Infection with B. anthracis
resulting in systemic disease is associated with high morality characterized by septicemia, toxemia, and meningitis 
. The presence of bacilli in brain autopsies indicates that vegetative bacteria are able to disseminate from the bloodstream to the CNS, however, the basic pathogenic mechanisms by which B. anthracis
penetrates the BBB have not been described. Using electron microscopy and an established in vitro
model of the BBB, we demonstrate here for the first time that B. anthracis
is capable of invading hBMEC, the single cell layer that comprises the BBB. Our observations extend recent studies reporting invasion of B. anthracis
into non-phagocytic fibroblasts and epithelial cell lines 
. Furthermore, our results suggest that uptake of B. anthracis
Sterne in hBMEC is specific and requires actin cytoskeleton rearrangements. Interestingly, a very recent report identified a pXO1-encoded adhesin, BslA important for adherence to keratinocytes and lung epithelial cells 
. Studies to identify and characterize additional factors involved in hBMEC adherence and invasion, including the BslA adhesin, are in progress.
We have used microarray analysis to examine the acute response of brain endothelium to infection with vegetative B. anthracis
Sterne. We have shown previously that the BBB plays an active role in initiating a very specific innate immune response to bacterial infection by inducing gene expression of factors promoting neutrophil recruitment 
. Most strikingly, B. anthracis
infection reduced steady-state expression of 270 genes by more than two-fold corresponding to 87% of all affected gene transcripts. This contrasts typical host cellular responses to microbial pathogens where the number of host genes induced by infection is significantly higher than the number of down-regulated genes 
. The majority of downregulated genes were related to transcription, signal transduction, stress, host immune response, and proliferation. As anthrax toxins are the major secreted B. anthracis
virulence factors, we also analyzed the gene expression profile of hBMEC upon infection with a strain lacking the pXO1 plasmid, ΔpXO1, which encodes both anthrax toxins. Ninety percent of affected genes upon B. anthracis
Sterne infection were differentially affected upon infection with ΔpXO1 bacteria, and in total only 31% of genes in ΔpXO1-infected cells were downregulated. Additionally, approximately 10% of genes were regulated independently of pXO1, suggesting possible involvement of B. anthracis
chromosomal factors to host response. Overall, these results suggest a major role for plasmid encoded factors and toxins in regulating the brain endothelial host response.
Of particular interest was the unambiguous effect on the expression levels of genes belonging to the CXC chemokine family, particularly the neutrophil chemotactic factors IL-8, CXCL1 and CXCL2 in response B. anthracis
Sterne infection. Notably the expression levels of other major pro-inflammatory mediators such as TNFα and IL-1 were not affected by B. anthracis
Sterne or ΔpXO1 infection. Neutrophil recruitment is thought to be part of the very first line of CNS defense against bacterial infection 
as many Gram-positive and Gram-negative meningeal pathogens induce expression of these genes in hBMEC 
, van Sorge et al. unpublished data). Active impairment of neutrophil recruitment could therefore benefit survival and proliferation of B. anthracis
, as both spores and vegetative bacteria are efficiently killed by human neutrophils 
. Our results clearly demonstrate that the suppression of CXCL1 and IL-8 expression is pXO1- and toxin-dependent, respectively. These data complement observations in recent studies where systemic infection with the encapsulated strain impaired production of cytokines in a toxin-dependent manner 
and purified LT reduced IL-8 production by the destabilization of IL-8 mRNA in HUVEC in vitro 
We hypothesized that altered chemokine expression would result in impaired neutrophil recruitment upon active infection with B. anthracis
Sterne. Using two independent in vivo
assays, we demonstrated that neutrophil chemotaxis was indeed reduced to the site of infection with the Sterne strain as compared to infection with the ΔLF/EF mutant. Similar observations were recently published in a systemic infection model using encapsulated WT B. anthracis
); host neutrophil recruitment in spleen and liver was significantly increased in the absence anthrax toxins compared to infection with the parent strain 
. In addition, purified LT has been shown to directly impair neutrophil motility 
. Toxin-mediated subversion of the innate immune system, specifically targeting neutrophils, may therefore contribute to unchecked bacterial replication and a more fulminent disease course.
Establishment of an anthrax meningitis model is critical to better understand disease pathogenesis. The current rabbit and rhesus monkey models of inhalation anthrax 
both report signs of meningitis in a subgroup of animals, however, a mouse model would be preferable due to availability, lower costs and well-characterized genetic systems. We found that intravenous injection of immunocompetent outbred CD-1 mice with B. anthracis
Sterne resulted in penetration of bacilli into the CNS. Microscopic analysis of brain sections confirmed the development of meningitis, showing inflammatory cell infiltration, hemorrhaging, thrombosis, edema and areas full of bacilli. While we did observe neutrophil infiltration in the brains of B. anthracis
Sterne infected mice at the time of death, we speculate that an initial reduction or delay in host neutrophilic response may promote acute unrestricted bacterial proliferation and further CNS dissemination ultimately responsible for the rapidly progressive deteriorating course associated with anthrax meningitis. These observations reflect autopsy findings in patients 
validating the utility of this newly developed mouse model of hematogenous anthrax meningitis. Finally, development of anthrax meningitis requires expression of anthrax toxins as no signs of disease developed in mice infected with the ΔLF/EF mutant strain. Additional in vitro
studies suggested that this could be due to a direct contribution of the toxins to penetration of brain endothelium; however, we cannot exclude the possibility that the lack of clinical symptoms observed during infection with the toxin-deficient mutant may partially reflect a generalized reduction in virulence.
In summary, our studies provide the first evidence that B. anthracis is capable of invading the human BBB. We have also demonstrated that diverse functional classes of genes, including chemokines involved in neutrophil recruitment and signaling, were downregulated in brain endothelium upon B. anthracis infection suggesting that the pathogen actively suppresses the BBB innate immune response. This signaling appears to be mediated largely by the bacterial pXO1-encoded toxins. Our in vivo studies indicate that the anthrax toxins contribute to impaired neutrophil recruitment and the development of anthrax meningitis. Additional studies aimed at further understanding the mechanisms governing the pathogenesis of anthrax meningitis should aid in the development of preventative therapies for this serious CNS infection.