The NKCC1 ion transporter contributes to the pathophysiology of common neurological disorders, but its function in microglia, the main inflammatory cells of the brain, has remained unclear to date. Therefore, we generated a novel transgenic mouse line in which microglial NKCC1 was deleted. We show that microglial NKCC1 shapes both baseline and reactive microglia morphology, process recruitment to the site of injury, and adaptation to changes in cellular volume in a cell-autonomous manner via regulating membrane conductance. In addition, microglial NKCC1 deficiency results in NLRP3 inflammasome priming and increased production of interleukin-1β (IL-1β), rendering microglia prone to exaggerated inflammatory responses. In line with this, central (intracortical) administration of the NKCC1 blocker, bumetanide, potentiated intracortical lipopolysaccharide (LPS)-induced cytokine levels. In contrast, systemic bumetanide application decreased inflammation in the brain. Microglial NKCC1 KO animals exposed to experimental stroke showed significantly increased brain injury, inflammation, cerebral edema and worse neurological outcome. Thus, NKCC1 emerges as an important player in controlling microglial ion homeostasis and inflammatory responses through which microglia modulate brain injury. The contribution of microglia to central NKCC1 actions is likely to be relevant for common neurological disorders.

All-trans retinoic acid (ATRA) is a derivative of vitamin A that has many important biological functions, including the modulation of immune responses. ATRA actions are mediated through the retinoic acid receptor that functions as a nuclear receptor, either regulating gene transcription in the nucleus or modulating signal transduction in the cytoplasm. NLRP3 inflammasome is a multiprotein complex that is activated by a huge variety of stimuli, including pathogen- or danger-related molecules. Activation of the inflammasome is required for the production of IL-1β, which drives the inflammatory responses of infectious or non-infectious sterile inflammation. Here, we showed that ATRA prolongs the expression of IL-6 and IL-1β following a 2-, 6-, 12-, and 24-h LPS (100ng/mL) activation in human monocyte-derived macrophages. We describe for the first time that ATRA modulates both priming and activation signals required for NLRP3 inflammasome function. ATRA alone induces NLRP3 expression, and enhances LPS-induced expression of NLRP3 and pro-IL-1β via the regulation of signal transduction pathways, like NF-κB, p38, and ERK. We show that ATRA alleviates the negative feedback loop effect of IL-10 anti-inflammatory cytokine on NLRP3 inflammasome function by inhibiting the Akt-mTOR-STAT3 signaling axis. We also provide evidence that ATRA enhances hexokinase 2 expression, and shifts the metabolism of LPS-activated macrophages toward glycolysis, leading to the activation of NLRP3 inflammasome.

Besides its well-known psychoactive effects, caffeine has a broad range of actions. It regulates several physiological mechanisms as well as modulates both native and adaptive immune responses by various ways. Although caffeine is assumed to be a negative regulator of inflammation, the effect on the secretion of pro- and anti-inflammatory cytokines is highly controversial. Macrophages are major mediators of inflammatory responses; however, the various subpopulations develop different effects ranging from the initiation to the resolution of inflammation. Here we report a comparative analysis of the effect of caffeine on two subpopulations of human monocyte-derived macrophages differentiated in the presence of macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF), resulting in M-MΦs and GM-MΦs, respectively. We showed that although TNF-α secretion was downregulated in both LPS-activated MΦ subtypes by caffeine, the secretion of IL-8, IL-6, and IL-1β as well as the expression of Nod-like receptors was enhanced in M-MΦs, while it did not change in GM-MΦs. We showed that caffeine (1) altered adenosine receptor expression, (2) changed Akt/AMPK/mTOR signaling pathways, and (3) inhibited STAT1/IL-10 signaling axis in M-MΦs. We hypothesized that these alterations play an important modulatory role in the upregulation of NLRP3 inflammasome-mediated IL-1β secretion in LPS-activated M-MΦs following caffeine treatment.

Syk is a non-receptor tyrosine kinase critically involved in signaling by various immunoreceptors including B-cell-receptors and activating Fc-receptors. We have previously shown that Syk also mediates immunoreceptor-like signals required for the in vitro development and function of osteoclasts. However, the perinatal lethality of Syk^−/− mice precluded the analysis of the role of Syk in in vivo bone metabolism. To overcome that problem, we generated mice with osteoclast-specific (Syk^ΔOC) or hematopoietic (Syk^ΔHaemo) Syk deficiency by conditional deletion of Syk using Cre recombinase expressed under the control of the Ctsk or Vav1 promoter, respectively. Micro-CT analysis revealed increased bone trabecular density in both Syk^ΔOC and Syk^ΔHaemo mice, although hematopoietic Syk deficiency caused a more severe phenotype than osteoclast-specific Syk deficiency. Osteoclast-specific Syk deficiency reduced, whereas hematopoietic Syk deficiency completely blocked in vitro development of osteoclasts. Both interventions inhibited the resorptive activity of osteoclasts and osteoclast-specific gene expression. Kinetic analysis of Syk protein levels, Cre expression and the genomic deletion of the Syk^flox allele revealed complete and early deletion of Syk from Syk^ΔHaemo osteoclasts whereas Syk was incompletely deleted at a later stage of osteoclast development from Syk^ΔOC cultures. Those results provide an explanation for the in vivo and in vitro difference between the Syk^ΔOC and Syk^ΔHaemo mutant strains and suggest late activation of, and incomplete target gene deletion upon, osteoclast-specific Cre expression driven by the Ctsk promoter. Taken together, our results indicate that Syk plays an indispensable role in osteoclast-mediated in vivo bone resorption and suggest that Syk-specific inhibitors may provide therapeutic benefit in inflammatory and other diseases characterized by excessive osteoclast-mediated bone resorption.

The NKCC1 ion transporter contributes to the pathophysiology of common neurological disorders, but its function in microglia, the main inflammatory cells of the brain, has remained unclear to date. Therefore, we generated a novel transgenic mouse line in which microglial NKCC1 was deleted. We show that microglial NKCC1 shapes both baseline and reactive microglia morphology, process recruitment to the site of injury, and adaptation to osmotic stress in a cell-autonomous manner via regulating membrane potential and chloride fluxes. In addition, microglial NKCC1 deficiency results in increased expression of the D subunit of volume regulated anion channel (VRAC), NLRP3 inflammasome priming and production of interleukin-1β (IL-1β), rendering microglia prone to exaggerated inflammatory responses. In line with this, central (intracortical) administration of the NKCC1 blocker, bumetanide, potentiated intracortical lipopolysaccharide (LPS)-induced cytokine levels, whereas systemic bumetanide application decreased inflammation in the brain. Microglial NKCC1 KO animals exposed to experimental stroke showed significantly increased brain injury, inflammation, cerebral edema and worse neurological outcome. Thus, NKCC1 emerges as an important player in controlling microglial ion homeostasis and inflammatory responses through which microglia modulate brain injury. The contribution of microglia to central NKCC1 actions is likely to be relevant for common neurological disorders.