Meijuan Zhou

IL-17C regulates the innate immune function of epithelial cells in an autocrine manner

Interleukin 17C (IL-17C) is a member of the IL-17 family that is selectively induced in epithelia by bacterial challenge and inflammatory stimuli. Here we show that IL-17C functioned in a unique autocrine manner, binding to a receptor complex consisting of the receptors IL-17RA and IL-17RE, which was preferentially expressed on tissue epithelial cells. IL-17C stimulated epithelial inflammatory responses, including the expression of proinflammatory cytokines, chemokines and antimicrobial peptides, which were similar to those induced by IL-17A and IL-17F. However, IL-17C was produced by distinct cellular sources, such as epithelial cells, in contrast to IL-17A, which was produced mainly by leukocytes, especially those of the TH17 subset of helper T cells. Whereas IL-17C promoted inflammation in an imiquimod-induced skin-inflammation model, it exerted protective functions in dextran sodium sulfate–induced colitis. Thus, IL-17C is an essential autocrine cytokine that regulates innate epithelial immune responses.

In vivo blockade of OX40 ligand inhibits thymic stromal lymphopoietin driven atopic inflammation

Thymic stromal lymphopoietin (TSLP) potently induces deregulation of Th2 responses, a hallmark feature of allergic inflammatory diseases such as asthma, atopic dermatitis, and allergic rhinitis. However, direct downstream in vivo mediators in the TSLP-induced atopic immune cascade have not been identified. In our current study, we have shown that OX40 ligand (OX40L) is a critical in vivo mediator of TSLP-mediated Th2 responses. Treating mice with OX40L-blocking antibodies substantially inhibited immune responses induced by TSLP in the lung and skin, including Th2 inflammatory cell infiltration, cytokine secretion, and IgE production. OX40L-blocking antibodies also inhibited antigen-driven Th2 inflammation in mouse and nonhuman primate models of asthma. This treatment resulted in both blockade of the OX40-OX40L receptor-ligand interaction and depletion of OX40L-positive cells. The use of a blocking, OX40L-specific mAb thus presents a promising strategy for the treatment of allergic diseases associated with pathologic Th2 immune responses.

Transcriptional programming of dendritic cells for enhanced MHC class II antigen presentation

CD11b+ dendritic cells (DCs) seem to be specialized for presenting antigens via major histocompatibility (MHC) class II complexes to stimulate helper T cells, but the genetic and regulatory basis for this is not established. Conditional deletion of Irf4 resulted in loss of CD11b+ DCs, impaired formation of peptide–MHC class II complexes and defective priming of helper T cells but not of cytotoxic T lymphocyte (CTL) responses. Gene expression and chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) analyses delineated an IRF4-dependent regulatory module that programs enhanced MHC class II antigen presentation. Expression of the transcription factor IRF4 but not of IRF8 restored the ability of IRF4-deficient DCs to efficiently process and present antigen to MHC class II–restricted T cells and promote helper T cell responses. We propose that the evolutionary divergence of IRF4 and IRF8 facilitated the specialization of DC subsets for distinct modes of antigen presentation and priming of helper T cell versus CTL responses.

IgE⁺ memory B cells and plasma cells generated through a germinal-center pathway.

Immunoglobulin E (IgE) antibodies are pathogenic in asthma and allergic diseases, but the in vivo biology of IgE-producing (IgE+) cells is poorly understood. A model of the differentiation of IgE+ B cells proposes that IgE+ cells develop through a germinal-center IgG1+ intermediate and that IgE memory resides in the compartment of IgG1+ memory B cells. Here we have used a reporter mouse expressing green fluorescent protein associated with membrane IgE transcripts (IgE-GFP) to assess in vivo IgE responses. In contrast to the IgG1-centered model of IgE switching and memory, we found that IgE+ cells developed through a germinal-center IgE+ intermediate to form IgE+ memory B cells and plasma cells. Our studies delineate a new model for the in vivo biology of IgE switching and memory.

Therapeutic antibodies reveal Notch control of transdifferentiation in the adult lung

Prevailing dogma holds that cell–cell communication through Notch ligands and receptors determines binary cell fate decisions during progenitor cell divisions, with differentiated lineages remaining fixed. Mucociliary clearance in mammalian respiratory airways depends on secretory cells (club and goblet) and ciliated cells to produce and transport mucus. During development or repair, the closely related Jagged ligands (JAG1 and JAG2) induce Notch signalling to determine the fate of these lineages as they descend from a common proliferating progenitor. In contrast to such situations in which cell fate decisions are made in rapidly dividing populations, cells of the homeostatic adult airway epithelium are long-lived, and little is known about the role of active Notch signalling under such conditions. To disrupt Jagged signalling acutely in adult mammals, here we generate antibody antagonists that selectively target each Jagged paralogue, and determine a crystal structure that explains selectivity. We show that acute Jagged blockade induces a rapid and near-complete loss of club cells, with a concomitant gain in ciliated cells, under homeostatic conditions without increased cell death or division. Fate analyses demonstrate a direct conversion of club cells to ciliated cells without proliferation, meeting a conservative definition of direct transdifferentiation. Jagged inhibition also reversed goblet cell metaplasia in a preclinical asthma model, providing a therapeutic foundation. Our discovery that Jagged antagonism relieves a blockade of cell-to-cell conversion unveils unexpected plasticity, and establishes a model for Notch regulation of transdifferentiation.

Regulation of T Cell Receptor Signaling by DENND1B in TH2 Cells and Allergic Disease

The DENN domain is an evolutionary conserved protein module found in all eukaryotes and serves as an exchange factor for Rab-GTPases to regulate diverse cellular functions. Variants in DENND1B are associated with development of childhood asthma and other immune disorders. To understand how DENND1B may contribute to human disease, Dennd1b(-/-) mice were generated and exhibit hyper-allergic responses following antigen challenge. Dennd1b(-/-) TH2, but not other TH cells, exhibit delayed receptor-induced T cell receptor (TCR) downmodulation, enhanced TCR signaling, and increased production of effector cytokines. As DENND1B interacts with AP-2 and Rab35, TH2 cells deficient in AP-2 or Rab35 also exhibit enhanced TCR-mediated effector functions. Moreover, human TH2 cells carrying asthma-associated DENND1B variants express less DENND1B and phenocopy Dennd1b(-/-) TH2 cells. These results provide a molecular basis for how DENND1B, a previously unrecognized regulator of TCR downmodulation in TH2 cells, contributes to asthma pathogenesis and how DENN-domain-containing proteins may contribute to other human disorders.

Addendum: IgE + memory B cells and plasma cells generated through a germinal-center pathway

Addendum: IgE + memory B cells and plasma cells generated through a germinal-center pathway

Depletion of major pathogenic cells in asthma by targeting CRTh2

Eosinophilic inflammation and Th2 cytokine production are central to the pathogenesis of asthma. Agents that target either eosinophils or single Th2 cytokines have shown benefits in subsets of biomarker-positive patients. More broadly effective treatment or disease-modifying effects may be achieved by eliminating more than one inflammatory stimulator. Here we present a strategy to concomitantly deplete Th2 T cells, eosinophils, basophils, and type-2 innate lymphoid cells (ILC2s) by generating monoclonal antibodies with enhanced effector function (19A2) that target CRTh2 present on all 4 cell types. Using human CRTh2 (hCRTh2) transgenic mice that mimic the expression pattern of hCRTh2 on innate immune cells but not Th2 cells, we demonstrate that anti-hCRTh2 antibodies specifically eliminate hCRTh2+ basophils, eosinophils, and ILC2s from lung and lymphoid organs in models of asthma and Nippostrongylus brasiliensis infection. Innate cell depletion was accompanied by a decrease of several Th2 cytokines and chemokines. hCRTh2-specific antibodies were also active on human Th2 cells in vivo in a human Th2-PBMC-SCID mouse model. We developed humanized hCRTh2-specific antibodies that potently induce antibody-dependent cell cytotoxicity (ADCC) of primary human eosinophils and basophils and replicated the in vivo depletion capacity of their murine parent. Therefore, depletion of hCRTh2+ basophils, eosinophils, ILC2, and Th2 cells with h19A2 hCRTh2-specific antibodies may be a novel and more efficacious treatment for asthma.

Polyclonal hyper-IgE mouse model reveals mechanistic insights into antibody class switch recombination

Significance Switch (S) regions are repetitive DNA sequences. During an immune response, one of several S regions recombine with a donor switch (Sμ) that is constitutively “on,” resulting in the production of antibodies with new functions. Donor Sμ is large and very repeat-rich, while another switch, Sε, is less than half its size with a low density of repeats. We replaced Sε with Sμ in mice. These mice switch to Sε more effectively and produce high levels of IgE antibodies implicated in asthma, making this a useful model to study disease. In addition, placing Sμ outside of its native context revealed insights into how switches work. Preceding antibody constant regions are switch (S) regions varying in length and repeat density that are targets of activation-induced cytidine deaminase. We asked how participating S regions influence each other to orchestrate rearrangements at the IgH locus by engineering mice in which the weakest S region, Sε, is replaced with prominent recombination hotspot Sμ. These mice produce copious polyclonal IgE upon challenge, providing a platform to study IgE biology and therapeutic interventions. The insertion enhances ε germ-line transcript levels, shows a preference for direct vs. sequential switching, and reduces intraswitch recombination events at native Sμ. These results suggest that the sufficiency of Sμ to mediate IgH rearrangements may be influenced by context-dependent cues.

Inhibition of the kinase ITK in a mouse model of asthma reduces cell death and fails to inhibit the inflammatory response

The kinase ITK is required for antigen-stimulated T cell death. Targeting ITK in asthma CD4+ T helper 2 (TH2) lymphocytes secrete the cytokines interleukin-4 (IL-4), IL-15, and IL-13, which are implicated in the pathogenesis of asthma. Antigen stimulation of T cells activates the kinase ITK, which is required for TH2-type cytokine production. ITK knockout mice are resistant to airway inflammation, which suggests that ITK inhibitors might be used to treat human asthma. However, Sun et al. found that a mouse model of asthma developed worse disease when treated with an ITK-specific inhibitor, exhibiting increased numbers of T cells and amounts of TH2-type cytokines in the airways. These effects were associated with a failure of ITK-inhibited T cells to undergo antigen-stimulated cell death. Together, these data suggest that targeting the kinase activity of ITK in human asthma may exacerbate disease. Interleukin-2 (IL-2)–inducible T cell kinase (ITK) mediates T cell receptor (TCR) signaling primarily to stimulate the production of cytokines, such as IL-4, IL-5, and IL-13, from T helper 2 (TH2) cells. Compared to wild-type mice, ITK knockout mice are resistant to asthma and exhibit reduced lung inflammation and decreased amounts of TH2-type cytokines in the bronchoalveolar lavage fluid. We found that a small-molecule selective inhibitor of ITK blocked TCR-mediated signaling in cultured TH2 cells, including the tyrosine phosphorylation of phospholipase C–γ1 (PLC-γ1) and the secretion of IL-2 and TH2-type cytokines. Unexpectedly, inhibition of the kinase activity of ITK during or after antigen rechallenge in an ovalbumin-induced mouse model of asthma failed to reduce airway hyperresponsiveness and inflammation. Rather, in mice, pharmacological inhibition of ITK resulted in T cell hyperplasia and the increased production of TH2-type cytokines. Thus, our studies predict that inhibition of the kinase activity of ITK may not be therapeutic in patients with asthma.