Diet-induced obesity and diabetes enhance mortality and reduce vaccine efficacy for SARS-CoV-2

Abstract

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused a wide spectrum of diseases in the human population, from asymptomatic infections to death. It is important to study the host differences that may alter the pathogenesis of this virus. One clinical finding in coronavirus disease 2019 (COVID-19) patients is that people with obesity or diabetes are at increased risk of severe illness from SARS-CoV-2 infection. We used a high-fat diet model in mice to study the effects of obesity and type 2 diabetes on SARS-CoV-2 infection as well as how these comorbidities alter the response to vaccination. We find that diabetic/obese mice have increased disease after SARS-CoV-2 infection and they have slower clearance of the virus. We find that the lungs of these mice have increased neutrophils and that removing these neutrophils protects diabetic/obese mice from disease. This demonstrates why these diseases have increased risk of severe disease and suggests specific interventions upon infection.

Keywords: coronavirus; diabetes; immunization.

Fig 1

A high-fat diet (HFD) induces obesity and diabetes in C57BL/6J mice. (A) Schematic overview illustrating diet schedule and infection for control and obese/diabetic mice. Briefly, at weaning (3 weeks), mice were placed on a normal diet (ND) or HFD. At 18 weeks of age, these mice were infected with either 1 × 10³ or 1 × 10⁵ pfu of SARS-CoV-2 Mouse Adaptive 10, monitored daily for weight changes, and a subset was euthanized at days 2, 4, and 7 to analyze lung viral titer and host immune response. The (B) fasted blood glucose concentration n = 13 mice/group, (C) the starting weight of ND and HFD mice n = 45 mice/group, and (D) ACE2 expression of ND and HFD mice n = 5 mice/group prior to infection. *P < 0.5 and ****P < 0.0001 as determined by an unpaired two-sided t-test. The data are presented as mean ± SD.

Fig 2

Enhanced mortality in obesity/diabetes mice after SARS-CoV-2 infection. The survival (A) percent weight loss for low-dose (B) high-dose (C) gram lost for low-dose (D) and high-dose (E) challenge. Viral lung titers were assessed for control and obese/diabetic mice throughout a SARS-CoV-2 MA10 infection (F) For (A). *P < 0.05 as determined by a Log-rank (Mantel-Cox) test for survival analysis between ND and HFD for low and high infection dose with the data presented as mean ± SD with n = 10 mice/group. In (G), lung cytokine, chemokine, and growth factors were assessed at 2, 4, and 7 dpi for the 1 × 10³ infectious dose using a mouse cytokine/chemokine array. The fold change was calculated compared to ND-infected mice (n = 3). For (B–D), the data are presented as mean ± SD with n = 3–5 mice/group.

Fig 3

Similar lung histology and viral clearance in control and obese/diabetic mice. Control and obese/diabetic mice were infected intranasally with either 1 × 10³ or 1 × 10⁵ pfu/ mouse. Lungs were collected on 2, 4, and 7 dpi and fixed in 10% neutral buffered formalin for greater than 24 hours. Tissue was embedded in paraffin. (A) 5 µm sections were cut and stained with hematoxylin and eosin and (B) immunostained with anti-SARS nucleocapsid protein antibody. Images are shown at original magnification ×10 and are representative of n= 3–5 mice/group.

Fig 4

Viral clearance is delayed in obese/diabetic vaccinated mice. (A) Schematic overview illustrating vaccination and challenge for ND and HFD mice. Briefly, at 18 weeks of age, mice were vaccinated as well as 14 days later. On day 28 post-vaccination, mice were bled to determine (B) serum-neutralizing antibodies prior to infection, n = 12–15 mice/group. On day 32, these mice were challenged with 1 × 10⁵ pfu of SARS-CoV-2 MA 10, monitored daily for weight changes, and a subset was euthanized on days 2 and 4 to analyze lung viral titer and host immune response. The (C) mortality, weight loss of (D) ND, (E) HFD, and (F) viral titer of sham and vaccinated mice after the SARS-2 MA10 challenge. n = 5–10 mice/group. For (D), *P < 0.5 and ***P < 0.001 as determined by a mixed-effect analysis followed by a Sidak test for multiple comparisons. For (F), P *** <0.001 and P **** <0.0001, data were log-transformed and analyzed by mixed-effect analysis followed by Sidik for multiple comparisons. The data are pooled from two independent experiments and presented as mean ± SD.

Fig 5

Increased neutrophil recruitment in the lungs of HFD mice after the SARS-CoV-2 challenge. After the SARS-2 MA10 challenge, lungs were harvested, cells were isolated, and stained for surface markers for (A) neutrophils, (B) CD11b⁺ DCs, (C) monocyte-derived DCs, (D) CD103⁺ cDCs, and (E) plasmacytoid DCs at days 2 and 4 post-challenge. *P < 0.05 and **P < 0.01 as data were analyzed by mixed-effect analysis followed by Tukey for multiple comparisons n = 3–5 mice/group.

Fig 6

T-cell dysregulation in the lungs of HFD mice after the SARS-CoV-2 challenge. After the SARS-2 MA10 challenge, lungs were harvested, cells were isolated, and stained for surface markers for (A) natural Killer cells, (B) B cells, (C) total T cells, (D) T-helper cells, and (E) cytotoxic T cells at days 2 and 4 post-challenge n = 3–5 mice/group. *P < 0.05 as data were analyzed by mixed-effect analysis followed by Tukey for multiple comparisons n = 3–5 mice/group (F) CD4⁺ SARS-CoV-2-specific T cells and (G) CD8⁺ SARS-CoV-2-specific T cells from naïve and vaccinated ND and HFD mice after ex vivo stimulation n = 6 mice/group. Cells were collected at day 28 after prime and boost vaccination in vaccine experiments. *P < 0.5 as determined by an unpaired two-sided t-test. The data are presented as mean ± SD.

Fig 7

Increased neutrophil recruitment and cytokine production in HFD-vaccinated mice after challenge. On days 2,4, and 7 post-infection, lungs were harvested and used in a Bio-plex pro-inflammatory mouse chemokine panel. The pg/mL was generated from a standard curve. *P < 0.05 and **P < 0.01 as analyzed by mixed-effect analysis followed by Sidak for multiple comparisons (n = 3–5 mice/group).

Fig 8

Increased HFD survival with neutrophil depletion after the challenge. The (A) survival and percent weight loss for (B) isotype antibody control or (C) anti-Ly6G antibody ND and HFD mice. *P < 0.05 as analyzed by Gehan-Breslow-Wilcoxon test for survival n = 5 mice/group.