Impact of age on pneumococcal colonization of the nasopharynx and oral cavity: an ecological perspective.

Pneumococcal carriage studies have suggested that pneumococcal colonization in adults is largely limited to the oral cavity and oropharynx. In this study, we used total abundance-based ß-diversity (dissimilarity) and ß-diversity components to characterize age-related differences in pneumococcal serotype composition of respiratory samples. quantitative PCR (qPCR) was applied to detect pneumococcal serotypes in nasopharyngeal samples collected from 946 toddlers and 602 adults, saliva samples collected from a subset of 653 toddlers, and saliva and oropharyngeal samples collected from a subset of 318 adults. Bacterial culture rates from nasopharyngeal samples were used to characterize age-related differences in rates of colonizing bacteria. Dissimilarity in pneumococcal serotype composition was low among saliva and nasopharyngeal samples from children. In contrast, respiratory samples from adults exhibited high serotype dissimilarity, which predominantly consisted of abundance gradients and was associated with reduced nasopharyngeal colonization. Age-related serotype dissimilarity was high among nasopharyngeal samples and relatively low for saliva samples. Reduced nasopharyngeal colonization by pneumococcal serotypes coincided with significantly reduced Moraxella catarrhalis and Haemophilus influenzae and increased Staphylococcus aureus nasopharyngeal colonization rates among adults. Findings from this study suggest that within-host environmental conditions, utilized in the upper airways by pneumococcus and other bacteria, undergo age-related changes. It may result in a host-driven ecological succession of bacterial species colonizing the nasopharynx and lead to competitive exclusion of pneumococcus from the nasopharynx but not from the oral habitat. This explains the poor performance of nasopharyngeal samples for pneumococcal carriage among adults and indicates that in adults saliva more accurately represents the epidemiology of pneumococcal carriage than nasopharyngeal samples.

Meningococcal carriage in children and young adults: a cross-sectional and longitudinal study, Iceland, 2019 to 2021.

BackgroundNeisseria meningitidis is a commensal bacterium which can cause invasive disease. Colonisation studies are important to guide vaccination strategies.AimThe study's aim was to determine the prevalence of meningococcal colonisation, duration of carriage and distribution of genogroups in Iceland.MethodsWe collected samples from 1 to 6-year-old children, 15-16-year-old adolescents and 18-20-year-old young adults. Carriers were sampled at regular intervals until the first negative swab. Conventional culture methods and qPCR were applied to detect meningococci and determine the genogroup. Whole genome sequencing was done on groupable meningococci.ResultsNo meningococci were detected among 460 children, while one of 197 (0.5%) adolescents and 34 of 525 young adults (6.5 %) carried meningococci. Non-groupable meningococci were most common (62/77 isolates from 26/35 carriers), followed by genogroup B (MenB) (12/77 isolates from 6/35 carriers). Genogroup Y was detected in two individuals and genogroup W in one. None carried genogroup C (MenC). The longest duration of carriage was at least 21 months. Serial samples from persistent carriers were closely related in WGS.ConclusionsCarriage of pathogenic meningococci is rare in young Icelanders. Non-groupable meningococci were the most common colonising meningococci in Iceland, followed by MenB. No MenC were found. Whole genome sequencing suggests prolonged carriage of the same strains in persistent carriers.

Surveillance of Neisseria meningitidis carriage four years after menACWY vaccine implementation in the Netherlands reveals decline in vaccine-type and rise in genogroup e circulation.

Carriage of Neisseria meningitidisis an accepted endpoint in monitoring meningococcal vaccine effects. We applied molecular methods to assess the impact of menACWY vaccine implementation on meningococcal carriage and genogroup-specific prevalence in young adults in Fall of 2022, four years after the introduction of the tetravalent vaccine in the Netherlands. The overall carriage rate of genogroupable meningococci was not significantly different compared to a pre-menACWY cohort investigated in 2018 (20.8 % or 125 of 601 versus 17.4 % or 52 of 299 individuals, p = 0.25). Of 125 carriers of genogroupable meningococci, 122 (97.6 %) were positive for either vaccine-types menC, menW, menY or genogroups, menB, menE, and menX, which are not targeted by the menACWY vaccine. Compared with a pre-vaccine-implementation cohort, there was 3.8-fold reduction (p < 0.001) in vaccine-type carriage rates and 9.0-fold increase (p < 0.0001) in non-vaccine type menE prevalence. We observe a reduction in menW and menY and an increase in menE, which suggest that implementation of menACWY vaccine affected carriage.

A spitting image: molecular diagnostics applied to saliva enhance detection of Streptococcus pneumoniae and pneumococcal serotype carriage.

Background: Despite strong historical records on the accuracy of saliva testing, oral fluids are considered poorly suited for pneumococcal carriage detection. We evaluated an approach for carriage surveillance and vaccine studies that increases the sensitivity and specificity of pneumococcus and pneumococcal serotype detection in saliva samples. Methods: Quantitative PCR (qPCR)-based methods were applied to detect pneumococcus and pneumococcal serotypes in 971 saliva samples collected from 653 toddlers and 318 adults. Results were compared with culture-based and qPCR-based detection in nasopharyngeal samples collected from children and in nasopharyngeal and oropharyngeal samples collected from adults. Optimal C q cut-offs for positivity in qPCRs were determined via receiver operating characteristic curve analysis and accuracy of different approaches was assessed using a composite reference for pneumococcal and for serotype carriage based on isolation of live pneumococcus from the person or positivity of saliva samples determined with qPCR. To evaluate the inter-laboratory reproducibility of the method, 229 culture-enriched samples were tested independently in the second center. Results: In total, 51.5% of saliva samples from children and 31.8% of saliva samples from adults were positive for pneumococcus. Detection of pneumococcus by qPCR in culture-enriched saliva exhibited enhanced sensitivity and higher agreement with a composite reference compared to diagnostic culture of nasopharyngeal samples in children (Cohen's κ: 0.69-0.79 vs. 0.61-0.73) and in adults (κ: 0.84-0.95 vs. 0.04-0.33) and culture of oropharyngeal samples in adults (κ: 0.84-0.95 vs. -0.12-0.19). Similarly, detection of serotypes with qPCR in culture-enriched saliva exhibited enhanced sensitivity and higher agreement with a composite reference compared to nasopharyngeal culture in children (κ: 0.73-0.82 vs. 0.61-0.73) and adults (κ: 0.90-0.96 vs. 0.00-0.30) and oropharyngeal culture in adults (κ: 0.90-0.96 vs. -0.13 to 0.30). However, results of qPCRs targeting serotype 4, 5, and 17F and serogroups 9, 12, and 35 were excluded due to assays' lack of specificity. We observed excellent quantitative agreement for qPCR-based detection of pneumococcus between laboratories. After exclusion of serotype/serogroup-specific assays with insufficient specificity, moderate agreement (κ 0.68, 95% CI 0.58-0.77) was observed. Conclusion: Molecular testing of culture-enriched saliva samples improves the sensitivity of overall surveillance of pneumococcal carriage in children and adults, but limitations of qPCR-based approaches for pneumococcal serotypes carriage detection should be considered.

Tracking Mycobacterium tuberculosis sequencing samples using unique spikes of random DNA.

In the Netherlands, local laboratories are involved in the primary diagnosis of tuberculosis. Positive Mycobacterium tuberculosis complex cultures are sent to the National Institute for Public Health and the Environment (RIVM) for species identification, epidemiological typing, and screening for resistance by Whole Genome Sequencing (WGS). Occasional sample-swaps and cross-contaminations are known to occur in the diagnostic procedures. Such errors may lead to incorrect diagnoses resulting in the unnecessary or sub-optimal treatment of patients. Internal controls throughout the process ideally allow the early detection of such mistakes.

It Takes Two to Tango: Combining Conventional Culture With Molecular Diagnostics Enhances Accuracy of Streptococcus pneumoniae Detection and Pneumococcal Serogroup/Serotype Determination in Carriage.

Background: The specificity of molecular methods for the detection of Streptococcus pneumoniae carriage is under debate. We propose a procedure for carriage surveillance and vaccine impact studies that increases the accuracy of molecular detection of live pneumococci in polymicrobial respiratory samples. Methods: Culture and qPCR methods were applied to detect pneumococcus and pneumococcal serotypes in 1,549 nasopharyngeal samples collected in the Netherlands (n = 972) and England (n = 577) from 946 toddlers and 603 adults, and in paired oropharyngeal samples collected exclusively from 319 Dutch adults. Samples with no live pneumococci isolated at primary diagnostic culture yet generating signal specific for pneumococcus in qPCRs were re-examined with a second, qPCR-guided culture. Optimal Cq cut-offs for positivity in qPCRs were determined via receiver operating characteristic (ROC) curve analysis using isolation of live pneumococci from the primary and qPCR-guided cultures as reference. Results: Detection of pneumococcus and pneumococcal serotypes with qPCRs in cultured (culture-enriched) nasopharyngeal samples exhibited near-perfect agreement with conventional culture (Cohen's kappa: 0.95). Molecular methods displayed increased sensitivity of detection for multiple serotype carriage, and implementation of qPCR-guided culturing significantly increased the proportion of nasopharyngeal and oropharyngeal samples from which live pneumococcus was recovered (p < 0.0001). For paired nasopharyngeal and oropharyngeal samples from adults none of the methods applied to a single sample type exhibited good agreement with results for primary and qPCR-guided nasopharyngeal and oropharyngeal cultures combined (Cohens kappa; 0.13-0.55). However, molecular detection of pneumococcus displayed increased sensitivity with culture-enriched oropharyngeal samples when compared with either nasopharyngeal or oropharyngeal primary cultures (p < 0.05). Conclusion: The accuracy of pneumococcal carriage surveillance can be greatly improved by complementing conventional culture with qPCR and vice versa, by using results of conventional and qPCR-guided cultures to interpret qPCR data. The specificity of molecular methods for the detection of live pneumococci can be enhanced by incorporating statistical procedures based on ROC curve analysis. The procedure we propose for future carriage surveillance and vaccine impact studies improves detection of pneumococcal carriage in adults in particular and enhances the specificity of serotype carriage detection.

High Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Household Transmission Rates Detected by Dense Saliva Sampling.

BACKGROUND: Understanding the dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) household transmission is important for adequate infection control measures in this ongoing pandemic. METHODS: Households were enrolled upon a polymerase chain reaction-confirmed index case between October and December 2020, prior to the coronavirus disease 2019 vaccination program. Saliva samples were obtained by self-sampling at days 1, 3, 5, 7, 10, 14, 21, 28, 35, and 42 from study inclusion. Nasopharyngeal swabs (NPS) and oropharyngeal swabs (OPS) were collected by the research team at day 7 and capillary blood samples at day 42. Household secondary attack rate (SAR) and per-person SAR were calculated based on at least 1 positive saliva, NPS, OPS, or serum sample. Whole genome sequencing was performed to investigate the possibility of multiple independent SARS-CoV-2 introductions within a household. RESULTS: Eighty-five households were included consisting of 326 (unvaccinated) individuals. Comparable numbers of secondary cases were identified by saliva (133/241 [55.2%]) and serum (127/213 [59.6%]). The household SAR was 88.2%. The per-person SAR was 64.3%. The majority of the secondary cases tested positive in saliva at day 1 (103/150 [68.7%]). Transmission from index case to household member was not affected by age or the nature of their relationship. Phylogenetic analyses suggested a single introduction for the investigated households. CONCLUSIONS: Households have a pivotal role in SARS-CoV-2 transmission. By repeated saliva self-sampling combined with NPS, OPS, and serology, we found the highest SARS-CoV-2 household transmission rates reported to date. Salivary (self-) sampling of adults and children is suitable and attractive for near real-time monitoring of SARS-CoV-2 transmission in this setting.

Detection of Neisseria meningitidis in saliva and oropharyngeal samples from college students.

Carriage of Neisseria meningitidis is an accepted endpoint in monitoring meningococcal vaccines effects. We have assessed N. meningitidis and vaccine-type genogroup carriage prevalence in college students at the time of MenACWY vaccine introduction in the Netherlands, and evaluated the feasibility of saliva sampling for the surveillance of carriage. For this, paired saliva and oropharyngeal samples collected from 299 students were cultured for meningococcus. The DNA extracted from all bacterial growth was subjected to qPCRs quantifying meningococcal and genogroup-specific genes presence. Samples negative by culture yet positive for qPCR were cultured again for meningococcus. Altogether 74 (25%) of students were identified as meningococcal carrier by any method. Sixty-one students (20%) were identified as carriers with qPCR. The difference between number of qPCR-positive oropharyngeal (n = 59) and saliva (n = 52) samples was not significant (McNemar's test, p = 0.07). Meningococci were cultured from 72 students (24%), with a significantly higher (p < 0.001) number of oropharyngeal (n = 70) compared with saliva (n = 54) samples. The prevalence of genogroups A, B, C, W, and Y was none, 9%, 1%, 1% and 6%, respectively, and 8% of students carried MenACWY vaccine-type genogroup meningococci. Saliva is easy to collect and when combined with qPCR detection can be considered for meningococcal carriage studies.

Increase in invasive disease caused by Haemophilus influenzae b, the Netherlands, 2020 to 2021.

The incidence of most respiratory-transmitted diseases decreased during the COVID-19 pandemic as a result of containment measures. In contrast, in the Netherlands we noted an increase in invasive disease caused by Haemophilus influenzae b (Hib) (from < 0.3/100,000 before 2019 to 0.39 and 0.33/100,000 in 2020 and 2021) in vaccinated and unvaccinated age groups. We did not find a change in vaccine effectiveness against Hib invasive disease (effectiveness > 90%). We discuss factors that may have contributed to this rise.

Naturally circulating pertactin-deficient Bordetella pertussis strains induce distinct gene expression and inflammatory signatures in human dendritic cells.

Respiratory infections caused by Bordetella pertussis are reemerging despite high pertussis vaccination coverage. Since the introduction of the acellular pertussis vaccine in the late twentieth century, circulating B. pertussis strains increasingly lack expression of the vaccine component pertactin (Prn). In some countries, up to 90% of the circulating B. pertussis strains are deficient in Prn. To better understand the resurgence of pertussis, we investigated the response of human monocyte-derived dendritic cells (moDCs) to naturally circulating Prn-expressing (Prn-Pos) and Prn-deficient (Prn-Neg) B. pertussis strains from 2016 in the Netherlands. Transcriptome analysis of moDC showed enriched IFNα response-associated gene expression after exposure to Prn-Pos B. pertussis strains, whereas the Prn-Neg strains induced enriched expression of interleukin- and TNF-signaling genes, as well as other genes involved in immune activation. Multiplex immune assays confirmed enhanced proinflammatory cytokine secretion by Prn-Neg stimulated moDC. Comparison of the proteomes from the Prn-Pos and Prn-Neg strains revealed, next to the difference in Prn, differential expression of a number of other proteins including several proteins involved in metabolic processes. Our findings indicate that Prn-deficient B. pertussis strains induce a distinct and stronger immune activation of moDCs than the Prn-Pos strains. These findings highlight the role of pathogen adaptation in the resurgence of pertussis as well as the effects that vaccine pressure can have on a bacterial population.

Regulatory KIR+ RA+ T cells accumulate with age and are highly activated during viral respiratory disease.

Severe respiratory viral infectious diseases such as influenza and COVID-19 especially affect the older population. This is partly ascribed to diminished CD8+ T-cell responses a result of aging. The phenotypical diversity of the CD8+ T-cell population has made it difficult to identify the impact of aging on CD8+ T-cell subsets associated with diminished CD8+ T-cell responses. Here we identify a novel human CD8+ T-cell subset characterized by expression of Killer-cell Immunoglobulin-like Receptors (KIR+ ) and CD45RA (RA+ ). These KIR+ RA+ T cells accumulated with age in the blood of healthy individuals (20-82 years of age, n = 50), expressed high levels of aging-related markers of T-cell regulation, and were functionally capable of suppressing proliferation of other CD8+ T cells. Moreover, KIR+ RA+ T cells were a major T-cell subset becoming activated in older adults suffering from an acute respiratory viral infection (n = 36), including coronavirus and influenza virus infection. In addition, older adults with influenza A infection showed that higher activation status of their KIR+ RA+ T cells associated with longer duration of respiratory symptoms. Together, our data indicate that KIR+ RA+ T cells are a unique human T-cell subset with regulatory properties that may explain susceptibility to viral respiratory disease at old age.

Influenza-like Illness Exacerbates Pneumococcal Carriage in Older Adults.

BACKGROUND: In older adults, pneumococcal disease is strongly associated with respiratory viral infections, but the impact of viruses on Streptococcus pneumoniae carriage prevalence and load remains poorly understood. Here, we investigated the effects of influenza-like illness (ILI) on pneumococcal carriage in community-dwelling older adults. METHODS: We investigated the presence of pneumococcal DNA in saliva samples collected in the 2014/2015 influenza season from 232 individuals aged ≥60 years at ILI onset, followed by sampling 2-3 weeks and 7-9 weeks after the first sample. We also sampled 194 age-matched controls twice 2-3 weeks apart. Pneumococcal DNA was detected with quantitative polymerase chain reaction assays targeting the piaB and lytA genes in raw and in culture-enriched saliva. Bacterial and pneumococcal abundances were determined in raw saliva with 16S and piaB quantification. RESULTS: The prevalence of pneumococcus-positive samples was highest at onset of ILI (42/232 [18%]) and lowest among controls (26/194 [13%] and 22/194 [11%] at the first and second samplings, respectively), though these differences were not significant. Pneumococcal carriage was associated with exposure to young children (odds ratio [OR], 2.71 [95% confidence interval {CI}, 1.51-5.02]; P < .001), and among asymptomatic controls with presence of rhinovirus infection (OR, 4.23 [95% CI, 1.16-14.22]; P < .05). When compared with carriers among controls, pneumococcal absolute abundances were significantly higher at onset of ILI (P < .01), and remained elevated beyond recovery from ILI (P < .05). Finally, pneumococcal abundances were highest in carriage events newly detected after ILI onset (estimated geometric mean, 1.21 × 10-5 [95% CI, 2.48 × 10-7 to 2.41 × 10-5], compared with preexisting carriage). CONCLUSIONS: ILI exacerbates pneumococcal colonization of the airways in older adults, and this effect persists beyond recovery from ILI.

Airborne particulate matter from goat farm increases acute allergic airway responses in mice.

Background: Inhalation exposure to biological particulate matter (BioPM) from livestock farms may provoke exacerbations in subjects suffering from allergy and asthma. The aim of this study was to use a murine model of allergic asthma to determine the effect of BioPM derived from goat farm on airway allergic responses.Methods: Fine (<2.5 µm) BioPM was collected from an indoor goat stable. Female BALB/c mice were ovalbumin (OVA) sensitized and challenged with OVA or saline as control. The OVA and saline groups were divided in sub-groups and exposed intranasally to different concentrations (0, 0.9, 3, or 9 µg) of goat farm BioPM. Bronchoalveolar lavage fluid (BALF), blood and lung tissues were collected.Results: In saline-challenged mice, goat farm BioPM induced 1) a dose-dependent increase in neutrophils in BALF and 2) production of macrophage inflammatory protein-3a. In OVA-challenged mice, BioPM induced 1) inflammatory cells in BALF, 2) OVA-specific Immunoglobulin (Ig)G1, 3) airway mucus secretion-specific gene expression. RNAseq analysis of lungs indicates that neutrophil chemotaxis and oxidation-reduction processes were the representative genomic pathways in saline and OVA-challenged mice, respectively.Conclusions: A single exposure to goat farm BioPM enhanced airway inflammation in both saline and OVA-challenged allergic mice, with neutrophilic response as Th17 disorder and eosinophilic response as Th2 disorder indicative of the severity of allergic responses. Identification of the mode of action by which farm PM interacts with airway allergic pathways will be useful to design potential therapeutic approaches.

Immunological effects of shift work in healthcare workers.

The immune system potentially plays an important mechanistic role in the relation between shift work and adverse health effects. To better understand the immunological effects of shift work, we compared numbers and functionality of immune cells between night-shift and non-shift workers. Blood samples were collected from 254 night-shift and 57 non-shift workers employed in hospitals. Absolute numbers of monocytes, granulocytes, lymphocytes, and T cell subsets were assessed. As read out of immune function, monocyte cytokine production and proliferative capacity of CD4 and CD8 T cells in response to various stimuli were analysed. The mean number of monocytes was 1.15 (95%-CI = 1.05-1.26) times higher in night-shift than in non-shift workers. Furthermore, night-shift workers who worked night shifts in the past three days had a higher mean number of lymphocytes (B = 1.12 (95%-CI = 1.01-1.26)), T cells (B = 1.16 (95%-CI = 1.03-1.31)), and CD8 T cells (B = 1.23 (95%-CI = 1.05-1.45)) compared to non-shift workers. No differences in functional parameters of monocytes and lymphocytes were observed. The differences in numbers of monocytes and T cells suggest that chronic exposure to night-shift work as well as recent night-shift work may influence the immune status of healthcare workers. This knowledge could be relevant for preventive initiatives in night-shift workers, such as timing of vaccination.

Microbiome composition of airborne particulate matter from livestock farms and their effect on innate immune receptors and cells.

Patients with respiratory diseases in rural areas have been reported to have enhanced responsiveness to ambient particulate matter (PM). In addition to the physical and chemical components, ambient PM can contain microorganisms or parts thereof, referred here as BioPM, that can also contribute to the adverse health effects. This study aimed to characterize the microbial composition of BioPM originating from livestock, and to investigate whether these BioPM can trigger the activation of innate receptors and cells. Coarse (PM2.5-10 µm) and fine (PM<2.5 µm) BioPM samples were collected from indoor chicken, pig and goat farms using the versatile aerosol concentration enrichment system (VACES) connected to a Biosampler. The fungal and bacterial communities were assessed with an amplicon based approach using Next Generation Sequencing (NGS). In parallel, HEK-Blue cells expressing different pattern recognition receptors (Toll like receptors (TLR) 2, 3, 4, 5, 7, 8, 9 and NOD 1, 2) and a human monocytic cell line (MM6) were exposed to BioPM samples from these sites. Distinct airborne microbiota profiles associated with the corresponding animal farm were observed. Moreover, the various BioPM contained mainly ligands for TLR2 and TLR4 resulting in a concentration-dependent increase of pro-inflammatory cytokine secreted by MM6 cells. In addition, we show for the first time that only the pig-derived BioPM induced TLR5 activation. These findings suggest that animal farm specific BioPM trigger distinct inflammatory responses, which may contribute to airway diseases in humans.

Activation of Human NK Cells by Bordetella pertussis Requires Inflammasome Activation in Macrophages.

Pertussis is a highly contagious respiratory infection caused by the bacterium Bordetella pertussis. Humans are the only known natural reservoir of B. pertussis. In mice, macrophages and NK cells have a key role in confining B. pertussis to the respiratory tract. However, the mechanisms underlying this process, particularly during human infections, remain unclear. Here we characterized the activation of human macrophages and NK cells in response to B. pertussis and unraveled the role of inflammasomes in this process. NLRP3 inflammasome activation by B. pertussis in human macrophage-like THP-1 cells and primary monocyte-derived macrophages (mo-MΦ) was shown by the visualization of ASC-speck formation, pyroptosis, and the secretion of caspase-mediated IL-1ß and IL-18. In contrast to macrophages, stimulation of human CD56+CD3- NK cells by B. pertussis alone did not result in activation of these cells. However, co-culture of B. pertussis-stimulated mo-MΦ and autologous NK cells resulted in high amounts of IFNγ secretion and an increased frequency of IL-2Rα+ and HLA-DR+ NK cells, indicating NK cell activation. This activation was significantly reduced upon inhibition of inflammasome activity or blocking of IL-18 in the mo-MΦ/NK cell co-culture. Furthermore, we observed increased secretion of proinflammatory cytokines in the B. pertussis-stimulated mo-MΦ/NK co-culture compared to the mo-MΦ single culture. Our results demonstrate that B. pertussis induces inflammasome activation in human macrophages and that the IL-18 produced by these cells is required for the activation of human NK cells, which in turn enhances the pro-inflammatory response to this pathogen. Our data provides a better understanding of the underlying mechanisms involved in the induction of innate immune responses against B. pertussis. These findings contribute to the knowledge required for the development of improved intervention strategies to control this highly contagious disease.

Transcriptomics in lung tissue upon respiratory syncytial virus infection reveals aging as important modulator of immune activation and matrix maintenance.

Aging poses an increased risk of severe infection by respiratory syncytial virus (RSV). The many different biological pathways comprising the response to infection in lungs that are influenced by aging are complex and remain to be defined more thoroughly. Towards finding new directions in research on aging, we aimed to define biological pathways in the acute response to RSV that are affected in the lungs by aging. We therefore profiled the full transcriptome of lung tissue of mice prior to and during RSV infection both at young and old age. In the absence of RSV, we found aging to downregulate genes that are involved in constitution of the extracellular matrix. Moreover, uninfected old mice showed elevated expression of pathways that resemble injury, metabolic aberrations, and disorders mediated by functions of the immune system that were induced at young age only by an exogenous trigger like RSV. Furthermore, infection by RSV mounted stronger activation of anti-viral type-I interferon pathways at old age. Despite such exaggerated anti-viral responses, old mice showed reduced control of virus. Altogether, our findings emphasize important roles in aging-related susceptibility to respiratory disease for extracellular matrix dysfunctions and dysregulated immune activation in lungs.

IBD risk loci are enriched in multigenic regulatory modules encompassing putative causative genes.

GWAS have identified >200 risk loci for Inflammatory Bowel Disease (IBD). The majority of disease associations are known to be driven by regulatory variants. To identify the putative causative genes that are perturbed by these variants, we generate a large transcriptome data set (nine disease-relevant cell types) and identify 23,650 cis-eQTL. We show that these are determined by ∼9720 regulatory modules, of which ∼3000 operate in multiple tissues and ∼970 on multiple genes. We identify regulatory modules that drive the disease association for 63 of the 200 risk loci, and show that these are enriched in multigenic modules. Based on these analyses, we resequence 45 of the corresponding 100 candidate genes in 6600 Crohn disease (CD) cases and 5500 controls, and show with burden tests that they include likely causative genes. Our analyses indicate that ≥10-fold larger sample sizes will be required to demonstrate the causality of individual genes using this approach.

Bordetella pertussis pertactin knock-out strains reveal immunomodulatory properties of this virulence factor.

Whooping cough, caused by Bordetella pertussis, has resurged and presents a global health burden worldwide. B. pertussis strains unable to produce the acellular pertussis vaccine component pertactin (Prn), have been emerging and in some countries represent up to 95% of recent clinical isolates. Knowledge on the effect that Prn deficiency has on infection and immunity to B. pertussis is crucial for the development of new strategies to control this disease. Here, we characterized the effect of Prn production by B. pertussis on human and murine dendritic cell (DC) maturation as well as in a murine model for pertussis infection. We incubated human monocyte-derived DCs (moDCs) with multiple isogenic Prn knockout (Prn-KO) and corresponding parental B. pertussis strains constructed either in laboratory reference strains with a Tohama I background or in a recently circulating clinical isolate. Results indicate that, compared to the parental strains, Prn-KO strains induced an increased production of pro-inflammatory cytokines by moDCs. This pro-inflammatory phenotype was also observed upon stimulation of murine bone marrow-derived DCs. Moreover, RNA sequencing analysis of lungs from mice infected with B. pertussis Prn-KO revealed increased expression of genes involved in cell death. These in vitro and in vivo findings indicate that B. pertussis strains which do not produce Prn induce a stronger pro-inflammatory response and increased cell death upon infection, suggesting immunomodulatory properties for Prn.

Fine-mapping inflammatory bowel disease loci to single-variant resolution.

Inflammatory bowel diseases are chronic gastrointestinal inflammatory disorders that affect millions of people worldwide. Genome-wide association studies have identified 200 inflammatory bowel disease-associated loci, but few have been conclusively resolved to specific functional variants. Here we report fine-mapping of 94 inflammatory bowel disease loci using high-density genotyping in 67,852 individuals. We pinpoint 18 associations to a single causal variant with greater than 95% certainty, and an additional 27 associations to a single variant with greater than 50% certainty. These 45 variants are significantly enriched for protein-coding changes (n = 13), direct disruption of transcription-factor binding sites (n = 3), and tissue-specific epigenetic marks (n = 10), with the last category showing enrichment in specific immune cells among associations stronger in Crohn's disease and in gut mucosa among associations stronger in ulcerative colitis. The results of this study suggest that high-resolution fine-mapping in large samples can convert many discoveries from genome-wide association studies into statistically convincing causal variants, providing a powerful substrate for experimental elucidation of disease mechanisms.