Natalie Stickel

Unraveling the Role of Podocyte Turnover in Glomerular Aging and Injury

Podocyte loss is a major determinant of progressive CKD. Although recent studies showed that a subset of parietal epithelial cells can serve as podocyte progenitors, the role of podocyte turnover and regeneration in repair, aging, and nephron loss remains unclear. Here, we combined genetic fate mapping with highly efficient podocyte isolation protocols to precisely quantify podocyte turnover and regeneration. We demonstrate that parietal epithelial cells can give rise to fully differentiated visceral epithelial cells indistinguishable from resident podocytes and that limited podocyte renewal occurs in a diphtheria toxin model of acute podocyte ablation. In contrast, the compensatory programs initiated in response to nephron loss evoke glomerular hypertrophy, but not de novo podocyte generation. In addition, no turnover of podocytes could be detected in aging mice under physiologic conditions. In the absence of podocyte replacement, characteristic features of aging mouse kidneys included progressive accumulation of oxidized proteins, deposits of protein aggregates, loss of podocytes, and glomerulosclerosis. In summary, quantitative investigation of podocyte regeneration in vivo provides novel insights into the mechanism and capacity of podocyte turnover and regeneration in mice. Our data reveal that podocyte generation is mainly confined to glomerular development and may occur after acute glomerular injury, but it fails to regenerate podocytes in aging kidneys or in response to nephron loss.

The Nlrp3 inflammasome regulates acute graft-versus-host disease.

Conditioning therapies before transplantation induce the release of uric acid, which triggers the NLRP3 inflammasome and IL-1β production contributing to graft-versus-host disease.

MiR-146a regulates the TRAF6/TNF-axis in donor T cells during GVHD.

Acute graft-versus-host disease (GVHD) limits the success of allogeneic hematopoietic cell transplantation (allo-HCT); therefore, a better understanding of its biology may improve therapeutic options. We observed miR-146a up-regulation in T cells of mice developing acute GVHD compared with untreated mice. Transplanting miR-146a(-/-) T cells caused increased GVHD severity, elevated tumor necrosis factor (TNF) serum levels, and reduced survival. TNF receptor-associated factor 6 (TRAF6), a verified target of miR-146a, was up-regulated in miR-146a(-/-) T cells following alloantigen stimulation. Higher TRAF6 levels translated into increased nuclear factor-κB activity and TNF production in miR-146a(-/-) T cells. Conversely, the detrimental effect of miR-146a deficiency in T cells was antagonized by TNF blockade, whereas phytochemical induction of miR-146a or its overexpression using a miR-146a mimic reduced GVHD severity. In humans, the minor genotype of the single nucleotide polymorphism rs2910164 in HCT donors, which reduces expression of miR-146a, was associated with severe acute GVHD (grade III/IV). We show that miR-146a functions as a negative regulator of donor T cells in GVHD by targeting TRAF6, leading to reduced TNF transcription. Because miR-146a expression can be exogenously enhanced, our results provide a novel targeted molecular approach to mitigate GVHD.

Fatal Eosinophilic Myocarditis Develops in the Absence of IFN-γ and IL-17A

CD4+ T cells play a central role in inflammatory heart disease, implicating a cytokine product associated with Th cell effector function as a necessary mediator of this pathophysiology. IFN-γ–deficient mice developed severe experimental autoimmune myocarditis (EAM), in which mice are immunized with cardiac myosin peptide, whereas IL-17A–deficient mice were protected from progression to dilated cardiomyopathy. We generated IFN-γ−/−IL-17A−/− mice to assess whether IL-17 signaling was responsible for the severe EAM of IFN-γ−/− mice. Surprisingly, IFN-γ−/−IL-17A−/− mice developed a rapidly fatal EAM. Eosinophils constituted a third of infiltrating leukocytes, qualifying this disease as eosinophilic myocarditis. We found increased cardiac production of CCL11/eotaxin, as well as Th2 deviation, among heart-infiltrating CD4+ cells. Ablation of eosinophil development improved survival of IFN-γ−/−IL-17A−/− mice, demonstrating the necessity of eosinophils in fatal heart failure. The severe and rapidly fatal autoimmune inflammation that developed in the combined absence of IFN-γ and IL-17A constitutes a novel model of eosinophilic heart disease in humans. This is also, to our knowledge, the first demonstration that eosinophils have the capacity to act as necessary mediators of morbidity in an autoimmune process.

The microenvironment differentially impairs passive and active immunotherapy in chronic lymphocytic leukaemia - CXCR4 antagonists as potential adjuvants for monoclonal antibodies.

Direct contact with stromal cells protects chronic lymphocytic leukaemia (CLL) B cells from chemotherapy‐induced apoptosis in vitro. Blockade of CXCR4 signalling antagonizes stroma‐mediated interactions and restores CLL chemosensitivity. In vivo, administration of CXCR4 antagonists effectively mobilizes haematopoietic progenitor cells. Therefore, combinations of CXCR4 blockade and cytoreductive treatment with selective activity on CLL cells may avoid potential haematotoxicity. Hence, we tested CXCR4 antagonists in the context of passive and active immunotherapeutic approaches. We evaluated how efficiently rituximab, alemtuzumab and cytotoxic T cells killed CLL cells cocultured with stromal cells in the presence and absence of a CXCR4 antagonist. Stromal cell contact attenuated rituximab‐ and alemtuzumab‐induced complement‐dependent cytotoxicity of CLL cells. Addition of CXCR4 antagonists abrogated the protective effect of stroma. In contrast, stromal cells did not impair antibody‐dependent cell‐mediated cytotoxicity and cytotoxicity induced by activated T cells. Destruction of microtubules in CLL target cells restored the protective effect of stroma coculture for CLL cells during Natural Killer cell attack by preventing mitochondrial relocalization towards the immunological synapse. Our data identify the combination of CXCR4 antagonists with passive ‐ but not active ‐ immunotherapy as a promising potential treatment concept in CLL.

Quantification of HLA class I molecules on renal cell carcinoma using Edman degradation

BackgroundUnimpaired HLA class I antigen presentation is a prerequisite for the recognition of tumor cells by cytotoxic T lymphocytes and thus essential for the success of anticancer immunotherapeutic concepts. Several approaches have been taken in the immunotherapy of metastatic renal cell carcinoma (RCC), however of limited success. HLA loss or down-regulation have often been reported and might interfere with immunotherapeutic approaches aimed at the recognition of HLA-presented peptides.MethodsWe employed a quantitative method of molecular analysis for the comparison of HLA amounts on primary tumor, normal kidney and metastases of RCC, using Edman degradation. We analyzed a series of 47 RCC samples including corresponding renal parenchyma, local lymph node metastases and distant metastases.ResultsResults of quantitative Edman degradation revealed significantly higher HLA yields on primary tumor and metastases compared to normal kidney tissue. This effect was shown not to result from infiltrating immune cells, since tumor-infiltrating lymphocytes had no influence on the overall HLA recovery from tumor tissue. Unexpectedly, we found a higher amount of HLA class I molecules on distant metastases compared to local lymph node metastases.ConclusionEdman degradation allows the direct quantitative comparison of HLA class I protein expression by tumor or normal tissue and metastases of RCC patients. Our results raise hopes for improving the success and effectiveness of future immunotherapeutic concepts for metastatic RCC.

MicroRNA-146a reduces MHC-II expression via targeting JAK/STAT signaling in dendritic cells after stem cell transplantation.

Acute Graft-versus-host disease (GVHD) is a major immunological complication after allogeneic hematopoietic cell transplantation and a better understanding of the molecular regulation of the disease could help to develop novel targeted therapies. Here we found that a G/C polymorphism within the human microRNA-146a (miR-146a) gene of transplant recipients, which causes reduced miR-146a levels, was strongly associated with the risk of developing severe acute GVHD (n=289). In mice, deficiency of miR-146a in the hematopoietic system or transfer of recipient-type miR-146a−/− dendritic cells (DCs) enhanced GVHD, while miR-146a mimic-transfected DCs ameliorated disease. Mechanistically, lack of miR-146a enhanced JAK2–STAT1 pathway activity, which led to higher expression of class II-transactivator (CIITA) and consecutively increased MHCII-levels on DCs. Inhibition of JAK1/2 or CIITA knockdown in DCs prevented miR-146a−/− DC-induced GVHD exacerbation. Consistent with our findings in mice, patients with the miR-146a polymorphism rs2910164 in hematopoietic cells displayed higher MHCII levels on monocytes, which could be targeted by JAK1/2 inhibition. Our findings indicate that the miR-146a polymorphism rs2910164 identifies patients at high risk for GVHD before allo-HCT. Functionally we show that miR-146a acts as a central regulator of recipient-type DC activation during GVHD by dampening the pro-inflammatory JAK–STAT/CIITA/MHCII axis, which provides a scientific rationale for early JAK1/2 inhibition in selected patients.

Activation loop phosphorylation regulates B-Raf in vivo and transformation by B-Raf mutants

Despite being mutated in cancer and RASopathies, the role of the activation segment (AS) has not been addressed for B‐Raf signaling in vivo. Here, we generated a conditional knock‐in mouse allowing the expression of the B‐RafAVKA mutant in which the AS phosphoacceptor sites T599 and S602 are replaced by alanine residues. Surprisingly, despite producing a kinase‐impaired protein, the BrafAVKA allele does not phenocopy the lethality of Braf‐knockout or paradoxically acting knock‐in alleles. However, BrafAVKA mice display abnormalities in the hematopoietic system, a distinct facial morphology, reduced ERK pathway activity in the brain, and an abnormal gait. This phenotype suggests that maximum B‐Raf activity is required for the proper development, function, and maintenance of certain cell populations. By establishing conditional murine embryonic fibroblast cultures, we further show that MEK/ERK phosphorylation and the immediate early gene response toward growth factors are impaired in the presence of B‐RafAVKA. Importantly, alanine substitution of T599/S602 impairs the transformation potential of oncogenic non‐V600E B‐Raf mutants and a fusion protein, suggesting that blocking their phosphorylation could represent an alternative strategy to ATP‐competitive inhibitors.

Complete Freund's adjuvant induces experimental autoimmune myocarditis by enhancing IL-6 production during initiation of the immune response

Complete Freunds Adjuvant (CFA) emulsified with an antigen is a widely used method to induce autoimmune disease in animal models, yet the contribution of CFA to the immune response is not well understood. We compared the effectiveness of CFA with Incomplete Freunds Adjuvant (IFA) or TiterMax Gold Adjuvant (TMax) in experimental autoimmune myocarditis (EAM) in male mice.

The microenvironment differentially impairs passive and active immunotherapy in chronic lymphocytic leukaemia - CXCR4 antagonists as potential adjuvants for monoclonal antibodies: Microenvironmental Impact on Immunotherapy in CLL

Direct contact with stromal cells protects chronic lymphocytic leukaemia (CLL) B cells from chemotherapy‐induced apoptosis in vitro. Blockade of CXCR4 signalling antagonizes stroma‐mediated interactions and restores CLL chemosensitivity. In vivo, administration of CXCR4 antagonists effectively mobilizes haematopoietic progenitor cells. Therefore, combinations of CXCR4 blockade and cytoreductive treatment with selective activity on CLL cells may avoid potential haematotoxicity. Hence, we tested CXCR4 antagonists in the context of passive and active immunotherapeutic approaches. We evaluated how efficiently rituximab, alemtuzumab and cytotoxic T cells killed CLL cells cocultured with stromal cells in the presence and absence of a CXCR4 antagonist. Stromal cell contact attenuated rituximab‐ and alemtuzumab‐induced complement‐dependent cytotoxicity of CLL cells. Addition of CXCR4 antagonists abrogated the protective effect of stroma. In contrast, stromal cells did not impair antibody‐dependent cell‐mediated cytotoxicity and cytotoxicity induced by activated T cells. Destruction of microtubules in CLL target cells restored the protective effect of stroma coculture for CLL cells during Natural Killer cell attack by preventing mitochondrial relocalization towards the immunological synapse. Our data identify the combination of CXCR4 antagonists with passive ‐ but not active ‐ immunotherapy as a promising potential treatment concept in CLL.