Fluvastatin enhances IL-33-mediated mast cell IL-6 and TNF production.

Statins are HMG-CoA reductase inhibitors prescribed for lowering cholesterol. They can also inhibit inflammatory responses by suppressing isoprenylation of small G proteins. Consistent with this, we previously found that fluvastatin suppresses IgE-mediated mast cell function. However, some studies have found that statins induced pro-inflammatory cytokines in macrophages and NK cells. In contrast to IgE signaling, we show that fluvastatin augments IL-33-induced TNF and IL-6 production by mast cells. This effect required the key mast cell growth factor, stem cell factor (SCF). Treatment of IL-33-activated mast cells with mevalonic acid or isoprenoids reduced fluvastatin effects, suggesting fluvastatin acts at least partly by reducing isoprenoid production. Fluvastatin also enhanced IL-33-induced NF-κB transcriptional activity and promoted neutrophilic peritonitis in vivo, a response requiring mast cell activation. Other statins tested did not enhance IL-33 responsiveness. Therefore, this work supports observations of unexpected pro-inflammatory effects of some statins and suggests mechanisms by which this may occur. Because statins are candidates for repurposing in inflammatory disorders, our work emphasizes the importance of understanding the pleiotropic and possible unexpected effects of these drugs.

Lactic acid suppresses IgE-mediated mast cell function in vitro and in vivo.

Mast cells have functional plasticity affected by their tissue microenvironment, which greatly impacts their inflammatory responses. Because lactic acid (LA) is abundant in inflamed tissues and tumors, we investigated how it affects mast cell function. Using IgE-mediated activation as a model system, we found that LA suppressed inflammatory cytokine production and degranulation in mouse peritoneal mast cells, data that were confirmed with human skin mast cells. In mouse peritoneal mast cells, LA-mediated cytokine suppression was dependent on pH- and monocarboxylic transporter-1 expression. Additionally, LA reduced IgE-induced Syk, Btk, and ERK phosphorylation, key signals eliciting inflammation. In vivo, LA injection reduced IgE-mediated hypothermia in mice undergoing passive systemic anaphylaxis. Our data suggest that LA may serve as a feedback inhibitor that limits mast cell-mediated inflammation.

Polymer scaffold architecture is a key determinant in mast cell inflammatory and angiogenic responses.

Implanted polymer scaffolds can induce inflammation leading to the foreign body response (FBR), fibrosis, and implant failure. Thus, it is important to understand how immune cells interact with scaffolds to mitigate inflammation and promote a regenerative response. We previously demonstrated that macrophage phenotype is modulated by fiber and pore diameters of an electrospun scaffold. However, it is unclear if this effect is consistent among other innate immune cells. Mast cells are inflammatory sentinels that play a vital role in the FBR of implanted biomaterials, as well as angiogenesis. We determined if altering electrospun scaffold architecture modulates mast cell responses, with the goal of promoting regenerative cell-scaffold interactions. Polydioxanone (PDO) scaffolds were made from 60 mg/mL or 140 mg/mL PDO solutions, yielding structures with divergent fiber and pore diameters. Mouse mast cells plated on these scaffolds were activated with IL-33 or lipopolysaccharide (LPS). Relative to the 60 mg/mL scaffold, 140 mg/mL scaffolds yielded less IL-6 and TNF, and greater VEGF secretion. Pores >4-6 µm elicited less IL-6 and TNF secretion. IL-33-induced VEGF regulation was more complex, showing effects of both pore size and fiber diameter. These data indicate parameters that can predict mast cell responses to scaffolds, informing biomaterial design to increase wound healing and diminish implant rejection. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 884-892, 2019.

Didox (3,4-dihydroxybenzohydroxamic acid) suppresses IgE-mediated mast cell activation through attenuation of NFκB and AP-1 transcription.

Mast cell activation via the high-affinity IgE receptor (FcεRI) elicits production of inflammatory mediators central to allergic disease. As a synthetic antioxidant and a potent ribonucleotide reductase (RNR) inhibitor, Didox (3,4-dihyroxybenzohydroxamic acid) has been tested in clinical trials for cancer and is an attractive therapeutic for inflammatory disease. We found that Didox treatment of mouse bone marrow-derived mast cells (BMMC) reduced IgE-stimulated degranulation and cytokine production, including IL-6, IL-13, TNF and MIP-1a (CCL3). These effects were consistent using BMMC of different genetic backgrounds and peritoneal mast cells. While the RNR inhibitor hydroxyurea had little or no effect on IgE-mediated function, high concentrations of the antioxidant N-acetylcysteine mimicked Didox-mediated suppression. Furthermore, Didox increased expression of the antioxidant genes superoxide dismutase and catalase, and suppressed DCFH-DA fluorescence, indicating reduced reactive oxygen species production. Didox effects were not due to changes in FcεRI expression or cell viability, suggesting it inhibits signaling required for inflammatory cytokine production. In support of this, we found that Didox reduced FcεRI-mediated AP-1 and NFκB transcriptional activity. Finally, Didox suppressed mast cell-dependent, IgE-mediated passive systemic anaphylaxis in vivo. These data demonstrate the potential use for Didox asa means of antagonizing mast cell responses in allergic disease.

Didox (3,4-dihydroxybenzohydroxamic acid) suppresses IL-33-induced cytokine production in primary mouse mast cells.

While IgE is considered the primary mediator of mast cell activation, IL-33 contributes substantially in asthma, allergic rhinitis, and atopic dermatitis. To develop effective treatments for allergic disease, it is important to understand the role of therapeutic agents on IL-33 activation. We examined the effect of Didox (3,4-dihydroxybenzohydroxamic acid), an antioxidant and ribonucleotide reductase (RNR) inhibitor, on IL-33-mediated mast cell activation. Didox suppressed IL-6, IL-13, TNF, and MIP-1α (CCL3) production in bone marrow derived mast cells following IL-33 activation. This suppression was observed in different genetic backgrounds and extended to peritoneal mast cells. The antioxidant N-acetylcysteine mimicked the suppression of Didox, albeit at a much higher dose, while the RNR inhibitor hydroxyurea had no effect. Didox substantially suppressed IL-33-mediated NFκB and AP-1 transcriptional activities. These results suggest that Didox attenuates IL-33-induced mast cell activation and should be further studied as a potential therapeutic agent for inflammatory diseases involving IL-33.

TGF-ß1 Suppresses IL-33-Induced Mast Cell Function.

TGF-ß1 is involved in many pathological conditions, including autoimmune disorders, cancer, and cardiovascular and allergic diseases. We have previously found that TGF-ß1 can suppress IgE-mediated mast cell activation of human and mouse mast cells. IL-33 is a member of the IL-1 family capable of inducing mast cell responses and enhancing IgE-mediated activation. In this study, we investigated the effects of TGF-ß on IL-33-mediated mast cell activation. Bone marrow-derived mast cells cultured in TGF-ß1, ß2, or ß3 showed reduced IL-33-mediated production of TNF, IL-6, IL-13, and MCP-1 in a concentration-dependent manner. TGF-ß1 inhibited IL-33-mediated Akt and ERK phosphorylation as well as NF-κB- and AP-1-mediated transcription. These effects were functionally important, as TGF-ß1 injection suppressed IL-33-induced systemic cytokines in vivo and inhibited IL-33-mediated cytokine release from human mast cells. TGF-ß1 also suppressed the combined effects of IL-33 and IgE-mediated activation on mouse and human mast cells. The role of IL-33 in the pathogenesis of allergic diseases is incompletely understood. These findings, consistent with our previously reported effects of TGF-ß1 on IgE-mediated activation, demonstrate that TGF-ß1 can provide broad inhibitory signals to activated mast cells.

Lactic Acid Suppresses IL-33-Mediated Mast Cell Inflammatory Responses via Hypoxia-Inducible Factor-1α-Dependent miR-155 Suppression.

Lactic acid (LA) is present in tumors, asthma, and wound healing, environments with elevated IL-33 and mast cell infiltration. Although IL-33 is a potent mast cell activator, how LA affects IL-33-mediated mast cell function is unknown. To investigate this, mouse bone marrow-derived mast cells were cultured with or without LA and activated with IL-33. LA reduced IL-33-mediated cytokine and chemokine production. Using inhibitors for monocarboxylate transporters (MCT) or replacing LA with sodium lactate revealed that LA effects are MCT-1- and pH-dependent. LA selectively altered IL-33 signaling, suppressing TGF-ß-activated kinase-1, JNK, ERK, and NF-κB phosphorylation, but not p38 phosphorylation. LA effects in other contexts have been linked to hypoxia-inducible factor (HIF)-1α, which was enhanced in bone marrow-derived mast cells treated with LA. Because HIF-1α has been shown to regulate the microRNA miR-155 in other systems, LA effects on miR-155-5p and miR-155-3p species were measured. In fact, LA selectively suppressed miR-155-5p in an HIF-1α-dependent manner. Moreover, overexpressing miR-155-5p, but not miR-155-3p, abolished LA effects on IL-33-induced cytokine production. These in vitro effects of reducing cytokines were consistent in vivo, because LA injected i.p. into C57BL/6 mice suppressed IL-33-induced plasma cytokine levels. Lastly, IL-33 effects on primary human mast cells were suppressed by LA in an MCT-dependent manner. Our data demonstrate that LA, present in inflammatory and malignant microenvironments, can alter mast cell behavior to suppress inflammation.

Closing the loop in legged neuromechanics: an open-source computer vision controlled treadmill.

The neural circuitry that controls legged locomotion is exquisitely state dependent. The response to a perturbation at one phase of the locomotor cycle is very different from another. As such, tools that enable closed loop experiments in which freely moving animals can be precisely perturbed, either neurally or mechanically, will improve our ability to probe the locomotor control architecture. Because locomotion emerges from the coupled interaction of the nervous system, musculoskeletal system, and physics of the moving body, ideally an experimenter could simultaneously perturb the nervous system, mechanics of the moving animal, or both, at precise moments during well defined locomotor behaviour. Combining the new technique of optogenetics with closed loop tracking of state and fast mechanical perturbations would make this possible. Here we present a computer vision feedback controlled treadmill designed to enable such perturbations. We show that insects (cockroaches Blaberus discoidalis) and mice (Mus mus; C57BL/6) run freely, with preferred speeds in agreement with the literature, over many strides, on the system. We demonstrate the utility of the closed loop system by automatically gathering high speed video (HSV) data when the running animal meets specific operational criteria; here, running within a restricted range of speeds near the midline of the treadmill for a minimum of 2s. We quantify the mouse's gait from HSV data with a phase-based approach, confirming a normal trotting gait. These results demonstrate the system's potential for dissecting the neuromechanical basis of legged locomotion through closed-loop experimental design.

A single muscle's multifunctional control potential of body dynamics for postural control and running.

A neuromechanical approach to control requires understanding how mechanics alters the potential of neural feedback to control body dynamics. Here, we rewrite activation of individual motor units of a behaving animal to mimic the effects of neural feedback without concomitant changes in other muscles. We target a putative control muscle in the cockroach, Blaberus discoidalis (L.), and simultaneously capture limb and body dynamics through high-speed videography and a micro-accelerometer backpack. We test four neuromechanical control hypotheses. We supported the hypothesis that mechanics linearly translates neural feedback into accelerations and rotations during static postural control. However, during running, the same neural feedback produced a nonlinear acceleration control potential restricted to the vertical plane. Using this, we reject the hypothesis from previous work that this muscle acts primarily to absorb energy from the body. The conversion of the control potential is paralleled by nonlinear changes in limb kinematics, supporting the hypothesis that significant mechanical feedback filters the graded neural feedback for running control. Finally, we insert the same neural feedback signal but at different phases in the dynamics. In this context, mechanical feedback enables turning by changing the timing and direction of the accelerations produced by the graded neural feedback.

Insects running on elastic surfaces.

In nature, cockroaches run rapidly over complex terrain such as leaf litter. These substrates are rarely rigid, and are frequently very compliant. Whether and how compliant surfaces change the dynamics of rapid insect locomotion has not been investigated to date largely due to experimental limitations. We tested the hypothesis that a running insect can maintain average forward speed over an extremely soft elastic surface (10 N m(-1)) equal to 2/3 of its virtual leg stiffness (15 N m(-1)). Cockroaches Blaberus discoidalis were able to maintain forward speed (mean +/- s.e.m., 37.2+/-0.6 cm s(-1) rigid surface versus 38.0+/-0.7 cm s(-1) elastic surface; repeated-measures ANOVA, P=0.45). Step frequency was unchanged (24.5+/-0.6 steps s(-1) rigid surface versus 24.7+/-0.4 steps s(-1) elastic surface; P=0.54). To uncover the mechanism, we measured the animal's centre of mass (COM) dynamics using a novel accelerometer backpack, attached very near the COM. Vertical acceleration of the COM on the elastic surface had a smaller peak-to-peak amplitude (11.50+/-0.33 m s(-2), rigid versus 7.7+/-0.14 m s(-2), elastic; P=0.04). The observed change in COM acceleration over an elastic surface required no change in effective stiffness when duty factor and ground stiffness were taken into account. Lowering of the COM towards the elastic surface caused the swing legs to land earlier, increasing the period of double support. A feedforward control model was consistent with the experimental results and provided one plausible, simple explanation of the mechanism.

Flexible multielectrodes can resolve multiple muscles in an insect appendage.

Research into the neuromechanical basis of behavior, either in biomechanics, neuroethology, or neuroscience, is frequently limited by methods of data collection. Two of the most pressing needs are for methods with which to (1) record from multiple neurons or muscles simultaneously and (2) perform this recording in intact, behaving animals. In this paper we present the fabrication and testing of flexible multielectrode arrays (fMEAs) that move us significantly towards these goals. The fMEAs were used to record the activity of several distinct units in the coxa of the cockroach Blaberus discoidalis. The devices fabricated here address the first goal in two ways: (1) their flexibility allows them to be inserted into an animal and guided through internal tissues in order to access distinct groups of neurons and muscles and (2) their recording site geometry has been tuned to suit the anatomy under study, yielding multichannel spike waveforms that are easily separable under conditions of spike overlap. The flexible nature of the devices simultaneously addresses the second goal, in that it is less likely to interfere with the natural movement of the animal.