Formylated Peptide Receptor-1-Mediated Gut Inflammation as a Therapeutic Target in Inflammatory Bowel Disease.

Background: Formylated peptide receptor (FPR)-1 is a G-coupled receptor that senses foreign bacterial and host-derived mitochondrial formylated peptides (FPs), leading to innate immune system activation. Aim: We sought to investigate the role of FPR1-mediated inflammation and its potential as a therapeutic target in inflammatory bowel disease (IBD). Methods: We characterized FPR1 gene and protein expression in 8 human IBD (~1000 patients) datasets with analysis on disease subtype, mucosal inflammation, and drug response. We performed in vivo dextran-sulfate sodium (DSS) colitis in C57/BL6 FPR1 knockout mice. In ex vivo studies, we studied the role of mitochondrial FPs and pharmacological blockade of FPR1 using cyclosporin H in human peripheral blood neutrophils. Finally, we assess mitochondrial FPs as a potential mechanistic biomarker in the blood and stools of patients with IBD. Results: Detailed in silico analysis in human intestinal biopsies showed that FPR1 is highly expressed in IBD (n = 207 IBD vs 67 non-IBD controls, P < .001), and highly correlated with gut inflammation in ulcerative colitis (UC) and Crohn's disease (CD) (both P < .001). FPR1 receptor is predominantly expressed in leukocytes, and we showed significantly higher FPR1+ve neutrophils in inflamed gut tissue section in IBD (17 CD and 24 UC; both P < .001). Further analysis in 6 independent IBD (data available under Gene Expression Omnibus accession numbers GSE59071, GSE206285, GSE73661, GSE16879, GSE92415, and GSE235970) showed an association with active gut inflammation and treatment resistance to infliximab, ustekinumab, and vedolizumab. FPR1 gene deletion is protective in murine DSS colitis with lower gut neutrophil inflammation. In the human ex vivo neutrophil system, mitochondrial FP, nicotinamide adenine dinucleotide dehydrogenase subunit-6 (ND6) is a potent activator of neutrophils resulting in higher CD62L shedding, CD63 expression, reactive oxygen species production, and chemotactic capacity; these effects are inhibited by cyclosporin H. We screened for mitochondrial ND6 in IBD (n = 54) using ELISA and detected ND6 in stools with median values of 2.2 gg/mL (interquartile range [IQR] 0.0-4.99; range 0-53.3) but not in blood. Stool ND6 levels, however, were not significantly correlated with paired stool calprotectin, C-reactive protein, and clinical IBD activity. Conclusions: Our data suggest that FPR1-mediated neutrophilic inflammation is a tractable target in IBD; however, further work is required to clarify the clinical utility of mitochondrial FPs as a potential mechanistic marker for future stratification.

Anthropogenic fragmentation may not alter pre-existing patterns of genetic diversity and differentiation in perennial shrubs.

Many plant species have pollination and seed dispersal systems and evolutionary histories that have produced strong genetic structuring. These genetic patterns may be consistent with expectations following recent anthropogenic fragmentation, making it difficult to detect fragmentation effects if no prefragmentation genetic data are available. We used microsatellite markers to investigate whether severe habitat fragmentation may have affected the structure and diversity of populations of the endangered Australian bird-pollinated shrub Grevillea caleyi R.Br., by comparing current patterns of genetic structure and diversity with those of the closely related G. longifolia R.Br. that has a similar life history but has not experienced anthropogenic fragmentation. Grevillea caleyi and G. longifolia showed similar and substantial population subdivision at all spatial levels (global F'ST  = 0.615 and 0.454; Sp  = 0.039 and 0.066), marked isolation by distance and large heterozygous deficiencies. These characteristics suggest long-term effects of inbreeding in self-compatible species that have poor seed dispersal, limited connectivity via pollen flow and undergo population bottlenecks because of periodic fires. Highly structured allele size distributions, most notably in G. caleyi, imply historical processes of drift and mutation were important in isolated subpopulations. Genetic diversity did not vary with population size but was lower in more isolated populations for both species. Through this comparison, we reject the hypothesis that anthropogenic fragmentation has impacted substantially on the genetic composition or structure of G. caleyi populations. Our results suggest that highly self-compatible species with limited dispersal may be relatively resilient to the genetic changes predicted to follow habitat fragmentation.

Tests for inbreeding and outbreeding depression and estimation of population differentiation in the bird-pollinated shrub Grevillea mucronulata.

BACKGROUND AND AIMS: Plants show patterns of spatial genetic differentiation reflecting gene flow mediated by pollen and seed dispersal and genotype × environment interactions. If patterns of genetic structure are determined largely by gene flow then they may be useful in predicting the likelihood of inbreeding or outbreeding depression but should be less useful if there is strong site-specific selection. For many Australian plants little is known about either their population genetics or the effects on mating systems of variation in pollen transfer distances. Experimental pollinations were used to compare the reproductive success of bird-adapted Grevillea mucronulata plants mated with individuals from a range of spatial scales. A hierarchical survey of microsatellite DNA variation was also conducted to describe the scale of population differentiation for neutral markers. METHODS: The effects of four pollen treatments on reproductive performance were compared. These treatments were characterized by transfer of pollen from (a) neighbouring adults; (b) an adjacent cluster of adults (30-50 m distant); (c) a distant cluster (>5 km distant); and (d) open pollination. Sets of 17·9 ± 3·3 leaves from each of 15 clusters of plants were genotyped and spatial autocorrelation and F statistics were used to describe patterns of genetic structure. KEY RESULTS: Grevillea mucronulata displayed evidence of both inbreeding and outbreeding depression, with 'intermediate' pollen producing consistently superior outcomes for most aspects of fitness including seed set, seed size, germination and seedling growth. Significant genotypic structuring was detected within clusters (spatial autocorrelation) and among adjacent clusters and clusters separated by >5 km distance (F(ST) = 0·07 and 0·10). CONCLUSIONS: The superior outcome of intermediate pollen transfer and genetic differentiation of adjacent clusters suggests that G. mucronulata selection disfavours matings among closely and distantly related neighbours. Moreover, the performance of open-pollinated seedlings was poor, implying that current mating patterns are suboptimal.

The importance of pre-mating barriers and the local demographic context for contemporary mating patterns in hybrid zones of Eucalyptus aggregata and Eucalyptus rubida.

The frequency of hybridization in plants is context dependent and can be influenced by the local mating environment. We used progeny arrays and admixture and pollen dispersal analyses to assess the relative importance of pre-mating reproductive barriers and the local demographic environment as explanations of variation in hybrid frequency in three mapped hybrid zones of Eucalyptus aggregata and E. rubida. A total of 731 open-pollinated progeny from 36 E. aggregata maternal parents were genotyped using six microsatellite markers. Admixture analysis identified substantial variation in hybrid frequency among progeny arrays (0-76.9%). In one hybrid zone, hybrid frequency was related to pre-mating barriers (degree of flowering synchrony) and demographic components of the local mating environment (decreasing population size, closer proximity to E. rubida and hybrid trees). At this site, average pollen dispersal distance was less and almost half (46%) of the hybrid progeny were sired by local E. rubida and hybrid trees. In contrast, at the other two sites, pre-mating and demographic factors were not related to hybrid frequency. Compared to the first hybrid zone where most of the E. rubida (76%) and all hybrids flowered, in the remaining sites fewer E. rubida (22-41%) and hybrid trees (0-50%) flowered and their reproductive success was lower (sired 0-23% of hybrids). As a result, most hybrids were sired by external E. rubida/hybrids located at least 2-3 km away. These results indicate that although pre-mating barriers and local demography can influence patterns of hybridization, their importance can depend upon the scale of pollen dispersal.

The birds and the bees: pollinator behaviour and variation in the mating system of the rare shrub Grevillea macleayana.

BACKGROUND AND AIMS: In Australia, honey-bees have invaded systems that evolved without social insect pollinators, where many plants are adapted to vertebrate pollination. Behavioural differences between pollinators are likely to influence mating patterns, but few studies have examined this empirically in long-lived, woody, perennials. It was shown previously that outcrossing rates in Grevillea macleayana vary among populations. Here tests were conducted to determine whether the behaviour of birds and honey-bees differed between a population previously found to be highly outcrossed and two inbreeding populations. METHODS: Visit frequencies and movement patterns of the visitors to inflorescences at three sites over two seasons were compared. A caging experiment was used to test the effects of excluding birds on pollen removal from newly opened flowers and on pollen deposition on stigmas that had been washed clean. KEY RESULTS: Honey-bees were the most frequent visitors overall, but honeyeaters were more frequent visitors in the population previously found to have a high outcrossing rate than they were in either of the other populations. More visits by honeyeaters were from distant plants. Pollen removal did not vary greatly among sites, and was not affected by bird exclusion; however, more pollen was deposited on the stigmas of cleaned pollen presenters in the population previously observed to be highly outcrossing than in the other two. This high level of pollen deposition was reduced by experimental bird exclusion. CONCLUSIONS: Honey-bees were the most frequent visitors, by an order of magnitude, and excluding vertebrates revealed that bees were removing most of the pollen but deposited fewer pollen grains on stigmas. Birds were more frequent visitors at the site previously found to be outcrossing than the other two sites, and they moved further between plants and visited fewer inflorescences on a plant during a foraging bout than bees did. These characteristics of bird visits to G. macleayana would be sufficient to produce significant variation in outcrossing rates among sites.

Urban plants as genetic reservoirs or threats to the integrity of bushland plant populations.

Remnant plants in urban fringes and native plants in gardens have the potential to contribute to the conservation of threatened plants by increasing genetic diversity, effective size of populations, and levels of genetic connectedness. But they also pose a threat through the disruption of locally adapted gene pools. At Hyams Beach, New South Wales, Australia, four bushland stands of the rare shrub, Grevillea macleayana McGillivray, surround an urban area containing remnant and cultivated specimens of this species. Numbers of inflorescences per plant, fruits per plant, and visits by pollinators were similar for plants in urban gardens and bushland. Urban plants represented a substantial but complex genetic resource, displaying more genetic diversity than bushland plants judged byH(e), numbers of alleles per locus, and number of private alleles. Of 27 private alleles in urban plants, 17 occurred in a set of 19 exotic plants. Excluding the exotic plants, all five stands displayed a moderate differentiation (F(ST)= 0.14 +/- 0.02), although the urban remnants clustered with two of the bushland stands. These patterns may be explained by high levels of selfing and inbreeding in this species and by long-distance dispersal (several seeds in the urban stand were fathered by plants in other stands). Genetic leakage (gene flow) from exotic plants to 321 seeds on surrounding remnant or bushland plants has not occurred. Our results demonstrate the conservation value of this group of urban plants, which are viable, productive, genetically diverse, and interconnected with bushland plants. Gene flow has apparently not yet led to genetic contamination of bushland populations, but high levels of inbreeding would make this a rare event and difficult to detect.

Dormancy and the fire-centric focus: germination of three Leucopogon species (Ericaceae) from South-eastern Australia.

BACKGROUND AND AIMS: Germination studies of species from fire-prone habitats are often focused on the role that fire plays in breaking dormancy. However, for some plant groups in these habitats, such as the genus Leucopogon (Ericaceae), dormancy of fresh seeds is not broken by fire cues. In the field, these same species display a flush of seedling emergence post-fire. Dormancy and germination mechanisms therefore appear complex and mostly unknown. This study aimed to identify these mechanisms by establishing dormancy class and testing the effects of a set of typical germination cues, including those directly related to fire and entirely independent of fire. METHODS: To classify dormancy, we assessed seed permeability and embryo morphology, and conducted germination experiments at seasonal temperatures in incubators. To test the effects of fire cues on germination, factorial combinations of smoke, heat and dark treatments were applied. Ageing treatments, using burial and seasonal incubation, were also tested. Germination phenology was established. KEY RESULTS: Seeds were dormant at release and had underdeveloped embryos. Primary dormancy of the study species was classified as morphophysiological. Seasonal temperature changes overcame primary dormancy and controlled timing of germination. Fire cues did not break primary dormancy, but there was a trend for smoke to enhance germination once this dormancy was overcome. CONCLUSIONS: Despite the fact that fire is a predominant disturbance and that many species display a flush of emergence post-fire, seasonal temperatures broke the primary physiological dormancy of the study species. It is important to distinguish between fire being responsible for breaking dormancy and solely having a role in enhancing levels of post-fire germination for seeds in which dormancy has been overcome by other factors. Biogeographical evidence suggests that morphological and physiological factors, and therefore seasonal temperatures, are likely to be important in controlling the dormancy and patterns of post-fire germination of many species in fire-prone regions.

Microsatellite diversity and genetic structure of fragmented populations of the rare, fire-dependent shrub Grevillea macleayana.

Recent habitat loss and fragmentation superimposed upon ancient patterns of population subdivision are likely to have produced low levels of neutral genetic diversity and marked genetic structure in many plant species. The genetic effects of habitat fragmentation may be most pronounced in species that form small populations, are fully self-compatible and have limited seed dispersal. However, long-lived seed banks, mobile pollinators and long adult lifespans may prevent or delay the accumulation of genetic effects. We studied a rare Australian shrub species, Grevillea macleayana (Proteaceae), that occurs in many small populations, is self-compatible and has restricted seed dispersal. However, it has a relatively long adult lifespan (c. 30 years), a long-lived seed bank that germinates after fire and is pollinated by birds that are numerous and highly mobile. These latter characteristics raise the possibility that populations in the past may have been effectively large and genetically homogeneous. Using six microsatellites, we found that G. macleayana may have relatively low within-population diversity (3.2-4.2 alleles/locus; Hexp = 0.420-0.530), significant population differentiation and moderate genetic structure (FST = 0.218) showing isolation by distance, consistent with historically low gene flow. The frequency distribution of allele sizes suggest that this geographical differentiation is being driven by mutation. We found a lack mutation-drift equilibrium in some populations that is indicative of population bottlenecks. Combined with evidence for large spatiotemporal variation of selfing rates, this suggests that fluctuating population sizes characterize the demography in this species, promoting genetic drift. We argue that natural patterns of pollen and seed dispersal, coupled with the patchy, fire-shaped distribution, may have restricted long-distance gene flow in the past.

Masting, seed dispersal and seed predation in the cycad Macrozamia communis.

Little is known about the adaptive value of mast seeding, a common phenomenon in temperate trees and shrubs. Masting is likely to affect both seed dispersal and seed predation. In systems where similar taxa of animals are involved in these two processes, the consequences of mast seeding are likely to be particularly complicated. This study examined the effects of mast seeding in a cycad, Macrozamia communis, on the dispersal of seeds, the pattern of dispersion of seeds and post-dispersal predation on seeds. Dispersal of seeds by possums was poorer from source plants in a masting population than from source plants in an adjacent, non-masting population. This resulted in fewer seeds per seeding female plant in the masting plot being dispersed to favourable sites. We conclude that this is caused by the feeding behaviour and movements of possums in the masting site. The abundance of seeds in this site did not satiate the post-dispersal predators, native rats. In fact, more seeds in this site were eaten than in the nonmasting site. We suggest that the mast seeding observed in M. communis may not be adaptive, but is more likely a consequence of other factors which synchromize flowering within local populations.