Comorbid diabetes results in immune dysregulation and enhanced disease severity following MERS-CoV infection

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2012 in Saudi Arabia and has caused over 2400 cases and more than 800 deaths. Epidemiological studies identified diabetes as the primary comorbidity associated with severe or lethal MERS-CoV infection. Understanding how diabetes affects MERS is important because of the global burden of diabetes and pandemic potential of MERS-CoV. We used a model in which mice were made susceptible to MERS-CoV by expressing human DPP4, and type 2 diabetes was induced by administering a high-fat diet. Upon infection with MERS-CoV, diabetic mice had a prolonged phase of severe disease and delayed recovery that was independent of virus titers. Histological analysis revealed that diabetic mice had delayed inflammation, which was then prolonged through 21 days after infection. Diabetic mice had fewer inflammatory monocyte/macrophages and CD4+ T cells, which correlated with lower levels of Ccl2 and Cxcl10 expression. Diabetic mice also had lower levels of Tnfa, Il6, Il12b, and Arg1 expression and higher levels of Il17a expression. These data suggest that the increased disease severity observed in individuals with MERS and comorbid type 2 diabetes is likely due to a dysregulated immune response, which results in more severe and prolonged lung pathology.

Keywords: Diabetes; Infectious disease; Mouse models; Virology.

Conflict of interest statement

Conflict of interest: Salary support for MBF is provided by Emergent BioSolutions and Regeneron, Inc.

Figures

Figure 1. Male DPP4H/M mice develop type 2 diabetes on an HFD.

DPP4H/M mice were split into age- and sex-matched cohorts at 4 to 6 weeks of age and maintained on either a high-fat chow (diabetic) or normal chow (control) diet. The (A) weight, (B) fasted blood glucose concentration, and (C) fasted serum insulin concentrations were determined. Glucose tolerance was determined using an intraperitoneal glucose tolerance test (IPGTT) in which 2 mg/kg of glucose was administered intraperitoneally and blood concentrations were measured every 30 minutes for 2 hours. The data for (D) male and (E) female mice were normalized to the baseline fasted glucose concentration, and (F) the area under the curve (AUC) was determined. Data for A, B, and D–F are pooled from 4 independent experiments with n = 37–53 mice/group presented as the mean ± SEM. ****P < 0.0001 as determined by 1-way ANOVA with Tukey’s posttest. Data for C are pooled from 2 independent experiments with n = 13–17 mice/group presented as the mean ± SEM. *P = 0.0256 as determined by 1-way ANOVA with Tukey’s posttest.

Figure 2. Diabetic male DPP4H/M mice exhibit prolonged severe disease following high-dose MERS-CoV infection.

Male diabetic and control and female HFD and control DPP4H/M mice were infected intranasally with 1.5 × 105 PFU of MERS-CoV Jordan. The (A and B) percentage of weight loss and (C and D) amount of weight lost were measured daily in (A and C) male and (B and D) female mice. Clinical signs of disease were also assessed daily in (E) male and (F) female mice. Clinical scores were determined on the following scale: 0 = healthy; 1 = slight ruffling of the fur, altered hind limb posture; 2 = mildly labored breathing, no lethargy, 3 = moderately labored breathing, lethargy; 4 = severely labored breathing, severe lethargy; and 5 = dead. Data are presented as the mean ± SEM and are pooled from 3 independent experiments with n = 12–17 mice/group. *P < 0.0239; **P < 0.01; ***P < 0.001; and ****P < 0.0001 as determined by 2-way ANOVA with Holm-Šídák posttest.

Figure 3. Diabetes does not alter virus replication and clearance within the lung or extrapulmonary virus dissemination.

Male diabetic and control and female HFD and control DPP4H/M were infected intranasally with 1.5 × 105 PFU of MERS-CoV Jordan. (A and D) Lungs were collected at days 2, 4, 7, 10, and 14 after infection from male (A) and female (D) mice and homogenized in PBS. Lung titers were determined by plaque assay. Data are pooled from 2 to 3 independent experiments with n = 3–8 mice/group. Data are presented as the mean ± SEM. No data were determined to be significant using a 2-way ANOVA with Holm-Šídák posttest. (B, C, E, and F) Lungs were collected at days 2, 4, 7, 10, 14, and 21 after infection and homogenized in TRIzol. RNA was isolated and levels of viral genomic RNA (B and E) and viral mRNA (C and F) were determined using qRT-PCR targeting UpE and M, respectively. Data were normalized to transferrin receptor protein 1 (TfRC) and then normalized to PBS controls and are presented as relative units. Data are pooled from 2 to 3 independent experiments with n = 3–11 mice/group and are presented as the mean ± SEM. (G–J) Lung, whole blood, brain, liver, kidney, and spleen from infected male (G and I) and female (H and J) mice 7 days after infection. Virus genomic RNA (G and H) and virus mRNA (I and J) were determined using qRT-PCR targeting UpE and M, respectively. Data were normalized to TfRC and then normalized to PBS controls and are presented as relative units. Data are from n = 4–11 mice/group and are presented as the mean ± SEM.

Figure 4. Lung histology shows delayed and unresolved inflammation in male diabetic mice infected with MERS-CoV.

Male diabetic and control DPP4H/M mice were infected intranasally with 1.5 × 105 PFU of MERS-CoV Jordan. (A) Lungs were collected at days 2, 4, 7, 10, 14, and 21 after infection and fixed in 10% neutral buffered formalin for more than 24 hours. Tissue was embedded in paraffin and 5-μm sections were cut and stained with hematoxylin and eosin. Blood vessels are marked by arrowheads, and airways are marked by an asterisk (*). Images are shown at original magnification ×10 and are representative of n = 5–11 mice/group from 2 to 3 independent experiments. (B) Overall lung inflammation, (C) bronchiolar inflammation, and (D) perivascular inflammation were scored by a board-certified veterinarian. The data are pooled from 2 to 3 independent experiments with n = 5–11 mice/group. Data are presented as the mean ± SEM. *P < 0.05; ***P < 0.001; and ****P < 0.0001 as determined by 2-way ANOVA with Holm-Šídák posttest.

Figure 5. Diabetic male mice have fewer CD4+ T cells and inflammatory monocyte/macrophages in the lungs at peak inflammation following MERS-CoV infection.

Male diabetic and DPP4H/M mice were infected intranasally with 1.5 × 105 PFU of MERS-CoV Jordan. Lungs were collected at days 4, 7, 14, and 21 after infection or from uninfected mice, and single cells were isolated from the tissue. Flow cytometric analysis was performed on the cells isolated from the lung. The numbers of (A) CD45+ cells, (B) inflammatory monocyte/macrophages, (C) CD3+ T cells, (D) CD4+ T cells, (E) CD8+ T cells, (F) B cells, (G) NK cells, and (H) neutrophils were determined. The data are pooled from 2 independent experiments with n = 3–9 mice/group. Data are presented as the mean ± SEM. *P < 0.05 as determined by 2-way ANOVA with Holm-Šídák posttest.

Figure 6. Cytokine and chemokine gene expression in the lungs is altered in male diabetic DPP4H/M mice following MERS-CoV infection.

Male diabetic and control DPP4H/M mice were infected intranasally with 1.5e5 PFU of MERS-CoV Jordan. Lung tissue was collected at days 2, 4, 7, 10, 14, and 21 after infection and from PBS-infected mice and was homogenized in TRIzol. RNA was isolated, cDNA was synthesized, and gene expression was determined using quantitative PCR. Gene expression was normalized to GAPDH, and fold change was calculated relative to PBS infected mice. Gene expression was determined for (A) Ccl2, (B) Tnfa, (C) Il6, (D) Il12b, (E) Nos2, (F) Arg1, (G) Cxcl10, (H) Il4, (I) Ifng, (J) Il17a, (K) Foxp3, and (L) Il10. Data are pooled from 2 to 3 independent experiments with n = 3–16 mice/group and are presented as the mean ± SEM. *P < 0.05; **P < 0.01; and ***P < 0.001 as determined by 2-way ANOVA with Holm-Šídák posttest.