John H. Kehrl, M.D.

Extracellular signals modulate and regulate the function of cells that participate in immune responses. Many of these signals activate pathways that utilize heterotrimeric G proteins to transduce signals. Our goals are to understand how G-protein-coupled receptors (GPCR) such as chemoattractant receptors transduce signals to downstream effectors in immune cells, to discern the mechanisms that control these responses, and to determine the physiologic consequences of G-protein activation for lymphocyte function. To do so, we are using genetically modified mice that have disruption of key genes in the chemokine receptor signaling pathway and sophisticated imaging techniques, including intravital two-photon laser scanning microscopy (TP-LSM). We also investigate the signaling pathway that triggers autophagy and inflammasome activation in macrophages and other cell types. Our COVID studies have focused on the cellular impact of several viral-derived proteins.

Discovery of RGS proteins

A major accomplishment of the B-Cell Molecular Immunology Section (BCMIS) was the identification of a family of proteins that regulate heterotrimeric G-protein signaling, which have been termed RGS proteins. RGS proteins are GTPase-activating proteins (GAPs) for Gα subunits of heterotrimeric G proteins. By shortening the duration that a Gα subunit is GTP bound, the RGS proteins curtail both Gα and Gβγ signaling. BCMIS is currently studying mice lacking various RGS proteins, as well as mice lacking specific GPCRs or other components in the GPCR signaling pathway. Because Rgs1, Rgs2, Rgs13, and Rgs18 are clustered together on mouse chromosome 1, we are using CRISPR-Cas9 to delete the entire cluster in mice.

Intravital imaging and leukocyte trafficking

BCMIS has employed intravital TP-LSM to study lymphocyte trafficking, examine the role of innate cells in B-cell responses, and study antigen processing in lymph nodes. We have 1) delineated how changes in chemokine and sphingosine-1 phosphate receptor 1 (S1PR1) expression and signaling coordinate the homeostatic trafficking of B cells through lymph nodes; 2) shown the importance of RGS proteins and Gαi2 in regulating B-lymphocyte chemotaxis and motility; 3) revealed how lymph node remodeling along with cell-intrinsic factors coordinate the trafficking of B cells into, thru, and out of lymph; 4) shown that RGS proteins are necessary to coordinate chemokine receptor sensitivity; 5) examined the interactions of neutrophils with B cells in immunized lymph nodes; 6) identified a network of cells in lymph nodes that uptake the envelope protein of HIV and deliver it to lymph node B cells; 7) characterized the role of Grk2 in regulating B-cell chemoattractant receptor signaling; 8) found that the loss of the small GTPase GAP Rasa3 in lymphocytes severely impairs lymphocyte trafficking; and 9) identified WNK1 as an early effector of Gαi signaling in B cells.

Cell stress-induced signaling pathways

BCMIS has 1) identified Toll-like receptors (TLRs) family engagement as a trigger for autophagy in macrophages; 2) shown that inflammasomes undergo ubiquitination and autophagic destruction, which can limit inflammasome activation; 3) determined how several SARS-CoV-derived proteins subvert cellular homeostasis and activate cellular defenses; 4) shown that cytochrome C negatively regulates NLRP3 inflammasomes; 5) identified the importance of Lrrk2 in CD38-mediated NAADP-Ca2+ signaling and the downstream activation of TFEB; and 6) provided evidence that AKT, Trim31, and PP2A together modulate NLRP3 protein levels and the tendency to oligomerize, thereby setting a threshold for NLRP3 activation. Currently, we are studying how activating mutations in ALPK1, the cause of ROSAH syndrome, affect inflammation and cell stress responses.