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The identification of lymphocyte subsets with non-overlapping effector functions has been pivotal to the development of targeted therapies in immune-mediated inflammatory diseases (IMIDs) 1 , 2 . However, it remains unclear whether fibroblast subclasses with non-overlapping functions also exist and are responsible for the wide variety of tissue-driven processes observed in IMIDs, such as inflammation and damage 3 , 4 – 5 . Here we identify and describe the biology of distinct subsets of fibroblasts responsible for mediating either inflammation or tissue damage in arthritis. We show that deletion of fibroblast activation protein-α (FAPα) + fibroblasts suppressed both inflammation and bone erosions in mouse models of resolving and persistent arthritis. Single-cell transcriptional analysis identified two distinct fibroblast subsets within the FAPα + population: FAPα + THY1 + immune effector fibroblasts located in the synovial sub-lining, and FAPα + THY1 − destructive fibroblasts restricted to the synovial lining layer. When adoptively transferred into the joint, FAPα + THY1 − fibroblasts selectively mediate bone and cartilage damage with little effect on inflammation, whereas transfer of FAPα + THY1 + fibroblasts resulted in a more severe and persistent inflammatory arthritis, with minimal effect on bone and cartilage. Our findings describing anatomically discrete, functionally distinct fibroblast subsets with non-overlapping functions have important implications for cell-based therapies aimed at modulating inflammation and tissue damage. Distinct subsets of fibroblasts, which differ in their expression of thymus cell antigen 1 (THY1), are responsible for inflammation and tissue damage in mouse models of arthritis.

We investigate the contribution of a candidate gene, fiz ( fezzik ), to complex polygenic adaptation to juvenile malnutrition in Drosophila melanogaster . Experimental populations maintained for >250 generations of experimental evolution to a nutritionally poor larval diet (Selected populations) evolved several-fold lower fiz expression compared to unselected Control populations. Here we show that this divergence in fiz expression is mediated by a cis -regulatory polymorphism. This polymorphism, originally sampled from a natural population in Switzerland, is distinct from a second cis -regulatory SNP previously identified in non-African D . melanogaster populations, implying that two independent cis -regulatory variants promoting high fiz expression segregate in non-African populations. Enzymatic analyses of Fiz protein expressed in E . coli demonstrate that it has ecdysone oxidase activity acting on both ecdysone and 20-hydroxyecdysone. Four of five fiz paralogs annotated to ecdysteroid metabolism also show reduced expression in Selected larvae, implying that malnutrition-driven selection favored general downregulation of ecdysone oxidases. Finally, as an independent test of the role of fiz in poor diet adaptation, we show that fiz knockdown by RNAi results in faster larval growth on the poor diet, but at the cost of greatly reduced survival. These results imply that downregulation of fiz in Selected populations was favored by selection on the nutritionally poor diet because of its role in suppressing growth in response to nutrient shortage. However, they suggest that fiz downregulation is only adaptive in combination with other changes evolved by Selected populations, which ensure that the organism can sustain the faster growth promoted by fiz downregulation.

While mortality is often the primary focus of pathogen virulence, non‐lethal consequences, particularly for male reproductive fitness, are less understood; however, they are essential for understanding how sexual selection contributes to promoting resistance. We investigated how the fungal pathogen Metarhizium brunneum affects mating ability, fertility, and seminal fluid protein (SFP) expression of male Drosophila melanogaster paired with highly receptive virgin females in non‐competitive settings. Depending on sex and dose, there was a 3–6‐day incubation period after infection, followed by an abrupt onset of mortality. Meanwhile, the immune response was strongly induced already 38 h after infection and continued to increase as infection progressed. Latency to mate somewhat increased during the incubation period compared to sham‐treated males, but even on Day 5 post infection >90% of infected males mated within 2 h. During the incubation period, M. brunneum infection reduced male reproductive potential (the number of offspring sired without mate limitation) by 11%, with no clear increase over time. Approaching the end of the incubation period, infected males had lower ability to convert number of mating opportunities into number of offspring. After repeated mating, infected males had lower SFP expression than sham controls, more so in males that mated with few mates 24 h earlier. Overall, despite strong activation of the immune response, males' mating ability and fertility remained surprisingly little affected by the fungal infection, even shortly before the onset of mortality. This suggests that the selection for resistance acts mainly through mortality, and the scope for fertility selection to enhance resistance in non‐competing settings is rather limited. This study investigates the effects of Metarhizium brunneum, a fungal pathogen, on the reproductive potential of male Drosophila melanogaster, covering both the pre‐copulatory and post‐copulatory aspects. We focused on scenarios where males paired with receptive virgin females in non‐competitive environments and measured male's fitness through the course of infection. Despite strong activation of the immune response, males' mating ability and fertility remained surprisingly little affected by the fungal infection, even shortly before the onset of mortality.

Juvenile undernutrition has lasting effects on adult metabolism of the affected individuals, but it is unclear how adult physiology is shaped over evolutionary time by natural selection driven by juvenile undernutrition. We combined RNAseq, targeted metabolomics, and genomics to study the consequences of evolution under juvenile undernutrition for metabolism of reproductively active adult females of Drosophila melanogaster . Compared to Control populations maintained on standard diet, Selected populations maintained for over 230 generations on a nutrient-poor larval diet evolved major changes in adult gene expression and metabolite abundance, in particular affecting amino acid and purine metabolism. The evolved differences in adult gene expression and metabolite abundance between Selected and Control populations were positively correlated with the corresponding differences previously reported for Selected versus Control larvae. This implies that genetic variants affect both stages similarly. Even when well fed, the metabolic profile of Selected flies resembled that of flies subject to starvation. Finally, Selected flies had lower reproductive output than Controls even when both were raised under the conditions under which the Selected populations evolved. These results imply that evolutionary adaptation to juvenile undernutrition has large pleiotropic consequences for adult metabolism, and that they are costly rather than adaptive for adult fitness. Thus, juvenile and adult metabolism do not appear to evolve independently from each other even in a holometabolous species where the two life stages are separated by a complete metamorphosis.

The geometric framework of nutrition predicts that populations restricted to a single imbalanced diet should evolve post-ingestive nutritional compensation mechanisms bringing the blend of assimilated nutrients closer to physiological optimum. The evolution of such nutritional compensation is thought to be mainly driven by the ratios of major nutrients rather than overall nutritional content of the diet. We report experimental evolution of divergence in post-ingestive nutritional compensation in populations of Drosophila melanogaster adapted to diets that contained identical imbalanced nutrient ratios but differed in total nutrient concentration. Larvae from ‘Selected’ populations maintained for over 200 generations on a nutrient-poor diet with a 1 : 13.5 protein : carbohydrate ratio showed enhanced assimilation of nitrogen from yeasts and reduced assimilation of carbon from sucrose than ‘Control’ populations evolved on a diet with the same nutrient ratio but fourfold greater nutrient concentration. Compared to the Controls, the Selected larvae also accumulated less triglycerides relative to protein. This implies that the Selected populations evolved a higher assimilation rate of amino acids from the poor imbalanced diet and a lower assimilation of carbohydrates than Controls. Thus, the evolution of nutritional compensation may be driven by changes in total nutrient abundance, even if the ratios of different nutrients remain unchanged.

The geometric framework of nutrition predicts that populations restricted to a single imbalanced diet should evolve post-ingestive nutritional compensation mechanisms bringing the blend of assimilated nutrients closer to physiological optimum. The evolution of such nutritional compensation is thought to be mainly driven by the ratios of major nutrients rather than overall nutritional content of the diet. We report experimental evolution of divergence in post-ingestive nutritional compensation in populations of Drosophila melanogaster adapted to diets that contained identical imbalanced nutrient ratios but differed in total nutrient concentration. Larvae from ‘Selected’ populations maintained for over 200 generations on a nutrient-poor diet with a 1 : 13.5 protein : carbohydrate ratio showed enhanced assimilation of nitrogen from yeasts and reduced assimilation of carbon from sucrose than ‘Control’ populations evolved on a diet with the same nutrient ratio but fourfold greater nutrient concentration. Compared to the Controls, the Selected larvae also accumulated less triglycerides relative to protein. This implies that the Selected populations evolved a higher assimilation rate of amino acids from the poor imbalanced diet and a lower assimilation of carbohydrates than Controls. Thus, the evolution of nutritional compensation may be driven by changes in total nutrient abundance, even if the ratios of different nutrients remain unchanged.

Shared developmental, physiological, and molecular mechanisms can generate strong genetic covariances across suites of traits, constraining genetic variability, and evolvability to certain axes in multivariate trait space (“variational modules” or “syndromes”). Such trait suites will not only respond jointly to selection; they will also covary across populations that diverged from one another by genetic drift. We report evidence for such a genetically correlated trait suite that links traits related to energy metabolism along a “power-endurance” axis in Drosophila melanogaster. The “power” pole of the axis is characterized by high potential for energy generation and expenditure—high expression of glycolysis and TCA cycle genes, high abundance of mitochondria, and high spontaneous locomotor activity. The opposite “endurance” pole is characterized by high triglyceride (fat) reserves, locomotor endurance, and starvation resistance (and low values of traits associated with the “power” pole). This trait suite also aligns with the first principal component of metabolome; the “power” direction is characterized by low levels of trehalose (blood sugar) and high levels of some amino acids and their derivatives, including creatine, a compound known to facilitate energy production in muscles. Our evidence comes from six replicate “Selected” populations adapted to a nutrient-poor larval diet regime during 250 generations of experimental evolution and six “Control” populations evolved in parallel on a standard diet regime. We found that, within each of these experimental evolutionary regimes, the above traits strongly covaried along this “power-endurance” axis across replicate populations which diversified by drift, indicating a shared genetic architecture. The two evolutionary regimes also drove divergence along this axis, with Selected populations on average displaced towards the “power” direction compared to Controls. Aspects of this “power-endurance” axis resemble the “pace of life” syndrome and the “thrifty phenotype”; it may have evolved as part of a coordinated organismal response to nutritional conditions.

Periodic food shortage is a common ecological stressor for animals, likely to drive physiological and metabolic adaptations to alleviate its consequences, particularly for juveniles that have no option but to continue to grow and develop despite undernutrition. Here we study changes in metabolism associated with adaptation to nutrient shortage, evolved by replicate Drosophila melanogaster populations maintained on a nutrient-poor larval diet for over 240 generations. In a factorial metabolomics experiment we showed that both phenotypic plasticity and genetically-based adaptation to the poor diet involved wide-ranging changes in metabolite abundance; however, the plastic response did not predict the evolutionary change. Compared to nonadapted larvae exposed to the poor diet for the first time, the adapted larvae showed lower levels of multiple free amino acids in their tissues—and yet they grew faster. By quantifying accumulation of the nitrogen stable isotope 15N we show that adaptation to the poor diet led to an increased use of amino acids for energy generation. This apparent “waste” of scarce amino acids likely results from the trade-off between acquisition of dietary amino acids and carbohydrates observed in these populations. The three branched-chain amino acids (leucine, isoleucine, and valine) showed a unique pattern of depletion in adapted larvae raised on the poor diet. A diet supplementation experiment demonstrated that these amino acids are limiting for growth on the poor diet, suggesting that their low levels resulted from their expeditious use for protein synthesis. These results demonstrate that selection driven by nutrient shortage not only promotes improved acquisition of limiting nutrients, but also has wide-ranging effects on how the nutrients are used. They also show that the abundance of free amino acids in the tissues does not, in general, reflect the nutritional condition and growth potential of an animal.

BackgroundStromal cells are key effector cells in tissue inflammation and damage. Despite their importance these cells are yet to be targeted therapeutically. We have previously identified a distinct subset of stromal cells defined by their expression of a cassette of cell surface proteins including Podoplanin/gp38 (PDPN), Fibroblast activation protein (FAP) and Cadherin-11. The aim of this study was to determine the pathogenic role of these cells in tissue inflammation.MethodsSalivary gland inflammation was induced by intra-ductal administration of a replication-deficient adenovirus resulting in tertiary lymphoid structure (TLS) formation as previously described.1 Polyarthritis was induced using the KRN serum transfer model as previously described.2 To delete FAP-expressing cells we used a model of conditional depletion of FAP-expressing cells, in which FAP-DTR mice were treated with Diphtheria Toxin Disease severity was assessed clinically, histologically and by MicroCT and a combination of immunofluorescence, quantitative RT-PCR and flow cytometry was performed on digested salivary glands and synovial membrane tissue.ResultsTissue resident stromal cells dynamically express FAP, PDPN and cadherin-11 during inflammation. This cassette of cell surface markers defined a population of stromal cells that expand during inflammation by in situ proliferation. In the salivary gland, these pathogeneic cells are found surrounding ectopic lymphoid aggregates where they produce lymphoid chemokines and survival factors. In the synovium, they are localised to the synovial lining layer and pannus tissue where they invade articular cartilage and bone.Selective deletion of FAP expressing cells resulted in a defect in lymphoid chemokine production, decreased number of infiltrating lymphocytes and severely inhibited TLO formation in the salivary gland. In the joint, therapeutic deletion significantly attenuated synovial inflammation, inflammatory cell infiltration, reduced the severity of arthritis and protected against joint damage.ConclusionsWe have identified a pathogenic subset of stromal cells defined by their expression of common cassette of cell surface markers. Whilst the phenotype of these cells is common to inflammation our data suggests their function is both tissue and disease specific.References1. Bombardieri, Barone, et al. JI. 2012.2. Huang, et al. Arthrits Rheumatol2014.

BackgroundStromal cells are key effector cells in tissue inflammation and damage. Despite their importance these cells are yet to be targeted therapeutically. We have previously identified a distinct subset of stromal cells defined by their expression of a cassette of cell surface proteins including Podoplanin/gp38 (PDPN), Fibroblast activation protein (FAP) and Cadherin-11. The aim of this study was to determine the pathogenic role of these cells in tissue inflammation.MethodsSalivary gland inflammation was induced by intra-ductal administration of a replication-deficient adenovirus resulting in tertiary lymphoid structure (TLS) formation as previously described.1 Polyarthritis was induced using the KRN serum transfer model as previously described.2 To delete FAP-expressing cells we used a model of conditional depletion of FAP-expressing cells, in which FAP-DTR mice were treated with Diphtheria Toxin Disease severity was assessed clinically, histologically and by MicroCT and a combination of immunofluorescence, quantitative RT-PCR and flow cytometry was performed on digested salivary glands and synovial membrane tissue.ResultsTissue resident stromal cells dynamically express FAP, PDPN and cadherin-11 during inflammation. This cassette of cell surface markers defined a population of stromal cells that expand during inflammation by in situ proliferation. In the salivary gland, these pathogeneic cells are found surrounding ectopic lymphoid aggregates where they produce lymphoid chemokines and survival factors. In the synovium, they are localised to the synovial lining layer and pannus tissue where they invade articular cartilage and bone.Selective deletion of FAP expressing cells resulted in a defect in lymphoid chemokine production, decreased number of infiltrating lymphocytes and severely inhibited TLO formation in the salivary gland. In the joint, therapeutic deletion significantly attenuated synovial inflammation, inflammatory cell infiltration, reduced the severity of arthritis and protected against joint damage.ConclusionsWe have identified a pathogenic subset of stromal cells defined by their expression of common cassette of cell surface markers. Whilst the phenotype of these cells is common to inflammation our data suggests their function is both tissue and disease specific.References1. Bombardieri, Barone, et al. JI. 2012.2. Huang, et al. Arthrits Rheumatol2014.