Interplay between liver and blood stages of Plasmodium infection dictates malaria severity via γδ T cells and IL-17-promoted stress erythropoiesis.

Plasmodium replicates within the liver prior to reaching the bloodstream and infecting red blood cells. Because clinical manifestations of malaria only arise during the blood stage of infection, a perception exists that liver infection does not impact disease pathology. By developing a murine model where the liver and blood stages of infection are uncoupled, we showed that the integration of signals from both stages dictated mortality outcomes. This dichotomy relied on liver stage-dependent activation of Vγ4+ γδ T cells. Subsequent blood stage parasite loads dictated their cytokine profiles, where low parasite loads preferentially expanded IL-17-producing γδ T cells. IL-17 drove extra-medullary erythropoiesis and concomitant reticulocytosis, which protected mice from lethal experimental cerebral malaria (ECM). Adoptive transfer of erythroid precursors could rescue mice from ECM. Modeling of γδ T cell dynamics suggests that this protective mechanism may be key for the establishment of naturally acquired malaria immunity among frequently exposed individuals.

Mapping the functional form of the trade-off between infection resistance and reproductive fitness under dysregulated immune signaling.

Immune responses benefit organismal fitness by clearing parasites but also exact costs associated with immunopathology and energetic investment. Hosts manage these costs by tightly regulating the induction of immune signaling to curtail excessive responses and restore homeostasis. Despite the theoretical importance of turning off the immune response to mitigate these costs, experimentally connecting variation in the negative regulation of immune responses to organismal fitness remains a frontier in evolutionary immunology. In this study, we used a dose-response approach to manipulate the RNAi-mediated knockdown efficiency of cactus (IκBα), a central regulator of Toll pathway signal transduction in flour beetles (Tribolium castaneum). By titrating cactus activity across four distinct levels, we derived the shape of the relationship between immune response investment and traits associated with host fitness, including infection susceptibility, lifespan, fecundity, body mass, and gut homeostasis. Cactus knock-down increased the overall magnitude of inducible immune responses and delayed their resolution in a dsRNA dose-dependent manner, promoting survival and resistance following bacterial infection. However, these benefits were counterbalanced by dsRNA dose-dependent costs to lifespan, fecundity, body mass, and gut integrity. Our results allowed us to move beyond the qualitative identification of a trade-off between immune investment and fitness to actually derive its functional form. This approach paves the way to quantitatively compare the evolution and impact of distinct regulatory elements on life-history trade-offs and fitness, filling a crucial gap in our conceptual and theoretical models of immune signaling network evolution and the maintenance of natural variation in immune systems.

Pleiotropy alleviates the fitness costs associated with resource allocation trade-offs in immune signalling networks.

Many genes and signalling pathways within plant and animal taxa drive the expression of multiple organismal traits. This form of genetic pleiotropy instigates trade-offs among life-history traits if a mutation in the pleiotropic gene improves the fitness contribution of one trait at the expense of another. Whether or not pleiotropy gives rise to conflict among traits, however, likely depends on the resource costs and timing of trait deployment during organismal development. To investigate factors that could influence the evolutionary maintenance of pleiotropy in gene networks, we developed an agent-based model of co-evolution between parasites and hosts. Hosts comprise signalling networks that must faithfully complete a developmental programme while also defending against parasites, and trait signalling networks could be independent or share a pleiotropic component as they evolved to improve host fitness. We found that hosts with independent developmental and immune networks were significantly more fit than hosts with pleiotropic networks when traits were deployed asynchronously during development. When host genotypes directly competed against each other, however, pleiotropic hosts were victorious regardless of trait synchrony because the pleiotropic networks were more robust to parasite manipulation, potentially explaining the abundance of pleiotropy in immune systems despite its contribution to life history trade-offs.

Pleiotropy promotes the evolution of inducible immune responses in a model of host-pathogen coevolution.

Components of immune systems face significant selective pressure to efficiently use organismal resources, mitigate infection, and resist parasitic manipulation. A theoretically optimal immune defense balances investment in constitutive and inducible immune components depending on the kinds of parasites encountered, but genetic and dynamic constraints can force deviation away from theoretical optima. One such potential constraint is pleiotropy, the phenomenon where a single gene affects multiple phenotypes. Although pleiotropy can prevent or dramatically slow adaptive evolution, it is prevalent in the signaling networks that compose metazoan immune systems. We hypothesized that pleiotropy is maintained in immune signaling networks despite slowed adaptive evolution because it provides some other advantage, such as forcing network evolution to compensate in ways that increase host fitness during infection. To study the effects of pleiotropy on the evolution of immune signaling networks, we used an agent-based modeling approach to evolve a population of host immune systems infected by simultaneously co-evolving parasites. Four kinds of pleiotropic restrictions on evolvability were incorporated into the networks, and their evolutionary outcomes were compared to, and competed against, non-pleiotropic networks. As the networks evolved, we tracked several metrics of immune network complexity, relative investment in inducible and constitutive defenses, and features associated with the winners and losers of competitive simulations. Our results suggest non-pleiotropic networks evolve to deploy highly constitutive immune responses regardless of parasite prevalence, but some implementations of pleiotropy favor the evolution of highly inducible immunity. These inducible pleiotropic networks are no less fit than non-pleiotropic networks and can out-compete non-pleiotropic networks in competitive simulations. These provide a theoretical explanation for the prevalence of pleiotropic genes in immune systems and highlight a mechanism that could facilitate the evolution of inducible immune responses.

The Impact of Coinfection Dynamics on Host Competition and Coexistence.

AbstractParasites can mediate competition among host species in an ecological community by differentially affecting key parameters that normally give one species a competitive edge. In nature, however, coinfecting parasites that antagonize or facilitate each other-for example, by altering cross-protective host immune responses-can modulate host infection outcomes and parasite transmission relative to a single infection. Under what conditions is coinfection likely to interfere with parasite-mediated apparent competition among hosts? To address this question, we created a model of two coinfected host species. Parasites could interact indirectly by affecting host reproduction or directly by modulating recovery and disease-induced mortality of each host species to a focal infection. We grounded our model with parameters from a classic apparent competition system but allowed for multiple parasite transmission modes and interaction scenarios. Our results suggest that infection-induced mortality has an outsized effect on competition outcomes relative to recovery but that coinfection-mediated modulation of mortality can produce a range of coexistence or competitive exclusion outcomes. Moreover, while infection prevalence is sensitive to variation in parasite transmission mode, host competitive outcomes are not. Our generalizable model highlights the influence of immunological variation and parasite ecology on community ecology.

Delayed puberty, gonadotropin abnormalities and subfertility in male Padi2/Padi4 double knockout mice.

BACKGROUND: Peptidylarginine deiminase enzymes (PADs) convert arginine residues to citrulline in a process called citrullination or deimination. Recently, two PADs, PAD2 and PAD4, have been linked to hormone signaling in vitro and the goal of this study was to test for links between PAD2/PAD4 and hormone signaling in vivo. METHODS: Preliminary analysis of Padi2 and Padi4 single knockout (SKO) mice did not find any overt reproductive defects and we predicted that this was likely due to genetic compensation. To test this hypothesis, we created a Padi2/Padi4 double knockout (DKO) mouse model and tested these mice along with wild-type FVB/NJ (WT) and both strains of SKO mice for a range of reproductive defects. RESULTS: Controlled breeding trials found that male DKO mice appeared to take longer to have their first litter than WT controls. This tendency was maintained when these mice were mated to either DKO or WT females. Additionally, unsexed 2-day old DKO pups and male DKO weanlings both weighed significantly less than their WT counterparts, took significantly longer than WT males to reach puberty, and had consistently lower serum testosterone levels. Furthermore, 90-day old adult DKO males had smaller testes than WT males with increased rates of germ cell apoptosis. CONCLUSIONS: The Padi2/Padi4 DKO mouse model provides a new tool for investigating PAD function and outcomes from our studies provide the first in vivo evidence linking PADs with hormone signaling.

Vector Immunity and Evolutionary Ecology: The Harmonious Dissonance.

Recent scientific breakthroughs have significantly expanded our understanding of arthropod vector immunity. Insights in the laboratory have demonstrated how the immune system provides resistance to infection, and in what manner innate defenses protect against a microbial assault. Less understood, however, is the effect of biotic and abiotic factors on microbial-vector interactions and the impact of the immune system on arthropod populations in nature. Furthermore, the influence of genetic plasticity on the immune response against vector-borne pathogens remains mostly elusive. Herein, we discuss evolutionary forces that shape arthropod vector immunity. We focus on resistance, pathogenicity and tolerance to infection. We posit that novel scientific paradigms should emerge when molecular immunologists and evolutionary ecologists work together.

Natural variation in the contribution of microbial density to inducible immune dynamics.

Immune responses evolve to balance the benefits of microbial killing against the costs of autoimmunity and energetic resource use. Models that explore the evolution of optimal immune responses generally include a term for constitutive immunity, or the level of immunological investment prior to microbial exposure, and for inducible immunity, or investment in immune function after microbial challenge. However, studies rarely consider the functional form of inducible immune responses with respect to microbial density, despite the theoretical dependence of immune system evolution on microbe- versus immune-mediated damage to the host. In this study, we analyse antimicrobial peptide (AMP) gene expression from seven wild-caught flour beetle populations (Tribolium spp.) during acute infection with the virulent bacteria Bacillus thuringiensis (Bt) and Photorhabdus luminescens (P.lum) to demonstrate that inducible immune responses mediated by the humoral IMD pathway exhibit natural variation in both microbe density-dependent and independent temporal dynamics. Beetle populations that exhibited greater AMP expression sensitivity to Bt density were also more likely to die from infection, while populations that exhibited higher microbe density-independent AMP expression were more likely to survive P. luminescens infection. Reduction in pathway signalling efficiency through RNAi-mediated knockdown of the imd gene reduced the magnitude of both microbe-independent and dependent responses and reduced host resistance to Bt growth, but had no net effect on host survival. This study provides a framework for understanding natural variation in the flexibility of investment in inducible immune responses and should inform theory on the contribution of nonequilibrium host-microbe dynamics to immune system evolution.

How Many Parameters Does It Take to Describe Disease Tolerance?

The study of infectious disease has been aided by model organisms, which have helped to elucidate molecular mechanisms and contributed to the development of new treatments; however, the lack of a conceptual framework for unifying findings across models, combined with host variability, has impeded progress and translation. Here, we fill this gap with a simple graphical and mathematical framework to study disease tolerance, the dose response curve relating health to microbe load; this approach helped uncover parameters that were previously overlooked. Using a model experimental system in which we challenged Drosophila melanogaster with the pathogen Listeria monocytogenes, we tested this framework, finding that microbe growth, the immune response, and disease tolerance were all well represented by sigmoid models. As we altered the system by varying host or pathogen genetics, disease tolerance varied, as we would expect if it was indeed governed by parameters controlling the sensitivity of the system (the number of bacteria required to trigger a response) and maximal effect size according to a logistic equation. Though either the pathogen or host immune response or both together could theoretically be the proximal cause of pathology that killed the flies, we found that the pathogen, but not the immune response, drove damage in this model. With this new understanding of the circuitry controlling disease tolerance, we can now propose better ways of choosing, combining, and developing treatments.

Tracking Resilience to Infections by Mapping Disease Space.

Infected hosts differ in their responses to pathogens; some hosts are resilient and recover their original health, whereas others follow a divergent path and die. To quantitate these differences, we propose mapping the routes infected individuals take through "disease space." We find that when plotting physiological parameters against each other, many pairs have hysteretic relationships that identify the current location of the host and predict the future route of the infection. These maps can readily be constructed from experimental longitudinal data, and we provide two methods to generate the maps from the cross-sectional data that is commonly gathered in field trials. We hypothesize that resilient hosts tend to take small loops through disease space, whereas nonresilient individuals take large loops. We support this hypothesis with experimental data in mice infected with Plasmodium chabaudi, finding that dying mice trace a large arc in red blood cells (RBCs) by reticulocyte space as compared to surviving mice. We find that human malaria patients who are heterozygous for sickle cell hemoglobin occupy a small area of RBCs by reticulocyte space, suggesting this approach can be used to distinguish resilience in human populations. This technique should be broadly useful in describing the in-host dynamics of infections in both model hosts and patients at both population and individual levels.

Dissecting the contributions of time and microbe density to variation in immune gene expression.

Widespread differential expression of immunological genes is a hallmark of the response to infection in almost all surveyed taxa. However, several challenges remain in the attempt to connect differences in gene expression with functional outcomes like parasite killing and host survival. For example, temporal gene expression patterns are not always monotonic (unidirectional slope), yielding results that qualitatively depend on the time point selected for analysis. They may also be correlated to microbe density, confounding the strength of an immune response and resistance to parasites. In this study, we analyse these relationships in an mRNA-seq time series of Tribolium castaneum infected with Bacillus thuringiensis Our results suggest that many extracellular immunological components with known roles in immunity, like antimicrobial peptides and recognition proteins, are highly correlated to microbe load. On the other hand, intracellular components of immunological signalling pathways overwhelmingly show non-monotonic temporal patterns of gene expression, despite the underlying assumption of monotonicity in most ecological and comparative transcriptomics studies that rely on cross-sectional analyses. Our results raise a host of new questions, including to what extent variation in host resistance, infection tolerance and immunopathology can be explained by variation in the slope or sensitivity of these newly characterized patterns.

The within-host dynamics of infection in trans-generationally primed flour beetles.

Many taxa exhibit plastic immune responses initiated after primary microbial exposure that provide increased protection against disease-induced mortality and the fitness costs of infection. In several arthropod species, this protection can even be passed from parents to offspring through a phenomenon called trans-generational immune priming. Here, we first demonstrate that trans-generational priming is a repeatable phenomenon in flour beetles (Tribolium castaneum) primed and infected with Bacillus thuringiensis (Bt). We then quantify the within-host dynamics of microbes and host physiological responses in infected offspring from primed and unprimed mothers by monitoring bacterial density and using mRNA-seq to profile host gene expression, respectively, over the acute infection period. We find that priming increases inducible resistance against Bt around a critical temporal juncture where host septicaemic trajectories, and consequently survival, may be determined in unprimed individuals. Our results identify a highly differentially expressed biomarker of priming, containing an EIF4-e domain, in uninfected individuals, as well as several other candidate genes. Moreover, the induction and decay dynamics of gene expression over time suggest a metabolic shift in primed individuals. The identified bacterial and gene expression dynamics are likely to influence patterns of bacterial fitness and disease transmission in natural populations.

Dynamic Patterns of Parasitism and Immunity across Host Development Influence Optimal Strategies of Resource Allocation.

The integration of physiological mechanisms into life-history theory is an emerging frontier in our understanding of the constraints and drivers of life-history evolution. Dynamic patterns of antagonism between developmental and immunological pathways in juvenile insects illustrate the importance of mechanisms for determining life-history strategy optima in the face of trade-offs. For example, developmental interference occurs when developmental processes transiently take priority over resources or pathway architecture, preventing allocation to immunity or other traits. We designed a within-host model of infected larval development to explore the impact of developmental dynamics on optimal resource mobilization and allocation strategies as well as on larval resistance and tolerance phenotypes. The model incorporates mechanism-inspired functional forms of developmental interference with immunity against parasites that attack specific larval stages. We find that developmental interference generally increases optimal investment in constitutive immunity and decreases optimal resource mobilization rates, but the results are sensitive to the developmental stage at first infection. Moreover, developmental interference reduces resistance but generally increases tolerance of infection. We demonstrate the potential impact of these dynamics on empirical estimates of host susceptibility and discuss the general implications of incorporating realistic physiological mechanisms and developmental dynamics for life-history theory in insects and other organisms.

Single-cell transcriptomics of the immune system in ME/CFS at baseline and following symptom provocation.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a serious and poorly understood disease. To understand immune dysregulation in ME/CFS, we use single-cell RNA sequencing (scRNA-seq) to examine immune cells in patient and control cohorts. Postexertional malaise (PEM), an exacerbation of symptoms following strenuous exercise, is a characteristic symptom of ME/CFS. To detect changes coincident with PEM, we applied scRNA-seq on the same cohorts following exercise. At baseline, ME/CFS patients display classical monocyte dysregulation suggestive of inappropriate differentiation and migration to tissue. We identify both diseased and more normal monocytes within patients, and the fraction of diseased cells correlates with disease severity. Comparing the transcriptome at baseline and postexercise challenge, we discover patterns indicative of improper platelet activation in patients, with minimal changes elsewhere in the immune system. Taken together, these data identify immunological defects present at baseline in patients and an additional layer of dysregulation in platelets.

Changes in clinical management and diagnosis following DaTscan SPECT imaging in patients with clinically uncertain parkinsonian syndromes: a 12-week follow-up study.

BACKGROUND: An accurate diagnosis is important for timely and adequate treatment in patients with clinically uncertain parkinsonian syndrome (CUPS). OBJECTIVE: The objective of this study was to assess safety and changes in clinical management, diagnosis and quality of life (QoL) at 4 and 12 weeks following DaTscan (ioflupane [(123)I] injection) imaging in patients with CUPS. METHODS: This randomized, open-label, single-dose, multicenter trial was carried out in patients with CUPS who were randomized to either a DaTscan imaging group or to a control group without imaging. The main outcome measures were the proportions of patients with changes in clinical management and diagnosis from baseline through to 12 weeks after DaTscan. A total of 19 university hospital centers in Europe and the USA participated in the study. There were 267 patients enrolled and randomized (131 DaTscan, 136 control). RESULTS: Significantly more DaTscan patients had changes in clinical management after 12 weeks (p = 0.004) compared to the control group, and significantly more DaTscan patients had changes in diagnosis at 4 weeks and at 12 weeks (both p < 0.001) compared to control patients. No significant difference in total score for QoL was observed between groups during the study duration. DaTscan was safe and well-tolerated. No deaths, serious adverse events (AEs) or withdrawals due to AEs occurred during the study. One patient had a headache following treatment with a suspected relationship to DaTscan. CONCLUSION: DaTscan imaging significantly affected the clinical management and diagnosis of patients with CUPS. DaTscan is safe and well-tolerated and is a useful adjunct to differential diagnosis of CUPS.

Pleiotropy alleviates the fitness costs associated with resource allocation trade-offs in immune signaling networks.

Many genes and signaling pathways within plant and animal taxa drive the expression of multiple organismal traits. This form of genetic pleiotropy instigates trade-offs among life-history traits if a mutation in the pleiotropic gene improves the fitness contribution of one trait at the expense of another. Whether or not pleiotropy gives rise to conflict among traits, however, likely depends on the resource costs and timing of trait deployment during organismal development. To investigate factors that could influence the evolutionary maintenance of pleiotropy in gene networks, we developed an agent-based model of co-evolution between parasites and hosts. Hosts comprise signaling networks that must faithfully complete a developmental program while also defending against parasites, and trait signaling networks could be independent or share a pleiotropic component as they evolved to improve host fitness. We found that hosts with independent developmental and immune networks were significantly more fit than hosts with pleiotropic networks when traits were deployed asynchronously during development. When host genotypes directly competed against each other, however, pleiotropic hosts were victorious regardless of trait synchrony because the pleiotropic networks were more robust to parasite manipulation, potentially explaining the abundance of pleiotropy in immune systems despite its contribution to life history trade-offs.

Mapping the functional form of the trade-off between infection resistance and reproductive fitness under dysregulated immune signaling.

Immune responses benefit organismal fitness by clearing parasites but also exact costs associated with immunopathology and energetic investment. Hosts manage these costs by tightly regulating the induction of immune signaling to curtail excessive responses and restore homeostasis. Despite the theoretical importance of turning off the immune response to mitigate these costs, experimentally connecting variation in the negative regulation of immune responses to organismal fitness remains a frontier in evolutionary immunology. In this study, we used a dose-response approach to manipulate the RNAi-mediated knockdown efficiency of cactus (IκBα), a central regulator of Toll pathway signal transduction in flour beetles (Tribolium castaneum). By titrating cactus activity along a continuous gradient, we derived the shape of the relationship between immune response investment and traits associated with host fitness, including infection susceptibility, lifespan, fecundity, body mass, and gut homeostasis. Cactus knock-down increased the overall magintude of inducible immune responses and delayed their resolution in a dsRNA dose-dependent manner, promoting survival and resistance following bacterial infection. However, these benefits were counterbalanced by dsRNA dose-dependent costs to lifespan, fecundity, body mass, and gut integrity. Our results allowed us to move beyond the qualitative identification of a trade-off between immune investment and fitness to actually derive its functional form. This approach paves the way to quantitatively compare the evolution and impact of distinct regulatory elements on life-history trade-offs and fitness, filling a crucial gap in our conceptual and theoretical models of immune signaling network evolution and the maintenance of natural variation in immune systems.

The Effect of Developmental Pleiotropy on the Evolution of Insect Immune Genes.

The pressure to survive ever-changing pathogen exposure explains the frequent observation that immune genes are among the fastest evolving in the genomes of many taxa, but an intriguing proportion of immune genes also appear to be under purifying selection. Though variance in evolutionary signatures of immune genes is often attributed to differences in gene-specific interactions with microbes, this explanation neglects the possibility that immune genes participate in other biological processes that could pleiotropically constrain adaptive selection. In this study, we analyzed available transcriptomic and genomic data from Drosophila melanogaster and related species to test the hypothesis that there is substantial pleiotropic overlap in the developmental and immunological functions of genes involved in immune signaling and that pleiotropy would be associated with stronger signatures of evolutionary constraint. Our results suggest that pleiotropic immune genes do evolve more slowly than those having no known developmental functions and that signatures of constraint are particularly strong for pleiotropic immune genes that are broadly expressed across life stages. These results support the general yet untested hypothesis that pleiotropy can constrain immune system evolution, raising new fundamental questions about the benefits of maintaining pleiotropy in systems that need to rapidly adapt to changing pathogen pressures.

Impact of DaTscan SPECT imaging on clinical management, diagnosis, confidence of diagnosis, quality of life, health resource use and safety in patients with clinically uncertain parkinsonian syndromes: a prospective 1-year follow-up of an open-label controlled study.

BACKGROUND: This study assessed the impact of DaTscan on clinical management, diagnosis, confidence of diagnosis (CoD), quality of life (QoL), health resource use (HRU) and safety during a 1-year follow-up in patients with clinically uncertain parkinsonian syndromes (CUPS). METHODS: A total of 19 university hospital centres in Europe and the USA participated in this open-label, single-dose, prospective, clinical trial in patients with CUPS who were randomised to a DaTscan imaging group or to a no-imaging (control) group. The proportion of patients with changes in clinical management, diagnosis, CoD, QoL and HRU from baseline through 1 year post-DaTscan was compared between groups. RESULTS: There were 273 patients randomised (135 DaTscan, 138 control). Significantly more patients in the DaTscan imaging group had at least one change in their actual clinical management after 12 weeks (p=0.002) and after 1 year (p<0.001) compared with patients in the control group. In addition, significantly more DaTscan patients had changes in diagnosis and an increased CoD at 4 weeks, 12 weeks and 1 year (all p<0.001) compared with control patients. No significant differences in total score for QoL or HRU were observed between groups during the 1-year follow-up period. DaTscan was safe and well tolerated. One patient in the imaging group had an adverse event (headache) with suspected relationship to DaTscan post-administration. CONCLUSIONS: DaTscan had a significant impact on clinical management, diagnosis and CoD in patients with CUPS. DaTscan is safe and well tolerated, and is a useful adjunct to differentiate a diagnosis of CUPS. Trial registration number http://ClinicalTrials.gov Identifier: NCT00382967.

Bet-hedging in innate and adaptive immune systems.

Immune system evolution is shaped by the fitness costs and trade-offs associated with mounting an immune response. Costs that arise mainly as a function of the magnitude of investment, including energetic and immunopathological costs, are well-represented in studies of immune system evolution. Less well considered, however, are the costs of immune cell plasticity and specialization. Hosts in nature encounter a large diversity of microbes and parasites that require different and sometimes conflicting immune mechanisms for defense, but it takes precious time to recognize and correctly integrate signals for an effective polarized response. In this perspective, we propose that bet-hedging can be a viable alternative to plasticity in immune cell effector function, discuss conditions under which bet-hedging is likely to be an advantageous strategy for different arms of the immune system, and present cases from both innate and adaptive immune systems that suggest bet-hedging at play.