B lymphocytes spread and extend membrane processes when searching for antigens and form immune synapses upon contacting cells that display antigens on their surface. Although these dynamic morphological changes facilitate B cell activation, the signaling pathways underlying these processes are not fully understood. We found that activation of the Rap GTPases was essential for these changes in B cell morphology. Rap activation was important for B cell receptor (BCR)- and lymphocyte-function-associated antigen-1 (LFA-1)-induced spreading, for BCR-induced immune-synapse formation, and for particulate BCR ligands to induce localized F-actin assembly and membrane-process extension. Rap activation and F-actin assembly were also required for optimal BCR signaling in response to particulate antigens but not soluble antigens. Thus by controlling B cell morphology and cytoskeletal organization, Rap might play a key role in the activation of B cells by particulate and cell-associated antigens.

mϕ are heterogeneous in their functions, and although it is clear that inflammatory mϕ contribute to inflammation in IBDs, multiple lines of evidence suggest that M2a mϕ may offer protection during intestinal inflammation. In vivo SHIP-deficient mouse mϕ are M2a so SHIP-deficient mice provide a unique genetic model of M2a mϕ. Based on this, this study tested the hypothesis that SHIP-deficient, M2a mϕ protect mice from intestinal inflammation. The objectives were to compare the susceptibility of SHIP+/+ and SHIP-/- littermates with DSS-induced intestinal inflammation and to determine whether protection was mϕ-mediated and whether protection could be transferred to a susceptible host. We have found that SHIP-/- mice are protected during DSS-induced intestinal inflammation. SHIP-/- mice have delayed rectal bleeding and reduced weight loss, disruption of intestinal architecture, and immune cell infiltration during DSS-induced colitis relative to their WT littermates. Using liposome depletion of mϕ, we found that SHIP-/- mouse protection was indeed mϕ-mediated. Finally, we determined that SHIP-/- mϕ-mediated protection could be conferred to susceptible WT mice by adoptive transfer of M2a mϕ derived ex vivo. This study supports our hypothesis by demonstrating that SHIP-deficient, M2a mϕ are protective in this murine model of acute intestinal inflammation. Adoptive transfer of M2a mϕ to patients with IBDs offers a promising, new strategy for treatment that may be particularly useful in patients who are otherwise refractory to conventional therapies.

Gram-negative bacterial infections, unlike viral infections, do not typically protect against subsequent viral infections. This is puzzling given that lipopolysaccharide (LPS) and double-stranded (ds) RNA both activate the TIR domain-containing adaptor-inducing interferon ␤ (TRIF) pathway and, thus, are both capable of eliciting an antiviral response by stimulating type I interferon (IFN) production. We demonstrate herein that SH2-containing inositol-5-phosphatase (SHIP) protein levels are dramatically increased in murine macrophages via the MyD88-dependent pathway, by up-regulating autocrine-acting transforming growth factor-␤ (TGF␤). The increased SHIP then mediates, via inhibition of the phosphatidylinositol-3-kinase (PI3K) pathway, cytosine-phosphate-guanosine (CPG)-and LPS-induced tolerance and cross-tolerance and restrains IFN-␤ production induced by a subsequent exposure to LPS or dsRNA. Intriguingly, we found, using isoform-specific PI3K inhibitors, that LPSor cytosine-phosphate-guanosine-induced interleukin-6 (IL-6) is positively regulated by p110␣, -␥, and -␦ but negatively regulated by p110␤. This may explain some of the controversy concerning the role of PI3K in Tolllike receptor-induced cytokine production. Consistent with our in vitro findings, SHIP ؊/؊ mice overproduce IFN-␤ in response to LPS, and this leads to antiviral hypothermia. Thus, up-regulation of SHIP in response to Gram-negative bacterial infections probably explains the inability of such infections to protect against subsequent viral infections. (Blood. 2009;113: 2945-2954) IntroductionWe reported in 2004 that the hematopoietic-restricted, SH2-containing inositol-5Ј-phosphatase (SHIP, also known as SHIP1) was markedly up-regulated in bone marrow-derived macrophages (BMms) and mast cells (BMMCs) on exposure to lipopolysaccharide (LPS, also known as endotoxin). 1 This increase in SHIP was mediated by the production of autocrine-acting transforming growth factor-␤ (TGF-␤) and blocked the production of proinflammatory cytokines and nitric oxide in response to a subsequent exposure to LPS. 1 Consistent with these results, we found that SHIP Ϫ/Ϫ BMms, BMMCs, and SHIP Ϫ/Ϫ mice did not display endotoxin tolerance. 1 Several questions arose as a result of this study, including whether SHIP plays a role in the tolerance induced by Toll-like receptors (TLRs) other than TLR4, whether TGF-␤ and SHIP are up-regulated via the MyD88-dependent or -independent pathway, and whether SHIP negatively regulates both of these pathways. To address these questions, we took advantage of differences in signaling initiated by TLR3, TLR4, and TLR9.TLR3, in response to binding double-stranded RNA (dsRNA) in endosomes, activates cells exclusively through MyD88-independent pathways by recruiting TRIF, which activates the IB kinase (IKK)-related kinases, TBK1 and IKK-⑀. These, together with the adaptors TANK and NAP1, phosphorylate the transcription factor IRF3, allowing it to dimerize and translocate to the nucleus where it collaborates with nuclear factor-B (NF-B) a...

Alternatively activated or M2 macrophages have been reported to protect mice from intestinal inflammation, but the mechanism of protection has not been elucidated. In this study, we demonstrate that mice deficient in the p110δ catalytic subunit activity of class I phosphatidylinositol 3-kinase (PI3Kp110δ) have increased clinical disease activity and histological damage during dextran sodium sulfate (DSS) induced colitis. Increased disease severity in PI3Kp110δ-deficient mice is dependent on professional phagocytes and correlates with reduced numbers of arginase I + M2 macrophages in the colon and increased production of inflammatory nitric oxide. We further demonstrate that PI3Kp110δ-deficient macrophages are defective in their ability to induce arginase I when skewed to an M2 phenotype with IL-4. Importantly, adoptive transfer of IL-4-treated macrophages derived from WT mice, but not those from PI3Kp110δ-deficient mice, protects mice during DSS-induced colitis. Moreover, M2 macrophages mediated protection is lost when mice are cotreated with inhibitors that block arginase activity or during adoptive transfer of arginase I deficient M2 macrophages. Taken together, our data demonstrate that arginase I activity is required for M2 macrophages mediated protection during DSS-induced colitis in PI3Kp110δ-deficient mice.Keywords: Alternatively activated macrophages r Arginase r Colitis r DSS-induced intestinal inflammation r PI3Kp110δAdditional supporting information may be found in the online version of this article at the publisher's web-site

Macrophages from SHIP-deficient mice have increased PI3Kp110α-mediated transcription of Il1b, which contributes to spontaneous ileal inflammation. SHIP levels and activity are lower in intestinal tissues and peripheral blood samples from patients with CD than controls. There is an inverse correlation between SHIP activity and induction of IL1β production by lipopolysaccharide and adenosine triphosphate in PBMCs. Strategies to reduce IL1B might be developed to treat patients with CD found to have low SHIP activity.

Crohn's disease (CD) is a polygenic immune-mediated disease characterized by gastrointestinal inflammation. Mice deficient in the hematopoietic-restricted SH2 domain-containing inositolpolyphosphate 5'-phosphatase (SHIP) develop spontaneous CD-like ileal inflammation. Intriguingly, SHIP mRNA is not upregulated in biopsies from patients with ileal CD despite immune cell infiltration, but SHIP's role in human CD remains unknown. We analyzed SHIP mRNA expression and activity in biopsies and peripheral blood mononuclear cells (PBMCs) from control and treatment-naive subjects with ileal CD, and demonstrated that SHIP mRNA and activity were lower in hematopoietic cells in ileal biopsies and PBMCs from subjects with CD. In all tissues from our patient cohort and in PBMCs from a second healthy control cohort, subjects homozygous for the autophagy-related 16-like protein (ATG16L1) CD-associated gene variant (rs2241880), had low SHIP mRNA expression and activity. SHIP protein expression increased during autophagy and SHIP upregulation was dependent on ATG16L1 and/or autophagy, as well as the ATG16L1 CD-associated gene variant. Finally, homozygosity for the ATG16L1 risk variant and low SHIP mRNA expression is inversely related to increased (LPS+ATP)-induced IL-1β production by PBMCs in our cohorts and was regulated by increased transcription of ILIB. These data suggest a novel mechanism by which the ATG16L1 CD-associated gene variant may predispose people to develop intestinal inflammation.

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