Apolipoprotein E controls Dectin-1-dependent development of monocyte-derived alveolar macrophages upon pulmonary ß-glucan-induced inflammatory adaptation.

The lung is constantly exposed to the outside world and optimal adaptation of immune responses is crucial for efficient pathogen clearance. However, mechanisms that lead to lung-associated macrophages' functional and developmental adaptation remain elusive. To reveal such mechanisms, we developed a reductionist model of environmental intranasal ß-glucan exposure, allowing for the detailed interrogation of molecular mechanisms of pulmonary macrophage adaptation. Employing single-cell transcriptomics, high-dimensional imaging and flow cytometric characterization paired with in vivo and ex vivo challenge models, we reveal that pulmonary low-grade inflammation results in the development of apolipoprotein E (ApoE)-dependent monocyte-derived alveolar macrophages (ApoE+CD11b+ AMs). ApoE+CD11b+ AMs expressed high levels of CD11b, ApoE, Gpnmb and Ccl6, were glycolytic, highly phagocytic and produced large amounts of interleukin-6 upon restimulation. Functional differences were cell intrinsic, and myeloid cell-specific ApoE ablation inhibited Ly6c+ monocyte to ApoE+CD11b+ AM differentiation dependent on macrophage colony-stimulating factor secretion, promoting ApoE+CD11b+ AM cell death and thus impeding ApoE+CD11b+ AM maintenance. In vivo, ß-glucan-elicited ApoE+CD11b+ AMs limited the bacterial burden of Legionella pneumophilia after infection and improved the disease outcome in vivo and ex vivo in a murine lung fibrosis model. Collectively these data identify ApoE+CD11b+ AMs generated upon environmental cues, under the control of ApoE signaling, as an essential determinant for lung adaptation enhancing tissue resilience.

Fluorite-related iridate Pr3IrO7: crystal growth, structure, magnetism, thermodynamic, and optical properties.

Spin-orbit coupling in heavy 5dmetal oxides, in particular, iridates have received tremendous interest in recent years due to the realization of exotic electronic and magnetic phases. Here, we report the synthesis, structural, magnetic, thermodynamic, and optical properties of the ternary iridate Pr3IrO7. Single crystals of Pr3IrO7have been grown by the KF flux method. Structural analysis shows that Pr3IrO7crystallizes in an orthorhombic phase withCmcmsymmetry. The electron energy loss spectroscopy study indicates that Pr is in a 3+ valence state, which implies a 5+ oxidation state of Ir. Magnetization data measured at high and low magnetic fields do not exhibit any bifurcation betweenMZFCandMFC, however, a weak hump inM(T) is observed atT∗∼10.4 K. The specific heat data reveal two maxima at ∼253 and ∼4.8 K. The optical conductivityσ1(ω)spectrum shows 24 infrared-active phonon modes and reveals an insulating behavior with an optical gapΔOPof size ∼500 meV. During cooling down, the temperature-dependent reflectivity spectrum reveals eight extra phonon modes below the structural phase transition (∼253 K). An anomaly is observed at aroundT∗in the temperature evolution of infrared-active mode frequencies suggesting the presence of significant spin-phonon coupling in the system.

Effects of continuous milking during a field trial on productivity, milk protein yield and health in dairy cows.

The objective of this field study with an automatic milking system was to evaluate the effects of omitting the dry period on health and productivity during the subsequent lactation in dairy cows. A total of 98 German Simmental cows of six Southern German farms were assigned randomly to two experimental groups: The first group was dried-off 56 days before calving (D for dried-off, n=49), and the second group was milked continuously during this period until calving (CM for continuous milking, n=49). From the latter a third group emerged, including cows that dried-off themselves spontaneously (DS for dried-off spontaneously, n=14). Blood serum values of glucose, ß-hydroxybutyrate (BHBA), non-esterified fatty acids (NEFA) and IGF-1 showed most pronounced fluctuations in D cows. Over the entire study period, the concentrations of BHBA and NEFA were markedly lower in the CM and DS groups. Furthermore, IGF-1 concentration was lowest for D cows and also decrease in back fat thickness was more pronounced. Mean concentration of milk protein was markedly higher in CM and DS cows (3.70% and 3.71%) compared with D cows (3.38%). Owing to the lower 305-day milk yield (-15.6%) and the lower total milk yield (-3.1%), the total amount of produced protein in the subsequent lactation was 2.5% (6.8 kg) lower, although the additional protein amount in CM cows from week -8 to calving was 35.7 kg. The greatest benefit resulted from positive effects on fertility and the lower incidence of diseases: CM cows had their first oestrus 1 week earlier compared with D cows, they also conceived earlier and showed a significantly lower risk of developing hypocalcaemia, ketosis and puerperal disorders. The present study showed that the costs of medical treatment and milk losses were twice as high in D cows, compared with CM and DS cows, and thus the reduced costs because of the more stable health outweighed the financial losses of milk yield by +18.49 € per cow and lactation.

Phase separation and disorder in doped nematic elastomers.

We formulate and analyse a model describing the combined effect of mechanical deformation, dynamics of the nematic order parameter, and concentration inhomogeneities in an elastomeric mixture of a mesogenic and an isotropic component. The uniform nematic state may exhibit a long-wave instability corresponding to nematic-isotropic demixing. Numerical simulations starting from either a perfectly ordered nematic state or a quenched isotropic state show that coupling between the mesogen concentration and the nematic order parameter influences the shape and orientation of the domains formed during the demixing process.

Allergic airway inflammation is exacerbated during acute influenza infection and correlates with increased allergen presentation and recruitment of allergen-specific T-helper type 2 cells.

BACKGROUND: Respiratory viral infections are a leading cause of the hospitalization of asthmatics, however, the cellular immunological interactions which underlie these two diseases remain elusive. OBJECTIVE: We sought to characterize the effect influenza viral infection has on allergic airway inflammation and to identify the cellular pathways involved. METHODS: We have used an ovalbumin (OVA) model of allergic airway inflammation, which involves sensitization of animals with OVA adsorbed in alum adjuvant followed by an intranasal challenge with OVA in phosphate-buffered saline. To study T cell recruitment into the lung, we adoptively transferred in vitro activated T cell receptor-transgenic T cells, which were subsequently identified by fluorescence-activated cell sorting (FACS) analysis. In addition, to study in vivo dendritic cell (DC) migration, we administered fluorescently labelled dextran and identified DCs that had phagocytosed it by FACS analysis. RESULTS: We found that different stages of influenza infection had contrasting effects upon the outcome of OVA-induced allergic airway inflammation. The allergic response against OVA was exacerbated during the acute stage of influenza infection; however, mice were protected against the development of airway eosinophilia at late time-points following infection. We investigated the mechanisms responsible for the virus-induced exacerbation and found that the response was partially independent of IL-4 and that there was increased delivery of inhaled allergens to the draining lymph node during the acute stage of the infection. In addition, virus-induced inflammation in the lung and draining lymph node resulted in the non-specific recruitment of circulating allergen-specific effector/memory cells. CONCLUSION: In addition to virus-mediated damage to the lung and airways, influenza viral infection can also enhance unrelated local allergic responses.

Dual role of the IL-12/IFN-gamma axis in the development of autoimmune myocarditis: induction by IL-12 and protection by IFN-gamma.

IL-12 and IFN-gamma positively regulate each other and type 1 inflammatory responses, which are believed to cause tissue damage in autoimmune diseases. We investigated the role of the IL-12/IFN-gamma (Th1) axis in the development of autoimmune myocarditis. IL-12p40-deficient mice on a susceptible background resisted myocarditis. In the absence of IL-12, autospecific CD4(+) T cells proliferated poorly and showed increased Th2 cytokine responses. However, IFN-gamma-deficient mice developed fatal autoimmune disease, and blockade of IL-4R signaling did not confer susceptibility to myocarditis in IL-12p40-deficient mice, demonstrating that IL-12 triggers autoimmunity by a mechanism independent of the effector cytokines IFN-gamma and IL-4. In conclusion, our results suggest that the IL-12/IFN-gamma axis is a double-edged sword for the development of autoimmune myocarditis. Although IL-12 mediates disease by induction/expansion of Th1-type cells, IFN-gamma production from these cells limits disease progression.

A high-throughput alphavirus-based expression cloning system for mammalian cells.

We have developed a widely applicable functional genomics strategy based on alphavirus expression vectors. The technology allows for rapid identification of genes encoding a functional activity such as binding of a defined ligand. Complementary DNA (cDNA) libraries were expressed in mammalian cells following infection with recombinant Sindbis virus (SIN replicon particles), a member of the Alphavirus genus. Virus-infected cells that specifically bound a ligand of choice were isolated using fluorescence-activated cell sorting (FACS). Replication-competent, infective SIN replicon particles harboring the corresponding cDNA were amplified in a next step. Within one round of selection, viral clones encoding proteins recognized by monoclonal antibodies or Fc-fusion molecules could be isolated and sequenced. Moreover, using the same viral libraries, a plaque-lift assay was established that allowed the identification of secreted, intracellular, and membrane proteins.

IL-5 deficiency abolishes aspects of airway remodelling in a murine model of lung inflammation.

BACKGROUND AND OBJECTIVES: Lung remodelling is a recognized feature of chronic asthma. In the present study, we have used IL-5-deficient mice to evaluate the role of this cytokine and eosinophilic inflammation in the initial stages of the structural changes occurring in the lung after antigen challenge. METHODS: Ovalbumin-sensitized wild type and IL-5-deficient mice were daily challenged for 5 consecutive days and killed 3 or 7 days after the last challenge to study the inflammatory and remodelling events, respectively. RESULTS: Wild type mice challenged with ovalbumin exhibited an accumulation of eosinophils in the bronchoalveolar lavage (BAL) fluid, associated with a production of BAL cellular fibronectin. Histological analysis also revealed an antigen-specific increase in epithelial and alveolar cell proliferation together with an increase in mucus producing epithelial cells. Eosinophilic infiltration and the associated lung remodelling were totally abrogated in IL-5-deficient mice. In wild type mice, treated intranasally with 1 microg of murine IL-5 for 5 consecutive days, no BAL eosinophilia and structural changes of the lungs could be observed. CONCLUSION: Our results demonstrate that eosinophil accumulation, but not IL-5 alone, plays a central role in the initial stages of the lung remodelling process and suggests that therapies directed at inhibiting eosinophilic inflammation may be beneficial in treating chronic asthma.

Retarded liver growth in interleukin-6-deficient and tumor necrosis factor receptor-1-deficient mice.

The liver size in adult mammals is tightly regulated in relation to body weight, but the hormonal control of this is largely unknown. We investigated the roles of interleukin-6 (IL-6) and tumor necrosis factor (TNF) receptor-1 in the regulation of intact liver weight in adult mice. The relative liver wet and dry weights of older adult (5- to 10-month-old) IL-6 knockout (IL-6(-/-)) mice were decreased by 22-28%, and total contents of DNA and protein were decreased compared with those in age-matched wild-type mice. Weights of other visceral organs were unaffected. Older adult (6- to 8-month-old) TNF receptor-1 knockout (TNFR1(-/-)) mice displayed decreased relative liver weight. Treatment with a single injection of IL-6 increased liver wet and dry weights in IL-6(-/-) and wild-type mice, but not TNFR1(-/-) mice. Treatment with TNFalpha enhanced liver weight and DNA synthesis of nonparenchymal liver cells at 24 h in wild-type, but not IL-6(-/-), mice. At 48 h, TNFalpha induced DNA synthesis in nonparenchymal cells and hepatocytes of both wild-type and IL-6(-/-) mice. In conclusion, TNF receptor-1 stimulation and IL-6 production are both necessary for normal liver weight gain in older adult mice. The results of TNFalpha and IL-6 treatment further indicate that the effects of TNF receptor-1 and IL-6 depend on each other for full stimulation of liver growth.

Normal pathogen-specific immune responses mounted by CTLA-4-deficient T cells: a paradigm reconsidered.

CTLA-4 is a critical negative regulator of T cell responses and CTLA-4-deficient (CTLA-4(-/-)) mice die of a lymphproliferative disease. Nevertheless, RAG-2-deficient mice reconstituted with a mixture of CTLA-4(-/-) and normal (CTLA-4(+/+)) bone marrow survive in the absence of any signs of disease, although 50% of their T cells do not express CTLA-4. Using such mixed chimeras, we analyzed the role of CTLA-4 in specific T cell responses to lymphocytic choriomeningitis virus, Leishmania major and mouse mammary tumor virus, which cause acute, chronic and persistent infections, respectively. The populations of antigen-specific CTLA-4(-/-)CD4(+) and CTLA-4(-/-)CD8(+) T cells became activated, expanded and contracted indistinguishably from CTLA-4(+/+)CD4(+) and CTLA-4(+/+)CD8(+) T cells after infection with all three pathogens. Thus, CTLA-4 is not involved in the down-regulation of specific T cell responses and peripheral deletion in a T cell-autonomous fashion.

Severe schistosomiasis in the absence of interleukin-4 (IL-4) is IL-12 independent.

An interleukin-4 (IL-4)-dependent Th2 response allows wild-type mice to survive infection with the parasite Schistosoma mansoni. In the absence of IL-4, infected mice mount a Th1-like proinflammatory response, develop severe disease, and succumb. Neither the Th1 response nor morbidity is IL-12 dependent in this system.

The antigen dose determines T helper subset development by regulation of CD40 ligand.

Although the amount of antigen and the strength of T cell stimulation have been suggested to regulate Th1 vs. Th2 polarization, it remains unclear how the antigen dose and the strength of signal is detected by the T cell and translated into differential cytokine production. Using co-cultures of dendritic cells (DC) and ovalbumin (OVA)-specific CD4+ T cells obtained from RAG-2)(-/-) DO11.10 mice, we show here that high-dose antigen induced Th1 development by up-regulation of CD40 ligand (CD40L), whereas low-dose antigen stimulation failed to induce CD40L and promoted Th2 development. CD40-CD40L interaction was essential for IL-12 production by DC. In the absence, de novo IL-4 production by T cells and autocrine Th2 development was induced. Furthermore, our results demonstrate that LFA-1/ ICAM interaction promotes Th1 differentiation by lowering the antigen dose required for CD40L up-regulation. Thus, we propose that (1) peptide-MHC density and (2) accessory molecules such as LFA-1 determine T helper polarization by regulation of CD40L.

Lipopolysaccharide-induced HIV-1 expression in transgenic mice is mediated by tumor necrosis factor-alpha and interleukin-1, but not by interferon-gamma nor interleukin-6.

BACKGROUND: As serum HIV-1 load correlates well with the prognosis of the disease, it is suggested that the viral load is one of the major determinants of the disease progression of AIDS. Accordingly, HIV-1 activation mechanisms were extensively studied in vitro, and involvement of cytokines including tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, IL-6 and interferon (IFN)-gamma has been suggested in this process. However, so far the roles of these cytokines in the HIV-1 expression in vivo have not been well elucidated because of the lack of appropriate animal disease models. OBJECTIVE: To elucidate the roles of cytokines in HIV-1 activation in vivo. DESIGN AND METHODS: Transgenic mice carrying a defective HIV-1 genome were used as a model for HIV-1 carriers. In order to examine the possible involvement of cytokines in HIV-1 expression, TNF-alpha-, IL-1-, IL-6- and IFN-gamma-deficient HIV-1 transgenic mice, were produced and HIV-1 expression was analyzed after activation with bacterial lipopolysaccharides (LPS). RESULTS: HIV-1 expression in the transgenic mouse spleen was activated 10- to 20-fold by LPS, and the serum p24 Gag protein levels reached 400 pg/ml, which is nearly equal to the levels that occur in AIDS patients. However, this augmentation was suppressed by 60% in TNF-alpha-deficient mice and by 40% in IL-1alpha/beta-deficient mice. In contrast, no suppression was observed in either IL-6-, IFN-gamma-, IL-1alpha, or IL-1beta-deficient mice. CONCLUSIONS: Results suggest that TNF-alpha and IL-1 play important roles in HIV-1 gene activation and selective suppression of these cytokines could improve clinical prognosis and potentially slow progression of the disease.

Inducible costimulator protein (ICOS) controls T helper cell subset polarization after virus and parasite infection.

It has been shown that certain pathogens can trigger efficient T cell responses in the absence of CD28, a key costimulatory receptor expressed on resting T cells. Inducible costimulator protein (ICOS) is an inducible costimulator structurally and functionally related to CD28. Here, we show that in the absence of CD28 both T helper cell type 1 (Th1) and Th2 responses were impaired but not abrogated after infection with lymphocytic choriomeningitis virus (LCMV), vesicular stomatitis virus (VSV), and the nematode Nippostrongylus brasiliensis. Inhibition of ICOS in CD28-deficient mice further reduced Th1/Th2 polarization. Blocking of ICOS alone had a limited but significant capacity to downregulate Th subset development. In contrast, cytotoxic T lymphocyte (CTL) responses, which are regulated to a minor and major extent by CD28 after LCMV and VSV infection, respectively, remained unaffected by blocking ICOS. Together, our results demonstrate that ICOS regulates both CD28-dependent and CD28-independent CD4(+) subset (Th1 and Th2) responses but not CTL responses in vivo.

Hapten-induced colitis associated with maintained Th1 and inflammatory responses in IFN-gamma receptor-deficient mice.

IFN-gamma is a potent pro-inflammatory cytokine thought to be involved in the pathogenesis of Crohn's disease. To further define the role of IFN-gamma in intestinal inflammation, we studied the effects of intra-colonic 2,4,6-trinitrobenzene sulfonic acid (TNBS) instillation in mice with a functionally inactivated IFN-gamma receptor 1 (IFN-gammaR1(- / -)). Our results indicate that IFN-gamma is not necessary for the induction of hapten-induced colitis: after TNBS administration both wild-type and IFN-gammaR1(- / -) mice lost body weight, and the histological features of TNBS-induced colitis were comparable. Colons of IFN-gammaR1(- / -) mice contained a greater number of cells, represented by macrophages and CD4(+) T cells; caudal lymph node cells produced more IFN-gamma and TNF-alpha upon stimulation in vitro. Moreover, IL-18 and IL-12 p40 RNA levels were comparably up-regulated after TNBS treatment in IFN-gammaR1(- / -) wild-type mice. These findings demonstrate that IFN-gamma is dispensable for the development of TNBS-induced colitis. Importantly, the production of Th1 cytokines (e. g. IFN-gamma and TNF-alpha) by caudal lymph node T lymphocytes was enhanced rather than decreased in IFNgammaR1(- / -) mice with no evidence for default Th2 development.

IL-4 and IL-10 antagonize IL-12-mediated protection against acute vaccinia virus infection with a limited role of IFN-gamma and nitric oxide synthetase 2.

Resistance or susceptibility to most infectious diseases is strongly determined by the balance of type 1 vs type 2 cytokines produced during infection. However, for viruses, this scheme may be applicable only to infections with some cytopathic viruses, where IFN-gamma is considered as mandatory for host defense with little if any participation of type 2 responses. We studied the role of signature Th1 (IL-12, IFN-gamma) and Th2 (IL-4, IL-10) cytokines for immune responses against vaccinia virus (VV). IL-12-/- mice were far more susceptible than IFN-gamma-/- mice, and primary CTL responses against VV were absent in IL-12-/- mice but remained intact in IFN-gamma-/- mice. Both CD4+ and CD8+ T cells from IL-12-/- mice were unimpaired in IFN-gamma production, although CD4+ T cells showed elevated Th2 cytokine responses. Virus replication was impaired in IL-4-/- mice and, even more strikingly, in IL-10-/- mice, which both produced elevated levels of the proinflammatory cytokines IL-1alpha and IL-6. Thus, IL-4 produced by Th2 cells and IL-10 produced by Th2 cells and probably also by macrophages counteract efficient anti-viral host defense. Surprisingly, NO production, which is considered as a major type 1 effector pathway inhibited by type 2 cytokines, appears to play a limited role against VV, because NO sythetase 2-deficient mice did not show increased viral replication. Thus, our results identify a new role for IL-12 in defense beyond the induction of IFN-gamma and show that IL-4 and IL-10 modulate host protective responses to VV.

Antifungal type 1 responses are upregulated in IL-10-deficient mice.

C57BL/6 mice are highly resistant to infections caused by Candida albicans and Aspergillus fumigatus. To elucidate the role of IL-10 produced by C57BL/6 mice during these infections, parameters of infection and immunity to it were evaluated in IL-10-deficient and wild-type mice with disseminated or gastrointestinal candidiasis or invasive pulmonary aspergillosis. Unlike parasitic protozoan infection, C. albicans or A. fumigatus infection did not induce significant acute toxicity in IL-10-deficient mice, who, instead, showed reduced fungal burden and fungal-associated inflammatory responses. The increased resistance to infections as compared to wild-type mice was associated with upregulation of innate and acquired antifungal Th1 responses, such as a dramatically higher production of IL-12, nitric oxide (NO) and TNF-alpha as well as IFN-gamma by CD4+ T cells. Pharmacological inhibition of NO production greatly reduced resistance to gastrointestinal candidiasis, thus pointing to the importance of IL-10-dependent NO regulation at mucosal sites in fungal infections. These results are reminiscent of those obtained in genetically susceptible mice, in which IL-10 administration increased, and IL-10 neutralization decreased, susceptibility to C. albicans and A. fumigatus infections. Collectively, these observations indicate that the absence of IL-10 augments innate and acquired antifungal immunity by upregulating type 1 cytokine responses. The resulting protective Th1 responses lead to a prompt reduction of fungal growth, thus preventing tissue destruction and lethal levels of proinflammatory cytokines.

CD2 sets quantitative thresholds in T cell activation.

It has been proposed that CD2, which is highly expressed on T cells, serves to enhance T cell-antigen presenting cell (APC) adhesion and costimulate T cell activation. Here we analyzed the role of CD2 using CD2-deficient mice crossed with transgenic mice expressing a T cell receptor specific for lymphocytic choriomeningitis virus (LCMV)-derived peptide p33. We found that absence of CD2 on T cells shifted the p33-specific dose-response curve in vitro by a factor of 3-10. In comparison, stimulation of T cells in the absence of lymphocyte function-associated antigen (LFA)-1-intercellular adhesion molecule (ICAM)-1 interaction shifted the dose-response curve by a factor of 10, whereas absence of both CD2-CD48 and LFA-1-ICAM-1 interactions shifted the response by a factor of approximately 100. This indicates that CD2 and LFA-1 facilitate T cell activation additively. T cell activation at low antigen density was blocked at its very first steps, as T cell APC conjugate formation, TCR triggering, and Ca(2+) fluxes were affected by the absence of CD2. In vivo, LCMV-specific, CD2-deficient T cells proliferated normally upon infection with live virus but responded in a reduced fashion upon cross-priming. Thus, CD2 sets quantitative thresholds and fine-tunes T cell activation both in vitro and in vivo.