Reduction of SIRT1 epigenetically upregulates NALP1 expression and contributes to neuropathic pain induced by chemotherapeutic drug bortezomib.

BACKGROUND: Bortezomib is a frequently used chemotherapeutic drug for the treatment of multiple myeloma and other nonsolid malignancies. Accumulating evidence has demonstrated that bortezomib-induced persistent pain serves as the most frequent reason for treatment discontinuation. METHODS: The von Frey test was performed to evaluate neuropathic pain behavior, and real-time quantitative reverse transcription polymerase chain reaction, chromatin immunoprecipitation, western blot, immunohistochemistry, and small interfering RNA were performed to explore the molecular mechanisms in adult male Sprague-Dawley rats. RESULTS: We found that application of bortezomib significantly increased the expression of NALP1 protein and mRNA levels in spinal dorsal horn neurons, and intrathecal application of NALP1 siRNA attenuated the bortezomib-induced mechanical allodynia. In addition, bortezomib also decreased the SIRT1 expression, and treatment with SIRT1 activator resveratrol ameliorated the NALP1 upregulation and mechanical allodynia induced by bortezomib. Meanwhile, knockdown of SIRT1 using the SIRT1 siRNA induced the NALP1 upregulation in dorsal horn and mechanical allodynia in normal animal. These results suggested that reduction of SIRT1 induced the NALP1 upregulation in dorsal horn neurons, and participated in bortezomib-induced mechanical allodynia. Importantly, we found that the binding of SIRT1 and NALP1 promoter region did not change before and after bortezomib treatment, but SIRT1 downregulation increased p-STAT3 expression. Furthermore, the activation of STAT3 enhanced the recruitment of p-STAT3 to the Nalp1 gene promoter, which increased the acetylation of histone H3 and H4 in NALP1 promoter regions and epigenetically upregulated NALP1 expression in the rodents with bortezomib treatment. CONCLUSION: These findings suggested a new epigenetic mechanism for NALP1 upregulation involving SIRT1 reduction and subsequent STAT3-mediated histone hyperacetylation in NALP1 promoter region in dorsal horn neurons, which contributed to the bortezomib-induced mechanical allodynia.

Early single Aspirin-triggered Lipoxin blocked morphine anti-nociception tolerance through inhibiting NALP1 inflammasome: Involvement of PI3k/Akt signaling pathway.

Clinical usage of opioids in pain relief is dampened by analgesic tolerance after chronic exposure, which is related to opioid-associated neuroinflammation. In the current study, which is based on a chronic morphine tolerance rat model and sustained morphine treatment on primary neuron culture, it was observed that Akt phosphorylation, cleaved-Caspase-1-dependent NALP1 inflammasome activation and IL-1ß maturation in spinal cord neurons were significantly enhanced by morphine. Moreover, treatment with LY294002, a specific inhibitor of PI3k/Akt signaling, significantly reduced Caspase-1 cleavage, NALP1 inflammasome activation and attenuated morphine tolerance. Tail-flick tests demonstrated that pharmacological inhibition on Caspase-1 activation or antagonizing IL-1ß dramatically blocked the development of morphine tolerance. The administration of an exogenous analogue of lipoxin, Aspirin-triggered Lipoxin (ATL), caused a decline in Caspase-1 cleavage, inflammasome activation and mature IL-1ß production and thus attenuated the development of morphine tolerance by inhibiting upstream Akt phosphorylation. Additionally, treatment with DAMGO, a selective µ-opioid receptor peptide, significantly induced Akt phosphorylation, Caspase-1 cleavage and anti-nociception tolerance, all of which were attenuated by ATL treatment. Taken together, the present study revealed the involvement of spinal NALP1 inflammasome activation in the development of morphine tolerance and the role of the µ-receptor/PI3k-Akt signaling/NALP1 inflammasome cascade in this process. By inhibiting this signaling cascade, ATL blocked the development of morphine tolerance.

Genetic analysis of long-lived families reveals novel variants influencing high density-lipoprotein cholesterol.

The plasma levels of high-density lipoprotein cholesterol (HDL) have an inverse relationship to the risks of atherosclerosis and cardiovascular disease (CVD), and have also been associated with longevity. We sought to identify novel loci for HDL that could potentially provide new insights into biological regulation of HDL metabolism in healthy-longevous subjects. We performed a genome-wide association (GWA) scan on HDL using a mixed model approach to account for family structure using kinship coefficients. A total of 4114 subjects of European descent (480 families) were genotyped at ~2.3 million SNPs and ~38 million SNPs were imputed using the 1000 Genome Cosmopolitan reference panel in MACH. We identified novel variants near-NLRP1 (17p13) associated with an increase of HDL levels at genome-wide significant level (p < 5.0E-08). Additionally, several CETP (16q21) and ZNF259-APOA5-A4-C3-A1 (11q23.3) variants associated with HDL were found, replicating those previously reported in the literature. A possible regulatory variant upstream of NLRP1 that is associated with HDL in these elderly Long Life Family Study (LLFS) subjects may also contribute to their longevity and health. Our NLRP1 intergenic SNPs show a potential regulatory function in Encyclopedia of DNA Elements (ENCODE); however, it is not clear whether they regulate NLRP1 or other more remote gene. NLRP1 plays an important role in the induction of apoptosis, and its inflammasome is critical for mediating innate immune responses. Nlrp1a (a mouse ortholog of human NLRP1) interacts with SREBP-1a (17p11) which has a fundamental role in lipid concentration and composition, and is involved in innate immune response in macrophages. The NLRP1 region is conserved in mammals, but also has evolved adaptively showing signals of positive selection in European populations that might confer an advantage. NLRP1 intergenic SNPs have also been associated with immunity/inflammasome disorders which highlights the biological importance of this chromosomal region.

Crystal structure of the leucine-rich repeat domain of the NOD-like receptor NLRP1: implications for binding of muramyl dipeptide.

The NOD-like receptor NLRP1 (NLR family, pyrin domain containing 1) senses the presence of the bacterial cell wall component l-muramyl dipeptide (MDP) inside the cell. We determined the crystal structure of the LRR domain of human NLRP1 in the absence of MDP to a resolution of 1.65Å. The fold of the structure can be assigned to the ribonuclease inhibitor-like class of LRR proteins. We compared our structure with X-ray models of the LRR domains of NLRX1 and NLRC4 and a homology model of the LRR domain of NOD2. We conclude that the MDP binding site of NLRP1 is not located in the LRR domain.

NLRP1 haplotypes associated with leprosy in Brazilian patients.

Polymorphisms in innate immunity genes are known to be involved in the multifactorial susceptibility to Mycobacterium leprae infection. M. leprae can downregulate IL-1ß secretion escaping monocyte digestion. The intracellular receptors NLRPs (NACHT, LRR and PYD domains-containing proteins) sense pathogen associated molecular patterns (PAMPs) activating caspase-1 and IL-1ß secretion in the context of inflammasome. Considering the possible role of inflammasome in the immune response against M. leprae, known single nucleotide polymorphisms (SNPs) in two NLRP genes, NLRP1 and NLRP3, were analyzed in Brazilian leprosy patients. Disease-associated SNPs (5 in NLRP1 and 2 in NLRP3), previously associated to infections and to immunologic disorders, were genotyped in 467 leprosy patients (327 multibacillary, MB; 96 paucibacillary, PB) and in 380 healthy controls (HC) from the state of Sao Paulo (Brazil), and in 183 patients (147MB; 64PB) and 186 HC from Mato Grosso (Brazil). Logistic regression analysis was performed considering susceptibility to leprosy di per se (leprosy versus HC) and clinical form (MB versus PB), adjusting for gender and ethnicity. Whereas none of the considered SNPs were statistically associated with leprosy, the NLRP1 combined haplotype rs2137722/G-rs12150220/T-rs2670660/G resulted significantly more frequent in patients than in HC as well as in PB than in MB. While both associations were lost after correction for gender and ethnicity, the NLRP1 combined haplotype rs2137722/G-rs12150220/A-rs2670660/G resulted strongly associated to PB. NLRP1 might be involved in the susceptibility to leprosy with particular emphasis for PB clinical form. Although preliminary, this is the first report linking NLRPs and inflammasome with leprosy: replication studies as well as functional assays are envisaged to deeper investigate the role of NLRP1 in M. leprae infection. Interestingly, NLRP1 SNPs were previously associated to susceptibility to Crohn disease, suggesting that NLRP1 could be a new modifier gene in common between leprosy and Crohn disease.

Involvement of the spinal NALP1 inflammasome in neuropathic pain and aspirin-triggered-15-epi-lipoxin A4 induced analgesia.

Neuroinflammation plays an important role in nerve-injury-induced neuropathic pain, but the explicit molecular mechanisms of neuroinflammation in neuropathic pain remain unclear. As one of the most critical inflammatory cytokines, interleukin-1ß (IL-1ß) has been regarded as broadly involved in the pathology of neuropathic pain. The inflammasome caspase-1 platform is one primary mechanism responsible for the maturation of IL-1ß. Lipoxins, a type of endogenous anti-inflammatory lipid, have proved to be effective in relieving neuropathic pain behaviors. The present study was designed to examine whether the inflammasome caspase-1 IL-1ß platform is involved in chronic constriction injury (CCI)-induced neuropathic pain and in lipoxin-induced analgesia. After rats were subjected to the CCI surgery, mature IL-1ß was significantly increased in the ipsilateral spinal cord, and the inflammasome platform consisting of NALP1 (NAcht leucine-rich-repeat protein 1), caspase-1 and ASC (apoptosis-associated speck-like protein containing a caspase-activating recruitment domain) was also activated in spinal astrocytes and neurons, especially at the superficial laminae of the spinal dorsal horn; The aspirin-triggered-15-epi-lipoxin A4 (ATL), which shares the potent actions of the endogenous lipoxins, was administered to the CCI rats. Repeated intrathecal injection with ATL markedly attenuated the CCI-induced thermal hyperalgesia and significantly inhibited NALP1 inflammasome activation, caspase-1 cleavage, and IL-1ß maturation. These results suggested that spinal NALP1 inflammasome was involved in the CCI-induced neuropathic pain and that the analgesic effect of ATL was associated with suppressing NALP1 inflammasome activation.

Genetic variations in nalp1 mRNA expressions in human vitiligo.

INTRODUCTION: Vitiligo is an acquired autoimmune disease of unknown etiology showing depigmentation of the skin due to the absence of melanocytes. Familial vitiligo suggests a genetic origin to this disease. Chromosome 17 was recently demonstrated to harbor the gene coding for NALP1. PATIENTS AND METHODS: A total of 18 patients of vitiligo were selected on the basis of clinical history. Group 1 (N=8) showing segmental or localized vitiligo with one or two macules on the body. Group 2 (N=10) with generalized or whole body vitiligo. A control group of 10 healthy individuals were selected from our laboratory persons with no history or any infections or skin disease. NALP1 gene expression was studied using RT-PCR assay and the bands quantitated as intensity using volume as measurement and comparison of results was done using SPSS 16 version for statistical analysis. NALP1 gene expression was observed in vitiligo patients with different intensities. RESULTS: Greater reduction in the intensity was seen in Group I, which was inversely proportional to the volume of the band. The intensity of the NALP1 and the GAPDH gene expression was more in Group 2 patients than that shown by Group 1. CONCLUSION: This study shows expression of NALP1 gene in patients as well as normals. NALP1 is widely expressed at low levels but is expressed at high levels in immune cells, particularly T cells and Langerhans cells, in which different patterns are seen that are consistent with the particular involvement of NALP1 in skin autoimmunity.

NLRP1 haplotypes associated with leprosy in Brazilian patients

Polymorphisms in innate immunity genes are known to be involved in the multifactorial susceptibility to Mycobacterium leprae infection. M. leprae can downregulate IL-1ß secretion escaping monocyte digestion. The intracellular receptors NLRPs (NACHT, LRR and PYD domains-containing proteins) sense pathogen associated molecular patterns (PAMPs) activating caspase-1 and IL-1ß secretion in the context of inflammasome. Considering the possible role of inflammasome in the immune response against M. leprae, known single nucleotide polymorphisms (SNPs) in two NLRP genes, NLRP1 and NLRP3, were analyzed in Brazilian leprosy patients. Disease-associated SNPs (5 in NLRP1 and 2 in NLRP3), previously associated to infections and to immunologic disorders, were genotyped in 467 leprosy patients (327 multibacillary, MB; 96 paucibacillary, PB) and in 380 healthy controls (HC) from the state of Sao Paulo (Brazil), and in 183 patients (147MB; 64PB) and 186 HC from Mato Grosso (Brazil). Logistic regression analysis was performed considering susceptibility to leprosy di per se (leprosy versus HC) and clinical form (MB versus PB), adjusting for gender and ethnicity. Whereas none of the considered SNPs were statistically associated with leprosy, the NLRP1 combined haplotype rs2137722/G-rs12150220/T-rs2670660/G resulted significantly more frequent in patients than in HC as well as in PB than in MB. While both associations were lost after correction for gender and ethnicity, the NLRP1 combined haplotype rs2137722/G-rs12150220/A-rs2670660/G resulted strongly associated to PB. NLRP1 might be involved in the susceptibility to leprosy with particular emphasis for PB clinical form. Although preliminary, this is the first report linking NLRPs and inflammasome with leprosy: replication studies as well as functional assays are envisaged to deeper investigate the role of NLRP1 in M. leprae infection. Interestingly, NLRP1 SNPs were previously associated to susceptibility to Crohn disease, suggesting that NLRP1 could be a new modifier gene in common between leprosy and Crohn disease.