A Promising New Approach for the Treatment of Inflammatory Pain: Transfer of Stem Cell-Derived Tyrosine Hydroxylase-Positive Cells.

OBJECTIVES: The appearance of endogenous tyrosine hydroxylase-positive cells (TH+ cells) in collagen-induced arthritis was associated with an anti-inflammatory effect. Here we investigated putative anti-inflammatory and antinociceptive effects of the transfer of induced, bone marrow stem cell-derived TH+ cells (iTH+ cells) on murine antigen-induced arthritis (AIA). METHODS: Bone marrow-derived stem cells were differentiated into iTH+ cells. These cells were transferred to mice immunized against methylated bovine serum albumin (mBSA) 2 days before AIA was induced by injection of mBSA into one knee joint. In AIA control mice and iTH+-treated mice the severity of AIA, pain-related behavior, humoral and cellular responses, and the invasion of macrophages into the dorsal root ganglia were assessed. RESULTS: The intravenous transfer of iTH+ cells before AIA induction did not cause a sustained suppression of AIA severity but significantly reduced inflammation-evoked pain-related behavior. The iTH+ cells used for transfer exhibited enormous production of interleukin-4. A major difference between AIA control mice and iTH+-treated AIA mice was a massive invasion of the dorsal root ganglia by iNOS-negative, arginine 1-positive macrophages corresponding to an M2 phenotype. The differences in other cellular and humoral immune parameters such as release of cytokines from stimulated lymphocytes between AIA control mice and iTH+-treated mice were small. CONCLUSIONS: The transfer of iTH+ cells may cause a long-lasting reduction of arthritis-induced pain even if it does not ameliorate inflammation. The invasion of M2 macrophages into the dorsal root ganglia is likely to be an important mechanism of antinociception.

Peripheral elimination of the sympathetic nervous system stimulates immunocyte retention in lymph nodes and ameliorates collagen type II arthritis.

OBJECTIVES: In collagen type II-induced arthritis (CIA), early activation of the sympathetic nervous system (SNS) is proinflammatory. Here, we wanted to find new target organs contributing to proinflammatory SNS effects. In addition, we wanted to clarify the importance of SNS-modulated immunocyte migration. METHODS: A new technique termed spatial energy expenditure configuration (SEEC) was developed to demonstrate bodily areas of high energy demand (to find new targets). We studied homing of labeled cells in vivo, lymphocyte expression of CCR7, supernatant concentration of CCL21, and serum levels of sphingosine-1-phosphate (S1P) in sympathectomized control/arthritic animals. RESULTS: During the course of arthritis, SEEC identified an early marked increase of energy expenditure in draining lymph nodes and spleen (nowhere else!). Although early sympathectomy ameliorated later disease, early sympathectomy increased energy consumption, organ weight, and cell numbers in arthritic secondary lymphoid organs, possibly a sign of lymphocyte retention (also in controls). Elimination of the SNS retained lymph node cells, elevated expression of CCR7 on lymph node cells, and increased CCL21. Serum levels of S1P, an important factor for lymphocyte egress, were higher in arthritic than control animals. Sympathectomy decreased S1P levels in arthritic animals to control levels. Transfer of retained immune cells from draining lymph nodes of sympathectomized donors to sympathectomized recipients markedly increased arthritis severity over weeks. CONCLUSIONS: By using the SEEC technique, we identified draining lymph nodes and spleen as major target organs of the SNS. The data show that the SNS increases egress of lymphocytes from draining lymph nodes to stimulate arthritic inflammation.

Catecholaminergic-to-cholinergic transition of sympathetic nerve fibers is stimulated under healthy but not under inflammatory arthritic conditions.

OBJECTIVE: Density of sympathetic nerve fibers decreases in inflamed arthritic tissue tested by immunoreactivity towards tyrosine-hydroxylase (TH, catecholaminergic key enzyme). Since sympathetic nerve fibers may change phenotype from catecholaminergic to cholinergic (example: sweat glands), loss of nerve fibers may relate to undetectable TH. We aimed to investigate possible catecholaminergic-to-cholinergic transition of sympathetic nerve fibers in synovial tissue of animals with arthritis, and patients with rheumatoid arthritis (RA) and osteoarthritis (OA), and we wanted to find a possible transition factor. METHODS: Nerve fibers were detected by immunofluorescence towards TH (catecholaminergic) and vesicular acetylcholine transporter (cholinergic). Co-culture experiments with sympathetic ganglia and lymphocytes or osteoclast progenitors were designed to find stimulators of catecholaminergic-to-cholinergic transition (including gene expression profiling). RESULTS: In mouse joints, an increased density of cholinergic relative to catecholaminergic nerve fibers appeared towards day 35 after immunization, but most nerve fibers were located in healthy joint-adjacent skin or muscle and almost none in inflamed synovial tissue. In humans, cholinergic fibers are more prevalent in OA synovial tissue than in RA. Co-culture of sympathetic ganglia with osteoclast progenitors obtained from healthy but not from arthritic animals induced catecholaminergic-to-cholinergic transition. Osteoclast mRNA microarray data indicated that leukemia inhibitory factor (LIF) is a candidate transition factor, which was confirmed in ganglia experiments, particularly, in the presence of progesterone. CONCLUSION: In humans and mice, catecholaminergic-to-cholinergic sympathetic transition happens in less inflamed tissue but not in inflamed arthritic tissue. Under healthy conditions, presence of cholinergic sympathetic nerve fibers may support the cholinergic anti-inflammatory influence recently described.

MHC/class-II-positive cells inhibit corticosterone of adrenal gland cells in experimental arthritis: a role for IL-1ß, IL-18, and the inflammasome.

In experimental arthritis, glucocorticoid secretion is inadequate relative to inflammation. We hypothesized that IL-1 is a key factor for inadequate glucocorticoid secretion in arthritic rats. Collagen type II-induced arthritis (CIA) in DA rats was the model to study effects of IL-1 on adrenal function. In the CIA model, an increase of intraadrenal MHCII-positive cells was observed. MHCII-positive cells or bone marrow-derived dendritic cells inhibited glucocorticoid secretion of adrenal gland cells. IL-1, but also IL-18 and the inflammasome were critical in glucocorticoid inhibition. Arthritic compared to control adrenal gland cells produced higher amounts of CXC chemokines from MHCII+ adrenal cells, particularly CINC-2, which is strongly dependent on presence of IL-1. In CIA, macrophages and/or dendritic cells inhibit glucocorticoid secretion via IL-1 in adrenal glands. These findings show that activated macrophages and/or dendritic cells inhibit glucocorticoid secretion in experimental arthritis and that IL-1ß is a decisive factor.

Learned Immunosuppressive Placebo Response Attenuates Disease Progression in a Rodent Model of Rheumatoid Arthritis.

OBJECTIVE: Patients with chronic inflammatory autoimmune diseases benefit from a broad spectrum of immunosuppressive and antiproliferative medication available today. However, nearly all of these therapeutic compounds have unwanted toxic side effects. Recent knowledge about the neurobiology of placebo responses indicates that associative learning procedures can be utilized for dose reduction in immunopharmacotherapy while simultaneously maintaining treatment efficacy. This study was undertaken to examine whether and to what extent a 75% reduction of pharmacologic medication in combination with learned immunosuppression affects the clinical outcome in a rodent model of type II collagen-induced arthritis. METHODS: An established protocol of taste-immune conditioning was applied in a disease model of chronic inflammatory autoimmune disease (type II collagen-induced arthritis) in rats, where a novel taste (saccharin; conditioned stimulus [CS]) was paired with an injection of the immunosuppressive drug cyclosporin A (CSA) (unconditioned stimulus [US]). Following conditioning with 3 CS/US pairings (acquisition), the animals were immunized with type II collagen and Freund's incomplete adjuvant. Fourteen days later, at the first occurrence of clinical symptoms, retrieval was started by presenting the CS together with low-dose CSA as reminder cues to prevent the conditioned response from being extinguished. RESULTS: This "memory-updating" procedure stabilized the learned immune response and significantly suppressed disease progression in immunized rats. Clinical arthritis score and histologic inflammatory symptoms (both P < 0.05) were significantly diminished by learned immunosuppression in combination with low-dose CSA (25% of the full therapeutic dose) via ß-adrenoceptor-dependent mechanisms, to the same extent as with full-dose (100%) pharmacologic treatment. CONCLUSION: These results indicate that learned immunosuppression appears to be mediated via ß-adrenoceptors and might be beneficial as a supportive regimen in the treatment of chronic inflammatory autoimmune diseases by diminishing disease exacerbation.

Cervical vagal nerve stimulation impairs glucose tolerance and suppresses insulin release in conscious rats.

Previously, we reported that cervical vagal nerve stimulation (VNS) increases blood glucose levels and inhibits insulin secretion in anesthetized rats through afferent signaling. Since afferent signaling is also thought to mediate the therapeutic effects of VNS in patients with therapy-refractory epilepsy and major depression, the question arises if patients treated with VNS develop impaired glucose tolerance. Thus, we hypothesized that cervical VNS impairs glucose tolerance in conscious rats. Rats (n = 7) were instrumented with telemetric blood pressure sensors and right- or left-sided cervical vagal nerve stimulators (3 V, 5 Hz, 1 msec pulse duration, 1 h on 1 h off). Glucose tolerance tests (GTTs, 1.5 g dextrose/kg BW, i.p.) were performed after overnight fasting with the stimulators on or off (sham stimulation) in randomized order separated by 3-4 days. Overnight VNS did not alter mean levels of blood pressure or heart rate, but increased fasted blood glucose levels (140 ± 13 mg/dL vs. 109 ± 8 mg/dL, P < 0.05). The area under the blood glucose concentration curves of the GTTs was larger during VNS than sham stimulation (3499 ± 211 mg/dL*h vs. 1810 ± 234 mg/dL*h, P < 0.05). One hour into the GTTs, the serum insulin concentrations had decreased during VNS (-0.57 ± 0.25 ng/mL, P < 0.05) and increased during sham stimulation (+0.71 ± 0.15 ng/mL, P < 0.05) compared to the fasted baseline levels. These results demonstrate that chronic cervical VNS elevates fasted blood glucose levels and impairs glucose tolerance likely through inhibition of glucose-induced insulin release in conscious rats. It remains to be determined if patients treated with VNS are at greater risk of developing glucose intolerance and type 2 diabetes.

Selective Activation of Tumor Necrosis Factor Receptor II Induces Antiinflammatory Responses and Alleviates Experimental Arthritis.

OBJECTIVE: Treg cells modulate immune responses and can suppress the development of autoimmune diseases. Tumor necrosis factor receptor II (TNFRII) has been recognized as a key receptor on these cells that facilitates expansion and stabilization of CD4+ Treg cells. The purpose of the present study was to investigate the therapeutic activity of a novel TNFRII agonist in experimental arthritis as well as the role of different Treg cell subsets. METHODS: A novel mouse TNFRII-selective fusion protein (EHD2-sc-mTNFR2 ) was generated by genetic engineering. Mouse T cells were incubated together with interleukin-2 and/or EHD2-sc-mTNFR2 , and the effects on Treg cells were analyzed by flow cytometry. Mice with collagen-induced arthritis (CIA) were treated with EHD2-sc-mTNFR2 or saline, and the therapeutic effects were monitored and characterized. RESULTS: Selective activation of TNFRII was found to expand both CD4+ and CD8+ Treg cells. Moreover, TNFRII activation elevated the number of CD4+CD25+ and CD8+CD25+ Treg cells and increased the number of FoxP3-expressing cells in CD8+, but not CD4+, Treg cells, indicating different mechanisms of TNFRII-induced expansion of diverse T cell subsets with suppressive activity. In the CIA model, we demonstrated that administration of the TNFRII agonist EHD2-sc-mTNFR2 led to the expansion of both CD4+ and CD8+ Treg cells in vivo and induced antiinflammatory responses that alleviated arthritis. CONCLUSION: Our findings support the use of TNFRII-selective therapeutics as an effective approach to the treatment of arthritic disease and possibly other inflammatory and autoimmune diseases.

Marbostat-100 Defines a New Class of Potent and Selective Antiinflammatory and Antirheumatic Histone Deacetylase 6 Inhibitors.

Epigenetic modifiers of the histone deacetylase (HDAC) family contribute to autoimmunity, cancer, HIV infection, inflammation, and neurodegeneration. Hence, histone deacetylase inhibitors (HDACi), which alter protein acetylation, gene expression patterns, and cell fate decisions, represent promising new drugs for the therapy of these diseases. Whereas pan-HDACi inhibit all 11 Zn2+-dependent histone deacetylases (HDACs) and cause a broad spectrum of side effects, specific inhibitors of histone deacetylase 6 (HDAC6i) are supposed to have less side effects. We present the synthesis and biological evaluation of Marbostats, novel HDAC6i that contain the hydroxamic acid moiety linked to tetrahydro-ß-carboline derivatives. Our lead compound Marbostat-100 is a more potent and more selective HDAC6i than previously established well-characterized compounds in vitro as well as in cells. Moreover, Marbostat-100 is well tolerated by mice and effective against collagen type II induced arthritis. Thus, Marbostat-100 represents a most selective known HDAC6i and the possibility for clinical evaluation of a HDAC isoform-specific drug.

Predominance of synovial sensory nerve fibers in arthrofibrosis following total knee arthroplasty compared to osteoarthritis of the knee.

BACKGROUND: So far, there exists no golden standard for the treatment of arthrofibrosis (AF) following total knee arthroplasty (TKA). Although pain is a hallmark of AF, nociceptive nerve fibers have never been investigated in affected joint tissue. METHODS: A total of 24 patients with osteoarthritis (OA) of the knee (n = 12) and post-TKA AF of the knee (n = 12) were included. Along evaluation of typical clinical signs and symptoms by using the Knee Society Clinical Rating System (KSS), the Knee Injury and Osteoarthritis Outcome Score (KOOS), and the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC index), the innervation of joint tissue was studied by semiquantitative immunofluorescence of nerve fibers. RESULTS: Patients with AF compared to OA had a lower KSS and lower KOOS. In all compartments (anterior, medial, and lateral recesses), the density of synovial sympathetic nerve fibers was significantly higher in OA compared to AF, which was also true for the density of sensory nerve fibers in the medial and lateral recesses. In synovial tissue of the anterior recess of patients with AF compared to OA, the density of nociceptive sensory nerve fibers was significantly higher relative to sympathetic nerve fibers. This was similarly observed in the neighboring infrapatellar fat pad of the knee. CONCLUSIONS: Similar as in many painful musculoskeletal diseases, this study indicates that patients with arthrofibrosis of the knee after TKA demonstrate a preponderance of profibrotic sensory nerve fibers over antifibrotic sympathetic nerve fibers. This could serve as a starting point for AF therapy with specific antifibrotic pain medication or regional anesthetic techniques.

Sympathetic Neurotransmitters Modulate Osteoclastogenesis and Osteoclast Activity in the Context of Collagen-Induced Arthritis.

Excessive synovial osteoclastogenesis is a hallmark of rheumatoid arthritis (RA). Concomitantly, local synovial changes comprise neuronal components of the peripheral sympathetic nervous system. Here, we wanted to analyze if collagen-induced arthritis (CIA) alters bone marrow-derived macrophage (BMM) osteoclastogenesis and osteoclast activity, and how sympathetic neurotransmitters participate in this process. Therefore, BMMs from Dark Agouti rats at different CIA stages were differentiated into osteoclasts in vitro and osteoclast number, cathepsin K activity, matrix resorption and apoptosis were analyzed in the presence of acetylcholine (ACh), noradrenaline (NA) vasoactive intestinal peptide (VIP) and assay-dependent, adenylyl cyclase activator NKH477. We observed modulation of neurotransmitter receptor mRNA expression in CIA osteoclasts without affecting protein level. CIA stage-dependently altered marker gene expression associated with osteoclast differentiation and activity without affecting osteoclast number or activity. Neurotransmitter stimulation modulated osteoclast differentiation, apoptosis and activity. VIP, NA and adenylyl cyclase activator NKH477 inhibited cathepsin K activity and osteoclastogenesis (NKH477, 10(-6) M NA) whereas ACh mostly acted pro-osteoclastogenic. We conclude that CIA alone does not affect metabolism of in vitro generated osteoclasts whereas stimulation with NA, VIP plus specific activation of adenylyl cyclase induced anti-resorptive effects probably mediated via cAMP signaling. Contrary, we suggest pro-osteoclastogenic and pro-resorptive properties of ACh mediated via muscarinic receptors.

Monoamine neurotransmitters as substrates for novel tick sulfotransferases, homology modeling, molecular docking, and enzyme kinetics.

Blacklegged ticks (Ixodes scapularis) transmit the causative agent of Lyme disease in the Northeastern United States. Current research focuses on elucidating biochemical pathways that may be disrupted to prevent pathogen transmission, thereby preventing disease. Genome screening reported transcripts coding for two putative sulfotransferases in whole tick extracts of the nymphal and larval stages. Sulfotransferases are known to sulfonate phenolic and alcoholic receptor agonists such as 17ß-estradiol, thereby inactivating the receptor ligands. We used bioinformatic approaches to predict substrates for Ixosc Sult 1 and Ixosc Sult 2 and tested the predictions with biochemical assays. Homology models of 3D protein structure were prepared, and visualization of the electrostatic surface of the ligand binding cavities showed regions of negative electrostatic charge. Molecular docking identified potential substrates including dopamine, R-octopamine and S-octopamine, which docked into Ixosc Sult 1 with favorable binding affinity and correct conformation for sulfonation. Dopamine, but not R- or S-octopamine, also docked into Ixosc Sult 2 in catalytic binding mode. The predictions were confirmed using cytosolic fractions of whole tick extracts. Dopamine was a good substrate (K(m) = 0.1-0.4 µM) for the native Ixodes scapularis sulfotransferases from larval and nymphal stages regardless of their fed/unfed status. Octopamine sulfonation was detected only after feeding when gene expression data suggests that Ixosc Sult 1 is present. Because dopamine is known to stimulate salivation in ticks through receptor stimulation, these results imply that the function(s) of Ixosc Sult 1 or 2 may include inactivation of the salivation signal via sulfonation of dopamine and/or octopamine.