Humoral and T-cell-mediated responses to an insect-specific flavivirus-based Zika virus vaccine candidate.

Flaviviruses represent a significant global health threat and relatively few licensed vaccines exist to protect against them. Insect-specific flaviviruses (ISFVs) are incapable of replication in humans and have emerged as a novel and promising tool for flavivirus vaccine development. ISFV-based flavivirus vaccines have shown exceptional safety, immunogenicity, and efficacy, however, a detailed assessment of the correlates of protection and immune responses induced by these vaccines are still needed for vaccine optimization. Here, we explore the mechanisms of protective immunity induced by a previously created pre-clinical Zika virus (ZIKV) vaccine candidate, called Aripo/Zika (ARPV/ZIKV). In brief, immunocompromised IFN-αßR-/- mice passively immunized with ARPV/ZIKV immune sera experienced protection after lethal ZIKV challenge, although this protection was incomplete. ARPV/ZIKV-vaccinated IFN-αßR-/- mice depleted of CD4+ or CD8+ T-cells at the time of ZIKV challenge showed no morbidity or mortality. However, the adoptive transfer of ARPV/ZIKV-primed T-cells into recipient IFN-αßR-/- mice resulted in a two-day median increase in survival time compared to controls. Altogether, these results suggest that ARPV/ZIKV-induced protection is primarily mediated by neutralizing antibodies at the time of challenge and that T-cells may play a comparatively minor but cumulative role in the protection observed. Lastly, ARPV/ZIKV-vaccinated Tcra KO mice, which are deficient in T-cell responses, experienced significant mortality post-challenge. These results suggest that ARPV/ZIKV-induced cell-mediated responses are critical for development of protective immune responses at vaccination. Despite the strong focus on neutralizing antibody responses to novel flavivirus vaccine candidates, these results suggest that cell-mediated responses induced by ISFV-based vaccines remain important to overall protective responses.

Cellular Context Dictates the Suppression or Augmentation of Triple-Negative Mammary Tumor Metastasis by NLRX1.

Prior studies have defined multiple, but inconsistent, roles for the enigmatic pattern recognition receptor NLRX1 in regulating several cancer-associated biological functions. In this study, we explore the role of NLRX1 in the highly metastatic murine 4T1 mammary tumor model. We describe a functional dichotomy of NLRX1 between two different cellular contexts: expression in healthy host cells versus expression in the 4T1 tumor cells. Using Nlrx1-/- mice engrafted with 4T1 tumors, we demonstrate that NLRX1 functions as a tumor suppressor when expressed in the host cells. Specifically, NLRX1 in healthy host cells attenuates tumor growth and lung metastasis through suppressing characteristics of epithelial-mesenchymal transition and the lung metastatic niche. Conversely, we demonstrate that NLRX1 functions as a tumor promoter when expressed in 4T1 tumor cells using gain- and loss-of-function studies both in vitro and in vivo. Mechanistically, NLRX1 in the tumor cells augments 4T1 aggressiveness and metastasis through regulating epithelial-mesenchymal transition hallmarks, cell death, proliferation, migration, reactive oxygen species levels, and mitochondrial respiration. Collectively, we provide critical insight into NLRX1 function and establish a dichotomous role of NLRX1 in the 4T1 murine mammary carcinoma model that is dictated by cellular context.

The innate immune sensor NLRC3 attenuates Toll-like receptor signaling via modification of the signaling adaptor TRAF6 and transcription factor NF-κB.

Several members of the NLR family of sensors activate innate immunity. In contrast, we found here that NLRC3 inhibited Toll-like receptor (TLR)-dependent activation of the transcription factor NF-κB by interacting with the TLR signaling adaptor TRAF6 to attenuate Lys63 (K63)-linked ubiquitination of TRAF6 and activation of NF-κB. We used bioinformatics to predict interactions between NLR and TRAF proteins, including interactions of TRAF with NLRC3. In vivo, macrophage expression of Nlrc3 mRNA was diminished by the administration of lipopolysaccharide (LPS) but was restored when cellular activation subsided. To assess biologic relevance, we generated Nlrc3(-/-) mice. LPS-treated Nlrc3(-/-) macrophages had more K63-ubiquitinated TRAF6, nuclear NF-κB and proinflammatory cytokines. Finally, LPS-treated Nlrc3(-/-) mice had more signs of inflammation. Thus, signaling via NLRC3 and TLR constitutes a negative feedback loop. Furthermore, prevalent NLR-TRAF interactions suggest the formation of a 'TRAFasome' complex.

A hepatitis B virus core antigen-based virus-like particle vaccine expressing SARS-CoV-2 B and T cell epitopes induces epitope-specific humoral and cell-mediated immune responses but confers limited protection against SARS-CoV-2 infection.

The hepatitis B virus core antigen (HBcAg) tolerates insertion of foreign epitopes and maintains its ability to self-assemble into virus-like particles (VLPs). We constructed a ∆HBcAg-based VLP vaccine expressing three predicted severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) B and T cell epitopes and determined its immunogenicity and protective efficacy. The recombinant ∆HBcAg-SARS-CoV-2 protein was expressed in Escherichia coli, purified, and shown to form VLPs. K18-hACE2 transgenic C57BL/6 mice were immunized intramuscularly with ∆HBcAg VLP control (n = 15) or ∆HBcAg-SARS-CoV-2 VLP vaccine (n = 15). One week after the 2nd booster and before virus challenge, five ∆HBcAg-SARS-CoV-2 vaccinated mice were euthanized to evaluate epitope-specific immune responses. There is a statistically significant increase in epitope-specific Immunoglobulin G (IgG) response, and statistically higher interleukin 6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) expression levels in ∆HBcAg-SARS-CoV-2 VLP-vaccinated mice compared to ∆HBcAg VLP controls. While not statistically significant, the ∆HBcAg-SARS-CoV-2 VLP mice had numerically more memory CD8+ T-cells, and 3/5 mice also had numerically higher levels of interferon gamma (IFN-γ) and tumor necrosis factor (TNF). After challenge with SARS-CoV-2, ∆HBcAg-SARS-CoV-2 immunized mice had numerically lower viral RNA loads in the lung, and slightly higher survival, but the differences are not statistically significant. These results indicate that the ∆HBcAg-SARS-CoV-2 VLP vaccine elicits epitope-specific humoral and cell-mediated immune responses but they were insufficient against SARS-CoV-2 infection.

Magic bubbles: utilizing histotripsy to modulate the tumor microenvironment and improve systemic anti-tumor immune responses.

Focused Ultrasound (FUS) is emerging as a promising primary and adjunct therapy for the treatment of cancer. This includes histotripsy, which is a noninvasive, non-ionizing, non-thermal ultrasound guided ablation modality. As histotripsy has progressed from bench-to-bedside, it has become evident that this therapy has benefits beyond local tumor ablation. Specifically, histotripsy has the potential to shift the local tumor microenvironment from immunologically 'cold' to 'hot'. This is associated with the production of damage associated molecular patterns, the release of a selection of proinflammatory mediators, and the induction of inflammatory forms of cell death in cells just outside of the treatment zone. In addition to the induction of this innate immune response, histotripsy can also improve engagement of the adaptive immune system and promote systemic anti-tumor immunity targeting distal tumors and metastatic lesions. These tantalizing observations suggest that, in settings of widely metastatic disease burden, selective histotripsy of a limited number of accessible tumors could be a means of maximizing responsiveness to systemic immunotherapy. More work is certainly needed to optimize treatment strategies that best synergize histotripsy parameters with innate and adaptive immune responses. Likewise, rigorous clinical studies are still necessary to verify the presence and repeatability of these phenomena in human patients. As this technology nears regulatory approval for clinical use, it is our expectation that the insights and immunomodulatory mechanisms summarized in this review will serve as directional guides for rational clinical studies to validate and optimize the potential immunotherapeutic role of histotripsy tumor ablation.

Investigating cell death responses associated with histotripsy ablation of canine osteosarcoma.

BACKGROUND: Osteosarcoma (OS) is the most frequently occurring primary bone tumor in dogs and people and innovative treatment options are profoundly needed. Histotripsy is an emerging tumor ablation modality, and it is essential for the clinical translation of histotripsy to gain knowledge about the outcome of nonablated tumor cells that could remain postablation. The objective of this study was to characterize the cell death genetic signature and proliferation response of canine OS cells post a near complete histotripsy ablation (96% ± 1.5) and to evaluate genetic cell death signatures associated with histotripsy ablation and OS in vivo. METHODS: In the current study, we ablated three canine OS cell lines with a histotripsy dose that resulted in near complete ablation to allow for a viable tumor cell population for downstream analyses. To assess the in vivo cell death genetic signature, we characterized cell death genetic signature in histotripsy-ablated canine OS tumors collected 24-h postablation. RESULTS: Differential gene expression changes observed in the 4% viable D17 and D418 cells, and histotripsy-ablated OS tumor samples, but not in Abrams cells, were associated with immunogenic cell death (ICD). The 4% viable OS cells demonstrated significantly reduced proliferation, compared to control OS cells, in vitro. CONCLUSION: Histotripsy ablation of OS cell lines leads to direct and potentially indirect cell death as evident by, reduced proliferation in remaining viable OS cells and cell death genetic signatures suggestive of ICD both in vitro and in vivo.

Ultrasound-guided noninvasive pancreas ablation using histotripsy: feasibility study in an in vivo porcine model.

Pancreatic cancer is a malignant disease associated with poor survival and nearly 80% present with unresectable tumors. Treatments such as chemotherapy and radiation therapy have shown overall improved survival benefits, albeit limited. Histotripsy is a noninvasive, non-ionizing, and non-thermal focused ultrasound ablation modality that has shown efficacy in treating hepatic tumors and other malignancies. In this novel study, we investigate histotripsy for noninvasive pancreas ablation in a pig model. In two studies, histotripsy was applied to the healthy pancreas in 11 pigs using a custom 32-element, 500 kHz histotripsy transducer attached to a clinical histotripsy system, with treatments guided by real-time ultrasound imaging. A pilot study was conducted in 3 fasted pigs with histotripsy applied at a pulse repetition frequency (PRF) of 500 Hz. Results showed no pancreas visualization on coaxial ultrasound imaging due to overlying intestinal gas, resulting in off-target injury and no pancreas damage. To minimize gas, a second group of pigs (n = 8) were fed a custard diet containing simethicone and bisacodyl. Pigs were euthanized immediately (n = 4) or survived for 1 week (n = 4) post-treatment. Damage to the pancreas and surrounding tissue was characterized using gross morphology, histological analysis, and CT imaging. Results showed histotripsy bubble clouds were generated inside pancreases that were visually maintained on coaxial ultrasound (n = 4), with 2 pigs exhibiting off-target damage. For chronic animals, results showed the treatments were well-tolerated with no complication signs or changes in blood markers. This study provides initial evidence suggesting histotripsy's potential for noninvasive pancreas ablation and warrants further evaluation in more comprehensive studies.

High intensity focused ultrasound for the treatment of solid tumors: a pilot study in canine cancer patients.

PURPOSE: To investigate the safety, feasibility, and outcomes of High-Intensity Focused Ultrasound (HIFU) for the treatment of solid tumors in a spontaneous canine cancer model. METHODS: Dogs diagnosed with subcutaneous solid tumors were recruited, staged and pretreatment biopsies were obtained. A single HIFU treatment was delivered to result in partial tumor ablation using a commercially available HIFU unit. Tumors were resected 3-6 days post HIFU and samples obtained for histopathology and immunohistochemistry. Total RNA was isolated from paired pre and post treated FFPE tumor samples, and quantitative gene expression analysis was performed using the nCounter Canine IO Panel. RESULTS: A total of 20 dogs diagnosed with solid tumors were recruited and treated in the study. Tumors treated included Soft Tissue Sarcoma (n = 15), Mast Cell Tumor (n = 3), Osteosarcoma (n = 1), and Thyroid Carcinoma (n = 1). HIFU was well tolerated with only 1 dog experiencing a clinically significant adverse event. Pathology confirmed the presence of complete tissue ablation at the HIFU targeted site and immunohistochemistry indicated immune cell infiltration at the treated/untreated tumor border. Quantitative gene expression analysis indicated that 28 genes associated with T-cell activation were differentially expressed post-HIFU. CONCLUSIONS: HIFU appears to be safe and feasible for the treatment of subcutaneous canine solid tumors, resulting in ablation of the targeted tissue. HIFU induced immunostimulatory changes, highlighting the canine cancer patient as an attractive model for studying the effects of focal ablation therapies on the tumor microenvironment.

Experimental and Numerical Investigation of Parameters Affecting High-Frequency Irreversible Electroporation for Prostate Cancer Ablation.

While the primary goal of focal therapy for prostate cancer (PCa) is conserving patient quality of life by reducing oncological burden, available modalities use thermal energy or whole-gland radiation which can damage critical neurovascular structures within the prostate and increase risk of genitourinary dysfunction. High-frequency irreversible electroporation (H-FIRE) is a promising alternative ablation modality that utilizes bursts of pulsed electric fields (PEFs) to destroy aberrant cells via targeted membrane damage. Due to its nonthermal mechanism, H-FIRE offers several advantages over state-of-the-art treatments, but waveforms have not been optimized for treatment of PCa. In this study, we characterize lethal electric field thresholds (EFTs) for H-FIRE waveforms with three different pulse widths as well as three interpulse delays in vitro and compare them to conventional irreversible electroporation (IRE). Experiments were performed in non-neoplastic and malignant prostate cells to determine the effect of waveforms on both targeted (malignant) and adjacent (non-neoplastic) tissue. A numerical modeling approach was developed to estimate the clinical effects of each waveform including extent of nonthermal ablation, undesired thermal damage, and nerve excitation. Our findings indicate that H-FIRE waveforms with pulse durations of 5 and 10 µs provide large ablations comparable to IRE with tolerable levels of thermal damage and minimized muscle contractions. Lower duration (2 µs) H-FIRE waveforms exhibit the least amount of muscle contractions but require increased voltages which may be accompanied by unwanted thermal damage.

NLRP12 suppresses colon inflammation and tumorigenesis through the negative regulation of noncanonical NF-κB signaling.

In vitro data suggest that a subgroup of NLR proteins, including NLRP12, inhibits the transcription factor NF-κB, although physiologic and disease-relevant evidence is largely missing. Dysregulated NF-κB activity is associated with colonic inflammation and cancer, and we found Nlrp12(-/-) mice were highly susceptible to colitis and colitis-associated colon cancer. Polyps isolated from Nlrp12(-/-) mice showed elevated noncanonical NF-κB activation and increased expression of target genes that were associated with cancer, including Cxcl13 and Cxcl12. NLRP12 negatively regulated ERK and AKT signaling pathways in affected tumor tissues. Both hematopoietic- and nonhematopoietic-derived NLRP12 contributed to inflammation, but the latter dominantly contributed to tumorigenesis. The noncanonical NF-κB pathway was regulated upon degradation of TRAF3 and activation of NIK. NLRP12 interacted with both NIK and TRAF3, and Nlrp12(-/-) cells have constitutively elevated NIK, p100 processing to p52 and reduced TRAF3. Thus, NLRP12 is a checkpoint of noncanonical NF-κB, inflammation, and tumorigenesis.

Cardiovascular function, pulmonary gas exchange and tissue oxygenation in isoflurane-anesthetized, mechanically ventilated Beagle dogs with four levels of positive end-expiratory pressure.

OBJECTIVES: To compare pulmonary gas exchange, tissue oxygenation and cardiovascular effects of four levels of end-expiratory pressure: no positive end-expiratory pressure (ZEEP), positive end-expiratory pressure (PEEP) of maximal respiratory system compliance (PEEPmaxCrs), PEEPmaxCrs + 2 cmH2O (PEEPmaxCrs+2), PEEPmaxCrs + 4 cmH2O (PEEPmaxCrs+4), in isoflurane-anesthetized dogs. STUDY DESIGN: Prospective randomized crossover study. ANIMALS: A total of seven healthy male Beagle dogs, aged 1 year and weighing 10.2 ± 0.7 kg (mean ± standard deviation). METHODS: The dogs were administered acepromazine and anesthesia was induced with propofol and maintained with isoflurane. Ventilation was controlled for 4 hours with ZEEP, PEEPmaxCrs, PEEPmaxCrs+2 or PEEPmaxCrs+4. Cardiovascular, pulmonary gas exchange and tissue oxygenation data were evaluated at 5, 60, 120, 180 and 240 minutes of ventilation and compared using a mixed-model anova followed by Bonferroni test. p < 0.05 was considered significant. RESULTS: Cardiac index (CI) and mean arterial pressure (MAP) were lower in all PEEP treatments at 5 minutes when compared with ZEEP. CI persisted lower throughout the 4 hours only in PEEPmaxCrs+4 with the lowest CI at 5 minutes (2.15 ± 0.70 versus 3.45 ± 0.94 L minute-1 m-2). At 180 and 240 minutes, MAP was lower in PEEPmaxCrs+4 than in PEEPmaxCrs, with the lowest value at 180 minutes (58 ± 7 versus 67 ± 7 mmHg). Oxygen delivery index (DO2I) was lower in PEEPmaxCrs+4 than in ZEEP at 5, 60, 120 and 180 minutes. Venous admixture was not different among treatments. CONCLUSION AND CLINICAL RELEVANCE: The use of PEEP caused a transient decrease in MAP and CI in lung-healthy dogs anesthetized with isoflurane, which improved after 60 minutes of ventilation in all levels of PEEP except PEEPmaxCrs+4. A clinically significant improvement in arterial oxygenation and DO2I was not observed with PEEPmaxCrs and PEEPmaxCrs+2 in comparison with ZEEP, whereas PEEPmaxCrs+4 decreased DO2I.

Targeted Delivery of Persulfides to the Gut: Effects on the Microbiome.

Persulfides (R-SSH) have been hypothesized as potent redox modulators and signaling compounds. Reported herein is the synthesis, characterization, and in vivo evaluation of a persulfide donor that releases N-acetyl cysteine persulfide (NAC-SSH) in response to the prokaryote-specific enzyme nitroreductase. The donor, termed NDP-NAC, decomposed in response to E. coli nitroreductase, resulting in release of NAC-SSH. NDP-NAC elicited gastroprotective effects in mice that were not observed in animals treated with control compounds incapable of persulfide release or in animals treated with Na2 S. NDP-NAC induced these effects by the upregulation of beneficial small- and medium-chain fatty acids and through increasing growth of Turicibacter sanguinis, a beneficial gut bacterium. It also decreased the populations of Synergistales bacteria, opportunistic pathogens implicated in gastrointestinal infections. This study reveals the possibility of maintaining gut health or treating microbiome-related diseases by the targeted delivery of reactive sulfur species.

NLRX1 protein attenuates inflammatory responses to infection by interfering with the RIG-I-MAVS and TRAF6-NF-κB signaling pathways.

The nucleotide-binding domain and leucine-rich-repeat-containing (NLR) proteins regulate innate immunity. Although the positive regulatory impact of NLRs is clear, their inhibitory roles are not well defined. We showed that Nlrx1(-/-) mice exhibited increased expression of antiviral signaling molecules IFN-ß, STAT2, OAS1, and IL-6 after influenza virus infection. Consistent with increased inflammation, Nlrx1(-/-) mice exhibited marked morbidity and histopathology. Infection of these mice with an influenza strain that carries a mutated NS-1 protein, which normally prevents IFN induction by interaction with RNA and the intracellular RNA sensor RIG-I, further exacerbated IL-6 and type I IFN signaling. NLRX1 also weakened cytokine responses to the 2009 H1N1 pandemic influenza virus in human cells. Mechanistically, Nlrx1 deletion led to constitutive interaction of MAVS and RIG-I. Additionally, an inhibitory function is identified for NLRX1 during LPS activation of macrophages where the MAVS-RIG-I pathway was not involved. NLRX1 interacts with TRAF6 and inhibits NF-κB activation. Thus, NLRX1 functions as a checkpoint of overzealous inflammation.

High-Frequency Irreversible Electroporation for Treatment of Primary Liver Cancer: A Proof-of-Principle Study in Canine Hepatocellular Carcinoma.

PURPOSE: To determine the safety and feasibility of percutaneous high-frequency irreversible electroporation (HFIRE) for primary liver cancer and evaluate the HFIRE-induced local immune response. MATERIALS AND METHODS: HFIRE therapy was delivered percutaneously in 3 canine patients with resectable hepatocellular carcinoma (HCC) in the absence of intraoperative paralytic agents or cardiac synchronization. Pre- and post-HFIRE biopsy samples were processed with histopathology and immunohistochemistry for CD3, CD4, CD8, and CD79a. Blood was collected on days 0, 2, and 4 for complete blood count and chemistry. Numeric models were developed to determine the treatment-specific lethal thresholds for malignant canine liver tissue and healthy porcine liver tissue. RESULTS: HFIRE resulted in predictable ablation volumes as assessed by posttreatment CT. No detectable cardiac interference and minimal muscle contraction occurred during HFIRE. No clinically significant adverse events occurred secondary to HFIRE. Microscopically, a well-defined ablation zone surrounded by a reactive zone was evident in the majority of samples. This zone was composed primarily of maturing collagen interspersed with CD3+/CD4-/CD8- lymphocytes in a proinflammatory microenvironment. The average ablation volumes for the canine HCC patients and the healthy porcine tissue were 3.89 cm3 ± 0.74 and 1.56 cm3 ± 0.16, respectively (P = .03), and the respective average lethal thresholds were 710 V/cm ± 28.2 and 957 V/cm ± 24.4 V/cm (P = .0004). CONCLUSIONS: HFIRE can safely and effectively be delivered percutaneously, results in a predictable ablation volume, and is associated with lymphocytic tumor infiltration. This is the first step toward the use of HFIRE for treatment of unresectable liver tumors.

Fabrication and characterization of PLGA nanoparticles encapsulating large CRISPR-Cas9 plasmid.

BACKGROUND: The clustered regularly interspaced short palindromic repeats (CRISPR) and Cas9 protein system is a revolutionary tool for gene therapy. Despite promising reports of the utility of CRISPR-Cas9 for in vivo gene editing, a principal problem in implementing this new process is delivery of high molecular weight DNA into cells. RESULTS: Using poly(lactic-co-glycolic acid) (PLGA), a nanoparticle carrier was designed to deliver a model CRISPR-Cas9 plasmid into primary bone marrow derived macrophages. The engineered PLGA-based carriers were approximately 160 nm and fluorescently labeled by encapsulation of the fluorophore 6,13-bis(triisopropylsilylethynyl) pentacene (TIPS pentacene). An amine-end capped PLGA encapsulated 1.6 wt% DNA, with an encapsulation efficiency of 80%. Release studies revealed that most of the DNA was released within the first 24 h and corresponded to ~ 2-3 plasmid copies released per nanoparticle. In vitro experiments conducted with murine bone marrow derived macrophages demonstrated that after 24 h of treatment with the PLGA-encapsulated CRISPR plasmids, the majority of cells were positive for TIPS pentacene and the protein Cas9 was detectable within the cells. CONCLUSIONS: In this work, plasmids for the CRISPR-Cas9 system were encapsulated in nanoparticles comprised of PLGA and were shown to induce expression of bacterial Cas9 in murine bone marrow derived macrophages in vitro. These results suggest that this nanoparticle-based plasmid delivery method can be effective for future in vivo applications of the CRISPR-Cas9 system.

Loss of NLRX1 Exacerbates Neural Tissue Damage and NF-κB Signaling following Brain Injury.

Traumatic and nontraumatic brain injury results from severe disruptions in the cellular microenvironment leading to massive loss of neuronal populations and increased neuroinflammation. The progressive cascade of secondary events, including ischemia, inflammation, excitotoxicity, and free-radical release, contribute to neural tissue damage. NLRX1 is a member of the NLR family of pattern recognition receptors and is a potent negative regulator of several pathways that significantly modulate many of these events. Thus, we hypothesized that NLRX1 limits immune system signaling in the brain following trauma. To evaluate this hypothesis, we used Nlrx1-/- mice in a controlled cortical impact (CCI) injury murine model of traumatic brain injury (TBI). In this article, we show that Nlrx1-/- mice exhibited significantly larger brain lesions and increased motor deficits following CCI injury. Mechanistically, our data indicate that the NF-κB signaling cascade is significantly upregulated in Nlrx1-/- animals. This upregulation is associated with increased microglia and macrophage populations in the cortical lesion. Using a mouse neuroblastoma cell line (N2A), we also found that NLRX1 significantly reduced apoptosis under hypoxic conditions. In human patients, we identify 15 NLRs that are significantly dysregulated, including significant downregulation of NLRX1 in brain injury following aneurysm. We further demonstrate a concurrent increase in NF-κB signaling that is correlated with aneurysm severity in these human subjects. Together, our data extend the function of NLRX1 beyond its currently characterized role in host-pathogen defense and identify this highly novel NLR as a significant modulator of brain injury progression.

NLRX1 Regulates Effector and Metabolic Functions of CD4+ T Cells.

Nucleotide oligomerization domain-like receptor X1 (NLRX1) has been implicated in viral response, cancer progression, and inflammatory disorders; however, its role as a dual modulator of CD4+ T cell function and metabolism has not been defined. The loss of NLRX1 results in increased disease severity, populations of Th1 and Th17 cells, and inflammatory markers (IFN-γ, TNF-α, and IL-17) in mice with dextran sodium sulfate-induced colitis. To further characterize this phenotype, we used in vitro CD4+ T cell-differentiation assays and show that NLRX1-deficient T cells have a greater ability to differentiate into an inflammatory phenotype and possess greater proliferation rates. Further, NLRX1-/- cells have a decreased responsiveness to immune checkpoint pathways and greater rates of lactate dehydrogenase activity. When metabolic effects of the knockout are impaired, NLRX1-deficient cells do not display significant differences in differentiation or proliferation. To confirm the role of NLRX1 specifically in T cells, we used an adoptive-transfer model of colitis. Rag2-/- mice receiving NLRX1-/- naive or effector T cells experienced increased disease activity and effector T cell populations, whereas no differences were observed between groups receiving wild-type or NLRX1-/- regulatory T cells. Metabolic effects of NLRX1 deficiency are observed in a CD4-specific knockout of NLRX1 within a Citrobacter rodentium model of colitis. The aerobic glycolytic preference in NLRX1-/- effector T cells is combined with a decreased sensitivity to immunosuppressive checkpoint pathways to provide greater proliferative capabilities and an inflammatory phenotype bias leading to increased disease severity.

Integrated STAT3 and Ikaros Zinc Finger Transcription Factor Activities Regulate Bcl-6 Expression in CD4+ Th Cells.

B cell lymphoma-6 (Bcl-6) is a transcriptional repressor that is required for the differentiation of T follicular helper (TFH) cell populations. Currently, the molecular mechanisms underlying the transcriptional regulation of Bcl-6 expression are unclear. In this study, we have identified the Ikaros zinc finger transcription factors Aiolos and Ikaros as novel regulators of Bcl-6. We found that increased expression of Bcl-6 in CD4+ Th cell populations correlated with enhanced enrichment of Aiolos and Ikaros at the Bcl6 promoter. Furthermore, overexpression of Aiolos or Ikaros, but not the related family member Eos, was sufficient to induce Bcl6 promoter activity. Intriguingly, STAT3, a known Bcl-6 transcriptional regulator, physically interacted with Aiolos to form a transcription factor complex capable of inducing the expression of Bcl6 and the TFH-associated cytokine receptor Il6ra Importantly, in vivo studies revealed that the expression of Aiolos was elevated in Ag-specific TFH cells compared with that observed in non-TFH effector Th cells generated in response to influenza infection. Collectively, these data describe a novel regulatory mechanism through which STAT3 and the Ikaros zinc finger transcription factors Aiolos and Ikaros cooperate to regulate Bcl-6 expression.

The NLRP3 inflammasome mediates in vivo innate immunity to influenza A virus through recognition of viral RNA.

The nucleotide-binding domain and leucine-rich-repeat-containing (NLR) family of pattern-recognition molecules mediate host immunity to various pathogenic stimuli. However, in vivo evidence for the involvement of NLR proteins in viral sensing has not been widely investigated and remains controversial. As a test of the physiologic role of the NLR molecule NLRP3 during RNA viral infection, we explored the in vivo role of NLRP3 inflammasome components during influenza virus infection. Mice lacking Nlrp3, Pycard, or caspase-1, but not Nlrc4, exhibited dramatically increased mortality and a reduced immune response after exposure to the influenza virus. Utilizing analogs of dsRNA (poly(I:C)) and ssRNA (ssRNA40), we demonstrated that an NLRP3-mediated response could be activated by RNA species. Mechanistically, NLRP3 inflammasome activation by the influenza virus was dependent on lysosomal maturation and reactive oxygen species (ROS). Inhibition of ROS induction eliminated IL-1beta production in animals during influenza infection. Together, these data place the NLRP3 inflammasome as an essential component in host defense against influenza infection through the sensing of viral RNA.

Design and Fabrication of Streptavidin-Functionalized, Fluorescently Labeled Polymeric Nanocarriers.

Targeted drug delivery has great potential for improving therapeutic outcomes for many diseases. Polymeric nanocarriers can improve the targeted delivery of insoluble and toxic drugs but, to achieve this, it is important to tailor the particle properties. In this study, nanoparticles comprised of poly(ethylene oxide)- b-poly(d,l-lactic acid) (PEO- b-PDLLA) were made by flash nanoprecipitation while varying the compositions of the additives poly(l-lactic acid) (PLLA), a fluorophore 6,13-bis(triisopropylsylylethynyl) (TIPS) pentacene, and poly(acrylic acid)- b-poly(d,l-lactic acid) (PAA- b-PDLLA) to characterize their effects on size, ζ potential, fluorescence, and surface functionalization. The particle size was readily increased by addition of PLLA homopolymer up to ∼40 wt % without significant change to the ζ potential. The maximum nanoparticle fluorescence was at 0.5 wt % TIPS based on the PDLLA core and exhibited quenching that could be described by Förster resonant energy transfer. The cores of the particles were coupled with streptavidin through 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide coupling chemistry. Even without the added carboxylate groups from the PAA, the base PEO- b-PDLLA nanoparticles were conjugated with streptavidin at comparable levels while retaining the functionality of streptavidin for further biotinylated ligand binding.