Lung Organoids Research Articles

Advance in Human Epithelial-Derived Organoids Research.

Organoids have complex three-dimensional structures that exhibit functionalities and feature architectures similar to those of in vivo organs and are developed from adult stem cells, embryonic stem cells, and pluripotent stem cells through a self-organization process. Organoids derived from adult epithelial stem cells are the most mature and extensive. In recent years, using organoid culture techniques, researchers have established various adult human tissue-derived epithelial organoids, including intestinal, colon, lung, liver, stomach, breast, and oral mucosal organoids, all of which exhibit strong research and application prospects. Studies have shown that epithelial organoids are mainly applied in drug discovery, personalized drug response testing, disease mechanism research, and regenerative medicine. In this review, we mainly discuss current organoid culture systems and potential applications of this technique with human epithelial tissue.

Discovery and Mechanism of SARS-CoV-2 Main Protease Inhibitors.

The emergence of a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), presents an urgent public health crisis. Without available targeted therapies, treatment options remain limited for COVID-19 patients. Using medicinal chemistry and rational drug design strategies, we identify a 2-phenyl-1,2-benzoselenazol-3-one class of compounds targeting the SARS-CoV-2 main protease (Mpro). FRET-based screening against recombinant SARS-CoV-2 Mpro identified six compounds that inhibit proteolysis with nanomolar IC50 values. Preincubation dilution experiments and molecular docking determined that the inhibition of SARS-CoV-2 Mpro can occur by either covalent or noncovalent mechanisms, and lead E04 was determined to inhibit Mpro competitively. Lead E24 inhibited viral replication with a nanomolar EC50 value (844 nM) in SARS-CoV-2-infected Vero E6 cells and was further confirmed to impair SARS-CoV-2 replication in human lung epithelial cells and human-induced pluripotent stem cell-derived 3D lung organoids. Altogether, these studies provide a structural framework and mechanism of Mpro inhibition that should facilitate the design of future COVID-19 treatments.

Gastric Organoids: Progress and Remaining Challenges.

The stomach is a complex and physiologically necessary organ, yet large differences in physiology between mouse and human stomachs have impeded translation of physiological discoveries and drug screens performed using murine gastric tissues. Gastric cancer (GC) is a global health threat, with a high mortality rate and limited treatment options. The heterogeneous nature of GC makes it poorly suited for current “one size fits all” standard treatments. In this review, we discuss the rapidly evolving field of gastric organoids, with a focus on studies expanding cultures from primary human tissues and describing the benefits of mouse organoid models. We introduce the differing methods for culturing healthy gastric tissue from adult tissues or pluripotent stem cells, discuss the promise these systems have for preclinical drug screens, and highlight applications of organoids for precision medicine. Finally, we discuss the limitations of these models and look to the future to present potential ways gastric organoids will advance treatment options for patients with GC.

Identifying FDA-approved drugs with multimodal properties against COVID-19 using a data-driven approach and a lung organoid model of SARS-CoV-2 entry

BackgroundVaccination programs have been launched worldwide to halt the spread of COVID-19. However, the identification of existing, safe compounds with combined treatment and prophylactic properties would be beneficial to individuals who are waiting to be vaccinated, particularly in less economically developed countries, where vaccine availability may be initially limited.MethodsWe used a data-driven approach, combining results from the screening of a large transcriptomic database (L1000) and molecular docking analyses, with in vitro tests using a lung organoid model of SARS-CoV-2 entry, to identify drugs with putative multimodal properties against COVID-19.ResultsOut of thousands of FDA-approved drugs considered, we observed that atorvastatin was the most promising candidate, as its effects negatively correlated with the transcriptional changes associated with infection. Atorvastatin was further predicted to bind to SARS-CoV-2’s main protease and RNA-dependent RNA polymerase, and was shown to inhibit viral entry in our lung organoid model.ConclusionsSmall clinical studies reported that general statin use, and specifically, atorvastatin use, are associated with protective effects against COVID-19. Our study corroborrates these findings and supports the investigation of atorvastatin in larger clinical studies. Ultimately, our framework demonstrates one promising way to fast-track the identification of compounds for COVID-19, which could similarly be applied when tackling future pandemics.

Development and Functional Characterization of Fetal Lung Organoids.

Preterm infants frequently suffer from pulmonary complications due to a physiological and structural lung immaturity resulting in significant morbidity and mortality. Novel in vitro and in vivo models are required to study the underlying mechanisms of late lung maturation and to facilitate the development of new therapeutic strategies. Organoids recapitulate essential aspects of structural organization and possibly organ function, and can be used to model developmental and disease processes. We aimed at generating fetal lung organoids (LOs) and to functionally characterize this in vitro model in comparison to primary lung epithelial cells and lung explants ex vivo. LOs were generated with alveolar and endothelial cells from fetal rat lung tissue, using a Matrigel-gradient and air-liquid-interface culture conditions. Immunocytochemical analysis showed that the LOs consisted of polarized epithelial cell adhesion molecule (EpCAM)-positive cells with the apical membrane compartment facing the organoid lumen. Expression of the alveolar type 2 cell marker, RT2-70, and the Club cell marker, CC-10, were observed. Na+ transporter and surfactant protein mRNA expression were detected in the LOs. First time patch clamp analyses demonstrated the presence of several ion channels with specific electrophysiological properties, comparable to vital lung slices. Furthermore, the responsiveness of LOs to glucocorticoids was demonstrated. Finally, maturation of LOs induced by mesenchymal stem cells confirmed the convenience of the model to test and establish novel therapeutic strategies. The results showed that fetal LOs replicate key biological lung functions essential for lung maturation and therefore constitute a suitable in vitro model system to study lung development and related diseases.

The Promise of Lung Organoids for Growth and Investigation of Pneumocystis Species.

Pneumocystis species (spp.) are host-obligate fungal parasites that colonize and propagate almost exclusively in the alveolar lumen within the lungs of mammals where they can cause a lethal pneumonia. The emergence of this pneumonia in non-HIV infected persons caused by Pneumocystis jirovecii (PjP), illustrates the continued importance of and the need to understand its associated pathologies and to develop new therapies and preventative strategies. In the proposed life cycle, Pneumocystis spp. attach to alveolar type 1 epithelial cells (AEC1) and prevent gas exchange. This process among other mechanisms of Pneumocystis spp. pathogenesis is challenging to observe in real time due to the absence of a continuous ex vivo or in vitro culture system. The study presented here provides a proof-of-concept for the development of murine lung organoids that mimic the lung alveolar sacs expressing alveolar epithelial type 1 cells (AEC1) and alveolar type 2 epithelial cells (AEC2). Use of these 3-dimensional organoids should facilitate studies of a multitude of unanswered questions and serve as an improved means to screen new anti- PjP agents.

Epithelial–stromal cell interactions and extracellular matrix mechanics drive the formation of airway-mimetic tubular morphology in lung organoids

SummaryComplex human airway cellular organization where extracellular matrix (ECM) and epithelial and stromal lineages interact present challenges for organ study in vitro. Current in vitro lung models that focus on the lung epithelium do not represent complex airway morphology and cell-ECM interactions seen in vivo. Models including stromal populations often separate them via a semipermeable barrier precluding cell–cell interaction or the effect of ECM mechanics. We investigated the effect of stromal cells on basal epithelial cell-derived bronchosphere structure and function through a triple culture of human bronchial epithelial, lung fibroblast, and airway smooth muscle cells. Epithelial–stromal cross-talk resulted in epithelial cell-driven branching tubules with stromal cells surrounding epithelial cells termed bronchotubules. Agarose– Matrigel scaffold (Agrigel) formed a mechanically tuneable ECM, with adjustable viscoelasticity and stiffness enabling long-term tubule survival. Bronchotubule models may enable research into how epithelial–stromal cell and cell–ECM communication drive tissue patterning, repair, and development of disease.

Adult stem cell-derived complete lung organoid models emulate lung disease in COVID-19.

SARS-CoV-2, the virus responsible for COVID-19, causes widespread damage in the lungs in the setting of an overzealous immune response whose origin remains unclear. We present a scalable, propagable, personalized, cost-effective adult stem cell-derived human lung organoid model that is complete with both proximal and distal airway epithelia. Monolayers derived from adult lung organoids (ALOs), primary airway cells, or hiPSC-derived alveolar type II (AT2) pneumocytes were infected with SARS-CoV-2 to create in vitro lung models of COVID-19. Infected ALO monolayers best recapitulated the transcriptomic signatures in diverse cohorts of COVID-19 patient-derived respiratory samples. The airway (proximal) cells were critical for sustained viral infection, whereas distal alveolar differentiation (AT2→AT1) was critical for mounting the overzealous host immune response in fatal disease; ALO monolayers with well-mixed proximodistal airway components recapitulated both. Findings validate a human lung model of COVID-19, which can be immediately utilized to investigate COVID-19 pathogenesis and vet new therapies and vaccines. This work was supported by the National Institutes for Health (NIH) grants 1R01DK107585-01A1, 3R01DK107585-05S1 (to SD); R01-AI141630, CA100768 and CA160911 (to PG) and R01-AI 155696 (to PG, DS and SD); R00-CA151673 and R01-GM138385 (to DS), R01- HL32225 (to PT), UCOP-R00RG2642 (to SD and PG), UCOP-R01RG3780 (to P.G. and D.S) and a pilot award from the Sanford Stem Cell Clinical Center at UC San Diego Health (P.G, S.D, D.S). GDK was supported through The American Association of Immunologists Intersect Fellowship Program for Computational Scientists and Immunologists. L.C.A's salary was supported in part by the VA San Diego Healthcare System. This manuscript includes data generated at the UC San Diego Institute of Genomic Medicine (IGC) using an Illumina NovaSeq 6000 that was purchased with funding from a National Institutes of Health SIG grant (#S10 OD026929).

A drug screen with approved compounds identifies amlexanox as a novel Wnt/β-catenin activator inducing lung epithelial organoid formation.

Background and Purpose:Emphysema is an incurable disease characterized by loss of lung tissue leading to impaired gas exchange. Wnt/β-catenin signalling is reduced in emphysema, and exogenous activation of the pathway in experimental models in vivo and in human ex vivo lung tissue improves lung function and structure. We sought to identify a pharmaceutical able to activate Wnt/β-catenin signalling and assess its potential to activate lung epithelial cells and repair.Experimental Approach:We screened 1216 human-approved compounds for Wnt/β-catenin signalling activation using luciferase reporter cells and selected candidates based on their computationally predicted protein targets. We further performed confirmatory luciferase reporter and metabolic activity assays. Finally, we studied the regenerative potential in murine adult epithelial cell-derived lung organoids and in vivo using a murine elastase-induced emphysema model.Key Results:The primary screen identified 16 compounds that significantly induced Wnt/β-catenin-dependent luciferase activity. Selected compounds activated Wnt/β-catenin signalling without inducing cell toxicity or proliferation. Two compounds were able to promote organoid formation, which was reversed by pharmacological Wnt/β-catenin inhibition, confirming the Wnt/β-catenin-dependent mechanism of action. Amlexanox was used for in vivo evaluation, and preventive treatment resulted in improved lung function and structure in emphysematous mouse lungs. Moreover, gene expression of Hgf, an important alveolar repair marker, was increased, whereas disease marker Eln was decreased, indicating that amlexanox induces proregenerative signalling in emphysema.Conclusion and Implications:Using a drug screen based on Wnt/β-catenin activity, organoid assays and a murine emphysema model, amlexanox was identified as a novel potential therapeutic agent for emphysema.

Airway and Alveoli Organoids as Valuable Research Tools in COVID-19.

The coronavirus disease 2019 (COVID-19), caused by the novel coronavirus, SARS-CoV-2, affects tissues from different body systems but mostly the respiratory system, and the damage evoked in the lungs may occasionally result in severe respiratory complications and eventually lead to death. Studies of human respiratory infections have been limited by the scarcity of functional models that mimic in vivo physiology and pathophysiology. In the last decades, organoid models have emerged as potential research tools due to the possibility of reproducing in vivo tissue in culture. Despite being studied for over one year, there is still no effective treatment against COVID-19, and investigations using pulmonary tissue and possible therapeutics are still very limited. Thus, human lung organoids can provide robust support to simulate SARS-CoV-2 infection and replication and aid in a better understanding of their effects in human tissue. The present review describes methodological aspects of different protocols to develop airway and alveoli organoids, which have a promising perspective to further investigate COVID-19.

SOX21 modulates SOX2-initiated differentiation of epithelial cells in the extrapulmonary airways.

SOX2 expression levels are crucial for the balance between maintenance and differentiation of airway progenitor cells during development and regeneration. Here, we describe patterning of the mouse proximal airway epithelium by SOX21, which coincides with high levels of SOX2 during development. Airway progenitor cells in this SOX2+/SOX21+ zone show differentiation to basal cells, specifying cells for the extrapulmonary airways. Loss of SOX21 showed an increased differentiation of SOX2+ progenitor cells to basal and ciliated cells during mouse lung development. We propose a mechanism where SOX21 inhibits differentiation of airway progenitors by antagonizing SOX2-induced expression of specific genes involved in airway differentiation. Additionally, in the adult tracheal epithelium, SOX21 inhibits basal to ciliated cell differentiation. This suppressing function of SOX21 on differentiation contrasts SOX2, which mainly drives differentiation of epithelial cells during development and regeneration after injury. Furthermore, using human fetal lung organoids and adult bronchial epithelial cells, we show that SOX2+/SOX21+ regionalization is conserved. Lastly, we show that the interplay between SOX2 and SOX21 is context and concentration dependent leading to regulation of differentiation of the airway epithelium.

Oncogenic potential of human pluripotent stem cell-derived lung organoids with HER2 overexpression.

Lung adenocarcinoma (LUAD) is the most common types among lung cancers generally arising from terminal airway and understanding of multistep carcinogenesis is crucial to develop novel therapeutic strategy for LUAD. Here we used human induced pluripotent stem cells (hiPSCs) to establish iHER2-hiPSCs in which doxycycline induced the expression of the oncoprotein human epidermal growth factor receptor 2 (HER2)/ERBB2. Lung progenitors that differentiated from iHER2-hiPSCs, which expressed NKX2-1/TTF-1 known as a lung lineage maker, were cocultured with human fetal fibroblast and formed human lung organoids (HLOs) comprising alveolar type 2-like cells. HLOs that overexpressed HER2 transformed to tumor-like structures similar to atypical adenomatous hyperplasia, which is known for lung precancerous lesion and upregulated the activities of oncogenic signaling cascades such as RAS/RAF/MAPK and PI3K/AKT/mTOR. The degree of morphological irregularity and proliferation capacity were significantly higher in HLOs from iHER2-hiPSCs. Moreover, the transcriptome profile of the HLOs shifted from a normal lung tissue-like state to one characteristic of clinical LUAD with HER2 amplification. Our results suggest that hiPSC-derived HLOs may serve as a model to recapitulate the early tumorigenesis of LUAD and would provide new insights into the molecular basis of tumor initiation and progression.

Identification of cross-reactive CD8+ T cell receptors with high functional avidity to a SARS-CoV-2 immunodominant epitope and its natural mutant variants

Despite the growing knowledge of T cell responses in COVID-19 patients, there is a lack of detailed characterizations for T cell-antigen interactions and T cell functions. Here, with a predicted peptide library from SARS-CoV-2 S and N proteins, we found that specific CD8+ T cell responses were identified in over 75% of COVID-19 convalescent patients (15/20) and an epitope from the N protein, N361-369 (KTFPPTEPK), was the most dominant epitope from our selected peptide library. Importantly, we discovered 2 N361-369-specific T cell receptors (TCRs) with high functional avidity that were independent of the CD8 co-receptor. These TCRs exhibited complementary cross-reactivity to several presently reported N361-369 mutant variants, as to the wild-type epitope. Further, the natural functions of these TCRs in the cytotoxic immunity against SARS-CoV-2 were determined with dendritic cells (DCs) and the lung organoid model. We found that the N361-369 epitope could be normally processed and endogenously presented by these different types of antigen presenting cells, to elicit successful activation and effective cytotoxicity of CD8+ T cells ex vivo. Our study evidenced potential mechanisms of cellular immunity to SARS-CoV-2, and illuminated potential ways of viral clearance in COVID-19 patients. These results indicate that utilizing CD8-independent TCRs against SARS-CoV-2-associated antigens may provide functional superiority that is beneficial for the adoptive cell immunotherapies based on natural or genetically engineered T cells. Additionally, this information is highly relevant for the development of the next-generation vaccines with protections against continuously emerged SARS-CoV-2 mutant strains.

#17: Disease and Immunoprophylaxis Model of Human Nose Organoids to Study SARS-CoV-2 and RSV Infection

BackgroundThere is a significant and unmet need for pre-clinical models to predict responsiveness of immunotherapies to both severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and respiratory syncytial virus (RSV) infection. Airway organoid models have been recently developed to study respiratory viruses; however, the current methods rely on invasive or biopsy derived samples to generate lung or airway organoids.ObjectiveTo establish human nose organoids (HNOs) as a model to study SARS-CoV-2 and RSV pathogenesis and test therapeutics.MethodsWe developed a non-invasive method to establish HNOs using stem cells isolated from nasal-wash and mid-turbinate samples. We made air liquid interface (ALI) cultures from undifferentiated 3-dimensional HNOs and differentiated for 21 days to form differentiated nasal epithelium. We inoculated the apical epithelium and assessed SARS-CoV-2 and RSV infection on the apical compartment using real time-polymerase chain reaction, plaque assays and immunofluorescence techniques. We then evaluated the feasibility of HNO-ALI model system to test the efficacy of serum antibodies to prevent SARS-CoV-2 infection and palivizumab monoclonal antibodies to prevent infection using palivizumab sensitive and resistant RSV strains. We introduced the antibodies in the basolateral compartment and monitored its neutralizing capacity on the apical side mimicking the neutralizing effects of antibodies in circulation.ResultsOur HNO-ALI cultures consist of well-differentiated, pseudostratified, ciliated, and mucosal respiratory epithelial cells and are susceptible to SARS-CoV-2, RSV A and B infection. SARS-CoV-2 and RSV replicates in the apical ciliated cells of the HNO-ALI cultures, peaks at 4 days, and plateaus at 8 days post infection. Infected HNO-ALI recapitulates aspects of SARS-CoV-2 and RSV disease, including viral shedding, asynchronous cilia beating/ciliary damage, and mucus hyper-secretion. Our model effectively showed protection to infection in a concentration dependent manner of the antibodies used.ConclusionWe established a non-invasive method to generate HNO-ALI epithelial model as an authentic and an alternative model to 1-D cell culture systems. Our ex-vivo HNO-ALI infection model provides a novel approach for testing therapeutic interventions.

RIP3-dependent necroptosis contributes to the pathogenesis of chronic obstructive pulmonary disease.

Necroptosis has emerged as a potential mechanism in the pathogenesis of chronic obstructive pulmonary disease (COPD). Here, we found that markers of necroptosis, including high mobility group box 1 release and phosphorylation of mixed lineage kinase domain-like protein (p-MLKL), were markedly induced in the late stage of cigarette smoking–induced (CS-induced) emphysema in mouse lung tissue as well as in lung epithelial cells and organoids with higher dosage of or more prolonged exposure to cigarette smoking extract (CSE). Apoptotic signals were also detected and maximally induced in the early stage of CS-exposed mice and CSE-treated epithelial cells. Inhibition of apoptosis by Z-VAD, a pan-caspase inhibitor, switched the cellular stress to enhanced necroptosis in lung epithelial cells and organoids treated with CSE. Depletion or inhibition of receptor-interacting protein kinase 3 (RIP3) or MLKL attenuated the CSE-induced cell death, suggesting that necroptosis contributes to CSE-induced cell death. Silencing or inhibition of RIP1 had no protective effect, indicating a RIP1-independent RIP3 activation pathway. CSE-induced necroptosis released more damage-associated molecular patterns and evoked greater engulfment but slower clearance by bone marrow–derived macrophages, leading to enhanced expression of proinflammatory cytokines Tnfα and Il6. Finally, our in vivo data verified that inhibition of necroptosis by RIP3 inhibitor GSK’872 protected mice from CS-induced emphysema and suppressed the lung inflammation. In conclusion, we provide evidence that necroptosis contributes to the pathogenesis of COPD. Targeting RIP3 and its downstream pathway may be an effective therapy for COPD.

New Insights into the Clinical Implications of Yes-Associated Protein in Lung Cancer: Roles in Drug Resistance, Tumor Immunity, Autophagy, and Organoid Development.

Simple SummaryInnovative advancements in lung cancer treatment have developed over the past decade with the advent of targeted and immune therapies. Yes-associated protein (YAP), an effector of the Hippo pathway, promotes the resistance of these targeted drugs and modulates tumor immunity in lung cancer. YAP is involved in autophagy in lung cancer and plays a prominent role in forming the tubular structure in lung organoids and alveolar differentiation. In this review, we discuss the central roles of YAP in lung cancer and present YAP as a novel target for treating resistance to targeted therapies and immunotherapies in lung cancer.Despite significant innovations in lung cancer treatment, such as targeted therapy and immunotherapy, lung cancer is still the principal cause of cancer-associated death. Novel strategies to overcome drug resistance and inhibit metastasis in cancer are urgently needed. The Hippo pathway and its effector, Yes-associated protein (YAP), play crucial roles in lung development and alveolar differentiation. YAP is known to mediate mechanotransduction, an important process in lung homeostasis and fibrosis. In lung cancer, YAP promotes metastasis and confers resistance against chemotherapeutic drugs and targeted agents. Recent studies revealed that YAP directly controls the expression of programmed death-ligand 1 (PD-L1) and modulates the tumor microenvironment (TME). YAP not only has a profound relationship with autophagy in lung cancer but also controls alveolar differentiation, and is responsible for tubular structure formation in lung organoids. In this review, we discuss the various roles and clinical implications of YAP in lung cancer and propose that targeting YAP can be a promising strategy for treating lung cancer.

SOX9 inactivation affects the proliferation and differentiation of human lung organoids

BackgroundThe regulation of the transcription factor sex-determining region Y-box transcription factor 9 (SOX9) in lung development has been described in mouse, but the same principles apply to human lung development is unknown due to a lack of appropriate experimental approaches and models.MethodsHere, we used gene editing technology to inactivate SOX9 in human embryonic stem cells that were then induced to differentiate into lung organoids to investigate the role of SOX9 in human lung epithelium development.ResultsComplete knockout of the transactivation domain of SOX9 by gene editing resulted in indels in both alleles of SOX9. SOX9−/− hESCs could be induced to differentiate into lung progenitor organoids. In vitro long-term expansion showed that SOX9 inactivation did not affect the differentiation of pulmonary epithelial cells, but promoted apoptosis and reduced proliferative capacity in the organoids. When lung progenitor organoids were transplanted under the kidney capsule of immunodeficient mice, expression of the club cell marker secretoglobin family 1A member 1 (SCGB1A1) was detected in SOX9−/− transplants but was absent in wild-type (WT) transplants. The maturation of goblet cells was also affected by SOX9 inactivation, as evidenced by the presence of mucin 5 AC (MUC5AC) in the cytoplasm of SOX9−/− grafts as compared to WT grafts in which most MUC5AC was secreted into the lumen. In vivo lung orthotopic transplantations showed that SOX9 inactivation had a limited effect on the differentiation of alveolar cells and lung regeneration in injured mice.ConclusionsSOX9 modulates the proliferative capacity of lung epithelium but is not an indispensable transcription factor in the regulation of human lung epithelium development.

Advancing lung organoids for COVID-19 research.

ABSTRACTThe COVID-19 pandemic has emphasised the need to develop effective treatments to combat emerging viruses. Model systems that poorly represent a virus' cellular environment, however, may impede research and waste resources. Collaborations between cell biologists and virologists have led to the rapid development of representative organoid model systems to study severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We believe that lung organoids, in particular, have advanced our understanding of SARS-CoV-2 pathogenesis, and have laid a foundation to study future pandemic viruses and develop effective treatments.

Defense of COVID-19 by Human Organoids.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has created an immense menace to public health worldwide, exerting huge effects on global economic and political conditions. Understanding the biology and pathogenesis mechanisms of this novel virus, in large parts, relies on optimal physiological models that allow replication and propagation of SARS-CoV-2. Human organoids, derived from stem cells, are three-dimensional cell cultures that recapitulate the cellular organization, transcriptional and epigenetic signatures of their counterpart organs. Recent studies have indicated their great values as experimental virology platforms, making human organoid an ideal tool for investigating host–pathogen interactions. Here, we summarize research developments for SARS-CoV-2 infection of various human organoids involved in multiple systems, including lung, liver, brain, intestine, kidney and blood vessel organoids. These studies help us reveal the pathogenesis mechanism of COVID-19, and facilitate the development of effective vaccines and drugs as well as other therapeutic regimes.

D-dopachrome tautomerase contributes to lung epithelial repair via atypical chemokine receptor 3-dependent Akt signaling.

BackgroundEmphysematous COPD is characterized by aberrant alveolar repair. Macrophage migration inhibitory factor (MIF) contributes to alveolar repair, but for its structural and functional homolog D-dopachrome tautomerase (DDT) this is unknown. MIF mediates its effects through CD74 and/or C-X-C chemokine receptors 2 (CXCR2), 4(CXCR4), and possibly 7 (ACKR3). DDT can also signal through CD74, but interactions with other receptors have not been described yet. We therefore aimed at investigating if and how DDT contributes to epithelial repair in COPD.MethodsWe studied effects of recombinant DDT on cell proliferation and survival by clonogenic assay and annexin V-PI staining respectively. DDT-induced signaling was investigated by Western blot. Effects on epithelial growth and differentiation was studied using lung organoid cultures with primary murine or human epithelial cells and incubating with DDT or an ACKR3-blocking nanobody. DDT-ACKR3 interactions were identified by ELISA and co-immunoprecipitation.FindingsWe found that DDT promoted proliferation of and prevented staurosporine-induced apoptosis in A549 lung epithelial cells. Importantly, DDT also stimulated growth of primary alveolar epithelial cells as DDT treatment resulted in significantly more and larger murine and human alveolar organoids compared to untreated controls. The anti-apoptotic effect of DDT and DDT-induced organoid growth were inhibited in the presence of an ACKR3-blocking nanobody. Furthermore, ELISA assay and co-immunoprecipitation suggested DDT complexes with ACKR3. DDT could activate the PI3K-Akt pathway and this activation was enhanced in ACKR3-overexpressing cells.InterpretationIn conclusion, DDT contributes to alveolar epithelial repair via ACKR3 and may thus augment lung epithelial repair in COPD.