Immunization Elicits Antigen-Specific Antibody Sequestration in Dorsal Root Ganglia Sensory Neurons

The immune and nervous systems are two major organ systems responsible for host defense and memory. Both systems achieve memory and learning that can be retained, retrieved, and utilized for decades. Here, we report the surprising discovery that peripheral sensory neurons of the dorsal root ganglia (DRGs) of immunized mice contain antigen-specific antibodies. Using a combination of rigorous molecular genetic analyses, transgenic mice, and adoptive transfer experiments, we demonstrate that DRGs do not synthesize these antigen-specific antibodies, but rather sequester primarily IgG1 subtype antibodies. As revealed by RNA-seq and targeted quantitative PCR (qPCR), dorsal root ganglion (DRG) sensory neurons harvested from either naïve or immunized mice lack enzymes (i.e., RAG1, RAG2, AID, or UNG) required for generating antibody diversity and, therefore, cannot make antibodies. Additionally, transgenic mice that express a reporter fluorescent protein under the control of Igγ1 constant region fail to express Ighg1 transcripts in DRG sensory neurons. Furthermore, neural sequestration of antibodies occurs in mice rendered deficient in neuronal Rag2, but antibody sequestration is not observed in DRG sensory neurons isolated from mice that lack mature B cells [e.g., Rag1 knock out (KO) or μMT mice]. Finally, adoptive transfer of Rag1-deficient bone marrow (BM) into wild-type (WT) mice or WT BM into Rag1 KO mice revealed that antibody sequestration was observed in DRG sensory neurons of chimeric mice with WT BM but not with Rag1-deficient BM. Together, these results indicate that DRG sensory neurons sequester and retain antigen-specific antibodies released by antibody-secreting plasma cells. Coupling this work with previous studies implicating DRG sensory neurons in regulating antigen trafficking during immunization raises the interesting possibility that the nervous system collaborates with the immune system to regulate antigen-mediated responses.

inTrODUcTiOn The mammalian immune system has acquired capacity to recall prior exposure to a vast number of potential antigens during the lifetime of the host. The development of this long-lasting immunity begins in a lymph node as an acute encounter between a naïve T cell and an antigen presenting cell. T and B lymphocytes recirculate between the lymphatic fluid, lymph node, and blood, entering the lymph node through high endothelial venules. Once inside the node, lymphocytes migrate through the T-cell zone and B-cell follicles until encountering an antigen presented by CD169+ macrophages or other antigen-presenting dendritic cells (1). The temporal and spatial control of antigen transport into the lymph node is dependent upon both the flow of isolated antigen via afferent lymphatic channels, and the trafficking into lymph nodes of intracellular antigen in antigen-presenting cells (APCs). Significant changes in lymph node architecture, inhibition of lymphocyte egress, and expansion of the lymph node stroma occur following antigen-mediated activation of intranodal immune responses.
We recently discovered that neural signals provide an essential mechanism in lymph nodes to retain antigen and lymphocytes (2). Lymph nodes are innervated by both sensory (afferent) and motor (efferent) neurons, with abundant innervation of the APCs in the T-cell zone, subsinoidal layer, and cortical extrafollicular zones (3,4). The neuronal density within the lymph nodes is dynamic, expanding significantly following antigenic stimulation of an early lymphocyte response (5,6). In naïve mice exposed to the antigen keyhole limpet hemocyanin (KLH) in the hind paw for the first time, we observed that KLH flowed rapidly from the popliteal lymph node (adjacent to the injection site) to the sciatic lymph node by traveling up the lymphatic chain. Surprisingly, prior exposure to KLH significantly impaired the flow of KLH from the popliteal to the sciatic lymph node during subsequent antigen exposure. This restriction of antigen flow from distal to proximal lymph nodes was antigen-specific and required functioning sensory neural signals, because inhibiting neural signals to the lymph node region restored antigen flow in immunized animals (2). The neural mechanism that restricted antigen flow is mediated by a subset of NaV1.8+ sensory neurons, which include nociceptors, because antigen failed to accumulate in the distal lymph node of animals rendered deficient in NaV1.8+ neurons (2). Early work had previously established that immune complexes can interact via Fc receptors expressed on sensory neurons to induce intracellular signaling mechanisms that require transient receptor potential canonical 3 (TRPC3) channel and the Syk-PLC-IP3 pathway (7,8) that mediates the release of calcitonin gene-related peptide (CGRP) and substance P (9). Immunohistochemistry using labeled antigen revealed colocalization of labeled antigen and FcγRI receptors in neurons at the site of antigen injection in the paw (2). Thus together, these studies implicate sensory neurons in regulating antigen trafficking during immunization through a pathway that requires NaV1. 8 and FcγR.
As we continued to explore the role of sensory neurons in immunization, we were very surprised to observe that sensory neurons obtained from the dorsal root ganglia (DRGs) of immunized animals contained abundant levels of antigen-specific antibodies. While others have reported the localization of antibodies to neurons in brain, to our knowledge, the localization of antibodies to sensory neurons in peripheral tissues has not been previously described. Rag2 expression has been identified in brain neurons, but convincing evidence that neurons can synthesize antigen-specific antibodies is lacking. Accordingly, here we used molecular genetic analyses, and transgenic and chimeric mice to show for the first time that dorsal root ganglion (DRG) sensory neurons in immunized animals accumulate lymphocyte-derived, antigen-specific IgG1.

generation of chimeric Mice
The recipient mice were sub-lethally irradiated with two split doses (WT: 5 Gy; Rag1 KO: 4 Gy) delivered 14 h apart, and their immune systems were rescued via BM transplantations from Rag1 KO (CD45.2) or WT (CD45.1) mice, respectively. Antibiotic gel diet (MediGel ® TMS) was given to animals before and after irradiation. Donor mice were euthanized by CO2 asphyxiation/cervical dislocation, and the femur and tibia were harvested. BM cells were collected by gently flushing the femurs and tibia and suspended at a concentration of 2 × 10 6 cells in 200 µL of sterile Hank's balanced salt solution (HBSS). Cells were administered intravenously (retro-orbital injection) to recipient mice 4-5 h after the second dose of irradiation. Reconstitution of the BM in recipient mice was confirmed after 2-4 weeks by assessing numbers of CD45.1 or CD45.2-positive cells in the peripheral blood by flow cytometry (reconstitution was >98%).

collection of Whole Drgs and isolation of sensory neuron Preparations
Dorsal root ganglia from thoracic and lumbar regions were harvested from mice immediately following euthanasia by CO2 asphyxiation. DRGs from the thoracic and lumbar regions were chosen because they have been shown to innervate the peritoneal cavity (10). DRGs were gently collected from the cavities along the lateral vertebral column as described previously (11) and enriched for neuronal populations, as described by Ref. (12). For the isolation of sensory neurons, DRGs were digested with 1 µg/ml collagenase/dispase (Roche Life Science, Germany) in neurobasal medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at 37°C on a rotator-shaker. Following digestion, DRGs were washed with HBSS (Gibco), triturated using fire-polished glass Pasteur pipettes (Fisher Scientific, Waltham, MA), and filtered through a 70-µm strainer. The cell suspension was layered onto 15% BSA in HBSS and centrifuged at ×129g for 20 min without brake. The cell pellet was re-suspended in neurobasal medium. B lymphocytes were depleted from cell preparations using Dynabeads™ mouse pan B purification kit (Invitrogen, USA), and cell purity was assessed by flow cytometry. The resulting sensory neuron preparation was resuspended in complete neurobasal medium [neurobasal™ medium supplemented with penicillin-streptomycin (Gibco), GlutaMax™ (Gibco), B-27 ® serum-free supplement (Gibco), and 50 ng/ml NGF (Sigma-Aldrich)].

immunohistochemical staining
Whole mount DRGs were harvested from mice immediately after euthanasia by CO2 asphyxiation as described above and fixed with 4% PFA for 2 h. Fixed specimens were incubated overnight in 30% sucrose/PBS, washed, embedded, and frozen in optimal cutting temperature compound (Tissue-TEK OCT, Electron Microscopy Sciences, VWR, Radnor, PA, USA). 14 µm thick frozen sections were mounted on the Superfrost Plus glass slides (Thermo Fisher Scientific). The tissue sections were blocked and permeabilized with blocking solution containing rabbit serum and 0.1% Triton X-100. For immunostaining, blocked/ permeabilized sections were stained with AlexaFluor 488-rabbit F(ab′)2 anti-mouse IgG (H + L) (1:1,000; Abcam, San Francisco, CA, USA), AlexaFluor 647-rabbit anti-NeuN (1:50; Abcam), and DAPI. Sections were mounted in ProLong anti-fade mounting medium (Thermo Fisher Scientific) and imaged using an LSM880 laser scanning confocal microscope (Carl Zeiss) using X40 Zeiss plan-apochromatic oil objective with Z stacks. The numbers of NeuN-positive and NeuN-positive IgG-positive neurons were quantified in five DRG sections pooled from three animals using ImageJ. The percentages of IgG-positive neurons were quantified by counting the number of neurons double positive for IgG and NeuN divided by the total NeuN-positive neurons in each DRG section and then multiplied by 100.

elispot assay
Dorsal root ganglion sensory neurons (isolated and prepared as described above) were cultured on anti-IgG or OVA-or KLHcoated ELISPOT plates and developed using the standardized ELISpot kit (Mabtech Inc., Cincinnati, OH, USA). Briefly, 96 well flat bottom multi-screen filter plates (Millipore, Billerica, MA, USA) were coated with 100 µL per well of KLH (50 µg/ml) or OVA (50 µg/ml) or anti-IgG antibody (15 µg/ml). The plates were incubated overnight at 4°C, washed with PBS, and blocked using complete neurobasal medium. DRG sensory neurons (isolated and prepared as described above from five mice per group for eight wells) suspended in complete neurobasal medium were cultured in the plates overnight at 37°C with 5% CO2. ELISpots were developed as per the manufacturer's protocol and scanned and analyzed at Cellular Technology Ltd. (Shaker Heights, OH, USA).

Fluorospot assay
Dorsal root ganglion sensory neurons (isolated and prepared as described above) were cultured in anti-mouse IgG-coated plates and incubated overnight at 37°C with 5% CO2. FluoroSpots were developed using the fluorescent tagged mouse IgG1, IgG2a, and IgG3 detection kit as per the manufacturer's protocol (Cellular Technology Ltd.) and scanned and analyzed at Cellular Technology Ltd.

In Vitro B cell and Drg neuron activation studies
Splenic B cells were purified from 7-to 10-week-old naïve C57BL/6 mice using the EasySep B Cell Isolation Kit (STEMCELL Technologies, Cambridge, MA, USA). B cell purity (CD19positive cells) was confirmed (>99%) using flow cytometry. Isolated B cells were cultured in complete DMEM containing penicillin, streptomycin, glutamine, β-mercaptoethanol (1× each) and 10% fetal bovine serum at 3 × 10 6 cells/ml per well (24 well plate) in duplicate. DRG neurons were isolated from naïve C57BL/6 mice and depleted of B cells, as described above. DRG neurons from five naïve mice were cultured in 0.25 ml per well (48 well plate) in duplicate per condition in complete neurobasal  48,72,96, and 120 h post stimulation, and markers of antibody production and the generation of plasmablasts/plasma cells were assessed by qPCR (e.g., Rag2, Aicda, and Prdm-1, as described below; see Table 1 for primers).

rna isolation
Naïve mouse spleens (positive control) and DRG neuron preparations isolated from naïve, alum-injected, and alum + KLHimmunized mice (n = 10 mice per condition, pooled) were lysed with Qiazol lysis reagent (Qiagen, Germantown, MD, USA) and stored at −80°C until RNA preparation (DRG neurons from 10 mice yielded approximately 50 µg RNA). Spleen and DRG RNA were isolated using the RNeasy Universal Kit (Qiagen). For the in vitro activation assays (described above), RNA was isolated from DRG neurons using the RNeasy Universal Kit (Qiagen) and from B cells using the RNeasy Mini Kit (Qiagen). For RNA-seq studies, RNA quality was analyzed using the Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA); RNA integrity numbers (or RIN values) were approximately 8.0. All RNA samples had OD260:OD280 ratios >1.8.

rna-seq and analysis
The sequencing mRNA libraries (for paired-end reads) were prepared using RNA isolated from DRG neurons purified from either alum-injected or alum + KLH-immunized C57BL/6J mice (n = 10 mice per group, pooled) using the TruSeq Stranded mRNA Sample Preparation Kit (Illumina, San Diego, CA, USA), according to the manufacturer's instructions. As an exploratory step for characterizing the DRG neuron preparations, deep RNA sequencing was performed on the Illumina NextSeq 500 platform. The read depth was at least 60 M reads per sample using 75 base pair paired-end reads for a total output of ~18 Gb. The raw image files from the NextSeq sequencer were de-multiplexed and converted to fastq files using the bcl2fastq BaseSpace App (Illumina). RNA-seq analysis was performed on the existing annotated reference transcriptome (mouse) -no alternate transcript discovery was performed. Adapter sequences and low-quality base reads were trimmed from fastq files using cutadapt. The fastq files were aligned to the iGenome mm10 reference sequence from Illumina, and gene expression was quantified using kallisto. 1 Data are reported as transcripts per million (TPM), which are considered more comparable between samples of different origins and composition, and the most frequently reported RNA-seq gene expression values (13). An expression value >3.0 TPM (transcripts per million) was used as a threshold for detectable expression. The RNA-seq data discussed here have been deposited in the NCBI Gene Expression Omnibus (GEO) (14) and are accessible through GEO Series accession number GSE108428. 2

rna-seq Deconvolution analysis
We utilized DeconRNASeq software (15) to perform RNAseq deconvolution analysis to infer cell type compositions of the neuronal preparations isolated from alum-injected and alum + KLH-immunized mice. DeconRNASeq, a freely available software package written in R, was downloaded from http:// bioconductor.org/packages. DeconRNASeq uses a globally optimized non-negative decomposition algorithm for estimating the relative proportions of distinctive cell types in a mixture sample.
High-quality pure cell-type signatures were derived from RNA sequencing data on single cells from the mouse lumbar DRGs (16). The data are available from the GEO (GSE59739), and the normalized data expressed as reads per million (RPM) were downloaded from http://linnarssonlab.org/drg/. This dataset contains single cell RNA-seq measures of 799 cells as well as clustering assignments of 731 of these cells by principal component analysis. The five principal cell classes included 109 non-neuronal (NoN) cells, 81 peptidergic nociceptor (PEP) cells, 169 non-pep (NP) cells, 139 neurofilament containing (NF) cells, and 233 tyrosine hydroxylases containing (TH) cells. We averaged the RNA-seq measures for each of the five cell classes. We excluded genes in which the mean RPKM (reads per kilobase of TPM mapped reads) value for the cells types was below a 0.1 cutoff value. There were 11,776 genes that passed this constraint and were used in the deconvolution analysis. Using the deconvolution analysis, we analyzed relative proportions of distinctive cell types in our groups. Based on pure cell-type signatures, approximately 73% of our DRG neuron preparations were neuronal ( Figure S1 in Supplementary Material). Of the cells with a neuronal origin, three different neuronal subtypes were identified: peptidergic nociceptor (PEP) cells, non-PEP (NP) cells, and NF (NF) cells.

Two step real-Time qPcr
Two step qPCR analysis was carried out to analyze the relative expression of target genes in B cell-depleted DRG neuron preparations isolated from naïve, alum-injected and alum + KLHimmunized mice, spleens and whole DRG preparations (without B cell depletion) isolated from naïve mice, as well as in vitro stimulated B cells and DRG neuron preparations (described above). First, cDNA synthesis was performed using ~1.5 μg of RNA using the ABI High-Capacity cDNA Reverse Transcription Kit (Thermo  resUlTs

Detection of igg in sensory neurons of Mouse Drg
To study the accumulation of IgG in DRG sensory neurons, we performed immunohistochemical analysis of whole mount DRGs collected from naïve (untreated), alum-injected, or alum + KLHimmunized mice using a rabbit F(ab′)2 anti-mouse IgG (H+L) antibody. The F(ab′)2 fragment against IgG has been used for immunohistochemical staining because it is small and can penetrate tissues/cells, and does not bind to the Fc receptors expressed on the target cells (18,19). Mice were subjected to either sham immunization, intraperitoneal injection with adjuvant (alum) or immunization with alum + KLH. Animals received an intraperitoneal booster dose on day 14, and whole mount lumbar and thoracic DRGs were isolated from naïve and immunized mice after 4 weeks of immunization. Neuronal populations from DRG sections were stained with labeled anti-NeuN antibodies that recognize the neuronal nuclear protein NeuN. While only sparse IgG-positive staining was observed within the NeuN-positive DRG sensory neurons from the naïve and alum-injected animals, a significant increase in the number of double IgG-positive NeuN-positive neurons was observed in DRGs collected from alum + KLH-immunized mice (Figures 1A-C). Quantitative analysis revealed that IgG-positive staining of the NeuN+ DRG neurons was similar in naïve (3.5% ± 1.8%,) and alum-injected mice (5.9% ± 4.1%) ( Figure 1D, a total of 824 and 1,343 NeuN+ neurons were analyzed from naive and alum-injected mice, respectively). By contrast, a significantly higher percentage of NeuN+ DRG neurons stained for IgG following alum + KLH immunization (23.8% ± 9.1%) ( Figure 1D, P < 0.05, a total of 814 NeuN+ neurons were analyzed). NeuN is most abundant in the neuronal nucleus, and IgG protein is primarily distributed in the cytoplasm of sensory neurons (Figures 1A-C).
sensory neurons From Drg release antigen-specific igg 1 To investigate the antigen-specificity of antibodies sequestered in neurons, DRG sensory neurons were cultured on ELISpot plates. A significant increase in the number of total IgG-releasing neurons was observed in DRGs isolated from alum + KLH-immunized mice as compared to alum-injected controls (Figures 2A,B). It is interesting to note that fewer neurons releasing IgG were detected in animals that received only alum (Figures 2A,B), which is in agreement with our immunohistochemical data showing low numbers of sensory neurons sequestering antibodies in DRGs of alum-injected controls ( Figure 1B). Similar to alum + KLHimmunization, a significant increase in number of neurons releasing total IgG and OVA-specific IgG was observed in DRGs harvested from alum + OVA-immunized mice (Figures 2C,D).
As alum + KLH-immunized mice exhibited robust responses, we utilized this model for our subsequent studies. The majority of these DRG sensory neurons from alum + KLH-immunized mice released IgG1 isotype (Figure 2E). To study the antigen specificity of the IgG associated with DRG sensory neurons, we next assessed the number of IgG-releasing neurons in a KLH-specific or an OVA-specific ELISpot assay. A significant increase in KLH-specific neurons was observed in animals immunized with alum + KLH as compared to alum-injected controls ( Figure 2F).
In contrast, no anti-OVA IgG-releasing neurons were detected in DRGs isolated from either alum-injected or alum + KLHimmunized mice (Figure 2F), indicating that antigen-specific IgG antibodies are associated with DRG sensory neurons in immunized animals.
Drg sensory neurons lack expression of essential enzymes required for igg synthesis To assess if sensory neurons from DRGs have the molecular machinery to synthesize antibodies and undergo class-switching, we analyzed the transcriptome of the neuronal population using RNA-seq. DRGs were harvested from animals immunized with alum + KLH (n = 10) or alum-injected controls (n = 10) and RNA-seq analysis was performed using the RNA prepared from pooled DRG neurons for downstream processing to maximize biological diversity. The RNA-seq analysis was primarily used to evaluate the overall transcriptome of the DRG neuron preparations and not to statistically compare the alum vs. alum + KLHimmunized groups. As an exploratory step for characterizing the DRG neuron preparations, RNA-seq was performed using the Illumina NextSeq platform. An average of 17,355 and 17,030 genes were detected (detection > 3 transcripts per million, TPM) in the two groups studied, alum-injected and alum + KLHimmunized, respectively. Further examination of the RNA-seq data revealed that the expression of neuronal and nociceptor-related gene expression was present in the alum-injected and alum + KLH-immunized groups ( Table 2). In contrast, the expression of B lymphocyte, T lymphocyte, mast cell, and basophil-related genes were not observed in either of the groups. Both neuronal preparations show a similar low level of positive regulatory domain zinc finger protein-1 (Prdm1) expression ( Table 2), which encodes Blimp1, a protein required for the differentiation of B cells into plasma cells (20). These data demonstrate that the enriched neuronal preparation in our study is devoid of B lymphocytes. We next evaluated whether the DRG sensory neuron populations express any of the essential genes required for antibody synthesis and class-switching. No expression of Rag1, Rag2, Aicda, or Ung genes was observed in either alum-injected or alum + KLH-immunized group (Table 2), indicating that DRG sensory neurons lack the genetic make-up to produce antibodies.
To confirm the RNA-seq data, we validated the expression of genes required for antibody synthesis and B cell differentiation into plasmablasts/plasma cells at different time points postimmunization. DRG neuronal preparations were isolated from animals (alum-injected controls or alum + KLH-immunized) at three different time points (n = 10 mice per group): postprimary immunization, post-first booster and post-second booster as described in the Section "Materials and Methods." The expression pattern of four genes related to antibody synthesis (Rag1, Rag2, Aicda, and Ung) and two genes corresponding to B lymphocyte/plasma cell markers (Cd19 and Prdm1) were validated by quantitative real-time RT-PCR. As expected, high levels of the B cell marker, Cd19, and low-moderate levels of Rag1, Rag2, Aicda, and Ung were observed in the naïve spleen. However, DRG sensory neurons failed to express Rag1, Aicda or Ung at any of the time points studied ( Table 3). Our preliminary RNA-seq data confirmed the expression of Il4ra (the gene that encodes the alpha chain of the IL-4 receptor, a type I transmembrane protein that can bind IL-4 and IL-13 to regulate IgG production) in DRG sensory neurons (TPM value ~5) and the expression of the Tlr4 gene (that encodes the receptor for LPS) (TPM value ~7). To address the possibility that the molecular events related to antibody synthesis are not operative during the DRG sample collection and analysis, but may be induced in a similar manner as activated B cells, we stimulated DRG sensory neurons (or splenic B cells as a positive control) in vitro and assessed the expression of Rag2 and Aicda at three different time points post-stimulation. We used two conditions for in vitro activation of DRG sensory neurons and B cells: LPS + IL-4 (T-independent) or anti-CD40 + IL-4 (T-dependent) and no treatment (vehicle). As reported previously (21,22), stimulation of mature mouse B cells with LPS + IL-4 resulted in the expression of both Rag2 and Aicda in vitro in a time-dependent manner (Figure 3). In contrast, activation of DRG sensory neurons with either T-independent (LPS + IL-4) or T-dependent (anti-CD40 + IL-4) stimuli failed to induce either Rag2 or Aicda gene expression (Figure 3). These data confirm that DRG sensory neurons lack the expression of essential enzymes required for IgG synthesis.
Transgenic Ighg1-tdTomato Mice Do not express tdTomato in Drg sensory neurons Casola et al (23) have produced a transgenic Ighg1-cre mouse that can be used in combination with a reporter gene to analyze the transcription of Igγ1 constant region gene segment (Cγ1) in antibody expressing cells. We utilized the Ighg1-cre mice to induce expression of a fluorescent reporter protein, tdTomato, in antibody-expressing cells because IgG1 was found to be the most prominent subtype in the DRG neurons ( Figure 2E). The elimination of the STOP cassette and subsequent tdTomato expression was achieved by crossing Ighg1-cre mice with B6.Cg-Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze /J mice, resulting in tdTomato expression mostly in cells expressing Cγ1 and hence, IgG1. Transgenic mice (carrying both cre and tdTomato) were indistinguishable from their WT littermate controls (carrying only cre or tdTomato) in terms of developmental features. tdTomato is one of the brightest among the red-fluorescent proteins and well-suited for immunohistochemical analysis. Accordingly, we found that the tdTomato reporter mice had increased numbers of tdTomato + cells in their spleens following immunization ( Figure 4A). However, no colocalization of tdTomato fluorescent signal and antibody staining was observed in sensory neurons following immunization ( Figure 4B). As tdTomato expression is driven by the transcription of Cγ1 (of IgG1), these data confirm that Ighg1 are not expressed in sensory neurons from DRG. Drg sensory neurons lacking neuronal Rag2 expression contain antigen-specific antibodies Because neuronal Rag2 expression has been implicated in previous work (24)(25)(26)(27), here we crossed floxed-Rag2 transgenic mice with synapsin-cre transgenic mice generating null mutants that selectively lack Rag2 expression in neurons (with abundant Rag2 expression in B cells). Analysis of immunoglobulin reactivity in the DRGs of null mutants revealed the presence of IgG-positive NeuN-positive neurons in both alum-injected ( Figure 5A) and alum + KLH-immunized (Figure 5B) mice. Similar to previous observations with WT mice (Figures 2A,B,F), significantly higher numbers of IgG-releasing and anti-KLH IgG-releasing DRG neurons were observed in the alum + KLH group when compared to the alum-injected group even in the absence of Rag2 expression (Figures 5C,D). Taken together, our results indicate that sensory neurons from DRGs lack the enzymes and cannot synthesize antibodies.
antibodies Found in Drg neurons are released by B cells Accordingly, we reasoned that the neurons from DRGs sequester and retain antibodies produced by plasma cells, and therefore, DRGs isolated from mice incapable of producing B cell-derived   antibodies would contain no antigen-specific antibodies. To address this question, we utilized mice with a homozygous targeted disruption of the membrane exon of the Ig mu-chain (μMT mice) that are deficient of mature peripheral B cells as well as Rag1 KO mice that do not produce mature T and B lymphocytes (28)(29)(30). Immunohistochemical analysis revealed that DRGs from either μMT or Rag1 KO mice do not contain antigen-specific antibodies ( Figures 6A,B). Next, we generated chimeric mice by transferring Rag1 KO BM to WT mice or WT BM to Rag1 KO mice.
After confirming the reconstitution of the recipient animals (data not shown), the chimeric mice received alum injections or alum + KLH immunizations as described. Immunohistochemical analysis of chimeric mice with Rag1-deficient BM (Rag1negative hematopoietic cells, but Rag1-positive DRG sensory (D) Quantification of anti-KLH-specific IgG immunospots from DRG neurons isolated from alum-injected and alum + KLH-immunized neuronal-specific Rag2 KO mice (mean ± SD, n = 4 per group) using the antigen-specific ELISpot assay *p < 0.05 vs. alum-injected (Mann-Whitney test). neurons) revealed that in the absence of antibody secreting plasma cells, no IgG reactivity is observed in sensory neurons from DRGs isolated from either alum-injected ( Figure 7A) or alum + KLH-immunized groups (Figures 7B,C). In contrast, IgG-positive neuronal populations were observed in DRGs from alum-injected chimeric mice with WT BM (Rag1-positive hematopoietic cells, Rag1-negative sensory neurons) ( Figure 7D). Furthermore, immunization with alum + KLH significantly increased the number of IgG-positive sensory neurons in the DRGs as observed with WT mice (Figures 7E,F). We next analyzed the immunoglobulin release by DRG sensory neurons from chimeric mice using both total IgG and KLHspecific IgG ELISpot assays. Increased IgG-positive neuronal cells are observed in the DRGs harvested from chimeric mice with WT BM, but not in the chimeric mice with Rag1-deficient BM (Figure 8A). Analysis of KLH-specific IgG-releasing cells revealed a significant increase in anti-KLH IgG-positive DRG sensory neurons in the DRGs isolated from alum + KLHimmunized chimeric mice with WT BM (Figure 8B). In contrast, no anti-KLH IgG-secreting DRG sensory neurons are observed in immunized chimeric mice with Rag1-deficient BM ( Figure 8B). Administration of alum alone also increased the number of total IgG-releasing DRG sensory neurons, but not anti-KLH IgG-positive neurons (Figures 8A,B). Together, these data suggest that DRG sensory neurons not only sequester and retain antibodies secreted by plasma B cells but also can release these antibodies.

DiscUssiOn
Here we demonstrate that antigen-specific antibodies are sequestered in mouse DRG sensory neurons. A significantly higher number of DRG sensory neurons from immunized animals showed sequestration of antigen-specific antibodies as compared to alum-injected control or naïve mice. RNA-seq analysis and qPCR of the neuronal transcripts revealed that sensory neurons from DRGs lack the genetic machinery to produce antibodies. Using transgenic and chimeric animals, we further confirmed that DRG sensory neurons do not synthesize antigen-specific antibodies, but sequester the antibodies released by plasma cells. As shown in Figure 1, DRG neurons isolated from naive mice and alum-injected mice had similar low percentages of IgG-containing NeuN+ neurons (~4-6%), while DRG neurons isolated from mice immunized with alum + KLH showed significantly higher percentages (~24%). Antibody localization appears intracellular. However, the subcellular localization of IgG within NeuN+ DRG neurons remains to be determined. All additional experiments included the alum-injected and alum + KLHimmunized groups. Although this study design limited our ability to compare DRG neurons obtained from naive vs. alum-treated mice, it focused our studies on assessing antigen-specific antibody responses in alum-injected vs. alum + KLH-immunized mice. The anti-KLH antibodies released by DRG neurons were primarily IgG1 (not IgG2 or IgG3) ( Figure 2D). We did not assay for IgA, IgE, or IgM antibodies. Future studies will examine what stimuli (e.g., antigen, capsaicin, or KCl) trigger IgG release by DRG neurons.
Antibodies are normally synthesized by mature plasma cells. However, recent accumulating evidence suggests that nonlymphoid cells, such as human sperm (31), hepatocytes (32), certain carcinoma cells (25,32,33), and neurons from the central nervous system (34)(35)(36)(37), also express genes required for antibody synthesis and produce antibodies. Here, we demonstrate that although DRG sensory neurons exhibit localization of antigenspecific antibodies, the neuronal cells do not express the essential enzymes required for generating antibody diversity. V(D)J recombination is tightly regulated during B cell differentiation by transcriptional regulation of lymphoid-specific recombinase genes encoding RAG1 and RAG2 enzymes (38)(39)(40)(41). In mature B cells, antigenic stimulation induces somatic hypermutation, gene conversion, and class switch recombination leading to generation of a secondary repertoire of high-affinity antigen-specific antibodies (42,43). Activation-induced cytidine deaminase (AID), an enzyme encoded by the Aicda gene, is responsible for regulating and coordinating these three genetic rearrangements in mature B cells (42,44). AID generates antibody diversity by converting cytosine to uracil within the immunoglobulin loci resulting in mismatch mutations. The uracil-guanine mismatch created by AID is repaired by uracil-DNA glycosylase (UNG), a DNA-repair enzyme encoded by the gene Ung, introducing base-pair changes resulting in antibody diversity (42). The DRG neuronal transcriptome analysis here revealed the absence of Rag1, Rag2, Aicda, and Ung transcripts. Furthermore, in vitro activation of DRG sensory neurons with LPS and IL-4 (to induce T-independent class switch recombination) or anti-CD40 and IL-4 (to induce T-dependent class switch recombination) did not induce significant increases in expression of either Rag1, Rag2, Aicda, or Ung genes. Moreover, a comprehensive transcriptome analysis of 622 hand-picked single neurons harvested from lumbar DRGs of naïve mice showed no detectable Rag1, Rag2, or Aicda expression (16).
An interesting finding in the current study is the marked discordance in the expression of Rag1 and Rag2 transcripts in DRG sensory neurons harvested from immunized mice. Amplification of the neuronal transcripts showed low levels of Rag2 but no Rag1 transcripts present in DRG sensory neurons. It is unlikely that DNA contamination contributed to the results, as all samples were treated with DNase before analysis, and intron spanning primers were used. These data rather suggest that Rag1 and Rag2 are differentially expressed in immunized mice. As discussed earlier, expression of Rag1 and Rag2 is tightly controlled and occurs primarily during the early developmental stages of T and B cells. A cis-regulatory element located in the RAG locus regulates coordinated expression of Rag1 and Rag2. However, a less-strict control of transcription within the RAG locus results in functionless expression of Rag2 transcript in the brain and other non-lymphoid tissues (27). Although expression of Rag1 and Rag2 transcripts have been reported in a variety of nonlymphoid cells, including different cancer cell types, epithelial cells and neurons (34,45,46), to our knowledge, no functional role for these enzymes have been identified in non-lymphoid cells.
In order to investigate the functional role of the RAG2 enzyme for antibody synthesis in neurons, we developed a neuronspecific Rag2 KO mouse to detect if antibodies are sequestered in neurons in the absence of neuronal Rag2 gene expression. We immunized the neuronal Rag2 KO mice with alum + KLH and observed a significant increase in antibody sequestration, total IgG and anti-KLH IgG-releasing neurons in DRGs from immunized mice even in the absence of Rag2 expression in neurons. Mice deficient in either RAG1 or RAG2 enzymes lack mature lymphocytes and fail to generate antibodies due to their inability to initiate V(D)J rearrangement (30,47). In the absence of V(D) J recombination and mature B cells, Rag1 KO mice completely lacked antibody localization in their DRG sensory neurons. Consistent with this observation, no localization of antibodies was observed in mice homozygous for an inactivating mutation of the membrane exon of their immunoglobulin μ chain gene (μMT) that results in mature B cell deficiency and no antibody production (29). Adoptive transfer of WT BM into Rag1-deficient mice restored antibody sequestration in DRGs. Together, these results indicate that DRG sensory neurons sequester antibodies released by mature B cells. Antibody accumulation has been described in brain tissues and cerebrospinal fluid in both healthy and pathological conditions (35,37,48,49). Antigen-antibody immune complexes directly activate sensory neurons resulting in an increase in intracellular calcium (9,50), initiation of action potentials (8,50), and the release of neurotransmitters from DRG neurons (9). The presence of antibodies in the nervous system could be attributed to receptor-mediated uptake of antibodies by neurons. Immunoglobulin G and its receptors have been observed in the human nervous system (34), rodent brains (9,35,37,51), and zebrafish sensory neurons (24,46). Expression of different subtypes of Fcγ receptors have been shown in Purkinje cells, parvalbumin neurons, primary neuronal cultures, and sensory neurons (52)(53)(54). While RNA-seq and qPCR (data not shown) failed to detect the expression of either Fcgr1 or Fcgr3 in DRG sensory neurons, the expression of one Fcgr2b variant and Fcgrt were detected ( Table 2). Consistent with our data, single cell analysis of the DRG neuron transcriptome revealed a lack of expression of Fcγ receptors 1 and 3 (16). However, the presence of FcRn, an MHC class I related antibody receptor, has been detected in sensory neurons (16). FcRn, a product of the Fcgrt gene, plays a pivotal role in passively transferring IgG within and across multiple cell types (55)(56)(57)(58)(59) and protecting IgG from degradation (59)(60)(61). Mice genetically deficient in Fcgrt (lacking FcRn) have significantly reduced plasma half-life of IgG and exhibit hypogammaglobulinemia (60)(61)(62). In the central nervous system, FcRn expressed by endothelial cells has been implicated in antibody transport across the blood brain barrier (63). Similar to the situation in the central nervous system, it is possible that in DRGs, FcRn mediates the transport of antibodies into sensory neurons and protects the IgGs from degradation within the neuronal cells. Further studies are required to address the functions of Fcγ receptors and FcRn in peripheral DRG sensory neurons. Future studies using in vitro and in vivo methods including single cell qPCR (to compare neurons that sequester IgG vs. those that do not) will provide insight on how and why antigen-specific antibodies are taken up by DRG neurons.
Several possibilities exist that may explain the accumulation of antibodies in sensory neurons. Antibody uptake by neurons may have a self-protecting effect. Using a murine stroke model, Arumugam et al have demonstrated that intravenously injected IgG protects the brain against neuronal death by neutralizing the effects of complement (64). In a mouse model of Alzheimer's disease, antibodies assisted clearance of amyloid β-peptide from the CNS into the circulation via FcRn (65). Antigen-specific antibodies also aid in clearance of neuronal-specific pathogens from neurons. Treatment of encephalitis virus-infected primary cultured neurons with virus-specific antibodies clears the infectious encephalitis virus from neurons by restricting viral gene expression (66). Accordingly, immunodeficient scid mice infected with the encephalitis virus develop persistent non-lethal central nervous system infection (66). It is also possible that invading pathogens get opsonized by the locally released antibodies and are subsequently phagocytosed by glial cells (67). In addition, antigen-antibody complexes may mediate hyperalgesia by activating peripheral sensory neurons. Numerous antigen-specific immune-related disorders are often accompanied by pain. These disorders include autoimmune diseases such as rheumatoid arthritis (68) and Guillain-Barre syndrome (69), viral reactivation syndromes such as herpes zoster (70,71), and allergic diseases such as atopic and allergic contact dermatitis (72,73). Elevated levels of antigen-specific antibodies, especially IgG, and immune complexes are found in the serum and affected tissues in these diseases. Immune complexes directly activate nociceptive DRG neurons in a TRPC3-receptor dependent manner, and increase neuronal excitability and thus, potentially contribute to pain (7,8). Recent studies have demonstrated that intradermal injection of immune complexes dose-dependently produces mechanical and thermal hyperalgesia in the hind paws of rats that can be alleviated by localized administration of non-specific antibody (74). Finally, it is important to note that our studies were confined to DRG sensory neurons. We did not study other sensory neurons (e.g., nodose or trigeminal ganglia).
Our studies demonstrate that peripheral DRG sensory neurons sequester antibodies made by plasma cells. In concluding that DRG neurons do not produce antibodies we are at risk of a type II error (in failing to reject a null hypothesis that is actually false). We have attempted to reduce the risk of this error by analyzing large numbers of DRG neurons from the mice and by testing the same hypothesis using multiple approaches and models [qPCR, RNA-seq, immunostaining, and both in vitro and in vivo models (using multiple genetically modified mice and BM transfer techniques)]. Therefore, we are confident that our results demonstrate that DRG neurons sequester antigen-specific antibodies. These antigen-specific antibodies may play an important role in protecting DRG neurons from pathogen-induced damage, or in inducing antigen-specific hyperalgesia. These findings, together with the previous studies, implicate immune complexes in mediating neuro-immune crosstalk between DRG sensory neurons and antibodies in both physiological and pathological conditions.

eThics sTaTeMenT
All animal experiments were performed in accordance with the National Institutes of Health Guidelines under protocols