TLR9 ligand sequestration by chemokine CXCL4 negatively affects central B cell tolerance.

Central B cell tolerance is believed to be regulated by B cell receptor signaling induced by the recognition of self-antigens in immature B cells. Using humanized mice with defective MyD88, TLR7, or TLR9 expression, we demonstrate that TLR9/MYD88 are required for central B cell tolerance and the removal of developing autoreactive clones. We also show that CXCL4, a chemokine involved in systemic sclerosis (SSc), abrogates TLR9 function in B cells by sequestering TLR9 ligands away from the endosomal compartments where this receptor resides. The in vivo production of CXCL4 thereby impedes both TLR9 responses in B cells and the establishment of central B cell tolerance. We conclude that TLR9 plays an essential early tolerogenic function required for the establishment of central B cell tolerance and that correcting defective TLR9 function in B cells from SSc patients may represent a novel therapeutic strategy to restore B cell tolerance.

Protection Elicited by Attenuated Live Yersinia pestis Vaccine Strains against Lethal Infection with Virulent Y. pestis.

The etiologic agent of plague, Yersinia pestis, is a globally distributed pathogen which poses both a natural and adversarial threat. Due largely to the rapid course and high mortality of pneumonic plague, vaccines are greatly needed. Two-component protein vaccines have been unreliable and potentially vulnerable to vaccine resistance. We evaluated the safety and efficacy of eight live Y. pestis strains derived from virulent strains CO92 or KIM6+ and mutated in one or more virulence-associated gene(s) or cured of plasmid pPst. Stringent, single-dose vaccination allowed down-selection of the two safest and most protective vaccine candidates, CO92 mutants pgm- pPst- and ΔyscN. Both completely protected BALB/c mice against subcutaneous and aerosol challenge with Y. pestis. Strain CD-1 outbred mice were more resistant to bubonic (but not pneumonic) plague than BALB/c mice, but the vaccines elicited partial protection of CD-1 mice against aerosol challenge, while providing full protection against subcutaneous challenge. A ΔyscN mutant of the nonencapsulated C12 strain was expected to display antigens previously concealed by the capsule. C12 ΔyscN elicited negligible titers to F1 but comparable antibody levels to whole killed bacteria, as did CO92 ΔyscN. Although one dose of C12 ΔyscN was not protective, vaccination with two doses of either CO92 ΔyscN, or a combination of the ΔyscN mutants of C12 and CO92, protected optimally against lethal bubonic or pneumonic plague. Protection against encapsulated Y. pestis required inclusion of F1 in the vaccine and was associated with high anti-F1 titers.

A TLR9-dependent checkpoint governs B cell responses to DNA-containing antigens.

Mature B cell pools retain a substantial proportion of polyreactive and self-reactive clonotypes, suggesting that activation checkpoints exist to reduce the initiation of autoreactive B cell responses. Here, we have described a relationship among the B cell receptor (BCR), TLR9, and cytokine signals that regulate B cell responses to DNA-containing antigens. In both mouse and human B cells, BCR ligands that deliver a TLR9 agonist induce an initial proliferative burst that is followed by apoptotic death. The latter mechanism involves p38-dependent G1 cell-cycle arrest and subsequent intrinsic mitochondrial apoptosis and is shared by all preimmune murine B cell subsets and CD27- human B cells. Survival or costimulatory signals rescue B cells from this fate, but the outcome varies depending on the signals involved. B lymphocyte stimulator (BLyS) engenders survival and antibody secretion, whereas CD40 costimulation with IL-21 or IFN-γ promotes a T-bet+ B cell phenotype. Finally, in vivo immunization studies revealed that when protein antigens are conjugated with DNA, the humoral immune response is blunted and acquires features associated with T-bet+ B cell differentiation. We propose that this mechanism integrating BCR, TLR9, and cytokine signals provides a peripheral checkpoint for DNA-containing antigens that, if circumvented by survival and differentiative cues, yields B cells with the autoimmune-associated T-bet+ phenotype.

Toll-Like Receptor-Dependent Immune Complex Activation of B Cells and Dendritic Cells.

High titers of autoantibodies reactive with DNA/RNA molecular complexes are characteristic of autoimmune disorders such as systemic lupus erythematosus (SLE). In vitro and in vivo studies have implicated the endosomal Toll-like receptor 9 (TLR9) and Toll-like receptor 7 (TLR7) in the activation of the corresponding autoantibody producing B cells. Importantly, TLR9/TLR7-deficiency results in the inability of autoreactive B cells to proliferate in response to DNA/RNA-associated autoantigens in vitro, and in marked changes in the autoantibody repertoire of autoimmune-prone mice. Uptake of DNA/RNA-associated autoantigen immune complexes (ICs) also leads to activation of dendritic cells (DCs) through TLR9 and TLR7. The initial studies from our lab involved ICs formed by a mixture of autoantibodies and cell debris released from dying cells in culture. To better understand the nature of the mammalian ligands that can effectively activate TLR7 and TLR9, we have developed a methodology for preparing ICs containing defined DNA fragments that recapitulate the immunostimulatory activity of the previous "black box" ICs. As the endosomal TLR7 and TLR9 function optimally from intracellular acidic compartments, we developed a facile methodology to monitor the trafficking of defined DNA ICs by flow cytometry and confocal microscopy. These reagents reveal an important role for nucleic acid sequence, even when the ligand is mammalian DNA and will help illuminate the role of IC trafficking in the response.

An unexpected role for RNA-sensing toll-like receptors in a murine model of DNA accrual.

OBJECTIVES: The goal of this study was to determine whether endosomal Toll-like receptors (TLRs) contribute to the clinical manifestation of systemic autoimmunity exhibited by mice that lack the lysosomal nuclease DNaseII. METHODS: DNaseII/IFNaR double deficient mice were intercrossed with Unc93b13d/3d mice to generate DNaseII-/-mice with non-functional endosomal TLRs. The resulting triple deficient mice were evaluated for arthritis, autoantibody production, splenomegaly, and extramedullary haematopoiesis. B cells from both strains were evaluated for their capacity to respond to endogenous DNA by using small oligonucleotide based TLR9D ligands and a novel class of bifunctional anti-DNA antibodies. RESULTS: Mice that fail to express DNaseII, IFNaR, and Unc93b1 still develop arthritis but do not make autoantibodies, develop splenomegaly, or exhibit extramedullary haematopoiesis. DNaseII-/- IFNaR-/- B cells can respond to synthetic ODNs, but not to endogenous dsDNA. CONCLUSIONS: RNA-reactive TLRs, presumably TLR7, are required for autoantibody production, splenomegaly, and extramedullary haematopoiesis in the DNaseII-/- model of systemic autoimmunity.

Cell-intrinsic expression of TLR9 in autoreactive B cells constrains BCR/TLR7-dependent responses.

Endosomal TLRs play an important role in systemic autoimmune diseases, such as systemic erythematosus lupus, in which DNA- and RNA-associated autoantigens activate autoreactive B cells through TLR9- and TLR7-dependent pathways. Nevertheless, TLR9-deficient autoimmune-prone mice develop more severe clinical disease, whereas TLR7-deficient and TLR7/9-double deficient autoimmune-prone mice develop less severe disease. To determine whether the regulatory activity of TLR9 is B cell intrinsic, we directly compared the functional properties of autoantigen-activated wild-type, TLR9-deficient, and TLR7-deficient B cells in an experimental system in which proliferation depends on BCR/TLR coengagement. In vitro, TLR9-deficient cells are less dependent on survival factors for a sustained proliferative response than are either wild-type or TLR7-deficient cells. The TLR9-deficient cells also preferentially differentiate toward the plasma cell lineage, as indicated by expression of CD138, sustained expression of IRF4, and other molecular markers of plasma cells. In vivo, autoantigen-activated TLR9-deficient cells give rise to greater numbers of autoantibody-producing cells. Our results identify distinct roles for TLR7 and TLR9 in the differentiation of autoreactive B cells that explain the capacity of TLR9 to limit, as well as TLR7 to promote, the clinical features of systemic erythematosus lupus.

Cutting Edge: DNase II deficiency prevents activation of autoreactive B cells by double-stranded DNA endogenous ligands.

In mice that fail to express the phagolysosomal endonuclease DNase II and the type I IFN receptor, excessive accrual of undegraded DNA results in a STING-dependent, TLR-independent inflammatory arthritis. These double-knockout (DKO) mice develop additional indications of systemic autoimmunity, including anti-nuclear autoantibodies and splenomegaly, that are not found in Unc93b1(3d/3d) DKO mice and, therefore, are TLR dependent. The DKO autoantibodies predominantly detect RNA-associated autoantigens, which are commonly targeted in TLR7-dominated systemic erythematosus lupus-prone mice. To determine whether an inability of TLR9 to detect endogenous DNA could explain the absence of dsDNA-reactive autoantibodies in DKO mice, we used a novel class of bifunctional autoantibodies, IgM/DNA dual variable domain Ig molecules, to activate B cells through a BCR/TLR9-dependent mechanism. DKO B cells could not respond to the IgM/DNA dual variable domain Ig molecule, despite a normal response to both anti-IgM and CpG ODN 1826. Thus, DKO B cells only respond to RNA-associated ligands because DNase II-mediated degradation of self-DNA is required for TLR9 activation.

A Yersinia pestis tat mutant is attenuated in bubonic and small-aerosol pneumonic challenge models of infection but not as attenuated by intranasal challenge.

Bacterial proteins destined for the Tat pathway are folded before crossing the inner membrane and are typically identified by an N-terminal signal peptide containing a twin arginine motif. Translocation by the Tat pathway is dependent on the products of genes which encode proteins possessing the binding site of the signal peptide and mediating the actual translocation event. In the fully virulent CO92 strain of Yersinia pestis, the tatA gene was deleted. The mutant was assayed for loss of virulence through various in vitro and in vivo assays. Deletion of the tatA gene resulted in several consequences for the mutant as compared to wild-type. Cell morphology of the mutant bacteria was altered and demonstrated a more elongated form. In addition, while cultures of the mutant strain were able to produce a biofilm, we observed a loss of adhesion of the mutant biofilm structure compared to the biofilm produced by the wild-type strain. Immuno-electron microscopy revealed a partial disruption of the F1 antigen on the surface of the mutant. The virulence of the ΔtatA mutant was assessed in various murine models of plague. The mutant was severely attenuated in the bubonic model with full virulence restored by complementation with the native gene. After small-particle aerosol challenge in a pneumonic model of infection, the mutant was also shown to be attenuated. In contrast, when mice were challenged intranasally with the mutant, very little difference in the LD50 was observed between wild-type and mutant strains. However, an increased time-to-death and delay in bacterial dissemination was observed in mice infected with the ΔtatA mutant as compared to the parent strain. Collectively, these findings demonstrate an essential role for the Tat pathway in the virulence of Y. pestis in bubonic and small-aerosol pneumonic infection but less important role for intranasal challenge.

Exposure to Bacillus anthracis capsule results in suppression of human monocyte-derived dendritic cells.

The antiphagocytic capsule of Bacillus anthracis is a major virulence factor. We hypothesized that it may also mediate virulence through inhibition of the host's immune responses. During an infection, the capsule exists attached to the bacterial surface but also free in the host tissues. We sought to examine the impact of free capsule by assessing its effects on human monocytes and immature dendritic cells (iDCs). Human monocytes were differentiated into iDCs by interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) over 7 days in the presence of capsule derived from wild-type encapsulated B. anthracis Ames (WT) or a control preparation from an isogenic B. anthracis Ames strain that produces only 2% of the capsule of the WT (capA mutant). WT capsule consistently induced release of IL-8 and IL-6 while the capA mutant control preparation elicited either no response or only a minimal release of IL-8. iDCs that were differentiated in the presence of WT capsule had increased side scatter (SSC), a measure of cellular complexity, when assessed by flow cytometry. iDCs differentiated in the presence of WT capsule also matured less well in response to subsequent B. anthracis peptidoglycan (Ba PGN) exposure, with reduced upregulation of the chemokine receptor CCR7, reduced CCR7-dependent chemotaxis, and reduced release of certain cytokines. Exposure of naive differentiated control iDCs to WT capsule did not alter cell surface marker expression but did elicit IL-8. These results indicate that free capsule may contribute to the pathogenesis of anthrax by suppressing the responses of immune cells and interfering with the maturation of iDCs.

Disrupting the luxS quorum sensing gene does not significantly affect Bacillus anthracis virulence in mice or guinea pigs.

Many bacterial species use secreted quorum-sensing autoinducer molecules to regulate cell density- and growth phase-dependent gene expression, including virulence factor production, as sufficient environmental autoinducer concentrations are achieved. Bacillus anthracis, the causative agent of anthrax, contains a functional autoinducer (AI-2) system, which appears to regulate virulence gene expression. To determine if the AI-2 system is necessary for disease, we constructed a LuxS AI-2 synthase-deficient mutant in the virulent Ames strain of B. anthracis. We found that growth of the LuxS-deficient mutant was inhibited and sporulation was delayed when compared with the parental strain. However, spores of the Ames luxS mutant remained fully virulent in both mice and guinea pigs.

Activation of autoreactive B cells by endogenous TLR7 and TLR3 RNA ligands.

The key step in the activation of autoreactive B cells is the internalization of nucleic acid containing ligands and delivery of these ligands to the Toll-like Receptor (TLR) containing endolysosomal compartment. Ribonucleoproteins represent a large fraction of autoantigens in systemic autoimmune diseases. Here we demonstrate that many uridine-rich mammalian RNA sequences associated with common autoantigens effectively activate autoreactive B cells. Priming with type I IFN increased the magnitude of activation, and the range of which RNAs were stimulatory. A subset of RNAs that contain a high degree of self-complementarity also activated B cells through TLR3. For the RNA sequences that activated predominantly through TLR7, the activation is proportional to uridine-content, and more precisely defined by the frequency of specific uridine-containing motifs. These results identify parameters that define specific mammalian RNAs as ligands for TLRs.

Opposing roles for membrane bound and soluble Fas ligand in glaucoma-associated retinal ganglion cell death.

Glaucoma, the most frequent optic neuropathy, is a leading cause of blindness worldwide. Death of retinal ganglion cells (RGCs) occurs in all forms of glaucoma and accounts for the loss of vision, however the molecular mechanisms that cause RGC loss remain unclear. The pro-apoptotic molecule, Fas ligand, is a transmembrane protein that can be cleaved from the cell surface by metalloproteinases to release a soluble protein with antagonistic activity. Previous studies documented that constitutive ocular expression of FasL maintained immune privilege and prevented neoangeogenesis. We now show that FasL also plays a major role in retinal neurotoxicity. Importantly, in both TNFα triggered RGC death and a spontaneous model of glaucoma, gene-targeted mice that express only full-length FasL exhibit accelerated RGC death. By contrast, FasL-deficiency, or administration of soluble FasL, protected RGCs from cell death. These data identify membrane-bound FasL as a critical effector molecule and potential therapeutic target in glaucoma.

The role of the phoPQ operon in the pathogenesis of the fully virulent CO92 strain of Yersinia pestis and the IP32953 strain of Yersinia pseudotuberculosis.

At the genomic level, Yersinia pestis and Yersinia pseudotuberculosis are nearly identical but cause very different diseases. Y. pestis is the etiologic agent of plague; whereas Y. pseudotuberculosis causes a gastrointestinal infection primarily after the consumption of contaminated food. In many gram-negative pathogenic bacteria, PhoP is part of a two-component global regulatory system in which PhoQ serves as the sensor kinase, and PhoP is the response regulator. PhoP is known to activate a number of genes in many bacteria related to virulence. To determine the role of the PhoPQ proteins in Yersinia infections, primarily using aerosol challenge models, the phoP gene was deleted from the chromosome of the CO92 strain of Y. pestis and the IP32953 strain of Y. pseudotuberculosis, leading to a polar mutation of the phoPQ operon. We demonstrated that loss of phoPQ from both strains leads to a defect in intracellular growth and/or survival within macrophages. These in vitro data would suggest that the phoPQ mutants would be attenuated in vivo. However, the LD(50) for the Y. pestis mutant did not differ from the calculated LD(50) for the wild-type CO92 strain for either the bubonic or pneumonic murine models of infection. In contrast, mice challenged by aerosol with the Y. pseudotuberculosis mutant had a LD(50) value 40× higher than the wild-type strain. These results demonstrate that phoPQ are necessary for full virulence by aerosol infection with the IP32953 strain of Y. pseudotuberculosis. However, the PhoPQ proteins do not play a significant role in infection with a fully virulent strain of Y. pestis.

Localization and assembly of proteins comprising the outer structures of the Bacillus anthracis spore.

Bacterial spores possess a series of concentrically arranged protective structures that contribute to dormancy, survival and, ultimately, germination. One of these structures, the coat, is present in all spores. In Bacillus anthracis, however, the spore is surrounded by an additional, poorly understood, morphologically complex structure called the exosporium. Here, we characterize three previously discovered exosporium proteins called ExsFA (also known as BxpB), ExsFB (a highly related paralogue of exsFA/bxpB) and IunH (similar to an inosine-uridine-preferring nucleoside hydrolase). We show that in the absence of ExsFA/BxpB, the exosporium protein BclA accumulates asymmetrically to the forespore pole closest to the midpoint of the sporangium (i.e. the mother-cell-proximal pole of the forespore), instead of uniformly encircling the exosporium. ExsFA/BxpB may also have a role in coat assembly, as mutant spore surfaces lack ridges seen in wild-type spores and have a bumpy appearance. ExsFA/BxpB also has a modest but readily detected effect on germination. Nonetheless, an exsFA/bxpB mutant strain is fully virulent in both intramuscular and aerosol challenge models in Guinea pigs. We show that the pattern of localization of ExsFA/BxpB-GFP is a ring, consistent with a location for this protein in the basal layer of the exosporium. In contrast, ExsFB-GFP fluorescence is a solid oval, suggesting a distinct subcellular location for ExsFB-GFP. We also used these fusion proteins to monitor changes in the subcellular locations of these proteins during sporulation. Early in sporulation, both fusions were present throughout the mother cell cytoplasm. As sporulation progressed, GFP fluorescence moved from the mother cell cytoplasm to the forespore surface and formed either a ring of fluorescence, in the case of ExsFA/BxpB, or a solid oval of fluorescence, in the case of ExsFB. IunH-GFP also resulted in a solid oval of fluorescence. We suggest the interpretation that at least some ExsFB-GFP and IunH-GFP resides in the region between the coat and the exosporium, called the interspace.

Characterization of a Bacillus anthracis spore coat-surface protein that influences coat-surface morphology.

Bacterial spores are encased in a multilayered proteinaceous shell, called the coat. In many Bacillus spp., the coat protects against environmental assault and facilitates germination. In Bacillus anthracis, the spore is the etiological agent of anthrax, and the functions of the coat likely contribute to virulence. Here, we characterize a B. anthracis spore protein, called Cotbeta, which is encoded only in the genomes of the Bacillus cereus group. We found that Cotbeta is synthesized specifically during sporulation and is assembled onto the spore coat surface. Our analysis of a cotbeta null mutant in the Sterne strain reveals that Cotbeta has a role in determining coat-surface morphology but does not detectably affect germination. In the fully virulent Ames strain, a cotbeta null mutation has no effect on virulence in a murine model of B. anthracis infection.

Bacillus anthracis spores of the bclA mutant exhibit increased adherence to epithelial cells, fibroblasts, and endothelial cells but not to macrophages.

Bacillus anthracis is the causative agent of anthrax, and the spore form of the bacterium represents the infectious particle introduced into a host. The spore is surrounded by an exosporium, a loose-fitting membrane composed of proteins and carbohydrates from which hair-like projections extend. These projections are composed mainly of BclA (Bacillus-collagen-like protein of B. anthracis). To date, exact roles of the exosporium structure and BclA protein remain undetermined. We examined differences in spore binding of wild-type Ames and a bclA mutant of B. anthracis to bronchial epithelial cells as well as to the following other epithelial cells: A549, CHO, and Caco-2 cells; the IMR-90 fibroblast line; and human umbilical vein vascular endothelium cells. The binding of wild-type Ames spores to bronchial epithelial cells appeared to be a dose-dependent, receptor-ligand-mediated event. There were similar findings for the bclA mutant, with an additional nonspecific binding component likely leading to significantly more adherence to all nonprofessional phagocytic cell types. In contrast, we detected no difference in adherence and uptake of spores by macrophages for either the wild-type Ames or the bclA mutant strain. These results suggest that one potential role of the BclA fibers may be to inhibit nonspecific interactions between B. anthracis spores with nonprofessional phagocytic cells and thus direct the spores towards uptake by macrophages during initiation of infection in mammals.

Morphogenesis of the Bacillus anthracis spore.

Bacillus spp. and Clostridium spp. form a specialized cell type, called a spore, during a multistep differentiation process that is initiated in response to starvation. Spores are protected by a morphologically complex protein coat. The Bacillus anthracis coat is of particular interest because the spore is the infective particle of anthrax. We determined the roles of several B. anthracis orthologues of Bacillus subtilis coat protein genes in spore assembly and virulence. One of these, cotE, has a striking function in B. anthracis: it guides the assembly of the exosporium, an outer structure encasing B. anthracis but not B. subtilis spores. However, CotE has only a modest role in coat protein assembly, in contrast to the B. subtilis orthologue. cotE mutant spores are fully virulent in animal models, indicating that the exosporium is dispensable for infection, at least in the context of a cotE mutation. This has implications for both the pathophysiology of the disease and next-generation therapeutics. CotH, which directs the assembly of an important subset of coat proteins in B. subtilis, also directs coat protein deposition in B. anthracis. Additionally, however, in B. anthracis, CotH effects germination; in its absence, more spores germinate than in the wild type. We also found that SpoIVA has a critical role in directing the assembly of the coat and exosporium to an area around the forespore. This function is very similar to that of the B. subtilis orthologue, which directs the assembly of the coat to the forespore. These results show that while B. anthracis and B. subtilis rely on a core of conserved morphogenetic proteins to guide coat formation, these proteins may also be important for species-specific differences in coat morphology. We further hypothesize that variations in conserved morphogenetic coat proteins may play roles in taxonomic variation among species.