Publications

Cyanotoxins at low doses induce apoptosis and inflammatory effects in murine brain cells: Potential implications for neurodegenerative diseases. 2016 Toxicology Reports doi:10.1016/j.toxrep

Takser L, Benachour N, Husk B, Cabana H, Gris D.

Abstract

Cyanotoxins have been shown to be highly toxic for mammalian cells, including brain cells. However, little is known about their effect on inflammatory pathways. This study investigated whether mammalian brain and immune cells can be a target of certain cyanotoxins, at doses approximating those in the guideline levels for drinking water, either alone or in mixtures. We examined the effects on cellular viability, apoptosis and inflammation signalling of several toxins on murine macrophage-like RAW264.7, microglial BV-2 and neuroblastoma N2a cell lines. We tested cylindrospermopsin (CYN), microcystin-LR (MC-LR), and anatoxin-a (ATX-a), individually as well as their mixture. In addition, we studied the neurotoxins ?-N-methylamino-l-alanine (BMAA) and its isomer 2,4-diaminobutyric acid (DAB), as well as the mixture of both. Cellular viability was determined by the MTT assay. Apoptosis induction was assessed by measuring the activation of caspases 3/7. Cell death and inflammation are the hallmarks of neurodegenerative diseases. Thus, our final step was to quantify the expression of a major proinflammatory cytokine TNF-? by ELISA. Our results show that CYN, MC-LR and ATX-a, but not BMAA and DAB, at low doses, especially when present in a mixture at threefold less concentrations than individual compounds are 3–15 times more potent at inducing apoptosis and inflammation. Our results suggest that common cyanotoxins at low doses have a potential to induce inflammation and apoptosis in immune and brain cells. Further research of the neuroinflammatory effects of these compounds in vivo is needed to improve safety limit levels for cyanotoxins in drinking water and food.

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The nod-like receptor, Nlrp12, plays an anti-inflammatory role in experimental autoimmune encephalomyelitis. J Neuroinflammation. 2015 Oct 31;12:198.

Gharagozloo M, Mahvelati TM, Imbeault E, Gris P, Zerif E, Bobbala D, Ilangumaran S, Amrani A, Gris D

Abstract

BACKGROUND:

Multiple sclerosis (MS) is an organ-specific autoimmune disease resulting in demyelinating plaques throughout the central nervous system. In MS, the exact role of microglia remains unknown. On one hand, they can present antigens, skew T cell responses, and upregulate the expression of pro-inflammatory molecules. On the other hand, microglia may express anti-inflammatory molecules and inhibit inflammation. Microglia express a wide variety of immune receptors such as nod-like receptors (NLRs). NLRs are intracellular receptors capable of regulating both innate and adaptive immune responses. Among NLRs, Nlrp12 is largely expressed in cells of myeloid origins. It plays a role in immune inflammatory responses by negatively regulating the nuclear factor-kappa B (NF-?B) pathway. Thus, we hypothesize that Nlrp12 suppresses inflammation and ameliorates the course of MS.

METHODS:

We used experimental autoimmune encephalomyelitis (EAE), a well-characterized mouse model of MS. EAE was induced in wild-type (WT) and Nlrp12 (-/-) mice with myelin oligodendrocyte glycoprotein (MOG):complete Freud's adjuvant (CFA). The spinal cords of healthy and immunized mice were extracted for immunofluorescence and pro-inflammatory gene analysis. Primary murine cortical microglia cell cultures of WT and Nlrp12 (-/-) were prepared with cortices of 1-day-old pups. The cells were stimulated with lipopolysaccharide (LPS) and analyzed for the expression of pro-inflammatory genes as well as pro-inflammatory molecule secretions.

RESULTS:

Over the course of 9 weeks, the Nlrp12 (-/-) mice demonstrated increased severity in the disease state, where they developed the disease earlier and reached significantly higher clinical scores compared to the WT mice. The spinal cords of immunized WT mice relative to healthy WT mice revealed a significant increase in Nlrp12 messenger ribonucleic acid (mRNA) expression at 1, 3, and 5 weeks post injection. A significant increase in the expression of pro-inflammatory genes Ccr5, Cox2, and IL-1? was found in the spinal cords of the Nlrp12 (-/-) mice relative to the WT mice (P?<?0.05). A significant increase in the level of gliosis was observed in the spinal cords of the Nlrp12 (-/-) mice compared to the WT mice after 9 weeks of disease (P?<?0.05). Primary Nlrp12 (-/-) microglia cells demonstrated a significant increase in inducible nitric oxide synthase (iNOS) expression (P?<?0.05) and secreted significantly (P?<?0.05) more tumor necrosis factor alpha (TNF?), interleukin-6 (IL-6), and nitric oxide (NO).

CONCLUSION:

Nlrp12 plays a protective role by suppressing inflammation during the development of EAE. The absence of Nlrp12 results in an increased inflammatory response.

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Nlrx1 regulates neuronal cell death. Mol Brain. 2014 Dec 24;7:90.

Imbeault E, Mahvelati TM, Braun R, Gris P, Gris D.

Abstract

BACKGROUND:

Regulation of cell death during neurodegeneration is one of the key factors that play a role in the speed at which a disease progresses. Out of several cellular pathways responsible for this progression, necrosis and apoptosis are situated on the opposite spectrum of cell death regulation. Necrosis produces an environment that promotes inflammation and cytotoxicity and apoptosis is a highly organized process that maintains tissue homeostasis. A recently discovered protein, Nlrx1, regulates inflammatory and cell death responses during infection.

FINDINGS:

Using transfections of N2A cell line, we demonstrate that Nlrx1 redirects cells away from necrosis and towards an apoptotic pathway following rotenone treatments. In addition, Nlrx1 promotes DRP1 phosphorylation and increases mitochondrial fission.

CONCLUSION:

Our results suggest a novel molecular pathway for regulating mitochondrial dynamics and neuronal death. Nlrx1 may play an important role in neurodegenerative diseases, where necrosis is a prominent factor.

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The nucleotide-binding leucine-rich repeat (NLR) family member NLRX1 mediates protection against experimental autoimmune encephalomyelitis and represses macrophage/microglia-induced inflammation. J Biol Chem. 2014 Feb 14;289(7):4173-9

Eitas TK1, Chou WC, Wen H, Gris D, Robbins GR, Brickey J, Oyama Y, Ting JP.

Abstract

The nucleotide binding domain and leucine-rich repeat-containing (NLR) family of proteins is known to activate innate immunity, and the inflammasome-associated NLRs are prime examples. In contrast, the concept that NLRs can inhibit innate immunity is still debated, and the impact of such inhibitory NLRs in diseases shaped by adaptive immune responses is entirely unexplored. This study demonstrates that, in contrast to other NLRs that activate immunity, NLRX1 plays a protective role in experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis. When compared with wild-type controls, Nlrx1(-/-) mice have significantly worsened clinical scores and heightened CNS tissue damage during EAE. NLRX1 does not alter the production of encephalitogenic T cells in the peripheral lymphatic tissue, but Nlrx1(-/-) mice are more susceptible to adoptively transferred myelin-reactive T cells. Analysis of the macrophage and microglial populations indicates that NLRX1 reduces activation during both active and passive EAE models. This work represents the first case of an NLR that attenuates microglia inflammatory activities and protects against a neurodegenerative disease model caused by autoreactive T cells.

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Involvement of neuronal IL-1b in acquired brain lesions in a rat model of neonatal encephalopathy. J Neuroinflammation. 2013 Sep 5;10:110

Savard A, Lavoie K, Brochu ME, Grbic D, Lepage M, Gris D, Sebire G.

Abstract

BACKGROUND:

Infection-inflammation combined with hypoxia-ischemia (HI) is the most prevalent pathological scenario involved in perinatal brain damage leading to life-long neurological disabilities. Following lipopolysaccharide (LPS) and/or HI aggression, different patterns of inflammatory responses have been uncovered according to the brain differentiation stage. In fact, LPS pre-exposure has been reported to aggravate HI brain lesions in post-natal day 1 (P1) and P7 rat models that are respectively equivalent - in terms of brain development - to early and late human preterm newborns. However, little is known about the innate immune response in LPS plus HI-induced lesions of the full-term newborn forebrain and the associated neuropathological and neurobehavioral outcomes.

METHODS:

An original preclinical rat model has been previously documented for the innate neuroimmune response at different post-natal ages. It was used in the present study to investigate the neuroinflammatory mechanisms that underline neurological impairments after pathogen-induced inflammation and HI in term newborns.

RESULTS:

LPS and HI exerted a synergistic detrimental effect on rat brain. Their effect led to a peculiar pattern of parasagittal cortical-subcortical infarcts mimicking those in the human full-term newborn with subsequent severe neurodevelopmental impairments. An increased IL-1b response in neocortical and basal gray neurons was demonstrated at 4 h after LPS + HI-exposure and preceded other neuroinflammatory responses such as microglial and astroglial cell activation. Neurological deficits were observed during the acute phase of injury followed by a recovery, then by a delayed onset of profound motor behavior impairment, reminiscent of the delayed clinical onset of motor system impairments observed in humans. Interleukin-1 receptor antagonist (IL-1ra) reduced the extent of brain lesions confirming the involvement of IL-1b response in their pathophysiology.

CONCLUSION:

In rat pups at a neurodevelopmental age corresponding to full-term human newborns, a systemic pre-exposure to a pathogen component amplified HI-induced mortality and morbidities that are relevant to human pathology. Neuronal cells were the first cells to produce IL-1b in LPS + HI-exposed full-term brains. Such IL-1b production might be responsible for neuronal self-injuries via well-described neurotoxic mechanisms such as IL-1b-induced nitric oxide production, or IL-1b-dependent exacerbation of excitotoxic damage.

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Assessment of oxidative metabolism. Methods Mol Biol. 2013;1031:51-7

Imbeault E, Gris D.

Abstract

Oxidative metabolism is one of the central physiological processes that regulate multiple functions in a cell including cell death and survival, proliferation, gene transcription, and protein modification. There are multitudes of techniques that are used to evaluate oxidative activity. Here, we summarize how to measure oxidative activity by flow cytometry. This versatile technique allows the evaluation of the level of oxidative activity within heterogeneous populations of cells and in cell culture. Flow cytometry is a quick method that yields highly reproducible results with small sample volumes. Therefore, it is an ideal technique for evaluating changes in oxidative activity in samples from mice.

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NLRX1 protein attenuates inflammatory responses to infection by interfering with the RIG-I-MAVS and TRAF6-NF-?B signaling pathways. Immunity. 2011 Jun 24;34(6):854-65.

Allen IC, Moore CB, Schneider M, Lei Y, Davis BK, Scull MA, Gris D, Roney KE, Zimmermann AG, Bowzard JB, Ranjan P, Monroe KM, Pickles RJ, Sambhara S, Ting JP.

Abstract

The nucleotide-binding domain and leucine-rich-repeat-containing (NLR) proteins regulate innate immunity. Although the positive regulatory impact of NLRs is clear, their inhibitory roles are not well defined. We showed that Nlrx1(-/-) mice exhibited increased expression of antiviral signaling molecules IFN-?, STAT2, OAS1, and IL-6 after influenza virus infection. Consistent with increased inflammation, Nlrx1(-/-) mice exhibited marked morbidity and histopathology. Infection of these mice with an influenza strain that carries a mutated NS-1 protein, which normally prevents IFN induction by interaction with RNA and the intracellular RNA sensor RIG-I, further exacerbated IL-6 and type I IFN signaling. NLRX1 also weakened cytokine responses to the 2009 H1N1 pandemic influenza virus in human cells. Mechanistically, Nlrx1 deletion led to constitutive interaction of MAVS and RIG-I. Additionally, an inhibitory function is identified for NLRX1 during LPS activation of macrophages where the MAVS-RIG-I pathway was not involved. NLRX1 interacts with TRAF6 and inhibits NF-?B activation. Thus, NLRX1 functions as a checkpoint of overzealous inflammation.

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Parkinson's disease-linked LRRK2 is expressed in circulating and tissue immune cells and upregulated following recognition of microbial structures. J Neural Transm. 2011 May;118(5):795-808. Epub 2011 May 7.

Hakimi M, Selvanantham T, Swinton E, Padmore RF, Tong Y, Kabbach G, Venderova K, Girardin SE, Bulman DE, Scherzer CR, LaVoie MJ, Gris D, Park DS, Angel JB, Shen J, Philpott DJ, Schlossmacher MG.

Abstract

Sequence variants at or near the leucine-rich repeat kinase 2 (LRRK2) locus have been associated with susceptibility to three human conditions: Parkinson's disease (PD), Crohn's disease and leprosy. As all three disorders represent complex diseases with evidence of inflammation, we hypothesized a role for LRRK2 in immune cell functions. Here, we report that full-length Lrrk2 is a relatively common constituent of human peripheral blood mononuclear cells (PBMC) including affinity isolated, CD14(+) monocytes, CD19(+) B cells, and CD4(+) as well as CD8(+) T cells. Up to 26% of PBMC from healthy donors and up to 43% of CD14(+) monocytes were stained by anti-Lrrk2 antibodies using cell sorting. PBMC lysates contained full-length (>260 kDa) and higher molecular weight Lrrk2 species. The expression of LRRK2 in circulating leukocytes was confirmed by microscopy of human blood smears and in sections from normal midbrain and distal ileum. Lrrk2 reactivity was also detected in mesenteric lymph nodes and spleen (including in dendritic cells), but was absent in splenic mononuclear cells from lrrk2-null mice, as expected. In cultured bone marrow-derived macrophages from mice we made three observations: (i) a predominance of higher molecular weight lrrk2; (ii) the reduction of autophagy marker LC3-II in (R1441C)lrrk2-mutant cells (fourfold) and protein after exposure to several microbial structures including bacterial lipopolysaccharide and lentiviral particles. We conclude that Lrrk2 is a constituent of many cell types in the immune system. Following the recognition of microbial structures, stimulated macrophages respond with altered lrrk2 gene expression. In the same cells, lrrk2 appears to co-regulate autophagy. A pattern recognition receptor-type function for LRRK2 could explain its locus' association with Crohn's disease and leprosy risk. We speculate that the role of Lrrk2 in immune cells may also be relevant to the susceptibility of developing PD or its progression.

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The NLR adaptor ASC/PYCARD regulates DUSP10, mitogen-activated protein kinase (MAPK), and chemokine induction independent of the inflammasome. J Biol Chem. 2011 Jun 3;286(22):19605-16. Epub 2011 Apr 12.

Taxman DJ, Holley-Guthrie EA, Huang MT, Moore CB, Bergstralh DT, Allen IC, Lei Y, Gris D, Ting JP.

Abstract

ASC/PYCARD is a common adaptor for a diverse set of inflammasomes that activate caspase-1, most prominently the NLR-based inflammasome. Mounting evidence indicates that ASC and these NLRs also elicit non-overlapping functions, but the molecular basis for this difference is unclear. To address this, we performed microarray and network analysis of ASC shRNA knockdown cells. In pathogen-infected cells, an ASC-dependent interactome is centered on the mitogen-activated protein kinase (MAPK) ERK and on multiple chemokines. ASC did not affect the expression of MAPK but affected its phosphorylation by pathogens and Toll-like receptor agonists via suppression of the dual-specificity phosphatase, DUSP10/MKP5. Chemokine induction, DUSP function, and MAPK phosphorylation were independent of caspase-1 and IL-1?. MAPK activation by pathogen was abrogated in Asc(-/-) but not Nlrp3(-/-), Nlrc4(-/-), or Casp1(-/-) macrophages. These results demonstrate a function for ASC that is distinct from the inflammasome in modulating MAPK activity and chemokine expression and further identify DUSP10 as a novel ASC target.

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Fatty acid-induced NLRP3-ASC inflammasome activation interferes with insulin signaling. Nat Immunol. 2011 May;12(5):408-15. Epub 2011 Apr 10.

Wen H, Gris D, Lei Y, Jha S, Zhang L, Huang MT, Brickey WJ, Ting JP.

Abstract

High-fat diet (HFD) and inflammation are key contributors to insulin resistance and type 2 diabetes (T2D). Interleukin (IL)-1? plays a role in insulin resistance, yet how IL-1? is induced by the fatty acids in an HFD, and how this alters insulin signaling, is unclear. We show that the saturated fatty acid palmitate, but not unsaturated oleate, induces the activation of the NLRP3-ASC inflammasome, causing caspase-1, IL-1? and IL-18 production. This pathway involves mitochondrial reactive oxygen species and the AMP-activated protein kinase and unc-51-like kinase-1 (ULK1) autophagy signaling cascade. Inflammasome activation in hematopoietic cells impairs insulin signaling in several target tissues to reduce glucose tolerance and insulin sensitivity. Furthermore, IL-1? affects insulin sensitivity through tumor necrosis factor-independent and dependent pathways. These findings provide insights into the association of inflammation, diet and T2D.

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The inflammasome sensor, NLRP3, regulates CNS inflammation and demyelination via caspase-1 and interleukin-18. J Neurosci. 2010 Nov 24;30(47):15811-20.

Jha S, Srivastava SY, Brickey WJ, Iocca H, Toews A, Morrison JP, Chen VS, Gris D, Matsushima GK, Ting JP.

Abstract

Inflammation is increasingly recognized as an important contributor to a host of CNS disorders; however, its regulation in the brain is not well delineated. Nucleotide-binding domain, leucine-rich repeat, pyrin domain containing 3 (NLRP3) is a key component of the inflammasome complex, which also includes ASC (apoptotic speck-containing protein with a card) and procaspase-1. Inflammasome formation can be triggered by membrane P2X(7)R engagement leading to cleavage-induced maturation of caspase-1 and interleukin-1? (IL-1?)/IL-18. This work shows that expression of the Nlrp3 gene was increased >100-fold in a cuprizone-induced demyelination and neuroinflammation model. Mice lacking the Nlrp3 gene (Nlrp3(-/-)) exhibited delayed neuroinflammation, demyelination, and oligodendrocyte loss in this model. These mice also showed reduced demyelination in the experimental autoimmune encephalomyelitis model of neuroinflammation. This outcome is also observed for casp1(-/-) and IL-18(-/-) mice, whereas IL-1?(-/-) mice were indistinguishable from wild-type controls, indicating that Nlrp3-mediated function is through caspase-1 and IL-18. Additional analyses revealed that, unlike the IL-1?(-/-) mice, which have been previously shown to show delayed remyelination, Nlrp3(-/-) mice did not exhibit delayed remyelination. Interestingly, IL-18(-/-) mice showed enhanced remyelination, thus providing a possible compensatory mechanism for the lack of a remyelination defect in Nlrp3(-/-) mice. These results suggest that NLRP3 plays an important role in a model of multiple sclerosis by exacerbating CNS inflammation, and this is partly mediated by caspase-1 and IL-18. Additionally, the therapeutic inhibition of IL-18 might decrease demyelination but enhance remyelination, which has broad implications for demyelinating diseases.

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Deletion of ripA alleviates suppression of the inflammasome and MAPK by Francisella tularensis. J Immunol. 2010 Nov 1;185(9):5476-85. Epub 2010 Oct 4.

Huang MT, Mortensen BL, Taxman DJ, Craven RR, Taft-Benz S, Kijek TM, Fuller JR, Davis BK, Allen IC, Brickey WJ, Gris D, Wen H, Kawula TH, Ting JP.

Abstract

Francisella tularensis is a facultative intracellular pathogen and potential biothreat agent. Evasion of the immune response contributes to the extraordinary virulence of this organism although the mechanism is unclear. Whereas wild-type strains induced low levels of cytokines, an F. tularensis ripA deletion mutant (LVS?ripA) provoked significant release of IL-1?, IL-18, and TNF-? by resting macrophages. IL-1? and IL-18 secretion was dependent on inflammasome components pyrin-caspase recruitment domain/apoptotic speck-containing protein with a caspase recruitment domain and caspase-1, and the TLR/IL-1R signaling molecule MyD88 was required for inflammatory cytokine synthesis. Complementation of LVS?ripA with a plasmid encoding ripA restored immune evasion. Similar findings were observed in a human monocytic line. The presence of ripA nearly eliminated activation of MAPKs including ERK1/2, JNK, and p38, and pharmacologic inhibitors of these three MAPKs reduced cytokine induction by LVS?ripA. Animals infected with LVS?ripA mounted a stronger IL-1? and TNF-? response than that of mice infected with wild-type live vaccine strain. This analysis revealed novel immune evasive mechanisms of F. tularensis.

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Cutting edge: NLRP12 controls dendritic and myeloid cell migration to affect contact hypersensitivity. J Immunol. 2010 Oct 15;185(8):4515-9. Epub 2010 Sep 22.

Arthur JC, Lich JD, Ye Z, Allen IC, Gris D, Wilson JE, Schneider M, Roney KE, O'Connor BP, Moore CB, Morrison A, Sutterwala FS, Bertin J, Koller BH, Liu Z, Ting JP.

Abstract

Nucleotide-binding domain leucine-rich repeat (NLR) proteins are regulators of inflammation and immunity. Although first described 8 y ago, a physiologic role for NLRP12 has remained elusive until now. We find that murine Nlrp12, an NLR linked to atopic dermatitis and hereditary periodic fever in humans, is prominently expressed in dendritic cells (DCs) and neutrophils. Nlrp12-deficient mice exhibit attenuated inflammatory responses in two models of contact hypersensitivity that exhibit features of allergic dermatitis. This cannot be attributed to defective Ag processing/presentation, inflammasome activation, or measurable changes in other inflammatory cytokines. Rather, Nlrp12(-/-) DCs display a significantly reduced capacity to migrate to draining lymph nodes. Both DCs and neutrophils fail to respond to chemokines in vitro. These findings indicate that NLRP12 is important in maintaining neutrophils and peripheral DCs in a migration-competent state.

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NLRP3 plays a critical role in the development of experimental autoimmune encephalomyelitis by mediating Th1 and Th17 responses. J Immunol. 2010 Jul 15;185(2):974-81. Epub 2010 Jun 23.

Gris D, Ye Z, Iocca HA, Wen H, Craven RR, Gris P, Huang M, Schneider M, Miller SD, Ting JP.

Abstract

The interplay between innate and adaptive immunity is important in multiple sclerosis (MS). The inflammasome complex, which activates caspase-1 to process pro-IL-1beta and pro-IL-18, is rapidly emerging as a pivotal regulator of innate immunity, with nucleotide-binding domain, leucine-rich repeat containing protein family, pyrin domain containing 3 (NLRP3) (cryopyrin or NALP3) as a prominent player. Although the role of NLRP3 in host response to pathogen associated molecular patterns and danger associated molecular patterns is well documented, its role in autoimmune diseases is less well studied. To investigate the role of NLRP3 protein in MS, we used a mouse model of MS, experimental autoimmune encephalomyelitis (EAE). Nlrp3 expression was elevated in the spinal cords during EAE, and Nlrp3(-/-) mice had a dramatically delayed course and reduced severity of disease. This was accompanied by a significant reduction of the inflammatory infiltrate including macrophages, dendritic cells, CD4, and CD8(+) T cells in the spinal cords of the Nlrp3(-/-) mice, whereas microglial accumulation remained the same. Nlrp3(-/-) mice also displayed improved histology in the spinal cords with reduced destruction of myelin and astrogliosis. Nlrp3(-/-) mice with EAE produced less IL-18, and the disease course was similar to Il18(-/-) mice. Furthermore, Nlrp3(-/-) and Il18(-/-) mice had similarly reduced IFN-gamma and IL-17 production. Thus, NLRP3 plays a critical role in the induction of the EAE, likely through effects on capase-1-dependent cytokines which then influence Th1 and Th17.

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A novel alternatively spliced isoform of the mu-opioid receptor: functional antagonism. Mol Pain. 2010 Jun 2;6:33.

Gris P, Gauthier J, Cheng P, Gibson DG, Gris D, Laur O, Pierson J, Wentworth S, Nackley AG, Maixner W, Diatchenko L.

Abstract

BACKGROUND: Opioids are the most widely used analgesics for the treatment of clinical pain. They produce their therapeutic effects by binding to mu-opioid receptors (MORs), which are 7 transmembrane domain (7TM) G-protein-coupled receptors (GPCRs), and inhibiting cellular activity. However, the analgesic efficacy of opioids is compromised by side-effects such as analgesic tolerance, dependence and opioid-induced hyperalgesia (OIH). In contrast to opioid analgesia these side effects are associated with cellular excitation. Several hypotheses have been advanced to explain these phenomena, yet the molecular mechanisms underlying tolerance and OIH remain poorly understood. RESULTS: We recently discovered a new human alternatively spliced isoform of MOR (MOR1K) that is missing the N-terminal extracellular and first transmembrane domains, resulting in a 6TM GPCR variant. To characterize the pattern of cellular transduction pathways activated by this human MOR1K isoform, we conducted a series of pharmacological and molecular experiments. Results show that stimulation of MOR1K with morphine leads to excitatory cellular effects. In contrast to stimulation of MOR1, stimulation of MOR1K leads to increased Ca2+ levels as well as increased nitric oxide (NO) release. Immunoprecipitation experiments further reveal that unlike MOR1, which couples to the inhibitory Galphai/o complex, MOR1K couples to the stimulatory Galphas complex. CONCLUSION: The major MOR1 and the alternative MOR1K isoforms mediate opposite cellular effects in response to morphine, with MOR1K driving excitatory processes. These findings warrant further investigations that examine animal and human MORK1 expression and function following chronic exposure to opioids, which may identify MOR1K as a novel target for the development of new clinically effective classes of opioids that have high analgesic efficacy with diminished ability to produce tolerance, OIH, and other unwanted side-effects.

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Staphylococcus aureus alpha-hemolysin activates the NLRP3-inflammasome in human and mouse monocytic cells. PLoS One. 2009 Oct 14;4(10):e7446.

Craven RR, Gao X, Allen IC, Gris D, Bubeck Wardenburg J, McElvania-Tekippe E, Ting JP, Duncan JA.

Abstract

Community Acquired Methicillin Resistant Staphylococcus aureus (CA-MRSA) causes severe necrotizing infections of the skin, soft tissues, and lungs. Staphylococcal alpha-hemolysin is an essential virulence factor in mouse models of CA-MRSA necrotizing pneumonia. S. aureus alpha-hemolysin has long been known to induce inflammatory signaling and cell death in host organisms, however the mechanism underlying these signaling events were not well understood. Using highly purified recombinant alpha-hemolysin, we now demonstrate that alpha-hemolysin activates the Nucleotide-binding domain and leucine-rich repeat containing gene family, pyrin domain containing 3 protein (NLRP3)-inflammasome, a host inflammatory signaling complex involved in responses to pathogens and endogenous danger signals. Non-cytolytic mutant alpha-hemolysin molecules fail to elicit NLRP3-inflammasome signaling, demonstrating that the responses are not due to non-specific activation of this innate immune signaling system by bacterially derived proteins. In monocyte-derived cells from humans and mice, inflammasome assembly in response to alpha-hemolysin results in activation of the cysteine proteinase, caspase-1. We also show that inflammasome activation by alpha-hemolysin works in conjunction with signaling by other CA-MRSA-derived Pathogen Associated Molecular Patterns (PAMPs) to induce secretion of pro-inflammatory cytokines IL-1beta and IL-18. Additionally, alpha-hemolysin induces cell death in these cells through an NLRP3-dependent program of cellular necrosis, resulting in the release of endogenous pro-inflammatory molecules, like the chromatin-associated protein, High-mobility group box 1 (HMGB1). These studies link the activity of a major S. aureus virulence factor to a specific host signaling pathway. The cellular events linked to inflammasome activity have clear relevance to the disease processes associated with CA-MRSA including tissue necrosis and inflammation.

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The systemic inflammatory response after spinal cord injury damages lungs and kidneys. Exp Neurol. 2008 May;211(1):259-70. Epub 2008 Mar 4.

Gris D, Hamilton EF, Weaver LC.

Abstract

Spinal cord injury (SCI) triggers a well characterized, acute, local inflammation leading to secondary damage at the lesion site. Another little recognized problem may be the activation of circulating inflammatory cells that potentially damage tissues outside the cord. We investigated this problem using severe clip-compression SCI in rats. We studied systemic inflammation after SCI and its effects on lungs and kidneys, as dysfunction of these organs is a frequent, early complication after SCI. From 2-24 h after SCI, the number of circulating neutrophils (especially immature cells) significantly increased by 3-10 fold. Flow cytometry experiments revealed that SCI transiently activates these neutrophils, causing increased oxidative responses to phorbolmyristic acid at 2 h after SCI; then, from 4-24 h, the neutrophils were less responsive. Neutrophil longevity was increased (30-50% decrease in apoptosis) at 2-8 h after SCI. Immunohistochemical analyses demonstrated the invasion of neutrophils into lungs and kidneys (2 h-7 d after SCI) and more phagocytic macrophages in lungs (12 h, 3 d after SCI). Myeloperoxidase and matrix metalloproteinase-9 activity in lung and kidney homogenates increased (12 h-7 d after SCI). Expression of COX-2 increased and lipid peroxidation also occurred within this time. Control experiments inducing local cord damage by excitotoxic quisqualate injection verified that SCI per se is sufficient to trigger systemic inflammation and organ damage. In summary, SCI mobilizes and activates neutrophils that then migrate into visceral organs, a phenomenon occurring in parallel with their well-known entry into the cord injury site. The systemic inflammatory response to SCI should be targeted in the development of new therapeutic strategies to treat SCI.

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Methylprednisolone causes minimal improvement after spinal cord injury in rats, contrasting with benefits of an anti-integrin treatment. J Neurotrauma. 2005 Dec;22(12):1375-87.

Weaver LC, Gris D, Saville LR, Oatway MA, Chen Y, Marsh DR, Hamilton EF, Dekaban GA.

Abstract

Spinal cord injury (SCI) leads to complex secondary events that expand and exacerbate the injury. Methylprednisolone (MP) has been considered a standard of care for acute SCI. The purpose of this study was to test the effects of MP, in severe and more moderate severe clip-compression models of SCI, on the measures of neurological function and lesion sparing that we used previously to assess a highly effective anti-inflammatory therapy, a monoclonal antibody (mAb) to the CD11d integrin. Intravenous treatment with the anti-CD11d mAb blocks the infiltration of leukocytes into the lesion, limits secondary cord damage, and improves neurological outcomes. We also undertook a 2- week study of effects of these two therapies in combination. To permit direct comparison, the new findings with MP are presented together with reference to the previously published effects of the mAb. The severe SCI was at the 4(th) thoracic segment (T4), causing extensive motor dysfunction; the more moderate SCI was at T12 and caused less locomotor loss but the induction of mechanical allodynia. Neither MP alone nor the combination treatment improved Basso, Beattie, and Bresnahan 21-point open-field locomotor scores at 2-12 weeks after SCI. These scores were ~4 points in the control, MP, and combination treatment groups, respectively, at 2 weeks after severe SCI at T4. By 6 weeks after T4 SCI, scores in the control and MP groups were ~7. At 12 weeks after the more moderate T12 injury, scores were ~8 in both control and MP treatment groups. MP treatment had no consistent effect on mechanical allodynia during 12 weeks after SCI. Control and MP-treated rats responded to approximately five of 10 stimuli to their backs and three of 10 stimuli to their hind paws. MP treatment increased areas of neurofilament and myelin near the injury site at T4 and T12. Thus, MP treatment spared tissue, but had no corresponding effect on neurological function. In contrast, the combination treatment did not spare myelin significantly. These neurological outcomes after treatment with MP contrast with the consistent and significant improvements after treatment with the anti-CD11d mAb. Effects of MP on the lesion were significant, but myelin sparing was less than that caused by the anti-CD11d mAb. The presence of MP in the combination therapy appeared to reverse the positive effects of the mAb. The poor neurological outcome after MP treatment may relate to the long-lasting reduction in hematogenous monocyte/macrophages within the injury site that it causes and to the prolongation of a neutrophil presence. These findings demonstrate that the non-selective and enduring effects of immunosuppressive therapy with MP not only fail to improve neurological outcomes, but also can block the beneficial actions of selective therapies such as the anti-CD11d mAb. Combination treatments that cause intense immunosuppression should be viewed with caution.

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Autonomic dysreflexia after spinal cord injury: central mechanisms and strategies for prevention. Prog Brain Res. 2006;152:245-63.

Weaver LC, Marsh DR, Gris D, Brown A, Dekaban GA.

Abstract

Spinal reflexes dominate cardiovascular control after spinal cord injury (SCI). These reflexes are no longer restrained by descending control and they can be impacted by degenerative and plastic changes within the injured cord. Autonomic dysreflexia is a condition of episodic hypertension that stems from spinal reflexes initiated by sensory input entering the spinal cord caudal to the site of injury. This hypertension greatly detracts from the quality of life for people with cord injury and can be life-threatening. Changes in the spinal cord contribute substantially to the development of this condition. Rodent models are ideal for investigating these changes. Within the spinal cord, injury-induced plasticity leads to nerve growth factor (NGF)-dependent enlargement of the central arbor of a sub-population of sensory neurons. This enlarged arbor can provide increased afferent input to the spinal reflex, intensifying autonomic dysreflexia. Treatments such as antibodies against NGF can limit this afferent sprouting, and diminish the magnitude of dysreflexia. To assess treatments, a compression model of SCI that leads to progressive secondary damage, and also to some white matter sparing, is very useful. The types of spinal reflexes that likely mediate autonomic dysreflexia are highly susceptible to inhibitory influences of bulbospinal pathways traversing the white matter. Compression models of cord injury reveal that treatments that spare white matter axons also markedly reduce autonomic dysreflexia. One such treatment is an antibody to the integrin CD11d expressed by inflammatory leukocytes that enter the cord acutely after injury and cause significant secondary damage. This antibody blocks integrin-mediated leukocyte entry, resulting in greatly reduced white-matter damage and decreased autonomic dysreflexia after cord injury. Understanding the mechanisms for autonomic dysreflexia will provide us with strategies for treatments that, if given early after cord injury, can prevent this serious disorder from developing.

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Comparison of effects of methylprednisolone and anti-CD11d antibody treatments on autonomic dysreflexia after spinal cord injury. Exp Neurol. 2005 Aug;194(2):541-9.

Gris D, Marsh DR, Dekaban GA, Weaver LC.

Abstract

Autonomic dysreflexia is a condition of episodic hypertension that develops after spinal cord injury (SCI). We previously showed that a two-day anti-inflammatory treatment with an anti-CD11d integrin monoclonal antibody (mAb), soon after SCI in rats, reduced the magnitude of dysreflexia for at least 6 weeks. Effects of methylprednisolone (MP), a commonly used neuroprotective treatment for SCI, on dysreflexia have never been examined. We compared the effects of a 2-day MP treatment and/or the anti-CD11d mAb on autonomic dysreflexia, elicited by colon distension, after clip-compression SCI at the 4th thoracic segment (T4) in rats. We assessed the effects of each treatment on the size of the calcitonin gene-related peptide (CGRP)-immunoreactive afferent arbour in the dorsal horn, as changes in this arbour can correlate with the development of dysreflexia. MP reduced autonomic dysreflexia by approximately 50% at 2 weeks after SCI, but this effect was lost by 6 weeks. At 2 weeks, the combined effects of MP and the mAb were not additive, reducing dysreflexia by approximately 50%. Neither MP nor the mAb treatment altered the area of CGRP-immunoreactive fibres in the lumbar cord, the crucial input region for dysreflexia initiated by colon distension. However, both treatments led to increased fibre areas in the T9 segment, correlated with greater tissue integrity and smaller lesions, delineated by inflammatory cells. In summary, MP only temporarily decreases autonomic dysreflexia after SCI. The early beneficial effects of both treatments on dysreflexia do not relate to changes in the CGRP-immunoreactive afferent arbour but may correlate with decreased lesion progression.

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Transient blockade of the CD11d/CD18 integrin reduces secondary damage after spinal cord injury, improving sensory, autonomic, and motor function. J Neurosci. 2004 Apr 21;24(16):4043-51.

Gris D, Marsh DR, Oatway MA, Chen Y, Hamilton EF, Dekaban GA, Weaver LC.

Abstract

The early inflammatory response to spinal cord injury (SCI) causes significant secondary damage. Strategies that nonselectively suppress inflammation have not improved outcomes after SCI, perhaps because inflammation has both adverse and beneficial effects after SCI. We have shown that the selective, time-limited action of a monoclonal antibody (mAb) to the CD11d subunit of the CD11d/CD18 integrin, delivered intravenously during the first 48 hr after SCI in rats, markedly decreases the infiltration of neutrophils and delays the entry of hematogenous monocyte-macrophages into the injured cord. We hypothesized that this targeted strategy would lead to neuroprotection and improved neurological outcomes. In this study the development of chronic pain was detected in rats by assessing mechanical allodynia on the trunk and hindpaws 2 weeks to 3 months after a clinically relevant clip-compression SCI at the twelfth thoracic segment. The anti-CD11d mAb treatment reduced this pain by half. Motor performance also improved as rats were able to plantar-place their hindpaws and use them for weight support instead of sweeping movements only. Improved cardiovascular outcome was shown after SCI at the fourth thoracic segment by significant decreases in autonomic dysreflexia. Locomotor performance was also improved. These functional changes correlated with significantly greater amounts and increased organization of myelin and neurofilament near the lesion. The improved neurological recovery after the specific reduction of early inflammation after SCI demonstrates that this selective strategy increases tissue at the injury site and improves its functional capacity. This early neuroprotective treatment would be an ideal foundation for building later cell-based therapies.

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