Britta Eiz-Vesper

Signaling to Heme Oxygenase-1 and its Anti-Inflammatory Therapeutic Potential

Heme oxygenase (HO)-1 is the inducible isoform of the first and rate-limiting enzyme of heme degradation. Induction of HO-1 protects against the cytotoxicity of oxidative stress and apoptotic cell death. More recently, HO-1 has been recognized to have major immunomodulatory and anti-inflammatory properties, which have been demonstrated in HO-1 knockout mice and a human case of genetic HO-1 deficiency. Beneficial protective effects of HO-1 in inflammation are not only mediated via enzymatic degradation of proinflammatory free heme, but also via production of the anti-inflammatory compounds bilirubin and carbon monoxide. The immunomodulatory role of HO-1 is associated with its cell type-specific functions in myeloid cells (eg. macrophages and monocytes) and in endothelial cells, as both cell types are crucially involved in the regulation of inflammatory responses. This review covers the molecular mechanisms and signaling pathways that are involved in HO-1 gene expression. In particular, it is discussed how redox-dependent transcriptional activators such as NF-E2 related factor 2 (Nrf2), NF-κB and AP-1 along with the transcription repressor BTB and CNC homologue 1 (Bach1) control the inducible HO-1 gene expression. The role of central pro- and anti-inflammatory cellular signaling cascades including p38 MAPK and phosphatidylinositol-3 kinase (PI3K)/Akt in HO-1 regulation is highlighted. Finally, emerging strategies that apply targeted pharmacological induction of HO-1 for therapeutic interventions in inflammatory conditions are summarized.

Evidence for a pathogenetic role of interleukin-18 in cutaneous lupus erythematosus.

OBJECTIVE Cutaneous manifestations are the most common clinical features of lupus erythematosus (LE). The aim of this study was to analyze differences in the inflammatory response of keratinocytes from patients with cutaneous LE (CLE) compared with healthy controls. METHODS Keratinocytes from LE patients and controls were cultured from epidermal stem cells of the hair follicle of anagen head hairs. Functional responses of keratinocytes to cytokine stimulation were determined by flow cytometry and enzyme-linked immunosorbent assay. Biopsy samples of lesional skin were analyzed by immunohistochemistry. RESULTS Keratinocytes from CLE patients expressed higher levels of IL-18 receptor on their cell surface in response to tumor necrosis factor alpha (TNFalpha) or interferon-gamma stimulation. In response to IL-18 stimulation, these cells produced large amounts of TNFalpha. Of note, in the presence of IL-18, CLE keratinocytes failed to express IL-12. IL-12 has previously been shown to protect keratinocytes from ultraviolet irradiation-induced apoptosis. Keratinocytes from LE patients were more prone to die upon exposure to IL-18, and this increased apoptosis was abrogated by blockade of endogenously produced TNFalpha as well as by the addition of exogenous IL-12. IL-18 was highly expressed in biopsy samples of lesional skin from CLE patients. CONCLUSION Our results demonstrate an intrinsic difference in the inflammatory response of keratinocytes and indicate an autocrine feedback loop involving TNFalpha, IL-18, and IL-12 family members. Our results suggest that IL-18 may occupy an important position in the cytokine hierarchy in CLE, indicating the potential benefit of a local agent that blocks IL-18 activity in the treatment of the manifestations of CLE.

Heat shock protein 70 (HSP70) induces cytotoxicity of T-helper cells

Heat shock protein 70 (HSP70) has gained plenty of attention because of its adjuvant capability to induce CD8(+) cytotoxic T lymphocyte and CD4(+) T-helper cell responses. We investigated the behavior of T-cell subsets stimulated with endotoxin-free HSP70 with respect to proliferation, cytokine expression, cytotoxicity against allogeneic B-lymphoblastoid cell line and K562 cells, as well as target-independent cytotoxicity. CD4(+) cells exhibited a strong increase in proliferation after stimulation with HSP70 (29%). In the presence of targets, a 35-fold up-regulation of granzyme B was observed after stimulation of CD4(+) T cells with HSP70 in combination with interleukin-7 (IL-7)/IL-12/IL-15. The target cell-independent secretion of granzyme B by CD4(+) cells was greatly augmented after stimulation with HSP70 plus IL-2 or IL-7/IL-12/IL-15. In this study, we showed that HSP70 is capable of inducing a cytotoxic response of T-helper cells in the absence of lipopolysaccharide. The granzyme B secretion and cytolytic activity of T-helper cells are induced in a target-independent way, whereas the cytotoxic activity of CD3(+) and CD8(+) T cells can be further enhanced in the presence of target cells. Our data provide novel insights into the role of extracellular HSP70 on T-cell immune response concerning the induction of target-independent T-helper cell cytotoxicity.

HSP70 enhances immunosuppressive function of CD4(+)CD25(+)FoxP3(+) T regulatory cells and cytotoxicity in CD4(+)CD25(-) T cells.

Human CD4+CD25+FoxP3+ T regulatory cells (Tregs) control effector T cells and play a central role in peripheral tolerance and immune homeostasis. Heat shock protein 70 (HSP70) is a major immunomodulatory molecule, but its effect on the functions of Tregs is not well understood. To investigate target-dependent and –independent Treg functions, we studied cytokine expression, regulation of proliferation and cytotoxicity after exposure of Tregs to HSP70. HSP70-treated Tregs significantly inhibited proliferation of CD4+CD25− target cells and downregulated the secretion of the proinflammatory cytokines IFN-γ and TNF-α. By contrast, HSP70 increased the secretion of Treg suppressor cytokines IL-10 and TGF-β. Treatment with HSP70 enhanced the cytotoxic properties of Tregs only to a minor extent (4-fold), but led to stronger responses in CD4+CD25− cells (42-fold). HSP70-induced modulation of T-cell responses was further enhanced by combined treatment with HSP70 plus IL-2. Treatment of Tregs with HSP70 led to phosphorylation of PI3K/AKT and the MAPKs JNK and p38, but not that of ERK1/2. Exposure of Tregs to specific inhibitors of PI3K/AKT and the MAPKs JNK and p38 reduced the immunosuppressive function of HSP70-treated Tregs as indicated by the modified secretion of specific target cell (IFN-γ, TNF-α) and suppressor cytokines (IL-10, TGF-β). Taken together, the data show that HSP70 enhances the suppressive capacity of Tregs to neutralize target immune cells. Thus HSP70-enhanced suppression of Tregs may prevent exaggerated immune responses and may play a major role in maintaining immune homeostasis.

Patient, Virus, and Treatment-Related Risk Factors in Pediatric Adenovirus Infection after Stem Cell Transplantation: Results of a Routine Monitoring Program

Human adenovirus (HAdV) infection after hematopoietic stem cell transplantation (HSCT) is associated with significant morbidity and mortality in children. The optimal surveillance and treatment strategies are under discussion. Here, we present data from 238 consecutive pediatric allogeneic HSCT recipients who underwent transplantation in a single center who were included in a prospective, weekly HAdV DNAemia monitoring program by quantitative PCR. HAdV loads >1000 copies/mL were detected in 15.5% of all patients. Despite a low mortality directly attributed to HAdV infection (2 patients, 0.84%), blood HAdV loads >10,000 copies/mL (6.7% of all patients) were significant and independent risk factors for poor survival. We searched for patient, virus, and treatment-related risk factors of HAdV DNAemia and disease. Detection of HAdV in blood before day 50 post transplantation was a major independent risk factor for the development of blood HAdV loads >10,000 copies/mL. HAdV typing revealed A31, C1, and C2 as the predominant pathogens among several other HAdV strains with type C species detected in most patients with severe HAdV disease. Stool HAdV loads were prospectively monitored in 111 patients and correlated with but did not significantly precede detection in blood. Treatment with cidofovir led to stable or reduced viral load in 70% of patients with blood HAdV loads >1000 copies/mL. Thus, early occurrence of HAdV-DNA in blood of pediatric HSCT recipients predisposes for development of high viral loads. Control of HAdV infections was attempted by preemptive cidofovir treatment of patients with high blood HAdV loads or with symptomatic organ infections and correlated with low HAdV-attributed mortality.

The impact of human leukocyte antigen (HLA) micropolymorphism on ligand specificity within the HLA-B*41 allotypic family

Background Polymorphic differences between human leukocyte antigen (HLA) molecules affect the specificity and conformation of their bound peptides and lead to differential selection of the T-cell repertoire. Mismatching during allogeneic transplantation can, therefore, lead to immunological reactions. Design and Methods We investigated the structure-function relationships of six members of the HLA-B*41 allelic group that differ by six polymorphic amino acids, including positions 80, 95, 97 and 114 within the antigen-binding cleft. Peptide-binding motifs for B*41:01, *41:02, *41:03, *41:04, *41:05 and *41:06 were determined by sequencing self-peptides from recombinant B*41 molecules by electrospray ionization tandem mass spectrometry. The crystal structures of HLA-B*41:03 bound to a natural 16-mer self-ligand (AEMYGSVTEHPSPSPL) and HLA-B*41:04 bound to a natural 11-mer self-ligand (HEEAVSVDRVL) were solved. Results Peptide analysis revealed that all B*41 alleles have an identical anchor motif at peptide position 2 (glutamic acid), but differ in their choice of C-terminal pΩ anchor (proline, valine, leucine). Additionally, B*41:04 displayed a greater preference for long peptides (>10 residues) when compared to the other B*41 allomorphs, while the longest peptide to be eluted from the allelic group (a 16mer) was obtained from B*41:03. The crystal structures of HLA-B*41:03 and HLA-B*41:04 revealed that both alleles interact in a highly conserved manner with the terminal regions of their respective ligands, while micropolymorphism-induced changes in the steric and electrostatic properties of the antigen-binding cleft account for differences in peptide repertoire and auxiliary anchoring. Conclusions Differences in peptide repertoire, and peptide length specificity reflect the significant functional evolution of these closely related allotypes and signal their importance in allogeneic transplantation, especially B*41:03 and B*41:04, which accommodate longer peptides, creating structurally distinct peptide-HLA complexes.

The endogenous danger signals HSP70 and MICA cooperate in the activation of cytotoxic effector functions of NK cells

Although natural killer (NK) cells are often described as first line defence against infected or malignant cells which act without the need of prior activation, it is known now that the NK cell activity is tightly regulated by other cells and soluble factors. We show here that the stress‐inducible heat shock protein (HSP) 70 activates human NK cells to kill target cells expressing major histocompatibility complex class I chain‐related molecule A (MICA) in a natural killer group 2 member D (NKG2D‐) dependent manner. The HSP70‐derived peptide TKD (TKDNNLLGRFELSG) was able to replace the full‐length HSP70 and to exert the same function. Interestingly, the expression of the cytotoxic effector protease granzyme B in NK cells was increased after TKD stimulation. When MICA and MICB expression was induced in human tumour cells by a histone deacetylase inhibitor and NK cells were activated by HSP70 or TKD, both treatments jointly improved the killing of the tumour cells. Thus, the synergistic activity of two stress‐inducible immunological danger signals, HSP70 and MICA/B, leads to activation and enhanced cytotoxicity of human NK cells against tumour cells.

Generation of HLA-deficient platelets from hematopoietic progenitor cells.

BACKGROUND: Exposure to allogeneic blood products often leads to the development of human leukocyte antigen (HLA) antibodies. Refractoriness to platelet (PLT) transfusion caused by alloimmunization against HLA Class I antigens constitutes a significant clinical problem.

A weak blood group A phenotype caused by a new mutation at the ABO locus

BACKGROUND: A number of alleles have been described for ABO encoding for common and rare ABO blood group phenotypes. Critical mutations in the coding sequence of ABO that may confer the different specificity and activity of the glycosyltransferases encoded by this gene locus have been identified.

Short-term in-vitro expansion improves monitoring and allows affordable generation of virus-specific T-cells against several viruses for a broad clinical application.

Adenoviral infections are a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT) in pediatric patients. Adoptive transfer of donor-derived human adenovirus (HAdV)-specific T-cells represents a promising treatment option. However, the difficulty in identifying and selecting rare HAdV-specific T-cells, and the short time span between patients at high risk for invasive infection and viremia are major limitations. We therefore developed an IL-15-driven 6 to 12 day short-term protocol for in vitro detection of HAdV-specific T cells, as revealed by known MHC class I multimers and a newly identified adenoviral CD8 T-cell epitope derived from the E1A protein for the frequent HLA-type A*02∶01 and IFN-γ. Using this novel and improved diagnostic approach we observed a correlation between adenoviral load and reconstitution of CD8+ and CD4+ HAdV-specific T-cells including central memory cells in HSCT-patients. Adaption of the 12-day protocol to good manufacturing practice conditions resulted in a 2.6-log (mean) expansion of HAdV-specific T-cells displaying high cytolytic activity (4-fold) compared to controls and low or absent alloreactivity. Similar protocols successfully identified and rapidly expanded CMV-, EBV-, and BKV-specific T-cells. Our approach provides a powerful clinical-grade convertible tool for rapid and cost-effective detection and enrichment of multiple virus-specific T-cells that may facilitate broad clinical application.