T lymphocyte priming by neutrophil extracellular traps links innate and adaptive immune responses

Polymorphonuclear neutrophils constitute the first line of defense against infections. Among their strategies to eliminate pathogens they release neutrophil extracellular traps (NETs), being chromatin fibers decorated with antimicrobial proteins. NETs trap and kill pathogens very efficiently, thereby minimizing tissue damage. Furthermore, NETs modulate inflammatory responses by activating plasmacytoid dendritic cells. In this study, we show that NETs released by human neutrophils can directly prime T cells by reducing their activation threshold. NETs-mediated priming increases T cell responses to specific Ags and even to suboptimal stimuli, which would not induce a response in resting T cells. T cell priming mediated by NETs requires NETs/cell contact and TCR signaling, but unexpectedly we could not demonstrate a role of TLR9 in this mechanism. NETs-mediated T cell activation adds to the list of neutrophil functions and demonstrates a novel link between innate and adaptive immune responses.     

Influenza neuraminidase inhibitors: structure-based design of a novel inhibitor series

Combinatorial and structure-based medicinal chemistry strategies were used together to advance a lead compound with an activity of K(i) = 58 microM via a potency enhancement of >70 000-fold to an analogue with an activity of K(i) = 0.8 nM against influenza neuraminidase (A/Tokyo/67). Lead optimization was initiated using molecular modeling and combinatorial chemistry. Protein crystal structures revealed that inconsistent structure-activity relationship (SAR) data resulted from different binding orientations of the inhibitor core five-membered rings from one series to another. Binding modes for a series of compounds showed up to a 180 degrees variation in orientation of the five-membered ring within the active site. Potent analogues were only achieved with chemical series that were observed to bind in the same orientation and yielded consistent SAR. In one series, consistent binding was obtained by an unprecedented occupation of a negatively charged binding pocket by a neutral methyl ester unit. The structural rationale for this novel SAR variation, based on protein crystallographic data, is given.     

Butterfly phenology in Mediterranean mountains using space‐for‐time substitution

Inferring species' responses to climate change in the absence of long‐term time series data is a challenge, but can be achieved by substituting space for time. For example, thermal elevational gradients represent suitable proxies to study phenological responses to warming. We used butterfly data from two Mediterranean mountain areas to test whether mean dates of appearance of communities and individual species show a delay with increasing altitude, and an accompanying shortening in the duration of flight periods. We found a 14‐day delay in the mean date of appearance per kilometer increase in altitude for butterfly communities overall, and an average 23‐day shift for 26 selected species, alongside average summer temperature lapse rates of 3°C per km. At higher elevations, there was a shortening of the flight period for the community of 3 days/km, with an 8.8‐day average decline per km for individual species. Rates of phenological delay differed significantly between the two mountain ranges, although this did not seem to result from the respective temperature lapse rates. These results suggest that climate warming could lead to advanced and lengthened flight periods for Mediterranean mountain butterfly communities. However, although multivoltine species showed the expected response of delayed and shortened flight periods at higher elevations, univoltine species showed more pronounced delays in terms of species appearance. Hence, while projections of overall community responses to climate change may benefit from space‐for‐time substitutions, understanding species‐specific responses to local features of habitat and climate may be needed to accurately predict the effects of climate change on phenology.     

Hematopoietic changes and altered reactivity to IL-17 in Syphacia obvelata-infected mice

Pinworm parasites commonly infect laboratory mice with high prevalence even in well-managed animal colonies. Although often considered as irrelevant, these parasites if undetected may significantly interfere with the experimental settings and alter the interpretation of final results. There are a few reports documenting the effects of pinworms on research and the effects of pinworms on the host hematopoiesis have not yet been investigated. In this study we examined the changes within various hematopoietic cell lineages in the bone marrow, spleen, peripheral blood and peritoneal space during naturally acquired Syphacia obvelata infection in inbred CBA mice. The data obtained showed significant hematopoietic alterations, characterized by increased myelopoiesis and erythropoiesis in S. obvelata-infected animals. In order to additionally evaluate if this pinworm infection modifies hematopoietic cells' reactivity, we examined the effect of murine interleukin-17, T cell-derived cytokine implicated in the regulation of hematopoiesis and inflammation, on the growth of bone marrow progenitor cells and demonstrated that bone marrow myeloid and erythroid progenitors from S. obvelata-infected mice displayed altered sensitivity to IL-17 when compared to non-infected controls. Taken together the alterations presented pointed out that this rodent pinworm is an important environmental agent that might significantly modify the hosts' hematopoietic response, and therefore interfere with the experimental settings and alter the interpretation of the final results. However, the results obtained also contributed new data concerning the activity of IL-17 on bone marrow hematopoietic cells, supporting our previous reports that depending on physiological/pathological status of the organism IL-17 exerts differential effects on the growth of progenitor cells.     

Hepsin regulates TGFβ signaling via fibronectin proteolysis

Transforming growth factor‐beta (TGFβ) is a multifunctional cytokine with a well‐established role in mammary gland development and both oncogenic and tumor‐suppressive functions. The extracellular matrix (ECM) indirectly regulates TGFβ activity by acting as a storage compartment of latent‐TGFβ, but how TGFβ is released from the ECM via proteolytic mechanisms remains largely unknown. In this study, we demonstrate that hepsin, a type II transmembrane protease overexpressed in 70% of breast tumors, promotes canonical TGFβ signaling through the release of latent‐TGFβ from the ECM storage compartment. Mammary glands in hepsin CRISPR knockout mice showed reduced TGFβ signaling and increased epithelial branching, accompanied by increased levels of fibronectin and latent‐TGFβ1, while overexpression of hepsin in mammary tumors increased TGFβ signaling. Cell‐free and cell‐based experiments showed that hepsin is capable of direct proteolytic cleavage of fibronectin but not latent‐TGFβ and, importantly, that the ability of hepsin to activate TGFβ signaling is dependent on fibronectin. Altogether, this study demonstrates a role for hepsin as a regulator of the TGFβ pathway in the mammary gland via a novel mechanism involving proteolytic downmodulation of fibronectin.     

Interstitial telomere sequences disrupt break-induced replication and drive formation of ectopic telomeres

Break-induced replication (BIR) is a mechanism used to heal one-ended DNA double-strand breaks, such as those formed at collapsed replication forks or eroded telomeres. Instead of utilizing a canonical replication fork, BIR is driven by a migrating D-loop and is associated with a high frequency of mutagenesis. Here we show that when BIR encounters an interstitial telomere sequence (ITS), the machinery frequently terminates, resulting in the formation of an ectopic telomere. The primary mechanism to convert the ITS to a functional telomere is by telomerase-catalyzed addition of telomeric repeats with homology-directed repair serving as a back-up mechanism. Termination of BIR and creation of an ectopic telomere is promoted by Mph1/FANCM helicase, which has the capacity to disassemble D-loops. Other sequences that have the potential to seed new telomeres but lack the unique features of a natural telomere sequence, do not terminate BIR at a significant frequency in wild-type cells. However, these sequences can form ectopic telomeres if BIR is made less processive. Our results support a model in which features of the ITS itself, such as the propensity to form secondary structures and telomeric protein binding, pose a challenge to BIR and increase the vulnerability of the D-loop to dissociation by helicases, thereby promoting ectopic telomere formation.     

The Arabidopsis Prohibitin Gene PHB3 Functions in Nitric Oxide–Mediated Responses and in Hydrogen Peroxide–Induced Nitric Oxide Accumulation

To discover genes involved in nitric oxide (NO) metabolism, a genetic screen was employed to identify mutants defective in NO accumulation after treatment with the physiological inducer hydrogen peroxide. In wild-type Arabidopsis thaliana plants, NO levels increase eightfold in roots after H₂O₂ treatment for 30 min. A mutant defective in H₂O₂-induced NO accumulation was identified, and the corresponding mutation was mapped to the prohibitin gene PHB3, converting the highly conserved Gly-37 to an Asp in the protein''s SPFH domain. This point mutant and a T-DNA insertion mutant were examined for other NO-related phenotypes. Both mutants were defective in abscisic acid–induced NO accumulation and stomatal closure and in auxin-induced lateral root formation. Both mutants were less sensitive to salt stress, showing no increase in NO accumulation and less inhibition of primary root growth in response to NaCl treatment. In addition, light-induced NO accumulation was dramatically reduced in cotyledons. We found no evidence for impaired H₂O₂ metabolism or signaling in the mutants as H₂O₂ levels and H₂O₂-induced gene expression were unaffected by the mutations. These findings identify a component of the NO homeostasis system in plants and expand the function of prohibitin genes to include regulation of NO accumulation and NO-mediated responses.