Cytokine expression in phytohaemagglutinin‐induced skin inflammation in a galliform bird

Researchers interested in ecological immunology face substantial methodological problems: 1) most immunological approaches are difficult to perform in free‐living animals, 2) in some of the applicable methods the immunological background of the test remains unclear. The latter is also true for the phytohaemagglutinin (PHA) skin‐swelling test, a trait of cell‐mediated immunity commonly measured in ecology. A lack of direct evidence documenting the immunological processes in the tissue limits our understanding of the mechanism triggering the response to PHA. Understanding of this mechanism is, nonetheless, crucial for us to uncover the nature of ecological costs and benefits of investments into the response. As knowledge of cytokine signalling in the tissue may clarify the response mechanism, in our study we investigated the association between the PHA‐induced skin‐swelling and tissue cytokine expression in males of grey partridge Perdix perdix. In PHA‐challenged birds we assessed expression of nine cytokines (IL‐1β, IL‐2, IL‐4, IL‐6, IL‐10, IL‐12, IL‐17, TGF‐β, IFN‐γ) in wing‐web skin during an early stage of the immune response. We examined the relationship between the magnitude of tissue swelling and cytokine expression. Contrary to some earlier expectations we did not find any differential expression of T‐cell growth factor, IL‐2, in the tissue. Hence, T‐cell proliferation at the time of the swelling measurement is unlikely. We detected differential expression in Th17 pro‐inflammatory (IL‐1β, IL‐6) and anti‐inflammatory (TGF‐β) cytokines. The PHA‐induced swelling response was only weakly linked to the expression of TGF‐β. We also found relationships between the PHA‐induced swelling response and phenotypic traits of the birds; the PHA swelling was positively associated with the extent of melanin‐based breast ornamentation and negatively related to body size. Our results might suggest that variation in swelling is influenced by total numbers of responding cells rather than by differences in signalling. Moreover, we revealed significant correlations in expression of IL‐1β, IL‐6 and TGF‐β. These findings are the first to show on the molecular level that the PHA skin‐swelling test actually measures inflammation process which is part of innate immune defence and not the adaptive immune response (as assumed if the test was the reflection of T‐cell proliferation).     

Histological assessment of cellular immune response to the phytohemagglutinin skin test in Brazilian free-tailed bats (Tadarida brasiliensis)

Bats are known reservoirs for numerous emerging infectious diseases, occupy unique ecological niches, and occur globally except for Antarctica. Given their impact on human and agricultural health, it is critical to understand the mechanisms underlying immunocompetence in this reservoir host. To date, few studies have examined immune function in the Order Chiroptera, particularly among natural colonies of bats. The phytohemagglutinin (PHA) skin test has been widely used to measure delayed-type cellular immune response in a wide variety of vertebrates, and has been routinely employed in immunoecological studies. Although this test is frequently described as a measure of T cell proliferation, recent studies indicate it may represent a combination of immune responses. In mammals, the immune response is differentially, temporally and spatially regulated, therefore, we characterized the infiltrating leukocyte response to the PHA skin test in bats by examining a time-series of histological sections from PHA and saline injection areas in 41 Brazilian free-tailed bats (Tadarida brasiliensis). Results suggest that bats exhibit diverse leukocyte traffic within 6 h, and up to 24 h following subcutaneous PHA injection. There was a significant presence of lymphocytes and neutrophils, as well as eosinophils, basophils, and macrophages observed in the PHA-injected tissues, compared with saline-injected control tissues. We observed a highly significant negative correlation between the number of lymphocytes and neutrophils in PHA-injected tissue, with peak lymphocyte response at 12 h, and peak neutrophil response at 24 h post-injection. These results indicate substantial variation in the immune response of individuals, and may aid our understanding of disease emergence in natural populations of bats.     

Secondary phytohaemagglutinin (PHA) swelling response is a good indicator of T‐cell‐mediated immunity in free‐living birds

The validity of using the phytohaemagglutinin (PHA) test to measure acquired immunity, one of the most widely used methods, is currently being debated due to new knowledge on the complex physiology of the process. As a greater secondary response to repeated challenges linked to increases of circulating lymphocyte levels would be indicative of a T‐cell‐mediated immune response, we performed for the first time an experiment under natural conditions with repeated PHA challenges in free‐living adult birds and chicks to shed light on this topic. We found significantly stronger secondary response to PHA injection independent of sex or age, while controlling for body condition, the second response being on average 90% larger than the first. Likewise, lymphocyte counts were significantly higher in the second PHA challenge, whereas no significant differences were found among untreated birds. Significant positive correlations between the PHA response and both lymphocyte counts and plasma protein levels (mainly albumin, globulin precursor) were recovered, whereas no significant differences were recovered in plasma protein levels between challenges. Our results are consistent with those from captive birds, supporting the validity of the PHA skin‐swelling test as an accurate gauge of acquired T‐cell‐mediated immunity in birds.     

Sex-specific effects of handling time on an index of immune activity in zebra finches

Recently, there has been considerable interest in the role of the immune system in shaping life-history evolution, sexual selection strategies, and indexes of individual quality. The most frequently used assay of immune function, particularly in avian field studies, is the phytohemagglunitin (PHA) skin test. PHA is injected subcutaneously into the wing web, and the magnitude of the resultant swelling has traditionally been interpreted as an index of an individual's cell-mediated immunocompetence. The test follows one of two protocols: the traditional two-wing injection protocol, with one wing web injected with PHA and the other with phosphate-buffered saline (PBS), or the simplified one-wing protocol that omits the PBS injection. In this technical comment, we alert researchers to the importance of considering handling time when performing the PHA test. We show that zebra finches (Taeniopygia guttata) subjected to the two-wing protocol had a lower wing-web swelling than individuals injected in one wing. In males, handling time explained over 50% of the variation in an individual's skin swelling response; females were relatively unaffected by handling time. We suggest that caution should be exercised when comparing the magnitude of wing-web swelling across studies in which the alternate protocol was followed. In addition, the recording of handling time, and its inclusion in subsequent statistical analyses, may aid in the detection of subtle differences across treatments.     

Phytohaemagglutinin injection has a long-lasting effect on immune cells

Measurement of phytohaemagglutinin (PHA)-induced skin swelling is the most popular assay of immune function in avian studies. The mechanisms causing swelling have been relatively well studied; however, very little is known about the potential long term physiological effects of PHA. Here we show that injection of PHA into patagium of captive greenfinches Carduelis chloris increases the concentration of heterophils (phagocytic cells of the innate immune response) in the peripheral blood for at least 30  days. Such long-term consequences should be taken into account when using PHA skin test in studies monitoring changes in individual physiological condition and/or immune status.     

不同繁殖状态雌性黑线仓鼠对植物血凝素的反应模式

由植物血凝素（Phytohemagglutinin,ＰＨＡ） 诱导产生的肿胀反应是生态免疫学中被广泛使用的免疫学参数,通过注射部位的肿胀程度大体上可反映细胞介导的免疫和天然免疫的综合变化情况。为探讨小型哺乳动物不同繁殖状态对ＰＨＡ 的反应模式,以非繁殖期、妊娠期、哺乳期和断乳期的雌性黑线仓鼠为研究对象,测定了注射ＰＨＡ 和生理盐水前（０ ｈ）及注射后６ ｈ、１２ ｈ 和２４ ｈ 注射部位皮肤组织的增厚程度。结果显示：（１）黑线仓鼠对ＰＨＡ 的反应有两种模式：非繁殖期、妊娠期和断乳期对ＰＨＡ 的反应模式相似,都在注射后６ ｈ 最高,１２ ｈ 和２４ ｈ 后逐渐下降;哺乳期对ＰＨＡ 的反应在注射后６ ｈ、１２ ｈ 和２４ ｈ 接近,彼此间的差异均不显著。（２）哺乳期黑线仓鼠的ＰＨＡ 反应高峰值有下降趋势,但与非繁殖期、妊娠期和断乳期高峰值之间的差异不显著。结果表明,黑线仓鼠在哺乳期对ＰＨＡ 的反应模式不同于其他繁殖阶段,且反应的高峰值被延迟,这对其存活可能有害,但有助于繁殖进程的延续和后代的生长发育。     

Low‐intensity pulsed ultrasound attenuates cardiac inflammation of CVB3‐induced viral myocarditis via regulation of caveolin‐1 and MAPK pathways

The aggressive immunological activity elicited by acute viral myocarditis contributes to a large amount of cardiomyocytes loss and poor prognosis of patients in clinic. Low‐intensity pulsed ultrasound (LIPUS), which is an effective treatment modality for osteoarthropathy, has been recently illustrated regulating the overactive inflammatory response in various diseases. Here, we aimed to investigate whether LIPUS could attenuate coxsackievirus B3 (CVB3) infection‐induced injury by coordinating the inflammatory response. Male BALB/c mice were inoculated intraperitoneally with CVB3 to establish the model of acute viral myocarditis. LIPUS treatment was given on Day 1, Day 1, 3 and Day 1, 3, 5 post‐inoculation, respectively. All mice were followed up for 14 days. Day 1, 3, 5 LIPUS treatment significantly improved the survival rate, attenuated the ventricular dysfunction and ameliorated the cardiac histopathological injury of CVB3‐infected mice. Western blotting analysis showed Day 1, 3, 5 LIPUS treatment decreased pro‐inflammatory cytokines, increased the activation of caveolin‐1 and suppressed p38 mitogen‐activated protein kinase (MAPK) and extracellular signal‐regulated kinase (ERK) signallings in heart tissue. RAW264.7 cells were treated with lipopolysaccharides (LPS) to simulate the augmented inflammatory response in vivo. LIPUS treatment on RAW264.7 inhibited the expression of pro‐inflammatory cytokines, activated caveolin‐1 and suppressed p38 MAPK and ERK signallings. Transfecting RAW264.7 with caveolin‐1 siRNA blunted the suppression of pro‐inflammatory cytokines and MAPK signallings by LIPUS treatment. Taken together, we demonstrated for the first time that LIPUS treatment attenuated the aggressive inflammatory response during acute viral myocarditis. The underlying mechanism may be activating caveolin‐1 and suppressing MAPK signallings.     

The perils and prospects of using phytohaemagglutinin in evolutionary ecology

Several techniques are available for quantifying the vertebrate immune response, information that is particularly useful for understanding the contribution of immunity to the evolution of life-history strategies. The most widely used is the phytohaemagglutinin (PHA) skin-swelling technique, which is usually regarded as an index of acquired immunity. However, our understanding of the effects of PHA in skin is poor, despite the fact that it has implications for what the test can tell us about immune activity. As we discuss here, a recent study by Martin and colleagues on the response to PHA at the cellular level in wild birds has highlighted the relative extent to which PHA-induced swelling, as most commonly applied, measures innate immunity versus acquired immunity.     

The PHA test reflects acquired T-cell mediated immunocompetence in birds

Background

cological immunology requires techniques to reliably measure immunocompetence in wild vertebrates. The PHA-skin test, involving subcutaneous injection of a mitogen (phytohemagglutinin, PHA) and measurement of subsequent swelling as a surrogate of T-cell mediated immunocompetence, has been the test of choice due to its practicality and ease of use in the field. However, mechanisms involved in local immunological and inflammatory processes provoked by PHA are poorly known, and its use and interpretation as an acquired immune response is currently debated.

Methodology

Here, we present experimental work using a variety of parrot species, to ascertain whether PHA exposure produces larger secondary than primary responses as expected if the test reflects acquired immunocompetence. Moreover, we simultaneously quantified T-lymphocyte subsets (CD4⁺, CD5⁺ and CD8⁺) and plasma proteins circulating in the bloodstream, potentially involved in the immunological and inflammatory processes, through flow cytometry and electrophoresis.

Principal Findings

Our results showed stronger responses after a second PHA injection, independent of species, time elapsed and changes in body mass of birds between first and second injections, thus supporting the adaptive nature of this immune response. Furthermore, the concomitant changes in the plasma concentrations of T-lymphocyte subsets and globulins indicate a causal link between the activation of the T-cell mediated immune system and local tissue swelling.

Conclusions/Significance

These findings justify the widespread use of the PHA-skin test as a reliable evaluator of acquired T-cell mediated immunocompetence in diverse biological disciplines. Further experimental research should be aimed at evaluating the relative role of innate immunocompetence in wild conditions, where the access to dietary proteins varies more than in captivity, and to ascertain how PHA responses relate to particular host-parasite interactions.     

Reduced naïve CD8+ T‐cell priming efficacy in elderly adults

Aging is associated with impaired vaccine efficacy and increased susceptibility to infectious and malignant diseases. CD8⁺ T‐cells are key players in the immune response against pathogens and tumors. In aged mice, the dwindling naïve CD8⁺ T‐cell compartment is thought to compromise the induction of de novo immune responses, but no experimental evidence is yet available in humans. Here, we used an original in vitro assay based on an accelerated dendritic cell coculture system in unfractioned peripheral blood mononuclear cells to examine CD8⁺ T‐cell priming efficacy in human volunteers. Using this approach, we report that old individuals consistently mount quantitatively and qualitatively impaired de novo CD8⁺ T‐cell responses specific for a model antigen. Reduced CD8⁺ T‐cell priming capacity in vitro was further associated with poor primary immune responsiveness in vivo. This immune deficit likely arises as a consequence of intrinsic cellular defects and a reduction in the size of the naïve CD8⁺ T‐cell pool. Collectively, these findings provide new insights into the cellular immune insufficiencies that accompany human aging.     

Cellular senescence impact on immune cell fate and function

Cellular senescence occurs not only in cultured fibroblasts, but also in undifferentiated and specialized cells from various tissues of all ages, in vitro and in vivo. Here, we review recent findings on the role of cellular senescence in immune cell fate decisions in macrophage polarization, natural killer cell phenotype, and following T‐lymphocyte activation. We also introduce the involvement of the onset of cellular senescence in some immune responses including T‐helper lymphocyte‐dependent tissue homeostatic functions and T‐regulatory cell‐dependent suppressive mechanisms. Altogether, these data propose that cellular senescence plays a wide‐reaching role as a homeostatic orchestrator.     

Mhc polymorphisms fail to explain the heritability of phytohaemagglutinin-induced skin swelling in a wild passerine

Genetic estimates of the variability of immune responses are rarely examined in natural populations because of confounding environmental effects. As a result, and because of the difficulty of pinpointing the genetic determinants of immunity, no study has to our knowledge examined the contribution of specific genes to the heritability of an immune response in wild populations. We cross-fostered nestling house sparrows to disrupt the association between genetic and environmental effects and determine the heritability of the response to a classic immunological test, the phytohaemagglutinin (PHA)-induced skin swelling. We detected significant heritability estimates of the response to PHA, of body mass and tarsus length when nestlings were 5 and 10 days old. Variation at Mhc genes, however, did not explain a significant portion of the genetic variation of nestling swelling to PHA. Our results suggest that while PHA-induced swelling is influenced by the nest of origin, the importance of additive genetic variation relative to non-additive genetic variation and the genetic factors that influence the former in wild populations still need to be identified for this trait.     

Significance of neutrophil microparticles in ischaemia‐reperfusion: Pro‐inflammatory effectors of endothelial senescence and vascular dysfunction

Endothelial senescence is an emerging cause of vascular dysfunction. Because microparticles are effectors of endothelial inflammation and vascular injury after ischaemia‐reperfusion, we examined leucocyte‐derived microparticles of spleen origin as possible contributors. Microparticles were generated from primary rat splenocytes by either lipopolysaccharide or phorbol‐myristate‐acetate/calcium ionophore, under conditions mimicking innate and adaptive immune responses. Incubation of primary porcine coronary endothelial cells with either type of microparticles, but not with those from unstimulated splenocytes, leads to a similar threefold raise in senescence‐associated β‐galactosidase activity within 48 hours, indicating accelerated senescence, to endothelial oxidative stress, and a fivefold and threefold increase in p21 and p16 senescence markers after 24 hours. After 12‐hour incubation, the endothelial‐dependent relaxation of coronary artery rings was reduced by 50%, at distinct optimal microparticle concentration. In vitro, microparticles were pro‐thrombotic by up‐regulating the local angiotensin system, by prompting tissue factor activity and a secondary generation of pro‐coagulant endothelial microparticles. They initiated an early pro‐inflammatory response by inducing phosphorylation of NF‐κB, MAP kinases and Akt after 1 hour, and up‐regulated VCAM‐1 and ICAM‐1 at 24 hours. Accordingly, VCAM‐1 and COX‐2 were also up‐regulated in the coronary artery endothelium and eNOS down‐regulated. Lipopolysaccharide specifically favoured the shedding of neutrophil‐ and monocyte‐derived microparticles. A 80% immuno‐depletion of neutrophil microparticles reduced endothelial senescence by 55%, indicating a key role. Altogether, data suggest that microparticles from activated splenocytes prompt early pro‐inflammatory, pro‐coagulant and pro‐senescent responses in endothelial cells through redox‐sensitive pathways. The control of neutrophil shedding could preserve the endothelium at site of ischaemia‐reperfusion–driven inflammation and delay its dysfunction.     

Variants of the IL‐10 gene associate with muscle strength in elderly from rural Africa: a candidate gene study

Recently, it has been shown that the capacity of the innate immune system to produce cytokines relates to skeletal muscle mass and strength in older persons. The interleukin‐10 (IL‐10) gene regulates the production capacities of IL‐10 and tumour necrosis factor‐α (TNF‐α). In rural Ghana, IL‐10 gene variants associated with different production capacities of IL‐10 and TNF‐α are enriched compared with Caucasian populations. In this setting, we explored the association between these gene variants and muscle strength. Among 554 Ghanaians aged 50 years and older, we determined 20 single nucleotide polymorphisms in the IL‐10 gene, production capacities of IL‐10 and TNF‐α in whole blood upon stimulation with lipopolysaccharide (LPS) and handgrip strength as a proxy for skeletal muscle strength. We distinguished pro‐inflammatory haplotypes associated with low IL‐10 production capacity and anti‐inflammatory haplotypes with high IL‐10 production capacity. We found that distinct haplotypes of the IL‐10 gene associated with handgrip strength. A pro‐inflammatory haplotype with a population frequency of 43.2% was associated with higher handgrip strength (P = 0.015). An anti‐inflammatory haplotype with a population frequency of 7.9% was associated with lower handgrip strength (P = 0.006). In conclusion, variants of the IL‐10 gene contributing to a pro‐inflammatory cytokine response associate with higher muscle strength, whereas those with anti‐inflammatory response associate with lower muscle strength. Future research needs to elucidate whether these effects of variation in the IL‐10 gene are exerted directly through its role in the repair of muscle tissue or indirectly through its role in the defence against infectious diseases.     

Fat tissue,aging, and cellular senescence

Fat tissue, frequently the largest organ in humans, is at the nexus of mechanisms involved in longevity and age‐related metabolic dysfunction. Fat distribution and function change dramatically throughout life. Obesity is associated with accelerated onset of diseases common in old age, while fat ablation and certain mutations affecting fat increase life span. Fat cells turn over throughout the life span. Fat cell progenitors, preadipocytes, are abundant, closely related to macrophages, and dysdifferentiate in old age, switching into a pro‐inflammatory, tissue‐remodeling, senescent‐like state. Other mesenchymal progenitors also can acquire a pro‐inflammatory, adipocyte‐like phenotype with aging. We propose a hypothetical model in which cellular stress and preadipocyte overutilization with aging induce cellular senescence, leading to impaired adipogenesis, failure to sequester lipotoxic fatty acids, inflammatory cytokine and chemokine generation, and innate and adaptive immune response activation. These pro‐inflammatory processes may amplify each other and have systemic consequences. This model is consistent with recent concepts about cellular senescence as a stress‐responsive, adaptive phenotype that develops through multiple stages, including major metabolic and secretory readjustments, which can spread from cell to cell and can occur at any point during life. Senescence could be an alternative cell fate that develops in response to injury or metabolic dysfunction and might occur in nondividing as well as dividing cells. Consistent with this, a senescent‐like state can develop in preadipocytes and fat cells from young obese individuals. Senescent, pro‐inflammatory cells in fat could have profound clinical consequences because of the large size of the fat organ and its central metabolic role.     

Esc‐1GN shows therapeutic potentials for acne vulgaris and inflammatory pain

The inflammatory response plays important roles in acne vulgaris and pain pathogenesis. In previous study, Esc‐1GN with anti‐inflammatory, antimicrobial, and lipopolysacchride (LPS) binding activity was identified from the skin of the frog Hylarana guentheri. Here, we report its therapeutic potentials for acne vulgaris and inflammatory pain. Esc‐1GN destroyed the cell membrane of Propionibacteria acnes in the membrane permeability assays. In addition, bacterial agglutination test suggested that Esc‐1GN triggered the agglutination of P. acnes, which was affected by LPS and Ca²⁺. Meanwhile, in vivo anti‐P. acnes and anti‐inflammatory effects of Esc‐1GN were confirmed by reducing the counts of P. acnes in mice ear, relieving P. acnes‐induced mice ear swelling, decreasing mRNA expression and the production of pro‐inflammatory cytokines, and attenuating the infiltration of inflammatory cells. Moreover, Esc‐1GN also displayed antinociceptive effect in mice induced by acetic acid and formalin. Therefore, Esc‐1GN is a promising candidate drug for treatment of acne vulgaris and inflammatory pain.     

WNT‐3A and WNT‐5A counteract lipopolysaccharide‐induced pro‐inflammatory changes in mouse primary microglia

Surveying microglia, the resident macrophage‐like cells in the central nervous system, continuously screen their surroundings to sense imbalance in tissue homeostasis. Their activity is tightly regulated in both a pro‐ and anti‐inflammatory manner. We have previously shown that the lipoglycoproteins WNT‐3A and WNT‐5A drive pro‐inflammatory transformation in primary mouse microglia cells, arguing that WNTs have a role in the modulation of the central nervous system immune response. In this study, we address the effects of recombinant WNT‐3A and WNT‐5A on lipopolysaccharide (LPS)‐activated mouse primary microglia to investigate the putative anti‐inflammatory modulation of microglia by WNTs. While both WNT‐3A and WNT‐5A alone induce an up‐regulation of cyclooxygenase 2 (COX2), a generic pro‐inflammatory microglia marker, LPS exceeds these effects dramatically. However, combination of LPS and WNTs results in a dose‐dependent decrease in LPS‐induced cyclooxygenase 2 protein and mRNA expression. In conclusion, our data suggest that WNTs have a dual and context‐dependent effect on microglia acting in a homeostatic pro‐ and anti‐inflammatory manner.