Optimization and qualification of an assay that demonstrates that a FimH vaccine induces functional antibody responses in women with histories of urinary tract infections.

Recurrent urinary tract infections (rUTI) are a serious disease associated with morbidities and mortality. Resistance to the standard of care antibiotics is now widespread because of the continued use of antibiotics among people who suffer from rUTI. We are therefore developing a vaccine to prevent recurrences among patients with rUTI. The antigen of the vaccine is FimH, a bacterial adhesin protein, and the vaccine is adjuvanted with a TLR-4 agonist. In a Phase 1 clinical study evaluating the vaccine, immunized individuals produced FimH-binding antibodies. Here we describe the optimization, qualification, and use of an assay to assess the functionality of these anti-FimH antibodies. The suitability of the assay for its intended purpose was demonstrated by selectivity, specificity, sensitivity, and intra-assay and inter-assay precision. The acceptance criteria were achieved for all parameters including intra-assay precision with ≤10% relative standard deviations and inter-assay precision with ≤25% relative standard deviations. The results presented herein suggest this functional assay will be important for supporting the vaccine's efficacy in future human studies. Furthermore and of great significance, these results prove that vaccine-induced functional antibodies can be elicited in rUTI patients against an essential virulence factor, FimH.

TAK1 kinase switches cell fate from apoptosis to necrosis following TNF stimulation.

TNF activates three distinct intracellular signaling cascades leading to cell survival, caspase-8-mediated apoptosis, or receptor interacting protein kinase 3 (RIPK3)-dependent necrosis, also called necroptosis. Depending on the cellular context, one of these pathways is activated upon TNF challenge. When caspase-8 is activated, it drives the apoptosis cascade and blocks RIPK3-dependent necrosis. Here we report the biological event switching to activate necrosis over apoptosis. TAK1 kinase is normally transiently activated upon TNF stimulation. We found that prolonged and hyperactivation of TAK1 induced phosphorylation and activation of RIPK3, leading to necrosis without caspase activation. In addition, we also demonstrated that activation of RIPK1 and RIPK3 promoted TAK1 activation, suggesting a positive feedforward loop of RIPK1, RIPK3, and TAK1. Conversely, ablation of TAK1 caused caspase-dependent apoptosis, in which Ripk3 deletion did not block cell death either in vivo or in vitro. Our results reveal that TAK1 activation drives RIPK3-dependent necrosis and inhibits apoptosis. TAK1 acts as a switch between apoptosis and necrosis.

Hexa-acylated lipid A is required for host inflammatory response to Neisseria gonorrhoeae in experimental gonorrhea.

Neisseria gonorrhoeae causes gonorrhea, a sexually transmitted infection characterized by inflammation of the cervix or urethra. However, a significant subset of patients with N. gonorrhoeae remain asymptomatic, without evidence of localized inflammation. Inflammatory responses to N. gonorrhoeae are generated by host innate immune recognition of N. gonorrhoeae by several innate immune signaling pathways, including lipooligosaccharide (LOS) and other pathogen-derived molecules through activation of innate immune signaling systems, including toll-like receptor 4 (TLR4) and the interleukin-1ß (IL-1ß) processing complex known as the inflammasome. The lipooligosaccharide of N. gonorrhoeae has a hexa-acylated lipid A. N. gonorrhoeae strains that carry an inactivated msbB (also known as lpxL1) gene produce a penta-acylated lipid A and exhibit reduced biofilm formation, survival in epithelial cells, and induction of epithelial cell inflammatory signaling. We now show that msbB-deficient N. gonorrhoeae induces less inflammatory signaling in human monocytic cell lines and murine macrophages than the parent organism. The penta-acylated LOS exhibits reduced toll-like receptor 4 signaling but does not affect N. gonorrhoeae-mediated activation of the inflammasome. We demonstrate that N. gonorrhoeae msbB is dispensable for initiating and maintaining infection in a murine model of gonorrhea. Interestingly, infection with msbB-deficient N. gonorrhoeae is associated with less localized inflammation. Combined, these data suggest that TLR4-mediated recognition of N. gonorrhoeae LOS plays an important role in the pathogenesis of symptomatic gonorrhea infection and that alterations in lipid A biosynthesis may play a role in determining symptomatic and asymptomatic infections.

Staphylococcus aureus Panton-Valentine leukocidin induces an inflammatory response in human phagocytes via the NLRP3 inflammasome.

The Staphylococcus aureus pore-forming toxin PVL is most likely causative for life-threatening necrotizing infections, which are characterized by massive tissue inflammation and necrosis. Whereas the cytotoxic action of PVL on human neutrophils is already well established, the PVL effects on other sensitive cell types, such as monocytes and macrophages, are less clear. In this study, we used different types of human leukocytes (neutrophils, monocytes, macrophages, lymphocytes) to investigate cell-specific binding of PVL subunits and subsequent proinflammatory and cytotoxic effects. In all PVL-sensitive cells, we identified the binding of the subunit LukS-PV as the critical factor for PVL-induced cytotoxicity, which was followed by binding of LukF-PV. LukS-PV binds to monocytes, macrophages, and neutrophils but not to lymphocytes. Additionally, we showed that PVL binding to monocytes and macrophages leads to release of caspase-1-dependent proinflammatory cytokines IL-1ß and IL-18. PVL activates the NLRP3 inflammasome, a signaling complex of myeloid cells that is involved in caspase-1-dependent IL-1ß processing in response to pathogens and endogenous danger signals. Specific inhibition of this pathway at several steps significantly reduced inflammasome activation and subsequent pyronecrosis. Furthermore, we found that PAMPs and DAMPs derived from dying neutrophils can dramatically enhance this response by up-regulating pro-IL-1ß in monocytes/macrophages. This study analyzes a specific host signaling pathway that mediates PVL-induced inflammation and cytotoxicity, which has high relevance for CA-MRSA-associated and PVL-mediated pathogenic processes, such as necrotizing infections.

Porphyromonas gingivalis mediates inflammasome repression in polymicrobial cultures through a novel mechanism involving reduced endocytosis.

The interleukin (IL)-1ß-processing inflammasome has recently been identified as a target for pathogenic evasion of the inflammatory response by a number of bacteria and viruses. We postulated that the periodontal pathogen, Porphyromonas gingivalis may suppress the inflammasome as a mechanism for its low immunogenicity and pathogenic synergy with other, more highly immunogenic periodontal bacteria. Our results show that P. gingivalis lacks signaling capability for the activation of the inflammasome in mouse macrophages. Furthermore, P. gingivalis can suppress inflammasome activation by another periodontal bacterium, Fusobacterium nucleatum. This repression affects IL-1ß processing, as well as other inflammasome-mediated processes, including IL-18 processing and cell death, in both human and mouse macrophages. F. nucleatum activates IL-1ß processing through the Nlrp3 inflammasome; however, P. gingivalis repression is not mediated through reduced levels of inflammasome components. P. gingivalis can repress Nlrp3 inflammasome activation by Escherichia coli, and by danger-associated molecular patterns and pattern-associated molecular patterns that mediate activation through endocytosis. However, P. gingivalis does not suppress Nlrp3 inflammasome activation by ATP or nigericin. This suggests that P. gingivalis may preferentially suppress endocytic pathways toward inflammasome activation. To directly test whether P. gingivalis infection affects endocytosis, we assessed the uptake of fluorescent particles in the presence or absence of P. gingivalis. Our results show that P. gingivalis limits both the number of cells taking up beads and the number of beads taken up for bead-positive cells. These results provide a novel mechanism of pathogen-mediated inflammasome inhibition through the suppression of endocytosis.

Staphylococcus aureus α-hemolysin mediates virulence in a murine model of severe pneumonia through activation of the NLRP3 inflammasome.

Staphylococcus aureus is a dangerous pathogen that can cause necrotizing infections characterized by massive inflammatory responses and tissue destruction. Staphylococcal α-hemolysin is an essential virulence factor in severe S. aureus pneumonia. It activates the nucleotide-binding domain and leucine-rich repeat containing gene family, pyrin domain containing 3 (NLRP3) inflammasome to induce production of interleukin-1ß and programmed necrotic cell death. We sought to determine the role of α-hemolysin-mediated activation of NLRP3 in the pathogenesis of S. aureus pneumonia. We show that α-hemolysin activates the NLRP3 inflammasome during S. aureus pneumonia, inducing necrotic pulmonary injury. Moreover, Nlrp3(-/-) mice have less-severe pneumonia. Pulmonary injury induced by isolated α-hemolysin or live S. aureus is independent of interleukin-1ß signaling, implicating NLRP3-induced necrosis in the pathogenesis of severe infection. This work demonstrates the exploitation of host inflammatory signaling by S. aureus and suggests the NLRP3 inflammasome as a potential target for pharmacologic interventions in severe S. aureus infections.

Transforming growth factor beta-activated kinase 1 (TAK1) kinase adaptor, TAK1-binding protein 2, plays dual roles in TAK1 signaling by recruiting both an activator and an inhibitor of TAK1 kinase in tumor necrosis factor signaling pathway.

Transforming growth factor beta-activated kinase 1 (TAK1) kinase is an indispensable signaling intermediate in tumor necrosis factor (TNF), interleukin 1, and Toll-like receptor signaling pathways. TAK1-binding protein 2 (TAB2) and its closely related protein, TAB3, are binding partners of TAK1 and have previously been identified as adaptors of TAK1 that recruit TAK1 to a TNF receptor signaling complex. TAB2 and TAB3 redundantly mediate activation of TAK1. In this study, we investigated the role of TAB2 by analyzing fibroblasts having targeted deletion of the tab2 gene. In TAB2-deficient fibroblasts, TAK1 was associated with TAB3 and was activated following TNF stimulation. However, TAB2-deficient fibroblasts displayed a significantly prolonged activation of TAK1 compared with wild type control cells. This suggests that TAB2 mediates deactivation of TAK1. We found that a TAK1-negative regulator, protein phosphatase 6 (PP6), was recruited to the TAK1 complex in wild type but not in TAB2-deficient fibroblasts. Furthermore, we demonstrated that both PP6 and TAB2 interacted with the polyubiquitin chains and this interaction mediated the assembly with TAK1. Our results indicate that TAB2 not only activates TAK1 but also plays an essential role in the deactivation of TAK1 by recruiting PP6 through a polyubiquitin chain-dependent mechanism.

TAB4 stimulates TAK1-TAB1 phosphorylation and binds polyubiquitin to direct signaling to NF-kappaB.

Responses to transforming growth factor beta and multiple cytokines involve activation of transforming growth factor beta-activated kinase-1 (TAK1) kinase, which activates kinases IkappaB kinase (IKK) and MKK3/6, leading to the parallel activation of NF-kappaB and p38 MAPK. Activation of TAK1 by autophosphorylation is known to involve three different TAK1-binding proteins (TABs). Here we report a protein phosphatase subunit known as type 2A phosphatase-interacting protein (TIP) that also acts as a TAB because it co-precipitates with and directly binds to TAK1, enhances TAK1 autophosphorylation at unique sites, and promotes TAK1 phosphorylation of IKKbeta and signaling to NF-kappaB. Mass spectrometry demonstrated that co-expression of TAB4 protein significantly increased phosphorylation of four sites in TAK1, in a linker region between the kinase and TAB2/3 binding domains, and two sites in TAB1. Recombinant GST-TAB4 bound in an overlay assay directly to inactive TAK1 and activated TAK1 but not TAK1 phosphorylated in the linker sites, suggesting a bind and release mechanism. In kinase assays using TAK1 immune complexes, added GST-TAB4 selectively stimulated IKK phosphorylation. TAB4 co-precipitated polyubiquitinated proteins dependent on a Phe-Pro motif that was required to enhance phosphorylation of TAK1. TAB4 mutated at Phe-Pro dominantly interfered with IL-1beta activation of NF-kappaB involving IKK-dependent but not p38 MAPK-dependent signaling. The results show that TAB4 binds TAK1 and polyubiquitin chains to promote specific sites of phosphorylation in TAK1-TAB1, which activates IKK signaling to NF-kappaB.