The olfactory organ is a unique site for resident neutrophils in the brain

For decades we have known that the brain “drains” through the subarachnoid space following a route that crosses the cribriform plate to the nasal mucosa and cervical lymph nodes. Yet little is known about the potential role of the olfactory epithelia and associated lymphatic vasculature in the immune response. To better understand the immune response in the olfactory organs we used cell-specific fluorescent reporter lines in dissected, intact adult brains to visualize blood-lymphatic vasculature and neutrophils in the olfactory sensory system. Here we show that the extensive blood vasculature of the olfactory organs is associated with a lymphatic cell type resembling high endothelial venules (HEVs) of the lymph nodes in mammals and a second resembling Mural Lymphatic Endothelial Cells (muLECs) that extended from the brain to the peripheral olfactory epithelia. Surprisingly, the olfactory organs contained the only neutrophil populations observed in the brain. Damage to the olfactory epithelia resulted in a rapid increase of neutrophils within the olfactory organs as well as the appearance of neutrophils in the brain suggesting that neutrophils enter the brain in response to damage. Analysis of cell division during and after damage showed an increase in BrdU labeling in the olfactory epithelia and a subset of the neutrophils. Our results reveal a unique population of neutrophils in the olfactory organs that are associated with an extensive lymphatic vasculature suggesting a dual olfactory-immune function for this unique sensory system. Highlights The olfactory organ is the only region of the brain that contains resident neutrophils in the adult animal. Damage to olfactory sensory neurons triggers a rapid mobilization of neutrophils within the olfactory organ and in the central nervous system. Two types of lymphatic vasculature resembling Mural Lymphatic Endothelial Cells (muLEC) and High Endothelial Venules (HEV) are present in the olfactory sensory system. Lymphatic vasculature resembling Mural Lymphatic Endothelial Cells (muLEC) wrap the olfactory bulbs and extend across the cribriform plate to olfactory epithelia.

2 Abstract 25 For decades we have known that the brain "drains" through the subarachnoid space 26 following a route that crosses the cribriform plate to the nasal mucosa and cervical 27 lymph nodes. Yet little is known about the potential role of the olfactory epithelia and 28 associated lymphatic vasculature in the immune response. To better understand the 29 immune response in the olfactory organs we used cell-specific fluorescent reporter lines 30 in dissected, intact adult brains to visualize blood-lymphatic vasculature and neutrophils 31 in the olfactory sensory system. Here we show that the extensive blood vasculature of 32 the olfactory organs is associated with a lymphatic cell type resembling high endothelial 33 venules (HEVs) of the lymph nodes in mammals and a second resembling Mural 34 Lymphatic Endothelial Cells (muLECs) that extended from the brain to the peripheral 35 olfactory epithelia. Surprisingly, the olfactory organs contained the only neutrophil 36 populations observed in the brain. Damage to the olfactory epithelia resulted in a rapid 37 increase of neutrophils within the olfactory organs as well as the appearance of 38 neutrophils in the brain suggesting that neutrophils enter the brain in response to 39 damage. Analysis of cell division during and after damage showed an increase in BrdU 40 labeling in the olfactory epithelia and a subset of the neutrophils. Our results reveal a 41 unique population of neutrophils in the olfactory organs that are associated with an 42 extensive lymphatic vasculature suggesting a dual olfactory-immune function for this 43 unique sensory system. 44 Introduction 66

The adult olfactory organ blood-lymphatic system 67
In vertebrates the olfactory sensory neurons (OSNs), a group of continually renewing 68 neurons located in the olfactory epithelium (OE), extend their axons across the 69 cribriform plate where they make their first synapses in the olfactory bulb (OB) (Sakano,70 2010; Whitlock, 2015). This connection between the OE and the OB is part of a complex 71 neural and immune interface that includes flow of cerebral spinal fluid (CSF) and 72 interstitial fluid (ISF) from the subarachnoid space toward the nasal mucosa. Evidence 73 supporting a connection between the subarachnoid space of the brain and cervical 74 lymph nodes via the nasal mucosa was first proposed over a century ago (for review 75 see: (Faber, 1937;Jackson et al., 1979). Subsequent studies in mammals using labeled 76 tracers confirmed a drainage route from the cranial subarachnoid space through the 77 olfactory pathway leaving the nasal mucosa via terminal lymphatics or into blood 78 capillaries (Cserr et al., 1992). Thus the potential for turnover of brain extracellular 79 fluids, via drainage to blood and deep cervical lymph, presented a system whereby 80 immunogenic material and immune cells from the central nervous system (CNS) could 81 pass to immune organs outside the brain via the olfactory epithelia. 82 The lymphatic system of vertebrates, composed of lymphatic vessels, lymphoid 83 organs/tissues and the circulating lymph fluid, is highly conserved at the functional level 84 (Boehm et al., 2012) and is suggested to have originated in teleost fishes where the 85 heart provided the energy to propel lymph through vessels associated with the primary 86 vasculature (Hedrick et al., 2013). Lymphocytes are generated in primary lymphoid 87 organs (thymus and bone marrow: mammals / thymus and kidney; teleost fishes) and 88

Neutrophil populations in the adult olfactory organ 271
Neutrophils, the most abundant leukocyte sub-types in adult zebrafish, are essential 272 players in the innate immune system and more recently have been shown to migrate 273 not only on BV but also LV. We used the Tg(OMP:RFP);Tg(mpx:GFP) animals to 274 visualize olfactory sensory neurons (red) and neutrophils (green), in fixed whole mount 275 brains. Surprisingly, we observed neutrophils only in the OO of adult brains (Fig. 4A, B  276 green). Neutrophils were localized in the fingerlike lamellae of the OE predominantly 277 associated with the EN wrapping around the OE (Fig 2A, B). The OMP:RFP + OSNs 278

Neutrophil response to damage in the adult olfactory sensory system 332
In order to investigate the neutrophil response to damage of the OE, we exposed After four hours of copper exposure, BrdU labeling showed significant increases in the 377 mr (Fig. 7B, white, arrow), and in the ns epithelia extending to the EN. In contrast, one 378 day post treatment (dpt) significant increases in BrdU labeling were observed in the ss 379 epithelia (Fig. 7C, F) consistent with the renewal of OSN in the OE after damage (Iqbal 380 and Byrd-Jacobs, 2010). Additionally the neutrophils now lined LOE (Fig. 7C, green) 381 possibly in association with the BV (Fig. 7D, green). The number of neutrophils showed 382 significant increases at 4 hours post-treatment (hpt) and remained high in the ss 383 epithelia one dpt (Fig, 7E; 444.67 ± 31.39 and 373.33 ± 32.32 neutrophils in 4 hpt, red, 384 and 1 dpt, green). Significant increases in BrdU labeling at both 4 hpt and 1 dpt were 385 observed only in the ss epithelia ( Fig. 7F; 480 ± 241.76 and 786 ± 211.6, respectively). 386 Analysis of cells expressing both mpx:GFP and BrdU showed a significant increase 387 compared to control animals (Fig. 7G, control: 9 ± 1, 4 hpt: 26 ± 6, 1 dpt: 22 ± 5.29). 388 The frequency of rounded neutrophils (see Fig. 4C, green, nt1; ci 0.7 or greater) and 389 amoeboid-like (see Fig. 4C, green, nt2; ci 0.4-0.6), potentially representing "resting" and 390 activated neutrophils, respectively, increased in the OE post-damage (Fig. 7H). The 391 columnar shaped cells (ci 0.1-0.3) increased in frequency at one dpt in the sensory 392 region (Fig. 7H, green, 0.2 -0.3 green bars) but remained as the least common 393 morphology. We found that damage to the OE resulted in an increased number of 394 rounded neutrophils and a small but significant number were double labeled for BrdU, 395 thus the majority of the increase in neutrophil number was likely due to migration as 396 opposed to proliferation. Future work using photoconvertible lineage tracers will allow us 397 to determine the exact contribution of local vs. immigrant neutrophils in the response to 398 damage in developing and adult brain.

DISCUSSION: 421
In this study we have shown that the olfactory sensory system has a unique "immune 422 architecture" where neutrophils permanently populate the olfactory sensory organs in 423 association with a complex network of BV-LV. These neutrophils mount a rapid 424 response to copper-induced damage to the OE populating not only the tissues of the 425 OE and associated EN, but also appearing in tracts extending posteriorly along the 426 ventral CNS. These data demonstrate a role for resident neutrophils in the olfactory 427 sensory system and suggest that the nasal lymphatic pathway may be a potential site of 428 entry for immune cells into the CNS. 429

Lymphatic Vasculature 430
The olfactory/nasal lymphatic route was first described using India ink to label CSF 431 drainage pathways from the brain where particles moved from cranial subarachnoid 432 space to lymphatic channels of the olfactory mucosa (Jackson et al., 1979). 433 Subsequently it was shown that while the subarachnoid space of the optic nerves and 434 cochlea region were labeled, the only direct connection between cranial CSF and 435 lymphatics was the nasal route (Kida et al., 1993;Kida et al., 1995;Koh et al., 2005) 436 passing through cribriform plate along perineural spaces near the olfactory nerves to the 437 nasal mucosa and cervical lymph nodes (Sun et al., 2018). With the re-discovery of the 438 brain lymphatics (Louveau et al., 2015) the relative importance of the drainage of CSF 439 via the meningeal LV versus olfactory/nasal LV is currently a subject of debate (see for 440 discussion (Dolgin, 2020). Attofluor Chamber for subsequent imaging (see below). 603

BrdU Labeling 604
For each experiment nine adult fish were first housed overnight in 1.5 liter tanks 605 containing 10 mM BrdU in system water. The next morning three fish were transferred 606 to a new 1.5-liter tank with system water (control) and six fish were transferred to a new 607 1.5 liter tank with system water containing 10 µM CuSO 4 , and allowed to swim freely (4 608 hours). All control fish (3) and half of copper-exposed fish (3) were then anesthetized, 609 sacrificed and heads fixed overnight in 4% PFA/1X PBS. The other half of copper-610 exposed fish (3) were transferred to a clean 1.5-liter tank, filled with system water, and 611 allowed to recover. The next day, these fish were anesthetized, sacrificed and fixed as 612 described above. After fixation, heads were incubated in EDTA (0.2 M, pH 7.5) for three 613 days at 4 ºC and brains dissected in sterile 1X PBS and pre treated in 2 M HCl for 30 614 minutes at 37 ºC. Immunocytochemistry was performed as described in 615 Immunocytochemistry & Cell Labeling. For imaging, whole adult brains were mounted 616 on 2% low melting temperature Agarose, and OE were mounted between coverslips, as 617 described above. The removal of brains from the skull with the OO still attached is a 618 difficult dissection because the OSN axons pass through the cribriform plate to arrive in 619 the OB. Therefore it was not always possible to have a preparation with both OE still 620 connected to the brain. 621

Cryosectioning 622
Fish were euthanized and heads were fixed overnight in 4% PFA at 4 ºC and decalcified 623 in EDTA (0.2 M, pH 7.6) for 3 days, and later embedded in 1.5% agarose/ 5% sucrose 624 blocks and submerged in 30% sucrose for 3 days at 4 ºC. Blocks were frozen (-20 ºC) 625 with O.C.T. Compound (Tissue Tek®) and sectioned (25 µm) using a cryostat. 626 For flat mounting of the olfactory epithelia, olfactory rosettes were dissected after 627 immunohistochemistry or staining, and mounted with the caudal side down on Poly-L-628 Lysine coated slides between triple 22x22 coverslip bridges and covered in 629 VECTASHIELD® Antifade Mounting Media (Vector laboratories). 630