The specific signals mediating the activation of microglia and astrocytes as a prelude to, or consequence of, CNS inflammation continue to be defined. We investigated TLRs as novel receptors mediating innate immune responses in human glial cells. We find that microglia express mRNA for TLRs 1–9, whereas astrocytes express robust TLR3, low-level TLR 1, 4, 5, and 9, and rare-to-undetectable TLR 2, 6, 7, 8, and 10 mRNA (quantitative real-time PCR). We focused on TLRs 3 and 4, which can signal through both the MyD88-dependent and -independent pathways, and on the MyD88-restricted TLR2. By flow cytometry, we established that microglia strongly express cell surface TLR2; TLR3 is expressed at higher levels intracellularly. Astrocytes express both cell surface and intracellular TLR3. All three TLRs trigger microglial activation upon ligation. TLR3 signaling induces the strongest proinflammatory polarizing response, characterized by secretion of high levels of IL-12, TNF-α, IL-6, CXCL-10, and IL-10, and the expression of IFN-β. CXCL-10 and IL-10 secretion following TLR4 ligation are comparable to that of TLR3; however, other responses were lower or absent. TLR2-mediated responses are dominated by IL-6 and IL-10 secretion. Astrocytes respond to TLR3 ligation, producing IL-6, CXCL-10, and IFN-β, implicating these cells as contributors to proinflammatory responses. Initial TLR-mediated glial activation also regulates consequent TLR expression; while TLR2 and TLR3 are subject to positive feedback, TLR4 is down-regulated in microglia. Astrocytes up-regulate all three TLRs following TLR3 ligation. Our data indicate that activation of innate immune responses in the CNS is not homogeneous but rather tailored according to cell type and environmental signal.

Oxidative burst, a critical antimicrobial mechanism of neutrophils, involves the rapid generation and release of reactive oxygen intermediates (ROIs) by the NADPH oxidase complex. Genetic mutations in an NADPH oxidase subunit, gp91 (also referred to as NOX2), are associated with chronic granulomatous disease (CGD), which is characterized by recurrent and life-threatening microbial infections. To combat such infections, ROIs are produced by neutrophils after stimulation by integrin-dependent adhesion to the ECM in conjunction with stimulation from inflammatory mediators, or microbial components containing pathogenassociated molecular patterns. In this report, we provide genetic evidence that both the Vav family of Rho GTPase guanine nucleotide exchange factors (GEFs) and phospholipase C-γ2 (PLC-γ2) are critical mediators of adhesion-dependent ROI production by neutrophils in mice. We also demonstrated that Vav was critically required for neutrophil-dependent host defense against systemic infection by Staphylococcus aureus and Pseudomonas aeruginosa, 2 common pathogens associated with fatal cases of hospital-acquired pneumonia. We identified a molecular pathway in which Vav GEFs linked integrin-mediated signaling with PLC-γ2 activation, release of intracellular Ca 2+ cations, and generation of diacylglycerol to control assembly of the NADPH oxidase complex and ROI production by neutrophils. Taken together, our data indicate that integrin-dependent signals generated during neutrophil adhesion contribute to the activation of NADPH oxidase by a variety of distinct effector pathways, all of which require Vav.

Class 1 phosphoinositide 3-kinases (PI3Ks), consisting of PI3K␣, ␤, ␥, and ␦, are a family of intracellular signaling molecules that play important roles in cellmediated immune responses. In thymocytes, however, their role is less clear, although PI3K␥ is postulated to partially contribute to pre-TCR-dependent differentiation. We now report that PI3K␦, in conjunction with PI3K␥, is required for thymocyte survival and ultimately for Tcell production. Surprisingly, genetic deletion of the p110␦ and p110␥ catalytic subunits resulted in a dramatic reduction in thymus size, cellularity, and lack of corticomedullary differentiation. Total thymocyte counts in these animals were 27-fold lower than in wild-type (WT) controls because of a diminished number of CD4 ؉ CD8 ؉ double-positive (DP) cells and were associated with T-cell depletion in blood and in secondary lymphoid organs. Moreover, this alteration in the DP population was intrinsic to thymocytes, because the reconstitution of p110␥␦ ؊/؊ animals with WT fetal liver cells restored the proportions of all thymocyte populations to those in WT controls. The observed defects were related to massive apoptosis in the DP population; TCRB expression, pre-TCR selection, and generation of DP cells appeared relatively unperturbed. Thus, class 1 PI3Ks work in concert to protect developing thymocytes from apoptosis. ( IntroductionThymocyte development relies on a series of intracellular signaling events that regulate cell differentiation, proliferation, and survival. This process can be followed based on the presence or absence of cell surface markers such as CD4, CD8, CD25, and CD44. [1][2][3] Early thymocyte progenitors lack CD4 and CD8 expression and are termed double-negative (DN) cells. The DN stage is subdivided into 4 categories. The DN1 stage is characterized by surface expression of CD44 (CD25 Ϫ CD44 ϩ ). Maturation of this earliest thymocyte subset then proceeds from the DN2 stage (CD25 ϩ CD44 ϩ ) to the DN3 stage (CD25 ϩ CD44 Ϫ ) and finally to the DN4 stage (CD25 Ϫ CD44 Ϫ ). The first regulatory checkpoint in thymocyte development, termed ␤-selection, occurs at the DN3 stage. This involves TCR␤ gene rearrangement and expression, which permits the subsequent formation of the pre-TCR complex. 4,5 Thymocytes unable to generate a functionally rearranged TCR␤ gene die by apoptosis. 6,7 Subsequently, signals provided by the pre-TCR and local microenvironment result in the proliferation and differentiation of DN thymocytes to the CD4 ϩ CD8 ϩ DP stage. A small subset of these cells ultimately bear a mature TCR␣␤ Ϫ CD3 complex and then further differentiate into CD4 ϩ or CD8 ϩ single-positive (SP) T cells.In addition to TCRB selection, thymocyte development is also shaped by the induction or inhibition of apoptosis. Although many different molecules can regulate this process, the proto-oncogene Bcl-2 appears to have a protective effect with regard to thymocyte survival. 8,9 This is supported by the observation that thymocytes in mice expressing a Bcl2 transgene are less pron...

The importance of reactive oxygen intermediate (ROI) production in antimicrobial responses is demonstrated in human patients who suffer from chronic granulomatous disease (CGD) due to defective NADPH oxidase function. Exactly how bacterial products activating Toll-like receptors (TLRs) induce oxidative burst is unknown. Here, we identify the Vav family of Rho guanine nucleotide exchange factors (GEFs) as critical mediators of LPSinduced MyD88-dependent activation of Rac2, NADPH oxidase, and ROI production using mice deficient in Vav1, Vav2, and Vav3. Vav proteins are also required for p38 MAPK activation and for normal regulation of proinflammatory cytokine production, but not for other MyD88- IntroductionMammalian Toll-like receptors (TLRs) initiate innate host defenses and regulate adaptive immune responses by virtue of their ability to recognize conserved microbial components. [1][2][3] Upon recognition of their ligands, TLRs transduce intracellular signals via associated Toll/interleukin-1 receptor (TIR) domain-containing adaptors, including MyD88, TIRAP, TRIF, and TRAM, which in turn recruit additional signaling molecules such as IRAK4, IRAK1, and TRAF6. 1,3 In this manner, recognition of LPS by TLR4, or peptidoglycan (PGN) by TLR2, on macrophages and neutrophils activates MyD88-dependent inflammatory responses. These include the production of cytokines and prostaglandins as well as oxidative burst, which generates reactive oxygen intermediates (ROIs). 4,5 The importance of oxidative burst for mounting effective antimicrobial responses is underscored by chronic granulomatous disease (CGD) in human patients bearing mutations in the NADPH oxidase complex. Phagocytes from these patients fail to efficiently generate superoxide (O 2 Ϫ) and secondary ROIs upon microbial challenge, thus rendering them susceptible to chronic and potentially life-threatening infections. However, excessive ROI generation in response to environmental stress or proinflammatory stimuli can cause widespread organ damage. 6,7 Generation of ROIs is dependent on assembly of the plasma membrane-associated NADPH oxidase complex that catalyzes electron transfer from NADPH to FAD, heme, and O 2 , resulting in the production of superoxide (O 2 Ϫ) and secondary ROIs, such as hydrogen peroxide, hydroxyl radical, and hypochlorous acid. Given the importance of ROIs in controlling bacterial infections, the mechanism of NADPH oxidase activation has been extensively examined (for reviews, see Bokoch 8 and Nathan 9 ). Thus, the activation of NADPH oxidase complex involves the assembly of cytosolic regulatory components p47 phox , p67 phox , p40 phox , and Rac2 and the recruitment of the transmembrane cytochrome b 558 complex composed of 2 subunits, gp91 phox and p22 phox . 8 In this regard, the activation of Rac2, which interacts with both the cytosolic p67 phox and the membrane-bound cytochrome b 558 , 8,10 is thought to be critical for NADPH oxidase induction. Accordingly, mutation of the RAC2 gene in humans leads to selective defects in neutrophil o...