Под управлением нейтрофилов
Aug. 11th, 2021 05:25 pm![[personal profile]](https://www.dreamwidth.org/img/silk/identity/user.png){kind=small}
chuka_lisРабота, показывающая на клеточной модели, что воспалительный ответ в дыхальных путях зависит от того, принимали ли участие в процессе нейтрофилы.
Там, где высокая инфильтрация эпителия нейтрофилами- при ковидной инфекции формировался про-воспалительный ответ, способствующий усилению инфекции и разрушению тканей. Количества нейтрофилов в тканях повышаются с возрастом, при ожирении, диабете, астме, ХОБЛ и других хронических легочных заболеваниях
А без них, все протекало заметно легче.
In response to viral infection, neutrophils release inflammatory mediators as part of the innate immune response, contributing to pathogen clearance through virus internalization and killing. Pre-existing co-morbidities, correlating to incidence of severe COVID-19, are associated with chronic airway neutrophilia and examination of COVID-19 lung tissue revealed a series of epithelial pathologies associated with infiltration and activation of neutrophils. To determine the impact of neutrophil-epithelial interactions on the infectivity and inflammatory response to SARS-CoV-2 infection, we developed a co-culture model of airway neutrophilia. We discovered that SARS-CoV-2 infection of the airway epithelium alone does not result in a notable release of pro-inflammatory cytokines, however in the presence of neutrophils, the inflammatory response is both polarized and significantly augmented, epithelial barrier integrity in impaired and viral load of the airway epithelium increased. Overall, study reveals a key role for neutrophil-epithelial interactions in determining inflammation, infectivity, and outcomes in response to SARS-CoV-2 infection.
In response to viral infection neutrophils are the first and predominant immune cells recruited to the respiratory tract(Pechous, 2017). Neutrophils release inflammatory mediators as part of the innate immune response and contribute to pathogen clearance through virus internalization and killing (Grommes and Soehnlein, 2011). While the protective versus pathological role of neutrophils in the airways during viral response is poorly understood, it has been shown that the number of neutrophils in the lower respiratory tract correlates to disease severity and is an early marker for COVID-19 (Munoz-Fontela et al., 2020; Song et al., 2020; Zhang et al., 2020a). Infiltration of neutrophils is also characteristic of other lung diseases associated with chronic infection and inflammation, such as asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF). All of these respiratory diseases have been associated with an increased risk of contracting severe COVID-19 (Aveyard et al., 2021) .Evaluating the relationship between SARS-CoV-2 infection and neutrophilia may provide critical insight into how host and viral factors contribute to disease severity Despite their importance in anti-viral immunity and response to viral pathogens, neutrophils have been somewhat overlooked for their role in the pathogenesis of SARS-CoV-2 infection (Hemmat et al., 2020; Shi et al., 2020; Veras et al., 2020a). It has been shown that the number of neutrophils in the lower respiratory tract correlates to disease severity inother viral infections, including influenza A infection (Radermecker et al., 2019) and, more recently, to also be a feature of COVID-19 pathology (Veras et al., 2020a). Several studies have highlighted the importance of neutrophils in the response to SARS-CoV-2 infection (Li et al., 2020; Shi et al., 2020; Tomar et al., 2020; Veras et al., 2020a) and clinically neutrophil-lymphocyte ratios (NLR) are becoming an important hallmark of severe COVID-19 (Qin et al., 2020). Furthermore, the expression of angiotensin converting enzyme 2 (ACE2)on neutrophils has also been demonstrated; (Arcanjo et al., 2020; Janiuk et al., 2021; Veras et al., 2020b) (Veras et al., 2020c). These studies, however, have primarily focused on the production of neutrophil extracellular traps and lack insights into other neutrophil functional responses such as inflammatory cytokine production and viral internalization in neutrophils. In this study, the relationship between SARS-CoV-2 infection and pre-existing airway neutrophilia in the presence of the lung airway epithelial niche was evaluated through the development of a co-culture infection model. Primary neutrophils were isolated from peripheral blood and co-cultured with differentiated primary tracheo-bronchial airway epithelium prior to infection with live SARS-CoV-2 virus.Changes in the inflammatory profile and epithelial response were comprehensively evaluated to determine the impact of neutrophils on the epithelial SARS-CoV-2 infection. Given the extensive infiltration of inflammatory cells, inclusive of neutrophils, we further evaluated the neutrophil- related tissue pathology in both patients. An array of airway tissue pathologies were evident in both tissuesincluding
1) basal cell hyperplasia and small airway occlusion(Fig. 1A-E)
2) epithelial damage and tissue remodeling of smaller ciliated airways (Fig. 1F)
3) epithelial shedding of large cartilaginous airways (Fig. 1G),
4) neutrophil invasion into the airway lumen(Fig.1H), and finally,
5) neutrophil invasion in the alveolar space with associated alveolar tissue damageand remodeling(Fig. 1I) In each of these examples found in post-mortem COVID-19 tissue, neutrophils were detected and frequently demonstrated increased neutrophil elastase (NE) activity, compared to neutrophils detected in tissue from no underlying respiratory disease. Interestingly,we noted that neutrophils strongly expressedACE2in these tissues.We confirmedACE2 expression in neutrophils present in human lung tissues by immuno fluorescence, comparing tissue from patients with no underlying respiratory disease and patients with 300CF(Supplemental Fig.S2A-B), a chronic lung disease where neutrophilia is a common hallmark. In these tissues, ACE2 expression was confirmed to co-localize with both NE and CD15 (Supplemental Fig.S2C-D). 302We further validated ACE2 expression inCD15+ purified neutrophils from peripheral human blood, purity of neutrophils was confirmed by FACS (>97%) Given the prevalence of neutrophilia in the airways of patients with chronic airway disease(Jasper et al., 2019) and its association with other SARS-CoV-2 co-morbidities, such as diabetes mellitus (Thomson et al., 1997) and hypertension (Florentin et al., 2021; Taylor et al., 2018), we proceeded to investigate the impact of 310neutrophilic airway inflammation in SARS-CoV-2 infection. We created a neutrophilic airway in vitro model by co-culturing CD15+peripheral blood polymorphonuclear leukocytes (PMNs) with primary human tracheo- bronchial airway epithelial cells (HBECs) differentiated at the air-liquid interface (ALI), and infected these cultures with live SARS-CoV-2 virus for 4 hours (Fig. 2A). This 4-hourtime point allows us to profile the acute phase cellular viral response, i.e.,degranulation of the neutrophils. The short time frame for analysis was chosen to both eliminate significant viral replication and thus anticipate any detectible viral load is as a result of initial infection(Zhu et al., 2020b), and to allow for optimal investigation into neutrophil function without loss of viability interfering with the assays due to there latively short half-life of neutrophils. Prior to infection we confirmed the expression ofACE2 and Transmembrane Serine Protease 2 (TMPRSS2in our in vitro airway epithelium models (Supplemental Fig.S3). While ACE2 RNA was relatively low in expression across basal, secretory and multiciliated cells (Supplemental Fig. S3B-C) at the protein level a predominant colocalization was detected with multiciliated cells in the airways Apical and basolateral cell culture supernatants were analyzed for secretion of inflammatory cytokines using the meso scale discovery (MSD) cytokine assay. As shown in Figure2C-Da differential inflammatory profile exists between theapicaland basolateral compartments.Surprisingly, in the absence of neutrophils,there was no significant difference in cytokine release from the airway epithelial cells upon SARS-CoV-2 infection, except forbasolateral interleukin-8 (IL-8) which significantly increased. As IL-8 is a major chemoattractant for neutrophils this suggests that the basolateral surface responds to viral infection with a predominant release of IL-8, likely in the attempt to recruit neutrophils to the epithelium (Baggiolini et al., 1989; Parsons et al., 1985; Yoshimura et al., 1987). This data suggests that the presence of neutrophilsatapical surface and infiltrating the airway epithelium stimulates the release of pro-inflammatory cytokinesfrom both the apical and basolateral faces of the epithelium, thus creating a pro-inflammatory niche in the co-cultures. In our model, this pro-inflammatory niche was used to recreate aspects of chronic airway inflammation in the human lung.Interestingly, the presence of neutrophils did not stimulate significant changes in IL-8 secretion from the basolateral surface supporting the role for IL-8 in the recruitment phase of airway neutrophilia which is already achieved in our model (Azevedo et al., 2021; Pease and Sabroe, 2002) This data indicates a significant augmentation of the inflammatory response when infection occurs in the presence of pre-existing airway neutrophilia.Importantly, this secretion profile closely reflects the cytokine biomarkers that have been clinically identified in patients hospitalized with severe COVID-19 disease (Del Valle et al., 2020; Han et al., 2020; Liu et al., 2020b), highlighting the importance of the co-culture models in recapitulating features associated with more severe responses to SARS-CoV-2. Increased in viral load of SARS-CoV-2 in epithelial cells is associated with disruption in epithelial barrier integrity.his indicates that in epithelium where chronic neutrophil infiltration is present there is a significant deterioration in epithelial barrier function which is associated with elevated cytokine levels and an increase in viral infection of the epithelium. Finally, to determine whether the expression ofACE2 protein in neutrophils has a significant impact in the response of neutrophils to SARS-CoV-2, we evaluated whether neutrophils were being actively infected via a physical interaction of ACE2 and SARS-CoV-2 or phagocytosing the SARS-CoV-2 virus. Infection, detected by RNA scope, occurred at a rate of 7.9±1% of neutrophils in culture. Disruption of the actin cytoskeleton, a core component of phagocytosis, therefore, significantly reduced viral uptake in neutrophils. This suggests the primary mechanism for SARS-CoV-2 internalization in neutrophils is phagocytosis Discussion It is well established that neutrophils are critical in the development of pathological inflammation which can result in both acute and chronic tissue damage. Upon evaluation of post-mortem COVID-19 tissues we observed significant neutrophil presence and activation in regions closely associated with pathogenic alterations in the airway epithelium. In addition, we know that many associated co-morbidities, including chronic airway disease (Gernez et al., 2010; Jasper et al., 2019), aging (Chen et al., 2014; Kulkarni et al., 2019; Sapey et al., 2017)and obesity (Kordonowy et al., 2012; Maia et al., 2019; Manicone et al., 2016)are also associated with chronic airway inflammation. In this study we, therefore, developed a model to evaluate neutrophil-epithelial interactions and how this may be critical in the progression to severe COVID-19. Using this model, we were able to conclude that the presence of neutrophils with the airway epithelium significantly augments the proinflammatory responses to SARS-CoV-2, increases airway viral load and decreases airway epithelial barrier integrity. Our data, therefore, supports a key role for neutrophil-epithelial interactions in determining the infectivity and outcome measures of COVID-19 n tissues from severe COVID-19 patients we observed a significant presence of neutrophils and neutrophil activity in and surrounding patient airways, including neutrophil invasion into the airway lumen, epithelial shedding, and large amounts of NE activity (Fig. 1and Supplemental Fig.S1-2). Highlighting a key role for neutrophils in the response to SARS-CoV-2 infection. Our model is designed to mimic neutrophilic airway inflammation associated with chronic lung disease, that has been linked with a predisposition to developing more severe COVID-19 disease. It is established that neutrophil phenotype and function, including those involved in resolving viral infections, is strongly regulated by signals received from their tissue micro-environment (Parkos, 2016), in our study we considered neutrophil responses in the presence of an epithelial micro-environment, a major advantage of our model for studying SARS-CoV-2 infection. Through paired comparisons to primary airway epithelial cells in monoculture, we were able to demonstrate key differences in the secretion of pro (IFNγ, IL1β, IL-6, IL-8 and TNFα) and anti-inflammatory (IL-4 and IL10) mediators, epithelial barrier integrity and infectivity of epithelial cells (Fig. 2), which would have been over-looked in monoculture experiments involving airway infection only. Importantly, the secretion of pro-inflammatory cytokines in our model is consistent with clinical studies that have reported an elevated inflammatory profile associated with severe COVID-19 disease. In patient peripheral blood samples, IL-6 (Godkin and Humphreys, 2020; Huang et al., 2020b; Liu et al., 2020a; Yang et al., 2020) IL-10 (Godkin and Humphreys, 2020; Liu et al., 2020a) are consistently higher in COVID- 19 patients and correlate with disease severity. Additionally, IL-6 and IL-8 are even higher in ICU than the IMU (Pandolfi et al., 2020). Our data also closely mimics responses observed in primate models of the disease (Fahlberg et al., 2020). Furthermore, the correlation of neutrophilic recruitment to the airways is also clinically elevant where substantially elevated neutrophil are observed in the peripheral blood of COVID-19 patients and neutrophil-to-lymphocyte ratios correlate with severe and fatal complications related to ARDS (Godkin and Humphreys, 2020; Huang et al., 2020b; Qun et al., 2020; Zhu et al., 2020a). Additionally, marked increases in neutrophils were also observed in bronchiolar alveolar lavage (BAL) fluids from patients admitted into the intensive care unit (ICU) compared to intermediary medical unit (IMU) patients correlating to disease severity (Pandolfi et al., 2020). The lack of robust inflammatory response of the epithelium alone may also provide rational for why some people are predisposed to more severe responses than others. In fact, our data evaluating the response of the more proximal, cartilaginous airways may highlight the importance of a robust proximal airway defense mechanism that controls the progression to severe COVID-19 associated with ARDS and distal airway dysfunction. The distinct polarized secretion of IL-8 in response to SARS-CoV-2 infection in the monocultured epithelial cells, indicates a signaling from the epithelium to recruit neutrophils,IL-8 is the core chemoattractant for neutrophils (Baggiolini et al., 1989; Kunkel et al., 1991; Parsons et al., 1985; Yoshimura et al., 1987). This response was not observed in the neutrophil-epithelial co-cultures and suggests 479that the epithelial cells can recognize neutrophils within their microenvironment. Pro-inflammatory cytokines, including IFNγ, IL1β, IL-6and TNFα, have extensively been shown to disrupt barrier integrity and permeability of the epithelium (Al-Sadi et al., 2009; Capaldo and Nusrat, 2009). This breakdown in barrier integrity exists to allow for leukocyte migration to sites of stress and infection. Theoretically, any tight-junction breakdown that allows for more leukocyte migration, would also allow for increased permeability for viral particles to sub-apical and sub-epithelial structures, thus increasing infectivity and cellular viral loads. In our data we show that neutrophils and cytomix both synonymously decrease barrier integrity (Fig.3). This association of epithelial barrier integrity with an increase in viral load suggests that,at least in part,the epithelial barrier integrity plays an important functional role in resisting SARS-CoV-2 infection. Finally, we addressed the key question of whether neutrophils are actively binding to and becoming infected with thevirus via ACE2 or, alternatively, as professional phagocytes (Silva and Correia-Neves, 2012; Uribe- Querol and Rosales, 2020), are engulfing the invading pathogen through innate recognition pathways. Our data supports a high level of expression of ACE2 at the protein level, but not the RNA level in neutrophils; an observation recently reported by Veras and colleagues (Veras et al.,2020c) Нowever,reports are emerging suggesting a significant role for cytoskeletal rearrangement inSARS-CoV-2 entry and, therefore,we cannot entirely rule out infection (Wen et al.,2020). The use of blocking antibodies has potential to elucidate the mechanisms of internalization, however, would pose significant challenges. Neutrophils famously express copious amounts of Fc-receptors (Wang and Jonsson, 2019). By including antibodies sensitive or otherwise to present antigens in neutrophil cultures would opsonize an present antigens and be recognized by the neutrophils eventually becoming internalized via opsonized mediated phagocytosis. Indeed,we might likely expect increased internalization with the presence of any antigens sensitive antibodies.
Там, где высокая инфильтрация эпителия нейтрофилами- при ковидной инфекции формировался про-воспалительный ответ, способствующий усилению инфекции и разрушению тканей. Количества нейтрофилов в тканях повышаются с возрастом, при ожирении, диабете, астме, ХОБЛ и других хронических легочных заболеваниях
А без них, все протекало заметно легче.
In response to viral infection, neutrophils release inflammatory mediators as part of the innate immune response, contributing to pathogen clearance through virus internalization and killing. Pre-existing co-morbidities, correlating to incidence of severe COVID-19, are associated with chronic airway neutrophilia and examination of COVID-19 lung tissue revealed a series of epithelial pathologies associated with infiltration and activation of neutrophils. To determine the impact of neutrophil-epithelial interactions on the infectivity and inflammatory response to SARS-CoV-2 infection, we developed a co-culture model of airway neutrophilia. We discovered that SARS-CoV-2 infection of the airway epithelium alone does not result in a notable release of pro-inflammatory cytokines, however in the presence of neutrophils, the inflammatory response is both polarized and significantly augmented, epithelial barrier integrity in impaired and viral load of the airway epithelium increased. Overall, study reveals a key role for neutrophil-epithelial interactions in determining inflammation, infectivity, and outcomes in response to SARS-CoV-2 infection.
In response to viral infection neutrophils are the first and predominant immune cells recruited to the respiratory tract(Pechous, 2017). Neutrophils release inflammatory mediators as part of the innate immune response and contribute to pathogen clearance through virus internalization and killing (Grommes and Soehnlein, 2011). While the protective versus pathological role of neutrophils in the airways during viral response is poorly understood, it has been shown that the number of neutrophils in the lower respiratory tract correlates to disease severity and is an early marker for COVID-19 (Munoz-Fontela et al., 2020; Song et al., 2020; Zhang et al., 2020a). Infiltration of neutrophils is also characteristic of other lung diseases associated with chronic infection and inflammation, such as asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF). All of these respiratory diseases have been associated with an increased risk of contracting severe COVID-19 (Aveyard et al., 2021) .Evaluating the relationship between SARS-CoV-2 infection and neutrophilia may provide critical insight into how host and viral factors contribute to disease severity Despite their importance in anti-viral immunity and response to viral pathogens, neutrophils have been somewhat overlooked for their role in the pathogenesis of SARS-CoV-2 infection (Hemmat et al., 2020; Shi et al., 2020; Veras et al., 2020a). It has been shown that the number of neutrophils in the lower respiratory tract correlates to disease severity inother viral infections, including influenza A infection (Radermecker et al., 2019) and, more recently, to also be a feature of COVID-19 pathology (Veras et al., 2020a). Several studies have highlighted the importance of neutrophils in the response to SARS-CoV-2 infection (Li et al., 2020; Shi et al., 2020; Tomar et al., 2020; Veras et al., 2020a) and clinically neutrophil-lymphocyte ratios (NLR) are becoming an important hallmark of severe COVID-19 (Qin et al., 2020). Furthermore, the expression of angiotensin converting enzyme 2 (ACE2)on neutrophils has also been demonstrated; (Arcanjo et al., 2020; Janiuk et al., 2021; Veras et al., 2020b) (Veras et al., 2020c). These studies, however, have primarily focused on the production of neutrophil extracellular traps and lack insights into other neutrophil functional responses such as inflammatory cytokine production and viral internalization in neutrophils. In this study, the relationship between SARS-CoV-2 infection and pre-existing airway neutrophilia in the presence of the lung airway epithelial niche was evaluated through the development of a co-culture infection model. Primary neutrophils were isolated from peripheral blood and co-cultured with differentiated primary tracheo-bronchial airway epithelium prior to infection with live SARS-CoV-2 virus.Changes in the inflammatory profile and epithelial response were comprehensively evaluated to determine the impact of neutrophils on the epithelial SARS-CoV-2 infection. Given the extensive infiltration of inflammatory cells, inclusive of neutrophils, we further evaluated the neutrophil- related tissue pathology in both patients. An array of airway tissue pathologies were evident in both tissuesincluding
1) basal cell hyperplasia and small airway occlusion(Fig. 1A-E)
2) epithelial damage and tissue remodeling of smaller ciliated airways (Fig. 1F)
3) epithelial shedding of large cartilaginous airways (Fig. 1G),
4) neutrophil invasion into the airway lumen(Fig.1H), and finally,
5) neutrophil invasion in the alveolar space with associated alveolar tissue damageand remodeling(Fig. 1I) In each of these examples found in post-mortem COVID-19 tissue, neutrophils were detected and frequently demonstrated increased neutrophil elastase (NE) activity, compared to neutrophils detected in tissue from no underlying respiratory disease. Interestingly,we noted that neutrophils strongly expressedACE2in these tissues.We confirmedACE2 expression in neutrophils present in human lung tissues by immuno fluorescence, comparing tissue from patients with no underlying respiratory disease and patients with 300CF(Supplemental Fig.S2A-B), a chronic lung disease where neutrophilia is a common hallmark. In these tissues, ACE2 expression was confirmed to co-localize with both NE and CD15 (Supplemental Fig.S2C-D). 302We further validated ACE2 expression inCD15+ purified neutrophils from peripheral human blood, purity of neutrophils was confirmed by FACS (>97%) Given the prevalence of neutrophilia in the airways of patients with chronic airway disease(Jasper et al., 2019) and its association with other SARS-CoV-2 co-morbidities, such as diabetes mellitus (Thomson et al., 1997) and hypertension (Florentin et al., 2021; Taylor et al., 2018), we proceeded to investigate the impact of 310neutrophilic airway inflammation in SARS-CoV-2 infection. We created a neutrophilic airway in vitro model by co-culturing CD15+peripheral blood polymorphonuclear leukocytes (PMNs) with primary human tracheo- bronchial airway epithelial cells (HBECs) differentiated at the air-liquid interface (ALI), and infected these cultures with live SARS-CoV-2 virus for 4 hours (Fig. 2A). This 4-hourtime point allows us to profile the acute phase cellular viral response, i.e.,degranulation of the neutrophils. The short time frame for analysis was chosen to both eliminate significant viral replication and thus anticipate any detectible viral load is as a result of initial infection(Zhu et al., 2020b), and to allow for optimal investigation into neutrophil function without loss of viability interfering with the assays due to there latively short half-life of neutrophils. Prior to infection we confirmed the expression ofACE2 and Transmembrane Serine Protease 2 (TMPRSS2in our in vitro airway epithelium models (Supplemental Fig.S3). While ACE2 RNA was relatively low in expression across basal, secretory and multiciliated cells (Supplemental Fig. S3B-C) at the protein level a predominant colocalization was detected with multiciliated cells in the airways Apical and basolateral cell culture supernatants were analyzed for secretion of inflammatory cytokines using the meso scale discovery (MSD) cytokine assay. As shown in Figure2C-Da differential inflammatory profile exists between theapicaland basolateral compartments.Surprisingly, in the absence of neutrophils,there was no significant difference in cytokine release from the airway epithelial cells upon SARS-CoV-2 infection, except forbasolateral interleukin-8 (IL-8) which significantly increased. As IL-8 is a major chemoattractant for neutrophils this suggests that the basolateral surface responds to viral infection with a predominant release of IL-8, likely in the attempt to recruit neutrophils to the epithelium (Baggiolini et al., 1989; Parsons et al., 1985; Yoshimura et al., 1987). This data suggests that the presence of neutrophilsatapical surface and infiltrating the airway epithelium stimulates the release of pro-inflammatory cytokinesfrom both the apical and basolateral faces of the epithelium, thus creating a pro-inflammatory niche in the co-cultures. In our model, this pro-inflammatory niche was used to recreate aspects of chronic airway inflammation in the human lung.Interestingly, the presence of neutrophils did not stimulate significant changes in IL-8 secretion from the basolateral surface supporting the role for IL-8 in the recruitment phase of airway neutrophilia which is already achieved in our model (Azevedo et al., 2021; Pease and Sabroe, 2002) This data indicates a significant augmentation of the inflammatory response when infection occurs in the presence of pre-existing airway neutrophilia.Importantly, this secretion profile closely reflects the cytokine biomarkers that have been clinically identified in patients hospitalized with severe COVID-19 disease (Del Valle et al., 2020; Han et al., 2020; Liu et al., 2020b), highlighting the importance of the co-culture models in recapitulating features associated with more severe responses to SARS-CoV-2. Increased in viral load of SARS-CoV-2 in epithelial cells is associated with disruption in epithelial barrier integrity.his indicates that in epithelium where chronic neutrophil infiltration is present there is a significant deterioration in epithelial barrier function which is associated with elevated cytokine levels and an increase in viral infection of the epithelium. Finally, to determine whether the expression ofACE2 protein in neutrophils has a significant impact in the response of neutrophils to SARS-CoV-2, we evaluated whether neutrophils were being actively infected via a physical interaction of ACE2 and SARS-CoV-2 or phagocytosing the SARS-CoV-2 virus. Infection, detected by RNA scope, occurred at a rate of 7.9±1% of neutrophils in culture. Disruption of the actin cytoskeleton, a core component of phagocytosis, therefore, significantly reduced viral uptake in neutrophils. This suggests the primary mechanism for SARS-CoV-2 internalization in neutrophils is phagocytosis Discussion It is well established that neutrophils are critical in the development of pathological inflammation which can result in both acute and chronic tissue damage. Upon evaluation of post-mortem COVID-19 tissues we observed significant neutrophil presence and activation in regions closely associated with pathogenic alterations in the airway epithelium. In addition, we know that many associated co-morbidities, including chronic airway disease (Gernez et al., 2010; Jasper et al., 2019), aging (Chen et al., 2014; Kulkarni et al., 2019; Sapey et al., 2017)and obesity (Kordonowy et al., 2012; Maia et al., 2019; Manicone et al., 2016)are also associated with chronic airway inflammation. In this study we, therefore, developed a model to evaluate neutrophil-epithelial interactions and how this may be critical in the progression to severe COVID-19. Using this model, we were able to conclude that the presence of neutrophils with the airway epithelium significantly augments the proinflammatory responses to SARS-CoV-2, increases airway viral load and decreases airway epithelial barrier integrity. Our data, therefore, supports a key role for neutrophil-epithelial interactions in determining the infectivity and outcome measures of COVID-19 n tissues from severe COVID-19 patients we observed a significant presence of neutrophils and neutrophil activity in and surrounding patient airways, including neutrophil invasion into the airway lumen, epithelial shedding, and large amounts of NE activity (Fig. 1and Supplemental Fig.S1-2). Highlighting a key role for neutrophils in the response to SARS-CoV-2 infection. Our model is designed to mimic neutrophilic airway inflammation associated with chronic lung disease, that has been linked with a predisposition to developing more severe COVID-19 disease. It is established that neutrophil phenotype and function, including those involved in resolving viral infections, is strongly regulated by signals received from their tissue micro-environment (Parkos, 2016), in our study we considered neutrophil responses in the presence of an epithelial micro-environment, a major advantage of our model for studying SARS-CoV-2 infection. Through paired comparisons to primary airway epithelial cells in monoculture, we were able to demonstrate key differences in the secretion of pro (IFNγ, IL1β, IL-6, IL-8 and TNFα) and anti-inflammatory (IL-4 and IL10) mediators, epithelial barrier integrity and infectivity of epithelial cells (Fig. 2), which would have been over-looked in monoculture experiments involving airway infection only. Importantly, the secretion of pro-inflammatory cytokines in our model is consistent with clinical studies that have reported an elevated inflammatory profile associated with severe COVID-19 disease. In patient peripheral blood samples, IL-6 (Godkin and Humphreys, 2020; Huang et al., 2020b; Liu et al., 2020a; Yang et al., 2020) IL-10 (Godkin and Humphreys, 2020; Liu et al., 2020a) are consistently higher in COVID- 19 patients and correlate with disease severity. Additionally, IL-6 and IL-8 are even higher in ICU than the IMU (Pandolfi et al., 2020). Our data also closely mimics responses observed in primate models of the disease (Fahlberg et al., 2020). Furthermore, the correlation of neutrophilic recruitment to the airways is also clinically elevant where substantially elevated neutrophil are observed in the peripheral blood of COVID-19 patients and neutrophil-to-lymphocyte ratios correlate with severe and fatal complications related to ARDS (Godkin and Humphreys, 2020; Huang et al., 2020b; Qun et al., 2020; Zhu et al., 2020a). Additionally, marked increases in neutrophils were also observed in bronchiolar alveolar lavage (BAL) fluids from patients admitted into the intensive care unit (ICU) compared to intermediary medical unit (IMU) patients correlating to disease severity (Pandolfi et al., 2020). The lack of robust inflammatory response of the epithelium alone may also provide rational for why some people are predisposed to more severe responses than others. In fact, our data evaluating the response of the more proximal, cartilaginous airways may highlight the importance of a robust proximal airway defense mechanism that controls the progression to severe COVID-19 associated with ARDS and distal airway dysfunction. The distinct polarized secretion of IL-8 in response to SARS-CoV-2 infection in the monocultured epithelial cells, indicates a signaling from the epithelium to recruit neutrophils,IL-8 is the core chemoattractant for neutrophils (Baggiolini et al., 1989; Kunkel et al., 1991; Parsons et al., 1985; Yoshimura et al., 1987). This response was not observed in the neutrophil-epithelial co-cultures and suggests 479that the epithelial cells can recognize neutrophils within their microenvironment. Pro-inflammatory cytokines, including IFNγ, IL1β, IL-6and TNFα, have extensively been shown to disrupt barrier integrity and permeability of the epithelium (Al-Sadi et al., 2009; Capaldo and Nusrat, 2009). This breakdown in barrier integrity exists to allow for leukocyte migration to sites of stress and infection. Theoretically, any tight-junction breakdown that allows for more leukocyte migration, would also allow for increased permeability for viral particles to sub-apical and sub-epithelial structures, thus increasing infectivity and cellular viral loads. In our data we show that neutrophils and cytomix both synonymously decrease barrier integrity (Fig.3). This association of epithelial barrier integrity with an increase in viral load suggests that,at least in part,the epithelial barrier integrity plays an important functional role in resisting SARS-CoV-2 infection. Finally, we addressed the key question of whether neutrophils are actively binding to and becoming infected with thevirus via ACE2 or, alternatively, as professional phagocytes (Silva and Correia-Neves, 2012; Uribe- Querol and Rosales, 2020), are engulfing the invading pathogen through innate recognition pathways. Our data supports a high level of expression of ACE2 at the protein level, but not the RNA level in neutrophils; an observation recently reported by Veras and colleagues (Veras et al.,2020c) Нowever,reports are emerging suggesting a significant role for cytoskeletal rearrangement inSARS-CoV-2 entry and, therefore,we cannot entirely rule out infection (Wen et al.,2020). The use of blocking antibodies has potential to elucidate the mechanisms of internalization, however, would pose significant challenges. Neutrophils famously express copious amounts of Fc-receptors (Wang and Jonsson, 2019). By including antibodies sensitive or otherwise to present antigens in neutrophil cultures would opsonize an present antigens and be recognized by the neutrophils eventually becoming internalized via opsonized mediated phagocytosis. Indeed,we might likely expect increased internalization with the presence of any antigens sensitive antibodies.
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