In this study, in line with previous work (24), we observed that anti-capsule response in the mouse model of colonization was extremely low, but antibody responses were induced to multiple protein antigens. nasopharyngeal colonization compared to untreated mice. Colonization induced antibody responses to protein rather than capsular antigen, and when measured using a protein array B cell depletion prior to colonization reduced serum levels of IgG to several protein antigens. However, B cell depleted colonized mice were still partially protected against both lung infection and septicemia when challenged with after reconstitution of their B cells. These data indicate that although B cell depletion markedly Rabbit Polyclonal to KCNJ9 impairs antibody recognition of in colonized mice, some protective immunity is maintained, perhaps mediated by cellular immunity. Keywords: is mediated through several mechanisms. The B1a Purpureaside C B cell subset produces natural IgM antibodies that are largely thought to target cell wall phosphocholine and improve complement-mediated systemic immunity against (23). Asymptomatic nasopharyngeal colonization with can induce antibody to both protein and/or capsular antigens (24C27). Recent data suggests anti-protein antibody probably forms the dominant component of naturally acquired IgG adaptive immunity against in humans (24, 28, 29), and have identified the range of antigens recognized in normal human sera (24, 30, 31). Despite the clinical importance of respiratory pathogens especially in immunosuppressed subjects, at present, there are limited data on the consequences of the different modalities of B cell depletion on antibody-mediated immunity to In this study, we have developed a mouse model of B cell depletion and tested the consequences of low levels of B cells on natural IgM and the development of colonization induced antibody mediated immunity to to subsequent pneumonia challenge. Materials and Methods Bacterial Strains strains D39, BHN418 6B, and TIGR4 were used for this study (capsular serotypes 2, 6B, and 4, respectively). All pneumococcal strains were cultured in Tryptic Soy Broth (TSB, Becton Dickinson) or on blood agar plates consisting of Columbia Agar (Becton Dickinson) supplemented with 3% v/v defibrinated horse blood at 37C in 5% CO2. Animal Models Five-week-old, female, inbred C57Bl/6 mice from Charles River (Margate, Kent CT9 4LT UK) were used in this study. Before use, mice were housed for at least 1 week under standard conditions, in the Biological Service animal facility at the University College of London, according to its guidelines for the maintenance of laboratory animals. No randomization or blinding was performed. All animal procedures were approved by the local ethical review process and conducted in accordance with the relevant, UK Home Office approved, project license (PPL70/6510). For the colonization model, mice were anaesthetized using isoflurane and then inoculated intranasally using a dose of 1 1 x 107 CFU Purpureaside C in 10 l volume (25, 32). For the Purpureaside C pneumonia with secondary septicemia model, mice were anaesthetized using isoflurane and then infected intranasally using a dose of 1 1 x 107 CFU in 50 l volume (25, 32). Mice were culled 24?h after infection. Mouse organs were homogenized in 1?ml of PBS for quantification of colony forming units (CFU) and flow cytometry analysis. Blood samples from mice were collected by tail bleeds or cardiac puncture under terminal anaesthesia, and treated with 100 U/ml of heparin (Sigma Aldrich, UK) to prevent blood coagulation. B Cell Depletion Treatment and Flow Cytometry Analysis of Splenocytes B cell depletion on mice was performed by IV or IP injection of aCD20 antibody (Rat IgG2b, , SA271G2, BioLegend) (33). Different doses were used depending on the route of injection (50C100 g for IV injection and 25C100 g for IP injections). Isotype control rat IgG was used as negative control. The effects Purpureaside C of B cell depletion treatment was analyzed using flow cytometry on splenocytes. Splenocytes were prepared by passing mouse spleens through a cell strainer to obtain single cell suspensions; red blood cells were removed using a.
In this study, in line with previous work (24), we observed that anti-capsule response in the mouse model of colonization was extremely low, but antibody responses were induced to multiple protein antigens