The lysine methyltransferase SMYD5 amplifies HIV-1 transcription and is post-transcriptionally upregulated by Tat and USP11.

A successful HIV-1 cure strategy may require enhancing HIV-1 latency to silence HIV-1 transcription. Modulators of gene expression show promise as latency-promoting agents in vitro and in vivo. Here, we identify Su(var)3-9, enhancer-of-zeste, and trithorax (SET) and myeloid, Nervy, and DEAF-1 (MYND) domain-containing protein 5 (SMYD5) as a host factor required for HIV-1 transcription. SMYD5 is expressed in CD4+ T cells and activates the HIV-1 promoter with or without the viral Tat protein, while knockdown of SMYD5 decreases HIV-1 transcription in cell lines and primary T cells. SMYD5 associates in vivo with the HIV-1 promoter and binds the HIV trans-activation response (TAR) element RNA and Tat. Tat is methylated by SMYD5 in vitro, and in cells expressing Tat, SMYD5 protein levels are increased. The latter requires expression of the Tat cofactor and ubiquitin-specific peptidase 11 (USP11). We propose that SMYD5 is a host activator of HIV-1 transcription stabilized by Tat and USP11 and, together with USP11, a possible target for latency-promoting therapy.

Digital Quantification of Tumor Cellularity as a Novel Prognostic Feature of Non-Small Cell Lung Carcinoma.

Non-small cell lung carcinoma is currently staged based on the size and involvement of other structures. Tumor size may be a surrogate measure of the total number of tumor cells. A recently revised reporting system for adenocarcinoma incorporates high-risk histologic patterns, which may have increased cellular density. Modern digital image analysis tools can be utilized to automate the quantification of cells. In this study, we tested the hypothesis that tumor cellularity can be used as a novel prognostic tool for lung cancer. Digital slides from The Cancer Genome Atlas lung adenocarcinoma (ADC) data set (n = 213) and lung squamous cell carcinoma (SCC) data set (n = 90) were obtained and analyzed using QuPath. The number of tumor cells was normalized with the surface area of the tumor to provide a measure of tumor cell density. Tumor cellularity was calculated by multiplying the size of the tumor with the cell density. Major histologic patterns and grade were compared with the tumor density of the lung ADC and lung SCC cases. The overall and progression-free survival were compared between groups of high and low tumor cellularity. High-grade histologic patterns in the ADC and SCC cases were associated with greater tumor densities compared with low-grade patterns. Cases with lower tumor cellularity had improved overall and progression-free survival compared with cases with higher cellularity. These results support tumor cellularity as a novel prognostic tool for non-small cell lung carcinoma that considers tumor stage and grade elements.

SIRT5 is a proviral factor that interacts with SARS-CoV-2 Nsp14 protein.

SARS-CoV-2 non-structural protein Nsp14 is a highly conserved enzyme necessary for viral replication. Nsp14 forms a stable complex with non-structural protein Nsp10 and exhibits exoribonuclease and N7-methyltransferase activities. Protein-interactome studies identified human sirtuin 5 (SIRT5) as a putative binding partner of Nsp14. SIRT5 is an NAD-dependent protein deacylase critical for cellular metabolism that removes succinyl and malonyl groups from lysine residues. Here we investigated the nature of this interaction and the role of SIRT5 during SARS-CoV-2 infection. We showed that SIRT5 interacts with Nsp14, but not with Nsp10, suggesting that SIRT5 and Nsp10 are parts of separate complexes. We found that SIRT5 catalytic domain is necessary for the interaction with Nsp14, but that Nsp14 does not appear to be directly deacylated by SIRT5. Furthermore, knock-out of SIRT5 or treatment with specific SIRT5 inhibitors reduced SARS-CoV-2 viral levels in cell-culture experiments. SIRT5 knock-out cells expressed higher basal levels of innate immunity markers and mounted a stronger antiviral response, independently of the Mitochondrial Antiviral Signaling Protein MAVS. Our results indicate that SIRT5 is a proviral factor necessary for efficient viral replication, which opens novel avenues for therapeutic interventions.

Viral E protein neutralizes BET protein-mediated post-entry antagonism of SARS-CoV-2.

Inhibitors of bromodomain and extraterminal domain (BET) proteins are possible anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) prophylactics as they downregulate angiotensin-converting enzyme 2 (ACE2). Here we show that BET proteins should not be inactivated therapeutically because they are critical antiviral factors at the post-entry level. Depletion of BRD3 or BRD4 in cells overexpressing ACE2 exacerbates SARS-CoV-2 infection; the same is observed when cells with endogenous ACE2 expression are treated with BET inhibitors during infection and not before. Viral replication and mortality are also enhanced in BET inhibitor-treated mice overexpressing ACE2. BET inactivation suppresses interferon production induced by SARS-CoV-2, a process phenocopied by the envelope (E) protein previously identified as a possible "histone mimetic." E protein, in an acetylated form, directly binds the second bromodomain of BRD4. Our data support a model where SARS-CoV-2 E protein evolved to antagonize interferon responses via BET protein inhibition; this neutralization should not be further enhanced with BET inhibitor treatment.

Cardiovascular Dysfunction in COVID-19: Association Between Endothelial Cell Injury and Lactate.

Coronavirus disease 2019 (COVID-19), an infectious respiratory disease propagated by a new virus known as Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), has resulted in global healthcare crises. Emerging evidence from patients with COVID-19 suggests that endothelial cell damage plays a central role in COVID-19 pathogenesis and could be a major contributor to the severity and mortality of COVID-19. Like other infectious diseases, the pathogenesis of COVID-19 is closely associated with metabolic processes. Lactate, a potential biomarker in COVID-19, has recently been shown to mediate endothelial barrier dysfunction. In this review, we provide an overview of cardiovascular injuries and metabolic alterations caused by SARS-CoV-2 infection. We also propose that lactate plays a potential role in COVID-19-driven endothelial cell injury.

SIRT5 is a proviral factor that interacts with SARS-CoV-2 Nsp14 protein.

SARS-CoV-2 non-structural protein Nsp14 is a highly conserved enzyme necessary for viral replication. Nsp14 forms a stable complex with non-structural protein Nsp10 and exhibits exoribonuclease and N7-methyltransferase activities. Protein-interactome studies identified human sirtuin 5 (SIRT5) as a putative binding partner of Nsp14. SIRT5 is an NAD-dependent protein deacylase critical for cellular metabolism that removes succinyl and malonyl groups from lysine residues. Here we investigated the nature of this interaction and the role of SIRT5 during SARS-CoV-2 infection. We showed that SIRT5 stably interacts with Nsp14, but not with Nsp10, suggesting that SIRT5 and Nsp10 are parts of separate complexes. We found that SIRT5 catalytic domain is necessary for the interaction with Nsp14, but that Nsp14 does not appear to be directly deacylated by SIRT5. Furthermore, knock-out of SIRT5 or treatment with specific SIRT5 inhibitors reduced SARS-CoV-2 viral levels in cell-culture experiments. SIRT5 knock-out cells expressed higher basal levels of innate immunity markers and mounted a stronger antiviral response. Our results indicate that SIRT5 is a proviral factor necessary for efficient viral replication, which opens novel avenues for therapeutic interventions.

Viral E Protein Neutralizes BET Protein-Mediated Post-Entry Antagonism of SARS-CoV-2.

Inhibitors of Bromodomain and Extra-terminal domain (BET) proteins are possible anti-SARS-CoV-2 prophylactics as they downregulate angiotensin-converting enzyme 2 (ACE2). Here, we show that BET proteins should not be inactivated therapeutically as they are critical antiviral factors at the post-entry level. Knockouts of BRD3 or BRD4 in cells overexpressing ACE2 exacerbate SARS-CoV-2 infection; the same is observed when cells with endogenous ACE2 expression are treated with BET inhibitors during infection, and not before. Viral replication and mortality are also enhanced in BET inhibitor-treated mice overexpressing ACE2. BET inactivation suppresses interferon production induced by SARS-CoV-2, a process phenocopied by the envelope (E) protein previously identified as a possible "histone mimetic." E protein, in an acetylated form, directly binds the second bromodomain of BRD4. Our data support a model where SARS-CoV-2 E protein evolved to antagonize interferon responses via BET protein inhibition; this neutralization should not be further enhanced with BET inhibitor treatment.

Genome Sequences of Akoni, Ashton, and Truong, Podoviridae Bacteriophages Isolated from Microbacterium foliorum.

Cluster EK2 Akoni, Ashton, and Truong are lytic Podoviridae actinobacteriophages that were isolated from soil in Florida using Microbacterium foliorum NRRL B-24224 as the host. The genomes are 54,307 bp, 54,560 bp, and 54,309 bp, respectively, and are 60% GC rich. Each genome contains a novel 13,464-bp gene that encompasses 25% of the genome.

CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes.

The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.

CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes.

The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.

DUSP11-mediated control of 5'-triphosphate RNA regulates RIG-I sensitivity.

Deciphering the mechanisms that regulate the sensitivity of pathogen recognition receptors is imperative to understanding infection and inflammation. Here we demonstrate that the RNA triphosphatase dual-specificity phosphatase 11 (DUSP11) acts on both host and virus-derived 5'-triphosphate RNAs rendering them less active in inducing a RIG-I-mediated immune response. Reducing DUSP11 levels alters host triphosphate RNA packaged in extracellular vesicles and induces enhanced RIG-I activation in cells exposed to extracellular vesicles. Virus infection of cells lacking DUSP11 results in a higher proportion of triphosphorylated viral transcripts and attenuated virus replication, which is rescued by reducing RIG-I expression. Consistent with the activity of DUSP11 in the cellular RIG-I response, mice lacking DUSP11 display lower viral loads, greater sensitivity to triphosphorylated RNA, and a signature of enhanced interferon activity in select tissues. Our results reveal the importance of controlling 5'-triphosphate RNA levels to prevent aberrant RIG-I signaling and demonstrate DUSP11 as a key effector of this mechanism.

The Role of Anterior Cervical Discectomy and Fusion on Relieving Axial Neck Pain in Patients With Single-Level Disease: A Systematic Review and Meta-Analysis.

STUDY DESIGN: Systematic review and meta-analysis. OBJECTIVES: This study aims to evaluate the effects of anterior cervical decompression and fusion (ACDF) on axial neck pain in adult patients receiving surgery for myelopathy, radiculopathy, or a combination of both. METHODS: Two independent reviewers completed a librarian-assisted search of 4 databases. Visual Analogue Scale (VAS) and Neck Disability Index (NDI) scores were extracted preoperatively and at 3, 6, 12, 24, 36, 48, and 48+ months postoperatively for ACDF groups and pooled using a random-effects model. RESULTS: Of 17 850 eligible studies, 37 were included for analysis, totaling 2138 patients analyzed with VAS and 2477 with NDI score. Individual VAS mean differences were reduced at 6 weeks (-2.5 [95% confidence interval (CI): -3.5 to -1.6]), 3 months (-2.9 [-3.7 to -2.2]), 6 months (-3.2 [-3.9 to -2.6]), 12 months (-3.7 [-4.3 to -3.1]), 24 months (-4.0 [-4.4 to -3.5]), 48 months (-4.6 [-5.5 to -3.8]), and >48 months (-4.7 [-5.8 to -3.6]) follow-up (P < .0001 for all endpoints). Individual NDI mean differences were reduced at 6 weeks (-26.7 [-30.9 to -22.6]), 3 months (-29.8 [-32.7 to -26.8]), 6 months (-31.2 [-35.5 to -26.8)], 12 months (-29.3 [-33.2 to -25.4]), 24 months (-28.9 [-32.6 to -25.2]), 48 months (-33.1 [-37.4 to -28.7]), and >48 months (-37.6 [-45.9 to -29.3]) follow-up (P < .0001 for all endpoints). CONCLUSIONS: ACDF is associated with a significant reduction in axial neck pain compared with preoperative values in patients being treated specifically for myelopathy or radiculopathy. This influences the preoperative discussions surgeons may have with patients regarding their expectations for surgery. The effects seen are stable over time and represent a clinically significant reduction in axial neck pain.

Acupuncture and related interventions for carpal tunnel syndrome: systematic review.

OBJECTIVE: To synthesize evidence on the effectiveness of acupuncture and related therapies for primary carpal tunnel syndrome (CTS) by conducting a systematic review of randomized controlled trials (RCTs). DATA SOURCES: Nine databases were searched for potential RCTs from their inception till July 2019. REVIEW METHODS: RCTs which reported at least one of the three outcomes were included: symptom severity, functional status and pain. Included RCTs were appraised using the Cochrane Risk of Bias Tool. RESULTS: A total of 10 RCTs (728 participants) were included. Majority were at high risk of bias for blinding of participants, personnel and outcome assessors. When compared to conventional medications, manual acupuncture showed significant superior effect in reducing symptom than ibuprofen (mean difference (MD) on Symptom Severity Scale (SSS)) = -5.80, 95% confidence interval (CI): -7.95 to -3.65) and prednisolone (MD = -6.50, 95% CI: -10.1, -2.86). Electroacupuncture plus splinting was more effective in reducing symptom severity than splinting alone (SSS score: MD = -0.20, 95% CI: -0.36 to -0.03). Manual acupuncture showed significantly superior effect than ibuprofen in improving functional status (Functional Status Scale (FSS): MD = -1.84, 95% CI: -2.66 to -1.02). The combination of electroacupuncture and splinting showed more improvement in functional status compared to splinting alone (FSS: MD = -6.22, 95%CI: -10.7 to -1.71). Triple treatment of acupuncture, magnetic spectrum heat lamp and splinting showed stronger pain relief than splinting alone. CONCLUSION: For both symptom relief and function improvement, manual acupuncture is superior to ibuprofen while electroacupuncture plus splinting outperforms splinting alone. Limited evidence showed electroacupuncture's potential role in pain reduction.

RNA triphosphatase DUSP11 enables exonuclease XRN-mediated restriction of hepatitis C virus.

Seventy percent of people infected with hepatitis C virus (HCV) will suffer chronic infection, putting them at risk for liver disease, including hepatocellular carcinoma. The full range of mechanisms that render some people more susceptible to chronic infection and liver disease is still being elucidated. XRN exonucleases can restrict HCV replication and may help to resolve HCV infections. However, it is unknown how 5' triphosphorylated HCV transcripts, primary products of the viral polymerase, become susceptible to attack by 5' monophosphate-specific XRNs. Here, we show that the 5' RNA triphosphatase DUSP11 acts on HCV transcripts, rendering them susceptible to XRN-mediated attack. Cells lacking DUSP11 show substantially enhanced HCV replication, and this effect is diminished when XRN expression is reduced. MicroRNA-122 (miR-122), a target of current phase II anti-HCV drugs, is known to protect HCV transcripts against XRNs. We show that HCV replication is less dependent on miR-122 in cells lacking DUSP11. Combined, these results implicate DUSP11 as an important component of XRN-mediated restriction of HCV.

Epidemiological characteristics and methodological quality of meta-analyses on diabetes mellitus treatment: a systematic review.

OBJECTIVE: Well-conducted meta-analyses (MAs) are considered as one of the best sources of clinical evidence for treatment decision. MA with methodological flaws may introduce bias and mislead evidence users. The aim of this study is to investigate the characteristics and methodological quality of MAs on diabetes mellitus (DM) treatments. DESIGN: Systematic review. METHODS: Cochrane Database of Systematic Review and Database of Abstract of Reviews of Effects were searched for relevant MAs. Assessing methodological quality of systematic reviews (AMSTAR) tool was used to evaluate the methodological quality of included MAs. Logistic regression analysis was used to identify association between characteristics of MA and AMSTAR results. RESULTS: A total of 252 MAs including 4999 primary studies and 13,577,025 patients were included. Over half of the MAs (65.1%) only included type 2 DM patients and 160 MAs (63.5%) focused on pharmacological treatments. About 89.7% MAs performed comprehensive literature search and 89.3% provided characteristics of included studies. Included MAs generally had poor performance on the remaining AMSTAR items, especially in assessing publication bias (39.3%), providing lists of studies (19.0%) and declaring source of support comprehensively (7.5%). Only 62.7% MAs mentioned about harm of interventions. MAs with corresponding author from Asia performed less well in providing MA protocol than those from Europe. CONCLUSIONS: Methodological quality of MA on DM treatments was unsatisfactory. There is considerable room for improvement, especially in assessing publication bias, providing lists of studies and declaring source of support comprehensively. Also, there is an urgent need for MA authors to report treatment harm comprehensively.

Single-laboratory validation of a method for the determination of vitamin D3 in dietary supplements by liquid chromatography with tandem mass spectrometry (LC-MS/MS).

A single-laboratory validation (SLV) for the analysis of vitamin D3 was performed in four types of dietary supplements (capsules, soft gels, syrups, and tablets) using LC-MS/MS. Samples were treated by alkaline saponification for oil-based soft gels and utilized EDTA solution for capsules, syrups, and tablets prior to n-hexane extraction. Vitamin D3 in sample extracts was separated on a reversed-phase C18 column (100 x 2.1 mm, 2.7 pm) using a mobile phase of a 95 + 5 (v/v) mixture of 5 mM ammonium formate in methanol containing 0.1% formic acid and 5 mM ammonium formate in 0.1% formic acid running at a flow rate of 0.2 mL/min. Vitamin D3 was confirmed by the presence of three fragment ions at m/z 107, 159, and 259 within a defined retention time window from the precursor ion at m/z 385. Quantitation was based on the peak area at m/z 367 to that of the internal standard (d3-vitamin D3) at m/z 370 with reference to the respective response ratios of the calibration standards. The linear response of vitamin D3 ranged from 0.10 to 6.29 mg/L and the correlation coefficient (r) of the six-point calibration curves was > 0.999. Accuracy, in terms of the spiked recoveries from blank syrup and starch powder at three different concentration levels, was 101-103%. Precision, determined by two different analysts over a period of 5 weeks, ranged from 2.7 to 7.0% for the four preparations. The SLV demonstrates the present LC-MS/MS method is reliable and robust for the determination of vitamin D3 in the studied dietary supplements. Considering the attainment of satisfactory SLV results, further validation through intra-laboratory collaborative study is recommended.

Single-Laboratory Validation of a Method for the Determination of Vitamin D3 in Dietary Supplements by Liquid Chromatography with Tandem Mass Spectrometry (LC-MS/MS).

A single-laboratory validation (SLV) for the analysis of vitamin D3 was performed in four types of dietary supplements (capsules, soft gels, syrups, and tablets) using LC-MS/MS. Samples were treated by alkaline saponification for oil-based soft gels and utilized EDTA solution for capsules, syrups, and tablets prior to n-hexane extraction. Vitamin D3 in sample extracts was separated on a reversed-phase C18 column (100 × 2.1 mm, 2.7 µm) using a mobile phase of a 95 + 5 (v/v) mixture of 5 mM ammonium formate in methanol containing 0.1% formic acid and 5 mM ammonium formate in 0.1% formic acid running at a flow rate of 0.2 mL/min. Vitamin D3 was confirmed by the presence of three fragment ions at m/z 107, 159, and 259 within a defined retention time window from the precursor ion at m/z 385. Quantitation was based on the peak area at m/z 367 to that of the internal standard (d3-vitamin D3) at m/z 370 with reference to the respective response ratios of the calibration standards. The linear response of vitamin D3 ranged from 0.10 to 6.29 mg/L and the correlation coefficient (r) of the six-point calibration curves was >0.999. Accuracy, in terms of the spiked recoveries from blank syrup and starch powder at three different concentration levels, was 101-103%. Precision, determined by two different analysts over a period of 5 weeks, ranged from 2.7 to 7.0% for the four preparations. The SLV demonstrates the present LC-MS/MS method is reliable and robust for the determination of vitamin D3 in the studied dietary supplements. Considering the attainment of satisfactory SLV results, further validation through intra-laboratory collaborative study is recommended.

Fecal microbiome and volatile organic compound metabolome in obese humans with nonalcoholic fatty liver disease.

BACKGROUND & AIMS: The histopathology of nonalcoholic fatty liver disease (NAFLD) is similar to that of alcoholic liver disease. Colonic bacteria are a source of many metabolic products, including ethanol and other volatile organic compounds (VOC) that may have toxic effects on the human host after intestinal absorption and delivery to the liver via the portal vein. Recent data suggest that the composition of the gut microbiota in obese human beings is different from that of healthy-weight individuals. The aim of this study was to compare the colonic microbiome and VOC metabolome of obese NAFLD patients (n = 30) with healthy controls (n = 30). METHODS: Multitag pyrosequencing was used to characterize the fecal microbiota. Fecal VOC profiles were measured by gas chromatography-mass spectrometry. RESULTS: There were statistically significant differences in liver biochemistry and metabolic parameters in NAFLD. Deep sequencing of the fecal microbiome revealed over-representation of Lactobacillus species and selected members of phylum Firmicutes (Lachnospiraceae; genera, Dorea, Robinsoniella, and Roseburia) in NAFLD patients, which was statistically significant. One member of phylum Firmicutes was under-represented significantly in the fecal microbiome of NAFLD patients (Ruminococcaceae; genus, Oscillibacter). Fecal VOC profiles of the 2 patient groups were different, with a significant increase in fecal ester compounds observed in NAFLD patients. CONCLUSIONS: A significant increase in fecal ester VOC is associated with compositional shifts in the microbiome of obese NAFLD patients. These novel bacterial metabolomic and metagenomic factors are implicated in the etiology and complications of obesity.

NLRP3 inflammasome plays a key role in the regulation of intestinal homeostasis.

BACKGROUND: Attenuated innate immune responses to the intestinal microbiota have been linked to the pathogenesis of Crohn's disease (CD). Recent genetic studies have revealed that hypofunctional mutations of NLRP3, a member of the NOD-like receptor (NLR) superfamily, are associated with an increased risk of developing CD. NLRP3 is a key component of the inflammasome, an intracellular danger sensor of the innate immune system. When activated, the inflammasome triggers caspase-1-dependent processing of inflammatory mediators, such as IL-1ß and IL-18. METHODS: In the current study we sought to assess the role of the NLRP3 inflammasome in the maintenance of intestinal homeostasis through its regulation of innate protective processes. To investigate this role, Nlrp3(-/-) and wildtype mice were assessed in the dextran sulfate sodium and 2,4,6-trinitrobenzenesulfonic acid models of experimental colitis. RESULTS: Nlrp3(-/-) mice were found to be more susceptible to experimental colitis, an observation that was associated with reduced IL-1ß, reduced antiinflammatory cytokine IL-10, and reduced protective growth factor TGF-ß. Macrophages isolated from Nlrp3(-/-) mice failed to respond to bacterial muramyl dipeptide. Furthermore, Nlrp3-deficient neutrophils exhibited reduced chemotaxis and enhanced spontaneous apoptosis, but no change in oxidative burst. Lastly, Nlrp3(-/-) mice displayed altered colonic ß-defensin expression, reduced colonic antimicrobial secretions, and a unique intestinal microbiota. CONCLUSIONS: Our data confirm an essential role for the NLRP3 inflammasome in the regulation of intestinal homeostasis and provide biological insight into disease mechanisms associated with increased risk of CD in individuals with NLRP3 mutations.