Walters, P.

ABSTRACT Myofibroblasts play key roles in wound healing and pathological fibrosis. Here, we used an RNAi screen to characterize myofibroblast regulatory genes, using a high-content imaging approach to quantify α-smooth muscle actin stress fibers in cultured human fibroblasts. Screen hits were validated on physiological compliance hydrogels, and selected hits tested in primary fibroblasts from patients with idiopathic pulmonary fibrosis. Our RNAi screen led to the identification of STAT3 as an essential mediator of myofibroblast activation and function. Strikingly, we found that STAT3 phosphorylation, while responsive to exogenous ligands on both soft and stiff matrices, is innately active on a stiff matrix in a ligand/receptor-independent, but ROCK- and JAK2-dependent fashion. These results demonstrate how a cytokine-inducible signal can become persistently activated by pathological matrix stiffening. Consistent with a pivotal role for this pathway in driving persistent fibrosis, a STAT3 inhibitor attenuated murine pulmonary fibrosis when administered in a therapeutic fashion after bleomycin injury. Our results identify novel genes essential for the myofibroblast phenotype, and point to STAT3 as an important target in pulmonary fibrosis and other fibrotic diseases.

Abstract B lymphocytes play an essential regulatory role in the adaptive immune response through Ab production during infection. A less known function of B lymphocytes is their ability to respond directly to infectious Ags through stimulation of pattern recognition receptors expressed on their surfaces. β-Glucans are carbohydrates present in the cell wall of many pathogenic fungi that can be detected in the peripheral blood of patients during infection. They have been shown to participate in the innate inflammatory response, as they can directly activate peripheral macrophages and dendritic cells. However, their effect as direct stimulators of B lymphocytes has not been yet fully elucidated. The aim of this study was to examine the molecular mechanisms and cytokine profiles generated following β-glucan stimulation of B lymphocytes, compared with the well-established TLR-9 agonist CpG oligodeoxynucleotide (CpG), and study the participation of β-glucan–stimulated B cells in the innate immune response. In this article, we demonstrate that β-glucan–activated B lymphocytes upregulate proinflammatory cytokines (TNF-α, IL-6, and IL-8). Of interest, β-glucan, unlike CpG, had no effect on B lymphocyte proliferation or IgM production. When compared with CpG (TLR9 agonist), β-glucan–activated cells secreted significantly higher levels of IL-8. Furthermore, IL-8 secretion was partially mediated by Dectin-1 and required SYK, MAPKs, and the transcription factors NF-κB and AP-1. Moreover, we observed that conditioned media from β-glucan–stimulated B lymphocytes elicited neutrophil chemotaxis. These studies suggest that β-glucan–activated B lymphocytes have an important and novel role in fungal innate immune responses.