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Table 1 Blood-based diagnostic biomarkers of inflammation in ALS

From: Blood-based biomarkers of inflammation in amyotrophic lateral sclerosis

Measured biomarker(s)

Tissue and detection method

Mean disease duration at sample donation

Number of samples

Potential Diagnostic Value

Study Reference Clinical Trial ID

Cell populations

 Monocyte subpopulations

Monocyte isolation kit from peripheral blood, and flow cytometry

Not reported

n = 68 ALS (1st cohort)

n = 55 controls (1st cohort)

n = 100 ALS (2nd cohort)

n = 60 controls (2nd cohort)

- CD14−/low/CD16+ monocytes decreased in ALS

Beers et al., 2020 [26]

 Subpopulations of T cells, B cells, natural killer cells, and antigen presenting cells

Peripheral blood and FACS

2.48 years

n = 73 ALS

n = 48 controls

- increased Th1 and Th17 cells in ALS

- decreased Th2 and Treg cells in ALS

- increased NK cells and monocytes in ALS

Jin et al., 2020 [27]

 neutrophil-to-lymphocyte ratio (NLR)

From whole blood

Not reported

n = 80 ALS

n = 80 matched controls(n = 41 ALS and n = 41 matched controls at follow-up 3-6 m after initial donation; n = 22 ALS patients and n = 22 matched controls at the third donation at least 3 months after the second)

- NLR was consistently elevated in ALS samples

Keizman et al., 2009 [28]

Candidate-based soluble factor analysis

 IL-2, IL-6, IL-10, IFN-γ, and TNF

Plasma, BioPlex

21.37 months (median)

n = 79 ALS

n = 79 controls

- all measured cytokines were increased in ALS

Tortelli et al., 2020 [29]

 CD14, LBP and CRP

Serum; ELISAs

Not reported

n = 68 ALS (1st cohort)

n = 55 controls (1st cohort)

n = 100 ALS (2nd cohort)

n = 60 controls (2nd cohort)

- soluble CD14 increased in ALS (both cohorts)

- LBP increased in ALS (both cohorts)

- CRP increased in ALS (both cohorts)

Beers et al., 2020 [26]

 IL-1b, IL-6, IL-10, IL-12, TNF,IFN-γ, IL17a, and IL-23

Serum; ELISAs

2.48 years

n = 73 ALS

n = 48 controls

- increased IL-1b, IL-6, and IFN-γ in ALS

- decreased IL-10 in ALS

- no difference in TNF, IL-12, IL-17a, and IL-23

Jin et al., 2020 [27]

 CD5L, Ficolin-3

Plasma; ELISAs

739.9 months (median)

n = 37 ALS

n = 30 controls

CD5L and Ficolin-3 are increased in ALS

Mohanty et al., 2020 [30]

 IL-2, IL-1b, TNF, IFN-γ and IL-4

Serum; ELISAs

40.4 months

n = 35 ALS

n = 30 controls

- increased IL-4 and IL-1b in ALS

- decreased IFN-γ in ALS

- no difference in IL-2 and TNF

Polverino et al., 2020 [31]

 MCP-1, eotaxin-1, IL-18, TNF, CRP, IL-1, sTREM2

Plasma; MSD assay

Not reported

n = 108 ALS

n = 41 controls

(n = 85 ALS with samples 2+ visits)

- MCP-1 and IL-18 are increased in ALS

- sTREM2 is increased in ALS

Huang et a;., 2020 [32] NCT01495390

 IL-6

Plasma; Chemokine assay

25.7 months

n = 82 ALS

n = 43 controls

Not upregulated in ALS (trend)

Pronto-Laborinho et al., 2019 [33]

 IP-10, MCP-1, MIG, RANTES, IL-2, IL-4, IL-6, IL-8, IL-10, IL-17a, TNF, IGF-g, sTNFR1, sTNFR2

Plasma; cytometric bead array and ELISA

3 years

n = 68 ALS

n = 62 controls over the first time point.

n = 24 ALS at the second time point (6–12 months later)

IL-6 + IL-8: upregulated in ALS

IL-2 (low) and IL-6 (high) predict ALS diagnosis

Prado et al., 2018 [34]

 Gene expression of 45 genes

Serum; individual RT-qPCRs

Within the first 2 months of diagnosis

n = 22 sALS

n = 13 controls

- ITGB2, INPP5D, SELL, ICAM1, MMP9 and TIMP2 are upregulated in ALS

- CCL5, CXC5R, IL10, TGFB2, IL10RA, IL-6, CD2 and TRBC1 are downregulated in ALS

Andres-Benito et al., 2017 [35]

 Gene expression of 37 brain-enriched and inflammation-associated microRNAs

Plasma; individual RT-qPCRs

1.8 years

n = 50 ALS

n = 50 FTD

n = 50 AD

n = 50 PD

n = 50 controls

miR-206/miR-31 and miR-206/ miR-125b and miR-99/ miR-338-3p most effectively differentiate between ALS and control

Sheinerman et al., 2017 [36]

 CC-16

Plasma; ELISA

27 months

n = 81 ALS

n = 30 controls

Upregulated in ALS

Pronto-Laborinho et al., 2017 [37]

 TNF, MCP-1, IL6, IL8, IL2, IFN-γ, IL1-beta, IL10, IL4, IL5, IL17, TNFR1, ELAM-1

Plasma; different per individual dataset (25 studies)

different per individual dataset (25 studies)

pooled n = 812 ALS

pooled n = 639 controls

TNF, TNFR-1, IL-6, IL-1β, and IL-8 levels were elevated in ALS.

Hu et al., 2017

 

[38]

 TNF, IL-8, IL-6, IL-10

Serum; multiplex assay

Not reported

n = 19 ALS

n = 10 controls

- IL-6 was increased in ALS.

- IL-8 was increased in ALS.

Blasco et al., 2016 [39]

 IL-1β, IL-18, IL-33, IL-37, IL-1Ra, sIL-1R2, IL-18BP, sIL-1R4

Serum; individual ELISAs

11.32 months

n = 144 sALS

n = 40 controls

- IL-18 was increased in ALS.

- IL-18BP was increased in ALS.

Italiani et al., 2014 [40]

 IL-33, soluble ST2

Serum; individual ELISAs

Not reported

n = 42 ALS

n = 38 controls

- IL-33 was increased in ALS

- soluble ST2 was decreased in ALS

Lin et al., 2012 [41]

 IL-17A

Serum; individual ELISA

23.4 months

n = 32 ALS

n = 14 controls

IL-17A was increased in ALS.

Fiala et al., 2010 [42]

 kynurenine pathway (tryptophan, picolinic acid)

Serum; HPLC, gas chromatography mass spectrometry

Not reported

n = 140 ALS

n = 35 controls

- TRP and KYN is increased ALS

- PIC is decreased in ALS

Chen et al., 2010 [43]

 eotaxin, eotaxin-3, IL-8, IP-10, MCP-1, MCP-4, MDC, MIP-1b, TARC

Serum; solid-phase sandwich immuno-assay

Not reported

n = 20 ALS

n = 20 controls (non-ALS neurologic)

No differences.

Kuhle et al., 2009 [44]

 TNF, IFN-γ, and NO

Serum; individual ELISAs and NO by determining nitrite and nitrate levels

12 months

n = 22 ALS

n = 20 controls

TNF, IFN-γ, and NO were all increased in ALS

Babu et al., 2008 [45]

 RANTES

Serum; individual ELISA

Not reported

n = 20 ALS

n = 14 NIND

n = 13 controls

RANTES was increased in ALS serum

Rentzos et al., 2008 [46]

 MCP-1

Serum; individual ELISA

19.4 months

n = 27 ALS

n = 30 NIND

MCP-1 was increased in ALS

Baron et al., 2005 [47]

 MCP-1

Serum; individual ELISA

8 months (median)

n = 29 ALS

n = 11 controls

MCP-1 was not altered in ALS

Wilms et al., 2005

[48]

 wide-range C-reactive protein (wrCRP)

From whole blood

Not reported

n = 80 ALS

n = 80 matched controls

- wrCRP was consistently elevated in ALS samples

Keizman et al., 2009 [28]

Unbiased analyses of serum or plasma

 miRNA gene expression

Plasma; next generation sequencing on neural-enriched extracellular vesicles

Not reported

n = 10 + 10 (replication set) ALS

n = 10 + 10 (replication set) controls

Eight miRNAs were differentially expressed between ALS samples and controls after replication. This included miR-146a-5p, which was upregulated in ALS samples and is associated with inflammation (monocytes) [49].

Banack et al.;. 2020 [50]Some samples from NCT03580616.

 Protein abundance detectable by mass spectrometry

Plasma; mass spectrometry

747 months (median)

n = 42 ALS

n = 18 controls

30 proteins are differentially detected between ALS and controls. IPA analysis identified two networks of interacting proteins that differ between ALS and controls; IL-1 and NFkB.

Xu et al., 2018 [51]

Blood-derived cells, in vitro assays

 Transcriptomic analysis

Gene expression of blood monocyte-derived macrophages, by RNAseq and RT-qPCR

Not reported

n = 5 controls

n = 5 sALS

n = 5 C9-ALS

- Increased type I interferon signature (pathway analysis)

- increased gene expression of MX1, OASL, OAS2, IF44L

McCauley et al., 2020 [52]

 Cell surface expression of VLA4, TLR4, CXCR3, CCR5, CXCR4, IFN-γ, CCR2, CD11B

Flow cytometry on blood-isolated T-cells, B-cells, monocytes, and NK cells

Not reported

n = 10 ALS

n = 10 controls

- CXCR4, CXCR3, CCR2, CCR5 increased on ALS T-cells.

- CD11B, CCR2 decreased on ALS monocytes

- The combination of the analyzed markers could significantly predict the categorization into ALS or healthy donors, with CXCR3 and CCR5 on T cells comprising the strongest predictors.

Perner et al., 2018 [53]

 Number of migrating cells

Boyden chamber. All cells migrated to the lower well after 2.5 h were stained using lineage antibody and counted by flow cytometry.

Not reported

n = 10 ALS

n = 10 controls

More ALS CD45+ cells chemotaxis with IP-10 chemoattractant.

Perner et al., 2018 [53]

 Frequency of myeloid dendritic cells

Flow cytometry (CD1chighCD19−)

Not calculated

n = 20 ALS

n = 10 healthy donors

- Less circulating myeloid dendritic cells in ALS.

- Increased CD62L expression on circulating myeloid dendritic cells in ALS.

Rusconi et al., 2017 [54]

 Concentrations of TNF, IL-1β, IL-6, IL-12p40, IL-8, CCL2 and IL-10 in BDCA1+ DC supernatants

Circulating myeloid dendritic (CD1chigh) cells stimulated with LPS.

Not calculated

n = 52 ALS

n = 36 healthy donors

n = 25 other neurological controls.

Higher levels of IL-8 and CCL-2 upon LPS- stimulation in ALS dendritic cells

Rusconi et al., 2017 [54]

 116 leukocyte populations and phenotypes from lymphocytes, monocytes, and granulocytes

Peripheral blood immunophenotyping by flow cytometry

21.6 months

n = 80 ALS

n = 50 controls

- 32 leukocyte phenotypes altered in ALS

- elevated cell counts of granulocytes, NK cells and T cells in ALS

- ALS patients were clustered into a profile distinct from controls primarily due to differences in multiple T cell phenotypes, CD3 CD56 T cells and HLA-DR on monocytes.

Gustafson et al., 2017 [55]

 Transcriptomic analysis

RNA sequencing of blood monocytes

Not reported

n = 43 ALS

n = 22 controls

ALS monocytes demonstrated a unique inflammation-related gene expression profile, the most prominent of which, including IL1B, IL8, FOSB, CXCL1, and CXCL2

Zhao et al., 2017 [56]

 IL1-b, IL-6, IL-8, IL-10, GM-CSF, and TNF and TGF-b1, −b2, and -b3

By Luminex xMAP on supernatants from PBMCs or macrophages cultured overnight (non-stimulated, and SOD1-stimulated)

Not reported

1 discordant twin pair

- In non-stimulated conditions the supernatants from the ALS PBMCs increased IL-6, TNF, and IL-1.

Lam et al., 2016 [57]

 Immune and cytokine profiling

freshly collected, un-stimulated cells by flow cytometry, on peripheral monocytes and T lymphocytes.

2.4 years

n = 24 ALS

n = 25 controls

Th1-, Th17-, and IL-6-driven inflammation increased in ALS.

Saresella et al., 2013 [58]

 90 inflammatory genes

qPCR analysis from isolated PBMCs

25.4 months

n = 10 ALS

n = 4 controls

- 50% of the ALS patients had ‘strong inflammation’ (upregulation of IL-1, IL-6, IL23a, PTGS2, MMP1, CCL20, CXCL3, CXCL5 and CXCR4; downregulation of CXCL9, CXCL10, and CXCL11), the other 50% had ‘weak inflammation’.

- all ALS patients had an ‘ALS signature’ with 4-fold increase of MMP1, CCL7, CCL13 and CCL24.

Fiala et al., 2013 [59] No NCT reported

 IL-1b, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IFN-γ, GM-CSF, TNF

PBMC cultures, non-stimulated and stimulated with SOD1 protein, supernatants analyzed by R&D Systems High Sensitivity Human Inflammation Multiplex-Kit

26.9 months

n = 8 sALS

n = 4 controls

n = 1 unaffected twin of sALS patient

- 4 sALS patients had increased expression of TLR2 and CD14; ALOX5, PTGS2 and MMP1; IL1α, IL1β, IL6, IL36G, IL8 and TNF; CCL3, CCL20, CXCL2, CXCL3 and CXCL5.

- 4 sALS patients had a decrease in the expression of PPARG, PPARA, RARG, HDAC4 and KAT2B; IL6R, IL6ST and ADAM17; TNFRSF11A; MGAT2 and MGAT3; PLCG1; CXCL3.

- Difference identified between rapid ALS and slow ALS or controls. No diff between slow ALS and controls.

Mizwicki et al., 2012 [60]

 monocyte and lymphocyte populations and activation

Surface expression, measured by flow cytometry from monocytes isolated from whole blood

4–93 months (range)

n = 38 sALS

n = 28 controls

- increased percentage of CD4+ cells in ALS

- increased mean CD14-HLD-DR expression in ALS

- increased percentage of CD14 and CD16+ cells in ALS

- increased serum IgG in ALS

- decreased serum IgM in ALS

Zhang et al., 2005 [61]

  1. AD Alzheimer’s Disease, ALS amyotrophic lateral sclerosis, ALSFRS-R ALS functional rating scale revised, C9-ALS ALS due to the harboring of the C9orf72 hexanucleotide repeat expansion, CC-16 club cell protein 16, CD14 cluster differentiation 14, CD5L cluster differentiation 5 ligand, CMAP compound muscle action potential, CRP c reactive protein, DC dendritic cells, ELISA enzyme-linked immunosorbent assay, FACS Fluorescence-activated cell sorting, FTD frontal temporal dementia, FoxP3 Forkhead Box P3, FVC forced vital capacity, HLA-DR Human Leukocyte Antigen – DR isotype, HPLC High Performance Liquid Chromatography, IFN-γ interferon gamma, IGF-g Insulin-like growth factor gamma, IgG Immunoglobulin G, IgM Immunoglobulin M, IL interleukin, IL-1RA interleukin 1 receptor agonist, IL-18BP interleukin 18 binding protein, IP-10 Interferon gamma-induced protein 10, LBP Lipopolysaccharide binding protein, LPS lipopolysaccharide, MIP-1β Macrophage inflammatory protein 1 beta, MCP-1 Monocyte Chemoattractant Protein 1, MIG monokine induced by gamma interferon, MMP Matrix Metalloproteinases, MSD Meso Scale Discovery (multiplexing), NFkB nuclear factor kappa-light-chain-enhancer of activated B cells, NIND non-inflammatory neurological disorder, NK natural killer cells, NLR neutrophil-to-lymphocyte ratio, NO nitric oxide, PBMC peripheral blood mononuclear cell, PD Parkinson’s Disease, RANTES Regulated upon Activation, Normal T Cell Expressed and Presumably Secreted, NA ribonucleic acid,
  2. RT-qPCR real time quantitative PCR, sALS sporadic amyotrophic lateral sclerosis, SOD1 super oxide dismutase 1, sTNFR soluble TNF receptor, sTREM2 soluble Triggering Receptor Expressed On Myeloid Cells 2, TNF tumor necrosis factor, TNFR tumor necrosis factor receptor, Tregs T regulatory cells, wrCRP wide-range c reactive protein