Leukocyte CD300a Contributes to the Resolution of Murine Allergic Inflammation.

CD300a is an inhibitory receptor for mast cells and eosinophils in allergic inflammation (AI); however, the spatiotemporal expression of CD300a and its potential roles in the resolution of AI are still to be determined. In this study, employing a mouse model of allergic peritonitis, we demonstrate that CD300a expression on peritoneal cells is regulated from inflammation to resolution. Allergic peritonitis-induced CD300a-/- mice had a rapid increase in their inflammatory cell infiltrates and tryptase content in the peritoneal cavity compared with wild type, and their resolution process was significantly delayed. CD300a-/- mice expressed lower levels of ALX/FPR2 receptor on peritoneal cells and had higher levels of LXA4 in the peritoneal lavage. CD300a activation on mouse bone marrow-derived mast cells regulated ALX/FPR2 expression levels following IgE-mediated activation. Together, these findings indicate a role for CD300a in AI and its resolution, in part via the specialized proresolving mediator LXA4 and ALX/FPR2 receptor pathway activation.

LL-37 opsonizes and inhibits biofilm formation of Aggregatibacter actinomycetemcomitans at subbactericidal concentrations.

Host defense peptides are immediate responders of the innate immunity that express antimicrobial, immunoregulatory, and wound-healing activities. Neutrophils are a major source for oral host defense peptides, and phagocytosis by neutrophils is a major mechanism for bacterial clearance in the gingival tissue. Dysfunction of or reduction in the numbers of neutrophils or deficiency in the LL-37 host defense peptide was each previously linked with proliferation of oral Aggregatibacter actinomycetemcomitans which resulted in an aggressive periodontal disease. Surprisingly, A. actinomycetemcomitans shows resistance to high concentrations of LL-37. In this study, we demonstrated that submicrocidal concentrations of LL-37 inhibit biofilm formation by A. actinomycetemcomitans and act as opsonins and agglutinins that greatly enhance its clearance by neutrophils and macrophages. Improved uptake of A. actinomycetemcomitans by neutrophils was mediated by their opsonization with LL-37. Enhanced phagocytosis and killing of A. actinomycetemcomitans by murine macrophage-like RAW 264.7 cells were dependent on their preagglutination by LL-37. Although A. actinomycetemcomitans is resistant to the bactericidal effect of LL-37, our results offer a rationale for the epidemiological association between LL-37 deficiency and the expansion of oral A. actinomycetemcomitans and indicate a possible therapeutic use of cationic peptides for host defense.

Single cell proteomics characterization of bone marrow hematopoiesis with distinct Ras pathway lesions.

Normal hematopoiesis requires constant prolific production of different blood cell lineages by multipotent hematopoietic stem cells (HSC). Stem- and progenitor- cells need to balance dormancy with proliferation. How genetic alterations impact frequency, lineage potential, and metabolism of HSC is largely unknown. Here, we compared induced expression of KRAS G12D or RasGRP1 to normal hematopoiesis. At low-resolution, both Ras pathway lesions result in skewing towards myeloid lineages. Single-cell resolution CyTOF proteomics unmasked an expansion of HSC- and progenitor- compartments for RasGRP1, contrasted by a depletion for KRAS G12D . SCENITH™ quantitates protein synthesis with single-cell precision and corroborated that immature cells display low metabolic SCENITH™ rates. Both RasGRP1 and KRAS G12D elevated mean SCENITH™ signals in immature cells. However, RasGRP1-overexpressing stem cells retain a metabolically quiescent cell-fraction, whereas this fraction diminishes for KRAS G12D . Our temporal single cell proteomics and metabolomics datasets provide a resource of mechanistic insights into altered hematopoiesis at single cell resolution.

Investigating increased hematopoietic stem cell fitness in a novel mouse model.

T-cell acute lymphoblastic leukaemia (T-ALL) is a bone marrow (BM) malignancy affecting children and adults. Typically treated with chemotherapy, leukaemia remains a major death cause in people under 20 years old. Understanding molecularly altered pathways in T-ALL may lead to new therapeutic avenues in the future. Ras pathway dysregulation is common in T-ALL. We have shown elevated expression levels of the Ras guanine nucleotide exchange factor RasGRP1 in T-ALL patients, which results in constant production of active Ras (RasGTP). When leukaemia cell lines are exposed to cytokines, RasGTP levels further increase in a RasGRP1-dependent manner. How overexpressed RasGRP1 may impact primary BM cells has remained unknown. We recently published a new RoLoRiG mouse model that allows for pIpC-induced overexpression of RasGRP1 in haematopoietic cells, which can be traced with an ires-EGFP cassette. This novel model revealed that RasGRP1 overexpression bestows a fitness advantage to haematopoietic stem cells (HSCs) over wild-type cells. Intriguingly, this increased fitness only manifests in native Hematopoiesis, and not in BM transplantation (BMT) assays. In this commentary, we summarize key features of our RoLoRiG model, elaborate on BM niche importance, and discuss differences between native Hematopoiesis and BMT in the context of stem cell metabolism.

Down-regulation of mast cell responses through ITIM containing inhibitory receptors.

The multiple cell types that comprise the immune system provide an efficient defense system against invading pathogens and micro-organisms. In general, immune cells are activated for disparate functions, such as proliferation, production and release of mediators and chemotaxis, as a result of interactions between ligands and their matching immunoreceptors. This in turn leads to the recruitment and activation of a cascade of second messengers, via their regulators/adaptors, that determine the net effect of the initial response. However, activation of cells of the immune system must be tightly regulated by a finely tuned interplay between activation and inhibition to avoid excessive or inappropriate responsiveness and to maintain homeostasis. Loss of inhibitory signals may disrupt this balance, leading to various pathological processes such as allergic and auto-immune diseases. In this chapter, we will discuss down-regulating mechanisms of mast cells focusing on immunoreceptor tyrosine-based inhibition motifs (ITIM)-containing inhibitory receptors (IR).

Increased baseline RASGRP1 signals enhance stem cell fitness during native hematopoiesis.

Oncogenic mutations in RAS genes, like KRASG12D or NRASG12D, trap Ras in the active state and cause myeloproliferative disorder and T cell leukemia (T-ALL) when induced in the bone marrow via Mx1CRE. The RAS exchange factor RASGRP1 is frequently overexpressed in T-ALL patients. In T-ALL cell lines overexpression of RASGRP1 increases flux through the RASGTP/RasGDP cycle. Here we expanded RASGRP1 expression surveys in pediatric T-ALL and generated a RoLoRiG mouse model crossed to Mx1CRE to determine the consequences of induced RASGRP1 overexpression in primary hematopoietic cells. RASGRP1-overexpressing, GFP-positive cells outcompeted wild type cells and dominated the peripheral blood compartment over time. RASGRP1 overexpression bestows gain-of-function colony formation properties to bone marrow progenitors in medium containing limited growth factors. RASGRP1 overexpression enhances baseline mTOR-S6 signaling in the bone marrow, but not in vitro cytokine-induced signals. In agreement with these mechanistic findings, hRASGRP1-ires-EGFP enhances fitness of stem- and progenitor- cells, but only in the context of native hematopoiesis. RASGRP1 overexpression is distinct from KRASG12D or NRASG12D, does not cause acute leukemia on its own, and leukemia virus insertion frequencies predict that RASGRP1 overexpression can effectively cooperate with lesions in many other genes to cause acute T-ALL.

High-Complexity shRNA Libraries and PI3 Kinase Inhibition in Cancer: High-Fidelity Synthetic Lethality Predictions.

Deregulated signal transduction is a cancer hallmark, and its complexity and interconnectivity imply that combination therapy should be considered, but large data volumes that cover the complexity are required in user-friendly ways. Here, we present a searchable database resource of synthetic lethality with a PI3 kinase signal transduction inhibitor by performing a saturation screen with an ultra-complex shRNA library containing 30 independent shRNAs per gene target. We focus on Ras-PI3 kinase signaling with T cell leukemia as a screening platform for multiple clinical and experimental reasons. Our resource predicts multiple combination-based therapies with high fidelity, ten of which we confirmed with small molecule inhibitors. Included are biochemical assays, as well as the IPI145 (duvelisib) inhibitor. We uncover the mechanism of synergy between the PI3 kinase inhibitor GDC0941 (pictilisib) and the tubulin inhibitor vincristine and demonstrate broad synergy in 28 cell lines of 5 cancer types and efficacy in preclinical leukemia mouse trials.

The immunoreceptor CD300a controls the intensity of inflammation and dysfunction in a model of Ag-induced arthritis in mice.

CD300a is an inhibitory immunoreceptor expressed in lymphoid and myeloid cells. This study evaluates whether CD300a plays a role in the control of joint inflammation in a model of Ag-induced arthritis (AIA) in mice. CD300a was found to be expressed mostly on neutrophils and its expression was enhanced on neutrophils that migrated to the inflamed synovial cavity. Joint inflammation, as characterized by neutrophil accumulation, was significantly greater in CD300a KO (CD300a-/- ) mice subjected to AIA, as compared to WT mice. This was associated with joint dysfunction, as measured by lower mechanical nociception threshold. There was greater production of the chemokine CXCL1 and the cytokine IL-6 in joints of CD300a-/- mice. In vitro, Mϕs from CD300a-/- mice released higher concentrations of CXCL1 and IL-6 in response to LPS. Splenocytes from immunized CD300a-/- mice produced increased levels of IFN-γ and IL-17 and lower levels of IL-10 when challenged with Ag than cells from WT mice. Neutrophils lacking the CD300a gene had greater chemotactic activity in response to fMLP, CXCL1, and LTB4 than WT neutrophils. In conclusion, these results reveal that the absence of CD300a promotes exacerbation of inflammation in a model of Ag-induced arthritis, suggesting that CD300a is an important receptor for negatively controlling the inflammatory response in this model. Mechanistically, CD300a seems to regulate the activity of various immune cells types involved in the process, including neutrophils, Mϕs, and lymphocytes.

All-trans-retinoic acid mediates changes in PI3K and retinoic acid signaling proteins of leiomyomas.

OBJECTIVE: To detect changes induced by all-trans-retinoic acid (ATRA) on the expression and activation of target proteins of the retinoic acid (RA) and PI3K/Akt pathways involved in leiomyoma growth. DESIGN: A study on human tissue cultures. SETTING: Hadassah University Hospital. PATIENT(S): Premenopausal women with uterine leiomyomas. INTERVENTION(S): Paired cultures of normal myometrium and leiomyomas, from women undergoing hysterectomy, were obtained. MAIN OUTCOME MEASURE(S): The effect of ATRA was examined on the expression and phosphorylation of relevant RA, PI3K/Akt, and Bcl2 proteins (immunochemical analysis), cell proliferation, cell cycle distribution, and apoptosis. RESULT(S): Applying our cell culture model, we demonstrated that ATRA induced changes in the expression and activation of the RA and PI3K/Akt pathway proteins in leiomyoma cells, with significant increases of alcohol dehydrogenase 1 and cyclin D2 protein levels. In part of the leiomyoma cells, ATRA induced a relative increase of Bax (proapoptotic) as well as a relative decrease of phosphorylated glycogen synthase kinase 3ß (proapoptotic). CONCLUSION(S): Our results highlight the involvement of ATRA in the RA and PI3K/Akt pathways, whose specific signaling products may influence the outcome of leiomyoma growth by regulating cell proliferation, apoptosis, and survival. These results might be useful for the on-going research into alternative methods for treating and preventing this disorder.

Changes related to phosphatidylinositol 3-kinase/Akt signaling in leiomyomas: possible involvement of glycogen synthase kinase 3alpha and cyclin D2 in the pathophysiology.

OBJECTIVE: To identify changes in the expression and phosphorylation of phosphatidylinositol 3-kinase (PI3K)/Akt protein kinases controlling survival and/or apoptosis of in vitro cell cultures of uterine leiomyomas. DESIGN: Establishment of paired cell cultures of leiomyoma and myometrial specimens. SETTING: Hadassah gynecology research laboratory. PATIENT(S): Eleven white premenopausal women, 35 to 50 years of age, undergoing hysterectomy because of symptomatic uterine leiomyomas. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Immunochemical analysis of expression and phosphorylation of relevant PI3K/Akt and BCL2 proteins. RESULT(S): Analysis of total phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and of nonphosphorylated and phosphorylated (p) PDK1, Akt, glycogen synthase kinase 3 (GSK3), FKHR, tuberin (TSC2) and hamartin (TSC1) complex, and cyclin D2 proteins indicated that [1] the level of pGSK3alpha and cyclin D2 proteins was elevated significantly in the leiomyoma compared with the normal myometrium, [2] there was a significant interaction between PTEN- PDK1 and between pAkt-pGSK3beta in the leiomyoma compared with the myometrial cells, and [3] there was a significant interaction between pAkt-pGSK3alpha in the paired leiomyoma and myometrial cultures. CONCLUSION(S): Our study suggests that the downstream signaling components of the PI3K/Akt pathway, GSK3 (a regulator of apoptosis), and cyclin D2 (a promoter of G1/S progression), as well as the significant interaction between PTEN-PDK and between pAkt-pGSK3beta, are involved in the survival and proliferation of leiomyomas.

Are we ready to downregulate mast cells?

Downregulation of mast cells (MCs) function and/or survival is warranted in allergic inflammation (AI), mastocytosis/MC leukemias and in other inflammatory diseases in which MCs have a central role. Human MCs (hMCs) have been recently shown to express the death receptor (DR) TRAIL and the inhibitory receptors (IRs) CD300a and Siglec-8. TRAIL is the only known DR functional on hMCs, and interestingly its function is upregulated by IgE-dependent MC activation. The newly described IRs, CD300a and Siglec-8, potently downregulate MC activation and survival in vitro and inhibit different IgE-mediated responses in vivo. Therefore a selective targeting of TRAIL and of IRs on MC could be a novel immunopharmacological way to downregulate MC-associated diseases.