Constitutive Immunity Research Articles

Constitutive activation of cellular immunity underlies the evolution of resistance to infection in Drosophila.

Organisms rely on inducible and constitutive immune defences to combat infection. Constitutive immunity enables a rapid response to infection but may carry a cost for uninfected individuals, leading to the prediction that it will be favoured when infection rates are high. When we exposed populations of Drosophila melanogaster to intense parasitism by the parasitoid wasp Leptopilina boulardi, they evolved resistance by developing a more reactive cellular immune response. Using single-cell RNA sequencing, we found that immune-inducible genes had become constitutively upregulated. This was the result of resistant larvae differentiating precursors of specialized immune cells called lamellocytes that were previously only produced after infection. Therefore, populations evolved resistance by genetically hard-wiring the first steps of an induced immune response to become constitutive.

Ontogeny of leukocyte profiles in a wild altricial passerine.

Ecophysiological studies have highlighted the relevance of the avian immune system in individual fitness prospects in the wild. However, studies on the ontogeny of avian immunity are scarce. We analyse age-related changes in the cellular constitutive immunity throughout nestling development, as well as its relationship with sex and brood size. We found that cellular constitutive immunity could be affected by age, sex, brood size, or daily rhythm. Early-stage nestlings relied more on cells of the innate immunity rather than on cells linked to the adaptive immune system. Cellular immunity may not be fully mature in fledglings, as reflected by differences in phagocytic cell counts with regard to adults. Beyond the age-dependent effects, agranulocyte cell counts were affected by sibling competition while granulocyte cell counts showed a daily rhythm. We also show that the heterophil to lymphocyte ratio was negatively related to body weight when nestlings become more independent. Our study contributes knowledge to the fields of developmental immunology and ecological immunology based on essential components of the cellular immune system.

Anti-microbial activity of whole blood and plasma collected from Anna's Hummingbirds (Calypte anna) against three different microbes.

Hummingbirds are essential pollinators in many ecosystems, making their conservation critical. As is the case with many species, hummingbirds are now facing a variety of challenges resulting from anthropogenic changes. As populations shift and species interactions change, disease is likely to pose a significant threat. There is a basic understanding of which pathogens currently affect a variety of hummingbird species, however there is a paucity of information about their immune systems capacity to kill pathogens and what specific factors may affect immunity. The objective of this study was to gain a basic understanding of the effect of age, sex, and molt on the constitutive innate immunity of hummingbirds. An in vitro assay was used to assess the microbiocidal capacity of the whole blood of Anna’s Hummingbirds (Calypte anna) against three different microbes: Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Candida albicans (C. albicans). The effect of age, sex and molt on anti-microbial capacity varied based on the microbe type. After-hatch-year birds tended to have better anti-microbial capacity compared to hatch-year birds. Male birds had higher anti-microbial activity than female birds, although this was not observed against C. albicans. Molting birds had a weaker antimicrobial activity against E. coli and S. aureus than birds that were not molting. These results represent an important first step towards defining the parameters of constitutive innate immunity of Anna’s Hummingbirds as well as providing important knowledge about factors that should be considered when evaluating the health of wild populations.

Plasma proteomic profiles differ between European and North American myotid bats colonized by Pseudogymnoascus destructans.

Emerging fungal diseases have become challenges for wildlife health and conservation. North American hibernating bat species are threatened by the psychrophilic fungus Pseudogymnoascus destructans (Pd) causing the disease called white-nose syndrome (WNS) with unprecedented mortality rates. The fungus is widespread in North America and Europe, however, disease is not manifested in European bats. Differences in epidemiology and pathology indicate an evolution of resistance or tolerance mechanisms towards Pd in European bats. We compared the proteomic profile of blood plasma in healthy and Pd-colonized European Myotis myotis and North American Myotis lucifugus in order to identify pathophysiological changes associated with Pd colonization, which might also explain the differences in bat survival. Expression analyses of plasma proteins revealed differences in healthy and Pd-colonized M.lucifugus, but not in M.myotis. We identified differentially expressed proteins for acute phase response, constitutive and adaptive immunity, oxidative stress defence, metabolism and structural proteins of exosomes and desmosomes, suggesting a systemic response against Pd in North American M.lucifugus but not European M.myotis. The differences in plasma proteomic profiles between European and North American bat species colonized by Pd suggest European bats have evolved tolerance mechanisms towards Pd infection.

Immune differences in captive and free-ranging zebras (Equus zebra and E. quagga)

Wild mammals in ex situ captivity experience substantially different environmental conditions compared to free-ranging conspecifics, e.g., in terms of diet, climatic conditions, social factors, movement space, and direct anthropogenic disturbance. Moreover, animals in captivity frequently undergo management interventions such as medical treatments which may influence pathogen pressure. Captivity is known to affect immunological responses in some terrestrial and marine mammals; however, it is unclear whether this can be generalized to other taxa. Furthermore, little is known about how energetically costly life history stages such as lactation influence the immune system in wildlife. We measured expression of components of the constitutive and induced innate immunity and of the adaptive immune system in plains and mountain zebras (Equus quagga and E. zebra), including lactating and non-lactating individuals. As a proxy for general immune function, we screened for lytic equine herpesvirus (EHV) infection, a common and often latent pathogen which is reactivated in response to stress and immune challenge. Both energetically cheap markers of the constitutive innate immunity were lower in captive than in wild zebras, whereas energetically costly markers of the induced innate immunity were more highly expressed in captive zebras. Lactation was associated with higher titers of natural antibodies and lysozyme. Lytic EHV infection was not significantly correlated with any of the measured immune markers. Our results suggest that captivity and lactation may influence immune functions in zebra mares.

Experimental facilitation of heat loss affects work rate and innate immune function in a breeding passerine bird.

The capacity to get rid of excess heat produced during hard work is a possible constraint on parental effort during reproduction [heat dissipation limit (HDL) theory]. We released hard-working blue tits (Cyanistes caeruleus) from this constraint by experimentally removing ventral plumage. We then assessed whether this changed their reproductive effort (feeding rate and nestling size) and levels of self-maintenance (change in body mass and innate immune function). Feather-clipped females reduced the number of feeding visits and increased levels of constitutive innate immunity compared with unclipped females but did not fledge smaller nestlings. Thus, they increased self-maintenance without compromising current reproductive output. In contrast, feather clipping did not affect the number of feeding visits or innate immune function in males, despite increased heat loss rate. Our results show that analyses of physiological parameters, such as constitutive innate immune function, can be important when trying to understand sources of variation in investment in self-maintenance versus reproductive effort and that risk of overheating can influence innate immune function during reproduction.

Continuous Agrochemical Treatments in Agroecosystems Can Modify the Effects of Pendimethalin-Based Herbicide Exposure on Immunocompetence of a Beneficial Ground Beetle

Herbicide application for pest control can negatively affect soil biodiversity, mainly acting on species that are involved in ecosystem service. In this study, field and laboratory trials were designed to assay herbicide exposure effects on the constitutive immunity of Harpalus (Pseudoophonus) rufipes (De Geer, 1774), a beneficial carabid species that inhabits croplands. The circulating hemocytes (THCs) and plasmatic levels of basal and total phenoloxidase (PO), as well as lysozyme-like enzyme activities, were measured as markers of exposure. In laboratory tests, the exposure to realistic field doses of pendimethalin-based herbicides for two, seven and 21 days caused a reduction in enzyme activities in beetles from organic crops. In beetles from conventional fields, the THCs and total PO activity decreased significantly at two and seven days after the initial exposure, though no effects were recorded on basal PO and lysozyme like-enzyme activities. These differences in enzyme activities and THCs indicate that the interference of pendimethalin with immune parameters clearly depends on both the different field conditions from which the population comes and the cumulative effects of repeated applications over the time.

Cross-Microbial Protection via Priming a Conserved Immune Co-Receptor through Juxtamembrane Phosphorylation in Plants

Living organisms can be primed for potentiated responses to recurring stresses based on prior experience. However, the molecular basis of immune priming remains elusive in plants that lack adaptive immunity. Here, we report that bacterial challenges can prepare plants for fungal attacks by inducing juxtamembrane phosphorylation of CERK1, the co-receptor indispensable for signaling in response to the fungal elicitor chitin. This phosphorylation is mediated by BAK1, a co-receptor for signaling in response to multiple elicitors. BAK1 interacts with CERK1, and loss of BAK1 reduces priming phosphorylation of CERK1. Juxtamembrane phosphomimetic mutations of CERK1 confer accelerated chitin responses and fortified fungal resistance without triggering constitutive immunity, whereas juxtamembrane phosphodeficient mutations diminish bacteria-induced protection against fungal infection. These findings reveal that crosstalk between cell-surface immune co-receptors can prime defense and demonstrate that juxtamembrane phosphorylation of plant receptor-like kinases can occur independent of kinase activation to place the protein into a prime state.

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.

Feeling the heat: Extreme temperatures compromise constitutive innate humoral immunity and skin color in a desert dwelling lizard.

Environmental temperature, particularly in habitats with extreme temperature fluctuations, may shape selection pressures on life history traits. Especially in ectotherms, temperature affects performance, physiology, and in some species, skin color. Skin color can be a sexual ornament signaling the bearer's ability to resist infections, when only high-quality individuals are able to invest both in high immune defense and elaborate ornament expression. However, how the information content of these sexual traits may vary with environmental conditions has been less studied. Dickerson's collared lizard (Crotaphytus dickersonae) males are blue and have a black and white collar. This conspicuous coloration signals performance and immune response, and is related to body temperature. Here, by maintaining males at higher, lower, and mean environmental temperatures we evaluated whether temperature variation influences color and constitutive innate humoral immunity (agglutination and lysis titers, estimated through hemolysis-hemagglutination assays), and whether extreme temperatures impose trade-offs between color and humoral immunity. We found that at low and high temperature treatments males had lower agglutination and lysis titers, and at low temperature, blue chroma from the dorsum declined and males became greener. Interestingly, at low and control temperature treatments, agglutination titer and blue coloration were positively correlated, whereas high temperatures revealed a trade-off between increasing agglutination titers and displaying bluer skin color. Our results suggest that in the Dickerson collared lizard even short-term variation of environmental temperature affects performance of constitutive innate humoral immunity and the brilliant blue skin color. Particularly, high temperatures may compromise some components of male's immunity and sexual signaling.

Constitutive innate immunity of tropical House Wrens varies with season and reproductive activity

AbstractIn lowland Neotropical regions, where air temperature and day length remain relatively constant year round, seasonality is determined primarily by changes in rainfall. The wet season triggers the start of breeding for many Neotropical birds but also alters the antigenic environment, likely increasing the risk of disease transmission. We explored 2 hypotheses about temporal variation in constitutive innate immunity of a Neotropical bird, the House Wren (Troglodytes aedon). The antigen response hypothesis proposes that Neotropical wrens upregulate their immune function in the wet season either in anticipation of or in response to vectors that become more prevalent. The resource constraint hypothesis proposes that during periods of putative high resource demand, such as when parents are feeding young, immune function should be compromised and downregulated. Controlling for reproductive stage, we found that microbicidal capacity of blood against Escherichia coli was higher in the wet than the dry season, consistent with the antigen response hypothesis. Phagocytosis of E. coli and Staphylococcus aureus did not differ between wet and dry seasons. Microbicidal capacity and H/L ratio of tropical House Wrens did not vary among reproductive stages, and our data offered no support for the idea that immune function is compromised during the period when parents are feeding young.

Kinase activity of SOBIR1 and BAK1 is required for immune signalling.

SummaryLeucine‐rich repeat‐receptor‐like proteins (LRR‐RLPs) and LRR‐receptor‐like kinases (LRR‐RLKs) trigger immune signalling to promote plant resistance against pathogens. LRR‐RLPs lack an intracellular kinase domain, and several of these receptors have been shown to constitutively interact with the LRR‐RLK Suppressor of BIR1‐1/EVERSHED (SOBIR1/EVR) to form signalling‐competent receptor complexes. Ligand perception by LRR‐RLPs initiates recruitment of the co‐receptor BRI1‐Associated Kinase 1/Somatic Embryogenesis Receptor Kinase 3 (BAK1/SERK3) to the LRR‐RLP/SOBIR1 complex, thereby activating LRR‐RLP‐mediated immunity. We employed phosphorylation analysis of in planta‐produced proteins, live cell imaging, gene silencing and co‐immunoprecipitation to investigate the roles of SOBIR1 and BAK1 in immune signalling. We show that Arabidopsis thaliana (At) SOBIR1, which constitutively activates immune responses when overexpressed in planta, is highly phosphorylated. Moreover, in addition to the kinase activity of SOBIR1 itself, kinase‐active BAK1 is essential for AtSOBIR1‐induced constitutive immunity and for the phosphorylation of AtSOBIR1. Furthermore, the defence response triggered by the tomato LRR‐RLP Cf‐4 on perception of Avr4 from the extracellular pathogenic fungus Cladosporium fulvum is dependent on kinase‐active BAK1. We argue that, in addition to the trans‐autophosphorylation of SOBIR1, it is likely that SOBIR1 and BAK1 transphosphorylate, and thereby activate the receptor complex. The signalling‐competent cell surface receptor complex subsequently activates downstream cytoplasmic signalling partners to initiate RLP‐mediated immunity.

Human iPSC-derived trigeminal neurons lack constitutive TLR3-dependent immunity that protects cortical neurons from HSV-1 infection

Herpes simplex virus type 1 (HSV-1) encephalitis (HSE) is the most common sporadic viral encephalitis in Western countries. Some HSE children carry inborn errors of the Toll-like receptor 3 (TLR3)-dependent IFN-α/β- and -λ-inducing pathway. Induced pluripotent stem cell (iPSC)-derived cortical neurons with TLR3 pathway mutations are highly susceptible to HSV-1, due to impairment of cell-intrinsic TLR3-IFN immunity. In contrast, the contribution of cell-intrinsic immunity of human trigeminal ganglion (TG) neurons remains unclear. Here, we describe efficient in vitro derivation and purification of TG neurons from human iPSCs via a cranial placode intermediate. The resulting TG neurons are of sensory identity and exhibit robust responses to heat (capsaicin), cold (icilin), and inflammatory pain (ATP). Unlike control cortical neurons, both control and TLR3-deficient TG neurons were highly susceptible to HSV-1. However, pretreatment of control TG neurons with poly(I:C) induced the cells into an anti-HSV-1 state. Moreover, both control and TLR3-deficient TG neurons developed resistance to HSV-1 following pretreatment with IFN-β but not IFN-λ. These data indicate that TG neurons are vulnerable to HSV-1 because they require preemptive stimulation of the TLR3 or IFN-α/β receptors to induce antiviral immunity, whereas cortical neurons possess a TLR3-dependent constitutive resistance that is sufficient to block incoming HSV-1 in the absence of prior antiviral signals. The lack of constitutive resistance in TG neurons in vitro is consistent with their exploitation as a latent virus reservoir in vivo. Our results incriminate deficiencies in the constitutive TLR3-dependent response of cortical neurons in the pathogenesis of HSE.

Coral tumor-like growth anomalies induce an immune response and reduce fecundity.

Coral growth anomalies (GAs) are chronic diseases that adversely affect organism health and fitness. We investigated immunity and fecundity within and among GA-affected and visually healthy control colonies of the reef-building coral Acropora hyacinthus. Compared to controls, GAs had higher activity of the key immunity enzyme phenoloxidase (PO), suggesting a localised immune response within the GA. Both GAs and healthy tissue of GA-affected colonies had significantly greater total potential PO (tpPO)-PO activity inclusive of the activated latent PO, prophenoloxidase-than control colonies. Higher tpPO activity in GA-affected corals suggests elevated constitutive immunity compared to visually healthy controls. Additionally, fewer GA-affected colonies produced gametes, fewer polyps had oocytes (p < 0.001) and the number of oocytes per polyp was lower. Therefore, GAs in A. hyacinthus might induce, or represent a shift in resource investment towards immunity and away from reproduction. While the effect on population growth is likely to be small, reduced fecundity in GA-affected colonies does suggest a selective pressure against GAs.

The effects of warm temperature acclimation on constitutive stress, immunity, and metabolism in white sturgeon (Acipenser transmontanus) of different ploidies

Previous studies suggest fish with additional copies of their genome (polyploids) underperform in suboptimal conditions and may be more susceptible to stress and disease. The objective of this study was to determine the role ploidy plays in the physiological response of white sturgeon to chronically elevated water temperatures. White sturgeon of two ploidies (8 N and 10 N) were acclimated to ambient (18 °C) and warm (22 °C) water. Bioindices of stress (plasma cortisol, glucose and lactate, total erythrocyte count, hematocrit, hemoglobin, mean erythrocyte volume, mean erythrocyte hemoglobin, and mean erythrocyte hemoglobin concentration), immunity (respiratory burst, plasma lysozyme, and total leukocyte count), and cellular metabolic capacity (lactate dehydrogenase and citrate synthase activity) were measured before and after a 6-week acclimation period. Both ploidies appear comparable in their constitutive immune and stress parameters and respond similarly to warming. Hematological indices suggest 8 N and 10 N sturgeon are similar in oxygen carrying capacity; however, differences in enzyme activity between ploidies indicate that 10 N sturgeon may have a lower cellular aerobic capacity. Our results have implications for the screening and management of ploidy on white sturgeon farms and hatcheries, as the differences between ploidies may affect 10 N sturgeon performance at elevated water temperatures. Further research is needed to elucidate the differences in inducible stress and immune responses and metabolism of white sturgeon of different ploidies.

Experimental increase in baseline corticosterone level reduces oxidative damage and enhances innate immune response.

Glucocorticoid (GC) hormones are significant regulators of homeostasis. The physiological effects of GCs critically depend on the time of exposure (short vs. long) as well as on their circulating levels (baseline vs. stress-induced). Previous experiments, in which chronic and high elevation of GC levels was induced, indicate that GCs impair both the activity of the immune system and the oxidative balance. Nonetheless, our knowledge on how mildly elevated GC levels, a situation much more common in nature, might influence homeostasis is limited. Therefore, we studied whether an increase in GC level within the baseline range suppresses or enhances condition (body mass, hematocrit and coccidian infestation) and physiological state (humoral innate immune system activity and oxidative balance). We implanted captive house sparrows Passer domesticus with either 60 days release corticosterone (CORT) or control pellets. CORT-treated birds had elevated baseline CORT levels one week after the implantation, but following this CORT returned to its pre-treatment level and the experimental groups had similar CORT levels one and two months following the implantation. The mass of tail feathers grown during the initial phase of treatment was smaller in treated than in control birds. CORT implantation had a transient negative effect on body mass and hematocrit, but both of these traits resumed the pre-treatment values by one month post-treatment. CORT treatment lowered oxidative damage to lipids (malondialdehyde) and enhanced constitutive innate immunity at one week and one month post-implantation. Our findings suggest that a relatively short-term (i.e. few days) elevation of baseline CORT might have a positive and stimulatory effect on animal physiology.

Assessment of Seasonal Metabolic Acclimation in Gopher Tortoises (Gopherus polyphemus): Does Dormancy Reduce Metabolic Rate?

Environmental selective pressures, and associated constraints (such as sub-optimal thermal environments), imposed on organisms can drive important changes in energy usage in organisms exposed to the environmental constraints. Gopherus polyphemus, Gopher Tortoises, significantly reduce constitutive immunity during winter dormancy, a pattern that has been observed across ectothermic vertebrates. Herein, we assessed the metabolic consequences of winter acclimation to determine if the previously documented seasonal immune reduction confers measurable energy conservation. We measured oxygen (O2) consumption, carbon dioxide (CO2) production, and calculated respiratory quotient in G. polyphemus at two seasonally relevant temperatures (12.5°C and 32.5°C) and two seasonal acclimation states (winter and summer). We found no effect of season on either O2 consumption or CO2 production but found that RQ was significantly elevated in animals at the colder temperature (P ...

Comparative genomics analysis of triatomines reveals common first line and inducible immunity-related genes and the absence of Imd canonical components among hemimetabolous arthropods

BackgroundInsects operate complex humoral and cellular immune strategies to fend against invading microorganisms. The majority of these have been characterized in Drosophila and other dipterans. Information on hemipterans, including Triatominae vectors of Chagas disease remains incomplete and fractionated.ResultsWe identified putative immune-related homologs of three Triatominae vectors of Chagas disease, Triatoma pallidipennis, T. dimidiata and T. infestans (TTTs), using comparative transcriptomics based on established immune response gene references, in conjunction with the predicted proteomes of Rhodnius prolixus, Cimex lecticularis and Acyrthosiphon pisum hemimetabolous. We present a compressive description of the humoral and cellular innate immune components of these TTTs and extend the immune information of other related hemipterans. Key homologs of the constitutive and induced immunity genes were identified in all the studied hemipterans.ConclusionsOur results in the TTTs extend previous observations in other hemipterans lacking several components of the Imd signaling pathway. Comparison with other hexapods, using published data, revealed that the absence of various Imd canonical components is common in several hemimetabolous species.

Metabolic Induction of Trained Immunity through the Mevalonate Pathway

Innate immune cells can develop long-term memory after stimulation by microbial products during infections or vaccinations. Here, we report that metabolic signals can induce trained immunity. Pharmacological and genetic experiments reveal that activation of the cholesterol synthesis pathway, but not the synthesis of cholesterol itself, is essential for training of myeloid cells. Rather, the metabolite mevalonate is the mediator of training via activation of IGF1-R and mTOR and subsequent histone modifications in inflammatory pathways. Statins, which block mevalonate generation, prevent trained immunity induction. Furthermore, monocytes of patients with hyper immunoglobulin D syndrome (HIDS), who are mevalonate kinase deficient and accumulate mevalonate, have a constitutive trained immunity phenotype at both immunological and epigenetic levels, which could explain the attacks of sterile inflammation that these patients experience. Unraveling the role of mevalonate in trained immunity contributes to our understanding of the pathophysiology of HIDS and identifies novel therapeutic targets for clinical conditions with excessive activation of trained immunity.

STING Senses Microbial Viability to Orchestrate Stress-Mediated Autophagy of the Endoplasmic Reticulum

Constitutive cell-autonomous immunity in metazoans predates interferon-inducible immunity and comprises primordial innate defense. Phagocytes mobilize interferon-inducible responses upon engagement of well-characterized signaling pathways by pathogen-associated molecular patterns (PAMPs). The signals controlling deployment of constitutive cell-autonomous responses during infection have remained elusive. Vita-PAMPs denote microbial viability, signaling the danger of cellular exploitation by intracellular pathogens. We show that cyclic-di-adenosine monophosphate in live Gram-positive bacteria is a vita-PAMP, engaging the innate sensor stimulator of interferon genes (STING) to mediate endoplasmic reticulum (ER) stress. Subsequent inactivation of themechanistic target of rapamycin mobilizes autophagy, which sequesters stressed ER membranes, resolves ER stress, and curtails phagocyte death. This vita-PAMP-induced ER-phagy additionally orchestrates an interferon response by localizing ER-resident STING to autophagosomes. Our findings identify stress-mediated ER-phagy as a cell-autonomous response mobilized by STING-dependent sensing of a specific vita-PAMP and elucidate how innate receptors engage multilayered homeostatic mechanisms topromote immunity and survival after infection.