Prevalence of human respiratory pathogens and associated mucosal cytokine levels in young children and adults: a cross-sectional observational study in the Netherlands during the winter of 2012/2013.

Respiratory pathogens can cause severe disease and even death, especially in the very young and very old. Studies investigating their prevalence often focus on individuals presenting to healthcare providers with symptoms. However, the design of prevention strategies, e.g. which target groups to vaccinate, will benefit from knowledge on the prevalence of, risk factors for and host response to these pathogens in the general population. In this study, upper respiratory samples (n = 1311) were collected cross-sectionally during winter from 11- and 24-month old children, their parents, and adults ≥60 years of age that were recruited irrespective of seeking medical care. Almost all children, approximately two-thirds of parents and a quarter of older adults tested positive for at least one pathogen, often in the absence of symptoms. Viral interference was evident for the combination of rhinovirus and respiratory syncytial virus. Attending childcare facilities and having siblings associated with increased pathogen counts in children. On average, children showed increased levels of mucosal cytokines compared to parents and especially proinflammatory molecules associated with the presence of symptoms. These findings may guide further research into transmission patterns of respiratory pathogens and assist in determining the most appropriate strategies for the prediction and prevention of disease.

Nontypeable Haemophilus influenzae Invasive Blood Isolates Are Mainly Phosphorylcholine Negative and Show Decreased Complement-Mediated Killing That Is Associated with Lower Binding of IgM and CRP in Comparison to Colonizing Isolates from the Oropharynx.

Nontypeable Haemophilus influenzae (NTHi) bacteria express various molecules that contribute to their virulence. The presence of phosphocholine (PCho) on NTHi lipooligosaccharide increases adhesion to epithelial cells and is an advantage for the bacterium, enabling nasopharyngeal colonization, as measured in humans and animal models. However, when PCho is expressed on the lipooligosaccharide, it is also recognized by the acute-phase protein C-reactive protein (CRP) and PCho-specific antibodies, both of which are potent initiators of the classical pathway of complement activation. In this study, we show that blood isolates, which are exposed to CRP and PCho-specific antibodies in the bloodstream, have a higher survival in serum than oropharyngeal isolates, which was associated with a decreased presence of PCho. PCholow strains showed decreased IgM, CRP, and complement C3 deposition, which was associated with increased survival in human serum. Consistent with the case for the PCholow strains, removal of PCho expression by licA gene deletion decreased IgM, CRP, and complement C3 deposition, which increased survival in human serum. Complement-mediated killing of PChohigh strains was mainly dependent on binding of IgM to the bacterial surface. These data support the hypothesis that a PCholow phenotype was selected in blood during invasive disease, which increased resistance to serum killing, mainly due to lowered IgM and CRP binding to the bacterial surface.

Increased carriage of non-vaccine serotypes with low invasive disease potential four years after switching to the 10-valent pneumococcal conjugate vaccine in The Netherlands.

The 7-valent pneumococcal conjugate vaccine (PCV7) was introduced in The Netherlands in 2006 and was replaced by PHiD-CV10 in 2011. Data on carriage prevalence of S. pneumoniae serotypes in children and invasive pneumococcal disease (IPD) in children and older adults were collected to examine the impact of PCVs on carriage and IPD in The Netherlands. Pneumococcal carriage prevalence was determined by conventional culture of nasopharyngeal swabs in 24-month-old children in 2015/2016. Data were compared to similar carriage studies in 2005 (pre-PCV7 introduction), 2009, 2010/2011 and 2012/2013. Invasive pneumococcal disease isolates from hospitalized children <5 years and adults >65 years (2004-2016) were obtained by sentinel surveillance. All isolates were serotyped by Quellung. Serotype invasive disease potential was calculated using carriage and nationwide IPD data in children. The overall pneumococcal carriage rate was 48% in 2015/2016, lower than in 2010/2011 (64%) and pre-vaccination in 2005 (66%). Carriage of the previously dominant non-vaccine serotypes 19A and 11A has declined since 2010/2011, from 14.2% to 4.6% and 4.2% to 2.7%, respectively, whereas carriage of serotypes 6C and 23B has increased (4.2% to 6.7% and 3.9% to 7.3%), making serotypes 6C and 23B the most prevalent carriage serotypes. IPD incidence declined in children (20/100,000 cases in 2004/2006 to 6/100,000 cases in 2015/2016) as well as in older adults (63/100,000 cases to 51/100,000 cases). Serotypes 6C, 23B and 11A have high carriage prevalence in children, but show low invasive disease potential. Serotype 8 is the main causative agent for IPD in older adults (11.3%). In conclusion, 10 years after the introduction of pneumococcal vaccination in children in The Netherlands shifts in carriage and disease serotypes are still ongoing. Surveillance of both carriage and IPD is important to assess PCV impact and to predict necessary future vaccination strategies in both children and older adults.

Haemophilus is overrepresented in the nasopharynx of infants hospitalized with RSV infection and associated with increased viral load and enhanced mucosal CXCL8 responses.

BACKGROUND: While almost all infants are infected with respiratory syncytial virus (RSV) before the age of 2 years, only a small percentage develops severe disease. Previous studies suggest that the nasopharyngeal microbiome affects disease development. We therefore studied the effect of the nasopharyngeal microbiome on viral load and mucosal cytokine responses, two important factors influencing the pathophysiology of RSV disease. To determine the relation between (i) the microbiome of the upper respiratory tract, (ii) viral load, and (iii) host mucosal inflammation during an RSV infection, nasopharyngeal microbiota profiles of RSV infected infants (< 6 months) with different levels of disease severity and age-matched healthy controls were determined by 16S rRNA marker gene sequencing. The viral load was measured using qPCR. Nasopharyngeal CCL5, CXCL10, MMP9, IL6, and CXCL8 levels were determined with ELISA. RESULTS: Viral load in nasopharyngeal aspirates of patients associates significantly to total nasopharyngeal microbiota composition. Healthy infants (n = 21) and RSV patients (n = 54) display very distinct microbial patterns, primarily characterized by a loss in commensals like Veillonella and overrepresentation of opportunistic organisms like Haemophilus and Achromobacter in RSV-infected individuals. Furthermore, nasopharyngeal microbiota profiles are significantly different based on CXCL8 levels. CXCL8 is a chemokine that was previously found to be indicative for disease severity and for which we find Haemophilus abundance as the strongest predictor for CXCL8 levels. CONCLUSIONS: The nasopharyngeal microbiota in young infants with RSV infection is marked by an overrepresentation of the genus Haemophilus. We present that this bacterium is associated with viral load and mucosal CXCL8 responses, both which are involved in RSV disease pathogenesis.

Siglec-1 inhibits RSV-induced interferon gamma production by adult T cells in contrast to newborn T cells.

Interferon gamma (IFN-γ) plays an important role in the antiviral immune response during respiratory syncytial virus (RSV) infections. Monocytes and T cells are recruited to the site of RSV infection, but it is unclear whether cell-cell interactions between monocytes and T cells regulate IFN-γ production. In this study, micro-array data identified the upregulation of sialic acid-binding immunoglobulin-type lectin 1 (Siglec-1) in human RSV-infected infants. In vitro, RSV increased expression of Siglec-1 on healthy newborn and adult monocytes. RSV-induced Siglec-1 on monocytes inhibited IFN-γ production by adult CD4+ T cells. In contrast, IFN-γ production by RSV in newborns was not affected by Siglec-1. The ligand for Siglec-1, CD43, is highly expressed on adult CD4+ T cells compared to newborns. Our data show that Siglec-1 reduces IFN-γ release by adult T cells possibly by binding to the highly expressed CD43. The Siglec-1-dependent inhibition of IFN-γ in adults and the low expression of CD43 on newborn T cells provides a better understanding of the immune response against RSV in early life and adulthood.

High pneumococcal density correlates with more mucosal inflammation and reduced respiratory syncytial virus disease severity in infants.

BACKGROUND: Respiratory syncytial virus (RSV) is an important cause of lower respiratory tract infections in infants. A small percentage of the infected infants develops a severe infection, while most of these severely ill patients were previously healthy. It remains unclear why these children develop severe RSV infections. In this study, we investigate whether pneumococcal nasopharyngeal carriage patterns correlate with mucosal inflammation and severity of disease. METHODS: In total, 105 infants hospitalized with RSV infection were included and recovery samples were taken from 42 patients. The presence and density of Streptococcus pneumoniae was determined by RT qPCR to study its relation to viral load, inflammation (MMP-9 and IL-6) and severity of RSV disease. RESULTS: We show that pneumococcal presence or absence in the nasopharynx does not correlate with viral load, inflammation or severity of disease. However, when pneumococcus is present in patients, a higher nasopharyngeal pneumococcal density was correlated with a higher RSV load, higher MMP-9 levels and a less severe course of disease. CONCLUSIONS: Our results show correlations between S. pneumoniae density and viral load, inflammation and disease severity, suggesting that pneumococcal density may be an indicator for severity in paediatric RSV disease.

Mucosal IgG Levels Correlate Better with Respiratory Syncytial Virus Load and Inflammation than Plasma IgG Levels.

Maternal vaccination is currently considered a strategy against respiratory syncytial virus (RSV) infections. In RSV-infected infants, high mucosal IgG levels correlated better with reduced RSV load and lower mucosal CXCL10 levels than plasma IgG levels. For future vaccination strategies against RSV, more focus should be on the mucosal humoral immune response.

Antibodies enhance CXCL10 production during RSV infection of infant and adult immune cells.

Respiratory syncytial virus (RSV) bronchiolitis is a major burden in infants below three months of age, when the primary immune response is mainly dependent on innate immunity and maternal antibodies. We investigated the influence of antibodies on innate immunity during RSV infection. PBMCs from infants and adults were stimulated with live RSV and inactivated RSV in combination with antibody-containing and antibody-depleted serum. The immune response was determined by transcriptome analysis and chemokine levels were measured using ELISA and flow cytometry. Microarray data showed that CXCL10 gene transcription was RSV dependent, whereas CXCL11 and IFNα were upregulated in an antibody-dependent manner. Although the presence of antibodies reduces RSV infection rate, it enhances the innate immune response. In adult immune cells, antibodies enhance CXCL10, CXCL11, IFNα and IFNγ production in response to RSV infection. Contrary, in infant immune cells only CXCL10 was enhanced in an antibody-dependent manner. Monocytes are the main source of CXCL10 and they produce CXCL10 in both an antibody- and virus-dependent manner. This study shows that antibodies enhance CXCL10 production in infant immune cells. CXCL10 has been implicated in exuberating the inflammatory response during viral infections and antibodies could therefore play a role in the pathogenesis of RSV infections.

Recognition of Streptococcus pneumoniae and muramyl dipeptide by NOD2 results in potent induction of MMP-9, which can be controlled by lipopolysaccharide stimulation.

Matrix metallopeptidase 9 (MMP-9) is a protease involved in the degradation of extracellular matrix collagen. Evidence suggests that MMP-9 is involved in pathogenesis during Streptococcus pneumoniae infection. However, not much is known about the induction of MMP-9 and the regulatory processes involved. We show here that the Gram-positive bacteria used in this study induced large amounts of MMP-9, in contrast to the Gram-negative bacteria that were used. An important pathogen-associated molecular pattern (PAMP) for Gram-positive bacteria is muramyl dipeptide (MDP). MDP is a very potent inducer of MMP-9 and showed a dose-dependent MMP-9 induction. Experiments using peripheral blood mononuclear cells (PBMCs) from Crohn's disease patients with nonfunctional NOD2 showed that MMP-9 induction by Streptococcus pneumoniae and MDP is NOD2 dependent. Increasing amounts of lipopolysaccharide (LPS), an important PAMP for Gram-negative bacteria, resulted in decreasing amounts of MMP-9. Moreover, the induction of MMP-9 by MDP could be counteracted by simultaneously adding LPS. The inhibition of MMP-9 expression by LPS was found to be regulated posttranscriptionally, independently of tissue inhibitor of metalloproteinase 1 (TIMP-1), an endogenous inhibitor of MMP-9. Collectively, these data show that Streptococcus pneumoniae is able to induce large amounts of MMP-9. These high MMP-9 levels are potentially involved in Streptococcus pneumoniae pathogenesis.

Bacterial lipopolysaccharide inhibits influenza virus infection of human macrophages and the consequent induction of CD8+ T cell immunity.

It is well established that infection with influenza A virus (IAV) facilitates secondary bacterial disease. However, there is a growing body of evidence that the microbial context in which IAV infection occurs can affect both innate and adaptive responses to the virus. To date, these studies have been restricted to murine models of disease and the relevance of these findings in primary human cells remains to be elucidated. Here, we show that pre-stimulation of primary human monocyte-derived macrophages (MDMs) with the bacterial ligand lipopolysaccharide (LPS) reduces the ability of IAV to infect these cells. The inhibition of IAV infection was associated with a reduced transcription of viral RNA and the ability of LPS to induce an anti-viral/type I interferon response in human MDMs. We demonstrated that this reduced rate of viral infection is associated with a reduced ability to present a model antigen to autologous CD8+ T cells. Taken together, these data provide the first evidence that exposure to bacterial ligands like LPS can play an important role in modulating the immune response of primary human immune cells towards IAV infection, which may then have important consequences for the development of the host's adaptive immune response.

Fc gamma receptors in respiratory syncytial virus infections: implications for innate immunity.

RSV infections are a major burden in infants less than 3 months of age. Newborns and infants express a distinct immune system that is largely dependent on innate immunity and passive immunity from maternal antibodies. Antibodies can regulate immune responses against viruses through interaction with Fc gamma receptors leading to enhancement or neutralization of viral infections. The mechanisms underlying the immunomodulatory effect of Fc gamma receptors on viral infections have yet to be elucidated in infants. Herein, we will discuss current knowledge of the effects of antibodies and Fc gamma receptors on infant innate immunity to RSV. A better understanding of the pathogenesis of RSV infections in young infants may provide insight into novel therapeutic strategies such as vaccination.

A Novel Method Linking Antigen Presentation by Human Monocyte-Derived Macrophages to CD8(+) T Cell Polyfunctionality.

To understand the interactions between innate and adaptive immunity, and specifically how virally infected macrophages impact T cell function, novel assays examining the ability of macrophages to present antigen to CD8(+) T cells are needed. In the present study, we have developed a robust in vitro assay to measure how antigen presentation by human monocyte-derived macrophages (MDMs) affects the functional capacity of autologous CD8(+) T cells. The assay is based on the polyfunctional characteristics of antigen-specific CD8(+) T cells, and is thus called a Mac-CD8 Polyfunctionality Assay. Following purification of monocytes and their maturation to MDMs, MDMs were pulsed with an antigenic peptide to be presented to CD8(+) T cells. Peptide-pulsed MDMs were then incubated with antigen-specific CD8(+) T cells in order to assess the efficacy of antigen presentation to T cells. CD8(+) T cell polyfunctionality was assessed by staining with mAbs to IFN-γ, TNF-α, and CD107a in a multi-color intracellular cytokine staining assay. To highlight the utility of the Mac-CD8 Polyfunctionality Assay, we assessed the effects of influenza infection on the ability of human macrophages to present antigen to CD8(+) T cells. We found that influenza infection of human MDMs can alter the effector efficacy of MDMs to activate more CD8(+) T cells with cytotoxic capacity. This has important implications for understanding how the virus-infected macrophages affect adaptive immunity at the site of infection.

An in vitro model to study immune responses of human peripheral blood mononuclear cells to human respiratory syncytial virus infection.

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology. Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

Respiratory syncytial virus infection augments NOD2 signaling in an IFN-ß-dependent manner in human primary cells.

Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in infants, with remarkable variability in disease severity. An exaggerated proinflammatory response and influx of leukocytes is part of the pathogenesis of severe RSV disease. Here, we show an increase in proinflammatory cytokine production by human immune cells after stimulation with RSV and muramyl dipeptide (MDP), which is recognized by nucleotide-binding oligomerization domain containing 2 (NOD2). PBMCs from Crohn's disease patients homozygous for the 3020insC mutation in the NOD2 gene did not show a synergistic response to stimulation with RSV and MDP, suggesting that NOD2 is essential for the observed synergy. Further experiments aimed at identifying the viral ligand indicated that viral RNA plays an essential role in the recognition of RSV. Stimulation with RSV or Poly(I:C) induced IFN-ß expression, which resulted in an increased expression of the viral receptors TLR3 and RIG-I, as well as an increased NOD2 expression. Our data indicate that IFN-ß induction by viral RNA is an essential first step in the increased proinflammatory response to MDP. We hypothesize that the enhanced proinflammatory response to MDP following RSV infection may be an important factor in determining the outcome of the severity of disease.