Philipp Yu

Endosomal Translocation of Vertebrate DNA Activates Dendritic Cells via TLR9-Dependent and -Independent Pathways

TLRs discriminate foreign from self via their specificity for pathogen-derived invariant ligands, an example being TLR9 recognizing bacterial unmethylated CpG motifs. In this study we report that endosomal translocation of CpG DNA via the natural endocytotic pathway is inefficient and highly saturable, whereas endosomal translocation of DNA complexed to the cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) is not. Interestingly, DOTAP-mediated enhanced endosomal translocation of otherwise nonstimulatory vertebrate DNA or of certain noncanonical CpG motifs triggers robust dendritic cell activation in terms of both up-regulation of CD40/CD69 and cytokine production, such as type I IFN and IL-6. We report that the stimulatory activity of phosphorothioated noncanonical CpG oligodeoxynucleotides is TLR9 dependent, whereas phosphodiester DNA, such as vertebrate DNA, in addition trigger TLR9-independent pathways. We propose that the inefficiency of the natural route for DNA internalization hinders low affinity TLR9 ligands in endosomes to reach threshold concentrations required for TLR9 activation. Endosomal compartmentalization of TLR9 may thus reflect an evolutionary strategy to avoid TLR9 activation by self-DNA.

An EF hand mutation in Stim1 causes premature platelet activation and bleeding in mice

Changes in cytoplasmic Ca2+ levels regulate a variety of fundamental cellular functions in virtually all cells. In nonexcitable cells, a major pathway of Ca2+ entry involves receptor-mediated depletion of intracellular Ca2+ stores followed by the activation of store-operated calcium channels in the plasma membrane. We have established a mouse line expressing an activating EF hand motif mutant of stromal interaction molecule 1 (Stim1), an ER receptor recently identified as the Ca2+ sensor responsible for activation of Ca2+ release-activated (CRAC) channels in T cells, whose function in mammalian physiology is not well understood. Mice expressing mutant Stim1 had macrothrombocytopenia and an associated bleeding disorder. Basal intracellular Ca2+ levels were increased in platelets, which resulted in a preactivation state, a selective unresponsiveness to immunoreceptor tyrosine activation motif-coupled agonists, and increased platelet consumption. In contrast, basal Ca2+ levels, but not receptor-mediated responses, were affected in mutant T cells. These findings identify Stim1 as a central regulator of platelet function and suggest a cell type-specific activation or composition of the CRAC complex.

Basophil-mediated protection against gastrointestinal helminths requires IgE-induced cytokine secretion

Significance Gastrointestinal worms (helminths) infect more than 2 billion people, and vaccines are not yet available. Helminths elicit a type 2 immune response characterized by high serum IgE levels and increased numbers of IL-4– or IL-13–secreting effector cells including Th2 cells, eosinophils, basophils, and type 2 innate lymphoid cells. We determined the mechanism by which basophils contribute to protection against secondary infections with gastrointestinal helminths. Here we demonstrate that basophils are recruited into the small intestine of infected mice and orchestrate the local type 2 immune response in this tissue. Basophil-mediated protection required the presence of IgE and the expression of activating Fc receptors and IL-4/IL-13 in basophils. These findings could help the development of new vaccination strategies against helminths. Basophils orchestrate protection against reinfections with gastrointestinal helminths and ticks, but the underlying mechanisms remain elusive. We investigated the role of Fc receptors on basophils, the antibody isotypes IgG1 and IgE, and basophil-derived IL-4/IL-13 during challenge infections with Heligmosomoides polygyrus and Nippostrongylus brasiliensis. Using mixed bone marrow chimeras, we found that activating Fc receptors on basophils were required for protective immunity but not for regulation of basophil homeostasis. Furthermore, rapid worm expulsion was impaired in IgE-deficient but not in IgG1-deficient mice. Basophils promoted the recruitment of other effector cells into the small intestine and induced expression of the antihelminthic proteins resistin-like molecule β and mucin 5ac. Selective deletion of IL-4/IL-13 in basophils resulted in impaired worm expulsion. Collectively, our results indicate that IgE-mediated activation of basophils and the release of basophil-derived IL-4/IL-13 are critical steps in protective immunity against helminths. Therefore, development of effective vaccines against helminths should consider boosting the IL-4/IgE/basophil axis of the immune system.

Characterization of phospholipase C gamma enzymes with gain-of-function mutations.

Phospholipase Cγ isozymes (PLCγ1 and PLCγ2) have a crucial role in the regulation of a variety of cellular functions. Both enzymes have also been implicated in signaling events underlying aberrant cellular responses. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we have recently identified single point mutations in murine PLCγ2 that lead to spontaneous inflammation and autoimmunity. Here we describe further, mechanistic characterization of two gain-of-function mutations, D993G and Y495C, designated as ALI5 and ALI14. The residue Asp-993, mutated in ALI5, is a conserved residue in the catalytic domain of PLC enzymes. Analysis of PLCγ1 and PLCγ2 with point mutations of this residue showed that removal of the negative charge enhanced PLC activity in response to EGF stimulation or activation by Rac. Measurements of PLC activity in vitro and analysis of membrane binding have suggested that ALI5-type mutations facilitate membrane interactions without compromising substrate binding and hydrolysis. The residue mutated in ALI14 (Tyr-495) is within the spPH domain. Replacement of this residue had no effect on folding of the domain and enhanced Rac activation of PLCγ2 without increasing Rac binding. Importantly, the activation of the ALI14-PLCγ2 and corresponding PLCγ1 variants was enhanced in response to EGF stimulation and bypassed the requirement for phosphorylation of critical tyrosine residues. ALI5- and ALI14-type mutations affected basal activity only slightly; however, their combination resulted in a constitutively active PLC. Based on these data, we suggest that each mutation could compromise auto-inhibition in the inactive PLC, facilitating the activation process; in addition, ALI5-type mutations could enhance membrane interaction in the activated state.

Tumor suppressor role of phospholipase C epsilon in Ras-triggered cancers

Significance Ras oncogens have been implicated in high proportion of cancers. However, selective treatment for Ras oncogenes is not yet available and components that could mediate Ras functions are being extensively assessed as alternative targets. For some potential targets, such as phospholipase Cε (PLCε), it is not clear how they contribute to tumor development. Here we provide several lines of experimental evidence that support a tumor suppressor function of PLCε, contrasting a positive role of several other well-defined effectors of Ras. Our results show a tumor suppressor role for this PLC in animal models and describe its impact on cell proliferation. Together with observations of marked down-regulation in several types of tumors, this study suggests its use as a biological marker in cancer. Phospholipase Cε (PLCε) has been characterized as a direct effector of Ras in vitro and in cellular systems; however, the role of PLCε in tumorigenesis and its link to Ras in this context remain unclear. To assess the role of PLCε in Ras-driven cancers, we generated two new mouse strains: one carrying a targeted deletion of Plce (Plce−/−) and the other carrying mutant alleles of Plce unable to bind to Ras (PlceRAm/RAm). The Plce−/− and, to a lesser degree, PlceRAm/RAm transgenic mice exhibited increased susceptibility to tumor formation in the two-stage skin carcinogenesis protocol, revealing a tumor suppressor function for this PLC. This result also suggests that in this context Ras binding in part regulates functions of PLCε. Although significant differences were not seen in the LSL-KrasG12D nonsmall cell lung carcinoma model, down-regulation of PLCε was found in animal tumors and in cellular systems following expression of the oncogenic Ras. An inhibitory impact of PLCε on cell growth requires intact lipase activity and is likely mediated by protein kinase C enzymes. Further cellular studies suggest involvement of histone deacetylase in the mechanism of PLCε down-regulation. Taken together, our results show a previously unidentified tumor suppressor role for this PLC in animal models and, together with observations of marked down-regulation in colorectal, lung, and skin tumors, suggest its use as a biological marker in cancer.

A novel N‐ethyl‐N‐nitrosourea–induced mutation in phospholipase Cγ2 causes inflammatory arthritis, metabolic defects, and male infertility in vitro in a murine model

OBJECTIVE It is difficult to identify a single causative factor for inflammatory arthritis because of the multifactorial nature of the disease. This study was undertaken to dissect the molecular complexity of systemic inflammatory disease, utilizing a combined approach of mutagenesis and systematic phenotype screening in a murine model. METHODS In a large-scale N-ethyl-N-nitrosourea mutagenesis project, the Ali14 mutant mouse strain was established because of dominant inheritance of spontaneous swelling and inflammation of the hind paws. Genetic mapping and subsequent candidate gene sequencing were conducted to find the causative gene, and systematic phenotyping of Ali14/+ mice was performed in the German Mouse Clinic. RESULTS A novel missense mutation in the phospholipase Cγ2 gene (Plcg2) was identified in Ali14/+ mice. Because of the hyperreactive external entry of calcium observed in cultured B cells and other in vitro experiments, the Ali14 mutation is thought to be a novel gain-of-function allele of Plcg2. Findings from systematic screening of Ali14/+ mice demonstrated various phenotypic changes: an abnormally high T cell:B cell ratio, up-regulation of Ig, alterations in body composition, and a reduction in cholesterol and triglyceride levels in peripheral blood. In addition, spermatozoa from Ali14/+ mice failed to fertilize eggs in vitro, despite the normal fertility of the Ali14/+ male mice in vivo. CONCLUSION These results suggest that the Plcg2-mediated pathways play a crucial role in various metabolic and sperm functions, in addition to initiating and maintaining the immune system. These findings may indicate the importance of the Ali14/+ mouse strain as a model for systemic inflammatory diseases and inflammation-related metabolic changes in humans.

Toll-like receptor 9 in murine lupus: more friend than foe!

The immune response induced by the pathogen-associated-pattern recognition receptor toll-like receptor 9 (TLR9) upon binding of CpG motif-containing DNA has been widely accepted as an important pathway in the immune defense against microbial pathogens. In contrast, the role of TLR9 in anti-DNA antibody generation and the pathogenesis of systemic lupus erythematosus (SLE) remains controversial. Indeed, the in vivo situation might consist of a delicate balance between B-cell receptor and DNA receptor signaling. Most surprisingly, TLR9 deletion does not ameliorate but rather exacerbates pathology in murine models. Such observations warrant caution with therapeutic efforts to treat autoimmune diseases, especially SLE, via TLR modulation.

The Extracellular Domains of IgG1 and T Cell-Derived IL-4/IL-13 Are Critical for the Polyclonal Memory IgE Response In Vivo

IgE-mediated activation of mast cells and basophils contributes to protective immunity against helminths but also causes allergic responses. The development and persistence of IgE responses are poorly understood, which is in part due to the low number of IgE-producing cells. Here, we used next generation sequencing to uncover a striking overlap between the IgE and IgG1 repertoires in helminth-infected or OVA/alum-immunized wild-type BALB/c mice. The memory IgE response after secondary infection induced a strong increase of IgE+ plasma cells in spleen and lymph nodes. In contrast, germinal center B cells did not increase during secondary infection. Unexpectedly, the memory IgE response was lost in mice where the extracellular part of IgG1 had been replaced with IgE sequences. Adoptive transfer studies revealed that IgG1+ B cells were required and sufficient to constitute the memory IgE response in recipient mice. T cell-derived IL-4/IL-13 was required for the memory IgE response but not for expansion of B cells from memory mice. Together, our results reveal a close relationship between the IgE and IgG1 repertoires in vivo and demonstrate that the memory IgE response is mainly conserved at the level of memory IgG1+ B cells. Therefore, targeting the generation and survival of allergen-specific IgG1+ B cells could lead to development of new therapeutic strategies to treat chronic allergic disorders.

IgE knock‐in mice suggest a role for high levels of IgE in basophil‐mediated active systemic anaphylaxis

Immunglobulin E (IgE) production is tightly regulated at the cellular and genetic levels and is believed to be central to allergy development. At least two cellular pathways exist that lead to systemic anaphylaxis reactions in vivo: IgE‐sensitized mast cells and IgG1‐sensitized basophils. Passive anaphylaxis, by application of allergen and allergen‐specific antibodies in mice, indicates a differential contribution of immunoglobulin isotypes to anaphylaxis. However, analysis of a dynamic immunization‐mediated antibody response in anaphylaxis is difficult. Here, we generated IgE knock‐in mice (IgEki), which express the IgE heavy chain instead of IgG1, in order to analyze the contribution of IgG1 and IgE to active anaphylaxis in vivo. IgEki mice display increased IgE production both in vitro and in vivo. The sensitization of IgEki mice by immunization followed by antigen challenge leads to increased anaphylaxis. Homozygous IgEki mice, which lack IgG1 due to the knock‐in strategy, are most susceptible to active systemic anaphylaxis. The depletion of basophils demonstrates their importance in IgE‐mediated anaphylaxis. Therefore, we propose that an enhanced, antigen‐specific, polyclonal IgE response, as is the case in allergic patients, is probably the most efficient way to sensitize basophils to contribute to systemic anaphylaxis in vivo.

A hypomorphic IgH-chain allele affects development of B-cell subsets and favours receptor editing

The quality and quantity of BCR signals impact on cell fate decisions of B lymphocytes. Here, we describe novel gene‐targeted mice, which in the context of normal VDJ recombination show hypomorphic expression of immunoglobulin μ heavy chain (μHC) mRNA levels and hence lower pre‐BCR and BCR levels. Hypomorphic expression of μHC leads to augmented selection processes at all stages of B‐cell development, noticeably at the expansion of pre‐B cells, the positive selection of immature B lymphocytes in the bone marrow and the selection of the follicular (FO), marginal zone (MZ) and B1 B‐lymphocyte compartment in peripheral lymphoid organs. Immature as well as mature FO and MZ B lymphocytes in the peripheral lymphoid organs express lower levels of the receptor for B‐cell activating factor (BAFF). In addition, hypomorphic expression of the BCR favours receptor editing. Together, our results highlight the critical importance of pre‐BCR and BCR receptor levels for the normal development of B‐lymphocyte subpopulations in the context of intact VDJ recombination and a diverse antibody repertoire.