Increases in biodiversity can result from an increase in species richness, as well as from a higher genetic diversity within species. Intraspecific genetic diversity, measured as the number of genotypes, can enhance plant primary productivity and have cascading effects at higher trophic levels, such as an increase in herbivore and predator richness. The positive effects of genotypic mixtures are not only determined by additive effects, but also by interactions among genotypes, such as facilitation or inhibition. However, so far there has been no effort to predict the extent of such effects. In this study, we address the question of whether the magnitude of the effect of genotype number on population performance can be explained by the extent of dissimilarity in key traits among genotypes in a mixture. We examine the relative contribution of genotype number and phenotypic dissimilarity among genotypes to population performance of the soil arthropod, Orchesella cincta. Nearly homogeneous genotypes were created from inbred isofemale lines. Phenotypic dissimilarity among genotypes was assessed in terms of three life-history traits that are associated with population growth rate, i.e., egg size, egg development time, and juvenile growth rate. A microcosm experiment with genotype mixtures consisting of one, two, four, and eight genotypes, showed that genotypic richness strongly increased population size and biomass production and was associated with greater net diversity effects. Most importantly, there was a positive log-linear relationship between phenotypic dissimilarity in a mixture and the net diversity effects for juvenile population size and total biomass. In other words, the degree of phenotypic dissimilarity among genotypes determined the magnitude of the genotypic richness effect, although this relationship leveled off at higher values of phenotypic dissimilarity. Although the exact mechanisms responsible for these effects are currently unknown, similar advantages of trait dissimilarity have been found among species. Hence, to better understand population performance, genotype number and phenotypic dissimilarity should be considered collectively.
Background and objectives The conversion of extracellular ATP (eATP) to adenosine is an important mechanism of immune suppression through the coordinated activity of two enzymes, CD39 and CD73. CD39 is a membrane bound ATPase, and CD73 is a membrane bound ectonucleotidase that converts AMP to adenosine. We have previously presented evidence that the balance between pro-inflammatory eATP and anti-inflammatory adenosine is skewed in the synovial compartment of rheumatoid arthritis patients. Here we investigated the efficacy of Adeno-associated viral type 5 (AAV5) vector mediated expression of CD39 and CD73 on in vitroand in vivoinflammation models. Materials and methods Adeno-associated viral (AAV) vectors expressing CD39 or CD73 were generated and used to transduce HEK 293 cells or RA fibroblast-like synoviocytes (FLS) and these transduced cells were co-cultured with LPS-activated human monocytes (THP-1) in the presence of ATP. Pro-inflammatory cytokine/chemokine (IL-6, CCL2) production was measured by ELISA. In vivo efficacy of AAV5-CD39-CD73 was investigated using the air pouch synovial inflammation (APSI) model in Balb/c mice using LPS as inflammatory stimulus. Pro-inflammatory cytokines (CCL2, IL-6) were measured by ELISA. Results HEK 293 cells and RA FLS cell lines transduced with CD39-and/or CD73-expressing AAV5 vectors demonstrated high CD39 and CD73 activity. THP-1 cells stimulated with LPS showed lower levels (>80% reduction p = <0.05) of IL-6 and CCL2 secretion when co-cultured with CD39 and CD73 expressing HEK293 cells or FLS cells in the presence of ATP. AAV mediated expression of CD39 and CD73 in the air pouch membrane resulted in significant decreases in pro-inflammatory cytokine levels at 24 h (˜35% reduced CCL2 p < 0.05) and 48 h (25% reduced CCL2 levels p < 0.05, 80% reduced IL-6 levels p < 0.001) post LPS administration. Conclusion Together, these data suggest that AAV mediated expression of CD39 and CD73 may be a novel strategy for the treatment of inflammatory disease, including RA.
Cell-surface receptors, which are either receptor-like proteins (RLPs) or receptor-like kinases (RLKs), form the first layer of the plant innate immune system. The LRR-RLKs SUPPRESSOR OF BIR1-1 (SOBIR1) and BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) are the common regulatory co-receptors of LRR-RLPs. The tomato LRR-RLP Cf-4, which specifically detects the apoplastic effector Avr4 that is secreted by the pathogenic intercellular fungus Cladosporium fulvum, requires SOBIR1 and BAK1 to mediate resistance against this fungus. It is proposed that phosphorylation events take place between the cytoplasmic kinase domains of SOBIR1 and BAK1, which lead to the initiation of various downstream signaling outputs such as the rapid phosphorylation of receptor-like cytoplasmic kinases (RLCKs), the accumulation of reactive oxygen species (ROS), the activation of mitogen-activated protein kinase (MAPK) cascades, and in some cases, the hypersensitive response (HR). Here, by performing in vitro phosphorylation assays, we show that SOBIR1 directly phosphorylates BAK1, whereas BAK1, in its turn, directly phosphorylates SOBIR1, which results in the full activation of the Cf-4-containing immune complex. We found that threonine 522, present in the activation segment of the SOBIR1 kinase domain of Nicotiana benthamiana (Nb), is required for its intrinsic kinase activity, and is essential for the Avr4/Cf-4/SOBIR1/BAK1-triggered ROS burst, MAPK activation and the HR. Tyrosine residue Y469 was found to be crucial for the Avr4/Cf-4-triggered activation of MAPKs and the HR, but not for ROS production and NbSOBIR1 intrinsic kinase activity. RLCKs are well-recognized to act as the initial cytoplasmic transducers, bridging cell-surface receptor complexes with their downstream signaling partners. By knocking out multiple genes belonging to different RLCK-VII subfamilies in N. benthamiana plants stably expressing Cf-4, we show that members of the RLCK-VII subfamilies 6, 7 and 8 are required for the Avr4/Cf-4-triggered ROS production. Typically, the Avr4 protein triggers a biphasic ROS burst in leaf discs obtained from N. benthamiana:Cf-4 plants, in which the first transitory response is followed by a second, sustained ROS burst. Interestingly, the intensities of the first and second phase of the ROS burst are affected in different ways in the various rlck-vii knock-out plants, indicating that there are different downstream ROS regulatory mechanisms, involving different RLCKs in N. benthamiana. Further studies show that members from RLCK-VII-6, 7 and 8 also play an essential role in regulating the ROS production triggered by flg22, chitin, and the nlp20/RLP23 and the pg13/RLP42 combinations.
Background and objectives Adenosine is an important regulator of immune responses. There is accumulating evidence that methotrexate, the anchor drug for rheumatoid arthritis (RA), mediates its anti-inflammatory effects by increasing adenosine levels at inflamed sites. Adenosine modulates (immune) cell responses through four receptors: ADORA1,-2A, -2B, and -3. ADORAs are linked to different intracellular signalling pathways: ADORA1/3 to pro-inflammatory and ADORA2A/2B to anti-inflammatory pathways. Here, we present data investigating the effects of adenosine and receptor expression in two of the major cell types involved in the pathogenesis of RA, monocytes and fibroblast-like synoviocytes (FLS). Materials and methods Primary monocytes were isolated from healthy donors blood (n = 3) and FLS from synovial biopsies of RA patients (n = 6). After stimulation with LPS or TNF/IL-1β, cells were lysed at several time points and ADORA expression was determined by QPCR. Supernatants were taken after O/N stimulation and cytokines (CCL2, IL-6 and TNF) were determined by ELISA. Results In resting cells, monocytes and FLS showed a distinct ADORA expression profile, with ADORA1/2B predominant on FLS, and ADORA3/2B predominant on monocytes. After stimulation with LPS or TNF/IL-1β, both cell types showed a striking upregulation of ADORA2A peaking at 4h (monocytes: 90-fold, FLS: 450 fold) but not of ADORA-1, -2B or -3. When monocytes were stimulated with LPS, co-incubation with adenosine significantly reduced pro-inflammatory cytokine production, resulting in a >70% decrease in CCL2 and TNF (p < 0.05). In similar studies using FLS, adenosine was found to have a much more modest effect, showing only a <20% decrease in CCL2 (p < 0.01) and no change in IL-6 production in TNF/IL-1β stimulated cells. In contrast, when no inflammatory stimuli were added to cell cultures, adenosine enhanced CCL2 and IL-6 production in both FLS and monocytes. In RA-FLS, the effect of adenosine was dependent on the dose of TNF/IL-1β. These results indicate that in a pro-inflammatory environment, adenosine will mainly signal through ADORA2A and repress immune responses. Interestingly, in the absence of inflammatory stimuli, adenosine will have pro-inflammatory properties, likely through A1/A3 signalling. Conclusion These data show that the primary cells involved in the pathogenesis of RA dynamically change their ADORA expression following inflammatory stimulus, and that this change is reflected in the cells response to adenosine.
Background and Objectives Adenosine and ATP are known to have important immunomodulatory properties. Extracellular ATP has multiple roles in inflammation and can act as a damageassociated molecular pattern (DAMP) that can activate the immune system. Conversely, adenosine is primarily anti-inflammatory and can inhibit the production of pro-inflammatory molecules by immune cells. The modulation of ATP and adenosine levels are an essential part of the induction and resolution of an inflammatory response. ENTPD1 (CD39) is a membrane-bound ectonucleoside triphosphate diphosphohydrolase enzyme that converts ATP and ADP to AMP 5NTE1 (CD73) is a 5′ ecto-nucleotidase that dephosphorylates AMP to form adenosine. We investigated the role of genes in the adenosine pathway in patients with rheumatoid arthritis (RA) and determined whether expression of CD39 and CD73 would have an effect in an in vitro inflammation model. Materials and MethodsGene expression analysis using 43k cDNA microarrays (Stanford Functional Genomics Facility) was performed on total RNA extracted from RA synovial tissues obtained by arthroscopy. Adenosine pathway gene expression was compared between high-inflammation versus low-inflammation tissue type synovial biopsies. ATPase levels were measured in synovial fluid (SF) from RA (n = 10) or osteoarthritis (OA) (n = 6) patients. Adeno-associated viral (AAV) vectors expressing CD39 or CD73 were generated and used to transduce HEK 293 cells or RA fibroblast-like synoviocytes (FLS) and these transduced cells were co-cultured with LPS-activated human monocytes (THP-1) in the presence of ATP. Pro-inflammatory cytokine/chemokine (IL-6, CCL2) production was measured by ELISA. Results Genes involved in the ATP:adenosine pathway, including CD73, were differentially expressed in high-inflammation synovial tissues, consistent with the hypothesis that there is skewing of the ATP:adenosine balance during inflammation. The half-life of ATP was significantly increased in SF from RA patients compared with OA (t 1/2 = 8.0 versus 4.5 min., p = <0.05), indicating that there was a significant decrease in ATPase activity in RA SF HEK 293 cells and RA FLS cell lines transduced with CD39-and/or CD73-expressing AAV5 vectors demonstrated high CD39 and CD73 activity. THP-1 cells stimulated with LPS showed lower levels (>80% reduction p = <0.05) of IL-6 and CCL2 secretion when co-cultured with CD39 and CD73 expressing HEK293 cells or FLS cells in the presence of ATP. Conclusions Together, these data suggest that synovial inflammation in RA is characterised by skewing of the ATP:adenosine balance. This could be reversed by overexpression of CD39 or CD73. Thus, these data show that the ATP:adenosine pathway may be a novel therapeutic target for the treatment of RA.
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