Mathieu Bangert

Controlled Human Infection and Rechallenge with Streptococcus pneumoniae Reveals the Protective Efficacy of Carriage in Healthy Adults

RATIONALE The immunological and protective role of pneumococcal carriage in healthy adults is not known, but high rates of disease and death in the elderly are associated with low carriage prevalence. OBJECTIVES We employed an experimental human pneumococcal carriage model to investigate the immunizing effect of a single carriage episode. METHODS Seventy healthy adults were challenged, and of those with carriage, 10 were rechallenged intranasally with live 6B Streptococcus pneumoniae up to 11 months after clearance of the first carriage episode. Serum and nasal wash antibody responses were measured before and after each challenge. MEASUREMENTS AND MAIN RESULTS A total of 29 subjects were experimentally colonized. No subjects were colonized by experimental rechallenge, demonstrating the protective effect of initial carriage against subsequent infection. Carriage increased both mucosal and serum IgG levels to pneumococcal proteins and polysaccharide, resulting in a fourfold increase in opsonophagocytic activity. Importantly, passive transfer of postcarriage sera from colonized subjects conferred 70% protection against lethal challenge by a heterologous strain in a murine model of invasive pneumococcal pneumonia. These levels were significantly higher than the protection conferred by either precarriage sera (30%) or saline (10%). CONCLUSIONS Experimental human carriage resulted in mucosal and systemic immunological responses that conferred protection against recolonization and invasive pneumococcal disease. These data suggest that mucosal pneumococcal vaccination strategies may be important for vulnerable patient groups, particularly the elderly, who do not sustain carriage.

Capsule type of Streptococcus pneumoniae determines growth phenotype.

The polysaccharide capsule of Streptococcus pneumoniae defines over ninety serotypes, which differ in their carriage prevalence and invasiveness for poorly understood reasons. Recently, an inverse correlation between carriage prevalence and oligosaccharide structure of a given capsule has been described. Our previous work suggested a link between serotype and growth in vitro. Here we investigate whether capsule production interferes with growth in vitro and whether this predicts carriage prevalence in vivo. Eighty-one capsule switch mutants were constructed representing nine different serotypes, five of low (4, 7F, 14, 15, 18C) and four of high carriage prevalence (6B, 9V, 19F, 23F). Growth (length of lag phase, maximum optical density) of wildtype strains, nontypeable mutants and capsule switch mutants was studied in nutrient-restricted Lacks medium (MLM) and in rich undefined brain heart infusion broth supplemented with 5% foetal calf serum (BHI+FCS). In MLM growth phenotype depended on, and was transferred with, capsule operon type. Colonization efficiency of mouse nasopharynx also depended on, and was transferred with, capsule operon type. Capsule production interfered with growth, which correlated inversely with serotype-specific carriage prevalence. Serotypes with better growth and higher carriage prevalence produced thicker capsules (by electron microscopy, FITC-dextran exclusion assays and HPLC) than serotypes with delayed growth and low carriage prevalence. However, expression of cpsA, the first capsule gene, (by quantitative RT-PCR) correlated inversely with capsule thickness. Energy spent for capsule production (incorporation of H3-glucose) relative to amount of capsule produced was higher for serotypes with low carriage prevalence. Experiments in BHI+FCS showed overall better bacterial growth and more capsule production than growth in MLM and differences between serotypes were no longer apparent. Production of polysaccharide capsule in S. pneumoniae interferes with growth in nutrient-limiting conditions probably by competition for energy against the central metabolism. Serotype-specific nasopharyngeal carriage prevalence in vivo is predicted by the growth phenotype.

Experimental Human Pneumococcal Carriage Augments IL-17A-dependent T-cell Defence of the Lung

Pneumococcal carriage is both immunising and a pre-requisite for mucosal and systemic disease. Murine models of pneumococcal colonisation show that IL-17A-secreting CD4+ T-cells (Th-17 cells) are essential for clearance of pneumococci from the nasopharynx. Pneumococcal-responding IL-17A-secreting CD4+ T-cells have not been described in the adult human lung and it is unknown whether they can be elicited by carriage and protect the lung from pneumococcal infection. We investigated the direct effect of experimental human pneumococcal nasal carriage (EHPC) on the frequency and phenotype of cognate CD4+ T-cells in broncho-alveolar lavage and blood using multi-parameter flow cytometry. We then examined whether they could augment ex vivo alveolar macrophage killing of pneumococci using an in vitro assay. We showed that human pneumococcal carriage leads to a 17.4-fold (p = 0.007) and 8-fold (p = 0.003) increase in the frequency of cognate IL-17A+ CD4+ T-cells in BAL and blood, respectively. The phenotype with the largest proportion were TNF+/IL-17A+ co-producing CD4+ memory T-cells (p<0.01); IFNγ+ CD4+ memory T-cells were not significantly increased following carriage. Pneumococci could stimulate large amounts of IL-17A protein from BAL cells in the absence of carriage but in the presence of cognate CD4+ memory T-cells, IL-17A protein levels were increased by a further 50%. Further to this we then show that alveolar macrophages, which express IL-17A receptors A and C, showed enhanced killing of opsonised pneumococci when stimulated with rhIL-17A (p = 0.013). Killing negatively correlated with RC (r = −0.9, p = 0.017) but not RA expression. We conclude that human pneumococcal carriage can increase the proportion of lung IL-17A-secreting CD4+ memory T-cells that may enhance innate cellular immunity against pathogenic challenge. These pathways may be utilised to enhance vaccine efficacy to protect the lung against pneumonia.

The Integrins Mac-1 and α4β1 Perform Crucial Roles in Neutrophil and T Cell Recruitment to Lungs during Streptococcus pneumoniae Infection

Neutrophils and T cells play an important role in host protection against pulmonary infection caused by Streptococcus pneumoniae. However, the role of the integrins in recruitment of these cells to infected lungs is not well understood. In this study we used the twin approaches of mAb blockade and gene-deficient mice to investigate the relative impact of specific integrins on cellular recruitment and bacterial loads following pneumococcal infection. We find that both Mac-1 (CD11b/CD18) and α4β1 (CD49d/CD29) integrins, but surprisingly not LFA-1 (CD11a/CD18), contribute to two aspects of the response. In terms of recruitment from the circulation into lungs, neutrophils depend on Mac-1 and α4β1, whereas the T cells are entirely dependent on α4β1. Second, immunohistochemistry results indicate that adhesion also plays a role within infected lung tissue itself. There is widespread expression of ICAM-1 within lung tissue. Use of ICAM-1−/− mice revealed that neutrophils make use of this Mac-1 ligand, not for lung entry or for migration within lung tissue, but for combating the pneumococcal infection. In contrast to ICAM-1, there is restricted and constitutive expression of the α4β1 ligand, VCAM-1, on the bronchioles, allowing direct access of the leukocytes to the airways via this integrin at an early stage of pneumococcal infection. Therefore, integrins Mac-1 and α4β1 have a pivotal role in prevention of pneumococcal outgrowth during disease both in regulating neutrophil and T cell recruitment into infected lungs and by influencing their behavior within the lung tissue itself.

A new protective role for S100A9 in regulation of neutrophil recruitment during invasive pneumococcal pneumonia

The S100A8/A9 heterodimer is abundantly expressed by myeloid cells, especially neutrophils, but its mechanism of action is only partially determined. In this study we investigated S100A8/A9 involvement in the host response to Streptococcus pneumoniae infection making use of S100a9–/– mice that lack heterodimer expression in myeloid cells. S100a9–/–mice that were infected intranasally with pneumococci rapidly succumbed, with 80% mortality after 48 h, whereas the majority of wild‐type mice recovered. Over this time period, S100a9–/– mice displayed an average 6‐fold reduction in circulating and lung‐recruited neutrophils. Taqman analysis of S100a9–/– lungs revealed decreased production of a dominant subset of 5 cytokines and chemokines associated with neutrophil recruitment. The greatest differential was with the cytokine granulocyte colony‐stimulating factor (G‐CSF) that causes bone marrow release of neutrophils into the circulation (1900‐fold difference at 48 h). Treating S100a9–/– mice with G‐CSF reversed their increased susceptibility to infection by enhancing both circulating neutrophils and neutrophil recruitment into infected lungs, by reducing pneumococcal colony forming units, and by elevation of chemokine CXCL1, cytokine IL‐6, and endogenous G‐CSF proteins. Thus S100A9, potentially with its partner S100A8, makes a major contribution in the host response to pneumococcal infection by increasing circulating neutrophils principally regulation of G‐CSF production.—De Filippo, K., Neill, D. R., Mathies, M., Bangert, M., McNeill, E., Kadioglu, A., Hogg, N. A new protective role for S100A9 in regulation of neutrophil recruitment during invasive pneumococcal pneumonia. FASEB J. 28, 3600–3608 (2014). www.fasebj.org

Airborne dust and high temperatures are risk factors for invasive bacterial disease

Background: The Sahel region of West Africa has the highest bacterial meningitis attack and case fatality rate in the world. The effect of climatic factors on patterns of invasive respiratory bacterial disease is not well documented. Objective: We aimed to assess the link between climatic factors and occurrence of invasive respiratory bacterial disease in a Sahel region of Niger. Methods: We conducted daily disease surveillance and climatic monitoring over an 8‐year period between January 1, 2003, and December 31, 2010, in Niamey, Niger, to determine risk factors for bacterial meningitis and invasive bacterial disease. We investigated the mechanistic effects of these factors on Streptococcus pneumoniae infection in mice. Results: High temperatures and low visibility (resulting from high concentrations of airborne dust) were identified as significant risk factors for bacterial meningitis. Dust inhalation or exposure to high temperatures promoted progression of stable asymptomatic pneumococcal nasopharyngeal carriage to pneumonia and invasive disease. Dust exposure significantly reduced phagocyte‐mediated bacterial killing, and exposure to high temperatures increased release of the key pneumococcal toxin pneumolysin through increased bacterial autolysis. Conclusion: Our findings show that climatic factors can have a substantial influence on infectious disease patterns, altering density of pneumococcal nasopharyngeal carriage, reducing phagocytic killing, and resulting in increased inflammation and tissue damage and consequent invasiveness. Climatic surveillance should be used to forecast invasive bacterial disease epidemics, and simple control measures to reduce particulate inhalation might reduce the incidence of invasive bacterial disease in regions of the world exposed to high temperatures and increased airborne dust.

P4-Mediated Antibody Therapy in an Acute Model of Invasive Pneumococcal Disease

New treatments against severe bacterial infections are needed because the response to antibiotic treatment is slow in acute settings and is becoming less effective owing to the emergence of antibiotic-resistant pathogens. P4-mediated antibody therapy offers a unique treatment strategy that combines exogenous immunoglobulin with the immunoactivating peptide P4. In an acute model of pneumococcal disease, mice were infected with Streptococcus pneumoniae and treated intravenously or intranasally with P4 and intravenous immunoglobulin (IVIG). Survival of P4-IVIG-treated mice increased from 0% to 60% among those that received intravenous treatment and from 0% to 100% among those that received intranasal treatment. Importantly, intranasal administration of P4 at an early stage of infection prevented the onset of bacteremia and sepsis. Increased survival was associated with reduced bacterial burden in affected tissues and with recruitment and activation of professional phagocytes, as manifested by increased expression of Fc-γ receptors. In vitro studies involving P4-stimulated alveolar, peritoneal, and J774.2 murine macrophages showed an increased ability of these immune cells to phagocytose pneumococci independent of capsule. The use of adjunct antibody therapies to treat infectious diseases shows promise.

Use of 2 pneumococcal common protein real-time polymerase chain reaction assays in healthy children colonized with Streptococcus pneumoniae.

Polymerase chain reaction (PCR) offers promise in pneumococcal diagnostics, but whether nasopharyngeal carriage causes false-positive results in people colonized with Streptococcus pneumoniae is unknown. We found in serum no positive samples for pneumococcal DNA in 100 carriers and noncarriers using 2 different real-time PCR assays targeting lytA and psaA genes.

Nicotinamide nucleotide transhydrogenase (NNT) acts as a novel modulator of macrophage inflammatory responses

Nicotinamide nucleotide transhydrogenase (NNT) is a mitochondrial redox‐driven proton pump that couples the production of NADPH to the mitochondrial metabolic rate. In this study, we demonstrated for the first time that NNT has a significant effect in the modulation of the immune response and host defense against pathogens. We found that NNT mRNA is enriched in immune system‐related tissues and regulated during macrophage activation. Overexpression of NNT in a macrophage cell‐line resulted in decreased levels of reactive oxygen species (ROS) and nitric oxide upon induction of the macrophage inflammatory responses. These cells failed to fully activate MAPK signaling pathways, resulting in defective secretion of proinflammatory cytokines in response to LPS, and were inefficient in clearance of intracellular bacteria. We have shown that C57BL/6J mice, which have a deletion in the Nnt gene, exhibited greater resistance to acute pulmonary infection with Streptococcus pneumoniae. Macrophages from these mice generated more ROS and established a stronger inflammatory response to this pathogen. Our results demonstrate a novel role for NNT as a regulator of macrophage‐mediated inflammatory responses.—Ripoll, V. M., Meadows, N. A., Bangert, M., Lee, A. W., Kadioglu, A., Cox, R. D. Nicotinamide nucleotide transhydrogenase (NNT) acts as a novel modulator of macrophage inflammatory responses. FASEB J. 26, 3550–3562 (2012). www.fasebj.org

Immunoactivating Peptide P4 Augments Alveolar Macrophage Phagocytosis in Two Diverse Human Populations

ABSTRACT New treatment strategies are urgently needed to overcome early mortality in acute bacterial infections. Previous studies have shown that administration of a novel immunoactivating peptide (P4) alongside passive immunotherapy prevents the onset of septicemia and rescues mice from lethal invasive disease models of pneumococcal pneumonia and sepsis. In this study, using two diverse populations of adult volunteers, we determined whether P4 treatment of human alveolar macrophages would upregulate phagocytic killing of Streptococcus pneumoniae ex vivo. We also measured macrophage intracellular oxidation, cytokine secretion, and surface marker expression following stimulation. Peptide treatment showed enhanced bacterial killing in the absence of nonspecific inflammation, consistent with therapeutic potential. This is the first demonstration of P4 efficacy on ex vivo-derived human lung cells.