Genetic factors regulating lung vasculature and immune cell functions associate with resistance to pneumococcal infection

Streptococcus pneumoniae is an important human pathogen responsible for high mortality and morbidity worldwide. The susceptibility to pneumococcal infections is controlled by as yet unknown genetic factors. To elucidate these factors could help to develop new medical treatments and tools to identify those most at risk. In recent years genome wide association studies (GWAS) in mice and humans have proved successful in identification of causal genes involved in many complex diseases for example diabetes, systemic lupus or cholesterol metabolism. In this study a GWAS approach was used to map genetic loci associated with susceptibility to pneumococcal infection in 26 inbred mouse strains. As a result four candidate QTLs were identified on chromosomes 7, 13, 18 and 19. Interestingly, the QTL on chromosome 7 was located within S. pneumoniae resistance QTL (Spir1) identified previously in a linkage study of BALB/cOlaHsd and CBA/CaOlaHsd F2 intercrosses. We showed that only a limited number of genes encoded within the QTLs carried phenotype-associated polymorphisms (22 genes out of several hundred located within the QTLs). These candidate genes are known to regulate TGFb signalling, smooth muscle and immune cells functions. Interestingly, our pulmonary histopathology and gene expression data demonstrated, lung vasculature plays an important role in resistance to pneumococcal infection. Therefore we concluded that the cumulative effect of these candidate genes on vasculature and immune cells functions as contributory factors in the observed differences in susceptibility to pneumococcal infection. We also propose that TGFbmediated regulation of fibroblast differentiation plays an important role in development of invasive pneumococcal disease.This work was supported by the European Union-funded Pneumopath Project HEALTH-F3-2009-222983. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer-reviewedPublisher Versio

Variation in Inflammatory Response during Pneumococcal Infection Is Influenced by Host-Pathogen Interactions but Associated with Animal Survival.

Inflammation is a crucial part of innate immune responses but, if imbalanced, can lead to serious clinical conditions or even death. Cytokines regulate inflammation, and studies report their impact on clinical outcome. However, host and pathogen genetic backgrounds influence cytokine production, making it difficult to evaluate which inflammatory profiles (if any) relate to improved prognosis.Streptococcus pneumoniaeis a common human pathogen associated with asymptomatic nasopharyngeal carriage. Infrequently, it can lead to a wide range of diseases with high morbidity and mortality rates. Studies show that both pneumococcal serotype and host genetic background affect the development of disease and contribute to variation in inflammatory responses. In this study, we investigated the impact of the host and pneumococcal genetic backgrounds on pulmonary cytokine responses and their relationship to animal survival. Two inbred mouse strains, BALB/c and CBA/Ca, were infected with 10 pneumococcal strains, and the concentrations of six pulmonary cytokines were measured at 6 h and 24 h postinfection. Collected data were analyzed by principal-component analysis to identify whether there is any pattern in the observed cytokine variation. Our results show that host-pneumococcus combination was at the core of observed variation in cytokine responses, yet the resulting cytokine profile discriminated only between survivors and fatalities but not mouse or pneumococcal strains used during infection. Therefore, our results indicate that although alternative inflammatory profiles are generated during pneumococcal infection, a common pattern emerged, which determined the clinical outcome of pneumococcal infections

Genetic factors regulating lung vasculature and immune cell functions associate with resistance to pneumococcal infection

Streptococcus pneumoniae is an important human pathogen responsible for high mortality and morbidity worldwide. The susceptibility to pneumococcal infections is controlled by as yet unknown genetic factors. To elucidate these factors could help to develop new medical treatments and tools to identify those most at risk. In recent years genome wide association studies (GWAS) in mice and humans have proved successful in identification of causal genes involved in many complex diseases for example diabetes, systemic lupus or cholesterol metabolism. In this study a GWAS approach was used to map genetic loci associated with susceptibility to pneumococcal infection in 26 inbred mouse strains. As a result four candidate QTLs were identified on chromosomes 7, 13, 18 and 19. Interestingly, the QTL on chromosome 7 was located within S. pneumoniae resistance QTL (Spir1) identified previously in a linkage study of BALB/cOlaHsd and CBA/CaOlaHsd F2 intercrosses. We showed that only a limited number of genes encoded within the QTLs carried phenotype-associated polymorphisms (22 genes out of several hundred located within the QTLs). These candidate genes are known to regulate TGFb signalling, smooth muscle and immune cells functions. Interestingly, our pulmonary histopathology and gene expression data demonstrated, lung vasculature plays an important role in resistance to pneumococcal infection. Therefore we concluded that the cumulative effect of these candidate genes on vasculature and immune cells functions as contributory factors in the observed differences in susceptibility to pneumococcal infection. We also propose that TGFbmediated regulation of fibroblast differentiation plays an important role in development of invasive pneumococcal disease

Phenotype-specific genetic polymorphisms within <i>S. pneumoniae</i> resistance QTLs.

<p>The number of phenotype-associated polymorphisms within the coding region of the QTLs and their consequences. The presented number of SNPs includes the SNPs identified in the EMMA mapping and in the analyses of the full-length sequences within the disease QTLs. No of SNPs– number of phenotype-associated SNPs identified within the gene; Chrom – chromosome, non-synon – SNP causing amino acid change, synon – SNP causing silent mutation, intron – SNP within intron, splicing – SNP within splicing site of the gene. EMMA – indicates whether the genome-wide significant SNPs (p-value< 5×10⁻⁸) were identified within the gene during the EMMA mapping: Y-yes, N-no.</p

Differentially expressed genes (DEGs) within candidate susceptibility QTL between mice intranasally infected with <i>S. pneumoniae</i>, D39 and control animals.

<p>Pulmonary gene expression 6 h post-infection is represented as fold change between infected and non-infected animals (BALB/c infection: BALB/c-infected <i>versus</i> PBS-treated, CBA/Ca infection: CBA/Ca infected <i>versus</i> PBS-treated), between PBS-treated control animals (BALB/CBA: BALB/c <i>versus</i> CBA/Ca PBS-treated) and between infected BALB/c and infected CBA/Ca (BALB/CBA infection). Chrom. - chromosome.</p

Survival of tested inbred mouse strains and disease phenotype after intranasal infection with <i>S. pneumoniae</i>, D39.

<p>SE – standard error.</p

Percentage of lung's blood vessel affected by inflammation within perivascular areas after infection with <i>S. pneumoniae</i> D39.

<p>Results are presented for 6-infection for BALB/cOlaHsd (black bars) and CBA/CaOlaHsd (open bars), from two independent experiments, with a total of 5 animals per group. ** p<0.001.</p

Change in gene expression 6-infection of BALB/cOlaHsd (resistant) and CBA/CaOlaHsd (susceptible) with <i>S. pneumoniae</i>, D39.

<p>Fold change is represented as compared to control animals (PBS-treated). Only the ten most up-regulated and ten most down-regulated genes are presented.</p

Inflammation pattern in lungs of BALB/cOlaHsd and CBA/CaOlaHsd mice after infection with <i>S. pneumoniae</i>, D39.

<p>Hematoxylin-and-eosin-stained paraffin wax lung sections from BALB/cOlaHsd (A, A1–C,C1) and CBA/CaOlaHsd (D,D1–F,F1). Panels A,A1 and D,D1 represent lungs of PBS-treated animals, panels B,B1–C,C1 and E,E1–F,F1 are lung sections at 24 h-post infection. Panels A–F represent magnification 40× and panels A1–F1 magnification 200×. Bar indicates 500 µm for 40× magnification and 100 µm for 200× magnification. Black arrows indicate perivascular areas. Yellow arrow in panel F1 indicates perivascular adipose tissue.</p

Differentially expressed genes during intranasal-infection with <i>S. pneumoniae</i>, D39 in both BALB/cOlaHsd and CBA/CaOlaHsd and showing at least two-fold change difference between the two strains during the infection.

<p>Differentially expressed genes during intranasal-infection with <i>S. pneumoniae</i>, D39 in both BALB/cOlaHsd and CBA/CaOlaHsd and showing at least two-fold change difference between the two strains during the infection.</p