Thoracic adipose tissue contributes to severe virus infection of the lung

Figures

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  Fig. 1. Obesity is associated with disease progression and high viral loads in influenza virus-infected mice. A Schematic overview of non-obese and diet-induced obese (DIO), female BALB/C mice with subsequent infection with IAV. B DIO mice (n = 64) were characterized by increased body weight and blood glucose, compared non-obese (n = 46). Also, relative mass of adipose tissue extracted from the abdomen were elevated, and relative lung weights decreased in obese (n = 8) compared to non-obese (n = 6). C Schematic overview of female C57BL/6 non-obese, heterozygous (ob/+), and homozygous obese mice with global leptin knockout (ob/ob) with subsequent infection with IAV. D obese ob/ob mice (n = 20) were characterized by increased body weigh tand blood glucose compared to non-obese ob/+ mice (n = 20). Also, relative mass of adipose tissue was elevated and relative lung weights decreased in ob/ob (n = 10) compared to non-obese ob/+ (n = 10) mice. E Relative body weight and overall scoring values after infection of DIO showed a significant temporal right shift and a slower recovery after 21 days p.i. F Infection of ob/ob mice led to lesser alteration in the body weight progression but equal…

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  Fig. 2. Thoracic adipose tissue adjacent to the lungs harbored influenza virus. A Image of the thorax lumen of uninfected DIO (left) and ob/ob mice (right) with H&E staining of a representative cryosection (4 µm thickness) of the extracted thoracic adipose tissue, Scale bar, 250 μm. B, C ob/ob (n = 5) mice showed increased thoracic adipose tissue mass (B) and elevated copies/mg of IAV within the extracted tissue day 4 p.i. compared to non-obese (n = 5). Extracted abdominal adipose tissue sections from obese ob/ob (D) and DIO mice (E) show upregulated mRNA levels of IL-6 and TNF-α compared to the corresponding non-obese mice. F IAV nucleoprotein (green) visualized by immunofluorescence staining detected at day 4 p.i. (arrowhead) within cryosections (4 µm) from lung and additionally extracted thoracic adipose tissue from representative ob/ob mice. Phalloidin (red) and DAPI (blue) staining were used to detect actin and the nuclei. Scale bar 100 μm. P calculated by Mann-Whitney-test (B, D, E). *P < 0.05**P < 0.01.

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  Fig. 3. Influenza virus replicated in human primary adipocytes. A Schematic overview of the experimental setup for the infection of primary derived adipocytes differentiated from preadipocytes and the subsequent infection with IAV (PR8) for 1 and 3 days with an MOI (Multiplicity of infection) of 1. B Active virus particles detected by standard plaque assay from supernatants of infected adipocytes were significantly increased after 1 and 3 days of infection with PR8 with an MOI of 1. C dsRNA (green) of IAV could be visualized by immune fluorescence staining within infected adipocytes day 3 p.i., mainly located at the lipid droplets and already damaged nucleus within the cells. Cytoplasm (red) was stained wheat germ agglutinin (WGA) and the nuclei (blue) with DAPI. Scale bars, 20 μm. D Infected adipocytes secreted significantly more IFN-γ, 1 day p.i. and more MCP-1, IP-10 3 days p.i. E Schematic illustration of the infection of primary lung fibroblasts co-cultivated in a trans-well system with adipocytes with IAV of an MOI of 1. F No differences in virus titer were determined via plaque assay in the co-culture compared to the mono-culture of solely lung fibroblasts. G Co-culture of…

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  Fig. 4. SARS-CoV-2 was detected in the thoracic adipose tissue of COVID-19 patients. A Schematic overview of the localization of the lung and adipose tissues biopsies taken from the thorax of five deceased COVID patients. B SARS-CoV-2 RNA was detected within different areas of the lung and in extracted thoracic adipose tissue (TAT) samples. C Virus particles were detected in cryosections from TAT (mediastinum) determined by immunofluorescence staining of the spike protein (green), actin (phalloidin, red) and nuclei (DAPI, blue) compared to the corresponding negative control (upper panel). Additional staining of CD68 from macrophages revealed the potential colocalization of macrophages and spike protein within TAT (mediastinum). Scale bar, 100 µm (lower panel). D Proinflammatory markers IL-6, G-CSF, MCP-1, and IP-10 were higher in homogenized tissue from TAT (mediastinum) compared to biopsies from the left inferior lung lobe (LIL). Data presented as Mean ± SD. P calculated by Ordinary one-way analysis of variance (ANOVA), multiple comparisons; **P < 0.01, ***P < 0.001.

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  Fig. 5. SARS-CoV-2 variants did not replicate in human primary adipocytes. A Schematic overview of the experimental setup for the infection of primary derived adipocytes differentiated from preadipocytes and the subsequent infection with the SARS-CoV-2 variants alpha, delta, and omicron for 1 and 3 days. B Active virus particles of the alpha and delta variant were detected 1 day p.i. but decreased until day 3 p.i. C dsRNA (green) of SARS-CoV-2 of all three variants were not visible within the infected adipocytes after day 3 determined by immunofluorescent staining of infected cells compared to uninfected mock cells. D mRNA expression of was not detected in human primary adipocytes compared to Calu-3 cells as positive control. E Western blot analysis of the ACE-2 protein revealed no expression in adipocytes compared to the Calu-3 positive control. Actin served as loading control. Data presented as Mean ± SD and presentative of two independent experiments for each variant; P calculated by two-way analysis of variance (ANOVA) test, Tukey’s multiple comparisons test (B) and Mann–Whitney test (D); ****P < 0.0001.

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  Fig. 6. Macrophages contributed to inflammation even in the absence of active viral replication. A Schematic overview of the experimental setup for the infection of macrophages derived from isolated PBMCs from a healthy donor with subsequent infection with IAV and SARS-CoV-2 (delta and omicron) for 8 and 24 h. B Active virus particles of IAV were detected after 8 and 24 h, whereas SARS-CoV-2 (delta) particles decreased from 8 to 24 h. C Viral RNA was determined extracellular (extra) and intracellular (intra) of infected macrophages. D dsRNA (green) of both SARS-CoV-2 and IAV detected by immune fluorescence staining within PBMCs stained with CD68 (red) and DAPI (blue) 24 h p.i. Scale bars 20 µm. E Proinflammatory markers MCP-1, IL-8, and IP-10 were significantly upregulated in supernatants of macrophages infected with the omicron variant. SARS-CoV-2 (omicron) and IAV led to increased relative cytotoxicity measured by Lactate dehydrogenase (LDH)-assay in cell supernatants 24 h p.i. Data presented as Mean ± SD and presentative of three independent experiments for each variant; P calculated by two-way analysis of variance (ANOVA) test, Tukey’s multiple comparisons test (B, E), Ordinary…