Murine Model Of Respiratory Infection Research Articles

Mechanisms of immunity to the respiratory pathogen Bordetella pertussis in normal and gene knockout mice: clearance of primary infection is not enhanced by therapeutic interleukin-12.

The current understanding of the function of CD4' T helper (Th) cells in immunity to infectious diseases is that antigen-specific Thl cells which secrete interferon (IFN)-y, interleukin (IL)-2 and tumour necrosis factor-/3 mediate cellular immunity, delayed type hypersensitivity and inflammatory responses, whereas Th2 cells which secrete IL-4, IL-5, and IL-6 are considered to be mainly responsible for the provision of specific T cell help for antibody production. Using a murine respiratory infection model we have demonstrated that CD4+ Thl cells play a key role in the protective immune response to B. pertussis generated by natural infection. [ 1, 21. We have also examined the immune responses and outcome of challenge following immunization with whole cell and acellular pertussis vaccines in our murine respiratory model. The rapid bacterial clearance observed in the lungs of mice which received the whole cell vaccine correlated with the induction of a Thl type response[3]. In contrast an acellular vaccine generated Th2 cells and was associated with delayed clearance. IL-12 is a key cytokine in the development of Thl type responses, and is secreted by activated macrophages, B cells and dendritic cells. It has been shown to exert pleiotropic effects on cells of the immune system. IL-12 can induce the secretion of IFN-y by natural killer cells and by CD4+ T cells and can promote the differentiation and development of Thl cells from Tho precursor populations [4]. Recently we demonstrated that intracellular infection of alveolar or other macrophages by B. pertussis or exposure to killed B. pertussis or purified LPS results in the production of IL-12 151. Furthermore the addition of IL-12 to an acellular vaccine switched the T cell response induced from Th2 to Th 1 and enhanced clearance of bacteria from the lungs of normal BALB/c mice [ 5 ] . In the present study we have examined the effect of therapeutic administration of IL-12 during primary respiratory infection of immunologically naive BALB/c mice. Mice which received a single intraperitoneal administration of recombinant murine IL-12 before aerosol challenge followed by a daily administration of 1.0 ng IL-12 showed no significant enhancement of bacterial clearance from the lungs (Fig. IA). Delaying administration of IL-12 until day 1 1 post aerosol challenge again resulted in no significant alteration in the kinetics of bacterial clearance (data not shown). Indeed administration of higher doses of IL-12 (50 ng) at longer time-intervals (Fig. 1B) gave rise to greater numbers of viable B. pertussis recovered from the lungs at early time points after challenge. Initial studies on the kinetics of pulmonary clearance of B. pertussis in gene knockout mice defective for the IFN-y receptor revealed increased pathology, but no difference in bacterial elimination from the lungs. Taken together these findings indicate that although 1L-12 may play an important role in the induction phase of vaccine mediated immunity against B. pertussis, the therapeutic administration of exogenous 11.-12 cannot improve the rate of bacterial clearance from the lungs of naive animals.

Bordetella pertussis respiratory infection in children is associated with preferential activation of type 1 T helper cells.

The mechanism of protective immunity against Bordetella pertussis generated following recovery from whooping cough in childhood has not yet been elucidated. Studies with a murine respiratory infection model have indicated that cellular immunity, mediated by Th1 cells, plays a role in the clearance of a primary infection with B. pertussis and in protection against subsequent challenge. In the present study, the induction of B. pertussis-specific Th cell subsets in children was examined. Peripheral blood mononuclear cells from B. pertussis-infected or convalescent children proliferated and secreted cytokines following antigen stimulation in vitro. In contrast, responses were weak or undetectable in the majority of children who had not been infected or vaccinated. In all cases, responding T cells produced interferon-gamma but low or undetectable interleukin-5. The findings suggest that Th1 cells may play a role in protective immunity generated following infection with B. pertussis in children.

Interleukin-12 is produced by macrophages in response to live or killed Bordetella pertussis and enhances the efficacy of an acellular pertussis vaccine by promoting induction of Th1 cells.

Using a murine respiratory infection model, we have demonstrated previously that infection with Bordetella pertussis or immunization with a whole-cell pertussis vaccine induced antigen-specific Th1 cells, which conferred a high level of protection against aerosol challenge. In contrast, immunization with an acellular vaccine, consisting of the B. pertussis components detoxified pertussis toxin, filamentous hemagglutinin, and pertactin adsorbed to alum, generated Th2 cells and was associated with delayed bacterial clearance following challenge. In this study, we demonstrated that addition of interleukin-12 (IL-12) either in vitro or in vivo enhanced type 1 T-cell cytokine responses induced with an acellular vaccine. Furthermore, the rate of bacterial clearance in mice coinjected with IL-12 and the acellular vaccine was similar to that observed following immunization with a potent whole-cell vaccine. Analysis of IL-12 secretion by murine macrophages suggested that this cytokine is produced in vivo following B. pertussis infection or immunization with the whole-cell vaccine. IL-12 was detected in the supernatants of lung, splenic, and peritoneal macrophages infected with live B. pertussis or stimulated with heat-killed whole B. pertussis or B. pertussis lipopolysaccharide. In contrast, IL-12 could not be detected following stimulation of macrophages with the bacterial antigens filamentous hemagglutinin, detoxified pertussis toxin, and pertactin, the components of acellular vaccines. Our findings suggest that induction of endogenous IL-12 may contribute to the high efficacy of pertussis whole-cell vaccines and also demonstrate that it is possible to attain these high levels of protection with a less reactogenic acellular vaccine incorporating IL-12 as an adjuvant.

In vivo and in vitro analysis of Bordetella pertussis catalase and Fe-superoxide dismutase mutants

Bordetella pertussis produces a catalase and a Fe-superoxide dismutase. The importance of these enzymes in virulence was investigated, in vitro as well as in vivo, by using mutants deficient in their production. The catalase-deficient mutant survived within polymorphonuclear leukocytes, killed J774A. 1 macrophages through apoptosis, and behaved as the parental strain in a murine respiratory infection model. These results suggest no direct role for catalase in B. pertussis virulence. The absence of expression of Fe-superoxide dismutase had profound effects on the bacterium including a reduced ability to express adenylate cyclase-hemolysin and pertactin, two factors important for B. pertussis pathogenesis. The Fe-superoxide dismutase-deficient mutant also had decreased abilities to colonize and persist in the murine respiratory infection model.

In vivo and in vitro analysis of Bordetella pertussis catalase and Fe-superoxide dismutase mutants.

Bordetella pertussis produces a catalase and a Fe-superoxide dismutase. The importance of these enzymes in virulence was investigated, in vitro as well as in vivo, by using mutants deficient in their production. The catalase-deficient mutant survived within polymorphonuclear leukocytes, killed J774A.1 macrophages through apoptosis, and behaved as the parental strain in a murine respiratory infection model. These results suggest no direct role for catalase in B. pertussis virulence. The absence of expression of Fe-superoxide dismutase had profound effects on the bacterium including a reduced ability to express adenylate cyclase-hemolysin and pertactin, two factors important for B. pertussis pathogenesis. The Fe-superoxide dismutase-deficient mutant also had decreased abilities to colonize and persist in the murine respiratory infection model.

Th1/Th2 cell dichotomy in acquired immunity to Bordetella pertussis: variables in the in vivo priming and in vitro cytokine detection techniques affect the classification of T‐cell subsets as Th1, Th2 or Th0

In studies of the mechanism of immunity to Bordetella pertussis in a murine respiratory infection model, we have previously demonstrated that natural infection of immunization with a whole cell vaccine induces a potent protective immune response, which is mediated by T-helper type-1 (Th1) cells. In contrast an acellular vaccine generates Th2 cells and is associated with delayed bacterial clearance following respiratory challenge. In the present study we have investigated the apparent Th1/Th2 cell dichotomy in acquired immunity and have examined the factors that affect their induction or detection. The cytokine profiles of B. pertussis-specific T cells in immune animals were determined using antigen-stimulated ex vivo spleen cells or CD4+ T-cell lines and clones established in the presence of interleukin-2 (IL-2) or IL-4. Antigen-specific T cells derived from mice immunized with the acellular vaccine were almost exclusively of the Th2 cell type. In contrast, T-cell lines and clones established following respiratory infection or immunization with the whole cell vaccine were predominantly of the Th1 type. However, a proportion of T cells from convalescent mice, especially when cultured in the presence of IL-4, secreted IL-4 and IL-5 with or without detectable IL-2 and interferon-gamma (IFN-gamma), suggesting that Th0 or Th2 cells were also primed during natural infection in vivo. Furthermore, when mice were assessed 6 months after infection, spleen cells produced significant levels of IL-4 and IL-5, which were not evident at 6 weeks. The route of immunization and the genetic background of the mice were also found to influence the preferential priming of Th1 cells, and this was directly related to the level of protection against respiratory or intracerebral (i.c.) challenge. Our findings underline the critical role of CD4+ Th1 cells in immunity to B. pertussis, but also demonstrate that a number of factors in the in vivo priming and in vitro restimulation can skew the apparent dominance of one Th cell type over another.

Cell-mediated immunity to Bordetella pertussis: role of Th1 cells in bacterial clearance in a murine respiratory infection model.

A murine respiratory infection model was used to study the mechanism of protective immunity to Bordetella pertussis. We found that nude mice, which are deficient in T cells, developed a persistent infection and failed to clear the bacteria after aerosol inoculation. In contrast, normal adult nonimmune mice cleared a respiratory infection approximately 35 days after challenge. Before bacterial clearance, antipertussis antibody levels in serum were low or undetectable, whereas consistent antigen-specific T-cell responses were demonstrated throughout the course of infection. The in vitro responses detected in immune spleen cells were mediated by a population of CD4+ major histocompatibility complex class II-restricted Th1-like cells that secreted interleukin-2 and gamma interferon but not interleukin-4. Adoptive transfer of immune spleen cells into nude or sublethally irradiated immunosuppressed mice before challenge resulted in bacterial clearance within 14 to 21 days. In contrast, injection of serum from convalescent mice before challenge only marginally reduced the bacterial load early in the course of infection. Furthermore, transfer of enriched T cells or purified CD4+ T cells but not CD8+ T cells from immune mice conferred a high level of protection. Recipients of CD4+ T cells cleared the bacteria from the lungs within 20 days of challenge, at which time B. pertussis-specific antibodies in the serum were undetectable. Although we do not rule out a contribution of mucosal immunoglobulin A, our findings suggest that cellular responses mediated by CD4+ Th1 cells play an important role in protective immunity to B. pertussis.