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Elevated temperature inhibits SARS-CoV-2 replication in respiratory epithelium independently of IFN-mediated innate immune defenses

['Vanessa Herder', 'Mrc-University Of Glasgow Centre For Virus Research', 'Cvr', 'Glasgow', 'Scotland United Kingdom', 'Kieran Dee', 'Joanna K. Wojtus', 'Ilaria Epifano', 'Daniel Goldfarb', 'Christoforos Rozario']

Date: 2022-01 

The pandemic spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiological agent of Coronavirus Disease 2019 (COVID-19), represents an ongoing international health crisis. A key symptom of SARS-CoV-2 infection is the onset of fever, with a hyperthermic temperature range of 38 to 41°C. Fever is an evolutionarily conserved host response to microbial infection that can influence the outcome of viral pathogenicity and regulation of host innate and adaptive immune responses. However, it remains to be determined what effect elevated temperature has on SARS-CoV-2 replication. Utilizing a three-dimensional (3D) air–liquid interface (ALI) model that closely mimics the natural tissue physiology of SARS-CoV-2 infection in the respiratory airway, we identify tissue temperature to play an important role in the regulation of SARS-CoV-2 infection. Respiratory tissue incubated at 40°C remained permissive to SARS-CoV-2 entry but refractory to viral transcription, leading to significantly reduced levels of viral RNA replication and apical shedding of infectious virus. We identify tissue temperature to play an important role in the differential regulation of epithelial host responses to SARS-CoV-2 infection that impact upon multiple pathways, including intracellular immune regulation, without disruption to general transcription or epithelium integrity. We present the first evidence that febrile temperatures associated with COVID-19 inhibit SARS-CoV-2 replication in respiratory epithelia. Our data identify an important role for tissue temperature in the epithelial restriction of SARS-CoV-2 independently of canonical interferon (IFN)-mediated antiviral immune defenses.

Funding: VH was funded by the German Research Foundation (Deutsche Forschungsgemeinschaft; project number 406109949) and the Federal Ministry of Food and Agriculture (BMEL; Förderkennzeichen: 01KI1723G). KD and PRM were funded by the Medical Research Council (MRC; MC_UU_12014/9 to PRM). JKW was funded by an MRC CVR DTA award (MC_ST_U18018). IE was funded by a CSO project grant (TCS/19/11). DG was funded by an MRC-DTP award (MR/R502327/1). CR was funded by a BBSRC-CTP award (BB/R505341/1). QG was funded by the MRC (MC_UU_12014/12). KN and ASF were funded by the MRC (MC_UU_12018/12). MES was funded by the MRC (MC PC 19026). AMS was funded by a UKRI/DHSC grant (BB/R019843/1 to Brian Willett, MRC-UoG CVR) and MRC CoV supplement grant (MC_PC_19026). RMP was funded by the MRC (MC_UU_12014/10). AMG was funded by studentship awards from the University of Glasgow School of Veterinary Medicine (Georgina D. Gardner, 145813; John Crawford, 123939). CB and SMF were funded by the MRC (MC_UU_12014/5 to CB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Utilizing a three-dimensional (3D) respiratory model that closely mimics the tissue physiology and cellular tropism of SARS-CoV-2 infection observed in the respiratory airway [ 16 – 24 ], we demonstrate temperature elevation (≥39°C) to restrict the replication and propagation of SARS-CoV-2 independently of the induction of IFN-mediated antiviral immune defenses. We show that respiratory epithelium remains permissive to SARS-CoV-2 infection at temperatures up to 40°C, but to restrict the initiation of viral transcription leading to reduced levels of viral RNA (vRNA) replication and apical shedding of infectious virus. We identify temperature to play an important role in the differential regulation of multiple epithelial host responses to SARS-CoV-2 infection, including epigenetic, long noncoding RNA (lncRNA), and immunity-related pathways. Our data identify an important role for tissue temperature in the epithelial restriction of SARS-CoV-2 replication independently of canonical IFN-mediated antiviral immune defenses previously reported to restrict SARS-CoV-2 infection.

With respect to COVID-19, up to 90% of hospitalized patients show low (44%) to moderate (13% to 34%) grade fever [ 4 – 8 ], with ICU patients presenting a 10% higher prevalence of fever relative to non-ICU patients [ 6 , 15 ]. In vitro studies have shown that SARS-CoV-2 replicates more efficiently at lower temperatures associated with the upper respiratory airway (33°C), which correlates with an overall weaker interferon (IFN)-mediated immune response to infection relative to lower respiratory airway (37°C) infection [ 16 ]. These data suggest that tissue temperature could be a significant factor in the host immune response to SARS-CoV-2 infection and COVID-19 disease progression. However, it remains to be determined what effect elevated temperature (≥37°C) has on SARS-CoV-2 replication. We therefore set out to determine the net effect of temperature elevation on SARS-CoV-2 infection within respiratory epithelial tissue.

The pandemic spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; [ 1 – 3 ]) is an ongoing international health crisis with over 245 million infections and 5 million reported deaths worldwide to date (WHO; https://covid19.who.int ; November 2021). The spectrum of SARS-CoV-2–related disease (Coronavirus Disease 2019 (COVID-19)) is highly variable, ranging from asymptomatic viral shedding to acute respiratory distress syndrome (ARDS), multiorgan failure, and death. Besides coughing, dyspnea, and fatigue, fever (also known as pyrexia) is one of the most frequently reported symptoms [ 4 – 8 ]. Fever is an evolutionarily conserved response to microbial infection and inflammation that can influence the regulation of multiple cellular processes, including host innate and adaptive immune responses [ 9 , 10 ]. Unlike hyperthermia or heat stroke, fever represents a controlled shift in body temperature regulation induced by the expression of exogenous (microbial) and endogenous (host) pyrogenic regulatory factors, including pathogen associated molecular patterns (PAMPs) and proinflammatory cytokines (e.g., interleukin 6 (IL-6)) [ 9 , 10 ]. Body temperature naturally varies throughout the day, with age, sex, and ethnic origin being contributing factors [ 9 , 10 ]. In healthy middle-aged adults, febrile temperatures range from 38 to 41°C (ΔT approximately 1 to 4°C above baseline), with low (38 to 39°C), moderate (39.1 to 40°C), high (40.1 to 41.1°C), and hyperpyrexia (>41.1°C) temperature ranges [ 10 ]. Temperature elevation is known to confer protection against a number of respiratory pathogens [ 9 , 11 ], with antipyretic drug treatment leading to increased mortality in intensive care unit (ICU) patients infected with influenza A virus (IAV) [ 12 – 14 ].

Ciliated respiratory cultures differentiated from primary HBEp cells isolated from 3 independent donors (donor 1, male Caucasian aged 63 years; donor 2, Hispanic male aged 62 years; donor 3, Caucasian female aged 16 years; all nonsmokers) were incubated at 37 or 40°C for 24 h prior to mock (media only) or SARS-CoV-2 (SCV2; MOI 0.05, 10 4 PFU/Tissue) infection. Tissues were incubated at their respective temperatures for 72 h prior to RNA extraction and RNA-Seq. (A) Total MRs (host + SARS-CoV-2) from infected tissues; means and SD shown; p-value shown, unpaired two-tailed t test. (B) SARS-CoV-2 MRs from infected tissues; means and SD shown. (C) % SARS-CoV-2 MRs of total MR count (human + SARS-CoV-2); means and SD shown. (B/C) Unpaired two-tailed t test, p-value shown. (D) Expression values (log2 MR) of SARS-CoV-2 ORFs. (E) Expression values (log2 sgRNA countnormalized to ORF1a) of SARS-CoV-2 sgRNAs. (D/E) Paired two-tailed t test, p-values shown. (A to E) RNA-Seq data derived from RNA isolated from 3 donors (donors 1 to 3) per sample condition derived from 3 independent experiments per donor. (F) Indirect immunofluorescence staining of tissue sections (donor 1) showing ISG Mx1 (green) and SARS-CoV-2 nucleocapsid (N, red) expression. Nuclei were stained with DAPI. Scale bars = 20 μm. (G) Quantitation of SARS-CoV-2 N foci in respiratory tissue sections (as in F). N = 6 tissue sections per sample condition. Black line, median; whisker, 95% confidence interval; all data points shown; p-value shown, unpaired two-tailed t test. Raw values presented in S9 Data . HBEp, human bronchiolar epithelial; ISG, IFN-stimulated gene; MR, mapped read; PFU, plaque-forming unit; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; sgRNA, subgenomic RNA.

We next investigated at which stage in the replication cycle SARS-CoV-2 became restricted at elevated temperature. RNA-Seq analysis of SARS-CoV-2–infected tissues demonstrated no difference in the total number of mapped reads (MRs; human + viral) between 37 and 40°C ( Fig 7A , p = 0.5690; S9 Data ). However, a significant decrease in the number of viral MRs spanning multiple SARS-CoV-2 ORFs was detected ( Fig 7B–7D ). Importantly, a substantial decrease in the expression of viral subgenomic RNAs (sgRNAs) was also detected at elevated temperature ( Fig 7E , p = 0.0018). As the expression of SARS-CoV-2 sgRNAs requires the onset of viral transcription [ 44 ], these data identify tissue temperature to play an important role in the regulation of viral transcription. Correspondingly, indirect immunofluorescence staining of tissue sections demonstrated significantly fewer SARS-CoV-2 nucleocapsid (N) positive foci in tissues infected at 40 relative to 37°C, confirming reduced levels of viral gene expression ( Fig 7F and 7G , p < 0.0001). Costaining of tissue sections for Mx1 expression, a well-established ISG product ( Fig 6D and 6F ), demonstrated Mx1 expression to be localized in proximity to SARS-CoV-2 infectious foci at 37°C, with little to no staining observed within infected respiratory epithelia at 40°C ( Fig 7F ). We conclude that infection of respiratory tissue at elevated temperature restricts SARS-CoV-2 replication through a mechanism that inhibits viral transcription independently of IFN-mediated antiviral immune defenses previously shown to restrict SARS-CoV-2 replication [ 37 , 38 ].

Ciliated respiratory cultures differentiated from primary HBEp cells isolated from 3 independent donors (donor 1, male Caucasian aged 63 years; donor 2, Hispanic male aged 62 years; donor 3, Caucasian female aged 16 years; all nonsmokers) were incubated at 37 or 40°C for 24 h prior to mock (media) or SARS-CoV-2 (SCV2; MOI 0.05, 10 4 PFU/Tissue) infection. Tissues were incubated at their respective temperatures for 72 h prior to RNA extraction and RNA-Seq or RT-qPCR. (A) Scatter plots showing high confidence (Q < 0.05) DEG transcripts identified between SARS-CoV-2–infected respiratory cultures at 37 or 40°C; up-regulated DEGs, red circles; down-regulated DEGs, blue circles. (B) Reactome pathway analysis of mapped down-regulated DEGs. Top 25 down-regulated (FDR < 0.05) pathways shown (blue bars; plotted as −log10 FDR). Dotted line, threshold of significance (−log10 FDR of 0.05). (C) Expression profile (log2 CPM) of down-regulated immune system DEGs (R-HSA168256; arrow in B) relative to expression levels in mock tissue at 37 or 40°C. Black line, median; dotted lines, fifth and 95th percentile range; p-values shown, one-way ANOVA (top), paired two-tailed t test (bottom). (D) Expression levels (log2 CPM) of immune system DEGs (identified in B, black arrow) relative to mock at 37 or 40°C. Every second gene labeled. Red arrows highlight genes chosen for RT-qPCR validation. (A to D) RNA-Seq data derived from RNA isolated from 3 donors (donors 1 to 3) per sample condition derived from 3 independent experiments per donor. (E/F) RT-qPCR (ΔΔCT) quantitation of IFN (IFNB1 and IFNL1) or ISG (Mx1, ISG15, and IFIT2) transcript levels within mock or SARS-CoV-2–infected respiratory cultures derived from 3 independent donors (transcript values normalized to GAPDH per sample condition). N ≥ 3 tissues per donor per condition derived from 3 independent biological experiments. Means and SD shown; all data points shown; p-values shown, one-way ANOVA. Individual ΔCT values per donor are shown in S6 Fig for donor comparison. (G) Respiratory cultures differentiated from donor 1 HBEp cells were pretreated with Ruxo (5 μM) or carrier control (DMSO) for 16 h prior to SARS-CoV-2 infection (MOI 0.05, 10 4 PFU/Tissue) and continued incubation at 37°C in the presence of inhibitor or carrier control. Apical washes were collected at the indicated times (h) and genome copies per ml determined RT-qPCR. N = 3 tissues per sample condition derived from 3 independent biological experiments. Means and SD shown; p-values shown, Mann–Whitney U test. Raw values presented in S8 and S9 Data. CPM, counts per million; DEG, differentially expressed gene; FDR, false discovery rate; HBEp, human bronchiolar epithelial; IFN, interferon; ISG, IFN-stimulated gene; PFU, plaque-forming unit; RT-qPCR, reverse transcription quantitative PCR; Ruxo, Ruxolitinib; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2.

Ciliated respiratory cultures differentiated from primary HBEp cells isolated from 3 independent donors (donor 1, male Caucasian aged 63 years; donor 2, Hispanic male aged 62 years; donor 3, Caucasian female aged 16 years; all nonsmokers) were incubated at 37 or 40°C for 24 h prior to mock (media only) or SARS-CoV-2 (SCV2; MOI 0.05, 10 4 PFU/Tissue) infection. Tissue were incubated at their respective temperatures for 72 h prior to RNA extraction and RNA-Seq. DEGs (Q < 0.05, up-regulated [top panels] or down-regulated [bottom panels]) were identified for each paired condition analyzed; blue ellipses, SARS-CoV-2 37°C/Mock 37°C (SCV37/Mock37); green ellipses, SARS-CoV-2 40°C/Mock 37°C (SCV40/Mock40); yellow ellipses, SARS-CoV-2 40°C/Mock 40°C (SCV40/Mock37); red ellipses, Mock 40°C/Mock 37°C (Mock40/Mock37). (A) Venn diagram showing the number of unique or shared DEGs between each paired condition analyzed. (B) Circos plot showing the proportion of unique (light orange inner circle) or shared (dark orange inner circle + purple lines) DEGs between each paired condition analyzed. (C) Metascape pathway analysis showing significant DEG enrichment p-value <0.05 (−log10 p-value shown) for each paired condition. Black arrow, immune system process pathway (GO:0002376). Gray boxes, p > 0.05. (A to C) RNA-Seq data derived from RNA isolated from 3 donors (donors 1 to 3) per sample condition derived from 3 independent experiments per donor. Raw values presented in S1 , S3 to S6 , and S9 Data . DEG, differentially expressed gene; HBEp, human bronchiolar epithelial; PFU, plaque-forming unit; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2.

Ciliated respiratory cultures differentiated from primary HBEp cells isolated from 3 independent donors (donor 1, male Caucasian aged 63 years; donor 2, Hispanic male aged 62 years; donor 3, Caucasian female aged 16 years; all nonsmokers) were incubated at 37 or 40°C for 24 h prior to SARS-CoV-2 (SCV2; MOI 0.05; 10 4 PFU/Tissue) infection and incubation at the indicated temperatures. Apical washes were collected over time (as indicated) and tissues harvested at 72 h for RNA extraction or fixed for ISH staining. (A/B) Genome copies per ml of SARS-CoV-2 in apical washes harvested over time as determined RT-qPCR. N = 9 tissues per condition derived from a minimum of 3 independent biological experiments. (A) Means and SD shown; p-values shown, Mann–Whitney U test between donor paired time points. (B) Black line, median; whisker, 95% confidence interval; all data points shown; p-values shown, one-way ANOVA Kruskal–Wallis test (top), Mann–Whitney U test (bottom). (C) Quantitation of genome copies per tissue of SARS-CoV-2 at 72 h by RT-qPCR using 2 independent primer probe sets (N and ORF1a). N = 6 tissues per sample condition derived from a minimum of 3 independent biological experiments. Medians and 95% confidence interval shown; p-values shown, Mann–Whitney U test. (D) Representative images of H&E and SARS-CoV-2 RNA ISH (red) stained sections. Hematoxylin was used as a counter stain. Scale bars = 20 μm. Raw values presented in S9 Data . HBEp, human bronchiolar epithelial; H&E, hematoxylin and eosin; ISH, in situ hybridization; PFU, plaque-forming unit; RT-qPCR, reverse transcription quantitative PCR; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; vRNA, viral RNA.

We next investigated whether this temperature-dependent restriction was donor dependent. Respiratory cultures were incubated at 37 or 40°C for 24 h prior to SARS-CoV-2 infection and continued incubation at their respective temperature. Measurement of genome copy titers in apical washes demonstrated viral titers were significantly reduced at 40°C in a donor-independent manner ( Fig 4A and 4B ). Notably, donor-dependent profiles of restriction were observed ( Fig 4B ), with respiratory tissue derived from donor 1 being the most refractory to SARS-CoV-2 replication at 40°C. We next examined if the decreased yield of infectious virus observed at elevated temperature occurred due to viral genetic mutation. Amplicon sequencing of vRNA isolated from donor 1 apical washes at 72 h identified a total of 7 unique single-nucleotide polymorphisms (SNPs) relative to input sequence with no indels being identified ( Table 1 and S2 Data ). SNPs were only identified in genomic sequences isolated from apical washes incubated at 37°C ( Table 1 ). Thus, the temperature-dependent restriction observed in SARS-CoV-2 replication at 40°C is not a consequence of viral mutation. As the generation of SNPs requires vRNA replication, the absence of genomic SNPs at 40°C may reflect a temperature-dependent block in vRNA replication. To investigate this, we quantified the intracellular levels of vRNA from within infected tissues. RT-qPCR analysis using 2 independent primer-probe sets (N and ORF1a) demonstrated significantly lower levels of intracellular vRNA in tissues incubated at 40°C relative to control samples ( Fig 4C ). ISH staining of tissue sections confirmed abundant levels of intraepithelial vRNA accumulation within all infected tissues incubated at 37°C, with little to no staining observed at 40°C ( Fig 4D ). Together, these data indicate that respiratory tissue remains permissive to SARS-CoV-2 infection (viral entry) but refractory to vRNA replication to levels sufficient for robust ISH detection.

Ciliated respiratory cultures differentiated from HBEp cells isolated from donor 1 (male Caucasian aged 63 years) were incubated at 37, 39, or 40°C for 24 h prior to SARS-CoV-2 (SCV2; MOI 0.05, 10 4 PFU/Tissue) infection and incubation at the indicated temperatures. Apical washes were collected over time (as indicated). (A) Genome copies per ml of SARS-CoV-2 in apical washes were determined RT-qPCR. N ≥ 11 tissues per condition derived from a minimum of 3 independent biological experiments. Black line, median; whisker, 95% confidence interval; all data points shown; p-values shown, one-way ANOVA Kruskal–Wallis test. (B) TCID 50 assay measuring infectious viral load in apical washes harvested from SARS-CoV-2–infected respiratory cultures. N ≥ 11 tissues per condition derived from a minimum of 3 independent biological experiments. Black line, median; whisker, 95% confidence interval; all data points shown; p-values shown, one-way ANOVA Kruskal–Wallis test. (C) Ciliated respiratory cultures were infected with SARS-CoV-2 (MOI 0.05, 10 4 PFU/Tissue) at 37°C and incubated for 24 h prior to continued incubation at 37 or temperature upshift to 40°C. Genome copies per ml of SARS-CoV-2 in apical washes collected at 72 h were determined RT-qPCR. N = 9 tissues per condition derived from a minimum of 3 independent biological experiments. Black line, median; whisker, 95% confidence interval; all data points shown; p-value shown, Mann–Whitney U test. Raw values presented in S9 Data . HBEp, human bronchiolar epithelial; PFU, plaque-forming unit; RT-qPCR, reverse transcription quantitative PCR; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2.

We next examined the effect of elevated temperature on SARS-CoV-2 replication. Respiratory cultures were incubated at 37, 39, or 40°C for 24 h prior to SARS-CoV-2 infection and continued incubation at their respective temperature. Measurement of genome copies and infectious virus from apical washes collected over time (24 to 72 h) demonstrated that extracellular viral titers were significantly decreased at both 39 and 40°C relative to 37°C ( Fig 3A and 3B ). Importantly, SARS-CoV-2 infection at 37°C for 24 h prior to temperature upshift also led to reduced genome copy titers in apical washes ( Fig 3C ; p = 0.0056). These data indicate that temperature elevation, either prior to or following SARS-CoV-2 infection, is inhibitory to viral replication and/or release of viral particles. These data identify febrile temperature ranges associated with low (39°C) to moderate (40°C) grade fever to restrict SARS-CoV-2 propagation in respiratory epithelia.

Ciliated respiratory cultures differentiated from HBEp cells isolated from 3 independent donors (donor 1, male Caucasian aged 63 years; donor 2, Hispanic male aged 62 years; donor 3, Caucasian female aged 16 years; all nonsmokers) were incubated at 37, 39, or 40°C for 72 h prior to fixation or RNA extraction and RNA-Seq. (A) Representative H&E stained tissue sections (donor 1) at 72 h. Scale bars = 20 μm. (B) TEER (Ohm per cm 2 ) measurements of respiratory epithelia integrity (donor 1) at 37 or 40°C. N = 3 tissues per condition; lines, mean; all data points shown; p-values shown, unpaired two-tailed t test. (C) ACE2 expression levels (CPM) in respiratory cultures incubated at 37 or 40°C; means and SD shown; p-value shown, unpaired two-tailed t test. (D) Expression values (log2 CPM) of a reference gene set (24 genes; [ 34 – 36 ]) in respiratory cultures incubated at 37 or 40°C; p-value shown, paired two-tailed t test. (E) Scatter plots showing high confidence (Q < 0.05) DEG transcripts identified between respiratory cultures incubated at 37 or 40°C; up-regulated DEGs, red circles; down-regulated DEGs, blue circles. (F) Reactome pathway analysis of mapped up-regulated DEGs. Top 30 pathways (FDR < 0.05; plotted as −log10 FDR) shown. Dotted line, threshold of significance (−log10 FDR of 0.05). (G) Expression values (log2 CPM) of DEGs associated with cellular response to heat stress pathway (R-HSA-3371556; arrow in F); p-value shown, paired two-tailed t test. (C to H) RNA-Seq data derived from RNA isolated from 3 donors (donors 1 to 3) per sample condition derived from 3 independent experiments per donor. Raw values presented in S1 and S9 Data. ACE2, angiotensin-converting enzyme 2; CPM, counts per million; DEG, differentially expressed gene; FDR, false discovery rate; HBEp, human bronchiolar epithelial; H&E, hematoxylin and eosin; TEER, transepithelial electrical resistance.

While the heat stress response has been extensively investigated in two-dimensional (2D) cell culture model systems [ 32 , 33 ], this pathway remains poorly characterized in 3D respiratory epithelia under air–liquid interface (ALI). We therefore investigated the influence of temperature on our 3D respiratory model. Mock-treated respiratory cultures were incubated at 37, 39, or 40°C (representative of core body temperature and low to moderate grade febrile temperatures, respectively) prior to tissue fixation or RNA extraction and RNA sequencing (RNA-Seq). H&E staining and transepithelial electrical resistance (TEER) measurements demonstrated no morphological or membrane integrity changes to the respiratory epithelium upon temperature elevation ( Fig 2A and 2B ). RNA-Seq analysis of respiratory epithelia demonstrated no difference in the expression level of ACE2 ( Fig 2C , p = 0.6753) or a reference set of 24 genes known to be constitutively expressed across a wide range of tissues and cell types ( Fig 2D , p = 0.2136; [ 34 – 36 ]) upon temperature elevation. These data indicate that general transcription remains largely unperturbed at elevated temperatures up to 40°C. Out of the 867 differentially expressed genes (DEGs) identified ( Fig 2E ; Q < 0.05), DEG enrichment was observed across multiple pathways in tissues incubated at 40 relative to 37°C, including cellular response to stress, RNA polymerase I promoter opening, and DNA methylation ( Fig 2F and S1 Data ). Significant DEG enrichment was also observed in cellular response to heat stress ( Fig 2F [arrow], 2G; S1 Data ). We conclude that our respiratory model induces a significant heat stress response at elevated temperature without visible damage to the epithelium or induction of innate immune defenses that may otherwise influence the interpretation of SARS-CoV-2 replication studies at elevated temperature.

Primary bronchial epithelial cells isolated from 3 independent donors (donor 1, male Caucasian aged 63 years; donor 2, Hispanic male aged 62 years; donor 3, Caucasian female aged 16 years; all nonsmokers) were seeded onto 6.5 mm transwells and differentiated under ALI conditions for ≥35 days. Ciliated respiratory cultures were mock treated (media only) or SARS-CoV-2 (SCV2; MOI 0.05, 10 4 PFU/Tissue) infected at 37°C for the indicated times (h). (A, B) Representative images of H&E, ACE2 IHC (brown), or SARS-CoV-2 RNA ISH (red) stained sections. Hematoxylin was used as a counter stain. Scale bars = 20 μm. (C) Genome copies per ml of SARS-CoV-2 in apical washes harvested over time as determined RT-qPCR. N ≥ 7 tissues per condition derived from a minimum of 3 independent biological experiments. Means and SD shown. (D) TCID 50 assay measuring infectious viral load in apical washes harvested from SARS-CoV-2 infected tissues over time. Means and SD shown. N = 6 tissues per condition derived from a minimum of 3 independent biological experiments. Raw values presented in S9 Data . ACE2, angiotensin-converting enzyme 2; ALI, air–liquid interface; H&E, hematoxylin and eosin; IHC, immunohistochemistry; ISH, in situ hybridization; PFU, plaque-forming unit; RT-qPCR, reverse transcription quantitative PCR; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; vRNA, viral RNA.

To establish a model suitable to study the effect of temperature on SARS-CoV-2 replication, we differentiated primary human bronchiolar epithelial (HBEp) cells isolated from 3 independent donors into stratified respiratory epithelium. Hematoxylin and eosin (H&E) and immunohistochemistry (IHC) staining of respiratory cultures demonstrated these tissues to contain a mixture of epithelial and goblet cells, with high levels of apical ciliation and angiotensin-converting enzyme 2 (ACE2) expression, the principal surface receptor of SARS-CoV-2 ( Fig 1A and 1B ) [ 25 – 28 ]. Infection of respiratory airway cultures with SARS-CoV-2 (England/02/2020; MOI 0.05, 10 4 plaque-forming unit (PFU)/Tissue) at 37°C demonstrated these tissues to support infection and viral replication, with intraepithelial and apical vRNA accumulation readily detected by in situ hybridization (ISH) by 72 h postinfection ( Fig 1A and 1B ). Notably, we observed discrete clusters of vRNA accumulation within respiratory epithelia ( Fig 1A , arrows), indicative of localized areas of intraepithelial infection, propagation, and spread [ 29 , 30 ]. The overall morphology of the respiratory epithelium remained largely intact, with little shedding of ciliated cells from the epithelial surface ( Fig 1A , 120 h). Measurement of genome copies by reverse transcription quantitative PCR (RT-qPCR) and infectious virus by TCID 50 in apical washes collected over time demonstrated the linear phase of virus release to occur between 48 and 120 h ( Fig 1C and 1D ). A substantial drop in apical infectious titers was observed at 144 h postinfection ( Fig 1D ), consistent with multiple waves of virus release over prolonged periods of incubation in primary airway cultures [ 31 ].

Discussion

A defining symptom of COVID-19 is the onset of fever with a febrile temperature range of 38 to 41°C [4–8]. However, the effect of elevated temperature on SARS-CoV-2 tissue tropism and replication has remained to be determined. Here, we identify a temperature-sensitive phenotype in SARS-CoV-2 replication in respiratory epithelia that occurs independently of the robust induction of IFN-mediated innate immune defenses known to restrict SARS-CoV-2 replication [37,38]. The differentiation of stratified respiratory epithelium has proven to be a valuable research tool to investigate the cellular tropism, replication kinetics, and immune regulation of SARS-CoV-2, as these tissue models mimic many aspects of infection observed in animal models and COVID-19 patients [16–24,29,45–49]. While the use of such 3D models represents an important advancement over traditional two-dimensional cell culture systems, the absence of circulating immune cells (e.g., macrophages, natural killer cells, dendritic cells, and neutrophils), which modulate fever and proinflammatory immune responses to infection is an important limiting factor [9,10,50]. Thus, we limit our conclusions to the effect of elevated temperature on SARS-CoV-2 infection and replication within respiratory epithelium.

Consistent with previous reports [16,17,19–21,30,38,49], SARS-CoV-2 infection of respiratory epithelium at 37°C induced a proinflammatory immune signature (S2D Fig; IFNβ and IFNλ1–3). While SARS-CoV-2 is known to induce a comparatively weak immune response relative to other respiratory viruses (e.g., IAV) [20,39], we observed localized areas of epithelial infection (Fig 1A, arrows) that were coincident with elevated levels of ISG expression by 72 h postinfection (Fig 7F, Mx1). These findings are consistent with reports that have shown localized areas of infection in the respiratory airway of infected ferrets and surface epithelium of organoid cultures [29–31,49]. Thus, the proportion of epithelial infection and/or rate of intraepithelial spread may account for the differences observed in immune signature reported between different respiratory pathogens [20,51]. As we observed localized areas of ISG induction, our data support a model of infection where cytokine (e.g., IFN) receptor-binding consumption kinetics may be a rate limiting factor in the protection of respiratory epithelia to SARS-CoV-2 infection [52,53]. Such observations warrant further investigation to determine whether these localized areas of epithelial immune protection are induced by specific cytokines (e.g., IFNβ or IFNλ1–3) and/or correlate with COVID-19 disease progression.

We demonstrate 3D respiratory epithelial cultures under ALI to induce a robust heat stress response upon temperature elevation without loss of epithelium integrity, disruption to general cellular transcription, or damage-associated molecular pattern (DAMP) activation of innate immune defenses (Figs 2 and S4). Temperature elevation alone was not sufficient to block SARS-CoV-2 entry (Figs 3, 4, and S7), but refractory to SARS-CoV-2 transcription leading to reduced levels of vRNA accumulation and apical shedding of infectious virus (Figs 3, 4, and 7). Thus, we identify febrile temperatures associated with COVID-19 to play an important role in limiting the epithelial replication of SARS-CoV-2 infection. Importantly, SARS-CoV-2 restriction occurred independently of the robust induction of type-I (IFNβ) or type-III (IFNλ) IFN-mediated immune defenses (Figs 5, 6, and S7), despite infected tissues having abundant levels of intracellular vRNA (Fig 4C). We posit that the lack of immune induction observed at 40°C is likely a consequence of diminished levels of vRNA replication, which has been shown to play an important role in the production of PAMPs (e.g., dsRNA intermediates) required for the activation of innate immune defenses to other respiratory pathogens [54–57]. However, PRR detection of PAMPs plays an important role in the induction of a proinflammatory cytokines (including IL-6) required to mount a fever response to microbial challenge in vivo [9,10]. Thus, additional animal studies will be required to determine whether the temperature-dependent restriction of SARS-CoV-2 occurs independently of an IFN response in vivo. While speculative, we posit that low to moderate grade fever may confer protection to respiratory tissue within SARS-CoV-2–infected individuals as a component of a homeostatically controlled non-hyperinflammatory immune response to infection.

While we identify tissue temperature to play an important role in the regulation of SARS-CoV-2 transcription and replication in respiratory epithelia (Fig 7D and 7E), the precise molecular mechanism(s) of restriction remain to be determined. The heat stress response has been shown to play both a positive (proviral) and negative (antiviral) role during virus replication [58–60]. Our transcriptomic analysis identified respiratory epithelial host responses to be differentially regulated in response to both temperature elevation and SARS-CoV-2 infection (Figs 5, 6, and S1–S5). Thus, SARS-CoV-2 infection of respiratory epithelia at elevated temperature elicits a distinct host response that may contribute to the restriction of SARS-CoV-2 replication at multiple stages of infection. For example, we identify lncRNAs to be differentially expressed in response to both SARS-CoV-2 infection and temperature (S5 Fig), which are known to influence the outcome of virus infection independently of IFN-mediated immune defenses [61]. Thus, multiple gene products and/or pathways may contribute to the sequential or accumulative restriction of SARS-CoV-2 replication within respiratory epithelia at elevated temperature.

We identify temperature elevation to correlate with lower levels of SARS-CoV-2 sgRNA expression (Fig 7E), identifying a temperature-dependent block in SARS-CoV-2 transcription. We hypothesize that this restriction may relate to an inhibition in SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) activity and/or binding affinity to vRNA, as the genomic replication activity of the IAV RdRp polymerase is known to be restricted at elevated temperature (41°C) [62]. Notably, avian IAV cold adaptation of RdRp is a host determinant of IAV zoonosis, which naturally replicates in the intestinal tract of birds at higher temperatures [63]. As circulating strains of SARS-CoV-2 are susceptible to nonsynonymous mutation within RdRp coding sequences (http://cov-glue.cvr.gla.ac.uk/#/home), molecular studies are warranted to determine if such amino acid substitutions might influence the thermal restriction of SARS-CoV-2 replication. While SARS-CoV-2 has been reported to replicate to higher viral titers at temperatures associated with the upper respiratory airway (33°C) relative to core body temperature (37°C) [16], the replication of coronaviruses at febrile body temperatures has remained poorly characterized. While temperature-sensitive (ts) coronavirus mutants have been identified for feline infectious peritonitis virus (tsFIPV) and murine hepatitis virus (MHV tsNC11), their parental wild-type derivatives were able to replicate at temperatures ≥39°C [64,65]. The replication defect for tsFIPV was attributed to a block in viral maturation that limited its replication in cats to the upper respiratory airway [64], whereas the growth defect in tsNC11 was attributed to a coding mutation within the macrodomain and papain-like protease 2 domain of the nonstructural protein 3 [65]. Thus, we present the first evidence demonstrating a circulating strain of coronavirus to be sensitive to temperature thermoregulation. Genomic analysis of SARS-CoV-2 isolated from apical washes failed to identify any unique SNPs from infected tissues at 40°C relative to input sequence. These data support the temperature-dependent block in SARS-CoV-2 propagation to occur prior to vRNA replication, a prerequisite requirement for genomic mutation. As such, heat adaptation gain of function experiments through serial passage of SARS-CoV-2 at elevated temperature (≥39°C) may shed light on whether the restriction observed is related to viral and/or cellular host factors. Importantly, however, appropriate levels of biosafety (ethical and genetic modification) should be considered prior to such experimentation.

In summary, we identify an important role for tissue temperature in the cellular restriction of SARS-CoV-2 during infection of respiratory epithelia that occurs independently of the robust induction of canonical IFN-mediated antiviral immune defenses known to restrict SARS-CoV-2. We demonstrate tissue temperature to significantly influence the differential regulation of epithelial host responses to SARS-CoV-2 infection and the progression of viral transcription upon tissue infection. Future investigation is warranted to determine the precise mechanism(s) of restriction, as this may uncover novel avenues for therapeutic intervention in the treatment of COVID-19.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001065

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