Functions of LynA and LynB kinases in dendritic-cell pro-inflammatory signaling and lupus

ABSTRACT The Src-family kinase Lyn is critical for maintaining immune homeostasis and protecting against autoimmunity, and deficits in Lyn function are linked to systemic lupus erythematosus (SLE). However, cell studies report con- flicting (paradoxical) inflammatory and immunosuppressive functions, varying by cell and perturbation. We dis-

covered that the two Lyn splice forms (A, B) are differentially regulated, suggesting non-overlapping interactions and roles, suggesting that the apparently paradoxical positive and negative effects might be explained by iso- form-specific functions that vary with expression, immune compartment, and environment. We generated single-

isoform LynAKO and LynBKO mice and discovered (1) a dominant role for LynB in protecting against autoimmunity and (2) a female-specific role for LynA in immune regulation. Our preliminary data point to overlapping roles of LynA and LynB in steady-state ITIM signaling but a specific role for LynB in inducing antimicrobial hemi-ITAM

signaling. To this toolkit we add a reagent that biases splicing toward increased LYNB production in human cells. We hypothesize that LynB forms unique interactions in ITAM and TLR complexes, while LynA uniquely sup- presses ER (estrogen-receptor) signaling, accounting for the sexual dimorphism. Alterations in LynA and LynB

expression will explain the apparently paradoxical observations of net positive and negative functions and sug- gest a path forward for therapeutic development. 1: Define roles of Lyn isoforms in ITAM, TLR, and ER signaling in human and murine myeloid cells. As myeloid cells drive autoimmunity and have been targets of

immunomodulatory therapies, they will be the focus of signaling studies. Candidate and unbiased kinase-sub- strate mapping and interactomics in macrophages and DCs will provide a comprehensive profile of LynA and LynB substrates and functions in receptor activation. Predictions: LynB will interact stably with ITAM and TLR

signaling complexes and downstream mediators, whereas LynA will uniquely suppress ER signaling. LynA and LynB will be found to localize differently at the cell membrane, explaining why upregulation of the other isoform fails to rescue signaling. 2: Define isoform- sex-, and cell-specific functions of LynA and LynB in lupus

progression. We will assess progression of lupus in spontaneous and inducible models in male and female mice from our Lyn knockout series. WT and LynKO experiments will be repeated mice with ovariectomy and with pharmacological antagonism/agonism of estrogen, progesterone, and TLRs to pinpoint contributors to sexual

dimorphism. We will assess cell-specific contributions to disease. Finally, we will test the ability of a splice- altering reagent to suppress disease. Predictions: Upregulation of a single isoform will only partially suppress lupus, cell-specificity will follow LynA/B expression patterns, sex hormones will drive lupus in females, and alter-

ing LynA/B balance will modulate disease. With our Lyn knockout series, we are poised to resolve longstanding paradoxes in Lyn signaling. Our Lyn splice reagent adds translational value, testing a new therapeutic avenue.