Background Janus kinases (JAKs) mediate cytokine signaling involved in inflammatory bowel disease. The pan-JAK inhibitor tofacitinib has shown efficacy in the treatment of ulcerative colitis. However, concerns regarding adverse events due to their wide spectrum inhibition fueled efforts to develop selective JAK inhibitors. Given the crucial role of myeloid cells in intestinal immune homeostasis, we evaluated the effect of pan-JAK and selective JAK inhibitors on pro- and anti-inflammatory macrophage polarization and function (M1/M2) and in experimental colitis. Methods Murine bone marrow–derived macrophages or human monocytes were treated using JAK1 and JAK3 selective inhibitors (JAK1i;JAK3i) and tofacitinib and were evaluated by transcriptional, functional, and metabolic analyses. In vivo, oral administration of JAK1i and tofacitinib (10 or 30 mg/kg) was tested in both acute and acute rescue dextran sodium sulfate (DSS) colitis. Results Both tofacitinib and JAK1i but not JAK3i effectively inhibited STAT1 phosphorylation and interferon gamma–induced transcripts in M1 polarized macrophages. Strikingly, transcriptional profiling suggested a switch from M1 to M2 type macrophages, which was supported by increased protein expression of M2-associated markers. In addition, both inhibitors enhanced oxidative phosphorylation rates. In vivo, JAK1i and tofacitinib did not protect mice from acute DSS-induced colitis but ameliorated recovery from weight loss and disease activity during acute rescue DSS-induced colitis at the highest dose. Conclusion JAK1i and tofacitinib but not JAK3i induce phenotypical and functional characteristics of anti-inflammatory macrophages, suggesting JAK1 as the main effector pathway for tofacitinib in these cells. In vivo, JAK1i and tofacitinib modestly affect acute rescue DSS-induced colitis.

Objectives:The esophageal mucosal integrity is impaired in eosinophilic esophagitis (EoE) and it has been suggested that the duodenal permeability is increased. The absence of food allergens may restore the integrity. The aims of this study were to assess duodenal permeability in EoE and to evaluate the effect of an elemental diet on the esophageal and duodenal integrity.Methods:In this prospective study 17 adult EoE patients and 8 healthy controls (HC) were included. Esophageal biopsy specimens were sampled before and after 4 weeks of elemental diet to measure eosinophil counts and gene expression of tight junction and barrier integrity proteins. Esophageal and duodenal impedance were measured by electrical tissue impedance spectroscopy and Ussing chambers were used to measure transepithelial resistance (TER) and transepithelial molecule flux. Small intestinal permeability was measured using a test, measuring lactulose/mannitol (L/M) ratios.Results:In EoE patients, the esophageal but not the duodenal integrity was impaired, compared with HC. We observed no significant difference between L/M ratios of HC and EoE patients. After diet, eosinophil counts decreased significantly, which was paralleled by normalization of esophageal impedance and transepithelial molecule flux. The esophageal TER improved significantly, but did not reach values seen in HC. Esophageal expression of genes encoding for barrier integrity proteins filaggrin and desmoglein-1 was impaired at baseline and restored after diet.Conclusions:An elemental diet restores esophageal integrity, suggesting that it is at least partly secondary to allergen exposure. Duodenal integrity seems not to be affected in EoE, and possibly plays a minor role in its pathophysiology.

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.

Background Vagus nerve stimulation has been suggested to affect immune responses, partly through a neuronal circuit requiring sympathetic innervation of the splenic nerve bundle and norepinephrine (NE) release. Molecular and cellular mechanisms of action remain elusive. Here, we investigated the therapeutic value of this neuromodulation in inflammatory bowel disease (IBD) by applying electrical splenic nerve bundle stimulation (SpNS) in mice with dextran sulfate sodium (DSS)-induced colitis. Methods Cuff electrodes were implanted around the splenic nerve bundle in mice, whereupon mice received SpNS or sham stimulation. Stimulation was applied 6 times daily for 12 days during DSS-induced colitis. Colonic and splenic tissues were collected for transcriptional analyses by qPCR and RNA-sequencing (RNA-seq). In addition, murine and human splenocytes were stimulated with lipopolysaccharide (LPS) in the absence or presence of NE. Single-cell RNA-seq data from publicly available data sets were analyzed for expression of β-adrenergic receptors (β-ARs). Results Colitic mice undergoing SpNS displayed reduced colon weight/length ratios and showed improved Disease Activity Index scores with reduced Tumor Necrosis Factor α mRNA expression in the colon compared with sham stimulated mice. Analyses of splenocytes from SpNS mice using RNA-seq demonstrated specific immune metabolism transcriptome profile changes in myeloid cells. Splenocytes showed expression of β-ARs in myeloid and T cells. Cytokine production was reduced by NE in mouse and human LPS-stimulated splenocytes. Conclusions Together, our results demonstrate that SpNS reduces clinical features of colonic inflammation in mice with DSS-induced colitis possibly by inhibiting splenic myeloid cell activation. Our data further support exploration of the clinical use of SpNS for patients with IBD.