Cross-reactivity of hantavirus antibodies after immunization with PUUV antigens.

Nephropathia epidemica (NE), caused by Puumala (PUUV) orthohantavirus, is endemic in the Republic of Tatarstan (RT). There are limited options for NE prevention in RT. Currently, available vaccines are made using Haantan (HNTV) orthohantavirus antigens. In this study, the efficacy of microvesicles (MVs) loaded with PUUV antigens to induce the humoral immune response in small mammals was analyzed. Additionally, the cross-reactivity of serum from immunized small mammals and NE patients with HNTV, Dobrava, and Andes orthohantaviruses was investigated using nucleocapsid (N) protein peptide libraries. Finally, the selected peptides were analyzed for allergenicity, their ability to induce an autoimmune response, and their interaction with Class II HLA. Several N protein peptides were found to be cross-reactive with serum from MVs immunized small mammals. These cross-reactive epitopes were located in oligomerization perinuclear targeting and Daxx-interacting domains. Most cross-reactive peptides lack allergenic and autoimmune reactivity. Molecular docking revealed two cross-reacting peptides, N6 and N19, to have good binding with three Class II HLA alleles. These peptides could be candidates for developing vaccines and therapeutics for NE.

Blocking the Hormone Receptors Modulates NLRP3 in LPS-Primed Breast Cancer Cells.

NOD-like receptor protein 3 (NLRP3) may contribute to the growth and propagation of breast cancer (BC). The effect of estrogen receptor-α (ER-α), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) on NLRP3 activation in BC remains unknown. Additionally, our knowledge of the effect of blocking these receptors on NLRP3 expression is limited. We used GEPIA, UALCAN, and the Human Protein Atlas for transcriptomic profiling of NLRP3 in BC. Lipopolysaccharide (LPS) and adenosine 5'-triphosphate (ATP) were used to activate NLRP3 in luminal A MCF-7 and in TNBC MDA-MB-231 and HCC1806 cells. Tamoxifen (Tx), mifepristone (mife), and trastuzumab (Tmab) were used to block ER-α, PR, and HER2, respectively, on inflammasome activation in LPS-primed MCF7 cells. The transcript level of NLRP3 was correlated with ER-ɑ encoding gene ESR1 in luminal A (ER-α+, PR+) and TNBC tumors. NLRP3 protein expression was higher in untreated and LPS/ATP-treated MDA-MB-231 cells than in MCF7 cells. LPS/ATP-mediated NLRP3 activation reduced cell proliferation and recovery of wound healing in both BC cell lines. LPS/ATP treatment prevented spheroid formation in MDA-MB-231 cells but did not affect MCF7. HGF, IL-3, IL-8, M-CSF, MCP-1, and SCGF-b cytokines were secreted in both MDA-MB-231 and MCF7 cells in response to LPS/ATP treatment. Tx (ER-α inhibition) promoted NLRP3 activation and increased migration and sphere formation after LPS treatment of MCF7 cells. Tx-mediated activation of NLRP3 was associated with increased secretion of IL-8 and SCGF-b compared to LPS-only-treated MCF7 cells. In contrast, Tmab (Her2 inhibition) had a limited effect on NLRP3 activation in LPS-treated MCF7 cells. Mife (PR inhibition) opposed NLRP3 activation in LPS-primed MCF7 cells. We have found that Tx increased the expression of NLRP3 in LPS-primed MCF7. These data suggest a link between blocking ER-α and activation of NLRP3, which was associated with increased aggressiveness of the ER-α+ BC cells.

Genetically Engineered Artificial Microvesicles Carrying Nerve Growth Factor Restrains the Progression of Autoimmune Encephalomyelitis in an Experimental Mouse Model.

Multiple sclerosis (MS) is an incurable, progressive chronic autoimmune demyelinating disease. Therapy for MS is based on slowing down the processes of neurodegeneration and suppressing the immune system of patients. MS is accompanied by inflammation, axon-degeneration and neurogliosis in the central nervous system. One of the directions for a new effective treatment for MS is cellular, subcellular, as well as gene therapy. We investigated the therapeutic potential of adipose mesenchymal stem cell (ADMSC) derived, cytochalasin B induced artificial microvesicles (MVs) expressing nerve growth factor (NGF) on a mouse model of multiple sclerosis experimental autoimmune encephalomyelitis (EAE). These ADMSC-MVs-NGF were tested using histological, immunocytochemical and molecular genetic methods after being injected into the tail vein of animals on the 14th and 21st days post EAE induction. ADMSC-MVs-NGF contained the target protein inside the cytoplasm. Their injection into the caudal vein led to a significant decrease in neurogliosis at the 14th and 21st days post EAE induction. Artificial ADMSC-MVs-NGF stimulate axon regeneration and can modulate gliosis in the EAE model.

Neutralizing Antibodies in COVID-19 Serum from Tatarstan, Russia.

The severity of COVID-19 is a result of the complex interplay between various branches of the immune system. However, our understanding of the role of neutralizing antibodies and the activation of cellular immune response in COVID-19 pathogenesis remains limited. In this study, we investigated neutralizing antibodies in patients with mild, moderate, and severe COVID-19, analyzing their cross-reactivity with the Wuhan and Omicron variants. We also assessed the activation of the immune response by measuring serum cytokines in patients with mild, moderate, and severe COVID-19. Our findings suggest the early activation of neutralizing antibodies in moderate COVID-19 compared to mild cases. We also observed a strong correlation between the cross-reactivity of neutralizing antibodies to the Omicron and Wuhan variants and the severity of the disease. In addition, we found that Th1 lymphocyte activation was present in mild and moderate cases, while inflammasomes and Th17 lymphocytes were activated in severe COVID-19. In conclusion, our data indicate that the early activation of neutralizing antibodies is evident in moderate COVID-19, and there is a strong correlation between the cross-reactivity of neutralizing antibodies and the severity of the disease. Our findings suggest that the Th1 immune response may play a protective role, while inflammasome and Th17 activation may be involved in severe COVID-19.

Seasonal Changes in Serum Metabolites in Multiple Sclerosis Relapse.

Multiple sclerosis (MS) is a debilitating chronic disease of unknown etiology. There are limited treatment options due to an incomplete understanding of disease pathology. The disease is shown to have seasonal exacerbation of clinical symptoms. The mechanisms of such seasonal worsening of symptoms remains unknown. In this study, we applied targeted metabolomics analysis of serum samples using LC-MC/MC to determine seasonal changes in metabolites throughout the four seasons. We also analyzed seasonal serum cytokine alterations in patients with relapsed MS. For the first time, we can demonstrate seasonal changes in various metabolites in MS compared to the control. More metabolites were affected in MS in the fall season followed by spring, while summer MS was characterized by the smallest number of affected metabolites. Ceramides were activated in all seasons, suggesting their central role in the disease pathogenesis. Substantial changes in glucose metabolite levels were found in MS, indicating a potential shift to glycolysis. An increased serum level of quinolinic acid was demonstrated in winter MS. Histidine pathways were affected, suggesting their role in relapse of MS in the spring and fall. We also found that spring and fall seasons had a higher number of overlapping metabolites affected in MS. This could be explained by patients having a relapse of symptoms during these two seasons.

Differential Cytokine Responses and the Clinical Severity of Adult and Pediatric Nephropathia Epidemica.

Nephropathia epidemica (NE), caused by the hantavirus infection, is endemic in Tatarstan Russia. The majority of patients are adults, with infection rarely diagnosed in children. This limited number of pediatric NE cases means there is an inadequate understanding of disease pathogenesis in this age category. Here, we have analyzed clinical and laboratory data in adults and children with NE to establish whether and how the disease severity differs between the two age groups. Serum cytokines were analyzed in samples collected from 11 children and 129 adult NE patients during an outbreak in 2019. A kidney toxicity panel was also used to analyze urine samples from these patients. Additionally, serum and urine samples were analyzed from 11 control children and 26 control adults. Analysis of clinical and laboratory data revealed that NE was milder in children than in adults. A variation in serum cytokine activation could explain the differences in clinical presentation. Cytokines associated with activation of Th1 lymphocytes were prominent in adults, while they were obscured in sera from pediatric NE patients. In addition, a prolonged activation of kidney injury markers was found in adults with NE, whilst only a short-lasting activation of these markers was observed in children with NE. These findings support previous observations of age differences in NE severity, which should be considered when diagnosing the disease in children.

Puumala Orthohantavirus Reassortant Genome Variants Likely Emerging in the Watershed Forests.

Hemorrhagic fever with renal syndrome (HFRS) remains a prevalent zoonosis in the Republic of Tatarstan (RT), Russian Federation. Puumala orthohantavirus (PUUV), carried by bank voles (Myodes glareolus), is the principal zoonotic pathogen of HFRS in the RT. In this study, we sought to demonstrate the similarity of the PUUV genetic sequences detected in HFRS case patients and bank vole samples previously collected in some areas of the RT. Furthermore, we intended to identify the reassortant PUUV genomes and locate a potential site for their emergence. During 2019 outbreaks, the PUUV genome sequences of the S and M segments from 42 HFRS cases were analysed and compared with the corresponding sequences from bank voles previously trapped in the RT. Most of the PUUV strains from HFRS patients turned out to be closely related to those isolated from bank voles captured near the site of the human infection. We also found possible reassortant PUUV genomes in five patients while they were absent in bank voles. The location of the corresponding HFRS infection sites suggests that reassortant PUUV genomes could emerge in the bank voles that inhabit the forests on the watershed between the Kazanka River and Myosha River. These findings could facilitate the search for the naturally occurring reassortants of PUUV in bank vole populations.

Azithromycin and Ceftriaxone Differentially Activate NLRP3 in LPS Primed Cancer Cells.

BACKGROUND: Cancer patients are prescribed antibiotics, such as macrolides and lactamides, for infection treatment. However, the effect of these antibiotics on NLRP3 activation remains largely unknown. METHOD: Lung cancer (A549) and prostate cancer (PC3) cell lines were primed with lipopolysaccharide (LPS) to activate NLRP3 transcription. Cells were then treated with azithromycin (Az) or ceftriaxone (Cf). NLRP3 activation was analyzed by qPCR, Western blot, and ELISA. Cell growth and viability were assessed by real-time cell analysis and Annexin V expression. Levels of 41 cytokines were also analyzed using a multiplex assay. RESULTS: LPS-Az activated transcription of NLRP3, Pro-CASP-1, and Pro-IL-1ß in A549 cells, while failing to upregulate NLRP3 and Pro-IL-1ß in PC3 cells. LPS-Az decreased the secretion of pro-inflammatory cytokines while it induced the pro-angiogenic factors in A549 and PC3 cells. In contrast, LPS-Cf suppressed the expression of NLRP3-associated genes, NLRP3 protein expression, the inflammatory cytokine secretion in A549 and PC3 cells. LPS-Az and LPS-Cf had a limited effect on cell growth and viability. DISCUSSION: Our data suggest that Cf could suppress LPS induced NLRP3, which should be considered when selecting antibiotics for cancer treatment. In contrast, the effect of Az on LPS primed NLRP3 and the inflammatory cytokines production appears to depend on the cancer cell origin. Therefore, these data indicate that considerations are required when selecting Az for the treatment of cancer patients.

Membrane Microvesicles as Potential Vaccine Candidates.

The prevention and control of infectious diseases is crucial to the maintenance and protection of social and public healthcare. The global impact of SARS-CoV-2 has demonstrated how outbreaks of emerging and re-emerging infections can lead to pandemics of significant public health and socio-economic burden. Vaccination is one of the most effective approaches to protect against infectious diseases, and to date, multiple vaccines have been successfully used to protect against and eradicate both viral and bacterial pathogens. The main criterion of vaccine efficacy is the induction of specific humoral and cellular immune responses, and it is well established that immunogenicity depends on the type of vaccine as well as the route of delivery. In addition, antigen delivery to immune organs and the site of injection can potentiate efficacy of the vaccine. In light of this, microvesicles have been suggested as potential vehicles for antigen delivery as they can carry various immunogenic molecules including proteins, nucleic acids and polysaccharides directly to target cells. In this review, we focus on the mechanisms of microvesicle biogenesis and the role of microvesicles in infectious diseases. Further, we discuss the application of microvesicles as a novel and effective vaccine delivery system.

Long Term Immune Response Produced by the SputnikV Vaccine.

SputnikV is a vaccine against SARS-CoV-2 developed by the Gamaleya National Research Centre for Epidemiology and Microbiology. The vaccine has been shown to induce both humoral and cellular immune responses, yet the mechanisms remain largely unknown. Forty SputnikV vaccinated individuals were included in this study which aimed to demonstrate the location of immunogenic domains of the SARS-CoV-2 S protein using an overlapping peptide library. Additionally, cytokines in the serum of vaccinated and convalescent COVID-19 patients were analyzed. We have found antibodies from both vaccinated and convalescent sera bind to immunogenic regions located in multiple domains of SARS-CoV-2 S protein, including Receptor Binding Domain (RBD), N-terminal Domain (NTD), Fusion Protein (FP) and Heptad Repeats (HRs). Interestingly, many peptides were recognized by immunized and convalescent serum antibodies and correspond to conserved regions in circulating variants of SARS-CoV-2. This breadth of reactivity was still evident 90 days after the first dose of the vaccine, showing that the vaccine has induced a prolonged response. As evidenced by the activation of T cells, cellular immunity strongly suggests the high potency of the SputnikV vaccine against SARS-CoV-2 infection.

Serum and Cerebrospinal Fluid Cytokine Biomarkers for Diagnosis of Multiple Sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a chronic debilitating disorder characterized by persisting damage to the brain caused by autoreactive leukocytes. Leukocyte activation is regulated by cytokines, which are readily detected in MS serum and cerebrospinal fluid (CSF). OBJECTIVE: Serum and CSF levels of forty-five cytokines were analyzed to identify MS diagnostic markers. METHODS: Cytokines were analyzed using multiplex immunoassay. ANOVA-based feature and Pearson correlation coefficient scores were calculated to select the features which were used as input by machine learning models, to predict and classify MS. RESULTS: Twenty-two and twenty cytokines were altered in CSF and serum, respectively. The MS diagnosis accuracy was ≥92% when any randomly selected five of these biomarkers were used. Interestingly, the highest accuracy (99%) of MS diagnosis was demonstrated when CCL27, IFN-γ, and IL-4 were part of the five selected cytokines, suggesting their important role in MS pathogenesis. Also, these binary classifier models had the accuracy in the range of 70-78% (serum) and 60-69% (CSF) to discriminate between the progressive (primary and secondary progressive) and relapsing-remitting forms of MS. CONCLUSION: We identified the set of cytokines from the serum and CSF that could be used for the MS diagnosis and classification.

Rab GTPase Mediating Regulation of NALP3 in Colorectal Cancer.

The NALP3 inflammasome signaling contributes to inflammation within tumor tissues. This inflammation may be promoted by the vesicle trafficking of inflammasome components and cytokines. Rab5, Rab7 and Rab11 regulate vesicle trafficking. However, the role of these proteins in the regulation of inflammasomes remains largely unknown. To elucidate the role of these Rab proteins in inflammasome regulation, HCT-116, a colorectal cancer (CRC) cell line expressing pDsRed-Rab5 wild type (WT), pDsRed-Rab5 dominant-negative (DN), pDsRed-Rab7 WT, pDsRed-Rab7 DN, pDsRed-Rab11 WT and pDsRed-Rab11 DN were treated with lipopolysaccharide (LPS)/nigericin. Inflammasome activation was analyzed by measuring the mRNA expression of NLRP3, Pro-CASP1, RAB39A and Pro-IL-1ß, conducting immunofluorescence imaging and western blotting of caspase-1 and analysing the secretion levels of IL-1ß using enzyme-linked immunosorbent assay (ELISA). The effects of Rabs on cytokine release were evaluated using MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel-Premixed 41 Plex. The findings showed that LPS/nigericin-treated cells expressing Rab5-WT indicated increased NALP3 expression and secretion of the IL-1ß as compared to Rab5-DN cells. Caspase-1 was localized in the nucleus and cytosol of Rab5-WT cells but was localized in the cytosol in Rab5-DN cells. There were no any effects of Rab7 and Rab11 expression on the regulation of inflammasomes. Our results suggest that Rab5 may be a potential target for the regulation of NALP3 in the treatment of the CRC inflammation.

The Interaction between Viral and Environmental Risk Factors in the Pathogenesis of Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic debilitating inflammatory disease of unknown ethology targeting the central nervous system (CNS). MS has a polysymptomatic onset and is usually first diagnosed between the ages of 20⁻40 years. The pathology of the disease is characterized by immune mediated demyelination in the CNS. Although there is no clinical finding unique to MS, characteristic symptoms include sensory symptoms visual and motor impairment. No definitive trigger for the development of MS has been identified but large-scale population studies have described several epidemiological risk factors for the disease. This list is a confusing one including latitude, vitamin D (vitD) levels, genetics, infection with Epstein Barr Virus (EBV) and endogenous retrovirus (ERV) reactivation. This review will look at the evidence for each of these and the potential links between these disparate risk factors and the known molecular disease pathogenesis to describe potential hypotheses for the triggering of MS pathology.

A Novel Receptor Tyrosine Kinase Switch Promotes Gastrointestinal Stromal Tumor Drug Resistance.

The fact that most gastrointestinal stromal tumors (GISTs) acquire resistance to imatinib (IM)-based targeted therapy remains the main driving force to identify novel molecular targets that are capable to increase GISTs sensitivity to the current therapeutic regimens. Secondary resistance to IM in GISTs typically occurs due to several mechanisms that include hemi- or homo-zygous deletion of the wild-type KIT allele, overexpression of focal adhesion kinase (FAK) and insulin-like growth factor receptor I (IGF-1R) amplification, BRAF mutation, a RTK switch (loss of c-KIT and gain of c-MET/AXL), etc. We established and characterized the IM-resistant GIST T-1 cell line (GIST T-1R) lacking secondary c-KIT mutations typical for the IM-resistant phenotype. The resistance to IM in GIST T-1R cells was due to RTK switch (loss of c-KIT/gain of FGFR2α). Indeed, we have found that FGFR inhibition reduced cellular viability, induced apoptosis and affected the growth kinetics of the IM-resistant GISTs in vitro. In contrast, IM-naive GIST T-1 parental cells were not susceptible to FGFR inhibition. Importantly, inhibition of FGF-signaling restored the susceptibility to IM in IM-resistant GISTs. Additionally, IM-resistant GISTs were less susceptible to certain chemotherapeutic agents as compared to parental IM-sensitive GIST cells. The chemoresistance in GIST T-1R cells is not due to overexpression of ABC-related transporter proteins and might be the result of upregulation of DNA damage signaling and repair (DDR) genes involved in DNA double-strand break (DSB) repair pathways (e.g., XRCC3, Rad51, etc.). Taken together, the established GIST T-1R cell subline might be used for in vitro and in vivo studies to examine the efficacy and prospective use of FGFR inhibitors for patients with IM-resistant, un-resectable and metastatic forms of GISTs with the type of RTK switch indicated above.

Serum Cytokine Profiles in Children with Crohn's Disease.

Crohn's disease (CD) is a chronic inflammatory bowel disease that can be diagnosed at any age. There are two major patient groups based on diagnosis of this disease, before or after the age of 20 (juvenile/adolescent or adult), with disease progression in adults usually milder than in juvenile CD patients. Immune mechanisms have been suggested to play an important role in CD pathogenesis, with cytokines governing the development of the immune response. Upregulation of inflammatory cytokines in serum of juvenile and adult CD patients has been documented; still little is known about age-dependent differences in serum cytokine profiles of CD patients. We applied multiplex technology to analyze serum levels of 12 cytokines in juveniles and adults. We show that during the acute stage of the disease all CD patients have high serum levels of CXCL10, which remains upregulated during remission. Increased serum levels of TNF-α and IL-6 during the acute stage was characteristic of juvenile CD patients, whereas adult CD patients had upregulated levels of GM-CSF and IFN-γ. Taken together, these results demonstrate age-dependent differences in cytokine profiles, which may affect the pathogenesis of CD in patients at different ages of disease onset.

Cytokine expression provides clues to the pathophysiology of Gulf War illness and myalgic encephalomyelitis.

Gulf War illness (GWI) is a chronic disease of unknown etiology characterized by persistent symptoms such as cognitive impairment, unexplained fatigue, pervasive pain, headaches, and gastrointestinal abnormalities. Current reports suggest that as many as 200,000 veterans who served in the 1990-1991 Persian Gulf War were afflicted. Several potential triggers of GWI have been proposed including chemical exposure, toxins, vaccines, and unknown infectious agents. However, a definitive cause of GWI has not been identified and a specific biological marker that can consistently delineate the disease has not been defined. Myalgic encephalomyelitis (ME) is a disease with similar and overlapping symptomology, and subjects diagnosed with GWI typically fit the diagnostic criteria for ME. For these reasons, GWI is often considered a subgroup of ME. To explore this possibility and identify immune parameters that may help to understand GWI pathophysiology, we measured 77 serum cytokines in subjects with GWI and compared these data to that of subjects with ME as well as healthy controls. Our analysis identified a group of cytokines that identified ME and GWI cases with sensitivities of 92.5% and 64.9%, respectively. The five most significant cytokines in decreasing order of importance were IL-7, IL-4, TNF-α, IL-13, and IL-17F. When delineating GWI and ME cases from healthy controls, the observed specificity was only 33.3%, suggesting that with respect to cytokine expression, GWI cases resemble control subjects to a greater extent than ME cases across a number of parameters. These results imply that serum cytokines are representative of ME pathology to a greater extent than GWI and further suggest that the two diseases have distinct immune profiles despite their overlapping symptomology.

Plasmacytoid dendritic cells, a role in neoplastic prevention and progression.

BACKGROUND: Plasmacytoid dendritic cells (pDCs) are multifunctional bone-marrow-derived immune cells that are key players in bridging the innate and adaptive immune systems. Activation of pDCs through toll-like receptor agonists has proven to be an effective treatment for some neoplastic disorders. MATERIALS AND METHODS: In this mini-review, we will explore the fascinating contribution of pDCs to neoplastic pathology and discuss their potential utilization in cancer immunotherapy. RESULTS: Current research suggests that pDCs have cytotoxic potential and can effectively induce apoptosis of tumour-derived cells lines. They are also reported to display tolerogenic function with the ability to suppress T-cell proliferation, analogous to regulatory T cells. In this capacity, they are critical in the suppression of autoimmunity but can be exploited by tumour cells to circumvent the expansion of tumour-specific T cells, thereby allowing tumours to persist. CONCLUSION: Several forms of skin cancer are successfully treated with the topical drug Imiquimod, which activates pDCs through toll-like receptor 7 engagement. Additionally, pDC-based anticancer vaccines have shown encouraging results for the treatment of melanoma in early trials. Future studies regarding the contributions of pDCs to malignancy will likely afford many opportunities for immunotherapy strategies.

Plasmacytoid dendritic cells of the gut: relevance to immunity and pathology.

Plasmacytoid dendritic cells (pDCs) are bone marrow-derived immune cells with the ability to express copious amounts of type I and III interferon (IFN) and can differentiate into antigen-presenting dendritic cells as a result of stimulation by pathogen-derived nucleic acid. These powerful combined functionalities allow pDCs to bridge the innate and adaptive immune systems resulting in a concerted pathogen response. The contribution of pDCs to gastrointestinal immunity is only now being elucidated and is proving to be a critical component in systemic immunity. This review will explore the immunology of pDCs and will discuss their involvement in human disease and tolerance with an emphasis on those in the gastrointestinal lymphoid tissue.

Differential immuno-reactivity to genomic DNA, RNA and mitochondrial DNA is associated with auto-immunity.

BACKGROUND: Circulating auto-reactive antibodies are hallmark features of auto-immune diseases, however little is known with respect to the specificity of such bio-markers. In the present study, we investigated the specificity of anti-nucleic acid antibodies in the blood of subjects with systemic lupus erythematosus (SLE) and healthy controls. METHODS: Sera from 12 SLE cases and 8 controls were evaluated for immuno-reactivity to purified RNA, DNA and mitochondrial DNA (mtDNA) by enzyme-linked immuno-sorbent assay (ELISA). RESULTS: As expected, immuno-reactivity to total nucleic acids was significantly higher in subjects with SLE when compared to healthy controls, however a clear distinction was observed among the various nucleic acid sub-types, with sera from SLE subjects displaying the greatest immuno-reactivity to RNA followed by mtDNA and then total DNA. CONCLUSION: The identification of auto-reactive antibodies can serve as highly sensitive biomarkers, although their specificity may not always allow diagnostic certainty. The knowledge that auto-antibodies in subjects with SLE display differential immuno-reactivity may help to improve existing diagnostics and may lead to a better understanding of the pathogenesis of auto-immune disorders.

Implications of NLRP3 Suppression Using Glibenclamide and miR-223 against Colorectal Cancer.

The NLR family pyrin domain containing 3 (NLRP3) promotes the growth of colorectal cancer (CRC). However, the therapeutic effect of NLRP3 inhibition on CRC cell progression is controversial. This study comparatively investigated the therapeutic effect of a pharmacological NLRP3 inhibitor, glibenclamide (gli), and the post-translational suppression of NLRP3 by miR-223 on CRC cell progression in HCT-116 and HCT-15 cells. LPS and ATP were used to activate Gli-treated and LSB-hsa-miR-223-3p (WTmiR-223)-expressing HCT-116 cells. NLRP3.AB.pCCL.sin.cPPT.U6.miR-223-Decoy.hPGK.GFP.WPRE plasmid (DmiR-223) was the negative control for miR-223 expression. NLRP3, gasdermin D, and BAX expressions were analyzed using western blotting. Real-time PCR detected the RNA expression of autophagy-related genes ATG5, BECN1, and miR-223 in non-transfected cells. ELISA analyzed IL-1ß and IL-18 in the medium. MTS-1, annexin V, wound-healing, and sphere-invasion assays were used to assess cell viability and progression. A multiplex cytokine assay detected proinflammatory cytokine secretion. LPS-ATP-activated NLRP3 produced gasdermin D cleavage, released IL-1b and IL-18, and activated cell migration and sphere invasion. In contrast, reduced cell growth, miR-223 expression, IFN-γ, CXCL10, and LIF secretion were found in cells after inflammasome activation. Both gli and WTmiR-223 induced autophagy genes ATG5 and BECN1 and reduced the NLRP3 activation and its downstream proteins. However, while gli had a limited effect on the production of IFN-γ, CXCL10, and LIF, WTmiR-223 increased the release of those cytokines. In addition, gli did not suppress cell growth, while WTmiR-223 promoted apoptosis. Notably, neither gli nor WTmiR-223 effectively prevented sphere invasion. These data suggest that, while WTmiR-223 could have a better anticancer effect in CRC compared to gli, the sole usage of miR-223-mediated NLRP3 suppression may not be sufficient to prevent CRC metastasis.