Activation induced cytidine deaminase (AID) is an enzyme essential for the generation of antibody diversity in B cells and is considered to be a general gene mutator. In addition, AID expression was also implicated in the pathogenesis of human B cell malignancies and associated with poor prognosis. Here we report that siRNA silencing of AID in plasmacytoma dramatically increased its susceptibility to immunotherapy by cytotoxic T lymphocytes. AID silencing did not decrease the mutation frequencies of tumor antigen gene P1A. Gene-array analysis showed dramatically altered expression of a number of genes in AID-silenced plasmacytoma cells and upregulation of CD200 was shown to be in favor of tumor eradication by CTL. Taken together, we demonstrate a novel function of AID in tumor evasion of CTL therapy and that targeting AID should be beneficial in the immunotherapy of AID positive tumors.
Activation induced cytidine deaminase; Plasmacytoma; Cytotoxic T Lymphocytes; Immune evasion
T Regulatory cells (Treg) play crucial roles in the regulation of cellular immunity. The development of Treg cells depends on signals from T cell receptors (TCR) and IL-2 receptors and is influenced by a variety of transcription factors. The basic helix-loop-helix (bHLH) proteins are known to influence TCR signaling thresholds. Whether this property impacts Treg differentiation is not understood. Here, we interrogated the role of bHLH proteins in the production of Treg cells using the CD4 promoter-driven Id1 transgene. We found that Treg cells continued to accumulate as Id1 transgenic mice aged, resulting in a significant increase in Treg cell counts in the thymus as well as in the periphery compared to wild type controls. Data from mixed-bone marrow assays suggest that Id1 acts intrinsically on developing Treg cells. We made a connection between Id1 expression and CD28 co-stimulatory signaling because Id1 transgene expression facilitated the formation of Treg precursors in CD28−/− mice and the in vitro differentiation of Treg cells on thymic dendritic cells despite the blockade of costimulation by anti-CD80/CD86. Id1 expression also allowed in vitro Treg differentiation without anti-CD28 co-stimulation, which was at least in part due to enhanced production of IL-2. Notably, with full strength of co-stimulatory signals, however, Id1 expression caused modest but significant suppression of Treg induction. Finally, we demonstrate that Id1 transgenic mice were less susceptible to the induction of experimental autoimmune encephalomyelitis (EAE), thus illustrating the impact of Id1-mediated augmentation of Treg cell levels on cellular immunity.
CD24 binds to and suppresses inflammation triggered by danger associated molecular patterns (DAMPS) such as heat-shock proteins (HSPs) and HMGB1. Paradoxically, CD24 has been shown to enhance autoimmune disease. Here we attempt to reconcile this paradox by deletion of CD24 (24KO) in a lupus-like disease model driven by forced expression of HSP gp96 at the cell surface (tm). As expected, tm24KO mice showed increased CD11c+ DC activation coupled to a significant increase in DC-specific IL-12 production compared to tm mice. However, tm24KO mice showed less CD4 T cell activation and peripheral inflammatory cytokine production in comparison to tm mice. We characterized an enhanced immune suppressive milieu in tm24KO mice distinguished by increased TGF-β and greater Treg suppressive capacity. We found greater absolute numbers of MDSCs in tm24KO mice and showed that the Ly6C+ MDSC subset had greater suppressive capacity from tm24KO mice. Deletion of CD24 in tm mice led to diminished lupus-like pathology as evidenced by anti-nuclear antibody deposition and glomerulonephritis. Finally, we show that expanded MDSC populations were mediated by increased free HMGB1 in tm24KO mice. Thus, the deletion of CD24 in an HSP-driven model of autoimmunity led to the unexpected development of Treg and MDSC populations that augmented immune tolerance. Further study of these populations as possible negative regulators of inflammation in the context of autoimmunity is warranted.
autoimmunity; MDSC; CD24; gp96; HMGB1
Exosomes released from different types of cells have been proposed to contribute to intercellular communication. We report that thymic exosome-like particles (ELPs) released from cells of the thymus can induce the development of Foxp3+ regulatory T (Treg) cells in the lung and liver. Thymic ELPs also induce the conversion of thymic CD4+CD25− T cells into Tregs. Tregs induced by thymic ELPs suppress the proliferation of CD4+CD25− T cells in vitro and in vivo. We further show that neutralization of TGF-β in ELPs partially reverses thymic ELP-mediated induction of CD4+Foxp3+ T cells in the lung and liver. This study demonstrates that thymic ELPs participate in the induction of Foxp3+ Tregs. Also, TGF-β of thymic ELPs might be required for the generation of Tregs in the peripheral tissues.
Chronic airway remodeling is a serious consequence of asthma, which is caused by complex but largely unknown mechanisms. Despite versatile functions, the role of Lyn in chronic airway remodeling remains undefined. Using Lyn−/− mice, we show that continual exposure (for 8 weeks) of house dust mite (HDM) extracts induced a severe phenotype of chronic airway remodeling including exacerbated mucus production, collagen deposition, dysregulated cytokine secretion, and elevated inflammation. Strikingly, a significant increase in TGF-β3 rather than TGF-β1 was observed in Lyn−/− mouse lungs compared to wild-type mice. Furthermore, TGF-β3 neutralizing antibodies not only inhibited the expression of STAT6 and Smad2/3, but also decreased phosphorylation of Smad2 and NFκB in Lyn−/− mouse lungs. In addition, both recombinant and adenoviral TGF-β3 significantly promoted epithelial to mesenchymal transition (EMT) and intensified collagen I production and MUC5AC expression. Further examining chronic asthma patients showed that a decreased Lyn correlated with the severity of airway inflammation and mucus hypersecretion. Finally, Lyn may critically regulate airway remodeling by directly interacting with TGF-β3. Collectively, these findings revealed that Lyn regulates TGF-β3 isoform and modulates the development of airway remodeling, which may have therapeutic indications for severe chronic asthma.
knockout mice; Th2 cytokines; HDM extracts; oxidative stress; airway remodeling; EMT
The airway epithelium is the first line of host defense against pathogens. The short PLUNC 1 (SPLUNC1) protein is secreted in respiratory tracts and a member of the BPI fold-containing (BPIF) protein family, which shares structural similarities with bactericidal/permeability-increasing (BPI)-like proteins. On the basis of its homology with BPIs and restricted expression of SPLUNC1 in serous cells of submucosal glands and surface epithelial cells of the upper respiratory tract, SPLUNC1 is thought to possess antimicrobial activity in host defense. SPLUNC1 is also reported to have surfactant properties, which may contribute to anti-biofilm defenses. The objective of this study was to determine the in vivo functions of SPLUNC1 following Pseudomonas aeruginosa infection and to elucidate the underlying mechanism, using a knockout (KO) mouse model with a genetic ablation of Splunc1. Splunc1 KO mice showed accelerated mortality and increased susceptibility to P. aeruginosa infection with significantly decreased survival rates, increased bacterial burdens, exaggerated tissue injuries, and elevated proinflammatory cytokine levels as compared to those of their wild-type (WT) littermates. Increased neutrophil infiltration in Splunc1 KO mice was accompanied by elevated chemokine levels including Cxcl1, Cxcl2, and Ccl20. Furthermore, the expression of several epithelial secretory proteins and antimicrobial molecules was considerably suppressed in the lungs of Splunc1 KO mice. The deficiencya of Splunc1 in mouse airway epithelium also results in increased biofilm formation of P. aeruginosa. Taken together, our results support that the ablation of Splunc1 in mouse airways affects the mucociliary clearance, resulting in decreased innate immune response during Pseudomonas-induced respiratory infection.
splunc1; epithelium; mucociliary clearance; Pseudomonas infection
Found in inflammatory zone (FIZZ) 2, also known as resistin-like molecule (RELM)-β, belongs to a novel cysteine-rich secreted protein family named FIZZ/RELM. Its function is unclear, but a closely related family member, FIZZ1, has profibrotic activities. The human ortholog of rodent FIZZ1 has not been identified, but human FIZZ2 has significant sequence homology to both rodent FIZZ2 (59%) and FIZZ1 (50%). Given the greater homology to rodent FIZZ2, analyzing the role of FIZZ2 in a rodent model of bleomycin-induced pulmonary fibrosis would be of greater potential relevance to human fibrotic lung disease. The results showed that FIZZ2 was highly induced in lungs of rodents with bleomycin-induced pulmonary fibrosis and of human patients with idiopathic pulmonary fibrosis. FIZZ2 expression was induced in rodent and human lung epithelial cells by Th2 cytokines, which was mediated via STAT6 signaling. The FIZZ2 induction in murine lungs was found to be essential for pulmonary fibrosis, as FIZZ2 deficiency significantly suppressed pulmonary fibrosis and associated enhanced extracellular matrix and cytokine gene expression. In vitro analysis indicated that FIZZ2 could stimulate type I collagen and α-smooth muscle actin expression in lung fibroblasts. Furthermore, FIZZ2 was shown to have chemoattractant activity for bone marrow (BM) cells, especially BM-derived CD11c+ dendritic cells. Notably, lung recruitment of BM-derived cells was impaired in FIZZ2 knockout mice. These findings suggest that FIZZ2 is a Th2-associated multifunctional mediator with potentially important roles in the pathogenesis of fibrotic lung diseases.
IL-35 is a member of the IL-12 family of cytokines consisting of IL-12 p35 subunit and IL-12 p40-related protein subunit, EBV-induced gene 3 (EBI3). IL-35 functions through IL-35R and has a potent immune suppressive activity. Although IL-35 has been demonstrated to be produced by regulatory T cells, gene expression analysis has revealed that IL-35 is likely to have wider distribution including expression in cancer cells. In this study we have demonstrated that IL-35 is produced in human cancer tissues such as large B cell lymphoma, nasopharyngeal carcinoma and melanoma. In order to determine the roles of tumor-derived IL-35 in tumorigenesis and tumor immunity, we generated IL-35 producing plasmacytoma J558 and B16 melanoma cells, and observed that the expression of IL-35 in cancer cells does not affect their growth and survival in vitro, but stimulates tumorigenesis in both immune competent and Rag1/2 deficient mice. Tumor-derived IL-35 increases CD11b+Gr1+ myeloid cell accumulation in tumor microenvironment, and thereby promotes tumor angiogenesis. In immune competent mice, spontaneous CTL responses to tumors are diminished. IL-35 does not directly inhibit tumor antigen specific CD8+ T cell activation, differentiation and effector functions. However, IL-35-treated cancer cells had increased expression of gp130 and reduced sensitivity to CTL destruction. Thus, our study indicates novel functions of IL-35 in promoting tumor growth via enhancing myeloid cell accumulation, tumor angiogenesis and suppression of tumor immunity.
Neutrophils kill bacteria generally through oxidative and nonoxidative mechanisms. Whereas much research has focused on the enzymes essential for neutrophil killing, little is known about the regulatory molecules responsible for such killing. In this study we investigated the role of olfactomedin 4 (OLFM4), an olfactomedin-related glycoprotein, in neutrophil bactericidal capability and host innate immunity. Neutrophils from OLFM4−/− mice have increased intracellular killing of Staphylococcus aureus and Escherichia coli in vitro. The OLFM4−/− mice have enhanced in vivo bacterial clearance and are more resistant to sepsis when challenged with S. aureus or E. coli by intraperitoneal injection. OLFM4 was found to interact with cathepsin C, a cysteine protease that plays an important role in bacterial killing and immune regulation. We demonstrated that OLFM4 inhibited cathepsin C activity in vitro and in vivo. The cathepsin C activity in neutrophils from OLFM4−/− mice was significantly higher than that in neutrophils from wild-type littermate mice. The activities of three serine proteases (neutrophil elastase, cathepsin G, and proteinase 3), which require cathepsin C activity for processing and maturity, were also significantly higher in OLFM4−/− neutrophils. The bacterial killing and clearance capabilities observed in OLFM4−/− mice that was enhanced relative to WT mice was significantly compromised by the additional loss of cathepsin C in mice with OLFM4 and cathepsin C double deficiency. These results indicate that OLFM4 is an important negative regulator of neutrophil bactericidal activity by restricting cathepsin C activity and its downstream granule-associated serine proteases.
B cells can mediate protective responses against nematode parasites by supporting Th2 cell development and/or by producing Abs. To examine this, B cell-deficient mice were inoculated with Nippostrongylus brasiliensis or Heligmosomoides polygyrus. B cell-deficient and wild type mice showed similar elevations in Th2 cytokines and worm expulsion after N. brasiliensis inoculation. Worm expulsion was inhibited in H. polygyrus-inoculated B cell-deficient mice, although Th2 cytokine elevations in mucosal tissues were unaffected. Impaired larval migration and development was compromised as early as day 4 after H. polygyrus challenge, and administration of immune serum restored protective immunity in B cell-deficient mice, indicating a primary role for Ab. Immune serum even mediated protective effects when administered to naive mice prior to inoculation. This study suggests variability in the importance of B cells in mediating protection against intestinal nematode parasites, and it indicates an important role for Ab in resistance to tissue-dwelling parasites.
Genome-wide association studies have identified lupus susceptibility genes such as IRF5 and PRDM1 (encoding for the IRF5 and Blimp-1) in the human genome. Accordingly, the murine Irf5 and Prdm1 genes have been shown to play a role in lupus susceptibility. However, it remains unclear how IRF5 and Blimp-1 (a transcriptional target of IRF5) contribute to lupus susceptibility. Given that the murine lupus susceptibility locus Nba2 includes the interferon-regulated genes Ifi202 (encoding for the p202 protein), Aim2 (encoding for the Aim2 protein), and Fcgr2b (encoding for the FcγRIIB receptor), we investigated whether the IRF5-Blimp-1 axis could regulate the expression of these genes. We found that an Irf5-deficiency in mice decreased the expression of Blimp-1 and reduced the expression of the Ifi202. However, the deficiency increased the expression of Aim2 and Fcgr2b. Correspondingly, increased expression of IRF5 in cells increased levels of Blimp-1 and p202 protein. Moreover, Blimp-1 expression increased the expression of Ifi202, whereas it reduced the expression of Aim2. Interestingly, an Aim2-deficiency in female mice increased the expression of IRF5. Similarly, the Fcgr2b-deficient mice expressed increased levels of IRF5. Moreover, increased expression of IRF5 and Blimp-1 in lupus-prone B6.Nba2, NZB, and B6.Sle123 female mice (as compared to age-matched C57BL/6 female mice) was associated with increased levels of the p202 protein. Together, our observations demonstrate that the IRF5-Blimp-1 axis differentially regulates the expression of Nba2 lupus susceptibility genes, and suggest an important role for the IRF5-Blimp-1-p202 axis in murine lupus susceptibility.
IRF5; Blimp-1; Nba2 locus; p202; interferon; autoimmunity; SLE
Commensal flora plays important roles in the regulation of the gene expression involved in many intestinal functions and the maintenance of immune homeostasis, as well as in the pathogenesis of inflammatory bowel diseases (IBD). The microRNAs (miRNAs), a class of small, non-coding RNAs, act as key regulators in many biological processes. The miRNAs are highly conserved among species and appear to play important roles in both innate and adaptive immunity, as they can control the differentiation of various immune cells as well as their functions. However, it is still largely unknown how microbiota regulates miRNA expression, thereby contributing to intestinal homeostasis and pathogenesis of IBD. In our current study, we found that microbiota negatively regulated intestinal miR-10a expression, in that the intestines, as well as intestinal epithelial cells and dendritic cells of specific pathogen-free (SPF) mice, expressed much lower levels of miR-10a compared to those in germ-free (GF) mice. Commensal bacteria downregulated DC miR-10a expression via TLR-TLR ligand interactions through a MyD88-dependent pathway. We identified IL-12/IL-23p40, a key molecule for innate immune responses to commensal bacteria, as a target of miR-10a. The ectopic expression of miR-10a precursor inhibited, whereas miR-10a inhibitor promoted, the expression of IL-12/IL-23p40 in DC. Mice with colitis expressing higher levels of IL-12/IL-23p40 exhibit lower levels of intestinal miR-10a compared to that in the control mice. Collectively, our data demonstrated that microbiota negatively regulates host miR-10a expression, which may contribute to the maintenance of intestinal homeostasis by targeting IL-12/IL-23p40 expression.
Naïve T cells receive stimulation from the positive selecting ligand in the periphery for their survival. This stimulation does not normally lead to overt activation of T cells, as the T cells remain largely quiescent until they receive either antigenic or lymphopenic stimuli. The underlying mechanism responsible for survival and quiescence of the naïve T cells remain largely unknown. Here we report that T cell-specific deletion of Tsc1, a negative regulator of mTOR, resulted in both spontaneous losses of quiescence and cellularity, especially within the CD8 subset. The Tsc1-deficient T cells have increased cell proliferation and apoptosis. Tsc1 deletion affects the survival and quiescence of T cells in the absence of antigenic stimulation. Loss of quiescence but not cellularity was inhibited by rapamycin. Our data demonstrate that TSC-mTOR maintains quiescence and survival of T cells.
Regulatory T lymphocytes (Treg) expressing the Forkhead Box Transcription Factor 3 (Foxp3) are critical modulators of autoimmunity. Foxp3+ Treg may develop in the thymus as a population distinct from conventional Foxp3− αβ T cells (Tconv). Alternatively, plasticity in Foxp3 expression may allow for the interconversion of mature Treg and Tconv. We examined >160,000 TCR sequences from Foxp3+ or Foxp3− populations in the spleens or CNS of wild type mice with experimental allergic encephalomyelitis (EAE) to determine their relatedness and identify distinguishing TCR features. Our results indicate that the CNS infiltrating Treg and Tconv arise predominantly from distinct sources. The repertoires of CNS Treg or Tconv TCR showed limited overlap with heterologous populations in either the CNS or spleen, indicating that they are largely unrelated. Indeed, Treg and Tconv TCR in the CNS were significantly less related than those populations in the spleen. In contrast, CNS Treg and Tconv repertoires strongly intersected those of the homologous cell type in the spleen. High frequency sequences more likely to be disease associated showed similar results, and some public TCR demonstrated Treg or Tconv-specific motifs. Different charge characteristics and amino acid use preferences were identified in the CDR3β of Treg and Tconv infiltrating the CNS, further indicating that their repertoires are qualitatively distinct. Therefore discrete populations of Treg and Tconv that do not substantially interconvert respond during EAE. Differences in sequence and physical characteristics distinguish Treg and Tconv TCR and imply dissimilar antigen recognition properties.
Infection with the parasitic nematode Nippostrongylus brasiliensis induces a potent Th2 response; however, little is known about early stages of the innate response that may contribute to protective immunity. To examine early events in this response, chemokine expression in the draining lymph node was examined after N. brasiliensis inoculation. Pronounced increases of several chemokines, including CCL2, were observed. Compared with wild-type mice, elevations in a Gr-1bright population in the draining lymph node was significantly decreased in CCL2−/− mice after N. brasiliensis inoculation. Further flow cytometric and immunofluorescent analysis showed that in wild-type mice, Gr-1+ cells transiently entered and exited the draining lymph node shortly after N. brasiliensis inoculation. The Gr-1bright population was comprised of neutrophils expressing TGF-β and TNF-α. Following Gr-1+ cell depletion, N. brasiliensis infection resulted in transient, but significantly increased levels of IFN-γ, increased serum IgG2a, reduced Th2 cytokines and serum IgE, greatly increased mortality, and delayed worm expulsion. Furthermore, bacteria were readily detected in vital organs. Infection of Gr-1+ cell-depleted mice with N. brasiliensis larvae that were pretreated with antibiotics prevented bacterial dissemination, Th1 inflammatory responses, and decreases in host survival. This study indicates that parasitic nematodes can be an important vector of potentially harmful bacteria, which is typically controlled by CCL2-dependent neutrophils that ensure the optimal development of Th2 immune responses and parasite resistance.
Previous studies have suggested that B cells promote Th2 cell development by inhibiting Th1 cell differentiation. To examine whether B cells are directly required for the development of IL-4-producing T cells in the lymph node during a highly polarized Th2 response, B cell-deficient and wild-type mice were inoculated with the nematode parasite, Nippostrongylus brasiliensis. On day 7, in the absence of increased IFN-γ, IL-4 protein and gene expression from CD4 T cells in the draining lymph nodes were markedly reduced in B cell-deficient mice and could not be restored by multiple immunizations. Using a DO11.10 T cell adoptive transfer system, OVA-specific T cell IL-4 production and cell cycle progression, but not cell surface expression of early activation markers, were impaired in B cell-deficient recipient mice following immunization with N. brasiliensis plus OVA. Laser capture microdissection and immunofluorescent staining showed that pronounced IL-4 mRNA and protein secretion by donor DO11.10 T cells first occurred in the T cell:B cell zone of the lymph node shortly after inoculation of IL-4−/− recipients, suggesting that this microenvironment is critical for initial Th2 cell development. Reconstitution of B cell-deficient mice with wild-type naive B cells, or IL-4−/− B cells, substantially restored Ag-specific T cell IL-4 production. However, reconstitution with B7-1/B7-2-deficient B cells failed to rescue the IL-4-producing DO11.10 T cells. These results suggest that B cells, expressing B7 costimulatory molecules, are required in the absence of an underlying IFN-γ-mediated response for the development of a polarized primary Ag-specific Th2 response in vivo.
The intestinal nematode parasite, Nippostrongylus brasiliensis, triggers potent type 2 immunity. Using OVA peptide as a model Ag, we have examined the adjuvant effects of this parasite on the in vivo development of Ag-specific Th2 cells from naive DO11.10 T cells. Our findings show that Th2 cells can develop from transferred naive OVA-specific DO11.10 T cells in recipient IL-4–/– mice inoculated with N. brasiliensis plus OVA. However, autocrine IL-4 is required for in situ Th2 cell differentiation since transferred IL-4Rα-deficient DO11.10 T cells showed greatly reduced Th2 cell development in inoculated IL-4–/– recipient mice. Surprisingly, we also found that IL-2 blockade promoted B7-dependent T cell cycling, but inhibited the development of OVA-specific Th2 cells. Furthermore, the effects of IL-2 occurred independently of CD25+ T regulatory cells. These studies establish a previously unrecognized requirement for autocrine IL-4 and IL-2 in Th2 responses elicited by nematode parasites.
Both commensal bacteria and infiltrating inflammatory cells play essential roles in the pathogenesis of inflammatory bowel disease. The molecular mechanisms whereby these pathogenic factors are regulated during the disease are not fully understood. We report here that a member of the TNFAIP8 (tumor necrosis factor-α-induced protein 8) family called TIPE2 (TNFAIP8-like 2, or TNFAIP8L2) plays a crucial role in regulating commensal bacteria dissemination and inflammatory cell function in experimental colitis induced by dextran sodium sulfate (DSS). Following DSS treatment, TIPE2-deficient mice, or chimeric mice that are deficient in TIPE2 only in their hematopoietic cells, lost less body weight and survived longer than wild type controls. Consistent with this clinical observation, TIPE2-deficient mice exhibited significantly less severe colitis and colonic damage. This was associated with a marked reduction in the colonic expression of inflammatory cytokines such as TNF-α, IL-6, and IL-12. Importantly, the ameliorated DSS-induced colitis in TIPE2−/− mice was also associated with reduced local dissemination of commensal bacteria and a weaker systemic inflammatory response. Combined with our previous report that TIPE2 is a negative regulator of anti-bacterial immunity, these results indicate that TIPE2 promotes colitis by inhibiting mucosal immunity to commensal bacteria.
Colitis; Inflammation; Innate immunity; TNFAIP8L2 (TIPE2)
Many members of the DEXD/H-box helicase family play important roles in the innate immune system against viral infection. We, therefore, isolated double-stranded RNA (dsRNA) complex in myeloid dendritic cells (mDCs). We found that DHX15, a DEXDc helicase family member, is one of the components of this complex. Knockdown of DHX15 expression by short hairpin RNA efficiently reduced the ability of mDCs to produce interferon beta (IFN-β), IL-6 and TNF-α in response to dsRNA and RNA virus. DHX15 specifically bound poly I:C via its HELICc domain. DHX15 interacted with MAVS and formed a complex following stimulation with poly I:C. The N-terminal domain containing a DEXDc motif in DHX15 bound the C-terminus of MAVS. DHX15 is required to activate IRF3 phosphorylation, as well as NF-κB and MAPK signaling during RNA virus infection. We, therefore, identified DHX15 as a new RNA virus sensor mediated by MAVS to activate the immune responses to RNA.
Dendritic cells; Cytokine Receptors; Signal transduction; Innate immunity; RNA virus
Scavenger receptor class B, member 2 (SCARB2) is essential for endosome biogenesis and reorganization and serves as a receptor for both β-glucocerebrosidase and enterovirus 71. However, little is known about its function in innate immune cells. In this study, we show that, among human peripheral blood cells, SCARB2 is most highly expressed in plasmacytoid dendritic cells (pDCs), and its expression is further upregulated by CpG oligodeoxynucleotide stimulation. Knockdown of SCARB2 in pDC cell line GEN2.2 dramatically reduces CpG-induced type I IFN production. Detailed studies reveal that SCARB2 localizes in late endosome/lysosome of pDCs, and knockdown of SCARB2 does not affect CpG oligodeoxynucleotide uptake but results in the retention of TLR9 in the endoplasmic reticulum and an impaired nuclear translocation of IFN regulatory factor 7. The IFN-I production by TLR7 ligand stimulation is also impaired by SCARB2 knockdown. However, SCARB2 is not essential for influenza virus or HSV-induced IFN-I production. These findings suggest that SCARB2 regulates TLR9-dependent IFN-I production of pDCs by mediating endosomal translocation of TLR9 and nuclear translocation of IFN regulatory factor 7.
Accumulating evidence suggests elements within tumors induce exhaustion of effector T cells and infiltration of immune-suppressive regulatory T cells (Tregs) thus preventing the development of durable anti-tumor immunity. Therefore, the discovery of agents that simultaneously block Treg suppressive function and reinvigorate effector function of lymphocytes is key to the development of effective cancer immunotherapy. Previous studies have shown that Toll-like receptor ligands (TLRL) could modulate the function of these T-cell targets; however, those studies relied on cell-free or accessory cell-based assay systems that do not accurately reflect in vivo responses. In contrast, we employed a human PBMC-based proliferation assay system to simultaneously monitor the effect of TLRLs on T cells (CD4+, CD8+, Tregs), B cells and NK cells, which gave different and even conflicting results. We found that the TLR7/8L:CL097 could simultaneously activate CD8+ T cells, B cells and NK cells plus block Treg suppression of T cells and B cells. The TLRLs TLR1/2L:Pam3CSK4, TLR5L:flagellin, TLR4L:LPS and TLR8/7L:CL075 also blocked Treg suppression of CD4+ or CD8+ T cell proliferation but not B cell proliferation. Besides CL097, TLR2L:PGN, CL075 and TLR9L:CpG-(A-C) were strong activators of NK cells. Importantly, we found that Pam3CSK4 could: 1) activate CD4+ T cells proliferation; 2) inhibit the expansion of IL-10+ nTregs and induction of IL-10+ CD4+ Tregs (Tr1); and 3) block nTreg suppressive function. Our results suggest these agents could serve as adjuvants to enhance the efficacy of current immunotherapeutic strategies in cancer patients.
Reactions to aspirin and non-steroidal anti-inflammatory drugs in patients with aspirin-exacerbated respiratory disease (AERD) are triggered when constraints upon activated eosinophils, normally supplied by PGE2, are removed secondary to cyclooxygenase-1 inhibition. However, the mechanism driving the concomitant cellular activation is unknown. We investigated the capacity of aspirin itself to provide this activation signal. Eosinophils were enriched from peripheral blood samples and activated with lysine ASA (LysASA). Parallel samples were stimulated with related non-steroidal anti-inflammatory drugs. Activation was evaluated as Ca+2 flux, secretion of cysteinyl leukotrienes (CysLT), and eosinophil derived neurotoxin (EDN) release. CD34+ progenitor-derived mast cells were also used to test the influence of aspirin on human mast cells with measurements of Ca+2 flux and PGD2 release. LysASA induced Ca+2 fluxes and EDN release, but not CysLT secretion from circulating eosinophils. There was no difference in the sensitivity or extent of activation between AERD and control subjects and sodium salicylate was without effect. Like eosinophils, aspirin was able to activate human mast cells directly through Ca+2 flux and PGD2 release. AERD is associated with eosinophils maturing locally in a high interferon (IFN)-γ milieu. As such, in additional studies, eosinophil progenitors were differentiated in the presence of IFN-γ prior to activation with aspirin. Eosinophils matured in the presence of IFN-γ displayed robust secretion of both EDN and CysLTs. These studies identify aspirin as the trigger of eosinophil and mast cell activation in AERD, acting in synergy with its ability to release cells from the anti-inflammatory constraints of PGE2.
eosinophil; aspirin exacerbated respiratory disease; leukotriene; cyclooxygenase; mast cell
While miRNAs have been shown to participate in innate immune responses, it is not completely understood how miRNAs regulate negative immuno-modulatory events. IL-10 is an important anti-inflammatory mediator that prevents excessive inflammation and associated immunological pathologies. Although the regulation of IL-10 expression has been well studied at both the transcriptional and translational levels, it is less clear how miRNAs control IL-10 expression during inflammation. In this study, we found that miR-27a is downregulated in macrophages following stimulation through TLR2 and TLR4, but not TLR3. Upregulation of miR-27a enhanced the expression of pro-inflammatory cytokines in TLR2/4 activated macrophages. Conversely, knockdown of miR-27a diminished cytokine expression. Mechanistically, we found that miR-27a negatively regulates IL-10 expression in that upregulation of miR-27a decreases, whereas downregulation of miR-27a increases IL-10 expression in activated macrophages. Likely due to the decreased expression of IL-10, upregulation of miR-27a diminished IL-10-dependent STAT3 phosphorylation in TLR4 activated macrophages. Consistent with IL-10 being a potential mediator for the role of miR-27a in immune response, blocking IL-10 abolished the enhancing effect of miR-27a on TLR4 activated inflammation. In conclusion, our study identified miR-27a downregulation as a negative regulatory mechanism that prevents overly exuberant TLR2 and TLR4 driven inflammatory responses.
miR-27a; IL-10; innate immunity; TLR4; TLR2
Acquired aplastic anemia (AA) is a potentially fatal bone marrow (BM) failure syndrome. IFN-γ-producing T helper (Th)1 CD4+ T cells mediate the immune destruction of hematopoietic cells, and are central to the pathogenesis. However, the molecular events that control the development of BM-destructive Th1 cells remain largely unknown. Ezh2 is a chromatin-modifying enzyme that regulates multiple cellular processes primarily by silencing gene expression. We recently reported that Ezh2 is crucial for inflammatory T cell responses after allogeneic BM transplantation. To elucidate whether Ezh2 mediates pathogenic Th1 responses in AA and the mechanism of Ezh2 action in regulating Th1 cells, we studied the effects of Ezh2 inhibition in CD4+ T cells using a mouse model of human AA. Conditionally deleting Ezh2 in mature T cells dramatically reduced the production of BM-destructive Th1 cells in vivo, decreased BM-infiltrating Th1 cells, and rescued mice from BM failure. Ezh2 inhibition resulted in significant decrease in the expression of Tbx21 and Stat4 (which encode transcription factors T-bet and STAT4, respectively). Introduction of T-bet but not STAT4 into Ezh2-deficient T cells fully rescued their differentiation into Th1 cells mediating AA. Ezh2 bound to the Tbx21 promoter in Th1 cells, and directly activated Tbx21 transcription. Unexpectedly, Ezh2 was also required to prevent proteasome-mediated degradation of T-bet protein in Th1 cells. Our results identify T-bet as the transcriptional and post-translational Ezh2 target that acts together to generate BM-destructive Th1 cells, and highlight the therapeutic potential of Ezh2 inhibition in reducing AA and other autoimmune diseases.
Mast cells play a central role in allergy through secretion of both preformed and newly synthesized mediators. Mast cell mediator secretion is controlled by a complex network of signaling events. Despite intensive studies, signaling pathways in the regulation of mast cell mediator secretion remain incompletely defined. Here, we examined the role of calpain in IgE-dependent mast cell activation. IgE-mediated activation of mouse bone marrow-derived mast cells (BMMCs) enhanced calpain activity. Inhibition of calpain activity by a number of calpain inhibitors reduced IgE-mediated mast cell degranulation both in vitro and in vivo. Calpain inhibitors blocked IgE-mediated TNF and IL-6 production in vitro and reduced late-phase allergic response in vivo. Importantly, mouse calpain-1 null BMMCs showed reduced IgE-mediated mast cell degranulation in vitro and in vivo, diminished cytokine and chemokine production in vitro, and impaired late-phase allergic response in vivo. Further studies revealed that calpain-1-deficiency led to specific attenuation of IκB-NF-κB pathway and IKK-SNAP23 pathway, while calcium flux, MAP kinases, Akt, and NFAT pathway proceed normally in IgE-activated calpain-1 null mast cells. Thus, calpain-1 is identified as a novel regulator in IgE-mediated mast cell activation and could serve as a potential therapeutic target for the management of allergic inflammation.