The nod-like receptor, Nlrp12, plays an anti-inflammatory role in experimental autoimmune encephalomyelitis.

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-κB) 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-1ß 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 (TNFα), 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.

Nlrx1 regulates neuronal cell death.

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.

Assessment of oxidative metabolism.

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.