In the absence of CD154, administration of interleukin-12 restores Th1 responses but not protective immunity to Schistosoma mansoni

The cytokine interplay during the development of protective immunity to the radiation-attenuated (RA) schistosome vaccine has been extensively characterized over recent years, yet the role of costimulatory molecules in the development of cell-mediated immunity is much less well understood. Here we demonstrate the importance of CD40/CD154 in vaccine-induced immunity, as CD154(-/-) mice exposed to RA schistosomes develop no protection to challenge infection. We showed that vaccinated CD154(-/-) mice have defective Th1-associated immune responses in the skin-draining lymph nodes and the lungs, with reduced or absent levels of interleukin-12p40 (IL-12p40), gamma interferon, and nitric oxide, but elevated levels of lung IL-4 and IL-5. The expression of major histocompatibility complex II (MHC-II) on antigen-presenting cells recovered from the lungs of vaccinated CD154(-/-) mice was also severely compromised. The administration of anti-CD40 monoclonal antibody (MAb) to CD154(-/-) mice did not reconstitute sustained Th1 responses in the lymph nodes or the lungs, nor did the MAb restore anti-parasite immunoglobulin G production or protective immunity. On the other hand, the administration of recombinant IL-12 (rIL-12) to CD154(-/-) mice shortly after vaccination caused elevated and sustained levels of Th1-associated cytokines, rescued MHC-II expression by lung CD11c(+) cells, and restored the appearance of inflammatory effector foci in the lungs. However, the treatment of CD154(-/-) mice with rIL-12 did not restore protection. We conclude that protective immunity to the RA schistosome vaccine is CD154 dependent but is independent of IL-12-orchestrated cellular immune mechanisms in the lungs

IL-10 production in macrophages is regulated by a TLR-driven CREB-mediated mechanism that is linked to genes involved in cell metabolism

IL-10 is produced by macrophages in diverse immune settings and is critical in limiting immune-mediated pathology. In helminth infections, macrophages are an important source of IL-10; however, the molecular mechanism underpinning production of IL-10 by these cells is poorly characterized. In this study, bone marrow–derived macrophages exposed to excretory/secretory products released by Schistosoma mansoni cercariae rapidly produce IL-10 as a result of MyD88-mediated activation of MEK/ERK/RSK and p38. The phosphorylation of these kinases was triggered by TLR2 and TLR4 and converged on activation of the transcription factor CREB. Following phosphorylation, CREB is recruited to a novel regulatory element in the Il10 promoter and is also responsible for regulating a network of genes involved in metabolic processes, such as glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation. Moreover, skin-resident tissue macrophages, which encounter S. mansoni excretory/secretory products during infection, are the first monocytes to produce IL-10 in vivo early postinfection with S. mansoni cercariae. The early and rapid release of IL-10 by these cells has the potential to condition the dermal microenvironment encountered by immune cells recruited to this infection site, and we propose a mechanism by which CREB regulates the production of IL-10 by macrophages in the skin, but also has a major effect on their metabolic state

Sm16, a major component of Schistosoma mansoni cercarial excretory/secretory products, prevents macrophage classical activation and delays antigen processing

Background: Schistosoma mansoni cercariae penetrate the skin by releasing excretory/secretory (E/S) products known as 0-3hRP, which are associated with immune modulation through Toll like receptor (TLR) signaling. Furthermore, these secretions contain Sm16, which when given to cells as a recombinant protein inhibits human monocyte derived cytokine responses to TLR4 and TLR3 ligands. Nonetheless, the extent and mechanism(s) of these inhibitory effects remain largely uncharacterized. Methods: Murine bone marrow derived macrophages were exposed to different fractions of 0-3hRP, obtained via ultracentrifugation, or recombinant Sm16. These cells were exposed to the parasite molecules in combination with different TLR ligands, or Interferon gamma, and tested for the production of the cytokines IL-10 and IL-12p40, and their ability to process antigen. Results: The immunomodulatory function of 0-3hRP is enriched predominantly in the pellet fraction, which contains a greater proportion of Sm16, also corroborating the ability of recombinant Sm16 to inhibit macrophage activation in response to TLR ligands. We further demonstrate that Sm16 blocks classical activation of macrophages to LPS or IFN-¿ stimulation in vitro, and that inhibition of macrophage classical activation is independent of TLR2 recognition. Finally we show that Sm16 shares the altered intracellular processing observed for 0-3hRP, and is able to delay antigen processing by macrophages. Conclusions: Collectively, our findings show that Sm16 is a major component of S. mansoni cercarial E/S products, and is partly responsible for its immune-regulatory properties. Moreover, we propose that the mechanism employed by Sm16 to exert its inhibitory function is likely to be linked with alteration of endosomal trafficking and is not dependent on particular TLR receptors. Finally, we suggest that accumulation of Sm16 in the skin after percutaneous infection with S. mansoni cercariae could contribute to limiting dermal inflammation

CD4+ T cell hyporesponsiveness after repeated exposure to Schistosoma mansoni larvae is dependent upon interleukin-10

The effect that multiple percutaneous exposures to Schistosoma larvae has on the development of early CD4+ lymphocyte reactivity is unclear, yet it is important in the context of humans living in areas where schistosomiasis is endemic. In a murine model of multiple infections, we show that exposure of mice to repeated doses (4×) of Schistosoma mansoni cercariae, compared to a single dose (1×), results in CD4+ T cell hyporesponsiveness within the skin-draining lymph nodes (sdLN), manifested as reduced CD4+ cell proliferation and cytokine production. FoxP3+ CD4+ regulatory T cells were present in similar numbers in the sdLN of 4× and 1× mice and thus are unlikely to have a role in effecting hyporesponsiveness. Moreover, anergy of the CD4+ cell population from 4× mice was slight, as proliferation was only partly circumvented through the in vitro addition of exogenous interleukin-2 (IL-2), and the in vivo blockade of the regulatory molecule PD1 had a minimal effect on restoring responsiveness. In contrast, IL-10 was observed to be critical in mediating hyporesponsiveness, as CD4+ cells from the sdLN of 4× mice deficient for IL-10 were readily able to proliferate, unlike those from 4× wild-type cohorts. CD4+ cells from the sdLN of 4× mice exhibited higher levels of apoptosis and cell death, but in the absence of IL-10, there was significantly less cell death. Combined, our data show that IL-10 is a key factor in the development of CD4+ T cell hyporesponsiveness after repeated parasite exposure involving CD4+ cell apoptosis

Fluorescent Imaging of Antigen Released by a Skin-Invading Helminth Reveals Differential Uptake and Activation Profiles by Antigen Presenting Cells

Infection of the mammalian host by the parasitic helminth Schistosoma mansoni is accompanied by the release of excretory/secretory molecules (ES) from cercariae which aid penetration of the skin. These ES molecules are potent stimulants of innate immune cells leading to activation of acquired immunity. At present however, it is not known which cells take up parasite antigen, nor its intracellular fate. Here, we develop a technique to label live infectious cercariae which permits the imaging of released antigens into macrophages (MΦ) and dendritic cells (DCs) both in vitro and in vivo. The amine reactive tracer CFDA-SE was used to efficiently label the acetabular gland contents of cercariae which are released upon skin penetration. These ES products, termed ‘0-3hRP’, were phagocytosed by MHC-II+ cells in a Ca+ and actin-dependent manner. Imaging of a labelled cercaria as it penetrates the host skin over 2 hours reveals the progressive release of ES material. Recovery of cells from the skin shows that CFDA-SE labelled ES was initially (3 hrs) taken up by Gr1+MHC-II− neutrophils, followed (24 hrs) by skin-derived F4/80+MHC-IIlo MΦ and CD11c+ MHC-IIhi DC. Subsequently (48 hrs), MΦ and DC positive for CFDA-SE were detected in the skin-draining lymph nodes reflecting the time taken for antigen-laden cells to reach sites of immune priming. Comparison of in vitro-derived MΦ and DC revealed that MΦ were slower to process 0-3hRP, released higher quantities of IL-10, and expressed a greater quantity of arginase-1 transcript. Combined, our observations on differential uptake of cercarial ES by MΦ and DC suggest the development of a dynamic but ultimately balanced response that can be potentially pushed towards immune priming (via DC) or immune regulation (via MΦ)

The Mannose Receptor (CD206) is an important pattern recognition receptor (PRR) in the detection of the infective stage of the helminth Schistosoma mansoni and modulates IFNγ production.

In this study, infective larvae of the parasitic helminth Schistosoma mansoni were shown to contain a large number of glycosylated components specific for the Mannose Receptor (MR; CD206), which is an important pattern recognition receptor (PRR) of the innate immune system. MR ligands were particularly rich in excretory/secretory (E/S) material released during transformation of cercariae into schistosomula, a process critical for infection of the host. E/S material from carboxyfluorescein diacetate succinimidyl ester (CFDA-SE)-labelled cercariae showed enhanced binding by cells lines that over-express the MR. Conversely, uptake was significantly lower by bone marrow-derived macrophages (MΦ) from MR(-/-) mice, although they were more active as judged by enhanced pro-inflammatory cytokine production and CD40 expression. After natural percutaneous infection of MR(-/-) mice with CFDA-SE-labelled parasites, there were fewer cells in the skin and draining lymph nodes that were CFDA-SE(+) compared with wild-type mice, implying reduced uptake and presentation of larval parasite antigen. However, antigen-specific proliferation of skin draining lymph node cells was significantly enhanced and they secreted markedly elevated levels of IFNγ but decreased levels of IL-4. In conclusion, we show that the MR on mononuclear phagocytic cells, which are plentiful in the skin, plays a significant role in internalising E/S material released by the invasive stages of the parasite which in turn modulates their production of pro-inflammatory cytokines. In the absence of the MR, antigen-specific CD4(+) cells are Th1 biased, suggesting that ligation of the MR by glycosylated E/S material released by schistosome larvae modulates the production of CD4(+) cell specific IFNγ

Helminth Infection and Commensal Microbiota Drive Early IL-10 Production in the Skin by CD4+ T Cells That Are Functionally Suppressive

The skin provides an important first line of defence and immunological barrier to invasive pathogens, but immune responses must also be regulated to maintain barrier function and ensure tolerance of skin surface commensal organisms. In schistosomiasis-endemic regions, populations can experience repeated percutaneous exposure to schistosome larvae, however little is known about how repeated exposure to pathogens affects immune regulation in the skin. Here, using a murine model of repeated infection with Schistosoma mansoni larvae, we show that the skin infection site becomes rich in regulatory IL-10, whilst in its absence, inflammation, neutrophil recruitment, and local lymphocyte proliferation is increased. Whilst CD4+ T cells are the primary cellular source of regulatory IL-10, they expressed none of the markers conventionally associated with T regulatory (Treg) cells (i.e. FoxP3, Helios, Nrp1, CD223, or CD49b). Nevertheless, these IL-10+ CD4+ T cells in the skin from repeatedly infected mice are functionally suppressive as they reduced proliferation of responsive CD4+ T cells from the skin draining lymph node. Moreover, the skin of infected Rag-/- mice had impaired IL-10 production and increased neutrophil recruitment. Finally, we show that the mechanism behind IL-10 production by CD4+ T cells in the skin is due to a combination of an initial (day 1) response specific to skin commensal bacteria, and then over the following days schistosome-specific CD4+ T cell responses, which together contribute towards limiting inflammation and tissue damage following schistosome infection. We propose CD4+ T cells in the skin that do not express markers of conventional T regulatory cell populations have a significant role in immune regulation after repeated pathogen exposure and speculate that these cells may also help to maintain skin barrier function in the context of repeated percutaneous insult by other skin pathogensy The Wellcome Trust, grant number: 092745/Z/10/Z

Alternatively Activated Mononuclear Phagocytes from the Skin Site of Infection and the Impact of IL-4Rα Signalling on CD4+T Cell Survival in Draining Lymph Nodes after Repeated Exposure to Schistosoma mansoni Cercariae

In a murine model of repeated exposure of the skin to infective Schistosoma mansoni cercariae, events leading to the priming of CD4 cells in the skin draining lymph nodes were examined. The dermal exudate cell (DEC) population recovered from repeatedly (4x) exposed skin contained an influx of mononuclear phagocytes comprising three distinct populations according to their differential expression of F4/80 and MHC-II. As determined by gene expression analysis, all three DEC populations (F4/80-MHC-IIhigh, F4/80+MHC-IIhigh, F4/80+MHC-IIint) exhibited major up-regulation of genes associated with alternative activation. The gene encoding RELMα (hallmark of alternatively activated cells) was highly up-regulated in all three DEC populations. However, in 4x infected mice deficient in RELMα, there was no change in the extent of inflammation at the skin infection site compared to 4x infected wild-type cohorts, nor was there a difference in the abundance of different mononuclear phagocyte DEC populations. The absence of RELMα resulted in greater numbers of CD4+ cells in the skin draining lymph nodes (sdLN) of 4x infected mice, although they remained hypo-responsive. Using mice deficient for IL-4Rα, in which alternative activation is compromised, we show that after repeated schistosome infection, levels of regulatory IL-10 in the skin were reduced, accompanied by increased numbers of MHC-IIhigh cells and CD4+ T cells in the skin. There were also increased numbers of CD4+ T cells in the sdLN in the absence of IL-4Rα compared to cells from singly infected mice. Although their ability to proliferate was still compromised, increased cellularity of sdLN from 4x IL-4RαKO mice correlated with reduced expression of Fas/FasL, resulting in decreased apoptosis and cell death but increased numbers of viable CD4+ T cells. This study highlights a mechanism through which IL-4Rα may regulate the immune system through the induction of IL-10 and regulation of Fas/FasL mediated cell death

Circulating CD14brightCD16+ 'intermediate' monocytes exhibit enhanced parasite pattern recognition in human helminth infection.

Circulating monocyte sub-sets have recently emerged as mediators of divergent immune functions during infectious disease but their role in helminth infection has not been investigated. In this study we evaluated whether 'classical' (CD14brightCD16-), 'intermediate' (CD14brightCD16+), and 'non-classical' (CD14dimCD16+) monocyte sub-sets from peripheral blood mononuclear cells varied in both abundance and ability to bind antigenic material amongst individuals living in a region of Northern Senegal which is co-endemic for Schistosoma mansoni and S. haematobium. Monocyte recognition of excretory/secretory (E/S) products released by skin-invasive cercariae, or eggs, of S. mansoni was assessed by flow cytometry and compared between S. mansoni mono-infected, S. mansoni and S. haematobium co-infected, and uninfected participants. Each of the three monocyte sub-sets in the different infection groups bound schistosome E/S material. However, 'intermediate' CD14brightCD16+ monocytes had a significantly enhanced ability to bind cercarial and egg E/S. Moreover, this elevation of ligand binding was particularly evident in co-infected participants. This is the first demonstration of modulated parasite pattern recognition in CD14brightCD16+ intermediate monocytes during helminth infection, which may have functional consequences for the ability of infected individuals to respond immunologically to infection

Analysis of ecological thresholds in a temperate forest undergoing dieback.

Positive feedbacks in drivers of degradation can cause threshold responses in natural ecosystems. Though threshold responses have received much attention in studies of aquatic ecosystems, they have been neglected in terrestrial systems, such as forests, where the long time-scales required for monitoring have impeded research. In this study we explored the role of positive feedbacks in a temperate forest that has been monitored for 50 years and is undergoing dieback, largely as a result of death of the canopy dominant species (Fagus sylvatica, beech). Statistical analyses showed strong non-linear losses in basal area for some plots, while others showed relatively gradual change. Beech seedling density was positively related to canopy openness, but a similar relationship was not observed for saplings, suggesting a feedback whereby mortality in areas with high canopy openness was elevated. We combined this observation with empirical data on size- and growth-mediated mortality of trees to produce an individual-based model of forest dynamics. We used this model to simulate changes in the structure of the forest over 100 years under scenarios with different juvenile and mature mortality probabilities, as well as a positive feedback between seedling and mature tree mortality. This model produced declines in forest basal area when critical juvenile and mature mortality probabilities were exceeded. Feedbacks in juvenile mortality caused a greater reduction in basal area relative to scenarios with no feedback. Non-linear, concave declines of basal area occurred only when mature tree mortality was 3-5 times higher than rates observed in the field. Our results indicate that the longevity of trees may help to buffer forests against environmental change and that the maintenance of old, large trees may aid the resilience of forest stands. In addition, our work suggests that dieback of forests may be avoidable providing pressures on mature and juvenile trees do not pass critical thresholds