Inflammation is a highly coordinated host response to infection, injury, or cell stress. In most instances, the inflammatory response is pro-survival and is aimed at restoring physiological tissue homeostasis and eliminating invading pathogens, although exuberant inflammation can lead to tissue damage and death. Intravascular injection of adenovirus (Ad) results in virus accumulation in resident tissue macrophages that trigger activation of CXCL1 and CXCL2 chemokines via the IL-1α-IL-1RI signaling pathway. However, the mechanistic role and functional significance of this pathway in orchestrating cellular inflammatory responses to the virus in vivo remain unclear. Resident metallophilic macrophages expressing macrophage receptor with collagenous structure (MARCO+) in the splenic marginal zone (MZ) play the principal role in trapping Ad from the blood. Here we show that intravascular Ad administration leads to the rapid recruitment of Ly-6G+7/4+ polymorphonuclear leukocytes (PMNs) in the splenic MZ, the anatomical compartment that remains free of PMNs when these cells are purged from the bone marrow via a non-inflammatory stimulus. Furthermore, PMN recruitment in the splenic MZ resulted in elimination of virus-containing cells. IL-1α-IL-1RI signaling is only partially responsible for PMN recruitment in the MZ and requires CXCR2, but not CXCR1 signaling. We further found reduced recruitment of PMNs in the splenic MZ in complement C3-deficient mice, and that pre-treatment of IL-1α-deficient, but not wild-type mice, with complement inhibitor CR2-Crry (inhibits all complement pathways at C3 activation) or CR2-fH (inhibits only the alternative complement activation pathway) prior to Ad infection, abrogates PMN recruitment to the MZ and prevents elimination of MARCO+ macrophages from the spleen. Collectively, our study reveals a non-redundant role of the molecular factors of innate immunity – the chemokine-activating IL-1α-IL-1RI-CXCR2 axis and complement – in orchestrating local inflammation and functional cooperation of PMNs and resident macrophages in the splenic MZ, which collectively contribute to limiting disseminated pathogen spread via elimination of virus-containing cells.

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Responding to systemic demands in producing and replenishing end-effector blood cells is predicated on the appropriate delivery and interpretation of extrinsic signals to the HSPCs. The data presented herein implicate the systemic, extracellular form of the glycosyltransferase ST6Gal-1 in the regulation of late-stage neutrophil development. ST6Gal-1 is typically a membrane-bound enzyme sequestered within the intracellular secretory apparatus, but an extracellular form is released into the blood from the liver. Both human and murine HSPCs, upon exposure to extracellular ST6Gal-1 ex vivo, exhibited decreased proliferation, diminished expression of the neutrophilic primary granule protein MPO, and decreased appearance of CD11b cells. HSPC suppression was preceded by decreased STAT-3 phosphorylation and diminished C/EBPα expression, without increased apoptosis, indicating attenuated G-CSF receptor signaling. A murine model to raise systemic ST6Gal-1 level was developed to examine the role of the circulatory enzyme in vivo. Our results show that systemic ST6Gal-1 modified the cell surface of the GMP subset of HSPCs and decreased marrow neutrophil reserves. Acute airway neutrophilic inflammation by LPS challenge was used to drive demand for new neutrophil production. Reduced neutrophil infiltration into the airway was observed in mice with elevated circulatory ST6Gal-1 levels. The blunted transition of GMPs into GPs in vitro is consistent with ST6Gal-1-attenuated granulopoiesis. The data confirm that circulatory ST6Gal-1 is a negative systemic regulator of granulopoiesis and moreover suggest a clinical potential to limit the number of inflammatory cells by manipulating blood ST6Gal-1 levels.

d Adenoviruses (Ads) are promising vectors for therapeutic interventions in humans. When injected into the bloodstream, Ad vectors can bind several vitamin K-dependent blood coagulation factors, which contributes to virus sequestration in the liver by facilitating transduction of hepatocytes. Although both coagulation factors FVII and FX bind the hexon protein of human Ad serotype 5 (HAdv5) with a very high affinity, only FX appears to play a role in mediating Ad-hepatocyte transduction in vivo. To understand the discrepancy between efficacy of FVII binding to hexon and its apparently poor capacity for supporting virus cell entry, we analyzed the HAdv5-FVII complex by using high-resolution cryo-electron microscopy (cryo-EM) followed by molecular dynamic flexible fitting (MDFF) simulations. The results indicate that although hexon amino acids T423, E424, and T425, identified earlier as critical for FX binding, are also involved in mediating binding of FVII, the FVII GLA domain sits within the surface-exposed hexon trimer depression in a different orientation from that found for FX. Furthermore, we found that when bound to hexon, two proximal FVII molecules interact via their serine protease (SP) domains and bury potential heparan sulfate proteoglycan (HSPG) receptor binding residues within the dimer interface. In contrast, earlier cryo-EM studies of the Ad-FX interaction showed no evidence of dimer formation. Dimerization of FVII bound to Ad may be a contributing mechanistic factor for the differential infectivity of Ad-FX and Ad-FVII complexes, despite high-affinity binding of both these coagulation factors to the virus. V iral vectors based on adenoviruses (Ads) specific to human and animal species have been adapted widely for gene transfer applications both in vitro and in vivo. Extensive in vitro analyses have revealed a model of Ad cell infection whereby the initial virus attachment to a plasma membrane-localized receptor via the fiber protein is followed by virus penton interaction with integrins that mediate virion internalization into the cell (1, 2). To date, a number of cellular proteins that serve as functional high-affinity virus attachment receptors have been identified. For the most common vectors, based on species C human adenovirus serotype 5 (HAdv5), as well as for HAdv of species A, D, E, and F, it was found that a tight junction protein designated coxsackie and adenovirus receptor (CAR) can serve as a high-affinity attachment receptor (3-5). The species B Ads may utilize CD46 and/or DSG2 proteins to gain entry into host cells (6-8), while several species D Ads may utilize CD46, sialic acid, or GD1a glycan to enter the cell (9, 10). It was also shown that HAdv can bind a variety of integrin classes that interact with a penton RGD amino acid motif to trigger internalization of cell-bound virus particles into the cell (2).Although this canonical pathway of HAdv cell entry operates efficiently in vitro and explains the topology and functional interdependence between viral capsid proteins (11, 12), ...

Humoral immunity is an effective but metabolically expensive defense mechanism. It is unclear whether systemic cues exist to communicate the dynamic need for antigen presentation and immunoglobulin production. Here, we report a novel role for the liver-produced, acute phase reactant ST6GAL1 in IgG production. B cell expression of ST6GAL1, a sialyltransferase mediating the attachment of α2,6-linked sialic acids on N-glycans, is classically implicated in the dysregulated B cell development and immunoglobulin levels of St6gal1-deficient mice. However, the blood-borne pool of ST6GAL1, upregulated during systemic inflammation, can also extrinsically modify leukocyte cell surfaces. We show that B cell independent, extracellular ST6GAL1 enhances B cell IgG production and increases blood IgG titers. B cells of mice lacking the hepatocyte specific St6gal1 promoter have reduced sialylation of cell surface CD22 and CD45 and produce less IgG upon stimulation. Sialylation of B cells by extracellular ST6GAL1 boosts expression of IgM, IgD, and CD86, proliferation, and IgG production in vitro. In vivo, elevation of blood ST6GAL1 enhances B cell development and systemic IgG in a CD22-dependent manner. Our data point to a function of an extracellular glycosyltransferase in promoting humoral immunity. Manipulation of systemic ST6GAL1 may represent an effective therapeutic approach for humoral insufficiency.