Use of adeno-associated viruses for transgenic modulation of microglia structure and function: A review of technical considerations and challenges.

Microglia play a central role in the etiology of many neuropathologies. Transgenic tools are a powerful experiment approach to gain reliable and specific control over microglia function. Adeno-associated virus (AAVs) vectors are already an indispensable tool in neuroscience research. Despite ubiquitous use of AAVs and substantial interest in the role of microglia in the study of central nervous system (CNS) function and disease, transduction of microglia using AAVs is seldom reported. This review explores the challenges and advancements made in using AAVs for expressing transgenes in microglia. First, we will examine the functional anatomy of the AAV capsid, which will serve as a basis for subsequent discussions of studies exploring the relationship between capsid mutations and microglia transduction efficacy. After outlining the functional anatomy of AAVs, we will consider the experimental evidence demonstrating AAV-mediated transduction of microglia and microglia-like cell lines followed by an examination of the most promising experimental approaches identified in the literature. Finally, technical limitations will be considered in future applications of AAV experimental approaches.

Microglia in neuroimmunopharmacology and drug addiction.

Drug addiction is a chronic and debilitating disease that is considered a global health problem. Various cell types in the brain are involved in the progression of drug addiction. Recently, the xenobiotic hypothesis has been proposed, which frames substances of abuse as exogenous molecules that are responded to by the immune system as foreign "invaders", thus triggering protective inflammatory responses. An emerging body of literature reveals that microglia, the primary resident immune cells in the brain, play an important role in the progression of addiction. Repeated cycles of drug administration cause a progressive, persistent induction of neuroinflammation by releasing microglial proinflammatory cytokines and their metabolic products. This contributes to drug addiction via modulation of neuronal function. In this review, we focus on the role of microglia in the etiology of drug addiction. Then, we discuss the dynamic states of microglia and the correlative and causal evidence linking microglia to drug addiction. Finally, possible mechanisms of how microglia sense drug-related stimuli and modulate the addiction state and how microglia-targeted anti-inflammation therapies affect addiction are reviewed. Understanding the role of microglia in drug addiction may help develop new treatment strategies to fight this devastating societal challenge.

Interleukin-1beta and inflammasome expression in spinal cord following chronic constriction injury in male and female rats.

Females represent a majority of chronic pain patients and show greater inflammatory immune responses in human chronic pain patient populations as well as in animal models of neuropathic pain. Recent discoveries in chronic pain research have revealed sex differences in inflammatory signaling, a key component of sensory pathology in chronic neuropathic pain, inviting more research into the nuances of these sex differences. Here we use the chronic constriction injury (CCI) model to explore similarities and differences in expression and production of Inflammatory cytokine IL-1beta in the lumbar spinal cord, as well as its role in chronic pain. We have discovered that intrathecal IL-1 receptor antagonist reverses established pain in both sexes, and increased gene expression of inflammasome NLRP3 is specific to microglia and astrocytes rather than neurons, while IL-1beta is specific to microglia in both sexes. We report several sex differences in the expression level of the genes coding for IL-1beta, as well as the four inflammasomes responsible for IL-1beta release: NLRP3, AIM2, NLRP1, and NLRC4 in the spinal cord. Total mRNA, but not protein expression of IL-1beta is greater in females than males after CCI. Also, while CCI increases all four inflammasomes in both sexes, there are sex differences in relative levels of inflammasome expression. NLRP3 and AIM2 are more highly expressed in females, whereas NLRP1 expression is greater in males.

Voluntary wheel running prevents formation of membrane attack complexes and myelin degradation after peripheral nerve injury.

Regular aerobic activity is associated with a reduced risk of chronic pain in humans and rodents. Our previous studies in rodents have shown that prior voluntary wheel running can normalize redox signaling at the site of peripheral nerve injury, attenuating subsequent neuropathic pain. However, the full extent of neuroprotection offered by voluntary wheel running after peripheral nerve injury is unknown. Here, we show that six weeks of voluntary wheel running prior to chronic constriction injury (CCI) reduced the terminal complement membrane attack complex (MAC) at the sciatic nerve injury site. This was associated with increased expression of the MAC inhibitor CD59. The levels of upstream complement components (C3) and their inhibitors (CD55, CR1 and CFH) were altered by CCI, but not increased by voluntary wheel running. Since MAC can degrade myelin, which in turn contributes to neuropathic pain, we evaluated myelin integrity at the sciatic nerve injury site. We found that the loss of myelinated fibers and decreased myelin protein which occurs in sedentary rats following CCI was not observed in rats with prior running. Substitution of prior voluntary wheel running with exogenous CD59 also attenuated mechanical allodynia and reduced MAC deposition at the nerve injury site, pointing to CD59 as a critical effector of the neuroprotective and antinociceptive actions of prior voluntary wheel running. This study links attenuation of neuropathic pain by prior voluntary wheel running with inhibition of MAC and preservation of myelin integrity at the sciatic nerve injury site.

Combining RNAscope and immunohistochemistry to visualize inflammatory gene products in neurons and microglia.

A challenge for central nervous system (CNS) tissue analysis in neuroscience research has been the difficulty to codetect and colocalize gene and protein expression in the same tissue. Given the importance of identifying gene expression relative to proteins of interest, for example, cell-type specific markers, we aimed to develop a protocol to optimize their codetection. RNAscope fluorescent in situ hybridization (FISH) combined with immunohistochemistry (IHC) in fixed (CNS) tissue sections allows for reliable quantification of gene transcripts of interest within IHC-labeled cells. This paper describes a new method for simultaneous visualization of FISH and IHC in thicker (14-µm), fixed tissue samples, using spinal cord sections. This method's effectiveness is shown by the cell-type-specific quantification of two genes, namely the proinflammatory cytokine interleukin-1beta (IL-1b) and the inflammasome NLR family pyrin domain containing 3 (NLRP3). These genes are challenging to measure accurately using immunohistochemistry (IHC) due to the nonspecificity of available antibodies and the hard-to-distinguish, dot-like visualizations of the labeled proteins within the tissue. These measurements were carried out in spinal cord sections after unilateral chronic constriction injury of the sciatic nerve to induce neuroinflammation in the spinal cord. RNAscope is used to label transcripts of genes of interest and IHC is used to label cell-type specific antigens (IBA1 for microglia, NeuN for neurons). This combination allowed for labeled RNA transcripts to be quantified within cell-type specific boundaries using confocal microscopy and standard image analysis methods. This method makes it easy to answer empirical questions that are intractable with standard IHC or in situ hybridization alone. The method, which has been optimized for spinal cord tissue and to minimize tissue preparation time and costs, is described in detail from tissue collection to image analysis. Further, the relative expression changes in inflammatory genes NLRP3 and IL-1b in spinal cord microglia vs. neurons of somatotopically relevant laminae are described for the first time.

Toll-like receptor 4 antagonists reduce cocaine-primed reinstatement of drug seeking.

RATIONALE: Cocaine can increase inflammatory neuroimmune markers, including chemokines and cytokines characteristic of innate inflammatory responding. Prior work indicates that the Toll-like receptor 4 (TLR4) initiates this response, and administration of TLR4 antagonists provides mixed evidence that TLR4 contributes to cocaine reward and reinforcement. OBJECTIVE: These studies utilize (+)-naltrexone, the TLR4 antagonist, and mu-opioid inactive enantiomer to examine the role of TLR4 on cocaine self-administration and cocaine seeking in rats. METHODS: (+)-Naltrexone was continuously administered via an osmotic mini-pump during the acquisition or maintenance of cocaine self-administration. The motivation to acquire cocaine was assessed using a progressive ratio schedule following either continuous and acute (+)-naltrexone administration. The effects of (+)-naltrexone on cocaine seeking were assessed using both a cue craving model and a drug-primed reinstatement model. The highly selective TLR4 antagonist, lipopolysaccharide from Rhodobacter sphaeroides (LPS-Rs), was administered into the nucleus accumbens to determine the effectiveness of TLR4 blockade on cocaine-primed reinstatement. RESULTS: (+)-Naltrexone administration did not alter the acquisition or maintenance of cocaine self-administration. Similarly, (+)-naltrexone was ineffective at altering the progressive ratio responding. Continuous administration of (+)-naltrexone during forced abstinence did not impact cued cocaine seeking. Acute systemic administration of (+)-naltrexone dose-dependently decreased cocaine-primed reinstatement of previously extinguished cocaine seeking, and administration of LPS-Rs into the nucleus accumbens shell also reduced cocaine-primed reinstatement of cocaine seeking. DISCUSSION: These results complement previous studies suggesting that the TLR4 plays a role in cocaine-primed reinstatement of cocaine seeking, but may have a more limited role in cocaine reinforcement.

Involvement of TLR2-TLR4, NLRP3, and IL-17 in pain induced by a novel Sprague-Dawley rat model of experimental autoimmune encephalomyelitis.

Up to 92% of patients suffering from multiple sclerosis (MS) experience pain, most without adequate treatment, and many report pain long before motor symptoms associated with MS diagnosis. In the most commonly studied rodent model of MS, experimental autoimmune encephalomyelitis (EAE), motor impairments/disabilities caused by EAE can interfere with pain testing. In this study, we characterize a novel low-dose myelin-oligodendrocyte-glycoprotein (MOG)-induced Sprague-Dawley (SD) model of EAE-related pain in male rats, optimized to minimize motor impairments/disabilities. Adult male SD rats were treated with increasing doses of intradermal myelin-oligodendrocyte-glycoprotein (MOG1-125) (0, 4, 8, and 16 µg) in incomplete Freund's adjuvant (IFA) vehicle to induce mild EAE. Von Frey testing and motor assessments were conducted prior to EAE induction and then weekly thereafter to assess EAE-induced pain and motor impairment. Results from these studies demonstrated that doses of 8 and 16 µg MOG1-125 were sufficient to produce stable mechanical allodynia for up to 1 month in the absence of hindpaw motor impairments/disabilities. In the follow-up studies, these doses of MOG1-125, were administered to create allodynia in the absence of confounded motor impairments. Then, 2 weeks later, rats began daily subcutaneous injections of the Toll-like receptor 2 and 4 (TLR2-TLR4) antagonist (+)-naltrexone [(+)-NTX] or saline for an additional 13 days. We found that (+)-NTX also reverses EAE-induced mechanical allodynia in the MOG-induced SD rat model of EAE, supporting parallels between models, but now allowing a protracted timecourse to be examined completely free of motor confounds. Exploring further mechanisms, we demonstrated that both spinal NOD-like receptor protein 3 (NLRP3) and interleukin-17 (IL-17) are necessary for EAE-induced pain, as intrathecal injections of NLRP3 antagonist MCC950 and IL-17 neutralizing antibody both acutely reversed EAE-induced pain. Finally, we show that spinal glial immunoreactivity induced by EAE is reversed by (+)-NTX, and that spinal demyelination correlates with the severity of motor impairments/disabilities. These findings characterize an optimized MOG-induced SD rat model of EAE for the study of pain with minimal motor impairments/disabilities. Finally, these studies support the role of TLR2-TLR4 antagonists as a potential treatment for MS-related pain and other pain and inflammatory-related disorders.

Mechanisms of microglia-mediated synapse turnover and synaptogenesis.

Microglia shape synaptic connections among neurons of the central nervous system (CNS) during development and adulthood. In this review, the actions by which they facilitate pruning, refinement, and new synaptic development throughout the lifespan are considered, along with the molecular mechanisms by which neurons and microglia communicate to guide these actions. Microglia survey neuronal activity and selectively modify synaptic connections at the level of individual dendrites and synapses. This is important given that microglia are necessary for a healthy nervous system capable of learning and other neural phenomena based on synaptic modifications and can also cause pathological synaptic disfunctions in immunologically driven neurodegenerative diseases. Understanding how microglia directly shape synaptic connections between neurons yields a more complete understanding of normal neuroplasticity and provides new routes for understanding disease states.

Preconditioning by voluntary wheel running attenuates later neuropathic pain via nuclear factor E2-related factor 2 antioxidant signaling in rats.

ABSTRACT: Animal and human studies have shown that exercise prior to nerve injury prevents later chronic pain, but the mechanisms of such preconditioning remain elusive. Given that exercise acutely increases the formation of free radicals, triggering antioxidant compensation, we hypothesized that voluntary running preconditioning would attenuate neuropathic pain by supporting redox homeostasis after sciatic nerve injury in male and female rats. We show that 6 weeks of voluntary wheel running suppresses neuropathic pain development induced by chronic constriction injury across both sexes. This attenuation was associated with reduced nitrotyrosine immunoreactivity-a marker for peroxynitrite-at the sciatic nerve injury site. Our data suggest that prior voluntary wheel running does not reduce the production of peroxynitrite precursors, as expression levels of inducible nitric oxide synthase and NADPH oxidase 2 were unchanged. Instead, voluntary wheel running increased superoxide scavenging by elevating expression of superoxide dismutases 1 and 2. Prevention of neuropathic pain was further associated with the activation of the master transcriptional regulator of the antioxidant response, nuclear factor E2-related factor 2 (Nrf2). Six weeks of prior voluntary wheel running increased Nrf2 nuclear translocation at the sciatic nerve injury site; in contrast, 3 weeks of prior wheel running, which failed to prevent neuropathic pain, had no effect on Nrf2 nuclear translocation. The protective effects of prior voluntary wheel running were mediated by Nrf2, as suppression was abolished across both sexes when Nrf2 activation was blocked during the 6-week running phase. This study provides insight into the mechanisms by which physical activity may prevent neuropathic pain. Preconditioning by voluntary wheel running, terminated prior to nerve injury, suppresses later neuropathic pain in both sexes, and it is modulated through the activation of Nrf2-antioxidant signaling.

T cell transgressions: Tales of T cell form and function in diverse disease states.

Insights into T cell form, function, and dysfunction are rapidly evolving. T cells have remarkably varied effector functions including protecting the host from infection, activating cells of the innate immune system, releasing cytokines and chemokines, and heavily contributing to immunological memory. Under healthy conditions, T cells orchestrate a finely tuned attack on invading pathogens while minimizing damage to the host. The dark side of T cells is that they also exhibit autoreactivity and inflict harm to host cells, creating autoimmunity. The mechanisms of T cell autoreactivity are complex and dynamic. Emerging research is elucidating the mechanisms leading T cells to become autoreactive and how such responses cause or contribute to diverse disease states, both peripherally and within the central nervous system. This review provides foundational information on T cell development, differentiation, and functions. Key T cell subtypes, cytokines that create their effector roles, and sex differences are highlighted. Pathological T cell contributions to diverse peripheral and central disease states, arising from errors in reactivity, are highlighted, with a focus on multiple sclerosis, rheumatoid arthritis, osteoarthritis, neuropathic pain, and type 1 diabetes.

Suppression of active phase voluntary wheel running in male rats by unilateral chronic constriction injury: Enduring therapeutic effects of a brief treatment of morphine combined with TLR4 or P2X7 antagonists.

The present series of studies examine the impact of systemically administered therapeutics on peripheral nerve injury (males; unilateral sciatic chronic constriction injury [CCI])-induced suppression of voluntary wheel running, across weeks after dosing cessation. Following CCI, active phase running distance and speed are suppressed throughout the 7-week observation period. A brief course of morphine, however, increased active phase running distance and speed throughout this same period, an effect apparent only in sham rats. For CCI rats, systemic co-administration of morphine with antagonists of either P2X7 (A438079) or TLR4 ((+)-naloxone) (receptors critical to the activation of NLRP3 inflammasomes and consequent inflammatory cascades) returned running behavior of CCI rats to that of shams through 5+ weeks after dosing ceased. This is a striking difference in effect compared to our prior CCI allodynia results using systemic morphine plus intrathecal delivery of these same antagonists, wherein a sustained albeit partial suppression of neuropathic pain was observed. This may point to actions of the systemic drugs at multiple sites along the neuraxis, modulating injury-induced, inflammasome-mediated effects at the injured sciatic nerve and/or dorsal root ganglia, spinal cord, and potentially higher levels. Given that our data to date point to morphine amplifying neuroinflammatory processes put into motion by nerve injury, it is intriguing to speculate that co-administration of TLR4 and/or P2X7 antagonists can intervene in these inflammatory processes in a beneficial way. That is, that systemic administration of such compounds may suppress inflammatory damage at multiple sites, rapidly and persistently returning neuropathic animals to sham levels of response.

Nicotine and its metabolite cotinine target MD2 and inhibit TLR4 signaling.

Nicotine is the principal alkaloid of tobacco often manufactured into cigarettes and belongs to a highly addictive class of drugs. Nicotine attenuates the neuroinflammation induced by microglial activation. However, the molecular target(s) underlying anti-inflammatory action of nicotine has not been fully understood. Considering the psychoactive substances morphine, cocaine, and methamphetamine act as xenobiotic-associated molecular patterns and can be specifically sensed by the innate immune receptor Toll-like receptor 4 (TLR4), here we sought to delineate whether nicotine and/or its metabolite cotinine may be recognized by the innate immune system via myeloid differentiation protein 2 (MD2), an accessory protein of TLR4 that is responsible for ligand recognition. MD2-intrinsic fluorescence titrations, surface plasmon resonance, and competitive displacement binding assays with curcumin (MD2 probe) demonstrated that both nicotine and cotinine targeted the lipopolysaccharide (LPS; TLR4 agonist) binding pocket of MD2 with similar affinities. The cellular thermal shift assay indicated that nicotine binding increased, while cotinine binding decreased, MD2 stability. These biophysical binding results were further supported by in silico simulations. In keeping with targeting MD2, both nicotine and cotinine inhibited LPS-induced production of nitric oxide and tumor necrosis factor alpha (TNF-α) and blocked microglial activation. Neither a pan nicotinic acetylcholine receptor (nAChR) inhibitor nor RNAi for nAChRs abolished the suppressive effect of nicotine- and cotinine-induced neuroinflammation. These data indicate that TLR4 inhibition by nicotine and cotinine at the concentrations tested in BV-2 cells is independent of classic neuronal nAChRs and validate that MD2 is a direct target of nicotine and cotinine in the inhibition of innate immunity.

Aging and miR-155 in mice influence survival and neuropathic pain after spinal cord injury.

Spinal cord injury (SCI) elicits chronic pain in 65% of individuals. In addition, SCI afflicts an increasing number of aged individuals, and those with SCI are predisposed to shorter lifespan. Our group previously identified that deletion of the microRNA miR-155 reduced neuroinflammation and locomotor deficits after SCI. Here, we hypothesized that aged mice would be more susceptible to pain symptoms and death soon after SCI, and that miR-155 deletion would reduce pain symptoms in adult and aged mice and improve survival. Adult (2 month-old) and aged (20 month-old) female wildtype (WT) and miR-155 knockout (KO) mice received T9 contusion SCI. Aged WT mice displayed reduced survival and increased autotomy - a symptom of spontaneous pain. In contrast, aged miR-155 KO mice after SCI were less susceptible to death or spontaneous pain. Evoked pain symptoms were tested using heat (Hargreaves test) and mechanical (von Frey) stimuli. At baseline, aged mice showed heightened heat sensitivity. After SCI, adult and aged WT and miR-155 KO mice all exhibited heat and mechanical hypersensitivity at all timepoints. miR-155 deletion in adult (but not aged) mice reduced mechanical hypersensitivity at 7 and 14 d post-SCI. Therefore, aging predisposes mice to SCI-elicited spontaneous pain and expedited mortality. miR-155 deletion in adult mice reduces evoked pain symptoms, and miR-155 deletion in aged mice reduces spontaneous pain and expedited mortality post-SCI. This study highlights the importance of studying geriatric models of SCI, and that inflammatory mediators such as miR-155 are promising targets after SCI for improving pain relief and longevity.

Autoimmune regulation of chronic pain.

Chronic pain is an unpleasant and debilitating condition that is often poorly managed by existing therapeutics. Reciprocal interactions between the nervous system and the immune system have been recognized as playing an essential role in the initiation and maintenance of pain. In this review, we discuss how neuroimmune signaling can contribute to peripheral and central sensitization and promote chronic pain through various autoimmune mechanisms. These pathogenic autoimmune mechanisms involve the production and release of autoreactive antibodies from B cells. Autoantibodies-ie, antibodies that recognize self-antigens-have been identified as potential molecules that can modulate the function of nociceptive neurons and thereby induce persistent pain. Autoantibodies can influence neuronal excitability by activating the complement pathway; by directly signaling at sensory neurons expressing Fc gamma receptors, the receptors for the Fc fragment of immunoglobulin G immune complexes; or by binding and disrupting ion channels expressed by nociceptors. Using examples primarily from rheumatoid arthritis, complex regional pain syndrome, and channelopathies from potassium channel complex autoimmunity, we suggest that autoantibody signaling at the central nervous system has therapeutic implications for designing novel disease-modifying treatments for chronic pain.

Experimental autoimmune encephalopathy (EAE)-induced hippocampal neuroinflammation and memory deficits are prevented with the non-opioid TLR2/TLR4 antagonist (+)-naltrexone.

Multiple sclerosis (MS) is associated with burdensome memory impairments and preclinical literature suggests that these impairments are linked to neuroinflammation. Previously, we have shown that toll-like receptor 4 (TLR4) antagonists, such as (+)-naltrexone [(+)-NTX], block neuropathic pain and associated spinal inflammation in rats. Here we extend these findings to first demonstrate that (+)-NTX blocks TLR2 in addition to TLR4. Additionally, we examined in two rat strains whether (+)-NTX could attenuate learning and memory disturbances and associated neuroinflammation using a low-dose experimental autoimmune encephalomyelitis (EAE) model of MS. EAE is the most commonly used experimental model for the human inflammatory demyelinating disease, MS. This low-dose model avoided motor impairments that would confound learning and memory measurements. Fourteen days later, daily subcutaneous (+)-NTX or saline injections began and continued throughout the study. Contextual and auditory-fear conditioning were conducted at day 21 to assess hippocampal and amygdalar function. With this low-dose model, EAE impaired long-term, but not short-term, contextual fear memory; both long-term and short-term auditory-cued fear memory were spared. This was associated with increased mRNA for hippocampal interleukin-1ß (IL-1ß), TLR2, TLR4, NLRP3, and IL-17 and elevated expression of the microglial marker Iba1 in CA1 and DG regions of the hippocampus, confirming the neuroinflammation observed in higher-dose EAE models. Importantly, (+)-NTX completely prevented the EAE-induced memory impairments and robustly attenuated the associated proinflammatory effects. These findings suggest that (+)-NTX may exert therapeutic effects on memory function by dampening the neuroinflammatory response in the hippocampus through blockade of TLR2/TLR4. This study suggests that TLR2 and TLR4 antagonists may be effective at treating MS-related memory deficits.

Postoperative cognitive dysfunction is made persistent with morphine treatment in aged rats.

Postoperative cognitive dysfunction (POCD) is the collection of cognitive impairments, lasting days to months, experienced by individuals following surgery. Persistent POCD is most commonly experienced by older individuals and is associated with a greater vulnerability to developing Alzheimer's disease, but the underlying mechanisms are not known. It is known that laparotomy (exploratory abdominal surgery) in aged rats produces memory impairments for 4 days. Here we report that postsurgical treatment with morphine extends this deficit to at least 2 months while having no effects in the absence of surgery. Indeed, hippocampal-dependent long-term memory was impaired 2, 4, and 8 weeks postsurgery only in aged, morphine-treated rats. Short-term memory remained intact. Morphine is known to have analgesic effects via µ-opioid receptor activation and neuroinflammatory effects through Toll-like receptor 4 activation. Here we demonstrate that persistent memory deficits were mediated independently of the µ-opioid receptor, suggesting that they were evoked through a neuroinflammatory mechanism and unrelated to pain modulation. In support of this, aged, laparotomized, and morphine-treated rats exhibited increased gene expression of various proinflammatory markers (IL-1ß, IL-6, TNFα, NLRP3, HMGB1, TLR2, and TLR4) in the hippocampus at the 2-week time point. Furthermore, central blockade of IL-1ß signaling with the specific IL-1 receptor antagonist (IL-1RA), at the time of surgery, completely prevented the memory impairment. Finally, synaptophysin and PSD95 gene expression were significantly dysregulated in the hippocampus of aged, laparotomized, morphine-treated rats, suggesting that impaired synaptic structure and/or function may play a key role in this persistent deficit. This instance of long-term memory impairment following surgery closely mirrors the timeline of persistent POCD in humans and may be useful for future treatment discoveries.

Toll-like receptor 2 and 4 antagonism for the treatment of experimental autoimmune encephalomyelitis (EAE)-related pain.

Neuropathic pain is a major symptom of multiple sclerosis (MS) with up to 92% of patients reporting bodily pain, and 85% reporting pain severe enough to cause functional disability. None of the available therapeutics target MS pain. Toll-like receptors 2 and 4 (TLR2/TLR4) have emerged as targets for treating a wide array of autoimmune disorders, including MS, as well as having demonstrated success at suppressing pain in diverse animal models. The current series of studies tested systemic TLR2/TLR4 antagonists in males and females in a low-dose Myelin oligodendrocyte glycoprotein (MOG) experimental autoimmune encephalomyelitis (EAE) model, with reduced motor dysfunction to allow unconfounded testing of allodynia through 50+ days post-MOG. The data demonstrated that blocking TLR2/TLR4 suppressed EAE-related pain, equally in males and females; upregulation of dorsal spinal cord proinflammatory gene expression for TLR2, TLR4, NLRP3, interleukin-1ß, IkBα, TNF-α and interleukin-17; and upregulation of dorsal spinal cord expression of glial immunoreactivity markers. In support of these results, intrathecal interleukin-1 receptor antagonist reversed EAE-induced allodynia, both early and late after EAE induction. In contrast, blocking TLR2/TLR4 did not suppress EAE-induced motor disturbances induced by a higher MOG dose. These data suggest that blocking TLR2/TLR4 prevents the production of proinflammatory factors involved in low dose EAE pathology. Moreover, in this EAE model, TLR2/TLR4 antagonists were highly effective in reducing pain, whereas motor impairment, as seen in high dose MOG EAE, is not affected.

Targeted interleukin-10 plasmid DNA therapy in the treatment of osteoarthritis: Toxicology and pain efficacy assessments.

Osteoarthritis results in chronic pain and loss of function. Proinflammatory cytokines create both osteoarthritis pathology and pain. Current treatments are poorly effective, have significant side effects, and have not targeted the cytokines central to osteoarthritis development and maintenance. Interleukin-10 is an anti-inflammatory cytokine that potently and broadly suppresses proinflammatory cytokine activity. However, interleukin-10 protein has a short half-life in vivo and poor joint permeability. For sustained IL-10 activity, we developed a plasmid DNA-based therapy that expresses a long-acting human interleukin-10 variant (hIL-10var). Here, we describe the 6-month GLP toxicology study of this therapy. Intra-articular injections of hIL-10var pDNA into canine stifle joints up to 1.5 mg bilaterally were well-tolerated and without pathologic findings. This represents the first long-term toxicologic assessment of intra-articular pDNA therapy. We also report results of a small double-blind, placebo-controlled study of the effect of intra-articular hIL-10var pDNA on pain measures in companion (pet) dogs with naturally occurring osteoarthritis. This human IL-10-based targeted therapy reduced pain measures in the dogs, based on veterinary and owner ratings, without any adverse findings. These results with hIL-10var pDNA therapy, well-tolerated and without toxicologic effects, establish the basis for clinical trials of a new class of safe and effective therapies for OA.

Acute stress induces the rapid and transient induction of caspase-1, gasdermin D and release of constitutive IL-1ß protein in dorsal hippocampus.

The proinflammatory cytokine interleukin (IL)-1ß plays a pivotal role in the behavioral manifestations (i.e., sickness) of the stress response. Indeed, exposure to acute and chronic stressors induces the expression of IL-1ß in stress-sensitive brain regions. Thus, it is typically presumed that exposure to stressors induces the extra-cellular release of IL-1ß in the brain parenchyma. However, this stress-evoked neuroimmune phenomenon has not been directly demonstrated nor has the cellular process of IL-1ß release into the extracellular milieu been characterized in brain. This cellular process involves a form of inflammatory cell death, termed pyroptosis, which involves: 1) activation of caspase-1, 2) caspase-1 maturation of IL-1ß, 3) caspase-1 cleavage of gasdermin D (GSDMD), and 4) GSDMD-induced permeability of the cell membrane through which IL-1ß is released into the extracellular space. Thus, the present study examined whether stress induces the extra-cellular release of IL-1ß and engages the above cellular process in mediating IL-1ß release in the brain. Male Sprague-Dawley rats were exposed to inescapable tailshock (IS). IL-1ß extra-cellular release, caspase-1 activity and cleavage of GSDMD were measured in dorsal hippocampus. We found that exposure to IS induced a transient increase in the release of IL-1ß into the extracellular space immediately after termination of the stressor. IS also induced a transient increase in caspase-1 activity prior to IL-1ß release, while activation of GSDMD was observed immediately after termination of the stressor. IS also increased mRNA and protein expression of the ESCRTIII protein CHMP4B, which is involved in cellular repair. The present results suggest that exposure to an acute stressor induces the hallmarks of pyroptosis in brain, which might serve as a key cellular process involved in the release of IL-1ß into the extracellular milieu of the brain parenchyma.

Acute stress induces chronic neuroinflammatory, microglial and behavioral priming: A role for potentiated NLRP3 inflammasome activation.

Prior exposure to acute and chronic stressors potentiates the neuroinflammatory and microglial pro-inflammatory response to subsequent immune challenges suggesting that stressors sensitize or prime microglia. Stress-induced priming of the NLRP3 inflammasome has been implicated in this priming phenomenon, however the duration/persistence of these effects has not been investigated. In the present study, we examined whether exposure to a single acute stressor (inescapable tailshock) induced a protracted priming of the NLRP3 inflammasome as well as the neuroinflammatory, behavioral and microglial proinflammatory response to a subsequent immune challenge in hippocampus. In male Sprague-Dawley rats, acute stress potentiated the neuroinflammatory response (IL-1ß, IL-6, and NFκBIα) to an immune challenge (lipopolysaccharide; LPS) administered 8 days after stressor exposure. Acute stress also potentiated the proinflammatory cytokine response (IL-1ß, IL-6, TNF and NFκBIα) to LPS ex vivo. This stress-induced priming of microglia also was observed 28 days post-stress. Furthermore, challenge with LPS reduced juvenile social exploration, but not sucrose preference, in animals exposed to stress 8 days prior to immune challenge. Exposure to acute stress also increased basal mRNA levels of NLRP3 and potentiated LPS-induction of caspase-1 mRNA and protein activity 8 days after stress. The present findings suggest that acute stress produces a protracted vulnerability to the neuroinflammatory effects of subsequent immune challenges, thereby increasing risk for stress-related psychiatric disorders with an etiological inflammatory component. Further, these findings suggest the unique possibility that acute stress might induce innate immune memory in microglia.