Causes and consequences of variation in nestling immune function

Defence against pathogens is a vital need of all living organisms that has led to the evolution of complex immune mechanisms. However, although immunocompetence the ability to resist pathogens and control infection has in recent decades become a focus for research in evolutionary ecology, the variation in immune function observed in natural populations is relatively little understood. This thesis examines sources of this variation (environmental, genetic and maternal effects) during the nestling stage and its fitness consequences in wild populations of passerines: the blue tit (Cyanistes caeruleus) and the collared flycatcher (Ficedula albicollis). A developing organism may face a dilemma as to whether to allocate limited resources to growth or to immune defences. The optimal level of investment in immunity is shaped inherently by specific requirements of the environment. If the probability of contracting infection is low, maintaining high growth rates even at the expense of immune function may be advantageous for nestlings, as body mass is usually a good predictor of post-fledging survival. In experiments with blue tits and haematophagous hen fleas (Ceratophyllus gallinae) using two methods, methionine supplementation (to manipulate nestlings resource allocation to cellular immune function) and food supplementation (to increase resource availability), I confirmed that there is a trade-off between growth and immunity and that the abundance of ectoparasites is an environmental factor affecting allocation of resources to immune function. A cross-fostering experiment also revealed that environmental heterogeneity in terms of abundance of ectoparasites may contribute to maintaining additive genetic variation in immunity and other traits. Animal model analysis of extensive data collected from the population of collared flycatchers on Gotland (Sweden) allowed examination of the narrow-sense heritability of PHA-response the most commonly used index of cellular immunocompetence in avian studies. PHA-response is not heritable in this population, but is subject to a non-heritable origin (presumably maternal) effect. However, experimental manipulation of yolk androgen levels indicates that the mechanism of the maternal effect in PHA-response is not in ovo deposition of androgens. The relationship between PHA-response and recruitment was studied for over 1300 collared flycatcher nestlings. Multivariate selection analysis shows that it is body mass, not PHA-response, that is under direct selection. PHA-response appears to be related to recruitment because of its positive relationship with body mass. These results imply that either PHA-response fails to capture the immune mechanisms that are relevant for defence against pathogens encountered by fledglings or that the selection pressure from parasites is not as strong as commonly assumed.

Anaesthetic impairment of immune function is mediated via GABA(A) receptors.

BACKGROUND: GABA(A) receptors are members of the Cys-loop family of neurotransmitter receptors, proteins which are responsible for fast synaptic transmission, and are the site of action of wide range of drugs. Recent work has shown that Cys-loop receptors are present on immune cells, but their physiological roles and the effects of drugs that modify their function in the innate immune system are currently unclear. We are interested in how and why anaesthetics increase infections in intensive care patients; a serious problem as more than 50% of patients with severe sepsis will die. As many anaesthetics act via GABA(A) receptors, the aim of this study was to determine if these receptors are present on immune cells, and could play a role in immunocompromising patients. PRINCIPAL FINDINGS: We demonstrate, using RT-PCR, that monocytes express GABA(A) receptors constructed of α1, α4, β2, γ1 and/or δ subunits. Whole cell patch clamp electrophysiological studies show that GABA can activate these receptors, resulting in the opening of a chloride-selective channel; activation is inhibited by the GABA(A) receptor antagonists bicuculline and picrotoxin, but not enhanced by the positive modulator diazepam. The anaesthetic drugs propofol and thiopental, which can act via GABA(A) receptors, impaired monocyte function in classic immunological chemotaxis and phagocytosis assays, an effect reversed by bicuculline and picrotoxin. SIGNIFICANCE: Our results show that functional GABA(A) receptors are present on monocytes with properties similar to CNS GABA(A) receptors. The functional data provide a possible explanation as to why chronic propofol and thiopental administration can increase the risk of infection in critically ill patients: their action on GABA(A) receptors inhibits normal monocyte behaviour. The data also suggest a potential solution: monocyte GABA(A) receptors are insensitive to diazepam, thus the use of benzodiazepines as an alternative anesthetising agent may be advantageous where infection is a life threatening problem.

Single cell proteomics microchip to profile immune function, with applications in stem cell biology, translational disease mechanism study and clinical therapeutics monitoring

In response to infection or tissue dysfunction, immune cells develop into highly heterogeneous repertoires with diverse functions. Capturing the full spectrum of these functions requires analysis of large numbers of effector molecules from single cells. However, currently only 3-5 functional proteins can be measured from single cells. We developed a single cell functional proteomics approach that integrates a microchip platform with multiplex cell purification. This approach can quantitate 20 proteins from >5,000 phenotypically pure single cells simultaneously. With a 1-million fold miniaturization, the system can detect down to ~100 molecules and requires only ~104 cells. Single cell functional proteomic analysis finds broad applications in basic, translational and clinical studies. In the three studies conducted, it yielded critical insights for understanding clinical cancer immunotherapy, inflammatory bowel disease (IBD) mechanism and hematopoietic stem cell (HSC) biology.

To study phenotypically defined cell populations, single cell barcode microchips were coupled with upstream multiplex cell purification based on up to 11 parameters. Statistical algorithms were developed to process and model the high dimensional readouts. This analysis evaluates rare cells and is versatile for various cells and proteins. (1) We conducted an immune monitoring study of a phase 2 cancer cellular immunotherapy clinical trial that used T-cell receptor (TCR) transgenic T cells as major therapeutics to treat metastatic melanoma. We evaluated the functional proteome of 4 antigen-specific, phenotypically defined T cell populations from peripheral blood of 3 patients across 8 time points. (2) Natural killer (NK) cells can play a protective role in chronic inflammation and their surface receptor – killer immunoglobulin-like receptor (KIR) – has been identified as a risk factor of IBD. We compared the functional behavior of NK cells that had differential KIR expressions. These NK cells were retrieved from the blood of 12 patients with different genetic backgrounds. (3) HSCs are the progenitors of immune cells and are thought to have no immediate functional capacity against pathogen. However, recent studies identified expression of Toll-like receptors (TLRs) on HSCs. We studied the functional capacity of HSCs upon TLR activation. The comparison of HSCs from wild-type mice against those from genetics knock-out mouse models elucidates the responding signaling pathway.

In all three cases, we observed profound functional heterogeneity within phenotypically defined cells. Polyfunctional cells that conduct multiple functions also produce those proteins in large amounts. They dominate the immune response. In the cancer immunotherapy, the strong cytotoxic and antitumor functions from transgenic TCR T cells contributed to a ~30% tumor reduction immediately after the therapy. However, this infused immune response disappeared within 2-3 weeks. Later on, some patients gained a second antitumor response, consisted of the emergence of endogenous antitumor cytotoxic T cells and their production of multiple antitumor functions. These patients showed more effective long-term tumor control. In the IBD mechanism study, we noticed that, compared with others, NK cells expressing KIR2DL3 receptor secreted a large array of effector proteins, such as TNF-α, CCLs and CXCLs. The functions from these cells regulated disease-contributing cells and protected host tissues. Their existence correlated with IBD disease susceptibility. In the HSC study, the HSCs exhibited functional capacity by producing TNF-α, IL-6 and GM-CSF. TLR stimulation activated the NF-κB signaling in HSCs. Single cell functional proteome contains rich information that is independent from the genome and transcriptome. In all three cases, functional proteomic evaluation uncovered critical biological insights that would not be resolved otherwise. The integrated single cell functional proteomic analysis constructed a detail kinetic picture of the immune response that took place during the clinical cancer immunotherapy. It revealed concrete functional evidence that connected genetics to IBD disease susceptibility. Further, it provided predictors that correlated with clinical responses and pathogenic outcomes.

UK Food Standards Agency Workshop Report: Diet and Immune Function

The UK Food Standards Agency convened a workshop on 13 May 2009 to discuss recently completed research on diet and immune function. The objective of the workshop was to review this research and to establish priorities for future research. Several of the trials presented at the workshop showed some effect of nutritional interventions (e.g. vitamin D, Zn, Se) on immune parameters. One trial found that increased fruit and vegetable intake may improve the antibody response to pneumococcal vaccination in older people. The workshop highlighted the need to further clarify the potential public health relevance of observed nutrition-related changes in immune function, e.g. susceptibility to infections and infectious morbidity.

Selection for cuticular melanism reveals immune function and life-history trade-offs in Spodoptera littoralis

Several insect species show an increase in cuticular melanism in response to high densities. In some species, there is evidence that this melanism is correlated with an up-regulation of certain immune system components, particularly phenoloxidase (PO) activity, and with the down-regulation of lysozyme activity, suggesting a trade-off between the two traits. As melanism has a genetic component, we selected both melanic and nonmelanic lines of the phase-polyphenic lepidopteran, Spodoptera littoralis, in order to test for a causative genetic link between melanism, PO activity and lysozyme activity, and to establish if there are any life-history costs associated with the melanic response. We found that, in fact, melanic lines had lower PO activity and higher lysozyme activity than nonmelanic lines, confirming a genetic trade-off between the two immune responses, but also indicating a genetic trade-off between melanism and PO activity. In addition, we found that lines with high PO activity had slower development rates suggesting that investment in PO, rather than in melanism, is costly.

Male morph predicts investment in larval immune function in the dung beetle, Onthophagus taurus

Investment in immunity is costly, so that resource-based trade-offs between immunity and sexually selected ornaments might be expected. The amount of resources that an individual can invest in each trait will be limited by the total resources available to them. It would therefore be informative to investigate how investment in immune function changes during growth or production of the sexual trait as resources are diverted to it. Using the dung beetle, Onthophagus taurus, which displays both sexual and male dimorphism in horn size, we examined changes in one measure of immune function, phenoloxidase (PO) activity, in the hemolymph of larvae prior to and during horn growth. We found that PO levels differed between small- and large-horned males throughout the final instar prior to the point where investment in horn growth was taking place. PO levels in females were intermediate to the 2 male morphs. These differences could not be accounted for by differences in condition, measured as hemolymph protein levels and weight. We suggest that the observed differences might be associated with sex- and morph-specific variation in juvenile hormone levels.

Density-dependent prophylaxis and condition-dependent immune function in Lepidopteran larvae: a multivariate approach

1. The risk of parasitism and infectious disease is expected to increase with population density as a consequence of positive density-dependent transmission rates. Therefore, species that encounter large fluctuations in population density are predicted to exhibit plasticity in their immune system, such that investment in costly immune defences is adjusted to match the probability of exposure to parasites and pathogens (i.e. density-dependent prophylaxis).

Heritability of immune function in the caterpillar Spodoptera littoralis

Phenoloxidase (PO) is believed to be a key mediator of immune function in insects and has been implicated both in non-self recognition and in resistance to a variety of parasites and pathogens, including baculoviruses and parasitoids. Using larvae of the Egyptian cotton leafworm, Spodoptera littoralis, we found that despite its apparent importance, haemolymph PO activity varied markedly between individuals, even amongst insects reared under apparently identical conditions. Sib-analysis methods were used to determine whether individuals varied genetically in their PO activity, and hence in one aspect of immune function. The heritability estimate of haemolymph PO activity was high (h 2 = 0.690 +/- 0.069), and PO activity in the haemolymph was strongly correlated with PO activity in both the cuticle and midgut; the sites of entry for most parasites and pathogens. Haemolymph PO activity was also strongly correlated with the degree to which a synthetic parasite (a small piece of nylon monofilament) was encapsulated and melanized (r = 0.622 +/- 0.142), suggesting that the encapsulation response is also heritable. The mechanism maintaining this genetic variation has yet to be elucidated.

Macronutrient balance mediates trade-offs between immune function and life history traits

1. Diet and health are intimately linked and recent studies have found that caloric restriction can affect immune function. However, when given a choice between diets that differ in their macronutrient composition, pathogen-infected individuals can select a diet that improves their survival, suggesting that the nutritional composition of the diet, as well as its calorie content, can play a role in defence against disease. Moreover, as individuals change their diet when infected, it suggests that a diet that is optimal for growth is not optimal for immunity, leading to trade-offs.
2. Currently, our knowledge of the effects of diet on immunity is limited because previous experiments have manipulated either single nutrients or the calorie content of the diet without considering their interactive effects. By simultaneously manipulating both the diet composition (quality) and its caloric density (quantity), in both naive and immune-challenged insects, we asked how do diet quality and quantity influence an individual's ability to mount an immune response? And to what extent are allocation trade-offs driven by quantity- versus quality-based constraints?
3. We restricted individuals to 20 diets varying in their protein and carbohydrate content and used 3D response surfaces to visualize dietary effects on larval growth and immune traits. Our results show that both constitutive and induced immune responses are not limited by the total quantity of nutrients consumed, but rather different traits respond differently to variation in the ratios of macronutrients (diet quality), and peak in different regions of macronutrient space. The preferred dietary composition therefore represents a compromise between the nutritional requirements of growth and immune responses. We also show that a non-pathogenic immune challenge does not affect diet choice, rather immune-challenged insects modify their allocation of nutrients to improve their immune response.
4. Our results indicate that immune traits are affected by the macronutrient content of the diet and that no diet can simultaneously optimize all components of the immune system. To date the emphasis has been on the effects of micronutrients in improving immunity, our findings indicate that this must be widened to include the neglected impact of macronutrients on defence against disease.

Effect of fruit and vegetable consumption on immune function in older people: A randomized controlled trial

Background: Fruit and vegetable (FV) intake, which is often low in older people, is associated with reduced chronic disease risk. Objective: We determined whether increased FV intake improves measures of immune function. Design: We conducted a randomized controlled trial (The Ageing and Dietary Intervention Trial) in 83 healthy volunteers aged 65-85 y with low FV intakes (=2 portions/d); 82 subjects completed the intervention. Participants were assigned to continue their normal diets or to consume =5 FV portions/d for 16 wk. At 12 wk, tetanus toxoid (0.5 mL intramuscular) and Pneumovax II vaccine (0.5 mL intramuscular; both vaccines from Sanofi Pasteur) were administered. FV intake was monitored by using diet histories, and biomarkers of nutritional status were assessed. The primary endpoint was the antibody response to vaccination. Specific antibodies binding to tetanus toxoid (total IgG) and pneumococcal capsular polysaccharide (total IgG and IgG2) were assessed at baseline and 16 wk. Participants were recruited between October 2006 and June 2008. Results: The change in FV consumption differed significantly between groups [mean change in number of portions (95% CI): in the 2-portion/d group, 0.4 portions/d (0.2, 0.7 portions/d); in the 5-portion/d group, 4.6 portions/d (4.1, 5.0 portions/d); P < 0.001)] and also in micronutrient status. Antibody binding to pneumococcal capsular polysaccharide (total IgG) increased more in the 5-portion/d group than in the 2-portion/d group [geometric mean (95% CI) of the week 16:baseline ratio: 3.1 (2.1, 4.4) and 1.7 (1.3, 2.1), respectively; P = 0.005)]. There was no significant difference in the increases in antibody binding to tetanus toxoid. Conclusion: Increased FV intake improves the Pneumovax II vaccination antibody response in older people, which links an achievable dietary goal with improved immune function. This trial was registered at clinicaltrials.gov as NCT00858728. © 2012 American Society for Nutrition.

Fatty acids and immune function: new insights into mechanisms

Fatty acids are known to play diverse roles in immune cells. They are important as a source of energy, as structural components of cell membranes, as signaling molecules and as precursors for the synthesis of eicosanoids and similar mediators. Recent research has suggested that the localization and organisation of fatty acids into distinct cellular pools has a direct influence on the behaviour of a number of proteins involved in immune cell activation, including those associated with T cell responses, antigen presentation and fatty acid-derived inflammatory mediator production. This article reviews these studies and places them in the context of existing literature in the field. These studies indicate the existence of several novel mechanisms by which altered fatty acid availability can modulate immune responses and impact upon clinical outcomes

Modulation of the fecal microflora profile and immune function by a novel trans-galactooligosaccharide mixture (B-GOS) in healthy elderly volunteers

Background: Aging is associated with reduced numbers of beneficial colonic bifidobacteria and impaired immunity. Galactooligosaccharides (GOSs) stimulate the growth of bifidobacteria in younger adults, but little is known about their effects in the elderly and their immunomodulatory capacity. Objective: We assessed the effect of a prebiotic GOS mixture (B-GOS) on immune function and fecal microflora composition in healthy elderly subjects. Design: In a double-blind, placebo-controlled, crossover study, 44 elderly subjects were randomly assigned to receive either a placebo or the B-GOS treatment (5.5 g/d). Subjects consumed the treatments for 10 wk, and then went through a 4-wk washout period, before switching to the other treatment for the final 10 wk. Blood and fecal samples were collected at the beginning, middle (5 wk), and end of the test period. Predominant bacterial groups were quantified, and phagocytosis, natural killer (NK) cell activity, cytokine production, plasma cholesterol, and HDL cholesterol were measured. Results: B-GOS significantly increased the numbers of beneficial bacteria, especially bifidobacteria, at the expense of less beneficial groups compared with the baseline and placebo. Significant increases in phagocytosis, NK cell activity, and the production of antiinflammatory cytokine interleukin-10 (IL-10) and significant reduction in the production of proinflammatory cytokines (IL-6, IL-1 beta , and tumor necrosis factor-alpha) were also observed. B-GOS exerted no effects on total cholesterol or HDL-cholesterol production, however. Conclusions: B-GOS administration to healthy elderly persons resulted in positive effects on both the microflora composition and the immune response. Therefore, B-GOS may be a useful dietary candidate for the enhancement of gastrointestinal health and immune function in elderly persons. Am J Clin Nutr 2008; 88: 1438-46.

Commentary on 'Prebiotics, immune function, infection and inflammation: a review of the evidence'

There is a growing awareness that the gut microbiota and an appropriately functioning immune system play an important role in maintaining human health. Recent population statistics have highlighted some worrying trends, specifically that there is a growing burden of immunological disease in Western populations, that Western populations are ageing, and that obesity, with its strong inflammatory component, is reaching epidemic proportions.