Primary Epithelial Cell Cultures Research Articles

Abstract P238: High Glucose Induces Interleukin-1β Synthesis In Renal Tubular Cells By Suppressing Autophagy

We showed that IL-1β derived from renal tubular epithelial cells initiates the inflammatory response that promotes salt sensitivity during diabetes. Yet, the intracellular mechanism that mediates IL-1β synthesis by tubular cells is unknown. We hypothesize that high glucose inhibits autophagy in tubular cells resulting in the accumulation of mitochondrial reactive oxygen spices (mtROS) that activate the NLRP3 inflammasome to produce IL-1β. Thirty-week-old db/db mice, a model of type 2 diabetes, and db/+ non-diabetic controls (n=6) were exposed to high salt diet (4% NaCl) for 4 weeks. At the end, mean arterial pressure was higher in db/db vs. db/+ mice (125±7 vs. 104±4 mmHg, P<0.05). db/db mice also displayed higher levels of IL-1β in tubular cells (E-cadherin+) compared to db/+ mice (22±4% vs. 7±2% of IL-1β + E-cadherin + cells, P<0.01 by flow cytometry). We measured two critical components of the autophagy machinery by Western blot. In db/db, we observed reduced LC3B-II expression (49% reduction, P<0.01) and increased p62 (2.7±0.3-fold increase, P<0.001) compared to db/+ mice indicating reduced autophagy. To investigate whether autophagy regulates IL-1β synthesis, we used a primary culture of tubular epithelial cells (TECs) (n=6). TECs exposed to high glucose (15mM) secreted higher levels of IL-1β (58±23 vs. 13±6 pg/ml, P>0.001) and displayed increased caspase-1 and NLRP3 expression compared to TECs exposed to low glucose (5mM). Autophagy analysis revealed low LCB3-II and high p62 suggesting that high glucose suppressed autophagy in tubular cells. This was associated with mtROS accumulation (1.9±0.2-fold increase, P<0.001 by MitoSox Red). We also analyzed mTOR, an inhibitor of autophagy. TECs exposed to high glucose showed higher levels of mTOR phosphorylated at Ser 2448 (a marker of activation) compared to low glucose-treated cells. Blocking mTOR with rapamycin (1μM) restored autophagy in high glucose-treated TECs, reduced caspase-1, NLRP3, and mitochondrial ROS and resulted in low IL-1β secretion. Thus, autophagy inhibition is a critical event behind the high glucose-mediated IL-1β secretion in tubular cells. Restoring autophagy might be a therapeutic target to blunt the inflammatory response that causes salt sensitivity during diabetes.

Carbohydrate-binding protein from stinging nettle as fusion inhibitor for SARS-CoV-2 variants of concern.

Urtica dioica agglutinin (UDA) is a carbohydrate-binding small monomeric protein isolated from stinging nettle rhizomes. It inhibits replication of a broad range of viruses, including coronaviruses, in multiple cell types, with appealing selectivity. In this work, we investigated the potential of UDA as a broad-spectrum antiviral agent against SARS-CoV-2. UDA potently blocks transduction of pseudotyped SARS-CoV-2 in A549.ACE2+-TMPRSS2 cells, with IC50 values ranging from 0.32 to 1.22 µM. Furthermore, UDA prevents viral replication of the early Wuhan-Hu-1 strain in Vero E6 cells (IC50 = 225 nM), but also the replication of SARS-CoV-2 variants of concern, including Alpha, Beta and Gamma (IC50 ranging from 115 to 171 nM). In addition, UDA exerts antiviral activity against the latest circulating Delta and Omicron variant in U87.ACE2+ cells (IC50 values are 1.6 and 0.9 µM, respectively). Importantly, when tested in Air-Liquid Interface (ALI) primary lung epithelial cell cultures, UDA preserves antiviral activity against SARS-CoV-2 (20A.EU2 variant) in the nanomolar range. Surface plasmon resonance (SPR) studies demonstrated a concentration-dependent binding of UDA to the viral spike protein of SARS-CoV-2, suggesting interference of UDA with cell attachment or subsequent virus entry. Moreover, in additional mechanistic studies with cell-cell fusion assays, UDA inhibited SARS-CoV-2 spike protein-mediated membrane fusion. Finally, pseudotyped SARS-CoV-2 mutants with N-glycosylation deletions in the S2 subunit of the spike protein remained sensitive to the antiviral activity of UDA. In conclusion, our data establish UDA as a potent fusion inhibitor for the current variants of SARS-CoV-2.

Time-resolved characterization of the innate immune response in the respiratory epithelium of human, porcine, and bovine during influenza virus infection.

Viral cross-species transmission is recognized to be a major threat to both human and animal health, however detailed information on determinants underlying virus host tropism and susceptibility is missing. Influenza C and D viruses (ICV, IDV) are two respiratory viruses that share up to 50% genetic similarity, and both employ 9-O-acetylated sialic acids to enter a host cell. While ICV infections are mainly restricted to humans, IDV possesses a much broader host tropism and has shown to have a zoonotic potential. This suggests that additional virus–host interactions play an important role in the distinct host spectrum of ICV and IDV. In this study, we aimed to characterize the innate immune response of the respiratory epithelium of biologically relevant host species during influenza virus infection to identify possible determinants involved in viral cross-species transmission. To this end, we performed a detailed characterization of ICV and IDV infection in primary airway epithelial cell (AEC) cultures from human, porcine, and bovine origin. We monitored virus replication kinetics, cellular and host tropism, as well as the host transcriptional response over time at distinct ambient temperatures. We observed that both ICV and IDV predominantly infect ciliated cells, independently from host and temperature. Interestingly, temperature had a profound influence on ICV replication in both porcine and bovine AEC cultures, while IDV replicated efficiently irrespective of temperature and host. Detailed time-resolved transcriptome analysis revealed both species-specific and species uniform host responses and highlighted 34 innate immune-related genes with clear virus-specific and temperature-dependent profiles. These data provide the first comprehensive insights into important common and species-specific virus-host dynamics underlying the distinct host tropism of ICV and IDV, as well as possible determinants involved in viral cross-species transmission.

Human Air-Liquid-Interface Organotypic Airway Cultures Express Significantly More ACE2 Receptor Protein and Are More Susceptible to HCoV-NL63 Infection than Monolayer Cultures of Primary Respiratory Epithelial Cells.

ABSTRACTHuman coronavirus NL63 (HCoV-NL63) is commonly associated with mild respiratory tract infections in infants, being that the respiratory epithelial cells are the main target for infection and initial replication of this virus. Standard immortalized cells are highly permissive to HCoV-NL63, and they are routinely used for isolation and propagation of the virus from clinical specimens. However, these cell lines are not the natural cell target of the virus and lack sufficient complexity to mimic the natural infection process in vivo. This study comparatively evaluated the differences on the susceptibility to HCoV-NL63 infection and virus replication efficiency of submerged monolayer cultures of LLC-MK2 and primary human respiratory epithelial cells (HRECs) and organotypic airway cultures of respiratory cells (ALI-HRECs). Productive viral infection and growth kinetics were assessed by morphologic examination of cytopathic effects, immunofluorescence, reverse transcription quantitative real-time PCR, and flow cytometry. Results from this study showed higher susceptibility to HCoV-NL63 infection and replication in LLC-MK2 cells followed by ALI-HRECs, with very low susceptibility and no significant virus replication in HRECs. This susceptibility was associated with the expression levels of angiontensin-converting enzyme 2 (ACE2) receptor protein in LLC-MK2, ALI-HRECs, and HRECs, respectively. Remarkably, organotypic ALI-HREC cultures expressed significantly more ACE2 receptor protein and were more susceptible to HCoV-NL63 infection than monolayer cultures of HREC. The ACE2 receptor is, therefore, a critical factor for susceptibility to HCoV-NL63 infection and replication, as is the type of culture used during infection studies.IMPORTANCE HCoV-NL63 is widespread globally, accounting for a significant number of respiratory infections in children and adults. HCoV-NL63 gains entrance into respiratory epithelial cells via the ACE2 receptor, the same cell receptor used by severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. Thus, HCoV-NL63 has been suggested as safe surrogate for studying disease mechanisms and therapeutic interventions against SARS-like CoVs, while working under BSL-2 conditions. The present study not only showed the critical role of ACE2 for effective HCoV-NL63 infection and replication, but also shed light on the need of more refined and complex in vitro organotypic models that recapitulate the proxy of air-liquid respiratory epithelia cell composition, structure, and functionality. These cultures have broaden virological studies toward improving our understanding of how coronaviruses cause disease and transmission not just within humans but also in animal populations.

Human papillomavirus targets the YAP1-LATS2 feedback loop to drive cervical cancer development.

Human papillomavirus (HPV) infection is very common in sexually active women, but cervical cancer only develops in a small fraction of HPV-infected women, suggesting that unknown intrinsic factors associated with the unique genetic/genomic background of the high-risk population play a critical role in cervical carcinogenesis. Although our previous studies have identified the hyperactivated YAP1 oncogene as a critical contributor to cervical cancer, the molecular mechanism by which YAP1 drives cervical cancer is unknown. In the present study, we found that although the hyperactivated YAP1 caused a malignant transformation of immortalized cervical epithelial cells, it induced cellular senescence in cultures of primary human cervical epithelial cells (HCvECs). However, the hyperactivated YAP1 induced malignant transformation of HCvECs in the presence of high-risk HPV E6/E7 proteins, suggesting that the hyperactivated YAP1 synergizes with HPV to initiate cervical cancer development. Our mechanistic studies demonstrate that YAP1, via up-regulating LATS2, formed a YAP1-LATS2 negative feedback loop in cervical epithelial cells to maintain homeostasis of cervical tissue. Intriguingly, we found that high-risk HPV targets LATS2 to disrupt the feedback loop leading to the malignant transformation of cervical epithelial cells. Finally, we report that mitomycin C, an FDA-approved drug that could upregulate LATS2 and drive cellular senescence in vitro and in vivo, induced a regression of cervical cancer in a pre-clinial animal model. Thus, high-risk HPV targeting the YAP1-LATS2 feedback loop represents a new mechanism of cervical cancer development.

Secretory Cells Are the Primary Source of pIgR in Small Airways.

Loss of secretory IgA (SIgA) is common in chronic obstructive pulmonary disease (COPD) small airways and likely contributes to disease progression. We hypothesized that loss of SIgA results from reduced expression of pIgR (polymeric immunoglobulin receptor), a chaperone protein needed for SIgA transcytosis, in the COPD small airway epithelium. pIgR-expressing cells were defined and quantified at single-cell resolution in human airways using RNA in situ hybridization, immunostaining, and single-cell RNA sequencing. Complementary studies in mice used immunostaining, primary murine tracheal epithelial cell culture, and transgenic mice with secretory or ciliated cell-specific knockout of pIgR. SIgA degradation by human neutrophil elastase or secreted bacterial proteases from nontypeable Haemophilus influenzae was evaluated invitro. We found that secretory cells are the predominant cell type responsible for pIgR expression in human and murine airways. Loss of SIgA in small airways was not associated with a reduction in secretory cells but rather a reduction in pIgR protein expression despite intact PIGR mRNA expression. Neutrophil elastase and nontypeable H. influenzae-secreted proteases are both capable of degrading SIgA invitro and may also contribute to a deficient SIgA immunobarrier in COPD. Loss of the SIgA immunobarrier in small airways of patients with severe COPD is complex and likely results from both pIgR-dependent defects in IgA transcytosis and SIgA degradation.

Tubular IL-1β Induces Salt Sensitivity in Diabetes by Activating Renal Macrophages.

Chronic renal inflammation has been widely recognized as a major promoter of several forms of high blood pressure including salt-sensitive hypertension. In diabetes, IL (interleukin)-6 induces salt sensitivity through a dysregulation of the epithelial sodium channel. However, the origin of this inflammatory process and the molecular events that culminates with an abnormal regulation of epithelial sodium channel and salt sensitivity in diabetes are largely unknown. Both in vitro and in vivo approaches were used to investigate the molecular and cellular contributors to the renal inflammation associated with diabetic kidney disease and how these inflammatory components interact to develop salt sensitivity in db/db mice. Thirty-four-week-old db/db mice display significantly higher levels of IL-1β in renal tubules compared with nondiabetic db/+ mice. Specific suppression of IL-1β in renal tubules prevented salt sensitivity in db/db mice. A primary culture of renal tubular epithelial cells from wild-type mice releases significant levels of IL-1β when exposed to a high glucose environment. Coculture of tubular epithelial cells and bone marrow-derived macrophages revealed that tubular epithelial cell-derived IL-1β promotes the polarization of macrophages towards a proinflammatory phenotype resulting in IL-6 secretion. To evaluate whether macrophages are the cellular target of IL-1β in vivo, diabetic db/db mice were transplanted with the bone marrow of IL-1R1 (IL-1 receptor type 1) knockout mice. db/db mice harboring an IL-1 receptor type 1 knockout bone marrow remained salt resistant, display lower renal inflammation and lower expression and activity of epithelial sodium channel compared with db/db transplanted with a wild-type bone marrow. Renal tubular epithelial cell-derived IL-1β polarizes renal macrophages towards a proinflammatory phenotype that promotes salt sensitivity through the accumulation of renal IL-6. When tubular IL-1β synthesis is suppressed or in db/db mice in which immune cells lack the IL-1R1, macrophage polarization is blunted resulting in no salt-sensitive hypertension.

Glycogene Expression Profile of Human Limbal Epithelial Cells with Distinct Clonogenic Potential.

Glycans function as valuable markers of stem cells but also regulate the ability of these cells to self-renew and differentiate. Approximately 2% of the human genome encodes for proteins that are involved in the biosynthesis and recognition of glycans. In the present study, we evaluated the expression of a small subset of glycogenes in human limbal epithelial cells with distinct clonogenic potential. Individual clones were classified as abortive or clonogenic, based on the fraction of the terminal colonies produced; clones leading exclusively to terminal colonies were referred to as abortive while those with half or fewer terminal colonies were referred to as clonogenic. An analysis of glycogene expression in clonogenic cultures revealed a high content of transcripts regulating the galactose and mannose metabolic pathways. Abortive clones were characterized by increased levels of GCNT4 and FUCA2, genes that are responsible for the branching of mucin-type O-glycans and the hydrolysis of fucose residues on N-glycans, respectively. The expansion of primary cultures of human limbal epithelial cells for 10 days resulted in stratification and a concomitant increase in MUC16, GCNT4 and FUCA2 expression. These data indicate that the clonogenic potential of human limbal epithelial cells is associated with specific glycosylation pathways. Mucin-type O-glycan branching and increased fucose metabolism are linked to limbal epithelial cell differentiation.

MO426: Elucidation of Mechanism of Low-Birth-Weight-Related Fsgs in Rats by Rna-Seq Analysis of Cultured Podocytes From Low-Birth-Weight Rats

Abstract BACKGROUND AND AIMS The pathogenesis of low-birth-weight (LBW)-related nephropathy has been considered to be the result of intra-glomerular hyperfiltration during growth due to the presence of low nephron numbers. On the other hand, we hypothesized that there may be pathogenic factor(s) other than low nephron number in LBW individuals [1]. We reported the results of proteomic analysis of the renal cortex of LBW rats to show that metabolic abnormalities should exist in the kidneys of LBW individuals [2]. However, our result of the previous study was obtained by analysis using the renal cortex, which did not allow us to interpret the mechanism of focal segmental glomerulosclerosis (FSGS) formation, the most typical pathological picture of LBW-related nephropathy. METHOD A total of 5 LBW rats (birth weight 4.38±0.08) were obtained by 55% caloric restriction to pregnant maternal rats. The normal-birth-weight (NBW) rats (birth weight 6.48±0.18) were obtained by feeding normal diets to pregnant maternal rats. Kidneys were removed at 4 weeks of age, and after isolation of the glomeruli by sieving of kidneys, primary culture of glomerular epithelial cells (podocytes) was performed. Then, mRNAs were extracted from the cultured podocytes after 7 days and RNA-seq analysis was performed using these mRNAs from LBW podocytes and NBW podocytes. RESULTS Gene expression of Nphs1 (nephrin), Nphs2 (podocin) Podxl (podocalyxin) was detected in all samples of both groups, confirming that podocytes were cultured. The number of transcripts detected was 17 967 genes. In addition, by comparison of mRNA expression levels between LBW podocytes and NBW podocytes, 107 genes (102 genes decreased in the LBW group and 5 genes increased in the LBW group) showed a False Discovery Rate (FDR) P -value (q-value) < 0.05 between the two groups. Furthermore, IPA (Ingenuity® Pathway Analysis) by using these 107 genes was performed. As a result, five candidates for the upstream master regulator were identified: TNF, SIRT1, SH2B1, C5 and GDF15 as shown in the Figure. The downstream of all these pathways contain TCS2. This gene has been implicated in FSGS formation, as we recently reported [3]. CONCLUSION It is possible that TCS2 is involved in the mechanism of FSGS formation in LBW-related nephropathy.

Renal Tubular IL‐1β Induces Salt Sensitivity in Diabetes by Activating Renal Macrophages

We recently showed that interleukin (IL)‐6 induces salt‐sensitivity in diabetic mice by dysregulating the epithelial sodium channel (ENaC). After 4 weeks on a high salt diet, male db/db mice displayed salt sensitivity, the accumulation of proinflammatory (CD80+) macrophages, and higher levels of IL‐6 and IL‐1β in the kidney compared to db/+ controls. However, the origin of this inflammatory response is unknown. We hypothesize that, during diabetes, renal tubular epithelial cells release IL‐1β which activates renal macrophages to produce IL‐6 and impair kidney function. Immunofluorescence analysis showed that the higher IL‐1β expression of db/db mice is exclusively localized in the renal tubules. Flow cytometry analysis of renal cells showed that db/db mice have higher IL‐1β expression in tubular (E‐cadherin+) cells (21 ± 2 vs. 8 ± 3 % of E‐cadherin+ cells, P<0.001) compared to db/+ controls. To evaluate whether high glucose triggers IL‐1β synthesis in tubular cells, a primary culture of renal tubular epithelial cells from wild‐type (WT) mice was exposed to either a low (LG, 5mM) or high (HG, 15mM) glucose media. HG‐treated cells released higher levels of IL‐1β (28 ± 12 vs. 8 ± 4 pg/ml, P<0.01) and displayed higher expression of NLRP3 (4 ± 1‐fold increase, P<0.001) and Caspase‐1 (2.1 ± 0.2‐fold increase, P<0.001) compared to LG‐treated cells. These are major components of the NLRP3 inflammasome that synthesizes IL‐1β. No equivalent effect was observed using mannitol. To evaluate whether tubular cells can modify macrophage phenotype through IL‐1β, we co‐cultured tubular cells with bone marrow‐derived macrophages. Flow cytometry revealed that HG‐treated tubular cells induced a pro‐inflammatory polarization of macrophages not observed with LG‐treated cells (50 ± 4 vs. 15 ± 2 CD80+ macrophages, P<0.001). This effect was blunted when macrophages were obtained from IL‐1 receptor KO mice (IL1RKO) confirming a role of IL‐1β. To evaluate whether IL‐1β‐mediated macrophage activation plays a role in vivo, 2‐mo‐old diabetic db/db mice (n=6) were transplanted with a bone marrow of IL1RKO mice. These mice, named db/db‐IL1RKO, cannot respond to IL‐1β. db/db receiving wild‐type (WT) bone marrow (db/db‐WT) were used as control. No differences in body weight and hyperglycemia were observed after transplantation. At 7 months, mice were exposed to high salt diet for 4 weeks. Compared to db/db‐WT, db/db‐IL1RKO did not develop salt‐sensitivity (blood pressure was 110 ± 7 vs. 122 ± 9 mmHg, P<0.05), have less renal CD80+ macrophages, (21 ± 4 vs. 54 ± 5 CD80+ macrophages, P<0.01), less renal IL‐6 (138 ± 35 vs. 263 ± 28 pg/mg kidney, P<0.05), and lower ENaC activity (65% decrease by amiloride test, P<0.05) despite an accumulation of tubular IL‐1β. In conclusion, our data show that tubular epithelial cells are a major source of IL‐1β under high glucose conditions. This cytokine promotes a pro‐inflammatory phenotype of macrophages resulting in higher renal IL‐6 levels which increases ENaC activity leading to salt sensitivity.

Chloride channel accessory 1 gene deficiency causes selective loss of mucus production in a new pig model.

Morbidity and mortality of respiratory diseases are linked to airway obstruction by mucus but there are still no specific, safe, and effective drugs to correct this phenotype. The need for better treatment requires a new understanding of the basis for mucus production. In that regard, studies of human airway epithelial cells in primary culture show that a mucin granule constituent known as chloride channel accessory 1 (CLCA1) is required for inducible expression of the inflammatory mucin MUC5AC in response to potent type 2 cytokines. However, it remained uncertain whether CLCLA1 is necessary for mucus production in vivo. Conventional approaches to functional biology using targeted gene knockout were difficult due to the functional redundancy of additional Clca genes in mice not found in humans. We reasoned that CLCA1 function might be better addressed in pigs that maintain the same four-member CLCA gene locus and the corresponding mucosal and submucosal populations of mucous cells found in humans. Here we develop to our knowledge the first CLCA1-gene-deficient (CLCA1-/-) pig and show that these animals exhibit loss of MUC5AC+ mucous cells throughout the airway mucosa of the lung without affecting comparable cells in the tracheal mucosa or MUC5B+ mucous cells in submucosal glands. Similarly, CLCA1-/- pigs exhibit loss of MUC5AC+ mucous cells in the intestinal mucosa without affecting MUC2+ mucous cells. These data establish CLCA1 function for controlling MUC5AC expression as a marker of mucus production and provide a new animal model to study mucus production at respiratory and intestinal sites.

An endogenously activated antiviral state restricts SARS-CoV-2 infection in differentiated primary airway epithelial cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the coronavirus disease-19 (COVID-19) pandemic, was identified in late 2019 and caused >5 million deaths by February 2022. To date, targeted antiviral interventions against COVID-19 are limited. The spectrum of SARS-CoV-2 infection ranges from asymptomatic to fatal disease. However, the reasons for varying outcomes to SARS-CoV-2 infection are yet to be elucidated. Here we show that an endogenously activated interferon lambda (IFNλ1) pathway leads to resistance against SARS-CoV-2 infection. Using a well-differentiated primary nasal epithelial cell (WD-PNEC) culture model derived from multiple adult donors, we discovered that susceptibility to SARS-CoV-2 infection, but not respiratory syncytial virus (RSV) infection, varied. One of four donors was resistant to SARS-CoV-2 infection. High baseline IFNλ1 expression levels and associated interferon stimulated genes correlated with resistance to SARS-CoV-2 infection. Inhibition of the JAK/STAT pathway in WD-PNECs with high endogenous IFNλ1 secretion resulted in higher SARS-CoV-2 titres. Conversely, prophylactic IFNλ treatment of WD-PNECs susceptible to infection resulted in reduced viral titres. An endogenously activated IFNλ response, possibly due to genetic differences, may be one explanation for the differences in susceptibility to SARS-CoV-2 infection in humans. Importantly, our work supports the continued exploration of IFNλ as a potential pharmaceutical against SARS-CoV-2 infection.

IDF21-0230 Anti-inflammatory activity of glyoxalase-1 inducer supports further evaluation for prevention of COVID-19

Background: Pre-clinical and clinical studies suggest an essential role of proteases, plasma membrane-associated transmembrane protease serine 2 (TMPRSS2) and furin, in virion fusion with lung alveolar epithelial cells for viral infectivity of coronavirus SARS-CoV-2. Angiotensin converting enzyme-2 (ACE2) and glucose regulated protein-78 (GRP78) are considered to be receptors for cell surface binding of the spike protein of SARS-CoV-2. Risk of progression of associated COVID-19 disease to severe symptoms and risk of mortality is linked to increased levels of inflammatory mediators, including monocyte chemoattract protein-1 (MCP-1) and interleukin-8 (IL8), which are also likely risk factors for severe, life-threatening disease. Increased MCP-1 and IL8 in obese subjects and patients with diabetes may contribute to poor clinical outcomes when infected with coronavirus. In a double blind placebo-controlled clinical crossover study in overweight and obese subjects, treatment with Glo1 inducer, trans-resveratrol and hesperetin combination (tRES-HESP), once daily by oral capsule dietary supplement for 8 weeks, decreased expression of MCP-1, IL8, the receptor for advanced glycation endproducts (RAGE) and cyclo-oxygenase-2 (COX-2) in peripheral blood mononuclear cells (PBMCs). Placebo had no effect. We hypothesized that tRES-HESP may decrease protease and inflammatory gene expression linked to SARS-CoV-2 infection and COVID-19 progression. If so, the anti-inflammatory activity of Glo1 inducer in PBMCs found in clinical trial may also be evidenced in human small airway alveolar epithelial cells in primary culture. Aim: The aim of this project was to assess the anti-inflammatory effect of tRES-HESP on human alveolar epithelial cells in primary culture, assessing effects on protease and inflammatory gene expression considered essential for SARS-CoV-2 infectivity and progression of COVID-19 severity. Method: Human small airway epithelial cells (SAECs) were purchased from Lonza. SAECs were cultured in Small Airway Growth Media with supplements, according to the supplier’s instructions. SAECs (10,000 cells/cm2) were cultured with and without 5 µM tRES-HESP and the effect of basal gene expression studied after 72 h for mRNA (qRT-PCR) and protein (Western blotting); and significance of effect assessed by Student’s t-test, n = 3 or 4. The concentration of glucose in the medium was 6 mM. Results: In SAEC primary cultures, tRES-HESP decreased basal protein levels of TMPRSS2 (- 58%; P<0.001) and furin protease (- 43%; P<0.01) after 72 h. tRES-HESP also decreased mRNA of TMPRSS2 (- 62%, P<0.01), GRP78 (- 67%, P<0.001), COX-2 (-34%, P<0.001) and RAGE mRNA (- 56%, P<.001) and decreased basal secretion of MCP-1 and IL8 (-29% and - 25%, P<0.001). Discussion: tRES-HESP decreased basal expression of proteases essential for SARS-CoV-2 infection, GRP78 and secretion of inflammatory mediator risk predictors of severe COVID-19. These effects are likely due to activation of Nrf2 by tRES-HESP and decrease of unfolded protein response activation by suppression of dicarbonyl stress. These outcomes suggest previous anti-inflammatory response of tRES-HESP in overweight and obese subjects found in PBMCs may also occur concomitantly in the lung. TMPRSS2 is an antioxidant response element-linked gene down regulated by activation of Nrf2. These anti-proteolytic and anti-inflammatory responses of tRES-HESP suggest it is suitable for further evaluation for prevention of COVID-19, particularly in obesity and diabetes.

SARS-CoV-2 infection of airway cells causes intense viral and cell shedding, two spreading mechanisms affected by IL-13

Muco-obstructive lung diseases are typically associated with high risks of COVID-19 severity; however, allergic asthma showed reduced susceptibility. To investigate viral spread, primary human airway epithelial (HAE) cell cultures were infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and host–virus interactions were examined via electron microscopy, immunohistochemistry, RNA in situ hybridization, and gene expression analyses. In HAE cell cultures, angiotensin-converting enzyme 2 (ACE2) expression governed cell tropism and viral load and was up-regulated by infection. Electron microscopy identified intense viral egress from infected ciliated cells and severe cytopathogenesis, culminating in the shedding of ciliated cells packed with virions, providing a large viral reservoir for spread and transmission. Intracellular stores of MUC5AC, a major airway mucin involved in asthma, were rapidly depleted, likely to trap viruses. To mimic asthmatic airways, HAE cells were treated with interleukin-13 (IL-13), which reduced viral titers, viral messenger RNA, and cell shedding, and significantly diminished the number of infected cells. Although mucus hyperproduction played a shielding role, IL-13–treated cells maintained a degree of protection despite the removal of mucus. Using Gene Expression Omnibus databases, bulk RNA-sequencing analyses revealed that IL-13 up-regulated genes controlling glycoprotein synthesis, ion transport, and antiviral processes (albeit not the typical interferon-induced genes) and down-regulated genes involved in cilial function and ribosomal processing. More precisely, we showed that IL-13 reduced ACE2 expression, intracellular viral load, and cell-to-cell transmission while increasing the cilial keratan sulfate coating. In conclusion, intense viral and cell shedding caused by SARS-CoV-2 infection was attenuated by IL-13, which affected viral entry, replication, and spread.

Molecular Genetics and Cytotoxic Responses to Titanium Diboride and Zinc Borate Nanoparticles on Cultured Human Primary Alveolar Epithelial Cells

Titanium diboride (TiB2) and zinc borate (Zn3BO6) have been utilized in wide spectrum industrial areas because of their favorable properties such as a high melting point, good wear resistance, high hardness and thermal conductivity. On the other hand, the biomedical potentials of TiB2 and Zn3BO6 are still unknown because there is no comprehensive analysis that uncovers their biocompatibility features. Thus, the toxicogenomic properties of TiB2 and Zn3BO6 nanoparticles (NPs) were investigated on human primary alveolar epithelial cell cultures (HPAEpiC) by using different cell viability assays and microarray analyses. Protein-Protein Interaction Networks Functional Enrichment Analysis (STRING) was used to associate differentially expressed gene probes. According to the results, up to 10 mg/L concentration of TiB2 and Zn3BO6 NPs application did not stimulate a cytotoxic effect on the HPAEpiC cell cultures. Microarray analysis revealed that TiB2 NPs exposure enhances cellular adhesion molecules, proteases and carrier protein expression. Furthermore, Zn3BO6 NPs caused differential gene expressions in the cell cycle, cell division and extracellular matrix regulators. Finally, STRING analyses put forth that inflammation, cell regeneration and tissue repair-related gene interactions were affected by TiB2 NPs application. Zn3BO6 NPs exposure significantly altered inflammation, lipid metabolism and infection response activator-related gene interactions. These investigations illustrated that TiB2 and Zn3BO6 NPs exposure may affect different aspects of cellular machineries such as immunogenic responses, tissue regeneration and cell survival. Thus, these types of cellular mechanisms should be taken into account before the use of the related NPs in further biomedical applications.

A Mammary Organoid Model to Study Branching Morphogenesis.

Branching morphogenesis is the process that gives rise to branched structures in several organs, such as the lung, the kidney, and the mammary gland. Although morphologically well described, the exact mechanisms driving branch elongation and bifurcation are still poorly understood. Signaling cues from the stroma and extracellular matrix have an important role in driving branching morphogenesis. Organoid models derived from primary mammary epithelial cells have emerged as a powerful tool to gain insight into branching morphogenesis of the mammary gland. However, current available mammary organoid culture protocols result in morphologically simple structures which do not resemble the complex branched structure of the in vivo mammary gland. Supplementation of growth factors to mammary organoids cultured in basement membrane extract or collagen I were shown to induce bud formation and elongation but are not sufficient to drive true branching events. Here, we present an improved culture approach based on 3D primary mammary epithelial cell culture to develop branched organoids with a complex morphology. By alternating the addition of fibroblast growth factor 2 and epidermal growth factor to mammary organoids cultured in a basement membrane extract matrix enriched with collagen type I fibers, we obtain complex mammary organoid structures with primary, secondary, and tertiary branches over a period of 15–20 days. Mammary organoid structures grow >1 mm in size and show an elongated and branched shape which resembles in vivo mammary gland morphology. This novel branched mammary organoid model offers many possibilities to study the mechanisms of branching in the developing mammary gland.

Impact of Air Pollution in Airway Diseases: Role of the Epithelial Cells (Cell Models and Biomarkers).

Biomedical research is multidisciplinary and often uses integrated approaches performing different experimental models with complementary functions. This approach is important to understand the pathogenetic mechanisms concerning the effects of environmental pollution on human health. The biological activity of the substances is investigated at least to three levels using molecular, cellular, and human tissue models. Each of these is able to give specific answers to experimental problems. A scientific approach, using biological methods (wet lab), cell cultures (cell lines or primary), isolated organs (three-dimensional cell cultures of primary epithelial cells), and animal organisms, including the human body, aimed to understand the effects of air pollution on the onset of diseases of the respiratory system. Biological methods are divided into three complementary models: in vitro, ex vivo, and in vivo. In vitro experiments do not require the use of whole organisms (in vivo study), while ex vivo experiments use isolated organs or parts of organs. The concept of complementarity and the informatic support are useful tools to organize, analyze, and interpret experimental data, with the aim of discussing scientific notions with objectivity and rationality in biology and medicine. In this scenario, the integrated and complementary use of different experimental models is important to obtain useful and global information that allows us to identify the effect of inhaled pollutants on the incidence of respiratory diseases in the exposed population. In this review, we focused our attention on the impact of air pollution in airway diseases with a rapid and descriptive analysis on the role of epithelium and on the experimental cell models useful to study the effect of toxicants on epithelial cells.

Entourage effect for phenolic compounds on production and metabolism of mammary epithelial cells

Primary culture of mammary epithelial cells (MEC) was exposed to ethyl-acetate, chloroform and hexane extracts of Pistacia lentiscus (lentisk). The hexane extract contained mainly ethyl gallate whereas the chloroform extract contained mainly ethyl-gallate with smaller amount of gallic acid, and the ethyl-acetate extract contained mainly rutin, gallic acid and myricetin. Ethyl acetate extract increased secretion of protein and fat and improved mitochondrial activity. The enhancing effect on protein production was attributed to myricetin, one of the polyphenols in the ethyl-acetate extract whereas gallic acid did not affect protein production or secretion. Interestingly, exposure to the isolated polyphenols did not improve mitochondrial productivity and activity as effectively as exposure to the complete plant extract. The results indicated that polyphenols improve production of milk constituents by MEC, through different modes of action for different polyphenols suggesting an additive or even synergistic effect on production traits of mammary cells.

Primary Nasal Epithelial Cells as a Surrogate Cell Culture Model for Type-II Alveolar Cells to Study ABCA-3 Deficiency.

ATP Binding Cassette Subfamily A Member 3 (ABCA-3) is a lipid transporter protein highly expressed in type-II alveolar (AT-II) cells. Mutations in ABCA3 can result in severe respiratory disease in infants and children. To study ABCA-3 deficiency in vitro, primary AT-II cells would be the cell culture of choice although sample accessibility is limited. Our aim was to investigate the suitability of primary nasal epithelial cells, as a surrogate culture model for AT-II cells, to study ABCA-3 deficiency. Expression of ABCA3, and surfactant protein genes, SFTPB and SFTPC, was detected in primary nasal epithelial cells but at a significantly lower level than in AT-II cells. ABCA-3, SP-B, and SP-C were detected by immunofluorescence microscopy in primary nasal epithelial cells. However, SP-B and SP-C were undetectable in primary nasal epithelial cells using western blotting. Structurally imperfect lamellar bodies were observed in primary nasal epithelial cells using transmission electron microscopy. Functional assessment of the ABCA-3 protein demonstrated that higher concentrations of doxorubicin reduced cell viability in ABCA-3 deficient nasal epithelial cells compared to controls in an assay-dependent manner. Our results indicate that there may be a role for primary nasal epithelial cell cultures to model ABCA-3 deficiency in vitro, although additional cell culture models that more effectively recapitulate the AT-II phenotype may be required.

Influence of culture expansion medium on human bronchial epithelium composition and function

Primary culture of human bronchial epithelial cells in air/liquid interface (ALI) is a validated in vitro model, which provides fully differentiated and pseudostratified bronchial epithelium and allows to analyze its functions in different respiratory diseases. However, the potential impact of the culture medium on the structure and the metabolism of this reconstituted epithelium is currently underestimated. Epithelial cells were grown either in PneumaCult Ex (PC Ex) or PneumaCult Ex+ (PC Ex + ) medium (StemCell, Vancouver, Canada). After the expansion phase, cells were differentiated in PneumaCult ALI medium. Analyses were performed during the expansion phase, and at day 0 (D0), D7, D14, D21 and D28 of the differentiation process. Pictures of the cell morphology were taken, immunostaining were carried out on cytocentrifuged cells, and culture supernatants were stored for subsequent analyses. At D28 of differentiation, bronchial epithelium was challenged with Poly I:C or TNF-α. The cytokine secretion in culture media or apical lavages, and the expression of mRNA and intra-cellular protein were assessed. PC Ex+ medium provided a higher cell yield than PC Ex. Morphology was found to be different, as PC Ex + -cultured cells formed clusters. At early and differentiated stage, cells cultured in PC Ex medium expressed specific epithelial and mesenchymal markers (pan-cytokeratin and vimentin, respectively), while cells in PC Ex+ mainly expressed the epithelial marker. Our preliminary results showed differences in terms of cellular yield and epithelial-mesenchymal cell features, depending on the culture medium used for cellular expansion. The influence of the expansion medium on cell subtypes during the differentiation steps is under investigation. In the near future, we will compare the release of cell mediators at steady state and after stimulation to better understand the effect of culture medium on the functional responses of epithelial cells.