The gut microbiota promotes distal tissue regeneration via RORγ+ regulatory T cell emissaries.

Specific microbial signals induce the differentiation of a distinct pool of RORγ+ regulatory T (Treg) cells crucial for intestinal homeostasis. We discovered highly analogous populations of microbiota-dependent Treg cells that promoted tissue regeneration at extra-gut sites, notably acutely injured skeletal muscle and fatty liver. Inflammatory meditators elicited by tissue damage combined with MHC-class-II-dependent T cell activation to drive the accumulation of gut-derived RORγ+ Treg cells in injured muscle, wherein they regulated the dynamics and tenor of early inflammation and helped balance the proliferation vs. differentiation of local stem cells. Reining in IL-17A-producing T cells was a major mechanism underlying the rheostatic functions of RORγ+ Treg cells in compromised tissues. Our findings highlight the importance of gut-trained Treg cell emissaries in controlling the response to sterile injury of non-mucosal tissues.

Aire Inhibits the Generation of a Perinatal Population of Interleukin-17A-Producing γδ T Cells to Promote Immunologic Tolerance.

Aire's primary mechanism of action is to regulate transcription of a battery of genes in medullary thymic epithelial cells (mTECs) and, consequently, negative selection of effector T cells and positive selection of regulatory T cells. We found that Aire-deficient mice had expanded thymic and peripheral populations of perinatally generated IL-17A+Vγ6+Vδ1+ T cells, considered to be "early responders" to tissue stress and drivers of inflammatory reactions. Aire-dependent control of Il7 expression in mTECs regulated the size of thymic IL-17A+Vγ6+Vδ1+ compartments. In mice lacking Aire and γδ T cells, certain tissues typically targeted in the "Aire-less" disease, notably the retina, were only minimally infiltrated. IL-17A+Vγ6+Vδ1+ cells were present in the retina of wild-type mice and expanded very early in Aire-deficient mice. A putatively parallel population of IL-17A+Vγ9+Vδ2+ T cells was increased in humans lacking Aire. Thus, Aire exerts multi-faceted autoimmune control that extends to a population of innate-like T cells.

PPARγ marks splenic precursors of multiple nonlymphoid-tissue Treg compartments.

Foxp3+CD4+ regulatory T cells (Tregs) regulate most types of immune response as well as several processes important for tissue homeostasis, for example, metabolism and repair. Dedicated Treg compartments-with distinct transcriptomes, T cell receptor repertoires, and growth/survival factor dependencies-have been identified in several nonlymphoid tissues. These Tregs are specifically adapted to function and operate in their home tissue-When, where, and how do they take on their specialized characteristics? We recently reported that a splenic Treg population expressing low levels of the transcription factor PPARγ (peroxisome proliferator-activated receptor gamma) contains precursors of Tregs residing in visceral adipose tissue. This finding made sense given that PPARγ, the "master regulator" of adipocyte differentiation, is required for the accumulation and function of Tregs in visceral adipose tissue but not in lymphoid tissues. Here we use single-cell RNA sequencing, single-cell Tcra and Tcrb sequencing, and adoptive-transfer experiments to show that, unexpectedly, the splenic PPARγlo Treg population is transcriptionally heterogeneous and engenders Tregs in multiple nonlymphoid tissues beyond visceral adipose tissue, such as skin and liver. The existence of a general pool of splenic precursors for nonlymphoid-tissue Tregs opens possibilities for regulating their emergence experimentally or therapeutically.

Secreted modular calcium-binding protein 1 binds and activates thrombin to account for platelet hyperreactivity in diabetes.

Secreted modular calcium-binding protein 1 (SMOC1) is an osteonectin/SPARC-related matricellular protein, whose expression is regulated by microRNA-223 (miR-223). Given that platelets are rich in miR-223, this study investigated the expression of SMOC1 and its contribution to platelet function. Human and murine platelets expressed SMOC1, whereas platelets from SMOC1+/- mice did not present detectable mature SMOC1 protein. Platelets from SMOC1+/- mice demonstrated attenuated responsiveness to thrombin (platelet neutrophil aggregate formation, aggregation, clot formation, Ca2+ increase, and ß3 integrin phosphorylation), whereas responses to other platelet agonists were unaffected. SMOC1 has been implicated in transforming growth factor-ß signaling, but no link to this pathway was detected in platelets. Rather, the SMOC1 Kazal domain directly bound thrombin to potentiate its activity in vitro, as well as its actions on isolated platelets. The latter effects were prevented by monoclonal antibodies against SMOC1. Platelets from miR-223-deficient mice expressed high levels of SMOC1 and exhibited hyperreactivity to thrombin that was also reversed by preincubation with monoclonal antibodies against SMOC1. Similarly, SMOC1 levels were markedly upregulated in platelets from individuals with type 2 diabetes, and the SMOC1 antibody abrogated platelet hyperresponsiveness to thrombin. Taken together, we have identified SMOC1 as a novel thrombin-activating protein that makes a significant contribution to the pathophysiological changes in platelet function associated with type 2 diabetes. Thus, strategies that target SMOC1 or its interaction with thrombin may be attractive therapeutic approaches to normalize platelet function in diabetes.

Minimal transmission in an influenza A (H3N2) human challenge-transmission model within a controlled exposure environment.

Uncertainty about the importance of influenza transmission by airborne droplet nuclei generates controversy for infection control. Human challenge-transmission studies have been supported as the most promising approach to fill this knowledge gap. Healthy, seronegative volunteer 'Donors' (n = 52) were randomly selected for intranasal challenge with influenza A/Wisconsin/67/2005 (H3N2). 'Recipients' randomized to Intervention (IR, n = 40) or Control (CR, n = 35) groups were exposed to Donors for four days. IRs wore face shields and hand sanitized frequently to limit large droplet and contact transmission. One transmitted infection was confirmed by serology in a CR, yielding a secondary attack rate of 2.9% among CR, 0% in IR (p = 0.47 for group difference), and 1.3% overall, significantly less than 16% (p<0.001) expected based on a proof-of-concept study secondary attack rate and considering that there were twice as many Donors and days of exposure. The main difference between these studies was mechanical building ventilation in the follow-on study, suggesting a possible role for aerosols.

Platelet-derived calpain cleaves the endothelial protease-activated receptor 1 to induce vascular inflammation in diabetes.

Diabetes mellitus is a major risk factor for cardiovascular disease. Platelets from diabetic patients are hyperreactive and release microparticles that carry activated cysteine proteases or calpains. Whether platelet-derived calpains contribute to the development of vascular complications in diabetes is unknown. Here we report that platelet-derived calpain1 (CAPN1) cleaves the protease-activated receptor 1 (PAR-1) on the surface of endothelial cells, which then initiates a signaling cascade that includes the activation of the tumor necrosis factor (TNF)-α converting enzyme (TACE). The latter elicits the shedding of the endothelial protein C receptor and the generation of TNF-α, which in turn, induces intracellular adhesion molecule (ICAM)-1 expression to promote monocyte adhesion. All of the effects of CAPN1 were mimicked by platelet-derived microparticles from diabetic patients or from wild-type mice but not from CAPN1-/- mice, and were not observed in PAR-1-deficient endothelial cells. Importantly, aortae from diabetic mice expressed less PAR-1 but more ICAM-1 than non-diabetic mice, effects that were prevented by treating diabetic mice with a calpain inhibitor as well as by the platelet specific deletion of CAPN1. Thus, platelet-derived CAPN1 contributes to the initiation of the sterile vascular inflammation associated with diabetes via the cleavage of PAR-1 and the release of TNF-α from the endothelial cell surface.

Dicer cleavage by calpain determines platelet microRNA levels and function in diabetes.

RATIONALE: MicroRNAs (miRNAs) are short noncoding RNA species generated by the processing of longer precursors by the ribonucleases Drosha and Dicer. Platelets contain large amounts of miRNA that are altered by disease, in particular diabetes mellitus. OBJECTIVE: This study determined why platelet miRNA levels are attenuated in diabetic individuals and how decreased levels of the platelet-enriched miRNA, miR-223, affect platelet function. METHODS AND RESULTS: Dicer levels were altered in platelets from diabetic mice and patients, a change that could be attributed to the cleavage of the enzyme by calpain, resulting in loss of function. Diabetes mellitus in human subjects as well as in mice resulted in decreased levels of platelet miR-142, miR-143, miR-155, and miR-223. Focusing on only 1 of these miRNAs, miR-223 deletion in mice resulted in modestly enhanced platelet aggregation, the formation of large thrombi and delayed clot retraction compared with wild-type littermates. A similar dysregulation was detected in platelets from diabetic patients. Proteomic analysis of platelets from miR-223 knockout mice revealed increased levels of several proteins, including kindlin-3 and coagulation factor XIII-A. Whereas, kindlin-3 was indirectly regulated by miR-223, factor XIII was a direct target and both proteins were also altered in diabetic platelets. Treating diabetic mice with a calpain inhibitor prevented loss of platelet dicer as well as the diabetes mellitus-induced decrease in platelet miRNA levels and the upregulation of miR-223 target proteins. CONCLUSIONS: Thus, calpain inhibition may be one means of normalizing platelet miRNA processing as well as platelet function in diabetes mellitus.

Dilution cultivation of marine heterotrophic bacteria abundant after a spring phytoplankton bloom in the North Sea.

The roles of individual bacterioplankton species in the re-mineralization of algal biomass are poorly understood. Evidence from molecular data had indicated that a spring diatom bloom in the German Bight of the North Sea in 2009 was followed by a rapid succession of uncultivated bacterioplankton species, including members of the genera Ulvibacter, Formosa, Polaribacter (class Flavobacteria) and Reinekea (class Gammaproteobacteria). We isolated strains from the same site during the diatom bloom in spring 2010 using dilution cultivation in an artificial seawater medium with micromolar substrate and nutrient concentrations. Flow cytometry demonstrated growth from single cells to densities of 10(4) -10(6) cells ml(-1) and a culturability of 35%. Novel Formosa, Polaribacter and Reinekea strains were isolated and had 16S rRNA gene sequence identities of > 99.8% with bacterioplankton in spring or summer 2009. Genomes of selected isolates were draft sequenced and used for read recruitment of metagenomes from bacterioplankton in 2009. Metagenome reads covered 93% of a Formosa clade B, 91% of a Reinekea and 74% of a Formosa clade A genome, applying a ≥ 94.5% nucleotide identity threshold. These novel strains represent abundant bacterioplankton species thriving on coastal phytoplankton blooms in the North Sea.

Halorhabdus tiamatea: proteogenomics and glycosidase activity measurements identify the first cultivated euryarchaeon from a deep-sea anoxic brine lake as potential polysaccharide degrader.

Euryarchaea from the genus Halorhabdus have been found in hypersaline habitats worldwide, yet are represented by only two isolates: Halorhabdus utahensis AX-2(T) from the shallow Great Salt Lake of Utah, and Halorhabdus tiamatea SARL4B(T) from the Shaban deep-sea hypersaline anoxic lake (DHAL) in the Red Sea. We sequenced the H. tiamatea genome to elucidate its niche adaptations. Among sequenced archaea, H. tiamatea features the highest number of glycoside hydrolases, the majority of which were expressed in proteome experiments. Annotations and glycosidase activity measurements suggested an adaptation towards recalcitrant algal and plant-derived hemicelluloses. Glycosidase activities were higher at 2% than at 0% or 5% oxygen, supporting a preference for low-oxygen conditions. Likewise, proteomics indicated quinone-mediated electron transport at 2% oxygen, but a notable stress response at 5% oxygen. Halorhabdus tiamatea furthermore encodes proteins characteristic for thermophiles and light-dependent enzymes (e.g. bacteriorhodopsin), suggesting that H. tiamatea evolution was mostly not governed by a cold, dark, anoxic deep-sea habitat. Using enrichment and metagenomics, we could demonstrate presence of similar glycoside hydrolase-rich Halorhabdus members in the Mediterranean DHAL Medee, which supports that Halorhabdus species can occupy a distinct niche as polysaccharide degraders in hypersaline environments.

The genome of the alga-associated marine flavobacterium Formosa agariphila KMM 3901T reveals a broad potential for degradation of algal polysaccharides.

In recent years, representatives of the Bacteroidetes have been increasingly recognized as specialists for the degradation of macromolecules. Formosa constitutes a Bacteroidetes genus within the class Flavobacteria, and the members of this genus have been found in marine habitats with high levels of organic matter, such as in association with algae, invertebrates, and fecal pellets. Here we report on the generation and analysis of the genome of the type strain of Formosa agariphila (KMM 3901(T)), an isolate from the green alga Acrosiphonia sonderi. F. agariphila is a facultative anaerobe with the capacity for mixed acid fermentation and denitrification. Its genome harbors 129 proteases and 88 glycoside hydrolases, indicating a pronounced specialization for the degradation of proteins, polysaccharides, and glycoproteins. Sixty-five of the glycoside hydrolases are organized in at least 13 distinct polysaccharide utilization loci, where they are clustered with TonB-dependent receptors, SusD-like proteins, sensors/transcription factors, transporters, and often sulfatases. These loci play a pivotal role in bacteroidetal polysaccharide biodegradation and in the case of F. agariphila revealed the capacity to degrade a wide range of algal polysaccharides from green, red, and brown algae and thus a strong specialization of toward an alga-associated lifestyle. This was corroborated by growth experiments, which confirmed usage particularly of those monosaccharides that constitute the building blocks of abundant algal polysaccharides, as well as distinct algal polysaccharides, such as laminarins, xylans, and κ-carrageenans.

Macrophages control pathological interferon responses during viral respiratory infection.

Antiviral immune mediators, including interferons and their downstream effectors, are critical for host defense yet can become detrimental when uncontrolled. Here, we identify a macrophage-mediated anti-inflammatory mechanism that limits type I interferon (IFN-I) responses. Specifically, we found that cellular stress and pathogen recognition induce Oncostatin M (OSM) production by macrophages. OSM-deficient mice succumbed to challenge with influenza or a viral mimic due to heightened IFN-I activation. Macrophage-derived OSM restricted excessive IFN-I production by lung epithelial cells following viral stimulation. Furthermore, reconstitution of OSM in the respiratory tract was sufficient to protect mice lacking macrophage-derived OSM against morbidity, indicating the importance of local OSM production. This work reveals a host strategy to dampen inflammation in the lung through the negative regulation of IFN-I by macrophages.

IL-17A-producing γδT cells promote muscle regeneration in a microbiota-dependent manner.

Subsequent to acute injury, skeletal muscle undergoes a stereotypic regenerative process that reestablishes homeostasis. Various types of innate and adaptive immunocytes exert positive or negative influences at specific stages along the course of muscle regeneration. We describe an unanticipated role for γδT cells in promoting healthy tissue recovery after injection of cardiotoxin into murine hindlimb muscle. Within a few days of injury, IL-17A-producing γδT cells displaying primarily Vγ6+ antigen receptors accumulated at the wound site. Punctual ablation experiments showed that these cells boosted early inflammatory events, notably recruitment of neutrophils; fostered the proliferation of muscle stem and progenitor cells; and thereby promoted tissue regeneration. Supplementation of mice harboring low numbers of IL-17A+ γδT cells with recombinant IL-17A largely reversed their inflammatory and reparative defects. Unexpectedly, the accumulation and influences of γδT cells in this experimental context were microbiota dependent, unveiling an orthogonal perspective on the treatment of skeletal muscle pathologies such as catastrophic wounds, wasting, muscular dystrophies, and myositides.

Folate-functionalized unimolecular micelles based on a degradable amphiphilic dendrimer-like star polymer for cancer cell-targeted drug delivery.

A folate-functionalized degradable amphiphilic dendrimer-like star polymer (FA-DLSP) with a well-defined poly(L-lactide) (PLLA) star polymer core and six hydrophilic polyester dendrons based on 2,2-bis(hydroxymethyl) propionic acid was successfully synthesized to be used as a nanoscale carrier for cancer cell-targeted drug delivery. This FA-DLSP hybrid formed unimolecular micelles in the aqueous solution with a mean particle size of ca. 15 nm as determined by dynamic light scattering and transmission electron microscopy. To study the feasibility of FA-DLSP micelles as a potential nanocarrier for targeted drug delivery, we encapsulated a hydrophobic anticancer drug, doxorubicin (DOX), in the hydrophobic core, and the loading content was determined by UV-vis analysis to be 4 wt %. The DOX-loaded FA-DLSP micelles demonstrated a sustained release of DOX due to the hydrophobic interaction between the polymer core and the drug molecules. The hydrolytic degradation in vitro was monitored by weight loss and proton nuclear magnetic resonance spectroscopy to gain insight into the degradation mechanism of the FA-DLSP micelles. It was found that the degradation was pH-dependent and started from the hydrophilic shell gradually to the hydrophobic core. Flow cytometry and confocal microscope studies revealed that the cellular binding of the FA-DLSP hybrid against human KB cells with overexpressed folate-receptors was about twice that of the neat DLSP (without FA). The in vitro cellular cytotoxicity indicated that the FA-DLSP micelles (without DOX) had good biocompatibility with KB cells, whereas DOX-loaded micelles exhibited a similar degree of cytotoxicity against KB cells as that of free DOX. These results clearly showed that the FA-DLSP unimolecular micelles could be a promising nanosize anticancer drug carrier with excellent targeting property.

Influenza A (H3) illness and viral aerosol shedding from symptomatic naturally infected and experimentally infected cases.

BACKGROUND: It has long been known that nasal inoculation with influenza A virus produces asymptomatic to febrile infections. Uncertainty persists about whether these infections are sufficiently similar to natural infections for studying human-to-human transmission. METHODS: We compared influenza A viral aerosol shedding from volunteers nasally inoculated with A/Wisconsin/2005 (H3N2) and college community adults naturally infected with influenza A/H3N2 (2012-2013), selected for influenza-like illness with objectively measured fever or a positive Quidel QuickVue A&B test. Propensity scores were used to control for differences in symptom presentation observed between experimentally and naturally infected groups. RESULTS: Eleven (28%) experimental and 71 (86%) natural cases shed into fine particle aerosols (P < .001). The geometric mean (geometric standard deviation) for viral positive fine aerosol samples from experimental and natural cases was 5.1E + 3 (4.72) and 3.9E + 4 (15.12) RNA copies/half hour, respectively. The 95th percentile shedding rate was 2.4 log10 greater for naturally infected cases (1.4E + 07 vs 7.4E + 04). Certain influenza-like illness-related symptoms were associated with viral aerosol shedding. The almost complete lack of symptom severity distributional overlap between groups did not support propensity score-adjusted shedding comparisons. CONCLUSIONS: Due to selection bias, the natural and experimental infections had limited symptom severity distributional overlap precluding valid, propensity score-adjusted comparison. Relative to the symptomatic naturally infected cases, where high aerosol shedders were found, experimental cases did not produce high aerosol shedders. Studying the frequency of aerosol shedding at the highest observed levels in natural infections without selection on symptoms or fever would support helpful comparisons.

Antigen-specific memory and naïve CD4+ T cells following secondary Chlamydia trachomatis infection.

Memory antigen-specific CD4+ T cells against Chlamydia trachomatis are necessary for protection against secondary genital tract infection. While it is known that naïve antigen-specific CD4+ T cells can traffic to the genital tract in an antigen-specific manner, these T cells are not protective during primary infection. Here, we sought to compare the differences between memory and naïve antigen-specific CD4+ T cells in the same mouse following secondary infection using transgenic CD4+ T cells (NR1 T cells). Using RNA sequencing, we found that there were subtle but distinct differences between these two T cell populations. Naïve NR1 T cells significantly upregulated cell cycle genes and were more proliferative than memory NR1 T cells in the draining lymph node. In contrast, memory NR1 T cells were more activated than naïve NR1 T cells and were enriched in the genital tract. Together, our data provide insight into the differences between memory and naïve antigen-specific CD4+ T cells during C. trachomatis infection.

Template-Free Orientation Selection of Rod-Like Molecular Semiconductors in Polycrystalline Films.

Many organic semiconductors (OSCs) feature strong optical anisotropy that can be exploited to increase the efficiency of optoelectronic devices. We demonstrate that for the technologically relevant, rod-like OSCs diindenoperylene (DIP), pentacene (PEN), and α-sexithiophene (6T) deposited on silicon oxide surfaces it is possible to prepare polycrystalline thin films in which the long molecular axis is oriented parallel to the substrate plane in a template-free fashion. In films grown by organic molecular beam deposition at room temperature or higher, the molecules are oriented upright standing (σ-orientation). Instead, the here-presented growth at low temperatures followed by slow annealing up to a temperature near molecular desorption has the effect of "freezing" the molecules with their long axis oriented parallel to the substrate plane (λ-orientation) while conferring them crystalline long-range order. We discuss the huge impact on the optical anisotropy of the films observed as a consequence of the orientation transition. Finally, we propose a mechanism for explaining the achieved λ-orientation, which is stable under environmental conditions.

Association between arginase-containing platelet-derived microparticles and altered plasma arginine metabolism in polycystic ovary syndrome.

BACKGROUND: Polycystic ovary syndrome (PCOS) is an endocrine and metabolic disease associated with insulin resistance and increased risk of cardiovascular diseases. However, a biomarker for potential cardiovascular disease in PCOS patients is not available. MATERIALS AND METHODS: Twenty-two patients with PCOS and 22 healthy controls were included in the present study and amino acid profiling was performed on fasting plasma samples. Circulating microparticles were characterized by FACS analysis and complemented with enzyme activity assays. RESULTS: The ratio of ornithine to arginine was significantly increased in plasma form PCOS patients and was associated with a significant increase in plasma arginase levels and activity. Platelet-derived microparticles were identified to be the main sources of the increased plasma arginase activity. CONCLUSIONS: Increased levels of arginase-bearing platelet-derived microparticles contribute to the alteration of the arginine metabolism in patients with polycystic ovary syndrome. Moreover, ornithine and arginine levels represent early biomarkers of potential cardiovascular disease in PCOS patients.

Deficient knowledge in adult Turner syndrome care as an incentive to found Turner centers in Germany.

OBJECTIVE: Turner syndrome (TS) is characterized by the complete or partial loss of the second sex chromosome and associated with a wide range of clinical manifestations. We aimed to assess the medical care of adult patients with TS in Germany. DESIGN: Retrospective multicenter observational study. METHODS: Data were collected from medical records of 258 women with TS treated between 2001 and 2017 in five non-university endocrinologic centers in Germany. RESULTS: Mean age was 29.8 ± 11.6 years, mean height 152 ± 7.7 cm, and mean BMI 26.6 ± 6.3 kg/m2. The karyotype was known in 50% of patients. Information on cholesterol state, liver enzymes, and thyroid status was available in 81-98% of women with TS; autoimmune thyroiditis was diagnosed in 37%. Echocardiography was performed in 42% and cardiac MRI in 8.5%, resulting in a diagnosis of cardiovascular disorder in 28%. Data on growth hormone therapy were available for 40 patients (15%) and data concerning menarche in 157 patients (61%). CONCLUSION: In 258 women with TS, retrospective analysis of healthcare data indicated that medical management was focused on endocrine manifestations. Further significant clinical features including cardiovascular disease, renal malformation, liver involvement, autoimmune diseases, hearing loss, and osteoporosis were only marginally if at all considered. Based on this evaluation and in accordance with recent guidelines, we compiled a documentation form facilitating the transition from pediatric to adult care and further medical management of TS patients. The foundation of Turner Centers in March 2019 will improve the treatment of TS women in Germany.