ABSTRACT
RATIONALE: Common human alphacoronaviruses and rhinoviruses (HRV) induce IFN I/III responses important to limiting viral propagation in the airway epithelium. In contrast, highly pathogenic human betacoronaviruses (e.g. SARS-CoV, circa 2002) may evade or antagonize RNA-induced IFN I/III responses. Aim: 1) Compare IFN I/III responses by bronchial AECs from children and adults between infection with SARS CoV2 and HRV-16. 2) Determine if pre-infection of AEC cultures with HRV-16, or pretreatment with IFN-β or IFN-λ, modifies SARS-CoV-2 replication. Methods: Bronchial AECs from children and adults (n=15;ages 8-75 yrs.) were differentiated ex vivo at an air-liquid interface to generate organotypic cultures. In a biosafety level 3 (BSL-3) facility, cultures were infected with SARS-CoV-2 isolate USA-WA1/2020 or HRV-16 at a multiplicity of infection (MOI) of 0.5. At 96 hrs. following infection, RNA and protein were isolated. Expression of IFNB1, IFNL2, and interferon stimulated genes (ISGs) IFITM1, IFITM3, and OAS1 was measured by qPCR in SARS-CoV-2-infected, HRV-16-infected, and uninfected AEC cultures. In additional experiments AECs were pre-infected with HRV-16 (MOI=0.5), or pre-treated with recombinant IFN-β1 (1ng/mL in media) or IFN-λ2 (10ng/mL), 72 hrs. before SARS-CoV-2 infection. Recombinant IFNs were refreshed with each media change. SARS-CoV-2 replication was assessed by qPCR, and quantified as viral copy number/ng RNA. Results: SARS-CoV-2 induced less robust increases then HRV-16 in expression of IFNB1 (median 1.4-fold increase, 95% CI 1.2-1.7, vs. 3.8-fold, 95% CI 2.3-6.6, p<0.01), IFNL2 (median 11-fold increase, 95% CI 3-17, vs. 23-fold, 95% CI 12-62, p<0.01), IFITM1 (median 3-fold increase, 95% CI 1.8-5.4, vs. 6.8-fold, 95% CI 3.9-16, p=0.001), IFITM3 (median 1.6-fold increase, 95% CI 1.2-2, vs. 2.5-fold, 95% CI 2.2-4, p=0.003), and OAS1 (median 1.9-fold increase, 95% CI 1.4-3.5, vs. 3.8-fold, 95% CI 2.8-7.8, p<0.001). SARS-CoV-2 replication was significantly reduced when AECs were pre-infected with HRV-16 (median 2126 viral copies/ng RNA, 95% CI 204-11,080, vs. 92 viral copies/ng RNA, 95% CI 24-289, p=0.002). SARS-CoV-2 replication was also significantly reduced when AECs were treated with recombinant IFN-β1 (median 2126 viral copies/ng RNA, 95% CI 204-11,080, vs. 81 viral copies/ng RNA, 95% CI 3-172, p=0.005) or IFN-λ2 (median 2126 viral copies/ng RNA, 95% CI 204-11,080, vs. 48 viral copies/ng RNA, 95% CI 26-143, p=0.001). Conclusion: SARS-CoV-2 elicits a less robust IFN I/III response by primary bronchial AECs than HRV-16. Pre-infection of AECs with HRV-16, or pre-treatment with recombinant IFN-β1 or IFN-λ2 markedly reduces SARS-CoV-2 replication.
ABSTRACT
RATIONALE: SARS-CoV-2 gains entrance to airway epithelial cells (AECs) through binding of the viral spike protein to the angiotensin-converting enzyme 2 (ACE2) on the cell surface. However, ACE-2 also converts angiotensin II into angiotensin-(1-7) and counterbalances the renin-angiotensin-aldosterone system, with resultant protective effects in the cardiovascular system. Some data suggest that two common antihypertension medications (angiotensin II receptor blockers, ARBs;and angiotensin-converting-enzyme inhibitors, AECi) may increase ACE-2 expression in heart and kidney cells, fueling debate about how these widely used medications may modulate SARS-CoV2 infectivity and risk of COVID-19. Aim: Determine whether exposure of bronchial AECs to the ARB losartan or the ACEi captopril modulate expression of ACE-2 by AECs and/or SARS CoV2 replication. Methods: Primary bronchial AECs from children and adults (n=18;ages 8-75 yrs.) were differentiated ex vivo at an air-liquid interface to generate organotypic cultures. Cultures were treated with captopril (1μM) or losartan (2μM) added to basolateral media with each culture media change starting 72 hrs. before infection with SARS-CoV-2. In a biosafety level 3 (BSL-3) facility, cultures were infected with SARS-CoV-2 isolate USA-WA1/2020 at a multiplicity of infection (MOI) of 0.5. At 96 hrs. following infection, RNA and protein were isolated from cultures. SARS-CoV-2 replication in cultures was assessed with quantitative PCR, and quantified as viral copy number/ng RNA. ACE-2 expression was assessed by qPCR. Results: Neither captopril nor losartan treatment significantly changed AEC-2 expression by AECs as compared to untreated AEC cultures or SARS-CoV-2 infected AEC cultures without captopril or losartan treatment. At 96 hrs. following infection, SARS-CoV-2 copy number/ng RNA was not significantly different between untreated AEC cultures (median 1752, 95% CI 213 - 8599), cultures treated with captopril (median 448.6, 95% CI 160 - 3051), or cultures treated with lorsartan (median 640, 95% CI 127 - 1610;Kruskal-Wallis ANOVA p=0.4). Conclusion: These findings suggest that at the level of the airway epithelium neither the ACEi captopril or ARB losartan significantly modify expression of the SARS-CoV-2 entry factor ACE-2, nor does either medication effect the replication of SARS-CoV-2. This ex vivo data is reassuring and is consistent with evolving clinical data suggesting ACEi and ARB medications do not increase the risk for poor prognosis with COVID-19, and may actually reduce the risk of COVID-19 disease.
ABSTRACT
RATIONALE: SARS-CoV-2 gains entrance to airway epithelial cells (AECs) via binding of the viral spike protein to the angiotensin-converting enzyme 2 (ACE2) on the cell surface, and the serine protease TMPRSS2 is thought to play an important role in facilitating SARS-CoV-2 entry by priming the spike protein. There is some data suggesting that ACE-2 expression by AECs is greater in adults than children, leading many to hypothesize that airway ACE-2 expression is a risk factor for SARS-CoV-2 replication and COVID-19 disease. Aim: Determine whether expression of ACE-2 and/or TMPRSS2 by bronchial AECs from children and adults is associated with SARS CoV2 replication. Methods: Primary bronchial AECs from children and adults (n=18;ages 8-75 yrs.) were differentiated ex vivo at an air-liquid interface to generate organotypic cultures. In a biosafety level 3 (BSL-3) facility, cultures were infected with SARS-CoV-2 isolate USA-WA1/2020 at a multiplicity of infection (MOI) of 0.5. At 96 hrs. following infection, RNA and protein were isolated from cultures. SARS-CoV-2 replication in cultures was assessed by PCR, and quantified as viral copy number/ng RNA. ACE-2 expression was assessed by qPCR in both SARS-CoV-2 infected AEC cultures and uninfected control cultures. In a subset of subjects (n=6), ACE-2 expression was measured in paired nasal and bronchial AEC cultures. Finally, we assessed the effect of apical treatment of AEC cultures with recombinant ACE-2 (rACE-2) prior to SARS-CoV-2 and once daily for 96rs. Results: In the primary bronchial AECs studied we observed marked between subject heterogeneity in ACE-2 expression (14-fold), TMPRSS2 expression (8-fold), and SARS-CoV-2 replication (range 167-89,040 copies/ng RNA). Baseline ACE-2 expression in uninfected AECs correlated with SARS-CoV-2 replication in infected AECs (Spearman r=0.6, p=0.02), whereas TMPRSS2 expression was not associated with viral replication (r=-0.2, p=0.5). In paired nasal and bronchial AEC cultures ACE-2 expression was strongly correlated (Pearson R2=0.66, p=0.05). Treatment of AECs with rACE-2 added apically immediately prior to infection and refreshed daily for 96 hrs. across a range of concentrations (0.1-1000 ng/mL rACE in 100μL of PBS;n=4 AEC primary lines) led to a marked reduction in SARS-CoV-2 replication (mean of 5040 viral copies/ng RNA in untreated AECs to 16 viral copies/ng RNA at 10ng/mL). Conclusion: Expression of ACE-2 by primary bronchial AECs from children and adults is heterogenous, and is associated with SARS-CoV-2 replication ex vivo. ACE-2 expression by AECs may partially explain the between subject variability in the risk and severity of COVID-19.
ABSTRACT
Aims: Covid-19 restrictions have impacted social interaction, work, education and care provision for young people with diabetes. Furthermore, people with diabetes have increased morbidity and mortality from covid-19. We aimed to explore the impact of the pandemic on young people's diabetes care and management;their psychosocial well-being;and identify preferences for future diabetes care. Methods: A survey was emailed to all patients aged 16-23 with diabetes attending two London (UK) hospital-based diabetes clinics. Descriptive and content analyses were conducted. Results: Response rate was 33% (n = 74/222). Respondents reflected clinic population in age, ethnicity and area-level deprivation, although males were underrepresented (34%, n = 25). Since the pandemic 55% (n = 41) felt well supported by their diabetes team;35% (n = 26) felt more confident and 40% (n = 29) more motivated to manage their diabetes. Conversely 22% (n = 16) felt less confident or motivated, which they attributed to decreased physical activity, disrupted diabetes routines and a lack of support. Mental well-being was reportedly negatively impacted in 57% (n = 42) of patients, with no differences by area-level deprivation or ethnicity. 31% (n = 23) and 41% (n = 30) of respondents felt more negatively about their diabetes and future health, respectively. Face-to- face care in the future remained the most popular option (69%, n = 51), despite increased virtual appointments during the pandemic. Conclusion: Negative impacts on mental well-being, feelings about diabetes and future health need to be considered when providing care for young people with diabetes. Variable experiences and views on care provision indicate the need for a flexible approach to future care delivery models.