Hanna Brauner

Nucleobindin-2/nesfatin in the endocrine pancreas: distribution and relationship to glycaemic state.

The protein nucleobindin-2 (NUCB2, also known as nesfatin) was recently implicated as a mediator of anorexia and catabolism in the central nervous system, and has been suggested to act as a cleaved and secreted messenger. Given the overlap of signalling molecules between the brain and pancreas, we have explored the presence of NUCB2 in the islets of Langerhans. We also performed an investigation of the dynamic regulation of pancreatic NUCB2 in different metabolic states. NUCB2-like immunoreactivity was detected by immunofluorescence in all human and rat islet beta-cells (as detected by co-localization with insulin), but not in other islet cells or in the exocrine pancreas. Islet NUCB2 content, as measured by enzyme immunoassay, did not change significantly following an overnight fast, but was substantially lower in islets isolated from an animal model of type 2 diabetes, the Goto-Kakizaki (GK) rats (48% of non-diabetic Wistar rat control). Serum levels, however, were not different between Wistar and GK rats. The release of NUCB2 from isolated rat islets was significantly elevated following glucose challenge (123%), but this effect was substantially lower than that observed for insulin (816%). In contrast, serum levels of NUCB2 showed a reversible decrease in an i.p. glucose tolerance test. These data suggest a role for NUCB2 in beta-cell function and a potential involvement in diabetic pathology. However, our findings, together with previous reports, appear more compatible with intracellular actions rather than with endocrine/paracrine communication, and suggest that NUCB2 in serum derives primarily from non-islet sources.

Vitamin D Induction of the Human Antimicrobial Peptide Cathelicidin in the Urinary Bladder

The urinary tract is frequently being exposed to potential pathogens and rapid defence mechanisms are therefore needed. Cathelicidin, a human antimicrobial peptide is expressed and secreted by bladder epithelial cells and protects the urinary tract from infection. Here we show that vitamin D can induce cathelicidin in the urinary bladder. We analyzed bladder tissue from postmenopausal women for expression of cathelicidin, before and after a three-month period of supplementation with 25-hydroxyvitamin D3 (25D3). Cell culture experiments were performed to elucidate the mechanisms for cathelicidin induction. We observed that, vitamin D per se did not up-regulate cathelicidin in serum or in bladder tissue of the women in this study. However, when the bladder biopsies were infected with uropathogenic E. coli (UPEC), a significant increase in cathelicidin expression was observed after 25D3 supplementation. This observation was confirmed in human bladder cell lines, even though here, cathelicidin induction occurred irrespectively of infection. Vitamin D treated bladder cells exerted an increased antibacterial effect against UPEC and colocalization to cathelicidin indicated the relevance of this peptide. In the light of the rapidly growing problem of resistance to common urinary tract antibiotics, we suggest that vitamin D may be a potential complement in the prevention of UTI.

Distinct Phenotype and Function of NK Cells in the Pancreas of Nonobese Diabetic Mice

Little is known about target organ-infiltrating NK cells in type 1 diabetes and other autoimmune diseases. In this study, we identified NK cells with a unique phenotype in the pancreas of NOD mice. Pancreatic NK cells, localized to the endocrine and exocrine parts, were present before T cells during disease development and did not require T cells for their infiltration. Furthermore, NK cells, or NK cell precursors, from the spleen could traffic to the pancreas, where they displayed the pancreatic phenotype. Pancreatic NK cells from other mouse strains shared phenotypic characteristics with pancreatic NK cells from NOD mice, but displayed less surface killer cell lectin-like receptor G1, a marker for mature NK cells that have undergone proliferation, and also did not proliferate to the same extent. A subset of NOD mouse pancreatic NK cells produced IFN-γ spontaneously, suggesting ongoing effector responses. However, most NOD mouse pancreatic NK cells were hyporesponsive compared with spleen NK cells, as reflected by diminished cytokine secretion and a lower capacity to degranulate. Interestingly, such hyporesponsiveness was not seen in pancreatic NK cells from the nonautoimmune strain C57BL/6, suggesting that this feature is not a general property of pancreatic NK cells. Based on our data, we propose that NK cells are sentinel cells in a normal pancreas. We further speculate that during inflammation, pancreatic NK cells initially mediate proinflammatory effector functions, potentially contributing to organ-specific autoimmunity, but later become hyporesponsive because of exhaustion or regulation.

Estrogen Supports Urothelial Defense Mechanisms

Estrogen supports urothelial defense against infection by increasing the expression of antimicrobial peptides and by protecting epithelial integrity. Urothelial Defense Tug-of-War It is well known that urinary tract infections (UTIs) are common in women, but which women are at more risk of UTIs is still far from certain, with some studies suggesting that it’s younger women, and others showing evidence of increased risk after menopause. Now, a study by Lüthje and coauthors suggests that both of those views may be partially correct and demonstrates the mechanisms for each. To understand how estrogen contributes to UTI pathogenesis, the authors examined cells from the urothelium (bladder lining) of menstruating women and older, postmenopausal subjects, studying the postmenopausal women before and after a 2-week period of estrogen supplementation. Then, to determine the mechanistic basis for their observations, they studied urothelial cells in an estrogen-depleted mouse model and in vitro. Thus, they demonstrated that estrogen has a protective effect on the urothelium, stimulating production of antimicrobial peptides and strengthening the attachment between urothelial cells. At the same time, the authors found that estrogen also increases the amount of bacteria taken up inside the urothelial cells. On the basis of the findings of Lüthje et al., one can conclude that young women may experience a greater incidence of UTIs because the high-estrogen environment increases the risk of bacterial invasion of the urothelium. Conversely, postmenopausal women may have more difficulty fighting off the infections because of their decreased production of antimicrobial peptides and diminished integrity of the urothelial lining. Additional studies of human and animal subjects will be needed to learn more about the molecular mechanism of estrogen’s effects on the urothelium and understand what determines the balance between its pro- and anti-UTI effects. However, this line of research does advance us closer to understanding and eventually helping prevent the problem of recurrent UTIs in women. Epidemiological data imply a role of estrogen in the pathogenesis of urinary tract infections (UTIs), although the underlying mechanisms are not well understood. However, it is thought that estrogen supplementation after menopause decreases the risk of recurrent infections. We sought to investigate the influence of estrogen on host-pathogen interactions and the consequences for UTI pathogenesis. We analyzed urothelial cells from menstruating and postmenopausal women before and after a 2-week period of estrogen supplementation, and also studied the influence of estradiol during Escherichia coli UTI in a mouse infection model. Important findings were confirmed in two human urothelial cell lines. We identified two epithelial defense mechanisms modulated by estrogen. Estrogen induced the expression of antimicrobial peptides, thereby enhancing the antimicrobial capacity of the urothelium and restricting bacterial multiplication. In addition, estrogen promoted the expression and redistribution of cell-cell contact–associated proteins, thereby strengthening the epithelial integrity and preventing excessive loss of superficial cells during infection. These two effects together may prevent bacteria from reaching deeper layers of the urinary tract epithelium and developing reservoirs that can serve as a source for recurrent infections. Thus, this study presents some underlying mechanisms for the beneficial effect of estradiol after menopause and supports the application of estrogen in postmenopausal women suffering from recurrent UTI.

Markers of innate immune activity in patients with type 1 and type 2 diabetes mellitus and the effect of the anti‐oxidant coenzyme Q10 on inflammatory activity

Major long‐term complications in patients with diabetes are related to oxidative stress, caused by the hyperglycaemia characteristic for diabetes mellitus. The anti‐oxidant coenzyme Q10 (CoQ10) has therefore been proposed as a beneficial supplement to diabetes treatment. Apart from its anti‐oxidative function, CoQ10 appears to modulate immune functions by largely unknown mechanisms. The aim of this study was therefore to investigate the effect of CoQ10 on antimicrobial peptides and natural killer (NK) cells, both innate immune components implicated in the pathogenesis of diabetes and diabetes‐associated long‐term complications such as cardiovascular disease. We determined serum levels of antimicrobial peptides and the phenotype of NK cells isolated from peripheral blood of patients with type 1 (T1DM) or type 2 diabetes mellitus (T2DM) and from healthy controls. In addition, the same parameters were determined in diabetic patients after a 12‐week period of CoQ10 supplementation. Two antimicrobial peptides, the human cathelicidin antimicrobial peptide (CAMP) and the human beta defensin 1 (hBD1), were reduced in serum from patients with T1DM. This defect was not reversible by CoQ10 supplementation. In contrast, CoQ10 reduced the levels of circulating hBD2 in these patients and induced changes in subset distribution and activation markers in peripheral NK cells. The results of the present study open up novel approaches in the prevention of long‐term complications associated to T1DM, although further investigations are needed.

AIRE deficiency leads to impaired iNKT cell development.

Autoimmune Polyendocrine Syndrome type I (APS I) is caused by mutations in the Autoimmune Regulator gene (AIRE), and results in the immunological destruction of endocrine organs. Herein we have characterized the CD1d-restricted invariant NKT cells (iNKT) and NK cells in APS I patients and Aire(-/-) mice, two cell populations known to play a role in the regulation of autoimmune disease. We show that the frequency of circulating iNKT cells is reduced in APS I patients compared to healthy controls. In accordance with this, iNKT cells are significantly reduced in the thymus and peripheral organs of Aire(-/-) mice. Bone marrow transfer from wild type donors into lethally irradiated Aire(-/-) recipients led to a decreased iNKT cell population in the liver, suggesting an impaired development of iNKT cells in the absence of Aire expression in radio-resistant cells. In contrast to the iNKT cells, both conventional NK cells and thymus-derived NK cells were unaffected by Aire deficiency and differentiated normally in Aire(-/-) mice. Our results show that expression of Aire in radio-resistant cells is important for the development of iNKT cells, whereas NK cell development and function does not depend on Aire.

IL-2 in the tumor microenvironment is necessary for Wiskott-Aldrich syndrome protein deficient NK cells to respond to tumors in vivo

To kill target cells, natural killer (NK) cells organize signaling from activating and inhibitory receptors to form a lytic synapse. Wiskott-Aldrich syndrome (WAS) patients have loss-of-function mutations in the actin regulator WASp and suffer from immunodeficiency with increased risk to develop lymphoreticular malignancies. NK cells from WAS patients fail to form lytic synapses, however, the functional outcome in vivo remains unknown. Here, we show that WASp KO NK cells had decreased capacity to degranulate and produce IFNγ upon NKp46 stimulation and this was associated with reduced capacity to kill MHC class I-deficient hematopoietic grafts. Pre-treatment of WASp KO NK cells with IL-2 ex vivo restored degranulation, IFNγ production, and killing of MHC class I negative hematopoietic grafts. Moreover, WASp KO mice controlled growth of A20 lymphoma cells that naturally produced IL-2. WASp KO NK cells showed increased expression of DNAM-1, LAG-3, and KLRG1, all receptors associated with cellular exhaustion and NK cell memory. NK cells isolated from WAS patient spleen cells showed increased expression of DNAM-1 and had low to negative expression of CD56, a phenotype associated with NK cells exhaustion. Finally, in a cohort of neuroblastoma patients we identified a strong correlation between WASp, IL-2, and patient survival.

Retuning of Mouse NK Cells after Interference with MHC Class I Sensing Adjusts Self-Tolerance but Preserves Anticancer Response

NK cell–based immunotherapy may be hampered by adaptation to reduced inhibitory input from MHC molecules on surrounding cells. However, while such readjustment of responsiveness leads to tolerance to healthy cells, reactivity to cancer cells remains. Natural killer (NK) cells are most efficient if their targets do not express self MHC class I, because NK cells carry inhibitory receptors that interfere with activating their cytotoxic pathway. Clinicians have taken advantage of this by adoptively transferring haploidentical NK cells into patients to mediate an effective graft-versus-leukemia response. With a similar rationale, antibody blockade of MHC class I–specific inhibitory NK cell receptors is currently being tested in clinical trials. Both approaches are challenged by the emerging concept that NK cells may constantly adapt or “tune” their responsiveness according to the amount of self MHC class I that they sense on surrounding cells. Hence, these therapeutic attempts would initially result in increased killing of tumor cells, but a parallel adaptation process might ultimately lead to impaired antitumor efficacy. We have investigated this question in two mouse models: inhibitory receptor blockade in vivo and adoptive transfer to MHC class I–disparate hosts. We show that changed self-perception via inhibitory receptors in mature NK cells reprograms the reactivity such that tolerance to healthy cells is always preserved. However, reactivity against cancer cells lacking critical MHC class I molecules (missing self-reactivity) still remains or may even be increased. This dissociation between activity against healthy cells and tumor cells may provide an answer as to why NK cells mediate graft-versus-leukemia effects without causing graft-versus-host disease and may also be utilized to improve immunotherapy. Cancer Immunol Res; 4(2); 113–23. ©2015 AACR.

Increased diabetes development and decreased function of CD4+CD25+ Treg in the absence of a functional DAP12 adaptor protein

Prior to the development of type 1 diabetes, T cells are primed in the pancreatic lymph nodes (PLN) where they interact with APC displaying β cell‐derived peptides. The details concerning the regulation of autoreactive T cell responses in the PLN are unclear. BDC2.5/B6g7 TCR transgenic mice represent a simplified model of type 1 diabetes, in which β cell‐specific CD4+ T cells expressing a diabetogenic transgenic TCR are first activated in the PLN and subsequently home to the pancreas where they mediate killing of β cells. DNAX‐activating protein of 12 kDa (DAP12) is an adaptor molecule carrying an ITAM motif. It associates with receptors on lymphoid and myeloid cells, including APC. We here show that introduction of a DAP12 null mutation in BDC2.5/B6g7 mice accelerated diabetes development and promoted an augmented activation state of PLN T cells expressing the transgenic TCR. Transferred BDC2.5 T cells proliferated more efficiently in the PLN of DAP12‐deficient B6g7 recipients, which correlated with a decreased impact of co‐transferred BDC2.5+CD4+CD25+ T cells. We propose that signaling through a DAP12‐associated receptor on APC facilitates activation of Treg in the PLN and by this contributes to the maintenance of peripheral tolerance to β cell‐derived antigens.

Depletion of IL-2 receptor β-positive cells protects from diabetes in non-obese diabetic mice

The destruction of β‐cells in type 1 diabetes (T1D) progresses silently until only a minor fraction of the β‐cells remain. A late acting therapy leading to the prevention of further β‐cell killing would therefore be desirable. CD122, the β chain of the interleukin‐2 receptor, is highly expressed on natural killer (NK) cells and on a subpopulation of CD8 T cells. In this study, we have treated non‐obese diabetic (NOD) mice with a depleting antibody against CD122. The treatment protected from diabetes, even when initiated just before disease onset. The degree of leukocyte infiltration into islets was unaffected by the treatment, further supporting effectiveness late in the disease process. It effectively removed all NK cells from the spleen, pancreas and pancreatic lymph nodes and abolished NK cell activity. Interestingly, despite the lack of CD122 expression on CD8 T cells in the pancreas, the overall frequency of CD8 cells decreased in this organ, whereas it was unaffected in the spleen. T cells were also still capable to respond against a foreign antigen. Conclusively, targeting of CD122+ cells could represent a novel treatment strategy against T1D.