Shanie Saghafian-Hedengren

Epstein-Barr Virus Coinfection in Children Boosts Cytomegalovirus-Induced Differentiation of Natural Killer Cells

ABSTRACT During childhood, infections with cytomegalovirus (CMV) and Epstein-Barr virus (EBV) can occur in close temporal proximity. Active, as well as latent, CMV infection is associated with enlarged subsets of differentiated natural killer (NK) and cytotoxic T cells. How EBV infection may influence CMV-driven immune differentiation is not known. We found that EBV coinfection selectively influenced the NK cell compartment of CMV-seropositive (CMV+) children. Coinfected children had significantly higher proportions of peripheral-blood NKG2C+ NK cells than CMV+ EBV− children. Ex vivo NK cell degranulation after target cell stimulation and plasma IL-15 levels were significantly higher in CMV+ children. EBV coinfection was related to the highest levels of plasma interleukin-15 (IL-15) and IL-12p70. Remarkably, in vitro EBV infection of peripheral blood mononuclear cells (PBMC) from EBV− CMV+ children increased NKG2C+ NK cell proportions. A similar tendency was seen in cocultures of PBMC with EBV+ lymphoblastoid B-cell lines (LCL) and IL-15. After K562 challenge, NKG2C+ NK cells excelled in regard to degranulation and production of gamma interferon, regardless of whether there was previous coculture with LCL. Taken together, our data suggest that dual latency with these herpesviruses during childhood could contribute to an in vivo environment supporting differentiation and maintenance of distinct NK cell populations. This viral imprint may affect subsequent immune responses through altered distributions of effector cells.

Early-life EBV infection protects against persistent IgE sensitization

BACKGROUND Infection with EBV has previously been implicated in influencing allergic disorders, but its precise role remains contradictory. The timing of primary infection may contribute to the discrepancies. OBJECTIVE This study aimed at investigating whether the time-point of primary EBV infection during childhood could be of importance in modulating the risk of developing IgE sensitization. METHODS A total of 219 Swedish infants were followed prospectively to 5 years of age with clinical examinations, skin prick testing, specific IgE analyses, and determination of serostatus against EBV. RESULTS After analysis of the childrens EBV serostatus, we found that 5-year-olds who were infected with EBV before the age of 2 years were at a significantly lower risk of being persistently IgE-sensitized-that is, sensitized at both 2 and 5 years of age (adjusted odds ratio, 0.34; 95% CI, 0.12-0.94). In contrast, contraction of EBV after 2 years of age was highly associated with late-onset IgE sensitization (adjusted odds ratio, 4.64; 95% CI, 1.57-13.69). Persistently sensitized 5-year-olds had higher specific-IgE levels than children with late-onset IgE sensitization (P < .01). CONCLUSION Our data support the value of early-life microbial exposure for protection against the development of IgE sensitization and underscore the proximate postnatal years as an important period during which EBV could contribute to an allergo-protective immune profile.

Early-life gut bacteria associate with IL-4-, IL-10- and IFN-γ production at two years of age.

Microbial exposure early in life influences immune maturation and potentially also the development of immune-mediated disease. Here we studied early-life gut colonization in relation to cytokine responses at two years of age. Fecal samples were collected from infants during the first two months of life. DNA was extracted from the fecal samples and Bifidobacterium (B.) adolescentis, B. breve, B. bifidum, a group of lactobacilli (L. casei, L. paracasei and L. rhamnosus) as well as Staphylococcus (S.) aureus were detected with real time PCR. Peripheral mononuclear cells were stimulated with phytohaemagglutinin (PHA) and numbers of IL-4−, IL-10− and IFN-γ secreting cells were evaluated using ELISpot. We further stimulated peripheral blood mononuclear cells with bacterial supernatants in vitro and assessed the IL-4−, IL-10− and IFN-γ inducing capacity by flow cytometry and ELISA. Early S. aureus colonization associated with higher numbers of IL-4− (p = 0.022) and IL-10 (p = 0.016) producing cells at two years of age. In contrast to colonization with S. aureus alone, co-colonization with lactobacilli associated with suppression of IL-4− (p = 0.004), IL-10− (p = 0.004) and IFN-γ (p = 0.034) secreting cells. In vitro stimulations of mononuclear cells with bacterial supernatants supported a suppressive role of L. rhamnosus GG on S. aureus-induced cytokine responses. We demonstrate that the early gut colonization pattern associates with the PHA-induced cytokine profile at two years of age and our in vitro findings support that specific bacterial species influence the T helper cell subsets. This suggests that dysbiosis in the early microbiota may modulate the risk of developing inflammatory conditions like allergy.

Impaired Toll-like receptor 2 signalling in monocytes from 5-year-old allergic children

The relative composition of the two major monocytic subsets CD14+CD16− and CD14+CD16+ is altered in some allergic diseases. These two subsets display different patterns of Toll‐like receptor levels, which could have implications for activation of innate immunity leading to reduced immunoglobulin E‐specific adaptive immune responses. This study aimed to investigate if allergic status at the age of 5 years is linked to differences in monocytic subset composition and their Toll‐like receptor levels, and further, to determine if Toll‐like receptor regulation and cytokine production upon microbial stimuli is influenced by the allergic phenotype. Peripheral blood mononuclear cells from 5‐year‐old allergic and non‐allergic children were stimulated in vitro with lipopolysaccharide and peptidoglycan. Cells were analysed with flow cytometry for expression of CD14, Toll‐like receptors 2 and 4 and p38‐mitogen‐activated protein kinase (MAPK). The release of cytokines and chemokines [tumour necrosis factor, interleukin (IL)‐1β, IL‐6, IL‐8, IL‐10, IL‐12p70] into culture supernatants was measured with cytometric bead array. For unstimulated cells there were no differences in frequency of the monocytic subsets or their Toll‐like receptor levels between allergic and non‐allergic children. However, monocytes from allergic children had a significantly lower up‐regulation of Toll‐like receptor 2 upon peptidoglycan stimulation. Further, monocytes from allergic children had a higher spontaneous production of IL‐6, but there were no differences between the two groups regarding p38‐MAPK activity or cytokine and chemokine production upon stimulation. The allergic subjects in this study have a monocytic population that seems to display a hyporesponsive state as implicated by impaired regulation of Toll‐like receptor 2 upon peptidoglycan stimulation.

Etiology of community acquired pneumonia among children in India: prospective, cohort study.

Background Childhood community acquired pneumonia (CAP) is a significant problem in developing countries, and confirmation of microbial etiology is important for individual, as well as public health. However, there is paucity of data from a large cohort, examining multiple biological specimens for diverse pathogens (bacteria and viruses). The Community Acquired Pneumonia Etiology Study (CAPES) was designed to address this knowledge gap. Methods We enrolled children with CAP (based on WHO IMCI criteria of tachypnea with cough or breathing difficulty) over 24 consecutive months, and recorded presenting symptoms, risk factors, clinical signs, and chest radiography. We performed blood and nasopharyngeal aspirate (NPA) bacterial cultures, and serology (Mycoplasma pneumoniae, Chlamydophila pneumoniae). We also performed multiplex PCR for 25 bacterial/viral species in a subgroup representing 20% of the cohort. Children requiring endotracheal intubation underwent culture and PCR of bronchoalveolar lavage (BAL) specimens. Findings We enrolled 2345 children. NPA and blood cultures yielded bacteria in only 322 (13.7%) and 49 (2.1%) children respectively. In NPA, Streptococcus pneumoniae (79.1%) predominated, followed by Haemophilus influenzae (9.6%) and Staphylococcus aureus (6.8%). In blood, S. aureus (30.6%) dominated, followed by S. pneumoniae (20.4%) and Klebsiella pneumoniae (12.2%). M. pneumoniae and C. pneumoniae serology were positive in 4.3% and 1.1% respectively. Multiplex PCR in 428 NPA specimens identified organisms in 422 (98.6%); of these 352 (82.2%) had multiple organisms and only 70 (16.4%) had a single organism viz. S. pneumoniae: 35 (50%), Cytomegalovirus (CMV): 13 (18.6%), Respiratory Syncytial Virus (RSV): 9 (12.9%), other viruses: 6 (8.7%), S. aureus: 5 (7.1%), and H. influenzae: 2 (2.9%). BAL PCR (n = 30) identified single pathogens in 10 (S. pneumoniae–3, CMV–3, S. aureus–2, H. influenzae–2) and multiple pathogens in 18 children. There were 108 (4.6%) deaths. The pattern of pathogens identified did not correlate with pneumonia severity or mortality. Conclusions The majority of children with CAP have multiple pathogens (bacteria and viruses). S. pneumoniae and S. aureus predominate in NPA and blood respectively. CMV and RSV were the dominant respiratory viruses in NPA and BAL. The presence of multiple pathogens, especially organisms associated with nasopharyngeal carriage, precludes confirmation of a causal relationship in most cases.

Herpesvirus Seropositivity in Childhood Associates with Decreased Monocyte-Induced NK Cell IFN-γ Production

EBV infection is inversely associated with IgE sensitization in children, and this association is further enhanced by CMV coinfection. In mice, herpesvirus latency causes systemic innate activation and protection from bacterial coinfection, implying the importance of herpesviruses in skewing immune responses during latent infection. Early control of viral infections depends on IFN-γ release by NK cells, which generally requires the presence of accessory cells. We investigated IFN-γ production by NK cells in PBMCs from children seropositive (SP) for EBV alone, for both EBV and CMV, or seronegative for both viruses. The ability of classical (CD14++CD16−) and proinflammatory (CD14+CD16+) monocytes to induce autologous NK cell IFN-γ was studied by coculture experiments with enriched CD3−CD56+ cells. Transwell experiments were used to evaluate how monocytes interact with NK cells to induce IFN-γ synthesis. SP children had a significantly reduced proportion of IFN-γ+ NK cells and cognate intracellular IFN-γ levels, which was more pronounced in CMV-coinfected subjects. Also, resting PBMCs of SP children displayed lower proportions of proinflammatory monocytes. IFN-γ production by NK cells was dependent on interactions with monocytes, with the proinflammatory subset inducing the highest IFN-γ. Finally, SP children had markedly lower levels of plasma IFN-γ, concurrent with in vitro findings. Herpesvirus infections could be one contributing factor for maturation toward balanced Th1-Th2 responses. Our data indicate that early infection by herpesviruses may affect NK cell and monocyte interactions and thereby also influence the development of allergies.

Cord blood monocyte subsets are similar to adult and show potent peptidoglycan-stimulated cytokine responses

Human monocytes can be divided into two major subpopulations, CD14++ CD16− and CD14+ CD16+ cells, which are suggested to play different roles in antimicrobial responses. In neonates, characteristics and functional responses of monocyte subsets have not previously been explored, and might contribute to the qualitative difference between neonatal and adult cytokine profiles. We report that at baseline, monocyte subsets in cord blood and adult peripheral blood are present in similar frequencies, and show similar expression of CD11c, CD80/CD86, CD163 and HLA‐DR. In response to the bacterial ligand peptidoglycan, cord blood monocytes had high inherent capacity for production of the early‐response cytokines with levels of tumour necrosis factor and interleukin‐12p70 exceeding adult levels, and also a higher phosphorylation of p38‐mitogen‐activated protein kinase. The CD14+ CD16+ cells expressed more interleukin‐12p70 than CD14++ CD16− cells and were present in a higher frequency in peptidoglycan‐stimulated cord blood mononuclear cell cultures. Together, the behaviour of cord blood CD14+ CD16+ cells following peptidoglycan stimulation might indicate a qualitative difference between the neonatal antimicrobial response and that of the adult. In addition we found that serum factors in cord blood and adult sera affected cytokine production similarly, with the exception of tumour necrosis factor, regardless of the source of serum or cells. Overall, our data provide new insights into monocyte heterogeneity in cord blood and monocyte subset responses to a bacterial ligand at birth.

Cytomegalovirus-Seropositive Children Show Inhibition of In Vitro EBV Infection That Is Associated with CD8+CD57+ T Cell Enrichment and IFN-γ

EBV, a human herpesvirus, is commonly acquired during childhood and persists latently in B cells. EBV seropositivity has been connected to immunomodulatory effects such as altered T and NK cell functional responses as well as protection against early IgE sensitization; however, owing to the asymptomatic presentation during childhood little is known regarding the infection process in children of different ages. In this study, we used mononuclear cells from cord blood and from 2- and 5-y-old EBV-naive children for in vitro EBV infection. We show that the degree of EBV-induced B cell activation and expansion differs between age groups and in particular in relationship to IFN-γ production capacity. EBV infection induced redistribution between B cell subsets with enrichment of IgD+CD27+ cells (commonly referred to as non–switched memory) in infected cord blood cell cultures, and of IgD−CD27+ cells (switched memory) in cell cultures from older children. We also related results to serostatus to CMV, a persistent herpesvirus that can affect differentiation status of T and NK cells. As compared with CMV− children, the EBV-induced enrichment of IgD−CD27+ B cells was significantly reduced in infected cell cultures from CMV+ children. This effect was associated with high levels of IFN-γ and frequencies of highly mature CD8+CD57+ T cells in CMV+ children. Our results demonstrate that both a child’s age and serostatus to CMV will have an impact on EBV-induced B cell activation and expansion, and they point to the ability of viruses with immunomodulatory functions, such as CMV, to affect immune responses within the host system.

Maternal allergy influences p38-mitogen-activated protein kinase activity upon microbial challenge in CD14+ monocytes from 2-year-old children

Background The development of allergic diseases is dependent on genetic and environmental factors. It has been shown previously that cord blood mononuclear cells (CBMCs) from infants with parental allergy have altered cytokine profiles upon bacterial encounter; it might be possible that such impairment persists during the early years of childhood.

Assessment of Cytokine and Chemokine Signatures as Potential Biomarkers of Childhood Community-acquired Pneumonia Severity: A Nested Cohort Study in India

Background: Pediatric community-acquired pneumonia (CAP) is a leading cause of childhood mortality in developing countries. In resource-poor settings, pneumonia diagnosis is commonly made clinically, based on World Health Organization guidelines, where breathing difficulty or cough and age-adjusted tachypnea suffice to establish diagnosis. Also, the severity of CAP is generally based on clinical features and existing biomarkers do not reliably correlate to either clinical severity or outcome. Here, we asked whether systemic immune and inflammatory mediators could act as biomarkers predicting CAP severity or outcome. Methods: Serum from a subset of a CAP cohort (n = 196), enrolled in India, classified according to World Health Organization criteria as having pneumonia or severe pneumonia, was used for simultaneous measurement of 21 systemic cytokines and chemokines. Results: We found significantly higher IL-6, IL-8, IL-13, IFN-&ggr; and lower CCL22 concentrations in patients with severe compared with mild CAP (P values: 0.019, 0.036, 0.006, 0.016 and 0.003, respectively). Based on higher MIP-1&agr;, IL-8, IL-17 or lower CCL22 response pattern at the time of enrolment, children with fatal outcome showed markedly different pattern of inflammatory response compared with children classified with the same disease severity, but with nonfatal outcome (P values: 0.043, 0.017, 0.008 and 0.020, respectively). Conclusions: Our results suggest a relation between an elevated mixed cytokine response and CAP severity on one hand, and a bias toward uncontrolled neutrophilic inflammation in subjects with fatal outcome on the other. Collectively our findings contribute to increased knowledge on new biomarkers that can potentially predict severity and outcome of childhood CAP in the future.