No abstract

phox -independent NADPH oxidase activation by prenylated Rac1 is inhibited by Rho GDP dissociation inhibitor and by phosphatidylcholine vesicles, both competing with membrane for prenylated Rac1. We conclude that, in vitro, targeting of Rac to the phagocyte membrane is sufficient for the induction of NADPH oxidase assembly, suggesting that the principal or, possibly, the only role of Rac is to recruit cytosolic p67 phox to the membrane environment, to be followed by the interaction of p67 phox with cytochrome b 559 .The production of reactive oxygen species represents the major microbicidal mechanism of professional phagocytes. The primordial oxygen radical is superoxide (O 2 . ), 1 and it is produced by the NADPH-derived one-electron reduction of molecular oxygen, by an enzyme complex known as the NADPH oxidase (referred to here as "oxidase"; reviewed in Refs. 1 and 2 phox , and Rac to a critical concentration of an anionic amphiphile (3, 4).Rac1 or -2 is absolutely required for oxidase assembly in the amphiphile-activated cell-free system (5, 6). It is also clearly involved in O 2. production in intact phagocytes, as shown by the inhibitory effect of Rac antisense oligonucleotides (7) and by selective defects in O 2 . production by neutrophils of Rac2-deficient mice (8) and of a patient with an inhibitory mutation in Rac2 (9). It is the consensus opinion that, in the course of oxidase assembly, Rac is translocated to the membrane (10), although lack of translocation (11) or the lack of relevance of translocation to assembly (12) was also claimed. In the cytosol, Rac is found as a C-terminally prenylated (geranylgeranylated) protein (13), forming a heterodimer with the regulatory protein GDP dissociation inhibitor for Rho (Rho GDI) (5). Dissociation of prenylated Rac from Rho GDI was proposed to be an obligatory step preceding translocation of Rac from the cytosol to the membrane (14 -16). The role of Rac in oxidase assembly was studied extensively in the semirecombinant amphiphile-activated cell-free system (4). Both nonprenylated (5, 17) and prenylated (17-19) Rac are capable of supporting oxidase activation in vitro. It was recently suggested (20) that, whereas membrane association of prenylated Rac (Rac1 or -2) is mediated principally by hydrophobic interaction between the geranylgeranyl tail and membrane lipids, nonprenylated Rac1 binds by electrostatic interaction between a C-terminal polybasic region (21) and negative charges on the membrane. We intended to test the hypothesis that binding of Rac to the membrane is a crucial event in the initiation of oxidase assembly. We found indeed that prenylated, but not nonprenylated, Rac1 initiates oxidase assembly and NADPH-dependent O 2 .production in a cell-free system, containing phagocyte membranes and p67 phox , in the absence of an amphiphilic activator

Immunotherapies targeting T lymphocytes are revolutionizing cancer therapy but only benefit a subset of patients, especially in colorectal cancer. Thus, additional insight into the tumor microenvironment (TME) is required. Eosinophils are bone marrow–derived cells that have been largely studied in the context of allergic diseases and parasite infections. Although tumor-associated eosinophilia has been described in various solid tumors including colorectal cancer, knowledge is still missing regarding eosinophil activities and even the basic question of whether the TME promotes eosinophil recruitment without additional manipulation (e.g., immunotherapy) is unclear. Herein, we report that eosinophils are recruited into developing tumors during induction of inflammation-induced colorectal cancer and in mice with the Apcmin/+ genotype, which develop spontaneous intestinal adenomas. Using adoptive transfer and cytokine neutralization experiments, we demonstrate that the TME supported prolonged eosinophil survival independent of IL5, an eosinophil survival cytokine. Tumor-infiltrating eosinophils consisted of degranulating eosinophils and were essential for tumor rejection independently of CD8+ T cells. Transcriptome and proteomic analysis revealed an IFNγ-linked signature for intratumoral eosinophils that was different from that of macrophages. Our data establish antitumorigenic roles for eosinophils in colorectal cancer. These findings may facilitate the development of pharmacologic treatments that could unleash antitumor responses by eosinophils, especially in colorectal cancer patients displaying eosinophilia.

Activation of the superoxide (O2(-))-generating NADPH oxidase of phagocytes is the consequence of the assembly of a membrane-associated flavocytochrome b(559) with the cytosolic proteins p47(phox) and p67(phox) and the small GTPase Rac (1 or 2). We proposed that Rac1 serves as a membrane-targeting molecule for p67(phox). This hypothesis was tested by constructing recombinant chimeric proteins, joining various functional domains of p67(phox) and Rac1, and expressing these in Escherichia coli. Chimeras were assayed for the ability to support O2(-) production by phagocyte membranes in an amphiphile-activated cell-free system in the presence or absence of p47(phox). A chimera consisting of p67(phox) truncated at residue 212 and fused to a full-length Rac1 [p67(phox)(1-212)-Rac1(1-192)] was a potent NADPH oxidase activator. A p67(phox)(1-212)-Rac1(178-192) chimera, to which Rac1 contributed only the C-terminal polybasic domain, was a weaker but consistent activator. Chimeras comprising the full length of Rac1 bound GTP/GDP, like bona fide GTPases. The activity of p67(phox)-Rac1 chimeras was dependent on the presence of the tetratricopeptide repeat and activation domains, in the p67(phox) segment, and on an intact polybasic region, at the C terminus of the Rac1 segment, but not on the insert region of Rac1. Partial activation by chimeras, in the GTP-bound form, was also possible in the absence of p47(phox). Evidence is offered in support of the proposal that the GTP- and GDP-bound forms of chimera p67(phox)(1-212)-Rac1(1-192) have distinct conformations, corresponding to the presence and absence of intrachimeric bonds, respectively.

Accumulating data have indicated a fundamental role of eosinophils in regulating adipose tissue homeostasis. Here, we performed whole-genome RNA sequencing of the small intestinal tract, which suggested the presence of impaired lipid metabolism in eosinophil-deficient ΔdblGATA mice. ΔdblGATA mice fed a high-fat diet (HFD) showed reduced body fat mass, impaired enlargement of adipocytes, decreased expression of adipogenic genes, and developed glucose intolerance. HFD induced accumulation of eosinophils in the perigonadal white adipose tissue. Concordantly, adipocyte-differentiated 3T3-L1 cells promoted the migration of eosinophils through the expression of CCL11 (eotaxin-1) and likely promoted their survival through the expression of interleukin (IL)-3, IL-5, and granulocyte-macrophage colony-stimulating factor. HFD-fed ΔdblGATA mice showed increased infiltration of macrophages, CD4+ T-cells, and B-cells, increased expression of interferon-γ, and decreased expression of IL-4 and IL-13 in white adipose tissue. Interferon-γ treatment significantly decreased lipid deposition in adipocyte-differentiated 3T3-L1 cells, while IL-4 treatment promoted lipid accumulation. Notably, HFD-fed ΔdblGATA mice showed increased lipid storage in the liver as compared with wild-type mice. We propose that obesity promotes the infiltration of eosinophils into adipose tissue that subsequently contribute to the metabolic homeostasis by promoting adipocyte maturation.