Nasal immunity is an ancient and conserved arm of the mucosal immune system in vertebrates. In teleost fish, we previously reported the presence of a nasopharynx-associated lymphoid tissue (NALT) characterized by scattered immune cells located in the trout olfactory lamellae. This diffuse NALT mounts innate and adaptive immune responses to nasal infection or vaccination. In mammals, lymphoid structures such as adenoids and tonsils support affinity maturation of the adaptive immune response in the nasopharyngeal cavity. These structures, known as organized NALT (O-NALT), have not been identified in teleost fish to date, but their evolutionary forerunners exist in sarcopterygian fish. In this study, we report that the rainbow trout nasal cavity is lined with a lymphoepithelium that extends from the most dorsal opening of the nares to the ventral nasal cavity. Within the nasal lymphoepithelium we found lymphocyte aggregates called O-NALT in this study that are composed of ∼ 56% CD4+, 24% IgM+, 16% CD8α+, and 4% IgT+ lymphocytes and that have high constitutive aicda mRNA expression. Intranasal (i.n.) vaccination with live attenuated infectious hematopoietic necrosis virus triggers expansions of B and T cells and aicda expression in response to primary i.n. vaccination. IgM+ B cells undergo proliferation and apoptosis within O-NALT upon prime but not boost i.n. vaccination. Our results suggest that novel mucosal microenvironments such as O-NALT may be involved in the affinity maturation of the adaptive immune response in early vertebrates.

Light-sheet fluorescence microscopy (LSFM) in conjunction with tissue clearing techniques enables morphological investigation of large tissues faster and with excellent optical sectioning. Recently, cleared tissue axially swept light-sheet microscope (ctASLM) demonstrated three-dimensional isotropic resolution in millimeter-scaled tissues. But ASLM based microscopes suffer from low detection signal and slow imaging speed. Here we report a simple and efficient imaging platform that employs precise control of two fixed distant light-sheet foci to carry out ASLM. This allowed us to carry out full field of view (FOV) imaging at 40 frames per second (fps) which is a four-fold improvement compared to the current state-of-the-art. In addition, in a particular frame rate, our method doubles the signal compared to the current ASLM technique. To augment the overall imaging performance, we also developed a deep learning based tissue information classifier that enables faster determination of tissue boundary. We demonstrated the performance of our imaging pipeline on various cleared tissue samples and demonstrated its robustness over a wide range of clearing protocols.

Light-sheet fluorescence microscopy (LSFM) in conjunction with tissue clearing techniques enables morphological investigation of large tissues faster and with excellent optical sectioning. Recently, cleared tissue axially swept light-sheet microscope (ctASLM) demonstrated three-dimensional isotropic resolution in millimeter-scaled tissues. But ASLM based microscopes suffer from low detection signal and slow imaging speed. Here we report a simple and efficient imaging platform that employs precise control of two fixed distant light-sheet foci to carry out ASLM. This allowed us to carry out full field of view (FOV) imaging at 40 frames per second (fps) which is a four-fold improvement compared to the current state-of-the-art. In addition, in a particular frame rate, our method doubles the signal compared to the current ASLM technique. To augment the overall imaging performance, we also developed a deep learning based tissue information classifier that enables faster determination of tissue boundary. We demonstrated the performance of our imaging platform on various cleared tissue samples and demonstrated its robustness over a wide range of clearing protocols.

The olfactory organ of all vertebrates is constantly exposed to pathogens. The nasal mucosa is therefore equipped with a local immune system that protects the host from infection. In teleost fish, we previously described the presence of a diffuse nasopharynx-associated lymphoid tissue (NALT) found in the olfactory lamellae. This diffuse NALT (d-NALT) consists of myeloid and lymphoid cells but lacks an organized structure. Rainbow trout d-NALT mounts quick innate immune responses as well as adaptive immune responses to nasal vaccines. Here we report the discovery of a novel lymphoid structure located in the inner epithelial lining of the nasal cavity, opposite to the tips of the olfactory lamellae, in rainbow trout. This lymphoid structure represents the first description of an organized NALT (O-NALT) in teleost fish. Rainbow trout O-NALT is rich in B and T lymphocytes at the steady state with a proportion of approximately 8:3:1 IgM+ B cells, CD8+ T cells and IgT+ cells, respectively. Using laser capture microdissection we show that compared to d-NALT, O-NALT is highly enriched in expression of CD4, CD8 and IgM but not IgT. Strikingly, O-NALT expresses 8-fold higher levels of activation-induced cytidine deaminase aidc mRNA compared to d-NALT. Following primary and secondary intranasal vaccination with a live attenuated viral vaccine against infectious hematopoietic necrosis (IHN), trout O-NALT was expanded with increased numbers of B and T cell lymphocytes. Our results indicate that teleost fish posses organized lymphoid structures in the nasal cavity that participate in the maturation of the adaptive immune response and that need to be considered as important players in the response to mucosal vaccines. This work was supported by USDA NIFA award # 2020-67015-31457

Olfactory sensory neurons (OSNs) are constantly exposed to pathogens, including viruses. However, serious infection of the brain by the olfactory route rarely occurs. When OSNs detect a virus, they coordinate local antiviral immune responses to stop virus progression into the brain. Despite effective immune control at the olfactory periphery, pathogen-triggered neuronal signals reach the CNS via initial outputs in the olfactory bulb (OB). We hypothesized that neuronal detection of a virus by OSNs initiates neuroimmune responses in the OB that prevent pathogen invasion. Using zebrafish (Danio rerio) as a model, we demonstrate viral-specific neuronal activation of OSNs projecting into the OB, indicating that OSNs are electrically activated by viruses. Further, behavioral changes are seen in both adult and larval zebrafish after viral exposure. By profiling the transcription of single cells in the OB after OSNs are exposed to virus, we found that both microglia and neurons enter a protective state. Cells with microglia and macrophage markers in the OB respond within minutes of nasal viral delivery followed by striking responses in neuronal clusters characterized by decreased expression of neuronal differentiation factors and enrichment of genes in the neuropeptide signaling pathway, especially the known antimicrobial pacap. We confirm that PACAP is antiviral in vitro and that PACAP expression increases in the OB 1 day post-viral treatment. Our work reveals how encounters with viruses in the olfactory periphery shape the vertebrate brain by inducing antimicrobial programs in neurons and by altering host behavior.