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.

The spleen is separated into two main compartments, namely the blood-containing red pulp (primarily for innate immunity) and the lymphoid cells-containing white pulp (primarily for adaptive immunity) by an interface called the marginal zone 10 . In the red pulp, macrophages efficiently remove pathogens, dead cells/cellular debris, and aging erythrocytes. In the white pulp, a highly-organized lymphoid structure is responsible for the local and systemic regulation of immunity 10 . Accumulating evidence suggests that an intricate communication exists between the PNS and the spleen, and that this crosstalk might play an essential role in the regulation of the immune response 9,[11][12][13][14][15][16][17] . Therefore, an understanding of the splenic innervation by both autonomic (efferent) and sensory (afferent) fibers is crucial for a better appreciation of the response of the spleen to immune challenge and tissue injury.
Sympathetic norepinephrine fibers enter the spleen via the splenic nerve, and much of the network is closely associated with the splenic artery and its branches into the spleen [18][19][20] . These sympathetic nerve fibers might have close associations with lymphocytes, macrophages, and DCs [14][15][16] . In addition, sensory innervation of the spleen has also been reported 11,13 . However, the suggestion of parasympathetic innervation and/or control of the spleen has continued to be controversial, ever since acetylcholine was first isolated from the spleen 11,13,20,21 .
The information on the innervation and nerve-immune interactions within the spleen remains very limited, given the studies described above. We have demonstrated in our previous study the existence of non-myelinating Schwann cells and Remak fibers (including small nociceptive (C-type) axons, postganglionic sympathetic axons, and some preganglionic sympathetic/parasympathetic fibers) inside the mouse spleen 22 . In the present study, a rabbit anti-neurofilament heavy (NF-H) antibody was utilized as a reliable marker to characterize the nerves/ nerve fibers inside the mouse spleen. Neurofilaments (NFs) are intermediate filaments particularly abundant in axons, where they are essential for the radial growth of axons during development, the maintenance of axon caliber, and the transmission of electrical impulses along axons 23 . NFs are composed of four subunits (neurofilament light (NF-L), neurofilament middle (NF-M), NF-H, and α-internexin (or peripherin)), each having different domain structures and functions. In addition, NFs might play a role in intracellular transport to axons and dendrites 23 . In the adult nervous system, NFs in small unmyelinated axons contain more peripherin and less NF-H, whereas NFs in large myelinated axons contain more NF-H and less peripherin.
NF-H has been utilized as a cellular marker for the characterization of nerve/nerve fibers in the lymphoid organs (e.g., thymus, lymph node, and spleen) across several species (e.g., human and rat) 19,22,24,25 . By using immunofluorescent staining and confocal microscopy/three-dimensional (3D) reconstruction, we have now investigated the distribution of nerve fibers and PNS-immune cell relationship in situ in the mouse spleen to improve our knowledge of the microanatomical basis of bi-directional communication of the PNS and secondary lymphoid tissue/organs (e.g., spleen, lymph nodes, and gut-associated lymphoid tissue).

Distribution of nerve fibers in the mouse spleen.
A rabbit anti-NF-H antibody was used as a reliable marker to label the nerve fibers inside the spleen. This antibody only recognized a protein of 220 KD, which is the mass of NF-H 26 . To validate this antibody, we also performed immunofluorescent staining on a few types of mouse tissues (e.g., brain, skin, liver, and small intestine) and observed brightly stained cells/fibers with clear morphology that is expected for the nerves/nerve fibers in these tissues ( Supplementary Fig. 1). For negative control experiments, no staining was observed when only three secondary antibodies were applied ( Supplementary  Fig. 2).
We found an extensive meshwork of nerve fibers in splenic compartments including the capsule, splenic nodules (B cell follicles), marginal zones, periarteriolar lymphoid sheath (PALS), and red pulps (Figs. 1 and 2). The intensity of nerve fibers varied in the various parts of the spleen. For example, if sectioned transversely, the middle portion of spleen had more innervation than other portions of the spleen (e.g., tips of the spleen, data not shown).
The splenic nodules ( Fig. 2A) had fewer nerve fibers compared with the PALS (Fig. 2B) and red pulp (Fig. 2C). The marginal zone ( Fig. 2A) contained extensive nerve fibers that were closely associated with marginal B cells and DCs. In the PALS (Fig. 2B), an extensive network of nerve fibers ran along the central artery, formed plexi around it, and extended into the PALS and splenic nodules. We also observed that many nerve fibers had close associations with B220 + B cells and with B220 -CD11c + /B220 + CD11c + DCs.
In the spleen two types of nerve-immune cell associations have been observed. The nerve fiber-immune cell association was regarded as the first, and nerve ending (appearing as small red dots)-immune cell association was the second (Fig. 2B). These two types of associations were also observed in the splenic red pulp (Fig. 2C,D).

Relationship of nerve fibers and immune cells in the mouse spleen.
We then investigated the distribution of nerve fibers, T helper cells (by anti-CD4 staining), and DCs inside the mouse spleen; the results are shown in Fig. 3 and Supplementary Fig. 3 (for an overview). Only some T helper cells were seen in the splenic nodules ( Supplementary Fig. 3). In the PALS, an extensive network of nerve fibers (plexus) was observed around the central arteries, and these nerve fibers exhibited close associations with many CD4 + T helper cells and CD4 -CD11c + /CD4 + CD11c + DCs (Fig. 3A,B). In addition, in the spleen red pulp, the nerve fibers exhibited close associations with CD4 + T helper cells and CD4 -CD11c + /CD4 + CD11c + DCs (Fig. 3C-E).
We also checked the distribution of nerve fibers, cytotoxic T cells (by anti-CD8a immunofluorescence), and DCs in the spleen. Only very few cytotoxic T cells were observed in splenic nodules (Fig. 4A). Extensive nerve fibers occurred in the splenic marginal zones, and some of these fibers exhibited close associations with marginal zone cytotoxic T cells and CD8a -CD11c + /CD8a + CD11c + DCs. In addition, the extensive nerve fibers around the central artery also exhibited close appositions to CD8a + cytotoxic T cells and CD8a -CD11c + /CD8a + CD11c + DCs (Fig. 4B,C) in the PALS.

Associations of nerve fibers with blood vessels in the mouse spleen. Splenic blood circulation is
open since afferent arterial blood ends in sinusoids surrounding the white pulp 10 . Blood flows into venous sinuses through sinusoidal spaces and red pulp, collecting into efferent splenic veins. To comprehend the connection between the nerve fibers and blood vessels/DCs, triple-immunolabelling with anti-CD31 (a blood vessel endothelial cell marker), anti-CD11c, and anti-NF-H antibodies was performed; the results are shown in Fig. 6. In splenic nodules and marginal zones, some blood vessels including the capillaries (containing pericytes) were closely associated with nerve fibers (Fig. 6A). Similar close associations of nerve fibers and blood vessels (including the splenic sinus) were also observed in the red pulp (Fig. 6B) indicating a neuronal control of blood flow inside the splenic red pulp. In addition, a close association of nerve fibers with blood vessels (including central arteries) was also found in the PALS (Fig. 6C).

Discussion
Branches from the celiac plexus, left celiac ganglion, and the right vagus nerve form the nerve plexi inside the spleen. At the hilum, nerve fibers from the splenic nerve enter the spleen around the splenic artery, travel with the vasculature in the plexi, continue into the spleen along the trabeculae with the trabecular plexi, and extend into the white pulp including the splenic nodules and PALS 18,22 .
In the present study, in the mouse spleen, we have observed an extensive meshwork of nerve fibers, which has not been reported before. Some previous reports have shown that sympathetic (norepinephrine) innervation is particularly rich in T cell zones and in areas of DCs/macrophages, whereas the follicular/nodular zones are poorly www.nature.com/scientificreports www.nature.com/scientificreports/ innervated 13,18,19,24 . In addition, in these reports, only scattered/sparse nerve fibers primarily associated with plexi along the trabeculae have been observed in the red pulp 13 . However, our study has demonstrated: (a) the presence of nerve fibers in each compartment of the spleen, including the splenic nodules, marginal zones, and red pulp, although the number of nerve fibers in the splenic nodules is much fewer compared with that of other compartments; (b) the intensity of nerve fibers in PALS and marginal zones is much higher than that of nerve fibers in some previous reports 11,[18][19][20] ; (c) the intensity of nerve fibers is similar to that of Remak fibers (indirectly demonstrated by non-myelinated Schwann cells) inside the spleen as shown in one of our previous studies 22 .
Since the NF-H is a non-specific neuronal/axonal marker, we should mention that we might see more nerve fibers (e.g., parasympathetic and visceral sensory fibers), except for the tyrosine hydroxylase positive sympathetic nerve fibers, inside the spleen. Additional cellular markers such as tyrosine hydroxylase (for sympathetic nerve fibers), choline acetyltransferase (for cholinergic fibers), calcitonin gene-related peptide (CGRP; for sensory www.nature.com/scientificreports www.nature.com/scientificreports/ fibers from dorsal root ganglion and motor fibers from anterior horn of spinal cord), and transient receptor potential cation channel subfamily V member 1 (TRPV1) and acetylcholinesterase can be used to identify specific nerve types inside the spleen.
Smooth muscles and pericytes are target cells of peripheral nerves and under the control of PNS. We have also shown that afferent nerve fibers are distributed through/along the trabeculae and closely associated with blood vessels with/without smooth muscles (e.g., the central arteries and their branches, penicillin arterioles, marginal sinuses, and splenic sinuses). The nerve fibers travel along blood vessels, form plexi around the blood vessels, and extend to the parenchyma of spleen. Therefore, the neuronal regulation of blood flow and vascular permeability of blood vessels might affect the subsequent dynamics of immune cells and the clearance of the blood-borne antigens/foreign materials. Some studies have revealed that sympathetic norepinephrine nerve fibers are associated with B/T lymphocytes within splenic white pulp 18,20 . However, these kinds of nerve-B/T cell associations have not been observed in several other studies 24 . We have seen intensive nerve fiber-B cell associations in the splenic nodules (including the germinal centers), PALS, marginal zones, and red pulp. These kinds of close associations show that B cell differentiation/maturation (inside the splenic nodules) and antigen presentation (in the marginal zones) may be regulated by the splenic innervation. We also observed close associations of nerve fibers with CD4 + T helper cells and CD8 + cytotoxic T cells, indicating a potential regulation of T cell response by the splenic innervation.
Mononuclear phagocytes (e.g., monocytes, macrophages, and DCs) protect the host by identifying pathogens/ foreign bodies, removing dead cells/ cell debris, and regulating tissue homeostasis and innate/adaptive immunity 10,28 . Close associations/interactions of nerve fibers and DCs have been observed inside the spleen in some reports 24 . In our previous study, we have also detected close association of a few subsets of DCs with Remak fibers in the splenic white pulp and red pulp 22 . In the present study, we have revealed intensive nerve-DC associations. A few interesting points should be mentioned concerning this type of nerve-DC associations. First, around the central arteries in the PALS, we have found clusters of DCs, many of which have close associations with nerve fibers. Second, nerve fibers are closely apposed to many DCs in PALS and marginal zones. Third, some nerve fibers are also closely apposed to DCs in the red pulp. Fourth, the phenotypes of DCs 28 associated with nerve fibers include B220 -CD11c + , B220 + CD11c + (plasmacytoid DCs),CD4 -CD11c + (lymphoid DCs), CD4 + CD11c + (lymphoid DCs), Mac1 low CD11c + , Mac1 + CD11c + , F4/80 -CD11c + , and F4/80 + CD11c + DCs in the spleen. Although further functional in vitro and in vivo studies need to be carried out, our findings provide a reliable microanatomical basis for the neural regulation/control of the functions of DCs including antigen presentation and cytokine production. www.nature.com/scientificreports www.nature.com/scientificreports/ Macrophages in the spleen have two main protective activities during infections of blood-borne pathogens or foreign materials. The first type of well-characterized macrophage is responsible for phagocytosis and the elimination of pathogens from the circulation 10 . The second type of macrophage is defined by the expression of CD markers (e.g., F4/80, Mac1 (CD11b), CD68), and these macrophages play a crucial role in the activation of the immune system 10 . Recent studies have demonstrated local associations of macrophages and nerve fibers in the spleen 18,22 . In our study, we have observed that some nerve fibers have close appositions with Mac1 + CD11c low and F4/80 + CD11cmacrophages in the red pulp, suggesting that splenic nerves might regulate the macrophage functions (e.g., antigen presentation and cytokine production).
Nerves and nerve fibers are comparatively static structures associated with blood vessels, while most immune cells are wandering cells inside tissues or organs. Therefore, this type of nerve-immune cell contacts or association, which may be defined as neuro-immune cell unit or neuro-immune synapse, should be dynamic 29,30 . This neuro-immune cell unit or synapse has some features of neurological and immune synapses 29,30 . Two types of cells can communicate either through direct ligand-receptor binding or through neurotransmitters or/and inflammatory mediators. Further functional studies are helpful in revealing the detailed mechanisms of this type of cell-to-cell communication.
Firstly, high-resolution confocal imaging/3D reconstruction, electron microscopy, and immune electron microscopy can be applied to confirm the presence of the "synapse" or synapse-like association if some critical requirements for a classical synapse are met 29,30 . In our previous study, we have analysed neuro-immune cell membrane-membrane contact by using high-resolution confocal imaging and quantitative colocalization analysis 31 . Some other studies utilizing electron microscopy have observed neuroimmune synapse with a synaptic cleft about 6nm 32 . Secondly, the effects of neurotransmitters or neuropeptides on immune cells should be studied. In our previous study, we have seen the expression of muscarinic acetylcholine receptor (subtype M2) on lymphocytes and DCs in mouse Peyers' patches 33 . Some other studies also showed that neurotransmitters such as acetylcholine and dopamine regulated the functions of B/T cells and DC/macrophages 34,35 . Some neurotransmitters are immunosuppressive, while others may stimulate and activate immunity 35,36 . Thirdly, inflammatory mediators such as pro-inflammatory cytokines, prostaglandins, serotonin, and histamine from immune cells (even from neural cells or glial cells) can have variable effects on nerves of PNS 37,38 .
In summary, our novel findings concerning extensive splenic innervation and its relationship with immune cells should shed some light on the microanatomical basis of the bi-directional crosstalk of the PNS and secondary lymphoid tissue/organs in health and diseases. Certainly, further in vivo and in vitro molecular and functional studies need to be carried out to undercover the mystery of this bi-directional communication 39 . Furthermore, chemical, pharmacological, electrical, or other manipulations of these neuroimmune interactions should benefit the development of potential practical therapeutic approaches for certain neurological, neuroimmunological, infectious, and immunological diseases [39][40][41][42][43] .

Methods
Animals and sectioning. Eight male C57BL/6 mice (age from 8-10 weeks) were bought from the Animal Resources Centre (Murdoch, Australia). All experiments were carried out in accordance with Australian national rules and approved by the Murdoch University Animal Ethics Committee (permit number: R2700/14). 20-µm-thick cryosections of mouse spleen, brain, skin, liver, and small intestine were produced and mounted on poly-L-lysine-coated microscope slides (Sigma, Castle Hill, Australia) as described 22 .