Combinatorial actions of IL-22 and IL-17 drive optimal immunity to oral candidiasis through SPRRs.

Oropharyngeal candidiasis (OPC) is the most common human fungal infection, arising typically from T cell immune impairments. IL-17 and IL-22 contribute individually to OPC responses, but here we demonstrate that the combined actions of both cytokines are essential for resistance to OPC. Mice lacking IL-17RA and IL-22RA1 exhibited high fungal loads in esophagus- and intestinal tract, severe weight loss, and symptoms of colitis. Ultimately, mice succumbed to infection. Dual loss of IL-17RA and IL-22RA impaired expression of small proline rich proteins (SPRRs), a class of antimicrobial effectors not previously linked to fungal immunity. Sprr2a1 exhibited direct candidacidal activity in vitro, and Sprr1-3a-/- mice were susceptible to OPC. Thus, cooperative actions of Type 17 cytokines mediate oral mucosal anti-Candida defenses and reveal a role for SPRRs.

Evolutionary innovation through transcription factor rewiring in microbes is shaped by levels of transcription factor activity, expression, and existing connectivity.

The survival of a population during environmental shifts depends on whether the rate of phenotypic adaptation keeps up with the rate of changing conditions. A common way to achieve this is via change to gene regulatory network (GRN) connections-known as rewiring-that facilitate novel interactions and innovation of transcription factors. To understand the success of rapidly adapting organisms, we therefore need to determine the rules that create and constrain opportunities for GRN rewiring. Here, using an experimental microbial model system with the soil bacterium Pseudomonas fluorescens, we reveal a hierarchy among transcription factors that are rewired to rescue lost function, with alternative rewiring pathways only unmasked after the preferred pathway is eliminated. We identify 3 key properties-high activation, high expression, and preexisting low-level affinity for novel target genes-that facilitate transcription factor innovation. Ease of acquiring these properties is constrained by preexisting GRN architecture, which was overcome in our experimental system by both targeted and global network alterations. This work reveals the key properties that determine transcription factor evolvability, and as such, the evolution of GRNs.

C/EBPδ Mediates Immunity to Renal Autoinflammatory Disorders in a Stage-specific Manner.

Kidney disease represents a major medical and economic burden for which improved treatments are urgently needed. Emerging data have implicated Th17 cells and IL-17 signaling in the underlying pathogenesis of autoantibody-induced glomerulonephritis (AGN). However, the downstream transduction pathways mediated by IL-17 in autoimmunity are not well defined. In this article, we show that CCAAT/enhancer-binding protein (C/EBP) δ is elevated in kidney biopsies from multiple manifestations of human AGN. C/EBPδ is similarly upregulated in a mouse model of anti-glomerular basement membrane protein-mediated kidney disease, and Cebpd-/- mice were fully refractory to disease. Although C/EBPδ is expressed in a variety of cell types, C/EBPδ was required only in the radioresistant compartment to drive GN pathology. C/EBPδ induced expression of several IL-17-induced kidney injury markers and cytokines implicated in disease, including Il6 and Lcn2. Because mouse AGN models do not progress to fibrosis, we employed a nephrotoxic injury model using aristolochic acid I to assess the contribution of the IL-17-C/EBPδ pathway to renal fibrotic events. Surprisingly, deficiency of either C/EBPδ or the IL-17 receptor caused kidney fibrosis to be enhanced. Thus, C/EBPδ and IL-17 play divergent and apparently stage-specific roles in the pathogenesis of kidney disease.

SARS-CoV-2 ORF8 Mediates Signals in Macrophages and Monocytes through MyD88 Independently of the IL-17 Receptor.

SARS-CoV-2 has caused an estimated 7 million deaths worldwide to date. A secreted SARS-CoV-2 accessory protein, known as open reading frame 8 (ORF8), elicits inflammatory pulmonary cytokine responses and is associated with disease severity in COVID-19 patients. Recent reports proposed that ORF8 mediates downstream signals in macrophages and monocytes through the IL-17 receptor complex (IL-17RA, IL-17RC). However, generally IL-17 signals are found to be restricted to the nonhematopoietic compartment, thought to be due to rate-limiting expression of IL-17RC. Accordingly, we revisited the capacity of IL-17 and ORF8 to induce cytokine gene expression in mouse and human macrophages and monocytes. In SARS-CoV-2-infected human and mouse lungs, IL17RC mRNA was undetectable in monocyte/macrophage populations. In cultured mouse and human monocytes and macrophages, ORF8 but not IL-17 led to elevated expression of target cytokines. ORF8-induced signaling was fully preserved in the presence of anti-IL-17RA/RC neutralizing Abs and in Il17ra-/- cells. ORF8 signaling was also operative in Il1r1-/- bone marrow-derived macrophages. However, the TLR/IL-1R family adaptor MyD88, which is dispensable for IL-17R signaling, was required for ORF8 activity yet MyD88 is not required for IL-17 signaling. Thus, we conclude that ORF8 transduces inflammatory signaling in monocytes and macrophages via MyD88 independently of the IL-17R.

Arid5a Mediates an IL-17-Dependent Pathway That Drives Autoimmunity but Not Antifungal Host Defense.

IL-17 contributes to the pathogenesis of certain autoimmune diseases, but conversely is essential for host defense against fungi. Ab-based biologic drugs that neutralize IL-17 are effective in autoimmunity but can be accompanied by adverse side effects. Candida albicans is a commensal fungus that is the primary causative agent of oropharyngeal and disseminated candidiasis. Defects in IL-17 signaling cause susceptibility to candidiasis in mice and humans. A key facet of IL-17 receptor signaling involves RNA-binding proteins, which orchestrate the fate of target mRNA transcripts. In tissue culture models we showed that the RNA-binding protein AT-rich interaction domain 5A (Arid5a) promotes the stability and/or translation of multiple IL-17-dependent mRNAs. Moreover, during oropharyngeal candidiasis, Arid5a is elevated within the oral mucosa in an IL-17-dependent manner. However, the contribution of Arid5a to IL-17-driven events in vivo is poorly defined. In this study, we used CRISPR-Cas9 to generate mice lacking Arid5a. Arid5a -/- mice were fully resistant to experimental autoimmune encephalomyelitis, an autoimmune setting in which IL-17 signaling drives pathology. Surprisingly, Arid5a -/- mice were resistant to oropharyngeal candidiasis and systemic candidiasis, similar to immunocompetent wild-type mice and contrasting with mice defective in IL-17 signaling. Therefore, Arid5a-dependent signals mediate pathology in autoimmunity and yet are not required for immunity to candidiasis, indicating that selective targeting of IL-17 signaling pathway components may be a viable strategy for development of therapeutics that spare IL-17-driven host defense.

Mutation bias and adaptation in bacteria.

Genetic mutation, which provides the raw material for evolutionary adaptation, is largely a stochastic force. However, there is ample evidence showing that mutations can also exhibit strong biases, with some mutation types and certain genomic positions mutating more often than others. It is becoming increasingly clear that mutational bias can play a role in determining adaptive outcomes in bacteria in both the laboratory and the clinic. As such, understanding the causes and consequences of mutation bias can help microbiologists to anticipate and predict adaptive outcomes. In this review, we provide an overview of the mechanisms and features of the bacterial genome that cause mutational biases to occur. We then describe the environmental triggers that drive these mechanisms to be more potent and outline the adaptive scenarios where mutation bias can synergize with natural selection to define evolutionary outcomes. We conclude by describing how understanding mutagenic genomic features can help microbiologists predict areas sensitive to mutational bias, and finish by outlining future work that will help us achieve more accurate evolutionary forecasts.

Mutational hotspots lead to robust but suboptimal adaptive outcomes in certain environments.

The observed mutational spectrum of adaptive outcomes can be constrained by many factors. For example, mutational biases can narrow the observed spectrum by increasing the rate of mutation at isolated sites in the genome. In contrast, complex environments can shift the observed spectrum by defining fitness consequences of mutational routes. We investigate the impact of different nutrient environments on the evolution of motility in Pseudomonas fluorescens Pf0-2x (an engineered non-motile derivative of Pf0-1) in the presence and absence of a strong mutational hotspot. Previous work has shown that this mutational hotspot can be built and broken via six silent mutations, which provide rapid access to a mutation that rescues swimming motility and confers the strongest swimming phenotype in specific environments. Here, we evolved a hotspot and non-hotspot variant strain of Pf0-2x for motility under nutrient-rich (LB) and nutrient-limiting (M9) environmental conditions. We observed the hotspot strain consistently evolved faster across all environmental conditions and its mutational spectrum was robust to environmental differences. However, the non-hotspot strain had a distinct mutational spectrum that changed depending on the nutrient environment. Interestingly, while alternative adaptive mutations in nutrient-rich environments were equal to, or less effective than, the hotspot mutation, the majority of these mutations in nutrient-limited conditions produced superior swimmers. Our competition experiments mirrored these findings, underscoring the role of environment in defining both the mutational spectrum and the associated phenotype strength. This indicates that while mutational hotspots working in concert with natural selection can speed up access to robust adaptive mutations (which can provide a competitive advantage in evolving populations), they can limit exploration of the mutational landscape, restricting access to potentially stronger phenotypes in specific environments.

Characteristics associated with poor COVID-19 outcomes in individuals with systemic lupus erythematosus: data from the COVID-19 Global Rheumatology Alliance.

AIM: To determine characteristics associated with more severe outcomes in a global registry of people with systemic lupus erythematosus (SLE) and COVID-19. METHODS: People with SLE and COVID-19 reported in the COVID-19 Global Rheumatology Alliance registry from March 2020 to June 2021 were included. The ordinal outcome was defined as: (1) not hospitalised, (2) hospitalised with no oxygenation, (3) hospitalised with any ventilation or oxygenation and (4) death. A multivariable ordinal logistic regression model was constructed to assess the relationship between COVID-19 severity and demographic characteristics, comorbidities, medications and disease activity. RESULTS: A total of 1606 people with SLE were included. In the multivariable model, older age (OR 1.03, 95% CI 1.02 to 1.04), male sex (1.50, 1.01 to 2.23), prednisone dose (1-5 mg/day 1.86, 1.20 to 2.66, 6-9 mg/day 2.47, 1.24 to 4.86 and ≥10 mg/day 1.95, 1.27 to 2.99), no current treatment (1.80, 1.17 to 2.75), comorbidities (eg, kidney disease 3.51, 2.42 to 5.09, cardiovascular disease/hypertension 1.69, 1.25 to 2.29) and moderate or high SLE disease activity (vs remission; 1.61, 1.02 to 2.54 and 3.94, 2.11 to 7.34, respectively) were associated with more severe outcomes. In age-adjusted and sex-adjusted models, mycophenolate, rituximab and cyclophosphamide were associated with worse outcomes compared with hydroxychloroquine; outcomes were more favourable with methotrexate and belimumab. CONCLUSIONS: More severe COVID-19 outcomes in individuals with SLE are largely driven by demographic factors, comorbidities and untreated or active SLE. Patients using glucocorticoids also experienced more severe outcomes.

Enantioselective Synthesis of Functionalized Arenes by Nickel-Catalyzed Site-Selective Hydroarylation of 1,3-Dienes with Aryl Boronates.

A catalytic method for the site-selective and enantioselective synthesis of functionalized arenes by the intermolecular hydroarylation of terminal and internal 1,3-dienes with aryl pinacolato boronates is reported. The reactions are promoted by 5.0 mol % of a readily available monodentate phosphoramidite-Ni complex in ethanol, affording a variety of enantioenriched products in up to 96 % yield and 99:1 er. Mechanistic studies indicate that Ni-allyl formation is irreversible and related to the nature of the arylboronate.

Ecological and demographic correlates of cooperation from individual to budding dispersal.

Identifying the ecological and demographic factors that promote the evolution of cooperation is a major challenge for evolutionary biologists. Explanations for the adaptive evolution of cooperation seek to determine which factors make reproduction in cooperative groups more favourable than independent breeding or other selfish strategies. A vast majority of the hypotheses posit that cooperative groups emerge in the context of philopatry, high costs of dispersal, high population density and environmental stability. This route to cooperation, however, fails to explain a growing body of empirical evidence in which cooperation is not associated with one or more of these predictors. We propose an alternative evolutionary path towards the emergence of cooperation that accounts for the disparities observed in the current literature. We find that when dispersal is mediated by a group mode of dispersal, commonly termed budding dispersal, our mathematical model reveals an association between cooperation and immigration, lower costs of dispersal, low population density and environmental variability. Furthermore, by studying the continuum from the individual to the partial and full budding mode of dispersal, we can explicitly explain why the correlates of cooperation change under budding. This enables us to outline a general model for the evolution of cooperation that accounts for a substantial amount of empirical evidence. Our results suggest that natural selection may have favoured two major contrasting pathways for the evolution of cooperation depending on a set of key ecological and demographic factors.

Resource competition promotes tumour expansion in experimentally evolved cancer.

BACKGROUND: Tumour progression involves a series of phenotypic changes to cancer cells, each of which presents therapeutic targets. Here, using techniques adapted from microbial experimental evolution, we investigate the evolution of tumour spreading - a precursor for metastasis and tissue invasion - in environments with varied resource supply. Evolutionary theory predicts that competition for resources within a population will select for individuals to move away from a natal site (i.e. disperse), facilitating the colonisation of unexploited resources and decreasing competition between kin. RESULTS: After approximately 100 generations in environments with low resource supply, we find that MCF7 breast cancer spheroids (small in vitro tumours) show increased spreading. Conversely, spreading slows compared to the ancestor where resource supply is high. Common garden experiments confirm that the evolutionary responses differ between selection lines; with lines evolved under low resource supply showing phenotypic plasticity in spheroid spreading rate. These differences in spreading behaviour between selection lines are heritable (stable across multiple generations), and show that the divergently evolved lines differ in their response to resource supply. CONCLUSIONS: We observe dispersal-like behaviour and an increased sensitivity to resource availability in our selection lines, which may be a response to selection, or alternatively may be due to epigenetic changes, provoked by prolonged resource limitation, that have persisted across many cell generations. Different clinical strategies may be needed depending on whether or not tumour progression is due to natural selection. This study highlights the effectiveness of experimental evolution approaches in cancer cell populations and demonstrates how simple model systems might enable us to observe and measure key selective drivers of clinically important traits.

Comparison of magnetic resonance feature tracking for systolic and diastolic strain and strain rate calculation with spatial modulation of magnetization imaging analysis.

PURPOSE: To compare cardiovascular magnetic resonance-feature tracking (CMR-FT) with spatial modulation of magnetization (SPAMM) tagged imaging for the calculation of short and long axis Lagrangian strain measures in systole and diastole. MATERIALS AND METHODS: Healthy controls (n = 35) and patients with dilated cardiomyopathy (n = 10) were identified prospectively and underwent steady-state free precession (SSFP) cine imaging and SPAMM imaging using a gradient-echo sequence. A timed offline analysis of images acquired at identical horizontal long and short axis slice positions was performed using CMR-FT and dynamic tissue-tagging (CIMTag2D). Agreement between strain and strain rate (SR) values calculated using these two different methods was assessed using the Bland-Altman technique. RESULTS: Across all participants, there was good agreement between CMR-FT and CIMTag for calculation of peak systolic global circumferential strain (-22.7 ± 6.2% vs. -22.5 ± 6.9%, bias 0.2 ± 4.0%) and SR (-1.35 ± 0.42 1/s vs. -1.22 ± 0.42 1/s, bias 0.13 ± 0.33 1/s) and early diastolic global circumferential SR (1.21 ± 0.44 1/s vs. 1.07 ± 0.30 1/s, bias -0.14 ± 0.34 1/s) at the subendocardium. There was satisfactory agreement for derivation of peak systolic global longitudinal strain (-18.1 ± 5.0% vs. -16.7 ± 4.8%, bias 1.3 ± 3.8%) and SR (-1.04 ± 0.29 1/s vs. -0.95 ± 0.32 1/s, bias 0.09 ± 0.26 1/s). The weakest agreement was for early diastolic global longitudinal SR (1.10 ± 0.40 1/s vs. 0.67 ± 0.32 1/s, bias -0.42 ± 0.40 1/s), although the correlation remained significant (r = 0.42, P < 0.01). CMR-FT generated these data over four times quicker than CIMTag. CONCLUSION: There is sufficient agreement between systolic and diastolic strain measures calculated using CMR-FT and myocardial tagging for CMR-FT to be considered as a potentially feasible and rapid alternative.

The biosurfactant viscosin produced by Pseudomonas fluorescens SBW25 aids spreading motility and plant growth promotion.

Food security depends on enhancing production and reducing loss to pests and pathogens. A promising alternative to agrochemicals is the use of plant growth-promoting rhizobacteria (PGPR), which are commonly associated with many, if not all, plant species. However, exploiting the benefits of PGPRs requires knowledge of bacterial function and an in-depth understanding of plant-bacteria associations. Motility is important for colonization efficiency and microbial fitness in the plant environment, but the mechanisms employed by bacteria on and around plants are not well understood. We describe and investigate an atypical mode of motility in Pseudomonas fluorescens SBW25 that was revealed only after flagellum production was eliminated by deletion of the master regulator fleQ. Our results suggest that this 'spidery spreading' is a type of surface motility. Transposon mutagenesis of SBW25ΔfleQ (SBW25Q) produced mutants, defective in viscosin production, and surface spreading was also abolished. Genetic analysis indicated growth-dependency, production of viscosin, and several potential regulatory and secretory systems involved in the spidery spreading phenotype. Moreover, viscosin both increases efficiency of surface spreading over the plant root and protects germinating seedlings in soil infected with the plant pathogen Pythium. Thus, viscosin could be a useful target for biotechnological development of plant growth promotion agents.

Pseudomonas aeruginosa's adaptive trajectory: diverse origins, convergent paths.

Does genetic background contribute to populations following the same or divergent adaptive trajectories? A recent study by Filipow et al. evolved multiple genetically distinct Pseudomonas aeruginosa strains to an artificial cystic fibrosis lung sputum media. The strains adapted at different rates but converged on similar phenotypes despite their initial diversity.

Conditions for the spread of CRISPR-Cas immune systems into bacterial populations.

Bacteria contain a wide variety of innate and adaptive immune systems which provide protection to the host against invading genetic material, including bacteriophages (phages). It is becoming increasingly clear that bacterial immune systems are frequently lost and gained through horizontal gene transfer. However, how and when new immune systems can become established in a bacterial population have remained largely unstudied. We developed a joint epidemiological and evolutionary model that predicts the conditions necessary for the spread of a CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) immune system into a bacterial population lacking this system. We found that whether bacteria carrying CRISPR-Cas will spread (increase in frequency) into a bacterial population depends on the abundance of phages and the difference in the frequency of phage resistance mechanisms between bacteria carrying a CRISPR-Cas immune system and those not (denoted as ${f}_{\Delta }$). Specifically, the abundance of cells carrying CRISPR-Cas will increase if there is a higher proportion of phage resistance (either via CRISPR-Cas immunity or surface modification) in the CRISPR-Cas-possessing population than in the cells lacking CRISPR-Cas. We experimentally validated these predictions in a model using Pseudomonas aeruginosa PA14 and phage DMS3vir. Specifically, by varying the initial ratios of different strains of bacteria that carry alternative forms of phage resistance, we confirmed that the spread of cells carrying CRISPR-Cas through a population can be predicted based on phage density and the relative frequency of resistance phenotypes. Understanding which conditions promote the spread of CRISPR-Cas systems helps to predict when and where these defences can become established in bacterial populations after a horizontal gene transfer event, both in ecological and clinical contexts.

The RNA binding protein Arid5a drives IL-17-dependent autoantibody-induced glomerulonephritis.

Autoantibody-mediated glomerulonephritis (AGN) arises from dysregulated renal inflammation, with urgent need for improved treatments. IL-17 is implicated in AGN and drives pathology in a kidney-intrinsic manner via renal tubular epithelial cells (RTECs). Nonetheless, downstream signaling mechanisms provoking kidney pathology are poorly understood. A noncanonical RNA binding protein (RBP), Arid5a, was upregulated in human and mouse AGN. Arid5a-/- mice were refractory to AGN, with attenuated myeloid infiltration and impaired expression of IL-17-dependent cytokines and transcription factors (C/EBPß, C/EBPδ). Transcriptome-wide RIP-Seq revealed that Arid5a inducibly interacts with conventional IL-17 target mRNAs, including CEBPB and CEBPD. Unexpectedly, many Arid5a RNA targets corresponded to translational regulation and RNA processing pathways, including rRNAs. Indeed, global protein synthesis was repressed in Arid5a-deficient cells, and C/EBPs were controlled at the level of protein rather than RNA accumulation. IL-17 prompted Arid5a nuclear export and association with 18S rRNA, a 40S ribosome constituent. Accordingly, IL-17-dependent renal autoimmunity is driven by Arid5a at the level of ribosome interactions and translation.

Multi-layered genome defences in bacteria.

Bacteria have evolved a variety of defence mechanisms to protect against mobile genetic elements, including restriction-modification systems and CRISPR-Cas. In recent years, dozens of previously unknown defence systems (DSs) have been discovered. Notably, diverse DSs often coexist within the same genome, and some co-occur at frequencies significantly higher than would be expected by chance, implying potential synergistic interactions. Recent studies have provided evidence of defence mechanisms that enhance or complement one another. Here, we review the interactions between DSs at the mechanistic, regulatory, ecological and evolutionary levels.

National Institutes of Health chronic graft-versus-host disease staging in severely affected patients: organ and global scoring correlate with established indicators of disease severity and prognosis.

Between 2004 and 2010, 189 adult patients were enrolled on the National Cancer Institute's cross-sectional chronic graft-versus-host disease (cGVHD) natural history study. Patients were evaluated by multiple disease scales and outcome measures, including the 2005 National Institutes of Health (NIH) Consensus Project cGVHD severity scores. The purpose of this study was to assess the validity of the NIH scoring variables as determinants of disease severity in severely affected patients in efforts to standardize clinician evaluation and staging of cGVHD. Out of 189 patients enrolled, 125 met the criteria for severe cGVHD on the NIH global score, 62 of whom had moderate disease, with a median of 4 (range, 1-8) involved organs. Clinician-assigned average NIH organ score and the corresponding organ scores assigned by subspecialists were highly correlated (r = 0.64). NIH global severity scores showed significant associations with nearly all functional and quality of life outcome measures, including the Lee Symptom Scale, Short Form-36 Physical Component Scale, 2-minute walk, grip strength, range of motion, and Human Activity Profile. Joint/fascia, skin, and lung involvement affected function and quality of life most significantly and showed the greatest correlation with outcome measures. The final Cox model with factors jointly predictive for survival included the time from cGVHD diagnosis (>49 versus ≤49 months, hazard ratio [HR] = 0.23; P = .0011), absolute eosinophil count at the time of NIH evaluation (0-0.5 versus >0.5 cells/µL, HR = 3.95; P = .0006), and NIH lung score (3 versus 0-2, HR = 11.02; P < .0001). These results demonstrate that NIH organs and global severity scores are reliable measures of cGVHD disease burden. The strong association with subspecialist evaluation suggests that NIH organ and global severity scores are appropriate for clinical and research assessments, and may serve as a surrogate for more complex subspecialist examinations. In this population of severely affected patients, NIH lung score is the strongest predictor of poor overall survival, both alone and after adjustment for other important factors.

Transient mutation bias increases the predictability of evolution on an empirical genotype-phenotype landscape.

Predicting how a population will likely navigate a genotype-phenotype landscape requires consideration of selection in combination with mutation bias, which can skew the likelihood of following a particular trajectory. Strong and persistent directional selection can drive populations to ascend toward a peak. However, with a greater number of peaks and more routes to reach them, adaptation inevitably becomes less predictable. Transient mutation bias, which operates only on one mutational step, can influence landscape navigability by biasing the mutational trajectory early in the adaptive walk. This sets an evolving population upon a particular path, constraining the number of accessible routes and making certain peaks and routes more likely to be realized than others. In this work, we employ a model system to investigate whether such transient mutation bias can reliably and predictably place populations on a mutational trajectory to the strongest selective phenotype or usher populations to realize inferior phenotypic outcomes. For this we use motile mutants evolved from ancestrally non-motile variants of the microbe Pseudomonas fluorescens SBW25, of which one trajectory exhibits significant mutation bias. Using this system, we elucidate an empirical genotype-phenotype landscape, where the hill-climbing process represents increasing strength of the motility phenotype, to reveal that transient mutation bias can facilitate rapid and predictable ascension to the strongest observed phenotype in place of equivalent and inferior trajectories. This article is part of the theme issue 'Interdisciplinary approaches to predicting evolutionary biology'.