Pulmonary SARS-CoV-2 infection leads to para-infectious immune activation in the brain

Abstract

Neurological complications, including encephalopathy and stroke, occur in a significant proportion of COVID-19 cases but viral protein is seldom detected in the brain parenchyma. To model this situation, we developed a novel low-inoculum K18-hACE2 mouse model of SARS-CoV-2 infection during which active viral replication was consistently seen in mouse lungs but not in the brain. We found that several mediators previously associated with encephalopathy in clinical samples were upregulated in the lung, including CCL2, and IL-6. In addition, several inflammatory mediations, including CCL4, IFNγ, IL-17A, were upregulated in the brain, associated with microglial reactivity. Parallel in vitro experiments demonstrated that the filtered supernatant from SARS-CoV-2 virion exposed brain endothelial cells induced activation of uninfected microglia. This model successfully recreates SARS-CoV-2 virus-associated para-infectious brain inflammation which can be used to study the pathophysiology of the neurological complications and the identification of potential immune targets for treatment.

Keywords: SARS-CoV-2; immunology; microglia; neurology; virology.

Figure 1

Low inoculum intranasal SARS-CoV-2 infection in human ACE2 transgenic C57BL/6 mice does not cause viral replication in the brain parenchyma. (A) Schema of K18 human-ACE2 transgenic C57B/6 mouse study randomised to no infection, 'low inoculum' infection at 1x10³ plaque-forming units (PFU) and 'high inoculum' infection at 1x10⁴ PFU, with endpoint at day 5 post-infection, showing one of two independent experiments, (n=4/group). (B) Real-time polymerase chain reaction (RT-QPCR) identifies SARS-CoV-2 N1 and (C) subgenomic E transcripts in lung homogenate confirming pulmonary infection and viral lytic replication at both inoculum of infection. (D) RT-QPCR of brain homogenate demonstrates that minimal SARS-CoV-2 N1 is present in the perfused brain parenchyma and there are no detectable SARS-CoV-2 subgenomic E transcripts (E) confirming the absence of lytic viral replication in the brain at low-dose infection. ND, not detected.

Figure 2

Spike protein is present in the lungs, but not in the brains of low-inoculum SARS-CoV-2 infected mice at day 5 post 1x10³ PFU infection. (A) Confocal microscopy of low-inoculum infection in this model confirms the presence of both SARS-CoV-2 spike protein (yellow) and monocyte lineage cells (red) in the lungs. (B) Despite the absence of spike protein (yellow) in the brain in the low-inoculum infection model, there are consistent large areas of accumulation of Iba1+ microglia (red) of perfused brain parenchyma.

Figure 3

Immune activation is present in the lungs of SARS-CoV-2 infected mice at day 5 post 1x10³ PFU infection. (A) Confocal microscopy of perfused organs in the low-inoculum SARS-CoV-2 infection model identifies the presence of CD45 positive leucocytes (yellow) and occasionally CD11b positive cells of macrophage/monocyte lineage (red), shown with DAPI (blue) in the lung. (B) No significant differences in CD45 and CD11b expression were found when comparing uninfected and infected brains.

Figure 4

RT-QPCR of perfused lung parenchyma in the low-inoculum infection model (day 5 post 1x10³ PFU infection) confirms upregulation of several inflammatory mediators. (A) RT-QPCR of six immune mediators of interest in perfused lung. (B) Luminex was used to check the same six immune mediators of interest in perfused lung. (C–F) In this low-inoculum infection model several pro-inflammatory mediators were found to be increased in the perfused brains, including (D) CCL4 by RT-QPCR and (F) IL-17A and IFNγ by Luminex. (G, H) In contrast, no statistically significant differences were seen in serum cytokine protein levels. *IL-RA undetectable by ELISA in all, but one uninfected serum sample (=3.5 pg/mL). (These data are from two independent experiments combined, n=7-8/group). Pairwise comparisons by Mann-Whitney U test (*p<0.05, **p<0.01).

Figure 5

Low inoculum intranasal SARS-CoV-2 infection in mice results in increased pro-inflammatory cytokines in the lung and brain and causes increased microglia reactivity in the brain. (A) Confocal microscopy of 30 μm sections of frontal lobe brain parenchyma following low-inoculum infection reveals reactive microglia with increased expression of Iba1 (red) with nuclei stained by DAPI (purple, 3 images/mouse brain at 40Х, n=2/group). (B) % area that was Iba1-positive, (C) Iba1+ cell counts, (D) Examples of microglia morphology Reactive Iba1+ microglia were quantified by (E) Iba1 fluorescence intensity, and (F) reactivation index between 0 and 1. Ac, cell area; Ap, projection area. Pairwise comparisons by Mann-Whitney U test (*p< 0.05, **p<0.01).

Figure 6

Viral effects on endothelial cells in vitro lead to pro-inflammatory cytokine production and subsequent microglia reactivation. (A, B) bEnd.3 cells were treated with heat and acid inactivated SARS-CoV-2 at a ratio of 0.01,0.1, or 1 virus copies per cell for 24 hours. At 24 hours concentrations of cytokines IL-6 and CCL2 were determined by ELISA and groups compared by ANOVA *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (C–E) Primary mouse microglia were treated with 20 nm filtered supernatant taken from bEnd. 3 cells incubated with virus at an MOI of 1, for 2 hours, before being washed and culture medium replaced. At 24 hours cells were fixed and immunostained for Ibal and CD45 and imaged by confocal microscopy. 6-7 separate wells were cultured for each treatment condition and 3-4 for each control condition, with 16 images taken per well at 25x magnification. Dots represent individual cells. Groups compared by Kruskal-Wallis *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.