In the host lung, the human fungal pathogen Cryptococcus neoformans undergoes a morphological switch from small haploid yeast to large polyploid titan cell, contributing to C. neoformans virulence. Titan cells are less readily phagocytosed and can survive host nitrosative and oxidative stresses. We and others previously showed that titanization is triggered by host-relevant signals including CO2 and lung-resident bacteria, and addition of these factor is sufficient to induce titan cells in vitro. Here we investigate the molecular mechanisms that drive this transition and demonstrate that host-derived immune signals can increase the degree and frequency of titanization. Specifically, host-relevant reactive nitrogen species increase the accumulation of endogenous superoxide within cryptococcal cells, particularly within nuclei, where it can cause genotoxic stress. Consistent with this, we observe the accumulation of Rad51 protein, a marker of the double strand break repair pathway, in titanizing cultures. Blocking superoxide accumulation inhibits titanization, yet titanization also requires superoxide detoxification through Superoxide Dismutase (SOD) activity. Loss of mitochondrial Sod2 activity locks cells in the yeast phase, while Sod1 is required for the production of viable titan daughter cells. We hypothesize that the redox responsive transcription factor Yap1 in part mediates this response by regulating SOD2/SOD1. In addition, we show that Sod1 translocates to the nucleus, where it is likely involved in the detoxification of genotoxic superoxide. Together, these findings reveal a major new regulatory mechanism for the yeast-to-titan transition.Author SummaryDuring fungal infection, host phagocytes produce reactive oxygen and nitrogen species (ROS/RNS), major determinants of infection outcome. Fungal pathogens have developed numerous strategies to neutralize and detoxify ROS, but RNS remain important effectors for infection control. In the lung, the human fungal pathogen Cryptococcus neoformans can undergo a morphological switch from small haploid yeast to large highly polyploid titan cells with increased ROS/RNS stress resistance, and the capacity to produce haploid or aneuploid daughters. Here, we report that RNS are a major signal driving the frequency and degree of titanization and act by increasing endogenous ROS within the fungus. We show that the accumulation of endogenous ROS is required for the yeast-to-titan transition, and is associated with increased genotoxic stress leading to polyploidy. Yet, failure to detoxify this ROS, either in mutants defective in Superoxide Dismutase activity or the oxidative stress response protein Yap1, impairs titan cell budding and reduces progeny viability. Therefore, the interface of exogenous RNS and endogenous ROS regulation during host-pathogen interaction represents an Achilles’ heel for this major human fungal pathogen.
Chronic inflammatory conditions such as Rheumatoid Arthritis are characterised by over expression of pro-inflammatory factors resulting in prolonged and destructive inflammation. Dysregulation of endogenous anti-inflammatory control mechanisms contribute to this aetiology. The mRNA destabilising protein Tristetraprolin (TTP) targets pro-inflammatory transcripts for destruction, limiting the production of key inflammatory factors. Expression and activity of TTP are controlled via phosphorylation of serines 52 and 178. MAPK p38-dependent phosphorylation of these residues stabilises and inactivates TTP, promoting expression of inflammatory mediators and driving the on-phase of an inflammatory response. As MAPK p38 activity declines, accumulated TTP is activated by PP2A mediated dephosphorylation, driving the off-phase of the inflammatory response. We investigated the role of the TTP phosphorylation switch in inflammatory arthritis. Immuno-staining of human RA synovial biopsy tissue revealed abnormally high expression of TTP protein compared to control tissues, with highest expression in synovial macrophages. We hypothesise that TTP protein accumulates in a phosphorylated, inactive form, contributing to sustained expression of inflammatory mediators. To assess the therapeutic potential of targeting TTP we generated TTPaa/aa mice in which the two key serine residues in endogenous TTP are substituted. These mice produced significantly lower amounts of pro-inflammatory factors after systemic LPS challenge, due to constitutive mRNA destabilising activity of the mutant form of TTP, yet were still able to generate a protective immune response to bacterial infection. Intriguingly TTPaa/aa mice were protected from K/BxN induced inflammatory arthrtitis with no adverse histological pathology or bone remodelling compared to WT mice. Heterozygote mice, in which 20% of endogenous TTP is mutant, also demonstrated significant protection from inflammatory arthritis. Therefore, therapeutically altering the balance of activation in the total TTP pool could lead to a significant anti-inflammatory effect. An experimental reagent that activates TTP decreased clinical score, joint inflammation and bone erosions in K/BxN induced arthritis. Taken together, these data suggest that targeting of the equilibrium between phosphorylated (inactive) and dephosphorylated (active) TTP may exert therapeutic effects in arthritis without compromising immune function.
Background and objectivesDuring synovial inflammation, platelets and their microparticles escape from the vasculature to fuel the synovial membrane with pro-inflammatory factors leading to the activation of synovial fibroblasts (SF) that actively contribute to joint damage.1 Patients with rheumatoid arthritis (RA) show an up-regulation of surface protein Podoplanin (PDPN) on SF.2,3 Although the function of PDPN is still poorly understood, recent data suggest that PDPN ligation to its ligand CLEC-2 can modulate cellular responses. Within the RA synovium, platelets are considered the sole source of CLEC-2.3 Despite these observations, clear experimental approaches that explore the role of PDPN/CLEC-2 interactions in RA are lacking.Materials and methodsPDPN expression by freshly isolated mouse synoviocytes was measured by flow cytometry during joint inflammation and after resolution. Tamoxifen-inducible Clec1b deletion mice (TIC mice) were used to assess the disease severity in absence of CLEC-2. CLEC-2 deletion was confirmed on circulating platelets by flow cytometry. Arthritis was induced by anti-collagen antibodies and LPS injections. The disease severity was monitored by body weight, clinical scores, ankle and paw thicknesses. Bone erosion and bone remodelling were studied by MicroCT scans and 3D reconstructions.ResultsJoint inflammation triggers PDPN up-regulation on SF and an accumulation of PDPN+ leucocytes in the synovium. These high levels of PDPN expression disappear when inflammation resolved. In absence of CLEC-2, arthritis is more severe, bone erosion and bone remodelling are more pronounced.ConclusionsIn this work, we provide the first in vivo evidence that PDPN/CLEC-2 interactions act to restrain arthritis by showing that ablation of CLEC-2 expression leads to worse arthritis, bone erosion and bone remodelling. These observations suggest that platelets, known for promoting joint inflammation, also contribute to the suppression of arthritis in a CLEC-2 dependent manner. The mechanisms underlying this anti-inflammatory process are currently under investigation.References Boilard E, et al. Nat Rev Rheumatol. 2012;8:534–542 Ekwall AK, et al. Arthritis Res Ther. 2011;13:R40 Del Rey MJ, et al. PLoS One. 2014;9:e0099607
Background Synovial fibroblasts (SF) are key cellular mediators of joint inflammation and destruction in rheumatoid arthritis (RA). RASF have the potential to migrate to distant cartilage sites where they attach, invade and degrade articular cartilage. Objectives Using novel markers of SF subsets to identify lining and sub-ling layer SF we investigated the ability of RASF to undergo self-assembly, transmigration and cartilage degradation in vivo. Methods Healthy human cartilage was co-implanted subcutaneously into SCID mice together with RASF. At the contralateral flank, cartilage was implanted without cells. After 60 days, implants and blood were removed and analyzed. For the detection of human cells, immunohistocytochemistry was performed with species-specific antibodies. For in vitro studies SF were isolated from patients with established RA and normal healthy controls under defined culture conditions and the expression of phenotypic markers analyzed. Results RASF at the ipsilateral implant differentiated into distinct fibroblast subsets in the presence of cartilage. Cells proximal to cartilage expressed markers of a lining layer phenotype (GP38, FAP, VCAM-1 and Cadherin-11). These cells attached to, invaded and degraded cartilage. Cells more distal to cartilage expressed sub-lining layer phenotype markers including CD248 and CD90. Cells expressing CD248 and CD90 were never observed in the lining layer (proximal to cartilage) and never invaded cartilage. The development of this stromal architecture was very similar to that observed in vivo in the inflamed synovial membrane. This stromal pattern of distinct lining layer and sub lining layer differentiation was completely recapitulated in the contra-lateral implant that contained only cartilage. In addition, we demonstrate that SF in vitro can be directed towards either a lining layer (GP38, FAP, VCAM-1 and Cadherin-11) or sub-lining layer phenotype (f CD248 and CD90) following treatment with various cytokines. The lining layer, but not sub lining cell phenotype is associated with increased cartilage degradation in vitro. Conclusions Our observations demonstrate that although RASF have an activated cell phenotype ex-vivo they also display a degree of plasticity with the capacity to differentiate into distinct fibroblast subsets associated with lining and sub-lining layer cell markers both in vitro and in vivo. Differentiation into distinct subsets of fibroblasts occurs locally at the site of engraftment following vascular transmigration and totally recapitulate the lining and sub lining anatomy observed at the site of origin. This plastic cell phenotype is dependent on local factors including proximity to damaged cartilage. The formation of such a pathogenic stromal architecture is required for cartilage destruction by RASF. We propose that cellular therapies targeting RASF specific subsets are a potentially important but unexplored therapeutic approach to reduce inflammation and joint damage in patients with RA. Disclosure of Interest None Declared
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