Inflammatory Response and Defects on Myelin Integrity in the Olfactory System of K18hACE2 Mice Infected with SARS-CoV-2

Molecular characterization of SARS-CoV-2–infected cells. A, Diagram illustrating the main cell types of the OE. B–b’’, IHC in the OE detecting the SC marker CK8 (b, green), SARS-CoV-2-NC (b’, red) and OSN marker OMP (b’’, magenta). SARS-CoV-2-NC⁺ cells colocalize with CK8 (B) but not with OMP (b’’, arrowheads). C–c’’, IHC in the OE staining for the macrophage marker IBA1 (c, green), SARS-CoV-2-NC (c’, red), and OMP (c’’, magenta), showing infected macrophages lying in the lamina propria (arrowheads). D, Diagram illustrating the layers and main cell types of the OB. E–e’, IHC in the OB staining for Reln (e, green) and SARS-CoV-2-NC (e’, red). All SARS-CoV-2-NC⁺ cells in the MCL express Reln (E, e), confirming their phenotype as infected mitral cells. F, SARS-CoV-2-NC⁺ labeling in blood vessels from the EPL. G–g’’, IHC staining for IBA1 (g, green), SARS-CoV-2-NC (g’, red), and Ctip2 (g’’, magenta), showing infected microglial cells in the ONL (G–g’, arrowheads). H, Diagram illustrating the layers and main cell types of the PC. I–i’’, IHC in PC staining for Tbr1 (i, green), SARS-CoV-2-NC (i’, red), and Ctip2 (i’’, magenta). All neurons from Layers 2 and 3 are found to coexpress SARS-CoV-2-NC⁺ with Tbr1 (I, i, dotted lines) and Ctip2 (I, i’’, dotted lines) confirming they are projection neurons. J–j’’, IHC staining to detect Ctip2 (g, green) and SARS-CoV-2-NC (g’, red) showing that Hc from the superficial Layer 1 are infected with SARS-CoV-2 (arrowheads). K, Diagram illustrating the layers and of the TuS. L–l’’, IHC in TuS staining for Tbr1 (l, green), SARS-CoV-2-NC (l’, red), and Ctip2 (l’’, magenta) showing the absence of Tbr1 expression in the entire TuS and coexpression of SARS-CoV-2-NC with Ctip2 highlighting the virus targeting projection neurons in the TuS. Nuclei counterstained with DAPI (blue). CK8, cytokeratine-8; Ctip2, COUP-TF–interacting protein 2; gl, glomeruli; Hc, horizontal cell; ic, island of Calleja; IHC, immunohistochemistry; LOT, lateral olfactory tract; MØ, microglia; Mc, mitral cells; OB, olfactory bulb; OE, olfactory epithelium; OMP, olfactory marker protein; OSN, olfactory sensory neurons; PC, piriform cortex; RMS, rostral migratory stream; SC, supporting/sustentacular cells; Tbr1, T-Box brain transcription Factor 1; TuS, tubular striatum. Scale bar, 50 µm.

Morphology of activated microglia. Immunostaining for the microglial marker IBA1 (green) with nuclei counterstained with DAPI (blue). A, a, Morphology of microglia in control animals. B, b, Morphology of microglia in infected animals. C, Microglia changes in morphology in response to SARS-CoV-2 infection to show a statistically significant increase in the fractal dimension in infected mice. Statistics: unpaired t test with ** = p < 0.01 (Table 2). Scale bar, 25 µm.

Microgliosis in the OE. Staining of the OE with the macrophage marker IBA1 (green), SARS-CoV-2 nucleocapsid (red), and active phagocytic macrophages Dectin-1 (Clec7A; magenta). Nuclei counterstained with DAPI (blue). A with insets, Representative images of the OE from control mice. B with insets, Representative images of the OE from SARS-CoV-2–infected mice. C, Quantification of total macrophage cells (IBA1⁺) and phagocytic macrophages (IBA1⁺/Clec7A⁺) in the medial wall (septum) and turbinates. The number of combined IBA1+ cells between the septum and turbinates shows an increase that was statistically significant only in the septum (C), suggesting a higher vulnerability of this region to the infection. Statistics: multiple unpaired t test with * = p < 0.05 (Table 2). Scale bars: A and B, 1 mm; septum and turbinate insets, 100 µm.

Microgliosis in the OB. Staining of microglia with IBA1 (green), SARS-CoV-2 nucleocapsid protein (red), and active phagocytic microglia with Dectin-1 (Clec7A; magenta). Nuclei counterstained with DAPI (blue). A, Representative images of the OE from control mice. B, Representative images of the OE from SARS-CoV-2–infected mice. Microglial cell bodies show a clear hypertrophy related to an activated response. Infected microglia are observed in the ONL (dotted lines). C, Quantification of the microglia (IBA1⁺) and phagocytic microglia (IBA1⁺/Clec7A⁺) in the OB. Total numbers of microglia (IBA⁺) are statistically significant higher numbers in infected animals, suggesting a strong inflammation. This effect is replicated on each individual layer excepting in the MCL-IPL, which is the layer less affected by the microgliosis. Numbers of phagocytic microglia (IBA1⁺/Clec7A⁺) are significantly higher only in the GL, indicating that this layer is the most affected by inflammation. EPL, external plexiform layer; GCL, granule cell layer; GL, glomerular layer; IPL, internal plexiform layer; MCL, mitral cell layer; OB, olfactory bulb; ONL, olfactory nerve layer; RMS, rostral migratory stream (bulbar part). Statistics: multiple unpaired t test with * = p < 0.05; ** = p < 0.01 (Table 2). Scale bars: A and B, 1 mm; high magnification insets, 200 µm.

Microgliosis in piriform cortex and TuS. Staining of microglia with IBA1 (green), SARS-CoV-2 nucleocapsid (red), and active phagocytic macrophages with Dectin-1 (Clec7A; magenta). Nuclei counterstained with DAPI (blue). A, Representative images of the PC and TuS from control mice. B, Representative images of the PC and TuS from SARS-CoV-2–infected mice, where clear hypertrophy is observed in all cell bodies extending not only into the PC and TuS but also across the entire brain. C, Quantification of microglia (IBA1⁺) and phagocytic microglia (IBA1⁺/Clec7A⁺) in PC. The numbers of microglia are statistically significantly higher in all layers of PC from infected animals proving the presence of strong inflammation. D, Quantification of microglia (IBA1⁺) and phagocytic microglia (IBA1⁺/Clec7A⁺) in TuS. The numbers of microglia (IBA1⁺) were significantly higher in Layers 2 and 3 highlighting that inflammation affected the projection neuron layers. Numbers of phagocytic microglia (IBA1⁺/Clec7A⁺) are similar in both controls and infected mice. LOT, lateral olfactory tract; PC, piriform cortex; TuS, tubular striatum. Statistics: multiple unpaired t test with * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (Table 2). Scale bars: A and B, 1 mm; high magnification insets, 200 µm.