Histone deacetylases (HDACs) are a group of enzymes that control histone deacetylation and bear potential to direct expression of large gene sets. We determined the effect of HDAC inhibitors (HDACi) on human monocytes and macrophages, with respect to their polarization, activation, and their capabilities of inducing endotoxin tolerance. To address the role for HDACs in macrophage polarization, we treated monocytes with HDAC3i, HDAC6i or pan-HDACi prior to polarization into M1 or M2 macrophages using IFNγ or IL-4 respectively. To study the HDAC inhibition effect on cytokine expression, macrophages were treated with HDACi prior to LPS-stimulation. TNFα, IL-6, and p40 were measured with ELISA, whereas modifications of Histone 3 and STAT1 were assessed using western blot. To address the role for HDAC3 in repeated LPS challenge induction, HDAC3i or HDAC3 siRNA was added to monocytes prior to incubation with IFNγ, which were then repeatedly challenged with LPS and analyzed by means of protein analyses and transcriptional profiling. Pan-HDACi and HDAC3i reduced cytokine secretion in monocytes and M1 macrophages, whereas HDAC6i yielded no such effect. Notably, neither pan-HDACi nor HDAC3i reduced cytokine secretion in M2 macrophages. In contrast to previous reports in mouse macrophages, HDAC3i did not affect macrophage polarization in human cells. Likewise, HDAC3 was not required for IFNγ signaling or IFNβ secretion. Cytokine and gene expression analyses confirmed that IFNγ-treated macrophages consistently develop a cytokine response after LPS repeated challenge, but pretreatment with HDAC3i or HDAC3 siRNA reinstates a state of tolerance reflected by general suppression of tolerizable genes, possibly through decreasing TLRs expression, and particularly TLR4/CD14. The development of endotoxin tolerance in macrophages is important to reduce exacerbated immune response and limit tissue damage. We conclude that HDAC3 is an attractive protein target to mediate macrophage reactivity and tolerance induction in inflammatory macrophages.

Crohn’s disease (CD) is a chronic inflammatory disorder belonging to the inflammatory bowel diseases (IBD). CD affects distinct parts of the gastrointestinal tract, leading to symptoms including diarrhea, fever, abdominal pain, weight loss, and anemia. The aim of this study was to assess whether the DNA methylome of peripheral blood cells can be associated with CD in women. Samples were obtained from 18 female patients with histologically confirmed ileal or ileocolic CD and 25 healthy age- and gender-matched controls (mean age and standard deviation: 30.5 ± 6.5 years for both groups). Genome-wide DNA methylation was determined using the Illumina HumanMethylation 450k BeadChip. Our analysis implicated 4287 differentially methylated positions (DMPs; corrected p < 0.05) that are associated to 2715 unique genes. Gene ontology enrichment analysis revealed significant enrichment of our DMPs in immune response processes and inflammatory pathways. Of the 4287 DMPs, 32 DMPs were located on chromosome X with several hits for MIR223 and PABPC5. Comparison with previously performed (epi)genome-wide studies revealed that we replicated 33 IBD-associated genes. In addition to DMPs, we found eight differentially methylated regions (DMRs). CD patients display a characteristic DNA methylation landscape, with the differentially methylated genes being implicated in immune response. Additionally, DMPs were found on chromosome X suggesting X-linked manifestations of CD that could be associated with female-specific symptoms.

Macrophages are heterogeneous leukocytes regulated in a tissue- and disease-specific context. While in vitro macrophage models have been used to study diseases empirically, a systematic analysis of the transcriptome thereof is lacking. Here, we acquired gene expression data from eight commonly-used in vitro macrophage models to perform a meta-analysis. Specifically, we obtained gene expression data from unstimulated macrophages (M0) and macrophages stimulated with lipopolysaccharides (LPS) for 2–4 h (M-LPS_early), LPS for 24 h (M-LPS_late), LPS and interferon-γ (M-LPS+IFNγ), IFNγ (M-IFNγ), interleukin-4 (M-IL4), interleukin-10 (M-IL10), and dexamethasone (M-dex). Our meta-analysis identified consistently differentially expressed genes that have been implicated in inflammatory and metabolic processes. In addition, we built macIDR, a robust classifier capable of distinguishing macrophage activation states with high accuracy (>0.95). We classified in vivo macrophages with macIDR to define their tissue- and disease-specific characteristics. We demonstrate that alveolar macrophages display high resemblance to IL10 activation, but show a drop in IFNγ signature in chronic obstructive pulmonary disease patients. Adipose tissue-derived macrophages were classified as unstimulated macrophages, but acquired LPS-activation features in diabetic-obese patients. Rheumatoid arthritis synovial macrophages exhibit characteristics of IL10- or IFNγ-stimulation. Altogether, we defined consensus transcriptional profiles for the eight in vitro macrophage activation states, built a classification model, and demonstrated the utility of the latter for in vivo macrophages.

Over the past decade, the DNA methylome has been increasingly studied in peripheral blood of inflammatory bowel disease [IBD] patients. However, a comprehensive summary and meta-analysis of peripheral blood leukocyte [PBL] DNA methylation studies has thus far not been conducted. Here, we systematically reviewed all available literature up to February 2022 and summarized the observations by means of meta-analysis. We conducted a systematic search and critical appraisal of IBD-associated DNA methylation studies in PBL using the biomarker-based cross-sectional studies [BIOCROSS] tool. Subsequently, we performed meta-analyses on the summary statistics obtained from epigenome-wide association studies [EWAS] that included patients with Crohn’s disease [CD], ulcerative colitis [UC] and/or healthy controls [HC]. Altogether, we included 15 studies for systematic review. Critical appraisal revealed large methodological and outcome heterogeneity between studies. Summary statistics were obtained from four studies based on a cumulative 552 samples [177 CD, 132 UC and 243 HC]. Consistent differential methylation was identified for 256 differentially methylated probes [DMPs; Bonferroni-adjusted p ≤ 0.05] when comparing CD with HC and 103 when comparing UC with HC. Comparing IBD [CD + UC] with HC resulted in 224 DMPs. Importantly, several of the previously identified DMPs, such as VMP1/TMEM49/MIR21 and RPS6KA2, were consistently differentially methylated across all studies. Methodological homogenization of IBD epigenetic studies is needed to allow for easier aggregation and independent validation. Nonetheless, we were able to confirm previous observations. Our results can serve as the basis for future IBD epigenetic biomarker research in PBL.

Patients with peritoneal metastasized colorectal cancer (PM-CRC) have a dismal prognosis. We hypothesized that an immunosuppressive environment in the peritoneal cavity underlies poor prognosis. We define the composition of the human peritoneal immune system (PerIS) using single-cell technologies in 18 patients with- and without PM-CRC, as well as in matched peritoneal metastases (n = 8). Here we show that the PerIS contains abundant immunosuppressive C1Q⁺VSIG4⁺ and SPP1⁺VSIG4⁺ peritoneal-resident macrophages (PRMs), as well as monocyte-like cavity macrophages (mono-CMs), which share features with tumor-associated macrophages, even in homeostasis. In PM-CRC, expression of immunosuppressive cytokines IL10 and VEGF increases, while simultaneously expression of antigen-presenting molecules decreases in PRMs. These intratumoral suppressive PRMs originate from the PerIS, and intraperitoneal depletion of PRMs in vivo using anti-CSF1R combined with anti-PD1 significantly reduces tumor burden and improves survival. Thus, PRMs define a metastatic site-specific immunosuppressive niche, and targeting PRMs is a promising treatment strategy for PM-CRC.

Background: Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by relapsing and remitting mucosal inflammation of the colon. While active UC mucosa is characterized by dysregulated B cell responses and increased B cell and IgG plasma cell populations, targeting CD20-expressing B cells in UC has proven ineffective. Methods: We conducted an exploratory single-cell transcriptomic analysis of colonic biopsies obtained from UC patients with (n = 5) or without (n = 5) active inflammation, and non-UC controls (n = 4). To explore whether B cells contribute to colitis severity, we transferred various ratios of spleen-derived naive B cells with CD45RBhigh T cells into severe combined immune deficient mice to induce colitis. Results: Our analysis identified a distinct subset of naive (MS4A1+CD27-IGHD+TCL1A+) B cells that are significantly enriched and present a more matured phenotype in inflamed compared to non-inflamed biopsies from UC patients. Cell-cell communication analysis indicated that naive B cells interacted predominantly with CD4+ T cell subsets. In the mice transfer colitis model, co-transfer of naive B cells at a ratio of 1-2 T and B cells, respectively, showed an increased maturation and activity, which led to exacerbation of colitis as measured by weight loss, increased colon density, and histological inflammation. Conclusion: Our findings suggest that naive B cells expand in actively inflamed mucosa from UC patients and play a pathogenic role in experimental colitis.