Highlights d HSCs permanently remodel the mitochondrial network after replicative stress d HSCs keep dysfunctional mitochondria because of Drp1 loss, causing functional decline d HSCs accumulate dysfunctional mitochondria through asymmetric division d HSC attrition is due to asynchrony in cell cycle and biosynthetic gene expression

Chemotaxis promotes neutrophil participation in cellular defense by enabling neutrophil migration to infected tissue and is controlled by persistent cell polarization. One long-standing question of neutrophil polarity has been how the pseudopod and the uropod are coordinated. In our previous report, we suggested that Rho GTPase Cdc42 controls neutrophil polarity through CD11b signaling at the uropod, albeit through an unknown mechanism. Here, we show that Cdc42 controls polarity, unexpectedly, via its effector WASp. Cdc42 controls WASp activation and its distant localization to the uropod. At the uropod, WASp regulates the reorganization of CD11b integrin into detergent resistant membrane domains; in turn, CD11b recruits the microtubule end binding protein EB1 to capture and stabilize microtubules at the uropod. This organization is necessary to maintain neutrophil polarity during migration and is critical for neutrophil emigration into inflamed lungs. These results suggest unrecognized mechanism of neutrophil polarity in which WASp mediates long-distance control of the uropod by Cdc42 to maintain a proper balance between the pseudopod and the uropod. IntroductionNeutrophils are the most abundant leukocyte and fastest moving cell in the body. Neutrophils play a central role in innate immunity as cellular defense against infecting microorganisms and inflammatory processes and can contribute to hyperinflammatory reactions causing tissue injury. Neutrophils move rapidly toward sites of infection through a multiple-step process that involves tethering, rolling, adhesion, and transmigration to reach the site of infection in the tissue. Moreover, a new step in the neutrophil extravasation cascade, so-called locomotion, has been recently identified where neutrophils crawl onto endothelium toward the nearest endothelial junctions before transmigrating into tissues. [1][2][3] Failure to regulate any of these events of neutrophil extravasation may lead to abnormal innate immune responses, including immunodeficiency or aberrant inflammatory reactions. Therefore, understanding the molecular mechanisms that control neutrophil migration is of significant therapeutic importance.Neutrophils are one of the fastest migrating cells in response to a shallow chemoattractant gradient. Migrating neutrophils are highly polarized cells, which enables them to persistently migrate along the chemotactic gradient. During this process, filamentous actin (F-actin) polymerizes asymmetrically at the cell leading edge, and provides the protrusive forces to propel the cell membrane forward. At the same time, lateral membrane protrusions (so-called secondary/abnormal protrusions) are inhibited by actomyosin contractile complexes forming along the cell sides and the trailing edge, or uropod. [4][5][6] Members of the Rho GTPase family, including Rho, Rac, and Cdc42, are key regulators of chemotaxis. They cycle between an inactive, GDP-bound and active, GTP-bound forms via guanine nucleotide exchange factors (GEFs) and GTPaseactivating proteins (GAP...

Mice lacking the small GTPase Rap1b exhibit enhanced neutrophil recruitment to inflamed lungs and susceptibility to endotoxin shock via enhance PI3K-Akt activation.

The mechanisms regulating hematopoietic stem and progenitor cell (HSPC) fate choices remain ill-defined. Here, we show that a signalling network of p190-B RhoGAP-ROS-TGF-β-p38MAPK balances HSPC self-renewal and differentiation. Upon transplantation, HSPCs express high amounts of bioactive TGF-β1 protein, which is associated with high levels of p38MAPK activity and loss of HSC self-renewal in vivo. Elevated levels of bioactive TGF-β1 are associated with asymmetric fate choice in vitro in single HSPCs via p38MAPK activity and this is correlated with the asymmetric distribution of activated p38MAPK. In contrast, loss of p190-B, a RhoGTPase inhibitor, normalizes TGF-β levels and p38MAPK activity in HSPCs and is correlated with increased HSC self-renewal in vivo. Loss of p190-B also promotes symmetric retention of multi-lineage capacity in single HSPC myeloid cell cultures, further suggesting a link between p190-B-RhoGAP and non-canonical TGF-β signalling in HSPC differentiation. Thus, intracellular cytokine signalling may serve as ‘fate determinants' used by HSPCs to modulate their activity.

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Hematopoiesis is regulated by components of the microenvironment, so-called niche. Here, we show that p190-B GTPase Activating Protein (p190-B) deletion in mice causes hematopoietic failure during ontogeny, in p190-B−/− fetal liver and bones, and in p190-B+/− adult bones and spleen. These defects are non-cell autonomous, since we previously showed that transplantation of p190-B−/− hematopoietic cells into wild-type hosts leads to normal hematopoiesis. Coculture of mesenchymal stem/progenitor cells (MSC) and wild-type bone marrow cells reveals that p190-B−/− MSCs are dysfunctional in supporting hematopoiesis due to impaired Wnt signaling. Furthermore, p190-B loss causes alteration in bone marrow niche composition, including abnormal CFU-fibroblast, CFU-adipocyte and CFU-osteoblast numbers. This is due to altered MSC lineage fate specification to osteoblast and adipocyte lineages. Thus, p190-B organizes a functional mesenchymal/microenvironment for normal hematopoiesis during development.

Bone marrow failure syndromes (BMF) are characterized by ineffective hematopoiesis due to impaired fitness of hematopoietic stem cells (HSC). BMFs can be acquired during bone marrow stress or innate are associated with driver genetic mutations. BMFs are at higher risks of developing secondary neoplasms, including myelodysplastic syndromes and leukemia. Despite the identification of genetic driver mutations, the hematopoietic presentation of the disease is quite heterogeneous raising the possibility that non-genetic factors contribute to the pathogenesis of the disease. The role of inflammation has emerged as an important contributing factors, but remain to be understood in detail. In this study, we examined the effect of increased TGFβ signaling in combination or not with an acute innate immune challenge using polyinosinc:polycytidilic acid (pIC) on the hematopoietic system without genetic mutations. We show that acute rounds of pIC alone drive a benign age-related myeloid cell expansion, increased TGFβ signaling alone causes a modest anemia on old mice. In sharp contrast, increased TGFβ signaling plus acute pIC challenge result in chronic pancytopenia, expanded hematopoietic stem and progenitor pools, and increased bone marrow dysplasia 3-4 months after stress, phenotypes similar to human bone marrow failure syndromes. Mechanistically, this disease phenotype is uniquely associated with increased mitochondrial content, increased reactive oxygen species and enhanced caspase-1 activity. Our results suggest that chronic increased TGFβ signaling modifies the memory of an acute immune response to drive bone marrow failure without the need for pre-existing genetic insult. Hence, non-genetic factors in combination are sufficient to drive bone marrow failure.

16 Neutrophils (PMNs) are the first line of immune defense by moving toward the infection site. A key event of cell migration is the maintenance of a polarized morphology characterized by a single protrusive leading edge (i.e. lamellipodia) and a contractile uropod. Using mice with a conditional Cdc42 (flox) allele, we have previously reported that the small Rho GTPase Cdc42 controls neutrophil polarity via CD11b integrin signaling at the uropod (Szczur; Blood 2009). In the present study, we seek to dissect the mechanism of Cdc42-CD11b axis in neutrophil polarity. We first examined the role of the Cdc42 effector Wiskott Aldrich Syndrome protein (WASp), as WASp−/− neutrophils showed defective neutrophil migration and integrin clustering (Yang; Immunity 2006). A Cdc42 mutant for WASp Binding (Cdc42S71P) was expressed in Cdc42−/− cells using retroviral transduction. Rescue of Cdc42 functions was analyzed compared to WT cells and Cdc42−/− cells transduced with an empty vector, and Cdc42−/− cells transduced with the wild type form of Cdc42 (wtCdc42). While expression of wtCdc42 rescued abnormal polarity and CD11b clustering in Cdc42−/− neutrophils to WT levels, expression of Cdc42S71P did not, suggesting that Cdc42 controls neutrophil polarity via WASp. We further confirmed the involvement of WASp in neutrophil polarity using WASp−/− neutrophils. WASp−/− neutrophils exhibited all characteristics of loss of polarity (ie frequent changes in direction during migration as assessed by video microscopy, multiple F-actin protrusions at the uropod). We next examined how Cdc42/WASp regulates neutrophil polarity and explored the roles of microtubules (MTs), since MTs are oriented toward the uropod in migrating WT neutrophils and Cdc42 is known to regulate MT polarity. Cdc42−/− as well as WASp−/− neutrophils showed loss of MT polarity with cells extending MT towards both the front and the uropod. They also exhibited less stabilized MTs (ie, defect in detyrosinated MT). Interestingly, MTs made contact with CD11b clusters at the uropod in WT, but not in Cdc42−/− and WASp−/− neutrophils. Enforcing CD11b clustering by CD11b antibody crosslinking in Cdc42−/− and WASp−/− neutrophils rescued polarity and MT capture at the plasma membrane to WT levels. Thus, Cdc42 regulates CD11b clustering via WASp, which, in turn, may capture and stabilize MTs at the uropod. To further examine this possibility, we examined in detail the plasma membrane of these cells. Indeed, on activation, the uropod of neutrophils reorganizes into detergent resistant membrane (DRM) domains – something, which is essential for polarity. Immunostaining with the DRM marker cholera toxin indicated loss of DRM assembly at the uropod of Cdc42−/− and WASp−/− neutrophils. Biochemistry analysis showed that DRM fraction of WT cells contained CD11b, WASp, tubulin and the microtubule end-binding protein EB1. In contrast, Cdc42−/− and WASp−/− DRMs had significantly lower amount of CD11b, WASp, tubulin, EB1 and CD11b, tubulin, EB1, respectively. Remarkably, CD11b crosslinking in WASp−/− cells rescued CD11b, tubulin and EB1 whereas CD11b crosslinking in Cdc42−/− cells rescued CD11b, tubulin and EB1 but not WASp, suggesting that Cdc42 and WASp controls DRM formation containing CD11b, and that CD11b recruits EB1 to capture and stabilize microtubules downstream of WASp. Together, our study uncovers a novel mechanism of neutrophil migration in which Cdc42 recruits WASp to the uropod to reorganize the plasma membrane into discrete DRM domains and induce CD11b clustering. CD11b clusters, in turn, capture and stabilize microtubules via EB1 – something that is critical for maintaining neutrophil polarity during directed migration. Our study thus reveals a new function for WASp in the control of neutrophil polarity via crosstalk of DRM/CD11b and microtubules. We used a 3-D migration model that mimics the extravasation cascade ex vivo to show that Cdc42/WASp/CD11b-dependent neutrophil polarity regulates the crawling step of neutrophils toward endothelial cell junction for efficient transmigration on HUVECs. Using a model of LPS-induced acute lung injury, we then show that Cdc42 loss dramatically reduced neutrophil recruitment to lung alveolar cavities as well as lung tissue inflammation – an indication for the patho-physiological importance of this pathway during the neutrophil extravasation cascade and subsequent inflammation in vivo. Disclosures: No relevant conflicts of interest to declare.