Accepted Manuscript Multiplex Analyte Assays to Characterise Different Dementias: Brain Inflammatory Cytokines in Post-Stroke and Other Dementias Aiqing Chen, Arthur E. Oakley, Maria Monteiro, Katri Tuomela, Louise M. Allan, Elizabeta B. Mukaetova-Ladinska, John T. O’Brien, Raj N. Kalaria PII:
S0197-4580(15)00533-3
DOI:
10.1016/j.neurobiolaging.2015.10.021
Reference:
NBA 9421
To appear in:
Neurobiology of Aging
Received Date: 27 May 2015 Revised Date:
20 October 2015
Accepted Date: 24 October 2015
Please cite this article as: Chen, A., Oakley, A.E., Monteiro, M., Tuomela, K., Allan, L.M., MukaetovaLadinska, E.B., O’Brien, J.T., Kalaria, R.N., Multiplex Analyte Assays to Characterise Different Dementias: Brain Inflammatory Cytokines in Post-Stroke and Other Dementias, Neurobiology of Aging (2015), doi: 10.1016/j.neurobiolaging.2015.10.021. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Chen A et al, Brain Cytokines in Dementia, NBA
ACCEPTED MANUSCRIPT
Multiplex Analyte Assays to Characterise Different Dementias: Brain Inflammatory Cytokines in Post-Stroke and Other Dementias
Aiqing Chen, Arthur E. Oakley, Maria Monteiro, Katri Tuomela, Louise M. Allan, Elizabeta
RI PT
B. Mukaetova-Ladinska, John T. O’Brien, Raj N. Kalaria† Neurovascular Research Group, Institute of Neuroscience, Newcastle University, Campus for Ageing & Vitality, Newcastle Upon Tyne NE4 5PL and Department of Psychiatry,
University of Cambridge School of Clinical Medicine, Cambridge Biomedical Campus,
SC
Cambridge CB2 0SP, United Kingdom
M AN U
Running Title: Interleukins in Dementia
Correspondence:
† Professor RN Kalaria or Dr Aiqing Chen
Newcastle University
TE D
Institute of Neuroscience
Campus for Ageing & Vitality
Newcastle upon Tyne, NE4 5PL, United Kingdom
EP
Tel: 0191 248 1352; Fax: 0191 248 1301 E-mail: [email protected]
AC C
[email protected]
Word count: Title: 111 characters; Abstract: 350 words; text: 6,105 words (28 pages); Tables: 2 Figures: 3; Supplementary Tables 2; References: 53; Total ms: 8,040 words.
1
Chen A et al, Brain Cytokines in Dementia, NBA
ACCEPTED MANUSCRIPT
Abstract Both the inflammatory potential and cognitive function decline during ageing. The association between the repertoire of inflammatory biomarkers and cognitive decline is unclear. Inflammatory cytokines have been reported to be increased, decreased, or unchanged
RI PT
in the cerebrospinal fluid and sera of subjects with dementia. We assessed 112 post-mortem brains from subjects diagnosed with post-stroke dementia (PSD), vascular dementia, mixed dementia and Alzheimer’s Disease (AD), comparing those to post-stroke non-demented
(PSND) subjects and age-matched controls. We analysed 5 brain regions including the grey
SC
and white matter from the frontal and temporal lobes for a panel of cytokine/chemokine
analytes using multiplex-array assays. Out of the 37 analytes, 14 were under or near the detection limits, 7 were close to the lowest detection level and 16 cytokines were within the
M AN U
linear range of the assay. We observed widely variable concentrations of C-reactive protein (CRP) and serum amyloid A (SAA) at the high end (1-150 ng/mg protein), whereas several of the interleukins (IL, interferon-gamma and tumour necrosis factor) at the low end (1-10 pg/mg). There were also regional variations; most notable being high concentrations of some cytokines (e.g. CRP and angiogenesis panel) in the frontal white matter. Overall, we found
TE D
decreased concentrations of several cytokines, including IL-1 beta (P=0.000), IL-6 (P=0.000), IL-7 (P=0.000), IL-8 (P=0.000), IL-16 (P=0.001), Interferon inducible protein (IP)-10 (0.044), SAA (P=0.011) and a trend in IL-1 alpha (P=0.084) across all dementia groups compared to non-demented controls. IL-6 and IL-8 were significantly lower in dementia
EP
subjects than non-demented subjects in every region. In particular, lower levels of IL-6 and IL-8 were notable in the PSD compared to PSND subjects. Since these two stroke groups had
AC C
comparable degree of vascular pathology, the lower production of IL-6 and IL-8 in PSD reaffirms a possible specific involvement of immunosenescence in dementia pathogenesis. In contrast, CRP was not altered between dementia and non-dementia subjects or between PSD and PSND. Our study provides evidence not only for the feasibility of tracking cytokines in post-mortem brain tissue but also suggests differentially impaired inflammatory mechanisms underlying dementia including AD. There was a diminished inflammatory response, possibly reflecting immunosenescence and cerebral atrophy, in all dementias. Strategies to enhance anti-inflammatory cytokines and boost the immune system of the brain may be beneficial for preventing cognitive dysfunction , especially after stroke.
2
Chen A et al, Brain Cytokines in Dementia, NBA
ACCEPTED MANUSCRIPT
Key words: Ageing, cognitive impairment, immunosenescence, inflammation, post-stroke
AC C
EP
TE D
M AN U
SC
RI PT
dementia, stroke, vascular dementia, white matter
3
Chen A et al, Brain Cytokines in Dementia, NBA
ACCEPTED MANUSCRIPT Introduction
During the last decade, there has been a surge of interest in whether inflammatory and immune responses contribute to ageing-related dementias. Both the inflammatory potential and the immune system may decline in tandem with cognitive function during ageing. However, it has been difficult to draw conclusions whether inflammation is a cause, a
RI PT
promoter, or simply a secondary phenomenon in dementing illness. A large number of studies focussed on Alzheimer’s disease (AD), as the most common form of dementia, indicate a direct role for inflammatory and immune processes in its pathogenesis [37,42,43,46,52,54].
dementias including vascular or stroke related dementias.
SC
However, much less is known on the modulation of inflammatory responses in other
The inflammatory response of the body is a dynamic process and the profile of
M AN U
cytokine responses may differ with the duration and severity of an illness [22,23,25]. Cytokines in blood and cerebrospinal fluid (CSF) have been reported to be increased, decreased, or unaltered in AD, vascular dementia (VaD) and ischemic stroke survivors [2,4,36,42,44,47,55]. Few population-based studies have shown that high levels of interleukin (IL) IL-6, IL-8 and C-reactive protein (CRP) are associated with poor cognitive
TE D
status , including poor performance in memory and processing speed, as well as cognitive decline [3,16,52,56]. In ischemic stroke, higher serum interleukins were associated with baseline cognitive impairment (IL-8) and subsequent cognitive decline (IL-12) [36]. Clinical trials have generally failed to demonstrate a robust beneficial effect of anti-inflammatory
EP
drugs in the progression of AD [20], implicating a complicated role of inflammation in the pathogenesis. In contrast, while most trials were negative in ischemic stroke injury, phase 2 trials with IL-1 receptor antagonist and the board spectrum anti-inflammatory agent
AC C
minocycline demonstrated improved outcomes [45]. The immune system is also a key player in central nervous system repair and
maintenance that undergoes a profound remodelling process over the lifetime, and has a major impact on individual’s health and survival [5,14,17]. Immunosenescence, or the aging immune system, is a constellation of age-related changes to the immune system, resulting in greater susceptibility to infection and reduced responses to infectious pathogen(s). The characteristics of immunosenescence include age-induced thymic atrophy, bone marrow decreased hematopoietic compartment and increased peripheral suppressor cell activity. It affects both innate and adaptive immune systems [17]. 4
Chen A et al, Brain Cytokines in Dementia, NBA
ACCEPTED MANUSCRIPT
Our aim was to investigate inflammatory cytokine profiles in extracts of post-mortem brain tissues from subjects with different dementias using multiplex immunoarrays. In addition to post-stroke demented (PSD) and post-stroke non-demented (PSND) subjects, we assessed brains from prospectively assessed dementia subjects diagnosed with VaD, mixed dementia and AD and from age-matched controls. With emphasis on PSD, we tested the
RI PT
hypothesis that different dementias have distinct profiles of cytokines compared to those without dementia.
SC
Methods: Subjects
M AN U
Brain tissues from a total of 112 subjects, including 21 post-stroke non-demented (PSND) and 20 post-stroke demented (PSD), 17 VaD, 18 mixed dementia, and 16 AD, were obtained from the Newcastle Brain Tissue Resources (NBTR). In addition, we analysed brain tissue from similar age controls (Table 1). For the post-stroke subjects, final MMSE and the highest and last revised Cambridge Cognition Examination (CAMCOG) battery scores of the relevant subjects were used to determine the cognitive profile of analysed subjects. Thus,
TE D
stroke survivors who did not meet DSM-IIIR or IV criteria for dementia and had MMSE scores >25 and CAMCOG scores >85, were designated as post-stroke survivors with no dementia. In the majority of cases, bronchopneumonia was recorded as the cause of death.
EP
Neuropathological assessment was carried out as described previously, using standardised protocols [1,24,27]. Macroscopical infarcts, detected by visual inspection while
AC C
dissecting the brain, were subsequently confirmed by light microscopy. Haematoxylin-eosin staining was used for assessment of structural integrity and infarcts, Nissl and luxol fast blue staining for cellular patterns and myelin loss, Bielschowsky’s silver impregnation and amyloid β for CERAD/Thal rating of neuritic plaques, Gallays for neuritic pathology, and tau immunohistochemistry for Braak staging of neurofibrillary tangles. Vascular pathology scores were determined in PSND and PSD cases and controls as described previously [9]. Pathological diagnosis of AD was consistent with the National Institute on AgingAlzheimer's Association guidelines for the neuropathological assessment of AD [34]. VaD was diagnosed as described previously [27]. In PSD cases, a definite diagnosis of VaD was made when there were multiple or cystic infarcts, lacunae, microinfarcts and small vessel 5
Chen A et al, Brain Cytokines in Dementia, NBA
ACCEPTED MANUSCRIPT
disease, and Braak stage
S0197-4580(15)00533-3
DOI:
10.1016/j.neurobiolaging.2015.10.021
Reference:
NBA 9421
To appear in:
Neurobiology of Aging
Received Date: 27 May 2015 Revised Date:
20 October 2015
Accepted Date: 24 October 2015
Please cite this article as: Chen, A., Oakley, A.E., Monteiro, M., Tuomela, K., Allan, L.M., MukaetovaLadinska, E.B., O’Brien, J.T., Kalaria, R.N., Multiplex Analyte Assays to Characterise Different Dementias: Brain Inflammatory Cytokines in Post-Stroke and Other Dementias, Neurobiology of Aging (2015), doi: 10.1016/j.neurobiolaging.2015.10.021. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Chen A et al, Brain Cytokines in Dementia, NBA
ACCEPTED MANUSCRIPT
Multiplex Analyte Assays to Characterise Different Dementias: Brain Inflammatory Cytokines in Post-Stroke and Other Dementias
Aiqing Chen, Arthur E. Oakley, Maria Monteiro, Katri Tuomela, Louise M. Allan, Elizabeta
RI PT
B. Mukaetova-Ladinska, John T. O’Brien, Raj N. Kalaria† Neurovascular Research Group, Institute of Neuroscience, Newcastle University, Campus for Ageing & Vitality, Newcastle Upon Tyne NE4 5PL and Department of Psychiatry,
University of Cambridge School of Clinical Medicine, Cambridge Biomedical Campus,
SC
Cambridge CB2 0SP, United Kingdom
M AN U
Running Title: Interleukins in Dementia
Correspondence:
† Professor RN Kalaria or Dr Aiqing Chen
Newcastle University
TE D
Institute of Neuroscience
Campus for Ageing & Vitality
Newcastle upon Tyne, NE4 5PL, United Kingdom
EP
Tel: 0191 248 1352; Fax: 0191 248 1301 E-mail: [email protected]
AC C
[email protected]
Word count: Title: 111 characters; Abstract: 350 words; text: 6,105 words (28 pages); Tables: 2 Figures: 3; Supplementary Tables 2; References: 53; Total ms: 8,040 words.
1
Chen A et al, Brain Cytokines in Dementia, NBA
ACCEPTED MANUSCRIPT
Abstract Both the inflammatory potential and cognitive function decline during ageing. The association between the repertoire of inflammatory biomarkers and cognitive decline is unclear. Inflammatory cytokines have been reported to be increased, decreased, or unchanged
RI PT
in the cerebrospinal fluid and sera of subjects with dementia. We assessed 112 post-mortem brains from subjects diagnosed with post-stroke dementia (PSD), vascular dementia, mixed dementia and Alzheimer’s Disease (AD), comparing those to post-stroke non-demented
(PSND) subjects and age-matched controls. We analysed 5 brain regions including the grey
SC
and white matter from the frontal and temporal lobes for a panel of cytokine/chemokine
analytes using multiplex-array assays. Out of the 37 analytes, 14 were under or near the detection limits, 7 were close to the lowest detection level and 16 cytokines were within the
M AN U
linear range of the assay. We observed widely variable concentrations of C-reactive protein (CRP) and serum amyloid A (SAA) at the high end (1-150 ng/mg protein), whereas several of the interleukins (IL, interferon-gamma and tumour necrosis factor) at the low end (1-10 pg/mg). There were also regional variations; most notable being high concentrations of some cytokines (e.g. CRP and angiogenesis panel) in the frontal white matter. Overall, we found
TE D
decreased concentrations of several cytokines, including IL-1 beta (P=0.000), IL-6 (P=0.000), IL-7 (P=0.000), IL-8 (P=0.000), IL-16 (P=0.001), Interferon inducible protein (IP)-10 (0.044), SAA (P=0.011) and a trend in IL-1 alpha (P=0.084) across all dementia groups compared to non-demented controls. IL-6 and IL-8 were significantly lower in dementia
EP
subjects than non-demented subjects in every region. In particular, lower levels of IL-6 and IL-8 were notable in the PSD compared to PSND subjects. Since these two stroke groups had
AC C
comparable degree of vascular pathology, the lower production of IL-6 and IL-8 in PSD reaffirms a possible specific involvement of immunosenescence in dementia pathogenesis. In contrast, CRP was not altered between dementia and non-dementia subjects or between PSD and PSND. Our study provides evidence not only for the feasibility of tracking cytokines in post-mortem brain tissue but also suggests differentially impaired inflammatory mechanisms underlying dementia including AD. There was a diminished inflammatory response, possibly reflecting immunosenescence and cerebral atrophy, in all dementias. Strategies to enhance anti-inflammatory cytokines and boost the immune system of the brain may be beneficial for preventing cognitive dysfunction , especially after stroke.
2
Chen A et al, Brain Cytokines in Dementia, NBA
ACCEPTED MANUSCRIPT
Key words: Ageing, cognitive impairment, immunosenescence, inflammation, post-stroke
AC C
EP
TE D
M AN U
SC
RI PT
dementia, stroke, vascular dementia, white matter
3
Chen A et al, Brain Cytokines in Dementia, NBA
ACCEPTED MANUSCRIPT Introduction
During the last decade, there has been a surge of interest in whether inflammatory and immune responses contribute to ageing-related dementias. Both the inflammatory potential and the immune system may decline in tandem with cognitive function during ageing. However, it has been difficult to draw conclusions whether inflammation is a cause, a
RI PT
promoter, or simply a secondary phenomenon in dementing illness. A large number of studies focussed on Alzheimer’s disease (AD), as the most common form of dementia, indicate a direct role for inflammatory and immune processes in its pathogenesis [37,42,43,46,52,54].
dementias including vascular or stroke related dementias.
SC
However, much less is known on the modulation of inflammatory responses in other
The inflammatory response of the body is a dynamic process and the profile of
M AN U
cytokine responses may differ with the duration and severity of an illness [22,23,25]. Cytokines in blood and cerebrospinal fluid (CSF) have been reported to be increased, decreased, or unaltered in AD, vascular dementia (VaD) and ischemic stroke survivors [2,4,36,42,44,47,55]. Few population-based studies have shown that high levels of interleukin (IL) IL-6, IL-8 and C-reactive protein (CRP) are associated with poor cognitive
TE D
status , including poor performance in memory and processing speed, as well as cognitive decline [3,16,52,56]. In ischemic stroke, higher serum interleukins were associated with baseline cognitive impairment (IL-8) and subsequent cognitive decline (IL-12) [36]. Clinical trials have generally failed to demonstrate a robust beneficial effect of anti-inflammatory
EP
drugs in the progression of AD [20], implicating a complicated role of inflammation in the pathogenesis. In contrast, while most trials were negative in ischemic stroke injury, phase 2 trials with IL-1 receptor antagonist and the board spectrum anti-inflammatory agent
AC C
minocycline demonstrated improved outcomes [45]. The immune system is also a key player in central nervous system repair and
maintenance that undergoes a profound remodelling process over the lifetime, and has a major impact on individual’s health and survival [5,14,17]. Immunosenescence, or the aging immune system, is a constellation of age-related changes to the immune system, resulting in greater susceptibility to infection and reduced responses to infectious pathogen(s). The characteristics of immunosenescence include age-induced thymic atrophy, bone marrow decreased hematopoietic compartment and increased peripheral suppressor cell activity. It affects both innate and adaptive immune systems [17]. 4
Chen A et al, Brain Cytokines in Dementia, NBA
ACCEPTED MANUSCRIPT
Our aim was to investigate inflammatory cytokine profiles in extracts of post-mortem brain tissues from subjects with different dementias using multiplex immunoarrays. In addition to post-stroke demented (PSD) and post-stroke non-demented (PSND) subjects, we assessed brains from prospectively assessed dementia subjects diagnosed with VaD, mixed dementia and AD and from age-matched controls. With emphasis on PSD, we tested the
RI PT
hypothesis that different dementias have distinct profiles of cytokines compared to those without dementia.
SC
Methods: Subjects
M AN U
Brain tissues from a total of 112 subjects, including 21 post-stroke non-demented (PSND) and 20 post-stroke demented (PSD), 17 VaD, 18 mixed dementia, and 16 AD, were obtained from the Newcastle Brain Tissue Resources (NBTR). In addition, we analysed brain tissue from similar age controls (Table 1). For the post-stroke subjects, final MMSE and the highest and last revised Cambridge Cognition Examination (CAMCOG) battery scores of the relevant subjects were used to determine the cognitive profile of analysed subjects. Thus,
TE D
stroke survivors who did not meet DSM-IIIR or IV criteria for dementia and had MMSE scores >25 and CAMCOG scores >85, were designated as post-stroke survivors with no dementia. In the majority of cases, bronchopneumonia was recorded as the cause of death.
EP
Neuropathological assessment was carried out as described previously, using standardised protocols [1,24,27]. Macroscopical infarcts, detected by visual inspection while
AC C
dissecting the brain, were subsequently confirmed by light microscopy. Haematoxylin-eosin staining was used for assessment of structural integrity and infarcts, Nissl and luxol fast blue staining for cellular patterns and myelin loss, Bielschowsky’s silver impregnation and amyloid β for CERAD/Thal rating of neuritic plaques, Gallays for neuritic pathology, and tau immunohistochemistry for Braak staging of neurofibrillary tangles. Vascular pathology scores were determined in PSND and PSD cases and controls as described previously [9]. Pathological diagnosis of AD was consistent with the National Institute on AgingAlzheimer's Association guidelines for the neuropathological assessment of AD [34]. VaD was diagnosed as described previously [27]. In PSD cases, a definite diagnosis of VaD was made when there were multiple or cystic infarcts, lacunae, microinfarcts and small vessel 5
Chen A et al, Brain Cytokines in Dementia, NBA
ACCEPTED MANUSCRIPT
disease, and Braak stage
