Summary Neutrophils are major inflammatory cells that rapidly infiltrate wounds to provide antimicrobial functions. Within the damaged tissue, neutrophil migration behavior often switches from exploratory patrolling to coordinated swarming, giving rise to dense clusters that further disrupt tissue architecture. This aggregation response is self-organized by neutrophil paracrine chemoattractant signaling (most notably of the inflammatory mediator leukotriene B4 [LTB4]). The coordination mechanism and possible evolutionary benefits of neutrophil swarms are elusive. Here, we show that neutrophil swarms require mutual reinforcement of damage signaling at the wound core. New biosensors and live imaging in zebrafish revealed that neutrophil chemoattractant synthesis is triggered by a sustained calcium flux upon contact with necrotic tissue that requires sensing of the damage signal ATP. This “calcium alarm” signal rapidly propagates in the nascent neutrophil cluster in a contact-dependent manner via connexin-43 (Cx43) hemichannels, which are mediators of active ATP release. This enhances chemoattractant biosynthesis in the growing cluster, which is instrumental for coordinated motion and swarming. Inhibition of neutrophil Cx43 compromises clearance of wound-colonizing P. aeruginosa bacteria and exacerbates infection-induced morbidity. Thus, cooperative production of alarm signals among pioneer clustering neutrophils fuels the growth of dense antimicrobial cell masses that effectively seal off breached tissue barriers from opportunistic pathogens.

Low CFTR mRNA expression due to nonsense-mediated mRNA decay (NMD) is a major hurdle in developing a therapy for cystic fibrosis (CF) caused by the W1282X mutation in the CFTR gene. CFTR-W1282X truncated protein retains partial function, so increasing its levels by inhibiting NMD of its mRNA will likely be beneficial. Because NMD regulates the normal expression of many genes, gene-specific stabilization of CFTR-W1282X mRNA expression is more desirable than general NMD inhibition. Synthetic antisense oligonucleotides (ASOs) designed to prevent binding of exon junction complexes (EJC) downstream of premature termination codons (PTCs) attenuate NMD in a gene-specific manner. We describe cocktails of three ASOs that specifically increase the expression of CFTR-W1282X mRNA and CFTR protein upon delivery into human bronchial epithelial cells. This treatment increases the CFTR-mediated chloride current. These results set the stage for clinical development of an allele-specific therapy for CF caused by the W1282X mutation.

The transcriptional effector SMAD4 is a core component of the TGF-β family signaling pathways. However, its role in vertebrate embryo development remains unresolved. To address this, we deleted Smad4 in zebrafish and investigated the consequences of this on signaling by the TGF-β family morphogens, BMPs and Nodal. We demonstrate that in the absence of Smad4, dorsal/ventral embryo patterning is disrupted due to the loss of BMP signaling. However, unexpectedly, Nodal signaling is maintained, but lacks robustness. This Smad4-independent Nodal signaling is sufficient for mesoderm specification, but not for optimal endoderm specification. Furthermore, using Optical Projection Tomography in combination with 3D embryo morphometry, we have generated a BMP morphospace and demonstrate that Smad4 mutants are morphologically indistinguishable from embryos in which BMP signaling has been genetically/pharmacologically perturbed. Smad4 is thus differentially required for signaling by different TGF-β family ligands, which has implications for diseases where Smad4 is mutated or deleted.

Low CFTR mRNA expression due to nonsense-mediated mRNA decay (NMD) is a major hurdle in developing a therapy for cystic fibrosis (CF) caused by the W1282X mutation in the CFTR gene. CFTR-W1282X truncated protein retains partial function, so increasing its levels by inhibiting NMD of its mRNA will likely be beneficial. Because NMD regulates the normal expression of many genes, gene-specific stabilization of CFTR-W1282X mRNA expression is more desirable than general NMD inhibition. Synthetic antisense oligonucleotides (ASOs) designed to prevent binding of exon junction complexes (EJC) downstream of premature termination codons (PTCs) attenuate NMD in a gene-specific manner. We developed a cocktail of three ASOs that specifically increases the expression of CFTR W1282X mRNA and CFTR protein in ASO-transfected human bronchial epithelial cells. This treatment increased the CFTR-mediated chloride current. These results set the stage for clinical development of an allele-specific therapy for CF caused by the W1282X mutation.