BCG vaccination alters the epigenetic landscape of progenitor cells in human bone marrow to influence innate immune responses.

Although the Bacille-Calmette-Guérin (BCG) vaccine is used to prevent tuberculosis, it also offers protection against a diverse range of non-mycobacterial infections. However, the underlying protective mechanisms in humans are not yet fully understood. Here, we surveyed at single-cell resolution the gene expression and chromatin landscape of human bone marrow, aspirated before and 90 days after BCG vaccination or placebo. We showed that BCG alters both the gene expression and epigenetic profiles of human hematopoietic stem and progenitor cells (HSPCs). Changes in gene expression occurred primarily within uncommitted stem cells. By contrast, changes in chromatin accessibility were most prevalent within differentiated progenitor cells at sites influenced by Kruppel-like factor (KLF) and early growth response (EGR) transcription factors and were highly correlated (r > 0.8) with the interleukin (IL)-1ß secretion capacity of paired peripheral blood mononuclear cells (PBMCs). Our findings shed light on BCG vaccination's profound and lasting effects on HSPCs and its influence on innate immune responses and trained immunity.

BCG Educates Hematopoietic Stem Cells to Generate Protective Innate Immunity against Tuberculosis.

The dogma that adaptive immunity is the only arm of the immune response with memory capacity has been recently challenged by several studies demonstrating evidence for memory-like innate immune training. However, the underlying mechanisms and location for generating such innate memory responses in vivo remain unknown. Here, we show that access of Bacillus Calmette-Guérin (BCG) to the bone marrow (BM) changes the transcriptional landscape of hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs), leading to local cell expansion and enhanced myelopoiesis at the expense of lymphopoiesis. Importantly, BCG-educated HSCs generate epigenetically modified macrophages that provide significantly better protection against virulent M. tuberculosis infection than naïve macrophages. By using parabiotic and chimeric mice, as well as adoptive transfer approaches, we demonstrate that training of the monocyte/macrophage lineage via BCG-induced HSC reprogramming is sustainable in vivo. Our results indicate that targeting the HSC compartment provides a novel approach for vaccine development.

Genetic Ancestry and Natural Selection Drive Population Differences in Immune Responses to Pathogens.

Individuals from different populations vary considerably in their susceptibility to immune-related diseases. To understand how genetic variation and natural selection contribute to these differences, we tested for the effects of African versus European ancestry on the transcriptional response of primary macrophages to live bacterial pathogens. A total of 9.3% of macrophage-expressed genes show ancestry-associated differences in the gene regulatory response to infection, and African ancestry specifically predicts a stronger inflammatory response and reduced intracellular bacterial growth. A large proportion of these differences are under genetic control: for 804 genes, more than 75% of ancestry effects on the immune response can be explained by a single cis- or trans-acting expression quantitative trait locus (eQTL). Finally, we show that genetic effects on the immune response are strongly enriched for recent, population-specific signatures of adaptation. Together, our results demonstrate how historical selective events continue to shape human phenotypic diversity today, including for traits that are key to controlling infection.

Evolution of immune genes is associated with the Black Death.

Infectious diseases are among the strongest selective pressures driving human evolution1,2. This includes the single greatest mortality event in recorded history, the first outbreak of the second pandemic of plague, commonly called the Black Death, which was caused by the bacterium Yersinia pestis3. This pandemic devastated Afro-Eurasia, killing up to 30-50% of the population4. To identify loci that may have been under selection during the Black Death, we characterized genetic variation around immune-related genes from 206 ancient DNA extracts, stemming from two different European populations before, during and after the Black Death. Immune loci are strongly enriched for highly differentiated sites relative to a set of non-immune loci, suggesting positive selection. We identify 245 variants that are highly differentiated within the London dataset, four of which were replicated in an independent cohort from Denmark, and represent the strongest candidates for positive selection. The selected allele for one of these variants, rs2549794, is associated with the production of a full-length (versus truncated) ERAP2 transcript, variation in cytokine response to Y. pestis and increased ability to control intracellular Y. pestis in macrophages. Finally, we show that protective variants overlap with alleles that are today associated with increased susceptibility to autoimmune diseases, providing empirical evidence for the role played by past pandemics in shaping present-day susceptibility to disease.

IL-15, gluten and HLA-DQ8 drive tissue destruction in coeliac disease.

Coeliac disease is a complex, polygenic inflammatory enteropathy caused by exposure to dietary gluten that occurs in a subset of genetically susceptible individuals who express either the HLA-DQ8 or HLA-DQ2 haplotypes1,2. The need to develop non-dietary treatments is now widely recognized3, but no pathophysiologically relevant gluten- and HLA-dependent preclinical model exists. Furthermore, although studies in humans have led to major advances in our understanding of the pathogenesis of coeliac disease4, the respective roles of disease-predisposing HLA molecules, and of adaptive and innate immunity in the development of tissue damage, have not been directly demonstrated. Here we describe a mouse model that reproduces the overexpression of interleukin-15 (IL-15) in the gut epithelium and lamina propria that is characteristic of active coeliac disease, expresses the predisposing HLA-DQ8 molecule, and develops villous atrophy after ingestion of gluten. Overexpression of IL-15 in both the epithelium and the lamina propria is required for the development of villous atrophy, which demonstrates the location-dependent central role of IL-15 in the pathogenesis of coeliac disease. In addition, CD4+ T cells and HLA-DQ8 have a crucial role in the licensing of cytotoxic T cells to mediate intestinal epithelial cell lysis. We also demonstrate a role for the cytokine interferon-γ (IFNγ) and the enzyme transglutaminase 2 (TG2) in tissue destruction. By reflecting the complex interaction between gluten, genetics and IL-15-driven tissue inflammation, this mouse model provides the opportunity to both increase our understanding of coeliac disease, and develop new therapeutic strategies.

Findings Associated With Nonaccidental Trauma in Children With Isolated Femoral Diaphyseal Fractures.

BACKGROUND: Pediatric patients with isolated femoral diaphyseal fractures are difficult to assess for nonaccidental trauma (NAT). The purpose of this study was to determine (1) if there are any demographic features of isolated femoral diaphyseal fractures associated with suspected NAT and (2) if there are clinical signs associated with isolated femoral diaphyseal fractures associated with suspected NAT. METHODS: All patients with femoral diaphyseal fractures from January 2010 to June 2018 were reviewed. We included patients younger than 4 years old with isolated femoral diaphyseal fractures. We excluded patients 4 years old and older, polytraumas, motor vehicle collisions, and patients with altered bone biology. Diagnosis of suspected NAT was determined by review of a documented social work assessment. We recorded fracture characteristics including location along femur as well as fracture pattern and presence of associated findings on NAT workup including the presence of retinal hemorrhage, subdural hematoma, evidence of prior fracture, or cutaneous lesions. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of these associated findings were calculated. RESULTS: Totally, 144 patients met the inclusion criteria. Social work was consulted on 50 patients (35%). Suspected NAT was diagnosed in 27 patients (19%). The average age of patients with suspected NAT was 0.82 and 2.25 years in patients without NAT ( P <0.01). The rate and type of skin lesions present on exam were not different between the 2 groups. Patients with suspected NAT had no findings of retinal hemorrhage or subdural hematoma, but 5 of 27 patients (19%) had evidence of prior fracture on skeletal survey. The sensitivities of retinal hemorrhage, subdural, and skeletal survey were 0%, 0%, and 19% and the specificities of all were 100%. The NPVs were 39%, 27%, and 63%, respectively. The PPV of skeletal survey was 100%. Since there were no patients in this study with positive findings of retinal hemorrhage or subdural hematoma, the PPV for these could not be assessed. CONCLUSIONS: In the current study, signs of NAT such as skin lesions, retinal hemorrhage, subdural hematoma, and evidence of prior fracture on skeletal survey may not be helpful to diagnosis suspected NAT in patients with an isolated femoral diaphyseal fracture. LEVEL OF EVIDENCE: Level III-diagnostic study.

Assessment and Validation of Anterior Humeral Line for Diagnosing Supracondylar Humerus Fractures in the Pediatric Population.

INTRODUCTION: Pediatric supracondylar humerus fractures are commonly evaluated using the anterior humeral line (AHL) on a lateral radiograph. Rotational variations in radiographic projection are common due to child discomfort and could lead to changes in management based on where the AHL intersects the capitellum. The purpose of this study was to establish whether rotational variations in elbow rotation leads to significant changes in AHL position and whether drawing the AHL based on the distal humerus versus shaft is more tolerant to rotation. METHODS: Fifty children with nonoperative supracondylar humerus fractures were identified with sub optimally positioned injury and well positioned follow-up lateral radiographs. The proportion of the bone anterior to the intersection of the AHL and the capitellum was measured using the humeral shaft versus distal humerus to guide position of the AHL. This process was repeated on ten pediatric humerus dry cadaveric specimens which were imaged in 5-degree rotational increments along the axis of the humeral shaft from -20 to +20 degrees. RESULTS: AHL position correlated poorly when measured on rotated lateral radiographs of clinical patients versus non-rotated lateral radiographs when using the distal humerus as a guide (intraclass correlation coefficient 0.14), compared with when using the humeral shaft as a guide (intraclass correlation coefficient 0.81). When assessing the pediatric humerus dry cadavers between the 2 techniques, there was greater statistically significant variation in rotated positions compared with the neutral position in the distal humerus AHL measurement approach compared with the humeral shaft AHL measurement approach, with the mean AHL within the central third of the capitellum for more rotational positions when using the shaft compared with the distal humerus. CONCLUSIONS: With rotated lateral elbow radiographs in supracondylar humerus fractures, utilizing the humeral shaft provides more consistent AHL measurements than utilizing the distal humerus, and thus drawing the line starting at the shaft of the humerus is recommended for surgical decision making.

Gene activation precedes DNA demethylation in response to infection in human dendritic cells.

DNA methylation is considered to be a relatively stable epigenetic mark. However, a growing body of evidence indicates that DNA methylation levels can change rapidly; for example, in innate immune cells facing an infectious agent. Nevertheless, the causal relationship between changes in DNA methylation and gene expression during infection remains to be elucidated. Here, we generated time-course data on DNA methylation, gene expression, and chromatin accessibility patterns during infection of human dendritic cells with Mycobacterium tuberculosis We found that the immune response to infection is accompanied by active demethylation of thousands of CpG sites overlapping distal enhancer elements. However, virtually all changes in gene expression in response to infection occur before detectable changes in DNA methylation, indicating that the observed losses in methylation are a downstream consequence of transcriptional activation. Footprinting analysis revealed that immune-related transcription factors (TFs), such as NF-κB/Rel, are recruited to enhancer elements before the observed losses in methylation, suggesting that DNA demethylation is mediated by TF binding to cis-acting elements. Collectively, our results show that DNA demethylation plays a limited role to the establishment of the core regulatory program engaged upon infection.

Use of Vancomycin Powder in Spinal Deformity Surgery in Cerebral Palsy Patients is Associated With Proteus Surgical Site Infections.

PURPOSE: Surgical site infection (SSI) rates in pediatric spinal deformity surgery for cerebral palsy (CP) patients are higher than that in idiopathic scoliosis. The use of vancomycin powder is associated with decreased risk of SSI in neuromuscular patients. Prior studies in adult and pediatric early-onset scoliosis patients have shown that vancomycin powder alters microbacterial profile in patients that develop SSI. However, the effects of topical vancomycin powder on microbiology in spinal deformity surgery for CP patients has not been studied. METHODS: An international multicenter database of CP neuromuscular scoliosis patients was used in this retrospective cohort study. All patients that underwent posterior spinal instrumented fusion for CP neuromuscular scoliosis from 2008 to 2019 were queried, and 50 cases complicated by postoperative SSI were identified. Intraoperative antibiotic details were documented in 49 cases (98.0%). Microbiology details were documented in 45 cases (91.8%). Microbiology for patients that received topical vancomycin powder were compared with patients that did not. A multivariate regression model was used to control for potential confounders. RESULTS: There were 45 patients included in this study. There were 27 males (60.0%) and 18 females (40.0%). Mean age at surgery was 14.8±2.4 years. There were 24 patients that received topical vancomycin powder (53.3%). The mean time from index surgery to SSI was 4.3±11.3 months.On univariate analysis of microbiology cultures by vancomycin powder cohort, there were no significant differences in culture types. Proteus spp. trended on significance with association with vancomycin powder use (P=0.078). When controlling for potential confounders on multivariate analysis, intraoperative topical vancomycin powder was associated with increased risk for proteus infection (adjusted odds ratio: 262.900, 95% confidence interval: 1.806-38,267.121, P=0.028). DISCUSSION: In CP patients undergoing pediatric spinal deformity surgery, the use of vancomycin powder was independently associated with increased risk for proteus infections. Further study into antibiotic regimens for spinal deformity surgery in the CP population should be performed. LEVEL OF EVIDENCE: Level III-retrospective cohort study.

When Will You Succeed Casting Patients With Early-onset Scoliosis? Prospective Evaluation of Predictive Radiographic Parameters.

BACKGROUND: In a recent retrospective study, in cast correction of the major curve correlated with final curve size in patients with early-onset scoliosis treated with casting. We therefore sought to perform a prospective study with controlled methodology to determine if there are parameters associated with reduction of coronal deformity. METHODS: A prospective, observational study was conducted between 2014 and 2019 at selected sites willing to comply with a standard radiographic and follow-up protocol. Radiographic data was collected at time points of precast, in traction, initial in-cast, and at minimum 1 year follow-up. Multivariate linear regression models were utilized to control for potential confounders using a stepwise procedure. Twenty-nine patients met inclusion criteria. RESULTS: On multivariate analysis, traction major curve (P=0.043) and initial in-cast (P=0.011) major curve Cobb angles were independently associated with final out of cast major curve Cobb angle. The only factor that was independently associated with failure to cure (<15-degree major curve) was traction major curve Cobb angle (P=0.046). A threshold traction major curve Cobb angle of 20 degrees was found to have good accuracy with 81% sensitivity and 73% specificity (receiver operator curve area: 0.869, P<0.001). A traction major curve Cobb angle over 20 degrees would accurately predict failure of casting treatment to cure scoliosis in 79% of cases. A threshold in-cast major curve Cobb angle of 21 degrees was found to have slightly less accuracy than traction with 69% sensitivity, 82% specificity, and 74% accuracy (receiver operator curve area: 0.830, P=0.004). CONCLUSIONS: Radiographic measurements in traction and initially in the cast are predictive of curve size at follow-up for children with early-onset scoliosis treated with casting. The standardization and utility of traction films should be further explored. LEVEL OF EVIDENCE: Level II.

Rel-Dependent Immune and Central Nervous System Mechanisms Control Viral Replication and Inflammation during Mouse Herpes Simplex Encephalitis.

Herpes simplex encephalitis (HSE), caused by HSV type 1 (HSV-1) infection, is an acute neuroinflammatory condition of the CNS and remains the most common type of sporadic viral encephalitis worldwide. Studies in humans have shown that susceptibility to HSE depends in part on the genetic make-up of the host, with deleterious mutations in the TLR3/type I IFN axis underlying some cases of childhood HSE. Using an in vivo chemical mutagenesis screen for HSV-1 susceptibility in mice, we identified a susceptible pedigree carrying a causal truncating mutation in the Rel gene (RelC307X ), encoding for the NF-κB transcription factor subunit c-Rel. Like Myd88-/- and Irf3-/- mice, RelC307X mice were susceptible to intranasal HSV-1 infection. Reciprocal bone marrow transfers into lethally irradiated hosts suggested that defects in both hematopoietic and CNS-resident cellular compartments contributed together to HSE susceptibility in RelC307X mice. Although the RelC307X mutation maintained cell-intrinsic antiviral control, it drove increased apoptotic cell death in infected fibroblasts. Moreover, reduced numbers of CD4+CD25+Foxp3+ T regulatory cells, and dysregulated NK cell and CD4+ effector T cell responses in infected RelC307X animals, indicated that protective immunity was also compromised in these mice. In the CNS, moribund RelC307X mice failed to control HSV-1 viral replication in the brainstem and cerebellum, triggering cell death and elevated expression of Ccl2, Il6, and Mmp8 characteristic of HSE neuroinflammation and pathology. In summary, our work implicates c-Rel in both CNS-resident cell survival and lymphocyte responses to HSV-1 infection and as a novel cause of HSE disease susceptibility in mice.

Preclinical in vitro and in vivo pharmacokinetic properties of danicamtiv, a new targeted myosin activator for the treatment of dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a disease of the myocardium defined by left ventricular enlargement and systolic dysfunction leading to heart failure. Danicamtiv, a new targeted myosin activator designed for the treatment of DCM, was characterised in in vitro and in vivo preclinical studies. Danicamtiv human hepatic clearance was predicted to be 0.5 mL/min/kg from in vitro metabolic stability studies in human hepatocytes. For human, plasma protein binding was moderate with a fraction unbound of 0.16, whole blood-to-plasma partitioning ratio was 0.8, and danicamtiv showed high permeability and no efflux in a Caco-2 cell line. Danicamtiv metabolism pathways in vitro included CYP-mediated amide-cleavage, N-demethylation, as well as isoxazole- and piperidine-ring-opening. Danicamtiv clearance in vivo was low across species with 15.5, 15.3, 1.6, and 5.7 mL/min/kg in mouse, rat, dog, and monkey, respectively. Volume of distribution ranged from 0.24 L/kg in mouse to 1.7 L/kg in rat. Oral bioavailability ranged from 26% in mouse to 108% in dog. Simple allometric scaling prediction of human plasma clearance, volume of distribution, and half-life was 0.64 mL/min/kg, 0.98 L/kg, and 17.7 h, respectively. Danicamtiv preclinical attributes and predicted human pharmacokinetics supported advancement toward clinical development.

Use of Vancomycin Powder in the Surgical Treatment of Early Onset Scoliosis Is Associated With Different Microbiology Cultures After Surgical Site Infection.

BACKGROUND: The use of vancomycin powder has been shown to decrease risk of surgical site infection (SSI) in early onset scoliosis (EOS). While there is potential benefit in SSI reduction, there is also theoretical risk in creating increased bacterial resistance to standard treatment regimens. However, the effects of topical vancomycin powder on microbiology in these patients has not been studied. METHODS: A multicenter database for EOS patients was retrospectively analyzed. All patients that underwent surgical treatment with traditional growing rods, magnetically controlled growing rods, vertical expandable prosthetic titanium rib, and Shilla for EOS performed after 2010 were identified (n=1115). Patients that sustained at least 1 SSI after guided growth surgery were assessed (n=104, 9.3%). Patients with culture and antibiotic details were included (n=55). Patients that received vancomycin powder at index surgery were compared with patients that did not. A multivariate regression model was used to control for potential confounders. RESULTS: There were 55 patients included in this study, including 26 males (47%) and 29 females (53%). Mean age at index surgery was 7.2±6.9 years. Vancomycin powder was utilized in 18 cases (33%). Mean time from index surgery to SSI was 2.0±1.3 years. There were 2 cases of wound dehiscence (4%), 7 cases of superficial infection (13%), and 46 cases of deep infection (84%).There were significant differences in overall microbiology results between vancomycin and no vancomycin cohorts (P=0.047). On univariate analysis, the vancomycin powder cohort had a significantly high incidence of cultures without growth (n=7, 39% vs. n=4, 11%, relative risk: 2.063, 95% confidence interval: 0.927-4.591, P=0.028). This association remained significant on multivariate analysis (adjusted odds ratio: 9.656, 95% confidence interval: 1.743-53.494, P=0.009). CONCLUSIONS: In EOS patients undergoing procedures complicated by SSI, the use of vancomycin powder was independently associated with increased risk of no culture growth. Surgeons and infectious disease physicians should be aware and adjust diagnostic and treatment strategies appropriately. LEVEL OF EVIDENCE: Level III-retrospective cohort study.

Postoperative Correction in Idiopathic Scoliosis: Which Preoperative Imaging Technique Is Most Predictive?

INTRODUCTION: Preoperative radiographic assessment of curve flexibility in patients with idiopathic scoliosis is important to determine Lenke classification, operative levels, and potential postoperative correction. However, no consensus exists regarding the optimal technique. We compared measurements from supine side bending (SB) and intraoperative traction radiographs under general anesthesia (TUGA) with actual postoperative correction followed for 1 year. METHODS: We identified 235 patients with idiopathic scoliosis who underwent posterior spinal fusion with pedicle screw instrumentation between 2010 and 2018 who had preoperative and postoperative radiographs including standing posterior-anterior (PA) and lateral radiographs, preoperative SB radiographs, and TUGA radiographs. Curves were categorized into proximal thoracic, main thoracic/thoracolumbar (MT), and distal thoracolumbar/lumbar (TL/L) curves. Flexibility was calculated from SB and TUGA radiographs. Correction rates were calculated from 1 month and 1 year radiographs postoperatively. Bending radiographs that correlated significantly with postoperative correction with P<0.10 were eligible for inclusion. Preoperative demographics, etiology, deformity details, and surgical details were included in the multivariate models. RESULTS: On univariate analysis, TUGA radiographs correlated with postoperative correction at 1 month and 1 year on MT curves (r=0.214, P=0.001; r=0.209, P=0.001) and TL/L curves (r=0.280, P<0.001; r=0.181, P=0.006). Supine SB radiographs did not correlate with postoperative correction on either MT or T/TL curves. On multivariate analysis, major curve TUGA radiographs were independently associated with postoperative MT curve correction at 1 month (beta: 0.158, 95% confidence interval: 0.035-0.280, P=0.012) and 1 year (beta: 0.195, 95% confidence interval: 0.049-0.340, P=0.009). MT curve SB radiographs were not associated with postoperative major curve correction at 1 month (P=0.088). CONCLUSIONS: TUGA radiographs independently correlated with postoperative main thoracic and distal thoracolumbar/lumbar curve correction at 1 month and 1 year postoperatively. SB radiographs independently correlated only with TL/L curve correction at 1 year postoperatively. However, this correlation was not as strong as TUGA correction (beta of 0.280 vs. beta of 0.092). TUGA radiographs appear superior to SB radiographs at predicting curve correction after surgery. LEVEL OF EVIDENCE: Level III.

Seromas Following Pediatric Spinal Deformity Surgery: Is Operative Management Necessary?

BACKGROUND: Seromas are known complications after pediatric spinal deformity surgery. Although many surgeons perform an early debridement to prevent deep surgical site infections (SSIs), a less invasive approach to seroma management has not been studied. We hypothesized that a conservative approach to seroma management would be safe and yield equivalent outcomes. METHODS: We performed a retrospective review of patients who developed a postoperative seroma with or without nonpurulent drainage. Inclusion criteria were patients below 21 years who underwent primary posterior spinal fusion from 1996 to 2016 and developed a postoperative wound seroma. Seromas were clinically defined as an afebrile patient with a fluid collection that was soft and nontender to palpation and without induration or erythema. Growing spine surgeries and revision procedures were excluded from this study. RESULTS: Twenty-five of 790 total patients with a mean follow-up of 57.8 months (±48.5 mo) developed a seroma. Seromas were identified at a mean of 13.6 days postoperatively and resolved after a mean of 12.2 days following the presentation. Seromas occurred in 12 patients with idiopathic scoliosis, 12 with neuromuscular scoliosis, and 1 patient with Scheuermann kyphosis. All cases were managed conservatively with monitoring of the incision without an operative procedure. In cases of spontaneous drainage, a sterile dressing was applied to the wound and changed as needed until drainage ceased. Two patients underwent bedside needle aspiration and 5 patients received prophylactic antibiotics at the treating surgeon's discretion. All cases resolved spontaneously without development of an acute SSI. Three cases subsequently developed a late SSI (range, 18 to 38 mo postoperatively). Two had idiopathic scoliosis and 1 had neuromuscular scoliosis. None of these seromas drained spontaneously. CONCLUSIONS: Conservative management of postoperative seromas after pediatric spinal deformity surgery is appropriate. It is unclear if seromas contributed to the development of the 3 late infections. Further studies are needed regarding the relationship of late infections in seroma patients. LEVEL OF EVIDENCE: Level IV-case series.

Widespread Shortening of 3' Untranslated Regions and Increased Exon Inclusion Are Evolutionarily Conserved Features of Innate Immune Responses to Infection.

The contribution of pre-mRNA processing mechanisms to the regulation of immune responses remains poorly studied despite emerging examples of their role as regulators of immune defenses. We sought to investigate the role of mRNA processing in the cellular responses of human macrophages to live bacterial infections. Here, we used mRNA sequencing to quantify gene expression and isoform abundances in primary macrophages from 60 individuals, before and after infection with Listeria monocytogenes and Salmonella typhimurium. In response to both bacteria we identified thousands of genes that significantly change isoform usage in response to infection, characterized by an overall increase in isoform diversity after infection. In response to both bacteria, we found global shifts towards (i) the inclusion of cassette exons and (ii) shorter 3' UTRs, with near-universal shifts towards usage of more upstream polyadenylation sites. Using complementary data collected in non-human primates, we show that these features are evolutionarily conserved among primates. Following infection, we identify candidate RNA processing factors whose expression is associated with individual-specific variation in isoform abundance. Finally, by profiling microRNA levels, we show that 3' UTRs with reduced abundance after infection are significantly enriched for target sites for particular miRNAs. These results suggest that the pervasive usage of shorter 3' UTRs is a mechanism for particular genes to evade repression by immune-activated miRNAs. Collectively, our results suggest that dynamic changes in RNA processing may play key roles in the regulation of innate immune responses.

A Multilevel Functional Study of a SNAP25 At-Risk Variant for Bipolar Disorder and Schizophrenia.

The synaptosomal-associated protein SNAP25 is a key player in synaptic vesicle docking and fusion and has been associated with multiple psychiatric conditions, including schizophrenia, bipolar disorder, and attention-deficit/hyperactivity disorder. We recently identified a promoter variant in SNAP25, rs6039769, that is associated with early-onset bipolar disorder and a higher gene expression level in human prefrontal cortex. In the current study, we showed that this variant was associated both in males and females with schizophrenia in two independent cohorts. We then combined in vitro and in vivo approaches in humans to understand the functional impact of the at-risk allele. Thus, we showed in vitro that the rs6039769 C allele was sufficient to increase the SNAP25 transcription level. In a postmortem expression analysis of 33 individuals affected with schizophrenia and 30 unaffected control subjects, we showed that the SNAP25b/SNAP25a ratio was increased in schizophrenic patients carrying the rs6039769 at-risk allele. Last, using genetics imaging in a cohort of 71 subjects, we showed that male risk carriers had an increased amygdala-ventromedial prefrontal cortex functional connectivity and a larger amygdala than non-risk carriers. The latter association has been replicated in an independent cohort of 121 independent subjects. Altogether, results from these multilevel functional studies are bringing strong evidence for the functional consequences of this allelic variation of SNAP25 on modulating the development and plasticity of the prefrontal-limbic network, which therefore may increase the vulnerability to both early-onset bipolar disorder and schizophrenia.SIGNIFICANCE STATEMENT Functional characterization of disease-associated variants is a key challenge in understanding neuropsychiatric disorders and will open an avenue in the development of personalized treatments. Recent studies have accumulated evidence that the SNARE complex, and more specifically the SNAP25 protein, may be involved in psychiatric disorders. Here, our multilevel functional studies are bringing strong evidence for the functional consequences of an allelic variation of SNAP25 on modulating the development and plasticity of the prefrontal-limbic network. These results demonstrate a common genetically driven functional alteration of a synaptic mechanism both in schizophrenia and early-onset bipolar disorder and confirm the shared genetic vulnerability between these two disorders.

Bacterial infection remodels the DNA methylation landscape of human dendritic cells.

DNA methylation is an epigenetic mark thought to be robust to environmental perturbations on a short time scale. Here, we challenge that view by demonstrating that the infection of human dendritic cells (DCs) with a live pathogenic bacteria is associated with rapid and active demethylation at thousands of loci, independent of cell division. We performed an integrated analysis of data on genome-wide DNA methylation, histone mark patterns, chromatin accessibility, and gene expression, before and after infection. We found that infection-induced demethylation rarely occurs at promoter regions and instead localizes to distal enhancer elements, including those that regulate the activation of key immune transcription factors. Active demethylation is associated with extensive epigenetic remodeling, including the gain of histone activation marks and increased chromatin accessibility, and is strongly predictive of changes in the expression levels of nearby genes. Collectively, our observations show that active, rapid changes in DNA methylation in enhancers play a previously unappreciated role in regulating the transcriptional response to infection, even in nonproliferating cells.

Specific dysregulation of IFNγ production by natural killer cells confers susceptibility to viral infection.

Natural Killer (NK) cells contribute to the control of viral infection by directly killing target cells and mediating cytokine release. In C57BL/6 mice, the Ly49H activating NK cell receptor plays a key role in early resistance to mouse cytomegalovirus (MCMV) infection through specific recognition of the MCMV-encoded MHC class I-like molecule m157 expressed on infected cells. Here we show that transgenic expression of Ly49H failed to provide protection against MCMV infection in the naturally susceptible A/J mouse strain. Characterization of Ly49H(+) NK cells from Ly49h-A transgenic animals showed that they were able to mount a robust cytotoxic response and proliferate to high numbers during the course of infection. However, compared to NK cells from C57BL/6 mice, we observed an intrinsic defect in their ability to produce IFNγ when challenged by either m157-expressing target cells, exogenous cytokines or chemical stimulants. This effect was limited to NK cells as T cells from C57BL/6 and Ly49h-A mice produced comparable cytokine levels. Using a panel of recombinant congenic strains derived from A/J and C57BL/6 progenitors, we mapped the genetic basis of defective IFNγ production to a single 6.6 Mb genetic interval overlapping the Ifng gene on chromosome 10. Inspection of the genetic interval failed to reveal molecular differences between A/J and several mouse strains showing normal IFNγ production. The chromosome 10 locus is independent of MAPK signalling or decreased mRNA stability and linked to MCMV susceptibility. This study highlights the existence of a previously uncovered NK cell-specific cis-regulatory mechanism of Ifnγ transcript expression potentially relevant to NK cell function in health and disease.

Viral MHC class I-like molecule allows evasion of NK cell effector responses in vivo.

The outcome of mouse CMV (MCMV) infection varies among different inbred mouse strains depending on NK cell effector functions governed through recognition receptor triggering. NK cells from different mouse strains possess diverse repertoires of activating or inhibitory Ly49 receptors, which share some of their polymorphic MHC class I (MHC-I) ligands. By examining the NK cell response to MCMV infection in novel BALB substrains congenic for different MHC (or H-2 in mice) haplotypes, we show that recognition of viral MHC-I-like protein m157 by inhibitory Ly49C receptor allows escape from NK cell control of viral replication. Dominant inhibition by Ly49C bound to self-H-2(b) encoded MHC-I molecules masks this effect, which only becomes apparent in distinct H-2 haplotypes, such as H-2(f). The recognition of m157-expressing cells by Ly49C resulted in both decreased NK cell killing in vitro and reduced rejection in vivo. Further, control of infection with m157-deletant (Δm157) MCMV was improved in mice carrying H-2 molecules unrecognized by Ly49C but allowing expansion of NK cell effectors expressing activating Ly49L receptors. Hence, our study is the first, to our knowledge, to demonstrate that MHC-I mimicry strategies used by MCMV to avoid NK cell control are biologically relevant during in vivo viral infection. Of value for human studies is that only a few genetic assortments conditional on the repertoires of viral MHC-I-like proteins/host NK receptors/MHC haplotypes should allow efficient protection against CMV infection.