The myeloid C-type lectin receptor Dectin-2 directs the generation of Th2 and Th17 immune responses to the house dust mite Dermatophagoides farinae (Df) through the generation of cysteinyl leukotrienes (cys-LTs) and pro-inflammatory cytokines, respectively, but a role for Dectin-2 in effector phase responses has not been described. Here, we demonstrate that administration of the Dectin-2 mAb solely at the time of Df challenge abrogated eosinophilic and neutrophilic inflammation in the bronchoalveolar lavage (BAL) fluid and Th1, Th2, and Th17 inflammation in the lung of previously sensitized mice. Furthermore, Dectin-2 null mice (Clec4n−/−) sensitized with the adoptive transfer of Df-pulsed wild-type (WT) bone marrow-derived DCs (BMDCs) also had less Df-elicited pulmonary inflammation, supporting an effector function for Dectin-2. The protection from pulmonary inflammation seen with the Dectin-2 mAb or in Clec4n−/− mice was associated with little or no reduction in lung-draining lymph node cells or their cytokine production, and with no reduction in serum IgE. WT and Clec4n−/− mice recipients, sensitized with Df-pulsed WT BMDCs, had comparable levels of Df-elicited IL-6, IL-23, TNF-α, and cys-LTs in the lung. By contrast, Df-elicited CCL4 and CCL8 production from pulmonary CD11c+CD11b+Ly6C+ and CD11c+CD11b+Ly6C−CD64+ monocyte-derived DCs was reduced in Clec4n−/− recipients. Addition of CCL8 at the time of Df challenge abrogated the protection from eosinophilic, neutrophilic, and Th2 pulmonary inflammation seen in Clec4n−/− recipients. Taken together, these results reveal that Dectin-2 regulates monocyte-derived DC function in the pulmonary microenvironment at Df challenge to promote the local inflammatory response.
Knockout mice; Dendritic Cells; Lipid mediators; Allergy; Inflammation
IL-17A stimulates cardiac fibroblasts to produce inflammatory mediators critical for the recruitment and differentiation of myeloid cells during inflammatory dilated cardiomyopathy.
Inflammatory dilated cardiomyopathy (DCMi) is a major cause of heart failure in individuals below the age of 40. We recently reported that IL-17A is required for the development of DCMi. We show a novel pathway connecting IL-17A, cardiac fibroblasts (CFs), GM-CSF, and heart-infiltrating myeloid cells with the pathogenesis of DCMi. Il17ra−/− mice were protected from DCMi, and this was associated with significantly diminished neutrophil and Ly6Chi monocyte/macrophage (MO/MΦ) cardiac infiltrates. Depletion of Ly6Chi MO/MΦ also protected mice from DCMi. Mechanistically, IL-17A stimulated CFs to produce key chemokines and cytokines that are critical downstream effectors in the recruitment and differentiation of myeloid cells. Moreover, IL-17A directs Ly6Chi MO/MΦ in trans toward a more proinflammatory phenotype via CF-derived GM-CSF. Collectively, this IL-17A–fibroblast–GM-CSF–MO/MΦ axis could provide a novel target for the treatment of DCMi and related inflammatory cardiac diseases.
In order to study regulatory T (Treg) cell control of chronic autoimmunity in a lymphoreplete host, we created and characterized a new model of autoimmune lung inflammation that targets the medium and small airways. We generated transgenic mice that express a chimeric membrane protein consisting of hen egg lysozyme (mHEL) and a hemoglobin (Hb) epitope tag under the control of the Clara cell secretory protein (CCSP) promoter, which largely limited transgene expression to the respiratory bronchioles. When CCSP-mHEL/Hb transgenic mice were crossed to N3.L2 TCR transgenic mice that recognize the Hb epitope, the bigenic progeny developed dense, pseudo-follicular lymphocytic peribronchiolar infiltrates that resembled the histological pattern of follicular bronchiolitis. Aggregates of activated IFN-γ- and IL-17A-secreting CD4+ T cells as well as B cells surrounded the airways. Lung pathology was similar in Ifng−/− and Il17a−/− mice, indicating that either cytokine is sufficient to establish chronic disease. A large number of antigen-specific Treg cells accumulated in the lesions and Treg cell-depletion in the affected mice led to an interstitial spread of the disease that ultimately proved fatal. Thus Treg cells act to restrain autoimmune responses, resulting in an organized and controlled chronic pathological process rather than a progressive disease.
Caspase recruitment domain-containing protein 9 (CARD9) is an adaptor molecule signal that is critical for NF-κB activation and is triggered through C-type lectin receptors (CLRs), which are pattern recognition receptors that recognize carbohydrate structures. Previous studies have reported that Cryptococcus neoformans, a fungal pathogen that causes meningoencephalitis in AIDS patients, is recognized through some CLRs, such as mannose receptors or DC-SIGN. However, the role of CARD9 in the host defense against cryptococcal infection remains to be elucidated. In the present study, we analyzed the role of CARD9 in the host defense against pulmonary infection with C. neoformans. CARD9 gene-disrupted (knockout [KO]) mice were highly susceptible to this infection, as shown by the reduced fungal clearance in the infected lungs of CARD9 KO mice, compared to that in wild-type (WT) mice. Gamma interferon (IFN-γ) production was strongly reduced in CARD9 KO mice during the innate-immunity phase of infection. Reduced IFN-γ synthesis was due to impaired accumulation of NK and memory phenotype T cells, which are major sources of IFN-γ innate-immunity-phase production; a reduction in the accumulation of these cells was correlated with reduced CCL4, CCL5, CXCL9, and CXCL10 synthesis. However, differentiation of Th17 cells, but not of Th1 cells, was impaired at the adaptive-immunity phase in CARD9 KO mice compared to WT mice, although there was no significant difference in the infection susceptibility between interleukin 17A (IL-17A) KO and WT mice. These results suggest that CARD9 KO mice are susceptible to C. neoformans infection probably due to the reduced accumulation of IFN-γ-expressing NK and memory phenotype T cells at the early stage of infection.
The eradication of tumor cells requires communication to and signaling by cells of the immune system. Natural killer (NK) cells are essential tumor-killing effector cells of the innate immune system; however, little is known about whether or how other immune cells recognize tumor cells to assist NK cells. Here, we show that the innate immune receptor Dectin-1 expressed on dendritic cells and macrophages is critical to NK-mediated killing of tumor cells that express N-glycan structures at high levels. Receptor recognition of these tumor cells causes the activation of the IRF5 transcription factor and downstream gene induction for the full-blown tumoricidal activity of NK cells. Consistent with this, we show exacerbated in vivo tumor growth in mice genetically deficient in either Dectin-1 or IRF5. The critical contribution of Dectin-1 in the recognition of and signaling by tumor cells may offer new insight into the anti-tumor immune system with therapeutic implications.
When cells in the body grow and divide uncontrollably, cancerous tumors can form. An individual's likelihood of recovering from cancer is highly variable and often depends on the type of cancer and the extent of the disease at the start of treatment. Researchers are therefore interested in discovering how the body responds against cancerous cells.
The first line of defense against infection and disease is the body's innate immune system, which includes a suite of immune cells known as white blood cells. These cells patrol the body's organs and tissues in an effort to immediately respond to pathogens and damaged, stressed or otherwise abnormal host cells. Among white blood cells, natural killer cells are involved in identifying and destroying tumor cells. However, it was unclear whether or how other immune cells might help natural killer cells to destroy tumors. In addition, although immune cells detect pathogens and injured cells by producing proteins called pattern recognition receptors, it was unknown whether these receptors also detect tumor cells.
Here, Chiba et al. reveal that two other types of immune cell—dendritic cells and macrophages—play essential roles in helping natural killer cells to prevent tumors from growing in mice. The dendritic cells and macrophages produce a pattern recognition receptor called Dectin-1 that recognizes a molecule found on the surface of some—but not all—types of tumor cell. In doing so, Dectin-1 activates a critical signaling pathway and directs the activity of the natural killer cells so that they can effectively kill tumor cells. Chiba et al. found that these tumors grew faster in mice that lack the Dectin-1 protein.
The findings of Chiba et al. may also help to explain the effectiveness of certain antibodies—proteins that recognize and neutralize foreign objects such as bacteria and viruses—in cancer therapy. In addition, the Dectin-1 pathway presents a new avenue of research that may offer new cancer treatments.
innate immunity; Dectin-1; NK cell; IRF; mouse
The skin is an immune organ that contains innate and acquired immune systems and thus is able to respond to exogenous stimuli producing large amount of proinflammatory cytokines including IL-1 and IL-1 family members. The role of the epidermal IL-1 is not limited to initiation of local inflammatory responses, but also to induction of systemic inflammation. However, association of persistent release of IL-1 family members from severe skin inflammatory diseases such as psoriasis, epidermolysis bullosa, atopic dermatitis, blistering diseases and desmoglein-1 deficiency syndrome with diseases in systemic organs have not been so far assessed. Here, we showed the occurrence of severe systemic cardiovascular diseases and metabolic abnormalities including aberrant vascular wall remodeling with aortic stenosis, cardiomegaly, impaired limb and tail circulation, fatty tissue loss and systemic amyloid deposition in multiple organs with liver and kidney dysfunction in mouse models with severe dermatitis caused by persistent release of IL-1s from the skin. These morbid conditions were ameliorated by simultaneous administration of anti-IL-1α and IL-1β antibodies. These findings may explain the morbid association of arteriosclerosis, heart involvement, amyloidosis and cachexia in severe systemic skin diseases and systemic autoinflammatory diseases, and support the value of anti-IL-1 therapy for systemic inflammatory diseases.
Obesity is associated with the development of asthma and considerable asthma-related healthcare utilization. To understand the immunological pathways that lead to obesity-associated asthma, we fed mice a high fat diet for 12 weeks, which resulted in obesity and the development of airway hyperreactivity (AHR), a cardinal feature of asthma. This AHR depended on innate immunity, since it occurred in obese Rag−/− mice, and on IL-17A and the NLRP3 inflammasome, since it did not develop in obese Il17−/− or Nlrp3−/− mice. The AHR was also associated with the presence in the lungs of CCR6+ innate lymphoid cells producing IL-17A (ILC3 cells), which could by themselves mediate AHR when adoptively transferred into Rag2−/−
Il2rγ−/− mice. IL-1β played an important role by expanding the ILC3 cells, and treatment to block the function of IL-1β abolished obesity-induced AHR. Since we found ILC3-like cells in the bronchoalveolar lavage fluid of human patients with asthma, we suggest that obesity-associated asthma is facilitated by inflammation mediated by NLRP3, IL-1β and ILC3 cells.
airway hyperreactivity; asthma; obesity; innate lymphoid cells; IL-17; NLRP3; ILC3
Disturbance of T cell homeostasis could lead to intestinal inflammation. Naïve CD4 T cells undergoing spontaneous proliferation, a robust proliferative response that occurs under severe lymphopenic conditions, differentiate into effector cells producing Th1 and/or Th17 type cytokines and induce a chronic inflammation in the intestine that resembles human inflammatory bowel disease. In this study, we investigated key properties of CD4 T cells necessary to induce experimental colitis. α4β7 upregulation was primarily induced by mLN resident CD11b+ dendritic cell subsets via TGFβ/retinoic acid-dependent mechanism. Interestingly, α4β7 expression was essential but not sufficient to induce inflammation. In addition to gut homing specificity, expression of gut Ag specificity was also crucial. T cell acquisition of the specificity was dramatically enhanced by the presence of γδ T cells, a population previously shown to exacerbate T cell mediated colitis. Importantly, IL-23-mediated γδ T cell stimulation was necessary to enhance colitogenicity but not gut antigen reactivity of proliferating CD4 T cells. These findings demonstrate that T cell colitogenicity is achieved through multiple processes, offering a therapeutic rationale by intervening these pathways.
α4β7 integrin; CD4 T cells; colitis; gut antigens; IBD
For patients with chronic lung diseases, such as chronic obstructive pulmonary disease (COPD), exacerbations are life-threatening events causing acute respiratory distress that can even lead to hospitalization and death. Although a great deal of effort has been put into research of exacerbations and potential treatment options, the exact underlying mechanisms are yet to be deciphered and no therapy that effectively targets the excessive inflammation is available. In this study, we report that interleukin-1β (IL-1β) and interleukin-17A (IL-17A) are key mediators of neutrophilic inflammation in influenza-induced exacerbations of chronic lung inflammation. Using a mouse model of disease, our data shows a role for IL-1β in mediating lung dysfunction, and in driving neutrophilic inflammation during the whole phase of viral infection. We further report a role for IL-17A as a mediator of IL-1β induced neutrophilia at early time points during influenza-induced exacerbations. Blocking of IL-17A or IL-1 resulted in a significant abrogation of neutrophil recruitment to the airways in the initial phase of infection or at the peak of viral replication, respectively. Therefore, IL-17A and IL-1β are potential targets for therapeutic treatment of viral exacerbations of chronic lung inflammation
Osteolysis of bone following total hip replacements is a major clinical problem. Examination of the areas surrounding failed implants has indicated an increase in the bone-resorption-inducing cytokine, interleukin 1β (IL-1β). NALP3, a NOD-like receptor protein located in the cytosol of macrophages, has been shown to signal the cleavage of pro-IL-1β into its mature, secreted form, IL-1β. Here we show that titanium particles stimulate the NALP3 inflammasome. We demonstrate that titanium induces IL-1β secretion from macrophages and this response is dependent on the expression of components of the NALP3 inflammasome, including NALP3, ASC, and Caspase-1. We also show that titanium particles trigger the recruitment of neutrophils and that this acute inflammatory response is dependent on the expression of the IL-1 receptor and IL-1α/β. Moreover, administration of the IL-1 receptor antagonist (IL-1Ra) diminished neutrophil recruitment in response to titanium particles. Together, these results suggest that titanium particle-induced acute inflammation is due to activation of the NALP3 inflammasome, which leads to increased IL-1β secretion and IL-1-associated signaling, including neutrophil recruitment. Efficacy of IL-1Ra treatment introduces the potential for antagonist based-therapies for implant osteolysis.
Titanium; inflammasome; neutrophils; IL-1; NALP3
Background and Aim
The etiology of post-inflammatory gastrointestinal (GI) motility dysfunction, after resolution of acute symptoms of inflammatory bowel diseases (IBD) and intestinal infection, is largely unknown, however, a possible involvement of T cells is suggested.
Using the mouse model of T cell activation-induced enteritis, we investigated whether enhancement of smooth muscle cell (SMC) contraction by interleukin (IL)-17A is involved in postinflammatory GI hypermotility.
Activation of CD3 induces temporal enteritis with GI hypomotility in the midst of, and hypermotility after resolution of, intestinal inflammation. Prolonged upregulation of IL-17A was prominent and IL-17A injection directly enhanced GI transit and contractility of intestinal strips. Postinflammatory hypermotility was not observed in IL-17A-deficient mice. Incubation of a muscle strip and SMCs with IL-17A in vitro resulted in enhanced contractility with increased phosphorylation of Ser19 in myosin light chain 2 (p-MLC), a surrogate marker as well as a critical mechanistic factor of SMC contractility. Using primary cultured murine and human intestinal SMCs, IκBζ- and p38 mitogen-activated protein kinase (p38MAPK)-mediated downregulation of the regulator of G protein signaling 4 (RGS4), which suppresses muscarinic signaling of contraction by promoting inactivation/desensitization of Gαq/11 protein, has been suggested to be involved in IL-17A-induced hypercontractility. The opposite effect of L-1β was mediated by IκBζ and c-jun N-terminal kinase (JNK) activation.
We propose and discuss the possible involvement of IL-17A and its downstream signaling cascade in SMCs in diarrheal hypermotility in various GI disorders.
Chemokine (C-C motif) receptor 8 (CCR8), the chemokine receptor for chemokine (C-C motif) ligand 1 (CCL1), is expressed in T-helper type-2 lymphocytes and peritoneal macrophages (PMφ) and is involved in various pathological conditions, including peritoneal adhesions. However, the role of CCR8 in inflammatory responses is not fully elucidated. To investigate the function of CCR8 in macrophages, we compared cytokine secretion from mouse PMφ or bone marrow-derived macrophages (BMMφ) stimulated with various Toll-like receptor (TLR) ligands in CCR8 deficient (CCR8-/-) and wild-type (WT) mice. We found that CCR8-/- PMφ demonstrated attenuated secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 when stimulated with lipopolysaccharide (LPS). In particular, LPS-induced IL-10 production absolutely required CCR8. CCR8-dependent cytokine secretion was characteristic of PMφ but not BMMφ. To further investigate this result, we selected the small molecule compound R243 from a library of compounds with CCR8-antagonistic effects on CCL1-induced Ca2+ flux and CCL1-driven PMφ aggregation. Similar to CCR8-/- PMφ, R243 attenuated secretion of TNF-α, IL-6, and most strikingly IL-10 from WT PMφ, but not BMMφ. CCR8-/- PMφ and R243-treated WT PMφ both showed suppressed c-jun N-terminal kinase activity and nuclear factor-κB signaling after LPS treatment when compared with WT PMφ. A c-Jun signaling pathway inhibitor also produced an inhibitory effect on LPS-induced cytokine secretion that was similar to that of CCR8 deficiency or R243 treatment. As seen in CCR8-/- mice, administration of R243 attenuated peritoneal adhesions in vivo. R243 also prevented hapten-induced colitis. These results are indicative of cross talk between signaling pathways downstream of CCR8 and TLR-4 that induces cytokine production by PMφ. Through use of CCR8-/- mice and the new CCR8 inhibitor, R243, we identified a novel macrophage innate immune response pathway that involves a chemokine receptor.
Infection by the human fungal pathogen Aspergillus fumigatus induces hypoxic microenvironments within the lung that can alter the course of fungal pathogenesis. How hypoxic microenvironments shape the composition and immune activating potential of the fungal cell wall remains undefined. Herein we demonstrate that hypoxic conditions increase the hyphal cell wall thickness and alter its composition particularly by augmenting total and surface-exposed β-glucan content. In addition, hypoxia-induced cell wall alterations increase macrophage and neutrophil responsiveness and antifungal activity as judged by inflammatory cytokine production and ability to induce hyphal damage. We observe that these effects are largely dependent on the mammalian β-glucan receptor dectin-1. In a corticosteroid model of invasive pulmonary aspergillosis, A. fumigatus β-glucan exposure correlates with the presence of hypoxia in situ. Our data suggest that hypoxia-induced fungal cell wall changes influence the activation of innate effector cells at sites of hyphal tissue invasion, which has potential implications for therapeutic outcomes of invasive pulmonary aspergillosis.
Aspergillus fumigatus; hypoxia; beta-glucan; fungal pathogenesis; cell wall
SDF-1/CXCR4 signalling plays an important role in neuronal cell migration and brain development. However, the impact of CXCR4 deficiency in the postnatal mouse brain is still poorly understood. Here, we demonstrate the importance of CXCR4 on cerebellar development and motor behaviour by conditional inactivation of Cxcr4 in the central nervous system. We found CXCR4 plays a key role in cerebellar development. Its loss leads to defects in Purkinje cell dentritogenesis and axonal projection in vivo but not in cell culture. Transcriptome analysis revealed the most significantly affected pathways in the Cxcr4 deficient developing cerebellum are involved in extra cellular matrix receptor interactions and focal adhesion. Consistent with functional impairment of the cerebellum, Cxcr4 knockout mice have poor coordination and balance performance in skilled motor tests. Together, these results suggest ectopic the migration of granule cells impairs development of Purkinje cells, causes gross cerebellar anatomical disruption and leads to behavioural motor defects in Cxcr4 null mice.
Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy that originates from leukemia-initiating cells (LICs). The identification of common mechanisms underlying LIC development will be important in establishing broadly effective therapeutics for AML. Constitutive NF-κB pathway activation has been reported in different types of AML; however, the mechanism of NF-κB activation and its importance in leukemia progression are poorly understood. Here, we analyzed myeloid leukemia mouse models to assess NF-κB activity in AML LICs. We found that LICs, but not normal hematopoietic stem cells or non-LIC fractions within leukemia cells, exhibited constitutive NF-κB activity. This activity was maintained through autocrine TNF-α secretion, which formed an NF-κB/TNF-α positive feedback loop. LICs had increased levels of active proteasome machinery, which promoted the degradation of IκBα and further supported NF-κB activity. Pharmacological inhibition of the proteasome complex markedly suppressed leukemia progression in vivo. Conversely, enhanced activation of NF-κB signaling expanded LIC frequency within leukemia cell populations. We also demonstrated a strong correlation between NF-κB activity and TNF-α secretion in human AML samples. Our findings indicate that NF-κB/TNF-α signaling in LICs contributes to leukemia progression and provide a widely applicable approach for targeting LICs.
Il1rn−/− mice spontaneously develop arthritis and aortitis by
an autoimmune mechanism and also develop dermatitis by an autoinflammatory mechanism.
Here, we show that Rag2−/−Il1rn−/−
mice develop spontaneous colitis with high mortality, making a contrast to the suppression
of arthritis in these mice. Enhanced IL-17A expression in group 3 innate lymphoid cells
(ILC3s) was observed in the colon of
IL-17A-deficiency prolonged the survival of
Rag2−/−Il1rn−/− mice, suggesting
a pathogenic role of this cytokine in the development of intestinal inflammation. Although
IL-17A-producing T cells were increased in Il1rn−/− mice,
these mice did not develop colitis, because CD4+Foxp3+ regulatory T
cell population was also expanded. Thus, excess IL-1 signaling and IL-1-induced IL-17A
from ILC3s cause colitis in Rag2−/−Il1rn−/− mice in
which Treg cells are absent. These observations suggest that the balance between
IL-17A-producing cells and Treg cells is important to keep the immune homeostasis of the
Colitis; IL-1; IL-17; innate lymphoid cells; regulatory Tcells
Attempts to create mouse models for AIDS have been hampered by species barriers in HIV-1 infection. We previously showed that the nuclear accumulation of HIV-1 preintegration complex (PIC) was suppressed in mouse cells. Lens epithelium-derived growth factor (LEDGF/p75) is a host factor identified as a binding partner of integrase (IN), and has been suggested to be involved in promoting viral integration by tethering PIC to the chromatin, which are observed as nuclear accumulation of IN by LEDGF/p75. Therefore, we here hypothesized that this host factor might act as one of the species-specific barriers in mouse cells. We generated transgenic (Tg) mice that constitutively express human (h) LEDGF/p75. The GFP-fused IN was efficiently accumulated into the nucleus of hLEDGF/p75 expressing Tg mouse embryonic fibroblast (MEF) cells in contrast to the control MEF cells. Importantly, hLEDGF/p75 Tg MEF cells were significantly more susceptible to HIV-1 infection. These results suggest that LEDGF/p75 is one of the host factors that constitute species barrier against HIV-1 in mouse cells.
HIV-1; LEDGF/p75; IN; transgenic mouse; species barrier
The protein-tyrosine phosphatase SHP-1 plays critical roles in immune signaling, but how mutations in SHP-1 cause inflammatory disease in humans remains poorly defined1. Mice homozygous for the Y208N amino acid substitution in the carboxy-terminus of SHP-1 (referred to as Ptpn6spin mice) spontaneously develop a severe inflammatory syndrome that resembles neutrophilic dermatosis in humans and is characterized by persistent footpad swelling and suppurative inflammation2,3. Here, we report that RIP1-regulated IL-1α production by hematopoietic cells critically mediates chronic inflammatory disease in Ptpn6spin mice, whereas inflammasome signaling and IL-1β-mediated events were dispensable. IL-1α was also critical for exacerbated inflammatory responses and unremitting tissue damage upon footpad microabrasion of Ptpn6spin mice. Intriguingly, pharmacological and genetic blockade of the kinase RIP1 protected against wound-induced inflammation and tissue damage in Ptpn6spin mice, whereas RIP3 deletion failed to do so. Moreover, RIP1-mediated inflammatory cytokine production was attenuated by NF-κB and ERK inhibition. Together, our results suggest that wound-induced tissue damage and chronic inflammation in Ptpn6spin mice are critically dependent on RIP1-mediated IL-1α production, whereas inflammasome signaling and RIP3-mediated necroptosis were dispensable. Thus, we have unravelled a novel inflammatory circuit in which RIP1-mediated IL-1α secretion in response to deregulated SHP-1 activity triggers an inflammatory destructive disease that proceeds independently of inflammasomes and programmed necrosis.
SHP-1; Ptpn6; inflammasome; NOD-like receptor; caspase; interleukin; RIP1; RIP3; cell death; inflammation
SMAP2 is an Arf GAP and modulates clathrin-coated vesicle formation. SMAP2-deficient male mice exhibited globozoospermia due to acrosome deformation. In SMAP2(−/−) spermatids, budding of proacrosomal vesicles from the TGN was distorted and clathrin traffic–related molecules such as CALM and syntaxin2 were mislocated.
The trans-Golgi network (TGN) functions as a hub organelle in the exocytosis of clathrin-coated membrane vesicles, and SMAP2 is an Arf GTPase-activating protein that binds to both clathrin and the clathrin assembly protein (CALM). In the present study, SMAP2 is detected on the TGN in the pachytene spermatocyte to the round spermatid stages of spermatogenesis. Gene targeting reveals that SMAP2-deficient male mice are healthy and survive to adulthood but are infertile and exhibit globozoospermia. In SMAP2-deficient spermatids, the diameter of proacrosomal vesicles budding from TGN increases, TGN structures are distorted, acrosome formation is severely impaired, and reorganization of the nucleus does not proceed properly. CALM functions to regulate vesicle sizes, and this study shows that CALM is not recruited to the TGN in the absence of SMAP2. Furthermore, syntaxin2, a component of the soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complex, is not properly concentrated at the site of acrosome formation. Thus this study reveals a link between SMAP2 and CALM/syntaxin2 in clathrin-coated vesicle formation from the TGN and subsequent acrosome formation. SMAP2-deficient mice provide a model for globozoospermia in humans.
It is unknown whether adipokines derived from adipose tissues modulate endoplasmic reticulum (ER) stress induced in obesity. Here, we show that visceral adipose tissue–derived serine protease inhibitor (vaspin) binds to cell-surface 78-kDa glucose-regulated protein (GRP78), which is recruited from ER to plasma membrane under ER stress. Vaspin transgenic mice were protected from diet-induced obesity, glucose intolerance, and hepatic steatosis, while vaspin-deficient mice developed glucose intolerance associated with upregulation of ER stress markers. With tandem affinity tag purification using HepG2 cells, we identified GRP78 as an interacting molecule. The complex formation of vaspin, GRP78, and murine tumor cell DnaJ-like protein 1 (MTJ-1) (DnaJ homolog, subfamily C, member 1) on plasma membrane was confirmed by cell-surface labeling with biotin and immunoprecipitation in liver tissues and H-4-II-E-C3 cells. The addition of recombinant human vaspin in the cultured H-4-II-E-C3 cells also increased the phosphorylation of Akt and AMP-activated protein kinase (AMPK) in a dose-dependent manner, and anti-GRP78 antibodies completely abrogated the vaspin-induced upregulation of pAkt and pAMPK. Vaspin is a novel ligand for cell-surface GRP78/MTJ-1 complex, and its subsequent signals exert beneficial effects on ER stress–induced metabolic dysfunctions.
TNF and IL-1 are major mediators involved in severe inflammatory diseases against which therapeutic neutralizing antibodies are developed. However, both TNF and IL-1 receptor pathways are essential for the control of Mycobacterium tuberculosis infection, and it is critical to assess the respective role of IL-1α, IL-1β, and TNF. Using gene-targeted mice we show that absence of both IL-1α and IL-1β recapitulates the uncontrolled M. tuberculosis infection with increased bacterial burden, exacerbated lung inflammation, high IFNγ, reduced IL-23 p19 and rapid death seen in IL-1R1-deficient mice. However, presence of either IL-1α or IL-1β in single-deficient mice is sufficient to control acute M. tuberculosis infection, with restrained bacterial burden and lung pathology, in conditions where TNF deficient mice succumbed within 4 weeks with overwhelming infection. Systemic infection by attenuated M. bovis BCG was controlled in the absence of functional IL-1 pathway, but not in the absence of TNF. Therefore, although both IL-1α and IL-1β are required for a full host response to virulent M. tuberculosis, the presence of either IL-1α or IL-1β allows some control of acute M. tuberculosis infection, and IL-1 pathway is dispensable for controlling M. bovis BCG acute infection. This is in sharp contrast with TNF, which is essential for host response to both attenuated and virulent mycobacteria and may have implications for anti-inflammatory therapy with IL-1β neutralizing antibodies.
Host response; IL-1β/IL-1α; M. bovis infection; M. tuberculosis; TNF
IL-17A, IL-17F and IL-25 are ligands for IL-17RA. In the present study, we demonstrated that IL-25-deficient mice, but not IL-17A-, IL-17F-, IL-17A/F-, IL-23p19- and ROR-γt-deficient mice, showed significant suppression of the number of eosinophils and the levels of proinflammatory mediators in bronchoalveolar lavage fluids, airway hyperresponsiveness to methacholine, or ovalbumin-specific IgG1 and IgE levels in the serum during ovalbumin-induced Th2-type/eosinophilic airway inflammation, without any effect on lung DC migration or antigen-specific memory-Th2-cell expansion during antigen sensitization. By adoptive transfer of either T cells, mast cells or bone marrow cells from IL-25-deficient mice, we found that IL-25 produced by airway structural cells such as epithelial cells—but not by such hematopoietic stem-cell-origin immune cells as T cells and mast cells—was indispensable for induction of Th2-type/eosinophilic airway inflammation by activating lung epithelial cells and eosinophils. Therefore, airway structural-cell-derived IL-25—rather than Th17-cell-derived IL-17A and IL-17F—is responsible for induction of local inflammation by promoting activation of lung epithelial cells and eosinophils in the elicitation phase—but is not required for antigen-specific Th2 cell differentiation in the sensitization phase—of Th2-type/eosinophilic airway inflammation.
Although thymus-independent type 2 antigens generally do not undergo Ig class switching from IgM to IgG, pneumococcal polysaccharide vaccine (PPV) induces the production of serotype-specific IgG. How this happens remains unclear, however. In the present study, PPV immunization induced production of IgG as well as IgM specific for a serotype 3-pneumococcal polysaccharide in the sera of wild-type (WT) mice, but this phenomenon was significantly reduced in Dectin-2 knockout (KO) mice. Immunization with PPV caused IL-12p40 production in WT mice, but this response was significantly reduced in Dectin-2KO mice. Likewise, immunization with PPV activated natural killer T (NKT) cells in WT mice but not in Dectin-2KO mice. Furthermore, administration of α-galactosylceramide, recombinant (r)IL-12 or rIFN-γ improved the reduced IgG levels in Dectin-2KO mice, and treatment with neutralizing anti-IFN-γ mAb resulted in the reduction of IgG synthesis in PPV-immunized WT mice. Transfer of spleen cells from PPV-immunized WT mice conferred protection against pneumococcal infection on recipient mice, whereas this effect was cancelled when the transferred spleen cells were harvested from PPV-immunized Dectin-2KO mice. These results suggest that the detection of PPV antigens via Dectin-2 triggers IL-12 production, which induces IFN-γ synthesis by NKT cells and subsequently the production of serotype-specific IgG.
The role of regulatory T cells (Tregs) in human colon cancer (CC) remains controversial: high densities of tumor-infiltrating Tregs can correlate with better or worse clinical outcomes depending on the study. In mouse models of cancer, Tregs have been reported to suppress inflammation and protect the host, suppress T cells and protect the tumor, or even have direct cancer-promoting attributes. These different effects may result from the presence of different Treg subsets. We report the preferential expansion of a Treg subset in human CC with potent T cell–suppressive, but compromised anti-inflammatory, properties; these cells are distinguished from Tregs present in healthy donors by their coexpression of Foxp3 and RORγt. Tregs with similar attributes were found to be expanded in mouse models of hereditary polyposis. Indeed, ablation of the RORγt gene in Foxp3+ cells in polyp-prone mice stabilized Treg anti-inflammatory functions, suppressed inflammation, improved polyp-specific immune surveillance, and severely attenuated polyposis. Ablation of interleukin-6 (IL-6), IL-23, IL-17, or tumor necrosis factor–α in polyp-prone mice reduced polyp number but not to the same extent as loss of RORγt. Surprisingly, loss of IL-17A had a dual effect: IL-17A–deficient mice had fewer polyps but continued to have RORγt+ Tregs and developed invasive cancer. Thus, we conclude that RORγt has a central role in determining the balance between protective and pathogenic Tregs in CC and that Treg subtype regulates inflammation, potency of immune surveillance, and severity of disease outcome.