Halina Machelska

Introduction: Knee osteoarthritis (OA) is a chronic, degenerative joint disorder characterized by cartilage deterioration and persistent pain. Currently, there are no disease-modifying drugs and pharmacological treatments are unsatisfactory for OA. Objective: To investigate the therapeutic effects of whole-body vibration (WBV), a nondemanding physical intervention, on pain behaviors and cartilage morphology in male and female mice with knee OA. Methods: A medial meniscal transection (MMT) was used as a model that mimics meniscal tear–induced knee OA in patients. Animals were exposed to WBV (15-Hz frequency, 332-µm amplitude, 0.3 g acceleration) for 15 minutes per day, 5 days per week, for 13 weeks, beginning 3 weeks after MMT, using a custom-made WBV device. Behavioral analysis included assessment of mechanical sensitivity (by von Frey filaments), the hind limb use (by dynamic weight-bearing), heat sensitivity (by Hargreaves test), locomotor activity (in open field boxes), and body weight. Microcomputed tomography was used to examine cartilage morphology. Results: Medial meniscal transection triggered mechanical hypersensitivity, reduced OA limb use, and induced cartilage damage in the medial and lateral compartments of femur and tibia, in both sexes. Whole-body vibration substantially alleviated mechanical hypersensitivity and improved limb use for up to 4 months after MMT, without affecting heat sensitivity, locomotor activity, or body weight. Furthermore, WBV considerably reduced the percentage of subchondral bone denuded of cartilage and increased cartilage thickness in the femur and tibia. Conclusion: These findings highlight the therapeutic potential of WBV as a nonpharmacological approach to relieve pain and restore cartilage integrity in knee OA.

Background, objectives and design: Arachidonic acid 15-lipoxygenase (ALOX15) has been implicated in the pathogenesis of inflammatory diseases but since pro- and anti-inflammatory roles have been suggested, the precise function of this enzyme is still a matter of discussion. To contribute to this discussion, we created transgenic mice, which express human ALOX15 under the control of the activating protein 2 promoter (aP2-ALOX15 mice) and compared the sensitivity of these gain-of-function animals in two independent mouse inflammation models with Alox15-deficient mice (loss-of-function animals) and wildtype control animals. Materials and methods: Transgenic aP2-ALOX15 mice were tested in comparison with Alox15 knockout mice (Alox15^−/−) and corresponding wildtype control animals (C57BL/6J) in the complete Freund’s adjuvant induced hind-paw edema model and in the dextran sulfate sodium induced colitis (DSS-colitis) model. In the paw edema model, the degree of paw swelling and the sensitivity of the inflamed hind-paw for mechanic (von Frey test) and thermal (Hargreaves test) stimulation were quantified as clinical readout parameters. In the dextran sodium sulfate induced colitis model the loss of body weight, the colon lengths and the disease activity index were determined. Results: In the hind-paw edema model, systemic inactivation of the endogenous Alox15 gene intensified the inflammatory symptoms, whereas overexpression of human ALOX15 reduced the degree of hind-paw inflammation. These data suggest anti-inflammatory roles for endogenous and transgenic ALOX15 in this particular inflammation model. As mechanistic reason for the protective effect downregulation of the pro-inflammatory ALOX5 pathways was suggested. However, in the dextran sodium sulfate colitis model, in which systemic inactivation of the Alox15 gene protected female mice from DSS-induced colitis, transgenic overexpression of human ALOX15 did hardly impact the intensity of the inflammatory symptoms. Conclusion: The biological role of ALOX15 in the pathogenesis of inflammation is variable and depends on the kind of the animal inflammation model.

Mammalian arachidonic acid lipoxygenases (ALOXs) have been implicated in the pathogenesis of inflammatory diseases, and its pro- and anti-inflammatory effects have been reported for different ALOX-isoforms. Human ALOX15B oxygenates arachidonic acid to its 15-hydroperoxy derivative, whereas the corresponding 8-hydroperoxide is formed by mouse Alox15b (Alox8). This functional difference impacts the biosynthetic capacity of the two enzymes for creating pro- and anti-inflammatory eicosanoids. To explore the functional consequences of the humanization of the reaction specificity of mouse Alox15b in vivo, we tested Alox15b knock-in mice that express the arachidonic acid 15-lipoxygenating Tyr603Asp and His604Val double mutant of Alox15b, instead of the arachidonic acid 8-lipoxygenating wildtype enzyme, in two different animal inflammation models. In the dextran sodium sulfate-induced colitis model, female Alox15b-KI mice lost significantly more bodyweight during the acute phase of inflammation and recovered less rapidly during the resolution phase. Although we observed significant differences in the colonic levels of selected pro- and anti-inflammatory eicosanoids during the time-course of inflammation, there were no differences between the two genotypes at any time-point of the disease. In Freund’s complete adjuvant-induced paw edema model, Alox15b-KI mice were less susceptible than outbred wildtype controls, though we did not observe significant differences in pain perception (Hargreaves-test, von Frey-test) when the two genotypes were compared. our data indicate that humanization of the reaction specificity of mouse Alox15b (Alox8) sensitizes mice for dextran sodium sulfate-induced experimental colitis, but partly protects the animals in the complete Freund’s adjuvant-induced paw edema model.

Arachidonic acid 5-lipoxygenase (ALOX5) is the key enzyme in the biosynthesis of pro-inflammatory leukotrienes. We recently created knock-in mice (Alox5-KI) which express an arachidonic acid 15-lipoxygenating Alox5 mutant instead of the 5-lipoxygenating wildtype enzyme. These mice were leukotriene deficient but exhibited an elevated linoleic acid oxygenase activity. Here we characterized the polyenoic fatty acid metabolism of these mice in more detail and tested the animals in three different experimental inflammation models. In experimental autoimmune encephalomyelitis (EAE), Alox5-KI mice displayed an earlier disease onset and a significantly higher cumulative incidence rate than wildtype controls but the clinical score kinetics were not significantly different. In dextran sodium sulfate-induced colitis (DSS) and in the chronic constriction nerve injury model (CCI), Alox5-KI mice performed like wildtype controls with similar genetic background. These results were somewhat surprising since in previous loss-of-function studies targeting leukotriene biosynthesis (Alox5^−/− mice, inhibitor studies), more severe inflammatory symptoms were observed in the EAE model but the degree of inflammation in DSS colitis was attenuated. Taken together, our data indicate that these mutant Alox5-KI mice respond differently in two models of experimental inflammation than Alox5^−/− animals tested previously in similar experimental setups.

Interleukin-4 (IL-4) is an anti-inflammatory cytokine, which can be protective in inflammatory and neurologic disorders, and can alleviate pain. Classically, IL-4 diminishes pain by blocking the production of proinflammatory cytokines. Here, we uncovered that IL-4 induces acute antinociception by IL-4 receptor α (IL-4Rα)-dependent release of opioid peptides from M1 macrophages at injured nerves. As a model of pathologic pain, we used a chronic constriction injury (CCI) of the sciatic nerve in male mice. A single application of IL-4 at the injured nerves (14 d following CCI) attenuated mechanical hypersensitivity evaluated by von Frey filaments, which was reversed by co-injected antibody to IL-4Rα, antibodies to opioid peptides such as Met-enkephalin (ENK), β-endorphin and dynorphin A 1–17, and selective antagonists of δ-opioid, µ-opioid, and κ-opioid receptors. Injured nerves were predominately infiltrated by proinflammatory M1 macrophages and IL-4 did not change their numbers or the phenotype, assessed by flow cytometry and qRT-PCR, respectively. Macrophages isolated from damaged nerves by immunomagnetic separation (IMS) and stimulated with IL-4 dose dependently secreted all three opioid peptides measured by immunoassays. The IL-4-induced release of ENK was diminished by IL-4Rα antibody, intracellular Ca²⁺chelator, and inhibitors of protein kinase A (PKA), phosphoinositide 3-kinase (PI3K), and ryanodine receptors. Together, we identified a new opioid mechanism underlying the IL-4-induced antinociception that involves PKA-mediated, PI3K-mediated, ryanodine receptor-mediated, and intracellular Ca²⁺-mediated release from M1 macrophages of opioid peptides, which activate peripheral opioid receptors in injured tissue.SIGNIFICANCE STATEMENTInterleukin-4 (IL-4) is an anti-inflammatory cytokine, which can ameliorate pain. The IL-4-mediated effects are considered to mostly result from the inhibition of the production of proinflammatory mediators (e.g., IL-1β, tumor necrosis factor, prostaglandin E2). Here, we found that IL-4 injected at the injured nerves attenuates pain by releasing opioid peptides from the infiltrating macrophages in mice. The opioids were secreted by IL-4 in the intracellular Ca²⁺-dependent manner and activated local peripheral opioid receptors. These actions represent a novel mode of IL-4 action, since its releasing properties have not been so far reported. Importantly, our findings suggest that the IL-4–opioid system should be targeted in the peripheral damaged tissue, since this can be devoid of central and systemic side effects.

The newly designed fentanyl derivative [( ±)-N-(3-fluoro-1-phenethylpiperidine-4-yl)-N-phenyl propionamide] (NFEPP) was recently shown to produce analgesia selectively via peripheral mu-opioid receptors (MOR) at acidic pH in rat inflamed tissues. Here, we examined the pH-dependency of NFEPP binding to brain MOR and its effects on bone cancer-induced pain in mice. The IC₅₀ of NFEPP to displace bound [³H]-DAMGO was significantly higher compared to fentanyl at pH 7.4, but no differences were observed at pH 5.5 or 6.5. Intravenous NFEPP (30–100 nmol/kg) or fentanyl (17–30 nmol/kg) inhibited heat hyperalgesia in mice inoculated with B16-F10 melanoma cells. The peripherally-restricted opioid receptor antagonist naloxone-methiodide reversed the effect of NFEPP (100 nmol/kg), but not of fentanyl (30 nmol/kg). The antihyperalgesic effect of NFEPP was abolished by a selective MOR- (cyprodime), but not delta- (naltrindole) or kappa- (nor-binaltorphimine) receptor antagonists. Ten-fold higher doses of NFEPP than fentanyl induced maximal antinociception in mice without tumors, which was reversed by the non-restricted antagonist naloxone, but not by naloxone-methiodide. NFEPP also reduced heat hyperalgesia produced by fibrosarcoma- (NCTC 2472) or prostate cancer-derived (RM1) cells. These data demonstrate the increased affinity of NFEPP for murine MOR at low pH, and its ability to inhibit bone cancer-induced hyperalgesia through peripheral MOR. In mice, central opioid receptors may be activated by ten-fold higher doses of NFEPP.

Immunohistochemical staining is widely used to identify opioid receptors in specific cell types throughout the nervous system. Opioid receptors are not restricted to the central nervous system, but are also present in peripheral sensory neurons, where their activation exerts analgesic effects without inducing centrally mediated side effects. Here, we describe immunohistochemical analysis of μ-opioid receptors in the peripheral sensory neuron cell bodies, along the axons and their peripheral endings in the hind paw skin, as well as in the spinal cord, under naïve and sciatic nerve damage conditions in mice. Importantly, we consider the ongoing debate on the specificity of antibodies.

Analgesia mediated by immune cell-derived opioid peptides (β-endorphin, enkephalins, dynorphin), which activate opioid receptors on peripheral sensory neurons in injured tissue, represents a powerful endogenous pain control that is devoid of side effects induced by currently available medications. This intrinsic pain inhibition has been discovered in animal models of pathological pain and translated to patients with postoperative pain (see Machelska and Celik, 2020 for a recent review {1}). The study by Aggarwal et al. extends this concept and elegantly shows that opioid peptide-containing immune cells play a role in chronic clinical pain in HIV-positive people. Specifically, the authors found that HIV-positive people with chronic widespread pain had substantially lower levels of β-endorphin in blood plasma and leukocytes compared to people without pain and to HIV-negative people with or without pain. These effects were associated with elevated cell-free heme in plasma. The authors then confirmed these findings in a mouse model of phenylhydrazine hydrochloride (PHZ)-induced hemolysis. In agreement with clinical findings, PHZ-treated mice had decreased concentrations of β-endorphin in plasma and leukocytes, and more severe mechanical pain compared to control, healthy mice.

In this interesting study, the authors generated and examined effects of morphine in humanized mice expressing human mu-opioid receptor. The authors found different expression levels of mu-receptors in various brain regions between humanized and wild-type mice, more potent and longer-lasting systemic morphine-induced antinociception, but comparable degree constipation. A particularly interesting finding is that morphine decreased locomotor activity suggesting sedation in humanized mice, which is similar to humans, but it contrasts hyperlocomotion observed in wild-type mice. Additionally, morphine caused less antinociceptive tolerance in humanized mice, which is in line with clinical data that analgesic tolerance might not be the major problem in humans. All experiments were performed in mice without tissue injury, and thus, it is important to test the humanized mice in models of pathological pain in the future.