Attenuation of clinical and immunological outcomes during SARS‐CoV‐2 infection by ivermectin
2021; Springer Nature; Volume: 13; Issue: 8 Linguagem: Inglês
10.15252/emmm.202114122
ISSN1757-4684
AutoresGuilherme Dias de Melo, Françoise Lazarini, Florence Larrous, Lena Feige, Étienne Kornobis, Sylvain Levallois, Agnès Marchio, Lauriane Kergoat, David Hardy, Thomas Cokelaer, Pascal Pineau, Marc Lecuit, Pierre‐Marie Lledo, Jean‐Pierre Changeux, Hervé Bourhy,
Tópico(s)COVID-19 and Mental Health
ResumoArticle12 July 2021Open Access Source DataTransparent process Attenuation of clinical and immunological outcomes during SARS-CoV-2 infection by ivermectin Guilherme Dias de Melo Guilherme Dias de Melo orcid.org/0000-0003-0747-7760 Lyssavirus Epidemiology and Neuropathology Unit, Institut Pasteur, Paris, France Search for more papers by this author Françoise Lazarini Françoise Lazarini orcid.org/0000-0001-5572-6982 Perception and Memory Unit, Institut Pasteur, CNRS UMR 3571, Paris, France Search for more papers by this author Florence Larrous Florence Larrous orcid.org/0000-0003-0881-4263 Lyssavirus Epidemiology and Neuropathology Unit, Institut Pasteur, Paris, France Search for more papers by this author Lena Feige Lena Feige Lyssavirus Epidemiology and Neuropathology Unit, Institut Pasteur, Paris, France Search for more papers by this author Etienne Kornobis Etienne Kornobis Biomics Technological Platform, Center for Technological Resources and Research (C2RT), Institut Pasteur, Paris, France Bioinformatics and Biostatistics Hub, Computational Biology Department, Institut Pasteur, Paris, France Search for more papers by this author Sylvain Levallois Sylvain Levallois orcid.org/0000-0002-5805-1092 Biology of Infection Unit, Institut Pasteur, Inserm U1117, Paris, France Search for more papers by this author Agnès Marchio Agnès Marchio Nuclear Organization and Oncogenesis Unit, Institut Pasteur, Paris, France Search for more papers by this author Lauriane Kergoat Lauriane Kergoat orcid.org/0000-0002-5609-4398 Lyssavirus Epidemiology and Neuropathology Unit, Institut Pasteur, Paris, France Search for more papers by this author David Hardy David Hardy orcid.org/0000-0001-5874-4377 Experimental Neuropathology Unit, Institut Pasteur, Paris, France Search for more papers by this author Thomas Cokelaer Thomas Cokelaer orcid.org/0000-0001-6286-1138 Biomics Technological Platform, Center for Technological Resources and Research (C2RT), Institut Pasteur, Paris, France Bioinformatics and Biostatistics Hub, Computational Biology Department, Institut Pasteur, Paris, France Search for more papers by this author Pascal Pineau Pascal Pineau Nuclear Organization and Oncogenesis Unit, Institut Pasteur, Paris, France Search for more papers by this author Marc Lecuit Marc Lecuit orcid.org/0000-0002-4491-1063 Biology of Infection Unit, Institut Pasteur, Inserm U1117, Paris, France Division of Infectious Diseases and Tropical Medicine, Institut Imagine, Université de Paris, Necker-Enfants Malades University Hospital, AP-HP, Paris, France Search for more papers by this author Pierre-Marie Lledo Pierre-Marie Lledo Perception and Memory Unit, Institut Pasteur, CNRS UMR 3571, Paris, France Search for more papers by this author Jean-Pierre Changeux Jean-Pierre Changeux orcid.org/0000-0003-0297-1583 Neuroscience Department, Institut Pasteur, Collège de France, Paris, France Search for more papers by this author Hervé Bourhy Corresponding Author Hervé Bourhy [email protected] orcid.org/0000-0002-2608-5589 Lyssavirus Epidemiology and Neuropathology Unit, Institut Pasteur, Paris, France Search for more papers by this author Guilherme Dias de Melo Guilherme Dias de Melo orcid.org/0000-0003-0747-7760 Lyssavirus Epidemiology and Neuropathology Unit, Institut Pasteur, Paris, France Search for more papers by this author Françoise Lazarini Françoise Lazarini orcid.org/0000-0001-5572-6982 Perception and Memory Unit, Institut Pasteur, CNRS UMR 3571, Paris, France Search for more papers by this author Florence Larrous Florence Larrous orcid.org/0000-0003-0881-4263 Lyssavirus Epidemiology and Neuropathology Unit, Institut Pasteur, Paris, France Search for more papers by this author Lena Feige Lena Feige Lyssavirus Epidemiology and Neuropathology Unit, Institut Pasteur, Paris, France Search for more papers by this author Etienne Kornobis Etienne Kornobis Biomics Technological Platform, Center for Technological Resources and Research (C2RT), Institut Pasteur, Paris, France Bioinformatics and Biostatistics Hub, Computational Biology Department, Institut Pasteur, Paris, France Search for more papers by this author Sylvain Levallois Sylvain Levallois orcid.org/0000-0002-5805-1092 Biology of Infection Unit, Institut Pasteur, Inserm U1117, Paris, France Search for more papers by this author Agnès Marchio Agnès Marchio Nuclear Organization and Oncogenesis Unit, Institut Pasteur, Paris, France Search for more papers by this author Lauriane Kergoat Lauriane Kergoat orcid.org/0000-0002-5609-4398 Lyssavirus Epidemiology and Neuropathology Unit, Institut Pasteur, Paris, France Search for more papers by this author David Hardy David Hardy orcid.org/0000-0001-5874-4377 Experimental Neuropathology Unit, Institut Pasteur, Paris, France Search for more papers by this author Thomas Cokelaer Thomas Cokelaer orcid.org/0000-0001-6286-1138 Biomics Technological Platform, Center for Technological Resources and Research (C2RT), Institut Pasteur, Paris, France Bioinformatics and Biostatistics Hub, Computational Biology Department, Institut Pasteur, Paris, France Search for more papers by this author Pascal Pineau Pascal Pineau Nuclear Organization and Oncogenesis Unit, Institut Pasteur, Paris, France Search for more papers by this author Marc Lecuit Marc Lecuit orcid.org/0000-0002-4491-1063 Biology of Infection Unit, Institut Pasteur, Inserm U1117, Paris, France Division of Infectious Diseases and Tropical Medicine, Institut Imagine, Université de Paris, Necker-Enfants Malades University Hospital, AP-HP, Paris, France Search for more papers by this author Pierre-Marie Lledo Pierre-Marie Lledo Perception and Memory Unit, Institut Pasteur, CNRS UMR 3571, Paris, France Search for more papers by this author Jean-Pierre Changeux Jean-Pierre Changeux orcid.org/0000-0003-0297-1583 Neuroscience Department, Institut Pasteur, Collège de France, Paris, France Search for more papers by this author Hervé Bourhy Corresponding Author Hervé Bourhy [email protected] orcid.org/0000-0002-2608-5589 Lyssavirus Epidemiology and Neuropathology Unit, Institut Pasteur, Paris, France Search for more papers by this author Author Information Guilherme Dias Melo1, Françoise Lazarini2, Florence Larrous1, Lena Feige1, Etienne Kornobis3,4, Sylvain Levallois5, Agnès Marchio6, Lauriane Kergoat1, David Hardy7, Thomas Cokelaer3,4, Pascal Pineau6, Marc Lecuit5,8, Pierre-Marie Lledo2, Jean-Pierre Changeux9 and Hervé Bourhy *,1 1Lyssavirus Epidemiology and Neuropathology Unit, Institut Pasteur, Paris, France 2Perception and Memory Unit, Institut Pasteur, CNRS UMR 3571, Paris, France 3Biomics Technological Platform, Center for Technological Resources and Research (C2RT), Institut Pasteur, Paris, France 4Bioinformatics and Biostatistics Hub, Computational Biology Department, Institut Pasteur, Paris, France 5Biology of Infection Unit, Institut Pasteur, Inserm U1117, Paris, France 6Nuclear Organization and Oncogenesis Unit, Institut Pasteur, Paris, France 7Experimental Neuropathology Unit, Institut Pasteur, Paris, France 8Division of Infectious Diseases and Tropical Medicine, Institut Imagine, Université de Paris, Necker-Enfants Malades University Hospital, AP-HP, Paris, France 9Neuroscience Department, Institut Pasteur, Collège de France, Paris, France *Corresponding author. Tel: +33 1 45 68 87 85; E-mail: [email protected] EMBO Mol Med (2021)13:e14122https://doi.org/10.15252/emmm.202114122 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The devastating pandemic due to SARS-CoV-2 and the emergence of antigenic variants that jeopardize the efficacy of current vaccines create an urgent need for a comprehensive understanding of the pathophysiology of COVID-19, including the contribution of inflammation to disease. It also warrants for the search of immunomodulatory drugs that could improve disease outcome. Here, we show that standard doses of ivermectin (IVM), an anti-parasitic drug with potential immunomodulatory activities through the cholinergic anti-inflammatory pathway, prevent clinical deterioration, reduce olfactory deficit, and limit the inflammation of the upper and lower respiratory tracts in SARS-CoV-2-infected hamsters. Whereas it has no effect on viral load in the airways of infected animals, transcriptomic analyses of infected lungs reveal that IVM dampens type I interferon responses and modulates several other inflammatory pathways. In particular, IVM dramatically reduces the Il-6/Il-10 ratio in lung tissue and promotes macrophage M2 polarization, which might account for the more favorable clinical presentation of IVM-treated animals. Altogether, this study supports the use of immunomodulatory drugs such as IVM, to improve the clinical condition of SARS-CoV-2-infected patients. SYNOPSIS COVID-19, caused by SARS-CoV-2, induces airways and pulmonary symptoms, and in severe cases can lead to respiratory distress and death. This study shows that the modulation of the host's inflammatory response using ivermectin as a repurposed drug, independently of the viral load, strongly diminished the clinical score and severity of the disease (including anosmia) observed in SARS-CoV-2-infected golden hamsters. This study brings the proof-of-concept that a chemical therapy using ivermectin can preserve the clinical condition by modulating the inflammatory response, even without antiviral activity. The clinical presentation is directly linked to inflammation and not necessarily to viral load. A chemical therapy by ivermectin induces a sex-dependent and compartmentalized response, preventing clinical deterioration and reducing olfactory deficit. Ivermectin limits the response of several signaling pathways related to that of type I/III interferon, cytokines activation and inflammatory cells population in infected lungs. A reduced Il-6/Il-10 ratio in the lung might account for a more favorable clinical presentation. Ivermectin-treated animals presented a M2 polarization of myeloid cells recruited to the lung. The paper explained Problem The current pandemic of COVID-19 has caused more than 3.5 million deaths and more than 150 million laboratory-confirmed cases worldwide since December 2019 (as of May 2021). COVID-19, caused by SARS-CoV-2, commonly brings about upper airways and pulmonary symptoms and in severe cases can lead to respiratory distress and death. Different therapeutic approaches have been proposed to fight this disease but comprehensive therapeutic studies are still lacking. Results We report that ivermectin, used at the standard anti-parasitic dose of 400 µg/kg, protects infected hamsters from developing clinical signs and from losing the sense of smell during SARS-CoV-2 infection. The treated animals exhibited a specific inflammatory response, presenting a reduced type I/III interferon stimulation and a modulation in several intracellular signaling pathways, with an important reduction of the Il-6/Il-10 ratio and promoting M2 polarization of myeloid cells recruited to the lung. These effects are strongly influenced by sex, with treated females exhibiting the best outcome. Surprisingly, ivermectin treatment did not limit viral replication, as treated and non-treated animals presented similar amounts of SARS-CoV-2 in the nasal cavity and in the lungs. Impact The results of this study establish that irrespective of viral load, the symptoms and severity of COVID-19 highlight the critical role played by host inflammatory response in COVID-19 severity and highlight that reduced type I/III interferon and Il-6/Il-10 and the presence of M2 macrophages might account for a more favorable clinical presentation, contributing to a better understanding of COVID-19 pathophysiology. Ivermectin might then be considered as promising therapeutic agent against COVID-19 with no impact on SARS-CoV-2 replication but alleviating inflammation and ensuing symptoms. Introduction Coronaviruses cause respiratory disease in a wide variety of hosts. During the ongoing pandemic of SARS-CoV-2 causing coronavirus disease 19 (COVID-19), clinical signs other than respiratory symptoms have been linked to infection, frequently associated with neurological symptoms such as anosmia and ageusia. These features have been related to an over-responsiveness of patients' immune system to SARS-CoV-2 (Bhaskar et al, 2020; Han et al, 2020; Qiu et al, 2020). Consequently, there is an urgent need to understand the hallmarks of this over-responsiveness and to find novel therapeutics or repurpose drugs to improve the clinical condition of COVID-19 patients (Batalha et al, 2021). Ivermectin (IVM), a macrocyclic lactone, is a commercially available anti-parasitic drug which prevents infection by a wide range of endo- and ectoparasites (Sajid et al, 2006; Heidary & Gharebaghi, 2020). IVM is an efficient positive allosteric modulator of the α-7 nicotinic acetylcholine receptor (nAChR) (Krause et al, 1998) and of several ligand-gated ion channels, including the muscle receptor for glutamate (GluCl) in worms (Hibbs & Gouaux, 2011). Furthermore, IVM has been shown to exert an immunomodulatory effect in humans and animals (Sajid et al, 2006; Heidary & Gharebaghi, 2020) under conditions that are known to involve the α-7 nAChR (Pavlov & Tracey, 2012), even though its underlying mechanisms are yet to be established (Laing et al, 2017). A direct or indirect interaction of SARS-CoV-2 with nAChR has also been hypothesized, in particular because of sequence homologies between SARS-CoV-2 spike proteins and nAChR ligands such as snake venom toxins (Changeux et al, 2020). IVM has been shown to be active beyond its anti-parasitic activity in a wide variety of pathologies, including cancer, allergy, and viral infections (Laing et al, 2017). Recently, IVM has been reported to reduce viral load and improve the clinical status of mice infected by an animal coronavirus, the mouse hepatitis virus (MHV) (Arévalo et al, 2021). In vitro inhibition of SARS-CoV-2 replication by IVM in Vero/hSLAM cells has also been reported (Caly et al, 2020), albeit at much higher concentrations (50- to 100-fold) than those clinically attainable in humans (150–400 µg/kg) (Guzzo et al, 2002; Bray et al, 2020; Chaccour et al, 2020). The aim of this study is to investigate the impact of IVM on the pathogenesis of COVID-19, in a SARS-CoV-2 infection model, the golden Syrian hamster. This species is naturally permissive to this virus and the most reliable and affordable animal model for COVID-19 (Chan et al, 2020; Muñoz-Fontela et al, 2020). Moreover, it was recently used to demonstrate the importance of lowering the inflammation with intranasal administration of type I IFN to prevent disease progression (Hoagland et al, 2020). Male and female adult golden Syrian hamsters were intranasally inoculated with 6 × 104 PFU of SARS-CoV-2 [BetaCoV/France/IDF00372/2020]. This inoculum size was selected as it invariably causes symptomatic infection in golden Syrian hamster, with a high incidence of anosmia and high viral loads in the upper and lower respiratory tracts within 4 days post-inoculation (dpi) (de Melo et al, 2021). At the time of infection, animals received a single subcutaneous injection of IVM at the anti-parasitic dose of 400 µg/kg, commonly used in human clinical setting, and were monitored over 4 days. Here, we show that the modulation of the host's inflammatory response using IVM as a repurposed drug strongly diminished the clinical score and severity of the disease (including anosmia) observed in these animals, although it has no impact on viral load. IVM-treated animals presented a strong modulation in several signaling pathways, including a significant reduction of the type I and III interferon response and of the Il-6/Il-10 ratio, along with the presence of M2 macrophages in the lung. These effects were mostly compartmentalized and sex-dependent, and treated infected females exhibited better clinical outcomes. Results and Discussion COVID-19 clinical outcome is attenuated by ivermectin In order to study the effects of IVM chemical therapy on clinical outcome, we assessed body weight, clinical score, and olfactory performance daily for 4 days post-infection. The golden hamster model reproduces a moderate-to-severe COVID-19 in humans, with a clinical phase lasting 5–6 days post-infection followed by a complete recovery by 2 weeks post-infection, with no death occurring. The viral titers and viral RNA loads in the airways of infected animals are elevated after 2–4 days post-infection, but they drastically drop by 7 days post-infection (Chan et al, 2020; Sia et al, 2020; de Melo et al, 2021). The IVM-treated infected animals, of both sexes, showed a decrease in body weight similar to that observed in saline-treated infected hamsters (Fig EV1A). However, IVM-treated infected animals exhibited a significant reduction in the severity of the clinical score, in a sex-dependent manner: Infected males presented a significantly reduced clinical score, and it fully returned to normal in infected females (Fig 1A). Remarkably, IVM treatment reduced the olfactory deficit in infected animals (Figs 1B and EV1B–D): 66.7% (12/18) of the saline-treated infected hamsters presented with hyposmia/anosmia, whereas only 22.2% (4/18) of IVM-treated infected hamsters presented with signs of olfactory dysfunction (Figs 1B and EV1B–D). The olfactory performance was also influenced by sex: 83.3% (10/12) of the saline-treated infected males presented with hyposmia/anosmia, against only 33.3% (4/12) of IVM-treated infected males (Figs 1B and EV1B–D). In females, while 33.3% (2/6) of saline-treated infected animals presented with hyposmia/anosmia, no olfactory deficit was observed in IVM-treated infected females (0/6; Figs 1B and EV1B–D). Click here to expand this figure. Figure EV1. Complementary clinical aspects and olfaction test in hamsters, infected or not by the SARS-CoV-2, with and without ivermectin treatment A. Progression of body weight in male and female hamsters, mock-infected or SARS-CoV-2-infected, treated with saline or with 400 µg/kg ivermectin. Symbols indicate the median ± interquartile range. B. Curves represent the percentage of animals that did not find the hidden (buried) food. C. Curves represent the percentage of animals that did not find the visible (unburied) food. D. Summary of olfactory status. Data information: Food finding assays were performed at 3 days post-infection. n = 6/group, except males CoV_saline and males CoV_ivermectin, where n = 12/group. The P value is indicated in bold when significant at a 0.05 threshold. Log-rank (Mantel–Cox) test (B, C) and Fisher's exact test (D). Data were obtained from three independent experiments for males and two independent experiments for females. The data for CoV_saline and CoV_ivermectin groups (B) are already presented in Fig 1B. Source data are available online for this figure. Download figure Download PowerPoint Figure 1. Clinical presentation, olfaction test, viral load and immune profile in the nasal turbinates of SARS-CoV-2-infected hamsters with and without ivermectin treatment A. Clinical signs in infected hamsters. The clinical score is based on a cumulative 0–4 scale: ruffled fur; slow movements; apathy; and absence of exploration activity. Symbols indicate the median ± interquartile range. B. Olfactory performance in infected hamsters. The olfaction test is based on the hidden (buried) food finding test. Curves represent the percentage of animals that did not find the buried food. Food finding assays were performed at 3 days post-infection (dpi). Data were obtained from three independent experiments for males and two independent experiments for females. C. Viral load in the nasal turbinates and in the lungs at 4 dpi. D. Ratio between the CPD (copy per droplets, normalized to γ-actin and Hprt reference gene relative expression) of structural [N, nucleocapsid] and non-structural [IP4: RdRp, RNA-dependent RNA polymerase] viral gene expression determined by digital droplet PCR (ddPCR) in the nasal turbinates and in the lungs at 4 dpi. E. Infectious viral titer in the lung at 4 dpi expressed as plaque-forming units (PFU)/g of tissue. F. Cytokine and chemokine transcripts in the nasal turbinates at 4 dpi in male and female SARS-CoV-2-infected hamsters, treated with saline or with 400 µg/kg ivermectin. Data information: Horizontal lines indicate medians. The P value is indicated in bold when significant at a 0.05 threshold. Mann–Whitney test (A, C–F) and log-rank (Mantel–Cox) test (B). M: male hamsters and F: female hamsters. Data were obtained from two independent experiments for each sex. See Figs EV1 and EV2 and Appendix Fig S1. Source data are available online for this figure. Source Data for Figure 1 [emmm202114122-sup-0004-SDataFig1.xlsx] Download figure Download PowerPoint Since males presented a higher incidence of anosmia/hyposmia, we subsequently performed a dose-response curve to test the effect of IVM on the clinical presentation and olfactory functions of infected males: Lower doses of IVM (100 or 200 µg/kg) elicited similar clinical outcomes as the anti-parasitic dose of 400 µg/kg (Fig EV2). As expected, no signs of olfactory deficit were observed in mock-infected hamsters (Fig EV1B–D). Click here to expand this figure. Figure EV2. Clinical aspects of SARS-CoV-2-infected male hamsters and treated with different doses of ivermectin A. Progression of body weight in male hamsters, treated with saline or with 400, 200, or 100 µg/kg ivermectin. Symbols indicate the median ± interquartile range. B. Clinical score based on a cumulative 0–4 scale: ruffled fur; slow movements; apathy; and absence of exploration activity. Symbols indicate the median ± interquartile range. C. Olfaction deficit based on the buried food finding test. Curves represent the percentage of animals that did not find the buried food. Food finding assays were performed at 3 days post-infection. Data information: n = 12/group (CoV_saline and CoV_ivermectin 400 µg/kg, as shown in Fig 1) or n = 4 (CoV_ivermectin 200 µg/kg and 100 µg/kg). The P value is indicated in bold when significant at a 0.05 threshold. Mann–Whitney test at 4 dpi (B) and log-rank (Mantel–Cox) test (C). The data for CoV_saline and CoV_ivermectin 400 µg/kg groups (B, C) are already presented in Fig 1B. Source data are available online for this figure. Download figure Download PowerPoint Ivermectin treatment does not influence SARS-CoV-2 load in the respiratory tract of infected hamsters To evaluate the effect of IVM treatment on the viral load in the respiratory tract, we tested the nasal turbinates and lungs of infected hamsters using both classical RT–qPCR (Fig 1C) and the highly sensitive technique of digital droplet PCR (Suo et al, 2020; Fig 1D, Appendix Fig S1). Surprisingly, the viral RNA load in the respiratory tract remained unaffected by IVM treatment in both samples in both sexes. Furthermore, IVM treatment did not influence viral replication rate, as evaluated by the ratio between structural and non-structural gene transcription (Fig 1D, Appendix Fig S1). Finally, IVM treatment did not alter infectious viral titers in the lungs (Fig 1E). These results illustrate that no antiviral activity of IVM is detected in vivo at the standard anti-parasitic dose of 400 µg/kg, in contrast to a previous report suggesting that IVM inhibits the replication of SARS-CoV-2 in vitro, albeit used at far higher concentrations (Caly et al, 2020). Therefore, the action of IVM on COVID-19 clinical signs of infected golden hamsters does not result from a decrease in viral replication. Ivermectin therapy modulates local immune responses in infected hamsters' nasal turbinates Anosmia is a typical symptom of COVID-19 in humans, with some sex-dependent differences (Han et al, 2020; Qiu et al, 2020; Xydakis et al, 2020). Inflammation in the nasal cavity, following olfactory sensory neurons infection and deciliation, has been shown to be an underlying factor for smell loss during SARS-CoV-2 infection (de Melo et al, 2021), and the chemokine Cxcl10 could be directly implicated due to its neurotoxic potential (Oliviero et al, 2020). We therefore tested a possible modulation by IVM of the local inflammatory response in hamsters and in particularly in the nasal turbinates, the primary target tissue of SARS-CoV-2 infection (de Melo et al, 2021), that could correlate its effect on the olfactory score. To this aim, a panel of cytokines (Il-6, Il-10, Il-1β, Tnf-α, Ifn-β, Ifn-γ, and Ifn-λ) and chemokines (Cxcl10 and Ccl5) were used to assess the impact of IVM treatment on immune responses in the nasal turbinates of SARS-CoV-2-infected hamsters at 4 dpi. Upon treatment with IVM, females presented a significant downregulation of Il-6, Il-10, and Tnf-α, which are key inflammatory mediators of prognostic value in COVID-19 patients (McElvaney et al, 2020a), and of Cxcl10 (Fig 1F), in line with their better olfactory performance observed in the food finding tests (Fig 1B). The differences between sex groups are illustrated by the increase in three pro-inflammatory mediators (Ifn-γ, Ifn-λ, and Ccl5) only in males (Fig 1F). No difference for the Il-6/Il-10 ratio was observed in the nasal turbinates. Lung immunometabolism is affected by SARS-CoV-2 infection and modulated by ivermectin IVM attenuates lung pathology and inflammation pathways, including cholinergic synapse-related genes In order to further study the mode of action of IVM on clinical signs, we performed at 4 dpi a comparative agnostic transcriptomic approach using RNA-seq in the lower respiratory tract in hamsters treated or not with IVM. In SARS-CoV-2-infected lungs, male and female hamsters exhibited an overall similar pattern, although there were slightly more KEGG pathways modulated by infection in males (Fig 2A). A high number of common dysregulated inflammatory and metabolic pathways in SARS-CoV-2-infected males and females (Fig EV3) were similar to those observed in lung cells from human COVID-19 patients, such as "cytokine-cytokine receptor interaction", "TNF signaling pathway", and "insulin resistance" (Dey et al, 2020; Islam et al, 2020) including many genes of the IFN response. The transcriptomic profiles observed in the lungs from IVM-treated hamsters compared to non-treated infected hamsters presented a striking sex difference: Lungs of IVM-treated females presented 1,206 downregulated genes and 1,428 upregulated genes, whereas only 36 downregulated and 51 upregulated genes were detected in the lungs of IVM-treated males. This sex difference is also illustrated by KEGG and GO enrichments representations (Fig EV4). Figure 2. Transcriptomic profile in the lung of SARS-CoV-2-infected hamsters with and without ivermectin treatment at 4 days post-infection A. Heatmaps showing the differentially expressed genes according to the selected KEGG pathways calculated in comparison with mock-infected hamsters. * indicates Benjamini–Hochberg-adjusted P-value < 0.05 in the comparison between saline and ivermectin within the same sex. Color gradient represents the transcription log2 fold change comparing infected and mock-infected. Complete analyses are listed in Dataset EV1. B. Validation targets in the lung at 4 dpi. Horizontal lines indicate medians. The P value is indicated in bold when significant at a 0.05 threshold. Mann–Whitney test. Data information: M: male hamsters and F: female hamsters. Data were obtained from two independent experiments for each sex. See Figs EV3-EV5. Source data are available online for this figure. Source Data for Figure 2 [emmm202114122-sup-0005-SDataFig2.xlsx] Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Transcriptomic aspects in the lung of SARS-CoV-2-infected hamsters compared to mock-infected hamsters at 4 days post-infection A. KEGG enrichment. B. GO enrichment analysis. Data information: Selected terms are based on the up- and downregulated genes between infected (CoV_saline) and mock-infected (mock_saline) samples. Only the 20 highest fold enrichments are plotted for the upregulated gene set. Circle sizes are proportional to the gene set size, which shows the total size of the gene set associated with GO terms. Circle color is proportional to the corrected P-values. Terms identified in both sexes are marked in bold. Complete analyses are listed in Dataset EV1. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Transcriptomic aspects in the lung of SARS-CoV-2-infected and ivermectin-treated hamsters compared to SARS-CoV-2-infected and saline-treated hamsters at 4 days post-infection A. KEGG enrichment. B. GO enrichment analysis. Data information: Selected terms are based on the up- and downregulated genes between IVM-treated (CoV_ivermectin) and saline-treated (CoV_saline) samples. Only the 20 highest fold enrichments are plotted for the upregulated gene set. Circle sizes are proportional to the gene set size, which shows the total size of the gene set associated with GO terms. Circle color is proportional to the corrected P-values. Complete analyses are listed in Dataset EV1. Source data are available online for this figure. Download figure Download PowerPoint Several KEGG pathways were significantly regulated in IVM-tr
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