X-linked adrenoleukodystrophy is caused by ATP-binding cassette transporter D1 (ABCD1) mutations and manifests by default as slowly progressive spinal cord axonopathy with associated demyelination (adrenomyloneuropathy). In 60% of male cases, however, X-linked adrenoleukodystrophy converts to devastating cerebral inflammation and demyelination (cerebral adrenoleukodystrophy) with infiltrating blood-derived monocytes and macrophages and cytotoxic T cells that can only be stopped by allogeneic haematopoietic stem cell transplantation or gene therapy at an early stage of the disease. Recently, we identified monocytes/macrophages but not T cells to be severely affected metabolically by ABCD1 deficiency. Here we found by whole transcriptome analysis that, although monocytes of patients with X-linked adrenoleukodystrophy have normal capacity for macrophage differentiation and phagocytosis, they are pro-inflammatory skewed also in patients with adrenomyloneuropathy in the absence of cerebral inflammation. Following lipopolysaccharide activation, the ingestion of myelin debris, normally triggering anti-inflammatory polarization, did not fully reverse the pro-inflammatory status of X-linked adrenoleukodystrophy macrophages. Immunohistochemistry on post-mortem cerebral adrenoleukodystrophy lesions reflected the activation pattern by prominent presence of enlarged lipid-laden macrophages strongly positive for the pro-inflammatory marker co-stimulatory molecule CD86. Comparative analyses of lesions with matching macrophage density in cases of cerebral adrenoleukodystrophy and acute multiple sclerosis showed a similar extent of pro-inflammatory activation but a striking reduction of anti-inflammatory mannose receptor (CD206) and haemoglobin-haptoglobin receptor (CD163) expression on cerebral adrenoleukodystrophy macrophages. Accordingly, ABCD1-deficiency leads to an impaired plasticity of macrophages that is reflected in incomplete establishment of anti-inflammatory responses, thus possibly contributing to the devastating rapidly progressive demyelination in cerebral adrenoleukodystrophy that only in rare cases arrests spontaneously. These findings emphasize monocytes/macrophages as crucial therapeutic targets for preventing or stopping myelin destruction in patients with X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD), the most frequent monogenetic disorder of brain white matter, is highly variable, ranging from slowly progressive adrenomyeloneuropathy (AMN) to life-threatening inflammatory brain demyelination (CALD). In this study involving 94 X-ALD patients and 55 controls, we tested whether plasma/serum neurofilament light chain protein (NfL) constitutes an early distinguishing biomarker. In AMN, we found moderately elevated NfL with increased levels reflecting higher grading of myelopathy-related disability. Intriguingly, NfL was a significant predictor to discriminate non-converting AMN from cohorts later developing CALD. In CALD, markedly amplified NfL levels reflected brain lesion severity. In rare cases, atypically low NfL revealed a previously unrecognized smoldering CALD disease course with slowly progressive myelin destruction. Upon halt of brain demyelination by hematopoietic stem cell transplantation, NfL gradually normalized. Together, our study reveals that blood NfL reflects inflammatory activity and progression in CALD patients, thus constituting a potential surrogate biomarker that may facilitate clinical decisions and therapeutic development.

Objective To identify a pharmacological compound targeting macrophages, the most affected immune cells in inflammatory X-linked adrenoleukodystrophy (cerebral X-ALD) caused by ABCD1 mutations and involved in the success of hematopoietic stem cell transplantation and gene therapy. Methods A comparative database analysis elucidated the epigenetic repressing mechanism of the related ABCD2 gene in macrophages and identified the histone deacetylase (HDAC) inhibitor Vorinostat as a compound to induce ABCD2 in these cells to compensate for ABCD1 deficiency. In these cells, we investigated ABCD2 and pro-inflammatory gene expression, restoration of defective peroxisomal beta-oxidation activity, accumulation of very long-chain fatty acids (VLCFAs) and their differentiation status. We investigated ABCD2 and pro-inflammatory gene expression, restoration of defective peroxisomal ss-oxidation activity, accumulation of very long-chain fatty acids (VLCFA) and differentiation status. Three advanced cerebral X-ALD patients received Vorinostat and CSF and MRI diagnostics was carried out in one patient after 80 days of treatment. Results Vorinostat improved the metabolic defects in X-ALD macrophages by stimulating ABCD2 expression, peroxisomal ss-oxidation, and ameliorating VLCFA accumulation. Vorinostat interfered with pro-inflammatory skewing of X-ALD macrophages by correcting IL12B expression and further reducing monocyte differentiation. Vorinostat normalized the albumin and immunoglobulin CSF-serum ratios, but not gadolinium enhancement upon 80 days of treatment. Interpretation The beneficial effects of HDAC inhibitors on macrophages in X-ALD and the improvement of the blood-CSF/blood-brain barrier are encouraging for future investigations. In contrast with Vorinostat, less toxic macrophage-specific HDAC inhibitors might improve also the clinical state of X-ALD patients with advanced inflammatory demyelination.

Objective: To assess class I‐histone deacetylase (HDAC) inhibition on formation of lipid‐accumulating, disease‐promoting phagocytes upon myelin load in vitro, relevant for neuroinflammatory disorders like multiple sclerosis (MS) and cerebral X‐linked adrenoleukodystrophy (X‐ALD).Methods: Immunohistochemistry on postmortem brain tissue of acute MS (n = 6) and cerebral ALD (n = 4) cases to analyze activation and foam cell state of phagocytes. RNA‐Seq of in vitro differentiated healthy macrophages (n = 8) after sustained myelin‐loading to assess the metabolic shift associated with foam cell formation. RNA‐Seq analysis of genes linked to lipid degradation and export in MS‐275‐treated human HAP1 cells and RT‐qPCR analysis of HAP1 cells knocked out for individual members of class I HDACs or the corresponding enzymatically inactive knock‐in mutants. Investigation of intracellular lipid/myelin content after MS‐275 treatment of myelin‐laden human foam cells. Analysis of disease characteristic very long‐chain fatty acid (VLCFA) metabolism and inflammatory state in MS‐275‐treated X‐ALD macrophages.Results: Enlarged foam cells coincided with a pro‐inflammatory, lesion‐promoting phenotype in postmortem tissue of MS and cerebral ALD patients. Healthy in vitro myelin laden foam cells upregulated genes linked to LXRα/PPARγ pathways and mimicked a program associated with tissue repair. Treating these cells with MS‐275, amplified this gene transcription program and significantly reduced lipid and cholesterol accumulation and, thus, foam cell formation. In macrophages derived from X‐ALD patients, MS‐275 improved the disease‐associated alterations of VLCFA metabolism and reduced the pro‐inflammatory status of these cells.Interpretation: These findings identify class I‐HDAC inhibition as a potential novel strategy to prevent disease promoting foam cell formation in CNS inflammation.

Elevated levels of saturated very long-chain fatty acids (VLCFAs) in cell membranes and secreted lipoparticles have been associated with neurotoxicity and, therefore, require tight regulation. Excessive VLCFAs are imported into peroxisomes for degradation by β-oxidation. Impaired VLCFA catabolism due to primary or secondary peroxisomal alterations is featured in neurodegenerative and neuroinflammatory disorders such as X-linked adrenoleukodystrophy and multiple sclerosis (MS). Here, we identified that healthy human macrophages upregulate the peroxisomal genes involved in β-oxidation during myelin phagocytosis and pro-inflammatory activation, and that this response is impaired in peripheral macrophages and phagocytes in brain white matter lesions in MS patients. The pharmacological targeting of VLCFA metabolism and peroxisomes in innate immune cells could be favorable in the context of neuroinflammation and neurodegeneration. We previously identified the epigenetic histone deacetylase (HDAC) inhibitors entinostat and vorinostat to enhance VLCFA degradation and pro-regenerative macrophage polarization. However, adverse side effects currently limit their use in chronic neuroinflammation. Here, we focused on tefinostat, a monocyte/macrophage-selective HDAC inhibitor that has shown reduced toxicity in clinical trials. By using a gene expression analysis, peroxisomal β-oxidation assay, and live imaging of primary human macrophages, we assessed the efficacy of tefinostat in modulating VLCFA metabolism, phagocytosis, chemotaxis, and immune function. Our results revealed the significant stimulation of VLCFA degradation with the upregulation of genes involved in peroxisomal β-oxidation and interference with immune cell recruitment; however, tefinostat was less potent than the class I HDAC-selective inhibitor entinostat in promoting a regenerative macrophage phenotype. Further research is needed to fully explore the potential of class I HDAC inhibition and downstream targets in the context of neuroinflammation.