Overview of the experiment, development of the infection, and the resulting snRNA-seq dataset.

A. Schematic representation of the experiment. B. Loss of body weight of mice during disease progression until full recovery (n = 5 per group). C. Viral M1 gene abundance in lung and olfactory bulb tissue at the 3 time points during disease progression and in controls. D. Location of microdissection punches of the hypothalamus mapped to the mouse brain atlas at distances from bregma of −1.22, –1.58 and −1.94. E. UMAP dimensional reduction of 30.452 cells, color-coded based on their sample group (different time points and control) membership (left) or their neuronal and non-neuronal identity (right). F. Bar graph showing the cell counts of neuronal and non-neuronal cells in the different sampling groups. G. Histograms depicting the distribution of transcript (lighter shading) and genes (solid shading) in all non-neuronal and neuronal cells. H. Normalized expression of neuronal marker genes (Snap25, Syp, Syt1, Elval2) in all cells shown on a UMAP plot.

Classification of non-neuronal cell types.

A. Normalized expression values of different neuronal and non-neuronal cell type markers (pan-neuronal marker - Snap25; Glutamatergic marker - Slc17a6; GABAergic markers - Slc32a1, Gad1, Gad2; Oligodendrocyte marker – Plp1; Astrocyte markers – Slc4a4; Oligodendrocyte precursor marker – Pdgfra; Cped1 – Vascular and leptomeningeal cell markers – Cped1, Slc47a1, Microglia marker – Inpp5d, Tanycytic marker – Col23a1, Pericyte marker – Flt1) in all non-neuronal cells (n = 12.940) mapped on a UMAP. B. Unsupervised clustering of non-neuronal cell types shown in a UMAP embedding and color-coded and annotated by potential cell type annotations. C. Heatmap of normalized expression values showing discriminatory cell type markers of 9 non-neuronal cell populations. D. Violin plots showing distribution of normalized expression values of neurotransmitters and best discriminatory cell type markers.

Classification of GABAergic and glutamatergic neuronal cell types in the hypothalamus.

A. Normalized expression values of different neurotransmitters (Glutamatergic marker - Slc17a6; GABAergic markers - Slc32a1, Gad1, Gad2) in all neuronal cells (left) and a color-coded UMAP projection based on their GABAergic or glutamatergic identity (right) (n = 17.512). B. Un-supervised clustering of GABAergic (upper, n = 6.032) and glutamatergic (lower, n = 11.481) cells in UMAP plots. Cell type clusters are color-coded and annotated with labels. C. Violin plots showing normalized expression values of neurotransmitters and discriminating marker genes of selected GABAergic (left) and glutamatergic (right) cell type clusters. D. Glutamatergic UMAP plots showing normalized expression values of distinct markers for hypothalamic neuron populations (HCRT neurons – upper left, PMCH neurons – lower left, GnRH neurons – upper right, Histaminergic neurons – lower right).

General changes in the transcriptomic landscape of the hypothalamus during peripheral IAV infections.

A. Principial component plot of combined counts per sample. Each sample snRNA-library were down-sampled to a total of 300 cells (100 non-neuronal, 100 GABAergic, 100 glutamatergic cells) before normalized counts were aggregated before principal component analysis. Samples are color-coded according to their group membership and the numbers correspond to the individual animal IDs. B. Violin plot showing the distribution of counts per cell at the different time points. The three points of infections are compared to the control group. C. Heatmap showing log-transformed log-fold changes of known immune-related genes. Black stars indicate significant expression (FDR <= 0.05).

Transcriptional changes in GABAergic and glutamatergic neurons of the hypothalamus during peripheral IAV infections.

A. Violin plots showing the log-transformed fold changes (logFC) per gene calculated for the GABAergic, glutamatergic, and non-neuronal cells at 3, 7, and 23 dpi. Dark-green dots represent significantly differential expressed genes (FDR <= 0.05) with a logFC greater than 1 or lower than - 1. Small grey dots show significantly expressed genes with a logFC between -1 and 1. B. Venn diagrams depicting the overlap of highly differential expressed genes (logFC > 1 or logFC < -1) between the three main cell type clusters at each time point. C. Gene ontology analysis showing the most significant ontology terms for the category of Biological Process (BP, top) and Molecular Function (MF, bottom). The two highest significant ontology’s for each time point and each cell type were chosen if it included more than 4 annotated genes. The size of the dots indicates the number of genes included in the ontology term and the colour show the cell type annotation.

Gene expression changes in different cell type clusters and microglia activation during peripheral IAV infections.

A. Bar plot showing the number of significantly differentially expressed genes in each cell type cluster at 3, 7 and 23 dpi. Dark solid blue and red bars show the number of highly differential expressed gene (FDR <= 0.05, logFC <= -1 or logFC >= 1) per cell type cluster and time point. Lighter shaded bars depict the number of all significant regulated genes (FDR <= 0.05, logFC >= 0 or logFC <= 0). Differentially expressed genes (DEGs) were only included for clusters containing at least 3 cells per sample and per time point in the cluster. A red cross indicates instances where these criteria were not met. B. Radar plot showing the number of significantly and strongly regulated genes (FDR <= 0.05, logFC >= 1 or logFC <= -1: n = 22) involved in immune processes (based on their gene ontology annotations) per cell cluster at the different time points (red: 3 dpi, yellow: 7 dpi, blue: 23 dpi). C. Percentage of the number of cells expressing distinct anti- or pro-inflammatory signature genes in the microglia cluster. Cells with a raw count of 0 for a gene were assumed as non-expressing, all other cells were assumed to express this gene. Shown are percentages of microglia cells expressing a gene compared to all microglia at each time point across all samples. D. Relative changes in expression levels of disease associated genes within the microglia cluster. Data is show as log-transformed fold changes (logFC) of each time point compared with control group. Black stars depict significant differentially expressed genes (FDR <= 0.05).

Identification of distinct oligodendrocyte and astrocyte sub-clusters at 7 dpi.

A. Combined UMAP plot of all cells of the oligodendrocyte (upper right, n = 5.023) and astrocyte (lower right, n = 4.542) cluster in controls and at different time points. B. Relative gene expression changes (in comparison of to the mock-infected group) of stress-related oligodendrocyte and astrocyte markers within the oligodendrocyte (NN_1) and astrocyte (NN_2) subcluster. Data is shown as log-transformed fold changes, an asterisks indicates statistical significance (FDR <= 0.05). C. Normalized expression values of selected marker genes at 7-dpi in UMAP plots of all oligodendrocytes or astrocytes. D. Number of differentially expressed genes (FDR <= 0.05, logFC <= -1 or logFC >= 1) annotated to gene ontologies associated with transport process in the oligodendrocytes at 3 and 7 dpi. E. Expression dynamics of differential expressed oxidoreductase genes in the astrocyte cluster at 3, 7 and 23 dpi. A black star indicated significant differential expression (FDR <= 0.05).

Highly enriched gene ontology categories in neuronal cell populations at different time points of infections.

Dot-plot depicting the number of neuronal cell populations significantly enriched gene ontology categories. Only cell cluster with at least 10 significantly differently expressed genes (logFC <= -1 or logFC >= 1, FDR <= 1) in all three analysed time points were included in the here presented plot. Neuronal cell clusters included are (GABA_2, 4, 6, 8, 10, 12; Glut_1, 3, 4, 6, 9, 11, 13, 15, 17). Only significantly enriched (Pelim <= 0.05) gene ontology terms were chosen and based for each cluster per time point the 10 most enriched GO terms were included.

Overview of molecular processes occurring in different cell types within the hypothalamus during a peripheral H1N1 IAV infection.

Schematic overview of the main molecular mechanisms of oligodendrocytes, astrocytes, microglia and neurons during acute to late immune responses of a peripheral IAV infection with the H1N1 pdm09 Influenza A virus.