Signal Transduction In B Lymphocytes and Macrophages: Identification Of Key Signaling Molecules

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

Abstract

The role of WNK kinases in immune cells remains poorly understood. The recognition that WNK1 inhibition caused rapid B cell depolarization and a cessation of cell motility suggested that constitutive WNK1 signaling maintained B cell size at physiological extracellular ion concentrations. Exposing B cells to hypertonic conditions recapitulated the loss of B cell polarity and motility. Wnk1 likely helps B cells negotiate the changing osmotic conditions they encounter as they migrate through lymphoid organs and inflammatory sites. To assess the impact of hypertonic stress on B cell function we exposed primary murine B cells to varying degrees of hyperosmolarity. We noted a progressive increase in cellular F-actin; an initial increase in cell motility followed by a severe reduction with higher extracellular ion concentrations; a strong increase in intracellular calcium insensitive to a phospholipase C inhibitor; rapid increases in serine/threonine and tyrosine phosphorylated proteins; and altered side and forward light scatter as assess by flow cytometry. RNA extraction from murine B cells subjected to 4 hours of moderate hypertonic stress and analysis by mRNA sequencing revealed a markedly altered gene expression profile in the B cells with a strong induction of stress related genes. To better understand the role of WNK1 in B cell function we crossed mb-1 Cre+/- and Wnk1flox/flox mice to delete WNK1 in developing B cells. We first assessed serum immunoglobulin levels in mice with WT WNK1 alleles and in mice with one or both WNK1 alleles disrupted. Surprisingly we found that the WNK1 B cell KO mice had a nearly complete loss of IgG isotypes, a 70% reduction in IgA, yet normal serum IgM levels. In contrast, the heterozygotic mice had levels of IgG2c, IgG3, and IgM 3-4 fold higher than the control mice but a 25% reduction in IgA. Assessing the B cell compartments of the WNK1 KO B cell mice revealed a near complete loss of B cells in the blood, spleen, lymph nodes, Peyer’s patches, thymus, and peritoneum. Analysis of B cell development revealed a marked loss of B cells after the pro-B cell stage consistent with the time of WNK1 deletion by mb-1 Cre. It remains unclear why the serum IgM levels were not decreased. Phenotyping of the heterozygotic mice to explore the source of elevated IgG isotypes and increased IgM is in progress. We also investigated how WNK kinase inhibition affects NK cells. In contrast to resting NK cells, Il-2 activated NK cells are large and highly motile cells. We utilized two WNK kinase inhibitors, WNK463 which inhibits all 4 WNK kinases and WNK-IN-11, which selectively inhibits WNK1. We found that either WNK463 or WNK-IN-11 dramatically decreased IL-2-activated NK-cell volume, motility, and cytolytic activity. Treatment of NK cells with these inhibitors induced autophagy by activating AMPK and inhibiting mTOR signaling. Moreover, WNK kinase inhibition increased Akt and c-Myc phosphorylation by misaligning activity of activating kinases and inhibitory phosphatases. Treatment of tumor-bearing mice with WNK463 impaired tumor metastasis control by adoptively transferred NK cells. Thus, the catalytic activity of WNK kinases has a critical role in multiple aspects of NK cell physiology and their pharmacologic inhibition negatively impacts NK cell function. The cytosolic alpha kinase-1 (ALPK1) senses microbial sugar metabolites augmenting ALPK1 catalytic activity. Activated ALPK1 phosphorylates TIFA (TRAF-interacting protein with FHA domain), which along with TRAFs proteins form TIFAsomes leading to nuclear factor-kB (NF-κB) activation. Gain- of-function mutations in ALPK1 cause auto-inflammatory disorders. Prior evaluations of primary patient samples and assays with mutated ALPK1 constructs reveal immune activation with increased NF-κB signaling, inappropriate STAT1 phosphorylation, and an interferon gene expression signature. This study sought further evidence that dysregulated interferon signaling contributes to ALPK1 mediated autoinflammatory diseases and to understand the underlying mechanism by examining whether ALPK1 activation affects STING signaling. We have shown that a subset of patients with ALPK1 mediated autoinflammatory disease exhibit premature intracranial mineralization, a radiological feature found in some patients with dysregulated interferon signaling. Mechanistically, we have shown that ALPK1 activation enhances canonical STING signaling, STING proton channel triggered LC3 lipidation, and NLRP3 inflammasome activation. Furthermore, ALPK1 signaling activates eIF2α, an effector of the integrated stress response, and enhances STING protein expression. Finally, STING activation increases ALPK1 protein amounts and triggers TIFA-Threonine 9 phosphorylation. Since pathogens typically engage multiple innate sensors, our data indicates that engagement of both ALPK1 and STING signaling amplifies and regulates both pathways. STING-mediated Golgi deacidification impacts Golgi transit and secretion for a subset of proteins and ArfGAP2 interacts with STING and is required for optimal STING proton channel activity. Deletion of ArfGAP2 in hematopoietic and endothelial cells markedly reduces STING-mediated cytokine and chemokine secretion, immune cell activation, and autoinflammatory pathology in SAVI mice (Cell 188, 1605-162, 2025). Treating THP-1 cells with ADP-heptose strongly upregulates ArfGAP2 expression. Suggesting that ALPK1 signaling affects ArfGAP2 activity, the expression and immunoprecipitation of the constitutively active forms of ALPK1, but not wild type ALPK1, pulled down ArfGAP2. Crossing the ALPK1 deficient mice to the SAVI mice may alleviate some of the autoinflammatory pathology found in the SAVI mice. We are exploring the signaling pathways induced by activation of ALPK1. Besides the activation of NF-κB, we have identified p38 and AMPK as targets of ALPK1 signaling. The activation of p38 and AMPK leads to a potent activation of TFEB, a known mediator of monocyte-to-macrophage differentiation. Studies are in progress to assess the role of ALPK1 in monocyte differentiation and macrophage polarization. To better assess TFEB activation we developed a HEK293 reporter cells line that responds to TFEB activation. Promoter analysis of a set of monocyte/macrophage genes revealed that many contained binding sites for NF-κB, TFEB, and SREBF1/2. ALPK1 signaling potently stimulates each of these transcriptional factors suggesting that it has an important functional role in the regulation of this set of genes.