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STAT6, NF-κB (p50) and C/EBPβ transcription factors (TF) were examined with respect to CD23 regulation. Electrophoretic mobility shift assay (EMSA), competition and supershift analysis demonstrated that STAT6 binds the CD23a promoter but with a lower affinity than the consensus site. STAT6-/- mice were analyzed for CD23 levels and showed reduced expression after CD40 ligand trimer (CD40LT) stimulation. However, normal CD23 expression and even some IgE production was induced in STAT6-/- mice with CD40LT/IL-4. EMSA analysis indicated that the CD23a STAT site was bound by a protein in nuclear extracts from CD40 ± IL-4-stimulated STAT6-/- B cells. Western blot analysis of these nuclear extracts demonstrated the presence of STAT3 and STAT5 suggesting that these STATs can induce CD23 in this situation. Further supporting evidence was obtained by showing that IL-2 and IL-4 both synergize with CD40 in an identical manner for CD23 induction on STAT6-/- B cells. EMSA analysis of the two putative NF-κB sites confirmed binding to both, although one site bound was a higher affinity than the second. Analysis of p50-/- mice indicated that this subunit was not necessary for CD23 induction or CD40/IL-4-induced IgE production. Finally, no role for C/EBP was observed in CD23 induction by EMSA or by CD23 induction analysis in C/EBPβ-/- mice, whereas the absence of C/EBPβ did have an effect on IgE production and lipopolysaccharide-induced B cell proliferation. Based on these data, a model is presented which suggests the CD23 superinduction results from STAT and NF-κ B interaction. [ABSTRACT FROM PUBLISHER]

Background: Immune activation, neuroinflammation, and cell death are the hallmarks of multiple sclerosis (MS), which is an autoimmune demyelinating disease of the central nervous system (CNS). It is well-documented that the cellular inhibitor of apoptosis 2 (cIAP2) is induced by inflammatory stimuli and regulates adaptive and innate immune responses, cell death, and the production of inflammatory mediators. However, the impact of cIAP2 on neuroinflammation associated with MS and disease severity remains unknown., Methods: We used experimental autoimmune encephalomyelitis (EAE), a widely used mouse model of MS, to assess the effect of cIAP2 deletion on disease outcomes. We performed a detailed analysis on the histological, cellular, and molecular levels. We generated and examined bone-marrow chimeras to identify the cIAP2-deficient cells that are critical to the disease outcomes., Results: cIAP2 -/- mice exhibited increased EAE severity, increased CD4 + T cell infiltration, enhanced proinflammatory cytokine/chemokine expression, and augmented demyelination. This phenotype was driven by cIAP2-deficient non-hematopoietic cells. cIAP2 protected oligodendrocytes from cell death during EAE by limiting proliferation and activation of brain microglia. This protective role was likely exerted by cIAP2-mediated inhibition of the non-canonical NLRP3/caspase-8-dependent myeloid cell activation during EAE., Conclusions: Our findings suggest that cIAP2 is needed to modulate neuroinflammation, cell death, and survival during EAE. Significantly, our data demonstrate the critical role of cIAP2 in limiting the activation of microglia during EAE, which could be explored for developing MS therapeutics in the future., (© 2022. The Author(s).)

Background: Hysterectomy is the most commonly performed benign gynaecological surgery. Recently, the rates of minimally invasive hysterectomy have fallen due to the banning of mechanical morcellation techniques that rendered minimal invasive gynaecology surgeons unable to extract large uteri from the relatively small colpotomy incisions., Aims: This study aims to share our experience in utilising Colpo-V incision to remove large uterine specimens transvaginally and report its success and complication rates to promote a minimal invasive approach in patients with large uteri without the need to perform large abdominal incisions or transabdominal morcellation., Methods: This is a prospective case series study in which women with large uteri and|or narrow vaginal canal underwent total laparoscopic hysterectomy and a subsequent posterior vaginal wall incision (Colpo-V) to facilitate the intact extraction of the uterus through the vagina. Patients were seen in the clinic six weeks after the surgery for post-operative assessment and documentation of late complications., Results: Seventeen women underwent the procedure, and the intact extraction of the specimen was successful in 16 out of the 17 cases (94%). No major complications were encountered during or after the procedure., Conclusion: Colpo-V incision is a simple and effective technique for the intact extraction of larger uterine specimens at hysterectomy., (© 2021 The Royal Australian and New Zealand College of Obstetricians and Gynaecologists.)

Anaplasma phagocytophilum causes granulocytic anaplasmosis, a debilitating infection that can be fatal in the immunocompromised. It also afflicts animals, including dogs, horses, and sheep. No granulocytic anaplasmosis vaccine exists. Because A. phagocytophilum is an obligate intracellular bacterium, inhibiting microbe-host cell interactions that facilitate invasion can disrupt infection. The binding domains of A. phagocytophilum adhesins A. phagocytophilum invasion protein A (AipA), A. phagocytophilum surface protein (Asp14), and outer membrane protein A (OmpA) are essential for optimal bacterial entry into host cells, but their relevance to infection in vivo is undefined. In this study, C57BL/6 mice were immunized with a cocktail of keyhole limpet hemocyanin-conjugated peptides corresponding to the AipA, Asp14, and OmpA binding domains in alum followed by challenge with A. phagocytophilum The bacterial peripheral blood burden was pronouncedly reduced in immunized mice compared to controls. Examination of pre- and postchallenge sera from these mice revealed that immunization elicited antibodies against AipA and Asp14 peptides but not OmpA peptide. Nonetheless, pooled sera from pre- and postchallenge groups, but not from control groups, inhibited A. phagocytophilum infection of HL-60 cells. Adhesin domain immunization also elicited interferon gamma (IFN-γ)-producing CD8-positive (CD8 + ) T cells. A follow-up study confirmed that immunization against only the AipA or Asp14 binding domain was sufficient to reduce the bacterial peripheral blood load in mice following challenge and elicit antibodies that inhibit A. phagocytophilum cellular infection in vitro These data demonstrate that AipA and Asp14 are critical for A. phagocytophilum to productively infect mice, and immunization against their binding domains elicits a protective immune response., (Copyright © 2020 American Society for Microbiology.)

The role of the inducible costimulatory of T cells (ICOS) has been shown to be important for many different T cell outcomes and is indispensable for follicular helper T cell (T FH ) responses. Since its discovery, there have been several studies on the regulation of ICOS at a transcriptional level. However, the post-translational regulation of ICOS has not been well characterized. Here, we demonstrate that ICOS is internalized following ligation. We show that costimulation with CD3 results in differential internalization and fate than stimulation of ICOS alone. Additionally, we show that ICOS internalization is PI3K and clathrin mediated. The studies presented here not only increase the mechanistic understanding of ICOS post-translational regulation but also give insight into the potential mechanisms by which T cells expressing high affinity receptors may be preferentially chosen to become T FH cells with increased ICOS levels., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Authors.)

Background: Myeloid derived suppressor cells (MDSCs) present a significant obstacle to cancer immunotherapy because they dampen anti-tumor cytotoxic T cell responses. Previous groups, including our own, have reported on the myelo-depletive effects of certain chemotherapy agents. We have shown previously that decitabine increased tumor cell Class I and tumor antigen expression, increased ability of tumor cells to stimulate T lymphocytes, depleted tumor-induced MDSC in vivo and augmented immunotherapy of a murine mammary carcinoma., Results: In this study, we expand upon this observation by testing a next-generation DNA methyltransferase inhibitor (DNMTi), guadecitabine, which has increased stability in the circulation. Using the 4 T1 murine mammary carcinoma model, in BALB/cJ female mice, we found that guadecitabine significantly reduces tumor burden in a T cell-dependent manner by preventing excessive myeloid proliferation and systemic accumulation of MDSC. The remaining MDSC were shifted to an antigen-presenting phenotype. Building upon our previous publication, we show that guadecitabine enhances the therapeutic effect of adoptively transferred antigen-experienced lymphocytes to diminish tumor growth and improve overall survival. We also show guadecitabine's versatility with similar tumor reduction and augmentation of immunotherapy in the C57BL/6 J E0771 murine breast cancer model., Conclusions: Guadecitabine depleted and altered MDSC, inhibited growth of two different murine mammary carcinomas in vivo, and augmented immunotherapeutic efficacy. Based on these findings, we believe the immune-modulatory effects of guadecitabine can help rescue anti-tumor immune response and contribute to the overall effectiveness of current cancer immunotherapies.

Diverse intracellular pathogens rely on eukaryotic cell surface disulfide reductases to invade host cells. Pharmacologic inhibition of these enzymes is cytotoxic, making it impractical for treatment. Identifying and mechanistically dissecting microbial proteins that co-opt surface reductases could reveal novel targets for disrupting this common infection strategy. Anaplasma phagocytophilum invades neutrophils by an incompletely defined mechanism to cause the potentially fatal disease granulocytic anaplasmosis. The bacterium's adhesin, Asp14, contributes to invasion by virtue of its C terminus engaging an unknown receptor. Yeast-two hybrid analysis identified protein disulfide isomerase (PDI) as an Asp14 binding partner. Coimmunoprecipitation confirmed the interaction and validated it to be Asp14 C terminus dependent. PDI knockdown and antibody-mediated inhibition of PDI reductase activity impaired A. phagocytophilum infection of but not binding to host cells. Infection during PDI inhibition was rescued when the bacterial but not host cell surface disulfide bonds were chemically reduced with tris(2-carboxyethyl)phosphine-HCl (TCEP). TCEP also restored bacterial infectivity in the presence of an Asp14 C terminus blocking antibody that otherwise inhibits infection. A. phagocytophilum failed to productively infect myeloid-specific-PDI conditional-knockout mice, marking the first demonstration of in vivo microbial dependency on PDI for infection. Mutational analyses identified the Asp14 C-terminal residues that are critical for binding PDI. Thus, Asp14 binds and brings PDI proximal to A. phagocytophilum surface disulfide bonds that it reduces, which enables cellular and in vivo infection. IMPORTANCE Anaplasma phagocytophilum infects neutrophils to cause granulocytic anaplasmosis, an emerging potentially fatal disease and the second-most common tick-borne illness in the United States. Treatment options are limited, and no vaccine exists. Due to the bacterium's obligatory intracellular lifestyle, A. phagocytophilum survival and pathogenesis are predicated on its ability to enter host cells. Understanding its invasion mechanism will yield new targets for preventing bacterial entry and, hence, disease. We report a novel entry pathway in which the A. phagocytophilum outer membrane protein Asp14 binds host cell surface protein disulfide isomerase via specific C-terminal residues to promote reduction of bacterial surface disulfide bonds, which is critical for cellular invasion and productive infection in vivo Targeting the Asp14 C terminus could be used to prevent/treat granulocytic anaplasmosis. Our findings have broad implications, as a thematically similar approach could be applied to block infection by other intracellular microbes that exploit cell surface reductases., (Copyright © 2020 Green et al.)

Background: The negative media attention surrounding vaginal mesh procedures has seen a rise in demand for minimally invasive non-mesh options for the treatment of stress urinary incontinence (SUI). The laparoscopic Burch colposuspension (LBC) is a non-mesh alternative to synthetic midurethral slings (MUS) with similar short-term outcomes. However, long-term outcomes are not well established., Aims: To evaluate the long-term outcomes of LBC for treatment of SUI in women., Material and Methods: One hundred and fifty-one cases of LBC were performed by a single surgeon over two private hospital settings between January 2010 and January 2016. Follow-up subjective outcomes were obtained in 137 cases (90.7%) utilising standardised questionnaires. Primary outcome was successful treatment of SUI, defined as subjective cure or significant improvement of stress incontinence symptoms. Secondary outcomes included new-onset or worsened symptoms of overactive bladder (OAB), voiding dysfunction, prolapse, and perioperative complications., Results: One hundred and thirty-seven patients were analysed with a mean follow-up of 50.6 months (range: 13-89 months). Primary outcome of successful treatment was achieved in 90.5% of women. New-onset or worsened symptoms of OAB was reported in 10.2%, with a further 8.8% of women experiencing symptomatic voiding dysfunction. Sixteen patients (11.7%) reported new-onset or worsening symptoms of prolapse. There were no major surgical complications., Conclusions: LBC is a safe and effective long-term treatment for SUI, with low failure rates and minimal adverse outcomes. It is a suitable alternative for women with contraindications to mesh or those having concomitant laparoscopic procedures., (© 2019 The Royal Australian and New Zealand College of Obstetricians and Gynaecologists.)

IL-9 is an important mediator of allergic disease that is critical for mast cell-driven diseases. IL-9 is produced by many cell types, including T cells, basophils, and mast cells. Yet, how IL-9 is regulated in mast cells or basophils is not well characterized. In this report, we tested the effects of deficiency of a mouse Il9 gene regulatory element ( Il9 CNS-25) in these cells in vivo and in vitro. In mast cells stimulated with IL-3 and IL-33, the Il9 CNS-25 enhancer is a potent regulator of mast cell Il9 gene transcription and epigenetic modification at the Il9 locus. Our data show preferential binding of STAT5 and GATA1 to CNS-25 over the Il9 promoter in mast cells and that T cells and mast cells have differing requirements for the induction of IL-9 production. Il9 CNS-25 is required for IL-9 production from T cells, basophils, and mast cells in a food allergy model, and deficiency in IL-9 expression results in decreased mast cell expansion. In a Nippostrongylus brasiliensis infection model, we observed a similar decrease in mast cell accumulation. Although decreased mast cells correlated with higher parasite egg burden and delayed clearance in vivo, T cell deficiency in IL-9 also likely contributes to the phenotype. Thus, our data demonstrate IL-9 production in mast cells and basophils in vivo requires Il9 CNS-25, and that Il9 CNS-25-dependent IL-9 production is required for mast cell expansion during allergic intestinal inflammation., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Immunoglobulin E (IgE), though constitutively present at low levels, is most commonly studied in atopic disease where it plays a vital role in mast cell degranulation and in initiating a T helper 2 (Th2) response. With the advent of better detection assays, however, researchers are discovering the importance of IgE in actively contributing to many disease states and pathologies. This review will discuss the latest findings in IgE beyond its role in allergies and recently discovered roles for IgE in its cell-bound form on FcεRI-expressing effector cells like monocytes and dendritic cells. In terms of parasites, we will discuss helminth-induced IgE that appears to protect the worms from immune recognition and a tick-borne illness that elicits an IgE response against red meat. Next, we describe recent findings of how auto-reactive IgE can contribute to the progression of lupus and induce organ damage. Finally, we summarize the emerging roles of IgE in tumor surveillance and antibody-dependent cytotoxicity. We additionally discuss recent or ongoing clinical trials that either target harmful IgE or use the unique characteristics of the isotype., Competing Interests: No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.

Group 2 innate lymphoid cells (ILC2s) play an important role in the initiation of type-2 immune responses. Numerous targets have been identified that may activate or repress ILC2 function, though few negative regulatory feedback pathways induced upon activation have been shown to be operative in ILC2s. Here we demonstrate that loss of ADAM17 from ILC2s results in a selective defect in IL-33 responsiveness, but not IL-25 responsiveness. We find that IL1R2 is significantly upregulated at both the transcript and protein level in IL-33 activated ILC2s. We are also able to demonstrate that ADAM17 regulates IL1R2 levels on ILC2s in both a constitutive and activation induced manner. Additionally, IL1R2 + ILC2s, a unique subset of ILC2s, have decreased Il5 and Il13 transcripts following IL-33 stimulation. Overall, these data suggest that the expression of IL1R2 may act as an activation-induced negative regulatory feedback mechanism to decrease ILC2 responsiveness to IL-33., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Intracellular bacteria that live in host cell-derived vacuoles are significant causes of human disease. Parasitism of low-density lipoprotein (LDL) cholesterol is essential for many vacuole-adapted bacteria. Acid sphingomyelinase (ASM) influences LDL cholesterol egress from the lysosome. Using functional inhibitors of ASM (FIASMAs), we show that ASM activity is key for infection cycles of vacuole-adapted bacteria that target cholesterol trafficking- Anaplasma phagocytophilum , Coxiella burnetii , Chlamydia trachomatis , and Chlamydia pneumoniae. Vacuole maturation, replication, and infectious progeny generation by A. phagocytophilum , which exclusively hijacks LDL cholesterol, are halted and C. burnetii , for which lysosomal cholesterol accumulation is bactericidal, is killed by FIASMAs. Infection cycles of Chlamydiae, which hijack LDL cholesterol and other lipid sources, are suppressed but less so than A. phagocytophilum or C. burnetii A. phagocytophilum fails to productively infect ASM -/- or FIASMA-treated mice. These findings establish the importance of ASM for infection by intracellular bacteria and identify FIASMAs as potential host-directed therapies for diseases caused by pathogens that manipulate LDL cholesterol., (© 2019 Cockburn et al.)

Orientia tsutsugamushi is an obligate intracellular bacterium that infects mononuclear and endothelial cells to cause the emerging global health threat scrub typhus. The ability of O. tsutsugamushi to survive in monocytes facilitates bacterial dissemination to endothelial cells, which can subsequently lead to several potentially fatal sequelae. As a strict intracellular pathogen that lives in the cytoplasm of host cells, O. tsutsugamushi has evolved to counter adaptive immunity. How the pathogen does so and the outcome of this strategy in monocytes versus endothelial cells are poorly understood. This report demonstrates that O. tsutsugamushi reduces cellular levels of NOD-, LRR-, and CARD-containing 5 (NLRC5), a recently identified specific transactivator of major histocompatibility complex class I (MHC-I) component gene expression, to inhibit MHC-I biosynthesis. Importantly, the efficacy of this approach varies with the host cell type infected. In nonprofessional antigen-presenting HeLa and primary human aortic endothelial cells, the O. tsutsugamushi -mediated reduction of NLRC5 results in lowered MHC-I component transcription and, consequently, lower total and/or surface MHC-I levels throughout 72 h of infection. However, in infected THP-1 monocytes, which are professional antigen-presenting cells, the reductions in NLRC5 and MHC-I observed during the first 24 h reverse thereafter. O. tsutsugamushi is the first example of a microbe that targets NLRC5 to modulate the MHC-I pathway. The differential ability of O. tsutsugamushi to modulate this pathway in nonprofessional versus professional antigen-presenting cells could influence morbidity and mortality from scrub typhus., (Copyright © 2019 American Society for Microbiology.)

The role of ICOS and its ligand (ICOSL) have both been shown to be essential for proper humoral responses as well as autoimmune Ab development in mouse models of lupus. In this paper, we report a specific role for the metalloprotease ADAM10 on B cells in regulating both ICOSL and ICOS in a mouse model of increased humoral immunity using B6 mir146a-/- mice and a model of lymphoproliferative disease using the well-characterized lpr model. B6 lpr mice lacking ADAM10 on B cells (A10B lpr ) have decreased nodal proliferation and T cell accumulation compared with control B6 lpr mice. Additionally, A10B lpr mice have a drastic reduction in autoimmune anti-dsDNA Ab production. In line with this, we found a significant reduction in follicular helper T cells and germinal center B cells in these mice. We also show that lymphoproliferation in this model is closely tied to elevated ICOS levels and decreased ICOSL levels. Overall, our data not only show a role of B cell ADAM10 in control autoimmunity but also increase our understanding of the regulation of ICOS and ICOSL in the context of autoimmunity., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Thermogenic fat is a promising target for new therapies in diabetes and obesity. Understanding how thermogenic fat develops is important to develop rational strategies to treat obesity. Previously, we have shown that Tyk2 and STAT3, part of the JAK-STAT pathway, are necessary for proper development of classical brown fat. Using primary preadipocytes isolated from newborn mice we demonstrate that STAT3 is required for differentiation and robust expression of Uncoupling Protein 1 (UCP1). We also confirm that STAT3 is necessary during the early induction stage of differentiation and is dispensable during the later terminal differentiation stage. The inability of STAT3 -/- preadipocytes to differentiate can be rescued using Wnt ligand secretion inhibitors when applied during the induction stage. Through chemical inhibition and RNAi, we show that it is the canonical β-catenin pathway that is responsible for the block in differentiation; inhibition or knockdown of β-catenin can fully rescue adipogenesis and UCP1 expression in the STAT3 -/- adipocytes. During the induction stage, Wnts 1, 3a, and 10b have increased expression in the STAT3 -/- adipocytes, potentially explaining the increased levels and activity of β-catenin. Our results for the first time point towards an interaction between the JAK/STAT pathway and the Wnt/β-catenin pathway during the early stages of in-vitro adipogenesis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Helminth infection is known for generating large amounts of poly-specific IgE. Here we demonstrate that innate-like B1 cells are responsible for this IgE production during infection with the nematode parasites Nippostrongylus brasiliensis and Heligmosomoides polygyrus bakeri. In vitro analysis of B1 cell immunoglobulin class switch recombination to IgE demonstrated a requirement for anti-CD40 and IL-4 that was further enhanced when IL-5 was added or when the B1 source was helminth infected mice. An IL-25-induced upregulation of IgE in B1 cells was also demonstrated. In T cell-reconstituted RAG1 -/- mice, N. brasiliensis clearance was enhanced with the addition of B2 cells in an IgE-dependent manner. This enhanced clearance was impeded by reconstitution with IgE sufficient B1 cells. Mucosal mast cells mediated the B2 cell enhancement of clearance in the absence of B1 cells. The data support B1 cell IgE secretion as a regulatory response exploited by the helminth., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Allergy and allergic asthma are significant health burdens in developed countries and are increasing in prevalence. Dendritic cells (DCs) initiate immune responses to common aeroallergens, and ADAM10 has been demonstrated to be important for the development of adaptive responses. This study's objective was to understand the role of ADAM10 on DCs in the development of allergic and anaphylactic responses., Methods: In this study, we used mouse models of allergic airway inflammation (house dust mice and Alternaria alternata) and OVA-induced models of active anaphylaxis to determine the DC-specific function of ADAM10 and Notch signaling. To examine T H 1 and T H 17 immunity infection with Anaplasma phagocytophilum and Citrobacter rodentium respectively, were used., Results: Mice, which have ADAM10 deleted from DCs, have dramatic reductions in IgE production and do not develop significant T H 2 immune responses. Further, ADAM10 DC -/- mice are resistant to IgE-mediated anaphylaxis. This response is selective for T H 2 immunity as T H 1 and T H 17 immunity is largely unaffected. Notch1, a known ADAM10 substrate, when knocked out of DCs (Notch1 DC -/- ) demonstrated a similar reduction in anaphylaxis and IgE. Without ADAM10 and Notch1 signaling, DCs were unable to make cytokines that stimulate T H 2 cells and cytokines. Anaphylaxis and allergic lung inflammation were restored in ADAM10 DC -/- with the overexpression of the Notch1-intracellular domain, confirming the role of Notch signaling., Conclusions: Targeting ADAM10 and Notch1 on DCs represent a novel strategy for modulating T H 2 immune responses and IgE production., (© 2017 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

The proper regulation of ICOS and ICOS ligand (ICOSL) has been shown to be essential for maintaining proper immune homeostasis. Loss of either protein results in defective humoral immunity, and overexpression of ICOS results in aberrant Ab production resembling lupus. How ICOSL is regulated in response to ICOS interaction is still unclear. We demonstrate that a disintegrin and metalloproteinase (ADAM)10 is the primary physiological sheddase of ICOSL in mice and humans. Using an in vivo system in which ADAM10 is deleted only on B cells, elevated levels of ICOSL were seen. This increase is also seen when ADAM10 is deleted from human B cell lines. Identification of the primary sheddase has allowed the characterization of a novel mechanism of ICOS regulation. In wild-type mice, interaction of ICOS/ICOSL results in ADAM10-induced shedding of ICOSL on B cells and moderate ICOS internalization on T cells. When this shedding is blocked, excessive ICOS internalization occurs. This results in severe defects in T follicular helper development and T H 2 polarization, as seen in a house dust mite exposure model. In addition, enhanced T H 1 and T H 17 immune responses are seen in experimental autoimmune encephalomyelitis. Blockade of ICOSL rescues T cell ICOS surface expression and rescues, at least in part, T follicular helper numbers and the abnormal Ab production previously reported in these mice. Overall, we propose a novel regulation of the ICOS/ICOSL axis, with ADAM10 playing a direct role in regulating ICOSL, as well as indirectly regulating ICOS, thus controlling ICOS/ICOSL-dependent responses., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Notch2 and B cell antigen receptor (BCR) signaling determine whether transitional B cells become marginal zone B (MZB) or follicular B (FoB) cells in the spleen, but it is unknown how these pathways are related. We generated Taok3 -/- mice, lacking the serine/threonine kinase Taok3, and found cell-intrinsic defects in the development of MZB but not FoB cells. Type 1 transitional (T1) B cells required Taok3 to rapidly respond to ligation by the Notch ligand Delta-like 1. BCR ligation by endogenous or exogenous ligands induced the surface expression of the metalloproteinase ADAM10 on T1 B cells in a Taok3-dependent manner. T1 B cells expressing surface ADAM10 were committed to becoming MZB cells in vivo, whereas T1 B cells lacking expression of ADAM10 were not. Thus, during positive selection in the spleen, BCR signaling causes immature T1 B cells to become receptive to Notch ligands via Taok3-mediated surface expression of ADAM10.

Rationale: Deficiency of secreted IgM (sIgM -/- ) accelerates atherosclerosis in Ldlr -/- mice. Several atheroprotective effects of increased levels of IgM antibodies have been suggested, including preventing inflammation induced by oxidized low-density lipoprotein and promoting apoptotic cell clearance. However, the mechanisms by which the lack of sIgM promotes lesion formation remain unknown., Objective: To identify the mechanisms by which sIgM deficiency accelerates atherosclerosis in mice., Methods and Results: We here show that both sIgM -/- and Ldlr -/- sIgM -/- mice develop increased plasma IgE titers because of impaired generation of B cells expressing the low-affinity IgE receptor CD23, which mediates the clearance of IgE antibodies. We further report that Ldlr -/- sIgM -/- mice exhibit increased numbers of activated mast cells and neutrophils in the perivascular area of atherosclerotic plaques. Treatment with an anti-IgE-neutralizing antibody fully reversed vascular inflammation and accelerated atherosclerotic lesion formation in cholesterol-fed Ldlr -/- sIgM -/- mice., Conclusions: Thus, our data identify a previously unsuspected mechanism by which sIgM deficiency aggravates atherosclerosis., (© 2016 American Heart Association, Inc.)

Infections with helminth parasites are endemic in the developing world and are a target for intervention with new therapies. Macrophage migration inhibitory factor (MIF) is a cytokine with pleiotropic effects in inflammation and immune responses. We investigated the role of MIF in a naturally cleared model of helminth infection in rodents, Nippostrongylus brasiliensis. At day 7 postinfection, MIF-deficient (MIF -/- ) mice had reduced parasite burden and mounted an enhanced type 2 immune response (Th2), including increased Gata3 expression and interleukin-13 (IL-13) production in the mesenteric lymph nodes (MLNs). Bone marrow reconstitution demonstrated that MIF produced from hematopoietic cells was crucial and Rag1 -/- reconstitution provided direct evidence that MIF -/- CD4 + T cells were responsible for the augmented parasite clearance. MIF -/- CD4 + T cells produced less IL-6 postinfection, which correlated with enhanced Th2 responses. MIF -/- CD4 + T cells exhibited lower nuclear factor-κB activation, potentially explaining the reduction in IL-6. Finally, we demonstrated enhanced clearance of the parasite and Th2 response in wild-type mice treated with the MIF tautomerase inhibitor, sulforaphane, a compound found naturally found in cruciferous vegetables. These results are the first to describe the importance of the tautomerase enzyme activity in MIF function in N. brasiliensis infection.

CD23 has been implicated as a negative regulator of IgE and IgG antibody responses. However, whether CD23 has any role in B-cell activation remains unclear. We examined the expression of CD23 in different subsets of peripheral B cells and the impact of CD23 expression on the early events of B-cell receptor (BCR) activation using CD23 knockout (KO) mice. We found that in addition to marginal zone B cells, mature follicular B cells significantly down regulate the surface expression level of CD23 after undergoing isotype switch and memory B-cell differentiation. Upon stimulation with membrane-associated antigen, CD23 KO causes significant increases in the area of B cells contacting the antigen-presenting membrane and the magnitude of BCR clustering. This enhanced cell spreading and BCR clustering is concurrent with increases in the levels of phosphorylation of tyrosine and Btk, as well as the levels of F-actin and phosphorylated Wiskott Aldrich syndrome protein, an actin nucleation promoting factor, in the contract zone of CD23 KO B cells. These results reveal a role of CD23 in the negative regulation of BCR signaling in the absence of IgE immune complex and suggest that CD23 down-regulates BCR signaling by influencing actin-mediated BCR clustering and B-cell morphological changes.

Sperm associated antigen 6 (SPAG6), a component of the central apparatus of the "9 + 2" axoneme, plays a central role in ciliary and flagellar motility; but, its contribution to adaptive immunity and immune system development is completely unknown. While immune cells lack a cilium, the immunological synapse is a surrogate cilium as it utilizes the same machinery as ciliogenesis including the nucleation of microtubules at the centrosome. This prompted our hypothesis that SPAG6 critically regulates the formation and function of immunological synapses. Using bone marrow reconstitution studies of adult WT mice, we demonstrate that SPAG6 is expressed in primary and secondary lymphoid tissues, is associated with the centrosome in lymphocytes, and its deficiency results in synapse disruption due to loss of centrosome polarization and actin clearance at the synaptic cleft. Improper synapse formation in Spag6KO mice was associated with defective CTL functions and impaired humoral immunity as indicated by reduced germinal centers reactions, follicular CD4 T cells, and production of class-switched antibody, together with expansion of B1 B cells. This novel report demonstrates the requirement of SPAG6 for optimal synapse formation and function, its direct role in immune cell function, and provides a novel mechanism for infertility disorders related to SPAG6.

The epithelial lining of the airway tract and allergen-specific IgE are considered essential controllers of inflammatory responses to allergens. The human low affinity IgE receptor, CD23 (FcɛRII), is capable of transporting IgE or IgE-allergen complexes across the polarized human airway epithelial cell (AEC) monolayer in vitro. However, it remains unknown whether the CD23-dependent IgE transfer pathway in AECs initiates and facilitates allergic inflammation in vivo, and whether inhibition of this pathway attenuates allergic inflammation. To this end, we show that in wild-type (WT) mice, epithelial CD23 transcytosed both IgE and ovalbumin (OVA)-IgE complexes across the airway epithelial barrier, whereas neither type of transcytosis was observed in CD23 knockout (KO) mice. In chimeric mice, OVA sensitization and aerosol challenge of WT/WT (bone-marrow transfer from the WT to WT) or CD23KO/WT (CD23KO to WT) chimeric mice, which express CD23 on radioresistant airway structural cells (mainly epithelial cells) resulted in airway eosinophilia, including collagen deposition and a significant increase in goblet cells, and increased airway hyperreactivity. In contrast, the absence of CD23 expression on airway structural or epithelial cells, but not on hematopoietic cells, in WT/CD23KO (the WT to CD23KO) chimeric mice significantly reduced OVA-driven allergic airway inflammation. In addition, inhalation of the CD23-blocking B3B4 antibody in sensitized WT mice before or during airway challenge suppressed the salient features of asthma, including bronchial hyperreactivity. Taken together, these results identify a previously unproven mechanism in which epithelial CD23 plays a central role in the development of allergic inflammation. Further, our study suggests that functional inhibition of CD23 in the airway is a potential therapeutic approach to inhibit the development of asthma.

Background: Asthma, a chronic inflammatory condition defined by episodic shortness of breath with expiratory wheezing and cough, is a serious health concern affecting more than 250 million persons. Genome-wide association studies have identified ORM (yeast)-like protein isoform 3 (ORMDL3) as a gene associated with susceptibility to asthma. Although its yeast ortholog is a negative regulator of de novo ceramide biosynthesis, how ORMDL3 contributes to asthma pathogenesis is not known., Objectives: We sought to decipher the molecular mechanism for the pathologic functions of ORMDL3 in asthma and the relationship to its evolutionarily conserved role in regulation of ceramide homeostasis., Methods: We determined the relationship between expression of ORMDL3 and ceramide in epithelial and inflammatory cells and in asthma pathogenesis in mice., Results: Although small increases in ORMDL3 expression decrease ceramide levels, remarkably, higher expression in lung epithelial cells and macrophages in vitro and in vivo increased ceramide production, which promoted chronic inflammation, airway hyperresponsiveness, and mucus production during house dust mite-induced allergic asthma. Moreover, nasal administration of the immunosuppressant drug FTY720/fingolimod reduced ORMDL3 expression and ceramide levels and mitigated airway inflammation and hyperreactivity and mucus hypersecretion in house dust mite-challenged mice., Conclusions: Our findings demonstrate that overexpression of ORMDL3 regulates ceramide homeostasis in cells in a complex manner and suggest that local FTY720 administration might be a useful therapeutic intervention for the control of allergic asthma., (Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

ADAM10, as the sheddase of the low affinity IgE receptor (CD23), promotes IgE production and thus is a unique target for attenuating allergic disease. Herein, we describe that B cell levels of ADAM10, specifically, are increased in allergic patients and Th2 prone WT mouse strains (Balb/c and A/J). While T cell help augments ADAM10 expression, Balb WT B cells exhibit increased ADAM10 in the naïve state and even more dramatically increased ADAM10 after anti-CD40/IL4 stimulation compared C57 (Th1 prone) WT B cells. Furthermore, ADAM17 and TNF are reduced in allergic patients and Th2 prone mouse strains (Balb/c and A/J) compared to Th1 prone controls. To further understand this regulation, ADAM17 and TNF were studied in C57Bl/6 and Balb/c mice deficient in ADAM10. C57-ADAM10B-/- were more adept at increasing ADAM17 levels and thus TNF cleavage resulting in excess follicular TNF levels and abnormal secondary lymphoid tissue architecture not noted in Balb-ADAM10B-/-. Moreover, the level of B cell ADAM10 as well as Th context is critical for determining IgE production potential. Using a murine house dust mite airway hypersensitivity model, we describe that high B cell ADAM10 level in a Th2 context (Balb/c WT) is optimal for disease induction including bronchoconstriction, goblet cell metaplasia, mucus, inflammatory cellular infiltration, and IgE production. Balb/c mice deficient in B cell ADAM10 have attenuated lung and airway symptoms compared to Balb WT and are actually most similar to C57 WT (Th1 prone). C57-ADAM10B-/- have even further reduced symptomology. Taken together, it is critical to consider both innate B cell levels of ADAM10 and ADAM17 as well as Th context when determining host susceptibility to allergic disease. High B cell ADAM10 and low ADAM17 levels would help diagnostically in predicting Th2 disease susceptibility; and, we provide support for the use ADAM10 inhibitors in treating Th2 disease.

IgE antigen complexes induce increased specific T cell proliferation and increased specific IgG production. Immediately after immunization, CD23(+) B cells capture IgE antigen complexes, transport them to the spleen where, via unknown mechanisms, dendritic cells capture the antigen and present it to T cells. CD23, the low affinity IgE receptor, binds IgE antigen complexes and internalizes them. In this study, we show that these complexes are processed onto B-cell derived exosomes (bexosomes) in a CD23 dependent manner. The bexosomes carry CD23, IgE and MHC II and stimulate antigen specific T-cell proliferation in vitro. When IgE antigen complex stimulated bexosomes are incubated with dendritic cells, dendritic cells induce specific T-cell proliferation in vivo, similar to IgE antigen complexes. This suggests that bexosomes can provide the essential transfer mechanism for IgE antigen complexes from B cells to dendritic cells.

It has been shown recently that MCs are required for differential regulation of the immune response by granulocytic versus monocytic MDSCs. Granulocytic MDSCs promoted parasite clearance, whereas monocytic MDSCs enhanced tumor progression; both activities were abrogated in MC-deficient mice. Herein, we demonstrate that the lack of MCs also influences MDSC trafficking. Preferential trafficking to the liver was not seen in MC-deficient mice. In addition, evidence that the MC mediator histamine was important in MDSC trafficking and activation is also shown. MDSCs express HR1-3. Blockade of these receptors by HR1 or HR2 antagonists reversed the histamine enhancement of MDSC survival and proliferation observed in cell culture. In addition, histamine differentially influenced Arg1 and iNOS gene expression in MDSCs and greatly enhanced IL-4 and IL-13 message, especially in granulocytic MDSCs. Evidence that histamine influenced activity seen in vitro translated to in vivo when HR1 and HR2 antagonists blocked the effect of MDSCs on parasite expulsion and tumor metastasis. All of these data support the MDSC-mediated promotion of Th2 immunity, leading to the suggestion that allergic-prone individuals would have elevated MDSC levels. This was directly demonstrated by looking at the relative MDSC levels in allergic versus control patients. Monocytic MDSCs trended higher, whereas granulocytic MDSCs were increased significantly in allergic patients. Taken together, our studies indicate that MCs and MC-released histamine are critical for MDSC-mediated immune regulation, and this interaction should be taken into consideration for therapeutic interventions that target MDSCs., (© 2014 Society for Leukocyte Biology.)

A Disintegrin and Metalloproteinase (ADAM)-10 plays critical roles in neuronal migration and distribution. Recently, ADAM10 deletion was shown to disrupt myelopoiesis. We found that inducible deletion of ADAM10 using Mx1-driven Cre recombinase for a period of three weeks resulted in mast cell hyperplasia in the skin, intestine and spleen. Mast cells express surface ADAM10 in vitro and in vivo, at high levels compared to other immune cells tested. ADAM10 is important for mast cell migration, since ADAM10-deficiency reduced c-Kit-mediated migration. As with some mast cell proteases, ADAM10 expression could be altered by the cytokine microenvironment, being inhibited by IL-10 or TGFβ1, but not by several other T cell-derived cytokines. Collectively these data show that the ADAM10 protease is an important factor in mast cell migration and tissue distribution, and can be manipulated by environmental cues., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Mast cells and MDSCs are increased by parasitic infection and tumor growth. We previously demonstrated that enhanced MDSC development in ADAM10 transgenic mice yielded resistance to Nb infection and that coculturing MDSCs and mast cells enhanced cytokine production. In the current work, we show that MDSC-mast cell coculture selectively enhances IgE-mediated cytokine secretion among mast cells, without increasing MDSC cytokine production. This effect was independent of cell contact and elicited by Ly6C(+) and Ly6C/G+ MDSC subsets. These interactions were functionally important. MDSC depletion with the FDA-approved drug gemcitabine exacerbated Nb or Trichinella spiralis infection and reduced mast cell-dependent AHR and lung inflammation. Adoptive transfer of MDSC worsened AHR in WT but not mast cell-deficient Wsh/Wsh mice. These data support the hypothesis that MDSCs enhance mast cell inflammatory responses and demonstrate that this interaction can be altered by an existing chemotherapeutic.

B cell A disintegrin and metalloproteinase 10 (ADAM10) is required for the development and maintenance of proper secondary lymphoid tissue architecture; however, the underlying mechanism remains unclear. In this study, we show disturbances in naive lymph node architecture from B cell-specific ADAM10-deficient mice (ADAM10(B-/-)) including loss of B lymphocyte/T lymphocyte compartmentalization, attenuation of follicular dendritic cell reticula, excessive collagen deposition, and increased high endothelial venule formation. Because TNF-α signaling is critical for secondary lymphoid tissue architecture, we examined compensatory changes in ADAM17 and TNF-α in ADAM10(B-/-) B cells. Surprisingly, defective follicular development in these mice was associated with increased rather than decreased TNF-α expression. In this article, we describe an increase in TNF-α message, mRNA stability, soluble protein release, and membrane expression in ADAM10(B-/-) B cells compared with wild type (WT), which coincides with increased ADAM17 message and protein. To assess the mechanistic contribution of excessive TNF-α to abnormal lymphoid architecture in ADAM10(B-/-) mice, we performed a bone marrow reconstitution study. Rectification of WT architecture was noted only in irradiated WT mice reconstituted with ADAM10(B-/-) + TNF knockout bone marrow because of normalization of TNF-α levels not seen in ADAM10(B-/-) alone. We conclude that ADAM17 overcompensation causes excessive TNF-α shedding and further upregulation of TNF-α expression, creating an aberrant signaling environment within B cell cortical regions of ADAM10(B-/-) lymph nodes, highlighting a key interplay between B cell ADAM10 and ADAM17 with respect to TNF-α homeostasis.

We previously demonstrated that TGF-β1 suppresses IgE-mediated signaling in human and mouse mast cells in vitro, an effect that correlated with decreased expression of the high-affinity IgE receptor, FcεRI. The in vivo effects of TGF-β1 and the means by which it suppresses mast cells have been less clear. This study shows that TGF-β1 suppresses FcεRI and c-Kit expression in vivo. By examining changes in cytokine production concurrent with FcεRI expression, we found that TGF-β1 suppresses TNF production independent of FcεRI levels. Rather, IgE-mediated signaling was altered. TGF-β1 significantly reduced expression of Fyn and Stat5, proteins critical for cytokine induction. These changes may partly explain the effects of TGF-β1, because Stat5B overexpression blocked TGF-mediated suppression of IgE-induced cytokine production. We also found that Stat5B is required for mast cell migration toward stem cell factor, and that TGF-β1 reduced this migration. We found evidence that genetic background may alter TGF responses. TGF-β1 greatly reduced mast cell numbers in Th1-prone C57BL/6, but not Th2-prone 129/Sv mice. Furthermore, TGF-β1 did not suppress IgE-induced cytokine release and did increase c-Kit-mediated migration in 129/Sv mast cells. These data correlated with high basal Fyn and Stat5 expression in 129/Sv cells, which was not reduced by TGF-β1 treatment. Finally, primary human mast cell populations also showed variable sensitivity to TGF-β1-mediated changes in Stat5 and IgE-mediated IL-6 secretion. We propose that TGF-β1 regulates mast cell homeostasis, and that this feedback suppression may be dependent on genetic context, predisposing some individuals to atopic disease.

The generation of antigen-specific antibodies and the development of immunological memory require collaboration between B and T cells. T cell-secreted IL-4 is important for B cell survival, isotype switch to IgG1 and IgE, affinity maturation, and the development of germinal centers (GC). Fyn, a member of the Src family tyrosine kinase, is widely expressed in many cell types, including lymphocytes. This kinase is known to interact with both the B cell and T cell receptor (BCR and TCR, respectively). While Fyn deletion does not impair the development of immature T cells and B cells, TCR signaling is altered in mature T cells. The current study demonstrates that Fyn deficient (KO) B cells have impaired IL-4 signaling. Fyn KO mice displayed low basal levels of IgG1, IgE and IgG2c, and delayed antigen-specific IgG1 and IgG2b production, with a dramatic decrease in antigen-specific IgG2c following immunization with a T-dependent antigen. Defects in antibody production correlated with significantly reduced numbers of GC B cells, follicular T helper cells (TFH), and splenic plasma cells (PC). Taken together, our data demonstrate that Fyn kinase is required for optimal humoral responses.

Immunoglobulins (Igs) are a critical component of the adaptive immune system of both man and mouse. The ability to detect and characterize Igs is an invaluable technique for immunology in either a research or a clinical setting. The advent of enzyme-linked immunosorbent assays (ELISAs) and monoclonal antibody technology has proven instrumental for advancing the science of Ig biology. IgE is of interest as it is the primary Ig responsible for allergic reactions ranging from allergic rhinitis to anaphylaxis. Here, we describe the history behind the IgE discovery and the protocol for purifying IgE and anti-IgE in the mouse. This is followed by our ELISA protocol for mouse IgE detection.

Background: Fullerenes are molecules being investigated for a wide range of therapeutic applications. We have shown previously that certain fullerene derivatives (FDs) inhibit mast cell (MC) function in vitro, and here we examine their in vivo therapeutic effect on asthma, a disease in which MCs play a predominant role., Objective: We sought to determine whether an efficient MC-stabilizing FD (C(70)-tetraglycolate [TGA]) can inhibit asthma pathogenesis in vivo and to examine its in vivo mechanism of action., Methods: Asthma was induced in mice, and animals were treated intranasally with TGA either simultaneously with treatment or after induction of pathogenesis. The efficacy of TGA was determined through the measurement of airway inflammation, bronchoconstriction, serum IgE levels, and bronchoalveolar lavage fluid cytokine and eicosanoid levels., Results: We found that TGA-treated mice have significantly reduced airway inflammation, eosinophilia, and bronchoconstriction. The TGA treatments are effective, even when given after disease is established. Moreover, we report a novel inhibitory mechanism because TGA stimulates the production of an anti-inflammatory P-450 eicosanoid metabolites (cis-epoxyeicosatrienoic acids [EETs]) in the lung. Inhibitors of these anti-inflammatory EETs reversed TGA inhibition. In human lung MCs incubated with TGA, there was a significant upregulation of CYP1B gene expression, and TGA also reduced IgE production from B cells. Lastly, MCs incubated with EET and challenged through FcεRI had a significant blunting of mediator release compared with nontreated cells., Conclusion: The inhibitory capabilities of TGA reported here suggest that FDs might be used a platform for developing treatments for asthma., (Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Myeloid-derived suppressor cells (MDSCs) are primarily recognized for their immunosuppressive properties in malignant disease. However, their interaction with other innate immune cells and their regulation of immune responses, such as in parasitic infection, necessitate further characterization. We used our previously published mouse model of MDSC accumulation to examine the immunoregulatory role of MDSCs in B16 melanoma metastasis and Nippostrongylus brasiliensis infection. In this study, we demonstrate that the activity of MDSCs is dependent on the immune stimuli and subset induced. Monocytic MDSCs predictably suppressed antitumor immune responses but granulocytic MDSCs surprisingly enhanced the clearance of N. brasiliensis infection. Intriguingly, both results were dependent on MDSC interaction with mast cells (MCs), as demonstrated by adoptive-transfer studies in MC-deficient (Kit(Wsh)(/)(Wsh)) mice. These findings were further supported by ex vivo cocultures of MCs and MDSCs, indicating a synergistic increase in cytokine production. Thus, MCs can enhance both immunosuppressive and immunosupportive functions of MDSCs.

Many chronic pulmonary diseases are associated with pulmonary hypertension (PH) and pulmonary vascular remodeling, which is a term that continues to be used to describe a wide spectrum of vascular abnormalities. Pulmonary vascular structural changes frequently increase pulmonary vascular resistance, causing PH and right heart failure. Although rat models had been standard models of PH research, in more recent years the availability of genetically engineered mice has made this species attractive for many investigators. Here we review a large amount of data derived from experimental PH reports published since 1996. These studies using wild-type and genetically designed mice illustrate the challenges and opportunities provided by these models. Hemodynamic measurements are difficult to obtain in mice, and right heart failure has not been investigated in mice. Anatomical, cellular, and genetic differences distinguish mice and rats, and pharmacogenomics may explain the degree of PH and the particular mode of pulmonary vascular adaptation and also the response of the right ventricle.

Anaphylaxis is a rapid, life-threatening hypersensitivity reaction. Until recently, it was mainly attributed to histamine released by mast cells activated by allergen crosslinking (XL) of FcεRI-bound allergen-specific IgE. However, recent reports established that anaphylaxis could also be triggered by basophil, macrophage, and neutrophil secretion of platelet-activating factor subsequent to FcγR stimulation by IgG/Ag complexes. We have investigated the contribution of Fyn and Lyn tyrosine kinases to FcγRIIb and FcγRIII signaling in the context of IgG-mediated passive systemic anaphylaxis (PSA). We found that mast cell IgG XL induced Fyn, Lyn, Akt, Erk, p38, and JNK phosphorylation. Additionally, IgG XL of mast cells, basophils, and macrophages resulted in Fyn- and Lyn-regulated mediator release in vitro. FcγR-mediated activation was enhanced in Lyn-deficient (knockout [KO]) cells, but decreased in Fyn KO cells, compared with wild-type cells. More importantly, Lyn KO mice displayed significantly exacerbated PSA features whereas no change was observed for Fyn KO mice, compared with wild-type littermates. Intriguingly, we establish that mast cells account for most serum histamine in IgG-induced PSA. Taken together, our findings establish pivotal roles for Fyn and Lyn in the regulation of PSA and highlight their unsuspected functions in IgG-mediated pathologies.

Periodontitis is the most common disease of microbial etiology in humans. Periopathogen survival is dependent upon evasion of complement-mediated destruction. Treponema denticola, an important contributor to periodontitis, evades killing by the alternative complement cascade by binding factor H (FH) to its surface. Bound FH is rapidly cleaved by the T. denticola protease, dentilisin. In this report, the structure of the T. denticola FH-binding protein, FhbB, was solved to 1.7 Å resolution. FhbB possesses a unique fold that imparts high thermostability. The kinetics of the FH/FhbB interaction were assessed using surface plasmon resonance. A K(D) value in the micromolar range (low affinity) was demonstrated, and rapid off kinetics were observed. Site-directed mutagenesis and sucrose octasulfate competition assays collectively indicate that the negatively charged face of FhbB binds within FH complement control protein module 7. This study provides significant new insight into the molecular basis of FH/FhbB interaction and advances our understanding of the role that T. denticola plays in the development and progression of periodontal disease.

Recognition of pathogen-associated molecular patterns by innate immune receptors is essential for host defense responses. Although extracellular stress proteins are considered as indicators of the stressful conditions (e.g., infection or cell injury), the exact roles of these molecules in the extracellular milieu remain less defined. We found that glucose-regulated protein 170 (Grp170), the largest stress protein and molecular chaperone, is highly efficient in binding CpG oligodeoxynucleotides (CpG-ODN), the microbial DNA mimetic sensed by toll-like receptor 9 (TLR9). Extracellular Grp170 markedly potentiates the endocytosis and internalization of CpG-ODN by mouse bone marrow-derived macrophages and directly interacts with endosomal TLR9 on cell entry. These molecular collaborations result in the synergistic activation of the MyD88-dependent signaling and enhanced production of proinflammatory cytokines and nitric oxide in mouse primary macrophages as well as human THP-1 monocyte-derived macrophages, suggesting that Grp170 released from injured cells facilitates the sensing of pathogen-associated "danger" signals by intracellular receptors. This CpG-ODN chaperone complex-promoted innate immunity confers increased resistance in mice to infection of Listeria monocytogenes compared with CpG-ODN treatment alone. Our studies reveal a previously unrecognized attribute of Grp170 as a superior DNA-binding chaperone capable of amplifying TLR9 activation on pathogen recognition, which provides a conceptual advance in understanding the dynamics of ancient chaperoning functions inside and outside the cell.

A disintegrin and metalloprotease 10 (ADAM10) is a key regulator of cellular processes by shedding extracellular domains of transmembrane proteins. We have previously demonstrated that deletion of B cell expressed ADAM10 results in changes in lymphoid tissue architecture and impaired germinal center (GC) formation. In this study, mice were generated in which ADAM10 is deleted in B cells following class switch recombination (ADAM10(Δ/Δ)IgG1-cre(+/-) mice). Despite normal GC formation, antibody responses were impaired in ADAM10(Δ/Δ)IgG1-cre(+/-) mice, implicating ADAM10 in post-GC and extrafollicular B cell terminal differentiation. Surprisingly, plasma cell (PC) numbers were normal in ADAM10(Δ/Δ)IgG1-cre(+/-) mice when compared to controls. However, PCs isolated from ADAM10(Δ/Δ)IgG1-cre(+/-) mice exhibited decreased expression of transcription factors important for PC function: Prdm1, Xbp1 and Irf4. Bcl6 is a GC transcriptional repressor that inhibits the PC transcriptional program and thus must be downregulated for PC differentiation to occur. Bcl6 expression was increased in PCs isolated from ADAM10(Δ/Δ)IgG1-cre(+/-) mice at both the mRNA and protein level. These results demonstrate that ADAM10 is required for proper transcription factor expression in PCs and thus, for normal PC function.