NLRX1 Expression Research Articles

IMMU-42. UNRAVELING THE ROLE OF NLRX1 IN GLIOBLASTOMA TUMOR MICROENVIRONMENT

Abstract •Gliomas are primary brain tumors that develop from glial cells within the central nervous system (CNS) and are among the deadliest human cancers. Glioblastoma (GBM) is the most aggressive glioma. Innate immune cells such as microglia and macrophages account for >50% of the cellular population within the GBM microenvironment. Innate immune cells express pattern recognition receptors (PRRs). NLRX1 is a PRR that regulates diverse signaling pathways and cellular processes including antiviral signaling, ROS production, apoptosis, autophagy, metabolic homeostasis, etc. Additionally, the tumor-suppressive and tumor-promoting functions of NLRX1 have been observed in many cancer types. For example, the tumor-suppressive role of NLRX1 has been observed in colitis-associated carcinogenesis, pancreatic cancer, and primary breast cancer. In contrast, the tumor-promoting role of NLRX1 was found in basal-like and metastatic breast carcinoma. Hence, the effect of NLRX1 on cancer may be context-dependent on cancer type or cell type aided by differences in the microenvironment. Further, how NLRX1 regulates GBM pathophysiology remains largely unexplored. This study aims to determine the expression pattern of NLRX1 in GBM cells and GBM-associated innate immune cells and investigate how NLRX1 expression regulates various cellular processes in GBM cell lines to regulate GBM pathophysiology. We report that NLRX1 is differentially expressed in GBM cell lines, GBM-associated innate immune cells, and glioma patient tissues. si-RNA-mediated silencing of Nlrx1 decreases the ability of the GBM cell line, LN-229 to proliferate and migrate. Further, Nlrx1-/- GBM cells show increased tunneling nanotube (TNT) formation capabilities. TNTs help in metabolic homeostasis maintenance by enabling the exchange of subcellular organelles such as mitochondria and endoplasmic reticulum. Nlrx1-/- GBM cells exhibit attenuated ability to form 3D spheroids. This research will aid the understanding of GBM pathophysiology, leading to novel diagnostic/prognostic markers and the discovery of signaling pathways that, in turn, may help create better GBM therapy approaches and improve overall survival.

NLRX1 and STING alleviate renal ischemia-reperfusion injury by regulating LC3 lipidation during mitophagy

Mitophagy significantly influences renal ischemia/reperfusion (I/R) injury and recovery. NLRX1 is recognized for its regulatory role in governing mitochondrial damage, autophagy, and the expression of pro-inflammatory factors. Despite the acknowledged involvement of NLRX1 in these crucial cellular processes, its specific function in renal I/R injury remains unclear. We detected the expression of NLRX1, the cGAS-STING pathway, and autophagy-related proteins using Western Blot analysis. RT-qPCR was utilized to measure the expression of NLRX1 mRNA and cytokines, and changes in mitochondrial DNA (mtDNA) within the cytoplasm. Immunofluorescence was applied to observe alterations in DNA distribution within the cytoplasm. The EtBr drug, which depletes mtDNA, and the Mdivi-1 mitophagy inhibitor, were used to verify the promotion of mitophagy by NLRX1. The results demonstrated that NLRX1 was downregulated after H/R injury, and there was an increase in cytoplasmic DNA. NLRX1 overexpression not only reduced IL-1β and IL-6 levels, but also decreased mtDNA in the cytoplasm. Additionally, NLRX1 further increases mitochondrial LC3 lipidation after H/R injury, and this effect is inhibited by Mdivi-1 drugs. The activation of the cGAS-STING pathway after H/R injury is inhibited by EtBr drugs and NLRX1. Co-immunoprecipitation results showed that NLRX1 could bind to STING. Moreover, inhibiting STING reversed NLRX1-induced mitochondrial LC3 lipidation. Our study reveals that NLRX1 can bind to STING to promote mitophagy and inhibits inflammation caused by mtDNA/cGAS/STING signaling.

Insights into the structure of NLR family member X1: Paving the way for innovative drug discovery

Nucleotide-binding oligomerization domain, leucine rich repeat containing X1 (NLRX1) is a negative regulator of the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) pathway, with a significant role in the context of inflammation. Altered expression of NLRX1 is prevalent in inflammatory diseases leading to interest in NLRX1 as a drug target. There is a lack of structural information available for NLRX1 as only the leucine-rich repeat domain of NLRX1 has been crystallised. This lack of structural data limits progress in understanding function and potential druggability of NLRX1. We have modelled full-length NLRX1 by combining experimental, homology modelled and AlphaFold2 structures. The full-length model of NLRX1 was used to explore protein dynamics, mutational tolerance and potential functions. We identified a new RNA binding site in the previously uncharacterized N-terminus, which served as a basis to model protein-RNA complexes. The structure of the adenosine triphosphate (ATP) binding domain revealed a potential catalytic functionality for the protein as a member of the ATPase Associated with Diverse Cellular Activity family of proteins. Finally, we investigated the interactions of NLRX1 with small molecule activators in development, revealing a binding site that has not previously been discussed in literature. The model generated here will help to catalyse efforts towards creating new drug molecules to target NLRX1 and may be used to inform further studies on functionality of NLRX1.

THE ONES AND TWOS: PATTERN RECOGNITION RECEPTOR NLRX1 IMPACTS VIRAL REPLICATION OF AND IMMUNE RESPONSE TO SARS-COV-2

Abstract When pandemic virus SARS-CoV-2 (SCV-2) emerged in 2020, research was quickly generated to investigate the virus and host immune responses to it. Much research has focused on the cytokine storm- when overzealous immune responses in severe COVID-19 disease causes rampant production of cytokines following signaling increased by the activation of pattern recognition receptors (PRRs). A unique PRR known as NLRX1 has many diverse roles, including regulation of key antiviral pathways- NF-kB and interferon- autophagy, and understudied roles in cellular metabolism. We show that changes in NLRX1 expression impacts immune responses to and viral growth of SCV-2 in cell culture and in mice. While Nlrx1-/- mice challenged with SCV-2 showed similar overall morbidity to WT counterparts, there were temporary increases in weight loss at multiple time points post infection. SCV-2 infection of Nlrx1-/- mice resulted in more airway inflammation, seemingly contrary to their also having increased viral titer at 2dpi. Data in cell culture report an enhanced replication ability of SCV-2 when NLRX1 expression is lower. Both cell culture and animal studies report differences in immune activation, with differences in cytokine production when NLRX1 expression changes. Together, these experiments: (1) support the enigmatic function of NLRX1 (2) suggest a possible role of NLRX1 in the pathobiology of SCV-2 infection and (3) add to the growing field exploring NLRX1 and its many curious roles.

NLRX1: a key regulator in mitochondrial respiration and colorectal cancer progression.

Colorectal cancer (CRC) is a prevalent and aggressive malignancy with high mortality rates and significant risks to human well-being. Population-wide screening for tumor suppressor genes and oncogenes shows promise for reducing the incidence and fatality of CRC. Recent studies have suggested that NLRX1, an innate immunity suppressor, may play a role in regulating chronic inflammation and tumorigenesis. However, further investigation is needed to understand the specific role of NLRX1 in CRC. To evaluate the impact of NLRX1 on migration, invasion, and metastasis, two human colon cancer cell lines were studied in vitro. Additionally, a knockout mouse tumor-bearing model was used to validate the inhibitory effect of NLRX1 on tumor emergence and progression. The Seahorse XF96 technology was employed to assess mitochondrial function and glycolysis in colorectal cancer cells overexpressing NLRX1. Moreover, public databases were consulted to analyze gene and protein expression levels of NLRX1. Finally, the results were validated using a series of CRC patient samples. Our findings demonstrate that downregulation of NLRX1 enhances proliferation, colony formation, and tumor-forming capacity in HCT116 and LoVo cells. Conversely, overexpression of NLRX1 negatively impacts basal respiration and mitochondrial ATP-linked respiration in both cell lines, resulting in a notable decrease in maximal respiration during the standard mitochondrial stress test. Furthermore, analysis of data from the TCGA database reveals a significant reduction in NLRX1 expression in colon and rectal cancer tissues compared to normal tissues. This result was validated using clinical samples, where immunohistochemistry staining and western blotting demonstrated a notable reduction in NLRX1 protein levels in CRC compared to adjacent normal tissues. The decreased expression of NLRX1 may serve as a significant prognostic indicator and diagnostic biomarker for CRC patients.

Bioinformatic Analysis of the Potential Common Pathogenic Mechanisms for Psoriasis and Metabolic Syndrome.

The pathogeneses of psoriasis and metabolic syndrome are closely related; however, the underlying biological mechanisms are yet to be clarified. A psoriasis training set was downloaded from the Gene Expression Omnibus database and analyzed to identify the differentially expressed genes (|logFC|> 1 and adjust P < 0.05). Differentially expressed genes for metabolic syndrome were obtained from the GeneCards, Online Mendelian Inheritance in Man, and DisGeNET databases, and crosstalk genes were obtained for multiple enrichment analysis after identifying the disease intersection. Characteristic crosstalk genes were screened using the least absolute shrinkage and selection operator regression model and random forest tree model, and the genes with area under the receiver operating characteristic curve > 0.7 were selected for validation by the two validation sets. Differential analyses of immune cell infiltration were performed on psoriasis lesion and control samples using the CIBERSORT and ImmuCellAI methods, and correlation analyses were performed between the screened signature crosstalk genes and immune cell infiltration. Significant crosstalk genes were analyzed based on the psoriasis area and severity index and on the responses to biological agents. We found five signature genes (NLRX1, KYNU, ABCC1, BTC, and SERPINB4) were screened based on two machine learning algorithms, and NLRX1 was validated. The infiltration of multiple immune cells in psoriatic lesions and non-lesions was associated with NLRX1 expression. NLRX1 was found to be associated with psoriasis severity and response rate after the use of biologics. NLRX1 could be a significant crosstalk gene for psoriasis and metabolic syndrome.

Calming the storm: Immunoregulatory receptor NLRX1 and its role in the immune response to SARS-CoV-2

Abstract One of the most characteristic clinical disease presentations of severe COVID-19 is the ‘Cytokine Storm,’ stemming from dysregulation of the immune response. This overproduction of proinflammatory cytokines in response to SARS-CoV-2 is responsible for Acute Respiratory Distress Syndrome (ARDS) in adult patients and the Multisystem Inflammatory Syndrome (MIS) of pediatric patients hospitalized with severe COVID-19. Pattern recognition receptor NLRX1 is a member of the NOD-like receptor family with complex involvement in antiviral immunity. It has been reported in the literature in many contexts to be anti-inflammatory by inhibiting NF-kB and interferon signaling-critical pathways in anti-SARS-CoV-2 immunity. Here we show that loss of NLRX1 impacts immune responses to SARS-CoV-2 both in cell culture and in mice. While Nlrx1 −/−mice showed similar overall morbidity, there were temporary increases in weight loss at multiple time points post infection. SARS-CoV-2 infection of Nlrx1 −/−mice also resulted in an increase in viral titer in the lungs at 2 dpi, despite having higher degrees of airway inflammation. Data in both cell culture and animal challenges report differences in immune activation, showing differences in cytokine production with changes in expression of NLRX1. Together, these experiments uncover another enigmatic function of NLRX1 while simultaneously offering pathways related to NLRX1 as possible therapeutic targets to help mitigate the effects of the ‘cytokine storm’ of severe COVID-19. Finally, these data also add to the growing field regarding SARS-CoV-2 and its pathobiology in an understudied context of immune regulation and continue to explore NLRX1 and its many curious roles in antiviral immunity.

P577 A Phase 1b Study to Evaluate Safety, Tolerability, Pharmacokinetics and Clinical Efficacy of the Nucleotide-binding oligomerization domain, Leucine rich Repeat containing X1 (NLRX1) agonist NX-13 in Ulcerative Colitis

Abstract Background NLRX1 is a mitochondrial membrane protein whose activation reduces oxidative stress and decreases effector cell differentiation and cytokine release. NX-13 is a first-in-class, orally active, gut selective NLRX1 agonist with low systemic exposure. In preclinical studies of IBD, NX-13 effectively reduced inflammatory responses and disease severity. A phase 1a study (healthy subjects) showed NX-13 to be well tolerated with low systemic drug exposure suggesting a gut-selective drug delivery. We report the results of a phase 1b study in patients with Ulcerative colitis (UC) on the safety, target engagement and clinical efficacy of NX-13. Methods In this double-blind trial, 36 patients with active UC (Total Mayo Score [MCS] 4-10; Mayo endoscopic subscore [MES] 2-3) were randomly assigned to NX-13 250mg Immediate Release (IR), 500mg IR, 500mg Modified Release (MR) or Placebo to be taken QD for 4 weeks (safety visit at week 5). Biologic exposed patients, stable 5’ASAs dose and corticosteroids (oral ≤20mg/day prednisone or equivalent) were permitted. IV or topical corticosteroids use was prohibited. Primary endpoints were safety and pharmacokinetic (PK) analysis. The study was not powered for clinical efficacy, however MCS, histopathologic Geboes score, and NLRX1, cytokine, and gene expression were obtained at baseline and day 28 in exploratory analyses (Table 1). Results During the 5-week observation period, no serious adverse events (AEs) were reported. All AEs were classified as mild or moderate and were most common in the 500mg MR group. One patient was withdrawn before week 5 due to a flare of UC. IR dose plasma PK levels peaked at 1hour, while the MR dose was absorbed later and maintained a low exposure longer. Qualitative immunohistochemical analysis showed target engagement as indicated by increase of NLRX1 at all doses. Symptomatic efficacy was seen in 8/11 patients in the 250mg group with a PRO (RB+SF) score of 0 at week 4. The decrease in MCS was 2.1-3.4 in the NX-13 arms vs 1 point with placebo (Fig 1A) while clinical response rates ranged from 27-72% amongst the active arms with no placebo responders, though no dose-response relationship was observed (Fig 1B). Endoscopic response ranged from 27-40% (Fig 1C), with 5/32 dosed patients being in endoscopic remission. Histologic remission largely paralleled endoscopic response and cytokine and mRNA levels support target engagement. Conclusion In patients with active UC, NX-13 was well-tolerated and target engagement was achieved. The exploratory efficacy endpoints indicated rapid clinical, endoscopic, and histologic improvement at week 4, relative to placebo. This novel mechanism of action will be further evaluated in a phase 2 study.

Neuroprotective effects of melatonin-mediated mitophagy through nucleotide-binding oligomerization domain and leucine-rich repeat-containing protein X1 in neonatal hypoxic-ischemic brain damage.

Hypoxia-ischemia (HI) is a major cause of brain damage in neonates. Mitochondrial dysfunction acts as a hub for a broad spectrum of signaling events, culminating in cell death triggered by HI. A neuroprotective role of melatonin (MT) has been proposed, and mitophagy regulation seems to be important for cell survival. However, the molecular mechanisms underlying MT-mediated mitophagy during HI treatment are poorly defined. Nucleotide-binding oligomerization domain and leucine-rich repeat-containing protein X1 (NLRX1) has emerged as a critical regulator of mitochondrial dynamics and neuronal death that participates in the pathology of diverse diseases. This study aimed to clarify whether NLRX1 participates in the regulation of mitophagy during MT treatment for hypoxic-ischemic brain damage (HIBD). We demonstrated that MT protected neonates from HIBD through NLRX1-mediated mitophagy in vitro and in vivo. Meanwhile, MT upregulated the expression of NLRX1, Beclin-1, and autophagy-related 7 (ATG7) but decreased the expression of the mammalian target of rapamycin (mTOR) and translocase of the inner membrane of mitochondrion 23 (TIM23). Moreover, the neuroprotective effects of MT were abolished by silencing NLRX1 after oxygen-glucose deprivation (OGD). In addition, the downregulation of mTOR and upregulation of Beclin-1 and ATG7 by MT were inhibited after silencing NLRX1 under OGD. In summary, MT modulates mitophagy induction through NLRX1 and plays a protective role in HIBD, providing insight into potential therapeutic targets for MT to exert neuroprotection.

Daming capsule protects against myocardial infarction by promoting mitophagy via the SIRT1/AMPK signaling pathway

Myocardial infarction (MI) is a myocardial injury caused by coronary thrombosis or persistent ischemia and hypoxia. Due to its high morbidity and mortality, a safer and more effective treatment strategy is urgently needed. Daming capsule (DMC), a hypolipidemic drug, reportedly exerts cardioprotective effects in clinical and basic research, although its protective mechanism remains unknown. To investigate the mechanism underlying DMC-mediated improvement of cardiac function post-MI, C57/BL6 mice subjected to coronary artery ligation were administered DMC for 4 weeks. Our data demonstrated that DMC significantly improved cardiac structure and function compared to the saline group. Moreover, DMC inhibited inflammatory response and oxidative stress and improved mitochondrial structure and function in MI mice and hypoxia-stressed cardiomyocytes. Next, our research proved that DMC increased the expression of mitophagy receptor NLRX1. Interestingly, with the administration of DMC and siNLRX1, NLRX1 expression, mitochondria and lysosome colocalization, and mitochondrial membrane potential decreased, while mitochondrial ROS accumulation increased, suggesting that DMC promoted mitophagy to improve mitochondrial function via NLRX1 regulation. Further analysis showed that DMC activated the SIRT1/AMPK signaling pathway in vivo and in vitro. Our data showed that SIRT1 knockdown downregulated NLRX1 expression, leading to structural damage and functional impairment in mitochondria, as well as increased oxidative stress, inflammatory response, and decreased cardiac function in MI mice. Collectively, our findings reveal that DMC improves cardiac function post-MI by increasing mitophagy and inhibiting oxidative stress and inflammotory response in cardiomyocytes through the SIRT1/AMPK signaling pathway.

LncRNA SNHG12 ameliorates bupivacaine-induced neurotoxicity by sponging miR-497-5p to upregulate NLRX1.

Long non-coding RNA (lncRNA) small nucleolar RNA host gene 12 (SNHG12) has been reported to participate in the regulation of various nervous system disorders. Bupivacaine (BV), a commonly used local anesthetic, could generate neurotoxicity in neurons. This work intended to investigate the role and specific mechanism of SNHG12 in BV-induced neurotoxicity. In this study, we established an in vitro cell model of BV-induced neurotoxicity by exposing human neuroblastoma cells (SH-SY5Y) to BV. It was found that SNHG12 and NLRX1 levels were gradually downregulated, while miR-497-5p enrichment was upregulated accordingly with the increase of BV concentration. As indicated by functional assays, SNHG12 overexpression promoted cell viability but inhibited cell apoptosis and oxidative stress in BV-treated SH-SY5Y cells. In addition, it was identified that SNHG12 directly targeted miR-497-5p and attenuated BV-induced neurotoxicity via interaction with miR-497-5p. Besides, it was confirmed that SNHG12 could upregulate NLRX1 expression by absorbing miR-497-5p. Moreover, miR-497-5p decreased cell viability and induced cell apoptosis and oxidative stress, which was partly reversed by NLRX1 upregulation. In conclusion, our findings indicated that SNHG12 might relieve BV-associated neurotoxicity by upregulating NLRX1 via miR-497-5p in vitro, providing novel clues and biomarkers for the treatment and prevention of BV-associated neurotoxicity.

NLRX1 can counteract innate immune response induced by an external stimulus favoring HBV infection by competitive inhibition of MAVS-RLRs signaling in HepG2-NTCP cells.

This study is aimed at the determination of the effect of the immune-regulatory factor NLRX1 on the antiviral activity of hepatocytes against an external stimuli favoring hepatitis B virus infection, and to explore its mechanism of action. A HepG2-NTCP model was established using the LV003 lentivirus. Cells were transfected using an overexpression vector and NLRX1 siRNA to achieve overexpression and interference of NLRX1 expression (OV-NLRX1, si-NLRX1). Levels of HBsAg and HBcAg were determined using Western blotting analysis and immunohistochemical analysis. The levels of hepatitis B virus DNA and hepatitis B virus cccDNA were determined by real-time quantitative polymerase chain reaction. The expression and transcriptional activity of IFN-α, IFN-β, and IL-6 were measured using real-time quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and promoter-luciferase reporter plasmids. Co-immunoprecipitation was used to determine the effect of NLRX1 on the interaction between MAVS and RIG-1. Western blotting was used to obtain the phosphorylation of essential proteins in the MAVS-RLRs signaling pathways. NLRX1 promoted HepG2-NTCP cell hepatitis B virus infection. Compared to the control group, the levels of HBsAg, HBcAg, hepatitis B virus cccDNA, and hepatitis B virus DNA increased in the OV-NLRX1 group and decreased in the si-NLRX1. Co-immunoprecipitation results showed that NLRX1 competitively inhibited the interaction between MAVS and RIG-1, and inhibited the phosphorylation of p65, IRF3, and IRF7. Additionally, NLRX1 reduced the transcription activity and expression levels of the final products: IFN-α, IFN-β, and IL-6. NLRX1 can counteract innate immune response induced by an external stimuli favoring hepatitis B virus infection by competitive inhibition of MAVS-RLRs signaling in HepG2-NTCP cells. Inhibition of the MAVS-RLR-mediated signaling pathways leads to a decline in the expression levels of I-IFN and IL-6.

Multi-omics analyses reveal that HIV-1 alters CD4+ T cell immunometabolism to fuel virus replication

Human Immunodeficiency Virus type-1 (HIV-1)-infected individuals show metabolic alterations of CD4 T cells through unclear mechanisms with undefined consequences. We analyzed the transcriptome of CD4 T cells from HIV-1 patients and revealed that elevated oxidative phosphorylation (OXPHOS) pathway is associated with poor outcomes. Inhibition of OXPHOS by the FDA-approved drug metformin, which targets mitochondrial respiratory chain complex I, suppresses HIV-1 replication in human CD4 T cells and humanized mice. In patients, HIV-1 peak viremia positively correlates with the expression of NLRX1, a mitochondrial innate immune receptor. Quantitative proteomics and metabolic analyses reveal that NLRX1 enhances OXPHOS and glycolysis during HIV-1-infection of CD4 T cells to promote viral replication. At the mechanistic level, HIV infection induces the association of NLRX1 with the mitochondrial protein, FASTKD5, to promote the expression of mitochondrial respiratory complex components. This study uncovers the OXPHOS pathway in CD4 T cells as a target for HIV-1 therapy.

MicroRNA-195 suppresses enterovirus A71-induced pyroptosis in human neuroblastoma cells through targeting NLRX1

Enterovirus A71 (EV-A71) emerged as a leading cause of virus derived infant encephalitis in most Asian countries. Some recent studies point out the critical role of microRNA (miRNA) in the regulation of pyroptosis. However, the role of miRNAs in the regulation of EV-A71 infection-induced pyroptosis was not previously explored. In this study, we utilized microRNA array and real-time PCR to verify that miR-195 significantly down-regulate in EV-A71-infected SH-SY5Y human neuroblastoma cells. An inverse correlation of NLRX1 with miR-195 expression in EV-A71-infected SH-SY5Y cells was found. Target prediction of miR-195 showed that NLRX1 could directly interact with miR-195. Results from luciferase reporter assays, qRT-PCR and western blotting demonstrated the negative regulation between miR-195 and NLRX1. Silencing NLRX1 expression with small interfering RNAs (siRNAs-NLRX1) and over-expression of miR-195 also attenuate the EV-A71 associated pyroptosis. Our findings provided evidence showed that miR-195 can regulate EV-A71 infection-induced pyroptosis, by directly targeting NLRX1.

Synergistic activation of p53 by actinomycin D and nutlin-3a is associated with the upregulation of crucial regulators and effectors of innate immunity

Actinomycin D and nutlin-3a (A + N) activate p53, partly through induction of phosphorylation on Ser392. The death of A549 cells induced by A + N morphologically resembles inflammation-inducing pyroptosis - cell destruction triggered by activated caspase-1. The treatment with A + N (or camptothecin) strongly upregulated caspase-1 and its two activators: IFI16 and NLRP1, however, caspase-1 activation was not detected. A549 cells may have been primed for pyroptosis, with the absence of a crucial trigger. The investigation of additional innate immunity elements revealed that A + N (or camptothecin) stimulated the expression of NLRX1, STING (stimulator of interferon genes) and two antiviral proteins, IFIT1 and IFIT3. IFI16 and caspase-1 are coded by p53-regulated genes which led us to investigate regulation of NLRP1, NLRX1, STING, IFIT1 and IFIT3 in p53-dependent mode. The upregulation of NLRP1, NLRX1 and STING was attenuated in p53 knockdown cells. The upsurge of the examined genes, and activation of p53, was inhibited by C16, an inhibitor of PKR kinase. PKR was tested due to its ability to phosphorylate p53 on Ser392. Surprisingly, C16 was active even in PKR knockdown cells. The ability of C16 to prevent activation of p53 and expression of innate immunity genes may be the source of its strong anti-inflammatory action. Moreover, cells exposed to A + N can influence neighboring cells in paracrine fashion, for instance, they shed ectodomain of COL17A1 protein and induce, in p53-dependent mode, the expression of gene for interleukin-7. Further, the activation of p53 also spurred the expression of SOCS1, an inhibitor of interferon triggered STAT1-dependent signaling. We conclude that, stimulation of p53 primes cells for the production of interferons (through upregulation of STING), and may activate negative-feedback within this signaling system by enhancing the production of SOCS1.

Nucleotide-binding oligomerization domain-like receptor X1 restricts porcine reproductive and respiratory syndrome virus-2 replication by interacting with viral Nsp9

Porcine reproductive and respiratory syndrome virus (PRRSV) causes one of the most economically important diseases of swine worldwide. Current antiviral strategies provide only limited protection. Nucleotide-binding oligomerization domain-like receptor (NLR) X1 is unique among NLR proteins in its functions as a pro-viral or antiviral factor to different viral infections. To date, the impact of NLRX1 on PRRSV infection remains unclear. In this study, we found that PRRSV infection promoted the expression of NLRX1 gene. In turn, ectopic expression of NLRX1 inhibited PRRSV replication in Marc-145 cells, whereas knockdown of NLRX1 enhanced PRRSV propagation in porcine alveolar macrophages (PAMs). Mechanistically, NLRX1 was revealed to impair intracellular viral subgenomic RNAs accumulation. Finally, Mutagenic analyses indicated that the LRR (leucine-rich repeats) domain of NLRX1 interacted with PRRSV Nonstructural Protein 9 (Nsp9) RdRp (RNA-dependent RNA Polymerase) domain and was necessary for antiviral activity. Thus, our study establishes the role of NLRX1 as a new host restriction factor in PRRSV infection.

NLRX1 regulates TNF-α-induced mitochondria-lysosomal crosstalk to maintain the invasive and metastatic potential of breast cancer cells

An increased level of proinflammatory cytokines, including TNF-α in tumor microenvironment regulates the bioenergetic capacity, immune evasion and survival of cancer cells. Emerging evidences suggest that mitochondrial immune signaling proteins modulates mitochondrial bioenergetic capacity, in addition to the regulation of innate immune response. The optimal oxidative phosphorylation (OxPhos) capacity is required for the maintenance of functional lysosomes and autophagy flux. NLRX1, a mitochondrial NOD family receptor protein, regulates mitochondrial function during apoptosis and tissue injury. However, its role in regulation of mitochondrial and lysosomal function to modulate autophagy flux during inflammatory conditions is not understood. In the current study, we investigated the role of NLRX1 in modulating TNF-α induced autophagy flux and mitochondrial turnover and its implication in regulating the invasive and metastatic capability of breast cancer cells. Expression analyses of clinical breast cancer samples and meta-analysis of multiple public databases revealed that NLRX1 expression is significantly increased in basal-like and metastatic breast carcinoma as compared to non-basal-like and primary breast cancer. Depletion of NLRX1 expression in triple-negative breast cancer cells, altered the organization and activity of OxPhos complexes in presence of TNF-α. NLRX1 depletion further impaired lysosomal function and hence the turnover of damaged mitochondria through mitophagy in presence of TNF-α. Importantly, loss of NLRX1 decreased OxPhos-dependent cell proliferation and migration ability of triple-negative breast cancer cells in presence of TNF-α. These evidences suggest an essential role of NLRX1 in maintaining the crosstalk of mitochondrial metabolism and lysosomal function to regulate invasion and metastasis capability of breast cancer cells.

Regulatory NLRs Control the RLR-Mediated Type I Interferon and Inflammatory Responses in Human Dendritic Cells.

Unique members of the nucleotide-binding domain leucine-rich repeat (NLR) family have been found to regulate intracellular signaling pathways initiated by other families of pattern recognition receptors (PRR) such as Toll-like receptors (TLRs) and retinoic-acid inducible gene I (RIG-I)-like receptors (RLRs). Plasmacytoid dendritic cells (pDCs), the most powerful type I interferon (IFN) producing cells, preferentially employ endosomal TLRs to elicit antiviral IFN responses. By contrast, conventional DCs (cDCs) predominantly use cytosolic RLRs, which are constitutively expressed in them, to sense foreign nucleic acids. Previously we have reported that, though RIG-I is absent from resting pDCs, it is inducible upon TLR stimulation. In the recent study we investigated the regulatory ability of NLRs, namely NLRC5 and NLRX1 directly associated with the RLR-mediated signaling pathway in DC subtypes showing different RLR expression, particularly in pDCs, and monocyte-derived DCs (moDCs). Here we demonstrate that similarly to RLRs, NLRC5 is also inducible upon TLR9 stimulation, whereas NLRX1 is constitutively expressed in pDCs. Inhibition of NLRC5 and NLRX1 expression in pDCs augmented the RLR-stimulated expression of type I IFNs but did not affect the production of the pro-inflammatory cytokines TNF, IL-6, and the chemokine IL-8. Further we show that immature moDCs constantly express RLRs, NLRX1 and NLRC5 that are gradually upregulated during their differentiation. Similarly to pDCs, NLRX1 suppression increased the RLR-induced production of type I IFNs in moDCs. Interestingly, RLR stimulation of NLRX1-silenced moDCs leads to a significant increase in pro-inflammatory cytokine production and IκBα degradation, suggesting increased NF-κB activity. On the contrary, NLRC5 does not seem to have any effect on the RLR-mediated cytokine responses in moDCs. In summary, our results indicate that NLRX1 negatively regulates the RLR-mediated type I IFN production both in pDCs and moDCs. Further we show that NLRX1 inhibits pro-inflammatory cytokine secretion in moDCs but not in pDCs following RLR stimulation. Interestingly, NLRC5 suppresses the RLR-induced type I IFN secretion in pDCs but does not appear to have any regulatory function on the RLR pathway in moDCs. Collectively, our work demonstrates that RLR-mediated innate immune responses are primarily regulated by NLRX1 and partly controlled by NLRC5 in human DCs.

Negative-Pressure Wound Therapy Promotes Wound Healing by Enhancing Angiogenesis Through Suppression of NLRX1 via miR-195 Upregulation.

Negative-pressure wound therapy (NPWT) is one of the most advanced therapeutic methods in the treatment of various hard-to-heal acute and refractory chronic wounds. Recent emerging evidence points to a role of the microRNA-mediated regulation of angiogenesis in ischemic tissues, and a series of microRNAs associated with angiogenesis have been successively identified. In this study, we found that miR-195 expression was significantly upregulated and the microvessel density (MVD) was increased in granulation tissue collected 7 days after NPWT compared with those in the pre-NPWT tissue. Moreover, the expression of NLRX1, the potential target gene of miR-195, was down-regulated in post-NPWT compared with that in pre-NPWT tissue. Significant negative correlations were detected between miR-195 and NLRX1 expression levels ( r = -.856, P < .001) and between NLRX1 expression and MVD ( r = -.618, P < .05), whereas miR-195 expression was positively correlated with MVD in the granulation tissue ( r = .630, P < .05). In summary, NPWT may suppress NLRX1 expression through the upregulation of miR-195 expression, thus efficaciously promoting angiogenesis in the granulation tissue to enhance wound healing.

Activation of NLRX1-mediated autophagy accelerates the ototoxic potential of cisplatin in auditory cells

To date, the mechanism (s) underlying the cisplatin-elicited ototoxicity has not been elucidated fully. Nucleotide-binding domain and leucine-rich-repeat-containing family member ×1 (NLRX1), a cytoplasmic pattern recognition receptor, is tightly related to mitochondrial function, reactive oxygen species (ROS) production, and autophagy. In this work, autophagy alteration, NLRX1 expression, ROS generation and cell injury were investigated correspondingly by immunofluorescence staining, western-blot, TEM, flow cytometry and MTT in HEI-OC1 cells of both NLRX1 overexpression and silencing in response to cisplatin stimulus. We found that NLRX1 expression was increased concurrent with the increase of autophagy activation in HEI-OC1 cells under the cisplatin insult. NLRX1 overexpression led to the amount of accumulation of autophagsomes in HEI-OC1 cells in normal condition and a higher activation of autophagy concurrent with cell injury in HEI-OC1 cells treated with cisplatin, whereas, NLRX1 silencing decreased the activation level of autophagy concurrent with increased cell viability in HEI-OC1 cells treated with cisplatin. Mechanistic studies showed that NLRX1 potentiated mitochondrial-derived ROS generation in response to cisplatin exposure. Inhibition of ROS generation significantly prevented autophagy activation and apoptosis both in HEI-OC1cells and cochlear explants treated with cisplatin. The findings from this work reveal that NLRX1 sensitizes auditory cells in vitro to cisplatin-induced ototoxity via autophagic cell death pathway, providing another strategy against cisplatin-induced ototoxity.