The B Cell Adaptor Molecule Bam32 Is Critically Important for Optimal Antibody Response and Resistance to Trypanosoma congolense Infection in Mice

Background

Bam32, a 32 kDa adaptor molecule, plays important role in B cell receptor signalling, T cell receptor signalling and antibody affinity maturation in germinal centres. Since antibodies against trypanosome variant surface glycoproteins (VSG) are critically important for control of parasitemia, we hypothesized that Bam32 deficient (Bam32-/-) mice would be susceptible to T. congolense infection.

Methodology/Principal Findings

We found that T. congolense-infected Bam32^-/- mice successfully control the first wave of parasitemia but then fail to control subsequent waves and ultimately succumb to their infection unlike wild type (WT) C57BL6 mice which are relatively resistant. Although infected Bam32^-/- mice had significantly higher hepatomegaly and splenomegaly, their serum AST and ALT levels were not different, suggesting that increased liver pathology may not be responsible for the increased susceptibility of Bam32^-/- mice to T. congolense. Using direct ex vivo flow cytometry and ELISA, we show that CD4+ T cells from infected Bam32^-/- mice produced significantly increased amounts of disease-exacerbating proinflammatory cytokines (including IFN-γ, TNF-α and IL-6). However, the percentages of regulatory T cells and IL-10-producing CD4+ cells were similar in infected WT and Bam32^-/- mice. While serum levels of parasite-specific IgM antibodies were normal, the levels of parasite-specific IgG, (particularly IgG1 and IgG2a) were significantly lower in Bam32^-/- mice throughout infection. This was associated with impaired germinal centre response in Bam32^-/- mice despite increased numbers of T follicular helper (Tfh) cells. Adoptive transfer studies indicate that intrinsic B cell defect was responsible for the enhanced susceptibility of Bam32^-/- mice to T. congolense infection.

Conclusions/Significance

Collectively, our data show that Bam32 is important for optimal anti-trypanosome IgG antibody response and suppression of disease-promoting proinflammatory cytokines and its deficiency leads to inability to control T. congolense infection in mice.     

Nucleotide-Oligomerization-Domain-2 Affects Commensal Gut Microbiota Composition and Intracerebral Immunopathology in Acute Toxoplasma gondii Induced Murine Ileitis

Background

Within one week following peroral high dose infection with Toxoplasma (T.) gondii, susceptible mice develop non-selflimiting acute ileitis due to an underlying Th1-type immunopathology. The role of the innate immune receptor nucleotide-oligomerization-domain-2 (NOD2) in mediating potential extra-intestinal inflammatory sequelae including the brain, however, has not been investigated so far.

Methodology/Principal Findings

Following peroral infection with 100 cysts of T. gondii strain ME49, NOD2^-/- mice displayed more severe ileitis and higher small intestinal parasitic loads as compared to wildtype (WT) mice. However, systemic (i.e. splenic) levels of pro-inflammatory cytokines such as TNF-α and IFN-γ were lower in NOD2^-/- mice versus WT controls at day 7 p.i. Given that the immunopathological outcome might be influenced by the intestinal microbiota composition, which is shaped by NOD2, we performed a quantitative survey of main intestinal bacterial groups by 16S rRNA analysis. Interestingly, Bifidobacteria were virtually absent in NOD2^-/- but not WT mice, whereas differences in remaining bacterial species were rather subtle. Interestingly, more distinct intestinal inflammation was accompanied by higher bacterial translocation rates to extra-intestinal tissue sites such as liver, spleen, and kidneys in T. gondii infected NOD2^-/- mice. Strikingly, intracerebral inflammatory foci could be observed as early as seven days following T. gondii infection irrespective of the genotype of animals, whereas NOD2^-/- mice exhibited higher intracerebral parasitic loads, higher F4/80 positive macrophage and microglia numbers as well as higher IFN-γ mRNA expression levels as compared to WT control animals.

Conclusion/Significance

NOD2 signaling is involved in protection of mice from T. gondii induced acute ileitis. The parasite-induced Th1-type immunopathology at intestinal as well as extra-intestinal sites including the brain is modulated in a NOD2-dependent manner.     

Expression and Function of S100A8/A9 (Calprotectin) in Human Typhoid Fever and the Murine Salmonella Model

Background

Typhoid fever, caused by the Gram-negative bacterium Salmonella enterica serovar Typhi, is a major cause of community-acquired bacteremia and death worldwide. S100A8 (MRP8) and S100A9 (MRP14) form bioactive antimicrobial heterodimers (calprotectin) that can activate Toll-like receptor 4, promoting lethal, endotoxin-induced shock and multi-organ failure. We aimed to characterize the expression and function of S100A8/A9 in patients with typhoid fever and in a murine invasive Salmonella model.

Methods and principal findings

S100A8/A9 protein levels were determined in acute phase plasma or feces from 28 Bangladeshi patients, and convalescent phase plasma from 60 Indonesian patients with blood culture or PCR-confirmed typhoid fever, and compared to 98 healthy control subjects. To functionally characterize the role of S100A8/A9, we challenged wildtype (WT) and S100A9^-/- mice with S. Typhimurium and determined bacterial loads and inflammation 2- and 5- days post infection. We further assessed the antimicrobial function of recombinant S100A8/A9 on S. Typhimurium and S. Typhi replication in vitro. Typhoid fever patients demonstrated a marked increase of S100A8/A9 in acute phase plasma and feces and this increases correlated with duration of fever prior to admission. S100A8/A9 directly inhibited the growth of S. Typhimurium and S. Typhi in vitro in a dose and time dependent fashion. WT mice inoculated with S. Typhimurium showed increased levels of S100A8/A9 in both the liver and the systemic compartment but S100A9^-/- mice were indistinguishable from WT mice with respect to bacterial growth, survival, and inflammatory responses, as determined by cytokine release, histopathology and organ injury.

Conclusion

S100A8/A9 is markedly elevated in human typhoid, correlates with duration of fever prior to admission and directly inhibits the growth of S. Typhimurium and S. Typhi in vitro. Despite elevated levels in the murine invasive Salmonella model, S100A8/A9 does not contribute to an effective host response against S. Typhimurium in mice.     

Extracellular Adenosine Formation by Ecto-5’-Nucleotidase (CD73) Is No Essential Trigger for Early Phase Ischemic Preconditioning

Background

Adenosine is a powerful trigger for ischemic preconditioning (IPC). Myocardial ischemia induces intracellular and extracellular ATP degradation to adenosine, which then activates adenosine receptors and elicits cardioprotection. Conventionally extracellular adenosine formation by ecto-5’-nucleotidase (CD73) during ischemia was thought to be negligible compared to the massive intracellular production, but controversial reports in the past demand further evaluation. In this study we evaluated the relevance of ecto-5’-nucleotidase (CD73) for infarct size reduction by ischemic preconditioning in in vitro and in vivo mouse models of myocardial infarction, comparing CD73^-/- and wild type (WT) mice.

Methods and Results

3x5 minutes of IPC induced equal cardioprotection in isolated saline perfused hearts of wild type (WT) and CD73^-/- mice, reducing control infarct sizes after 20 minutes of ischemia and 90 minutes of reperfusion from 46 ± 6.3% (WT) and 56.1 ± 7.6% (CD73^-/-) to 26.8 ± 4.7% (WT) and 25.6 ± 4.7% (CD73^-/-). Coronary venous adenosine levels measured after IPC stimuli by high-pressure liquid chromatography showed no differences between WT and CD73^-/- hearts. Pharmacological preconditioning of WT hearts with adenosine, given at the measured venous concentration, was evenly cardioprotective as conventional IPC. In vivo, 4x5 minutes of IPC reduced control infarct sizes of 45.3 ± 8.9% (WT) and 40.5 ± 8% (CD73^-/-) to 26.3 ± 8% (WT) and 22.6 ± 6.6% (CD73^-/-) respectively, eliciting again equal cardioprotection. The extent of IPC-induced cardioprotection in male and female mice was identical.

Conclusion

The infarct size limiting effects of IPC in the mouse heart in vitro and in vivo are not significantly affected by genetic inactivation of CD73. The ecto-5’-nucleotidase derived extracellular formation of adenosine does not contribute substantially to adenosine’s well known cardioprotective effect in early phase ischemic preconditioning.     

Topical flagellin protects the injured corneas from Pseudomonas aeruginosa infection

Among bacterial pathogens, Pseudomonas (P.) aeruginosa infection is the most sight threatening. The corneal innate immune responses are key mediators of the host’s defense to P. aeruginosa. Using a mouse model of Pseudomonas keratitis, we evaluated the protective effects of topical application of flagellin, a ligand for Toll-Like receptor 5 (TLR5), on the development of Pseudomonas keratitis and elucidated the underlying mechanisms. Topical application of purified flagellin 6 and 24 h prior to P. aeruginosa inoculation on injured mouse corneas significantly attenuated clinical symptoms of P. aeruginosa keratitis, decreased bacterial burden, and suppressed infection induced inflammation in the B6 mouse cornea. Topical application of flagellin on wounded cornea induced PMN infiltration and markedly upregulated cathelicidin-related antimicrobial peptide (CRAMP) expression. In PMN depleted mice, flagellin promoted bacterial clearance in the cornea compared to that of the PBS treated mice, but was unable to prevent corneal perforation and systemic bacterial dissemination and sepses. Deletion of CRAMP increased corneal susceptibility to P. aeruginosa and abolished flagellin-induced protection in B6 mice. Our findings illustrate the profound protective effect of flagellin on the cornea innate defense, a response that can be exploited for prophylactic purposes to prevent contact lens associated Pseudomonas keratitis.     

Airway Surface Dehydration Aggravates Cigarette Smoke-Induced Hallmarks of COPD in Mice

Introduction

Airway surface dehydration, caused by an imbalance between secretion and absorption of ions and fluid across the epithelium and/or increased epithelial mucin secretion, impairs mucociliary clearance. Recent evidence suggests that this mechanism may be implicated in chronic obstructive pulmonary disease (COPD). However, the role of airway surface dehydration in the pathogenesis of cigarette smoke (CS)-induced COPD remains unknown.

Objective

We aimed to investigate in vivo the effect of airway surface dehydration on several CS-induced hallmarks of COPD in mice with airway-specific overexpression of the β-subunit of the epithelial Na⁺ channel (βENaC).

Methods

βENaC-Tg mice and wild-type (WT) littermates were exposed to air or CS for 4 or 8 weeks. Pathological hallmarks of COPD, including goblet cell metaplasia, mucin expression, pulmonary inflammation, lymphoid follicles, emphysema and airway wall remodelling were determined and lung function was measured.

Results

Airway surface dehydration in βENaC-Tg mice aggravated CS-induced airway inflammation, mucin expression and destruction of alveolar walls and accelerated the formation of pulmonary lymphoid follicles. Moreover, lung function measurements demonstrated an increased compliance and total lung capacity and a lower resistance and hysteresis in βENaC-Tg mice, compared to WT mice. CS exposure further altered lung function measurements.

Conclusions

We conclude that airway surface dehydration is a risk factor that aggravates CS-induced hallmarks of COPD.     

Delayed Wound Healing in Heat Stable Antigen (HSA/CD24)-Deficient Mice

Background

Healthy individuals rarely have problems with wound healing. Most skin lesions heal rapidly and efficiently within one to two weeks. However, many medical and surgical complications can be attributed to deficiencies in wound repair. Open wounds have lost the barrier that protects tissues from bacterial invasion and allows the escape of vital fluids. Without expeditious healing, infections become more frequent. The CD24 gene encodes a heavily-glycosylated cell surface protein anchored to the membrane by phosphatidylinositol. CD24 plays an important role in the adaptive immune response and controls an important genetic checkpoint for homeostasis and autoimmune diseases in both mice and humans. We have previously shown that overexpression of CD24 results in increased proliferation and migration rates.

Aim

To examine the role of CD24 in the wound healing process.

Methods

An excisional model of wound healing was used and delayed wound healing was studied in genetically modified heat stable antigen (HSA/CD24)-deficient mice (HSA ^-/-) compared to wild-type (WT) mice.

Results

Large full-thickness skin wounds, excised on the back of mice, exhibited a significant delay in the formation of granulation tissue, and in wound closure when compared to their WTHSA ^+/+ littermates. Wounds were histologically analyzed and scored, based on the degree of cellular invasion, granulation tissue formation, vascularity, and re-epithelialization. Additionally, in stitched wounds, the HSA ^-/- mice failed to maintain their stitches; they did not hold and fell already 24 hours, revealing erythematous wound fields. Re-expression of HSA, delivered by lentivirus, restored the normal healing phenotype, within 24 hours post-injury, and even improved the healing in WT, and in BalbC mice.

Conclusions

Delayed wound-healing in the absence of HSA/CD24 suggests that CD24 plays an important role in this process. Increased expression of CD24, even in the normal state, may be used to enhance wound repair.     

Invariant NKT Cells Act as an Adjuvant to Enhance Th2 Inflammatory Response in an OVA-Induced Mouse Model of Asthma

Background

Invariant natural killer T cells (iNKT cells) are a unique subset of T lymphocytes and are considered to play an important role in the development of allergic bronchial asthma. Recently, iNKT cells were shown to play an immunoregulatory role in CD4⁺ and CD8⁺ T cell-mediated adaptive immune response. Allergen-specific Th2 inflammatory responses are an important part of the adaptive immune response in asthma. However, the regulatory functions of the Th2 inflammatory response in asthma have not been studied in detail.

Method

In this study, we have investigated the regulatory functions of iNKT cells on the Th2 inflammatory response in an ovalbumin (OVA)-induced murine model of asthma.

Results

Our results demonstrate that α-Galactosylceramide (α-GalCer) administration activated iNKT cells but could not induce the Th2 inflammatory response in wild-type (WT) mice. In the OVA-induced asthma model, α-GalCer administration and adoptive transfer of iNKT cells significantly augmented the Th2 inflammatory responses, including elevated inflammatory cell infiltration in the lung and bronchoalveolar lavage fluid (BALF); increased levels of IL-4, IL-5, and IL-13 in the BALF and splenocyte culture supernatant; and increased serum levels of OVA-specific IgE and IgG1. In addition, the Th2 inflammatory response was reduced, but not completely abrogated in CD1d^-/- mice immunized and challenged with OVA, compared with WT mice.

Conclusion

These results suggest that iNKT cells may serve as an adjuvant to enhance Th2 inflammatory response in an OVA-induced murine model of asthma.     

Airway hyperresponsiveness is associated with airway remodeling but not inflammation in aging Cav1-/- mice

Background

Airway inflammation and airway remodeling are the key contributors to airway hyperresponsiveness (AHR), a characteristic feature of asthma. Both processes are regulated by Transforming Growth Factor (TGF)-β. Caveolin 1 (Cav1) is a membrane bound protein that binds to a variety of receptor and signaling proteins, including the TGF-β receptors. We hypothesized that caveolin-1 deficiency promotes structural alterations of the airways that develop with age will predispose to an increased response to allergen challenge.

Methods

AHR was measured in Cav1-deficient and wild-type (WT) mice 1 to 12 months of age to examine the role of Cav1 in AHR and the relative contribution of inflammation and airway remodeling. AHR was then measured in Cav1^-/- and WT mice after an ovalbumin-allergen challenge performed at either 2 months of age, when remodeling in Cav1^-/- and WT mice was equivalent, and at 6 months of age, when the Cav1^-/- mice had established airway remodeling.

Results

Cav1^-/- mice developed increased thickness of the subepithelial layer and a correspondingly increased AHR as they aged. In addition, allergen-challenged Cav1^-/- mice had an increase in AHR greater than WT mice that was largely independent of inflammation. Cav1^-/- mice challenged at 6 months of age have decreased AHR compared to those challenged at 2 months with correspondingly decreased BAL IL-4 and IL-5 levels, inflammatory cell counts and percentage of eosinophils. In addition, in response to OVA challenge, the number of goblet cells and α-SMA positive cells in the airways were reduced with age in response to OVA challenge in contrast to an increased collagen deposition further enhanced in absence of Cav1.

Conclusion

A lack of Cav1 contributed to the thickness of the subepithelial layer in mice as they aged resulting in an increase in AHR independent of inflammation, demonstrating the important contribution of airway structural changes to AHR. In addition, age in the Cav1^-/- mice is a contributing factor to airway remodeling in the response to allergen challenge.     

Cardiovascular Patterning as Determined by Hemodynamic Forces and Blood Vessel Genetics

Background

Vascular patterning depends on coordinated timing of arteriovenous specification of endothelial cells and the concomitant hemodynamic forces supplied by the onset of cardiac function. Using a combination of 3D imaging by OPT and embryo registration techniques, we sought to identify structural differences between three different mouse models of cardiovascular perturbation.

Results

Endoglin mutant mice shared a high degree of similarity to Mlc2a mutant mice, which have been shown to have a primary developmental heart defect causing secondary vessel remodeling failures. Dll4 mutant mice, which have well-characterized arterial blood vessel specification defects, showed distinct differences in vascular patterning when compared to the disruptions seen in Mlc2a ^-/- and Eng ^-/- models. While Mlc2a ^-/- and Eng ^-/- embryos exhibited significantly larger atria than wild-type, Dll4 ^-/- embryos had significantly smaller hearts than wild-type, but this quantitative volume decrease was not limited to the developing atrium. Dll4 ^-/- embryos also had atretic dorsal aortae and smaller trunks, suggesting that the cardiac abnormalities were secondary to primary arterial blood vessel specification defects.

Conclusions

The similarities in Eng ^-/- and Mlc2a ^-/- embryos suggest that Eng ^-/- mice may suffer from a primary heart developmental defect and secondary defects in vessel patterning, while defects in Dll4 ^-/- embryos are consistent with primary defects in vessel patterning.     

By Regulating Mitochondrial Ca2+-Uptake UCP2 Modulates Intracellular Ca2+

Introduction

The possible role of UCP2 in modulating mitochondrial Ca²⁺-uptake (mCa²⁺-uptake) via the mitochondrial calcium uniporter (MCU) is highly controversial.

Methods

Thus, we analyzed mCa²⁺-uptake in isolated cardiac mitochondria, MCU single-channel activity in cardiac mitoplasts, dual Ca²⁺-transients from mitochondrial ((Ca²⁺)m) and intracellular compartment ((Ca²⁺)c) in the whole-cell configuration in cardiomyocytes of wild-type (WT) and UCP2^-/- mice.

Results

Isolated mitochondria showed a Ru360 sensitive mCa²⁺-uptake, which was significantly decreased in UCP2^-/- (229.4±30.8 FU vs. 146.3±23.4 FU, P<0.05). Single-channel registrations confirmed a Ru360 sensitive voltage-gated Ca²⁺-channel in mitoplasts, i.e. mCa1, showing a reduced single-channel activity in UCP2^-/- (Po,total: 0.34±0.05% vs. 0.07±0.01%, P<0.05). In UCP2^-/- cardiomyocytes (Ca²⁺)m was decreased (0.050±0.009 FU vs. 0.021±0.005 FU, P<0.05) while (Ca²⁺)c was unchanged (0.032±0.002 FU vs. 0.028±0.004 FU, P>0.05) and transsarcolemmal Ca²⁺-influx was inhibited suggesting a possible compensatory mechanism. Additionally, we observed an inhibitory effect of ATP on mCa²⁺-uptake in WT mitoplasts and (Ca²⁺)m of cardiomyocytes leading to an increase of (Ca²⁺)c while no ATP dependent effect was observed in UCP2^-/-.

Conclusion

Our results indicate regulatory effects of UCP2 on mCa²⁺-uptake. Furthermore, we propose, that previously described inhibitory effects on MCU by ATP may be mediated via UCP2 resulting in changes of excitation contraction coupling.     

TLR4/NF-κB Signaling Contributes to Chronic Unpredictable Mild Stress-Induced Atherosclerosis in ApoE-/- Mice

Objective

Chronic stress is an important risk factor for atherosclerotic diseases. Our previous studies have shown that chronic unpredictable mild stress (CUMS) accelerates atherosclerosis and up-regulates TLR4/NF-κB expression in apoE^-/- mice. However, TLR4/NF-κB signaling whether directly contributes to the development of atherosclerosis in CUMS mice is unclear. We hypothesized that the interference of TLR4/NF-κB can ameliorate CUMS-induced inflammation and atherosclerosis in apoE^-/- mice.

Methods

ApoE^-/- mice were exposed to 12 weeks CUMS. Ad-siRNA TLR4 was given by tail vein injection (10 μl/mouse, every 5 days), and PDTC (an inhibitor of NF-κB) was given by intraperitoneal injection (60 mg/kg, once a day). Plasma corticosterone concentrations were determined by solid-phase ¹²⁵I radioimmunoassay. Atherosclerosis lesions in aortic sinuses were evaluated and quantified by IMAGEPRO PLUS. Western blotting was used to detect the expression of TLR4, NF-κB, and IL-1β in aortas of the mice. Plasma lipid profiles, IL-1β, TNF-α, and MCP-1 were measured by ELISA.

Results

Our results indicated that CUMS apoE^-/- mice treatment with siRNA TLR4 significantly decreased atherosclerosis and down-regulated TLR4, NF-κB, and inflammatory cytokines. PDTC also remarkably reduced atherosclerosis and the levels of IL-1β, TNF-α and MCP-1 in plasma. However, Treatment with siRNA TLR4 or PDTC had no effect on plasma corticosterone levels, and lipid profiles.

Conclusions

TLR4/NF-κB pathway may participate in CUMS-induced atherosclerosis through activation of proinflammatory cytokines in apoE^-/- mice. Our data may provide a new potential therapeutic target for prevention of CUMS -induced atherosclerosis.     

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.     

Pseudomonas aeruginosa LPS or Flagellin Are Sufficient to Activate TLR-Dependent Signaling in Murine Alveolar Macrophages and Airway Epithelial Cells

Background

The human lung is exposed to a large number of airborne pathogens as a result of the daily inhalation of 10,000 liters of air. Innate immunity is thus essential to defend the lungs against these pathogens. This defense is mediated in part through the recognition of specific microbial ligands by Toll-like receptors (TLR) of which there are at least 10 in humans. Pseudomonas aeruginosa is the main pathogen that infects the lungs of cystic fibrosis patients. Based on whole animal experiments, using TLR knockout mice, the control of this bacterium is believed to occur by the recognition of LPS and flagellin by TLRs 2,4 and 5, respectively.

Methodology/Principal Findings

In the present study, we investigated in vitro the role of these same TLR and ligands, in alveolar macrophage (AM) and epithelial cell (EC) activation. Cellular responses to P. aeruginosa was evaluated by measuring KC, TNF-α, IL-6 and G-CSF secretion, four different markers of the innate immune response. AM and EC from WT and TLR2, 4, 5 and MyD88 knockout mice for were stimulated with the wild-type P. aeruginosa or with a mutant devoid of flagellin production.

Conclusions/Significance

The results clearly demonstrate that only two ligand/receptor pairs are necessary for the induction of KC, TNF-α, and IL-6 synthesis by P. aeruginosa-activated cells, i.e. TLR2,4/LPS and TLR5/flagellin. Either ligand/receptor pair is sufficient to sense the bacterium and to trigger cell activation, and when both are missing lung EC and AM are unable to produce such a response as were cells from MyD88^−/− mice.     

Modification of β-Defensin-2 by Dicarbonyls Methylglyoxal and Glyoxal Inhibits Antibacterial and Chemotactic Function In Vitro

Background

Beta-defensins (hBDs) provide antimicrobial and chemotactic defense against bacterial, viral and fungal infections. Human β-defensin-2 (hBD-2) acts against gram-negative bacteria and chemoattracts immature dendritic cells, thus regulating innate and adaptive immunity. Immunosuppression due to hyperglycemia underlies chronic infection in Type 2 diabetes. Hyperglycemia also elevates production of dicarbonyls methylgloxal (MGO) and glyoxal (GO).

Methods

The effect of dicarbonyl on defensin peptide structure was tested by exposing recombinant hBD-2 (rhBD-2) to MGO or GO with subsequent analysis by MALDI-TOF MS and LC/MS/MS. Antimicrobial function of untreated rhBD-2 vs. rhBD-2 exposed to dicarbonyl against strains of both gram-negative and gram-positive bacteria in culture was determined by radial diffusion assay. The effect of dicarbonyl on rhBD-2 chemotactic function was determined by chemotaxis assay in CEM-SS cells.

Results

MGO or GO in vitro irreversibly adducts to the rhBD-2 peptide, and significantly reduces antimicrobial and chemotactic functions. Adducts derive from two arginine residues, Arg²² and Arg²³ near the C-terminus, and the N-terminal glycine (Gly¹). We show by radial diffusion testing on gram-negative E. coli and P. aeruginosa, and gram-positive S. aureus, and a chemotaxis assay for CEM-SS cells, that antimicrobial activity and chemotactic function of rhBD-2 are significantly reduced by MGO.

Conclusions

Dicarbonyl modification of cationic antimicrobial peptides represents a potential link between hyperglycemia and the clinical manifestation of increased susceptibility to infection, protracted wound healing, and chronic inflammation in undiagnosed and uncontrolled Type 2 diabetes.     

Pharmacological Treatment with Annexin A1 Reduces Atherosclerotic Plaque Burden in LDLR-/- Mice on Western Type Diet

Objective

To investigate therapeutic effects of annexin A1 (anxA1) on atherogenesis in LDLR^-/- mice.

Methods

Human recombinant annexin A1 (hr-anxA1) was produced by a prokaryotic expression system, purified and analysed on phosphatidylserine (PS) binding and formyl peptide receptor (FPR) activation. Biodistribution of ^99mTechnetium-hr-anxA1 was determined in C57Bl/6J mice. 12 Weeks old LDLR^-/- mice were fed a Western Type Diet (WTD) during 6 weeks (Group I) or 12 weeks (Group P). Mice received hr-anxA1 (1 mg/kg) or vehicle by intraperitoneal injection 3 times per week for a period of 6 weeks starting at start of WTD (Group I) or 6 weeks after start of WTD (Group P). Total aortic plaque burden and phenotype were analyzed using immunohistochemistry.

Results

Hr-anxA1 bound PS in Ca²⁺-dependent manner and activated FPR2/ALX. It inhibited rolling and adherence of neutrophils but not monocytes on activated endothelial cells. Half lives of circulating ^99mTc-hr-anxA1 were <10 minutes and approximately 6 hours for intravenously (IV) and intraperitoneally (IP) administered hr-anxA1, respectively. Pharmacological treatment with hr-anxA1 had no significant effect on initiation of plaque formation (-33%; P = 0.21)(Group I) but significantly attenuated progression of existing plaques of aortic arch and subclavian artery (plaque size -50%, P = 0.005; necrotic core size -76% P = 0.015, hr-anxA1 vs vehicle) (Group P).

Conclusion

Hr-anxA1 may offer pharmacological means to treat chronic atherogenesis by reducing FPR-2 dependent neutrophil rolling and adhesion to activated endothelial cells and by reducing total plaque inflammation.     

Intestine-Specific Deletion of Microsomal Triglyceride Transfer Protein Increases Mortality in Aged Mice

Background

Mice with conditional, intestine-specific deletion of microsomal triglyceride transfer protein (Mttp-IKO) exhibit a complete block in chylomicron assembly together with lipid malabsorption. Young (8–10 week) Mttp-IKO mice have improved survival when subjected to a murine model of Pseudomonas aeruginosa-induced sepsis. However, 80% of deaths in sepsis occur in patients over age 65. The purpose of this study was to determine whether age impacts outcome in Mttp-IKO mice subjected to sepsis.

Methods

Aged (20–24 months) Mttp-IKO mice and WT mice underwent intratracheal injection with P. aeruginosa. Mice were either sacrificed 24 hours post-operatively for mechanistic studies or followed seven days for survival.

Results

In contrast to young septic Mttp-IKO mice, aged septic Mttp-IKO mice had a significantly higher mortality than aged septic WT mice (80% vs. 39%, p = 0.005). Aged septic Mttp-IKO mice exhibited increased gut epithelial apoptosis, increased jejunal Bax/Bcl-2 and Bax/Bcl-X_L ratios yet simultaneously demonstrated increased crypt proliferation and villus length. Aged septic Mttp-IKO mice also manifested increased pulmonary myeloperoxidase levels, suggesting increased neutrophil infiltration, as well as decreased systemic TNFα compared to aged septic WT mice.

Conclusions

Blocking intestinal chylomicron secretion alters mortality following sepsis in an age-dependent manner. Increases in gut apoptosis and pulmonary neutrophil infiltration, and decreased systemic TNFα represent potential mechanisms for why intestine-specific Mttp deletion is beneficial in young septic mice but harmful in aged mice as each of these parameters are altered differently in young and aged septic WT and Mttp-IKO mice.     

Hepatic Overexpression of Soluble Urokinase Receptor (uPAR) Suppresses Diet-Induced Atherosclerosis in Low-Density Lipoprotein Receptor-Deficient (LDLR-/-) Mice

Objective

Atherosclerosis, a chronic inflammatory disease, arises from metabolic disorders and is driven by inappropriate recruitment and proliferation of monocytes / macrophages and vascular smooth-muscle-cells. The receptor for the urokinase-type plasminogen activator (uPAR, Plaur) regulates the proteolytic activation of plasminogen. It is also a coactivator of integrins and facilitates leukocyte-endothelial interactions and vascular smooth-muscle-cell migration. The role of uPAR in atherogenesis remains elusive.

Methods and Results

We generated C57Bl6/J low-density lipoprotein receptor (LDL) and uPAR double knockout (uPAR^-/-/LDLR^-/-) mice to test the role of uPAR in two distinct atherosclerosis models. In LDLR^-/- mice, hepatic overexpression following hydrodynamic transfection of soluble uPAR that competes with endogenous membrane-bound uPAR was performed as an interventional strategy. Aortic root atherosclerotic lesions induced by feeding a high-fat diet were smaller and comprised less macrophages and vascular smooth-muscle-cells in double knockout mice and animals overexpressing soluble uPAR when compared to controls. In contrast, lesion size, lipid-, macrophage-, and vascular smooth muscle cell content of guide-wire-induced intima lesions in the carotid artery were not affected by uPAR deficiency. Adhesion of uPAR^-/--macrophages to TNFα-stimulated endothelial cells was decreased in vitro accompanied by reduced VCAM-1 expression on primary endothelial cells. Hepatic overexpression of soluble full-length murine uPAR in LDLR^-/- mice led to a reduction of diet-induced atherosclerotic lesion formation and monocyte recruitment into plaques. Ex vivo incubation with soluble uPAR protein also inhibited adhesion of macrophages to TNFα-stimulated endothelial cells in vitro.

Conclusion

uPAR-deficiency as well as competitive soluble uPAR reduced diet-promoted but not guide-wire induced atherosclerotic lesions in mice by preventing monocyte recruitment and vascular smooth-muscle-cell infiltration. Soluble uPAR may represent a therapeutic tool for the modulation of hyperlipidemia-associated atherosclerotic lesion formation.     

Deletion of Fibrinogen-like Protein 2 (FGL-2), a Novel CD4+ CD25+ Treg Effector Molecule,Leads to Improved Control of Echinococcus multilocularis Infection in Mice

Background

The growth potential of the tumor-like Echinococcus multilocularis metacestode (causing alveolar echinococcosis, AE) is directly linked to the nature/function of the periparasitic host immune-mediated processes. We previously showed that Fibrinogen-like-protein 2 (FGL2), a novel CD4⁺CD25⁺ Treg effector molecule, was over-expressed in the liver of mice experimentally infected with E. multilocularis. However, little is known about its contribution to the control of this chronic helminth infection.

Methods/Findings

Key parameters for infection outcome in E. multilocularis-infected fgl2^-/- (AE-fgl2^-/-) and wild type (AE-WT) mice at 1 and 4 month(s) post-infection were (i) parasite load (i. e. wet weight of parasitic metacestode tissue), and (ii) parasite cell proliferation as assessed by determining E. multilocularis 14-3-3 gene expression levels. Serum FGL2 levels were measured by ELISA. Spleen cells cultured with ConA for 48h or with E. multilocularis Vesicle Fluid (VF) for 96h were analyzed ex-vivo and in-vitro. In addition, spleen cells from non-infected WT mice were cultured with rFGL2/anti-FGL2 or rIL-17A/anti-IL-17A for further functional studies. For Treg-immune-suppression-assays, purified CD4⁺CD25⁺ Treg suspensions were incubated with CD4⁺ effector T cells in the presence of ConA and irradiated spleen cells as APCs. Flow cytometry and qRT-PCR were used to assess Treg, Th17-, Th1-, Th2-type immune responses and maturation of dendritic cells. We showed that AE-fgl2^-/- mice exhibited (as compared to AE-WT-animals) (a) a significantly lower parasite load with reduced proliferation activity, (b) an increased T cell proliferative response to ConA, (c) reduced Treg numbers and function, and (d) a persistent capacity of Th1 polarization and DC maturation.

Conclusions

FGL2 appears as one of the key players in immune regulatory processes favoring metacestode survival by promoting Treg cell activity and IL-17A production that contributes to FGL2-regulation. Prospectively, targeting FGL2 could be an option to develop an immunotherapy against AE and other chronic parasitic diseases.