Targeting BMI-1 to deplete antibody-secreting cells in autoimmunity

Objectives. B cells drive the production of autoreactive antibodysecreting cells, ASCs, in autoimmune diseases such as Systemic Lupus Erythematosus, SLE, and Sjogren, s syndrome, causing long-, term organ damage. Current treatments for antibody-mediated autoimmune diseases target B cells or broadly suppress the immune system. However, pre-existing long-lived ASCs are often refractory to treatment, leaving a reservoir of autoreactive cells that continue to produce antibodies. Therefore, the development of novel treatment methods targeting ASCs is vital to improve patient outcomes. Our objective was to test whether targeting the epigenetic regulator BMI-, could deplete ASCs in autoimmune conditions in vivo and in vitro. Methods. Use of a BMI-, inhibitor in both mouse and human autoimmune settings was investigated. Lyn, mice, a model of SLE, were treated with the BMI-, small molecule inhibitor PTC-, before assessment of ASCs, serum antibody and immune complexes. To examine human ASC survival, a novel human fibroblast-based assay was established, and the impact of PTC-, on ASCs derived from Sjogren, s syndrome, patients was evaluated. Results. BMI-, inhibition significantly decreased splenic and bone marrow ASCs in Lyn, mice. The decline in ASCs was linked to aberrant cell cycle gene expression and led to a significant decrease in serum IgG, immune complexes and anti-DNA IgG. PTC-, was also efficacious in reducing ex vivo plasma cell survival from both Sjogren, s syndrome, patients and age-matched healthy donors. Conclusion. These data provide evidence that inhibiting BMI-, can deplete ASC in a variety of contexts and thus BMI-, is a viable therapeutic target for antibody-mediated autoimmune diseases.Jack Polmear, Lauren Hailes, Moshe Olshansky, Maureen Rischmueller, Elan L'Estrange-Stranieri, Anne L Fletcher, Margaret L Hibbs, Vanessa L Bryant, Kim L Good-Jacobso

Csk-binding protein mediates sequential enzymatic down-regulation and degradation of Lyn in erythropoietin-stimulated cells

We have shown previously that the Src family kinase Lyn is involved in differentiation signals emanating from activated erythropoietin (Epo) receptors. The importance of Lyn to red cell maturation has been highlighted by Lyn -/- mice developing anemia. Here we show that Lyn interacts with C-terminal Src kinase-binding protein (Cbp), an adaptor protein that recruits negative regulators C-terminal Src kinase (Csk)/Csk-like protein-tyrosine kinase (Ctk). Lyn phosphorylated Cbp on several tyrosine residues, including Tyr 314, which recruited Csk/Ctk to suppress Lyn kinase activity. Intriguingly, phosphorylated Tyr314 also bound suppressor of cytokine signaling 1 (SOCS1), another well characterized negative regulator of cell signaling, resulting in elevated ubiquitination, and degradation of Lyn. In Epo-responsive primary cells and cell lines, Lyn rapidly phosphorylated Cbp, suppressing Lyn kinase activity via Csk/Ctk within minutes of Epo stimulation; hours later, SOCS1 bound to Cbp and was involved in the ubiquitination and turnover of Lyn protein. Thus, a single phosphotyrosine residue on Cbp coordinates a two-phase process involving distinct negative regulatory pathways to inactivate, then degrade, Lyn

The lyn tyrosine kinase is essential for erythropoietin induced erythroid differentiation and specifically interacts with lckbp-1/hs1 and several novel molecules

Erythropoietin (EPO) stimulates the immature erythroid J2E cell line to terminally di.fferentiate, proliferate and maintains their viability in the absence of serum. In contrast, a mutant J2E clone (J2E-NR) fails to mature in response to the hormone which we have shown is due to a very low expression level of the Lyn tyrosine kinase. Co-immunoprecipitation and yeast two-hybrid analysis indicates that Lyn directly associates with the EPO-receptor complex. Using the yeast two hybrid system we have identified LckBP-1/HS1 and several novel molecules as Lyn interactors. LckBP-1/HS1 has been shown to bind to the SH3 domain of Lck and contains four tandem helix-turn-helix motifs, a proline rich region, a proline and glutamine rich segment, and an SH3 domain. The importance of this interaction in EPO-induced signalling through Lyn is currently be!ng investigated. Three novel molecules were also identified in the two-hybrid screen as specifically interacting with Lyn. One of these has an ankyrin repeat most closely related to a K+ channel. Another novel protein bound specifically to a kinase inactive mutant of Lyn where tyrosine 397 had been mutated to phenyalanine. Full-length clones of these novel molecules are currently being isolated and their involvement in erythroid development will be analysed

Gain-of-function Lyn induces anemia: appropriate Lyn activity is essential for normal erythropoiesis and Epo receptor signaling

Lyn is involved in erythropoietin (Epo)-receptor signaling and erythroid homeostasis. Downstream pathways influenced following Lyn activation and their significance to erythropoiesis remain unclear. To address this, we assessed a gain-of-function Lyn mutation (Lynup/up) on erythropoiesis and Epo receptor signaling. Adult Lynup/up mice were anemic, with dysmorphic red cells (spherocyte-like, acanthocytes) in their circulation, indicative of hemolytic anemia and resembling the human disorder chorea acanthocytosis. Heterozygous Lyn+/up mice became increasingly anemic with age, indicating that the mutation was dominant. In an attempt to overcome this anemia, extramedullary erythropoiesis was activated. As the mice aged, the levels of different immature erythroid populations changed, indicating compensatory mechanisms to produce more erythrocytes were dynamic. Changes in Epo signaling were observed in Lyn+/up erythroid cell lines and primary CD71+ Lynup/up erythroblasts, including significant alterations to the phosphorylation of Lyn, the Epo receptor, Janus kinase 2, Signal Transducer and Action of Transcription-5, GRB2-associated- binding protein-2, Akt, and Forkhead box O3. As a consequence of altered Lyn signaling, Lyn+/up cells remained viable in the absence of Epo but displayed delayed Epo-induced differentiation. These data demonstrate that Lyn gene dosage and activity are critical for normal erythropoiesis; constitutively active Lyn alters Epo signaling, which in turn produces erythroid defects

Lyn kinase plays important roles in erythroid expansion, maturation and erythropoietin receptor signalling by regulating inhibitory signalling pathways that control survival

Erythroid homoeostasis is primarily controlled by Epo (erythropoietin) receptor signalling; however, the Lyn tyrosine kinase plays an important subsidiary role in regulating the erythroid compartment. Nonetheless, specific erythroid pathways that require Lyn activity and their biological significance remain unclear. To address this, we asked what consequence loss of Lyn had on the ex vivo expansion and maturation of splenic erythroid progenitors and Epo receptor signalling. Pharmacological inhibition of Lyn with PP2 inhibited the survival of terminally differentiated erythroblasts. Less committed erythroid progenitors expanded well, whereas early splenic Lyn-/- erythroblasts had attenuated ex vivo expansion, and late stage Lyn-/-erythroblasts were retarded in completing morphological maturation ex vivo. Furthermore, immortalized Lyn-/-erythroblasts were slower growing, less viable and inhibited in their differentiation. Signalling studies showed that Lyn was required for both positive GAB2/Akt/FoxO3 (forkhead box O3) survival signals as well as negative feedback of JAK2 (Janus kinase 2)/STAT5 (signal transducer and activator of transcription 5) and ERK1/2 (extracellular-signal-regulated kinase 1/2) signals via SHP-1 (Src homology 2 domain-containing protein tyrosine phosphatase 1). During differentiation, Lyn controls survival and cell cycle exit as demonstrated by reduced STAT5 and FoxO3/GSKa/ß (glycogen synthase kinase a/ß) phosphorylation and diminished p27Kip1 induction in Lyn-deficient erythroblasts. Lyn deficiency alters the balance of pro-and anti-apoptotic molecules (BAD and BclXL), thereby reducing survival and preventing cell cycle exit. Consequently, Lyn facilitates normal erythrocyte production by influencing different stages of erythroid progenitor expansion, and mature cell development and survival signalling

β-glucan receptors on IL-4 activated macrophages are required for hookworm larvae recognition and trapping.

Recent advances in the field of host immunity against parasitic nematodes have revealed the importance of macrophages in trapping tissue migratory larvae. Protective immune mechanisms against the rodent hookworm Nippostrongylus brasiliensis (Nb) are mediated, at least in part, by IL-4-activated macrophages that bind and trap larvae in the lung. However, it is still not clear how host macrophages recognize the parasite. We utilized an in vitro co-culture system of bone marrow-derived macrophages and Nb infective larvae to screen for the possible ligand-receptor pair involved in macrophage attack of larvae. Competitive binding assays revealed an important role for β-glucan recognition in the process. We further identified a role for CD11b and the non-classical pattern recognition receptor ephrin-A2 (EphA2), but not the highly expressed β-glucan dectin-1 receptor, in this process of recognition. This work raises the possibility that parasitic nematodes synthesize β-glucans and identifies CD11b and Ephrin-A2 as important pattern recognition receptors involved in the host recognition of these evolutionary old pathogens. To our knowledge, this is the first time that EphA2 has been implicated in immune responses to a helminth

IL-33-mediated mast cell activation promotes gastric cancer through macrophage mobilization

The contribution of mast cells in the microenvironment of solid malignancies remains controversial. Here we functionally assess the impact of tumor-adjacent, submucosal mast cell accumulation in murine and human intestinal-type gastric cancer. We find that genetic ablation or therapeutic inactivation of mast cells suppresses accumulation of tumor-associated macrophages, reduces tumor cell proliferation and angiogenesis, and diminishes tumor burden. Mast cells are activated by interleukin (IL)-33, an alarmin produced by the tumor epithelium in response to the inflammatory cytokine IL-11, which is required for the growth of gastric cancers in mice. Accordingly, ablation of the cognate IL-33 receptor St2 limits tumor growth, and reduces mast cell-dependent production and release of the macrophage-attracting factors Csf2, Ccl3, and Il6. Conversely, genetic or therapeutic macrophage depletion reduces tumor burden without affecting mast cell abundance. Therefore, tumor-derived IL-33 sustains a mast cell and macrophage-dependent signaling cascade that is amenable for the treatment of gastric cancer.Moritz F. Eissmann, Christine Dijkstra, Andrew Jarnicki, Toby Phesse, Jamina Brunnberg, Ashleigh R. Poh, Nima Etemadi, Evelyn Tsantikos, Stefan Thiem, Nicholas D. Huntington, Margaret L. Hibbs, Alex Boussioutas, Michele A. Grimbaldeston, Michael Buchert, Robert J.J. O’Donoghue, Frederick Masson, Matthias Erns