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.

Glucocorticoids attenuate acute graft-versus-host disease by suppressing the cytotoxic capacity of CD8(+) T cells.

Glucocorticoids (GCs) are released from the adrenal gland during inflammation and help to keep immune responses at bay. Owing to their potent anti-inflammatory activity, GCs also play a key role in controlling acute graft-versus-host disease (aGvHD). Here we demonstrate that mice lacking the glucocorticoid receptor (GR) in T cells develop fulminant disease after allogeneic bone marrow transplantation. In a fully MHC-mismatched model, transfer of GR-deficient T cells resulted in severe aGvHD symptoms and strongly decreased survival times. Histopathological features were aggravated and infiltration of CD8(+) T cells into the jejunum was increased when the GR was not expressed. Furthermore, serum levels of IL-2, IFNγ, and IL-17 were elevated and the cytotoxicity of CD8(+) T cells was enhanced after transfer of GR-deficient T cells. Short-term treatment with dexamethasone reduced cytokine secretion but neither impacted disease severity nor the CTLs' cytolytic capacity. Importantly, in an aGvHD model in which disease development exclusively depends on the presence of CD8(+) T cells in the transplant, transfer of GR-deficient T cells aggravated clinical symptoms and reduced survival times as well. Taken together, our findings highlight that suppression of CD8(+) T-cell function is a crucial mechanism in the control of aGvHD by endogenous GCs.

Acid sphingomyelinase is required for protection of effector memory T cells against glucocorticoid-induced cell death.

The activity of acid sphingomyelinase (aSMase) was previously reported to be involved in glucocorticoid-induced cell death (GICD) of T lymphocytes. This mechanism in turn is believed to contribute to the therapeutic efficacy of glucocorticoids (GCs) in the treatment of inflammatory diseases. In this study, we reassessed the role of aSMase in GICD by using aSMase knockout mice. The absence of aSMase largely abolished the partial protection that effector memory CD4(+) T cells in wild-type mice possess against GICD. Reduced IL-2 secretion by aSMase-deficient CD4(+) T cells suggested that a lack of this important survival factor might be the cause of these cells' enhanced susceptibility to GICD. Indeed, addition of IL-2 restored the protection against GICD, whereas neutralization of IL-2 abrogated the otherwise protective effect seen in wild-type effector memory CD4(+) T cells. The therapeutic implications of the altered sensitivity of aSMase-deficient T cells to GICD were assessed in models of inflammatory disorders; namely, experimental autoimmune encephalomyelitis and acute graft-versus-host disease. Surprisingly, GC treatment was equally efficient in both models in terms of ameliorating the diseases, regardless of the genotype of the T cells. Thus, our data reveal a hitherto unrecognized contribution of aSMase to the sensitivity of effector memory CD4(+) T cells to GICD and call into question the traditionally attributed importance of GICD of T cells to the treatment of inflammatory diseases by GCs.

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.

Characterization of testicular macrophage subpopulations in mice.

Testis is an immune privileged site, a feature that prevents germ cells from eliciting an autoimmune response. Macrophages contribute to this state of tolerance by adopting an immunoregulatory phenotype. Here, we further characterized their features in mice by analyzing surface markers, anatomic localization as well as morphology and function. Testicular macrophages (TMΦ) were stained for various surface receptors, and MHCII and CD206 were found to be most suitable to discriminate between two subpopulations. Our immunohistochemical analysis further confirmed a predominant localization of CD206+ cells in the interstitial space. Imaging flow cytometry revealed that both subtypes of TMΦ differed in size and contrast, and to some extent also in their ability to engulf high-molecular dextran. To investigate whether the polarization of the immune system had any influence on the phenotype of TMΦ, we compared C57BL/6 and BALB/c mice. Importantly, our analysis revealed that the abundance of cells expressing either MHCII or any of the scavenger receptors CD206, CD163 and CD71 differed between both mouse strains. In addition, the presence of the glucocorticoid receptor in macrophages affected the ratio between individual subpopulations, which is consistent with a crucial role of glucocorticoids in macrophage polarization. Collectively, our results indicate that TMΦ are composed in a variable ratio of distinct subsets with characteristic features, which may shape the immune privilege of the testis also in humans.

A flow cytometric approach to study glucocorticoid receptor expression in immune cell subpopulations of genetically engineered mice.

Glucocorticoids (GCs) constitute one of the most powerful classes of anti-inflammatory agents and are used for the treatment of a plethora of diseases related to autoimmunity, allergy, cancer, and infection. In the last two decades, multiple studies using genetically engineered mice with targeted deletions of the GC receptor (GR) in individual cell types have provided insights into the mechanisms of GCs in the control of the immune system. The characterization of GR expression in these mouse models, however, mostly relied on the analysis of mRNA expression or reporter gene activity. In contrast, approaches directly detecting the GR protein on a cellular level are scarce. Thus, we here used a flow cytometric method to analyze mice in which the GR gene locus was disrupted with the help of a Cre recombinase expressed under the control of either the lck or the lysM promoter. Measuring GR protein expression in immune cell subpopulations unveiled an efficient and highly selective depletion in both strains of knock-out mice in accordance with the expected cellular specificity of the employed promoters for T cells or myeloid cells, respectively. The flow cytometric data were well in line with those from the analysis of GR mRNA expression in magnetically sorted immune cell subpopulations but they could be obtained much more quickly. In summary, our data indicate that flow cytometry is a powerful tool with which to define GR protein content at a single cell level when studying the function of GCs in the immune system.

The Glucocorticoid Receptor in Intestinal Epithelial Cells Alleviates Colitis and Associated Colorectal Cancer in Mice.

BACKGROUND & AIMS: Inflammatory bowel disease is commonly treated by administration of glucocorticoids. While the importance of intestinal epithelial cells for the pathogenesis of this disorder is widely accepted, their role as target cells for glucocorticoids has not been explored. To address this issue, we induced colonic inflammation in GRvillin mice, which carry an inducible deletion of the glucocorticoid receptor in intestinal epithelial cells. METHODS: Colitis and colitis-associated colorectal cancer were induced by administration of dextran sulfate sodium and azoxymethane in mice. Clinical parameters, epithelial permeability and tumor development were monitored during disease progression. Colon tissue, lamina propria cells and intestinal epithelial cells were examined by gene expression analyses, flow cytometry, histopathology, and immunohistochemistry. RESULTS: The absence of the intestinal epithelial glucocorticoid receptor aggravated clinical symptoms and tissue damage, and compromised epithelial barrier integrity during colitis. Gene expression of chemokines, pattern recognition receptors and molecules controlling epithelial permeability was dysregulated in intestinal epithelial cells of GRvillin mice, leading to a reduced recruitment and a hyperactivation of leukocytes in the lamina propria of the colon. Importantly, the exaggerated inflammatory response in GRvillin mice also enhanced associated tumorigenesis, resulting in a higher number and larger size of tumors in the colon. CONCLUSIONS: Our results reveal an important role of intestinal epithelial cells as targets of glucocorticoid action in inflammatory bowel disease and suggest that the efficacy with which colitis is kept at bay directly affects the progression of colorectal cancer.

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.

Glucocorticoid resistance of allogeneic T cells alters the gene expression profile in the inflamed small intestine of mice suffering from acute graft-versus-host disease.

Glucocorticoids (GCs) play an important role in controlling acute graft-versus-host disease (aGvHD), a frequent complication of allogeneic hematopoietic stem cell transplantation. The anti-inflammatory activity of GCs is mainly ascribed to the modulation of T cells and macrophages, for which reason a genetically induced GC resistance of either of these cell types causes aggravated aGvHD. Since only a few genes are currently known that are differentially regulated under these conditions, we analyzed the expression of 54 candidate genes in the inflamed small intestine of mice suffering from aGvHD when either allogeneic T cells or host myeloid cells were GC resistant using a microfluidic dynamic array platform for high-throughput quantitative PCR. The majority of genes categorized as cytokines (e.g. Il2, Il6), chemokines (e.g. Ccl2, Cxcl1), cell surface receptors (e.g. Fasl, Ctla4) and intracellular molecules (e.g. Dusp1, Arg1) were upregulated in mice transplanted with GC resistant allogeneic T cells. Moreover, the expression of several genes linked to energy metabolism (e.g. Glut1) was altered. Surprisingly, mice harboring GC resistant myeloid cells showed almost no changes in gene expression despite their fatal disease course after aGvHD induction. To identify additional genes in the inflamed small intestine that were affected by a GC resistance of allogeneic T cells, we performed an RNAseq analysis, which uncovered more than 500 differentially expressed transcripts (e.g. Cxcr6, Glut3, Otc, Aoc1, Il1r1, Sphk1) that were enriched for biological processes associated with inflammation and tissue disassembly. The changes in gene expression could be confirmed during full-blown disease but hardly any of them in the preclinical phase using high-throughput quantitative PCR. Further analysis of some of these genes revealed a highly selective expression pattern in T cells, intestinal epithelial cells and macrophages, which correlated with their regulation during disease progression. Collectively, we identified an altered gene expression profile caused by GC resistance of transplanted allogeneic T cells, which could help to define new targets for aGvHD therapy.

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

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 GRlysM 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 GRflox mice, the disease further aggravated in GRlysM 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 GRlysM 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.

The glucocorticoid receptor in recipient cells keeps cytokine secretion in acute graft-versus-host disease at bay.

Graft-versus-host disease (GvHD) is a life-threatening complication of hematopoietic stem cell transplantation (HSCT), which is caused by allogeneic T cells recognizing molecules of the recipient as foreign. Endogenous glucocorticoids (GC) released from the adrenal gland are crucial in regulating such inflammatory diseases. Here we demonstrate that genetically engineered mice, that are largely unresponsive to GC, suffer from aggravated clinical symptoms and increased mortality after HSCT, effects that could be tempered by neutralization of IL-6. Interestingly, selective ablation of the GC receptor (GR) in recipient myeloid cells resulted in fulminant disease as well. While histopathological analysis of the jejunum failed to reveal any differences between sick mice of both genotypes, systemic IL-6 and TNFα secretion was strongly increased in transplanted mice lacking the GR in myeloid cells briefly before the majority of them succumbed to the disease. Collectively, our findings reveal an important role of the GR in recipient cells in limiting the cytokine storm caused by GvHD induction.

Targeted delivery of glucocorticoids to macrophages in a mouse model of multiple sclerosis using inorganic-organic hybrid nanoparticles.

Glucocorticoids (GC) are widely used to treat acute relapses in multiple sclerosis (MS) patients, but their application is accompanied by side effects due to their broad spectrum of action. Here, we report on the therapeutic option to apply GC via inorganic-organic hybrid nanoparticles (IOH-NP) with the composition [ZrO]2+[(BMP)0.9(FMN)0.1]2- (designated BMP-NP with BMP: betamethasone phosphate; FMN: flavinmononucleotide). We found that these BMP-NP have an increased cell type-specificity compared to free GC while retaining full therapeutic efficacy in a mouse model of MS. BMP-NP were preferentially taken up by phagocytic cells and modulated macrophages in vivo more efficiently than T cells. When GC were applied in the form of BMP-NP, treatment of neuroinflammatory disease in mice exclusively depended on the control of macrophage function whereas effects on T cells and brain endothelial cells were dispensable for therapeutic efficacy. Importantly, BMP-NP were not only active in mice but also showed strong activity towards monocytes isolated from healthy human volunteers. We conclude that application of GC via IOH-NP has the potential to improve MS therapy in the future.

T cell abundance in blood predicts acute organ toxicity in chemoradiotherapy for head and neck cancer.

Treatment of head and neck squamous cell carcinoma (HNSCC) by chemoradiotherapy (CRT) often results in high-grade acute organ toxicity (HGAOT). As these adverse effects impair the patients' quality of life and the feasibility of the planned therapy, we sought to analyze immunological parameters in tumor material and blood samples obtained from 48 HNSCC patients in order to assess the potential to predict the individual acute organ toxicity. T cells in the tumor stroma were enriched in patients developing HGAOT whereas levels of soluble factors in the plasma and gene expression in whole blood did not coincide with the occurrence of acute organ toxicity. In contrast, the frequency and absolute numbers of selected leukocyte subpopulations measured in samples of peripheral blood mononuclear cells (PBMCs) directly before the beginning of CRT were significantly different in patients with HGAOT as compared to those without. When we validated several potential markers including the abundance of T cells in a small prospective study with 16 HNSCC patients, we were able to correctly predict acute organ toxicity in up to 81% of the patients. We conclude that analysis of PBMCs by fluorescence-activated cell sorting (FACS) might be a convenient strategy to identify patients at risk of developing HGAOT caused by CRT, which might allow to adapt the treatment regimen and possibly improve disease outcome.

Glucocorticoids induce gastroparesis in mice through depletion of l-arginine.

Glucocorticoids (GCs) constitute a highly pleiotropic class of drugs predominantly employed in the treatment of inflammatory diseases. In our search for new mechanisms of action, we identified a hitherto unknown effect of GCs in the gastrointestinal tract. We found that oral administration of dexamethasone (Dex) to mice caused an enlargement of the stomach due to the induction of gastroparesis and that this effect was abolished in GR(dim) mice carrying the A458T mutation in the GC receptor (GR). Gastroparesis was unrelated to the enhanced gastric acid secretion observed after Dex treatment, although both effects were mediated by the same molecular mechanism of the GR. Using conditional GR-knockout mice, we could further rule out that GC effects on enterocytes or myeloid cells were involved in the induction of gastroparesis. In contrast, we found that Dex upregulated arginase 2 (Arg2) in the stomach both at the mRNA and protein level. This suggests that GC treatment leads to a depletion of l-arginine thereby impeding the production of nitric oxide (NO), which is required for gastric motility. We tested this hypothesis by supplementing the drinking water of the mice with exogenous l-arginine to compensate for the presumed shortage of this major substrate of NO synthases. Importantly, this measure completely prevented both the enlargement of the stomach and the induction of gastroparesis after Dex treatment. Our findings raise considerations of combining orally applied GCs with l-arginine to improve tolerability of GC treatment and provide a possible explanation for the antiemetic effects of GCs widely exploited in chemotherapy.

Glucocorticoids enhance intestinal glucose uptake via the dimerized glucocorticoid receptor in enterocytes.

Glucocorticoid (GC) treatment of inflammatory disorders, such as inflammatory bowel disease, causes deranged metabolism, in part by enhanced intestinal resorption of glucose. However, the underlying molecular mechanism is poorly understood. Hence, we investigated transcriptional control of genes reported to be involved in glucose uptake in the small intestine after GC treatment and determined effects of GC on electrogenic glucose transport from transepithelial currents. GR(villinCre) mice lacking the GC receptor (GR) in enterocytes served to identify the target cell of GC treatment and the requirement of the GR itself; GR(dim) mice impaired in dimerization and DNA binding of the GR were used to determine the underlying molecular mechanism. Our findings revealed that oral administration of dexamethasone to wild-type mice for 3 d increased mRNA expression of serum- and GC-inducible kinase 1, sodium-coupled glucose transporter 1, and Na(+)/H(+) exchanger 3, as well as electrogenic glucose transport in the small intestine. In contrast, GR(villinCre) mice did not respond to GC treatment, neither with regard to gene activation nor to glucose transport. GR(dim) mice were also refractory to GC, because dexamethasone treatment failed to increase both, gene expression and electrogenic glucose transport. In addition, the rise in blood glucose levels normally observed after GC administration was attenuated in both mutant mouse strains. We conclude that enhanced glucose transport in vivo primarily depends on gene regulation by the dimerized GR in enterocytes, and that this mechanism contributes to GC-induced hyperglycemia.

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(T(reg)) cells may contribute to the immunosuppressive effects of glucocorticoids (GCs). OBJECTIVE: We readdressed the influence of GC therapy on T(reg) cells in immunocompetent human subjects and naïve mice. METHODS: Mice were treated with increasing doses of intravenous dexamethasone followed by oral taper, and T(reg) 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 T(reg) 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 T(reg) cells in blood (100 mg dexamethasone/kg body weight: 2.8±1.8×10(4) cells/ml vs. 33±11×10(4) in control mice) and spleen (dexamethasone: 2.8±1.9×10(5)/spleen vs. 95±22×10(5)/spleen in control mice), which slowly recovered after 14 days taper in spleen but not in blood. The relative frequency of FOXP3(+) T(reg) 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 T(reg) 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 T(reg) cells in a relevant manner, although there was some variation depending on the definition of the T(reg) cells (FOXP3(+): 4.0±1.5% vs 3.4±1.5%*; AITR(+): 0.6±0.4 vs 0.5±0.3%, CD127(low): 4.0±1.3 vs 5.0±3.0%* and CTLA4+: 13.8±11.5 vs 15.6±12.5%; * p<0.05). CONCLUSION: Short-term GC therapy does not induce the hitherto supposed increase in circulating T(reg) cell frequency, neither in immunocompetent humans nor in mice. Thus, it is questionable that the clinical efficacy of GCs is achieved by modulating T(reg) cell numbers.