Therapy of autoimmune inflammation in sporadic amyotrophic lateral sclerosis: Dimethyl fumarate and H-151 downregulate inflammatory cytokines in the cGAS-STING pathway

Abstract

In sporadic amyotrophic lateral sclerosis (sALS), IL-17A- and granzyme-positive cytotoxic T lymphocytes (CTL), IL-17A-positive mast cells, and inflammatory macrophages invade the brain and spinal cord. In some patients, the disease starts following a trauma or a severe infection. We examined cytokines and cytokine regulators over the disease course and found that, since the early stages, peripheral blood mononuclear cells (PBMC) exhibit increased expression of inflammatory cytokines IL-12A, IFN-γ, and TNF-α, as well as granzymes and the transcription factors STAT3 and STAT4. In later stages, PBMCs upregulated the autoimmunity-associated cytokines IL-23A and IL-17B, and the chemokines CXCL9 and CXCL10, which attract CTL and monocytes into the central nervous system. The inflammation is fueled by the downregulation of IL-10, TGFβ, and the inhibitory T-cell co-receptors CTLA4, LAG3, and PD-1, and, in vitro, by stimulation with the ligand PD-L1. We investigated in two sALS patients the regulation of the macrophage transcriptome by dimethyl fumarate (DMF), a drug approved against multiple sclerosis and psoriasis, and the cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway inhibitor H-151. Both DMF and H-151 downregulated the expression of granzymes and the pro-inflammatory cytokines IL-1β, IL-6, IL-15, IL-23A, and IFN-γ, and induced a pro-resolution macrophage phenotype. The eicosanoid epoxyeicosatrienoic acids (EET) from arachidonic acid was anti-inflammatory in synergy with DMF. H-151 and DMF are thus candidate drugs targeting the inflammation and autoimmunity in sALS via modulation of the NFκB and cGAS/STING pathways.

Keywords: IFNγ; IL-17B; IL-1β; TNFα; H-151; autoimmunity; cGAS-STING pathway; dimethyl fumarate; sporadic amyotrophic lateral sclerosis.

Fig. 1.. IL-17A- and granzyme B-positive CD3 cells invade the ALS spinal cord gray matter.

Immunohistochemistry (A) and immunofluorescence (B, C) demonstrate (A) IL-17A-positive cells (indicated by *) in the gray matter; (B) Granzyme B-positive CD3 cells (red/green); (C) IL-17A-positive CD3 cells (red/green) (sporadic ALS patient #8; reprinted from Fiala, M. et al JNI 2010).

Fig. 2.. Study populations.

Transcriptomic analysis of PBMCs from 1) 4 controls and 8 ALS patients over the disease course, 2) one ALS-discordant pair, or 3) two ALS patients’ PBMCs and macrophages treated in vitro with candidate drug combinations.

Fig. 3.. ALS PBMC transcriptome over the disease course:

A Cytokines and cytokine regulators. The transcriptome of ALS patients is shown over the disease course; the transcriptome of controls is shown at baseline (data on the ALS patients #1 to #8 according to the time after onset). B Transcription factors and granzymes. C Co-receptors

Fig. 3.. ALS PBMC transcriptome over the disease course:

A Cytokines and cytokine regulators. The transcriptome of ALS patients is shown over the disease course; the transcriptome of controls is shown at baseline (data on the ALS patients #1 to #8 according to the time after onset). B Transcription factors and granzymes. C Co-receptors

Fig. 3.. ALS PBMC transcriptome over the disease course:

A Cytokines and cytokine regulators. The transcriptome of ALS patients is shown over the disease course; the transcriptome of controls is shown at baseline (data on the ALS patients #1 to #8 according to the time after onset). B Transcription factors and granzymes. C Co-receptors

Fig. 4.. Natural killer (NK) cell proteome in the twin-ship:

Ratio of the inflammatory proteins in the ALS twin vs. the healthy twin. Note the overexpression of granzymes, RAC, MAPK signaling, cell adhesion, immunophilins, and apoptotic proteins in the ALS twin.

Fig. 5.. Aberrant paradoxical effect of the checkpoint blockade modulation in ALS macrophages.

PDL1 and PD1 are pro-inflammatory, while DMF and PD1 antibody are anti-inflammatory.

Fig. 6.

Dimethyl fumarate (DMF) down-regulates inflammatory cytokines (except TNF), granzymes, and NFκB target genes in macrophages of both ALS patients

Fig. 7.

The STING inhibitor H-151 down-regulates in a concentration-dependent fashion the inflammatory cytokines, granzymes, and the transcription factor NFκB targets in ALS macrophages of the slowly progressing patient #10.

Fig. 8

Biological and treatment replicates

Fig. 9. PCA of DMF and H-151 effects:

The unsupervised analysis shows stronger down regulation of inflammatory chemokines (CXCL9 and CCL15), inflammatory cytokine (IFN), granzymes, and IGFBP5 by H-151 in comparison to DMF.