Role of negative regulation of immune signaling pathways in neutrophil function

Polymorphonuclear neutrophils (PMNs) play a critical role in host defense against infection and in the resolution of inflammation. However, immune responses mediated by PMN must be tightly regulated to facilitate elimination of invading pathogens without inducing detrimental inflammation and host tissue damage. Specific engagement of cell surface immunoreceptors by a diverse range of extracellular signals regulates PMN effector functions through differential activation of intracellular signaling cascades. Although mechanisms of PMN activation mediated via cell signaling pathways have been well described, less is known about negative regulation of PMN function by immune signaling cascades. Here, we provide an overview of immunoreceptor‐mediated negative regulation of key PMN effector functions including maturation, migration, phagocytosis, reactive oxygen species release, degranulation, apoptosis, and NET formation. Increased understanding of mechanisms of suppression of PMN effector functions may point to possible future therapeutic targets for the amelioration of PMN‐mediated autoimmune and inflammatory diseases.Review on how PMN functions are negatively regulated by immune signaling pathways.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/145227/1/jlb10023.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/145227/2/jlb10023_am.pd

Microtubules regulate disassembly of epithelial apical junctions

BACKGROUND: Epithelial tight junction (TJ) and adherens junction (AJ) form the apical junctional complex (AJC) which regulates cell-cell adhesion, paracellular permeability and cell polarity. The AJC is anchored on cytoskeletal structures including actin microfilaments and microtubules. Such cytoskeletal interactions are thought to be important for the assembly and remodeling of apical junctions. In the present study, we investigated the role of microtubules in disassembly of the AJC in intestinal epithelial cells using a model of extracellular calcium depletion. RESULTS: Calcium depletion resulted in disruption and internalization of epithelial TJs and AJs along with reorganization of perijunctional F-actin into contractile rings. Microtubules reorganized into dense plaques positioned inside such F-actin rings. Depolymerization of microtubules with nocodazole prevented junctional disassembly and F-actin ring formation. Stabilization of microtubules with either docetaxel or pacitaxel blocked contraction of F-actin rings and attenuated internalization of junctional proteins into a subapical cytosolic compartment. Likewise, pharmacological inhibition of microtubule motors, kinesins, prevented contraction of F-actin rings and attenuated disassembly of apical junctions. Kinesin-1 was enriched at the AJC in cultured epithelial cells and it also accumulated at epithelial cell-cell contacts in normal human colonic mucosa. Furthermore, immunoprecipitation experiments demonstrated association of kinesin-1 with the E-cadherin-catenin complex. CONCLUSION: Our data suggest that microtubules play a role in disassembly of the AJC during calcium depletion by regulating formation of contractile F-actin rings and internalization of AJ/TJ proteins

Regulation of neutrophil function by selective targeting of glycan epitopes expressed on the integrin CD11b/CD18

Polymorphonuclear neutrophils (PMNs) play a critical role in the innate immune response to invading pathogens. However, dysregulated mucosal trafficking of PMNs and associated epithelial tissue damage is a pathological hallmark of numerous inflammatory conditions including inflammatory bowel disease. The glycoprotein CD11b/CD18 plays a well‐described role in regulating PMN transepithelial migration and PMN inflammatory functions. Previous studies have demonstrated that targeting of the N‐linked glycan Lewis X on CD11b blocks PMN transepithelial migration (TEpM). Given evidence of glycosylation‐dependent regulation of CD11b/CD18 function, we performed MALDI TOF Mass Spectrometry (MS) analyses on CD11b/CD18 purified from human PMNs. Unusual glycan epitopes identified on CD11b/CD18 included high Mannose oligosaccharides recognized by the Galanthus Nivalis lectin and biantennary galactosylated N‐glycans recognized by the Phaseolus Vulgaris erythroagglutinin lectin. Importantly, we show that selective targeting of glycans on CD11b with such lectins results in altered intracellular signaling events that inhibit TEpM and differentially affect key PMN inflammatory functions including phagocytosis, superoxide release and apoptosis. Taken together, these data demonstrate that discrete glycan motifs expressed on CD11b/CD18 such as biantennary galactose could represent novel targets for selective manipulation of CD11b function and reduction of PMN‐associated tissue damage in chronic inflammatory diseases.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154461/1/fsb220152-sup-0003-FigS3.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154461/2/fsb220152_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154461/3/fsb220152-sup-0004-TableS1.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154461/4/fsb220152-sup-0001-FigS1.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154461/5/fsb220152.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154461/6/fsb220152-sup-0002-FigS2.pd

Expression of Lewis‐a glycans on polymorphonuclear leukocytes augments function by increasing transmigration

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141391/1/jlb0753.pd

Myosin II regulates the shape of three-dimensional intestinal epithelial cysts.

The development of luminal organs begins with the formation of spherical cysts composed of a single layer of epithelial cells. Using a model three-dimensional cell culture, this study examines the role of a cytoskeletal motor, myosin II, in cyst formation. Caco-2 and SK-CO15 intestinal epithelial cells were embedded into Matrigel, and myosin II was inhibited by blebbistatin or siRNA-mediated knockdown. Whereas control cells formed spherical cysts with a smooth surface, inhibition of myosin II induced the outgrowth of F-actin-rich surface protrusions. The development of these protrusions was abrogated after inhibition of F-actin polymerization or of phospholipase C (PLC) activity, as well as after overexpression of a dominant-negative ADF/cofilin. Surface protrusions were enriched in microtubules and their formation was prevented by microtubule depolymerization. Myosin II inhibition caused a loss of peripheral F-actin bundles and a submembranous extension of cortical microtubules. Our findings suggest that inhibition of myosin II eliminates the cortical F-actin barrier, allowing microtubules to reach and activate PLC at the plasma membrane. PLC-dependent stimulation of ADF/cofilin creates actin-filament barbed ends and promotes the outgrowth of F-actin-rich protrusions. We conclude that myosin II regulates the spherical shape of epithelial cysts by controlling actin polymerization at the cyst surface

Deposition of microparticles by neutrophils onto inflamed epithelium: a new mechanism to disrupt epithelial intercellular adhesions and promote transepithelial migration

Neutrophil [polymorphonuclear leukocyte (PMN)] transepithelial migration (TEM) is a hallmark of inflammatory mucosal disorders. PMN TEM is associated with epithelial injury; however, mechanisms involved in this process are not well defined. The current work describes a new mechanism whereby deposition of PMN membranederived microparticles (PMNâ MPs) onto intestinal epithelial cells (IECs) during TEM leads to loss of epithelial cadherins, thus promoting epithelial injury and increased PMN recruitment. PMNâ MPs secreted by activated PMNs during TEM displayed a high level of enzymatically activematrixmetalloproteinase 9 (MMPâ 9), and were capable of mediating potent effects on IEC integrity. Isolated PMNâ MPs efficiently bound to IEC monolayers and induced cleavage of desmogleinâ 2 (DSGâ 2) but not Eâ cadherin, leading to disruption of IEC intercellular adhesions. Furthermore, PMNâ MP binding to intestinal epithelium in vitro in transwell assays and in vivo in ligated intestinal loop preparations facilitated increases in PMN TEM. These effects were MMPâ 9 dependent and were reversed in the presence of specific pharmacological inhibitors. Finally, we demonstrated that IEC Dsgâ 2 serves as a barrier for migrating PMNs, and its removal by PMNâ MPâ associated MMPâ 9 facilitates PMNtrafficking across epithelial layers. Our findings thus implicate PMNâ MPs in PMNâ mediated inflammation and epithelial damage, as observed in inflammatory disorders ofmucosal surfaces.â Butinâ Israeli, V., Houser, M.C., Feng, M., Thorp, E. B., Nusrat, A., Parkos, C. A, Sumagin, R. Deposition of microparticles by neutrophils onto inflamed epithelium: anewmechanism to disrupt epithelial intercellular adhesions and promote transepithelialmigration. FASEB J. 30, 4007â 4020 (2016). www.fasebj.orgPeer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154234/1/fsb2fasebj30120734r.pd

Antiadhesive Role of Apical Decay-accelerating Factor (CD55) in Human Neutrophil Transmigration across Mucosal Epithelia

Neutrophil migration across mucosal epithelium during inflammatory episodes involves the precise orchestration of a number a cell surface molecules and signaling pathways. After successful migration to the apical epithelial surface, apically localized epithelial proteins may serve to retain PMN at the lumenal surface. At present, identification of apical epithelial ligands and their PMN counter-receptors remain elusive. Therefore, to define the existence of apical epithelial cell surface proteins involved in PMN–epithelial interactions, we screened a panel of antibodies directed against epithelial plasma membranes. This strategy identified one antibody (OE-1) that both localized to the apical cell membrane and significantly inhibited PMN transmigration across epithelial monolayers. Microsequence analysis revealed that OE-1 recognized human decay-accelerating factor (DAF, CD55). DAF is a highly glycosylated, 70–80-kD, glycosyl-phosphatidyinositol–linked protein that functions predominantly as an inhibitor of autologous complement lysis. DAF suppression experiments using antisense oligonucleotides or RNA interference revealed that DAF may function as an antiadhesive molecule promoting the release of PMN from the lumenal surface after transmigration. Similarly, peptides corresponding to the antigen recognition domain of OE-1 resulted in accumulation of PMN on the apical epithelial surface. The elucidation of DAF as an apical epithelial ligand for PMN provides a target for novel anti-inflammatory therapies directed at quelling unwanted inflammatory episodes

Western diet‐induced increase in colonic bile acids compromises epithelial barrier in nonalcoholic steatohepatitis

There is compelling evidence implicating intestinal permeability in the pathogenesis of nonalcoholic steatohepatitis (NASH), but the underlying mechanisms remain poorly understood. Here we examined the role of bile acids (BA) in western diet (WD)‐induced loss of colonic epithelial barrier (CEB) function in mice with a genetic impairment in intestinal epithelial barrier function, junctional adhesion molecule A knockout mice, F11r−/−. WD‐fed knockout mice developed severe NASH, which was associated with increased BA concentration in the cecum and loss of CEB function. Analysis of cecal BA composition revealed selective increases in primary unconjugated BAs in the WD‐fed mice, which correlated with increased abundance of microbial taxa linked to BA metabolism. In vitro permeability assays revealed that chenodeoxycholic acid (CDCA), which was elevated in the cecum of WD‐fed mice, increased paracellular permeability, while the BA‐binding resin sevelamer hydrochloride protected against CDCA‐induced loss of barrier function. Sequestration of intestinal BAs by in vivo delivery of sevelamer to WD‐fed knockout mice attenuated colonic mucosal inflammation and improved CEB. Sevelamer also reduced hepatic inflammation and fibrosis, and improved metabolic derangements associated with NASH. Collectively, these findings highlight a hitherto unappreciated role for BAs in WD‐induced impairment of the intestinal epithelial barrier in NASH.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/155502/1/fsb220488.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/155502/2/fsb220488_am.pd

Gab2 and Gab3 Redundantly Suppress Colitis by Modulating Macrophage and CD8+ T-Cell Activation

Inflammatory Bowel Disease (IBD) is a multi-factorial chronic inflammation of the gastrointestinal tract prognostically linked to CD8+ T-cells, but little is known about their mechanism of activation during initiation of colitis. Here, Grb2-associated binding 2/3 adaptor protein double knockout mice (Gab2/3−/−) were generated. Gab2/3−/− mice, but not single knockout mice, developed spontaneous colitis. To analyze the cellular mechanism, reciprocal bone marrow (BM) transplantation demonstrated a Gab2/3−/− hematopoietic disease-initiating process. Adoptive transfer showed individual roles for macrophages and T-cells in promoting colitis development in vivo. In spontaneous disease, intestinal intraepithelial CD8+ but much fewer CD4+, T-cells from Gab2/3−/− mice with rectal prolapse were more proliferative. To analyze the molecular mechanism, reduced PI3-kinase/Akt/mTORC1 was observed in macrophages and T-cells, with interleukin (IL)-2 stimulated T-cells showing increased pSTAT5. These results illustrate the importance of Gab2/3 collectively in signaling responses required to control macrophage and CD8+ T-cell activation and suppress chronic colitis