IFN-γ Drives Human Monocyte Differentiation into Highly Proinflammatory Macrophages That Resemble a Phenotype Relevant to Psoriasis.

As key cells of the immune system, macrophages coordinate the activation and regulation of the immune response. Macrophages present a complex phenotype that can vary from homeostatic, proinflammatory, and profibrotic to anti-inflammatory phenotypes. The factors that drive the differentiation from monocyte to macrophage largely define the resultant phenotype, as has been shown by the differences found in M-CSF- and GM-CSF-derived macrophages. We explored alternative inflammatory mediators that could be used for in vitro differentiation of human monocytes into macrophages. IFN-γ is a potent inflammatory mediator produced by lymphocytes in disease and infections. We used IFN-γ to differentiate human monocytes into macrophages and characterized the cells at a functional and proteomic level. IFN-γ alone was sufficient to generate macrophages (IFN-γ Mϕ) that were phagocytic and responsive to polarization. We demonstrate that IFN-γ Mϕ are potent activators of T lymphocytes that produce IL-17 and IFN-γ. We identified potential markers (GBP-1, IP-10, IL-12p70, and IL-23) of IFN-γ Mϕ and demonstrate that these markers are enriched in the skin of patients with inflamed psoriasis. Collectively, we show that IFN-γ can drive human monocyte to macrophage differentiation, leading to bona fide macrophages with inflammatory characteristics.

Phase 1 and preclinical profiling of ESM-HDAC391, a myeloid-targeted histone deacetylase inhibitor, shows enhanced pharmacology and monocytopaenia.

AIMS: To improve the tolerability and therapeutic application of histone deacetylase inhibitors (HDACi), by application of an esterase-sensitive motif (ESM), to target pharmacological activity directly to mononuclear myeloid cells expressing the processing enzyme carboxylesterase-1 (CES1). METHODS: This first-in-human study comprised single and multiple ascending dose cohorts to determine safety and tolerability. Pharmacodynamic parameters included acetylation, cytokine inhibition and intracellular concentrations of processed acid metabolite in isolated monocytes. Mechanistic work was conducted in vitro and in a CES1/Es1elo mouse strain. RESULTS: ESM-HDAC391 showed transient systemic exposure (plasma half-life of 21-30 min) but selective retention of processed acid for at least 12 hours, resulting in robust targeted mechanistic engagement (increased acetylation in monocytes plus inhibition of ex vivo stimulated cytokine production). ESM-HDAC391 was well tolerated and clinical toxicities common to non-targeted HDACi were not observed. ESM-HDAC391 treatment was accompanied by the novel finding of a dose-dependent monocyte depletion that was transient and reversible and which plateaued at 0.06 × 109 monocytes/L after repeat dosing with 20 or 40 mg. Characterisation of monocyte depletion in transgenic mice (CES1/Es1elo ) suggested that colony stimulating factor 1 receptor loss on circulating cells contributed to ESM-HDAC-mediated depletion. Further mechanistic investigations using human monocytes in vitro demonstrated HDACi-mediated change in myeloid fate through modulation of colony stimulating factor 1 receptor and downstream effects on cell differentiation. CONCLUSION: These findings demonstrate selective targeting of monocytes in humans using the ESM approach and identify monocytopaenia as a novel outcome of ESM-HDACi treatment, with implications for potential benefit of these molecules in myeloid-driven diseases.

Whole-Genome DNA Methylation Profiling of CD14+ Monocytes Reveals Disease Status and Activity Differences in Crohn's Disease Patients.

Crohn's disease (CD) is a multifactorial incurable chronic disorder. Current medical treatment seeks to induce and maintain a state of remission. During episodes of inflammation, monocytes infiltrate the inflamed mucosa whereupon they differentiate into macrophages with a pro-inflammatory phenotype. Here, we sought to characterize the circulating monocytes by profiling their DNA methylome and relate it to the level of CD activity. We gathered an all-female age-matched cohort of 16 CD patients and 7 non-CD volunteers. CD patients were further subdivided into 8 CD patients with active disease (CD-active) and 8 CD patients in remission (CD-remissive) as determined by the physician global assessment. We identified 15 and 12 differentially methylated genes (DMGs) when comparing CD with non-CD and CD-active with CD-remissive, respectively. Differential methylation was predominantly found in the promoter regions of inflammatory genes. Comparing our observations with gene expression data on classical (CD14++CD16-), non-classical (CD14+CD16++) and intermediate (CD14++CD16+) monocytes indicated that while 7 DMGs were differentially expressed across the 3 subsets, the remaining DMGs could not immediately be associated with differences in known populations. We conclude that CD activity is associated with differences in DNA methylation at the promoter region of inflammation-associated genes.

Pharmacological validation of targets regulating CD14 during macrophage differentiation.

The signalling receptor for LPS, CD14, is a key marker of, and facilitator for, pro-inflammatory macrophage function. Pro-inflammatory macrophage differentiation remains a process facilitating a broad array of disease pathologies, and has recently emerged as a potential target against cytokine storm in COVID19. Here, we perform a whole-genome CRISPR screen to identify essential nodes regulating CD14 expression in myeloid cells, using the differentiation of THP-1 cells as a starting point. This strategy uncovers many known pathways required for CD14 expression and regulating macrophage differentiation while additionally providing a list of novel targets either promoting or limiting this process. To speed translation of these results, we have then taken the approach of independently validating hits from the screen using well-curated small molecules. In this manner, we identify pharmacologically tractable hits that can either increase CD14 expression on non-differentiated monocytes or prevent CD14 upregulation during macrophage differentiation. An inhibitor for one of these targets, MAP2K3, translates through to studies on primary human monocytes, where it prevents upregulation of CD14 following M-CSF induced differentiation, and pro-inflammatory cytokine production in response to LPS. Therefore, this screening cascade has rapidly identified pharmacologically tractable nodes regulating a critical disease-relevant process.

A distinct epigenetic profile distinguishes stenotic from non-inflamed fibroblasts in the ileal mucosa of Crohn's disease patients.

BACKGROUND: The chronic remitting and relapsing intestinal inflammation characteristic of Crohn's disease frequently leads to fibrosis and subsequent stenosis of the inflamed region. Approximately a third of all Crohn's disease patients require resection at some stage in their disease course. As the pathogenesis of Crohn's disease associated fibrosis is largely unknown, a strong necessity exists to better understand the pathophysiology thereof. METHODS: In this study, we investigated changes of the DNA methylome and transcriptome of ileum-derived fibroblasts associated to the occurrence of Crohn's disease associated fibrosis. Eighteen samples were included in a DNA methylation array and twenty-one samples were used for RNA sequencing. RESULTS: Most differentially methylated regions and differentially expressed genes were observed when comparing stenotic with non-inflamed samples. By contrast, few differences were observed when comparing Crohn's disease with non-Crohn's disease, or inflamed with non-inflamed tissue. Integrative methylation and gene expression analyses revealed dysregulation of genes associated to the PRKACA and E2F1 network, which is involved in cell cycle progression, angiogenesis, epithelial to mesenchymal transition, and bile metabolism. CONCLUSION: Our research provides evidence that the methylome and the transcriptome are systematically dysregulated in stenosis-associated fibroblasts.

Potential novel biomarkers of disease activity in rheumatoid arthritis patients: CXCL13, CCL23, transforming growth factor alpha, tumor necrosis factor receptor superfamily member 9, and macrophage colony-stimulating factor.

OBJECTIVE: To determine whether the plasma levels of a range of inflammatory proteins have utility as biomarkers of disease activity in rheumatoid arthritis (RA) patients. METHODS: Plasma proteins (n = 163) were profiled in 44 patients with RA diagnosed according to the American College of Rheumatology 1987 criteria (22 with active and 22 with quiescent disease) and in 16 age- and sex-matched healthy controls. The utility of a subset of differentially expressed proteins as predictors of RA disease activity was investigated using partial least-squares discriminant analysis, and their response to therapeutic intervention was evaluated in plasma from an additional cohort of 16 patients with active RA treated with anti-tumor necrosis factor alpha (anti-TNFalpha). RESULTS: The protein profiling study identified 25 proteins that were differentially expressed in plasma samples from patients with active RA (P for the false discovery rate < or = 0.01) compared with those with quiescent RA, including the previously described interleukin-6 (IL-6), oncostatin M, and IL-2, and the 5 less-established markers macrophage colony-stimulating factor (M-CSF), tumor necrosis factor receptor superfamily member 9, CCL23, transforming growth factor alpha, and CXCL13. Systemic levels of these 5 markers correlated with the C-reactive protein level, erythrocyte sedimentation rate, rheumatoid factor level, tender joint count in 68 joints, and Disease Activity Score in 28 joints (DAS28), and their combined plasma levels were shown to be good predictors of disease activity (kappa = 0.64). In anti-TNFalpha-treated RA patients, plasma levels of CXCL13 were reduced after 1 and 7 days of therapy, and levels of CCL23, M-CSF, and CXCL13 showed a statistically significant positive correlation with the DAS28 score. CONCLUSION: This exploratory study for biomarker discovery led to the identification of several proteins predictive of RA disease activity that may be useful in the definition of disease subphenotypes and in the measurement of response to therapy in clinical studies.