Epicutaneous model of community-acquired Staphylococcus aureus skin infections.

Staphylococcus aureus is one of the most common etiological agents of community-acquired skin and soft tissue infection (SSTI). Although the majority of S. aureus community-acquired SSTIs are uncomplicated and self-clearing in nature, some percentage of these cases progress into life-threatening invasive infections. Current animal models of S. aureus SSTI suffer from two drawbacks: these models are a better representation of hospital-acquired SSTI than community-acquired SSTI, and they involve methods that are difficult to replicate. For these reasons, we sought to develop a murine model of community-acquired methicillin-resistant S. aureus SSTI (CA-MRSA SSTI) that can be consistently reproduced with a high degree of precision. We utilized this model to begin to characterize the host immune response to this type of infection. We infected mice via epicutaneous challenge of the skin on the outer ear pinna using Morrow-Brown allergy test needles coated in S. aureus USA300. When mice were challenged in this model, they developed small, purulent, self-clearing lesions with predictable areas of inflammation that mimicked a human infection. CFU in the ear pinna peaked at day 7 before dropping by day 14. The T(h)1 and T(h)17 cytokines gamma interferon (IFN-γ), interleukin-12 (IL-12) p70, tumor necrosis factor alpha (TNF-α), IL-17A, IL-6, and IL-21 were all significantly increased in the draining lymph node of infected mice, and there was neutrophil recruitment to the infection site. In vivo neutrophil depletion demonstrated that neutrophils play a protective role in preventing bacterial dissemination and fatal invasive infection.

Dissemination bottleneck in a murine model of inhalational anthrax.

Inhalational anthrax is caused by the sporulating bacterium Bacillus anthracis. A current model for progression in mammalian hosts includes inhalation of bacterial spores, phagocytosis of spores in the nasal mucosa-associated lymphoid tissue (NALT) and lungs by macrophages and dendritic cells, trafficking of phagocytes to draining lymph nodes, germination of spores and multiplication of vegetative bacteria in the NALT and lymph nodes, and dissemination of bacteria via the bloodstream to multiple organs. In previous studies, the kinetics of infection varied greatly among mice, leading us to hypothesize the existence of a bottleneck past which very few spores (perhaps only one) progress to allow the infection to proceed. To test this hypothesis, we engineered three strains of B. anthracis Sterne, each marked with a different fluorescent protein, enabling visual differentiation of strains grown on plates. Mice were infected with a mixture of the three strains, the infection was allowed to proceed, and the strains colonizing the organs were identified. Although the inoculum consisted of approximately equal numbers of each of the three strains, the distal organs were consistently colonized by a majority of only one of the three strains, with the dominant strain varying among animals. Such dominance of one strain over the other two was also found at early time points in the cervical lymph nodes but not in the mediastinal lymph nodes. These results support the existence of a bottleneck in the infectious process.

Role of anthrax toxins in dissemination, disease progression, and induction of protective adaptive immunity in the mouse aerosol challenge model.

Anthrax toxins significantly contribute to anthrax disease pathogenesis, and mechanisms by which the toxins affect host cellular responses have been identified with purified toxins. However, the contribution of anthrax toxin proteins to dissemination, disease progression, and subsequent immunity after aerosol infection with spores has not been clearly elucidated. To better understand the role of anthrax toxins in pathogenesis in vivo and to investigate the contribution of antibody to toxin proteins in protection, we completed a series of in vivo experiments using a murine aerosol challenge model and a collection of in-frame deletion mutants lacking toxin components. Our data show that after aerosol exposure to Bacillus anthracis spores, anthrax lethal toxin was required for outgrowth of bacilli in the draining lymph nodes and subsequent progression of infection beyond the lymph nodes to establish disseminated disease. After pulmonary exposure to anthrax spores, toxin expression was required for the development of protective immunity to a subsequent lethal challenge. However, immunoglobulin (immunoglobulin G) titers to toxin proteins, prior to secondary challenge, did not correlate with the protection observed upon secondary challenge with wild-type spores. A correlation was observed between survival after secondary challenge and rapid anamnestic responses directed against toxin proteins. Taken together, these studies indicate that anthrax toxins are required for dissemination of bacteria beyond the draining lymphoid tissue, leading to full virulence in the mouse aerosol challenge model, and that primary and anamnestic immune responses to toxin proteins provide protection against subsequent lethal challenge. These results provide support for the utility of the mouse aerosol challenge model for the study of inhalational anthrax.

Anamnestic protective immunity to Bacillus anthracis is antibody mediated but independent of complement and Fc receptors.

The threat of bioterrorist use of Bacillus anthracis has focused urgent attention on the efficacy and mechanisms of protective immunity induced by available vaccines. However, the mechanisms of infection-induced immunity have been less well studied and defined. We used a combination of complement depletion along with immunodeficient mice and adoptive transfer approaches to determine the mechanisms of infection-induced protective immunity to B. anthracis. B- or T-cell-deficient mice lacked the complete anamnestic protection observed in immunocompetent mice. In addition, T-cell-deficient mice generated poor antibody titers but were protected by the adoptive transfer of serum from B. anthracis-challenged mice. Adoptively transferred sera were protective in mice lacking complement, Fc receptors, or both, suggesting that they operate independent of these effectors. Together, these results indicate that antibody-mediated neutralization provides significant protection in B. anthracis infection-induced immunity.

Protective-antigen (PA) based anthrax vaccines confer protection against inhalation anthrax by precluding the establishment of a systemic infection.

An intense effort has been launched to develop improved anthrax vaccines that confer rapid, long lasting protection preferably with an extended stability profile amenable for stockpiling. Protective antigen (PA)-based vaccines are most favored as immune responses directed against PA are singularly protective, although the actual protective mechanism remains to be unraveled. Herein we show that contrary to the prevailing view, an efficacious PA-based vaccine confers protection against inhalation anthrax by preventing the establishment of a toxin-releasing systemic infection. Equally importantly, antibodies measured by the in vitro lethal toxin neutralization activity assay (TNA) that is considered as a reliable correlate of protection, especially for PA protein-based vaccines adjuvanted with aluminum salts appear to be not absolutely essential for this protective immune response.

Murine aerosol challenge model of anthrax.

The availability of relevant and useful animal models is critical for progress in the development of effective vaccines and therapeutics. The infection of rabbits and non-human primates with fully virulent Bacillus anthracis spores provides two excellent models of anthrax disease. However, the high cost of procuring and housing these animals and the specialized facilities required to deliver fully virulent spores limit their practical use in early stages of product development. Conversely, the small size and low cost associated with using mice makes this animal model more practical for conducting experiments in which large numbers of animals are required. In addition, the availability of knockout strains and well-characterized immunological reagents makes it possible to perform studies in mice that cannot be performed easily in other species. Although we, along with others, have used the mouse aerosol challenge model to examine the outcome of B. anthracis infection, a detailed characterization of the disease is lacking. The current study utilizes a murine aerosol challenge model to investigate disease progression, innate cytokine responses, and histological changes during the course of anthrax after challenge with aerosolized spores. Our results show that anthrax disease progression in a complement-deficient mouse after challenge with aerosolized Sterne spores is similar to that described for other species, including rabbits and non-human primates, challenged with fully virulent B. anthracis. Thus, the murine aerosol challenge model is both useful and relevant and provides a means to further investigate the host response and mechanisms of B. anthracis pathogenesis.

MyD88-dependent signaling contributes to protection following Bacillus anthracis spore challenge of mice: implications for Toll-like receptor signaling.

Bacillus anthracis is a spore-forming, gram-positive organism that is the causative agent of the disease anthrax. Recognition of Bacillus anthracis by the host innate immune system likely plays a key protective role following infection. In the present study, we examined the role of TLR2, TLR4, and MyD88 in the response to B. anthracis. Heat-killed Bacillus anthracis stimulated TLR2, but not TLR4, signaling in HEK293 cells and stimulated tumor necrosis factor alpha (TNF-alpha) production in C3H/HeN, C3H/HeJ, and C57BL/6J bone marrow-derived macrophages. The ability of heat-killed B. anthracis to induce a TNF-alpha response was preserved in TLR2-/- but not in MyD88-/- macrophages. In vivo studies revealed that TLR2-/- mice and TLR4-deficient mice were resistant to challenge with aerosolized Sterne strain spores but MyD88-/- mice were as susceptible as A/J mice. We conclude that, although recognition of B. anthracis occurs via TLR2, additional MyD88-dependent pathways contribute to the host innate immune response to anthrax infection.

Cytokine response to infection with Bacillus anthracis spores.

Bacillus anthracis, the etiological agent of anthrax, is a gram-positive, spore-forming bacterium. The inhalational form of anthrax is the most severe and is associated with rapid progression of the disease and the outcome is frequently fatal. Transfer from the respiratory epithelium to regional lymph nodes appears to be an essential early step in the establishment of infection. This transfer is believed to occur by means of carriage within alveolar macrophages following phagocytosis. Therefore, the ability of B. anthracis to transit through the host macrophage or dendritic cell appears to be an early and critical step in B. anthracis pathogenesis. In this work, we examined the cytokine responses to spore infection in mouse primary peritoneal macrophages, in primary human dendritic cells, and during a spore aerosol infection model utilizing the susceptible A/J mouse strain. We demonstrated that both mouse peritoneal macrophages and human dendritic cells exhibited significant intracellular bactericidal activity during the first hours following uptake, providing the necessary time to mount a cytokine response prior to cell lysis. Strong tumor necrosis factor (TNF-alpha) and interleukin-6 (IL-6) responses were seen in mouse peritoneal macrophages. In addition to TNF-alpha and IL-6, human dendritic cells produced the cytokines IL-1beta, IL-8, and IL-12. A mixture of Th1 and Th2 cytokines were detected in sera obtained from infected animals. In this study, we provide further evidence of an acute cytokine response when cells in culture and mice are infected with B. anthracis spores.