Nod2 Mutation Enhances NF-kappaB Activity and Bacterial Killing Activity of Macrophages

NOD2, an intracellular sensor of bacteria, are linked to increased susceptibility to bacteria in Crohn’s disease (CD). The NOD2 protein is expressed mainly by macrophages and dendritic cells. This study is to examine the role of NOD2 in the innate response of macrophages to bacterial challenge. First, peritoneal macrophages and alveolar macrophages were harvested from WT, Nod2^2939iC, as well as TLR4^−/− mice and incubated with E. coli or P. aeruginosa. Bacterial killing activity; IL-1β and TLR4 protein expression; NF-κB DNA binding activity assay; as well as IL-1β, TNFα, TLR2, TLR4 and TLR9 mRNA expression of macrophages were examined. We found that alveolar macrophages and peritoneal macrophages of Nod2^2939iC mice but not WT mice or TLR4^−/− mice demonstrated a significant increase of E. coli killing activity. Bacterial challenge also induced a significant increase of pro-IL-1β protein expression; NF-κB DNA binding activity; as well as IL-1β and TNFα mRNA expression of the peritoneal macrophages in Nod2^2939iC mice. Collectively, the increase of bacterial killing activity, IL-1β expression, and NF-κB DNA binding activity of macrophages in Nod2^2939iC mice suggests that NOD2 is a positive regulator of NF-κB/IL-1β-mediated innate response to bacteria challenge in Crohn’s disease.     

Type I Interferons Attenuate T Cell Activating Functions of Human Mast Cells by Decreasing TNF-α Production and OX40 Ligand Expression While Increasing IL-10 Production

Recent studies have demonstrated that mast cells not only mediate inflammatory reactions in type I allergy but also play an important role in adaptive immunity. In the present study, we investigated the effects of interferon-α, which shares the same receptor as IFN-β, on human cord blood-derived mast cells. Mast cells produced TNF-α, and IL-10, and expressed OX40 ligand upon activation by crosslinking of FcɛRI. When treated with interferon-α, TNF-α production was decreased while IL-10 and TGF-β productions were increased. Furthermore, flow cytometric analysis revealed that interferon-α downregulated expression OX40 ligand on mast cells which is crucial for mast cell-T cell interaction. We confirmed that the viability of mast cells was not affected by interferon-α treatment. Accordingly, interferon-α-treated mast cells induced lower levels of CD4⁺ T cell proliferation compared with those without interferon-α treatment. These results suggest that type I interferons suppress T cell immune responses through their regulatory effects on mast cells.     

Lipoteichoic acid improves the capability of mast cells in the host defense system against bacteria

Objectives and design

We investigated the effects of microbial components on the uptake of microbes by mast cells (MCs), and studied the change in cytokine production in MCs after bacterial uptake.

Material or subjects

LAD2 human mast cells, cord-blood and peripheral-blood derived MCs were employed to analyze their surface molecule expression and cytokine generation by flow cytometry. Bacterial internalization in these MCs was observed by confocal microscopy and flow cytometry.

Results

Complement receptor 3 expression was augmented by LTA but not by PGN or 3CpG-oligodeoxynucleotide. LTA also enhanced the uptake of opsonized bacteria (over twofold augmentation). After bacterial uptake, MCs augmented the production of chemoattractant cytokines for neutrophils, while Th1 and Th2 cytokine production showed little or no change.

Conclusions

LTA increases the capability of the MC as a sentinel in the host immune response, and some bacterial components direct human MC function towards innate immunity after pathogen infection.     

Comparison of Pulmonary Inflammatory and Antioxidant Responses to Intranasal Live and Heat-Killed Streptococcus pneumoniae in Mice

Inflammatory and antioxidant responses, in male C57Bl6J mice, to single intranasal inoculations with live or heat-killed Streptococcus pneumoniae were studied in order to tease out differences in responses. Heat-killed bacteria elicited weak lung neutrophil infiltration and raised concentrations (peak 6–8 h), in serum or lung tissue, of CXCL1 and 2, tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), and granulocyte-macrophage-colony stimulating factor, with later increases in CCL2 and IL-1β. Live bacteria induced profound pulmonary neutrophil infiltration and acute chemokine/cytokine elevations. After 72–96 h, live S. pneumoniae induced a delayed rise in chemokines CXCL2 and CCL2, preceded by increases in TNFα, IL-1β, and IL-6 and mononuclear infiltration of lungs. With both live and heat-killed bacteria, alveolar epithelial type II cells and alveolar macrophages were the main sources of TNFα and IL-1β. Only live bacteria caused an acute decrease in lung glutathione peroxidase, an increase in superoxide dismutase, and a sustained increase in serum amyloid protein A. Acute innate immune responses to live and heat-killed S. pneumoniae are similar. In response to live bacteria, inflammation is greater, accompanied by changes in antioxidant enzymes and has an additional, later mononuclear component.     

IL-9 Contributes to Immunosuppression Mediated by Regulatory T Cells and Mast Cells in B-Cell Non-Hodgkin’s Lymphoma

It has been known that regulatory T (Treg) cells and mast cells (MCs) are involved in tumor immunity regulation, but the exact roles and mechanisms of Treg cells and MCs in B-cell non-Hodgkin’s lymphoma (NHL) are incompletely defined. In the present study, we found that the number of Foxp3⁺ Treg cells and CD117⁺ MCs increased in B-cell NHL patients. Concomitantly, a high level of interleukin (IL)-9 was observed in the sera from B-cell NHL patients. Neutralizing IL-9 significantly inhibited tumor growth in the lymphoma model of murine, and this process was associated with down-regulation of Treg cells and MCs. Furthermore, IL-9 was also demonstrated to induce expression of MC-related genes and proliferation of MCs from the bone marrow stem cells. Collectively, our results indicate that Treg cell and MCs are involved in immunosuppression in B-cell NHL, and IL-9 is a key mediator of Treg cells and MCs in that process. These findings provide novel insight for the pathogenesis and possible therapeutic strategy of B-cell NHL.     

Opiate abuse, innate immunity, and bacterial infectious diseases

The first line of defense against invading bacteria is provided by the innate immune system. Morphine and other opiates can immediately disrupt the body’s first line of defense against harmful external bacteria. Opiate, for example morphine, abuse degrades physical and physiologic barriers, and modulates phagocytic cells (macrophages, neutrophils) and, nonspecific cytotoxic T cells (γδ T), natural killer cells, and dendritic cells, that are functionally important for carrying out a rapid immune reaction to invading pathogens. In vitro studies with innate immune cells from experimental animals and humans and in vivo studies with animal models have shown that opiate abuse impairs innate immunity and is responsible for increased susceptibility to bacterial infection. However, to better understand the complex interactions between opiates, innate immunity, and bacterial infection and develop novel approaches to treat and even prevent bacterial infection in the opiate-abuse population, there is an urgent need to fill the numerous gaps in our understanding of the cellular and molecular mechanisms by which opiate abuse increases susceptibility to bacterial infection. Received: 2008.05.23, Accepted: 2008.07.28     

Low interleukin-10 production is associated with diabetes in HIV-infected patients undergoing antiviral therapy

Reduced interleukin-10 (IL-10) production is associated with type 2 diabetes in elderly individuals. Antiviral therapy (ARV)-induced immune modulation results in diminished IL-10 production, and diabetes can be observed in ARV-treated human immunodeficiency virus (HIV)-infected individuals. We analyzed, in a cross-sectional pilot study, HIV-antigen-stimulated IL-10 and tumor necrosis factor alpha (TNFα) production, and intracellular concentration (ICC), as well as B7-H1 expression, a marker preferentially presented by IL-10-producing cells, in 20 ARV-treated individuals in whom diabetes did (n=10; diabetes mellitus, DM) or did not (n=10; controls) develop. Pre-ARV glucose, cholesterol, and triglycerides levels, duration of HIV infection and of therapy, exposure to protease inhibitors (PI), HIV plasma viremia, CD4 counts, and nadir were similar in DM and control patients. Results showed that: (1) IL-10 production was lower; (2) IL-10 ICC was reduced; (3) B7-H1-expressing CD19+ cells were diminished; and (4) TNFα production and ICC by CD4+ T cells was augmented in DM patients. Development of diabetes in HIV infected, ARV-treated individuals could be a response to therapy. Similar to what is observed in elderly individuals, low IL-10 production is associated with diabetes in antiviral-treated HIV infection. Further studies will be necessary to clarify whether low IL-10 is a risk factor for, or a consequence of, diabetes.     

Etanercept Impairs Maturation of Human Monocyte-Derived Dendritic Cells by Inhibiting the Autocrine TNFα-mediated Signaling

The success of anti-tumor necrosis factor alpha (TNFα) therapies has led to increased interest as to the mechanisms and consequences of TNFα blockade. The aim of the study was to examine the effects of TNFα blockade by etanercept on lipopolysaccharide (LPS) or peptidoglycan (PG)-induced maturation of human monocyte-derived dendritic cells (MDDCs). MDDCs grown from peripheral blood of healthy donors were stimulated by LPS or PG with/without the presence of etanercept. Concentrations of TNFα in cell supernatants were assessed by ELISA, while the cells were stained with monoclonal antibodies to CD83, CD80, CD86, CD11c, CD40, HLA-DR, and annexin-V and acquired using a flow cytometer. Etanercept significantly decreased the stimulated cell surface expression of HLA-DR, CD80, CD86, CD40 and CD83 on MDDCs in all examined samples. Etanercept in the same dose, but denatured to loss of specificity for TNFα, failed to change any of the aforementioned markers. In the presence of etanercept, concentrations of TNFα in cell supernatants were decreased by 53% on average, with a range of 25%–87%. Etanercept impaired the stimulated maturation of MDDCs by neutralizing the induced TNFα, produced by the same MDDCs after antigenic stimulation. The reported data confirms that TNFα blockade may have a direct effect on DCs, with a wide spectrum of potential secondary effects downstream. The data also suggests the presence of TNFα-mediated autocrine signaling, serving to accelerate or catalyze the maturation process of MDDCs.     

Does interleukin-1 mediate tumour necrosis factor alpha-induced fever in rabbits?

We investigated the humoral mechanisms involved in tumour necrosis factor alpha (TNFα)-induced fever in rabbits. No change in lymphocyte-activating factor activity was detected in serum drawn during TNFα-induced fever. The pyrogenic activity of recombinant rabbit interleukin-1β (IL-1β) was entirely abolished by pre-incubation with anti-IL-1β antiserum from the goat. Fever induced by intravenous (i.v.) injection of IL-1β was significantly diminished by i.v. infusion of the antiserum. However, i.v. infusion of the antiserum for 1 h did not affect fever induced by i.v. injection of TNFα, when the antiserum infusion began either simultaneously with, or 2 h after, the injection of TNFα. Furthermore, intracerebroventricular injection of the antiserum did not affect TNFα-induced fever. The intracerebroventricular administration of naloxone (an opioid receptor antagonist) significantly diminished TNFα-induced fever. The results suggest that IL-1, both in the blood circulation and in the brain, may not be involved in TNFα-induced fever. Similar to the contribution of eicosanoids, the opioid system in the brain seems some-how to contribute to the mechanism of the development of fever induced by TNFα in rabbits.     

Virtual memory CD8 T cells expanded by helminth infection confer broad protection against bacterial infection

Epidemiological data and animal studies suggest that helminth infection exerts potent immunomodulatory effects that dampen host immunity against unrelated pathogens. Despite this notion, we unexpectedly discovered that prior helminth infection resulted in enhanced protection against subsequent systemic and enteric bacterial infection. A population of virtual memory CD8 T (CD8 T_VM) cells underwent marked expansion upon infection with the helminth Heligmosomoides polygurus by an IL-4-regulated, antigen-independent mechanism. CD8 T_VM cells disseminated to secondary lymphoid organs and established a major population of the systemic CD8 T cell pool. IL-4 production elicited by protein immunization or selective activation of natural killer T cells also results in the expansion of CD8 T_VM cells. Notably, CD8 T_VM cells expanded by helminth infection are sufficient to transfer innate non-cognate protection against bacteria to naïve animals. This innate non-cognate “collateral protection” mediated by CD8 T_VM might provide parasitized animals an advantage against subsequent unrelated infections, and represents a potential novel strategy for vaccination.     

Neutrophil Migration Induced by IL-1β Depends upon LTB4 Released by Macrophages and upon TNF-α and IL-1β Released by Mast Cells

In the present study, we investigate whether mast cells and macrophages are involved in the control of IL-1β-induced neutrophil migration, as well as the participation of chemotactic mediators. IL-1β induced a dose-dependent neutrophil migration to the peritoneal cavity of rats which depends on LTB₄, PAF and cytokines, since the animal treatment with inhibitors of these mediators (MK 886, PCA 4248 and dexamethasone respectively) inhibited IL-1β-induced neutrophil migration. The neutrophil migration induced by IL-1β is dependent on mast cells and macrophages, since depletion of mast cells reduced the process whereas the increase of macrophage population enhanced the migration. Moreover, mast cells or macrophages stimulated with IL-1β released a neutrophil chemotactic factor, which mimicked the neutrophil migration induced by IL-1β. The chemotactic activity of the supernatant of IL-1β-stimulated macrophages is due to the presence of LTB₄, since MK 886 inhibited its release. Moreover, the chemotactic activity of IL-1β-stimulated mast cells supernatant is due to the presence of IL-1β and TNF-α, since antibodies against these cytokines inhibited its activity. Furthermore, significant amounts of these cytokines were detected in the supernatant. In conclusion, our results suggest that neutrophil migration induced by IL-1β depends upon LTB₄ released by macrophages and upon IL-1β and TNFα released by mast cells.     

Anti-tumoral capabilities of effector cells after IFN-α or CpG-motif treatment of cocultured dendritic cells

Introduction Ex vivo expansion of monocyte-derived dendritic cells (mDCs) and subsequent coculture with autologous cytokine-induced killer (CIK) cells is an established system to create specific and non-specific anti-tumoral immunity. mDCs constitute the most frequently applied DC subset in clinical studies. One recently published approach to optimize the immunological functions of the DC/CIK cell system is the replacement of interleukin (IL)-4 by interferon (IFN)-α in the maturation process of the DCs. Materials and Methods The expressions of relevant surface antigens of IL-4-DCs and IFNα-DCs by flow cytometry and the anti-tumoral activation of effector cells cocultured with both types of DCs using cytotoxicity assays were compared. In addition, short-term coculture experiments with both types of DCs and IFNγ-LAK effector cells were performed and compared with standard CIK cell coculture experiments. Results Regarding the expressions of functionally relevant surface markers, no differences could be detected for CD80, CD83, and HLA-DR between IFNα-DCs and IL-4-DCs, whereas the mean fluorescence intensities of CD40, CD86, CD54, and HLA-ABC were decreased and the expression of CD14 was increased for IFNγ-DCs. Moreover, no enhancement of cytotoxicity of cocultured CIK cells against tumor cell lines (A498 and SW480) was detected by the use of IFNα-DCs. Additionally, coculture experiments with IFNγ-LAK cells were performed and unexpectedly higher lysis rates in comparison with the established IL-4-DC/CIK coculture model was observed. Early incubation of the mDCs with several CpG-ODNs failed to increase the anti-tumoral cytotoxicity of the cocultured IFNγ-LAK cells. Conclusions These results demonstrate that in the mDC/CIK cell system, IFNα-DCs are not superior in inducing anti-tumoral cytotoxicity and even moderately inferior regarding the expression of functionally relevant surface markers compared with IL-4-DCs. Michael Erhardt and Ingo G. H. Schmidt-Wolf contributed equally.     

Bacterial components regulate the expression of Toll-like receptor 4 on human mast cells

Objectives and design: The aim of this study was to investigate whether the exposure of mast cells (MCs) to bacterial components affects the expression of Toll-like receptor (TLR) 4, and to elucidate the behavior of MCs during the early response to infection. Materials: Two human MC lines, HMC-1 and LAD2, were employed. Messenger RNA expression was observed by RT and real-time PCR. TLR4 expression was determined by Western blotting. TNF-α secretion was analyzed with ELISA. The degranulation ratio was measured with betahexosaminidase assay. Results: Although bacterial components increased TLR4 mRNA, only lipopolysaccharide (LPS) augmented the TLR4 protein expression. LAD2 pre-treated with LPS for 8 h resulted in 2-fold increased TNF-α secretion on LPS restimulation. Conclusion: These results suggest that the exposure of MCs to LPS may reinforce the innate immune system due to up-regulation of MC TLR4, followed by increased TNF-α release. Received 20 April 2006; returned for revision 14 July 2006; accepted by G. Wallace 11 August 2006     

CD28 and IL-4: two heavyweights controlling the balance between immunity and inflammation

The costimulatory receptor CD28 and IL-4Rα-containing cytokine receptors play key roles in controlling the size and quality of pathogen-specific immune responses. Thus, CD28-mediated costimulation is needed for effective primary T-cell expansion and for the generation and activation of regulatory T-cells (Treg cells), which protect from immunopathology. Similarly, IL-4Rα signals are required for alternative activation of macrophages, which counteract inflammation by type 1 responses. Furthermore, immune modulation by CD28 and IL-4 is interconnected through the promotion of IL-4 producing T-helper 2 cells by CD28 signals. Using conditionally IL-4Rα and CD28 deleting mice, as well as monoclonal antibodies, which block or stimulate CD28, or mAb that deplete Treg cells, we have studied the roles of CD28 and IL-4Rα in experimental mouse models of virus (influenza), intracellular bacteria (L. monocytogenes, M. tuberculosis), and parasite infections (T. congolense, L. major). We observed that in some, but not all settings, Treg cells and type 2 immune deviation, including activation of alternative macrophages can be manipulated to protect the host either from infection or from immunopathology with an overall beneficial outcome. Furthermore, we provide direct evidence that secondary CD8 T-cell responses to i.c. bacteria are dependent on CD28-mediated costimulation.     

Mast cells: beyond IgE

Mast cells, historically known for their involvement in type I hypersensitivity, also serve critical protective and homeostatic functions. They directly recognize the products of bacterial infection through several surface receptor proteins, releasing proteases, cytokines, and eicosanoid mediators that recruit neutrophils, limit the spread of bacterial infection, and facilitate subsequent tissue repair. In vitro studies suggest that the spectrum of microbes capable of initiating mast cell activation is broad and extends to common respiratory viruses, mycoplasma, and even products of tissue injury, such as nucleotides. TH2-polarized inflammation elicits a reactive hyperplasia of mast cells at the involved mucosal surfaces in both mice and human subject. Several recombinant TH2 cytokines (IL-3, IL-4, IL-5, and IL-9) act synergistically with stem cell factor to facilitate proliferation of nontransformed human mast cells in vitro. IL-4 induces the expression of critical inflammation-associated genes by human mast cells, such as those encoding leukotriene C4 synthase, Fc(epsilon)RI, and several cytokines. Consequently, priming with IL-4 not only amplifies classical Fc(epsilon)RI-dependent mast cell activation but also dramatically alters the product profile of mast cells activated by innate signals and by chemical mediators of inflammation. Strikingly, IL-4 induces an activation response by mast cells to cysteinyl leukotrienes, which act through a receptor shared with uridine diphosphate to induce cytokine generation without exocytosis. It Is possible that alterations in mast cell phenotype by the TH2 milieu of allergy permits otherwise trivial infections or homeostatic chemical signals to initiate harmful inflammatory cascades and sustain tissue pathology. Drug development must take these nonclassical mast cell activation pathways into account without compromising the beneficial and protective functions of mast cells.     

The role of mast cells in autoinflammation

The concept of autoinflammation was proposed to define a new class of immune disorders categorized by self-directed inflammation that is driven via activation of innate immune pathways. Within innate immunity, inflammasomes serve as intracellular signaling platforms to endogenous danger molecules and pathogens. Their key function is the cleavage of pro-interleukin-1β (pro-IL-1β) into its active form to promote inflammation and programmed cell death. A growing number of inflammasome sensors were described, among which NLR family pyrin domain containing 3 (NLRP3) is the best-studied sensor. Besides macrophages, monocytes, and other innate immune cells, mast cells (MCs) were shown to express functional inflammasomes too. Also, MCs are both, a source and target of IL-1β. Here we review the functional relevance and role of MC inflammasomes and MC-derived IL-1β in contributing to the inflammation at the skin, joints, and central nervous system in rare monogenic autoinflammatory conditions and also common inflammatory and degenerative diseases.     

Hemodynamic Regulation of Inflammation at the Endothelial–Neutrophil Interface

Arterial shear stress can regulate endothelial phenotype. The potential for anti-inflammatory effects of shear stress on TNFα-activated endothelium was tested in assays of cytokine expression and neutrophil adhesion. In cultured human aortic endothelial cells (HAEC), arterial shear stress of 10 dyne/cm² blocked by >80% the induction by 5 ng/mL TNFα of interleukin-8 (IL-8) and IL-6 secretion (50 and 90% reduction, respectively, in the presence of nitric oxide synthase antagonism with 200 μM nitro-l-arginine methylester, l-NAME). Exposure of TNFα-stimulated HAEC to arterial shear stress for 5 h also reduced by 60% (p < 0.001) the conversion of neutrophil rolling to firm arrest in a venous flow assay conducted at 1 dyne/cm². Also, neutrophil rolling lengths at 1 dyne/cm² were longer when TNFα-stimulated HAEC were presheared for 5 h at arterial stresses. In experiments with a synthetic promoter that provides luciferase induction to detect cis interactions of glucocorticoid receptor (GR) and NFκB, shear stress caused a marked 40-fold induction of luciferase in TNFα-treated cells, suggesting a role for GR pathways in the anti-inflammatory actions of fluid shear stress. Hemodynamic force exerts anti-inflammatory effects on cytokine-activated endothelium by attenuation of cytokine expression and neutrophil firm arrest.     

CD252 regulates mast cell mediated,CD1d‐restricted NKT‐cell activation in mice

The interaction between tissue‐resident mast cells (MCs) and recruited immune cells contributes to tissue immunosurveillance. However, the cells, mechanisms, and receptors involved in this crosstalk remain ill defined. Invariant natural killer T (iNKT) cells are CD1d‐restricted innate lymphocytes that recognize glycolipid antigens and have emerged as critical players in immunity. Here, we show that primary mouse peritoneal MCs express surface CD1d, which is upregulated in vivo following administration of alpha‐galactosylceramide. In contrast, in BM‐derived MCs CD1d was found to be stored intracellularly and to relocate at the cell surface upon IgE‐mediated degranulation. Activated BM‐derived MCs expressing surface CD1d and loaded with alpha‐galactosylceramide were found to induce iNKT‐cell proliferation and the release of IFN‐γ, IL‐13, and IL‐4 in a CD1d‐restricted manner. Moreover, the costimulatory molecules CD48, CD137L, CD252, CD274, and CD275 affected MC‐induced IFN‐γ release and iNKT‐cell proliferation. Interestingly, among the costimulatory molecules, CD48 and CD252 exhibited a distinctly regulatory activity on iNKT‐cell release of both IFN‐γ and IL‐13. In conclusion, we demonstrate that the crosstalk between MCs and iNKT cells may regulate inflammatory immune responses.     

Directing traffic: IL-17 and IL-22 coordinate pulmonary immune defense

Respiratory infections and diseases are among the leading causes of death worldwide, and effective treatments probably require manipulating the inflammatory response to pathogenic microbes or allergens. Here, we review mechanisms controlling the production and functions of interleukin-17 (IL-17) and IL-22, cytokines that direct several aspects of lung immunity. Innate lymphocytes (γδ T cells, natural killer cells, innate lymphoid cells) are the major source of IL-17 and IL-22 during acute infections, while CD4⁺ T-helper 17 (Th17) cells contribute to vaccine-induced immunity. The characterization of dendritic cell (DC) subsets has revealed their central roles in T-cell activation. CD11b⁺ DCs stimulated with bacteria or fungi secrete IL-1β and IL-23, potent inducers of IL-17 and IL-22. On the other hand, recognition of viruses by plasmacytoid DCs inhibits IL-1β and IL-23 release, increasing susceptibility to bacterial superinfections. IL-17 and IL-22 primarily act on the lung epithelium, inducing antimicrobial proteins and neutrophil chemoattractants. Recent studies found that stimulation of macrophages and DCs with IL-17 also contributes to antibacterial immunity, while IL-22 promotes epithelial proliferation and repair following injury. Chronic diseases such as asthma and chronic obstructive pulmonary disease have been associated with IL-17 and IL-22 responses directed against innocuous antigens. Future studies will evaluate the therapeutic efficacy of targeting the IL-17/IL-22 pathway in pulmonary inflammation.