Although primary and memory responses against bacteria and viruses have been studied extensively, T helper type 2 (T H 2) effector mechanisms leading to host protection against helminthic parasites remain elusive 1 . Examination of the intestinal epithelial submucosa of mice after primary and secondary infections by a natural gastrointestinal parasite revealed a distinct immune-cell infiltrate after challenge, featuring interleukin-4-expressing memory CD4 + T cells that induced IL-4 receptor hi (IL-4R hi ) CD206 + alternatively activated macrophages 2 . In turn, these alternatively activated macrophages (AAMacs) functioned as important effector cells of the protective memory response contributing to parasite elimination, demonstrating a previously unknown mechanism for host protection against intestinal helminths.Productive adaptive immune responses result in CD4 + T-cell polarization into effector phenotypes defined by differing cytokine milieus 3 . Helminth parasites and allergens induce T H 2 responses, including CD4 + T-cell interleukin (IL)-4 production promoting arginase-1 expression by alternatively activated macrophages (AAMacs) 4 . Although it is known that these AAMacs accumulate during asthmatic inflammation 5 and helminth parasite infections 2,4,6, 7 , and downregulate type 1 inflammation 2,4,6 , a protective role for them remains undefined.Infection of mice with the natural mouse gastrointestinal helminth parasite Heligmosomoides polygyrus triggers a highly polarized T H 2 response 1 . H. polygyrus infection is chronic with established adult worms; if parasites are cleared from the host's intestinal lumen, a rapid, protective T H 2 memory response operates against challenge infections 8 . Our studies examined early events in this memory response to H. polygyrus larvae developing in the intestinal submucosa and indicated that AAMacs have an important role in parasite expulsion. Fig. 1 online), with tissue-invading larvae developing into adults and migrating into the lumen at day 8 after inoculation ( Supplementary Fig. 1). To examine which stages of the protective memory response required CD4 + T cells, we administered CD4-specific antibody to mice to deplete CD4 + cells at specific intervals after secondary infection (Fig. 1a). Administration at early time points (days 0 and 7) resulted in increased worm burden (Fig. 1b), day 7 treatment had intermediate effects, and later treatments (days 9 or 11) had marginal effects, indicating that CD4 + T cells are required at early stages of a secondary infection for effective parasite expulsion. This implicated the adaptive immune response during larval development in the intestinal tissue as crucial for host protection. To further confirm that the memory response affects invasive larvae, we recovered muscularis-residing larvae from infected small intestines by using a Baermann apparatus 9 , which provokes premature larval evacuation of the tissue, as an indicator of health and mobility. Significantly fewer larvae were recovered from the tissues of m...

Parasitic helminth infection has been shown to modulate pathological inflammatory responses in allergy and autoimmune disease. The aim of this study was to examine the effects of infection with a helminth parasite, Heligmosomoides polygyrus, on type 1 diabetes (T1D) in nonobese diabetic (NOD) mice and to elucidate the mechanisms involved in this protection. H. polygyrus inoculation at 5 weeks of age protected NOD mice from T1D until 40 weeks of age and also inhibited the more aggressive cyclophosphamide-induced T1D. Moreover, H. polygyrus inoculation as late as 12 weeks of age reduced the onset of T1D in NOD mice. Following H. polygyrus inoculation of NOD mice, pancreatic insulitis was markedly inhibited. Interleukin-4 (IL-4), IL-10, and IL-13 expression and the frequency of CD4 ؉ CD25 ؉ FoxP3 ؉ regulatory T cells were elevated in mesenteric and pancreatic lymph nodes. Depletion of CD4 ؉ CD25 ؉ T cells in vivo did not abrogate H. polygyrus-induced T1D protection, nor did anti-IL-10 receptor blocking antibody. These findings suggest that infection with H. polygyrus significantly inhibits T1D in NOD mice through CD25-and IL-10-independent mechanisms and also reduces the severity of T1D when administered late after the onset of insulitis.

We sought to visualize the site of Bacillus anthracis spore germination in vivo. For that purpose, we constructed a reporter plasmid with the lux operon under control of the spore small acid-soluble protein B (sspB) promoter. In B. subtilis, sspB-driven synthesis of luciferase during sporulation results in incorporation of the enzyme in spores. We observed that B. anthracis Sterne transformed with our sspBp::lux plasmid was only luminescent during germination. In contrast, Sterne transformed with a similarly constructed plasmid with lux expression under control of the protective antigen promoter displayed luminescence only during vegetative growth. We then infected A/J mice intranasally with spores that harbored the germination reporter. Mice were monitored for up to 14 days with the Xenogen In Vivo Imaging System. While luminescence only became evident in live animals at 18 h, dissection after sacrificing infected mice at earlier time points revealed luminescence in lung tissue at 30 min after intranasal infection. Microscopic histochemical and immunofluorescence studies on luminescent lung sections and imprints revealed that macrophages were the first cells in contact with the B. anthracis spores. By 6 h after infection, polymorphonuclear leukocytes with intracellular spores were evident in the alveolar spaces. After 24 h, few free spores were observed in the alveolar spaces; most of the spores detected by immunofluorescence were in the cytoplasm of interstitial macrophages. In contrast, mediastinal lymph nodes remained nonluminescent throughout the infection. We conclude that in this animal system, the primary site of B. anthracis spore germination is the lungs.

Based on previous studies showing that host chemokines exert antimicrobial activities against bacteria, we sought to determine whether the interferon-inducible Glu-Leu-Arg-negative CXC chemokines CXCL9, CXCL10, and CXCL11 exhibit antimicrobial activities against Bacillus anthracis. In vitro analysis demonstrated that all three CXC chemokines exerted direct antimicrobial effects against B. anthracis spores and bacilli including marked reductions in spore and bacillus viability as determined using a fluorometric assay of bacterial viability and CFU determinations. Electron microscopy studies revealed that CXCL10-treated spores failed to undergo germination as judged by an absence of cytological changes in spore structure that occur during the process of germination. Immunogold labeling of CXCL10-treated spores demonstrated that the chemokine was located internal to the exosporium in association primarily with the spore coat and its interface with the cortex. To begin examining the potential biological relevance of chemokine-mediated antimicrobial activity, we used a murine model of inhalational anthrax. Upon spore challenge, the lungs of C57BL/6 mice (resistant to inhalational B. anthracis infection) had significantly higher levels of CXCL9, CXCL10, and CXCL11 than did the lungs of A/J mice (highly susceptible to infection). Increased CXC chemokine levels were associated with significantly reduced levels of spore germination within the lungs as determined by in vivo imaging. Taken together, our data demonstrate a novel antimicrobial role for host chemokines against B. anthracis that provides unique insight into host defense against inhalational anthrax; these data also support the notion for an innovative approach in treating B. anthracis infection as well as infections caused by other spore-forming organisms.Bacillus anthracis is a gram-positive, spore-forming bacterium that causes the disease anthrax. The infectious B. anthracis spore is a dormant, metabolically inactive form of the organism made up of distinct, concentric layers that collectively provide a highly structured casing capable of protecting the spore core from high temperature, UV irradiation, lytic digestion, and numerous reactive agents (31, 59). Spore germination is initiated through receptor-mediated interactions between soluble germinant molecules (typically nutrients such as single amino acids, sugars, or purine nucleosides) and germinant receptors located at the inner membrane of the dormant spore (20,36). Although the molecular mechanism(s) linking germinant binding to the loss of dormancy is undefined, germinant receptor engagement initiates a cascade of processes, including dipicolinic acid (DPA) release, that promote core rehydration and result in the controlled degradation of the protective spore structures; as germination concludes, metabolic activity resumes, and vegetative outgrowth is initiated (58). Fully virulent B. anthracis bacilli generate several virulence factors including an antiphagocytic, poly-D-glutamic acid capsule encode...

The Bacillus anthracis genome encodes four superoxide dismutases (SODs), enzymes capable of detoxifying oxygen radicals. That two of these SODs, SOD15 and SODA1, are present in the outermost layers of the B. anthracis spore is indicated by previous proteomic analyses of the exosporium. Given the requirement that spores must survive interactions with reactive oxygen species generated by cells such as macrophages during infection, we hypothesized that SOD15 and SODA1 protect the spore from oxidative stress and contribute to the pathogenicity of B. anthracis. To test these theories, we constructed a double-knockout (⌬sod15 ⌬sodA1) mutant of B. anthracis Sterne strain 34F2 and assessed its lethality in an A/J mouse intranasal infection model. The 50% lethal dose of the ⌬sod15 ⌬sodA1 strain was similar to that of the wild type (34F2), but surprisingly, measurable whole-spore SOD activity was greater than that in 34F2. A quadruple-knockout strain (⌬sod15 ⌬sodA1 ⌬sodC ⌬sodA2) was then generated, and as anticipated, spore-associated SOD activity was diminished. Moreover, the quadruple-knockout strain, compared to the wild type, was attenuated more than 40-fold upon intranasal challenge of mice. Spore resistance to exogenously generated oxidative stress and to macrophagemediated killing correlated with virulence in A/J mice. Allelic exchange that restored sod15 and sodA1 to their wild-type state restored wild-type characteristics. We conclude that SOD molecules within the spore afford B. anthracis protection against oxidative stress and enhance the pathogenicity of B. anthracis in the lung. We also surmise that the presence of four SOD alleles within the genome provides functional redundancy for this key enzyme.