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Humoral immune responses are maintained with age in a long-lived ectotherm, the red-eared slider turtle

Humoral immune responses are maintained with age in a long-lived ectotherm, the red-eared slider... The Journal of Experimental Biology 216, 633-640 © 2013. Published by The Company of Biologists Ltd doi:10.1242/jeb.078832 RESEARCH ARTICLE Humoral immune responses are maintained with age in a long-lived ectotherm, the red-eared slider turtle Laura M. Zimmerman*, Sandrine G. Clairardin, Ryan T. Paitz, Justin W. Hicke, Katie A. LaMagdeleine, Laura A. Vogel and Rachel M. Bowden School of Biological Sciences, Illinois State University, Normal, IL 61790, USA *Author for correspondence ([email protected]) SUMMARY Aging is typically associated with a decrease in immune function. However, aging does not affect each branch of the immune system equally. Because of these varying effects of age on immune responses, aging could affect taxa differently based on how the particular taxon employs its resources towards different components of immune defense. An example of this is found in the humoral immune system. Specific responses tend to decrease with age while non-specific, natural antibody responses increase with age. Compared with mammals, reptiles of all ages have a slower and less robust humoral immune system. Therefore, they may invest more in non-specific responses and thus avoid the negative consequences of age on the immune system. We examined how the humoral immune system of reptiles is affected by aging and investigated the roles of non-specific, natural antibody responses and specific responses by examining several characteristics of antibodies against lipopolysaccharide (LPS) in the red-eared slider turtle. We found very little evidence of immunosenescence in the humoral immune system of the red-eared slider turtle, Trachemys scripta, which supports the idea that non-specific, natural antibody responses are an important line of defense in reptiles. Overall, this demonstrates that a taxonʼs immune strategy can influence how the immune system is affected by age. Key words: eco-immunology, reptile, Trachemys scripta. Received 1 August 2012; Accepted 10 October 2012 INTRODUCTION less robust (reviewed by Zimmerman et al., 2010a). Like mammals, Immunosenescence, which is a decrease in immune function with reptiles produce antibodies after a lag time of about one week, but increasing age, is a common observation in vertebrates. This process production often does not peak until 6–8 weeks later. If exposed for can have important consequences for an organism; for example, an a second time to the same antigen, reptiles do not exhibit a memory increase in mortality and morbidity in elderly humans is commonly response typical of mammals. While the lag time is reduced, titers attributed to a decrease in immune function (Dicarlo et al., 2009). are not increased compared with the primary response. It is generally However, the aging process does not affect every branch of the considered that affinity maturation does not occur in ectotherms, immune system in the same manner (Palacios et al., 2007; Ujvari including reptiles, but a small increase in affinity has been found and Madsen, 2011). This is highlighted in the humoral immune in response to immunization in the rainbow trout, Oncorhynchus system, where it has been found in a number of taxa that specific mykiss (Cain et al., 2002). B cell subsets have not yet been antibody production decreases with age, while non-specific, identified in reptiles due to a lack of specific reagents. polyreactive antibodies increase with age (Frasca et al., 2008). Given the less robust antibody response of reptiles, they may rely Because of these varying effects of age on immune responses, aging instead on production of non-specific antibodies in the form of could affect taxa differently based on how the particular taxon natural antibodies (NAbs). While NAbs have been identified in a employs its resources towards different components of immune variety of taxa, including reptiles, most available information comes defense. from studies of mammals. Natural antibodies are produced by a An example of how taxa can vary their utilization of immune subset of B cells known as B-1 cells and can be produced defenses is found in the differences in timing of the humoral response constitutively at low levels in the absence of antigen or be induced to antigen exposure in mammals and reptiles. In mammals, the in response to certain antigens including microbial components such specific humoral response is produced by a subset of B cells called as lipopolysaccharide (LPS) and phosphocoline (Baumgarth et al., B-2 cells. When a mammal is exposed to an antigen, it will produce 2005; Yang et al., 2007), although the antibody produced in antigen-specific antibodies of moderate affinity after a lag period response to antigen stimulation is of lower affinity than that of approximately 1 week and peak production at 2 weeks after produced by B-2 cells. Natural antibodies are polyreactive to exposure. After this first exposure to an antigen, memory cells are evolutionarily conserved components of pathogens, have low formed, and the lag time to antibody production is reduced upon a binding affinities and play active roles in triggering both innate and second exposure to the antigen, and antibody titers and affinity are adaptive immunity (Ochsenbein and Zinkernagel, 2000). They also increased (Coico et al., 2003). Compared with their mammalian bind to a variety of self-antigens, which allows them to play counterparts, the specific humoral system of reptiles is slower and housekeeping roles such as the clearing of apoptotic cells (Binder THE JOURNAL OF EXPERIMENTAL BIOLOGY 634 The Journal of Experimental Biology 216 (4) Fig. 1. Modes of B cell activation induced by A B lipopolysaccharide (LPS) in mammals. (A) LPS is a B cell mitogen that acts through TLR4 and CD14 to induce B cell antibody secretion independent of B cell antigen specificity. This results in the activation of a B cell A large number of B cells and polyclonal B cell B cell B immunoglobulin (Ig) secretion. In this situation, B cells A, B and C would secrete antibody. (B) LPS can also activate B cells in the traditional manner by binding directly to antigen-specific surface Ig. In this B cell l B cell case, only LPS-specific B cells (B-cell B) are activated to produce Ig. (C) Natural antibodies can be secreted in the absence of antigen stimulation. In this situation, B-cell D would spontaneously secrete low-affinity antibodies with the ability to bind LPS. B cell Antibodies with different LPS TLR4 CD14 B-1 B cell B-2 B cell antigen specificities et al., 2008) and may even act to reduce the risk of autoimmune determine how many cells are producing antibodies and how many diseases (Boes et al., 2000). Although they can be of the antibodies are being produced by each cell. These characteristics immunoglobulin A (IgA) or immunoglobulin G (IgG) isotype, most are measured frequently in traditional immunology, but thus far have NAbs are of the immunoglobulin M (IgM) isotype and are secreted not been examined in any eco-immunology study and can provide as pentamers. The pentameric structure allows them to be a strong a fuller picture of humoral immune functioning. activator of complement and permits more efficient production of We examined how the humoral immune system of reptiles is antigen–antibody complexes, which helps target antigens to affected by aging and investigated the roles of natural/B-1-like lymphatic tissues and improve adaptive responses (Boes, 2000). antibody and specific/B-2-like responses by examining several Both mammals and reptiles show an age-related decrease in specific, characteristics of antibodies against LPS. Lipopolysaccharide, which B-2-cell-produced antibodies and an increase in non-specific, B-1- is a component of Gram-negative bacteria, is a T-independent cell-produced NAbs. In humans, this reduction in specific antibody antigen, meaning that a B cell can respond to the antigen without responses has been viewed as a contributor to the rise in mortality direct contact with a T cell. In mammals, the humoral immune and morbidity with increasing age and, although there is also an system responds to LPS with (1) NAbs produced in the absence of increase in NAbs with age in humans, they do not appear to be able antigen stimulation by B-1 cells, (2) antibodies produced as a direct to compensate for the loss of the specific antibodies (McGlauchlen result of antigen stimulation by LPS binding directly to an antigen- and Vogel, 2003). Interestingly, the less-robust specific immune specific membrane Ig by B-1 or B-2 cells or (3) polyclonal antibody response of reptiles of all ages, combined with the increase in NAbs production through a Toll-like receptor pathway by B-1 or B-2 cells with age, may constitute an overall positive change in immune (Fig. 1). All receptors are constitutively expressed, so a B cell can defense (Ujvari and Madsen, 2011). However, little is known about be activated by any mechanism at any time. The isotypes of both antibody production in reptiles beyond quantity. Examining other specific antibodies and NAbs that bind to LPS include IgA, IgG antibody characteristics in addition to quantity would help us to and IgM, although NAbs are predominantly IgM. Although germinal decipher if changes in humoral immune responses with age centers typically do not form in response to LPS, specific antibodies differentially affect taxa with varying immune strategies. One still have a higher affinity to LPS than NAbs (Bruderer et al., 1992). important characteristic to consider is strength of binding. Affinity In mice, an age-associated decrease in antibody responses to T- is the measure of how well one particular antibody-combining site independent antigens, including LPS, has been reported (Chelvarajan binds to a single antigenic epitope, while avidity is a measure of et al., 2005). A similar result was found in a natural population of how well the total antibody population binds to the entire multivalent tree swallows (Tachycineta bicolor), where young and mid-age antigen. Thus, avidity can be measured from a plasma sample females injected with LPS mounted a specific antibody response, containing antibodies with different specificities. Knowing the while the oldest individuals did not (Palacios et al., 2011). avidity of an immune response can help determine if the antibodies The present study was conducted on a natural population of red- −3 −7 –1 are NAbs (affinity ranges from 10 to 10 mol l ) (Notkins, 2004) eared slider turtles (Trachemys scripta Schoepff). The slider is an or specific antibodies produced in response to antigen stimulation excellent model for examining humoral responses to LPS with age −10 –1 (affinity averages 10 mol l ) (Foote and Eisen, 1995). Another for several reasons. Sliders grow throughout their lifetime, so important characteristic that has been understudied in reptilian plastron length can be used as a proxy for age (Ernst et al., 1994). immunology is antibody production by individual B cells. Measuring Due to the challenges of following a turtle population over several the function of antibody secreting cells (AbSCs) allows us to decades, it is difficult to give an average life span for the slider THE JOURNAL OF EXPERIMENTAL BIOLOGY Immunity and aging in an ectotherm 635 turtle, although it is generally estimated to be around 30–40 years IL, USA on a natural population of red-eared sliders from May to (Ernst et al., 1994). Studies in the closely related painted turtle found August 2011 (IDNR permit NH11.2084). Adult male and female that they could live upwards of 60 years (Congdon et al., 2003), so turtles were trapped at three points in the active season: early May, it is possible that 40 years is a conservative estimate for sliders. We late June and late August. At time of capture, any unmarked have previously reported an increase in total Ig levels with age in individuals were uniquely marked, and plastron length was measured the slider (Zimmerman et al., 2010b). In addition, sliders are likely to the nearest 0.1 mm. No individual was used in more than one to be exposed to LPS in their natural environment, with likelihood collection period. Approximately 1 ml of blood was taken from the of exposure varying seasonally. A previous study in our population caudal vein using an EDTA-coated syringe. Plasma was separated has found a high frequency of Salmonella prevalence in adult turtles, from 500 μl of blood by centrifugation (2000 g, 3 min) at the field with prevalence increasing as temperatures increase and remaining site and kept on ice until it was taken to Illinois State University high through the remainder of the active season (Holgersson, 2009). and stored at −20°C. A blood smear was made using 7 μl of whole It is reasonable to suggest that other bacteria may also increase in blood. The remaining blood was diluted ~1:2 with a 75% RPMI- prevalence as temperatures increase. Because of this, and the fact 25% EDTA mixture for use in the ELISpot. that we have also found significant seasonal variation in a number of immune measures (Zimmerman et al., 2010b), we sampled ELISA immune responses at three points throughout the active season. An ELISA was used to measure total Ig and anti-LPS Abs Importantly, we have now validated an ELISpot assay to examine (Zimmerman et al., 2010b). Polystyrene 96-well plates (Costar, –1 the properties of AbSCs, along with an avidity assay to measure Tewksbury, MA, USA) were coated with either 100 μl of a 25 μg ml antibody binding. These new assays, along with the ELISA that we dilution of unlabeled anti-turtle light chain antibody (HL673; –1 have previously validated and the common practice of using blood University of Florida Hybridoma Facility) or 100 μl of a 20 μg ml smears to examine leukocyte populations, will allow us to gain a solution of LPS (from Salmonella enterica serotype typhimurium; fuller understanding of humoral immune responses of sliders. Sigma, St Louis, MO, USA) in phosphate-buffered saline (PBS) A wide range of studies in a variety of vertebrate taxa, including and incubated overnight at 4°C. Plates were washed three times for mammals, birds and reptiles, have found that specific antibodies 3 min with 200 μl per well of PBS–1% BSA–0.05% Tween buffer decrease with age while NAbs increase with age (Candore et al., (PBS-BSA-T), which also serves as a blocking step. Plasma samples 1997; Parmentier et al., 2004; Ujvari and Madsen, 2005; Lavoie, were diluted 1:1000 for total Ig and 1:50 for LPS-specific Abs in 2006; Benatuil et al., 2008; Frasca et al., 2008; Sparkman and PBS-BSA-T. Goat serum was added as a negative control. Plates Palacios, 2009; Ujvari and Madsen, 2011). This pattern, along with were incubated at room temperature for at least 1 h, then washed as the typically slow and less-robust specific responses of reptiles, leads before. One hundred microliters of a 1:500 dilution of anti-turtle us to hypothesize that red-eared sliders utilize a predominantly NAb- antibody conjugated to biotin was added to each well, and plates based response, and thus would not show a deficit in humoral were incubated and washed as before. One hundred microliters of immune defenses with age. Because both NAbs and specific streptavidin-HRP (Southern Biotech, Birmingham, AL, USA; antibodies can be produced in response to antigenic stimulation, we diluted 1:1000 in PBS-BSA-T) was added to each well, and plates also hypothesized that the humoral response would change across were incubated and washed as before. Wells were washed once with the active season as the animals naturally encounter LPS in the 100 μl double distilled water before 100 μl of ABTS (Southern environment, regardless of whether the responses were Biotech) substrate powder dissolved in ABTS solution was added predominantly NAb-based or specific. If natural antibodies/B-1-cell- to each well. The plate was read at 17 min after adding substrate like immunity are predominant in sliders, we expect immune using a Powerwave 340 plate reader (BioTek Inc., Winooski, VT, measures to increase or show no change in function with age. Thus, USA) at 405 nm. we would predict that the amount and avidity of antigen-specific antibody, as well as total Ig and AbSC number and function, should Avidity be preserved. Alternatively, if specific responses are predominant Avidity was measured using a competitive ELISA. Fifty microliters in sliders, we would expect immune measures to decrease with age. of diluted test serum (1:50) was added to six 50 μl aliquots of serial –1 Thus, we would predict that AbSCs would show a reduced response diluted LPS (0.08–5 mg ml ) and two tubes of 50 μl of PBS only, to LPS stimulation and that avidity, the amount of antibodies that and incubated for 18 h at room temperature in glass tubes. An ELISA can bind to LPS (LPS-Abs) and the percentage of lymphocytes was then run as described above with the plates coated with LPS. would decrease with age. We also predict that LPS-Abs, total Ig, Avidity values were calculated from the absorbances according to avidity, the number of AbSCs, the amount of antibody produced previously described methods (Friguet et al., 1985). Thus, for the by each cell, and the percentage of lymphocytes would increase individual turtle plasma sample, six values (one from each tube used through the active season as the turtles are more likely to be exposed in the serial dilution) were obtained by subtracting the absorbance to LPS. To examine our hypotheses, we determined both the quantity in a well from the average absorbance of the wells that came from and quality of LPS humoral responses in the slider by measuring the tubes that contained no antigen and then dividing that number LPS-Abs, total Ig, antibody avidity to LPS, the properties of AbSCs by the molarity of the antigen present in the glass tube during and the percentage of leukocytes that are lymphocytes. While the incubation. Those six values were then plotted; a linear regression assays used in this study examine variation in both NAbs and specific was used and the slope of the line was the dissociation constant. antibodies, the prediction for the pattern of responses with age and Avidity was then recorded as the reciprocal of the dissociation across the active season differs and thus allows us to distinguish constant. between the two types of responses. ELISpot MATERIALS AND METHODS Leukocytes were isolated over a Percoll gradient (Harms et al., This study was conducted under IACUC approval (protocol 04- 2000). Wells of MultiScreen-IP ELISpot plates (Millipore, Billerica, –1 2010) at Banner Marsh State Fish and Wildlife Area, Fulton Co., MA, USA) were coated with a 20 μg ml dilution of unlabeled anti- THE JOURNAL OF EXPERIMENTAL BIOLOGY 636 The Journal of Experimental Biology 216 (4) turtle light chain overnight at 4°C. The wells were then washed with RESULTS PBS and blocked for 2 h at 37°C with RPMI (Hyclone, Logan, UT, First, LPS binding antibody concentrations were examined (N=56). USA) supplemented with 5% fetal bovine serum, 1% All animals tested produced detectable anti-LPS antibodies as penicillin/streptomycin/glutamine, 0.5% 2-mercaptoethanol and measured by ELISA. As animals were not deliberately immunized, 0.5% sodium pyruvate (cRPMI). A known number of leukocytes either pre-existing polyreactive NAbs were detected or animals had in 100 μl cRPMI was added to each well. The number of leukocytes naturally encountered the antigen. Quantity of LPS-Abs significantly 5 6 –1 plated ranged from 10 to 10 cells well . An additional 100 μl of increased with plastron length (Fig. 2A; F =5.09, P=0.029) and 1,49 –1 cRPMI or cRPMI supplemented with 40 μg ml LPS was added to across the active season (Fig. 3A; F =3.34, P=0.044). There was 2,49 each well to observe spontaneous antibody production and to no correlation with sex on LPS-Abs (F =0.17, P=0.69) and the 1,49 stimulate antibody production, respectively. Cells from each turtle date by sex interaction was not significant (F =1.39, P=0.26). The 1,49 were given each treatment in duplicate wells. The cells were then avidity of these anti-LPS antibodies was also measured by ELISA incubated at 31°C in 5% CO for five days. The cell remains as described (N=48). Avidity did not vary with plastron length stationary, while any antibody produced remains attached to the (Fig. 2B; F =1.69, P=0.20), date (Fig. 3B; F =1.40, P=0.26) or 1,41 2,41 capture antibody. After five days, wells were washed with sex (F =3.05, P=0.09). The date by sex interaction was also not 1,41 PBS–0.01% Tween to remove the cells. The antibody remains significant (F =1.22, P=0.31). 2,41 attached and was detected with anti-turtle light chain conjugated to Next, total Ig levels were examined (N=55). Total Ig levels ranged –1 biotin, followed by streptavidin-HRP. The wells were developed from 0 to 9.7 mg ml . Total Ig varied significantly by date (Fig. 3C; with AEC substrate (BD Biosciences, San Jose, CA, USA), which F =7.68, P=0.0013) and sex (F =4.26, P=0.045), with males 2,48 1,48 leaves a red spot where antibodies have been detected, with each having significantly higher total Ig levels than females. There was spot representing one cell. The size of spot indicates how much no correlation with plastron length (Fig. 2C; F =1.87, P=0.18), and 1,48 antibody was produced by each cell. The number of spots was the date by sex interaction was not significant (F =0.25, P=0.78). 1,48 counted to determine the number of AbSCs per 10 cells, based on The number of spontaneous AbSCs in the blood ranged from 0 the number of cells added to the well. Size of spots was determined to 13.17 cells per 10 leukocytes (N=40), and the number of LPS- using ImageJ software (National Institutes of Health, Bethesda, MD, stimulated AbSC ranged from 0 to 22.73 cells per 10 leukocytes USA). (N=44). For cells cultured in media alone in the ELISpot, the number of AbSCs and average spot size did not vary with plastron length Blood smears (Fig. 2D; F =0.02, P=0.88; F =0.25, P=0.62, respectively), date 1,34 1,27 Previous studies in a number of vertebrates have shown that (Fig. 3D; F =0.57, P=0.57; F =0.44, P=0.65, respectively) or 2,34 2,27 leukocyte ratios, specifically heterophil:lymphocyte ratio, can sex (F =1.45, P=0.24; F =0.87, P=0.36, respectively). The date 1,34 1,27 change significantly as a result of handling stress (reviewed by by sex interaction was also not significant (F =0.83, P=0.37; 1,34 Davis et al., 2008). A previous study in our lab demonstrated that F =2.24, P=0.15). For the cells cultured with LPS in the ELISpot, 1,27 heterophil:lymphocyte ratio did not vary from baseline after the average spot size was significantly larger in wells that we turtles were restrained for 2 h (K.A.L. and L.M.Z., unpublished stimulated with LPS than in wells that received media only (LPS data). All blood samples used in this study were taken within mean ± s.e.m.=35.43±2.18 pixels, media-only mean ± s.e.m.= 90 min of the turtle being removed from the trap. Blood smears 28.68±1.79 pixels; T =3.49, P=0.001), and the total number of Ig- were fixed in methanol and stained with Wright-Giemsa (Sigma- secreting cells was increased by LPS (LPS mean ± s.e.m.= Aldrich, St Louis, MO, USA. The leukocyte profile was 3.62±0.66 pixels, media-only mean ± s.e.m.=2.74±0.46 pixels; determined by counting a total of 100 leukocytes and calculating T =2.345, P=0.024). However, the number of LPS-stimulated the percentage of lymphocytes, basophils, eosinophils, monocytes AbSCs and spot size did not vary with plastron length (Fig. 2E; and heterophils. The number of white blood cells per 10,000 red F =0.00, P=0.96; F =0.06, P=0.81), date (Fig. 3E; F =1.12, 1,38 1,30 2,38 blood cells was determined as a proxy for white blood cell count P=0.34; F =1.30, P=0.29, respectively) or sex (F =1.85, 2,30 1,38 (WBC) (Norte et al., 2008). P=0.18; F =0.57, P=0.46, respectively). The date by sex 1,30 interaction was also not significant (F =1.14, P=0.29; F =0.31, 1,38 1,30 Statistical analysis P=0.58, respectively). The effect of date of sampling, sex and plastron length on number Finally, WBC differential counts were examined (N=54). Total of AbSCs when stimulated and cultured in media only, spot size WBC number significantly decreased with increasing plastron for both conditions, avidity, LPS-Abs, total Ig, and WBC was length (Fig. 2F; F =4.45, P=0.0399) but did not vary with sex 4,49 examined using ANCOVAs. ANCOVA was chosen rather than (F =3.42, P=0.07) or date (Fig. 3F; F =0.76, P=0.48). A 4,49 4,49 MANCOVA because sample size did not permit the inclusion of significant effect of date on leukocyte profile was detected (Pillai’s all immune measures and we had no a priori reasons to group trace: F =3.23, P=0.001). A significant axis was found (Table 1; 10,92 certain immune measures. The number of AbSCs for stimulated eigenvalue=0.7858, P=0.0006) and it explained 90.07% of the and media-only conditions and total Ig were square root variation among dates. This was mostly driven by eosinophils and transformed while avidity was log transformed. All interactions heterophils and, to a lesser extent, monocytes (Tables 1, 2). were tested, and non-significant interactions involving plastron Lymphocytes decreased across the active season but did not explain length were removed from the model. Tukey’s post hoc tests were much of the variation. There was no significant effect of sex (Pillai’s conducted. For the leukocyte profile, a MANOVA was run trace: F =0.1.81, P=0.13) or plastron length (Pillai’s trace: 5,45 including the proportions of each cell type as dependent variables. F =0.25, P=0.94) on the leukocyte profile. 5,45 Date, sex and plastron length were included as main effects. All interactions were tested and found to be non-significant so they DISCUSSION were removed from the models. A paired t-test was used to Aging negatively affects many aspects of the immune response, compare spot size and number of AbSCs between stimulated and including the specific antibody response. However, some immune media-only wells. measures are not negatively affected by age, and may even increase, THE JOURNAL OF EXPERIMENTAL BIOLOGY Immunity and aging in an ectotherm 637 AB C 1.2 1.8 1.6 1.4 0.8 1.2 8 0.6 0.8 0.4 0.6 4 0.4 0.2 0.2 0 0 0 DE F 14 800 4 500 2 450 100 125 150 175 200 225 250 275 100 125 150 175 200 225 250 275 100 125 150 175 200 225 250 275 Plastron (mm) Fig. 2. Relationship between plastron length and (A) the amount of antibodies that can bind to lipopolysaccharide (LPS), (B) avidity, (C) total 5 5 cells cultured in media only, (E) number of spots per 10 cells cultured in the presence of LPS and (F) white immunoglobulin, (D) number of spots per 10 blood cell count (WBC). such as the non-specific NAb response (Frasca et al., 2008). In Abs significantly increased with age. In addition, the avidity of reptiles of all ages, the specific antibody response is slower and less antibodies to LPS was in the range of NAbs (Ochsenbein and robust than its mammalian counterpart (Zimmerman et al., 2010a). Zinkernagel, 2000). Thus, we are likely to be measuring This may force them to rely more heavily on NAbs to deal with predominantly NAbs that are binding to LPS with a low avidity, pathogens. Thus, an increase in NAbs with age may be viewed as rather than measuring highly specific antibodies that would bind to a positive change in immunity with age in reptiles (Ujvari and LPS with a high avidity. In addition, the number of AbSCs that Madsen, 2011). responded to LPS stimulation did not vary with age, nor did the The primary goal of the current study was to examine the effect amount of antibody produced by each cell. Thus, our turtles seem of age on humoral immune responses to LPS in a long-lived reptile, to have an increase in NAb, B-1-like immunity with age, but the the red-eared slider turtle. We hypothesized that red-eared sliders specific, B-2-like response to LPS was not significantly impacted utilize a predominantly NAb response, and thus would be less by age. While an increase in NAbs with age has been reported in affected by changes in humoral immune defenses with age. LPS- mammals, birds and reptiles (Candore et al., 1997; Parmentier et 0.9 Fig. 3. Mean ± s.e.m. during May bb AB C 0.8 6 (N=26), late June (N=22) and August 0.7 0.7 (N=17) for (A) the amount of a,b 0.6 5 a antibodies that can bind to 0.6 0.5 lipopolysaccharide (LPS), (B) avidity, 0.5 0.4 (C) total immunoglobulin, (D) number 0.4 0.3 of spots per 10 cells cultured in 0.3 media only, (E) number of spots per 0.2 0.2 1 10 cells cultured in the presence of 0.1 0.1 LPS and (F) white blood cell count 0 0 (WBC). Sampling dates with different 4.5 620 4.0 letters are significantly different DE F 4.0 3.5 (P<0.05). 3.5 3.0 3.0 580 2.5 2.5 2.0 560 2.0 1.5 1.5 540 1.0 1.0 0.5 0.5 0 0 May June August May June August May June August THE JOURNAL OF EXPERIMENTAL BIOLOGY LPS-Abs (O.D.) Unstimulated spots per 10 cells Unstimulated spots per 10 cells LPS-Abs (O.D.) –4 log (Avidity10 ) Stimulated spots per 10 cells 5 –4 Stimulated spots per 10 cells log (Avidity10 ) WBC –1 Total Ig (mg ml ) WBC –1 Total Ig (mg ml ) 638 The Journal of Experimental Biology 216 (4) Table 1. MANOVA summary statistics for effect of date on leukocyte profile Standardized canonical coefficients Variance accounted d.f. FP Lymphocyte Heterophil Basophil Eosinophil Monocyte 90.07 10 3.54 0.0006 −0.432 −0.922 −0.462 −1.215 −0.732 al., 2004; Benatuil et al., 2008; Sparkman and Palacios, 2009; Ujvari fewer negative changes with age. Therefore, we hypothesize that the and Madsen, 2011), studies in these same taxa have also reported antibody produced in response to LPS is polyreactive, of a low affinity a decrease in specific antibodies with age (Ujvari and Madsen, 2005; and produced by a B-1-like cell (Fig. 4). Although we are unable to Lavoie, 2006; Frasca et al., 2008; Ujvari and Madsen, 2011). These determine through what pathway the B cells are activated (see Fig. 1), studies on specific immune responses include a wide range of both we hypothesize that it is through the Toll-like receptor pathway. It is T-dependent and T-independent antigens, including LPS. Thus, the possible that the B cell could be activated through an antigen-specific red-eared slider immune system appears to be unusual in that there pathway, but we feel that is unlikely because studies in mammals is no evidence of declining humoral responses during aging. have demonstrated that signaling through surface Ig fails to activate However, it should be noted that many of these studies measured B-1 cells (Berland and Wortis, 2002). antibody production in vivo after immunization while our study We did find a decrease in WBC count with age, and this could utilized in vitro measures. Further work is currently being conducted potentially negatively affect other types of immune responses by to examine the effect of age on antibody production in vivo in red- directly limiting the cells available for innate responses and also by eared sliders. having fewer cells available to produce cytokines that direct immune So what could be allowing the sliders to maintain immune responses. However, in a previous study on the same population of responsiveness to LPS as they age? In mammals, there is an age- turtles, we found no effect of age on bactericidal capacity or the related shift from a prominently B-2 response to a B-1 response. In delayed hypersensitivity response to the mitogen some animals, such as rabbits, all B cells are B-1 cells. In general, phytohemagglutinin (Zimmerman et al., 2010b), again suggesting B-1 cells are thought to maintain their function with increasing age, limited impacts of age on immune responses of sliders. Importantly, while B-2 cells do not. For example, a previous study in mice found although WBC count decreased with age, the distribution of that a subset of B-1 cells known as CD5+ B-1 cells maintained their leukocytes did not change with age. ability to respond to antigen with age while CD5– cells did not (Hu We have previously reported an increase in total Ig levels with et al., 1993). Thus, it is possible that if all of the turtle B cells were age in red-eared sliders (Zimmerman et al., 2010b) but did not find CD5+ B-1 cells, their response to LPS would not change significantly the same pattern in this study. At present, we cannot provide an during aging. Currently, cell markers do not exist that would allow explanation for the variation in our two datasets from the same us to determine what type of B cells are present in red-eared sliders, population but are continuing to monitor total Ig levels. There may but several lines of evidence suggest they are B-1-like. First is the be year-to-year variation in the levels of Igs produced, which could increase in total Ig reported previously and LPS-Abs reported here. coincide with local pathogen pressures. −4 −6 The avidity for LPS reported in this study (10 to 10 ) is within A secondary goal of this study was to examine seasonal variation −3 −7 range of the avidity for natural antibodies (10 to 10 ) (Notkins, in the humoral immune response. We predicted all immune measures 2004). In addition, we have also found an increase in antibodies to would increase across the active season. Response to stimulation several antigens that the turtles should not have been previously with LPS and antibody avidity did not vary across dates, while LPS- exposed to, including keyhole limpet hemocyanin, ovalbumin, and Abs and total Ig increased. NAbs can be produced as a result of hen egg white lysozyme. These levels also increase with age (L.M.Z., stimulation by antigens, so the increase in total Ig and LPS-Abs unpublished data). Recently, it has been reported that B-1 cells of could have resulted from an increase in exposure to potential mice are capable of phagocytosing antigen while B-2 cells did not pathogens across the active season. This idea of increased pathogen have this ability (Parra et al., 2011). We have previously described exposure is supported by the changes in leukocyte profiles across phagocytic B cells in red-eared sliders, indicating that these may be the active season. The proportion of cells that were heterophils, of the B-1 type (Zimmerman et al., 2010c). In mice, the phagocytic eosinophils and monocytes increased throughout the active season. B cells were located only in the peritoneal cavity, but the phagocytic These cell types are involved in the innate immune response to B cells of the turtles were circulating in the periphery, which suggests bacteria and parasites (Zimmerman et al., 2010a). Alternatively, that the B cells used in the current study were B-1-like cells as well. because of the slow humoral immune response of reptiles, the Many of the negative effects of age on the humoral immune system increase in antibodies could be a result of a delay in response to are attributed to the shift from predominately B-2 cells to B-1 cells antigen exposures early in the active season. (Weksler and Szabo, 2000). Thus, if all B cells in the red-eared slider The leukocyte profile (Table 2) showed several differences from are B-1-like, then their humoral immune responses would demonstrate published hematological values for captive red-eared sliders. A Table 2. Leukocyte profile for each sampling period Cell type May Late June Late August Lymphocyte 44.55±0.73 (39–50.49) 43.66±1.29 (40.19–56.19) 38.31±0.66 (34.95–42.86) Heterophil 37.48±0.73 (30.1–44.55) 38.4±0.81 (33.03–42.16) 38.7±0.56 (33.64–42.57) Basophil 5.6±0.27 (3.7–8.4) 5.25±0.23 (3.81–6.93) 6.6±0.22 (5.61–8.91) Eosinophil 5.49±0.25 (2.94–7.69) 6.21±0.33 (3.67–7.92) 7.93±0.46 (5–11.54) Monocyte 6.92±0.39 (3.77–10.78) 7.23±0.57 (4.76–11.43) 8.46±0.53 (4.85–12.15) Values represent the average percentage ± s.e.m. The range of percentages for each sampling period is in parentheses. THE JOURNAL OF EXPERIMENTAL BIOLOGY Immunity and aging in an ectotherm 639 Fig. 4. Hypothesized response to LPS in red- AB eared slider turtles. (A) In the absence of antigen stimulation, some turtle B cells secrete antibodies, as demonstrated in the media-only wells of the ELISpot. In this case, B-cells D B cell B and F are secreting antibodies. All B cells B cell l produce a membrane-bound antibody, but B- cell E is not secreting antibodies. (B) When stimulated with antigen, as demonstrated in the B cell wells of the ELISpot that were cultured in the presence of lipopolysaccharide (LPS), more B B cell cells produce antibodies and each cell secretes more antibodies compared with the B cell unstimulated wells. So, in this case, B-cells D, B B cell E and F all produce antibodies. We hypothesize that the antibody produced from all B cells is a natural antibody (NAb)-like antibody that is polyreactive and of low affinity. Although we cannot determine the mode of activation at this point, we also hypothesize that the B cell is most likely activated via the Toll-like receptor (TLR) pathway as shown in Unstimulated Stimulated with LPS B-cell E. LPS TLR4 CD14 B-1 B cell B-2 B cell Polyreactive natural antibodies previous study found that basophils and monocytes were the most a recent study has suggested that estrogens enhance T-dependent common leukocytes while lymphocytes accounted for around 14% humoral responses but do not affect T-independent responses of the leukocyte population (Taylor and Kaplan, 1961). We found (Adori et al., 2010). For the other immune measures, including the heterophils and lymphocytes to be the most common cell types, with number of spontaneously secreting AbSCs and total Ig, we would lymphocytes accounting for an average of 42% of the leukocyte expect greater responses in females (Verthelyi and Ahmed, 1998; population. The previous report is similar to other counts of captive Ahmed et al., 1999). turtles of various species (Metin et al., 2006; Metin et al., 2008), while In conclusion, this study supports the idea that NAbs are an our higher number is similar to what is found in most species of lizards, important line of defense in reptiles (Zimmerman et al., 2010a; where heterophils and lymphocytes are the most common cell types Ujvari and Madsen, 2011). LPS-Abs increased with age while (Fisse et al., 2004). It is unclear what could explain the different results, response to stimulation with LPS did not vary with age. Thus, the but many factors are known to alter leukocyte counts, including humoral immune system of sliders may not change as dramatically nutritional status, parasite load, and season (Fisse et al., 2004). with age as that of mammals. An increase in NAbs with age may As mentioned earlier, previous studies in a number of also be viewed as a positive change in immune function. This vertebrates have shown that capture stress can affect immune contrasts sharply to mammals, where a shift in humoral responses measures, including leukocyte ratios (Davis et al., 2008). A to a predominantly B-1 response is viewed negatively and in humans previous study in our lab demonstrated that corticosterone levels is considered a factor in the increase in morbidity and mortality in did not rise until after 60 min of restraint and that leukocyte the elderly (Dicarlo et al., 2009). We also identified seasonal changes distribution, including the heterophil:lymphocyte ratio, did not in immunity that could be a result of increased pathogen pressure vary from baseline even after 2 h of restraint (K.A.L. and L.M.Z., across the active season. Further, we validate the use of both avidity unpublished data). All blood samples used in this study were taken and ELISpot assays for assessing characteristics of antibodies in within 90 min of the turtle being removed from the trap and thus the red-eared slider turtle. we do not expect variation in leukocyte profiles to be affected by capture stress. We believe our other immune measures are ACKNOWLEDGEMENTS unlikely to be affected by capture stress as well because antibody We would like to thank Steve Juliano and Ebony Murrell for statistical advice. We would also like to thank the Illinois Department of Natural Resources for allowing measures examine antibodies that are already circulating in the access to Banner Marsh. animal and are unlikely to change in the short time period we held the turtles before sample collection. 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Humoral immune responses are maintained with age in a long-lived ectotherm, the red-eared slider turtle

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The Journal of Experimental Biology 216, 633-640 © 2013. Published by The Company of Biologists Ltd doi:10.1242/jeb.078832 RESEARCH ARTICLE Humoral immune responses are maintained with age in a long-lived ectotherm, the red-eared slider turtle Laura M. Zimmerman*, Sandrine G. Clairardin, Ryan T. Paitz, Justin W. Hicke, Katie A. LaMagdeleine, Laura A. Vogel and Rachel M. Bowden School of Biological Sciences, Illinois State University, Normal, IL 61790, USA *Author for correspondence ([email protected]) SUMMARY Aging is typically associated with a decrease in immune function. However, aging does not affect each branch of the immune system equally. Because of these varying effects of age on immune responses, aging could affect taxa differently based on how the particular taxon employs its resources towards different components of immune defense. An example of this is found in the humoral immune system. Specific responses tend to decrease with age while non-specific, natural antibody responses increase with age. Compared with mammals, reptiles of all ages have a slower and less robust humoral immune system. Therefore, they may invest more in non-specific responses and thus avoid the negative consequences of age on the immune system. We examined how the humoral immune system of reptiles is affected by aging and investigated the roles of non-specific, natural antibody responses and specific responses by examining several characteristics of antibodies against lipopolysaccharide (LPS) in the red-eared slider turtle. We found very little evidence of immunosenescence in the humoral immune system of the red-eared slider turtle, Trachemys scripta, which supports the idea that non-specific, natural antibody responses are an important line of defense in reptiles. Overall, this demonstrates that a taxonʼs immune strategy can influence how the immune system is affected by age. Key words: eco-immunology, reptile, Trachemys scripta. Received 1 August 2012; Accepted 10 October 2012 INTRODUCTION less robust (reviewed by Zimmerman et al., 2010a). Like mammals, Immunosenescence, which is a decrease in immune function with reptiles produce antibodies after a lag time of about one week, but increasing age, is a common observation in vertebrates. This process production often does not peak until 6–8 weeks later. If exposed for can have important consequences for an organism; for example, an a second time to the same antigen, reptiles do not exhibit a memory increase in mortality and morbidity in elderly humans is commonly response typical of mammals. While the lag time is reduced, titers attributed to a decrease in immune function (Dicarlo et al., 2009). are not increased compared with the primary response. It is generally However, the aging process does not affect every branch of the considered that affinity maturation does not occur in ectotherms, immune system in the same manner (Palacios et al., 2007; Ujvari including reptiles, but a small increase in affinity has been found and Madsen, 2011). This is highlighted in the humoral immune in response to immunization in the rainbow trout, Oncorhynchus system, where it has been found in a number of taxa that specific mykiss (Cain et al., 2002). B cell subsets have not yet been antibody production decreases with age, while non-specific, identified in reptiles due to a lack of specific reagents. polyreactive antibodies increase with age (Frasca et al., 2008). Given the less robust antibody response of reptiles, they may rely Because of these varying effects of age on immune responses, aging instead on production of non-specific antibodies in the form of could affect taxa differently based on how the particular taxon natural antibodies (NAbs). While NAbs have been identified in a employs its resources towards different components of immune variety of taxa, including reptiles, most available information comes defense. from studies of mammals. Natural antibodies are produced by a An example of how taxa can vary their utilization of immune subset of B cells known as B-1 cells and can be produced defenses is found in the differences in timing of the humoral response constitutively at low levels in the absence of antigen or be induced to antigen exposure in mammals and reptiles. In mammals, the in response to certain antigens including microbial components such specific humoral response is produced by a subset of B cells called as lipopolysaccharide (LPS) and phosphocoline (Baumgarth et al., B-2 cells. When a mammal is exposed to an antigen, it will produce 2005; Yang et al., 2007), although the antibody produced in antigen-specific antibodies of moderate affinity after a lag period response to antigen stimulation is of lower affinity than that of approximately 1 week and peak production at 2 weeks after produced by B-2 cells. Natural antibodies are polyreactive to exposure. After this first exposure to an antigen, memory cells are evolutionarily conserved components of pathogens, have low formed, and the lag time to antibody production is reduced upon a binding affinities and play active roles in triggering both innate and second exposure to the antigen, and antibody titers and affinity are adaptive immunity (Ochsenbein and Zinkernagel, 2000). They also increased (Coico et al., 2003). Compared with their mammalian bind to a variety of self-antigens, which allows them to play counterparts, the specific humoral system of reptiles is slower and housekeeping roles such as the clearing of apoptotic cells (Binder THE JOURNAL OF EXPERIMENTAL BIOLOGY 634 The Journal of Experimental Biology 216 (4) Fig. 1. Modes of B cell activation induced by A B lipopolysaccharide (LPS) in mammals. (A) LPS is a B cell mitogen that acts through TLR4 and CD14 to induce B cell antibody secretion independent of B cell antigen specificity. This results in the activation of a B cell A large number of B cells and polyclonal B cell B cell B immunoglobulin (Ig) secretion. In this situation, B cells A, B and C would secrete antibody. (B) LPS can also activate B cells in the traditional manner by binding directly to antigen-specific surface Ig. In this B cell l B cell case, only LPS-specific B cells (B-cell B) are activated to produce Ig. (C) Natural antibodies can be secreted in the absence of antigen stimulation. In this situation, B-cell D would spontaneously secrete low-affinity antibodies with the ability to bind LPS. B cell Antibodies with different LPS TLR4 CD14 B-1 B cell B-2 B cell antigen specificities et al., 2008) and may even act to reduce the risk of autoimmune determine how many cells are producing antibodies and how many diseases (Boes et al., 2000). Although they can be of the antibodies are being produced by each cell. These characteristics immunoglobulin A (IgA) or immunoglobulin G (IgG) isotype, most are measured frequently in traditional immunology, but thus far have NAbs are of the immunoglobulin M (IgM) isotype and are secreted not been examined in any eco-immunology study and can provide as pentamers. The pentameric structure allows them to be a strong a fuller picture of humoral immune functioning. activator of complement and permits more efficient production of We examined how the humoral immune system of reptiles is antigen–antibody complexes, which helps target antigens to affected by aging and investigated the roles of natural/B-1-like lymphatic tissues and improve adaptive responses (Boes, 2000). antibody and specific/B-2-like responses by examining several Both mammals and reptiles show an age-related decrease in specific, characteristics of antibodies against LPS. Lipopolysaccharide, which B-2-cell-produced antibodies and an increase in non-specific, B-1- is a component of Gram-negative bacteria, is a T-independent cell-produced NAbs. In humans, this reduction in specific antibody antigen, meaning that a B cell can respond to the antigen without responses has been viewed as a contributor to the rise in mortality direct contact with a T cell. In mammals, the humoral immune and morbidity with increasing age and, although there is also an system responds to LPS with (1) NAbs produced in the absence of increase in NAbs with age in humans, they do not appear to be able antigen stimulation by B-1 cells, (2) antibodies produced as a direct to compensate for the loss of the specific antibodies (McGlauchlen result of antigen stimulation by LPS binding directly to an antigen- and Vogel, 2003). Interestingly, the less-robust specific immune specific membrane Ig by B-1 or B-2 cells or (3) polyclonal antibody response of reptiles of all ages, combined with the increase in NAbs production through a Toll-like receptor pathway by B-1 or B-2 cells with age, may constitute an overall positive change in immune (Fig. 1). All receptors are constitutively expressed, so a B cell can defense (Ujvari and Madsen, 2011). However, little is known about be activated by any mechanism at any time. The isotypes of both antibody production in reptiles beyond quantity. Examining other specific antibodies and NAbs that bind to LPS include IgA, IgG antibody characteristics in addition to quantity would help us to and IgM, although NAbs are predominantly IgM. Although germinal decipher if changes in humoral immune responses with age centers typically do not form in response to LPS, specific antibodies differentially affect taxa with varying immune strategies. One still have a higher affinity to LPS than NAbs (Bruderer et al., 1992). important characteristic to consider is strength of binding. Affinity In mice, an age-associated decrease in antibody responses to T- is the measure of how well one particular antibody-combining site independent antigens, including LPS, has been reported (Chelvarajan binds to a single antigenic epitope, while avidity is a measure of et al., 2005). A similar result was found in a natural population of how well the total antibody population binds to the entire multivalent tree swallows (Tachycineta bicolor), where young and mid-age antigen. Thus, avidity can be measured from a plasma sample females injected with LPS mounted a specific antibody response, containing antibodies with different specificities. Knowing the while the oldest individuals did not (Palacios et al., 2011). avidity of an immune response can help determine if the antibodies The present study was conducted on a natural population of red- −3 −7 –1 are NAbs (affinity ranges from 10 to 10 mol l ) (Notkins, 2004) eared slider turtles (Trachemys scripta Schoepff). The slider is an or specific antibodies produced in response to antigen stimulation excellent model for examining humoral responses to LPS with age −10 –1 (affinity averages 10 mol l ) (Foote and Eisen, 1995). Another for several reasons. Sliders grow throughout their lifetime, so important characteristic that has been understudied in reptilian plastron length can be used as a proxy for age (Ernst et al., 1994). immunology is antibody production by individual B cells. Measuring Due to the challenges of following a turtle population over several the function of antibody secreting cells (AbSCs) allows us to decades, it is difficult to give an average life span for the slider THE JOURNAL OF EXPERIMENTAL BIOLOGY Immunity and aging in an ectotherm 635 turtle, although it is generally estimated to be around 30–40 years IL, USA on a natural population of red-eared sliders from May to (Ernst et al., 1994). Studies in the closely related painted turtle found August 2011 (IDNR permit NH11.2084). Adult male and female that they could live upwards of 60 years (Congdon et al., 2003), so turtles were trapped at three points in the active season: early May, it is possible that 40 years is a conservative estimate for sliders. We late June and late August. At time of capture, any unmarked have previously reported an increase in total Ig levels with age in individuals were uniquely marked, and plastron length was measured the slider (Zimmerman et al., 2010b). In addition, sliders are likely to the nearest 0.1 mm. No individual was used in more than one to be exposed to LPS in their natural environment, with likelihood collection period. Approximately 1 ml of blood was taken from the of exposure varying seasonally. A previous study in our population caudal vein using an EDTA-coated syringe. Plasma was separated has found a high frequency of Salmonella prevalence in adult turtles, from 500 μl of blood by centrifugation (2000 g, 3 min) at the field with prevalence increasing as temperatures increase and remaining site and kept on ice until it was taken to Illinois State University high through the remainder of the active season (Holgersson, 2009). and stored at −20°C. A blood smear was made using 7 μl of whole It is reasonable to suggest that other bacteria may also increase in blood. The remaining blood was diluted ~1:2 with a 75% RPMI- prevalence as temperatures increase. Because of this, and the fact 25% EDTA mixture for use in the ELISpot. that we have also found significant seasonal variation in a number of immune measures (Zimmerman et al., 2010b), we sampled ELISA immune responses at three points throughout the active season. An ELISA was used to measure total Ig and anti-LPS Abs Importantly, we have now validated an ELISpot assay to examine (Zimmerman et al., 2010b). Polystyrene 96-well plates (Costar, –1 the properties of AbSCs, along with an avidity assay to measure Tewksbury, MA, USA) were coated with either 100 μl of a 25 μg ml antibody binding. These new assays, along with the ELISA that we dilution of unlabeled anti-turtle light chain antibody (HL673; –1 have previously validated and the common practice of using blood University of Florida Hybridoma Facility) or 100 μl of a 20 μg ml smears to examine leukocyte populations, will allow us to gain a solution of LPS (from Salmonella enterica serotype typhimurium; fuller understanding of humoral immune responses of sliders. Sigma, St Louis, MO, USA) in phosphate-buffered saline (PBS) A wide range of studies in a variety of vertebrate taxa, including and incubated overnight at 4°C. Plates were washed three times for mammals, birds and reptiles, have found that specific antibodies 3 min with 200 μl per well of PBS–1% BSA–0.05% Tween buffer decrease with age while NAbs increase with age (Candore et al., (PBS-BSA-T), which also serves as a blocking step. Plasma samples 1997; Parmentier et al., 2004; Ujvari and Madsen, 2005; Lavoie, were diluted 1:1000 for total Ig and 1:50 for LPS-specific Abs in 2006; Benatuil et al., 2008; Frasca et al., 2008; Sparkman and PBS-BSA-T. Goat serum was added as a negative control. Plates Palacios, 2009; Ujvari and Madsen, 2011). This pattern, along with were incubated at room temperature for at least 1 h, then washed as the typically slow and less-robust specific responses of reptiles, leads before. One hundred microliters of a 1:500 dilution of anti-turtle us to hypothesize that red-eared sliders utilize a predominantly NAb- antibody conjugated to biotin was added to each well, and plates based response, and thus would not show a deficit in humoral were incubated and washed as before. One hundred microliters of immune defenses with age. Because both NAbs and specific streptavidin-HRP (Southern Biotech, Birmingham, AL, USA; antibodies can be produced in response to antigenic stimulation, we diluted 1:1000 in PBS-BSA-T) was added to each well, and plates also hypothesized that the humoral response would change across were incubated and washed as before. Wells were washed once with the active season as the animals naturally encounter LPS in the 100 μl double distilled water before 100 μl of ABTS (Southern environment, regardless of whether the responses were Biotech) substrate powder dissolved in ABTS solution was added predominantly NAb-based or specific. If natural antibodies/B-1-cell- to each well. The plate was read at 17 min after adding substrate like immunity are predominant in sliders, we expect immune using a Powerwave 340 plate reader (BioTek Inc., Winooski, VT, measures to increase or show no change in function with age. Thus, USA) at 405 nm. we would predict that the amount and avidity of antigen-specific antibody, as well as total Ig and AbSC number and function, should Avidity be preserved. Alternatively, if specific responses are predominant Avidity was measured using a competitive ELISA. Fifty microliters in sliders, we would expect immune measures to decrease with age. of diluted test serum (1:50) was added to six 50 μl aliquots of serial –1 Thus, we would predict that AbSCs would show a reduced response diluted LPS (0.08–5 mg ml ) and two tubes of 50 μl of PBS only, to LPS stimulation and that avidity, the amount of antibodies that and incubated for 18 h at room temperature in glass tubes. An ELISA can bind to LPS (LPS-Abs) and the percentage of lymphocytes was then run as described above with the plates coated with LPS. would decrease with age. We also predict that LPS-Abs, total Ig, Avidity values were calculated from the absorbances according to avidity, the number of AbSCs, the amount of antibody produced previously described methods (Friguet et al., 1985). Thus, for the by each cell, and the percentage of lymphocytes would increase individual turtle plasma sample, six values (one from each tube used through the active season as the turtles are more likely to be exposed in the serial dilution) were obtained by subtracting the absorbance to LPS. To examine our hypotheses, we determined both the quantity in a well from the average absorbance of the wells that came from and quality of LPS humoral responses in the slider by measuring the tubes that contained no antigen and then dividing that number LPS-Abs, total Ig, antibody avidity to LPS, the properties of AbSCs by the molarity of the antigen present in the glass tube during and the percentage of leukocytes that are lymphocytes. While the incubation. Those six values were then plotted; a linear regression assays used in this study examine variation in both NAbs and specific was used and the slope of the line was the dissociation constant. antibodies, the prediction for the pattern of responses with age and Avidity was then recorded as the reciprocal of the dissociation across the active season differs and thus allows us to distinguish constant. between the two types of responses. ELISpot MATERIALS AND METHODS Leukocytes were isolated over a Percoll gradient (Harms et al., This study was conducted under IACUC approval (protocol 04- 2000). Wells of MultiScreen-IP ELISpot plates (Millipore, Billerica, –1 2010) at Banner Marsh State Fish and Wildlife Area, Fulton Co., MA, USA) were coated with a 20 μg ml dilution of unlabeled anti- THE JOURNAL OF EXPERIMENTAL BIOLOGY 636 The Journal of Experimental Biology 216 (4) turtle light chain overnight at 4°C. The wells were then washed with RESULTS PBS and blocked for 2 h at 37°C with RPMI (Hyclone, Logan, UT, First, LPS binding antibody concentrations were examined (N=56). USA) supplemented with 5% fetal bovine serum, 1% All animals tested produced detectable anti-LPS antibodies as penicillin/streptomycin/glutamine, 0.5% 2-mercaptoethanol and measured by ELISA. As animals were not deliberately immunized, 0.5% sodium pyruvate (cRPMI). A known number of leukocytes either pre-existing polyreactive NAbs were detected or animals had in 100 μl cRPMI was added to each well. The number of leukocytes naturally encountered the antigen. Quantity of LPS-Abs significantly 5 6 –1 plated ranged from 10 to 10 cells well . An additional 100 μl of increased with plastron length (Fig. 2A; F =5.09, P=0.029) and 1,49 –1 cRPMI or cRPMI supplemented with 40 μg ml LPS was added to across the active season (Fig. 3A; F =3.34, P=0.044). There was 2,49 each well to observe spontaneous antibody production and to no correlation with sex on LPS-Abs (F =0.17, P=0.69) and the 1,49 stimulate antibody production, respectively. Cells from each turtle date by sex interaction was not significant (F =1.39, P=0.26). The 1,49 were given each treatment in duplicate wells. The cells were then avidity of these anti-LPS antibodies was also measured by ELISA incubated at 31°C in 5% CO for five days. The cell remains as described (N=48). Avidity did not vary with plastron length stationary, while any antibody produced remains attached to the (Fig. 2B; F =1.69, P=0.20), date (Fig. 3B; F =1.40, P=0.26) or 1,41 2,41 capture antibody. After five days, wells were washed with sex (F =3.05, P=0.09). The date by sex interaction was also not 1,41 PBS–0.01% Tween to remove the cells. The antibody remains significant (F =1.22, P=0.31). 2,41 attached and was detected with anti-turtle light chain conjugated to Next, total Ig levels were examined (N=55). Total Ig levels ranged –1 biotin, followed by streptavidin-HRP. The wells were developed from 0 to 9.7 mg ml . Total Ig varied significantly by date (Fig. 3C; with AEC substrate (BD Biosciences, San Jose, CA, USA), which F =7.68, P=0.0013) and sex (F =4.26, P=0.045), with males 2,48 1,48 leaves a red spot where antibodies have been detected, with each having significantly higher total Ig levels than females. There was spot representing one cell. The size of spot indicates how much no correlation with plastron length (Fig. 2C; F =1.87, P=0.18), and 1,48 antibody was produced by each cell. The number of spots was the date by sex interaction was not significant (F =0.25, P=0.78). 1,48 counted to determine the number of AbSCs per 10 cells, based on The number of spontaneous AbSCs in the blood ranged from 0 the number of cells added to the well. Size of spots was determined to 13.17 cells per 10 leukocytes (N=40), and the number of LPS- using ImageJ software (National Institutes of Health, Bethesda, MD, stimulated AbSC ranged from 0 to 22.73 cells per 10 leukocytes USA). (N=44). For cells cultured in media alone in the ELISpot, the number of AbSCs and average spot size did not vary with plastron length Blood smears (Fig. 2D; F =0.02, P=0.88; F =0.25, P=0.62, respectively), date 1,34 1,27 Previous studies in a number of vertebrates have shown that (Fig. 3D; F =0.57, P=0.57; F =0.44, P=0.65, respectively) or 2,34 2,27 leukocyte ratios, specifically heterophil:lymphocyte ratio, can sex (F =1.45, P=0.24; F =0.87, P=0.36, respectively). The date 1,34 1,27 change significantly as a result of handling stress (reviewed by by sex interaction was also not significant (F =0.83, P=0.37; 1,34 Davis et al., 2008). A previous study in our lab demonstrated that F =2.24, P=0.15). For the cells cultured with LPS in the ELISpot, 1,27 heterophil:lymphocyte ratio did not vary from baseline after the average spot size was significantly larger in wells that we turtles were restrained for 2 h (K.A.L. and L.M.Z., unpublished stimulated with LPS than in wells that received media only (LPS data). All blood samples used in this study were taken within mean ± s.e.m.=35.43±2.18 pixels, media-only mean ± s.e.m.= 90 min of the turtle being removed from the trap. Blood smears 28.68±1.79 pixels; T =3.49, P=0.001), and the total number of Ig- were fixed in methanol and stained with Wright-Giemsa (Sigma- secreting cells was increased by LPS (LPS mean ± s.e.m.= Aldrich, St Louis, MO, USA. The leukocyte profile was 3.62±0.66 pixels, media-only mean ± s.e.m.=2.74±0.46 pixels; determined by counting a total of 100 leukocytes and calculating T =2.345, P=0.024). However, the number of LPS-stimulated the percentage of lymphocytes, basophils, eosinophils, monocytes AbSCs and spot size did not vary with plastron length (Fig. 2E; and heterophils. The number of white blood cells per 10,000 red F =0.00, P=0.96; F =0.06, P=0.81), date (Fig. 3E; F =1.12, 1,38 1,30 2,38 blood cells was determined as a proxy for white blood cell count P=0.34; F =1.30, P=0.29, respectively) or sex (F =1.85, 2,30 1,38 (WBC) (Norte et al., 2008). P=0.18; F =0.57, P=0.46, respectively). The date by sex 1,30 interaction was also not significant (F =1.14, P=0.29; F =0.31, 1,38 1,30 Statistical analysis P=0.58, respectively). The effect of date of sampling, sex and plastron length on number Finally, WBC differential counts were examined (N=54). Total of AbSCs when stimulated and cultured in media only, spot size WBC number significantly decreased with increasing plastron for both conditions, avidity, LPS-Abs, total Ig, and WBC was length (Fig. 2F; F =4.45, P=0.0399) but did not vary with sex 4,49 examined using ANCOVAs. ANCOVA was chosen rather than (F =3.42, P=0.07) or date (Fig. 3F; F =0.76, P=0.48). A 4,49 4,49 MANCOVA because sample size did not permit the inclusion of significant effect of date on leukocyte profile was detected (Pillai’s all immune measures and we had no a priori reasons to group trace: F =3.23, P=0.001). A significant axis was found (Table 1; 10,92 certain immune measures. The number of AbSCs for stimulated eigenvalue=0.7858, P=0.0006) and it explained 90.07% of the and media-only conditions and total Ig were square root variation among dates. This was mostly driven by eosinophils and transformed while avidity was log transformed. All interactions heterophils and, to a lesser extent, monocytes (Tables 1, 2). were tested, and non-significant interactions involving plastron Lymphocytes decreased across the active season but did not explain length were removed from the model. Tukey’s post hoc tests were much of the variation. There was no significant effect of sex (Pillai’s conducted. For the leukocyte profile, a MANOVA was run trace: F =0.1.81, P=0.13) or plastron length (Pillai’s trace: 5,45 including the proportions of each cell type as dependent variables. F =0.25, P=0.94) on the leukocyte profile. 5,45 Date, sex and plastron length were included as main effects. All interactions were tested and found to be non-significant so they DISCUSSION were removed from the models. A paired t-test was used to Aging negatively affects many aspects of the immune response, compare spot size and number of AbSCs between stimulated and including the specific antibody response. However, some immune media-only wells. measures are not negatively affected by age, and may even increase, THE JOURNAL OF EXPERIMENTAL BIOLOGY Immunity and aging in an ectotherm 637 AB C 1.2 1.8 1.6 1.4 0.8 1.2 8 0.6 0.8 0.4 0.6 4 0.4 0.2 0.2 0 0 0 DE F 14 800 4 500 2 450 100 125 150 175 200 225 250 275 100 125 150 175 200 225 250 275 100 125 150 175 200 225 250 275 Plastron (mm) Fig. 2. Relationship between plastron length and (A) the amount of antibodies that can bind to lipopolysaccharide (LPS), (B) avidity, (C) total 5 5 cells cultured in media only, (E) number of spots per 10 cells cultured in the presence of LPS and (F) white immunoglobulin, (D) number of spots per 10 blood cell count (WBC). such as the non-specific NAb response (Frasca et al., 2008). In Abs significantly increased with age. In addition, the avidity of reptiles of all ages, the specific antibody response is slower and less antibodies to LPS was in the range of NAbs (Ochsenbein and robust than its mammalian counterpart (Zimmerman et al., 2010a). Zinkernagel, 2000). Thus, we are likely to be measuring This may force them to rely more heavily on NAbs to deal with predominantly NAbs that are binding to LPS with a low avidity, pathogens. Thus, an increase in NAbs with age may be viewed as rather than measuring highly specific antibodies that would bind to a positive change in immunity with age in reptiles (Ujvari and LPS with a high avidity. In addition, the number of AbSCs that Madsen, 2011). responded to LPS stimulation did not vary with age, nor did the The primary goal of the current study was to examine the effect amount of antibody produced by each cell. Thus, our turtles seem of age on humoral immune responses to LPS in a long-lived reptile, to have an increase in NAb, B-1-like immunity with age, but the the red-eared slider turtle. We hypothesized that red-eared sliders specific, B-2-like response to LPS was not significantly impacted utilize a predominantly NAb response, and thus would be less by age. While an increase in NAbs with age has been reported in affected by changes in humoral immune defenses with age. LPS- mammals, birds and reptiles (Candore et al., 1997; Parmentier et 0.9 Fig. 3. Mean ± s.e.m. during May bb AB C 0.8 6 (N=26), late June (N=22) and August 0.7 0.7 (N=17) for (A) the amount of a,b 0.6 5 a antibodies that can bind to 0.6 0.5 lipopolysaccharide (LPS), (B) avidity, 0.5 0.4 (C) total immunoglobulin, (D) number 0.4 0.3 of spots per 10 cells cultured in 0.3 media only, (E) number of spots per 0.2 0.2 1 10 cells cultured in the presence of 0.1 0.1 LPS and (F) white blood cell count 0 0 (WBC). Sampling dates with different 4.5 620 4.0 letters are significantly different DE F 4.0 3.5 (P<0.05). 3.5 3.0 3.0 580 2.5 2.5 2.0 560 2.0 1.5 1.5 540 1.0 1.0 0.5 0.5 0 0 May June August May June August May June August THE JOURNAL OF EXPERIMENTAL BIOLOGY LPS-Abs (O.D.) Unstimulated spots per 10 cells Unstimulated spots per 10 cells LPS-Abs (O.D.) –4 log (Avidity10 ) Stimulated spots per 10 cells 5 –4 Stimulated spots per 10 cells log (Avidity10 ) WBC –1 Total Ig (mg ml ) WBC –1 Total Ig (mg ml ) 638 The Journal of Experimental Biology 216 (4) Table 1. MANOVA summary statistics for effect of date on leukocyte profile Standardized canonical coefficients Variance accounted d.f. FP Lymphocyte Heterophil Basophil Eosinophil Monocyte 90.07 10 3.54 0.0006 −0.432 −0.922 −0.462 −1.215 −0.732 al., 2004; Benatuil et al., 2008; Sparkman and Palacios, 2009; Ujvari fewer negative changes with age. Therefore, we hypothesize that the and Madsen, 2011), studies in these same taxa have also reported antibody produced in response to LPS is polyreactive, of a low affinity a decrease in specific antibodies with age (Ujvari and Madsen, 2005; and produced by a B-1-like cell (Fig. 4). Although we are unable to Lavoie, 2006; Frasca et al., 2008; Ujvari and Madsen, 2011). These determine through what pathway the B cells are activated (see Fig. 1), studies on specific immune responses include a wide range of both we hypothesize that it is through the Toll-like receptor pathway. It is T-dependent and T-independent antigens, including LPS. Thus, the possible that the B cell could be activated through an antigen-specific red-eared slider immune system appears to be unusual in that there pathway, but we feel that is unlikely because studies in mammals is no evidence of declining humoral responses during aging. have demonstrated that signaling through surface Ig fails to activate However, it should be noted that many of these studies measured B-1 cells (Berland and Wortis, 2002). antibody production in vivo after immunization while our study We did find a decrease in WBC count with age, and this could utilized in vitro measures. Further work is currently being conducted potentially negatively affect other types of immune responses by to examine the effect of age on antibody production in vivo in red- directly limiting the cells available for innate responses and also by eared sliders. having fewer cells available to produce cytokines that direct immune So what could be allowing the sliders to maintain immune responses. However, in a previous study on the same population of responsiveness to LPS as they age? In mammals, there is an age- turtles, we found no effect of age on bactericidal capacity or the related shift from a prominently B-2 response to a B-1 response. In delayed hypersensitivity response to the mitogen some animals, such as rabbits, all B cells are B-1 cells. In general, phytohemagglutinin (Zimmerman et al., 2010b), again suggesting B-1 cells are thought to maintain their function with increasing age, limited impacts of age on immune responses of sliders. Importantly, while B-2 cells do not. For example, a previous study in mice found although WBC count decreased with age, the distribution of that a subset of B-1 cells known as CD5+ B-1 cells maintained their leukocytes did not change with age. ability to respond to antigen with age while CD5– cells did not (Hu We have previously reported an increase in total Ig levels with et al., 1993). Thus, it is possible that if all of the turtle B cells were age in red-eared sliders (Zimmerman et al., 2010b) but did not find CD5+ B-1 cells, their response to LPS would not change significantly the same pattern in this study. At present, we cannot provide an during aging. Currently, cell markers do not exist that would allow explanation for the variation in our two datasets from the same us to determine what type of B cells are present in red-eared sliders, population but are continuing to monitor total Ig levels. There may but several lines of evidence suggest they are B-1-like. First is the be year-to-year variation in the levels of Igs produced, which could increase in total Ig reported previously and LPS-Abs reported here. coincide with local pathogen pressures. −4 −6 The avidity for LPS reported in this study (10 to 10 ) is within A secondary goal of this study was to examine seasonal variation −3 −7 range of the avidity for natural antibodies (10 to 10 ) (Notkins, in the humoral immune response. We predicted all immune measures 2004). In addition, we have also found an increase in antibodies to would increase across the active season. Response to stimulation several antigens that the turtles should not have been previously with LPS and antibody avidity did not vary across dates, while LPS- exposed to, including keyhole limpet hemocyanin, ovalbumin, and Abs and total Ig increased. NAbs can be produced as a result of hen egg white lysozyme. These levels also increase with age (L.M.Z., stimulation by antigens, so the increase in total Ig and LPS-Abs unpublished data). Recently, it has been reported that B-1 cells of could have resulted from an increase in exposure to potential mice are capable of phagocytosing antigen while B-2 cells did not pathogens across the active season. This idea of increased pathogen have this ability (Parra et al., 2011). We have previously described exposure is supported by the changes in leukocyte profiles across phagocytic B cells in red-eared sliders, indicating that these may be the active season. The proportion of cells that were heterophils, of the B-1 type (Zimmerman et al., 2010c). In mice, the phagocytic eosinophils and monocytes increased throughout the active season. B cells were located only in the peritoneal cavity, but the phagocytic These cell types are involved in the innate immune response to B cells of the turtles were circulating in the periphery, which suggests bacteria and parasites (Zimmerman et al., 2010a). Alternatively, that the B cells used in the current study were B-1-like cells as well. because of the slow humoral immune response of reptiles, the Many of the negative effects of age on the humoral immune system increase in antibodies could be a result of a delay in response to are attributed to the shift from predominately B-2 cells to B-1 cells antigen exposures early in the active season. (Weksler and Szabo, 2000). Thus, if all B cells in the red-eared slider The leukocyte profile (Table 2) showed several differences from are B-1-like, then their humoral immune responses would demonstrate published hematological values for captive red-eared sliders. A Table 2. Leukocyte profile for each sampling period Cell type May Late June Late August Lymphocyte 44.55±0.73 (39–50.49) 43.66±1.29 (40.19–56.19) 38.31±0.66 (34.95–42.86) Heterophil 37.48±0.73 (30.1–44.55) 38.4±0.81 (33.03–42.16) 38.7±0.56 (33.64–42.57) Basophil 5.6±0.27 (3.7–8.4) 5.25±0.23 (3.81–6.93) 6.6±0.22 (5.61–8.91) Eosinophil 5.49±0.25 (2.94–7.69) 6.21±0.33 (3.67–7.92) 7.93±0.46 (5–11.54) Monocyte 6.92±0.39 (3.77–10.78) 7.23±0.57 (4.76–11.43) 8.46±0.53 (4.85–12.15) Values represent the average percentage ± s.e.m. The range of percentages for each sampling period is in parentheses. THE JOURNAL OF EXPERIMENTAL BIOLOGY Immunity and aging in an ectotherm 639 Fig. 4. Hypothesized response to LPS in red- AB eared slider turtles. (A) In the absence of antigen stimulation, some turtle B cells secrete antibodies, as demonstrated in the media-only wells of the ELISpot. In this case, B-cells D B cell B and F are secreting antibodies. All B cells B cell l produce a membrane-bound antibody, but B- cell E is not secreting antibodies. (B) When stimulated with antigen, as demonstrated in the B cell wells of the ELISpot that were cultured in the presence of lipopolysaccharide (LPS), more B B cell cells produce antibodies and each cell secretes more antibodies compared with the B cell unstimulated wells. So, in this case, B-cells D, B B cell E and F all produce antibodies. We hypothesize that the antibody produced from all B cells is a natural antibody (NAb)-like antibody that is polyreactive and of low affinity. Although we cannot determine the mode of activation at this point, we also hypothesize that the B cell is most likely activated via the Toll-like receptor (TLR) pathway as shown in Unstimulated Stimulated with LPS B-cell E. LPS TLR4 CD14 B-1 B cell B-2 B cell Polyreactive natural antibodies previous study found that basophils and monocytes were the most a recent study has suggested that estrogens enhance T-dependent common leukocytes while lymphocytes accounted for around 14% humoral responses but do not affect T-independent responses of the leukocyte population (Taylor and Kaplan, 1961). We found (Adori et al., 2010). For the other immune measures, including the heterophils and lymphocytes to be the most common cell types, with number of spontaneously secreting AbSCs and total Ig, we would lymphocytes accounting for an average of 42% of the leukocyte expect greater responses in females (Verthelyi and Ahmed, 1998; population. The previous report is similar to other counts of captive Ahmed et al., 1999). turtles of various species (Metin et al., 2006; Metin et al., 2008), while In conclusion, this study supports the idea that NAbs are an our higher number is similar to what is found in most species of lizards, important line of defense in reptiles (Zimmerman et al., 2010a; where heterophils and lymphocytes are the most common cell types Ujvari and Madsen, 2011). LPS-Abs increased with age while (Fisse et al., 2004). It is unclear what could explain the different results, response to stimulation with LPS did not vary with age. Thus, the but many factors are known to alter leukocyte counts, including humoral immune system of sliders may not change as dramatically nutritional status, parasite load, and season (Fisse et al., 2004). with age as that of mammals. An increase in NAbs with age may As mentioned earlier, previous studies in a number of also be viewed as a positive change in immune function. This vertebrates have shown that capture stress can affect immune contrasts sharply to mammals, where a shift in humoral responses measures, including leukocyte ratios (Davis et al., 2008). A to a predominantly B-1 response is viewed negatively and in humans previous study in our lab demonstrated that corticosterone levels is considered a factor in the increase in morbidity and mortality in did not rise until after 60 min of restraint and that leukocyte the elderly (Dicarlo et al., 2009). We also identified seasonal changes distribution, including the heterophil:lymphocyte ratio, did not in immunity that could be a result of increased pathogen pressure vary from baseline even after 2 h of restraint (K.A.L. and L.M.Z., across the active season. Further, we validate the use of both avidity unpublished data). All blood samples used in this study were taken and ELISpot assays for assessing characteristics of antibodies in within 90 min of the turtle being removed from the trap and thus the red-eared slider turtle. we do not expect variation in leukocyte profiles to be affected by capture stress. We believe our other immune measures are ACKNOWLEDGEMENTS unlikely to be affected by capture stress as well because antibody We would like to thank Steve Juliano and Ebony Murrell for statistical advice. We would also like to thank the Illinois Department of Natural Resources for allowing measures examine antibodies that are already circulating in the access to Banner Marsh. animal and are unlikely to change in the short time period we held the turtles before sample collection. Conceivably, short-term FUNDING B cell function, as measured by ELISpot, could be affected by This research was supported by a Weigel Grant from the Beta Lambda chapter of capture stress, but we believe this is unlikely because the cells Phi Sigma to L.M.Z. and NSF grant IOS-0748505 to R.M.B. and L.A.V. are washed prior to culturing for five days, and any hormones present in the sample should have been removed. REFERENCES Interestingly, only total Ig varied between sexes, with males Adori, M., Kiss, E., Barad, Z., Barabás, K., Kiszely, E., Schneider, A., Kövesdi, D., Sziksz, E., Abrahám, I. M., Matkó, J. et al. (2010). Estrogen augments the T cell- having higher levels. However, we have not found sex differences dependent but not the T-independent immune response. Cell. Mol. Life Sci. 67, in any immune response measured in this study or a previous study 1661-1674. Ahmed, S. A., Hissong, B. 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Published: Feb 15, 2013

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