Andreas Spittler

Vitamin D3 down-regulates monocyte TLR expression and triggers hyporesponsiveness to pathogen-associated molecular patterns.

Toll‐like receptors (TLR) represent an ancient front‐line defence system that enables the host organism to sense the presence of microbial components within minutes. As inducers of inflammation, TLR act as important triggers of distinct entities such as sepsis or autoimmune disease exacerbation. We report here that vitamin D3 [1α,25‐dihydroxycholecalciferol, 1,25(OH)2D3] suppresses the expression of TLR2 and TLR4 protein and mRNA in human monocytes in a time‐ and dose‐dependent fashion. Despite 1,25(OH)2D3‐induced up‐regulation of CD14, challenge of human monocytes with either LPS or lipoteichoic acid resulted in impaired TNF‐α and procoagulatory tissue factor (CD142) production, emphasizing the critical role of TLR in the induction of inflammation. Moreover, reduced TLR levels in 1,25(OH)2D3‐treated phagocytes were accompanied by impaired NF‐κB/RelA translocation to the nucleus and by reduced p38 and p42/44 (extracellular signal‐regulated kinase 1/2) phosphorylation upon TLR‐ligand engagement. Both TLR down‐regulation and CD14 up‐regulation were substantially inhibited by the vitamin D receptor (VDR) antagonist ZK 159222, indicating that the immunomodulatory effect of 1,25(OH)2D3 on innate immunity receptors requires VDR transcription factor activation. Our data provide strong evidence that 1,25(OH)2D3 primes monocytes to respond less effectively to bacterial cell wall components in a VDR‐dependent mechanism, most likely due to decreased levels of TLR2 and TLR4.

Immaturity of Infection Control in Preterm and Term Newborns Is Associated with Impaired Toll-Like Receptor Signaling

The impaired infection control related to the functional immaturity of the neonatal immune system is an important cause of infection in preterm newborns. We previously reported that constitutive Toll-like receptor (TLR) 4 expression and cytokine secretion on lipopolysaccharide (LPS) stimulation increases with gestational age. Here, we analyzed constitutive monocyte TLR2 expression and evaluated the expression profiles of the proximal downstream adapter molecule myeloid differentiation factor 88 (MyD88). We further investigated activation of protein kinases p38 and extracellular regulated kinsase (ERK) 1/2 in CD14 monocytes after ex vivo stimulation with bacterial TLR ligands (LPS and lipoteichoic acid [LTA]). The functional outcome of the stimulation was determined by cytokine secretion. Monocytes from 31 preterm newborns (<30 weeks of gestation, n=16; 30-37 weeks of gestation, n=15), 10 term newborns, and 12 adults were investigated. In contrast to TLR4 expression, TLR2 levels did not differ between age groups. However, MyD88 levels were significantly lower in preterm newborns. Activation of p38 and ERK1/2 was impaired in all newborn age groups after stimulation with TLR-specific ligands. Accordingly, after LTA stimulation, the levels of interleukin (IL)-1 beta , IL-6, and IL-8 cytokine production were substantially lower (P<.001) in preterm newborns than in adults. The reduced functional response to bacterial cell wall components appears to be part of the functional immaturity of the neonatal immune system and might predispose premature newborns to bacterial infection.

Monocyte Toll-Like Receptor 4 Expression and LPS-Induced Cytokine Production Increase during Gestational Aging

Premature newborns are highly susceptible to severe bacterial infections. This is partially due to their immature innate immune system, characterized by decreased neutrophil and monocyte activity as well as by reduced concentrations of complement factors. However, additional mechanisms might be important for innate immunity and are still the subject of considerable debate. The importance of pattern recognition domains such as Toll-like receptors (TLR) has been fully acknowledged within the last few years. Therefore, we investigated age-related monocyte TLR4 expression and lipopolysaccharide-induced cytokine secretion from very low birth weight infants (VLBWI) and from newborns after wk 30 of gestation in comparison to healthy adults. In VLBWI, expression of TLR4 surface protein, detected by flow cytometry, and TLR4-specific mRNA, quantified by real time-PCR, were significantly reduced in comparison to mature infants and to adults. Reduced TLR4 expression was paralleled by significantly diminished ex vivo LPS stimulated IL-1β, IL-6, and tumor necrosis factor-α secretion into whole blood. We conclude that, in VLBWI, the minimized expression of TLR4 contributes to the susceptibility of VLBWI to infections with Gram-negative bacteria due to the lack of cytokines to boost initial immune response.

Oral Feeding With Glutamine Prevents Lymphocyte and Glutathione Depletion of Peyer’s Patches in Endotoxemic Mice

ObjectiveTo determine the effect of oral glutamine feeding on lymphocyte subpopulations and glutathione metabolism in Peyer’s patches (PPs) of healthy and endotoxemic mice. Summary Background DataRecent data indicate that nutrients both maintain nitrogen and energy balances and modulate cell and organ function. In particular, glutamine has an impact on gut and immune function. This is of special importance in the perioperative phase. MethodsFemale Balb/c mice were fed a glutamine-enriched diet or a control diet for 10 days. On day 7 25 &mgr;g lipopolysaccharide (LPS) or saline was injected. On day 3 after the challenge, mice were killed, total cell yield was determined, and lymphocyte subpopulations (total T cells, CD4+, CD8+ cells, and B cells) were analyzed by flow cytometry. One experimental group was treated with buthionine sulfoximine, a specific inhibitor of glutathione synthesis. The glutathione content in PPs was measured by high-performance liquid chromatography. ResultsGlutamine administration led to a significant increase in total cell yield, including T and B cells, in PPs. The LPS-induced reduction of T cells (−45%) and of B cells (−30%) was significantly lower in glutamine-treated mice. Endotoxemia caused a 42% decrease of glutathione in control animals, but not in glutamine-treated animals. As with LPS, buthionine sulfoximine also lowered lymphocyte numbers and glutathione content of the PPs. ConclusionsAdministration of glutamine prevents LPS-stimulated lymphocyte atrophy in PPs, possibly by increasing the glutathione content in the PPs. Therefore, oral glutamine supply seems to be a suitable approach for improving intestinal immunity in immunocompromised patients.

Relationship Between Interleukin-6 Plasma Concentration in Patients with Sepsis, Monocyte Phenotype, Monocyte Phagocytic Properties, and Cytokine Production

Monocyte phenotype, their phagocytic capacity as well as the cytokine production from 10 patients with sepsis with low interleukin-6 (IL-6) serum concentrations (<1000 pg/mL) and 8 patients with sepsis with high IL-6 (> or = 1000 pg/mL) plasma concentrations were investigated within 24 hours of fulfilling the criteria for sepsis. Monocytes from patients with high IL-6 levels had higher levels of human leukocyte antigen (HLA)-DR, HLA-ABC, CD64, and CD71, and the production of tumor necrosis factor-alpha (TNF-alpha) and IL-8, as well as the capacity of monocytes to phagocytose, was significantly elevated. Of 8 patients with high levels of plasma IL-6, 4 patients died. In contrast, all 10 patients with low plasma IL-6 concentrations survived until day 28. Patients who died had constant high IL-6 concentrations during the first 3 days, whereas IL-6 levels in patients who survived decreased by 88%. Our data indicate that IL-6 levels are a better prognostic parameter in the early phase of sepsis than the monocyte HLA-DR expression.

Artemisinins Target GABAA Receptor Signaling and Impair α Cell Identity

Summary Type 1 diabetes is characterized by the destruction of pancreatic β cells, and generating new insulin-producing cells from other cell types is a major aim of regenerative medicine. One promising approach is transdifferentiation of developmentally related pancreatic cell types, including glucagon-producing α cells. In a genetic model, loss of the master regulatory transcription factor Arx is sufficient to induce the conversion of α cells to functional β-like cells. Here, we identify artemisinins as small molecules that functionally repress Arx by causing its translocation to the cytoplasm. We show that the protein gephyrin is the mammalian target of these antimalarial drugs and that the mechanism of action of these molecules depends on the enhancement of GABAA receptor signaling. Our results in zebrafish, rodents, and primary human pancreatic islets identify gephyrin as a druggable target for the regeneration of pancreatic β cell mass from α cells.

Immunomodulatory effects of glycine on LPS-treated monocytes: reduced TNF-α production and accelerated IL-10 expression

Cytokines play a pivotal role in the pathogenesis of septic shock. Proinflammatory cytokines such as tumor necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) stimulate the progression of septic shock whereas the anti‐inflammatory cytokine IL‐10 has counterregulative potency. The amino acid glycine (GLY) has been shown to protect against endotoxin shock in the rat by inhibiting TNF‐α production. In the current study we investigated the role of GLY on lipopolysaccharide (LPS) ‐induced cell surface marker expression, phagocytosis, and cytokine production on purified monocytes from healthy donors. GLY did not modulate the expression of HLA‐DR and CD64 on monocytes, whereas CD11b/CD18 expression (P<0.05) and E. coli phagocytosis (P<0.05) decreased significantly. GLY decreased LPS‐induced TNF‐α production (P<0.01) and increased IL‐10 expression of purified monocytes. Similarly, in a whole blood assay, GLY reduced TNF‐α (P< 0.0001) and IL‐1β (P<0.0001) synthesis and increased IL‐10 expression (P<0.05) in a dose‐dependent manner. The inhibitory effects of GLY were neutralized by strychnine, and the production of IL‐10 and TNF‐α was augmented by anti‐IL‐10 antibodies. Furthermore, GLY decreased the amount of IL‐1β and TNF‐α‐specific mRNA. Our data indicate that GLY has a potential to be used as an additional immunomodulatory tool in the early phase of sepsis and in different pathophysiological situations related to hypoxia and reperfusion.—Spittler, A., Reissner, C. M., Oehler, R., Gornikiewicz, A., Gruenberger, T., Manhart, N., Brodowicz, T., Mittlboeck, M., Boltz‐Nitulescu, G., Roth, E. Immunomodulatory effects of glycine on LPS‐treated monocytes: reduced TNF‐α production and accelerated IL‐10 expression. FASEB J. 13, 563–571 (1999)

GREEN TEA POLYPHENOL EXTRACT ATTENUATES ZYMOSAN-INDUCED NON-SEPTIC SHOCK IN MICE

ABSTRACT Multiple-organ failure (MOF) is defined as the progressive deterioration in function which occurs in several organs or systems in patients with septic shock, multiple trauma, severe burns, or pancreatitis. In the present study, we have investigated the effects of Green Tea extract (GTE) on the development of general inflammation caused by zymosan (500 mg/kg, administered i.p. as a suspension in saline) in mice. Organ failure and systemic inflammation in mice was assessed 18 hours after administration of zymosan and/or GTE and monitored for 12 days (for loss of body weight and mortality). Treatment of mice with GTE (25 mg/kg i.p., 1 and 6 hours after zymosan) attenuated the peritoneal exudation and the migration of polymorphonuclear cells (PMNs) caused by zymosan, GTE also attenuated the lung, liver and pancreatic injury and renal dysfunction caused by zymosan as well as the increase in myeloperoxidase (MPO) activity caused by zymosan in the lung and intestine. Immunohistochemical analysis for inducible nitric oxide synthase (iNOS), nitrotyrosine and poly(ADP-ribose) (PAR) revealed positive staining in lung and intestine tissues obtained from zymosan-treated mice. The degree of staining for nitrotyrosine, iNOS and PAR were markedly reduced in tissue sections obtained from zymosan-treated mice, which received GTE. In conclusion this study provides evidence, for the first time, that GTE attenuates the degree of zymosan induced generalized inflammation in mice.

A Th17- and Th2-skewed Cytokine Profile in Cystic Fibrosis Lungs Represents a Potential Risk Factor for Pseudomonas aeruginosa Infection

RATIONALE Cystic fibrosis (CF) is characterized by progressive pulmonary inflammation that is infection-triggered. Pseudomonas aeruginosa represents a risk factor for deterioration of lung function and reduced life expectancy. OBJECTIVES To assess T-cell cytokine/chemokine production in clinically stable children with CF and evaluate the association between T-cell subtypes and susceptibility for infection with P. aeruginosa. METHODS T-cell cytokine/chemokine profiles were measured in bronchoalveolar lavage fluid (BALF) from children with CF (n = 57; 6.1 ± 5.9 yr) and non-CF control subjects (n = 18; 5.9 ± 4.3 yr). Memory responses to Aspergillus fumigatus and P. aeruginosa were monitored. High-resolution computed tomography-based Helbich score was assessed. In a prospective observational trial the association between BALF cytokine/chemokine profiles and subsequent infection with P. aeruginosa was studied. MEASUREMENTS AND MAIN RESULTS Th1- (INF-γ), Th2- (IL-5, IL-13), Th17- (IL-17A), and Th17-related cytokines (IL-1β, IL-6) were significantly up-regulated in airways of patients with CF. IL-17A, IL-13, and IL-5 were significantly higher in BALF of symptomatic as compared with clinically asymptomatic patients with CF. IL-17A and IL-5 correlated with the percentage of neutrophils in BALF (r = 0.41, P < 0.05 and r = 0.46, P < 0.05, respectively). Th17- (IL-17A, IL-6, IL-1β, IL-8) and Th2-associated cytokines and chemokines (IL-5, IL-13, TARC/CCL17), but not IFN-γ levels, significantly correlated with high-resolution computed tomography changes (Helbich score; P < 0.05). P. aeruginosa- and A. fumigatus-specific T cells from patients with CF displayed significantly higher IL-5 and IL-17A mRNA expression. IL-17A and TARC/CCL17 were significantly augmented in patients that developed P. aeruginosa infection within 24 months. CONCLUSIONS We propose a role for Th17 and Th2 T cells in chronic inflammation in lungs of patients with CF. High concentrations of these cytokines/chemokines in CF airways precede infection with P. aeruginosa.

Single-cell transcriptomes reveal characteristic features of human pancreatic islet cell types

Pancreatic islets of Langerhans contain several specialized endocrine cell types, which are commonly identified by the expression of single marker genes. However, the established marker genes cannot capture the complete spectrum of cellular heterogeneity in human pancreatic islets, and existing bulk transcriptome datasets provide averages across several cell populations. To dissect the cellular composition of the human pancreatic islet and to establish transcriptomes for all major cell types, we performed single‐cell RNA sequencing on 70 cells sorted from human primary tissue. We used this dataset to validate previously described marker genes at the single‐cell level and to identify specifically expressed transcription factors for all islet cell subtypes. All data are available for browsing and download, thus establishing a useful resource of single‐cell expression profiles for endocrine cells in human pancreatic islets.