52(2014), (2015),••–••. 333–341. Periodicals, Inc. Printed the USA. Psychophysiology, •• WileyWiley Periodicals, Inc. Printed in the in USA. C 2014 Society for Psychophysiological V Psychophysiological Research Research Copyright © 10.1111/psyp.12360 DOI: 10.1111/psyp.12360

Induced mild systemic inflammation is associated with impaired ability to improve cognitive task performance by practice

NICOLA J. PAINE,a,b JOS A. BOSCH,a,c,d CHRISTOPHER RING,a MARK T. DRAYSON,e and JET J. C. S. VELDHUIJZEN VAN ZANTENa a

School of Sport, Exercise and Rehabilitation Sciences, College of Life and Environmental Sciences, University of Birmingham, Birmingham, UK Department of Psychiatry and Behavioral Sciences, Duke University Medical Center, Durham, North Carolina, USA c Department of Clinical Psychology, University of Amsterdam, Amsterdam, The Netherlands d Mannheim Institute of Public Health, Social and Preventive Medicine (MIPH), Mannheim Medical Faculty, University of Heidelberg, Heidelberg, Germany e School of Immunity and Infection, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK b

Abstract Elevated inflammatory levels are linked to poorer cognition, but experimental confirmation is lacking. This report examined associations between cognitive performance and inflammation induced by exercise and vaccination. Thirty-six (exercise N = 18, vaccination N = 18) healthy males completed a paced auditory serial addition test (PASAT), which is a multifaceted measure of cognitive function. The task was completed in placebo and elevated inflammation states. Improvements in PASAT performance were related to inflammation. In the exercise study, IL-6 during the first PASAT negatively correlated with PASAT improvement (p = .022). In the vaccination study, increases in C-reactive protein between PASATs correlated with reduced PASAT improvement (p < .001). Inflammation was linked to reduced improvements in cognitive performance. Further research should identify the specific cognitive functions affects and the underlying mechanisms. Descriptors: Cognitive function, Inflammation, Vaccination, Eccentric exercise, PASAT (Lupien, McEwen, Gunnar, & Heim, 2009), or comorbid factors, including fatigue and depressive symptoms (Chang et al., 2012; Krogh et al., 2014). Therefore, an experimental approach investigating the effects of inflammation on cognition in otherwise healthy individuals can help elucidate the unique role of inflammation in cognitive function. The systemic elevation of inflammatory cytokines has been proposed to have central nervous system effects, which may impair cognitive functioning. Circulating cytokines and inflammatory cytokines that are released by activated innate immune cells can access the brain and hypothalamus and induce increases in localized inflammation, leading to neurodegeneration in the hypothalamus (Miller & Spencer, 2014). This can lead to altered cognitive function through areas of the brain including the hippocampus and amygdala, as well as further production of cytokines by activated glial cells in the central nervous system (for review, see Castanon, Lasselin, & Capuron, 2014; Miller & Spencer, 2014). To date, very few experimental studies have been performed utilizing endotoxin as an inflammatory stimulus (Grigoleit et al., 2010; Reichenberg, 2001), with inconsistent findings. Endotoxin is a highly potent inflammatory stimulus that can also induce changes in variables, such as mood and physical discomfort, which could potentially confound the associations between cognition and inflammation (Reichenberg, 2001). Therefore, the current investigation aims to examine the effects of two different inflammatory challenges on cognitive performance that induce acute increases in inflammation while not altering the aforementioned variables, which could confound the results.

Inflammation is implicated in the development and presence of adverse physical health outcomes, such as atherosclerosis and cardiovascular diseases (Hansson, 2005; Ó Hartaigh et al., 2013), as well as mental health problems, including depression (Baune et al., 2012). Further, evidence has emerged that inflammation may also have detrimental effects on cognitive function (Sartori, Vance, Slater, & Crowe, 2012). For example, cross-sectional studies of middle-aged and older adults have shown negative associations between cognitive function and plasma levels of inflammatory markers, such as C-reactive protein (CRP) and interleukin-6 (IL-6; Gimeno, Marmot, & Singh-Manoux, 2008; Luciano, Marioni, Gow, Starr, & Deary, 2009; Marsland et al., 2006; Mooijaart et al., 2011, 2013; Roberts et al., 2009; Schram et al., 2007; Trollor et al., 2012). Longitudinal studies tend to replicate these findings (Komulainen et al., 2007; Rafnsson, Deary, Smith, Whiteman, & Fowkes, 2007), although not consistently (Dik et al., 2005; van den Biggelaar et al., 2007), which has created some uncertainty regarding the causal role of inflammation. Further supporting evidence comes from studies of clinical populations with inflammatory diseases, which also show associations between inflammation and impaired cognitive function (Kuo et al., 2005; Whitmer, 2007). However, these studies might be confounded by other influences that can impair cognition, such as glucocorticoid treatment

Address correspondence to: Nicola J. Paine, Ph.D., Box 3119, Department of Psychiatry and Behavioral Sciences, Duke University Medical Center, Durham, NC 27710, USA. E-mail: [email protected]

333 1

N.J. N.J. Paine Paine et et al.

334 2 The present report employed two methods to induce inflammation in healthy young adults. The first method employed a bout of acute eccentric exercise (Study 1). Eccentric exercise causes an increase in IL-6 within 1 to 2 hr, presumably as a result of skeletal muscle damage and glycogen depletion (Febbraio & Pedersen, 2002; Steensberg et al., 2002). These increases in IL-6 are still evident between 6 to 12 hr postexercise (Jackman, Witard, Jeukendrup, & Tipton, 2010; Paine, Ring, Aldred et al., 2013; Willoughby, McFarlin, & Bois, 2003). Given the time frame for the paced addition serial auditory test (PASAT) in relation to the exercise protocol, and the mechanism through which eccentric exercise can induce increases in inflammation, granulocyte counts were assessed as they tend to precede the appearance of other inflammatory markers such as IL-6 (but also TNF-α, IL-1) in the blood (Ekstrom, Eriksson, & Tornvall, 2008; Medzhitov, 2008; Möller & Villiger, 2006). The second method involved the administration of a Salmonella typhi vaccination (Study 2). This vaccination method has been successfully utilized to induce acute increases in inflammatory markers over a 24-hr period (Padfield et al., 2010; Paine, Ring, Bosch, Drayson, & Veldhuijzen van Zanten, 2013). It appears to elicit a broader range of inflammatory cytokines than eccentric exercise, including elevations in TNF-α, CRP, and fibrinogen (Padfield et al., 2010; Paine, Ring, Bosch et al., 2013). The primary aim of this report was to assess the relationship between inflammation and cognitive performance in a secondary analysis of data in a sample where inflammation was acutely induced in an otherwise healthy population. It was hypothesized that increases in inflammatory markers would be associated with poorer cognitive performance. Based on the known practice effect of the PASAT task, the improvements on task performance were deemed an appropriate assessment of cognitive function. Cognitive performance was assessed by the PASAT. Whereas in psychophysiological studies the PASAT has been used as a mild cognitive stressor (e.g., Paine, Ring, Bosch, McIntyre, & Veldhuijzen van Zanten, 2013), it is normally regarded as a “multifaceted measure” of cognitive performance, which incorporates aspects of cognitive function such as processing speed, divided attention, and working memory (Tombaugh, 2006). It has been used previously to assess various aspects of cognitive functioning, including measures of executive function, working memory, and information processing speed (Shucard, Gaines, Ambrus, & Shucard, 2007). The study by Shucard et al. (2007) is of impor-

tance as it focused on the relationship between CRP in patients with systemic lupus erythematosus and PASAT performance; increased CRP was associated with poorer performance on the PASAT. For each method (i.e., eccentric exercise and vaccination) that was used to induce inflammation, the PASAT was administered twice, and, therefore, it was possible to test if inflammatory activity was associated with the expected improvement with repeated task performance (Tombaugh, 2006). Study 1—Eccentric Exercise Method Recruitment and instructions. Study participants were recruited from the University of Birmingham. None were suffering from an acute illness or infection, reported a history of inflammatory, cardiovascular, or autoimmune disorders, or had taken any medication in the preceding 4 weeks. Participants were instructed to refrain from vigorous exercise for at least 24 hr, from alcohol for at least 12 hr, and food or caffeine in the 2 hr prior to testing. The study was approved by the local Research Ethics Committees and conducted in line with the Helsinki Declaration (2000). All participants gave written informed consent. Participants. Eighteen male participants were recruited (mean age ± SD = 20.4 ± 1.2 years, mean body mass index (BMI) ± SD = 23.7 ± 0.6 kg/m2). Participants who undertook regular resistance exercise were excluded, as greater amounts of muscle damage and inflammation have been demonstrated in those who are unaccustomed to eccentric exercise (Sorichter et al., 2006). Participants undertaking the exercise intervention completed two PASAT sessions (one in a placebo condition, the other in an inflammation condition) scheduled 7 days apart (Figure 1). These sessions were completed in a counterbalanced order. Participants completed the PASAT in the afternoon (between 2–6 pm) in both conditions, with either 25 min of rest (completing a questionnaire pack) or the exercise task completed 6 hr before the PASAT. The timing of the exercise task was chosen so the PASAT would coincide with the peak of the IL-6 response to eccentric exercise (Willoughby et al., 2003). The PASAT was undertaken as part of a stress-reactivity study, and the possible confounding aspects of this study (such as stress-related changes in cognitive function) may have been

Figure 1. Study design of the eccentric exercise study, completed in a counterbalanced order (N = 18).

Inflammation in cognitive cognitive task task Inflammation and and reduced practice effect in present. However, given that this potential confounding aspect was present for all participants, (across both Study 1 and Study 2), the possible impact of this confounding factor is standardized. All testing was performed in a temperature-controlled laboratory (18°C). Eccentric exercise task. The eccentric exercise task was undertaken using a Cybex leg extension machine (Cybex International, Medway, MA), further details of which can be found elsewhere (Paine, Ring, Aldred et al., 2013). First, each participant’s one repetition maximum (1RM) of their nondominant leg was determined, then the eccentric exercise task was explained before participants completed one set at a reduced weight (50% 1RM) to ensure understanding of the action. Participants were seated with their nondominant leg positioned at 90 degrees to their torso. Two experimenters lifted the weight to its starting position (with the exercising leg extended to approximately 15 degrees of flexion), and the participant lowered the weight until their knee was flexed at approximately 110 degrees. Participants lowered the weight slowly over a 4-s period, resisting the weight at all times. If this was not achieved, the weight was replaced at the starting position and the action repeated. Participants completed 12 sets of five eccentric repetitions at 120% of their concentric 1RM. Five seconds of rest separated each repetition, with a 1-min rest at the end of each set. PASAT session. All participants returned to the laboratory between 2–6 pm to complete the PASAT. An 18-gauge cannula (Insyte, Becton Dickinson, UK) was inserted into an antecubital vein of the dominant arm of each participant. The participant rested for 20 min (baseline rest period) while watching a nature documentary. A resting blood sample was taken at the end of the baseline rest period. The PASAT was then completed. PASAT protocol. The PASAT is a serial addition task, where numbers are audibly presented to the participant via a CD player. The objective is to remember and retain the last number that was heard by the participant and add it to the next number that is presented on the CD (Gronwall, 1977; Tombaugh, 2006). The PASAT has been used as a test of cognition when assessing brain injury and subsequent recovery, as several cognitive functions are necessary for the successful completion of the PASAT: sustained attention, working memory, and simultaneously performing several cognitive tasks under specific time constraints (Madigan, DeLuca, Diamond, Tramontano, & Averill, 2000). Studies that have used the PASAT to examine cognitive function have found that in individuals with mild traumatic brain injury (and impaired cognition) lower PASAT scores were evident in contrast to a control group without concussion (Gronwall, 1977; Gronwall & Wrightson, 1981). The task lasted 16 min, split into two 8-min tasks separated by a 1-min rest. This version of the PASAT has been adapted from the original version of Gronwall (1977), which presented numbers in blocks of 60 numbers. This adaptation was made because of previous data that showed the majority of the participants from this population would achieve close to maximum scores on the test. Each answer given by the participant was either marked as correct or incorrect. For an answer to be considered incorrect, either no answer was given before the next number was presented or an incorrect answer was given. The number of correct answers was summed for the entire PASAT. The numbers of correct answers were calculated from both of the 8-min tasks. The numbers were delivered in four 2-min sections, with the numbers presented every 3.2 s, 2.8 s, 2.4 s, and 2.0 s for the first task, and every 2.4 s, 2.0 s,

3353 1.6 s, and 1.2 s for the second task. This resulted in a progressive increase in task difficulty. The experimenter, who sat 1 m adjacent to the participant, checked their responses against the correct answers. Verbal encouragement and monetary reward were utilized to encourage the participants to complete the task to the best of their ability (Veldhuijzen van Zanten et al., 2004). All participants were given the opportunity to familiarize themselves with the task before it began (for both conditions), which involved completing a practice consisting of 10 numbers of the PASAT paced at the slowest number delivery. Participants were told that a £10 gift voucher would be awarded for the best performance on the task and a leader board with the highest five scores achieved by the participants was displayed, to compare participant scores. Another £10 voucher was given to the participant who recorded the greatest improvement in score between the two sessions. However, the latter of these rewards was only revealed before the start of the test in the intervention condition, to eliminate the risk of a poor performance in the placebo condition. The monetary reward was given to encourage the participants to perform to the best of their ability. Further, verbal encouragement was given whenever a participant looked as though they were struggling with the demands of the PASAT; examples included “keep going,” “you’re doing well,” and “keep concentrating.” Blood sampling. Blood samples were taken at the end of the baseline period and were collected into three 6-ml and one 2-ml vacutainers containing potassium ethylene diaminetetraacetic acid (K3EDTA; Becton, Dickinson). The 6-ml samples were stored on ice until centrifugation (1,500 g for 10 min at 4°C) and plasma was stored at −80°C for later assessment of IL-6. The 2-ml samples were analyzed for full differential blood cell count (Coulter Analyzer, Beckman Coulter, Inc.). Assays. Plasma IL-6 was measured in duplicate using high sensitivity ELISA (Quantikine HS Human IL-6 ELISA, R&D Systems, UK) in accordance with the manufacturer’s instructions. The reported limit of detection of the assays was 0.039 pg/ml, with recorded intraassay and interassay variations < 10%. Data reduction and analysis. A series of two condition (placebo, inflammation) repeated measures analyses of variance (ANOVAs) were conducted to examine differences in inflammatory markers between conditions, as indexed by white blood cells, granulocytes, lymphocytes, and IL-6. Two condition (placebo, inflammation) repeated measures ANOVAs were conducted to examine differences in PASAT performance between conditions. Subsequently, in order to account for the counterbalanced design of the study and the order in which the participants undertook the two conditions, further two time (first PASAT, second PASAT) ANOVAs were undertaken to examine improvements between assessments and any possible practice effect. For all ANOVAs, eta squared (η2) was used as a measure of effect size. The changes in basal inflammation between conditions were calculated as the difference between baseline inflammation in the inflammation condition minus baseline inflammation in the placebo condition. In order to determine the relationship between markers of inflammation and cognitive function, a series of Pearson’s correlations were conducted. Relationships between inflammation and cognitive performance were examined using baseline levels of inflammation, as well as the changes in inflammation between conditions. As a result, correlations were undertaken between the differences in inflammation between the two testing sessions (regardless of the order in which

N.J. N.J. Paine Paine et et al.

336 4 Table 1. Mean (SD) Inflammatory Levels Before the Start of Each PASAT in the Eccentric Exercise Study

Eccentric exercise study

Placebo condition

Inflammation condition

White blood cells (109/L) Granulocytes (109/L) IL-6 (pg/mL)

5.97 (0.74) 3.67 (0.94) 0.63 (0.38)

6.35 (1.49) 4.16 (1.28) 2.49 (3.40)*

Note. N = 17. *p < .05.

they were completed) and measures of cognitive performance. Occasional missing data are reflected in the reported degrees of freedom. All data were screened for statistical outliers and removed where any value was greater than 3 SD from the mean. Statistical significance was set at p < .05.

were also undertaken using log-transformed IL-6 values. This still revealed evidence of an association between baseline IL-6 during the inflammation condition and changes in PASAT performance, r(16) = −.45, p = .07. Subsequent analyses tested if there was an order effect (i.e., to determine if inflammation during the first or during the second performance is predictive of lower improvement). Analysis revealed the level of IL-6 during the first PASAT test to be negatively correlated with the change in the number of correct answers between the first and second completion of PASAT, r(16) = −.55, p = .022. Finally, correlation analyses revealed no association between inflammation (IL-6, white blood cells, granulocytes, or lymphocytes) and the number of correct answers given during the PASAT as a composite score, the number of correct answers given during each 2-min section of number pacing, or as a percentage of correct answers given during each 2-min section of number pacing, which was apparent in either a placebo or inflammation condition, regardless of order.

Results

Study 2—Vaccination

Differences in inflammatory levels and PASAT performance between conditions. Differences in inflammatory markers between the two conditions are displayed in Table 1. As expected, ANOVAs confirmed elevations in IL-6 in the inflammation condition, in comparison to the placebo condition, F(1,15) = 5.32, p = .036, η2 = .26. Marginal increases in granulocytes were also evident in the inflammation condition, F(1,16) = 3.30, p = .088, η2 = .17, but no increases were found for total white blood cells or lymphocytes. Table 2 shows the changes in the PASAT scores between both the placebo and inflammation conditions. A two condition (placebo, inflammation) repeated measures ANOVA showed no differences (mean ± SD) in the performance between the placebo (327 ± 58 [out of 513]) and inflammation condition (338 ± 72 [out of 513]), F(1,17) = 0.70, p > .20, η2 = .04. However, further analyses, aimed to assess the improvements in PASAT performance between the first and second sessions, demonstrated that regardless of the order of the condition performance improved (1st PASAT, 317 ± 62; 2nd PASAT, 349 ± 65; F(1,17) = 7.90, p = .012, η2 = .32). Subsequently, correlation analyses were conducted to examine the relationship between inflammatory markers and performance improvement. These analyses showed that IL-6 levels in the inflammation condition (regardless of the order in which the conditions were completed) negatively correlated with task improvement, r(16) = −.501, p = .048, indicating that elevated baseline IL-6 was associated with a smaller increase in correct answers (see Figure 2a). To examine whether this association was driven by the two participants with high levels of IL-6, analyses

Method Rationale and recruitment. Because the results of the previous study indicated that inflammation might attenuate a potential practice effect during the second completion of the PASAT, participants completed two cognitive test sessions in noncounterbalanced order: the first session was in a placebo condition and the second in an inflammation condition, induced by typhoid vaccination, scheduled 7 days apart (Figure 3). As with Study 1, participants were recruited from the University of Birmingham, in line with the inclusion criteria described above. Participants also adhered to the pretesting adherence criteria described above. The study was approved by the local Research Ethics Committees and conducted in line with the Helsinki Declaration (2000). All participants gave written informed consent. Participants and study design. Participants were eighteen male university students (mean age ± SD = 19.5 ± 0.9 years, mean BMI ± SD = 24.6 ± 2.8 kg/m2). None reported vaccine-related allergies or had received a typhoid vaccination in the last 12 months. The order in which the conditions were completed was fixed to determine if the extent of inflammation impaired the improvement in task performance. Both conditions were identical except that, in the inflammation condition, a typhoid vaccination was given 6 hr before the start of the PASAT session to ascertain that the PASAT took place at the peak IL-6 response (Padfield et al., 2010; Paine, Ring, Bosch et al., 2013). All testing was performed in a temperature-controlled laboratory (18°C). The PASAT

Table 2. Mean (SD) PASAT Scores in the Placebo and Inflammation Condition, Plus the Mean Change in PASAT Scores Between Conditions

PASAT scores

Placebo condition

Inflammation condition

Change in score between condition

Study 1 (exercise) Study 1 (exercise): Order 1 (placebo–inflammation) Study 1 (exercise): Order 2 (inflammation–placebo) Study 2 (vaccination)

327 (58) 320 (30) 315 (85) 285 (66)

338 (72) 363 (49)* 335 (79) 352 (66)*

11 (57) 43 (51) 20 (44) 67 (38)

Note. N = 18 for both studies. *significantly different between conditions (p < .05).

Inflammation in cognitive cognitive task task Inflammation and and reduced practice effect in

3375 b

120

140

100

120

Change in PASAT performance

Change in PASAT performance

a

80 60 40 20 0 -20 -40 -60

100 80 60 40 20 0 -20 -40

-80 -100 0

2

4

6

8

10

12

Baseline IL-6 during 1st PASAT task (pg/ml)

-60 -0.15

-0.10

-0.05

0.00

0.05

0.10

0.15

0.20

Change in CRP between condition (mg/L)

Figure 2. Scattergrams illustrating the relationship between changes in (a) PASAT performance and IL-6 (Study 1), and (b) PASAT performance and CRP (Study 2).

session took place in the afternoon and began at the same time for each participant, starting between 2–5 pm. Vaccination administration. Participants received a 0.5-ml S. typhi capsular polysaccharide vaccine (0.025 mg in 0.5 ml, Typhim Vi, Sanofi Pasteur, UK) via intramuscular injection into the deltoid muscle of the nondominant arm, by a registered nurse. All participants remained in the laboratory for 20 min postinjection for observation and then returned to the laboratory 6 hr later to complete the cognitive function session. PASAT session. Similar to the eccentric exercise paradigm, all participants came to the laboratory between 2–6 pm to complete the PASAT session, which was identical to the one completed within the eccentric exercise study. PASAT. As with the eccentric exercise study, the cognitive function assessment was a 16-min version of the PASAT. The timing of the number delivery was the same as in the eccentric exercise study.

Blood sampling. The blood sampling techniques, preparation, and timing of the samples were completed as described in the eccentric exercise study. In addition to IL-6, assessment of TNF-α, CRP, and fibrinogen was completed from the plasma, which was stored at −80°C. Assays. Plasma IL-6 and TNF-α were measured in duplicate using high sensitivity ELISA (Quantikine HS Human IL-6 ELISA and Quantikine HS Human TNF-α ELISA, both R&D Systems) in accordance with the manufacturer’s instructions. The reported limit of detection of the assays was 0.039 and 0.106 pg/ml, respectively. Analysis of high sensitivity C-reactive protein was undertaken at a commercial laboratory (Synlab, Leinfelden, Germany) by immunonephelometry using a Behring Nephelometer II. The detection limit for CRP was 0.015 mg/L (high sensitivity CRP, Dade Behring). Analysis of fibrinogen was also conducted at a commercial laboratory (Synlab) using the Clauss method. The detection range was 190 mg/dL. All assays yielded with intraassay and interassay coefficient of variation (CV)% of < 10%.

Figure 3. Study design of the vaccination study (N = 18).

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338 6 Table 3. Mean (SD) Inflammatory Levels at the Start of Each PASAT in the Vaccination Study

Vaccination study White blood cells (109/L) Granulocytes (109/L) IL-6 (pg/mL) TNF-α (pg/mL) CRP (mg/L) Fibrinogen (mg/L)

Placebo condition

Inflammation condition

6.08 (1.25) 3.92 (1.16) 0.68 (0.50) 4.93 (4.13) 0.05 (0.04) 241.13 (25.80)

9.29 (1.39)** 6.89 (1.23)** 2.97 (1.57)** 18.15 (8.96)** 0.06 (0.04) 229.50 (40.33)

Note. N = 18. **p < .01.

Data reduction and analysis. A series of two condition (placebo, inflammation) repeated measures ANOVAs were conducted to examine differences in inflammatory markers between conditions, as indexed by TNF-α, IL-6, CRP, and fibrinogen. Further, two condition (placebo, inflammation) repeated measures ANOVAs were conducted to examine differences in cognitive performance. For all ANOVAs, eta squared (η2) was used as a measure of effect size. The changes in baseline inflammation between conditions were calculated as the difference (delta) between baseline inflammation levels in the inflammation condition minus baseline inflammation levels in the placebo condition. In order to determine the relationship between markers of inflammation and cognitive function, a series of Pearson’s correlations were conducted. Relationships between inflammation and cognitive performance were examined using baseline levels of inflammation, as well as the changes in inflammation between conditions. Occasional missing data are reflected in the reported degrees of freedom. All data were screened for statistical outliers and removed where any value was greater than 3 SD from the mean. Statistical significance was set at p < .05. Results Differences in inflammatory levels and PASAT performance between conditions. A series of two condition (placebo, inflammation) repeated measures ANOVAs were conducted to examine the differences in inflammatory markers between the two conditions, which is displayed in Table 3. These ANOVAs yielded increase in WBC, F(1,17) = 94.59, p < .001, η2 = .85; granulocytes, F(1,17) = 106.53, p < .001, η2 = .86; TNF-α, F(1,15) = 27.90, p = .001, η2 = .65; and IL-6, F(1,14) = 19.25, p = .001, η2 = .58, between conditions. No overall changes in CRP or fibrinogen were observed (ps > .20). Table 2 illustrates the changes in PASAT scores between conditions. A two condition (placebo, inflammation) repeated measures ANOVA also showed improvements in the mean performance score from the placebo condition (286 ± 66 [out of 513]) to the inflammation condition (352 ± 66 [out of 513]), F(1,17) = 54.28, p < .001, η2 = .76, replicating the practice effect seen in the exercise study. Pearson correlations were again calculated to examine the relationships between inflammatory markers and PASAT improvement. CRP level in the inflammatory condition was negatively correlated with the changes in performance between the two sessions, r(17) = −.703, p = .002. Further, examination of changes in CRP between the two conditions revealed negative correlations with the change in CRP between conditions and the difference in the number of correct answers given, r(17) = −.882, p < .001, indi-

cating that an increase in CRP was associated with a poorer improvement in PASAT performance (see Figure 2b). A similar correlation was also observed for the change in fibrinogen between conditions, showing negative association with the change in the number of correct answers between PASAT administrations, r(17) = −.508, p = .045. Results further showed a trend indicating a weak negative association between basal CRP in the inflammation condition with PASAT performance in the inflammation condition, r(17) = −.466, p = .069, such that higher CRP was linked to a poorer performance. No significant correlations were found between basal levels of other markers of inflammation (IL-6, TNF-α, WBC, granulocytes, or lymphocytes) and PASAT performance (as a composite score, the number of correct answers given during each 2-min section of number pacing, or as a percentage of correct answers given during each 2-min section of number pacing), in either control or inflammation conditions, regardless of order. Discussion We examined the association between cognitive function and acute inflammation in a healthy male population. In two independent studies, utilizing exercise and vaccination as methods to induce release of inflammatory markers, it was consistently observed that inflammation was associated with attenuated improvement in cognitive performance normal to repeated PASAT performance (Tombaugh, 2006). These observations thus suggest that inflammation may impair the ability to improve cognitive performance through practice. To the best of our knowledge, this is the first experimental human study to demonstrate that inflammation may impair this aspect of cognitive performance. In the initial exercise study, it was observed that higher inflammatory levels (e.g., IL-6) during the first PASAT were negatively correlated with an improvement in performance during the second PASAT, which indicated that inflammation may reduce any potential practice effect that is sometimes observed with repeated testing. Based on this observation, Study 2 (vaccination) was designed to directly study the potential effect of inflammation on performance improvement, aiming to replicate this practice effect using an established paradigm for inflammation induction. The negative association between cognitive functioning and inflammation is consistent with epidemiological findings (Gimeno et al., 2008; Komulainen et al., 2007; Luciano et al., 2009; Marioni et al., 2011; Marsland et al., 2006; Mooijaart et al., 2011, 2013; Quinn et al., 2011; Rafnsson et al., 2007; Schram et al., 2007; Shucard et al., 2007). Of particular note is the study by Shucard and colleagues (2007) who found that systemic lupus erythematosus (SLE) patients with elevated CRP levels performed worse on the PASAT than a control group with normal CRP levels. This finding is in line with the results of Study 2 (vaccination) in that CRP was linked to reduced PASAT performance. These previous studies examined middle-aged and older populations, and the current study extends these findings to a young population with no underlying pathologies, applying an experimental design. No direct associations were seen between inflammation and the number of correct answers during the PASAT in the inflammation condition, for either study. This might be because our acute inflammatory paradigm elevates inflammatory markers but does not mimic the chronic levels of inflammation observed during cognitive dysfunction. Further, it may be that inflammation might only influence the practice effect observed (i.e., the ability to adapt to the task), such that only certain aspects of cognitive function are

Inflammation in cognitive cognitive task task Inflammation and and reduced practice effect in altered. The PASAT reflects working memory and processing speed (Tombaugh, 2006), so examination of other cognitive constructs (assessed by additional neuropsychological tests) is an important consideration for future research. Human studies designed to elucidate the mechanisms through which inflammation may alter cognitive function have provided inconsistent findings. Some have demonstrated changes in memory function in response to inflammation (Krabbe et al., 2005), whereas others have not (Grigoleit et al., 2010; Holden et al., 2008). There appears to be more consistent evidence towards no effect of inflammation on cognitive function, but these studies utilized alternative methods to increase inflammatory markers, such as lipopolysaccharide administration, which in addition to enhancing inflammatory levels can also induce other effects including fever, sickness, and malaise (Grigoleit et al., 2010; Holden et al., 2008; Krabbe et al., 2005). However, others have revealed that IL-6 increases can lead to slower reaction time (Brydon, Harrison, Walker, Steptoe, & Critchley, 2008), which might be a reason for a decline in the number of correct answers during the second completion of the PASAT in these studies. Another study found negative associations between IL-6 and hippocampal gray matter using fMRI (Marsland, Gianaros, Abramowitch, Manuck, & Hariri, 2008), such that increased IL-6 was associated with reduced hippocampal gray matter. This is interesting given that the hippocampus plays an integral role in memory formation (Squire & Zola-Morgan, 1991), and increased IL-6 can reduce hippocampaldependent learning (Gibertini, Newton, Friedman, & Klein, 1995; Heyser, Masliah, Samimi, Campbell, & Gold, 1997; Pugh et al., 1998). However, given that the current methodology did not utilize techniques such as fMRI to examine brain activity, the implications of these findings must remain speculative. The mechanisms underlying the present observations will need further elucidation. A vast body of work has examined changes in IL-6 and the effect on the brain and neurogenesis (McAfoose & Baune, 2009), specifically the hippocampus (memory), cognitive function, reaction time (Brydon et al., 2008), and psychomotor function (Felger & Miller, 2012). Experimental animal studies have found that elevations in inflammatory markers are associated with decreased neuroplasticity, which affects cognitive function and hippocampal-dependent learning (McAfoose & Baune, 2009). Further work has demonstrated that inflammatory cytokines can lead to alterations in basal ganglia and dopamine function, which in turn lead to psychomotor slowing (Felger & Miller, 2012). To the best of our knowledge, work examining the role of inflammation on brain activity and learning has focused on IL-6 and other markers such as TNF-α, but not CRP. CRP is produced in response to IL-6 (Ekstrom et al., 2008; Möller & Villiger, 2006), and appears in the blood later than IL-6 (Zakynthinos & Pappa, 2009). Therefore, given the relationship between CRP and IL-6, it is plausible that CRP might also influence brain activity as has been demonstrated with IL-6. However, it should be conceded that no changes in CRP were observed as a result of the vaccination paradigm, but this is more likely due to the time course of the CRP response to vaccination (Paine, Ring, Bosch et al., 2013) and the time point that we conducted the PASAT tests in relation to the vaccination (i.e., 6 hr postvaccination), rather than to the lack of a change in inflammatory activity. It is notable that the increases in IL-6 produced in response to both vaccination and exercise, while modest, are comparable with those found in clinical populations such as coronary artery disease (Kop et al., 2008) or myocardial infarction (Ridker, Rifai, Stampfer, & Hennekens, 2000) patients. Importantly, these levels

3397 are also comparable to those in studies that have established associations between inflammation and cognitive function (Gimeno et al., 2008; Luciano et al., 2009; Schram et al., 2007). A main difference, however, is that the elevated inflammation in the current study was acute and transient. It remains possible, therefore, that different mechanisms may explain the associations with cognitive performance compared to those observed with aging or chronic illness. However, utilization of an experimental design to induce inflammation in a healthy population does provide us with preliminary evidence of a direct relationship between inflammation and cognitive function. Indeed, aside from the elevations in inflammation, the population studied was healthy, and the observed relationships remained when controlling for factors such as BMI, limiting alternative explanations for the observed associations. Future work should look to expand on these initial findings. The most obvious limitations regarding the study are related to the sample. Studying a young, male, student population leads to issues with the ability to generalize the results, as does the relatively small sample size utilized in the current study. For example, both responsiveness of the immune system and cognitive performance changes with increasing age, and it thus remains to be determined if similar associations can be observed in older populations (Chung, Kim, Kim, & Yu, 2001). Also, the participants were university students and therefore likely to have an average IQ higher than the general population. This might be important for interpreting our results, as PASAT performance is related to IQ (Tombaugh, 2006). Further confirmation of these results is required in a larger and mixed gender sample. However, neither Study 1 nor Study 2 contains a control for the improvement in PASAT as a result of task repetition (i.e., second administration of the PASAT without an inflammation induction earlier in the day), and therefore future studies including a control group who undertake the PASAT twice, both times without an inflammation induction, is needed. While the PASAT does examine executive function and is a test of working memory and attention, it does not reflect a more typical set of cognitive functions that other neuropsychological tests examine, nor does it assess other aspects of executive function such as inhibition or planning (Marsland et al., 2006). However, the PASAT tests multiple aspects of cognitive function such as information processing, memory, and attentional capabilities (Tombaugh, 2006). Future work should aim to use a wider range of neuropsychological tests to cover a broader range of cognitive assessments. Additionally, while CRP and fibrinogen did not show an average increase in response to the vaccination, analyses of individual differences showed that changes in each marker were associated with task improvement. Further work may also examine CRP and fibrinogen at a time when levels would be expected to peak after exercise and vaccination (i.e, 24 hr postparadigm), and the relationship to PASAT performance and other neurocognitive tests. Based on our initial findings, future work may wish to examine the effect of inflammation on a clinical neuropsychological level, for example, to determine whether recent exercise or receiving a vaccination may impair neuropsychological assessment during cognitively demanding training situations. In addition, this task was conducted as part of a stress-reactivity study, and was as a result conducted in a setting with a strong emphasis on performance. Future work should consider addressing the issues of sample, alternative cognitive tests and other markers of inflammation and their relation to cognitive function. To our knowledge, this is the first study to induce increases in inflammation via vaccination and exercise paradigms to explore the relationships between inflammation and cognitive function,

N.J. N.J. Paine Paine et et al.

340 8 specifically the effects of inflammation on a potential practice effect. Basal levels of CRP and fibrinogen were negatively associated with aspects of cognitive function when utilizing a vaccination

model to increase inflammatory markers. Further, the exercise study revealed preliminary evidence for the role of elevated IL-6 in reduced cognitive ability.

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