Rapid Host Defense against Aspergillus fumigatus Involves Alveolar Macrophages with a Predominance of Alternatively Activated Phenotype

The ubiquitous fungus Aspergillus fumigatus is associated with chronic diseases such as invasive pulmonary aspergillosis in immunosuppressed patients and allergic bronchopulmonary aspergillosis (ABPA) in patients with cystic fibrosis or severe asthma. Because of constant exposure to this fungus, it is critical for the host to exercise an immediate and decisive immune response to clear fungal spores to ward off disease. In this study, we observed that rapidly after infection by A. fumigatus, alveolar macrophages predominantly express Arginase 1 (Arg1), a key marker of alternatively activated macrophages (AAMs). The macrophages were also found to express Ym1 and CD206 that are also expressed by AAMs but not NOS2, which is expressed by classically activated macrophages. The expression of Arg1 was reduced in the absence of the known signaling axis, IL-4Rα/STAT6, for AAM development. While both Dectin-1 and TLR expressed on the cell surface have been shown to sense A. fumigatus, fungus-induced Arg1 expression in CD11c+ alveolar macrophages was not dependent on either Dectin-1 or the adaptor MyD88 that mediates intracellular signaling by most TLRs. Alveolar macrophages from WT mice efficiently phagocytosed fungal conidia, but those from mice deficient in Dectin-1 showed impaired fungal uptake. Depletion of macrophages with clodronate-filled liposomes increased fungal burden in infected mice. Collectively, our studies suggest that alveolar macrophages, which predominantly acquire an AAM phenotype following A. fumigatus infection, have a protective role in defense against this fungus

Cell surface exposure and immunogenicity of foreign epitopes in outer membrane proteins.

To evaluate the accessibility of epitopes to the extracellular environment, bacteria expressing LamB:V3 or OmpA:TOP chimeric protein were fluorescently labeled with mouse anti-gp120 (V3), and analyzed by cytofluorimetry. Clones with the highest fluorescence intensity were selected for further studies.To investigate how the exposure of an epitope on the bacterial surface influences the immunogenicity of the epitope, mice were immunized with live attenuated Salmonella typhimurium expressing the selected fusion proteins with various degrees of epitope exposure. The humoral response to Salmonella typhimurium/chimeric proteins was analyzed by ELISA and Western immunoblot. The data showed strong correlation between the exposure of the epitopes to the extracellular environment, and the immunogenicity in mice.To display antigenic epitopes on the bacterial surface, LamB and OmpA, two outer membrane proteins from E. coli, were used as carriers for two heterologous epitopes: the V3 loop (residues 293 to 334) of gp120 from HIV-1 and the TOP epitope, a smaller part of the V3 loop (residues 309 to 320). In order to optimize the exposure of the heterologous epitopes on the cell surface, three amino acids were introduced into the immediate upstream and downstream junction regions flanking the epitopes. PCR mutagenesis was utilized to randomly mutate these amino acids, creating chimeric protein libraries: each member of the libraries had unique flanking sequences. Because bends and turns in proteins derive from four sequential amino acids, the individual chimeric proteins projected the epitope in different ways

Hyperresponse to T-Cell Receptor Signaling and Apoptosis of Id1 Transgenic Thymocytes

The basic helix-loop-helix transcription factors, E2A and HEB, play important roles in T-cell development at multiple checkpoints. Expression of their inhibitor, Id1, abolishes the function of both transcription factors in a dose-dependent manner. The Id1 transgenic thymus is characterized by an accumulation of CD4(−) CD8(−) CD44(+) CD25(−) thymocytes, a dramatic reduction of CD4(+) CD8(+) thymocytes, and an abundance of apoptotic cells. Here we show that these apoptotic cells carry functional T-cell receptors (TCRs), suggesting that apoptosis occurs during T-cell maturation. In contrast, viable Id1 transgenic CD4 single positive T cells exhibit costimulation-independent proliferation upon treatment with anti-CD3 antibody, probably due to a hyperresponse to TCR signaling. Furthermore, Id1 expression causes apoptosis of CD4 and CD8 double- or single-positive thymocytes in HY- or AND-TCR transgenic mice under conditions that normally support positive selection. Collectively, these results suggest that E2A and HEB proteins are crucial for controlling the threshold for TCR signaling, and Id1 expression lowers the threshold, resulting in apoptosis of developing thymocytes

Electrical Treeing and Partial Discharges in Double Layer Printed Circuit Board

With the development of power devices, the demand to improve insulation performance of printed circuit boards (PCBs) is increasing. PCB faces great challenges in its insulation failure caused by long-term ageing when subject to intensive electric fields. It is observed that failures are commonly detected between layers. Therefore, this paper designed and made V-shaped double layer samples to test the partial discharge and electrical tree growth. Based on the method of pulse sequence analysis and phase-revolved partial discharge pattern, the insulation deterioration in layers is analyzed. It was found that the electrical tree grew into branched electrical trees. Based on the PSA, the growth of tree conforms the characteristic of non-conductive electrical tree. The partial discharges inception and extinction voltage decrease at first as the tree grows. But as the electric tree grows, their trend becomes very complex. The apparent charge followed almost linear relationship with the maximum length of electrical tree in the first 5 minutes.</p

STAT6 Activation Confers upon T Helper Cells Resistance to Suppression by Regulatory T Cells

Recent studies have highlighted characteristics of T regulatory cells (Tregs) that underlie their suppressive function. However, mechanisms that override their suppressive function in the context of an adaptive immune response are not well understood. In the lungs of mice undergoing allergic inflammation, appreciable numbers of Tregs were identified that possessed suppressive function when assayed ex vivo. We investigated whether the Th2-promoting cytokine IL-4 played a permissive role that superseded Treg function, thereby allowing the development of allergic inflammation. IL-4 signaling via the IL-4R alpha-STAT6 axis was required to maintain Foxp3 expression in Tregs and promote their proliferation. However, the results of both in vivo experiments involving adoptive transfer of Tregs into Ag-sensitized vs naive animals and in vitro suppression assays performed with or without exogenous IL-4 showed the ability of IL-4 to compromise Treg-mediated suppression. Use of retrovirally expressed, constitutively active STAT6 revealed that the underlying mechanism was not IL-4-mediated dysfunction of Tregs but involved the resistance of Th cells to Treg-mediated suppression that would permit the development of an adaptive immune response. Our data suggest that infectious tolerance, mediated by membrane-bound TGF-beta expressed by Tregs, is compromised by the competing effects of IL4-induced signaling in naive CD4(+) Th cells. The Journal of Immunology, 2009, 183: 155-163

Autoantibody levels in myositis patients correlate with clinical response during B cell depletion with rituximab

To determine the longitudinal trends in serum levels of four myositis-associated autoantibodies: anti-Jo-1, -transcription intermediary factor 1 γ (TIF1-γ), -signal recognition particle (SRP) and -Mi-2, after B cell depletion with rituximab, and to determine the longitudinal association of these autoantibody levels with disease activity as measured by myositis core-set measures (CSMs). Treatment-resistant adult and pediatric myositis subjects (n = 200) received rituximab in the 44-week Rituximab in Myositis Trial. CSMs [muscle enzymes, manual muscle testing (MMT), physician and patient global disease activity, HAQ, and extramuscular disease activity] were evaluated monthly and anti-Jo-1 (n = 28), -TIF1-γ (n = 23), -SRP (n = 25) and -Mi-2 (n = 26) serum levels were measured using validated quantitative ELISAs. Temporal trends and the longitudinal relationship between myositis-associated autoantibodies levels and CSM were estimated using linear mixed models. Following rituximab, anti-Jo-1 levels decreased over time (P < 0.001) and strongly correlated with all CSMs (P < 0.008). Anti-TIF1-γ levels also decreased over time (P < 0.001) and were only associated with HAQ, MMT and physician and patient global disease activity. Anti-SRP levels did not change significantly over time, but were significantly associated with serum muscle enzymes. Anti-Mi-2 levels significantly decreased over time and were associated with muscle enzymes, MMT and the physician global score. Anti-Jo-1, anti-TIF1-γ and anti-Mi-2 levels in myositis subjects decreased after B cell depletion and were correlated with changes in disease activity, whereas anti-SRP levels were only associated with longitudinal muscle enzyme levels. The strong association of anti-Jo-1 levels with clinical outcomes suggests that anti-Jo-1 autoantibodies may be a good biomarker for disease activity

Macrophages: Regulators of Sex Differences in Asthma?

Females are more susceptible to development of asthma than are males. In a mouse model of ovalbumin-induced airway inflammation, with aggravated disease in females compared with males, we studied interactions between immune and resident lung cells during asthma development to elucidate which processes are affected by sex. We studied numbers of regulatory T cells (Tregs), effector T cells, myeloid dendritic cells (mDCs), and alternatively activated macrophages (AAMΦ), and their functional capabilities. Male and female mice had comparable Treg numbers in lung tissue and comparable Treg function, but effector T cells had expanded to a greater extent in lungs of females after ovalbumin exposure. This difference in T cell expansion was therefore not the result of lack of Treg control, but appeared to be driven by a greater number of inflammatory mDCs migrating from the lungs to lymph nodes in females. Resident lung cells can influence mDC migration, and AAMΦ in lung tissue were found to be involved. Artificially elevating the number of AAMΦ in lung tissue increased the migration of mDCs and airway inflammation. We found greater numbers of AAMΦ in female lungs than in males; we therefore postulate that AAMΦ are involved in increased airway inflammation found in female mice