridging the gap between simple in vitro and complex in vivo systems (24), by recapitulating pathophysiological conditions and local inflammatory responses, that are believed to occur in vivo (28). The advantage of PCLS compared to lung explants is to ensure homogeneity and reproducibility, thus reducing the degree of variability between donors (29,30). ...
The recent coronavirus disease 2019 (COVID-19) outbreak revealed the susceptibility of elderly patients to respiratory virus infections, showing cell senescence or subclinical persistent inflammatory profiles and favouring the development of severe pneumonia. In our study, we evaluated the potential influence of lung aging on the efficiency of replication of influenza A virus (IAV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV 2), as well as determined the pro-inflammatory and antiviral responses of the distal lung tissue. Using precision-cut lung slices (PCLS) from donors of different ages, we found that pandemic H1N1 and avian H5N1 IAV replicated in the lung parenchyma with high efficacy. In contrast to these IAV strains, SARS-CoV-2 early isolate and Delta variant of concern (VOC) replicated less efficiently in PCLS. Interestingly, both viruses showed reduced replication in PCLS from older compared to younger donors, suggesting that aged lung tissue represents a sub optimal environment for viral replication. Regardless of the age-dependent viral loads, PCLS responded to infection with both viruses by an induction of IL-6 and IP-10/CXCL10 mRNAs, being highest for H5N1. Finally, while SARS-CoV-2 infection was not causing detectable cell death, IAV infection caused significant cytotoxicity and induced significant early interferon responses. In summary, our findings suggest that aged lung tissue might not favour viral dissemination, pointing to a determinant role of dysregulated immune mechanisms in the development of severe disease.
... Precision-cut tissue slices (PCTS) fill a gap between such in vitro and in vivo models and mimic a mini-organ model whilst preserving the tissue architecture and extracellular matrix. 5,6 The development of tissue slicers, e.g. vibratomes, 7 has allowed the generation of thinner slices with better preserved structural integrity. ...
Background
In academic research and the pharmaceutical industry, in vitro cell lines and in vivo animal models are considered as gold standards in modelling diseases and assessing therapeutic efficacy. However, both models have intrinsic limitations, whilst the use of precision-cut tissue slices can bridge the gap between these mainstream models. Precision-cut tissue slices combine the advantage of high reproducibility, studying all cell sub-types whilst preserving the tissue matrix and extracellular architecture, thereby closely mimicking a mini-organ. This approach can be used to replicate the biological phenotype of liver monogenic diseases using mouse models.
Methods
Here, we describe an optimised and easy-to-implement protocol for the culture of sections from mouse livers, enabling its use as a reliable ex-vivo model to assess the therapeutic screening of inherited metabolic diseases
Results
We show that precision-cut liver sections can be a reliable model for recapitulating the biological phenotype of inherited metabolic diseases, exemplified by common urea cycle defects such as citrullinemia type 1 and argininosuccinic aciduria, caused by argininosuccinic synthase (ASS1) and argininosuccinic lyase (ASL) deficiencies respectively.
Conclusions
Therapeutic response to gene therapy such as messenger RNA replacement delivered via lipid nanoparticles can be monitored, demonstrating that precision-cut liver sections can be used as a preclinical screening tool to assess therapeutic response and toxicity in monogenic liver diseases.
... To test this hypothesis, we performed a multicycle growth curve in a relevant tissue model; human precision cut lung slices (PCLS). These volumedefined tissue discs cut from healthy tumor bystander tissue of fresh lung resections, preserve the natural cell composition and 3D-architecture of lung tissue, and constitute, thus, a highly relevant preclinical model (24). PCLS from tissue of three human patients (Table S1) were infected with VN/1203 WT virus or the VN/ 1203 PB1-F2-ILVF. ...
Influenza A virus infection activates the NLRP3 inflammasome, a multiprotein signaling complex responsible for the proteolytic activation and release of the proinflammatory cytokine IL-1β from monocytes and macrophages. Some influenza A virus (IAV) strains encode a short 90-amino acid peptide (PB1-F2) on an alternative open reading frame of segment 2, with immunomodulatory activity. We recently demonstrated that contemporary IAV PB1-F2 inhibits the activation of NLRP3, potentially by NEK7-dependent activation. PB1-F2 binds to NLRP3 with its C-terminal 50 amino acids, but the exact binding motif was unknown. On the NLRP3 side, the interface is formed through the leucine-rich-repeat (LRR) domain, potentially in conjunction with the pyrin domain. Here, we took advantage of PB1-F2 sequences from IAV strains with either weak or strong NLRP3 interaction. Sequence comparison and structure prediction using Alphafold2 identified a short four amino acid sequence motif (TQGS) in PB1-F2 that defines NLRP3-LRR binding. Conversion of this motif to that of the non-binding PB1-F2 suffices to lose inhibition of NLRP3 dependent IL-1β release. The TQGS motif further alters the subcellular localization of PB1-F2 and its colocalization with NLRP3 LRR and pyrin domain. Structural predictions suggest the establishment of additional hydrogen bonds between the C-terminus of PB1-F2 and the LRR domain of NLRP3, with two hydrogen bonds connecting to threonine and glutamine of the TQGS motif. Phylogenetic data show that the identified NLRP3 interaction motif in PB1-F2 is widely conserved among recent IAV-infecting humans. Our data explain at a molecular level the specificity of NLRP3 inhibition by influenza A virus.
IMPORTANCE
Influenza A virus infection is accompanied by a strong inflammatory response and high fever. The human immune system facilitates the swift clearance of the virus with this response. An essential signal protein in the proinflammatory host response is IL-1b. It is released from inflammatory macrophages, and its production and secretion depend on the function of NLRP3. We had previously shown that influenza A virus blocks NLRP3 activation by the expression of a viral inhibitor, PB1-F2. Here, we demonstrate how this short peptide binds to NLRP3 and provide evidence that a four amino acid stretch in PB1-F2 is necessary and sufficient to mediate this binding. Our data identify a new virus-host interface required to block one signaling path of the innate host response against influenza A virus.
... Indeed, in several recent studies, PCLS from a variety of species, including human, have been used to elucidate the adherence of other microbial pathogens and local cytokine responses against these pathogens (21)(22)(23)(24). Therefore, from a clinical perspective, this new paper adds to the recent successful application of ex vivo PCLS systems for modeling lung disorders (25,26) and for studies of asthma (27), chronic obstructive pulmonary disease (COPD) (28), and idiopathic pulmonary fibrosis (29). Organotypic brain slice cultures have also been used successfully to study neuroimmune responses to brain infections with Cryptococcus neoformans (30), a fungal pathogen that usually infects the lung initially but later disseminates to cause injury to the brain. ...
Pneumocystis jirovecii is a major fungal pathogen of humans that causes life-threatening lung infections in immunocompromised individuals. Despite its huge global impact upon human health, our understanding of the pathobiology of this deadly fungus remains extremely limited, largely because it is not yet possible to cultivate Pneumocystis in vitro, independently of the host. However, a recent paper by Munyonho et al. offers a major step forward (F. T. Munyonho, R. D. Clark, D. Lin, M. S. Khatun, et al., 2023, mBio 15:e01464-23, https://doi.org/10.1128/mbio.01464-23 ). They show that it is possible to maintain both the trophozoite and cyst forms of the mouse pathogen, Pneumocystis murina, in precision-cut lung slices for several weeks. Furthermore, they demonstrate that this offers the exciting opportunity to examine potential virulence factors such as possible biofilm formation as well as antifungal drug responses in the lung.
... Then, PCLSs were incubated in Dulbecco's Modified Eagle Media (DMEM) supplemented with 10% FBS, 1% L-Glutamine, 1% NEAA, 1% Gentamicin and 1% Penicillin/Streptomycin at 37 • C, 5% CO 2 humidified incubator (Henjakovic et al., 2008). All these procedures made it possible for the PCLS tissue culture to remain viable, maintain normal metabolic activity, tissue homeostasis, structural integrity, and generate immune responses after viral infection (Lam et al., 2023;Nußbaum et al., 2022;Viana et al., 2022). ...
... Precision-cut lung slices (PCLS), uniformly sliced human or animal lung tissues generated by a vibrating microtome, have recently emerged as a novel ex vivo organo typic system for modeling lung inflammatory diseases (12,13). Several studies have reported the successful utilization of PCLS as an ex vivo platform to model various lung disorders such as asthma, chronic obstructive pulmonary disease, and idiopathic pulmonary fibrosis (14)(15)(16). ...
... Several studies have reported the successful utilization of PCLS as an ex vivo platform to model various lung disorders such as asthma, chronic obstructive pulmonary disease, and idiopathic pulmonary fibrosis (14)(15)(16). PCLSs have the main advantage of retaining a phenomenal cellular complexity and lung architecture, which provides a platform to study respira tory pathogens in an environment that closely mimic the in vivo lung conditions (12). Moreover, the PCLS model allows the generation of large numbers of uniformly cut tissue sections from a single lung tissue enabling multiple variables to be tested simultane ously. ...
... Moreover, the PCLS model allows the generation of large numbers of uniformly cut tissue sections from a single lung tissue enabling multiple variables to be tested simultane ously. This reduces the number of animals and their discomfort as well as the time needed for infection studies (12,15). Furthermore, PCLSs are reproducible and allow for high-resolution imaging of the lung tissue and cellular functions in three dimensions (17,18). ...
Pneumocystis pneumonia (PCP) is a serious fungal lung infection caused by Pneumocystis jirovecii in immunosuppressed individuals. The lack of viable in vitro/ex vivo PCP models has greatly hindered the progress in studying the biology of these fungi, the host/pathogen interactions, as well as antifungal susceptibility testing. In this study, we show the utility of precision-cut lung slices (PCLS) to support the survival of Pneumocystis murina in vitro . We cultured PCLS tissue derived from wild type and immunocompromised mice with a P. murina inoculum in submerged or air-liquid interface models for up to 14 days. We isolated total RNA from the cultured lung tissues at days 0, 3, 7, 10, and 14 and analyzed for the expression of host lung genes and P. murina genes ( Gsc1 for asci and Sp for trophs) by real-time quantitative PCR. When cultured in media alone, P. murina died gradually within a few days. However, when cultured on PCLS, both the troph and ascus forms survived throughout the incubation period of 2 weeks. Moreover, immunohistochemistry staining of P. murina inoculated PCLS sections using polyclonal anti- Pneumocystis sera and showed evidence of fungal aggregation and possible biofilm formation. Additionally, in vitro (PCLS) antibiotic susceptibility testing using commonly used antifungal drugs confirmed successful targeting of the troph and ascus forms by trimethoprim sulfamethoxazole and the ascus form by echinocandins.
IMPORTANCE
Our study reveals the potential of precision-cut lung slices as an ex vivo platform to study the growth/survival of Pneumocystis spp. that can facilitate the development of new anti-fungal drugs.
... In the present study, we investigated the transcriptional host response in a porcine PCLS model during infection with three S. suis strains of varying virulence potential: the highly virulent serotype 2 strain S10, the non-virulent serotype 2 strain T15, and the moderately virulent serotype 9 strain 8067. Despite the fact that there are limitations of PCLS, such as lack of cellular infiltration and adaptive immunity [41], the model proved to be a suitable tool for investigation of the pathogenic effect of S. suis in the porcine lung, evident from the observed induction of a host innate immune response mirroring findings from in vivo experimental challenge studies, including the transcriptional upregulation of IL1B, IL6, IL18, TNF, CXCL8, SAA, SELE, and NFKBIA [17,23]. ...
Streptococcus suis is a porcine and zoonotic pathogen in the upper respiratory tract, expressing different capsular serotypes and virulence-associated factors. Given its genomic and phenotypic diversity, the virulence potential of S. suis cannot be attributed to a single factor. Since strong in-flammatory response is a hallmark of S. suis infection, the objective of this study was to investigate the differences in transcriptional host responses to two serotype 2 and one serotype 9 strains. Both serotypes are frequently found in clinical isolates. We infected porcine precision-cut lung slices (PCLSs) with two serotype 2 strains of high (strain S10) and low (strain T15) virulence, and a serotype 9 strain 8067 of moderate virulence. We observed higher expression of inflammation-related genes during early infection with strains T15 and 8067, in contrast to infection with strain 10, whose expression peaked late. In addition, bacterial gene expression from infected PCLSs revealed differences, mainly of metabolism-related and certain virulence-associated bacterial genes amongst these strains. We conclude that the strain-and time-dependent induction of genes involved in innate immune response might reflect clinical outcomes of infection in vivo, implying rapid control of infection with less virulent strains compared to the highly virulent strain S10.
... Precision-cut tissue slices (PCTS) fill a gap between such in vitro and in vivo models and mimic a mini-organ model whilst preserving the tissue architecture and extracellular matrix. 5,6 The development of tissue slicers, e.g. vibratomes, 7 has allowed the generation of thinner slices with better preserved structural integrity. ...
Background In academic research and the pharmaceutical industry, in vitro cell lines and in vivo animal models are considered as gold standards in modelling diseases and assessing therapeutic efficacy. However, both models have intrinsic limitations, whilst the use of precision-cut tissue slices can bridge the gap between these mainstream models. Precision-cut tissue slices combine the advantage of high reproducibility, studying all cell sub-types whilst preserving the tissue matrix and extracellular architecture, thereby closely mimicking a mini-organ. This approach can be used to replicate the biological phenotype of liver monogenic diseases using mouse models. Methods Here, we describe an optimised and easy-to-implement protocol for the culture of sections from mouse livers, enabling its use as a reliable ex-vivo model to assess the therapeutic screening of inherited metabolic diseases Results We show that precision-cut liver sections can be a reliable model for recapitulating the biological phenotype of inherited metabolic diseases, exemplified by common urea cycle defects such as citrullinemia type 1 and argininosuccinic aciduria, caused by argininosuccinic synthase (ASS1) and argininosuccinic lyase (ASL) deficiencies respectively. Conclusions Therapeutic response to gene therapy such as messenger RNA replacement delivered via lipid nanoparticles can be monitored, demonstrating that precision-cut liver sections can be used as a preclinical screening tool to assess therapeutic response and toxicity in monogenic liver diseases.
... While precision-cut lung slices can preserve the threedimensional architecture and native environment of the lung 26 , it is important to note that generating PCLS is a complex and time-consuming process. The success of generating lung tissue slices greatly depends on the efficiency of agarose filling, which, in turn, is influenced by the presence of intact pleura and viable injection sites in the obtained tissue. ...
