Neuroanatomy of the spleen: Mapping the relationship between sympathetic neurons and lymphocytes

Abstract

The nervous system plays a profound regulatory role in maintaining appropriate immune responses by signaling to immune cells. These immune cells, including B- and T-cells, can further act as intermediary messengers, with subsets of B- and T-cells expressing choline acetyltransferase (ChAT), the enzyme required for acetylcholine (ACh) synthesis. Neural control of ACh release from ChAT+ T-cells can have powerful immune implications, regulating lymphocyte trafficking, inflammation, and prevent death due to experimental septic shock. Although ACh release from T-cells has been proposed to occur following norepinephrine (NE) released from sympathetic nerve terminals in the spleen, it is unknown how this communication occurs. While it was proposed that tyrosine hydroxylase (TH+) axons form synapse-like structures with ChAT+ T-cells, there is scant evidence to support or refute this phenomenon. With this in mind, we sought to determine the relative abundance of ChAT+ B- and T-cells in close proximity to TH+ axons, and determine what factors contribute to their localization in the spleen. Using confocal microscopy of tissue sections and three-dimensional imaging of intact spleen, we confirmed that ChAT+ B-cells exceed the number of ChAT+ T-cells, and overall few ChAT+ B- or T-cells are located close to TH+ fibers compared to total numbers. The organized location of ChAT+ lymphocytes within the spleen suggested that these cells were recruited by chemokine gradients. We identified ChAT+ B- and T-cells express the chemokine receptor CXCR5; indicating that these cells can respond to CXCL13 produced by stromal cells expressing the β2 adrenergic receptor in the spleen. Our findings suggest that sympathetic innervation contributes to organization of ChAT+ immune cells in the white pulp of the spleen by regulating CXCL13. Supporting this contention, chemical sympathectomy significantly reduced expression of this chemokine. Together, we demonstrated that there does not appear to be a basis for synaptic neuro-immune communication, and that sympathetic innervation can modulate immune function through altering stromal cell chemokine production.

Fig 1. Interactions between splenic ChAT⁺ lymphocytes and TH⁺ axons are rare events.

Confocal microscopy was conducted on spleens from ChAT-GFP reporter mice with immunostaining for CD3, ChAT, TH (A), or B220, ChAT, TH (B). Quantification of the total number of ChAT⁺ and ChAT^- B-cells (C) and T-cells (D) with in 1.5 cell diameters. ** P<0.001, *** P<0.0001.

Fig 2. Three-dimensional reconstruction of splenic neuro-immune interactions by CLARITY.

Spleens from ChAT-GFP mice were subjected to CLARITY and imaged by two-photon microscopy (A) allowing for individual cells and neural surfaces to be identified and modelled (B). Distances of ChAT⁺ lymphocytes to the nearest TH⁺ axons was determined and quantified (C). Representative images from n = 6 animals.

Fig 3. ChAT⁺ B- and T-cells express CXCR5.

Splenocytes isolated from ChAT-GFP reporter mice were subjected to flow cytometry to assess CXCR5 expression on ChAT⁺ and ChAT- B^- (A), and T-cells (B). **P<0.01, n = 6 mice.

Fig 4. Splenic stromal cells express β2AR and CXCL13.

The expression of β2AR and CXCL13 in stromal cells isolated from C57BL/6 mice were characterized by flow cytometry. Stromal cells were identified using the gating strategy depicted to include single cells, CD45^- (top panels). Stromal cells were then identified based on lack of CD31, with β2AR⁺ (R1) and β2AR^- (R3) populations assessed for CXCL13. Data are representative from 4 mice, mean ± SD.

Fig 5. Splenic sympathetic innervation regulates CXCL13 expression.

Spleen from control mice or mice subjected to chemical sympathectomy by 6-OHDA administration were assessed for expression of TH and CXCL13 by confocal microscopy (A) and quantified (B). Expression of Th, Ccl19, Ccl21 and Cxcl13 expression by qRT-PCR. *P<0.05, *** P<0.001.