Subhendu Basu

Murine Macrophages Kill the Vegetative Form of Bacillus anthracis

ABSTRACT Anti-protective antigen antibody was reported to enhance macrophage killing of ingested Bacillus anthracis spores, but it was unclear whether the antibody-mediated macrophage killing mechanism was directed against the spore itself or the vegetative form emerging from the ingested and germinating spore. To address this question, we compared the killing of germination-proficient (gp) and germination-deficient (ΔgerH) Sterne 34F2 strain spores by murine peritoneal macrophages. While macrophages similarly ingested both spores, only gp Sterne was killed at 5 h (0.37 log kill). Pretreatment of macrophages with gamma interferon (IFN-γ) or opsonization with immunoglobulin G (IgG) isolated from a subject immunized with an anthrax vaccine enhanced the killing of Sterne to 0.49 and 0.73 log, respectively, but the combination of IFN-γ and IgG was no better than either treatment alone. Under no condition was there killing of ΔgerH spores. To examine the ability of the exosporium to protect spores from macrophages, we compared the macrophage-mediated killing of nonsonicated (exosporium+) and sonicated (exosporium−) Sterne 34F2 spores. More sonicated spores than nonsonicated spores were killed at 5 h (0.98 versus 0.37 log kill, respectively). Pretreatment with IFN-γ increased the sonicated spore killing to 1.39 log. However, the opsonization with IgG was no better than no treatment or pretreatment with IFN-γ. We conclude that macrophages appear unable to kill the spore form of B. anthracis and that the exosporium may play a role in the protection of spores from macrophages.

Differential Involvement of BB Loops of Toll-IL-1 Resistance (TIR) Domain-Containing Adapter Proteins in TLR4- versus TLR2-Mediated Signal Transduction

TLRs sense pathogens and transmit intracellular signals via the use of specific adapter proteins. We designed a set of “blocking peptides” (BPs) comprised of the 14 aa that correspond to the sequences of the BB loops of the four known Toll-IL-1 resistance (TIR) domain-containing adapter proteins (i.e., MyD88, TIR domain-containing adapter inducing IFN-β (TRIF), TRIF-related adapter molecule (TRAM), and TIR-domain containing adapter protein (TIRAP)) linked to the cell-penetrating segment of the antennapedia homeodomain. LPS (TLR4)-mediated gene expression, as well as MAPK and transcription factor activation associated with both MyD88-dependent and -independent signaling pathways, were disrupted by all four BPs (TRAM ≈ MyD88 > TRIF > TIRAP), but not by a control peptide. In contrast, none of the BPs inhibited TLR2-mediated activation of MAPKs. Only the MyD88 BP significantly blocked Pam3Cys-induced IL-1β mRNA; however, the inhibitory effect was much less than observed for LPS. Our data suggest that the interactions required for a fully functional TLR4 signaling “platform” are disrupted by these BPs, and that the adapter BB loops may serve distinct roles in TLR4 and TLR2 signalosome assembly.

Bacillus anthracis spores and lethal toxin induce IL‐1β via functionally distinct signaling pathways

Previous reports suggested that lethal toxin (LT)‐induced caspase‐1 activity and/or IL‐1β accounted for Bacillus anthracis (BA) infection lethality. In contrast, we now report that caspase‐1‐mediated IL‐1β expression in response to BA spores is required for anti‐BA host defenses. Caspase‐1–/– and IL‐1β–/– mice are more susceptible than wild‐type (WT) mice to lethal BA infection, are less able to kill BA both in vivo and in vitro, and addition of rIL‐1β to macrophages from these mice restored killing in vitro. Non‐germinating BA spores induced caspase‐1 activity, IL‐1β and nitric oxide, by which BA are killed in WT but not in caspase‐1–/– mice, suggesting that the spore itself stimulated inflammatory responses. While spores induced IL‐1β in LT‐susceptible and ‐resistant macrophages, LT induced IL‐1β only in LT‐susceptible macrophages. Cooperation between MyD88‐dependent and ‐independent signaling pathways was required for spore‐induced, but not LT‐induced, IL‐1β. While both spores and LT induced caspase‐1 activity and IL‐1β, LT did not induce IL‐1β mRNA, and spores did not induce cell death. Thus different components of the same bacterium each induce IL‐1β by distinct signaling pathways. Whereas the spore‐induced IL‐1β limits BA infection, LT‐induced IL‐1β enables BA to escape host defenses.

Role of Bacillus anthracis Spore Structures in Macrophage Cytokine Responses

ABSTRACT The innate immune response of macrophages (Mφ) to spores, the environmentally acquired form of Bacillus anthracis, is poorly characterized. We therefore examined the early Mφ cytokine response to B. anthracis spores, before germination. Mφ were exposed to bacilli and spores of Sterne strain 34F2 and its congenic nongerminating mutant (ΔgerH), and cytokine expression was measured by real-time PCR and an enzyme-linked immunosorbent assay. The exosporium spore layer was retained (exo+) or removed by sonication (exo−). Spores consistently induced a strong cytokine response, with the exo− spores eliciting a two- to threefold-higher response than exo+ spores. The threshold for interleukin-1β (IL-1β) production by wild-type Mφ was significantly lower than that required for tumor necrosis factor alpha expression. Cytokine production was largely dependent on MyD88, suggesting Toll-like receptor involvement; however, the expression of beta interferon in MyD88−/− Mφ suggests involvement of a MyD88-independent pathway. We conclude that (i) the B. anthracis spore is not immunologically inert, (ii) the exosporium masks epitopes recognized by the Mφ, (iii) the Mφ cytokine response to B. anthracis involves multiple pattern recognition receptors and signaling pathways, and (iv) compared to other cytokines, IL-1β is expressed at a lower spore concentration.

Antibody feedback and somatic mutation in B cells: regulation of mutation by immune complexes with IgG antibody.

Summary: In response to an appropriate antigenic stimulus, and with help from T lymphocytes, naive B cells differentiate into plasmacytes which produce the primary (germline‐encoded) IgM and IgG antibody with low affinity for the antigen. The isotype switch from IgM to IgG coincides with the burst of germinal center reaction and the onset of “somatic hypermutation. Here we propose that formation of immune complexes between the residual antigen and the primary IgG antibody, which activate complement and localize specifically in the network of follicular dendritic cells, provides an important signal for triggering the mutation mechanism in germinal center B cells. This hypothesis has been supported by studies on immunogenicity of immune complexes in vivo. The experiments have included an immunization with pre‐formed antigen/IgG antibody complex and/or an administration of IgG and body shortly after the antigen injection. Either of these strategies, which are known to augment the germinal center formation, resulted in earlier onset of somatic mutation and increased mutation frequency in VDJ rearrangements in antigen‐reactive B cells, provided that help from T cells was also present. It is presumed that the antigen/antibody/complement complex is able to deliver this important signal by cross‐linking of antigen receptor with the CD21/CD19/CD81 molecules on B cells. As a corollary, the signaling by immune complexes may lower the threshold of cell activation determined by receptor affinity for antigen and stimulate diverse V‐gene repertoire of B‐cell clones in germinal centers.

Regulation of VH gene repertoire and somatic mutation in germinal centre B cells by passively administered antibody

Immunization with T‐dependent antigens induces a rapid differentiation of B cells to plasmacytes that produce the primary immunoglobulin M (IgM) and IgG antibodies with low affinities for the immunogen. It is proposed that the IgG antibody forms immune complexes with the residual antigen which provide an important stimulus for the formation of germinal centres (GC) and the activation of somatic mutation. This hypothesis was tested by passive administration of hapten‐specific antibody into mice shortly after the immunization with nitrophenyl (NP) coupled to chicken gamma globulin (NP‐CGG) in an environment of limited T‐cell help. Athymic mice that received normal T helper cells at 72 hr after the administration of antigen produced low levels of anti‐NP antibody and the splenic GC formation was delayed until day 12 after the antigen administration. The analysis of VDJ segments from NP‐reactive GC B cells showed very few mutations in the VH genes. Passive injection of anti‐NP IgG1 monoclonal antibody – but, not IgM – stimulated the GC formation up to normal levels and the somatic mutation activity in the GC B cells was fully restored. In addition, GC B cells in the recipients of IgG1 antibody demonstrated a change in the usage of germline‐encoded VH genes which was not apparent among the primary antibody‐forming cells. These results suggest the existence of a specific feedback mechanism whereby the IgG antibody regulates the GC formation, clonotypic repertoire and somatic mutation in GC B cells.

Enhanced antibody responses to a detoxified lipopolysaccharide-group B meningococcal outer membrane protein vaccine are due to synergistic engagement of Toll-like receptors

When given passively or elicited actively, antibodies induced by a detoxified Escherichia coli Rc chemotype (J5) mutant lipopolysaccharide (J5dLPS)—group B meningococcal outer membrane protein (OMP) complex vaccine protected animals from lethal sepsis. The protection from sepsis is believed to be dependent on high levels of antibodies against the core glycolipid (CGL), a region of LPS that is rather conserved among Enterobacteriaceae. The addition of unmethylated deoxycytidyl-deoxyguanosine dinucleotide (CpG)-containing oligodeoxynucleotides (ODN) was used as an immuno-adjuvant to improve antibody responses. In preparation for a Phase I human trial, we elucidated potential contributions by which the sepsis vaccine (J5dLPS—OMP) and CpG ODN might enhance the antibody response and provide evidence that the generation of immune responses is Toll-like receptor (TLR) dependent. Toll-like receptor 2, TLR4, and TLR9 were each essential for generating robust cytokine and antibody responses. The signature cytokine of dendritic cells, interleukin-12, was one of the cytokines that demonstrated synergy with the optimal TLR ligand/ engagement combination. We conclude that the involvement of multiple TLRs upon immunization was critical for the generation of optimal antibody responses. These observations provide further evidence for the inclusion of innate immune-based adjuvants during the development of next-generation vaccines.

The IL‐23/Th17 axis is involved in the adaptive immune response to Bacillus anthracis in humans

The neutralization of toxins is considered essential for protection against lethal infection with Bacillus anthracis (BA), a select agent and bioterrorism threat. However, toxin‐neutralizing activity alone would not be expected to provide sterile immunity. Therefore, we hypothesized that the development of an adaptive immune response against BA is required for bacterial clearance. We found that human monocyte‐derived dendritic cells (hDCs) kill germinated BA bacilli, but not nongerminated BA spores. hDCs produce IL‐1β, IL‐6, IL‐12, and IL‐23, and these cytokines are differentially regulated by germination‐proficient versus germination‐deficient BA spores. Moreover, the IL‐23 response to BA spores is regulated by IL‐1R‐mediated signaling. hDCs infected with germinating BA spores stimulated autologous CD4+ T cells to secrete IL‐17A and IFN‐γ in a contact‐dependent and antigen‐specific manner. The T‐cell response to BA spores was not recapitulated by hDCs infected with germination‐deficient BA spores, implying that the germination of spores into replicating bacilli triggers the proinflammatory cytokine response in hDCs. Our results provide primary evidence that hDCs can generate a BA‐specific Th17 response, and help elucidate the mechanisms involved. These novel findings suggest that the IL‐23/Th17 axis is involved in the immune response to anthrax in humans.