Granzyme A Required for Regulatory T-Cell Mediated Prevention of Gastrointestinal Graft-versus-Host Disease

In our previous work we could identify defects in human regulatory T cells (Tregs) likely favoring the development of graft-versus-host disease (GvHD) following allogeneic stem cell transplantation (SCT). Treg transcriptome analyses comparing GvHD and immune tolerant patients uncovered regulated gene transcripts highly relevant for Treg cell function. Moreover, granzyme A (GZMA) also showed a significant lower expression at the protein level in Tregs of GvHD patients. GZMA induces cytolysis in a perforin-dependent, FAS- FASL independent manner and represents a cell-contact dependent mechanism for Tregs to control immune responses. We therefore analyzed the functional role of GZMA in a murine standard model for GvHD. For this purpose, adoptively transferred CD4+CD25+ Tregs from gzmA-/- mice were analyzed in comparison to their wild type counterparts for their capability to prevent murine GvHD. GzmA-/- Tregs home efficiently to secondary lymphoid organs and do not show phenotypic alterations with respect to activation and migration properties to inflammatory sites. Whereas gzmA-/- Tregs are highly suppressive in vitro, Tregs require GZMA to rescue hosts from murine GvHD, especially regarding gastrointestinal target organ damage. We herewith identify GZMA as critical effector molecule of human Treg function for gastrointestinal immune response in an experimental GvHD model

Impact of the rpoS genotype for acid resistance patterns of pathogenic and probiotic Escherichia coli

BACKGROUND: Enterohemorrhagic E. coli (EHEC), a subgroup of Shiga toxin (Stx) producing E. coli (STEC), may cause severe enteritis and hemolytic uremic syndrome (HUS) and is transmitted orally via contaminated foods or from person to person. The infectious dose is known to be very low, which requires most of the bacteria to survive the gastric acid barrier. Acid resistance therefore is an important mechanism of EHEC virulence. It should also be a relevant characteristic of E. coli strains used for therapeutic purposes such as the probiotic E. coli Nissle 1917 (EcN). In E. coli and related enteric bacteria it has been extensively demonstrated, that the alternative sigma factor σ(S), encoded by the rpoS gene, acts as a master regulator mediating resistance to various environmental stress factors. METHODS: Using rpoS deletion mutants of a highly virulent EHEC O26:H11 patient isolate and the sequenced prototype EHEC EDL933 (ATCC 700927) of serotype O157:H7 we investigated the impact of a functional rpoS gene for orchestrating a satisfactory response to acid stress in these strains. We then functionally characterized rpoS of probiotic EcN and five rpoS genes selected from STEC isolates pre-investigated for acid resistance. RESULTS: First, we found out that ATCC isolate 700927 of EHEC EDL933 has a point mutation in rpoS, not present in the published sequence, leading to a premature stop codon. Moreover, to our surprise, one STEC strain as well as EcN was acid sensitive in our test environment, although their cloned rpoS genes could effectively complement acid sensitivity of an rpoS deletion mutant. CONCLUSION: The attenuation of sequenced EHEC EDL933 might be of importance for anyone planning to do either in vitro or in vivo studies with this prototype strain. Furthermore our data supports recently published observations, that individual E. coli isolates are able to significantly modulate their acid resistance phenotype independent of their rpoS genotype

The host response to the probiotic Escherichia coli strain Nissle 1917: Specific up-regulation of the proinflammatory chemokine MCP-1

BACKGROUND: The use of live microorganisms to influence positively the course of intestinal disorders such as infectious diarrhea or chronic inflammatory conditions has recently gained increasing interest as a therapeutic alternative. In vitro and in vivo investigations have demonstrated that probiotic-host eukaryotic cell interactions evoke a large number of responses potentially responsible for the effects of probiotics. The aim of this study was to improve our understanding of the E. coli Nissle 1917-host interaction by analyzing the gene expression pattern initiated by this probiotic in human intestinal epithelial cells. METHODS: Gene expression profiles of Caco-2 cells treated with E. coli Nissle 1917 were analyzed with microarrays. A second human intestinal cell line and also pieces of small intestine from BALB/c mice were used to confirm regulatory data of selected genes by real-time RT-PCR and cytometric bead array (CBA) to detect secretion of corresponding proteins. RESULTS: Whole genome expression analysis revealed 126 genes specifically regulated after treatment of confluent Caco-2 cells with E. coli Nissle 1917. Among others, expression of genes encoding the proinflammatory molecules monocyte chemoattractant protein-1 ligand 2 (MCP-1), macrophage inflammatory protein-2 alpha (MIP-2α) and macrophage inflammatory protein-2 beta (MIP-2β) was increased up to 10 fold. Caco-2 cells cocultured with E. coli Nissle 1917 also secreted high amounts of MCP-1 protein. Elevated levels of MCP-1 and MIP-2α mRNA could be confirmed with Lovo cells. MCP-1 gene expression was also up-regulated in mouse intestinal tissue. CONCLUSION: Thus, probiotic E. coli Nissle 1917 specifically upregulates expression of proinflammatory genes and proteins in human and mouse intestinal epithelial cells

The host response to the probiotic <it>Escherichia coli </it>strain Nissle 1917: Specific up-regulation of the proinflammatory chemokine MCP-1

Abstract Background The use of live microorganisms to influence positively the course of intestinal disorders such as infectious diarrhea or chronic inflammatory conditions has recently gained increasing interest as a therapeutic alternative. In vitro and in vivo investigations have demonstrated that probiotic-host eukaryotic cell interactions evoke a large number of responses potentially responsible for the effects of probiotics. The aim of this study was to improve our understanding of the E. coli Nissle 1917-host interaction by analyzing the gene expression pattern initiated by this probiotic in human intestinal epithelial cells. Methods Gene expression profiles of Caco-2 cells treated with E. coli Nissle 1917 were analyzed with microarrays. A second human intestinal cell line and also pieces of small intestine from BALB/c mice were used to confirm regulatory data of selected genes by real-time RT-PCR and cytometric bead array (CBA) to detect secretion of corresponding proteins. Results Whole genome expression analysis revealed 126 genes specifically regulated after treatment of confluent Caco-2 cells with E. coli Nissle 1917. Among others, expression of genes encoding the proinflammatory molecules monocyte chemoattractant protein-1 ligand 2 (MCP-1), macrophage inflammatory protein-2 alpha (MIP-2α) and macrophage inflammatory protein-2 beta (MIP-2β) was increased up to 10 fold. Caco-2 cells cocultured with E. coli Nissle 1917 also secreted high amounts of MCP-1 protein. Elevated levels of MCP-1 and MIP-2α mRNA could be confirmed with Lovo cells. MCP-1 gene expression was also up-regulated in mouse intestinal tissue. Conclusion Thus, probiotic E. coli Nissle 1917 specifically upregulates expression of proinflammatory genes and proteins in human and mouse intestinal epithelial cells.</p

Granzyme A Is Required for Regulatory T-Cell Mediated Prevention of Gastrointestinal Graft-versus-Host Disease

<div><p>In our previous work we could identify defects in human regulatory T cells (Tregs) likely favoring the development of graft-versus-host disease (GvHD) following allogeneic stem cell transplantation (SCT). Treg transcriptome analyses comparing GvHD and immune tolerant patients uncovered regulated gene transcripts highly relevant for Treg cell function. Moreover, granzyme A (GZMA) also showed a significant lower expression at the protein level in Tregs of GvHD patients. GZMA induces cytolysis in a perforin-dependent, FAS-FASL independent manner and represents a cell-contact dependent mechanism for Tregs to control immune responses. We therefore analyzed the functional role of GZMA in a murine standard model for GvHD. For this purpose, adoptively transferred CD4⁺CD25⁺ Tregs from <i>gzmA</i>^-/- mice were analyzed in comparison to their wild type counterparts for their capability to prevent murine GvHD. <i>GzmA</i>^-/- Tregs home efficiently to secondary lymphoid organs and do not show phenotypic alterations with respect to activation and migration properties to inflammatory sites. Whereas <i>gzmA</i>^-/- Tregs are highly suppressive <i>in vitro</i>, Tregs require GZMA to rescue hosts from murine GvHD, especially regarding gastrointestinal target organ damage. We herewith identify GZMA as critical effector molecule of human Treg function for gastrointestinal immune response in an experimental GvHD model.</p></div

<i>GzmA</i>^<i>-/-</i> Tregs are required for prevention of gastrointestinal GvHD.

<p>Lethally irradiated BALB/c mice (n = 9) received T cell depleted (TCD) bone marrow cells (BM) together with CD4⁺CD25^- T effector cells (squares, n = 3) and CD4⁺CD25⁺ WT Treg cells (circle, n = 3) or CD4⁺CD25⁺<i>gzmA</i>^<i>-/-</i> Treg cells (triangle, n = 3) from WT mice. (A) Body weight of the recipients was monitored daily. (B) Histopathological investigations of liver, small and large intestine of mice sacrified after 3 weeks. (C) Histopathological GvHD scoring 3 weeks after transplantation were graded for severity of inflammation and degree of organ destruction resulting in an organ-specific cumulative pathology score. For statistical analysis student`s t-test was performed and p values <0.05 (*) were considered significant, while p<0.01 (**) and p<0.001 (**) were considered highly significant.</p

<i>GzmA</i>^<i>-/-</i> Tregs show no phenotypic alteration with respect to activation and migration.

<p>WT Tregs (white bars) and <i>gzmA</i>^<i>-/-</i> Tregs (grey bars) were isolated from spleen (A) and peripheral lymph nodes (B) and analyzed by FACS. Expression values were calculated as a percentage of the CD4⁺ T lymphocyte population and mean values are presented as bar graphs. Students t-test did not reveal any significant differences between WT and <i>gzmA</i>^<i>-/-</i> Tregs. Results are shown for n = 3 WT and <i>gzmA</i>^<i>-/-</i> mice each.</p

<i>GzmA</i>^<i>-/-</i> Tregs home efficiently to secondary lymphoid organs.

<p>CD4⁺ (A) and CD4⁺CD25⁺ Tregs (B) were isolated from spleen and peripheral lymph node from wildtype (WT; n = 3) and <i>gzmA</i>^<i>-/-</i> mice (n = 3) and analyzed by FACS. Expression values were calculated as a percentage of the T lymphocyte population and mean values are presented as bar graphs. Students t-test did not reveal any significant differences between WT and <i>gzmA</i>^<i>-/-</i> Tregs.</p

Human Regulatory T Cells of G-CSF Mobilized Allogeneic Stem Cell Donors Qualify for Clinical Application

<div><p>Recent clinical studies demonstrate the high potency of regulatory T cells (Tregs) to control graft-versus-host disease in hematopoietic stem cell transplantation (SCT). However, the adoptive transfer of Tregs is limited by their low frequency in unstimulated donors and considerable concerns that G-CSF induced SC mobilization might have negative effects on the stability and function of Tregs. The isolation of Tregs from the G-CSF mobilized SC grafts would extend this novel strategy for tolerance induction to the unrelated setting and simplify global clinical application. We characterized CD4⁺CD25^highCD127⁻ Tregs from SC donors before and after G-CSF mobilization for their phenotype, function, and stability. After G-CSF application the Treg cell yield increased significantly. Donor Tregs retained their cytokine profile, phenotypic characteristics and <em>in vitro</em> expansion capacity after SC mobilization. Most importantly, <em>in vivo</em> G-CSF stimulated Tregs remained highly suppressive on the proliferation of effector T cells, also after <em>in vitro</em> expansion, and displayed a stable phenotype in epigenetic studies. The surface expression of CXCR3 is transiently reduced. However, donor-derived Tregs maintain their migratory properties after G-CSF stimulation. Therefore, the adoptive transfer of Tregs from G-CSF mobilized SC donors seems to be a feasible and safe strategy for clinical application in allogeneic SCT.</p> </div