Nicolas Charles

Regulation of microRNA Expression and Abundance during Lymphopoiesis

Although the cellular concentration of miRNAs is critical to their function, how miRNA expression and abundance are regulated during ontogeny is unclear. We applied miRNA-, mRNA-, and ChIP-Seq to characterize the microRNome during lymphopoiesis within the context of the transcriptome and epigenome. We show that lymphocyte-specific miRNAs are either tightly controlled by polycomb group-mediated H3K27me3 or maintained in a semi-activated epigenetic state prior to full expression. Because of miRNA biogenesis, the cellular concentration of mature miRNAs does not typically reflect transcriptional changes. However, we uncover a subset of miRNAs for which abundance is dictated by miRNA gene expression. We confirm that concentration of 5p and 3p miRNA strands depends largely on free energy properties of miRNA duplexes. Unexpectedly, we also find that miRNA strand accumulation can be developmentally regulated. Our data provide a comprehensive map of immunitys microRNome and reveal the underlying epigenetic and transcriptional forces that shape miRNA homeostasis.

Basophils and the T helper 2 environment can promote the development of lupus nephritis

In systemic lupus erythematosus (SLE), self-reactive antibodies can target the kidney (lupus nephritis), leading to functional failure and possible mortality. We report that activation of basophils by autoreactive IgE causes their homing to lymph nodes, promoting T helper type 2 (TH2) cell differentiation and enhancing the production of self-reactive antibodies that cause lupus-like nephritis in mice lacking the Src family protein tyrosine kinase Lyn (Lyn−/− mice). Individuals with SLE also have elevated serum IgE, self-reactive IgEs and activated basophils that express CD62 ligand (CD62L) and the major histocompatibility complex (MHC) class II molecule human leukocyte antigen-DR (HLA-DR), parameters that are associated with increased disease activity and active lupus nephritis. Basophils were also present in the lymph nodes and spleen of subjects with SLE. Thus, in Lyn−/− mice, basophils and IgE autoantibodies amplify autoantibody production that leads to lupus nephritis, and in individuals with SLE IgE autoantibodies and activated basophils are factors associated with disease activity and nephritis.

Lyn Kinase Controls Basophil GATA-3 Transcription Factor Expression and Induction of Th2 Cell Differentiation

T helper 1 (Th1)-Th2 cell balance is key to host defense and its dysregulation has pathophysiological consequences. Basophils are important in Th2 cell differentiation. However, the factors controlling the onset and extent of basophil-mediated Th2 cell differentiation are unknown. Here, we demonstrate that Lyn kinase dampened basophil expression of the transcription factor GATA-3 and the initiation and extent of Th2 cell differentiation. Lyn-deficient mice had a marked basophilia, a constitutive Th2 cell skewing that was exacerbated upon in vivo challenge of basophils, produced antibodies to a normally inert antigen, and failed to appropriately respond to a Th1 cell-inducing pathogen. The Th2 cell skewing was dependent on basophils, immunoglobulin E, and interleukin-4, but was independent of mast cells. Our findings demonstrate that basophil-expressed Lyn kinase exerts regulatory control on Th2 cell differentiation and function.

Cutting Edge: Genetic Variation Influences FcεRI-Induced Mast Cell Activation and Allergic Responses

Mast cell responses are influenced by a diverse array of environmental factors, but little is known about the effect of genetic background. In this study, we report that 129/Sv mice had high levels of circulating IgE, increased expression of the high-affinity receptor for IgE (FcεRI), and greater sensitivity to anaphylaxis when compared with C57BL/6 mice. Bone marrow-derived mast cells (BMMCs) from 129/Sv mice showed more robust degranulation upon the engagement of FcεRI. Deficiency of the Src family kinase Lyn enhanced degranulation in 129/Sv BMMCs but inhibited this response in C57BL/6 cells. C57BL/6 lyn−/− BMMCs had reduced expression of the Src family kinase Fyn, and increasing its expression markedly enhanced degranulation. In human mast cells the silencing of Lyn or Fyn expression resulted in hyperdegranulation or hypodegranulation, respectively. The findings demonstrate a genetic influence on the extent of a mast cell’s response and identify Fyn kinase as a contributory determinant.

Evidence for neuronal expression of functional Fc (ε and γ) receptors

To the Editor: The Fc receptor family plays a key role in adaptive immunity through the binding of immunoglobulin antibodies that recognize an immune insult and elicit an inflammatory response leading to its clearance. Dysregulation of this receptor family may have untoward consequences that result in autoimmune and allergic diseases. Many of these diseases seem to involve the nervous system and are exacerbated by stress or other neurological challenges. Recently, the presence of Fc receptors was uncovered on dorsal root ganglion neurons and suggested an IgG and possibly IgE-mediated activation of neurons 1–3. We set out to more extensively explore which Fc receptors might be expressed in neurons, and whether they were functional and able to transmit signals to interconnected neurites in vitro and in vivo Messenger RNA was isolated from a highly pure culture of mouse superior cervical ganglion (SCG) neurons 4 and expression of Fc receptor transcripts assessed by reverse transcriptase-polymerase chain reactions (RT-PCR) using specific primers for Fcγ and Fce family members. Fig. 1,a demonstrates the presence of transcripts for the immunoglobulin-binding α chain of FcγRI, II, III, and IV in three individual SCG neuron mRNA preparations. A small amount of the transcript for the low affinity IgE receptor (FceRII or CD23) was also detected relative to that seen in B cells, known to express this receptor. FcγRI transcripts were detected in bone marrow derived mouse mast cells but these levels were less than seen in the neurons. This observation and the inability to detect mMCP-6mRNA in either Balb/c or Bl6 mice, together with the absence of CD23 transcripts in both neurons and mast cells provided confidence that the observed Fc receptor transcripts in neurons was not a result of mast cell contamination of cultures. Figure 1 mRNA expression of the subunits of FceRI, FcγRI-IV, and CD23 in SCG neurons. mRNA was collected from three (1–3) SCG neuronal cultures and expression of the indicated mRNA was measured by RT-PCR. (a) Expression of the α ... We also unexpectedly observed the presence of transcripts for the α, β, and γ chains of the high affinity IgE receptor (FceRI) (Fig. 1,b). While the trimeric form (αγ2) of this receptor has been described in cells other than mast cells or basophils (such as in human Langerhans cells 5), the expression of the tetrameric form (αβγ2) was previously thought to be limited to these pro-inflammatory cells. The trimeric FceRI shows weak calcium signals when compared to the tetrameric form due to the absence of the FceRIβ in the former 6. To determine if the FceRI was expressed on the cell surface of SCG neurons, cells were incubated with IgE and with an antibody to the neuronal specific protein gene product (PGP) 9.5 (which encodes a neuronal ubiquitin C-terminal hydrolase not found on glia) and binding visualized with a fluorescent secondary antibody. IgE was detected on the neuronal cell surface (Fig. 2,a). While the presence of PGP9.5 was most evident in the cell body, IgE binding was detected in plasma membrane of both the cell body and neurite extensions. As shown in Figs. 2,b–d, the expression of FceRI was further confirmed by detection of the α, β, and γ chains of this receptor. Figure 2 Neurons were sensitized with IgE and incubated overnight with antibody against IgE (a, left panel) or with an antibody of unknown specificity (a, right panel). Staining with the neuron specific marker PGP9.5 (b–c, left panel in red) and FceRIα ... Scorpion venom is a known potent selective activator of neurons and elicits a rapid rise in intracellular Ca2+ (Fig. 3,a). To test the functionality of FceRI expressed on neurons, SCG neurons sensitized with DNP-specific IgE were challenged with DNP-HSA (Ag) and a rapid rise in intracellular Ca2+ was observed (Fig. 3,a). No changes in intracellular Ca2+ were observed when serum albumin alone was used as Ag or when cells were not sensitized with IgE (data not shown). FcγRIV was recently described to bind the IgEb allotype but does not recognize the IgEa allotype 7, whereas FceRI binds both allotypes. We excluded that IgE/Ag-mediated calcium responses might occur through FcγRIV, by use of both a IgEa and IgEb allotypes. Both IgE allotypes similarly elicited calcium responses (Fig. 3,a). Addition of Ag elicited a relatively uniform Ca2+ responses in the stimulated cell population (Fig. 3,b). . Based on the high affinity binding of monomeric IgE (was not removed by washing the cells) and the dose response to Ag (Fig. 3, D), we could also exclude the involvement of CD23. Figure 3 (a) Kinetics of [Ca2+]i increase upon stimulation with scorpion venom (SV, Control) or with anti-DNP IgEa,/b sensitized neurons following Ag stimulation (10 ng/ml). The arrows indicate the time of addition of the stimulus. (b) Confocal image of calcium ... Thus, the findings demonstrate the presence of functional high affinity Fce receptors on SCG neurons. Since FcγRIII is known to activate mast cells, its functionality was also tested. SCG neurons sensitized with DNP-specific IgG1, which preferentially binds FcγRII and III but weakly to FcγRI and not to FcγRIV 8, showed modest increases in intracellular Ca2+ that increased with a large dose of Ag (Fig. 3,c). Both IgE and IgG-mediated responses were concentration dependent and a Ca2+ response (Fig. 3,d) was not elicited in all challenged neurons. Moreover, as expected (given the weak binding of monomeric IgG1 to Fcγ receptors), increased responsiveness via IgG required much higher concentrations of Ag than for IgE. To test if the Ca2+ signals elicited by FceRI stimulation could be transferred to interconnected neurites, we explored whether Ca2+ rises might be elicited in neighboring neurites after Ag challenge of an IgE sensitized cell body or neurite. Using a spritzer micro-pipette, antigen was puffed directly onto a neuronal cell body, causing an instantaneous (<5 sec) increased fluorescence in that cell body which moved from there to the connected neurites and propagated to the neighboring cell bodies and neurites (Fig. 4). These, findings showed that FceRI stimulation causes communication among interconnected neurites. To extend these findings to a more physiological setting, we explored whether neurons from the highly innervated intact jejunum 9 would respond to an FceRI stimulus. After placement of a micropipette spritzer (dotted lines shown in Fig. 5,a and g) on a large myenteric plexus ganglion neuron (plain lines shown in Fig. 5,a and g), the anti-DNP IgE sensitized plexus was challenged with Ag. Challenge with a spritz of 1 μg of Ag gave robust calcium responses in adjacent neurons along the nerve fiber in sensitized (2/2) but not in non-sensitized mice (0/3). Repeated spritzes of non-conjugated HSA at this concentration elicited no responses (0/2) (data not shown). To exclude possible mast cell involvement in the transmission of these robust signals, we conducted similar experiments in mast cell deficient W/Wv mice and in their wild type control WBB6F1. In W/Wv mice (3/3) detectable intracellular calcium increases were observed upon Ag challenge (Fig. 5,a–c). No calcium signal was seen when the same ganglion was first challenged with HSA alone (Fig. 5,d–f). The wild type littermates (WBB6F1) responded positively (3/3), upon challenge with specific Ag (Fig. 5,g–i) but gave no response to HSA alone. These findings confirm that the observed signal transmission by FceRI was not likely caused by mast cells and demonstrate the in vivo presence of functional FceRI on jejunal neurons, since sham sensitization in vivo prior to an ex vivo challenge yielded no response to Ag challenge. Figure 4 Activation of interconnected neurites by FceRI stimulation of a single neuronal cell body. (a) Bright field image of neuron network showing relation of Ag-containing spritzer to neuronal cell body sensitized with IgE anti-DNP. (b) At time zero, ... Figure 5 (A–I) Myenteric ganglion calcium imaging. Spritzer (internal bore, 40 μm) is indicated by dotted lines and myenteric plexus by solid lines. (a–c) Anti-DNP IgE sensitized myenteric neurons were imaged in mast cell-deficient W/W ... It is well known that sensory nerves may participate in hypersensitivity reactions, a process known as neurogenic inflammation and several lines of evidence support the notion that sensory nerves may play an important role in cutaneous, lung, GI and joint inflammatory diseases. Here we now demonstrate that functionally active FceRI is expressed on SCG and myenteric plexus neurons. The discovery of functional Fce and Fcγ receptors on nerves clearly shows that this biological compartment is able to respond to the direct stimulus of antibody-antigen interactions. Our findings define an independent neuronal (non-mast cell/non-basophil) compartment with probable involvement in allergic and possibly other diseases.

Naive T cells sense the cysteine protease allergen papain through protease-activated receptor 2 and propel TH2 immunity.

BACKGROUND Sensitization to protease allergens, such as papain, or helminth infection is associated with basophil recruitment to draining lymph nodes (LNs). Basophils have the capacity to present antigen to naive T cells and promote T(H)2 differentiation directly or indirectly through IL-4 production. OBJECTIVE We studied how papain induces basophil migration to LNs and the contribution of various leukocytes to papain-induced immune responses. METHODS We immunized mice in the footpad with papain and studied leukocyte recruitment and inflammatory cytokine and chemokine production in the draining popliteal LNs. RESULTS Papain directly activated naive T cells through protease-activated receptor (PAR) 2 to initiate a chemokine/cytokine program that includes CCL17, CCL22, and IL-4. Papain-triggered innate immune responses were dependent on both CD4 T cells and PAR2 and were strongly reduced in the absence of CCR4, the primary receptor for CCL17/CCL22. CONCLUSION These results elucidate a novel innate allergen-recognition pathway mediated by naive T cells through PAR2, which provide an immediate source of chemokines and IL-4 upstream of basophils and antigen-restricted T(H)2 differentiation. PAR2 antagonism might thus hold promise for the treatment of allergic disease.

Basophils and Autoreactive IgE in the Pathogenesis of Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a heterogeneous disease that can affect multiple organs. A hallmark of this disease, as is the case for other autoimmune diseases, is the presence of large numbers of autoantibodies. As such, SLE is considered to be a B-cell disease perpetuated by the expansion of autoreactive T and B cells. The T cells involved have long been considered to be T-helper type 1 (Th1) and Th17 cells, as these potent proinflammatory cells can be found in the tissues of SLE patients. Recent advances point to a role for the Th2 environment in contributing to SLE through promotion of autoantibody production. Here we describe the recent work focusing on autoreactive IgE and the activation of basophils as promoting the production of autoantibodies in SLE. The findings, both in a murine model of SLE and in humans with SLE, support the concept that the activation of the basophil by autoreactive IgE-containing immune complexes serves to amplify the production of autoantibodies and contributes to the pathogenesis of disease. We propose that therapeutic targeting of this amplification loop by reducing the levels of circulating autoreactive IgE may have benefit in SLE.

Ablation of Tumor Progression Locus 2 Promotes a Type 2 Th Cell Response in Ovalbumin-Immunized Mice

The protein kinase encoded by the Tpl2 proto-oncogene regulates ERK activation and cytokine gene expression in macrophages in response to LPS and TNF-α. In this study we show that OVA-immunized Tpl2−/− mice express high levels of IgE and develop more severe bronchoalveolar eosinophilic inflammation than Tpl2+/+ controls, when challenged with OVA intranasally. Bronchoalveolar exudates and supernatants of OVA-stimulated splenocytes from immunized Tpl2−/− mice express elevated levels of IL-4 and IL-5, suggesting that Tpl2 ablation promotes the Th2 polarization of the T cell response. Anti-CD3 stimulation of CD4+ T cells of wild-type and Tpl2 knockout mice revealed that Tpl2 ablation gives rise to a cell autonomous T cell defect that is primarily responsible for the Th2 polarization of the T cell response to Ag. This observation was further supported by experiments addressing the expression of Th1 and Th2 cytokines in OVA-stimulated mixed cultures of CD4+ T cells from Tpl2+/+/OT2 or Tpl2−/−/OT2 mice and dendritic cells from Tpl2+/+ or Tpl2−/− mice. Further studies revealed that Th1 cells express significantly higher levels of Tpl2 than Th2 cells. As a result, Tpl2−/− Th1 cells exhibit a stronger defect in ERK activation by anti-CD3 than Th2 cells and express low levels of T-bet. Given that the development of Th1 and Th2 cells depends on positive feedback signals from the T cells, themselves, the functional defect of the Tpl2−/− Th1 cells provides a mechanistic explanation for the T cell autonomous Th2 polarization in Tpl2−/− mice.

PTEN deficiency in mast cells causes a mastocytosis-like proliferative disease that heightens allergic responses and vascular permeability

Kit regulation of mast cell proliferation and differentiation has been intimately linked to the activation of phosphatidylinositol 3-OH kinase (PI3K). The activating D816V mutation of Kit, seen in the majority of mastocytosis patients, causes a robust activation of PI3K signals. However, whether increased PI3K signaling in mast cells is a key element for their in vivo hyperplasia remains unknown. Here we report that dysregulation of PI3K signaling in mice by deletion of the phosphatase and tensin homolog (Pten) gene (which regulates the levels of the PI3K product, phosphatidylinositol 3,4,5-trisphosphate) caused mast cell hyperplasia and increased numbers in various organs. Selective deletion of Pten in the mast cell compartment revealed that the hyperplasia was intrinsic to the mast cell. Enhanced STAT5 phosphorylation and increased expression of survival factors, such as Bcl-XL, were observed in PTEN-deficient mast cells, and these were further enhanced by stem cell factor stimulation. Mice carrying PTEN-deficient mast cells also showed increased hypersensitivity as well as increased vascular permeability. Thus, Pten deletion in the mast cell compartment results in a mast cell proliferative phenotype in mice, demonstrating that dysregulation of PI3K signals is vital to the observed mast cell hyperplasia.

Cutting Edge: Persistence of Increased Mast Cell Numbers in Tissues Links Dermatitis to Enhanced Airway Disease in a Mouse Model of Atopy

The development of chronic allergic dermatitis in early life has been associated with increased onset and severity of allergic asthma later in life. However, the mechanisms linking these two diseases are poorly understood. In this study, we report that the development of oxazolone-induced chronic allergic dermatitis, in a mouse model, caused enhanced OVA-induced allergic asthma after the resolution of the former disease. Our findings show that oxazolone-induced dermatitis caused a marked increase in tissue mast cells, which persisted long after the resolution of this disease. Subsequent OVA sensitization and airway challenge of mice that had recovered from dermatitis resulted in increased allergic airway hyperreactivity. The findings demonstrate that the accumulation of mast cells during dermatitis has the detrimental effect of increasing allergic airway hypersensitivity. Importantly, our findings also show that exposure to a given allergen can modify the immune response to an unrelated allergen.