Transcriptomic Profiling of Influenza A Virus-Infected Mouse Lung at Recovery Stage Using RNA Sequencing.

Influenza A virus (IAV) is known to cause mild to severe respiratory illness. Under some conditions, the infection can lead to pneumonia (viral or bacterial), acute respiratory distress syndrome, and other complications that can be fatal, especially in vulnerable populations such as the elderly, young children, and individuals with underlying health conditions. Despite previous studies, little is known about the host immune response and neuroimmune interactions in IAV infection. Using RNA sequencing, we performed transcriptomic analysis of murine lung tissue 21 days post infection (dpi) with IAV (H1N1) in order to find the differentially expression genes (DEGs) related to the host immune response and neuroimmune interactions inside the lung during recovery. Among 792 DEGs, 434 genes were up-regulated, whereas 358 genes were down-regulated. The most prominent molecular functions of the up-regulated genes were related to the immune response and tissue repair, whereas a large proportion of the down-regulated genes were associated with neural functions. Although further molecular/functional studies need to be performed for these DEGs, our results facilitate the understanding of the host response (from innate immunity to adaptive immunity) and neuroimmune interactions in infected lungs at the recovery stage of IAV infection. These genes might have potential uses as mechanistic/diagnostic biomarkers and represent possible targets for anti-IAV therapies.

Systemic perturbations in amino acids/amino acid derivatives and tryptophan pathway metabolites associated with murine influenza A virus infection.

BACKGROUND: Influenza A virus (IAV) is the only influenza virus causing flu pandemics (i.e., global epidemics of flu disease). Influenza (the flu) is a highly contagious disease that can be deadly, especially in high-risk groups. Worldwide, these annual epidemics are estimated to result in about 3 to 5 million cases of severe illness and in about 290,000 to 650,000 respiratory deaths. We intend to reveal the effect of IAV infection on the host's metabolism, immune response, and neurotoxicity by using a mouse IAV infection model. METHODS: 51 metabolites of murine blood plasma (33 amino acids/amino acid derivatives (AADs) and 18 metabolites of the tryptophan pathway) were analyzed by using Ultra-High-Performance Liquid Chromatography-Mass Spectrometry with Electrospray Ionization at the acute (7 days post-infection (dpi)), resolution (14 dpi), and recovery (21 dpi) stages of the virus infection in comparison with controls. RESULTS: Among the 33 biogenic amino acids/AADs, the levels of five amino acids/AADs (1-methylhistidine, 5-oxoproline, α-aminobutyric acid, glutamine, and taurine) increased by 7 dpi, whereas the levels of ten amino acids/AADs (4-hydroxyproline, alanine, arginine, asparagine, cysteine, citrulline, glycine, methionine, proline, and tyrosine) decreased. By 14 dpi, the levels of one AAD (3-methylhistidine) increased, whereas the levels of five amino acids/AADs (α-aminobutyric acid, aminoadipic acid, methionine, threonine, valine) decreased. Among the 18 metabolites from the tryptophan pathway, the levels of kynurenine, quinolinic acid, hydroxykynurenine increased by 7 dpi, whereas the levels of indole-3-acetic acid and nicotinamide riboside decreased. CONCLUSIONS: Our data may facilitate understanding the molecular mechanisms of host responses to IAV infection and provide a basis for discovering potential new mechanistic, diagnostic, and prognostic biomarkers and therapeutic targets for IAV infection.

Distribution of nerve fibers and nerve-immune cell association in mouse spleen revealed by immunofluorescent staining.

The central nervous system regulates the immune system through the secretion of hormones from the pituitary gland and other endocrine organs, while the peripheral nervous system (PNS) communicates with the immune system through local nerve-immune cell interactions, including sympathetic/parasympathetic (efferent) and sensory (afferent) innervation to lymphoid tissue/organs. However, the precise mechanisms of this bi-directional crosstalk of the PNS and immune system remain mysterious. To study this kind of bi-directional crosstalk, we performed immunofluorescent staining of neurofilament and confocal microscopy to reveal the distribution of nerve fibers and nerve-immune cell associations inside mouse spleen. Our study demonstrates (i) extensive nerve fibers in all splenic compartments including the splenic nodules, periarteriolar lymphoid sheath, marginal zones, trabeculae, and red pulp; (ii) close associations of nerve fibers with blood vessels (including central arteries, marginal sinuses, penicillar arterioles, and splenic sinuses); (iii) close associations of nerve fibers with various subsets of dendritic cells, macrophages (Mac1+ and F4/80+), and lymphocytes (B cells, T helper cells, and cytotoxic T cells). Our data concerning the extensive splenic innervation and nerve-immune cell communication will enrich our knowledge of the mechanisms through which the PNS affects the cellular- and humoral-mediated immune responses in healthy and infectious/non-infectious states.

Innervation and nerve-immune cell contacts in mouse Peyer's patches.

Neural regulation of the function of the gastrointestinal tract (GIT) relies on a delicate balance of the two divisions of its nervous system, namely, the intrinsic and extrinsic divisions. The intrinsic innervation is provided by the enteric nervous system (ENS), whereas the extrinsic innervation includes sympathetic/parasympathetic nerve fibers and extrinsic sensory nerve fibers. In the present study, we used immunofluorescent staining of neurofilament-heavy (NF-H) to reveal the distribution of nerve fibers and their associations with immune cells inside mouse Peyer's patches (PP), an essential part of gut-associated lymphoid tissue (GALT). Our results demonstrate (1) the presence of an extensive meshwork of NF-H-immunoreactive presumptive nerve fibers in all PP compartments including the lymphoid nodules, interfollicular region, follicle-associated epithelium, and subepithelial dome; (2) close associations/contacts of nerve fibers with blood vessels including high endothelial venules, indicating neural control of blood flow and immune cell dynamics inside the PP; (3) close contacts between nerve fibers/endings and B/T cells and various subsets of dendritic cells ( e.g., B220⁻, B220⁺, CD4⁻, CD4⁺, CD8⁻, and CD8⁺). Our novel findings concerning PP innervation and nerve-immune-cell contacts in situ should facilitate our understanding of bi-directional communications between the PNS and GALT. Since the innervation of the gut, including PP, might be important in the pathogenesis and progression of some neurological, infectious, and autoimmune diseases, e.g., prion diseases and inflammatory bowel disease, better knowledge of PNS-immune system interactions in the GALT (including PP) should benefit the development of potential treatments for these diseases via neuroimmune manipulations.

Immunofluorescent characterization of innervation and nerve-immune cell neighborhood in mouse thymus.

The central nervous system impacts the immune system mainly by regulating the systemic concentration of humoral substances, whereas the peripheral nervous system (PNS) communicates with the immune system specifically according to local "hardwiring" of sympathetic/parasympathetic (efferent) and sensory (afferent) nerves to the primary and secondary lymphoid tissue/organs (e.g., thymus spleen and lymph nodes). In the present study, we use immunofluorescent staining of neurofilament-heavy to reveal the distribution of nerve fibers and the nerve-immune cell neighborhood inside the mouse thymus. Our results demonstrate (a) the presence of an extensive meshwork of nerve fibers in all thymic compartments, including the capsule, subcapsular region, cortex, cortico-medullary junction and medulla; (b) close associations of nerve fibers with blood vessels (including the postcapillary venules), indicating the neural control of blood circulation and immune cell dynamics inside the thymus; (c) the close proximity of nerve fibers to various subsets of thymocytes (e.g., CD4+, CD8+ and CD4+CD8+), dendritic cells (e.g., B220+, CD4+, CD8+ and F4/80+), macrophages (Mac1+ and F4/80+) and B cells. Our novel findings concerning thymic innervation and the nerve-immune cell neighborhood in situ should facilitate the understanding of bi-directional communications between the PNS and primary lymphoid organs. Since the innervation of lymphoid organs, including the thymus, may play essential roles in the pathogenesis and progression of some neuroimmune, infectious and autoimmune diseases, better knowledge of PNS-immune system crosstalk should benefit the development of potential therapies for these diseases.