Azithromycin Augments Bacterial Uptake and Anti-Inflammatory Macrophage Polarization in Cystic Fibrosis.

BACKGROUND: Azithromycin (AZM) is widely being used for treating patients with cystic fibrosis (pwCF) following clinical trials demonstrating improved lung function and fewer incidents of pulmonary exacerba-tions. While the precise mechanisms remain elusive, immunomodulatory actions are thought to be involved. We previously reported impaired phagocytosis and defective anti-inflammatory M2 macrophage polarization in CF. This study systematically analyzed the effect of AZM on the functions of unpolarized and M1/M2 polarized macrophages in CF. METHODS: Monocytes, isolated from the venous blood of patients with CF (pwCF) and healthy controls (HCs), were differentiated into monocyte-derived macrophages (MDMs) and subsequently infected with P. aeruginosa. P. aeruginosa uptake and killing by MDMs in the presence or absence of AZM was studied. M1 and M2 macrophage polarizations were induced and their functions and cytokine release were analyzed. RESULTS: Following AZM treatment, both HC and CF MDMs exhibited a significant increase in P. aeruginosa uptake and killing, however, lysosomal acidification remained unchanged. AZM treatment led to higher activation of ERK1/2 in both HC and CF MDMs. Pharmacological inhibition of ERK1/2 using U0126 significantly reduced P. aeruginosa uptake in HC MDMs. M1 macrophage polarization remained unaffected; however, AZM treatment led to increased IL-6 and IL-10 release in both HC and CF M1 macrophages. AZM also significantly increased the phagocytic index for both pHrodo E. coli and S. aureus in CF M1 macrophages. In CF, AZM treatment promoted anti-inflammatory M2 macrophage polarization, with an increased percentage of CD209+ M2 macrophages, induction of the M2 gene CCL18, along with its secretion in the culture supernatant. However, AZM d'd not restore endocytosis in CF, another essential feature of M2 macrophages. CONCLUSIONS: This study highlights the cellular functions and molecular targets of AZM which may involve an improved uptake of both Gram-positive and Gram-negative bacteria, restored anti-inflammatory macrophage polarization in CF. This may in turn shape the reduced lung inflammation observed in clinical trials. In addition, we confirmed the role of ERK1/2 activation for bacterial uptake.

The transmembrane adapter SCIMP recruits tyrosine kinase Syk to phosphorylate Toll-like receptors to mediate selective inflammatory outputs.

Innate immune signaling by Toll-like receptors (TLRs) involves receptor phosphorylation, which helps to shape and drive key inflammatory outputs, yet our understanding of the kinases and mechanisms that mediate TLR phosphorylation is incomplete. Spleen tyrosine kinase (Syk) is a nonreceptor protein tyrosine kinase, which is known to relay adaptive and innate immune signaling, including from TLRs. However, TLRs do not contain the conserved dual immunoreceptor tyrosine-based activation motifs that typically recruit Syk to many other receptors. One possibility is that the Syk-TLR association is indirect, relying on an intermediary scaffolding protein. We previously identified a role for the palmitoylated transmembrane adapter protein SCIMP in scaffolding the Src tyrosine kinase Lyn, for TLR phosphorylation, but the role of SCIMP in mediating the interaction between Syk and TLRs has not yet been investigated. Here, we show that SCIMP recruits Syk in response to lipopolysaccharide-mediated TLR4 activation. We also show that Syk contributes to the phosphorylation of SCIMP and TLR4 to enhance their binding. Further evidence pinpoints two specific phosphorylation sites in SCIMP critical for its interaction with Syk-SH2 domains in the absence of immunoreceptor tyrosine-based activation motifs. Finally, using inhibitors and primary macrophages from SCIMP-/- mice, we confirm a functional role for SCIMP-mediated Syk interaction in modulating TLR4 phosphorylation, signaling, and cytokine outputs. In conclusion, we identify SCIMP as a novel, immune-specific Syk scaffold, which can contribute to inflammation through selective TLR-driven inflammatory responses.

SCIMP is a spatiotemporal transmembrane scaffold for Erk1/2 to direct pro-inflammatory signaling in TLR-activated macrophages.

Immune cells are armed with Toll-like receptors (TLRs) for sensing and responding to pathogens and other danger cues. The role of extracellular-signal-regulated kinases 1/2 (Erk1/2) in TLR signaling remains enigmatic, with both pro- and anti-inflammatory functions described. We reveal here that the immune-specific transmembrane adaptor SCIMP is a direct scaffold for Erk1/2 in TLR pathways, with high-resolution, live-cell imaging revealing that SCIMP guides the spatial and temporal recruitment of Erk2 to membrane ruffles and macropinosomes for pro-inflammatory TLR4 signaling. SCIMP-deficient mice display defects in Erk1/2 recruitment to TLR4, c-Fos activation, and pro-inflammatory cytokine production, with these effects being phenocopied by Erk1/2 signaling inhibition. Our findings thus delineate a selective role for SCIMP as a key scaffold for the membrane recruitment of Erk1/2 kinase to initiate TLR-mediated pro-inflammatory responses in macrophages.

Live Fluorescence, Inverse Imaging of Cell Ruffling, and Macropinocytosis.

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase nutrients and other material in cells. The dramatic extension of cell surface ruffles, their closure to form macropinosomes, and the maturation of internalized macropinosomes are key events in this pathway that can be difficult to capture using conventional confocal imaging based on tracking a bolus of fluorescent cargo. Fluorescent dextrans are commonly used experimentally as fluid phase markers for macropinosomes and for other endocytic pathways. A method the lab has adopted to optimize the imaging of dextran uptake involves using live imaging of cells bathed in high concentrations of fluorescent dextran in the medium, with the unlabeled cells appearing in relief (as black). The cell ruffles are highlighted to visualize ruffle closure, and internalized macropinosomes appear as fluorescent vacuoles in the cell interior. This method is optimal for visualizing macropinosome features and allows for easy segmentation and quantification. This paper describes dual-labeling of pathways with different sized dextrans and the co-expression of lipid probes and fluorescent membrane proteins to demark macropinosomes and other endosomes. The detection of internalized dextran at an ultrastructural level using correlative light and electron microscopy (CLEM) is also demonstrated. These cell processes can be imaged using multiple live imaging modalities, including in 3D. Taken together, these approaches optimize macropinosome imaging for many different settings and experimental systems.

SCIMP is a universal Toll-like receptor adaptor in macrophages.

In innate immune cells, pathogens and danger signals activate TLRs, unleashing potent and tailored inflammatory responses. Previously, we reported that an immune-specific transmembrane adaptor, SLP adaptor and CSK interacting membrane protein (SCIMP), interacts with TLR4 via direct binding to its cytoplasmic TIR domain. SCIMP scaffolds a Src family kinase, Lyn, for TLR4 phosphorylation and activation. Consequently, SCIMP is able to direct selective production of the proinflammatory cytokines IL-6 and IL-12p40 downstream of TLR4 in macrophages. Here, we set out to investigate whether SCIMP also acts as an adaptor for other TLR family members. We report here that SCIMP is phosphorylated and activated in response to agonists of multiple TLRs, including TLR2, TLR3, TLR4, and TLR9. SCIMP also interacts with TLRs that are known to signal from both the cell surface and endosomal compartments. In so doing, this transmembrane adaptor presents Lyn, along with other effectors such as Grb2, Csk, and SLP65, to multiple TLRs during cellular activation. CRISPR-mediated knockout or silencing of SCIMP in macrophages alters TLR signaling outputs and the production of IL-6 and IL-12p40 downstream of multiple TLRs, and upon challenge with live bacteria. Furthermore, the selectivity in cytokine responses is preserved downstream of TLR3, with inducible expression of Il-12p40 and IL-6, but not IFNß, being SCIMP dependent. SCIMP is thus a universal TLR adaptor for scaffolding the Lyn tyrosine kinase and its effectors to enable responses against a wide range of danger signals.