Increased Cytokines Response in Patients with Tuberculosis Complicated with Chronic Obstructive Pulmonary Disease

Objectives

To explore the change and its significance of cytokines in patients with pulmonary tuberculosis complicated with COPD.

Methods

The immune function of 152 cases of pulmonary tuberculosis with COPD was detected to compare with 150 cases of patients with pulmonary tuberculosis, 157 cases of patients with COPD and 50 cases of healthy volunteers who were in the hospital during the same period. T lymphocyte cell population in peripheral blood was detected by flow cytometry. The serum levels of sIL-2R, IL-6, IFN-γ, TNF-α were measured using ELISA.

Results

The percentage of CD4+ T cells in TB patients with or without COPD and COPD patients without TB was significantly lower than that in control group. The percentage of CD4+ T cells in patients with TB and COPD was significantly lower than that in the non-COPD TB patients. The percentage of CD8+ T cells was higher in the TB patients group than that in control group. The CD4+/CD8+ ratio in the TB patients group was significantly lower than that in control group. The concentrations of sIL-2R, IL-6, TNF-α, IFN-γ in TB patients with or without COPD and COPD patients without TB were significantly higher than those in control group. In addition, sIL-2R, IL-6, TNF-α concentrations in the patients with TB and COPD were higher than those in the non-COPD TB patients. The concentrations of sIL-2R, IL-6, TNF-α, IFN-γ in COPD patients with TB were significantly higher than those in COPD patients without TB. There was a significant negative correlation between serum levels of TNF-α, IL-6 and FEV1 (%, predicted) in COPD without TB group.

Conclusions

The patients with pulmonary tuberculosis complicated with COPD were impaired in cellular immunity, and its extent of immune impairment is more serious than those of the patients with pulmonary tuberculosis and the patients with COPD.     

Respiratory Syncytial Virus Induced Type I IFN Production by pDC Is Regulated by RSV-Infected Airway Epithelial Cells,RSV-Exposed Monocytes and Virus Specific Antibodies

Innate immune responses elicited upon virus exposure are crucial for the effective eradication of viruses, the onset of adaptive immune responses and for establishing proper immune memory. Respiratory syncytial virus (RSV) is responsible for a high disease burden in neonates and immune compromised individuals, causing severe lower respiratory tract infections. During primary infections exuberant innate immune responses may contribute to disease severity. Furthermore, immune memory is often insufficient to protect during RSV re-exposure, which results in frequent symptomatic reinfections. Therefore, identifying the cell types and pattern recognition receptors (PRRs) involved in RSV-specific innate immune responses is necessary to understand incomplete immunity against RSV. We investigated the innate cellular response triggered upon infection of epithelial cells and peripheral blood mononuclear cells. We show that CD14⁺ myeloid cells and epithelial cells are the major source of IL-8 and inflammatory cytokines, IL-6 and TNF-α, when exposed to live RSV Three routes of RSV-induced IFN-α production can be distinguished that depend on the cross-talk of different cell types and the presence or absence of virus specific antibodies, whereby pDC are the ultimate source of IFN-α. RSV-specific antibodies facilitate direct TLR7 access into endosomal compartments, while in the absence of antibodies, infection of monocytes or epithelial cells is necessary to provide an early source of type I interferons, required to engage the IFN-α,β receptor (IFNAR)-mediated pathway of IFN-α production by pDC. However, at high pDC density infection with RSV causes IFN-α production without the need for a second party cell. Our study shows that cellular context and immune status are factors affecting innate immune responses to RSV. These issues should therefore be addressed during the process of vaccine development and other interventions for RSV disease.     

Regulation of Porcine Plasmacytoid Dendritic Cells by Cytokines

Plasmacytoid dendritic cells (pDC) are the most potent producers of type-I interferon (IFN) and represent the main interferon (IFN)-α source in response to many viruses. Considering the important roles played by type I IFN’s, not only as antiviral effectors but also as potent alarming cytokine of the immune system, we investigated how such responses are regulated by various cytokines. To this end, we stimulated enriched pDC in the presence or absence of particular cytokines with a strong activator, CpG DNA, or a weak activator of pDC, foot-and-mouth disease virus (FMDV). Alternatively, we pre-incubated pDC for 16 h before stimulation. The pro-inflammatory cytokines tested Interleukin (IL)-6, IL17A, tumour necrosis factor (TNF)-α did not influence IFN-α responses except TNF-α, which promoted responses induced by FMDV. The haematopoietic cytokines Fms-related tyrosine kinase 3 ligand (Flt3-L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) had enhancing effects on pDC activation at least in one of the protocols tested. IFN-β and IFN-γ were the most potent at enhancing FMDV-induced IFN-α, up to 10-fold. Interestingly, also the Th2 cytokine IL-4 was an efficient promoter of pDC activity, while IL-10 was the only negative regulator of IFN-α in pDC identified. The cytokines enhancing IFN-α responses also promoted pDC survival in cell culture with the exception of GM-CSF. Taken together this work illustrates how the cytokine network can influence pDC activation, a knowledge of relevance for improving vaccines and therapeutic interventions during virus infections, cancers and autoimmune diseases in which pDC play a role.     

Role of aberrant metalloproteinase activity in the pro-inflammatory phenotype of bronchial epithelium in COPD

Background

Cigarette smoke, the major risk factor for COPD, is known to activate matrix metalloproteinases in airway epithelium. We investigated whether metalloproteinases, particularly A Disintegrin and Metalloproteinase (ADAM)17, contribute to increased pro-inflammatory epithelial responses with respect to the release of IL-8 and TGF-α, cytokines implicated in COPD pathogenesis.

Methods

We studied the effects of cigarette smoke extract (CSE) and metalloproteinase inhibitors on TGF-α and IL-8 release in primary bronchial epithelial cells (PBECs) from COPD patients, healthy smokers and non-smokers.

Results

We observed that TGF-α was mainly shed by ADAM17 in PBECs from all groups. Interestingly, IL-8 production occurred independently from ADAM17 and TGF-α shedding, but was significantly inhibited by broad-spectrum metalloproteinase inhibitor TAPI-2. CSE did not induce ADAM17-dependent TGF-α shedding, while it slightly augmented the production of IL-8. This was accompanied by reduced endogenous inhibitor of metalloproteinase (TIMP)-3 levels, suggesting that CSE does not directly but rather indirectly alter activity of ADAM17 through the regulation of its endogenous inhibitor. Furthermore, whereas baseline TGF-α shedding was lower in COPD PBECs, the early release of IL-8 (likely due to its shedding) was higher in PBECs from COPD than healthy smokers. Importantly, this was accompanied by lower TIMP-2 levels in COPD PBECs, while baseline TIMP-3 levels were similar between groups.

Conclusions

Our data indicate that IL-8 secretion is regulated independently from ADAM17 activity and TGF-α shedding and that particularly its early release is differentially regulated in PBECs from COPD and healthy smokers. Since TIMP-2-sensitive metalloproteinases could potentially contribute to IL-8 release, these may be interesting targets to further investigate novel therapeutic strategies in COPD.     

Cigarette smoke induces IL-8, but inhibits eotaxin and RANTES release from airway smooth muscle

Background

Cigarette smoke is the leading risk factor for the development of chronic obstructive pulmonary disease (COPD) an inflammatory condition characterised by neutrophilic inflammation and release of proinflammatory mediators such as interleukin-8 (IL-8). Human airway smooth muscle cells (HASMC) are a source of proinflammatory cytokines and chemokines. We investigated whether cigarette smoke could directly induce the release of chemokines from HASMC.

Methods

HASMC in primary culture were exposed to cigarette smoke extract (CSE) with or without TNFα. Chemokines were measured by enzyme-linked immunosorbent assay (ELISA) and gene expression by real time polymerase chain reaction (PCR). Data were analysed using one-way analysis of variance (ANOVA) followed by Bonferroni''s t test

Results

CSE (5, 10 and 15%) induced IL-8 release and expression without effect on eotaxin or RANTES release. At 20%, there was less IL-8 release. TNFα enhanced CSE-induced IL-8 release and expression. However, CSE (5–30%) inhibited TNFα-induced eotaxin and RANTES production. The effects of CSE on IL-8 release were inhibited by glutathione (GSH) and associated with the induction of the oxidant sensing protein, heme oxygenase-1.

Conclusion

Cigarette smoke may directly cause the release of IL-8 from HASMC, an effect enhanced by TNF-α which is overexpressed in COPD. Inhibition of eotaxin and RANTES by cigarette smoke is consistent with the predominant neutrophilic but not eosinophilic inflammation found in COPD.     

Zearalenone Mycotoxin Affects Immune Mediators,MAPK Signalling Molecules,Nuclear Receptors and Genome-Wide Gene Expression in Pig Spleen

The toxicity of zearalenone (ZEA) was evaluated in swine spleen, a key organ for the innate and adaptative immune response. Weaned pigs were fed for 18 days with a control or a ZEA contaminated diet. The effect of ZEA was assessed on wide genome expression, pro- (TNF-α, IL-8, IL-6, IL-1β, IFN-γ) and anti-inflammatory (IL-10, IL-4) cytokines, other molecules involved in inflammatory processes (MMPs/TIMPs), as well as signaling molecules, (p38/JNK1/JNK2-MAPKs) and nuclear receptors (PPARγ/NFkB/AP-1/STAT3/c-JUN). Microarray analysis showed that 46% of total number of differentially expressed genes was involved in cellular signaling pathway, 13% in cytokine network and 10% in the inflammatory response. ZEA increased expression and synthesis of pro- inflammatory (TNF-α, IL-8, IL-6, IL-1β) and had no effect on IFN-γ, IL-4 and IL-10 cytokines in spleen. The inflammatory stimulation might be a consequence of JNK pathway activation rather than of p-38MAPK and NF-kB involvement whose gene and protein expression were suppressed by ZEA action. In summary, our findings indicated the role of ZEA as an immune disruptor at spleen level.     

TLR7/TLR8 Activation Restores Defective Cytokine Secretion by Myeloid Dendritic Cells but Not by Plasmacytoid Dendritic Cells in HIV-Infected Pregnant Women and Newborns

Mother-to-child transmission (MTCT) of HIV-1 has been significantly reduced with the use of antiretroviral therapies, resulting in an increased number of HIV-exposed uninfected infants. The consequences of HIV infection on the innate immune system of both mother-newborn are not well understood. In this study, we analyzed peripheral blood and umbilical cord blood (CB) collected from HIV-1-infected and uninfected pregnant women. We measured TNF-α, IL-10 and IFN-α secretion after the stimulation of the cells with agonists of both extracellular Toll-like receptors (TLRs) (TLR2, TLR4 and TLR5) and intracellular TLRs (TLR7, TLR7/8 and TLR9). Moreover, as an indicator of the innate immune response, we evaluated the responsiveness of myeloid dendritic cells (mDCs) and plasmacytoid DCs (pDCs) to TLRs that are associated with the antiviral response. Our results showed that peripheral blood mononuclear cells (PBMCs) from HIV-1-infected mothers and CB were defective in TNF-α production after activation by TLR2, TLR5, TLR3 and TLR7. However, the TNF-α response was preserved after TLR7/8 (CL097) stimulation, mainly in the neonatal cells. Furthermore, only CL097 activation was able to induce IL-10 and IFN-α secretion in both maternal and CB cells in the infected group. An increase in IFN-α secretion was observed in CL097-treated CB from HIV-infected mothers compared with control mothers. The effectiveness of CL097 stimulation was confirmed by observation of similar mRNA levels of interferon regulatory factor-7 (IRF-7), IFN-α and TNF-α in PBMCs of both groups. The function of both mDCs and pDCs was markedly compromised in the HIV-infected group, and although TLR7/TLR8 activation overcame the impairment in TNF-α secretion by mDCs, such stimulation was unable to reverse the dysfunctional type I IFN response by pDCs in the HIV-infected samples. Our findings highlight the dysfunction of innate immunity in HIV-infected mother-newborn pairs. The activation of the TLR7/8 pathway could function as an adjuvant to improve maternal-neonatal innate immunity.     

Acute Systemic Infection with Dengue Virus Leads to Vascular Leakage and Death through Tumor Necrosis Factor-α and Tie2/Angiopoietin Signaling in Mice Lacking Type I and II Interferon Receptors

Severe dengue is caused by host responses to viral infection, but the pathogenesis remains unknown. This is, in part, due to the lack of suitable animal models. Here, we report a non-mouse-adapted low-passage DENV-3 clinical isolate, DV3P12/08, derived from recently infected patients. DV3P12/08 caused a lethal systemic infection in type I and II IFN receptor KO mice (IFN-α/β/γR KO mice), which have the C57/BL6 background. Infection with DV3P12/08 induced a cytokine storm, resulting in severe vascular leakage (mainly in the liver, kidney and intestine) and organ damage, leading to extensive hemorrhage and rapid death. DV3P12/08 infection triggered the release of large amounts of TNF-α, IL-6, and MCP-1. Treatment with a neutralizing anti-TNF-α antibody (Ab) extended survival and reduced liver damage without affecting virus production. Anti-IL-6 neutralizing Ab partly prolonged mouse survival. The anti-TNF-α Ab suppressed IL-6, MCP-1, and IFN-γ levels, suggesting that the severe response to infection was triggered by TNF-α. High levels of TNF-α mRNA were expressed in the liver and kidneys, but not in the small intestine, of infected mice. Conversely, high levels of IL-6 mRNA were expressed in the intestine. Importantly, treatment with Angiopoietin-1, which is known to stabilize blood vessels, prolonged the survival of DV3P12/08-infected mice. Taken together, the results suggest that an increased level of TNF-α together with concomitant upregulation of Tie2/Angiopoietin signaling have critical roles in severe dengue infection.     

Pro-inflammatory mechanisms of muscarinic receptor stimulation in airway smooth muscle

Background

Acetylcholine, the primary parasympathetic neurotransmitter in the airways, plays an important role in bronchoconstriction and mucus production. Recently, it has been shown that acetylcholine, by acting on muscarinic receptors, is also involved in airway inflammation and remodelling. The mechanism(s) by which muscarinic receptors regulate inflammatory responses are, however, still unknown.

Methods

The present study was aimed at characterizing the effect of muscarinic receptor stimulation on cytokine secretion by human airway smooth muscle cells (hASMc) and to dissect the intracellular signalling mechanisms involved. hASMc expressing functional muscarinic M₂ and M₃ receptors were stimulated with the muscarinic receptor agonist methacholine, alone, and in combination with cigarette smoke extract (CSE), TNF-α, PDGF-AB or IL-1β.

Results

Muscarinic receptor stimulation induced modest IL-8 secretion by itself, yet augmented IL-8 secretion in combination with CSE, TNF-α or PDGF-AB, but not with IL-1β. Pretreatment with GF109203X, a protein kinase C (PKC) inhibitor, completely normalized the effect of methacholine on CSE-induced IL-8 secretion, whereas PMA, a PKC activator, mimicked the effects of methacholine, inducing IL-8 secretion and augmenting the effects of CSE. Similar inhibition was observed using inhibitors of IκB-kinase-2 (SC514) and MEK1/2 (U0126), both downstream effectors of PKC. Accordingly, western blot analysis revealed that methacholine augmented the degradation of IκBα and the phosphorylation of ERK1/2 in combination with CSE, but not with IL-1β in hASMc.

Conclusions

We conclude that muscarinic receptors facilitate CSE-induced IL-8 secretion by hASMc via PKC dependent activation of IκBα and ERK1/2. This mechanism could be of importance for COPD patients using anticholinergics.     

Effects of the Commercial Flame Retardant Mixture DE-71 on Cytokine Production by Human Immune Cells

IntroductionAlthough production of polybrominated diphenyl ethers (PBDEs) is now banned, release from existing products will continue for many years. The PBDEs are assumed to be neurotoxic and toxic to endocrine organs at low concentrations. Their effect on the immune system has not been investigated thoroughly. We aimed to investigate the influence of DE-71 on cytokine production by peripheral blood mononuclear cells (PBMCs) stimulated with Escherichia Coli lipopolysaccharide (LPS) or phytohaemagglutinin-L (PHA-L).ResultsAt non-cytotoxic concentrations (0.01–10 μg/mL), DE-71 significantly enhanced secretion of IL-1β, IL-6, CXCL8, IL-10, and TNF-α (p<0.001–0.019; n = 6) from LPS-stimulated PBMCs. IFN-γ, TNF-α, IL-17A, and IL-17F (p = <0.001–0.043; n = 6) secretion were enhanced from PHA-L-stimulated PBMCs as well. Secretion of IL-1β, IL-2, IL-10, IL-8 and IL-6 was not significantly affected by DE-71.ConclusionsWe demonstrate an enhancing effect of DE-71 on cytokine production by normal human PBMCs stimulated with LPS or PHA-L ex vivo.     

Immunogenicity without Efficacy of an Adenoviral Tuberculosis Vaccine in a Stringent Mouse Model for Immunotherapy during Treatment

To investigate if bacterial persistence during TB drug treatment could be overcome by modulation of host immunity, we adapted a clinically-relevant model developed for the evaluation of new drugs and examined if immunotherapy with two adenoviral vaccines, Ad35-TBS (AERAS-402) and Ad26-TBS, could shorten therapy in mice. Even though immunotherapy resulted in strong splenic IFN-γ responses, no effect on bacterial replication in the lungs was seen. Multiplex assay analysis of lung samples revealed the absence of cytokine augmentation such as IFN-γ, TNF-α and IL-2, suggesting that immunization failed to induce immunity in the lungs. In this model, we show that IFN-γ levels were not associated with protection against disease relapse. The results obtained from our study raise questions regarding the traits of protective TB immunity that are relevant for the development of future immunotherapeutic and post-exposure vaccination strategies.     

Innate Immune Responses and Rapid Control of Inflammation in African Green Monkeys Treated or Not with Interferon-Alpha during Primary SIVagm Infection

Chronic immune activation (IA) is considered as the driving force of CD4⁺ T cell depletion and AIDS. Fundamental clues in the mechanisms that regulate IA could lie in natural hosts of SIV, such as African green monkeys (AGMs). Here we investigated the role of innate immune cells and IFN-α in the control of IA in AGMs. AGMs displayed significant NK cell activation upon SIVagm infection, which was correlated with the levels of IFN-α. Moreover, we detected cytotoxic NK cells in lymph nodes during the early acute phase of SIVagm infection. Both plasmacytoid and myeloid dendritic cell (pDC and mDC) homing receptors were increased, but the maturation of mDCs, in particular of CD16⁺ mDCs, was more important than that of pDCs. Monitoring of 15 cytokines showed that those, which are known to be increased early in HIV-1/SIVmac pathogenic infections, such as IL-15, IFN-α, MCP-1 and CXCL10/IP-10, were significantly increased in AGMs as well. In contrast, cytokines generally induced in the later stage of acute pathogenic infection, such as IL-6, IL-18 and TNF-α, were less or not increased, suggesting an early control of IA. We then treated AGMs daily with high doses of IFN-α from day 9 to 24 post-infection. No impact was observed on the activation or maturation profiles of mDCs, pDCs and NK cells. There was also no major difference in T cell activation or interferon-stimulated gene (ISG) expression profiles and no sign of disease progression. Thus, even after administration of high levels of IFN-α during acute infection, AGMs were still able to control IA, showing that IA control is independent of IFN-α levels. This suggests that the sustained ISG expression and IA in HIV/SIVmac infections involves non-IFN-α products.     

Pro- and Anti-Inflammatory Cytokines against Rv2031 Are Elevated during Latent Tuberculosis: A Study in Cohorts of Tuberculosis Patients,Household Contacts and Community Controls in an Endemic Setting

Tuberculosis (TB) is among the leading causes of morbidity and mortality. The causative agent, Mycobacterium tuberculosis (Mtb), has evolved virulent factors for entry, survival, multiplication and immune evasion. Rv2031 (also called alpha crystallin, hspX, 16-kDa antigen), one of the most immunogenic latency antigens, is believed to play a key role in long-term viability of Mtb. Here, we report the dynamics of pro-inflammatory (IFN-γ, TNF-α) and anti-inflammatory (IL-10) cytokines against Rv2031 using whole blood assay in human cohorts in a TB endemic setting. Cytokine responses to ESAT-6-CFP-10 were also measured for comparison. Blood samples were collected from smear positive pulmonary TB patients and their contacts at baseline, 6 and 12 months, and from community controls at entry. At baseline, 54.4% of controls and 73.2% of contacts were QFT-GIT test positive. Baseline IFN-γ, TNF-α and IL-10 responses to Rv2031 were significantly higher in controls compared to contacts and untreated patients (p<0.001). Furthermore, untreated patients had significantly higher TNF-α and IL-10 responses to Rv2031 compared to contacts (p<0.001). In contacts and treated patients, IFN-γ, TNF-α and IL-10 responses to Rv2031 significantly increased over 12 months (p<0.0001) and became comparable with the corresponding levels in controls. There was a positive and significant correlation between Rv2031 and ESAT-6-CFP-10 specific cytokine responses in each study group. The fact that the levels of IFN-γ, TNF-α and IL-10 against Rv2031 were highest during latent TB infection may indicate their potential as markers of protection against TB. Taken together, the findings of this study suggest the potential of IFN-γ, TNF-α and IL-10 against Rv2031 as biomarkers of the host response to Mtb during convalescence from, and the absence of, active tuberculosis.     

HBsAg Inhibits IFN-α Production in Plasmacytoid Dendritic Cells through TNF-α and IL-10 Induction in Monocytes

Type I Interferon (IFN) is one of the first lines of defense against viral infection. Plasmacytoid dendritic cells (pDCs) are professional IFN-α-producing cells that play an important role in the antiviral immune response. Previous studies have reported that IFN-α production is impaired in chronic hepatitis B (CHB) patients. However, the mechanisms underlying the impairment in IFN-α production are not fully understood. Here, we report that plasma-derived hepatitis B surface antigen (HBsAg) and HBsAg expressed in CHO cells can significantly inhibit toll like receptor (TLR) 9-mediated Interferon-α (IFN-α) production in peripheral blood mononuclear cells (PBMCs) from healthy donors. Further analysis indicated that monocytes participate in the inhibitory effect of HBsAg on pDCs through the secretion of TNF-α and IL-10. Furthermore, TLR9 expression on pDCs was down-regulated by TNF-α, IL-10 and HBsAg treatment. This down-regulation may partially explain the inhibition of IFN-α production in pDCs. In conclusion, we determined that HBsAg inhibited the production of IFN-α by pDCs through the induction of monocytes that secreted TNF-α and IL-10 and through the down-regulation of TLR9 expression on pDCs. These data may aid in the development of effective antiviral treatments and lead to the immune control of the viral infections.     

Blocking TLR7- and TLR9-mediated IFN-α Production by Plasmacytoid Dendritic Cells Does Not Diminish Immune Activation in Early SIV Infection

Persistent production of type I interferon (IFN) by activated plasmacytoid dendritic cells (pDC) is a leading model to explain chronic immune activation in human immunodeficiency virus (HIV) infection but direct evidence for this is lacking. We used a dual antagonist of Toll-like receptor (TLR) 7 and TLR9 to selectively inhibit responses of pDC but not other mononuclear phagocytes to viral RNA prior to and for 8 weeks following pathogenic simian immunodeficiency virus (SIV) infection of rhesus macaques. We show that pDC are major but not exclusive producers of IFN-α that rapidly become unresponsive to virus stimulation following SIV infection, whereas myeloid DC gain the capacity to produce IFN-α, albeit at low levels. pDC mediate a marked but transient IFN-α response in lymph nodes during the acute phase that is blocked by administration of TLR7 and TLR9 antagonist without impacting pDC recruitment. TLR7 and TLR9 blockade did not impact virus load or the acute IFN-α response in plasma and had minimal effect on expression of IFN-stimulated genes in both blood and lymph node. TLR7 and TLR9 blockade did not prevent activation of memory CD4+ and CD8+ T cells in blood or lymph node but led to significant increases in proliferation of both subsets in blood following SIV infection. Our findings reveal that virus-mediated activation of pDC through TLR7 and TLR9 contributes to substantial but transient IFN-α production following pathogenic SIV infection. However, the data indicate that pDC activation and IFN-α production are unlikely to be major factors in driving immune activation in early infection. Based on these findings therapeutic strategies aimed at blocking pDC function and IFN-α production may not reduce HIV-associated immunopathology.     

Campylobacter fetus Induced Proinflammatory Response in Bovine Endometrial Epithelial Cells

Campylobacter fetus subsp. fetus is the causal agent of sporadic abortion in bovines and infertility that produces economic losses in livestock. In many infectious diseases, the immune response has an important role in limiting the invasion and proliferation of bacterial pathogens. Innate immune sensing of microorganisms is mediated by pattern-recognition receptors (PRRs) that identify pathogen-associated molecular patterns (PAMPs) and induces the secretion of several proinflammatory cytokines, like IL-1β, TNF-α, and IL-8. In this study, the expression of IL-1β, TNF-α, IL-8, and IFN-γ in bovine endometrial epithelial cells infected with C. fetus and Salmonella Typhimurium (a bacterial invasion control) was analyzed. The results showed that expression levels of IL-1β and IL-8 were high at the beginning of the infection and decreased throughout the intracellular period. Unlike in this same assay, the expression levels of IFN-γ increased through time and reached the highest peak at 4 hours post infection. In cells infected with S. Typhimurium, the results showed that IL8 expression levels were highly induced by infection but not IFN-γ. In cells infected with S. Typhimurium or C. fetus subsp. fetus, the results showed that TNF-α expression did not show any change during infection. A cytoskeleton inhibition assay was performed to determine if cytokine expression was modified by C. fetus subsp. fetus intracellular invasion. IL-1β and IL-8 expression were downregulated when an intracellular invasion was avoided. The results obtained in this study suggest that bovine endometrial epithelial cells could recognize C. fetus subsp. fetus resulting in early proinflammatory response.     

ApoE Production in Human Monocytes and Its Regulation by Inflammatory Cytokines

The apoE production by tissue macrophages is crucial for the prevention of atherosclerosis and the aim of this study was to further elucidate how this apolipoprotein is regulated by cytokines present during inflammation. Here we studied apoE production in peripheral blood mononuclear cells (PBMC) and analysis was made with a newly developed apoE ELISpot assay. In PBMC, apoE secretion was restricted to monocytes with classical (CD14⁺⁺CD16⁻) and intermediate (CD14⁺CD16⁺) monocytes being the main producers. As earlier described for macrophages, production was strongly upregulated by TGF-β and downregulated by bacterial lipopolysaccharide (LPS) and the inflammatory cytokines IFN-γ, TNF-α and IL-1β. We could here show that a similar down-regulatory effect was also observed with the type I interferon, IFN-α, while IL-6, often regarded as one of the more prominent inflammatory cytokines, did not affect TGF-β-induced apoE production. The TNF-α inhibitor Enbrel could partly block the down-regulatory effect of IFN-γ, IFN-α and IL-1β, indicating that inhibition of apoE by these cytokines may be dependent on or synergize with TNF-α. Other cytokines tested, IL-2, IL-4, IL-12, IL-13, IL-17A and IL-23, had no inhibitory effect on apoE production. In contrast to the effect on monocytes, apoE production by primary hepatocytes and the hepatoma cell line HepG2 was more or less unaffected by treatment with cytokines or LPS.     

Increased Inflammatory Response in Cytomegalovirus Seropositive Patients with Alzheimer’s Disease

Alzheimer’s disease (AD) has been associated with increased local inflammation in the affected brain regions, and in some studies also with elevated levels of proinflammatory cytokines in peripheral blood. Cytomegalovirus (CMV) is known to promote a more effector-oriented phenotype in the T-cell compartment, increasing with age. The aim of this study was to investigate the inflammatory response of peripheral blood mononuclear cells (PBMCs) from AD patients and non-demented (ND) controls. Using a multiplex Luminex xMAP assay targeting GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IP-10 and TNF-α, cytokine profiles from PBMCs were analysed after stimulation with anti-CD3/CD28 beads, CMV pp65 peptide mix or amyloid β (Aβ) protofibrils, respectively. CMV seropositive AD subjects presented with higher IFN-γ levels after anti-CD3/CD28 and CMV pp65 but not after Aβ stimulation, compared to CMV seropositive ND controls. When analysing IFN-γ response to anti-CD3/CD28 stimulation on a subgroup level, CMV seropositive AD subjects presented with higher levels compared to both CMV seronegative AD and CMV seropositive ND subjects. Taken together, our data from patients with clinically manifest AD suggest a possible role of CMV as an inflammatory promoter in AD immunology. Further studies of AD patients at earlier stages of disease, could provide better insight into the pathophysiology.     

Neonatal Plasmacytoid Dendritic Cells (pDCs) Display Subset Variation but Can Elicit Potent Anti-Viral Innate Responses

Neonates are highly susceptible to infectious diseases and defective antiviral pDC immune responses have been proposed to contribute to this phenomenon. Isolated cord blood pDCs innately responded to a variety of TLR7 and TLR9 dependent viruses, including influenza A virus (IAV), human immunodeficiency virus (HIV) or herpes-simplex virus (HSV) by efficiently producing IFN-α, TNF-α as well as chemokines. Interestingly, following activation by CpGA, but not viruses, cord pDCs tend to survive less efficiently. We found that a hallmark of pDCs in neonates is an extended CD2+pDCs compartment compared to adult pDCs without affecting the antiviral IFN-α response. Within CD2+pDCs, we identified a subpopulation expressing CD5 and responsible for IL-12p40 production, however this population is significantly decreased in cord blood compared to adult blood. Therefore, neonatal pDCs clearly display variation in phenotype and subset composition, but without major consequences for their antiviral responses.     

Attenuated expression of tenascin-c in ovalbumin-challenged STAT4-/- mice

Background

Asthma leads to structural changes in the airways, including the modification of extracellular matrix proteins such as tenascin-C. The role of tenascin-C is unclear, but it might act as an early initiator of airway wall remodelling, as its expression is increased in the mouse and human airways during allergic inflammation. In this study, we examined whether Th1 or Th2 cells are important regulators of tenascin-C in experimental allergic asthma utilizing mice with impaired Th1 (STAT4-/-) or Th2 (STAT6-/-) immunity.

Methods

Balb/c wildtype (WT), STAT4-/- and STAT6-/- mice were sensitized with intraperitoneally injected ovalbumin (OVA) followed by OVA or PBS airway challenge. Airway hyperreactivity (AHR) was measured and samples were collected. Real time PCR and immunohistochemistry were used to study cytokines and differences in the expression of tenascin-C. Tenascin-C expression was measured in human fibroblasts after treatment with TNF-α and IFN-γ in vitro.

Results

OVA-challenged WT mice showed allergic inflammation and AHR in the airways along with increased expression of TNF-α, IFN-γ, IL-4 and tenascin-C in the lungs. OVA-challenged STAT4-/- mice exhibited elevated AHR and pulmonary eosinophilia. The mRNA expression of TNF-α and IFN-γ was low, but the expression of IL-4 was significantly elevated in these mice. OVA-challenged STAT6-/- mice had neither AHR nor pulmonary eosinophilia, but had increased expression of mRNA for TNF-α, IFN-γ and IL-4. The expression of tenascin-C in the lungs of OVA-challenged STAT4-/- mice was weaker than in those of OVA-challenged WT and STAT6-/- mice suggesting that TNF-α and IFN-γ may regulate tenascin-C expression in vivo. The stimulation of human fibroblasts with TNF-α and IFN-γ induced the expression of tenascin-C confirming our in vivo findings.

Conclusions

Expression of tenascin-C is significantly attenuated in the airways of STAT4-/- mice, which may be due to the impaired secretion of TNF-α and IFN-γ in these mice.