Impact of immunosuppressive therapy on brain derived cytokines after liver transplantation

Journal Pre-proof Impact of immunosuppressive therapy on brain derived cytokines after liver transplantation

Meike Dirks, Henning Pflugrad, Anita B. Tryc, Anna-Kristina Schrader, Xiaoqi Ding, Heinrich Lanfermann, Elmar Jäckel, Harald Schrem, Jan Beneke, Hannelore Barg-Hock, Jürgen Klempnauer, Christine S. Falk, Karin Weissenborn PII:

S0966-3274(19)30066-8

DOI:

https://doi.org/10.1016/j.trim.2019.101248

Reference:

TRIM 101248

To appear in:

Transplant Immunology

Received date:

20 May 2019

Accepted date:

27 September 2019

Please cite this article as: M. Dirks, H. Pflugrad, A.B. Tryc, et al., Impact of immunosuppressive therapy on brain derived cytokines after liver transplantation, Transplant Immunology(2019), https://doi.org/10.1016/j.trim.2019.101248

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© 2019 Published by Elsevier.

Journal Pre-proof

Impact of immunosuppressive therapy on brain derived cytokines after liver transplantation

Meike Dirks1,2,1,* [email protected], Henning Pflugrad1,2,1, Anita B. Tryc1,2, Anna-Kristina Schrader1,2 , Xiaoqi Ding4, Heinrich Lanfermann4, Elmar

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Jäckel2,5, Harald Schrem2,6, Jan Beneke2, Hannelore Barg-Hock6, Jürgen

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Klempnauer2,6, Christine S. Falk2,3,1, Karin Weissenborn1,2,1

1

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Department of Neurology, Hannover Medical School, Hannover, Germany

2

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Integrated Research and Treatment Centre Transplantation (IFB-Tx), Hannover

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Medical School, Hannover, Germany 3

Institute of Transplant Immunology, Hannover Medical School, Hannover, Germany

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4

Institute of Diagnostic and Interventional Neuroradiology, Hannover Medical School,

Hannover, Germany 5

Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical

School, Hannover, Germany 6

General, Visceral and Transplant Surgery, Hannover Medical School, Hannover,

Germany

1

shared authorship: Meike Dirks, Henning Pflugrad, Christine Falk and Karin

Weissenborn all contributed equally

Journal Pre-proof *Corresponding author at: Department of Neurology, Hannover Medical School, Carl-

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Neuberg-Str. 1, 30625 Hannover, Germany, Email:

Journal Pre-proof Abstract Background: While acute neurotoxic side effects of calcineurin inhibitors (CNI) are well-known, data upon long-term effects on brain structure and function are sparse. We hypothesize that long-term CNI therapy affects the neuroimmune system, thereby, increasing the risk of neurodegeneration. Here, we measured the impact of CNI therapy on plasma levels of brain- and T cell-derived cytokines in a cohort of

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patients after liver transplantation (LT).

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Methods: Levels of T cell-mediated cytokines (e.g. Interferon-IFN-) and brain-

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derived cytokines (e.g. brain derived neurotrophic factor (BDNF), platelet derived growth factor (PDGF)) were measured by multiplex assays in plasma of 82 patients

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about 10 years after LT (17 with CNI free, 35 with CNI low dose, 30 with standard

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dose CNI immunosuppression) and 33 healthy controls. Data were related to

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psychometric test results and parameters of cerebral magnetic resonance imaging. Results: IFN- levels were significantly higher in the CNI free LT patient group

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(p=0.027) compared to healthy controls. BDNF levels were significantly lower in LT patients treated with CNI (CNI low: p<0.001; CNI standard: p=0.016) compared to controls. PDGF levels were significantly lower in the CNI low dose group (p=0.004) and for PDGF-AB/BB also in the CNI standard dose group (p=0.029) compared to controls. BDNF and PDGF negatively correlated with cognitive function and brain volume (p<0.05) in the CNI low dose group. Conclusion: Our results imply that long-term treatment with CNI suppresses BDNF and PDGF expression, both crucial for neuronal signaling, cell survival and synaptic plasticity and thereby may lead to cognitive dysfunction and neurodegeneration.

Journal Pre-proof Keywords: Immunosuppression, brain derived cytokines, cognitive function, brain, imaging

Abbreviations: ALT

Alanine aminotransferase

AST

Aspartate transaminase

calcineurin inhibitors

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CNI

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-p

CNS Central nervous system Cyclosporine A

interquartile

IL

Interleukin

IFN

Interferon

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IQ

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ɣ-GT ɣ-Glutamyl transferase

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CsA

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BDNF brain derived neurotrophic factor

MPA mycophenolic acid MRI

magnetic resonance imaging

NFAT nuclear factor of activated T cells PDGF platelet derived growth factor RANTES

regulated on activation normal T-cell expressed and secreted

RBANS

Repeatable Battery for the Assessment of Neuropsychological Status

Journal Pre-proof sNCAM Tac

soluble neural cell adhesion molecule

tacrolimus

TNF-α tumor necrosis factor-α TrkB tyrosine kinase receptor B Ventricular width at the level of the caudate nucleus

VWSC

Ventricular width at the level of the semioval centre

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VWCN

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WMH white matter hyperintensities

Journal Pre-proof 1. Introduction The use of cyclosporine and tacrolimus, both calcineurin inhibitors (CNI), led to a significantly increased survival rate after liver transplantation (LT). The standard immunosuppression consists of a combination of CNI, mycophenolic acid (MPA) and/or steroids 1,2. It is well established that CNI can induce long term side effects such as renal dysfunction, malignancy and cardiovascular diseases 3 but also

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neurotoxicity. In the acute phase after LT, some patients develop neurological side effects like disorientation, hallucinations, alterations of consciousness and seizures

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4,5

. Studies regarding long-term neurotoxicity of CNI show cognitive impairment and

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brain atrophy in patients who underwent liver transplantation 4,6-9. However, data are

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rather limited while different potential pathophysiological mechanisms regarding the

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influence of long-term CNI therapy on the central nervous system (CNS) are discussed. One possible cause for brain dysfunction due to CNI might be an

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alteration of the cerebral immune system with consecutive neurodegeneration 4.

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The mode of action of CNI as specific T and NK cell inhibitor and suppressor of cellmediated immune reactions is well known. After diffusing into immune cells, cyclosporine binds to cyclophilin to inhibit the calcium/calmodulin-dependent phosphatase complex calcineurin. This prevents nuclear factor of activated T cells (NFAT) from translocation into the nucleus and its activity as transcription factor for Interleukin-2 (IL-2) and other proinflammatory cytokines. Tacrolimus also inhibits calcineurin through binding to FK506 binding protein (FKBP12) resulting in a similar suppression of cytokine expression 1,5. Several studies address the various effects of immunosuppressants on peripheral cytokines (e.g. IFN- and Tumor necrosis factor-α (TNF-α)) 6-8. However, there is only

Journal Pre-proof sparse information about the effect of CNI therapy on the regulation of cerebral function and its brain derived markers

9-11

. So far, it is not fully understood how CNI

affect signaling pathways modulating microglia activity and the permeability of the blood-brain-barrier.

2. Objective

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Since alteration of the cerebral immune system with consecutive neurodegeneration

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might be a potential mechanism of long-term neurotoxicity of CNI, we aimed to

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evaluate the impact of CNI on the levels of brain-derived cytokines and growth

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factors in parallel to classical T and NK cell-derived cytokines. These cytokines and

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growth factors are crucial for neuronal plasticity and cell survival though involved in different signaling networks. In addition, we investigated whether the plasma levels of

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dosage of the patients.

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these soluble markers correlate with clinical and imaging data of the brain and CNI

3. Materials and Methods

The data presented here are part of a comprehensive study aiming to analyze neurological sequelae and possible mechanisms of brain injury in patients on longterm CNI therapy after liver transplantation. Patients underwent cognitive testing, magnetic resonance tomography and -spectroscopy. Furthermore, blood plasma of these patients was analyzed to assess inter alia brain- and T-cell derived cytokines. Neuropsychological test results and MRI findings have been described elsewhere in

Journal Pre-proof detail 12. The present paper focusses on the data concerning brain and T-cell derived cytokines and growth factors and their relationship to brain function and structure. Liver transplanted patients and healthy control individuals: The patient cohort was selected from the database of liver transplanted patients at Hannover Medical School with inclusion criteria of LT more than 2 years ago, age between 18 and 80 years and stable immunosuppressive therapy. Exclusion criteria

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were additional transplantation of other organs, liver re-transplantation (more than 3

brain

function,

acute

transplant-rejection

or

acute

infection

and

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affecting

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months after first LT), neurological or psychiatric disorders, current medication

decompensated heart-, liver- or kidney function. Based on these criteria, 375 of 1045

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patients registered in the database remained available for study participation, 51

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were on a CNI free immunosuppression and 20 agreed to participate, three had to be excluded from further analysis in this paper because of damaged blood samples. The

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study group was completed with 35 patients on low dose CNI therapy (stable

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tacrolimus trough levels below 5 µg/liter or stable Cyclosporine A (CsA) trough levels below 50 µg/liter) and 30 patients on standard dose CNI therapy (stable tacrolimus trough levels above 5 µg/liter or stable CsA trough levels above 50 µg/liter). All patients had undergone liver transplantation about 10 years ago (median; 25 th/75th percentiles: 10.0 years; 8.0-13.3) and were comparable considering age and education (Table 1). Liver enzymes (ALT, AST, ɣGT) in all patients were in normal range or slightly elevated.

Journal Pre-proof Table 1 Demographic and clinical characteristics (1) CNI free

(2) CNI low

(3) CNI standard

(4) controls

n=17

n=35

n=30

n=33

p-value

age (years)

60.7 ± 7.6

59.6 ± 9.5

54.8 ± 10.1

58.6 ± 7.8

0.10

sex (m/f)

12/5

23/12

18/12

15/18

education in years

10.0 (4;9.5-12.0)

9.0 (1; 9.0-10.0)

10.0 (3; 9.0-12.3)

10.0 (2; 10.0-12.0)

0.09

years since LT (median)

11 (3; 10-13)

10 (7; 8-15)

10 (9; 4.8-14)

n.a.

0.19

years on standard dose CNI

4.0 (8; 0.5-8.0)

4.0 (6; 2.0-8.0)

10 (9; 4.8-14)

n.a.

0.001 1 vs 3

101.6 ± 12.3

92.6 ± 13.3

96.7 ± 14.7

re

-p

RBANS Total scale

ro

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p=0.003

Immunosuppression

0

lP

CsA CsA + Prednisolon

4

0

4

10

2

8

6

1

0

1

3

0

4

10

3

Tac + MPA + Prednisolone

5

2

Tac + Azathioprin + Prednisolone

0

2

CsA + MPA

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CsA + MPA + Prednisolone

Tac Tac + Prednisolone Tac + MPA

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CsA+ Azathioprin + Prednisolone

Aetiology of liver disease autoimmune diseases

2

16

11

HCV

1

0

0

HBV

2

3

6

Alcohol

1

1

1

ALF

2

2

2

Others

9

13

10

2 vs 3 p=0.02 102.9 ± 13.7

0.01 2 vs 4 p=0.02

Journal Pre-proof Normally distributed values in mean ± standard deviation. Not normally distributed values in median th

th

(Inter-quartile range; 25 -75 percentile. Median/ mean and significant p-values in bold LT, liver transplantation, CNI, calcineurininhibitor, RBANS, Repeatable Battery for the Assessement of Neuropsychological Status, CsA, cyclosporine, Tac, tacrolimus, MPA, mycophenolic acid, HCV, hepatitis C virus, HBV, hepatitis B virus, ALF, acute liver failure, n.a, not applicable

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For the patients with CNI free immunosuppression, the maintenance therapy consisted of

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Sirolimus and MPA (n=1), Sirolimus and Prednisolone (n=2), Sirolimus, MPA and Prednisolone (n=2), Everolimus (n=1), Everolimus and MPA (n=1), MPA and Prednisolone

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(n=9) or MPA (n=1). The immunosuppressive treatment of the 65 patients treated with

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CNI is displayed in Table 1. The patients currently on CNI free immunosuppression

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were treated for 4.0 (0.5-8.0) years with CNI after LT and were CNI free for 7.0 (5.510.5) years at the time of the study. The main reason for the reduction or termination

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of CNI therapy in the past had been CNI induced kidney toxicity. After adjustment of

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the treatment regimen kidney function recovered (Table 1). 33 healthy controls (age 58.6 ± 7.9 years, n=15 (45.5%) male) adjusted for age, sex and education served as control group. All subjects gave written informed consent. The study was approved by the local ethics committee (MHH No. 6525) and performed according to the World Medical Association Declaration of Helsinki 1975 (revised in 2008). Neurological examinations Patients underwent a neurological examination by an experienced neurologist and the following data were assessed from the patients anamnesis and clinical data base: age, sex, etiology of liver disease, medication, years of education, years since LT, glomerular filtration rate, years on standard dose CNI, total CNI dosages and CNI

Journal Pre-proof trough level (for tacrolimus and cyclosporine, respectively) at each visit at the outpatient clinic. Mean CNI trough levels and total CNI dosage for each patient were calculated with last observations carried forward between each measuring point between LT and the study examination date (for further details see Pflugrad 2018 et al.

12

). In 3 patients, calculation of the mean CNI trough level and in 2 patients,

calculation of the CNI total dose was impossible. imaging (MRI)

and

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Patients and controls underwent magnetic resonance

psychometric testing using the Repeatable Battery for the Assessment of

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Neuropsychological Status (RBANS) 13,14.

The total scale of the RBANS as representative of cognitive function, the ventricular

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width at the level of the caudate nucleus (VWCN) and the semioval centre (VWSC)

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as estimates of brain atrophy as well as the extent of white matter hyperintensities (WMH), assessed according to the Scheltens scale

15

were related to plasma levels

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of brain and T cell-derived cytokines and growth factors.

Luminex-based multiplex measurements of cytokines and growth factors EDTA blood samples were taken from patients and controls before starting the neurological examinations. Samples were centrifuged for 8 min at 2100 rpm, plasma was stored at -80°C until further analysis. Plasma protein levels of analytes in the Human CD8+ T Cell Panel (Cat. No. HCD8MAG-15K) and Human Neurodegenerative Disease Panel 3 (Cat. No. HNDG3MAG-36K, both Merck/Millipore Darmstadt, Germany) were measured according to the manufacturer’s instructions: IFN-, TNF-α, soluble CD137 (4-1BB), Granzyme A (GzmA), soluble Fas (CD95

Journal Pre-proof death receptor; sFas), IL-6 and perforin and six brain-derived cytokines, brain derived neurotrophic factor (BDNF), neural cell adhesion molecule (sNCAM), platelet derived growth factor (PDGF-AA and AB/BB variants), Cathepsin D and the chemokine regulated on activation normal T-cell expressed and secreted (RANTES, CCL5) were measured. All concentrations are given in pg/ml. Statistical analysis

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Statistical analysis was done using SPSS Version 24. The Kolmogorov-Smirnov test

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was used to explore for normality of distribution. Normally, i.e. parametric distributed

-p

values were evaluated by Analysis of variance (ANOVA) and the Bonferroni post-hoc test. Significant group differences for not normally distributed values were evaluated

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with the Kruskal-Wallis-Test and the Mann-Whitney-U test. If data followed a

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Gaussian parametric distribution, values were given as mean and standard deviations. Non-parametric data were given as median and 25th-75th percentiles. A p-

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value <0.05 was considered as significant. The Spearmen rank test was used for

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correlation analyses between plasma levels of cytokines, brain derived biomarkers, age, RBANS Total Scale, MRI data and the tacrolimus and cyclosporine total dose and mean trough level. Correlation coefficients r and p-values are displayed. Pvalues <0.01 were considered as significant.

4. Results LT patients show impaired neurological performance and alterations in MRI Neurological performance and MRI alterations of patients and controls from our study are described and discussed in detail by Pflugrad et al.

12

. Since in this study, 3

Journal Pre-proof subjects have to be removed due to missing samples, the demographic data of the subjects included into this study are presented in Table 1. The neurological status was normal in all subjects. In accordance with

12

, the 3 patient groups showed worse

results than the control group in the RBANS Total scale with significant differences only for patients on low dose CNI therapy. MRI data were available for 16 patients in the CNI free group, 35 in the low dose group, 30 in the standard dose group and 32 controls included in this study. MRI showed significantly more WMH in the temporal

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regions in the patients (n=81) compared to controls and patients showed a

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significantly broader ventricular width at the level of the semioval centre (p=0.013),

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though without significant differences between patient groups.

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LT patients show higher T cell-mediated and lower brain-derived cytokine

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plasma levels

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Plasma levels of the pro-inflammatory Th1 cytokine IFN- were increased in LT patients compared to control individuals, but significance was reached only for the

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CNI free patient group (p=0.03; Figure 1A). Soluble CD137 levels, another indicator of T cell activation, were twice as high in all three patient groups compared to controls (Figure 1B, p=0.001). The effector cytokine TNF-α was also elevated in all patient groups (p=0.001) compared to controls (Figure 1C). In addition, the level of the T and NK cell cytotoxin, Granzyme A, was significantly upregulated in the CNI standard dose group compared to the control group (p=0.02, Figure 1D). Moreover, the levels of the soluble death receptor sFas (sCD95) were significantly higher in CNI free and CNI low dose patients compared to controls (p≤0.001) and also compared to CNI standard dose patients (p=0.02 and p=0.003, Figure 1E). Levels of the pro-inflammatory cytokine IL-6 were upregulated in the CNI free

Journal Pre-proof (p=0.03) and low dose (p=0.001) patient groups in comparison to controls (Figure 1F). Perforin and cathepsin D levels, two effector proteases released by T and NK cells, did not differ between the four groups indicating that not all T cell-mediated cytokines and cytotoxins are differentially regulated in LT patients (Table 2, Figure

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1G,H)

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Figure 1 Results for the level of the different T cell-mediated cytokines for the three patient groups

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and the control group.

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Box plots with mean and 25% - 75% quartiles are shown for plasma concentrations of IFN-y (A), sCD137 (B), TNF-a (C), Granzyme A (D), sFAS (sCD95, E), IL-6 (F), perforin (G), Cathepsin D (H); all

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given in pg/ml.

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For A: One outlier of the CNI standard group was rejected for the boxplot, but not for the analysis (IFN-y level= 3378.60 pg/ml)

Table 2 Level of plasma proteins between the different patient groups and control individuals (1)

(2)

(3)

control

CNI

CNI

CNI

s

free

low

stand

n=17

n=35

n=30

P-value

n=33

medi

IQ-range; 25-

medi

IQ-range;

media

IQ-range;

an

75th

an

25-75th

n

25-75th

25-75th

percentile

percentile

percentile

percentile

median

IQ-range;

Inflammatory markers IFN-γ

50.8

98; 33.08-

(pg/ml)

2

131.22

P=0.

44.71

82; 21.15102.72

51.52

80; 22.36101.99

25.97

37; 12.0649.24

0.01

Journal Pre-proof 027 sCD137

23.9

16; 19.51-

(pg/ml)

1

35.26

P<0.

27.14

27.15

21;20.68-

56; 16.02-

41.21

13.88

71.52

P<0.0

P<0.0

01

01

6; 11.56-

0.001

17.73

001 Granzyme

436.

594;329.61-

470.5

241; 310.40-

612.7

1870;

A (pg/ml)

04

923.77

5

551.68

6

259.71-

329.61

343; 204.49-

0.03

547.10

2130.04 P=0.0 20 sFas

1602

7144;12038.

1475

7881;

1072

5238;

9824.1

4355;

(pg/ml)

8.60

86-19183.25

2.55

11999.06-

7.15

7907.90-

5

7798.37-

001

01

13146.00

12152.98

(pg/ml)

P=0.

P=0.0

025

01

(3) P=0.003

8.50

(pg/ml)

P<0.

4;6.45-10.66

5.61

6.17

6; 3.94-9.51

5.38

5; 6.17-11.29

P<0.0

7.02

For (2) vs. (3)

8; 2.55-10.41

3.01

4; 1.72-5.77

0.01

4; 5.32-9.60

4.71

3; 3.13-5.84

0.001

0.125

P<0.0

lP

TNF-α

4;4.76-8.32

-p

6.99

re

IL-6

01

BDNF

3842

7946;1763.7

3673.

4506;

3441.

7785;

769

12581; 5360.05-

(pg/ml)

.70

5-9710.15

58

1484.20-

94

1871.73-

7.13

17940.80

001

01

Perforin

4526

3685;

4535.

(pg/ml)

.74

3135.66-

40

5225.

2580;

5436.8

2038;

2891.60-

83

4147.80-

3

4488.27-

5934.31

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Neuronal function markers

3042;

na

6820.94

6727.74

5990.46

6526.13

P<0.0

P=0.0

01

16

7048

297526;6140

6287

255487;

6511

165173;

724

2561970;

(pg/ml)

19.4

23.84-

53.47

548694.51-

68.47

589151.66-

968.

596500.53-

5

911550.71

754324.85

61

852697.57

RANTES

4871

112532;

5336

43683;

7830

84720;

124

104240;

(CCL5)

5.16

25271.49-

9.73

31802.40-

0.70

37578.50-

366.

69427.10-

122298.64

49

173667.19

804181.21

137803.75

0.001

9656.85

sNCAM

(pg/ml)

P=0.02 for (1) vs

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P<0.0

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19879.68 P=0.

0.001

75485.73

0.284

0.001

P<0.0 01 Endothelial activation markers PDGF-AA

2455

4346;

1895.

2150;

1868.

2399;

3459.7

4893;

(pg/ml)

.80

1047.73-

60

918.10-

80

1284.31-

4

1998.08-

5393.62

3068.57

3683.49

0.006

6891.47

P=0.0 04 PDGF-

5712

12303;

3018.

4787;

3766.

5988;

8412.9

11947;

0.004

Journal Pre-proof AB/BB

.71

(pg/ml)

1973.90-

77

14276.85

25

1824.79-

1824.79-

6612.24

9

7812.84

P=0.0

P=0.0

04

29

4664.1416610.92

Coagulation marker Cathepsin-

4160

168630;

3661

168476;

3782

223094;

416388

183106;

D (pg/ml)

89.9

361604.88-

70.74

287677.35-

39.44

283736.85-

.27

349714.16-

9

530235.13

456153.08

506831.30

0.073

532820.07

th

Values are given in median and IQR; 25 -75th percentile; Kruskal-Wallis-Test was performed for group differences. Significant p-values are in bold.

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Abbreviations: IFN-y, Interferon-y, IL, Interleukin, TNF, tumor necrosis factor; BDNF, brain derived

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neurotrophic factor; sNCAM, soluble neural cell adhesion molecule; RANTES, regulated on activation

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lP

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normal T-cell expressed and secreted; PDGF, platelet derived growth factor

Journal Pre-proof LT patients show lower levels of BDNF and PDGF compared to healthy controls and CNI trough levels and steroids may influence the neuroimmunological communication The BDNF levels were significantly lower in patients treated with CNI (CNI low: p=0.001 and CNI standard: p=0.016) compared to healthy control individuals (Figure 2A, Table 2). The growth factor variants PDGF-AA and PDGF-AB/BB were detected

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at significantly lower concentrations in the CNI low dose group (p=0.004) and PDGF-

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AB/BB also in the CNI standard dose group (p=0.03) compared to controls (Table 2, Figure 2B, C). The chemokine RANTES (CCL5) showed also significantly lower

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plasma levels in the CNI low dose patient group compared to controls (p=0.001,

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Figure 2D). In contrast to these neurotropic factors and chemokines, sNCAM plasma

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concentrations showed no differences between the four groups (Figure 2E). When patients were divided according to absence or presence of current prednisolone

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therapy irrespective of the CNI therapy, significant differences for both, T and NK cell

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and brain-derived cytokines, were found between both patient groups and controls but not between the patient groups. This distinction indicates that steroids may not have a major impact on the alterations observed between LT and control individuals. Since for perforin, sNCAM and cathepsin D, no significant differences could be found over all groups (for further details see Table 3), these proteins seem to be less dysregulated in LT patients in general and not influenced by steroid treatment.

Journal Pre-proof Figure 2 Results for the level of the brain-derived cytokines for the three patient groups and the

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control group.

Box plots with mean and 25% - 75% quartiles are shown for plasma concentrations of BDNF (A), PDGF-AA (B), PDGF-AA/AB (C), RANTES (D), sNCAM (E ) For C: One outlier of the control group was rejected for the boxplot, but not for the analysis (PDGFAA/AB level= 58422.9 pg/ml).

Journal Pre-proof Table 3 Patients with and without current prednisolone therapy vs. controls (1) Patients without

(2) Patients with

prednisolone therapy

prednisolone therapy

n=37

n=45

median

th

25/75

median

percentiles

controls

Mann-Whitney Test

n=33

p-value

25/75th

media

25/75th

controls

controls

percentiles

n

percentiles

vs. (1)

vs. (2)

Inflammatory markers sCD137

29.30

18.62-43.93

23.91

18.98-41.75

13.88

11.56-17.73

0.000

0.000

IFN-γ

46.24

22.76-86.27

40.86

21.96-113.23

25.97

12.06-49.24

0.028

0.006

Granzy

489.45

257.82-547.40

534.12

320.01-

329.61

204.49-547.10

n.s.

0.011

9748.59-

9824.1

7798.37-

0.000

0.006

17221.80

5

12152.98

3.01

1.72-5.77

0.007

0.005

4.71

3.13-5.84

0.000

0.000

5436.8

4488.27-

n.s.

n.s.

3

6526.13

1825.54-

7697.1

5360.05-

0.001

0.000

7207.49

3

17940.80

575600.38-

n.s.

n.s.

0.003

0.002

0.004

0.018

0.006

0.010

n.s.

n.s.

13105.16

10683.05-

12522.18

19658.59 IL-6

6.39

4.21-9.00

5.46

3.53-9.47

TNF-α

8.88

6.77-11.95

7.52

5.67-9.26

Perforin

4997.11

3479.75-

4535.40

3345.51-

Neuronal function markers BDNF

3850.04

1413.66-

2946.98

7798.29 681731.53

569961.02-

669383.14

788144.93 64747.79

S

32354.99-

Endothelial activation markers 1837.89

AA PDGF-

3527.45 3915.82

AB/BB

1973.90-

2108.73

3616.68

7062.24

Coagulation marker Cathepsi

828.28-

Jo ur

PDGF-

60179.81

98029.88

na

RANTE

366170.74

n-D

289433.23-

72496

596500.53-

814407.18

8.61

852697.57

29227.03-

12436

69427.10-

108029.37

6.49

173667.19

1217.79-

3459.7

1998.08-

3286.25

4

6891.47

1824.79-

8412.9

4664.14-

8112.97

9

16610.92

317451.41-

41638

349714.16-

464283.03

8.27

532820.07

lP

sNCAM

-p

6543.03

re

6031.73

376651.21

523144.46

of

sFAS

1433.18

ro

me A

th

Values are given in median with 25 -75th percentile; Kruskal-Wallis-Test was performed for group differences Abbreviations: IFN-y, Interferon-y; sFAS, IL, Interleukin; TNF, tumor necrosis factor; BDNF, brain derived neurotrophic factor; sNCAM, soluble neural cell adhesion molecule; RANTES, regulated on activation normal T-cell expressed and secreted; PDGF, platelet derived growth factor

Journal Pre-proof Correlations between neurological measures and soluble plasma factors indicate a pathophysiological link between CNI treatment and neurocognitive function For the whole patient group (CNI treated and CNI free patients, n=81) a positive association was found between age and VWCN (r=0.504, p<0.001, Figure 3A), VWSC (r=0.368, p<0.001) and WMH (r=0.407, p<0.001) implying that brain atrophy

of

and white matter hyperintensities increase with age (Table 4, see bottom of the file).

ro

Of note, age was negatively correlated with Granzyme A level (r=-0.373, p=0.001, Figure 3B). CsA mean trough levels showed a positive correlation with white matter

-p

hyperintensities (r=0.399, p=0.005, Figure 3C). RANTES correlated positively with

re

CsA total dose (r=0.542, p=0.001, Figure 3D) while Granzyme A levels correlated

lP

positively with Tacrolimus total dose (r=0.483, p=0.003, Figure 3E), whereas sFas correlated negatively with CsA mean trough levels (r=-0.444, p=0.001, Figure 3F).

na

Accordingly, in the control group (n=33), age correlated positively with VWCN

Jo ur

(r=0.547, p=0.001, Figure 4A) and WMH (r=0.680, p=0.001). With regard to the brain-derived cytokines, here sNCAM correlated positively with VWSC (r=0.594, p=0.001, Figure 4B). In addition, BDNF and PDGF, though significantly decreased compared to healthy controls (Table 2), correlated negatively with cognitive function and brain volume (p<0.05) in the CNI low dose group but not the CNI free group, the CNI standard dose group or the controls (BDNF/RBANS total scale: r=-0.339, p=0.47; BDNF/VWCN: r=0.410, p=0.015; BDNF/VWCS: r=0.431, p=0.01) (PDGFAA/RBANS total scale: r=-0.394, p=0.019; PDGF-AA/VWCN: r=0.518, p=0.001; PDGF-AA/VWSC: r=0.535, p=0.001), (PDGF-AB/RBANS total scale: r=-0.418, p=0.012; PDGF-AB/VWCN: r=0.445, p=0.007; PDGF-AB/VWSC: r=0.477; p=0.004).

Journal Pre-proof

na

lP

re

-p

ro

of

Figure 3 Correlation analysis for all patients.

Linear correlation analyses are shown for age and VWCN, n=81(A), age and Granzyme A, n= 82 (B),

Jo ur

CsA mean trough level and WMH, n=48 (C), RANTES and CsA total dose, n=49 (D), Granzyme A and Tacrolimus total dose, n=36 (E), sFAS and CsA mean trough level, n=49 (F). VWCN= ventricular width at level of caudate nucleus; WMH= white matter hyperintensities; CsA= Cyclosporine A

Journal Pre-proof

of

Figure 4 Correlation analysis for controls

ro

age and VWCN, n=32 (A), sNCAM and VWSC, n=32 (B). VWCN= ventricular width at level of

Tacrolimus

and

na

5. Discussion

lP

re

-p

caudate nucleus; VWSC= ventricular width at level of semioval centre.

cyclosporine,

both

calcineurin

inhibitors,

are

crucial

as

Jo ur

immunosuppressive agents to prevent allograft rejection after liver transplantation. The major effect of these CNI on T and NK cells is blocking of the phosphatase calcineurin, which prevents NFAT activation and subsequent expression of NFATdependent cytokines such as IL-2, IFN-γ, and GM- CSF

16-19

. However, CNI have

been shown to have cytotoxic potential for kidney and brain tissue. Therefore, our aim of the study was to link the levels of NFAT-dependent immune- and brainmediators with cognitive function of liver transplant recipients. As first step, we measured the cytokine levels linked to T cell activation and observed increased levels of pro-inflammatory cytokines in transplant patients irrespective of their immunosuppressive therapy indicating sub-maximal T cell inhibition. In vitro,

Journal Pre-proof expression of IFN-γ as proinflammatory Th1-cytokine is blocked during CNI treatment 7. In our study, the levels of IFN-γ were twice as high in the patient groups compared to controls, indicating some degree of immune activation due to LT. Our findings are in line with a study of Alvares-de-Silva et al. 20 who measured the level of inflammatory cytokines and endothelial biomarkers in patients after LT, non-alcoholic steatohepatitis (NASH) patients and healthy controls. One year after LT, NASH and other patients showed higher IFN-γ levels compared to controls suggesting an

of

increased inflammatory reaction. The fact that IFN-γ is lower in our patient groups

ro

treated with CNI than in CNI-free patients implies that CNI suppress IFN-γ

-p

expression although not sufficiently to reach normal levels. Similar results could be

re

shown for the levels of sCD137 which were significantly higher in all patient groups compared to controls. The co-stimulatory molecule CD137, member of the TNF

lP

receptor family, is associated with T cell activation, proliferation, apoptosis and 21

), released upon T cell stimulation

22

and

na

cytokine production (for review see elevated in autoimmune patients

23

. Hence, elevated sCD137 levels in LT patients

Jo ur

argue for sustained T cell activation despite immunosuppression. Our results are in line with Melendreras et al.

24

who showed higher sCD137 levels in patients before

kidney transplantation with decreasing concentrations upon immunosuppressive therapy with prednisolone, CNI in combination with MPA or mTOR inhibitors (Sirolimus or Everolimus), which were still higher compared to healthy individuals. Thus, it is conceivable that higher sCD137 levels imply a continuous, subclinical T cell activation in liver transplanted patients. The generally low IL-6 concentrations in LT patients together with the low TNF-α levels argue for the absence of a continuous inflammatory process in the liver which is supported by the normal range of liver enzymes.

Journal Pre-proof Molecules associated with cytolysis and proinflammatory immune responses (see review

25

), like Granzyme A, perforin and sFAS displayed also higher plasma

concentrations in LT patients. sFAS was upregulated in all LT patients, reaching significance for CNI free and CNI low groups and correlation analysis showed that higher CsA mean trough levels were associated with lower level of sFAS suggesting that shedding of FAS from different cell types including hepatocytes may be sensitive to CNI treatment. Our results are in agreement with a study of van de Wetering et al. who demonstrated in kidney recipients an influence of CNI withdrawal on

of

26

ro

expression of a number of regulatory genes at the mRNA level, including Granzyme

-p

A and perforin. Taken together, the results of T cell-derived cytokines are in line with

re

the current knowledge about the NFAT-dependent mechanism of action of CNI.

lP

With respect to neuronal function, recent studies suggest that CNI do not only affect signaling pathways in lymphocytes but also in microglia, the immune defense of the 9,10

. Therefore, an interesting finding of our study are lower

na

CNS, and other glial cells

Jo ur

BDNF levels in LT patients treated with CNI (low and standard dose) compared to controls. The neurotrophin BDNF regulates neuronal differentiation in developing brains and is expressed and released by glial cells and neurons. BDNF and its receptor tyrosine kinase receptor B (TrkB) are essential for neuronal signaling, cell survival and regulation of synaptic plasticity

27

. Kingsbury et al.

28

showed that

depolarization–induced calcium influx stimulates CREB-dependent BDNF and TrkB expression in embryonic cortical neurons of mice, which was decreased in the presence of tacrolimus. Accordingly Chen et al.

9

demonstrated in a rat model

decreased BDNF and TrkB mRNA and protein levels in hippocampus and midbrain but not in the cortex upon chronic CsA administration. Suppressed BDNF levels seemed also to correlate with altered behavior under CsA administration. NFATc4, a

Journal Pre-proof member of the NFAT family, expressed in the adult dentate gyrus can be activated by BDNF and NMDA in primary neurons

29

and, hence may represent a suitable CsA

target. These results, which are in line with our findings, point towards decreased calcineurin activity supporting neurodegeneration in long-term treatment of LT patients with CNI. However correlation analysis between BDNF levels and MRI as well as cognition data of the whole patient group showed no significant results and, hence, would argue for a minor or rather indirect impact of reduced BDNF levels on

ro

of

brain function.

Similar to BDNF, PDGF- AA and AB/BB levels were downregulated in patients

-p

treated with CNI. In the CNS, PDGF is expressed in neurons, astrocytes,

re

oligodendrocytes and vascular cells and involved in the interaction between CNS and

lP

vascular cells to control maintenance of the blood-brain-barrier. During injury or stress, it is important for neuronal excitability and affecting synaptic plasticity. The

na

source for systemic PDGF cannot be directly assigned to these cells since

11

Jo ur

hematopoietic cells also contribute to altered PDGF levels in the blood. Savikko et al. showed in a rat renal transplant model suppressive effects of Tac rather than CsA

on PDGF expression associated with normal histology of the kidney grafts in longterm follow-up indicating that higher PDGF levels might induce chronic allograft injury. Since similar to BDNF, no significant correlations were seen between PDGF levels and cognition and MRI data, reduced systemic PDGF levels do not seem to impair the interaction between cells of the CNS and vasculature. In parallel, the chemokine RANTES (CCL5) was also reduced in CNI low dose patients compared to controls. Since lower RANTES levels correlated with higher CsA total dosis, systemic RANTES secretion also seems to be sensitive to CNI although it is impossible to determine its cellular source in this LT setting. RANTES

Journal Pre-proof regulates inflammatory processes by mediating migration of immune cells, primarily memory T cells and monocytes via CCR5 into inflamed tissues. Activated T cells are perceived as a major source of RANTES and, thus, it is mainly discussed as one promoting factor of acute allograft rejection

25,30

. However, RANTES seems to play

also a role in activation and migration of microglia

31

and, thus, may represent an

important link between immune activation and brain function. In our cohort, reduced

of

systemic RANTES concentrations do not seem to impair cognition.

ro

Different from the suppressive impact of CNI on BDNF, PDGF and RANTES, the concentrations of NCAM and Cathepsin D did not vary in the different groups.

-p

Although NCAM was originally identified in the nervous system, it represents also a

re

marker for human NK cells 8. Thus, both brain-derived and hematopoietic cells can

lP

serve as source of its soluble form which is obviously insensitive to CNI treatment. However, in the control group the correlation analysis showed that higher sNCAM

Jo ur

integrity.

na

levels were associated with more brain atrophy indicating a contribution to brain

Taken together, the most unexpected finding was that the CNI low dose group represented the only patient group with significantly decreased cognitive function compared to controls and that BDNF as well as the PDGF levels correlated negatively with both, RBANS and brain volume. This underlines the recently discussed hypothesis that patients who presented earlier with increased CNI nephrotoxicity might differ also in regard to CNI effects of BDNF and/or PDGFmediated regulation of neuronal signaling and synaptic plasticity. Our study has certain limitations: We were not able to differentiate between treatment with Tacrolimus and cyclosporine due to the limited statistical power of the subgroups

Journal Pre-proof supposing that the two drugs have different effects from the calcineurin pathway. Additionally, the levels of the different proteins were only measured in EDTA blood samples and, therefore, our results should be verified by using cerebrospinal fluid. And– as usual in clinical conditions – the patients also received co-medication like prednisolone and MPA and it is not clear how the combination of the different agents influence the cytokine profiles.

of

Finally, our results show that CNI do not only influence signaling pathways in

ro

lymphocytes, but seem to play a role in the regulation of the cerebral immune system

-p

in vivo as well. CNI are likely to suppress distinct brain derived growth factors as BDNF and PDGF, both crucial for neuronal signaling, cell survival and synaptic

re

plasticity. Modification of these pathways may lead to altered behavior and

Jo ur

Acknowledgement:

na

lP

neurodegeneration in patients undergoing long-term CNI treatment.

An official German translation of RBANS was kindly provided by Dr. Chris Randolph (Loyala University Medical Center, Maywood, IL). We would like to thank Drs. Hans Messner, Alois Gratwohl and David Gjertson – all three members of the external advisory board of the IFB Tx at MHH - for scientific advice and statistical support and Kerstin Daemen and Jana Keil for excellent technical assistance. Declaration of interest: none Funding: This study was supported by a grant from the German Federal Ministry of Education and Research (reference number: 01EO1302)

Journal Pre-proof References

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1. Choudhary NS, Saigal S, Shukla R, et al. Current status of immunosuppression in liver transplantation. Journal of clinical and experimental hepatology. 2013;3(2):150-158. 2. Herzer K, Strassburg CP, Braun F, et al. Selection and use of immunosuppressive therapies after liver transplantation: current German practice. Clinical transplantation. 2016;30(5):487-501. 3. Aberg F, Isoniemi H, Hockerstedt K. Long-term results of liver transplantation. Scandinavian journal of surgery : SJS : official organ for the Finnish Surgical Society and the Scandinavian Surgical Society. 2011;100(1):14-21. 4. Klawitter J, Gottschalk S, Hainz C, et al. Immunosuppressant neurotoxicity in rat brain models: oxidative stress and cellular metabolism. Chemical research in toxicology. 2010;23(3):608-619. 5. Geissler EK, Schlitt HJ. Immunosuppression for liver transplantation. Gut. 2009;58(3):452-463. 6. Liu Z, Yuan X, Luo Y, et al. Evaluating the effects of immunosuppressants on human immunity using cytokine profiles of whole blood. Cytokine. 2009;45(2):141147. 7. Jiang H, Yang X, Soriano RN, et al. Distinct patterns of cytokine gene suppression by the equivalent effective doses of cyclosporine and tacrolimus in rat heart allografts. Immunobiology. 2000;202(3):280-292. 8. Hoffmann U, Neudorfl C, Daemen K, et al. NK Cells of Kidney Transplant Recipients Display an Activated Phenotype that Is Influenced by Immunosuppression and Pathological Staging. PloS one. 2015;10(7):e0132484. 9. Chen CC, Hsu LW, Huang LT, et al. Chronic administration of cyclosporine A changes expression of BDNF and TrkB in rat hippocampus and midbrain. Neurochemical research. 2010;35(7):1098-1104. 10. Zawadzka M, Kaminska B. Immunosuppressant FK506 affects multiple signaling pathways and modulates gene expression in astrocytes. Molecular and cellular neurosciences. 2003;22(2):202-209. 11. Savikko J, Teppo AM, Taskinen E, et al. Different effects of tacrolimus and cyclosporine on PDGF induction and chronic allograft injury: evidence for improved kidney graft outcome. Transplant immunology. 2014;31(3):145-151. 12. Pflugrad H, Schrader AK, Tryc AB, et al. Longterm calcineurin inhibitor therapy and brain function in patients after liver transplantation. Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society. 2018;24(1):56-66. 13. Weissenborn K. Psychometric tests for diagnosing minimal hepatic encephalopathy. Metabolic brain disease. 2013;28(2):227-229. 14. Goldbecker A, Weissenborn K, Hamidi Shahrezaei G, et al. Comparison of the most favoured methods for the diagnosis of hepatic encephalopathy in liver transplantation candidates. Gut. 2013;62(10):1497-1504. 15. Scheltens P, Barkhof F, Leys D, et al. A semiquantative rating scale for the assessment of signal hyperintensities on magnetic resonance imaging. Journal of the neurological sciences 1993;114(1):7-12. 16. Giese T, Zeier M, Schemmer P, et al. Monitoring of NFAT-regulated gene expression in the peripheral blood of allograft recipients: a novel perspective toward

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individually optimized drug doses of cyclosporine A. Transplantation. 2004;77(3):339344. 17. Hermann-Kleiter N, Baier G. NFAT pulls the strings during CD4+ T helper cell effector functions. Blood. 2010;115(15):2989-2997. 18. Panicker AK, Buhusi M, Thelen K, et al. Cellular signalling mechanisms of neural cell adhesion molecules. Frontiers in bioscience : a journal and virtual library. 2003;8:d900-911. 19. Ke H, Huai Q. Structures of calcineurin and its complexes with immunophilinsimmunosuppressants. Biochemical and biophysical research communications. 2003;311(4):1095-1102. 20. Alvares-da-Silva MR, de Oliveira CP, Stefano JT, et al. Pro-atherosclerotic markers and cardiovascular risk factors one year after liver transplantation. World journal of gastroenterology. 2014;20(26):8667-8673. 21. Watts TH. TNF/TNFR family members in costimulation of T cell responses. Annual review of immunology. 2005;23:23-68. 22. Shao Z, Sun F, Koh DR, et al. Characterisation of soluble murine CD137 and its association with systemic lupus. Molecular immunology. 2008;45(15):3990-3999. 23. Michel J, Langstein J, Hofstadter F, et al. A soluble form of CD137 (ILA/41BB), a member of the TNF receptor family, is released by activated lymphocytes and is detectable in sera of patients with rheumatoid arthritis. European journal of immunology. 1998;28(1):290-295. 24. Melendreras SG, Martinez-Camblor P, Menendez A, et al. Soluble cosignaling molecules predict long-term graft outcome in kidney-transplanted patients. PloS one. 2014;9(12):e113396. 25. Wensink AC, Hack CE, Bovenschen N. Granzymes regulate proinflammatory cytokine responses. J Immunol. 2015;194(2):491-497. 26. van de Wetering J, Koumoutsakos P, Peeters A, et al. Discontinuation of calcineurin inhibitors treatment allows the development of FOXP3+ regulatory T-cells in patients after kidney transplantation. Clinical transplantation. 2011;25(1):40-46. 27. Webster MJ, Herman MM, Kleinman JE, et al. BDNF and trkB mRNA expression in the hippocampus and temporal cortex during the human lifespan. Gene expression patterns : GEP. 2006;6(8):941-951. 28. Kingsbury TJ, Bambrick LL, Roby CD, et al. Calcineurin activity is required for depolarization-induced, CREB-dependent gene transcription in cortical neurons. Journal of neurochemistry. 2007;103(2):761-770. 29. Vashishta A, Habas A, Pruunsild P, et al. Nuclear factor of activated T-cells isoform c4 (NFATc4/NFAT3) as a mediator of antiapoptotic transcription in NMDA receptor-stimulated cortical neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2009;29(48):15331-15340. 30. Friedman BH, Wolf JH, Wang L, et al. Serum cytokine profiles associated with early allograft dysfunction in patients undergoing liver transplantation. Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society. 2012;18(2):166-176. 31. Louboutin JP, Strayer DS. Relationship between the chemokine receptor CCR5 and microglia in neurological disorders: consequences of targeting CCR5 on neuroinflammation, neuronal death and regeneration in a model of epilepsy. CNS & neurological disorders drug targets. 2013;12(6):815-829.

Journal Pre-proof Table 4 Correlation analysis for all patients (CNI free, CNI treated n=82)

CsA total dose

Tacrolimu s total dose

RANTES

Granzym eA

sFas

Pvalu e n r Pvalu e n r Pvalu e n r Pvalu e n

81 -0.158 0,159

0.842 0.001

81 0.407 0.001

81 0.075 0.505

81 0.212 0.057

0.165 0.141

81 0.093 0.524

81 -0.054 0.714

81 0.105 0.476

81 0.145 0.326

49 0.111 0.447

49 -0.171 0.241

48 0.189 0.199

49 0.102 0.556

49 -0.347

Tacrolimu s mean trough level

Tacrolimu s total dose

of

81 0.386 0.001

CsA total dose

ro

-0.161 0.152

CsA mean troug h level

-p

0.504 0.001

WMH total

0.399 0.005

re

CsA mean trough level

VWC S

48 0.228 0.120

48 0.216 0.141

48 0.008 0.962

48 0.220

0.038

48 0.037 0.832

36 0.027

36 0.047

36 0.144

36 0.059

0.807

0.677

0.200

82 0.373 0.001

82 0.077

lP

WMH total

VWC N

0.631 0.001

49 0.300 0.624

0.400 0.505

0.626

5 0.291

5 0.542

35 0.107

-0.046

0.602

36 0.054 0.635

0.042

0.001

0.541

0.792

81 0.133 0.236

81 0.058 0.606

49 0.115

36 0.483

0.431

49 0.039 0.788

35 0.277

0.494

81 0.147 0.189

0.108

0.003

82 0.070

82 0.017

81 0.030 0.793

49 0.444 0.001

49 0.222 0.125

36 0.099

0.880

81 0.116 0.302

35 -0.245

0.530

81 0.030 0.793

0.156

0.566

82

82

81

81

81

49

49

35

36

na

VWSC

r Pvalu e n r Pvalu e n r Pvalu e n r Pvalu e n r Pvalu e n r

RBAN S Total Scale

Jo ur

VWCN

age

0.198

0.001

Journal Pre-proof The results only for significant correlations are shown. r, correlation coefficient; n, number of patients; VWCN, Ventricular width at the level of the caudate nucleus; VWSC, Ventricular width at the level of the semioval centre; WMH, white matter hyperintensities; CsA, Cyclosporine A; RBANS, Repeatable Battery for the Assessment of Neuropsychological Status; RANTES, regulated on activation normal T-cell expressed and secreted; P-values are two-sided.

Highlights Calcineurininhibitors (CNI) may induce long term neurological side effects



After liver transplantation CNI influence the level of T- cell mediated cytokines

of



-p

CNI suppress BDNF and PDGF expression, both crucial for neuronal signaling

na

lP

re

and synaptic plasticity

Jo ur



ro

and brain derived cytokines

Figure 1

Figure 2

Figure 3

Figure 4