Physiological and molecular features of glucocorticoid actions in the gastrointestinal tract

Ever since their first successful application in the treatment of RA patients in the late 1950, GCs have been the gold standard for the treatment of multiple inflammatory and neoplastic diseases. There are, however, also severe adverse effects that denote restrictions upon the use of GCs. In particular oral application of GCs can lead to gastrointestinal complications that may severely affect the patient’s quality of life and lead to a reduced tolerability of the therapy. In this work I have described and characterized gastroparesis as a so far unrecognized effect of GCs in the gastrointestinal tract that is mediated via trans-activation of genes. Changes in gene expression characteristic for M2 macrophage polarization proved to be unrelated to gastroparesis. Similarly, an involvement of two genes specifically expressed in the gastrointestinal tract could be ruled out. In contrast, I could confirm that genes related to the regulation of NO production contribute to gastroparesis. A decrease in iron availability through up-regulation of Lcn2 was found to partially impact on gastric motility whereas reduced substrate availability for NO synthesis through up-regulation of Arg2 proved to be responsible for impaired gastric emptying. Hence, GC therapy causes gastroparesis by increasing gene expression in the stomach in a DNA-binding-dependent manner thereby diminishing the availability of NO required for gastric motility. Complete prevention of gastroparesis was achieved by an exogenous supply of L-arginine thus providing a means to overcome the observed effect with the help of a dietary supplement. My study also offers a possible explanation for the anti-emetic effect of GCs that has been used for long to interfere with CINV without knowing the underlying mechanism. Now it appears likely that reducing NO availability in the stomach is the way how GCs counteract CINV, which is accompanied by increased NO production. Unfortunately, further elucidation of this process is impossible in rodent models due to the inability of mice and rats to vomit. Taken together, the identification and characterization of GC-induced gastroparesis sheds new light on both adverse and beneficial activities of GCs in the stomach and may help to optimize therapy in the future for the patients’ benefit

Glucocorticoids delivered by inorganic‐organic hybrid nanoparticles mitigate acute graft‐versus‐host disease and sustain graft‐versus‐leukemia activity

Glucocorticoids (GCs) are widely used to treat acute graft‐versus‐host disease (aGvHD) due to their immunosuppressive activity, but they also reduce the beneficial graft‐versus‐leukemia (GvL) effect of the allogeneic T cells contained in the graft. Here, we tested whether aGvHD therapy could be improved by delivering GCs with the help of inorganic–organic hybrid nanoparticles (IOH‐NPs) that preferentially target myeloid cells. IOH‐NPs containing the GC betamethasone (BMP‐NPs) efficiently reduced morbidity, mortality, and tissue damage in a totally MHC mismatched mouse model of aGvHD. Therapeutic activity was lost in mice lacking the GC receptor (GR) in myeloid cells, confirming the cell type specificity of our approach. BMP‐NPs had no relevant systemic activity but suppressed cytokine and chemokine gene expression locally in the small intestine, which presumably explains their mode of action. Most importantly, BMP‐NPs delayed the development of an adoptively transferred B cell lymphoma better than the free drug, although the overall incidence was unaffected. Our findings thus suggest that employing IOH‐NPs could diminish the risk of relapse associated with GC therapy of aGvHD patients while still allowing to efficiently ameliorate the disease

Influence of Short-Term Glucocorticoid Therapy on Regulatory T Cells In Vivo

Background: Pre- and early clinical studies on patients with autoimmune diseases suggested that induction of regulatory T(Treg) cells may contribute to the immunosuppressive effects of glucocorticoids(GCs). Objective: We readdressed the influence of GC therapy on Treg cells in immunocompetent human subjects and naı¨ve mice. Methods: Mice were treated with increasing doses of intravenous dexamethasone followed by oral taper, and Treg cells in spleen and blood were analyzed by FACS. Sixteen patients with sudden hearing loss but without an inflammatory disease received high-dose intravenous prednisolone followed by stepwise dose reduction to low oral prednisolone. Peripheral blood Treg cells were analyzed prior and after a 14 day GC therapy based on different markers. Results: Repeated GC administration to mice for three days dose-dependently decreased the absolute numbers of Treg cells in blood (100 mg dexamethasone/kg body weight: 2.861.86104 cells/ml vs. 336116104 in control mice) and spleen (dexamethasone: 2.861.96105/spleen vs. 956226105/spleen in control mice), which slowly recovered after 14 days taper in spleen but not in blood. The relative frequency of FOXP3+ Treg cells amongst the CD4+ T cells also decreased in a dose dependent manner with the effect being more pronounced in blood than in spleen. The suppressive capacity of Treg cells was unaltered by GC treatment in vitro. In immunocompetent humans, GCs induced mild T cell lymphocytosis. However, it did not change the relative frequency of circulating Treg cells in a relevant manner, although there was some variation depending on the definition of the Treg cells (FOXP3+: 4.061.5% vs 3.461.5%*; AITR+: 0.660.4 vs 0.560.3%, CD127low: 4.061.3 vs 5.063.0%* and CTLA4+: 13.8611.5 vs 15.6612.5%; * p,0.05). Conclusion: Short-term GC therapy does not induce the hitherto supposed increase in circulating Treg cell frequency, neither in immunocompetent humans nor in mice. Thus, it is questionable that the clinical efficacy of GCs is achieved by modulating Treg cell numbers

Phenotype and gene signature of testicular tumors in 129.MOLF‐Chr19 mice resemble human teratomas

Abstract Background Testicular germ cell tumor (TGCT) is the most common type of tumor in young men. Type II germ cell tumors including postpubertal‐type teratomas are derived from the germ cell neoplasia in situ (GCNIS), whereas prepubertal‐type teratomas arise independently of the GCNIS. The consomic mouse strain 129.MOLF‐Chr19 (M19) is a suitable murine model of such tumors, but its characterization remains incomplete. Objective Here, we interrogated the suitability of testicular tumors in M19 mice as a model of human TGCT by analyzing their histological features and gene expression signature. Material and methods Testes collected from M19 mice of different ages were categorized by macroscopic appearance based on size and the degree of suspected tumorigenesis. Histological sections from selected tumors were stained with Hematoxylin and Eosin, and expression of genes associated with tumorigenesis was determined in frozen tissue samples from a large range of tumors of different subclasses using RT‐qPCR and Fluidigm Dynamic Arrays. Results Macroscopically, testicular specimens appeared very heterogeneous concerning size and signs indicating the presence of a tumor. Histological analysis confirmed the development of teratomas with areas of cells corresponding to all three germ cell layers. Gene expression analyses indicated upregulation of markers related to proliferation, vascular invasive potential and pluripotency, and revealed a strong correlation of gene expression with tumor size and a significant intercorrelation of individual genes. Discussion and conclusion TGCT in M19 mice is reminiscent of human testicular teratomas presenting with areas of cells derived from all germ layers and showing a typical gene signature. We thus confirm that these mice can serve as a suitable murine model of pure teratomas for preclinical research.Deutsche Forschungsgemeinschaft https://doi.org/10.13039/50110000165

The Subtype Identity of Testicular Cancer Cells Determines Their Immunostimulatory Activity in a Coculture Model

Testicular germ cell cancer (TGCC) is subdivided into several subtypes. While seminomatous germ cell tumors (SGCT) are characterized by an intensive infiltration of immune cells which constitute a pro-inflammatory tumor micromilieu (TME), immune cells in non-seminomatous germ cell tumors (NSGCT) are differently composed and less abundant. Previously, we have shown that the seminomatous cell line TCam-2 promotes T cell and monocyte activation in a coculture model, resulting in mutual interactions between both cell types. Here we set out to compare this feature of TCam-2 cells with the non-seminomatous cell line NTERA-2. Peripheral blood T cells or monocytes cocultured with NTERA-2 cells failed to secrete relevant amounts of pro-inflammatory cytokines, and significantly downregulated the expression of genes encoding activation markers and effector molecules. In contrast, immune cells cocultured with TCam-2 cells produced IL-2, IL-6 and TNFα, and strongly upregulated the expression of multiple pro-inflammatory genes. Furthermore, the expression of genes involved in proliferation, stemness and subtype specification remained unaltered in NTERA-2 cells during coculture with T cells or monocytes, indicating the absence of mutual interactions. Collectively, our findings uncover fundamental differences between SGCT and NSGCT in their capability to generate a pro-inflammatory TME, which possibly impacts the clinical features and prognosis of both TGCC subtypes.Deutsche ForschungsgemeinschaftDeutsche ForschungsgemeinschaftOpen Access Publication Funds of the University of GöttingenOpen-Access-Publikationsfonds 202

The Role of Glucocorticoids in Inflammatory Diseases

For more than 70 years, glucocorticoids (GCs) have been a powerful and affordable treatment option for inflammatory diseases. However, their benefits do not come without a cost, since GCs also cause side effects. Therefore, strong efforts are being made to improve their therapeutic index. In this review, we illustrate the mechanisms and target cells of GCs in the pathogenesis and treatment of some of the most frequent inflammatory disorders affecting the central nervous system, the gastrointestinal tract, the lung, and the joints, as well as graft-versus-host disease, which often develops after hematopoietic stem cell transplantation. In addition, an overview is provided of novel approaches aimed at improving GC therapy based on chemical modifications or GC delivery using nanoformulations. GCs remain a topic of highly active scientific research despite being one of the oldest class of drugs in medical use

Impaired resolution of DSS-induced colitis in mice lacking the glucocorticoid receptor in myeloid cells

<div><p>Inflammatory bowel disease (IBD) is a highly prevalent intestinal disorder for which no cure exists. Currently, the standard first-line treatment of IBD consists of systemic glucocorticoid (GC) application, even though therapy can be complicated by unresponsiveness or adverse effects. In view of the importance of macrophages and neutrophils for the pathogenesis of IBD we set out to define the relevance of these cell types as targets of GC using the mouse model of DSS-induced colitis. We found that the disease did not resolve in GR^lysM mice lacking the GC receptor (GR) in myeloid cells after removal of the chemical insult. While clinical symptoms and tissue damage in the colon ameliorated again in GR^flox mice, the disease further aggravated in GR^lysM littermates. The observed difference coincided with an increased abundance of macrophages in inflammatory infiltrates in the colon of mutant mice whereas neutrophil and T cell numbers were similar. Concomitantly, systemic IL-6 secretion and mRNA levels of pro-inflammatory cytokines in the colon were elevated in GR^lysM mice and gene expression of scavenger receptors and IL-10 was diminished. Taken together, our results reveal an important role of myeloid cells as targets of GC in DSS-induced colitis and probably in IBD in humans as well.</p></div

The Subtype Identity of Testicular Cancer Cells Determines Their Immunostimulatory Activity in a Coculture Model

Testicular germ cell cancer (TGCC) is subdivided into several subtypes. While seminomatous germ cell tumors (SGCT) are characterized by an intensive infiltration of immune cells which constitute a pro-inflammatory tumor micromilieu (TME), immune cells in non-seminomatous germ cell tumors (NSGCT) are differently composed and less abundant. Previously, we have shown that the seminomatous cell line TCam-2 promotes T cell and monocyte activation in a coculture model, resulting in mutual interactions between both cell types. Here we set out to compare this feature of TCam-2 cells with the non-seminomatous cell line NTERA-2. Peripheral blood T cells or monocytes cocultured with NTERA-2 cells failed to secrete relevant amounts of pro-inflammatory cytokines, and significantly downregulated the expression of genes encoding activation markers and effector molecules. In contrast, immune cells cocultured with TCam-2 cells produced IL-2, IL-6 and TNFα, and strongly upregulated the expression of multiple pro-inflammatory genes. Furthermore, the expression of genes involved in proliferation, stemness and subtype specification remained unaltered in NTERA-2 cells during coculture with T cells or monocytes, indicating the absence of mutual interactions. Collectively, our findings uncover fundamental differences between SGCT and NSGCT in their capability to generate a pro-inflammatory TME, which possibly impacts the clinical features and prognosis of both TGCC subtypes

Systemic IL-6 secretion during DSS-induced colitis in GR^flox and GR^lysM mice.

<p>Mice received 2% DSS in the drinking water for 8 days and were sacrificed on day 10 or 12. Mice receiving tap water served as controls. Blood was collected through cardiac puncture and serum IL-6 levels were determined by ELISA. GR^flox mice: N = 5/3/4 (con/10/12), GR^lysM mice: N = 6/7/5 (con/10/12). Statistical analysis was performed by unpaired two-tailed Student’s t test (*: p <0.05, **: p <0.01).</p

Immunohistochemical analysis of the colon during the resolution phase of DSS-induced colitis in GR^flox and GR^lysM mice.

<p>Mice received 2% DSS in the drinking water for 8 days and were sacrificed on day 12. Mice receiving tap water served as controls. The colons were flushed, opened longitudinally and rolled up from distal to proximal to obtain a “swiss-role” for histological analysis. Representative photomicrographs from 2 μm colonic tissue sections incubated with an (A) anti-CD3 or (B) anti-GR1 antibody at 20x (upper panel) or 40x (lower panel) magnification. Photomicrographs of DSS-treated mice in both panels are representative of heavily inflamed areas of the colon. Scale bars correspond to 200 μm and 100 μm, respectively.</p