Longitudinal Multi-omics Analyses Identify Responses of Megakaryocytes, Erythroid Cells, and Plasmablasts as Hallmarks of Severe COVID-19.

Temporal resolution of cellular features associated with a severe COVID-19 disease trajectory is needed for understanding skewed immune responses and defining predictors of outcome. Here, we performed a longitudinal multi-omics study using a two-center cohort of 14 patients. We analyzed the bulk transcriptome, bulk DNA methylome, and single-cell transcriptome (>358,000 cells, including BCR profiles) of peripheral blood samples harvested from up to 5 time points. Validation was performed in two independent cohorts of COVID-19 patients. Severe COVID-19 was characterized by an increase of proliferating, metabolically hyperactive plasmablasts. Coinciding with critical illness, we also identified an expansion of interferon-activated circulating megakaryocytes and increased erythropoiesis with features of hypoxic signaling. Megakaryocyte- and erythroid-cell-derived co-expression modules were predictive of fatal disease outcome. The study demonstrates broad cellular effects of SARS-CoV-2 infection beyond adaptive immune cells and provides an entry point toward developing biomarkers and targeted treatments of patients with COVID-19.

Analysis of single nucleotide polymorphisms in chronic beryllium disease.

Sarcoidosis and chronic beryllium disease (CBD) are phenocopies, however the latter one has a clear trigger factor that is beryllium exposure. This study analyses single nucleotide polymorphisms (SNPs) in a large cohort for beryllium-exposed persons. SNPs were chosen for their relevance in sarcoidosis. Even though one of largest cohorts of beryllium-exposed persons was analysed, no statistically relevant association between any SNP and CBD could be verified. Notably, some SNPs exhibit inverse OR for beryllium sensitization and CBD with nominally statistical significance, which allows hypothesizing about pathophysiological role of genes for the disease triggering and development.

IL23R on myeloid cells is involved in murine pulmonary granuloma formation.

PURPOSE OF THE STUDY: The involvement of the IL-23/IL23R pathway is well known in the disease pathogenesis of sarcoidosis and other inflammatory diseases. To date, the pathogenic mechanism of IL-23 is most notably described on CD4+ Th17 lymphocytes. However, the function of the IL23R on myeloid cells in sarcoidosis is poorly understood. Thus, the aim of the study is to investigate the role of the IL23R on myeloid cell in pulmonary granuloma formation. Methods: We generated IL23RLysMCre mice lacking the IL23R gene in myeloid cells. The importance of IL23R in myeloid cells for the development of sarcoidosis was studied in a mouse model of inflammatory lung granuloma formation through embolization of PPD from Mycobacterium bovis-coated Sepharose beads into previously PPD-immunized mice. In addition the function of IL23R on myeloid cells was studied in LPS or IFNγ stimulated BMDMs and BMDCs. The mRNA and protein expression levels of relevant cytokines were analyzed by RT-PCR (TaqMan) and ELISA. The composition of immune cells in BALF was quantified by flow cytometry and alteration in granuloma sizes were observed by H&E stained lung sections. Results: Mycobacterium Ag-elicted pulmonary granulomas tend to be smaller in IL23RLysMCre mice and NF-κB dependent Th1 cytokines in the murine lungs are reduced compared to wildtype mice. In line, we observed that IL23R-deficient bone marrow-derived macrophages show a reduced production of Th1 cytokines after LPS stimulation. Conclusion: We here for the first time demonstrate a role for IL23R on myeloid cells in pulmonary inflammation and granuloma formation. Our findings provide essential insights in the pathogenesis of inflammatory lung diseases like sarcoidosis, which might be useful for the development of novel therapeutics targeting distinct immunological pathways like IL-23/IL23R.

Oral glutamine supplementation improves intestinal permeability dysfunction in a murine acute graft-vs.-host disease model.

Although a profound barrier dysfunction has been reported, little is known about the pathophysiological mechanism evoking gastrointestinal graft-vs.-host disease (GI-GvHD) and apparent therapeutic options. The aim of this study was to evaluate the influence of oral glutamine on the course of GI-GvHD in an acute semiallogenic graft-vs.-host disease (GvHD) in irradiated B6D2F1 mice. An acute semiallogenic GvHD was induced by intraperitoneal injection of lymphocytes from C57BL/6 mice to irradiated B6D2F1 mice. Half of the GvHD animals received oral glutamine supplementation for 6 days started at the time of lymphocyte transfer. Six days after induction of the semiallogenic GvHD, jejunum specimens were prepared. The expression of the proinflammatory cytokine TNF-α and the tight junction protein occludin was investigated by PCR. Histological changes along with the apoptotic response were evaluated and intestinal permeability was assessed. Animals with GvHD showed a strong increase in paracellular permeability as a sign of the disturbed barrier function. TNF-α expression was significantly increased and the expression of the tight junction protein occludin decreased. GvHD led to mucosal atrophy, crypt hyperplasia, crypt apoptosis, and a disintegration of the tight junctions. Glutamine-treated mice showed reduced expression of TNF-α, increased occludin expression, fewer histological changes in the jejunum, smaller number of apoptotic cells in the crypt, and reduced gastrointestinal permeability. In conclusion, oral glutamine seems to have beneficial effects on the severity of inflammatory changes in the course of GvHD and might be a therapeutic option.

Increased intestinal permeability and tight junction disruption by altered expression and localization of occludin in a murine graft versus host disease model.

BACKGROUND: Hematopoietic stem cell transplantation is increasingly performed for hematologic diseases. As a major side effect, acute graft versus host disease (GvHD) with serious gastrointestinal symptoms including diarrhea, gastrointestinal bleeding and high mortality can be observed. Because surveillance and biopsies of human gastrointestinal GvHD are difficult to perform, rare information of the alterations of the gastrointestinal barrier exists resulting in a need for systematic animal models. METHODS: To investigate the effects of GvHD on the intestinal barrier of the small intestine we utilized an established acute semi allogenic GvHD in C57BL/6 and B6D2F1 mice. RESULTS: By assessing the differential uptake of lactulose and mannitol in the jejunum, we observed an increased paracellular permeability as a likely mechanism for disturbed intestinal barrier function. Electron microscopy, immunohistochemistry and PCR analysis indicated profound changes of the tight-junction complex, characterized by downregulation of the tight junction protein occludin without any changes in ZO-1. Furthermore TNF-α expression was significantly upregulated. CONCLUSIONS: This analysis in a murine model of GvHD of the small intestine demonstrates serious impairment of intestinal barrier function in the jejunum, with an increased permeability and morphological changes through downregulation and localization shift of the tight junction protein occludin.

Anti-TNF-α antibodies improve intestinal barrier function in Crohn's disease.

BACKGROUND AND AIMS: Intestinal barrier function in Crohn's disease patients and their first degree healthy relatives is impaired. The increased intestinal permeability may result in an enhanced mucosal immune response and thereby aggravate intestinal inflammation. Humanised anti-TNF-α antibodies have been shown to be effective in the treatment of active Crohn's disease and in the treatment of entero-cutaneous fistula. The aim of the present study was to investigate the influence of anti-TNF-α antibody (infliximab) treatment on the intestinal barrier function of patients with active Crohn's disease. METHODS: The differential intestinal uptake of lactulose and mannitol was measured to quantify intestinal permeability in patients with long standing active Crohn's disease (n=17) directly before and seven days after treatment with infliximab (5 mg/kg bodyweight). In parallel, intestinal permeability was studied in a healthy control group (n=20). Serum samples were analysed with pulsed amperometric detection after separation on an anion exchange column. RESULTS: Intestinal permeability was significantly increased in all patients with Crohn's disease (L/M ratio 0.24±0.17) prior to infliximab treatment compared to the control group (L/M ratio 0.01±0.02; p-value <1×10(-7)). Treatment of patients with infliximab resulted in a marked decrease of intestinal permeability as measured by L/M ratio from 0.24±0.17 before to 0.02±0.02 (p-value <1×10(-7)) seven days after infliximab application. CONCLUSIONS: Treatment with anti-TNF-α antibodies improved impaired intestinal barrier function in patients with Crohn's disease. This effect may correlate to the well documented anti-inflammatory effect of TNF-α blockade in this intestinal disease.

The role of tissue transglutaminase (transglutaminase type II) for the intestinal manifestations of murine semi-allogenic graft-versus-host disease.

The intestinal manifestation of acute murine semi-allogenic graft-versus-host (GvH) disease is characterized by the occurrence of lymphocytic infiltrates in the lamina propria, by crypt hyperplasia and villous atrophy. In a histological respect, this animal model resembles human celiac disease. Tissue transglutaminase (tTG) (transglutaminase type II) has been identified to be the major B cell autoantigen in celiac disease. Furthermore, tissue transglutaminase has been implicated to be involved in its pathogenesis. Therefore, we aimed to investigate whether tissue transglutaminase is expressed in the intestines of GvH animals and whether its inhibition has any effect on the intestinal histology. Sera of patients with celiac disease and anti-tTG antibodies were purified. These antibodies were used for immuno-histochemistry of jejunal cryosection from GvH and syngenic control animals at day 6 after lymphocyte transfer. Furthermore, GvH mice were treated with antitTG antibodies and with the inhibitor of tissue transglutaminase monodansyl-cadaverine. The effect of this treatment on the development of crypt hyperplasia and villous atrophy were examined by light microscopy of hematoxylin-eosin (H&E) stained jejunal paraffin sections. We found a strong subepithelial expression of tissue transglutaminase in GvH animals but not in syngenic control mice. The localization of tTG seemed to be associated with the extracellular matrix (ECM). However, neither the treatment of GvH animals with anti-tTG antibodies nor the application of mono-dansyl-cadaverine prevented the development of crypt hyperplasia and villous atrophy. Similar to the situation in human celiac disease tissue, transglutaminase is highly expressed in the intestine of animals undergoing a semi-allogenic graft-versus-host reaction. However, this enzyme is probably not involved in the development of crypt hyperplasia and villous atrophy in this animal model.