The Role of Interchain Heterodisulfide Formation in Activation of the Human Common β and Mouse βIL-3 Receptors

The cytokines, interleukin-3 (IL-3), interleukin-5 (IL-5), and granulocyte-macrophage colony-stimulating factor (GM-CSF), exhibit overlapping activities in the regulation of hematopoietic cells. In humans, the common β (βc) receptor is shared by the three cytokines and functions together with cytokine-specific α subunits in signaling. A widely accepted hypothesis is that receptor activation requires heterodisulfide formation between the domain 1 D-E loop disulfide in human βc (hβc) and unidentified cysteine residues in the N-terminal domains of the α receptors. Since the development of this hypothesis, new data have been obtained showing that domain 1 of hβc is part of the cytokine binding epitope of this receptor and that an IL-3Rα isoform lacking the N-terminal Ig-like domain (the “SP2” isoform) is competent for signaling. We therefore investigated whether distortion of the domain 1-domain 4 ligand-binding epitope in hβc and the related mouse receptor, β_IL-3, could account for the loss of receptor signaling when the domain 1 D-E loop disulfide is disrupted. Indeed, mutation of the disulfide in hβc led to both a complete loss of high affinity binding with the human IL-3Rα SP2 isoform and of downstream signaling. Mutation of the orthologous residues in the mouse IL-3-specific receptor, β_IL-3, not only precluded direct binding of mouse IL-3 but also resulted in complete loss of high affinity binding and signaling with the mouse IL-3Rα SP2 isoform. Our data are most consistent with a role for the domain 1 D-E loop disulfide of hβc and β_IL-3 in maintaining the precise positions of ligand-binding residues necessary for normal high affinity binding and signaling.     

CSL311, a novel,potent, therapeutic monoclonal antibody for the treatment of diseases mediated by the common β chain of the IL-3, GM-CSF and IL-5 receptors

The β common-signaling cytokines interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-5 stimulate pro-inflammatory activities of haematopoietic cells via a receptor complex incorporating cytokine-specific α and shared β common (β_c, CD131) receptor. Evidence from animal models and recent clinical trials demonstrate that these cytokines are critical mediators of the pathogenesis of inflammatory airway disease such as asthma. However, no therapeutic agents, other than steroids, that specifically and effectively target inflammation mediated by all 3 of these cytokines exist. We employed phage display technology to identify and optimize a novel, human monoclonal antibody (CSL311) that binds to a unique epitope that is specific to the cytokine-binding site of the human β_c receptor. The binding epitope of CSL311 on the β_c receptor was defined by X-ray crystallography and site-directed mutagenesis. CSL311 has picomolar binding affinity for the human β_c receptor, and at therapeutic concentrations is a highly potent antagonist of the combined activities of IL-3, GM-CSF and IL-5 on primary eosinophil survival in vitro. Importantly, CSL311 inhibited the survival of inflammatory cells present in induced sputum from human allergic asthmatic subjects undergoing allergen bronchoprovocation. Due to its high potency and ability to simultaneously suppress the activity of all 3 β common cytokines, CSL311 may provide a new strategy for the treatment of chronic inflammatory diseases where the human β_c receptor is central to pathogenesis. The coordinates for the β_c/CSL311 Fab complex structure have been deposited with the RCSB Protein Data Bank (PDB 5DWU).     

Absence of the Common Gamma Chain (γc), a Critical Component of the Type I IL-4 Receptor,Increases the Severity of Allergic Lung Inflammation

The T_H2 cytokines, IL-4 and IL-13, play critical roles in inducing allergic lung inflammation and drive the alternative activation of macrophages (AAM). Although both cytokines share receptor subunits, IL-4 and IL-13 have differential roles in asthma pathogenesis: IL-4 regulates T_H2 cell differentiation, while IL-13 regulates airway hyperreactivity and mucus production. Aside from controlling T_H2 differentiation, the unique contribution of IL-4 signaling via the Type I receptor in airway inflammation remains unclear. Therefore, we analyzed responses in mice deficient in gamma c (γ_c) to elucidate the role of the Type I IL-4 receptor. OVA primed CD4⁺ OT-II T cells were adoptively transferred into RAG2^−/− and γ_c ^−/− mice and allergic lung disease was induced. Both γ_c ^−/− and γ_cxRAG2^−/− mice developed increased pulmonary inflammation and eosinophilia upon OVA challenge, compared to RAG2^−/− mice. Characteristic AAM proteins FIZZ1 and YM1 were expressed in lung epithelial cells in both mouse strains, but greater numbers of FIZZ1+ or YM1+ airways were present in γ_c ^−/− mice. Absence of γ_c in macrophages, however, resulted in reduced YM1 expression. We observed higher T_H2 cytokine levels in the BAL and an altered DC phenotype in the γ_c ^−/− recipient mice suggesting the potential for dysregulated T cell and dendritic cell (DC) activation in the γ_c-deficient environment. These results demonstrate that in absence of the Type I IL-4R, the Type II R can mediate allergic responses in the presence of T_H2 effectors. However, the Type I R regulates AAM protein expression in macrophages.     

IL-36α Exerts Pro-Inflammatory Effects in the Lungs of Mice

Interleukin (IL-) 36 cytokines (previously designated as novel IL-1 family member cytokines; IL-1F5– IL-1F10) constitute a novel cluster of cytokines structurally and functionally similar to members of the IL-1 cytokine cluster. The effects of IL-36 cytokines in inflammatory lung disorders remains poorly understood. The current study sought to investigate the effects of IL-36α (IL-1F6) and test the hypothesis that IL-36α acts as a pro-inflammatory cytokine in the lung in vivo. Intratracheal instillation of recombinant mouse IL-36α induced neutrophil influx in the lungs of wild-type C57BL/6 mice and IL-1αβ^−/− mice in vivo. IL-36α induced neutrophil influx was also associated with increased mRNA expression of neutrophil-specific chemokines CXCL1 and CXCL2 in the lungs of C57BL/6 and IL-1αβ^−/− mice in vivo. In addition, intratracheal instillation of IL-36α enhanced mRNA expression of its receptor IL-36R in the lungs of C57BL/6 as well as IL-1αβ^−/− mice in vivo. Furthermore, in vitro incubation of CD11c⁺ cells with IL-36α resulted in the generation of neutrophil-specific chemokines CXCL1, CXCL2 as well as TNFα. IL-36α increased the expression of the co-stimulatory molecule CD40 and enhanced the ability of CD11c⁺ cells to induce CD4⁺ T cell proliferation in vitro. Furthermore, stimulation with IL-36α activated NF-κB in a mouse macrophage cell line. These results demonstrate that IL-36α acts as a pro-inflammatory cytokine in the lung without the contribution of IL-1α and IL-1β. The current study describes the pro-inflammatory effects of IL-36α in the lung, demonstrates the functional redundancy of IL-36α with other agonist cytokines in the IL-1 and IL-36 cytokine cluster, and suggests that therapeutic targeting of IL-36 cytokines could be beneficial in inflammatory lung diseases.     

Reciprocal Regulation of Development of Neutrophil-Dendritic Cell Hybrids in Mice by IL-4 and Interferon-Gamma

Neutrophils contribute to innate host immunity by functioning as professional phagocytes, whereas dendritic cells (DCs) are prototypic antigen presenting cells (APCs) responsible for the induction of adaptive immune responses. We have demonstrated recently that neutrophils trans-differentiate into a unique population, termed “neutrophil-DC hybrids,” expressing surface markers of both neutrophils and DCs and exhibiting dual functionality of both phagocytes and APCs. Although the hybrid cells emerged in significant numbers in murine bone marrow (BM) culture in the presence of GM-CSF, mechanisms regulating their development remained mostly unknown. In this study, we tested a total of 61 cytokines for their potentials to regulate neutrophil-DC hybrid formation using a newly developed BM micro-culture system combined with semi-automated FACS analysis. Several cytokines including GM-CSF were found to promote the generation of neutrophil-DC hybrids defined by the phenotype of CD11c⁺/MHC II⁺/Ly6G⁺. When tested in the presence of GM-CSF, hybrid cell development was enhanced by IL-4 and suppressed by interferon-γ (IFNγ) in dose-dependent fashions. We next determined in vivo impacts of IL-4 and IFNγ on the development of neutrophil-DC hybrids in thioglycollate-induced peritonitis lesions. Intraperitoneal administrations of IL-4/anti-IL-4 antibody complex (IL-4C) significantly increased the number of hybrids recovered from the lesions. By contract, recovery of hybrids was reduced by recombinant IFNγ. With regard to function, those hybrid cells recovered from IL-4C-treated mice and IFNγ-treated mice showed potent abilities to capture E.coli. These observations imply that emergence of neutrophil-DC hybrids in inflammatory sites is tightly regulated by local cytokine milieus.     

Type II-Activated Murine Macrophages Produce IL-4

Background

Type II activation of macrophages is known to support Th2 responses development; however, the role of Th2 cytokines (esp. IL-4) on type II activation is unknown. To assess whether the central Th2 cytokine IL-4 can alter type II activation of macrophages, we compared the ability of bone marrow-derived macrophages from wild type (WT) and IL-4Rα-deficient mice to be classically or type II-activated in vitro.

Results

We found that although both WT and IL-4Rα-deficient macrophages could be classically activated by LPS or type II activated by immune complexes plus LPS, IL-4Rα-deficient macrophages consistently produced much higher levels of IL-12p40 and IL-10 than WT macrophages. Additionally, we discovered that type II macrophages from both strains were capable of producing IL-4; however, this IL-4 was not responsible for the reduced IL-12p40 and IL-10 levels produced by WT mice. Instead, we found that derivation culture conditions (GM-CSF plus IL-3 versus M-CSF) could explain the different responses of BALB/c and IL-4Rα−/− macrophages, and these cytokines shaped the ensuing macrophage such that GM-CSF plus IL-3 promoted more IL-12 and IL-4 while M-CSF led to higher IL-10 production. Finally, we found that enhanced IL-4 production is characteristic of the type II activation state as other type II-activating products showed similar results.

Conclusions

Taken together, these results implicate type II activated macrophages as an important innate immune source of IL-4 that may play an important role in shaping adaptive immune responses.     

Lack of IL-6 during Coxsackievirus Infection Heightens the Early Immune Response Resulting in Increased Severity of Chronic Autoimmune Myocarditis

Background

Chronic myocarditis is often initiated by viral infection, the most common of which is coxsackievirus infection. The precise mechanism by which viral infection leads to chronic autoimmune pathology is poorly understood, however it is clear that the early immune response plays a critical role. Previous results have shown that the inflammatory cytokine interleukin (IL)-6 is integral to the development of experimental-induced autoimmune myocarditis. However, the function of IL-6 during viral-mediated autoimmunity has yet to be elucidated.

Methods and Results

To address the requirement of IL-6 during disease induction, IL-6 deficient mice were infected with coxsackievirus B3 (CB3). Following infection, mice lacking IL-6 developed increased chronic autoimmune disease pathology compared to wild type controls without a corresponding change in the level of viral replication in the heart. This increase in disease severity was accompanied by elevated levels of TNF-α, MCP-1, IL-10, activated T cells and cardiac infiltrating macrophage/monocytes. Injection of recombinant IL-6 early following infection in the IL-6 deficient mice was sufficient to lower the serum cytokines TNF-α and IL-10 as well as the serum chemokines MCP-1, MIP-1β, RANTES and MIG with a corresponding decrease in the chronic disease pathology strongly suggests an important regulatory role for IL-6 during the early response.

Conclusions

While IL-6 plays a pathogenic role in experimental-induced autoimmune disease, its function following viral-induced autoimmunity is not reprised. By regulating the early immune response and thereby controlling the severity of chronic disease, IL-6 directs the outcome of chronic autoimmune myocarditis.     

High Pulmonary Levels of IL-6 and IL-1β in Children with Chronic Suppurative Lung Disease Are Associated with Low Systemic IFN-γ Production in Response to Non-Typeable Haemophilus influenzae

Non-typeable Haemophilus influenzae (NTHi) is commonly associated with chronic suppurative lung disease in children. We have previously shown that children with chronic suppurative lung disease have a reduced capacity to produce IFN-γ in response to NTHi compared with healthy control children. The aim of this study was to determine if deficient NTHi-specific IFN-γ production is associated with heightened systemic or airway inflammation. We measured a panel of cytokines (IFN-γ, IL-1β, IL-6, IL-8, IL-12 p70), antimicrobial proteins (LL-37, IP-10) as well as cellular and clinical factors associated with airway and systemic inflammation in 70 children with chronic suppurative lung disease. IFN-γ was measured in peripheral blood mononuclear cells challenged in vitro with live NTHi. Regression analysis was used to assess the association between the systemic and airway inflammation and the capacity to produce IFN-γ. On multivariate regression, NTHi-specific IFN-γ production was significantly negatively associated with the BAL concentrations of the inflammatory cytokines IL-6 (β=-0.316; 95%CI -0.49, -0.14; p=0.001) and IL-1β (β=-0.023; 95%CI -0.04, -0.01; p=0.001). This association was independent of bacterial or viral infection, BAL cellularity and the severity of bronchiectasis (using modified Bhalla score on chest CT scans). We found limited evidence of systemic inflammation in children with chronic suppurative lung disease. In summary, increased local airway inflammation is associated with a poorer systemic cell-mediated immune response to NTHi in children with chronic suppurative lung disease. These data support the emerging body of evidence that impaired cell-mediated immune responses and dysregulated airway inflammation may be linked and contribute to the pathobiology of chronic suppurative lung disease.     

IL-12 Can Target Human Lung Adenocarcinoma Cells and Normal Bronchial Epithelial Cells Surrounding Tumor Lesions

Background

Non small cell lung cancer (NSCLC) is a leading cause of cancer death. We have shown previously that IL-12rb2 KO mice develop spontaneously lung adenocarcinomas or bronchioalveolar carcinomas.Aim of the study was to investigate i) IL-12Rβ2 expression in human primary lung adenocarcinomas and in their counterparts, i.e. normal bronchial epithelial cells (NBEC), ii) the direct anti-tumor activity of IL-12 on lung adenocarcinoma cells in vitro and vivo, and the mechanisms involved, and iii) IL-12 activity on NBEC.

Methodology/Principal Findings

Stage I lung adenocarcinomas showed significantly (P = 0.012) higher frequency of IL-12Rβ2 expressing samples than stage II/III tumors. IL-12 treatment of IL-12R⁺ neoplastic cells isolated from primary adenocarcinoma (n = 6) inhibited angiogenesis in vitro through down-regulation of different pro-angiogenic genes (e.g. IL-6, VEGF-C, VEGF-D, and laminin-5), as assessed by chorioallantoic membrane (CAM) assay and PCR array. In order to perform in vivo studies, the Calu6 NSCLC cell line was transfected with the IL-12RB2 containing plasmid (Calu6/β2). Similar to that observed in primary tumors, IL-12 treatment of Calu6/β2⁺ cells inhibited angiogenesis in vitro. Tumors formed by Calu6/β2 cells in SCID/NOD mice, inoculated subcutaneously or orthotopically, were significantly smaller following IL-12 vs PBS treatment due to inhibition of angiogenesis, and of IL-6 and VEGF-C production. Explanted tumors were studied by histology, immuno-histochemistry and PCR array. NBEC cells were isolated and cultured from lung specimens of non neoplastic origin. NBEC expressed IL-12R and released constitutively tumor promoting cytokines (e.g. IL-6 and CCL2). Treatment of NBEC with IL-12 down-regulated production of these cytokines.

Conclusions

This study demonstrates that IL-12 inhibits directly the growth of human lung adenocarcinoma and targets the adjacent NBEC. These novel anti-tumor activities of IL-12 add to the well known immune-modulatory properties of the cytokine and may provide a rational basis for the development of a clinical trial.     

Deletion of IL-33R (ST2) Abrogates Resistance to EAE in BALB/C Mice by Enhancing Polarization of APC to Inflammatory Phenotype

The administration of interleukin 33 and deletion of IL-33 receptor, ST2 molecule, affects the induction of autoimmunity in different experimental models of human autoimmune diseases. The aim of this study was to analyze the effect of ST2 deletion on the induction of experimental autoimmune encephalomyelitis (EAE) in resistant BALB/c mice. Mice were immunized with MOG_35–55 peptide or disease was induced by passive transfer of encephalitogenic singenic cells and EAE was clinically and histologically evaluated. Expression of intracellular inflammatory cytokines, markers of activation and chemokine receptors on lymphoid tissue and CNS infiltrating mononuclear cells was analyzed by flow cytometry. We report here that deletion of ST2^−/− molecule abrogates resistance of BALB/c mice to EAE induction based on clinical and histopathological findings. Brain and spinal cord infiltrates of ST2^−/− mice had significantly higher number of CD4⁺ T lymphocytes containing inflammatory cytokines compared to BALB/c WT mice. Adoptive transfer of ST2^−/− primed lymphocytes induced clinical signs of the disease in ST2^−/− as well as in WT mice. MOG_35–55 restimulated ST2^−/− CD4⁺ cells as well as ex vivo analyzed lymph node cells had higher expression of T-bet and IL-17, IFN-γ, TNF-α and GM-CSF in comparison with WT CD4⁺ cells. ST2^−/− mice had higher percentages of CD4⁺ cells expressing chemokine receptors important for migration to CNS in comparison with WT CD4⁺ cells. Draining lymph nodes of ST2^−/− mice contained higher percentage of CD11c⁺CD11b⁺CD8⁻ cells containing inflammatory cytokines IL-6 and IL-12 with higher expression of activation markers. Transfer of ST2^−/− but not WT dendritic cells induced EAE in MOG_35–55 immunized WT mice. Our results indicate that ST2 deficiency attenuates inherent resistance of BALB/c mice to EAE induction by enhancing differentiation of proinflammatory antigen presenting cells and consecutive differentiation of encephalitogenic T cells in the draining lymph node rather than affecting their action in the target tissue.     

Deletion of IL-4 Receptor Alpha on Dendritic Cells Renders BALB/c Mice Hypersusceptible to Leishmania major Infection

In BALB/c mice, susceptibility to infection with the intracellular parasite Leishmania major is driven largely by the development of T helper 2 (Th2) responses and the production of interleukin (IL)-4 and IL-13, which share a common receptor subunit, the IL-4 receptor alpha chain (IL-4Rα). While IL-4 is the main inducer of Th2 responses, paradoxically, it has been shown that exogenously administered IL-4 can promote dendritic cell (DC) IL-12 production and enhance Th1 development if given early during infection. To further investigate the relevance of biological quantities of IL-4 acting on DCs during in vivo infection, DC specific IL-4Rα deficient (CD11c^creIL-4Rα^-/lox) BALB/c mice were generated by gene targeting and site-specific recombination using the cre/loxP system under control of the cd11c locus. DNA, protein, and functional characterization showed abrogated IL-4Rα expression on dendritic cells and alveolar macrophages in CD11c^creIL-4Rα^-/lox mice. Following infection with L. major, CD11c^creIL-4Rα^-/lox mice became hypersusceptible to disease, presenting earlier and increased footpad swelling, necrosis and parasite burdens, upregulated Th2 cytokine responses and increased type 2 antibody production as well as impaired classical activation of macrophages. Hypersusceptibility in CD11c^creIL-4Rα^-/lox mice was accompanied by a striking increase in parasite burdens in peripheral organs such as the spleen, liver, and even the brain. DCs showed increased parasite loads in CD11c^creIL-4Rα^-/lox mice and reduced iNOS production. IL-4Rα-deficient DCs produced reduced IL-12 but increased IL-10 due to impaired DC instruction, with increased mRNA expression of IL-23p19 and activin A, cytokines previously implicated in promoting Th2 responses. Together, these data demonstrate that abrogation of IL-4Rα signaling on DCs is severely detrimental to the host, leading to rapid disease progression, and increased survival of parasites in infected DCs due to reduced killing effector functions.     

Long-Term Dietary Supplementation of Pomegranates,Figs and Dates Alleviate Neuroinflammation in a Transgenic Mouse Model of Alzheimer’s Disease

Alzheimer’s disease (AD) is a devastating age-related neurodegenerative disease with no specific treatment at present. The APPsw/Tg2576 mice exhibit age-related deterioration in memory and learning as well as amyloid-beta (Aβ) accumulation, and this mouse strain is considered an effective model for studying the mechanism of accelerated brain aging and senescence. The present study was aimed to investigate the beneficial effects of dietary supplements pomegranate, figs, or the dates on suppressing inflammatory cytokines in APPsw/Tg2576 mice. Changes in the plasma cytokines and Aβ, ATP, and inflammatory cytokines were investigated in the brain of transgenic mice. Significantly enhanced levels of inflammatory cytokines IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, TNF-α and Eotaxin activity were decreased by administration of the diet supplements containing pomegranates, figs, or dates. In addition, putative delays in the formation of senile plaques, as indicated by a decreasing tendency of brain Aβ1–40 and Aβ1–42 contents, were observed. Thus, novel results mediated by reducing inflammatory cytokines during aging may represent one mechanism by which these supplements exert their beneficial effects against neurodegenerative diseases such as AD.     

An Evaluation of the Effects of Cytokines on Intracellular Oxidative Production in Normal Neutrophils by Flow Cytometry

To evaluate the effects of inflammatory cytokines on oxidative production in normal neutrophils, seven kinds of cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colonystimulating factor (G-CSF), interleukin-2 (IL-2), IL-6, IL-1α IL-1β, and interferon-β (IFN-β) were tested. The intracellular hydrogen peroxide (H₂O₂) in individual cells was determined by flow cytometry. According to the levels of intracellular H₂O₂ enhanced by cytokines, these seven cytokines were classified into three types: (1) prominently effective—GM-CSF; (2) moderately effective—G-CSF, IL-6, and IL-2; (3) weakly or ineffective—IFN-β, IL-1α, and IL-1β. Changes in cell size and cell surface structure after stimulation of those seven cytokines were simultaneously measured by flow cytometry. The most prominently effective cytokine, GM-CSF, initially caused enlargement of cell size and irregularity of the cell surface and subsequently increased H₂O₂ production by neutrophils. In contrast, the weakly or ineffective cytokines, like IL-1β, had no effects on cell size or cell surface. Our study indicates that some kinds of cytokines enhance oxidetive production and cause morphological changes in neutrophils.     

Elevation of IL-6 in the allergic asthmatic airway is independent of inflammation but associates with loss of central airway function

Background

Asthma is a chronic inflammatory disease of the airway that is characterized by a Th2-type of immune response with increasing evidence for involvement of Th17 cells. The role of IL-6 in promoting effector T cell subsets suggest that IL-6 may play a functional role in asthma. Classically IL-6 has been viewed as an inflammatory marker, along with TNFα and IL-1β, rather than as regulatory cytokine.

Objective

To investigate the potential relationship between IL-6 and other proinflammatory cytokines, Th2/Th17 cytokines and lung function in allergic asthma, and thus evaluate the potential role of IL-6 in this disease.

Methods

Cytokine levels in induced sputum and lung function were measured in 16 healthy control and 18 mild-moderate allergic asthmatic subjects.

Results

The levels of the proinflammatory biomarkers TNFα and IL-1β were not different between the control and asthmatic group. In contrast, IL-6 levels were specifically elevated in asthmatic subjects compared with healthy controls (p < 0.01). Hierarchical regression analysis in the total study cohort indicates that the relationship between asthma and lung function could be mediated by IL-6. Among Th2 cytokines only IL-13 (p < 0.05) was also elevated in the asthmatic group, and positively correlated with IL-6 levels (r_S = 0.53, p < 0.05).

Conclusions

In mild-moderate asthma, IL-6 dissociates from other proinflammatory biomarkers, but correlates with IL-13 levels. Furthermore, IL-6 may contribute to impaired lung function in allergic asthma.     

IL-4 Receptor-Alpha-Dependent Control of Cryptococcus neoformans in the Early Phase of Pulmonary Infection

Cryptococcus neoformans is an opportunistic fungal pathogen that causes lung inflammation and meningoencephalitis in immunocompromised people. Previously we showed that mice succumb to intranasal infection by induction of pulmonary interleukin (IL)-4Rα–dependent type 2 immune responses, whereas IL-12-dependent type 1 responses confer resistance. In the experiments presented here, IL-4Rα^−/− mice unexpectedly show decreased fungal control early upon infection with C. neoformans, whereas wild-type mice are able to control fungal growth accompanied by enhanced macrophage and dendritic cell recruitment to the site of infection. Lower pulmonary recruitment of macrophages and dendritic cells in IL-4Rα^−/− mice is associated with reduced pulmonary expression of CCL2 and CCL20 chemokines. Moreover, IFN-γ and nitric oxide production are diminished in IL-4Rα^−/− mice compared to wild-type mice. To directly study the potential mechanism(s) responsible for reduced production of IFN-γ, conventional dendritic cells were stimulated with C. neoformans in the presence of IL-4 which results in increased IL-12 production and reduced IL-10 production. Together, a beneficial role of early IL-4Rα signaling is demonstrated in pulmonary cryptococcosis, which contrasts with the well-known IL-4Rα-mediated detrimental effects in the late phase.     

Estradiol Enhances CD4+ T-Cell Anti-Viral Immunity by Priming Vaginal DCs to Induce Th17 Responses via an IL-1-Dependent Pathway

Clinical and experimental studies have shown that estradiol (E2) confers protection against HIV and other sexually transmitted infections. Here, we investigated the underlying mechanism. Better protection in E2-treated mice, immunized against genital HSV-2, coincided with earlier recruitment and higher proportions of T_h1 and T_h17 effector cells in the vagina post-challenge, compared to placebo-treated controls. Vaginal APCs isolated from E2-treated mice induced 10-fold higher T_h17 and T_h1 responses, compared to APCs from progesterone-treated, placebo-treated, and estradiol-receptor knockout mice in APC-T cell co-cultures. CD11c⁺ DCs in the vagina were the predominant APC population responsible for priming these T_h17 responses, and a potent source of IL-6 and IL-1β, important factors for T_h17 differentiation. T_h17 responses were abrogated in APC-T cell co-cultures containing IL-1β KO, but not IL-6 KO vaginal DCs, showing that IL-1β is a critical factor for T_h17 induction in the genital tract. E2 treatment in vivo directly induced high expression of IL-1β in vaginal DCs, and addition of IL-1β restored T_h17 induction by IL-1β KO APCs in co-cultures. Finally, we examined the role of IL-17 in anti-HSV-2 memory T cell responses. IL-17 KO mice were more susceptible to intravaginal HSV-2 challenge, compared to WT controls, and vaginal DCs from these mice were defective at priming efficient T_h1 responses in vitro, indicating that IL-17 is important for the generation of efficient anti-viral memory responses. We conclude that the genital mucosa has a unique microenvironment whereby E2 enhances CD4⁺ T cell anti-viral immunity by priming vaginal DCs to induce T_h17 responses through an IL-1-dependent pathway.     

IL-17A Modulates Oxidant Stress-Induced Airway Hyperresponsiveness but Not Emphysema

IL-17A induces the release of pro-inflammatory cytokines and of reactive oxygen species which could lead to neutrophilic inflammation. We determined the role of IL-17 receptor (IL-17R) signalling in oxidant-induced lung emphysema and airway hyperresponsiveness. IL-17R^−/− and wild-type C57/BL6 mice were exposed to ozone (3 ppm; 3 hours) for 12 times over 6 weeks. Bronchial responsiveness to acetylcholine was measured, and lungs were retrieved. Mean linear intercept (L_m) and isometric contractile responses of intrapulmonary airways to acetylcholine were determined. In wild-type mice but not in IL-17R^−/−, chronic ozone exposure caused airway hyperresponsiveness. The increase in L_m after chronic ozone exposure of wild-type mice was also observed in IL-17R^−/− mice. The increased maximal contractile response to acetylcholine seen in airways of wild-type mice exposed to ozone was abolished in IL-17R^−/− mice. p38-mitogen-activated protein kinase (MAPK) and dexamethasone-dependent increase in contractile response was reduced in airways from IL-17R^−/− ozone-exposed mice. Lung inflammation scores were not altered in IL-17R^−/− mice exposed to ozone compared to wild-type mice. The increased release of IL-17 and IL-1β, and the activation of p38 MAPK in the lungs of ozone-exposed mice was reduced in IL-17R^−/− mice. IL-17R signalling underlies the increase in airway hyperresponsiveness seen after ozone exposure, mediated by the increased contractility of airway smooth muscle. The emphysema and lung inflammation induced by ozone is not dependent on IL-17.     

Prostaglandin E2 Production and T Cell Function in Mouse Adenovirus Type 1 Infection following Allogeneic Bone Marrow Transplantation

Adenovirus infections are important complications of bone marrow transplantation (BMT). We demonstrate delayed clearance of mouse adenovirus type 1 (MAV-1) from lungs of mice following allogeneic BMT. Virus-induced prostaglandin E₂ (PGE₂) production was greater in BMT mice than in untransplanted controls, but BMT using PGE₂-deficient donors or recipients failed to improve viral clearance, and treatment of untransplanted mice with the PGE₂ analog misoprostol did not affect virus clearance. Lymphocyte recruitment to the lungs was not significantly affected by BMT. Intracellular cytokine staining of lung lymphocytes demonstrated impaired production of INF-γ and granzyme B by cells from BMT mice, and production of IFN-γ, IL-2, IL-4, and IL-17 following ex vivo stimulation was impaired in lymphocytes obtained from lungs of BMT mice. Viral clearance was not delayed in untransplanted INF-γ-deficient mice, suggesting that delayed viral clearance in BMT mice was not a direct consequence of impaired IFN-γ production. However, lung viral loads were higher in untransplanted CD8-deficient mice than in controls, suggesting that delayed MAV-1 clearance in BMT mice is due to defective CD8 T cell function. We did not detect significant induction of IFN-β expression in lungs of BMT mice or untransplanted controls, and viral clearance was not delayed in untransplanted type I IFN-unresponsive mice. We conclude that PGE₂ overproduction in BMT mice is not directly responsible for delayed viral clearance. PGE₂-independent effects on CD8 T cell function likely contribute to the inability of BMT mice to clear MAV-1 from the lungs.     

IL-21R Signaling Suppresses IL-17+ Gamma Delta T Cell Responses and Production of IL-17 Related Cytokines in the Lung at Steady State and After Influenza A Virus Infection

Influenza A virus (IAV) infection of the respiratory tract elicits a robust immune response, which is required for efficient virus clearance but at the same time can contribute to lung damage and enhanced morbidity. IL-21 is a member of the type I cytokine family and has many different immune-modulatory functions during acute and chronic virus infections, although its role in IAV infection has not been fully evaluated. In this report we evaluated the contributions of IL-21/IL-21 receptor (IL-21R) signaling to host defense in a mouse model of primary IAV infection using IL-21R knock out (KO) mice. We found that lack of IL-21R signaling had no significant impact on virus clearance, adaptive T cell responses, or myeloid cell accumulations in the respiratory tract. However, a subset of inflammatory cytokines were elevated in the bronchoalveolar lavage fluid of IL-21R KO mice, including IL-17. Although there was only a small increase in Th17 cells in the lungs of IL-21R KO mice, we observed a dramatic increase in gamma delta (γδ) T cells capable of producing IL-17 both after IAV infection and at steady state in the respiratory tract. Finally, we found that IL-21R signaling suppressed the accumulation of IL-17⁺ γδ T cells in the respiratory tract intrinsically. Thus, our study reveals a previously unrecognized role of IL-21R signaling in regulating IL-17 production by γδ T cells.     

Salmonella Induced IL-23 and IL-1β Allow for IL-12 Production by Monocytes and M?1 through Induction of IFN-γ in CD56+ NK/NK-Like T Cells

Background

The type-1 cytokine pathway plays a pivotal role in immunity against intracellular bacterial pathogens such as Salmonellae and Mycobacteria. Bacterial stimulation of pattern recognition receptors on monocytes, macrophages and dendritic cells initiates this pathway, and results in the production of cytokines that activate lymphocytes to produce interferon (IFN)-γ. Interleukin (IL)-12 and IL-23 are thought to be the key cytokines required for initiating a type-1 cytokine immune response to Mycobacteria and Salmonellae. The relative contribution of IL-23 and IL-12 to this process is uncertain.

Methodology/Principal Findings

We show that various TLR agonists induce the production of IL-23 but not IL-12 in freshly isolated human monocytes and cultured human macrophages. In addition, type 1 pro-inflammatory macrophages (Mϕ1) differentiated in the presence of GM-CSF and infected with live Salmonella produce IL-23, IL-1β and IL-18, but not IL-12. Supernatants of Salmonella-infected Mϕ1 contained more IL-18 and IL-1β as compared with supernatants of Mϕ1 stimulated with isolated TLR agonists, and induced IFN-γ production in human CD56⁺ cells in an IL-23 and IL-1β-dependent but IL-12-independent manner. In addition, IL-23 together with IL-18 or IL-1β led to the production of GM-CSF in CD56⁺ cells. Both IFN-γ and GM-CSF enhanced IL-23 production by monocytes in response to TLR agonists, as well as induced IL-12 production.

Conclusions/Significance

The findings implicate a positive feedback loop in which IL-23 can enhance its release via induction of IFN-γ and GM-CSF. The IL-23 induced cytokines allow for the subsequent production of IL-12 and amplify the IFN-γ production in the type-1 cytokine pathway.