The optimal gestation for pertussis vaccination during pregnancy: a prospective cohort study.

BACKGROUND: There is an increasing incidence of pertussis infection in infants too young to be adequately protected via vaccination. Maternal pertussis vaccination during the third trimester of pregnancy is a new strategy to provide protection to newborn infants. OBJECTIVE: This study sought to determine the optimal gestational window for vaccination in the third trimester. STUDY DESIGN: This prospective study recruited 3 groups of women: an early vaccination group, vaccinated between 28-32 weeks' gestation; a late vaccination group, vaccinated between 33-36 weeks' gestation; and an unvaccinated control group. Maternal venous blood was taken prior to pertussis vaccination. At birth, infant cord blood was collected to determine antibody levels to pertussis toxin (PT), pertactin (PRN), and filamentous hemagglutinin (FHA). RESULTS: In all, 154 women were recruited from April through September 2014. There was no significant difference between maternal PRN and FHA antibody levels among the 3 groups, however, PT was higher in the early compared to late vaccination group (P = .05). Cord blood antibody levels to PT, PRN, and FHA were significantly higher in those born to vaccinated women compared with unvaccinated controls (P < .001, P = .001, and P < .001, respectively). Vaccination between 28-32 weeks' gestation resulted in significantly higher cord blood PT (4.18.0 vs 3.50 IU/mL, P = .009), PRN (5.83 vs 5.31 IU/mL, P = .03), and FHA (5.56 vs 5.03 IU/mL, P = .03) antibody levels than vaccination between 33-36 weeks' gestation. When adjusted for maternal prevaccination antibody levels, PT levels in early vs late vaccination approached significance (P = .06). PRN levels were significantly higher in the early vaccination group (P = .003). There was no significant difference for FHA antibody levels between the 2 groups (P = .16). CONCLUSION: Maternal vaccination during the third trimester is effective in affording higher levels of pertussis antibody protection to the newborn infant. Vaccination early in the third trimester appears more effective than later in pregnancy.

Neutrophil myeloperoxidase regulates T-cell-driven tissue inflammation in mice by inhibiting dendritic cell function.

Myeloperoxidase (MPO) is important in intracellular microbial killing by neutrophils but extracellularly causes tissue damage. Its role in adaptive immunity and T-cell-mediated diseases is poorly understood. Here, T-cell responses in lymph nodes (LNs) were enhanced by MPO deletion or in vivo inhibition, causing enhanced skin delayed-type hypersensitivity and antigen (Ag)-induced arthritis. Responses of adoptively transferred OT-II T cells were greater in MPO-deficient than wild-type (WT) recipients. MPO, deposited by neutrophils in LNs after Ag injection, interacted with dendritic cells (DCs) in vivo. Culture of murine or human DCs with purified MPO or neutrophil supernatant showed that enzymatically dependent MPO-mediated inhibition of DC activation occurs via MPO-generated reactive intermediates and involves DC Mac-1. Transfer of DCs cultured with WT, but not MPO-deficient, neutrophil supernatant attenuated Ag-specific immunity in vivo. MPO deficiency or in vivo inhibition increased DC activation in LNs after immunization. Studies with DQ-ovalbumin showed that MPO inhibits Ag uptake/processing by DCs. In vivo DC transfer and in vitro studies showed that MPO inhibits DC migration to LNs by reducing their expression of CCR7. Therefore, MPO, via its catalytic activity, inhibits the generation of adaptive immunity by suppressing DC activation, Ag uptake/processing, and migration to LNs to limit pathological tissue inflammation.

Activin A contributes to the development of hyperoxia-induced lung injury in neonatal mice.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is one of the leading causes of morbidity and mortality in babies born prematurely, yet there is no curative treatment. In recent years, a number of inhibitors against TGFß signaling have been tested for their potential to prevent neonatal injury associated with hyperoxia, which is a contributing factor of BPD. In this study, we assessed the contribution of activin A-a member of the TGFß superfamily-to the development of hyperoxia-induced lung injury in neonatal mice. METHODS: We placed newborn C57Bl6 mouse pups in continuous hyperoxia (85% O2) to mimic many aspects of BPD including alveolar simplification and pulmonary inflammation. The pups were administered activin A receptor type IIB-Fc antagonist (ActRIIB-Fc) at 5 mg/kg or follistatin at 0.1 mg/kg on postnatal days 4, 7, 10, and 13. RESULTS: Treatment with ActRIIB-Fc and follistatin protected against hyperoxia-induced growth retardation. ActRIIB-Fc also reduced pulmonary leukocyte infiltration, normalized tissue: airspace ratio and increased septal crest density. These findings were associated with reduced phosphorylation of Smad3 and decreased matrix metalloproteinase (MMP)-9 activity. CONCLUSION: This study suggests that activin A signaling may contribute to the pathology of bronchopulmonary dysplasia.

Trefoil factor-2 reverses airway remodeling changes in allergic airways disease.

Trefoil factor 2 (TFF2) is a small peptide with an important role in mucosal repair. TFF2 is up-regulated in asthma, suggesting a role in asthma pathogenesis. Given its known biological role in promoting epithelial repair, TFF2 might be expected to exert a protective function in limiting the progression of airway remodeling in asthma. The contribution of TFF2 to airway remodeling in asthma was investigated by examining the expression of TFF2 in the airway and lung, and evaluating the effects of recombinant TFF2 treatment on established airway remodeling in a murine model of chronic allergic airways disease (AAD). BALB/c mice were sensitized and challenged with ovalbumin (OVA) or saline for 9 weeks, whereas mice with established OVA-induced AAD were treated with TFF2 or vehicle control (intranasally for 14 d). Effects on airway remodeling, airway inflammation, and airway hyperresponsiveness were then assessed, whereas TFF2 expression was determined by immunohistochemistry. TFF2 expression was significantly increased in the airways of mice with AAD, compared with expression levels in control mice. TFF2 treatment resulted in reduced epithelial thickening, subepithelial collagen deposition, goblet-cell metaplasia, bronchial epithelium apoptosis, and airway hyperresponsiveness (all P < 0.05, versus vehicle control), but TFF2 treatment did not influence airway inflammation. The increased expression of endogenous TFF2 in response to chronic allergic inflammation is insufficient to prevent the progression of airway inflammation and remodeling in a murine model of chronic AAD. However, exogenous TFF2 treatment is effective in reversing aspects of established airway remodeling. TFF2 has potential as a novel treatment for airway remodeling in asthma.

Mast cells contribute to peripheral tolerance and attenuate autoimmune vasculitis.

Mast cells contribute to the modulation of the immune response, but their role in autoimmune renal disease is not well understood. Here, we induced autoimmunity resulting in focal necrotizing GN by immunizing wild-type or mast cell-deficient (Kit(W-sh/W-sh)) mice with myeloperoxidase. Mast cell-deficient mice exhibited more antimyeloperoxidase CD4+ T cells, enhanced dermal delayed-type hypersensitivity responses to myeloperoxidase, and more severe focal necrotizing GN. Furthermore, the lymph nodes draining the sites of immunization had fewer Tregs and reduced production of IL-10 in mice lacking mast cells. Reconstituting these mice with mast cells significantly increased the numbers of Tregs in the lymph nodes and attenuated both autoimmunity and severity of disease. After immunization with myeloperoxidase, mast cells migrated from the skin to the lymph nodes to contact Tregs. In an ex vivo assay, mast cells enhanced Treg suppression through IL-10. Reconstitution of mast cell-deficient mice with IL-10-deficient mast cells led to enhanced autoimmunity to myeloperoxidase and greater disease severity compared with reconstitution with IL-10-intact mast cells. Taken together, these studies establish a role for mast cells in mediating peripheral tolerance to myeloperoxidase, protecting them from the development of focal necrotizing GN in ANCA-associated vasculitis.

Interleukin-17A promotes early but attenuates established disease in crescentic glomerulonephritis in mice.

T helper (Th)17 cells might contribute to immune-mediated renal injury. Thus, we sought to define the time course of IL-17A-induced kidney damage and examined the relation between Th17 and Th1 cells in a model of crescentic anti-glomerular basement membrane glomerulonephritis. Renal injury and immune responses were assessed in wild-type and in IL-17A-deficient mice on days 6, 14, and 21 of disease development. On day 6, when mild glomerulonephritis developed, IL-17A-deficient mice were protected from renal injury. On day 14, when more severe disease developed, protection from renal injury due to IL-17A deficiency was less evident. On day 21, when crescentic glomerulonephritis was fully established, disease was enhanced in IL-17A(-/-) mice, with increased glomerular T-cell accumulation and fibrin deposition, and augmented Th1 responses. Mice lacking the Th17-promoting cytokine, IL-23 (p19), also developed more severe disease than wild-type animals on day 21. In contrast, mice deficient in the key Th1-promoting cytokine, IL-12 (p35), had decreased Th1 and increased Th17 responses and developed less severe crescentic glomerulonephritis than wild-type animals. These studies show that IL-17A contributes to early glomerular injury, but it attenuates established crescentic glomerulonephritis by suppressing Th1 responses. They provide further evidence that Th1 cells mediate crescentic injury in this model and that Th1 and Th17 cells counterregulate each other during disease development.

Trefoil factor 2 regulates airway remodeling in animal models of asthma.

BACKGROUND: Epithelial denudation and metaplasia are important in the pathogenesis of airway remodeling and asthma. Trefoil factor 2 (TFF2) is a member of a family of peptides involved in protection and healing of the gastrointestinal epithelium but which are also secreted in the airway mucosa. METHODS: We investigated the role of TFF2 in airway remodeling by histological and morphometric analysis of lung tissue from TFF2-deficient mice subjected to two relevant animal models of asthma: an ovalbumin model of allergic airways disease and an Aspergillus fumigatus antigen sensitization model. RESULTS: In the ovalbumin model TFF2-deficient mice had increased goblet cell hyperplasia, but not epithelial thickening compared to wild-type (WT) counterparts. In the Aspergillus model TFF2-deficient mice also had increased goblet cell hyperplasia, and epithelial thickness was also increased in the Aspergillus-sensitized mice compared to WT controls. TFF2 deficiency was also associated with increased subepithelial collagen layer thickness. DISCUSSION: The current study demonstrates a role of TFF2 in airway remodeling in mouse models of airway disease. Further studies into the mechanisms of action of TFF2 and its role in asthma are warranted.

Human amnion epithelial cells rescue cell death via immunomodulation of microglia in a mouse model of perinatal brain injury.

BACKGROUND: Human amnion epithelial cells (hAECs) are clonogenic and have been proposed to reduce inflammatory-induced tissue injury. Perturbation of the immune response is implicated in the pathogenesis of perinatal brain injury; modulating this response could thus be a novel therapy for treating or preventing such injury. The immunomodulatory properties of hAECs have been shown in other animal models, but a detailed investigation of the effects on brain immune cells following injury has not been undertaken. Here, we investigate the effects of hAECs on microglia, the first immune responders to injury within the brain. METHODS: We generated a mouse model combining neonatal inflammation and perinatal hyperoxia, both of which are risk factors associated with perinatal brain injury. On embryonic day 16 we administered lipopolysaccharide (LPS), or saline (control), intra-amniotically to C57Bl/6 J mouse pups. On postnatal day (P)0, LPS pups were placed in hyperoxia (65% oxygen) and control pups in normoxia for 14 days. Pups were given either hAECs or saline intravenously on P4. RESULTS: At P14, relative to controls, LPS and hyperoxia pups had reduced body weight, increased density of apoptotic cells (TUNEL) in the cortex, striatum and white matter, astrocytes (GFAP) in the white matter and activated microglia (CD68) in the cortex and striatum, but no change in total microglia density (Iba1). hAEC administration rescued the decreased body weight and reduced apoptosis and astrocyte areal coverage in the white matter, but increased the density of total and activated microglia. We then stimulated primary microglia (CD45lowCD11b+) with LPS for 24 h, followed by co-culture with hAEC conditioned medium for 48 h. hAEC conditioned medium increased microglial phagocytic activity, decreased microglia apoptosis and decreased M1 activation markers (CD86). Stimulating hAECs for 24 h with LPS did not alter release of cytokines known to modulate microglia activity. CONCLUSIONS: These data demonstrate that hAECs can directly immunomodulate brain microglia, probably via release of trophic factors. This observation offers promise that hAECs may afford therapeutic utility in the management of perinatal brain injury.

Amnion Epithelial Cells Promote Lung Repair via Lipoxin A4.

Human amnion epithelial cells (hAECs) have been shown to possess potent immunomodulatory properties across a number of disease models. Recently, we reported that hAECs influence macrophage polarization and activity, and that this step was dependent on regulatory T cells. In this study, we aimed to assess the effects of hAEC-derived proresolution lipoxin-A4 (LXA4) on T-cell, macrophage, and neutrophil phenotype and function during the acute phase of bleomycin-induced lung injury. Using C57Bl6 mice, we administered 4 million hAECs intraperitoneally 24 hours after bleomycin challenge. Outcomes were measured at days 3, 5, and 7. hAEC administration resulted in significant changes to T-cell, macrophage, dendritic cell, and monocyte/macrophage infiltration and phenotypes. Endogenous levels of lipoxygenases, LXA4, and the lipoxin receptor FPR2 were elevated in hAEC-treated animals. Furthermore, we showed that the effects of hAECs on macrophage phagocytic activity and T-cell suppression are LXA4 dependent, whereas the inhibition of neutrophil-derived myleoperoxidase by hAECs is independent of LXA4. This study provides the first evidence that lipid-based mediators contribute to the immunomodulatory effects of hAECs and further supports the growing body of evidence that LXA4 is proresolutionary in lung injury. This discovery of LXA4-dependent communication between hAECs, macrophages, T cells, and neutrophils is important to the understanding of hAEC biodynamics and would be expected to inform future clinical applications. Stem Cells Translational Medicine 2017;6:1085-1095.

Human amnion cells reverse acute and chronic pulmonary damage in experimental neonatal lung injury.

BACKGROUND: Despite advances in neonatal care, bronchopulmonary dysplasia (BPD) remains a significant contributor to infant mortality and morbidity. While human amnion epithelial cells (hAECs) have shown promise in small and large animal models of BPD, there is scarce information on long-term benefit and clinically relevant questions surrounding administration strategy remain unanswered. In assessing the therapeutic potential of hAECs, we investigated the impact of cell dosage, administration routes and timing of treatment in a pre-clinical model of BPD. METHODS: Lipopolysaccharide was introduced intra-amniotically at day 16 of pregnancy prior to exposure to 65% oxygen (hyperoxia) at birth. hAECs were administered either 12 hours (early) or 4 days (late) after hyperoxia commenced. Collective lung tissues were subjected to histological analysis, multikine ELISA for inflammatory cytokines, FACS for immune cell populations and 3D lung stem cell culture at neonatal stage (postnatal day 7 and 14). Invasive lung function test and echocardiography were applied at 6 and 10 weeks of age. RESULTS: hAECs improved the tissue-to-airspace ratio and septal crest density in a dose-dependent manner, regardless of administration route. Early administration of hAECs, coinciding with the commencement of postnatal hyperoxia, was associated with reduced macrophages, dendritic cells and natural killer cells. This was not the case if hAECs were administered when lung injury was established. Fittingly, early hAEC treatment was more efficacious in reducing interleukin-1ß, tumour necrosis factor alpha and monocyte chemoattractant protein-1 levels. Early hAEC treatment was also associated with reduced airway hyper-responsiveness and normalisation of pressure-volume loops. Pulmonary hypertension and right ventricle hypertrophy were also prevented in the early hAEC treatment group, and this persisted until 10 weeks of age. CONCLUSIONS: Early hAEC treatment appears to be advantageous over late treatment. There was no difference in efficacy between intravenous and intratracheal administration. The benefits of hAEC administration resulted in long-term improvements in cardiorespiratory function.

Resveratrol mitigates trophoblast and endothelial dysfunction partly via activation of nuclear factor erythroid 2-related factor-2.

INTRODUCTION: Maternal endothelial dysfunction underlying preeclampsia arises from excessive placental release of anti-angiogenic factors, such as soluble fms-like tyrosine kinase-1 (sFlt1), soluble endoglin (sEng) and activin A. Resveratrol, an activator of the nuclear factor erythroid 2-related factor-2 (Nrf2) transcription factor, mediates the gene expression of antioxidant and vasoprotective factors that may counter the endothelial damage imposed by these anti-angiogenic factors. The objective of this study was to assess whether resveratrol could reduce placental oxidative stress and production of anti-angiogenic factors in vitro and/or improve in vitro markers of endothelial dysfunction via Nrf2 activation. METHOD: We used in vitro term placental explants to assess the effects of resveratrol on placental oxidative stress and production of sFlt1, sEng and activin A. Using human umbilical vein endothelial cells we investigated the effects of resveratrol on markers of in vitro endothelial dysfunction, including the expression of intercellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1), E-selectin and endothelin-1, and endothelial permeability. To confirm that resveratrol mediated its effects via Nrf2, we examined the impact of resveratrol on the same in vitro markers of endothelial and placental dysfunction following Nrf2 knockdown. RESULTS: Resveratrol significantly decreased placental oxidative stress and the production of sFlt1 and activin A. Resveratrol significantly mitigated tumor necrosis factor-α stimulated endothelial expression of ICAM1, VCAM1, E-selectin and endothelin-1 and prevented an increase in endothelial monolayer permeability. Nrf2 knockdown abolished some of the protective effects of resveratrol on endothelial cells, but not in primary trophoblast cells. CONCLUSION: Features of placental and endothelial dysfunction characteristic of preeclampsia are improved by resveratrol in vitro, partially via the modulation of Nrf2.

Evaluating the Impact of Human Amnion Epithelial Cells on Angiogenesis.

The effects of human amnion epithelial cells (hAECs) on angiogenesis remain controversial. It is yet unknown if the presence of inflammation and/or gestational age of hAEC donors have an impact on angiogenesis. In this study, we examined the differences between term and preterm hAECs on angiogenesis in vitro and in vivo. Conditioned media from term hAECs induced the formation of longer huVEC tubules on Matrigel. Both term and preterm hAECs expressed VEGFA, PDGFB, ANGPT1, and FOXC1, which significantly increased after TNFα and IFNγ stimulation. In the presence of TNFα and IFNγ, coculture with term hAECs reduced gene transcription of Tie-2 and Foxc1 in huVECs, while coculture with preterm hAECs increased gene transcription of PDGFRα and PDGFRß and reduced gene transcription of FOXC1 in huVECs. In vivo assessment of angiogenesis using vWF immunostaining revealed that hAEC treatment decreased angiogenesis in a bleomycin model of lung fibrosis but increased angiogenesis in a neonatal model of hyperoxia-induced lung injury. In summary, our findings suggested that the impact of hAECs on angiogenesis may be influenced by the presence of inflammation and underlying pathology.

Suppression of Autoimmunity and Renal Disease in Pristane-Induced Lupus by Myeloperoxidase.

OBJECTIVE: Myeloperoxidase (MPO) locally contributes to organ damage in various chronic inflammatory conditions by generating reactive intermediates. The contribution of MPO in the development of experimental lupus is unknown. The aim of this study was to define the role of MPO in murine lupus nephritis (LN). METHODS: LN was induced in C57BL/6 wild-type (WT) and MPO knockout (MPO(-/-) ) mice by intraperitoneal injection of pristane. Autoimmunity and glomerulonephritis were assessed 20 and 40 weeks after pristane administration. Cell apoptosis, leukocyte accumulation, and cytokine levels in the peritoneal cavity of WT and MPO(-/-) mice were assessed 3 or 6 days after pristane injection. RESULTS: MPO(-/-) mice developed more severe nephritis than did WT mice 20 and 40 weeks after pristane injection, despite having reduced glomerular deposition of antibody and complement and diminished levels of markers of oxidative stress (oxidized DNA and glutathione sulfonamide). Enhancement of renal disease in MPO-deficient mice correlated with increased accumulation of CD4+ T cells and macrophages in glomeruli, which, in turn, was associated with augmented generation of CD4+ T cell responses and increased activation and migration of dendritic cells in secondary lymphoid organs. In addition, the enhanced renal injury in MPO(-/-) mice was associated with increased glomerular accumulation of neutrophils and deposition of neutrophil extracellular traps. MPO deficiency also increased early cell apoptosis, leukocyte accumulation, and proinflammatory cytokine expression in the peritoneum. CONCLUSION: MPO attenuates pristane-induced LN by inhibiting early inflammatory responses in the peritoneum and limiting the generation of CD4+ T cell autoimmunity in secondary lymphoid organs.

Endogenous myeloperoxidase is a mediator of joint inflammation and damage in experimental arthritis.

OBJECTIVE: Myeloperoxidase (MPO) is implicated as a local mediator of tissue damage when released extracellularly in many chronic inflammatory diseases. The purpose of this study was to explore the role of endogenous MPO in experimental rheumatoid arthritis (RA). METHODS: K/BxN serum-transfer arthritis was induced in C57BL/6 wild-type (WT) and MPO knockout (MPO(-/-) ) mice, and disease development was assessed. MPO activity was measured in joint tissues from mice with or without K/BxN arthritis. Collagen-induced arthritis (CIA) was induced in WT and MPO(-/-) mice, and disease development and immune responses were examined. MPO expression was assessed in synovial biopsy samples from patients with active RA, and the effect of MPO on synovial fibroblasts was tested in vitro. RESULTS: MPO was up-regulated in the joints of mice with K/BxN arthritis, and MPO deficiency attenuated the severity of the disease without affecting circulating cytokine levels. In CIA, MPO(-/-) mice had enhanced CD4+ T cell responses and reduced frequency of regulatory T cells in the lymph nodes and spleen, as well as augmented interleukin-17A and diminished interferon-γ secretion by collagen-stimulated splenocytes, without an effect on circulating anticollagen antibody levels. Despite enhanced adaptive immunity in secondary lymphoid organs, CIA development was attenuated in MPO(-/-) mice. Intracellular and extracellular MPO was detected in the synovium of patients with active RA, and human MPO enhanced the proliferation and decreased the apoptosis of synovial fibroblasts in vitro. CONCLUSION: MPO contributes to the development of arthritis despite suppressing adaptive immunity in secondary lymphoid organs. This suggests distinct effects of local MPO on arthritogenic effector responses.