Tolerance of infections.

A host has two methods to defend against pathogens: It can clear the pathogens or reduce their impact on health in other ways. The first, resistance, is well studied. Study of the second, which ecologists call tolerance, is in its infancy. Tolerance measures the dose response curve of a host's health in reaction to a pathogen and can be studied in a simple quantitative manner. Such studies hold promise because they point to methods of treating infections that put evolutionary pressures on microbes different from antibiotics and vaccines. Studies of tolerance will provide an improved foundation to describe our interactions with all microbes: pathogenic, commensal, and mutualistic. One obvious mechanism affecting tolerance is the intensity of an immune response; an overly exuberant immune response can cause collateral damage through immune effectors and because of the energy allocated away from other physiological functions. There are potentially many other tolerance mechanisms, and here we systematically describe tolerance using a variety of animal systems.

A Macrophage Colony-Stimulating-Factor-Producing γδ T Cell Subset Prevents Malarial Parasitemic Recurrence.

Despite evidence that γδ T cells play an important role during malaria, their precise role remains unclear. During murine malaria induced by Plasmodium chabaudi infection and in human P. falciparum infection, we found that γδ T cells expanded rapidly after resolution of acute parasitemia, in contrast to αß T cells that expanded at the acute stage and then declined. Single-cell sequencing showed that TRAV15N-1 (Vδ6.3) γδ T cells were clonally expanded in mice and had convergent complementarity-determining region 3 sequences. These γδ T cells expressed specific cytokines, M-CSF, CCL5, CCL3, which are known to act on myeloid cells, indicating that this γδ T cell subset might have distinct functions. Both γδ T cells and M-CSF were necessary for preventing parasitemic recurrence. These findings point to an M-CSF-producing γδ T cell subset that fulfills a specialized protective role in the later stage of malaria infection when αß T cells have declined.

Western diet regulates immune status and the response to LPS-driven sepsis independent of diet-associated microbiome.

Sepsis is a deleterious immune response to infection that leads to organ failure and is the 11th most common cause of death worldwide. Despite plaguing humanity for thousands of years, the host factors that regulate this immunological response and subsequent sepsis severity and outcome are not fully understood. Here we describe how the Western diet (WD), a diet high in fat and sucrose and low in fiber, found rampant in industrialized countries, leads to worse disease and poorer outcomes in an LPS-driven sepsis model in WD-fed mice compared with mice fed standard fiber-rich chow (SC). We find that WD-fed mice have higher baseline inflammation (metaflammation) and signs of sepsis-associated immunoparalysis compared with SC-fed mice. WD mice also have an increased frequency of neutrophils, some with an "aged" phenotype, in the blood during sepsis compared with SC mice. Importantly, we found that the WD-dependent increase in sepsis severity and higher mortality is independent of the microbiome, suggesting that the diet may be directly regulating the innate immune system through an unknown mechanism. Strikingly, we could predict LPS-driven sepsis outcome by tracking specific WD-dependent disease factors (e.g., hypothermia and frequency of neutrophils in the blood) during disease progression and recovery. We conclude that the WD is reprogramming the basal immune status and acute response to LPS-driven sepsis and that this correlates with alternative disease paths that lead to more severe disease and poorer outcomes.

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.

Uncovering drivers of dose-dependence and individual variation in malaria infection outcomes.

To understand why some hosts get sicker than others from the same type of infection, it is essential to explain how key processes, such as host responses to infection and parasite growth, are influenced by various biotic and abiotic factors. In many disease systems, the initial infection dose impacts host morbidity and mortality. To explore drivers of dose-dependence and individual variation in infection outcomes, we devised a mathematical model of malaria infection that allowed host and parasite traits to be linear functions (reaction norms) of the initial dose. We fitted the model, using a hierarchical Bayesian approach, to experimental time-series data of acute Plasmodium chabaudi infection across doses spanning seven orders of magnitude. We found evidence for both dose-dependent facilitation and debilitation of host responses. Most importantly, increasing dose reduced the strength of activation of indiscriminate host clearance of red blood cells while increasing the half-life of that response, leading to the maximal response at an intermediate dose. We also explored the causes of diverse infection outcomes across replicate mice receiving the same dose. Besides random noise in the injected dose, we found variation in peak parasite load was due to unobserved individual variation in host responses to clear infected cells. Individual variation in anaemia was likely driven by random variation in parasite burst size, which is linked to the rate of host cells lost to malaria infection. General host vigour in the absence of infection was also correlated with host health during malaria infection. Our work demonstrates that the reaction norm approach provides a useful quantitative framework for examining the impact of a continuous external factor on within-host infection processes.

What Can Vampires Teach Us about Immunology?

Speculative fiction examines the leading edge of science and can be used to introduce ideas into the classroom. For example, most students are already familiar with the fictional infectious diseases responsible for vampire and zombie outbreaks. The disease dynamics of these imaginary ailments follow the same rules we see for real diseases and can be used to remind students that they already understand the basic rules of disease ecology and immunology. By engaging writers of this sort of fiction in an effort to solve problems in immunology we may be able to perform a directed evolution experiment where we follow the evolution of plots rather than genetic traits.

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.

The ubiquitin ligase parkin mediates resistance to intracellular pathogens.

Ubiquitin-mediated targeting of intracellular bacteria to the autophagy pathway is a key innate defence mechanism against invading microbes, including the important human pathogen Mycobacterium tuberculosis. However, the ubiquitin ligases responsible for catalysing ubiquitin chains that surround intracellular bacteria are poorly understood. The parkin protein is a ubiquitin ligase with a well-established role in mitophagy, and mutations in the parkin gene (PARK2) lead to increased susceptibility to Parkinson's disease. Surprisingly, genetic polymorphisms in the PARK2 regulatory region are also associated with increased susceptibility to intracellular bacterial pathogens in humans, including Mycobacterium leprae and Salmonella enterica serovar Typhi, but the function of parkin in immunity has remained unexplored. Here we show that parkin has a role in ubiquitin-mediated autophagy of M. tuberculosis. Both parkin-deficient mice and flies are sensitive to various intracellular bacterial infections, indicating parkin has a conserved role in metazoan innate defence. Moreover, our work reveals an unexpected functional link between mitophagy and infectious disease.

Immunity in society: diverse solutions to common problems.

Understanding how organisms fight infection has been a central focus of scientific research and medicine for the past couple of centuries, and a perennial object of trial and error by humans trying to mitigate the burden of disease. Vaccination success relies upon the exposure of susceptible individuals to pathogen constituents that do not cause (excessive) pathology and that elicit specific immune memory. Mass vaccination allows us to study how immunity operates at the group level; denser populations are more prone to transmitting disease between individuals, but once a critical proportion of the population becomes immune, "herd immunity" emerges. In social species, the combination of behavioural control of infection--e.g., segregation of sick individuals, disposal of the dead, quality assessment of food and water--and aggregation of immune individuals can protect non-immune members from disease. While immune specificity and memory are well understood to underpin immunisation in vertebrates, it has been somewhat surprising to find similar phenomena in invertebrates, which lack the vertebrate molecular mechanisms deemed necessary for immunisation. Indeed, reports showing alternative forms of immune memory are accumulating in invertebrates. In this issue of PLoS Biology, Konrad et al. present an example of fungus-specific immune responses in social ants that lead to the active immunisation of nestmates by infected individuals. These findings join others in showing how organisms evolved diverse mechanisms that fulfil common functions, namely the discrimination between pathogens, the transfer of immunity between related individuals, and the group-level benefits of immunisation.

How the fly balances its ability to combat different pathogens.

Health is a multidimensional landscape. If we just consider the host, there are many outputs that interest us: evolutionary fitness determining parameters like fecundity, survival and pathogen clearance as well as medically important health parameters like sleep, energy stores and appetite. Hosts use a variety of effector pathways to fight infections and these effectors are brought to bear differentially. Each pathogen causes a different disease as they have distinct virulence factors and niches; they each warp the health landscape in unique ways. Therefore, mutations affecting immunity can have complex phenotypes and distinct effects on each pathogen. Here we describe how two components of the fly's immune response, melanization and phagocytosis, contribute to the health landscape generated by the transcription factor ets21c (CG2914) and its putative effector, the signaling molecule wntD (CG8458). To probe the landscape, we infect with two pathogens: Listeria monocytogenes, which primarily lives intracellularly, and Streptococcus pneumoniae, which is an extracellular pathogen. Using the diversity of phenotypes generated by these mutants, we propose that survival during a L. monocytogenes infection is mediated by a combination of two host mechanisms: phagocytic activity and melanization; while survival during a S. pneumoniae infection is determined by phagocytic activity. In addition, increased phagocytic activity is beneficial during S. pneumoniae infection but detrimental during L. monocytogenes infection, demonstrating an inherent trade-off in the immune response.

Tracing personalized health curves during infections.

It is difficult to describe host-microbe interactions in a manner that deals well with both pathogens and mutualists. Perhaps a way can be found using an ecological definition of tolerance, where tolerance is defined as the dose response curve of health versus parasite load. To plot tolerance, individual infections are summarized by reporting the maximum parasite load and the minimum health for a population of infected individuals and the slope of the resulting curve defines the tolerance of the population. We can borrow this method of plotting health versus microbe load in a population and make it apply to individuals; instead of plotting just one point that summarizes an infection in an individual, we can plot the values at many time points over the course of an infection for one individual. This produces curves that trace the course of an infection through phase space rather than over a more typical timeline. These curves highlight relationships like recovery and point out bifurcations that are difficult to visualize with standard plotting techniques. Only nine archetypical curves are needed to describe most pathogenic and mutualistic host-microbe interactions. The technique holds promise as both a qualitative and quantitative approach to dissect host-microbe interactions of all kinds.

Reciprocal analysis of Francisella novicida infections of a Drosophila melanogaster model reveal host-pathogen conflicts mediated by reactive oxygen and imd-regulated innate immune response.

The survival of a bacterial pathogen within a host depends upon its ability to outmaneuver the host immune response. Thus, mutant pathogens provide a useful tool for dissecting host-pathogen relationships, as the strategies the microbe has evolved to counteract immunity reveal a host's immune mechanisms. In this study, we examined the pathogen Francisella novicida and identified new bacterial virulence factors that interact with different parts of the Drosophila melanogaster innate immune system. We performed a genome-wide screen to identify F. novicida genes required for growth and survival within the fly and identified a set of 149 negatively selected mutants. Among these, we identified a class of genes including the transcription factor oxyR, and the DNA repair proteins uvrB, recB, and ruvC that help F. novicida resist oxidative stress. We determined that these bacterial genes are virulence factors that allow F. novicida to counteract the fly melanization immune response. We then performed a second in vivo screen to identify an additional subset of bacterial genes that interact specifically with the imd signaling pathway. Most of these mutants have decreased resistance to the antimicrobial peptide polymyxin B. Characterization of a mutation in the putative transglutaminase FTN_0869 produced a curious result that could not easily be explained using known Drosophila immune responses. By using an unbiased genetic screen, these studies provide a new view of the Drosophila immune response from the perspective of a pathogen. We show that two branches of the fly's immunity are important for fighting F. novicida infections in a model host: melanization and an imd-regulated immune response, and identify bacterial genes that specifically counteract these host responses. Our work suggests that there may be more to learn about the fly immune system, as not all of the phenotypes we observe can be readily explained by its interactions with known immune responses.

The role of anorexia in resistance and tolerance to infections in Drosophila.

Most infections induce anorexia but its function, if any, remains unclear. Because this response is common among animals, we hypothesized that infection-induced diet restriction might be an adaptive trait that modulates the host's ability to fight infection. Two defense strategies protect hosts against infections: resistance, which is the ability to control pathogen levels, and tolerance, which helps the host endure infection-induced pathology. Here we show that infected fruit flies become anorexic and that diet restriction alters defenses, increasing the fly's tolerance to Salmonella typhimurium infections while decreasing resistance to Listeria monocytogenes. This suggests that attempts to extend lifespan through diet restriction or the manipulation of pathways mimicking this process will have complicated effects on a host's ability to fight infections.

Resilience integrates concepts in aging research.

Aging research is unparalleled in the breadth of disciplines it encompasses, from evolutionary studies examining the forces that shape aging to molecular studies uncovering the underlying mechanisms of age-related functional decline. Despite a common focus to advance our understanding of aging, these disciplines have proceeded along distinct paths with little cross-talk. We propose that the concept of resilience can bridge this gap. Resilience describes the ability of a system to respond to perturbations by returning to its original state. Although resilience has been applied in a few individual disciplines in aging research such as frailty and cognitive decline, it has not been explored as a unifying conceptual framework that is able to connect distinct research fields. We argue that because a resilience-based framework can cross broad physiological levels and time scales it can provide the missing links that connect these diverse disciplines. The resulting framework will facilitate predictive modeling and validation and influence targets and directions in research on the biology of aging.

A signaling protease required for melanization in Drosophila affects resistance and tolerance of infections.

Organisms evolve two routes to surviving infections-they can resist pathogen growth (resistance) and they can endure the pathogenesis of infection (tolerance). The sum of these two properties together defines the defensive capabilities of the host. Typically, studies of animal defenses focus on either understanding resistance or, to a lesser extent, tolerance mechanisms, thus providing little understanding of the relationship between these two mechanisms. We suggest there are nine possible pairwise permutations of these traits, assuming they can increase, decrease, or remain unchanged in an independent manner. Here we show that by making a single mutation in the gene encoding a protease, CG3066, active in the melanization cascade in Drosophila melanogaster, we observe the full spectrum of changes; these mutant flies show increases and decreases in their resistance and tolerance properties when challenged with a variety of pathogens. This result implicates melanization in fighting microbial infections and shows that an immune response can affect both resistance and tolerance to infections in microbe-dependent ways. The fly is often described as having an unsophisticated and stereotypical immune response where single mutations cause simple binary changes in immunity. We report a level of complexity in the fly's immune response that has strong ecological implications. We suggest that immune responses are highly tuned by evolution, since selection for defenses that alter resistance against one pathogen may change both resistance and tolerance to other pathogens.

WntD is a feedback inhibitor of Dorsal/NF-kappaB in Drosophila development and immunity.

Regulating the nuclear factor-kappaB (NF-kappaB) family of transcription factors is of critical importance to animals, with consequences of misregulation that include cancer, chronic inflammatory diseases and developmental defects. Studies in Drosophila melanogaster have proved fruitful in determining the signals used to control NF-kappaB proteins, beginning with the discovery that the Toll/NF-kappaB pathway, in addition to patterning the dorsal-ventral axis of the fly embryo, defines a major component of the innate immune response in both Drosophila and mammals. Here, we characterize the Drosophila wntD (Wnt inhibitor of Dorsal) gene. We show that WntD acts as a feedback inhibitor of the NF-kappaB homologue Dorsal during both embryonic patterning and the innate immune response to infection. wntD expression is under the control of Toll/Dorsal signalling, and increased levels of WntD block Dorsal nuclear accumulation, even in the absence of the IkappaB homologue Cactus. The WntD signal is independent of the common Wnt signalling component Armadillo (beta-catenin). By engineering a gene knockout, we show that wntD loss-of-function mutants have immune defects and exhibit increased levels of Toll/Dorsal signalling. Furthermore, the wntD mutant phenotype is suppressed by loss of zygotic dorsal. These results describe the first secreted feedback antagonist of Toll signalling, and demonstrate a novel Wnt activity in the fly.

Multi-start Evolutionary Nonlinear OpTimizeR (MENOTR): A hybrid parameter optimization toolbox.

Parameter optimization or "data fitting" is a computational process that identifies a set of parameter values that best describe an experimental data set. Parameter optimization is commonly carried out using a computer program utilizing a non-linear least squares (NLLS) algorithm. These algorithms work by continuously refining a user supplied initial guess resulting in a systematic increase in the goodness of fit. A well-understood problem with this class of algorithms is that in the case of models with correlated parameters the optimized output parameters are initial guess dependent. This dependency can potentially introduce user bias into the resultant analysis. While many optimization programs exist, few address this dilemma. Here we present a data analysis tool, MENOTR, that is capable of overcoming the initial guess dependence in parameter optimization. Several case studies with published experimental data are presented to demonstrate the capabilities of this tool. The results presented here demonstrate how to effectively overcome the initial guess dependence of NLLS leading to greater confidence that the resultant optimized parameters are the best possible set of parameters to describe an experimental data set. While the optimization strategies implemented within MENOTR are not entirely novel, the application of these strategies to optimize parameters in kinetic and thermodynamic biochemical models is uncommon. MENOTR was designed to require minimal modification to accommodate a new model making it immediately accessible to researchers with a limited programming background. We anticipate that this toolbox can be used in a wide variety of data analysis applications. Prototype versions of this toolbox have been used in a number of published investigations already, as well as ongoing work with chemical-quenched flow, stopped-flow, and molecular tweezers data sets. STATEMENT OF SIGNIFICANCE: Non-linear least squares (NLLS) is a common form of parameter optimization in biochemistry kinetic and thermodynamic investigations These algorithms are used to fit experimental data sets and report corresponding parameter values. The algorithms are fast and able to provide good quality solutions for models involving few parameters. However, initial guess dependence is a well-known drawback of this optimization strategy that can introduce user bias. An alternative method of parameter optimization are genetic algorithms (GA). Genetic algorithms do not have an initial guess dependence but are slow at arriving at the best set of fit parameters. Here, we present MENOTR, a parameter optimization toolbox utilizing a hybrid GA/NLLS algorithm. The toolbox maximizes the strength of each strategy while minimizing the inherent drawbacks.

Metabolomic Analysis of Diverse Mice Reveals Hepatic Arginase-1 as Source of Plasma Arginase in Plasmodium chabaudi Infection.

Infections disrupt host metabolism, but the factors that dictate the nature and magnitude of metabolic change are incompletely characterized. To determine how host metabolism changes in relation to disease severity in murine malaria, we performed plasma metabolomics on eight Plasmodium chabaudi-infected mouse strains with diverse disease phenotypes. We identified plasma metabolic biomarkers for both the nature and severity of different malarial pathologies. A subset of metabolic changes, including plasma arginine depletion, match the plasma metabolomes of human malaria patients, suggesting new connections between pathology and metabolism in human malaria. In our malarial mice, liver damage, which releases hepatic arginase-1 (Arg1) into circulation, correlated with plasma arginine depletion. We confirmed that hepatic Arg1 was the primary source of increased plasma arginase activity in our model, which motivates further investigation of liver damage in human malaria patients. More broadly, our approach shows how leveraging phenotypic diversity can identify and validate relationships between metabolism and the pathophysiology of infectious disease. IMPORTANCE Malaria is a severe and sometimes fatal infectious disease endemic to tropical and subtropical regions. Effective vaccines against malaria-causing Plasmodium parasites remain elusive, and malaria treatments often fail to prevent severe disease. Small molecules that target host metabolism have recently emerged as candidates for therapeutics in malaria and other diseases. However, our limited understanding of how metabolites affect pathophysiology limits our ability to develop new metabolite therapies. By providing a rich data set of metabolite-pathology correlations and by validating one of those correlations, our work is an important step toward harnessing metabolism to mitigate disease. Specifically, we showed that liver damage in P. chabaudi-infected mice releases hepatic arginase-1 into circulation, where it may deplete plasma arginine, a candidate malaria therapeutic that mitigates vascular stress. Our data suggest that liver damage may confound efforts to increase levels of arginine in human malaria patients.

Psidin is required in Drosophila blood cells for both phagocytic degradation and immune activation of the fat body.

Phagocytic blood cells are critical to innate immune defense: They internalize and destroy microbial invaders and produce signals that trigger other immune responses. Despite this central role, the in vivo contributions of phagocytosis to systemic immune activation are not well understood. Drosophila has proven a fruitful model for the investigation of evolutionarily conserved innate immune mechanisms, including NF-kappaB-dependent transcriptional induction, RNAi in antiviral responses, and phagocytosis. The phagocytes of Drosophila encounter bacterial invaders early in infection and contribute to survival of infection. Phagocytosis in flies and mammals is highly homologous: Both rely on scavenger receptors, opsonins, and actin rearrangements for engulfment; have phagosomal cysteine proteases active at low pH; and can be subverted by similar intracellular pathogens. Although the role of Drosophila phagocytes in the activation of other immune tissues has not been clear, we show that induction of the antibacterial-peptide gene Defensin in the fat body during infection requires blood-cell contributions. We identify a gene, psidin, that encodes a lysosomal protein required in the blood cells for both degradation of engulfed bacteria and activation of fat-body Defensin. These data establish a role for the phagocytic blood cells of Drosophila in detection of infection and activation of the humoral immune response.