A complement-microglial axis drives synapse loss during virus-induced memory impairment.

Over 50% of patients who survive neuroinvasive infection with West Nile virus (WNV) exhibit chronic cognitive sequelae. Although thousands of cases of WNV-mediated memory dysfunction accrue annually, the mechanisms responsible for these impairments are unknown. The classical complement cascade, a key component of innate immune pathogen defence, mediates synaptic pruning by microglia during early postnatal development. Here we show that viral infection of adult hippocampal neurons induces complement-mediated elimination of presynaptic terminals in a murine WNV neuroinvasive disease model. Inoculation of WNV-NS5-E218A, a WNV with a mutant NS5(E218A) protein leads to survival rates and cognitive dysfunction that mirror human WNV neuroinvasive disease. WNV-NS5-E218A-recovered mice (recovery defined as survival after acute infection) display impaired spatial learning and persistence of phagocytic microglia without loss of hippocampal neurons or volume. Hippocampi from WNV-NS5-E218A-recovered mice with poor spatial learning show increased expression of genes that drive synaptic remodelling by microglia via complement. C1QA was upregulated and localized to microglia, infected neurons and presynaptic terminals during WNV neuroinvasive disease. Murine and human WNV neuroinvasive disease post-mortem samples exhibit loss of hippocampal CA3 presynaptic terminals, and murine studies revealed microglial engulfment of presynaptic terminals during acute infection and after recovery. Mice with fewer microglia (Il34(-/-) mice with a deficiency in IL-34 production) or deficiency in complement C3 or C3a receptor were protected from WNV-induced synaptic terminal loss. Our study provides a new murine model of WNV-induced spatial memory impairment, and identifies a potential mechanism underlying neurocognitive impairment in patients recovering from WNV neuroinvasive disease.

West Nile Virus Infection Blocks Inflammatory Response and T Cell Costimulatory Capacity of Human Monocyte-Derived Dendritic Cells.

West Nile virus (WNV) is a neurotropic flavivirus and the leading cause of mosquito-borne encephalitis in the United States. Recent studies in humans have found that dysfunctional T cell responses strongly correlate with development of severe WNV neuroinvasive disease. However, the contributions of human dendritic cells (DCs) in priming WNV-specific T cell immunity remains poorly understood. Here, we demonstrate that human monocyte derived DCs (moDCs) support productive viral replication following infection with a pathogenic strain of WNV. Antiviral effector gene transcription was strongly induced during the log phase of viral growth, while secretion of type I interferons (IFN) occurred with delayed kinetics. Activation of RIG-I like receptor (RLR) or type I IFN signaling prior to log phase viral growth significantly diminished viral replication, suggesting that early activation of antiviral programs can block WNV infection. In contrast to the induction of antiviral responses, WNV infection did not promote transcription or secretion of proinflammatory (interleukin-6 [IL-6], granulocyte-macrophage colony-stimulating factor [GM-CSF], CCL3, CCL5, and CXCL9) or T cell modulatory (IL-4, IL-12, and IL-15) cytokines. There was also minimal induction of molecules associated with antigen presentation and T cell priming, including the costimulatory molecules CD80, CD86, and CD40. Functionally, WNV-infected moDCs dampened allogenic CD4 and CD8 T cell activation and proliferation. Combining these observations, we propose a model whereby WNV subverts human DC activation to compromise priming of WNV-specific T cell immunity.IMPORTANCE West Nile virus (WNV) is an encephalitic flavivirus that remains endemic in the United States. Previous studies have found dysfunctional T cell responses correlate to severe disease outcomes during human WNV infection. Here, we sought to better understand the ability of WNV to program human dendritic cells (DCs) to prime WNV-specific T cell responses. While productive infection of monocyte-derived DCs activated antiviral and type I interferon responses, molecules associated with inflammation and programming of T cells were minimally induced. Functionally, WNV-infected DCs dampened T cell activation and proliferation during an allogeneic response. Combined, our data support a model whereby WNV infection of human DCs compromises WNV-specific T cell immunity.

STAT5: a Target of Antagonism by Neurotropic Flaviviruses.

Flaviviruses are a diverse group of arthropod-borne viruses responsible for numerous significant public health threats; therefore, understanding the interactions between these viruses and the human immune response remains vital. West Nile virus (WNV) and Zika virus (ZIKV) infect human dendritic cells (DCs) and can block antiviral immune responses in DCs. Previously, we used mRNA sequencing and weighted gene coexpression network analysis (WGCNA) to define molecular signatures of antiviral DC responses following activation of innate immune signaling (RIG-I, MDA5, or type I interferon [IFN] signaling) or infection with WNV. Using this approach, we found that several genes involved in T cell cosignaling and antigen processing were not enriched in DCs during WNV infection. Using cis-regulatory sequence analysis, STAT5 was identified as a regulator of DC activation and immune responses downstream of innate immune signaling that was not activated during either WNV or ZIKV infection. Mechanistically, WNV and ZIKV actively blocked STAT5 phosphorylation downstream of RIG-I, IFN-ß, and interleukin-4 (IL-4), but not granulocyte-macrophage colony-stimulating factor (GM-CSF), signaling. Unexpectedly, dengue virus serotypes 1 to 4 (DENV1 to DENV4) and the yellow fever 17D vaccine strain (YFV-17D) did not antagonize STAT5 phosphorylation. In contrast to WNV, ZIKV inhibited JAK1 and TYK2 phosphorylation following type I IFN treatment, suggesting divergent mechanisms used by these viruses to inhibit STAT5 activation. Combined, these findings identify STAT5 as a target of antagonism by specific pathogenic flaviviruses to subvert the immune response in infected DCs.IMPORTANCE Flaviviruses are a diverse group of insect-borne viruses responsible for numerous significant public health threats. Previously, we used a computational biology approach to define molecular signatures of antiviral DC responses following activation of innate immune signaling or infection with West Nile virus (WNV). In this work, we identify STAT5 as a regulator of DC activation and antiviral immune responses downstream of innate immune signaling that was not activated during either WNV or Zika virus (ZIKV) infection. WNV and ZIKV actively blocked STAT5 phosphorylation downstream of RIG-I, IFN-ß, and IL-4, but not GM-CSF, signaling. However, other related flaviviruses, dengue virus serotypes 1 to 4 and the yellow fever 17D vaccine strain, did not antagonize STAT5 phosphorylation. Mechanistically, WNV and ZIKV showed differential inhibition of Jak kinases upstream of STAT5, suggesting divergent countermeasures to inhibit STAT5 activation. Combined, these findings identify STAT5 as a target of antagonism by specific pathogenic flaviviruses to subvert antiviral immune responses in human DCs.

Zika Virus Antagonizes Type I Interferon Responses during Infection of Human Dendritic Cells.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that is causally linked to severe neonatal birth defects, including microcephaly, and is associated with Guillain-Barre syndrome in adults. Dendritic cells (DCs) are an important cell type during infection by multiple mosquito-borne flaviviruses, including dengue virus, West Nile virus, Japanese encephalitis virus, and yellow fever virus. Despite this, the interplay between ZIKV and DCs remains poorly defined. Here, we found human DCs supported productive infection by a contemporary Puerto Rican isolate with considerable variability in viral replication, but not viral binding, between DCs from different donors. Historic isolates from Africa and Asia also infected DCs with distinct viral replication kinetics between strains. African lineage viruses displayed more rapid replication kinetics and infection magnitude as compared to Asian lineage viruses, and uniquely induced cell death. Infection of DCs with both contemporary and historic ZIKV isolates led to minimal up-regulation of T cell co-stimulatory and MHC molecules, along with limited secretion of inflammatory cytokines. Inhibition of type I interferon (IFN) protein translation was observed during ZIKV infection, despite strong induction at the RNA transcript level and up-regulation of other host antiviral proteins. Treatment of human DCs with RIG-I agonist potently restricted ZIKV replication, while type I IFN had only modest effects. Mechanistically, we found all strains of ZIKV antagonized type I IFN-mediated phosphorylation of STAT1 and STAT2. Combined, our findings show that ZIKV subverts DC immunogenicity during infection, in part through evasion of type I IFN responses, but that the RLR signaling pathway is still capable of inducing an antiviral state, and therefore may serve as an antiviral therapeutic target.

Sonographic Assessment of Hip Swaddling Techniques in Infants With and Without DDH.

BACKGROUND: The purpose of this single-examination pilot study was to confirm the ability to perform hip sonography while swaddled and to ascertain whether the various swaddling techniques influenced hip position and dynamics. METHODS: Dynamic sonography was used to evaluate 30 infants in both swaddled and unswaddled positions who were being seen in clinic for suspected or documented developmental dysplasia of the hip. A "treatment group" of 16 infants (32 hips) treated in a Pavlik harness and a "nontreatment group" of 14 untreated infants (28 hips) were studied.Criteria for comparing sonographic results between swaddled and unswaddled hip positions included femoral head position, instability, and range-of-motion restriction. RESULTS: Tight swaddling with a blanket was applied in 11 "nontreatment group" cases (20 hips; in 2 cases, only 1 hip studied) and produced limited flexion and abduction. One unstable left hip dislocated when tightly swaddled. Safe swaddling technique in 12 cases (24 hips) showed no limitation of flexion and abduction of the legs and no change in stability by sonography. Commercial swaddling products appeared to mildly restrict leg motion in 14 hips, but there was no change in hip position in the "nontreatment group." However, the commercial swaddling products changed the hip position in 3 Pavlik harness cases. CONCLUSIONS: Swaddling techniques that allow a free range of leg motion may not affect hip stability in normal infants or those being treated with Pavlik harness. Swaddling with restricted leg motion increases potential for hip instability. Tight swaddling dislocated 1 unstable hip, and commercial swaddling products judged to apply only mild restriction of leg motion negatively impacted 3 cases being treated for developmental dysplasia of the hip with Pavlik harness. On the basis of this pilot study, we advise caution when swaddling infants, especially with techniques that restrict leg motion. Further study of the long-term effects of swaddling is warranted. LEVEL OF EVIDENCE: Level II.

Comparative effectiveness research using electronic health records: impacts of oral antidiabetic drugs on the development of chronic kidney disease.

PURPOSE: Little is known about the comparative effects of common oral antidiabetic drugs ([OADs] metformin, sulfonylureas, or thiazolidinediones [THZs]) on chronic kidney disease (CKD) outcomes in patients newly diagnosed with type 2 diabetes (T2DM) and followed in community primary care practices. Electronic health records (EHRs) were used to evaluate the relationships between OAD class use and incident proteinuria and prevention of glomerular filtration rate decline. METHODS: A retrospective cohort study on newly diagnosed T2D cases requiring OADs documented in the EHRs of two primary care networks between 1998 and 2009 was conducted. CKD outcomes were new-onset proteinuria and estimated GFR (eGFR) falling below 60 ml/min/1.73 m(2). OAD exposures defined cohorts. Hazard ratios represent differential CKD outcome risk per year of OAD class use. RESULTS: A total of 798 and 977 patients qualified for proteinuria and eGFR outcome analyses, respectively. With metformin as the reference group, sulfonylurea exposure trended toward association with an increased risk of developing proteinuria ([adjusted hazard ratio; 95% CI] 1.27; 0.93, 1.74); proteinuria risk associated with THZ exposure (1.00; 0.70, 1.42) was similar to metformin. Compared with metformin, sulfonylurea exposure was associated with an increased risk of eGFR reduction to <60 ml/min/1.73 m(2) (1.41; 1.05, 1.91). THZ exposure (1.04; 0.71, 1.50) was not associated with change in the risk of eGFR decline. CONCLUSIONS: In a primary care population, metformin appeared to decrease the risk of CKD development compared with sulfonlyureas; risks of CKD development between metformin and THZs were similar. EHR use in pharmacotherapy comparative effectiveness research creates specific challenges and study limitations.

Urinary detection of early responses to checkpoint blockade and of resistance to it via protease-cleaved antibody-conjugated sensors.

Immune checkpoint blockade (ICB) therapy does not benefit the majority of treated patients, and those who respond to the therapy can become resistant to it. Here we report the design and performance of systemically administered protease activity sensors conjugated to anti-programmed cell death protein 1 (αPD1) antibodies for the monitoring of antitumour responses to ICB therapy. The sensors consist of a library of mass-barcoded protease substrates that, when cleaved by tumour proteases and immune proteases, are released into urine, where they can be detected by mass spectrometry. By using syngeneic mouse models of colorectal cancer, we show that random forest classifiers trained on mass spectrometry signatures from a library of αPD1-conjugated mass-barcoded activity sensors for differentially expressed tumour proteases and immune proteases can be used to detect early antitumour responses and discriminate resistance to ICB therapy driven by loss-of-function mutations in either the B2m or Jak1 genes. Biomarkers of protease activity may facilitate the assessment of early responses to ICB therapy and the classification of refractory tumours based on resistance mechanisms.

The amphibian peptide Yodha is virucidal for Zika and dengue viruses.

Zika virus (ZIKV) has emerged as a serious health threat in the Americas and the Caribbean. ZIKV is transmitted by the bite of an infected mosquito, sexual contact, and blood transfusion. ZIKV can also be transmitted to the developing fetus in utero, in some cases resulting in spontaneous abortion, fetal brain abnormalities, and microcephaly. In adults, ZIKV infection has been correlated with Guillain-Barre syndrome. Despite the public health threat posed by ZIKV, neither a vaccine nor antiviral drugs for use in humans are currently available. We have identified an amphibian host defense peptide, Yodha, which has potent virucidal activity against ZIKV. It acts directly on the virus and destroys Zika virus particles within 5 min of exposure. The Yodha peptide was effective against the Asian, African, and South American Zika virus strains and has the potential to be developed as an antiviral therapeutic in the fight against Zika virus. The peptide was also effective against all four dengue virus serotypes. Thus, Yodha peptide could potentially be developed as a pan-therapeutic for Zika and dengue viruses.

Strong TH1-biased CD4 T cell responses are associated with diminished SIV vaccine efficacy.

Activated CD4 T cells are a major target of HIV infection. Results from the STEP HIV vaccine trial highlighted a potential role for total activated CD4 T cells in promoting HIV acquisition. However, the influence of vaccine insert-specific CD4 T cell responses on HIV acquisition is not known. Here, using the data obtained from four macaque studies, we show that the DNA prime/modified vaccinia Ankara boost vaccine induced interferon γ (IFNγ+) CD4 T cells [T helper 1 (TH1) cells] rapidly migrate to multiple tissues including colon, cervix, and vaginal mucosa. These mucosal TH1 cells persisted at higher frequencies and expressed higher density of CCR5, a viral coreceptor, compared to cells in blood. After intravaginal or intrarectal simian immunodeficiency virus (SIV)/simian-human immunodeficiency virus (SHIV) challenges, strong vaccine protection was evident only in animals that had lower frequencies of vaccine-specific TH1 cells but not in animals that had higher frequencies of TH1 cells, despite comparable vaccine-induced humoral and CD8 T cell immunity in both groups. An RNA transcriptome signature in blood at 7 days after priming immunization from one study was associated with induction of fewer TH1-type CD4 cells and enhanced protection. These results demonstrate that high and persisting frequencies of HIV vaccine-induced TH1-biased CD4 T cells in the intestinal and genital mucosa can mitigate beneficial effects of protective antibodies and CD8 T cells, highlighting a critical role of priming immunization and vaccine adjuvants in modulating HIV vaccine efficacy.

Taking the defensive: Immune control of Zika virus infection.

ZIKV is a neurotropic mosquito-borne flavivirus that has recently emerged in the Americas and is a pathogen of significant public health concern across the world. ZIKV was first isolated in Uganda in 1947 and remained dormant in Africa and Asia for decades, with sporadic outbreaks characterized by a mild self-limiting disease in humans. The emergence of ZIKV in the Americas corresponded with enhanced disease severity and congenital Zika syndrome, a phenotype characterized by severe microcephaly, brain anomalies, ocular anomalies, congenital contractures and neurological impairments. In less than two years, a collective effort led by the scientific research community has uncovered many new facets to the once rarely discussed ZIKV. In this review, we highlight the known immune parameters that correlate with protective immunity to ZIKV infection, including pattern recognition receptors, interferons, humoral and cell-mediated responses, as well as countermeasures utilized by ZIKV to inhibit host antiviral immune responses.

Systems biology: A tool for charting the antiviral landscape.

The host antiviral programs that are initiated following viral infection form a dynamic and complex web of responses that we have collectively termed as "the antiviral landscape". Conventional approaches to studying antiviral responses have primarily used reductionist systems to assess the function of a single or a limited subset of molecules. Systems biology is a holistic approach that considers the entire system as a whole, rather than individual components or molecules. Systems biology based approaches facilitate an unbiased and comprehensive analysis of the antiviral landscape, while allowing for the discovery of emergent properties that are missed by conventional approaches. The antiviral landscape can be viewed as a hierarchy of complexity, beginning at the whole organism level and progressing downward to isolated tissues, populations of cells, and single cells. In this review, we will discuss how systems biology has been applied to better understand the antiviral landscape at each of these layers. At the organismal level, the Collaborative Cross is an invaluable genetic resource for assessing how genetic diversity influences the antiviral response. Whole tissue and isolated bulk cell transcriptomics serves as a critical tool for the comprehensive analysis of antiviral responses at both the tissue and cellular levels of complexity. Finally, new techniques in single cell analysis are emerging tools that will revolutionize our understanding of how individual cells within a bulk infected cell population contribute to the overall antiviral landscape.

Zika Virus Infects Human Placental Macrophages.

The recent Zika virus (ZIKV) outbreak in Brazil has been directly linked to increased cases of microcephaly in newborns. Current evidence indicates that ZIKV is transmitted vertically from mother to fetus. However, the mechanism of intrauterine transmission and the cell types involved remain unknown. We demonstrate that the contemporary ZIKV strain PRVABC59 (PR 2015) infects and replicates in primary human placental macrophages, called Hofbauer cells, and to a lesser extent in cytotrophoblasts, isolated from villous tissue of full-term placentae. Viral replication coincides with induction of type I interferon (IFN), pro-inflammatory cytokines, and antiviral gene expression, but with minimal cell death. Our results suggest a mechanism for intrauterine transmission in which ZIKV gains access to the fetal compartment by directly infecting placental cells and disrupting the placental barrier.

Distinct Sources of Hematopoietic Progenitors Emerge before HSCs and Provide Functional Blood Cells in the Mammalian Embryo.

Hematopoietic potential arises in mammalian embryos before adult-repopulating hematopoietic stem cells (HSCs). At embryonic day 9.5 (E9.5), we show the first murine definitive erythro-myeloid progenitors (EMPs) have an immunophenotype distinct from primitive hematopoietic progenitors, maturing megakaryocytes and macrophages, and rare B cell potential. EMPs emerge in the yolk sac with erythroid and broad myeloid, but not lymphoid, potential. EMPs migrate to the fetal liver and rapidly differentiate, including production of circulating neutrophils by E11.5. Although the surface markers, transcription factors, and lineage potential associated with EMPs overlap with those found in adult definitive hematopoiesis, they are present in unique combinations or proportions that result in a specialized definitive embryonic progenitor. Furthermore, we find that embryonic stem cell (ESC)-derived hematopoiesis recapitulates early yolk sac hematopoiesis, including primitive, EMP, and rare B cell potential. EMPs do not have long-term potential when transplanted in immunocompromised adults, but they can provide transient adult-like RBC reconstitution.

Asymptomatic deep vein thrombosis in patients undergoing screening duplex ultrasonography.

BACKGROUND: Because of concerns for propagating clots into pulmonary emboli by the placement of pneumatic compression boots (PCBs), the standard of care at our institution was to perform a duplex Doppler ultrasound with compression (DUSC) before applying PCBs. We sought to determine the rate of asymptomatic preexisting deep vein thrombosis (DVT) in hospitalized patients who underwent DUSC before PCB. METHODS: We evaluated consecutive patients who underwent lower extremity DUSC within 48 hours of admission. All patients were assessed for DVT risk factors using the American College of Chest Physicians' criteria (American College of Chest Physicians Conference on Antithrombotic/Thrombolytic Therapy: Evidence-Based Guidelines, 9th Edition). A t test, Wilcoxon rank sum test, and χ(2) or Fisher exact test were used to compare patients characteristics according to DVT status. Logistic regression was used to determine the importance of each risk factor on the risk of DVT. RESULTS: DUSC was performed during 1136 hospitalizations; 1071 patients were included in the dataset. Of those, 19 patients (1.8%) had asymptomatic DVT and had at least 1 risk factor; 16 (84.2%) had more than 1 risk factor. The only risk factors that were statistically significant were ambulatory dysfunction and thromboembolic disease history. CONCLUSION: Few patients have asymptomatic DVT upon admission; all of these patients have at least 1 predisposing risk factor. There appears to be no need for DUSC prior to initiation of PCBs. DUSC evaluation for DVT may be of value if there is a history of previous DVT, ambulatory dysfunction, or more than 3 risk factors, as the information may change therapeutic approaches.