Bacterial colonization and TH17 immunity are shaped by intestinal sialylation in neonatal mice.

Interactions between the neonate host and its gut microbiome are central to the development of a healthy immune system. However, the mechanisms by which animals alter early colonization of microbiota for their benefit remain unclear. Here, we investigated the role of early-life expression of the α2,6-sialyltransferase ST6GAL1 in microbiome phylogeny and mucosal immunity. Fecal, upper respiratory, and oral microbiomes of pups expressing or lacking St6gal1 were analyzed by 16S rRNA sequencing. At weaning, the fecal microbiome of St6gal1-KO mice had reduced Clostridiodes, Coprobacillus, and Adlercreutzia, but increased Helicobacter and Bilophila. Pooled fecal microbiomes from syngeneic donors were transferred to antibiotic-treated wild-type mice, before analysis of recipient mucosal immune responses by flow cytometry, RT-qPCR, microscopy, and ELISA. Transfer of St6gal1-KO microbiome induced a mucosal Th17 response, with expression of T-bet and IL-17, and IL-22-dependent gut lengthening. Early life intestinal sialylation was characterized by RT-qPCR, immunoblot, microscopy, and sialyltransferase enzyme assays in genetic mouse models at rest or with glucocorticoid receptor modulators. St6gal1 expression was greatest in the duodenum, where it was mediated by the P1 promoter and efficiently inhibited by dexamethasone. Our data show that the inability to produce α2,6-sialyl ligands contributes to microbiome-dependent Th17 inflammation, highlighting a pathway by which the intestinal glycosylation regulates mucosal immunity.

The sialyltransferase ST6GAL1 protects against radiation-induced gastrointestinal damage.

High-dose irradiation poses extreme risk of mortality from acute damage to the hematopoietic compartment and gastrointestinal tract. While bone marrow transplantation can reestablish the hematopoietic compartment, a more imminent risk of death is posed by gastrointestinal acute radiation syndrome (GI-ARS), for which there are no FDA-approved medical countermeasures. Although the mechanisms dictating the severity of GI-ARS remain incompletely understood, sialylation by ST6GAL1 has been shown to protect against radiation-induced apoptosis in vitro. Here, we used a C57BL/6 St6gal1-KO mouse model to investigate the contribution of ST6GAL1 to susceptibility to total body irradiation in vivo. Twelve gray total body ionizing γ-irradiation (TBI) followed by bone marrow transplant is not lethal to wild-type mice, but St6gal1-KO counterparts succumbed within 7 d. Both St6gal1-KO and wild-type animals exhibited damage to the GI epithelium, diarrhea and weight loss, but these symptoms became progressively more severe in the St6gal1-KO animals while wild-type counterparts showed signs of recovery by 120 h after TBI. Increased apoptosis in the GI tracts of St6gal1-KO mice and the absence of regenerative crypts were also observed. Together, these observations highlight an important role for ST6GAL1 in protection and recovery from GI-ARS in vivo.

IL-1α and complement cooperate in triggering local neutrophilic inflammation in response to adenovirus and eliminating virus-containing cells.

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.

Coagulation factor binding orientation and dimerization may influence infectivity of adenovirus-coagulation factor complexes.

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.

Blood-Borne ST6GAL1 Regulates Immunoglobulin Production in B Cells.

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.

Macrophages Expressing GALC Improve Peripheral Krabbe Disease by a Mechanism Independent of Cross-Correction.

Many therapies for lysosomal storage disorders rely on cross-correction of lysosomal enzymes. In globoid cell leukodystrophy (GLD), mutations in GALC cause psychosine accumulation, inducing demyelination, a neuroinflammatory "globoid" reaction and neurodegeneration. The efficiency of GALC cross-correction in vivo, the role of the GALC substrate galactosylceramide, and the origin of psychosine are poorly understood. Using a novel GLD model, we show that cross-correction does not occur efficiently in vivo and that Galc-deficient Schwann cells autonomously produce psychosine. Furthermore, macrophages require GALC to degrade myelin, as Galc-deficient macrophages are transformed into globoid cells by exposure to galactosylceramide and produce a more severe GLD phenotype. Finally, hematopoietic stem cell transplantation in patients reduces globoid cells in nerves, suggesting that the phagocytic response of healthy macrophages, rather than cross-correction, contributes to the therapeutic effect. Thus, GLD may be caused by at least two mechanisms: psychosine-induced demyelination and secondary neuroinflammation from galactosylceramide storage in macrophages.

B cells suppress medullary granulopoiesis by an extracellular glycosylation-dependent mechanism.

The immune response relies on the integration of cell-intrinsic processes with cell-extrinsic cues. During infection, B cells vacate the marrow during emergency granulopoiesis but return upon restoration of homeostasis. Here we report a novel glycosylation-mediated crosstalk between marrow B cells and hematopoietic progenitors. Human B cells secrete active ST6GAL1 sialyltransferase that remodels progenitor cell surface glycans to suppress granulopoiesis. In mouse models, ST6GAL1 from B cells alters the sialylation profile of bone marrow populations, and mature IgD+ B cells were enriched in sialylated bone marrow niches. In clinical multiple myeloma, ST6GAL1 abundance in the multiple myeloma cells negatively correlated with neutrophil abundance. These observations highlight not only the ability of medullary B cells to influence blood cell production, but also the disruption to normal granulopoiesis by excessive ST6GAL1 in malignancy.

Systemic ST6Gal-1 Is a Pro-survival Factor for Murine Transitional B Cells.

Humoral immunity depends on intrinsic B cell developmental programs guided by systemic signals that convey physiologic needs. Aberrant cues or their improper interpretation can lead to immune insufficiency or a failure of tolerance and autoimmunity. The means by which such systemic signals are conveyed remain poorly understood. Hence, further insight is essential to understanding and treating autoimmune diseases and to the development of improved vaccines. ST6Gal-1 is a sialyltransferase that constructs the α2,6-sialyl linkage on cell surface and extracellular glycans. The requirement for functional ST6Gal-1 in the development of humoral immunity is well documented. Canonically, ST6Gal-1 resides within the intracellular ER-Golgi secretory apparatus and participates in cell-autonomous glycosylation. However, a significant pool of extracellular ST6Gal-1 exists in circulation. Here, we segregate the contributions of B cell intrinsic and extrinsic ST6Gal-1 to B cell development. We observed that B cell-intrinsic ST6Gal-1 is required for marginal zone B cell development, while B cell non-autonomous ST6Gal-1 modulates B cell development and survival at the early transitional stages of the marrow and spleen. Exposure to extracellular ST6Gal-1 ex vivo enhanced the formation of IgM-high B cells from immature precursors, and increased CD23 and IgM expression. Extrinsic sialylation by extracellular ST6Gal-1 augmented BAFF-mediated activation of the non-canonical NF-kB, p38 MAPK, and PI3K/AKT pathways, and accelerated tyrosine phosphorylation after B cell receptor stimulation. in vivo, systemic ST6Gal-1 did not influence homing of B cells to the spleen but was critical for their long-term survival and systemic IgG levels. Circulatory ST6Gal-1 levels respond to inflammation, infection, and malignancy in mammals, including humans. In turn, we have shown previously that systemic ST6Gal-1 regulates inflammatory cell production by modifying bone marrow myeloid progenitors. Our data here point to an additional role of systemic ST6Gal-1 in guiding B cell development, which supports the concept that circulating ST6Gal-1 is a conveyor of systemic cues to guide the development of multiple branches of immune cells.

The blood-borne sialyltransferase ST6Gal-1 is a negative systemic regulator of granulopoiesis.

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.