Attenuation of clinical and immunological outcomes during SARS-CoV-2 infection by ivermectin

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.

Keywords: SARS-CoV-2; coronavirus; inflammation; ivermectin; viral infections.

Figure EV1. Complementary clinical aspects and olfaction test in hamsters, infected or not by the SARS‐CoV‐2, with and without ivermectin treatment

1. 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.

2. B

   Curves represent the percentage of animals that did not find the hidden (buried) food.

3. C

   Curves represent the percentage of animals that did not find the visible (unburied) food.

4. 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.

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

1. 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.

2. 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.

3. C

   Viral load in the nasal turbinates and in the lungs at 4 dpi.

4. 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.

5. E

   Infectious viral titer in the lung at 4 dpi expressed as plaque‐forming units (PFU)/g of tissue.

6. 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.

Figure EV2. Clinical aspects of SARS‐CoV‐2‐infected male hamsters and treated with different doses of ivermectin

1. 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.

2. 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.

3. 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.

Figure 2. Transcriptomic profile in the lung of SARS‐CoV‐2‐infected hamsters with and without ivermectin treatment at 4 days post‐infection

1. 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.

2. 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, EV4, EV5. Source data are available online for 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

1. A

   KEGG enrichment.

2. 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.

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

1. A

   KEGG enrichment.

2. 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.

Figure EV5. Transcriptomic profile in the lung of SARS‐CoV‐2‐infected hamsters with and without ivermectin treatment at 4 days post‐infection

1. 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.

2. 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. Source data are available online for this figure.

Figure 3. Identification of macrophages in the lung of SARS‐CoV‐2‐infected hamsters with and without ivermectin treatment and characterization of their transcriptomic profile related to M1/M2 polarization

1. A

   Representative histopathology photomicrographies of lungs according to the different groups: mock_saline, CoV_saline, and CoV_ivermectin. Top panels: whole lung sections. Bottom panels: high magnification. CoV_saline section exhibits important congestion (*), edema associated with few mononuclear cells (white arrowheads). Note the thickening of the alveolar walls. CoV_ivermectin section exhibits important amounts of mononuclear cells (black arrowheads) and less marked signs of congestion or edema. Hematoxylin and eosin. Scale bars = 1 mm (top panels) and 20 μm (bottom panels).

2. B

   Representative immunofluorescence photomicrographies of neutrophils (Ly‐6G), monocytes/macrophages (Iba1), M2 macrophages (Arg1), and SARS‐CoV‐2 (NP) in the lung. Scale bars = 50 μm.

3. C

   Quantification of Iba1⁺ cells, Arg1⁺ cells, and Iba1⁺Arg1⁺ cells in the lungs. mock_saline n = 3 (males), mock_ivermectin n = 4 (males), CoV_saline n = 9 (6 males and 3 females), and CoV_ivermectin n = 6 (4 males and 2 females).

4. D

   Heatmaps showing the differentially expressed genes related to the M1/M2 polarization 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.

5. E

   Lung weight‐to‐body weight ratio in the different groups (n = 4/sex/group).

Data information: M: male hamsters and F: female hamsters. Horizontal lines indicate medians. The P value is indicated in bold when significant at a 0.05 threshold. Mann–Whitney test (C, E). Source data are available online for this figure.