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.

Toll-like receptor driven B cell activation in the induction of systemic autoimmunity.

Studies over the past decade have demonstrated a key role for pattern recognition receptors in the activation of autoreactive B cells. Self reactive B cells that manage to escape negative selection often express relatively low affinity receptors for self antigens (ignorant B cells), and can only be activated by integrating a relatively weak BCR signal with signals from additional receptors. Members of the toll-like receptor (TLR) gene family, and especially the nucleic acid binding receptors TLR 7, 8 and 9, appear to play a key role in this regard and promote the production of autoantibodies reactive with DNA- or RNA-associated autoantigens. These autoantibodies are able to form immune complexes with soluble or cell-bound ligands, and these immune complexes can in turn activate a second round of proinflammatory cells that further contribute to the autoimmune disease process. Recent data have emerged showing a pathogenic role for TLR7, with an opposing, protective role for TLR9. Targeting these disregulated pathways offers a therapeutic opportunity to treat autoimmune diseases without crippling the entire immune system. Further understanding of the role of specific receptors, cell subsets, and inhibitory signals that govern these TLR-associated pathways will enable future therapeutics to be tailored to specific categories of autoimmune disease.

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.

Long-term antibiotic exposure promotes mortality after systemic fungal infection by driving lymphocyte dysfunction and systemic escape of commensal bacteria.

Antibiotics are a modifiable iatrogenic risk factor for the most common human nosocomial fungal infection, invasive candidiasis, yet the underlying mechanisms remain elusive. We found that antibiotics enhanced the susceptibility to murine invasive candidiasis due to impaired lymphocyte-dependent IL-17A- and GM-CSF-mediated antifungal immunity within the gut. This led to non-inflammatory bacterial escape and systemic bacterial co-infection, which could be ameliorated by IL-17A or GM-CSF immunotherapy. Vancomycin alone similarly enhanced the susceptibility to invasive fungal infection and systemic bacterial co-infection. Mechanistically, vancomycin reduced the frequency of gut Th17 cells associated with impaired proliferation and RORγt expression. Vancomycin's effects on Th17 cells were indirect, manifesting only in vivo in the presence of dysbiosis. In humans, antibiotics were associated with an increased risk of invasive candidiasis and death after invasive candidiasis. Our work highlights the importance of antibiotic stewardship in protecting vulnerable patients from life-threatening infections and provides mechanistic insights into a controllable iatrogenic risk factor for invasive candidiasis.

Murine B cell response to TLR7 ligands depends on an IFN-beta feedback loop.

Type I IFNs play an important, yet poorly characterized, role in systemic lupus erythematosus. To better understand the interplay between type I IFNs and the activation of autoreactive B cells, we evaluated the effect of type I IFN receptor (IFNAR) deficiency in murine B cell responses to common TLR ligands. In comparison to wild-type B cells, TLR7-stimulated IFNAR(-/-) B cells proliferated significantly less well and did not up-regulate costimulatory molecules. By contrast, IFNAR1(-/-) B cells did not produce cytokines, but did proliferate and up-regulate activation markers in response to other TLR ligands. These defects were not due to a difference in the distribution of B cell populations or a failure to produce a soluble factor other than a type I IFN. Instead, the compromised response pattern reflected the disruption of an IFN-beta feedback loop and constitutively low expression of TLR7 in the IFNAR1(-/-) B cells. These results highlight subtle differences in the IFN dependence of TLR7 responses compared with other TLR-mediated B cell responses.

Autoreactive B cells discriminate CpG-rich and CpG-poor DNA and this response is modulated by IFN-alpha.

Autoreactive B cells are activated by DNA, chromatin, or chromatin-containing immune complexes (ICs) through a mechanism dependent on dual engagement of the BCR and TLR9. We examined the contribution of endogenous DNA sequence elements to this process. DNA sequence can determine both recognition by the BCR and by TLR9. DNA fragments containing CpG islands, a natural source of unmethylated CpG dinucleotides, promote the activation of DNA-reactive B cells derived from BCR transgenic mice as well as DNA-reactive B cells present in the normal repertoire. ICs containing these CpG island fragments are potent ligands for AM14 IgG2a-reactive B cells. In contrast, ICs containing total mammalian DNA, or DNA fragments lacking immunostimulatory motifs, fail to induce B cell proliferation, indicating that BCR crosslinking alone is insufficient to activate low-affinity autoreactive B cells. Importantly, priming B cells with IFN-alpha lowers the BCR activation threshold and relaxes the selectivity for CpG-containing DNA. Taken together, our findings underscore the importance of endogenous CpG-containing DNAs in the TLR9-dependent activation of autoreactive B cells and further identify an important mechanism through which IFN-alpha can contribute to the pathogenesis of systemic lupus erythematosus.

CXCR1-mediated neutrophil degranulation and fungal killing promote Candida clearance and host survival.

Systemic Candida albicans infection causes high morbidity and mortality and is now the leading cause of nosocomial bloodstream infection in the United States. Neutropenia is a major risk factor for poor outcome in infected patients; however, the molecular factors that mediate neutrophil trafficking and effector function during infection are poorly defined. Using a mouse model of systemic candidiasis, we found that the neutrophil-selective CXC chemokine receptor Cxcr1 and its ligand, Cxcl5, are highly induced in the Candida-infected kidney, the target organ in the model. To investigate the role of Cxcr1 in antifungal host defense in vivo, we generated Cxcr1(-/-) mice and analyzed their immune response to Candida. Mice lacking Cxcr1 exhibited decreased survival with enhanced Candida growth in the kidney and renal failure. Increased susceptibility of Cxcr1(-/-) mice to systemic candidiasis was not due to impaired neutrophil trafficking from the blood into the infected kidney but was the result of defective killing of the fungus by neutrophils that exhibited a cell-intrinsic decrease in degranulation. In humans, the mutant CXCR1 allele CXCR1-T276 results in impaired neutrophil degranulation and fungal killing and was associated with increased risk of disseminated candidiasis in infected patients. Together, our data demonstrate a biological function for mouse Cxcr1 in vivo and indicate that CXCR1-dependent neutrophil effector function is a critical innate protective mechanism of fungal clearance and host survival in systemic candidiasis.

CX3CR1-dependent renal macrophage survival promotes Candida control and host survival.

Systemic Candida albicans infection causes high morbidity and mortality and is associated with neutropenia; however, the roles of other innate immune cells in pathogenesis are poorly defined. Here, using a mouse model of systemic candidiasis, we found that resident macrophages accumulated in the kidney, the main target organ of infection, and formed direct contacts with the fungus in vivo mainly within the first few hours after infection. Macrophage accumulation and contact with Candida were both markedly reduced in mice lacking chemokine receptor CX3CR1, which was found almost exclusively on resident macrophages in uninfected kidneys. Infected Cx3cr1-/- mice uniformly succumbed to Candida-induced renal failure, but exhibited clearance of the fungus in all other organs tested. Renal macrophage deficiency in infected Cx3cr1-/- mice was due to reduced macrophage survival, not impaired proliferation, trafficking, or differentiation. In humans, the dysfunctional CX3CR1 allele CX3CR1-M280 was associated with increased risk of systemic candidiasis. Together, these data indicate that CX3CR1-mediated renal resident macrophage survival is a critical innate mechanism of early fungal control that influences host survival in systemic candidiasis.

LcrV plague vaccine with altered immunomodulatory properties.

Yersinia pestis, the causative agent of plague, secretes LcrV (low-calcium-response V or V antigen) during infection. LcrV triggers the release of interleukin 10 (IL-10) by host immune cells and suppresses proinflammatory cytokines such as tumor necrosis factor alpha and gamma interferon as well as innate defense mechanisms required to combat the pathogenesis of plague. Although immunization of animals with LcrV elicits protective immunity, the associated suppression of host defense mechanisms may preclude the use of LcrV as a human vaccine. Here we show that short deletions within LcrV can reduce its immune modulatory properties. An LcrV variant lacking amino acid residues 271 to 300 (rV10) elicited immune responses that protected mice against a lethal challenge with Y. pestis. Compared to full-length LcrV, rV10 displayed a reduced ability to release IL-10 from mouse and human macrophages. Furthermore, the lipopolysaccharide-stimulated release of proinflammatory cytokines by human or mouse macrophages was inhibited by full-length LcrV but not by the rV10 variant. Thus, it appears that LcrV variants with reduced immune modulatory properties could be used as a human vaccine to generate protective immunity against plague.