IL-12Rbeta2-deficient mice of a genetically resistant background are susceptible to Leishmania major infection and develop a parasite-specific Th2 immune response

The cytokine IL-12 plays a critical role in inducing the production of IFN-gamma from T and NK cells and in the polarization of T cells towards the Th1 phenotype. IL-12 is comprised of two subunits (IL-12p40 and IL-12p35) that together form the biologically active p70 molecule, and IL-12 functions via binding to a heterodimeric receptor (IL-12Rbeta1 and IL-12Rbeta2). Previous studies utilizing mice deficient for either the IL-12 cytokine or the IL-12-induced signaling molecule STAT4 have established a critical role for IL-12 during infection with Leishmania major. However, these studies warrant careful re-interpretation in light of the recent discovery of the IL-12-related cytokine, IL-23, which utilizes the IL-12p40 chain in combination with an IL-12p35-related molecule, called p19, and a receptor comprised of the IL-12Rbeta1 chain plus a unique chain referred to as IL-23R. We analyzed the course of L. major infection in mice deficient for the IL-12-specific IL-12Rbeta2 subunit in order to assess the role of IL-12 signaling without disruption of the IL-23 pathway. After infection with L. major, IL-12Rbeta2KO mice of a resistant background (C57Bl/6) developed large cutaneous lesions similar to those developed by susceptible BALB/c mice. Draining lymph node cells from L. major-infected IL-12Rbeta2KO mice released the Th2 cytokines IL-4 and IL-5 after in vitro stimulation with Leishmania lysate but were completely devoid of IFN-gamma, consistent with a default towards a strong parasite-specific Th2 response. L. major-infected IL-12Rbeta2KO mice were also devoid of parasite-specific IgG2a antibodies, and interestingly, their footpad lesions ulcerated earlier than those of susceptible BALB/c mice.     

Differentiation of murine NK cells into distinct subsets based on variable expression of the IL-12R beta 2 subunit

The cytokine IL-12 manifests its biological activity via interaction with a heterodimeric receptor (IL-12R) present on activated T and NK cells. The cDNAs for two IL-12R subunits have been cloned from human and mouse and designated IL-12Rbeta1 and IL-12Rbeta2. The expression of IL-12Rbeta2 on T cells is influenced by cytokines, particularly IL-4, IL-12, and IFN-gamma; however, little is known regarding regulation of IL-12R expression on NK cells. In this study we show that murine NK cells differentiate into IL-12Rbeta2(low) and IL-12Rbeta2(high) subsets after in vitro stimulation with IL-2 in the absence of exogenous polarizing cytokines. Subset development occurs gradually as NK cells expand in vitro and is generally complete by 8-12 days of culture. Once established, IL-12Rbeta2(low) and IL-12Rbeta2(high) subsets are highly stable in vitro and can be maintained for at least 20 days after FACS sorting. Formation of these NK subsets appears to be strain independent. Flow cytometric analyses demonstrate that both subsets express a number of NK-associated markers, including NK1.1, DX-5, Ly-49A, and Ly-49C, but that the Ly-49G2 class I inhibitory receptor is expressed predominantly on the IL-12Rbeta2(high) population. Both IL-12Rbeta2(low) and IL-12Rbeta2(high) NK cells respond to exogenous IL-12 by rapid production of high levels of IFN-gamma and increased lytic activity against NK-sensitive YAC-1 target cells. Analyses of cytokine gene expression by RNase protection assay indicated that similar to the recently described human NK1 subset, both IL-12Rbeta2(high) and IL-12Rbeta2(low) murine NK subsets expressed high levels of IFN-gamma, whereas neither subset expressed mRNA for the NK2-associated cytokines IL-5 and IL-13.     

Comparative Life Histories and Ecophysiology of Drosophila melanogaster and D. simulans

Numerous laboratory investigations have compared Drosophila melanogaster and D. simulans for various life history traits and fitness related ecophysiological parameters. From presently available information, it is however difficult to get a general comparative pattern describing the divergence of their ecological niches and understanding their demographic success. Two environmental factors seem however to have played a major role: temperature and alcoholic resources. From an ecophysiological approach, D. simulans may be described as generally more sensitive to stresses; other results point to this species as more cold adapted than its sibling; in some cases, however, D. simulans may appear as better adapted to a warm environment. When investigated, ecophysiological traits show a lesser geographic variability in D. simulans than in D. melanogaster. Presently available information does not explain the ecological prevalence of D. simulans in many places with a mild temperate or subtropical climate. This is presumably due to the fact that most comparisons have been done at a single, standard temperature of 25 degrees C. Comparative studies should be undertaken, spanning the thermal ranges of the two species, and the phenotypic plasticity of ecophysiological traits should now be considered.     

Pentraxin 3 (PTX3) expression in allergic asthmatic airways: role in airway smooth muscle migration and chemokine production

Background

Pentraxin 3 (PTX3) is a soluble pattern recognition receptor with non-redundant functions in inflammation and innate immunity. PTX3 is produced by immune and structural cells. However, very little is known about the expression of PTX3 and its role in allergic asthma.

Objectives and Methods

We sought to determine the PTX3 expression in asthmatic airways and its function in human airway smooth muscle cells (HASMC). In vivo PTX3 expression in bronchial biopsies of mild, moderate and severe asthmatics was analyzed by immunohistochemistry. PTX3 mRNA and protein were measured by real-time RT-PCR and ELISA, respectively. Proliferation and migration were examined using ³H-thymidine incorporation, cell count and Boyden chamber assays.

Results

PTX3 immunoreactivity was increased in bronchial tissues of allergic asthmatics compared to healthy controls, and mainly localized in the smooth muscle bundle. PTX3 protein was expressed constitutively by HASMC and was significantly up-regulated by TNF, and IL-1β but not by Th2 (IL-4, IL-9, IL-13), Th1 (IFN-γ), or Th-17 (IL-17) cytokines. In vitro, HASMC released significantly higher levels of PTX3 at the baseline and upon TNF stimulation compared to airway epithelial cells (EC). Moreover, PTX3 induced CCL11/eotaxin-1 release whilst inhibited the fibroblast growth factor-2 (FGF-2)-driven HASMC chemotactic activity.

Conclusions

Our data provide the first evidence that PTX3 expression is increased in asthmatic airways. HASMC can both produce and respond to PTX3. PTX3 is a potent inhibitor of HASMC migration induced by FGF-2 and can upregulate CCL11/eotaxin-1 release. These results raise the possibility that PTX3 may play a dual role in allergic asthma.     

Osmotic and Atmospheric Dehydration Effects in the Lichens Hypogymnia Physodes,Lobaria Pulmonaria,and Peltigera Aphthosa: An in vivo Study of the Chlorophyll Fluorescence Induction

Inactivation of photosynthesis during atmospheric and osmotic (highly concentrated NaCl or sucrose solutions) dehydration was monitored by measurement of chlorophyll fluorescence induction (OIP-phase, Kautsky-curves) in three lichen species. The induction curves were changed in a very similar way by all three treatments. All dehydration effects were rapidly reversible after rehydration. At relatively mild water stress, the rise time to the transient peak F_p was prolonged, and the variable part of fluorescence was diminished. In addition, at severe water stress, a considerable decline of the F₀ value was observed. For NaCl treatment this effect started at water potentials <-8.5 MPa in P. aphthosa, <-12 MPa in H. physodes, and <-21 MPa in L. pulmonaria. Above these water potentials, our observations are in agreement with values from desiccation-tolerant algae, higher plants, and lichens, where an inactivation on the photosystem 2 (PS2) donor side has been postulated. At very low water potentials, the decrease in F₀ probably monitors changes in the organization of the antenna apparatus of PS2. This revised version was published online in June 2006 with corrections to the Cover Date.     

Male sterility at extreme temperatures: a significant but neglected phenomenon for understanding Drosophila climatic adaptations

The thermal range for viability is quite variable among Drosophila species and it has long been known that these variations are correlated with geographic distribution: temperate species are on average more cold tolerant but more heat sensitive than tropical species. At both ends of their viability range, sterile males have been observed in all species investigated so far. This symmetrical phenomenon restricts the temperature limits within which permanent cultures can be kept in the laboratory. Thermal heat sterility thresholds are very variable across species from 23 degrees C in heat sensitive species up to 31 degrees C in heat tolerant species. In Drosophila melanogaster, genetic variations are observed among geographic populations. Tropical populations are more tolerant to heat induced sterility and recover more rapidly than temperate ones. A genetic analysis revealed that about 50% of the difference observed between natural populations was due to the Y chromosome. Natural populations have not reached a selection limit, however: thermal tolerance was still increased by keeping strains at a high temperature, close to the sterility threshold. On the low temperature side, a symmetrical reverse phenomenon seems to exist: temperate populations are more tolerant to cold than tropical ones. Compared to Mammals, drosophilids exhibit two major differences: first, male sterility occurs not only at high temperature, but also at a low temperature; second, sterility thresholds are not evolutionarily constrained, but highly variable. Altogether, significant and sometimes major genetic variations have been observed between species, between geographic races of the same species, and even between strains kept in the laboratory under different thermal regimes. In each case, it is easily argued that the observed variations correspond to adaptations to climatic conditions, and that male sterility is a significant component of fitness and a target of natural selection.     

CCR3 expression and function in asthmatic airway smooth muscle cells

Asthma is characterized by an increase in airway smooth muscle mass and a decreased distance between the smooth muscle layer and the epithelium. Furthermore, there is evidence to indicate that airway smooth muscle cells (ASMC) express a wide variety of receptors involved in the immune response. The aims of this study were to examine the expression of CCR3 on ASMC, to compare this expression between asthmatic and nonasthmatic subjects, and to determine the implications of CCR3 expression in the migration of ASMC. We first demonstrated that ASMC constitutively express CCR3 at both mRNA and protein levels. Interestingly, TNF-alpha increases ASMC surface expression of CCR3 from 33 to 74%. Furthermore, using FACS analysis, we found that ASMC CCR3 is expressed to a greater degree in asthmatic vs control subjects (95 vs 75%). Functionality of the receptor was demonstrated by calcium assay; the addition of CCR3 ligand eotaxin to ASMC resulted in an increase in intracellular calcium production. Interestingly, ASMC was seen to demonstrate a positive chemotactic response to eotaxin. Indeed, ASMC significantly migrated toward 100 ng/ml eotaxin (2.2-fold increase, compared with control). In conclusion, the expression of CCR3 by ASMC is increased in asthmatics, and our data show that a CCR3 ligand such as eotaxin induces migration of ASMC in vitro. These results may suggest that eotaxin could be involved in the increased smooth muscle mass observed in asthmatics through the activation of CCR3.     

Co-Culture of Human Bronchial Fibroblasts and CD4+ T Cells Increases Th17 Cytokine Signature

Background

Airway inflammation is an important characteristic of asthma and has been associated with airway remodelling and bronchial hyperreactivity. The mucosal microenvironment composed of structural cells and highly specialised extracellular matrix is able to amplify and promote inflammation. This microenvironment leads to the development and maintenance of a specific adaptive response characterized by Th2 and Th17. Bronchial fibroblasts produce multiple mediators that may play a role in maintaining and amplifying this response in asthma.

Objective

To investigate the role of bronchial fibroblasts obtained from asthmatic subjects and healthy controls in regulating Th17 response by creating a local micro-environment that promotes this response in the airways.

Methods

Human bronchial fibroblasts and CD4⁺T cells were isolated from atopic asthmatics and non-atopic healthy controls. CD4⁺T were co-cultured with bronchial fibroblasts of asthmatic subjects and healthy controls. RORc gene expression was detected by qPCR. Phosphorylated STAT-3 and RORγt were evaluated by western blots. Th17 phenotype was measured by flow cytometry. IL-22, IL17, IL-6 TGF-β and IL1-β were assessed by qPCR and ELISA.

Results

Co-culture of CD4⁺T cells with bronchial fibroblasts significantly stimulated RORc expression and induced a significant increase in Th17 cells as characterized by the percentage of IL-17⁺/CCR6⁺ staining in asthmatic conditions. IL-17 and IL-22 were increased in both normal and asthmatic conditions with a significantly higher amount in asthmatics compared to controls. IL-6, IL-1β, TGF-β and IL-23 were significantly elevated in fibroblasts from asthmatic subjects upon co-culture with CD4⁺T cells. IL-23 stimulates IL-6 and IL-1β expression by bronchial fibroblasts.

Conclusion

Interaction between bronchial fibroblasts and T cells seems to promote specifically Th17 cells profile in asthma. These results suggest that cellular interaction particularly between T cells and fibroblasts may play a pivotal role in the regulation of the inflammatory response in asthma.