key: cord-0736549-m1zw7j27
authors: Surendran, Harshini; Kumar, Saurabh; Narasimhaiah, Swathi; Ananthamurthy, Anuradha; Varghese, PS; D'Souza, George A.; Medigeshi, Guruprasad; Pal, Rajarshi
title: SARS‐CoV‐2 infection of human‐induced pluripotent stem cells‐derived lung lineage cells evokes inflammatory and chemosensory responses by targeting mitochondrial pathways
date: 2022-04-23
journal: J Cell Physiol
DOI: 10.1002/jcp.30755
sha: 35e9622abc70e5606685c60ebfbb1491d230cc35
doc_id: 736549
cord_uid: m1zw7j27

The COVID‐19 disease caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) primarily affects the lung, particularly the proximal airway and distal alveolar cells. NKX2.1+ primordial lung progenitors of the foregut (anterior) endoderm are the developmental precursors to all adult lung epithelial lineages and are postulated to play an important role in viral tropism. Here, we show that SARS‐CoV‐2 readily infected and replicated in human‐induced pluripotent stem cell‐derived proximal airway cells, distal alveolar cells, and lung progenitors. In addition to the upregulation of antiviral defense and immune responses, transcriptomics data uncovered a robust epithelial cell‐specific response, including perturbation of metabolic processes and disruption in the alveolar maturation program. We also identified spatiotemporal dysregulation of mitochondrial heme oxygenase 1 (HMOX1), which is associated with defense against antioxidant‐induced lung injury. Cytokines, such as TNF‐α, INF‐γ, IL‐6, and IL‐13, were upregulated in infected cells sparking mitochondrial ROS production and change in electron transport chain complexes. Increased mitochondrial ROS then activated additional proinflammatory cytokines leading to an aberrant cell cycle resulting in apoptosis. Notably, we are the first to report a chemosensory response resulting from SARS‐CoV‐2 infection similar to that seen in COVID‐19 patients. Some of our key findings were validated using COVID‐19‐affected postmortem lung tissue sections. These results suggest that our in vitro system could serve as a suitable model to investigate the pathogenetic mechanisms of SARS‐CoV‐2 infection and to discover and test therapeutic drugs against COVID‐19 or its consequences.

The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to over 6.0 million deaths worldwide as of April 2022. It is primarily transmitted via respiratory droplets and the lungs are the most important organ involved (Pei et al., 2021) . It causes fever, shortness of breath, fatigue, diarrhea, and loss of smell and taste. The infection is initiated in the proximal airways and the alveolar type 2 (AT2) cells of the distal lung (Mulay et al., 2021) . The damage to the alveolar cells is associated with inflammation, which triggers focal capillary micro-thrombus formation causing pulmonary parenchymal fibrosis and the eventual death of the patient (Kommoss et al., 2020) .

Researchers are still trying to understand how COVID-19 infection leads to acute respiratory distress syndrome (ARDS), severe pneumonia, and multiple organ failure all of which contribute to high patient mortality.

In addition, the consequences of "lung Covid syndrome" have created a substantial societal burden. Even though vaccines such as an inactivated virus or a modified version of the virus have been developed, potential therapeutic drugs in the form of antivirals etc. are still lacking. One major obstacle in developing effective treatment is the lack of understanding of viral pathogenesis due to the unavailability of a proper model system.

Using patient cells is advantageous, but there is an acute shortage of patient tissues, particularly from the early stages of the disease . Further, the inability to grow and expand these primary cells for extended periods of time, compared to immortalized cell lines, has made them difficult to use. Animal models like mice, rats, hamsters etc., help achieve a better understanding of in vivo tissue-specific and systemic host−pathogen interactions. However, the genetic and tissue-level variations among species are a major drawback in extrapolating such results to the human system. Nevertheless, cell lines can be used for in vitro studies as a viable model system to investigate the cellular and molecular effects of viral infection and also for evaluating a potential therapeutic drug (Pei et al., 2021) . Although various SARS-CoV-2 infection studies have used multiple cell lines, such as Caco-2, Calu-3, HEK 293T, and Huh7 etc., there are limitations such as low viral titers, lack of appropriate immune responses, cytogenetic instability, insufficient physiological relevance, and low and inconsistent expression of ACE2 and TMPRSS2 .

The foundation of our model lies in a differentiation protocol that generates biopotential lung progenitor cells eventually giving rise to both airway and alveolar cells. Employing this 2D model, we sought to understand the underlying mechanism involved in SARS-CoV-2 infection and the pathogenesis of the severe disease. First, we tested the permissiveness of this model system by infecting the cultures at various multiplicities of infection (MOI) and progressive time points. Following infection, among other parameters, we observed the presence of viral spike protein inside the cell establishing its permissibility. We then performed bulk RNA sequencing to study the molecular changes and compared the observed cellular responses with published reports using primary cells and patient samples. We noticed massive inflammation, fibrosis, and cell death sequentially, reminiscent of COVID-19 pathology.

A deeper analysis of the transcriptomics data helped identify the possible role of mitochondria in COVID-19. Modulation of electron transport chain (ETC) complexes postinfection marked by heme oxygenase 1 (HMOX1) dysregulation and other indicators pointed toward mitochondrial damage as a key factor in the pathogenesis of severe infection. Our model suggests that the cells manifest changes that may be relevant to alterations in sensory functions (taste change) in COVID-19 patients, thus highlighting its physiological relevance when compared to other in vitro models in use. Lastly, we validated some of our key findings in lung tissue samples from COVID-19 patients.

All stem cell-related experiments were approved by the Institutional Committee for Stem Cell Research (ICSCR), Indian Council of Medical Research (ICMR), New Delhi. Lung differentiation was carried out using a previously established protocol (Banerjee et al., 2018; Surendran et al., 2019) . In principle, undifferentiated iPSCs were converted to desired lung lineages via the formation of definitive endoderm and anterior foregut endoderm (AFE) thus simulating the in vivo events of lung development. De novo-generated cells were cryopreserved at the progenitor stage (Day 14) and proximal airway and distal alveolar cell stages (Day 21) after adapting them to air−liquid interface (ALI) cultures for 7−14 days. Live cells were washed twice with 1X DPBS and incubated for 3 min at 37°C with prewarmed Accutase (Gibco). Cells were flushed from the wells using 1 ml pipette tips and centrifuged at 1000 rpm for 2 min at room temperature (RT). The supernatant was discarded and the cell pellet was resuspended in CryoStor CS10 (Stem Cell Technologies) freezing medium at 1 million cells per vial. It was then frozen and stored in liquid nitrogen (LN2) until further use. Each batch of cells was characterized using stage-specific markers to test their identity, purity, and potency.

Cells were partially thawed in a 37°C water bath and immediately transferred to a 15 ml falcon tube containing a sterile medium, allowing the cells to completely thaw in the presence of a prewarmed medium. The cells were pelleted at 1000 rpm for 2 min at RT and the pellet was resuspended in an appropriate culture medium. Cells were then seeded onto 48-well tissue culture plates at a density of 

Cells were lysed in Trizol and total RNA was extracted using the conventional phenol-chloroform method. Viral RNA PCR was carried out using the 2019-nCoV CDC Probe and Primer kit for SARS-CoV-2 (Biosearch Technologies) to detect the N gene using real-time RT-PCR following the manufacturer's instruction. TaqMan™ RNase P assay kit (Thermo Fisher Scientific) was used as an endogenous control for normalization. Fold change was calculated using ∆∆ 2 C t method with uninfected control and RNAseP. The RNA was converted to cDNA using the RevertAid reverse transcription kit (Thermo Fisher Scientific) as per the manufacturer's protocol, and quantitative PCR was performed for human genes using the QuantStudio5 Real Time PCR system (Applied Biosystems). Fold change estimations were based on double normalization with β-actin and undifferentiated iPSC. The human-specific primers used for PCR are listed in Table S1 .

SARS-CoV-2 titer was determined by plaque assay using a 10-fold serial dilution method and was performed on the Vero/E6 monolayer.

Briefly, 150 K Vero/E6 cells were seeded in DMEM (Himedia) supplemented with 10% FBS, 1X penicillin-streptomycin antibiotic solution (Himedia). 24 h after seeding of cells, the monolayer was washed with PBS. One hundred and twenty-five microlitres of 10fold serially diluted virus inoculum was added and incubated for 1 h at 37°C in a CO 2 incubator. The plate was moved slightly every 10 min so that monolayer was covered by the inoculum and did not dry.

After 1 h of incubation, the inoculum was removed and CMC was overlaid. The plate was left undisturbed in the CO 2 incubator for 48 h. Plates were fixed using 3.7% formaldehyde solution for 30 min inside BSL3 after aspirating the overlay medium followed by staining with crystal violet solution. Plates were washed using tap water and dried at RT. The plaque number was counted manually.

Infected cultures fixed in chilled methanol were placed on ice for 10 min.

Cells were washed twice with PBS, once with ice-cold PBS, and then once with PBS at RT followed by blocking with 0.2% BSA-PBS for 10 min at RT. Cells were incubated with the (Genscript) antibody at 1:1000 for 1 h at RT and counterstained with secondary antibody-goat anti-mouse AF488 (Life Technologies) at 1:500 dilution for 30 min in the dark at RT. Cells were washed with PBS three times and stained with 4ʹ,6-diamidino-2-phenylindole (DAPI) (molecular probes) at 1:10,000 dilution for 10 min.

Cells were washed with PBS and images were captured using an Olympus DP80 microscope. Differentiated cells for immunocytochemistry were fixed with 4% paraformaldehyde and stained using a previously described protocol (Surendran et al., 2019) . Images were captured using Olympus fluorescent microscope CKX53. Image analysis was performed using ImageJ (NIH) and graphics editing software (Photoshop, Adobe, https:// www.adobe.com). The antibodies used for staining are listed in Table S2 . Microsoft excel and a protein network was made using the string protein interaction database. The DEGs were further validated by qPCR using SYBR green reaction dye using Quantstudio5 (Applied Biosystems). Fold change was calculated by ∆∆ 2 C t method using uninfected cells as control after normalization with β-actin.

Results are represented as mean ± standard error (SE) with technical triplicates. Statistical significance was calculated using the Student's t-test -95% confidence interval and p ≤ 0.05 was considered statistically significant. The RNAseq data have been deposited in Gene Expression Omnibus and are accessible through accession number GSE190193.

Postmortem specimens of lung tissue from five COVID-19 positive cases were received in 10% neutral buffered formalin after obtaining Institutional Ethics Committee (IEC) approval. Representative blocks were taken for further processing based on clinical descriptions. The H&E stained slides were examined and a report was generated in each of the cases. Representative blocks were selected from the five cases and 3−4 µm-thick sections were cut on charged slides. The primary antibodies tested were CC10, SPC, IL-6, and IL-13. The immunohistochemistry was done by the peroxidase-conjugated polymer method, including antigen retrieval using the automated method-Ventana Benchmark GX machine.

Universal DAB was the detection kit with hematoxylin counterstain.

Before running the IHC on the COVID-19 positive lung blocks, the markers were standardized on normal lung tissue sections.

We generated biopotential lung progenitor cells from iPSC, which after adapting to ALI were further differentiated to distal alveolar and fold change >2 and <−2 in comparison to respective uninfected controls). Proximal epithelium-specific transcription factor SOX2, which regulates the basal cells of the airway was significantly downregulated across all infected samples. Contrastingly, distal epithelium-specific transcription factor, SOX9, which controls proliferation and differentiation of alveolar cells was increased in infected samples (Figure 3a,b) . Similarly, we observed a decrease in other airway-specific genes TP63, FOXJ1, and surfactant protein SFTPC.

Likewise, Tenascin C (TNC), which is associated with tissue repair, and HOPX, which is involved in alveolar homeostasis, were downregulated in proximal cells but slightly upregulated in distal cells 

Existing literature suggests that oxidative stress is an acute response to COVID-19 infection because the pathophysiology is very similar to that seen in ALI. ACE2 also affects mitochondrial functions and low levels of ACE2 are associated with decreased ATP production and altered activation of NADPH oxidase 4 (NOX4) in the mitochondria (K. K. Singh et al., 2020) . Interestingly, we detected decreased levels of ACE2 immediately after infection across the cell types, but we are not sure of the underlying mechanism. We then tested the levels of NOX4 in the infected cells and found it decreasing in progenitor and airway cell types but increasing in alveolar cells (Figure 4a ). This could have been driven by NOX-induced TGF-β1-mediated conversion of fibroblasts into myofibroblasts, leading to fibrosis in alveolar cells (Amara et al., 2010) . This was clearly demonstrated in our model marked by high TGF-β1 and collagen deposition. Figure 4b ). Further, we tested the members of the NADH ubiquinone oxidoreductase (NDUF) gene family, which is crucial for initiating the ETC for ATP production inside the cell. A similar pattern of dysregulation was also seen in MRPS genes (Figure 4c ). In addition, we found that some of the reactive oxygen species (ROS) related showed a similar expression trend to sequencing data (Figure 4f ).

Heme oxygenase has been identified to play a vital role as a defense molecule activated in injured lung tissue against injury to prevent antiinflammatory and antiapoptotic effects. HMOX1 is the most inducible isoform involved in preventing vascular inflammation and is used as a therapeutic target for acute lung injury in multiple pulmonary disease models (Fredenburgh et al., 2007) . To understand the cytoprotective effects of HMOX1, we created a protein network using the STRING database with network edges, indicating the predicted mode of action between two proteins. We included HMOX1 and key inflammatory and apoptotic genes to illustrate their interaction in the normal state ( Figure 4g) . Some of the direct interactions include HMOX1 and antiinflammatory molecule IL-10, HMOX1, and VEGF mutually activating each other, and proinflammatory cytokine IL-13 inhibits HMOX1. These findings also support the known role of HMOX1 in preventing inflammation and promoting angiogenesis upon lung damage.

We further checked the presence of HMOX1 in our in vitro differentiated cells and fluorescent microscopy showed considerable levels of protein expression (Figure 4h) . NGS data showed that HMOX1 was slightly downregulated at 24 hpi, which could be due to a sudden spike in the inflammatory cascade after infection (Figure 4i ).

At 72 hpi, HMOX1 was highly upregulated across all lung cell types, something that has been demonstrated previously in ARDS patients' lung tissue (Fredenburgh et al., 2007) . The levels of HMOX2, which is a constitutive form, were observed to be consistent irrespective of infection and HMOX3 was not present (Figure 4i ). Lastly, we verified the mRNA levels of HMOX1 by qPCR and found them to be matching the FPKM values (Figure 4j ). Upregulation of HMOX1 in the face of inflammatory response and apoptosis concurs with its established role as a defense molecule activated for damage control. clearly indicates that this pandemic is far from over. In fact, it is predicted that more variants may emerge to trigger additional pandemic waves in the future. Understanding the cellular processes and molecular interactions impacted in the host cells responding to a virus infection will be necessary to understand the virus pathogenesis and to develop better intervention strategies.

With respect to viral cell tropism, besides corroborating the previously reported ciliated and AT2 cells as target cells (Djidrovski et al., 2021; Huang et al., 2020) , lung progenitor cells from the AFE were also found to be permissive to SARS-CoV-2 infection. This is a key finding since progenitor cells found in the adult lung are involved in lung branching morphogenesis, cell growth, maturation, injury, and repair (Kotton & Morrisey, 2014) . Furthermore, it was reported that lung stem or progenitor cells could be infected by SARS-CoV-2, which may lead to defects in regeneration capacity, partially accounting for the severity of SARS-CoV-2 infection (Valyaeva et al., 2020) .

Once inside the cell, SARS-CoV-2 prompts a massive inflammatory response, which is similar to a cell-autonomous or ''epithelial only" host response to pathogens . Our model responded to SARS-CoV-2 infection within 24 h and dysregulation in NF-κΒ signaling is prominent across the airway, alveolar, and lung progenitor cells. The aberrant gene expression observed in relation to inflammation, host defense, lung development, surfactant production, apoptosis, fibrosis, and tissue repair is clinically relevant. Like others Katsura et al., 2020) we witnessed perturbation in IFN-γ signaling presenting significant differential expression across three cell types after SARS-CoV-2 infection at 24 and 72 h compared to noninfected cells (Figure 2g ). Our finding shows that the type-II IFN pathway is activated and contradicts the F I G U R E 5 Disruption of alveolar spaces and inflammation in COVID-19 infected lung tissues support in vitro findings. (a, b) Hematoxylin and eosin-stained COVID lung section showing (a) ARDS like pathology with edema and hyaline membrane (b) features of pneumonia (c−f) Lung surfactant-SPC, secretory protein CC10 highlights the alveolar lining cells in normal lung and disrupted staining along the alveolar walls with no proper air spaces (g−j) Inflammatory cytokines -IL-6, IL-13 stained macrophages in normal lung and massive cellular infiltration in COVID lung. Scale bars represent 100 µm more prevalent activation of the IFN-I and IFN-III pathways in cells due to viral infection. However, our data is consistent with earlier studies, showing delayed host innate immune responses after SARS-CoV (2003) infection (Menachery et al., 2014) . At the same time, it underscores the need for dynamic analyses of host responses using multiple MOIs and at different times after infection. After innate immunity is triggered upon viral infection, cytokines, such as TNF-α, . are activated in infected cells, causing an increase in mitochondrial ROS production through gene expression upregulation and ETC modulation (Saleh et al., 2020) . Mitochondrial ROS then stimulates additional proinflammatory cytokine production as the virus persists leading to a "cytokine storm." This immune response could also prompt the mitochondria to deviate from ATP production toward ROS production, which can harm the mitochondria, leading to apoptosis (Saleh et al., 2020) .

We have demonstrated that ACE2 and TMPRSS2 are robust but heterogeneously expressed, which is similar to what was observed in adult human AT2s in vivo . Entry of SARS-CoV-2 inside the cell using the ACE2 receptor results in a failure of ACE2 conversion of angiotensin II to angiotensin. This excess angiotensin II stimulates NADPH oxidase thus generating high levels of ROS. As a result, cortisol stimulates the release of ATP via activation of mineralocorticoid receptor (MR), which then acts on purinergic receptors leading to an increase in intracellular calcium (Edwards et al., 2021) . Purinergic receptors are known to play an important role in both taste and smell (Eddy et al., 2009; Hegg et al., 2003) and ATP signaling has been shown to be crucial for communication from taste buds to gustatory nerves (Finger et al., 2005) .

In our model, ACE2 decreased significantly from 24 to 72 h and calcium signaling genes like CAMKK, RAC1, and TG2 (data not shown) increased considerably after SARS-CoV-2 infection. Therefore, it is likely that the SARS-CoV-2 virus might well activate ATP-mediated odor suppression as a novel protective mechanism.

Mitochondria play a key role in the host's response to viral infection and immunity (Takumi Koshiba, 2013) . Using machine learning models, Wu et al. determined that the SARS-CoV-2 RNA genome and all subgenomic RNAs were enriched in the host mitochondria and nucleolus (Wu et al., 2020) , and investigations into the SARS-CoV-2 hijacking of host mitochondria may lead to novel approaches to prevent and treat COVID-19 (K. K. Singh et al., 2020) . We observed that across cell types, SARS-CoV-2 reduced nuclear-encoded mitochondrial (NEM) gene host response related to cellular respiration and Complex I. Complex I is one of the main contributors to ROS production and downregulation of many NDUF and MRPS family of genes after SARS-CoV-2 infection could be behind lower ROS production, thus facilitating SARS-CoV-2 propagation.

SARS-CoV-2 ORF9c has been previously reported to interact with mitochondrial NDUFAF1, NDUFB9, MRPS2, MRPS5, MRPS25, and MRPS27 (Gordon et al., 2020) . This could explain the direct interaction between SARS-CoV-2 and these Complex I and mitochondrial ribosome proteins observed in our transcriptomic data after SARS-CoV-2 infection.

HMOX1 is a cytoprotective enzyme that plays a crucial role in the defense against oxidant and inflammation-induced lung injury during ARDS and idiopathic pulmonary fibrosis (Fredenburgh et al., 2007) . Among the three isoforms, HO-1 is the inducible isoform and is thought to be an oxidative stress-responsive protein (Morse & Choi, 2005) . Likewise, the dysregulation of HMOX1 and not HMOX2 noticed in proximal airway cells after SARS-CoV-2 infection could result from oxidant−antioxidant imbalance, thus contributing to the pathogenesis of lung fibrosis in COVID-19. This is a significant finding as modulation of HO-1 has been hypothesized as a potential therapeutic target for COVID-19 via suppression of viral replication by increasing IFNs (K. K. Singh et al., 2020) .

There are many other interesting and novel observations from this study that provide critical insights for understanding the pathophysiology of SARS-CoV-2 infection. Airway secretory mucin gene (muc5ac) was downregulated, which could lead to neutrophil trafficking into the lungs (Koeppen et al., 2013) . Aquaporins regulate the osmotic pressure during normal lung functioning (Wittekindt & Dietl, 2019) and the water channel protein AQP5, which maintains the alveolar epithelial barrier was significantly downregulated upon SARS-CoV-2 infection. Tissue repair gene TNC was upregulated, which has been shown to induce lung injury due to activation of multiple inflammatory cytokines, such as pathogens and damage-associated molecular patterns (PAMP and DAMP) (Midwood & Orend, 2009 ). Further, downregulation of BCL2 inhibits the intrinsic pathway and PAMP-induced apoptosis (Leibowitz & Yu, 2010) . EMT features transdifferentiation of AT1/AT2 to fibroblast/myofibroblast, resulting in increased MMPs and collagen deposition. Likewise, increased expression of MMPs and collagen was observed in distal alveolar cells following SARS-CoV-2 infection. Since SARS-CoV-2 infection eventually leads to pulmonary fibrosis, HOPX upregulation could be a potential indicator of progression to pulmonary fibrosis in our model (Ota et al., 2018) . Persistent alveolar activation of TGF-β, the release of PDGF and IL-6 from alveolar epithelial cells, immune cells, and myofibroblasts leads to the proliferation of myofibroblasts and the development of fibrosis (John et al., 2021) . Imbalance in the expression of SOX2/SOX9 and PITX2/CTNNB1 indicates perturbed proximal-distal lung patterning (Banerjee et al., 2018) .

Most COVID-19 patients suffer from sensory dysfunction (anosmia), which starts with their sense of smell, but because the smell is necessary to taste the flavor, the symptoms are often connected (Hannum et al., 2021) . Since ACE2 receptors are not found in the olfactory nerves and taste buds, the high incidence of anosmia and ageusia caused by SARS-CoV-2 is without directly infecting these cell types. Further, it is well known that taste receptors are expressed far beyond the tongue, from the airway and gastrointestinal epithelia to the pancreas and brain (Yamamoto & Ishimaru, 2013) . Bitter taste receptors (TAS2Rs) are G-protein coupled receptors divided into 36 subunits, depending on the species (over 25 subunits in humans). With respect to the lung, the presence of TAS2Rs has been shown in airway ciliated cells (Shah et al., 2009) , sinonasal epithelial cells (Lee et al., 2012) , solitary chemosensory cells (Gulbransen et al., 2008) , and bronchial smooth muscle cells (Robinett et al., 2014) . Interestingly, GO analysis identified the expression of a subset of TAS2R bitter receptor genes but not TAS1R sweet receptor genes, which was further confirmed by qPCR and immunostaining. However, we failed to detect many other TAS2Rs, possibly because their expression level is below the detection limit or because these receptors are only weakly expressed in the iPSC-derived lung epithelial lineages. Olfactory receptors in the airway were first identified in PNECs, a rare yet multifunctional epithelial cells and suggested that they act as intrapulmonary chemosensors (Gu et al., 2014) . Hor et al. (2020) successfully differentiated human iPSCs to iPNECs with a gene expression profile like that of primary fetal PNECs. Interestingly, they adapted this iPNEC differentiation protocol from their previously established airway epithelial differentiation protocol (Firth et al., 2014) , which was quite similar to our lung differentiation protocol (Banerjee et al., 2018; Surendran et al., 2019) . Based on this information, we established the expression of major PNEC marker genes, including SYP, UCHL1, DLL3, NCAM1, BDNF, and ENO2, consistent with the phenotype of primary human PNECs (Branchfield et al., 2016) . We also demonstrated the presence of rare TUJ1 + /P63 + positive solitary PNEC in our airway cultures, which is in line with a recent study showing the ability of airway basal stem cells to generate TUBB3 + PNEC (Mou et al., 2021) . Within the lung milieu, activation of proneural transcription factor ASCL1 is required for cells to form the pulmonary neuroendocrine lineage (Linnoila, 2006) Nonetheless, with <1% of the cellular composition of the adult lung being PNECs and given the heterogeneity in iPSC-based differentiation it is not ideal to make head to head comparisons with respect to quantification based on in vitro results.

In the current study, we focused on the transcriptional response F I G U R E 6 A schematic representing the working model of our study, starting from entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inside the lung epithelial cell types, triggering an inflammatory response, chemosensory changes, and impaired proximal-distal lung patterning. These alterations eventually resulted in mitochondrial dysfunction and pulmonary fibrosis via specific cellular and molecular events. HMOX1, heme oxygenase 1; iPSC, induced pluripotent stem cells.

Human iPSC-derived alveolar and airway epithelial cells can be cultured at air-liquid interface and express SARS-CoV-2 host factors. bioRxiv: The Preprint Server for Biology

NOX4/NADPH oxidase expression is increased in pulmonary fibroblasts from patients with idiopathic pulmonary fibrosis and mediates TGF-B1-induced fibroblast differentiation into myofibroblasts

Resolution of viral load in mild COVID-19 patients is associated with both innate and adaptive immune responses

Long noncoding RNA RP11-380D23.2 drives distal-proximal patterning of the lung by regulating PITX2 expression

Pulmonary neuroendocrine cells function as airway sensors to control lung immune response

COVID-19 and the chemical senses: Supporting players take center stage

SARS-CoV-2 infects an upper airway model derived from induced pluripotent stem cells

Double P2X2/P2X3 purinergic receptor knockout mice do not taste NaCl or the artificial sweetener SC45647

Follow your nose: A key clue to understanding and treating COVID-19

ATP signaling is crucial for communication from taste buds to gustatory nerves

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A SARS-CoV-2 protein interaction map reveals targets for drug repurposing

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Ascl1 and Neurog2 form novel complexes and regulate Delta-like3 (Dll3) expression in the neural tube

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SARS-CoV-2 infection of pluripotent stem cellderived human lung alveolar type 2 cells elicits a rapid epithelialintrinsic inflammatory response

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Heme oxygenase-1: From bench to bedside

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Dynamic expression of HOPX in alveolar epithelial cells reflects injury and repair during the progression of pulmonary fibrosis

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SARS-CoV-2 infection of human induced pluripotent stem cells-derived lung lineage cells evokes inflammatory and chemosensory responses by targeting mitochondrial pathways

The authors declare no conflicts of interest.

http://orcid.org/0000-0001-6334-4562