The ability of an individual to sense pain is fundamental for its capacity to adapt to its environment and to avoid damage. The sensation of pain can be enhanced by acute or chronic inflammation. In the present study, we have investigated whether inflammatory pain, as measured by hypernociceptive responses, was modified in the absence of the microbiota. To this end, we evaluated mechanical nociceptive responses induced by a range of inflammatory stimuli in germ-free and conventional mice. Our experiments show that inflammatory hypernociception induced by carrageenan, lipopolysaccharide, TNF-α, IL-1β, and the chemokine CXCL1 was reduced in germ-free mice. In contrast, hypernociception induced by prostaglandins and dopamine was similar in germ-free or conventional mice. Reduction of hypernociception induced by carrageenan was associated with reduced tissue inflammation and could be reversed by reposition of the microbiota or systemic administration of lipopolysaccharide. Significantly, decreased hypernociception in germ-free mice was accompanied by enhanced IL-10 expression upon stimulation and could be reversed by treatment with an anti-IL-10 antibody. Therefore, these results show that contact with commensal microbiota is necessary for mice to develop inflammatory hypernociception. These findings implicate an important role of the interaction between the commensal microbiota and the host in favoring adaptation to environmental stresses, including those that cause pain.

Objective: Deposition of monosodium urate monohydrate (MSU) crystals in the joints promotes an intense inflammatory response and joint dysfunction. This study evaluated the role of the NLRP3 inflammasome and 5‐lipoxygenase (5‐LOX)–derived leukotriene B₄(LTB₄) in driving tissue inflammation and hypernociception in a murine model of gout.Methods: Gout was induced by injecting MSU crystals into the joints of mice. Wild‐type mice and mice deficient in NLRP3, ASC, caspase 1, interleukin‐1β (IL‐1β), IL‐1 receptor type I (IL‐1RI), IL‐18R, myeloid differentiation factor 88 (MyD88), or 5‐LOX were used. Evaluations were performed to assess neutrophil influx, LTB₄activity, cytokine (IL‐1β, CXCL1) production (by enzyme‐linked immunosorbent assay), synovial microvasculature cell adhesion (by intravital microscopy), and hypernociception. Cleaved caspase 1 and production of reactive oxygen species (ROS) were analyzed in macrophages by Western blotting and fluorometric assay, respectively.Results: Injection of MSU crystals into the knee joints of mice induced neutrophil influx and neutrophil‐dependent hypernociception. MSU crystal–induced neutrophil influx was CXCR2‐dependent and relied on the induction of CXCL1 in an NLRP3/ASC/caspase 1/IL‐1β/MyD88–dependent manner. LTB₄was produced rapidly after injection of MSU crystals, and this was necessary for caspase 1–dependent IL‐1β production and consequent release of CXCR2‐acting chemokines in vivo. In vitro, macrophages produced LTB₄after MSU crystal injection, and LTB₄was relevant in the MSU crystal–induced maturation of IL‐1β. Mechanistically, LTB₄drove MSU crystal–induced production of ROS and ROS‐dependent activation of the NLRP3 inflammasome.Conclusion: These results reveal the role of the NLRP3 inflammasome in mediating MSU crystal–induced inflammation and dysfunction of the joints, and highlight a previously unrecognized role of LTB₄in driving NLRP3 inflammasome activation in response to MSU crystals, both in vitro and in vivo.

Morphine is one of the most prescribed and effective drugs used for the treatment of acute and chronic pain conditions. In addition to its central effects, morphine can also produce peripheral analgesia. However, the mechanisms underlying this peripheral action of morphine have not yet been fully elucidated. Here, we show that the peripheral antinociceptive effect of morphine is lost in neuronal nitric-oxide synthase null mice and that morphine induces the production of nitric oxide in primary nociceptive neurons. The activation of the nitric-oxide pathway by morphine was dependent on an initial stimulation of PI3Kγ/AKT protein kinase B (AKT) and culminated in increased activation of K _ATP channels. In the latter, this intracellular signaling pathway might cause a hyperpolarization of nociceptive neurons, and it is fundamental for the direct blockade of inflammatory pain by morphine. This understanding offers new targets for analgesic drug development.

Activation of the renin-angiotensin (Ang) system induces inflammation via interaction between Ang II and type 1 receptor on leukocytes. The relevance of the new arm of the renin-Ang system, namely Ang-converting enzyme-2/Ang-(1–7)/Mas receptor, for inflammatory responses is not known and was investigated in this study. For this purpose, two experimental models were used: Ag-induced arthritis (AIA) in mice and adjuvant-induced arthritis (AdIA) in rats. Male C57BL/6 wild-type or Mas^−/− mice were subjected to AIA and treated with Ang-(1–7), the Mas agonist AVE 0991, or vehicle. AdIA was performed in female rats that were given AVE 0991 or vehicle. In wild-type mice, Mas protein is expressed in arthritic joints. Administration of AVE 0991 or Ang-(1–7) decreased AIA-induced neutrophil accumulation, hypernociception, and production of TNF-α, IL-1β, and CXCL1. Histopathological analysis showed significant reduction of inflammation. Mechanistically, AVE 0991 reduced leukocyte rolling and adhesion, even when given after Ag challenge. Mas^−/− mice subjected to AIA developed slightly more pronounced inflammation, as observed by greater neutrophil accumulation and cytokine release. Administration of AVE 0991 was without effect in Mas^−/− mice subjected to AIA. In rats, administration of AVE 0991 decreased edema, neutrophil accumulation, histopathological score, and production of IL-1β and CXCL1 induced by AdIA. Therefore, activation of Mas receptors decreases neutrophil influx and cytokine production and causes significant amelioration of arthritis in experimental models of arthritis in rats and mice. This approach might represent a novel therapeutic opportunity for arthritis.

The final step in the direct restoration of the nociceptor threshold by peripheral administration of morphine and dipyrone was recently suggested to result from the opening of ATP-sensitive K ⁺ channels ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{equation*}{\mathrm{K}}_{{\mathrm{ATP}}}^{+}\end{equation*} ). This channel is known to be open either directly by cGMP or indirectly via protein kinase G (PKG) stimulation. In the present study, it was shown that the blockade was caused by a specific PKG inhibitor (KT5823) of the antinociceptive effect of morphine and dipyrone on acute hypernociception and of dipyrone on persistent hypernociception. It was also shown that, in both models, KT5823 prevented the peripheral antinociceptive effect of an analogue of cGMP, the nitric oxide (NO) donor ( S -nitroso- n -acetyl- d , l -penicilamine). However, in acute hypernociception, KT5823 did not prevent the peripheral antinociceptive effect of diazoxide (a direct \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{equation*}{\mathrm{K}}_{{\mathrm{ATP}}}^{+}\end{equation*} opener). In persistent hypernociception, the sensitization plateau was induced by daily injections of prostaglandin E ₂ (PGE ₂ , 100 ng) into the rat paw for 14 days. After cessation of PGE ₂ injections, the pharmacological blockade of persistent hypernociception led to a quiescent phase in which a rather small stimulus restored the hypernociceptive plateau. In this phase, glibenclamide (which specifically closes \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{equation*}{\mathrm{K}}_{{\mathrm{ATP}}}^{+}\end{equation*} ) fully restored persistent hypernociception, as did injection of PGE ₂ . Thus, the activation of the arginine/NO/cGMP pathway causes direct blockade of acute and persistent hypernociception by opening \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{equation*}{\mathrm{K}}_{{\mathrm{ATP}}}^{+}\end{equation*} via the stimulation of PKG. Analgesic stimulators of the neuronal arginine/NO/cGMP/PKG/ \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{equation*}{\mathrm{K}}_{{\mathrm{ATP}}}^{+}\end{equation*} pathway constitute a previously undescribed well defined class of peripheral analgesics with a mechanism of action different from either glucocorticoids or inhibitors of cyclooxygenases.

Objective: The chemokine receptors CXCR1 and CXCR2 play a role in mediating neutrophil recruitment and neutrophil‐dependent injury in several models of inflammation. We undertook this study to investigate the role of these receptors in mediating neutrophil adhesion, subsequent migration, and neutrophil‐dependent hypernociception in a murine model of monarticular antigen‐induced arthritis (AIA).Methods: AIA was induced by administration of antigen into the knee joint of previously immunized mice. Intravital microscopy studies were performed to assess leukocyte rolling and adhesion. Mechanical hypernociception was investigated using an electronic pressure meter. Neutrophil accumulation in the tissue was measured by counting neutrophils in the synovial cavity and assaying myeloperoxidase activity. Levels of tumor necrosis factor α (TNFα) and the chemokines CXCL1 and CXCL2 were quantified by enzyme‐linked immunosorbent assay. Histologic analysis was performed to evaluate the severity of arthritis and leukocyte infiltration.Results: Antigen challenge in immunized mice induced production of TNFα, CXCL1, and CXCL2 and also resulted in neutrophil recruitment, leukocyte rolling and adhesion, and hypernociception. Treatment with reparixin or DF2162 (allosteric inhibitors of CXCR1/CXCR2) decreased neutrophil recruitment, an effect that was associated with marked inhibition of neutrophil adhesion. Drug treatment also inhibited TNFα production, hypernociception, and the overall severity of the disease in the tissue.Conclusion: Blockade of CXCR1/CXCR2 receptors inhibits neutrophil recruitment by inhibiting the adhesion of neutrophils to synovial microvessels. As a consequence, there is decreased local cytokine production and reduced hypernociception, as well as ameloriation of overall disease in the tissue. These studies suggest a potential therapeutic role for the modulation of CXCR1/CXCR2 receptor signaling in the treatment of arthritis.