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. Author manuscript; available in PMC: 2021 Aug 1.
Published in final edited form as: Brain Behav Immun. 2020 May 12;88:725–734. doi: 10.1016/j.bbi.2020.05.029

Germinal Center Formation, Immunoglobulin Production and Hindlimb Nociceptive Sensitization after Tibia Fracture

Wen-Wu Li a,b, Yang Yang d, Xiao-you Shi a,b, Tian-Zhi Guo a,c, Qin Guang d, Wade S Kingery a,c, Leonore A Herzenberg d, J David Clark a,b
PMCID: PMC7416484  NIHMSID: NIHMS1595204  PMID: 32413559

Abstract

Emerging evidence suggests that Complex Regional Pain Syndrome (CRPS) is in part a post-traumatic autoimmune disease mediated by an adaptive immune response after limb injuries. We previously observed in a murine tibial fracture model of CRPS that pain-related behaviors were dependent upon adaptive immune mechanisms including the neuropeptide-dependent production of IgM for 5 months after injury. However, the time course of induction of this immune response and the demonstration of germinal center formation in lymphoid organs has not been evaluated. Using the murine fracture model, we employed behavioral tests of nociceptive sensitization and limb dysfunction, serum passive transfer techniques, western blot analysis of IgM accumulation, fluorescence-activated cell sorting (FACS) of lymphoid tissues and immunohistochemistry to follow the temporal activation of the adaptive immune response over the first 3 weeks after fracture. We observed that: 1) IgM protein levels in the skin of the fractured mice were elevated at 3 weeks post fracture, but not at earlier time points, 2) serum from fracture mice at 3 weeks, but not 1 and 2 weeks post fracture, had pro-nociceptive effects when passively transferred to fractured muMT mice lacking B cells, 3) fracture induced popliteal lymphadenopathy occurred ipsilateral to fracture beginning at 1 week and peaking at 3 weeks post fracture, 4) a germinal center reaction was detected by FACS analysis in the popliteal lymph nodes from injured limbs by 3 weeks post fracture but not in other lymphoid tissues, 5) germinal center formation was characterized by the induction of T follicular helper cells (Tfh) and germinal center B cells in the popliteal lymph nodes of the injured but not contralateral limbs, and 6) fracture mice treated with the Tfh signaling inhibitor FK506 had impaired germinal center reactions, reduced IgM levels, reduced nociceptive sensitization, and no pronociceptive serum effects after administration to fractured muMT mice. Collectively these data demonstrate that tibia fracture induces an adaptive autoimmune response characterized by popliteal lymph node germinal center formation and Tfh cell dependent B cell activation, resulting in nociceptive sensitization within 3 weeks.

Keywords: Fracture, Complex regional pain syndrome, Pain, Autoimmunity, Germinal center, Follicular B cells, T follicular helper cells, FK506

Introduction

The transition from acute to chronic pain after surgery and other forms of trauma is a major source of chronic pain and disability (Correll, 2017; Stamer et al., 2019). In fact, chronic postsurgical and posttraumatic pain have been added to the recently approved eleventh version of the International Classification of Diseases (ICD-11) (Schug et al., 2019). Although a range of causes of chronic posttraumatic pain have been described, Complex Regional Pain Syndrome (CRPS) is one well-documented often severe chronic pain outcome of surgery and limb injury. Despite improvements in CRPS symptoms over the first several months in some CRPS patients, symptoms often fail to fully resolve, and disability is found in most patients with CRPS lasting more than 1 year (Bean et al., 2016; Subbarao and Stillwell, 1981). The mechanistic underpinnings of CRPS remain enigmatic, and we have no broadly effective therapies for established disease.

Recent reports from patients and laboratory models suggest that dysfunction of the adaptive immune system may occur early after the appearance of the CRPS syndrome and support the transition of CRPS to its chronic phase (Cuhadar et al., 2019; David Clark et al., 2018; Guo et al., 2017). For example, genetic studies have linked CRPS to specific HLA loci such as HLADQ8 and HLA-B62 (van Rooijen et al., 2012), and the density of Langerhans antigen presenting cells in the skin of CRPS patients is increased early in the course of the disease (Li et al., 2018). Moreover, recent evidence shows that IgM class antibodies isolated from CRPS model animals and both IgM and IgG class antibodies from some patients can cause nociceptive sensitization, limb dysfunction and vascular changes in the limbs of recipient laboratory animals (Cuhadar et al., 2019; Li et al., 2018; Li et al., 2014). Production of these autoantibodies appears to be reliant upon injury-induced neuropeptide release from peripheral nerve fibers (Li et al., 2018), and pain-related targets are located in both peripheral and spinal cord tissue (Guo et al., 2020). Although the targets of CRPS-related autoantibodies have not been searched for comprehensively, keratin, tubulin and histone proteins are likely candidates (Tajerian et al., 2017), and the enhanced expression of at least one of these targets, keratin 16, in a mouse CRPS model is again dependent on neuropeptide signaling (Li et al., 2018). Immunotherapy for CRPS using low dose IVIG failed in a clinical trial although plasmapheresis has shown promise (Aradillas et al., 2015; Goebel et al., 2017).

Autoantibodies that drive the pathogenesis of diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and Guillain-Barré syndrome (GBS) are initially generated in specialized microanatomical compartments in secondary lymphoid tissues termed germinal centers described more than 125 years ago (Victora and Nussenzweig, 2012; Vinuesa et al., 2009). It has become clear that follicular B cells, the predominant population participating in the germinal center response, along with antigen-specific helper T cells referred to as T follicular helper cells (Tfh) colocalize in germinal centers (Crotty, 2011; Nutt et al., 2015). The formation of such germinal centers has not been evaluated in models of CRPS or CRPS patients. We therefore designed a series of studies to explore the hypotheses that germinal center formation in regional lymph nodes would be observed in the injured limb of the tibia fracture mouse model of CRPS (Birklein et al., 2018), and that medications that interrupt early steps in the adaptive immune response would prevent the formation of pain-related autoantibodies.

2. Materials and methods

2.1. Animals

All animal experiment protocols were approved by the Veterans Affairs Palo Alto Health Care System Institutional Animal Care and Use Committee (Palo Alto, CA, USA) and followed the animal subjects guidelines of the International Association for the Study of Pain. Male C57BL/6J mice and muMT mice in the same background strain were obtained from The Jackson Laboratory (Sacramento, CA, USA) at 8–12 weeks of age. Experiments were done after a 7–10-day acclimation period in our animal care facility. The animals were housed 4 per cage under pathogen-free conditions with soft bedding and were given food and water ad libitum, with a 12:12 light:dark cycle. The animals were fed Teklad lab rodent diet 2018 (Teklad Diets; Harlan Laboratories, Indianapolis, IN, USA), which contains 1.0% calcium, 0.7% phosphorus, and 1.5 IU/g vitamin D3. Data collection and analysis was conducted blind to group assignment, and ARRIVE guidelines were followed.

2.2. Tibia fracture surgery

The mouse fracture model was used as described previously (Li et al., 2014). Briefly, under isoflurane anesthesia, a hemostat was used to make a closed fracture of the right tibia just distal to the middle of the tibia. The hind limb was then wrapped in casting tape (Delta-Lite; BSN Medical, Hamburg, Germany) so the hip, knee, and ankle were all fixed. The cast extended from the metatarsals of the hind paw up to a spica formed around the abdomen. A window was left open over the dorsal paw and ankle to prevent constriction if post fracture edema developed. After fracture and casting, the mice were given, subcutaneously, 2 days of buprenorphine (0.05 mg/kg) and baytril (5 mg/kg), as well as 1.0 mL of normal saline to maintain hydration. At 3 weeks after surgery, the mice were anesthetized with isoflurane and the cast removed. All mice had union at the fracture site, as assessed using manual inspection.

2.3. Hind paw nociceptive testing

To measure mechanical allodynia in the mice, an up-down von Frey testing paradigm was used as previously described (Li et al., 2018; Li et al., 2014). Briefly, mice were placed on wire mesh platforms in clear cylindrical plastic enclosures 10 cm in diameter and 40 cm in height, and after 15 minutes of acclimation, von Frey fibers of sequentially increasing stiffness were applied against the hind paw plantar skin at approximately midsole, taking care to avoid the tori pads, and pressed upward to cause a slight bend in the fiber and left in place for 5 seconds. Withdrawal of or licking the hind paw after fiber application was scored as a response. When no response was obtained, the next stiffest fiber in the series was applied to the same paw; if a response was obtained, a less stiff fiber was applied. Testing proceeded in this manner until 4 fibers had been applied. Estimation of the mechanical withdrawal threshold by data-fitting algorithm permitted the use of parametric statistics. Hind paw mechanical nociceptive thresholds were analyzed as the difference between the fractured side (right hind paw) and the contralateral unfractured side (left hind paw).

An incapacitance device (IITC Inc. Life Science, Woodland Hills, CA, USA) was used to measure hind paw unweighting. The mice were manually held in a vertical position over the apparatus with the hind paws resting on separate metal scale plates, and the entire weight of the mouse was supported on the hind paws. The duration of each measurement was 6 seconds, and 6 consecutive measurements were taken at 10-second intervals. All 6 readings were averaged to calculate the bilateral hind paw weight-bearing values. Right hindlimb weight-bearing data (the tibia fracture limb) were analyzed as a ratio between the right hindlimb weight bearing and the sum of right and left hindlimb values using the formula: [2R/(R+L)]×100%.

2.4. Passive transfer experiments in the fracture mouse CRPS model

As described previously (Guo et al., 2017; Li et al., 2018), to prepare serum for passive transfer, blood was collected by transcardial puncture in isoflurane anesthetized mice. The blood was left undisturbed at room temperature for 60 min to allow clotting, then the blood samples were centrifuged at 1,500g for 20 min at 4°C and the serum supernatants were aliquoted and frozen at − 80°C.

Serum recipient muMT mice underwent tibia fracture and casting 3 weeks before use with casts removed one day before serum injection. These mice were injected with serum (500 ul/mouse, i.p.) originating from the various experimental groups. Nociceptive behavioral testing was performed at 1, 7, 14, and 21 days after injection.

2.5. Western blot analysis

As described previously (Guo et al., 2017; Li et al., 2018), mouse hind paw skin and spinal cord tissue were harvested and stored at −80 C. Tissues were then homogenized in ice-cold Tris buffer with 0.7% (v/v) β-mercaptoethanol and 10% glycerol. Lysates were centrifuged at 13,000g for 15 minutes at 4 °C, and the protein concentration of the supernatant was measured by a Bio-Rad DC protein assay (Bio-Rad). Equal amounts of protein (50 μg) were size fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane. The blots were blocked overnight with 5% normal serum in Tris-buffered saline with 0.5% Tween-20, and incubated with primary antibodies against immunoglobulin M (IgM) or β-actin (Santa Cruz Biotechnology, Dallas, TX, USA) for 1 hour on a rocking platform at room temperature. After 3 washes, the blots were incubated with secondary antibody for 1 hour at room temperature. The membrane was then washed again, and proteins were detected using ECL chemiluminescence reagent (GE Healthcare, Pittsburgh, PA, USA). The band intensities were quantified using ImageJ (National Institutes of Health, Bethesda, MD, USA), IgM/Actin band intensity ratio was calculated to demonstrate the changes in skin IgM levels at various time points after fracture.

2.6. Popliteal lymph node dissection and size measurement

This protocol quantified the size of popliteal lymph nodes after fracture. The popliteal lymph node is embedded in the adipose tissue of the popliteal fossa. To isolate nodes, mice were euthanized by carbon dioxide asphyxiation followed by cervical dislocation. The skin over the distal injured limb covering the popliteal nodes was incised, and the subcutaneous tissue, fat, and fascia were carefully dissected under a low-power microscope. The lymph node sizes (diameters) were measured in millimeters (mm) using a caliper (Li et al., 2018), with the average diameter for each lymph node defined as [(short-axis diameter + long-axis diameter)/2].

2.7. Fluorescence-activated cell sorting (FACS) analysis

FACS analysis was performed as previously described (Yang et al., 2012). In brief, single cell suspensions were prepared from the spleen, bilateral popliteal lymph nodes and inguinal lymph nodes of wild-type nonfracture mice, wild-type mice at 1, 2, and 3 weeks after fracture, and wild-type 3-week post fracture mice treated with FK506 or vehicle. About 1.25 × 106 cells were incubated with LIVE/DEAD Aqua (Invitrogen), washed, and incubated with unconjugated anti-CD16/CD32 (FcγRII/III) mAb to block Fc-receptors. Cells were then stained on ice for 20 minutes with a “cocktail” of fluorochrome-conjugated antibodies. After washing, 0.1–0.3 × 106 cells were analyzed on Aria (BD Bioscience) at Herzenberg laboratory at Stanford University. Data were analyzed with FlowJo (TreeStar).

Fluorochrome-conjugated antibodies include: anti-CD38-Alexa488 (clone 90, Biolegend), anti-CD43-PE (clone S11, Biolegend), anti-CD5-PE-Cy5 (clone 53–7.3, Biolegend), anti-CD19-PECy5.5 (clone ID3, Invitrogen, cat#35-0193-82), anti-IgG1-PE-Cy7(clone RMG1–1, Biolegend), anti-IgM-APC (clone RMM1, Biolegend), anti-IgD-APC-Cy7(clone 11–26c.2a, Biolegend), anti-CD95-Qdot605 (clone SA367H8, Biolegend), anti-CD11b-PB (clone M1/70, Biolegend), anti-Gr-1-PB(clone RB6–8C5, Biolegend), anti-TCRαβ-PB(clone H57, Invitrogen), anti-CD11c-PB (clone N418, Biolegend), anti-CD3ε-PB (clone 145–2C11, Biolegend), anti-F4/80-PB (clone BM8, Biolegend). Dead, myeloid, and T cells were gated out and live CD19 positive B cells were further characterized (live myeloid− CD3− CD19+) to reveal CD95 and CD38 surface expression. Germinal center B cells were defined as CD19+ CD38− CD95+.

2.8. Tissue processing and immunofluorescence confocal microscopy

Confocal microscopy was performed to detect germinal center formation, Tfh and germinal center B cells in the popliteal lymph nodes. Mice were euthanized and immediately perfused with 4% paraformaldehyde (PFA) in PBS, pH 7.4, via the ascending aorta; the popliteal lymph nodes were removed and postfixed in 4% PFA for 4 hours. The tissues were then treated with 10%, 20%, and 30% sucrose in PBS at 4 °C for 30 min respectively followed by 30% sucrose at 4 °C overnight. The tissue was embedded in optimum cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA, USA), and 6-μm thick slices were made using a cryostat, mounted onto Superfrost microscope slides (Fisher Scientific, Pittsburgh, PA, USA), and stored at −80 °C.

Frozen sections were permeabilized and blocked with “immunomix” staining solution (i.e., 0.3% Triton X-100, 0.2% bovine serum albumin, and 0.1% sodium azide in 1X PBS) containing 5% normal donkey serum and followed by incubated with rat anti-mouse CD16/CD32 (clone 2.4G2, mouse BD Fc block) before primary antibody incubation. Sections were incubated in primary antibody cocktail, i.e., FITC conjugated rat anti-mouse T- and B-cell activation antigen (Clone GL7), 1:100 (BD Pharmingen, USA), Alexa Flour 647 anti-mouse CD279 (PD-1, clone 29F.1A12), 1:100 (BioLegend), and PE rat anti-mouse IgD (clone 11–26c), 1:100 (eBioscience™, USA) diluted in imunomix containing 1% donkey serum at 4 °C overnight. After washing in PBS, the sections were counterstained with DAPI (diluted 1:3000; Thermo Scientific, Waltham, MA, USA) to locate nuclei. After 3 washes, the sections were mounted with anti-fade mounting medium (Invitrogen, Life Technologies). Images were visualized and captured using a confocal microscope (Zeiss LSM710, Carl Zeiss, Jena, Germany). Control experiments included incubation of slices in primary and secondary antibody-free solutions, both of which led to low-intensity nonspecific staining patterns in preliminary experiments.

2.9. Chronic administration of the Tfh cell inhibitor FK506

The calcineurin inhibitor tacrolimus (FK506, Tocris Bioscience) was used to identify the contributions of Tfh cells to the post fracture immune response. Tacrolimus specifically suppresses both lymph node Tfh cells and circulating Tfh cells, but not their regulatory counterparts or other CD4 T cell subsets (Wallin et al., 2018). In the current study fracture mice were treated with FK506 (5 mg/kg) or vehicle via subcutaneous injection 3 times a week (once every other day from Monday to Friday) for 3 weeks, followed by cast removal, nociceptive testing and tissue harvesting. This dosage was derived from prior literature demonstrating FK506 efficacy in reducing autoimmune dermatitis in transgenic mice with mixed connective tissue disease (Loser et al., 2006).

2.10. Statistical analysis

We used 1-way analysis of variance with Bonferroni corrections (Figure 2, 3, 4, 6, 7, and 8), some data were analyzed using 2-way repeated-measures analysis of variance followed by a Holm-Sidak test for post hoc contrasts as detailed in the figure legends (Figure 1 and 9). All data are presented as the mean ± SEM, and differences are considered significant at a P value <0.05 (Prism 6, GraphPad Software, La Jolla, CA, USA).

Figure 2.

Figure 2.

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Changes in Immunoglobulin M (IgM) protein levels in hind paw skin and spinal cord tissue from control mice (NonFX) and 1, (1wkFX), 2 (2wkFX), and 3 weeks (3wkFX) after fracture (FX). IgM protein levels were determined by Western blot analysis. Compared to control no fracture mice, both hindlimb skin and spinal cord IgM levels were not changed at 1 and 2 weeks after fracture, while both were dramatically increased at 3 weeks after fracture. Data were analyzed using a one-way analysis of variance (ANOVA) with Bonferroni correction test for post hoc contrasts. Data are expressed means ± SEM, n = 8 per cohort. ***P < 0.001 vs. control NonFX.

Figure 3.

Figure 3.

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Changes in size of the popliteal lymph node at 1, 2, and 3 weeks after fracture. Post fracture popliteal lymphadenopathy were observed at 1, 2, and 3 weeks after fracture with highest enlargement at 3 weeks after fracture. Data were analyzed using a one-way analysis of variance with Bonferroni correction test for post hoc contrasts, error bars indicate SEM, n = 10 per cohort. ***P < 0.001 vs. NonFX, ###P < 0.001 for ipsilateral (ipsi) to fracture popliteal lymph node vs. contralateral (contra), $$P < 0.01 and $$$P < 0.001 vs. the 2-week and 1-week ipsilateral popliteal lymph node.

Figure 4.

Figure 4.

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Germinal center (GC) B cells start to appear in popliteal lymph nodes of mice around 2 weeks post fracture and further increase 3 weeks post fracture. (A) FACS plots showing GC B cells in popliteal lymph nodes of control mice or mice at 1, 2, 3 weeks after fracture. Live T and myeloid cells-negative, CD19-positive cells (myeloid− CD3− CD19+) are gated to reveal CD95 and CD38 surface expression. GC B cells are defined as (CD19+CD38−CD95+). (B) Absolute numbers of GC B cells in popliteal lymph nodes of control mice or mice at 1, 2, 3 weeks after fracture are shown. Data were analyzed using a one-way analysis of variance with Bonferroni correction test for post hoc contrasts, error bars indicate SEM. ***P < 0.001 for 3wkFX (n=6) vs. control NonFX (n=10), 1wkFX (n=6), or 2wkFX (n=6) values respectively.

Figure 6.

Figure 6.

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FK506 treatment impaired lymph node germinal center (GC) response 3 weeks after fracture. (A) FACS plots showing GC B cells in popliteal lymph nodes of control mice, FX mice treated with vehicle or FK506 for 3 weeks after fracture. Live T and myeloid cells-negative, CD19-positive cells (myeloid− CD3− CD19+) are gated to reveal CD95 and CD38 surface expression. GC B cells are defined as (CD19+CD38+CD95). (B) Absolute numbers of GC B cells in popliteal lymph nodes of control mice or FX mice treated with vehicle or FK506 for 3 weeks after fracture are shown. Data were analyzed using a one-way analysis of variance with Bonferroni correction test for post hoc contrasts, error bars indicate SEM. **P < 0.01 for FX-vehicle (n=5) vs. control-NO FX (n=5), ##P < 0.01 for FX-vehicle (n=5) vs. FX-FK506 (n=4) values.

Figure 7.

Figure 7.

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FK506 treatment reduced immunoglobulin M (IgM) protein levels in mouse hindpaw skin, and spinal cord 3 weeks after fracture (FX). Western blot analysis was performed. Compared with control intact mice treated with vehicle DMSO NonFX, IgM protein levels were greatly increased in skin (A), and spinal cord (B) in wild-type mice ipsilateral to the fracture (3wkFX+Vehicle), but dramatically reduced in 3-week fracture mice treated with FK506 (3wkFX+FK506). Data were analyzed using a one-way analysis of variance with Bonferroni correction for post hoc contrasts. ***P<0.001, for 3wkFX+Vehicle (n=4) vs control. ###P<0.001 for 3wkFX+FK506 (n=4) vs 3wkFX+Vehicle (n=4). Data are expressed as mean values ± SEM.

Figure 8.

Figure 8.

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FK506 (Tacrolimus) treatment alleviated CRPS-like nociceptive changes after fracture (FX).

Mice underwent distal tibia FX with hind limb casting for 3 weeks, then the cast was removed. Behavioral testing performed prior to FX (baseline, BL) and at 1 day after cast removal. One group of fracture mice was treated with vehicle, another fracture group was treated with FK506 to selectively suppress T follicular helper cells for 3 weeks following the fracture. At 3 weeks post-FX the mice treated with vehicle had developed hind paw von Frey allodynia (A), unweighting (B), but the mice treated with FK506 exhibited attenuated allodynia (A) and unweighting (B). A one-way analysis of variance was performed with Bonferroni correction for post hoc contrasts. Data are expressed as mean values ± SEM. ***P<0.001, for 3wkFX+Vehicle (n=12) or 3wkFX+FK506 (n=13) vs its baseline respectively. ###P<0.001 for 3wkFX+FK506 (n=13) vs. 3wkFX+Vehicle (n=12).

Figure 1.

Figure 1.

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Serum from wildtype (WT) 3-week fracture (FX) mice had pronociceptive effects in B cell deficient (muMT) FX mice. When serum from 3-week post-FX WT (3wkFX-serum) mice was injected (500 ul, i.p.) into 3-week post-FX muMT mice, the mice gradually developed increased hindpaw von Frey allodynia (A) and unweighting (B) over the ensuing week, and consistent with the half-life of immunoglobulin, these pronociceptive effects resolved by 2 weeks post-injection. Injecting serum from wildtype control non-FX (NonFX-serum), wildtype 1 (1wkFX-serum) and 2-week FX serum (2wkFX-serum) did not alter hindpaw von Frey allodynia (A) and unweighting (B) in the muMT FX mice. The pronociceptive effects of the 3wkFX-serum were restricted to the FX limb and not observed in nonfractured mice (data not shown). A 2-way repeated measures analysis of variance was performed followed by a Holm-Sidak test for post hoc contrasts. Data are expressed as mean values ± SEM. N=8 per cohort. ###P < 0.001 for 3wkFX-serum vs. NonFX-serum.

Figure 9.

Figure 9.

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Serum from wildtype (WT) fracture (FX) mice treated with FK506 had no pronociceptive effects in B cell deficient (muMT) FX mice. Serum from 3-week post-FX (3wkFX) mice treated with FK506 or vehicle were injected into 3wkFX muMT mice lacking B cells. Injection of sera (500 ul, ip) from 3wkFX WTmice that had vehicle treatment caused increased hindpaw allodynia (A) and unweighting (B) in 3wkFX muMT mice, but sera from 3wkFX mice with FK506 treatment had no effect on allodynia and unweighting in 3wkFX muMT mice. A 2-way repeated measures analysis of variance was performed followed by a Holm-Sidak test for post hoc contrasts. Data are expressed as mean values ± SEM. ##P<0.01 and ###P<0.001 for 3wkFX muMT+3wkFX+FK506 serum (n=8/cohort) vs 3wkFX muMT+3wkFX+vehicle serum (n=7/cohort).

3. Results

3.1. The pronociceptive effects of fracture mouse serum gradually developed over 3 weeks post fracture.

Previously we observed that serum or purified IgM from wildtype mice had pronociceptive effects when passively transferred to B cell deficient (muMT) fracture mice (Guo et al., 2017), an effect observed using material from mice 3 to 18 weeks after fracture. To define the development of the pronociceptive effects, sera from 1, 2, and 3 weeks post fracture wild-type mice were injected into 3 weeks post fracture muMT mice. As shown in Figure 1, the muMT fracture mice receiving 3 weeks wild-type fracture serum, gradually developed increased hindpaw mechanical allodynia and unweighting over the ensuing week. Consistent with the serum half-life of IgM and our previous observations, these effects resolved by 2 weeks post-injection. However, sera from 1- and 2-week post fracture mice had no pronociceptive effects, indicating that a 3-week latency was required for the development of post fracture serum effects.

3.2. Post fracture IgM deposition in the injured hindpaw skin required 3 weeks to develop.

We used western blot analysis to measure the post fracture accumulation of IgM in the hindpaw skin and spinal cord tissue of the injured limb, an index of pain-related autoimmune activity in this model (Guo et al., 2019b). IgM, and not IgG, was previously identified as the pronociceptive immunoglobulin isotype in the serum of fracture mice and in patients with early phase (less than 12 months duration) CRPS (Guo et al., 2019b). As illustrated in Figure 2, IgM levels were increased in fracture limb hindpaw skin at 3 weeks after fracture, but not at 1 or 2 weeks, consistent with the serum transfer studies. These data indicate that 3 weeks are required to fully mount the pronociceptive IgM response after limb fracture.

3.3. Popliteal lymph nodes progressively hypertrophy after tibia fracture.

Lymph node adenopathy suggests activation of immune activity in those structures. We recently observed popliteal lymph node enlargement 3 weeks after fracture in mice (Li et al., 2018). Popliteal and iliac lymph node enlargement has also been observed at 7 days after tibia fracture and casting in rats, and for several months after tibia fracture in man (Szczesny et al., 2007; Szczesny et al., 2004). In this study, as shown in Figure 3, tibia fracture was accompanied by gradual and significant enlargement of popliteal lymph nodes. The average diameter of popliteal lymph nodes was increased by 134%, 175%, and 206% at 1, 2, and 3 weeks after fracture, respectively. These findings are consistent with an early and progressive immune response initiated by limb trauma.

3.4. Fluorescence-activated cell sorting (FACS) analysis revealed a germinal center response in the popliteal lymph nodes of injured limbs.

Germinal centers are secondary follicles that are formed within peripheral lymphoid organs such as lymph nodes in which B cells proliferate and differentiate (Victora and Nussenzweig, 2012). These B cells form antibody-secreting plasma cells and memory B cells thus playing an important role in adaptive immunity. We hypothesized that a germinal center response would be detected in regional lymph nodes after limb fracture. Using fluorescence-activated cell sorting (FACS) analysis, we studied germinal center B cells in lymph node and splenic tissue at 1, 2, and 3 weeks after tibia fracture. FACS analysis showed that germinal center B cells are rare in popliteal lymph nodes of uninjured control mice and in 1-week post fracture mice. In contrast, germinal center B cells started to appear in the ipsilateral popliteal lymph nodes at 2 weeks post fracture and further increased at 3 weeks post fracture (Figure 4A). The absolute number of germinal center B cells (CD38−CD95+) in 3-week fracture mice showed an increase by about 85-fold compared to the intact control values (Figure 4B). Additional studies showed the germinal center response to be limited to the ipsilateral popliteal and to a lesser extent inguinal lymph nodes. No germinal center formation was observed in the spleen (data not shown). Collectively, these data demonstrate that a robust germinal center response is induced in the regional lymph nodes following the limb fracture.

3.5. Immunohistochemical analysis of the popliteal lymph node demonstrated post-fracture germinal center formation.

Immunohistochemical techniques were utilized to examine popliteal lymph nodes at 3 weeks post fracture, the time point of maximal germinal center formation per FACS analysis. Figure 5 illustrates that both germinal center cell types, i.e., the Tfh cells defined as cells expressing high levels of the programmed cell death-1 marker (PD-1) and the germinal center B cells defined as expressing the GL7 marker, were induced in the popliteal lymph nodes. These popliteal lymph node germinal centers were surrounded by immature B cells defined by their expression of IgD. Only rare germinal center cell B cells or Tfh cells were identified in the popliteal lymph nodes of control animals (Figure 5).

Figure 5.

Figure 5.

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Representative fluorescent photomicrographs of immunostaining for DAPI (counterstain to show DNA content and nuclei, Blue) GL7 (a murine germinal center (GC) B-cell marker, green), IgD1 (immature B-cell marker, yellow), and PD1 (T follicular helper cell (Tfh) marker, red) in the popliteal lymph node at 3 weeks post fracture (FX). Top panels are from a popliteal lymph node contralateral to FX of 3-week FX mouse, bottom panels are from the ipsilateral popliteal lymph node 3 weeks after FX. Triple labelling presents activated B-cell (GC B-cell) and Tfh cells surrounded by immature B cells in the ipsilateral to fracture popliteal lymph node 3 weeks after fracture. The GC B-cells associated intimately with Tfh cells. This image demonstrates that fracture induced germinal center formation. Scale bar = 50 um.

3.6. Chronic administration of the Tfh cell inhibitor FK506 impairs the germinal center response and IgM production after tibia fracture.

T follicular helper (Tfh) cells play a central role in the development of the germinal center, where antigen-specific B cells undergo affinity maturation and selection (Crotty, 2011). Tacrolimus (FK506), is a calcineurin inhibitor that selectively disrupts Tfh activity by inhibiting the production of cytokines required for B cell maturation (Wallin et al., 2018). As shown in Figure 6, when fracture mice were treated with FK506 for 3 weeks beginning immediately after injury, they fail to generate germinal center responses in popliteal lymph nodes. In addition, IgM content in skin and spinal cord tissue 3 weeks after injury was profoundly reduced compared to samples harvested from vehicle treated mice (Figure 7).

3.7. Chronic administration of FK506 attenuates fracture-induced nociceptive behavior.

The finding of an impaired germinal center response and reduced tissue accumulation of IgM in FK506 treated fracture mice prompted us to determine what effect this drug had on post-fracture nociceptive changes. As shown in Figure 8, vehicle-treated mice developed hindpaw mechanical allodynia and unweighting at 3 weeks post fracture. However, FK506 treatment started immediately after fracture and continuing to the time of cast removal (3wks) significantly attenuated these nociceptive changes.

3.8. Sera from fracture mice treated with FK506 fail to support nociceptive sensitization in recipient fractured muMT mice.

Having observed that FK506 impairs the germinal center response and reduces both IgM accumulation and nociceptive sensitization in fracture mice, we sought to determine whether FK506 also eliminated the pronociceptive activity of fracture mouse serum. To address this hypothesis, sera were collected from wild-type fracture mice treated for 3 weeks with FK506 or vehicle. These sera were passively transferred to muMT 3-week fracture mice lacking B cells, and nociceptive behavioral tests were followed for 21 days. Consistent with our previous findings, the muMT fracture mice that received serum from vehicle treated wild-type fracture mice gradually developed increased hindpaw von Frey allodynia (Figure 9A) and unweighting (Figure 9B) over the ensuing week, effects that resolved 14–21 days post injection. However, muMT fracture mice receiving sera from FK506 treated wild-type fracture mice did not demonstrate sensitization. Collectively, these FK506 data support the hypothesis that Tfh cells are required for inducing B cell proliferation and maturation into cells capable of producing IgM autoantibodies that in turn support the pain-related changes in the murine fracture model of post-traumatic pain and CRPS.

Discussion

Complex Regional Pain Syndrome (CRPS) is an enigmatic syndrome most often initiated by limb injury or surgery that can result in persistent pain and disability. The mechanisms underlying CRPS are poorly understood and treatment failure is common (Zyluk and Puchalski, 2018). Accumulating evidence suggests that CRPS may involve both autoinflammatory and autoimmune components under control of peripheral neuropeptide and sympathetic signaling (Clark et al., 2018; Goebel, 2016), possibly involving the innervation of lymph nodes by peptidergic sensory neurons and sympathetic fibers (Fink and Weihe, 1988; Huang et al., 2013). We previously observed that B cells are required for the full expression of CRPS-like changes in a tibia fracture mouse model of CRPS (Birklein et al., 2018) and that the passive transfer of purified IgM antibodies (or whole serum) from tibia fracture mice (Guo et al., 2017) or from CRPS patients (Guo et al., 2019b) can produce CRPS-like signs in recipient muMT fracture mice lacking mature B cells. The current study examined the temporal development, localization, and cellular mechanisms supporting injury induced chronic pronociceptive autoimmune responses. Our primary findings included; 1) production of pain-promoting IgM requires approximately 3 weeks to develop after tibia fracture in a laboratory model, 2) regionally restricted lymph node hypertrophy and the formation of germinal centers also occurs over the 3 weeks following limb injury in the model animals, and 3) the pharmacologic disruption of T follicular helper (Tfh)-B cell interactions using the Tfh inhibitor FK506 (tacrolimus) attenuates post-fracture nociceptive sensitization, prevents the formation of germinal B cells, reduces IgM accumulation in the injured limbs, and renders serum from drug treated animals ineffective in transmitting the CRPS pain-related phenotype. Thus, the data collected are all consistent with the notion that a germinal center response requiring about 3 weeks to mature is necessary in order for pain-supporting antibodies to be formed, although this may not be the only inflammatory response supporting pain after limb fracture (Clark et al., 2018). The pronociceptive adaptive immune response from inciting injury to autoantibody formation and resulting persistent pain sensitization is novel and opens new avenues of treatment for CRPS and possibly other post-traumatic pain syndromes.

The expected initial response to major injuries is a strong activation of the innate system of immunity characterized by cytokine and inflammatory mediator generation, and a compensatory suppression of the adaptive system of immunity including components such as B and specific T cell subsets (Hotchkiss et al., 2013; Thompson et al., 2019). Indeed, major trauma is associated with B cell dysfunction and suppressed production of IgM lasting 3 weeks or more (Faist et al., 1989a; Faist et al., 1989b). However, CRPS is most commonly seen not after major trauma, but rather sprains, soft tissue trauma, minor hand and foot surgeries and distal limb fractures such as radial fractures (Corradini et al., 2015; Li et al., 2010). Even minor tissue injury, however, could lead to the production or exposure of neoantigens (Rosen and Casciola-Rosen, 2009). The local production of neoantigens is in keeping with our observations that only ipsilateral lymph node hypertrophy and germinal center formation was seen in our model. Our previous studies have shown the expression of neoantigens such as keratin 16 is enhanced in the ipsilateral limbs of CRPS model fracture mice and IgM accumulation occurs in the ipsilateral but not contralateral limbs of these animals. Importantly, neuropeptide (substance P and CGRP) signaling is required for neoantigen expression and pain-related IgM formation (Li et al., 2018). The innervation of lymph nodes by neuropeptide containing fibers is believed to be a pathway for neuro-immune signaling (Fink and Weihe, 1988; Huang et al., 2013). We also observed that sensitization after serum injection takes about one week to manifest indicating that sufficient accumulation of IgM at its pain-promoting target sites might take some time, and/or that secondary effector pathways such as the activation of complement may require time to reach the level where pain sensitivity is altered.

In addition, fracture or ischemia from immobilization may cause localized oxidative stress that is also linked to autoimmune phenomena through the production of protein adducts, e.g. modification with oxidation-related malondialdehyde (MDA) (Wang et al., 2013). In fact, the production of MDA in the affected limbs has been demonstrated in various laboratory models of CRPS, and measures limiting oxidative stress reduce nociceptive sensitization (Guo et al., 2018; Muthuraman et al., 2010; Salgado et al., 2019). Translational studies involving CRPS patients show evidence of oxidative stress including MDA production lasting several weeks after the inciting limb injuries (Eisenberg et al., 2008). While the aforementioned conditions may explain persistent pain in injured limbs, CRPS occasionally spreads to non-injured tissues. It is conceivable that this is due to the eventual expression of CRPS-related neoepitopes in those limbs that would be accessible to circulating IgM.

Our studies not only delineate the time course of pain-related autoantibody production after tibia fracture, but also suggest germinal center formation might be a target for therapeutic agents. For example, staining of lymph nodes from tibia fracture mice revealed the presence of Tfh cells. These CD4+ T cells assist in the maturation of B cells within the germinal centers, including in autoimmune responses (Seth and Craft, 2019). In order to probe the function of these cells in the autoimmune response to tibia fracture, we used the immunosuppressive drug FK-506, a well-characterized calcineurin inhibitor. Specifically, this drug interacts with an intracellular FK binding protein that in turn inhibits calcineurin’s phosphatase activity preventing activation of the transcription factor NFAT (nuclear factor of activated T cells). Without activation of NFAT, T cells, particularly Tfh cells, lose function (Reynolds and Al-Daraji, 2002). As hypothesized, administration of FK-506 to the fractured mice substantially reduced post fracture pain sensitization, germinal center formation, IgM accumulation and pain-supporting activity of passively transferred IgM from the FK-506 treated wild-type fracture mice to muMT fracture mice. It should be noted that while these data are encouraging regarding the therapeutic potential of FK-506 for CRPS, additional studies involving the timing of drug administration, the required duration of therapy, and the risks of immunosuppression after injuries will need to be undertaken. Furthermore, we focused on the analysis of secondary lymphoid structures (lymph nodes), and cannot exclude contributions of FK506 on circulating Tfh, or Tfh cells in tertiary lymphoid tissues not yet identified in this model; it is known that B cells accumulate in the fracture callus and can be found there weeks after injury (Konnecke et al., 2014).

Our studies focused on the adverse consequences of pain and limb dysfunction supported by IgM autoantibodies after limb injury. In fact, IgM-mediated processes may exacerbate damage after other injuries such as spinal cord injury (Narang et al., 2017) or ischemia/reperfusion (Marshall et al., 2018). However, IgM autoantibodies are abundant and contribute in positive ways to normal physiological functioning. For example, IgM autoantibodies have been shown to enhance the functional recovery from nerve injuries by speeding the clearance of damaged myelin (Vargas et al., 2010). IgM has other useful roles such as in reducing the progression of atherosclerosis by binding to oxidatively-modified low-density lipoprotein (LDL) (Lewis et al., 2009). In fact, the IgM-mediated clearance of the oxidized products of damaged or dying cells has broad value to maintaining homeostasis (Binder, 2010). Thus, the particular disease setting is critical to the contributions of IgM to positive versus adverse outcomes.

The findings of these studies provide new insights into post-traumatic autoimmunity and the mechanisms supporting CRPS and chronic post-traumatic pain. However, there are several limitations that should be recognized. For example, these studies used a single strain of mice, and only male subjects. Sex differences in Tfh cells have been shown to govern the severity of collagen-induced arthritis (Dimitrijevic et al., 2020). Substantial diversity in the functioning of the adaptive immune system is attributable to genetics and sex (Laffont and Guery, 2019), and in fact our own recent work suggest that we might find a more delayed development of adaptive immune changes in female mice (Guo et al., 2019a). The work also leaves unanswered whether and how other mechanisms such as autonomic and sensory neural activation linked to CRPS-related immune responses (David Clark et al., 2018) might control germinal center formation and autoantibody production. In addition, our work has focused on the IgM-related responses characteristic of the first year of CRPS, but has not addressed antibody class switching possibly required for more chronic CRPS IgG-related phenomena (Cuhadar et al., 2019). Indeed, mechanisms such as neuroplastic changes within the CNS may supplant immunological pronociceptive mechanisms in the most chronic phases of the syndrome (Marinus et al., 2011). Regardless of these caveats, the data from our previous post traumatic adaptive immunity studies and the results of the current investigation provide translational support for future immunotherapy trials with calcineurin inhibitors such as FK506, B cell depletion with rituximab, or perhaps even immunological checkpoint therapy with PD-1 ligands for the treatment of post traumatic pain conditions.

Conclusions

The current observations in the context of accumulating experimental evidence suggest that post traumatic pronociceptive adaptive immune responses could potentially play a crucial role in the perpetuation of chronic pain. Understanding the localization, mechanisms and time course of these post traumatic adaptive immune responses could potentially identify unique cellular and molecular targets, thus generating novel approaches for treating chronic pain.

Highlights.

  1. Limb fracture in mice caused pain sensitization accompanied by regional lymph node hypertrophy.

  2. Germinal center formation paralleled IgM accumulation in skin and spinal cord tissue during the 3 weeks after fracture.

  3. The calcineurin inhibitor FK506 blocked the changes in regional lymph nodes, IgM accumulation in skin and spinal cord tissue and pain sensitization.

Funding support:

This work was supported by NIH grant R01 NS094438 (WSK, JDC), VA Merit Review award I01RX001475 (JDC) and R01 AI128839-01(LAH)

Footnotes

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Declaration of Interest: The authors have no disclosures relevant to this work.

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