Cord Blood Adductomics Reveals Oxidative Stress Exposure Pathways of Bronchopulmonary Dysplasia.

Fetal and neonatal exposures to perinatal oxidative stress (OS) are key mediators of bronchopulmonary dysplasia (BPD). To characterize these exposures, adductomics is an exposure science approach that captures electrophilic addition products (adducts) in blood protein. Adducts are bound to the nucleophilic cysteine loci of human serum albumin (HSA), which has a prolonged half-life. We conducted targeted and untargeted adductomics to test the hypothesis that adducts of OS vary with BPD. We studied 205 preterm infants (≤28 weeks) and 51 full-term infants from an ongoing birth cohort. Infant plasma was collected at birth (cord blood), 1-week, 1-month, and 36-weeks postmenstrual age. HSA was isolated from plasma, trypsin digested, and analyzed using high-performance liquid chromatography-mass spectrometry to quantify previously annotated (known) and unknown adducts. We identified 105 adducts in cord and postnatal blood. A total of 51 known adducts (small thiols, direct oxidation products, and reactive aldehydes) were increased with BPD. Postnatally, serial concentrations of several known OS adducts correlated directly with supplemental oxygen exposure. The application of large-scale adductomics elucidated OS-mediated pathways of BPD. This is the first study to investigate the "neonatal-perinatal exposome" and to identify oxidative stress-related exposure biomarkers that may inform antioxidant strategies to protect the health of future generations of infants.

Placental dysfunction influences fetal monocyte subpopulation gene expression in preterm birth.

The placenta is the primary organ for immune regulation, nutrient delivery, gas exchange, protection against environmental toxins, and physiologic perturbations during pregnancy. Placental inflammation and vascular dysfunction during pregnancy are associated with a growing list of prematurity-related complications. The goal of this study was to identify differences in gene expression profiles in fetal monocytes - cells that persist and differentiate postnatally - according to distinct placental histologic domains. Here, by using bulk RNA-Seq, we report that placental lesions are associated with gene expression changes in fetal monocyte subsets. Specifically, we found that fetal monocytes exposed to acute placental inflammation upregulate biological processes related to monocyte activation, monocyte chemotaxis, and platelet function, while monocytes exposed to maternal vascular malperfusion lesions downregulate these processes. Additionally, we show that intermediate monocytes might be a source of mitogens, such as HBEGF, NRG1, and VEGFA, implicated in different outcomes related to prematurity. This is the first study to our knowledge to show that placental lesions are associated with unique changes in fetal monocytes and monocyte subsets. As fetal monocytes persist and differentiate into various phagocytic cells following birth, our study may provide insight into morbidity related to prematurity and ultimately potential therapeutic targets.

Placental vascular maldevelopment, intrauterine growth restriction, and pulmonary hypertension.

A 33-year-old gravida 2, para 1 woman was noted to have early intrauterine growth restriction at 22 weeks gestation and subsequently developed severe pre-eclampsia. She delivered a 460 g male neonate at 28 weeks. The infant was managed on non-invasive ventilatory support and was gaining weight on enteral feeds for the first eight weeks of life, at which point he developed necrotizing enterocolitis. He then developed severe pulmonary hypertension that was refractory to maximal medical management. He died at 10 weeks of life due to hypoxemic respiratory and heart failure. Placental pathology revealed a constellation of findings consistent with maternal vascular malperfusion. Lung autopsy revealed muscularized and hypertrophied pulmonary arterioles consistent with severe pulmonary hypertension. Von Willebrand factor immunofluorescent staining of autopsy specimens suggest parallels in extent of endothelial injury. This case study illustrates our evolving knowledge of the fetal origins of neonatal lung diseases.

The impact of placental pathology discordance in multiple gestation pregnancies on bronchopulmonary dysplasia-associated pulmonary hypertension.

Bronchopulmonary dysplasia-associated pulmonary hypertension (BPD-PH) may either be concordant or discordant between multiple gestation births. Abnormal placental development, particularly maternal vascular malperfusion, may account for discordance in BPD-PH through fetal programming mechanisms. Maternal vascular malperfusion is a placental histologic lesion associated with intrauterine growth restriction and BPD-PH. We conducted a retrospective longitudinal cohort study of infants born <29 weeks gestation with available placental histology at Prentice Women's Hospital in Chicago from 2005-2012. The primary outcome was discordant BPD-PH associated with placental maternal vascular malperfusion. We secondarily assessed whether the risk of BPD-PH and placental lesions was different among infants of multiple (compared to singleton) gestations. The cohort consisted of 135 multiple gestation infants and 355 singletons. In a separate cohort of 39 singletons and 35 multiples, associations between 12 cytokines and angiogenic growth factors in cord blood plasma for biomarker discordance, maternal vascular malperfusion, and bronchopulmonary dysplasia were explored. Among multiples, discordant maternal vascular malperfusion was not associated with BPD-PH (OR = 1.9 (0.52, 6.9); p = 0.33) in infants exposed to placental maternal vascular malperfusion. However, singleton infants were more likely to develop BPD-PH (compared to multiples) after adjusting for mode of delivery, chorioamnionitis, chronic hypertension, placental abruption, small-for-gestational age birth weight, and gestational age (aOR = 2.7 (1.2, 5.8); p = 0.038). Singletons were more likely to be small-for-gestational age (11% vs 4%, p = 0.025) and have placental lesions compared to their multiple-gestation counterparts (96% vs 81%, p < 0.001), principally severe maternal vascular malperfusion (17% vs 4%, p < 0.001) and chronic inflammation (32% vs 11%, p < 0.001). Increased risk of BPD-PH in singleton pregnancies <29 weeks gestation compared to multiples may be related to increased frequency of these histologic lesions. Placental pathology in singleton and multiple gestation pregnancies may serve as an early biomarker to predict BPD-PH.

Variations in Umbilical Cord Hematopoietic and Mesenchymal Stem Cells With Bronchopulmonary Dysplasia.

Objective: To test the hypothesis that umbilical cord blood-derived CD34+ hematopoietic stem cells (HPSC), cord tissue-derived CD90+ and CD105+ mesenchymal stem cells (MSC) vary with bronchopulmonary dysplasia (BPD). Methods: We conducted a prospective longitudinal study at a large birth center (Prentice Women's Hospital in Chicago, IL). Premature infants (N = 200) were enrolled in 2:1:1 ratio based on gestational age (GA): mildly preterm (31-32 weeks), moderately preterm (29-30 weeks), and extremely preterm (23-28 weeks). Cord blood (CB) and cord tissues (CT) were collected at birth using commercial banking kits, and analyzed for collection blood volume, tissue mass, CD34+, CD90+, CD105+ counts, and concentrations. Multiplex immunoassay was used to measure 12 cytokines and growth factors in CB plasma of 74 patients. BPD severity was defined according to NIH consensus definitions. Univariate and multivariate regression models were used to identify perinatal covariates and assess associations between stem cell concentrations, cytokines, and BPD outcomes. Results: Of 200 patients enrolled (mean GA = 30 ± 2 weeks), 30 developed mild, 24 moderate, and 19 severe BPD. Concentrations of HPSC and MSC, as measured by %CD34+, %CD90+, and %CD105+ of total cells, increased with degree of prematurity. Collection parameters varied with GA, birth weight (BW), gender, prolonged rupture of membranes, mode of delivery, chorioamnionitis, and multiple gestation. Moderate-severe BPD or death was increased with lower GA, BW, Apgar scores, and documented delayed cord clamping. %CD34+ and %CD90+ were increased with BPD and directly correlated with BPD severity. Severe BPD was positively associated with %CD34+ (beta-coefficient = 0.9; 95% CI = 0.4-1.5; P < 0.01) and %CD90+ (beta-coefficient = 0.4; 95% CI = 0.2-0.6; P < 0.001) after adjustment for covariates. CB plasma granulocyte-colony stimulating factor (G-CSF) was inversely associated with %CD90+, and decreased with BPD. Below median G-CSF combined with elevated %CD90+ predicted BPD (positive predictive value = 100%). Conclusions: CB and CT collections yielded high concentrations of HPSCs and MSCs in BPD infants, accompanied by low circulating G-CSF. These variations suggest possible mechanisms by which stem cell differentiation and function predict BPD.

Cord blood alpha klotho is decreased in small for gestational age preterm infants with placental lesions of accelerated aging.

BACKGROUND: Intrauterine growth restriction is often accompanied by placental vascular disease, of which histologic maternal vascular malperfusion is prominent. Maternal vascular malperfusion is characterized by accelerated villous maturation consistent with placental aging. Alpha klotho is an anti-aging protein produced by the placenta. We hypothesize that cord blood alpha klotho varies with maternal vascular malperfusion and small for gestational age infants through dysregulated angiogenesis. METHODS: Nested case-control study of 54 preterm infants (N = 22 small for gestational age infants, 32 appropriate for gestational age infants, mean gestational age = 33.7 ±â€¯2.7 weeks) and validation sample (N = 39) from a longitudinal birth cohort at Prentice Women's Hospital, Chicago, IL. Cord blood alpha klotho was measured via enzyme-linked immunoassay; concentrations were linked to multiplex data of cord blood angiogenic growth factors. RESULTS: Median cord blood alpha klotho was decreased in small for gestational age infants (1200 [859, 2083] pg/mL) versus controls (3193 [1703, 3963] pg/mL; p < 0.01) and with severe maternal vascular malperfusion (1170 [760, 2645] pg/mL; P < 0.01), consistent with validation sample. Alpha klotho was decreased with maternal vascular malperfusion sublesions signifying accelerated villous maturation, including increased syncytial knots (1230 [805, 3606] pg/mL; p < 0.05) and distal villous hypoplasia (1170 [770, 3390] pg/mL; p < 0.05). Among 15 angiogenic markers, alpha klotho correlated directly with angiopoietin-2 (beta-coefficient = 2.6, p = 0.01). CONCLUSIONS: Cord blood alpha klotho is decreased with small for gestational infants and maternal vascular malperfusion sublesions of accelerated placental villous maturation, and correlated with angiopoietin-2. Alpha klotho may play a role in vascular-mediated accelerated placental aging leading to intrauterine growth restriction.

The Role of Macrophages in the Response to TNF Inhibition in Experimental Arthritis.

The reduction of synovial tissue macrophages is a reliable biomarker for clinical improvement in patients with rheumatoid arthritis (RA), and macrophages are reduced in synovial tissue shortly after initiation of TNF inhibitors. The mechanism for this initial response is unclear. These studies were performed to identify the mechanisms responsible for the initial reduction of macrophages following TNF inhibition, positing that efflux to draining lymph nodes was involved. RA synovial tissue and synovial fluid macrophages expressed CCR7, which was increased in control macrophages following incubation with TNF-α. Human TNF transgenic (hTNF-Tg) mice were treated with infliximab after development of arthritis. Ankles were harvested and examined by histology, immunohistochemistry, quantitative RT-PCR, ELISA, and flow cytometry. hTNF-Tg mice treated with infliximab demonstrated significant clinical and histologic improvement 3 d after the initiation of therapy, at which time Ly6C+ macrophages were significantly reduced in the ankles. However, no evidence was identified to support a role of macrophage efflux to draining lymph nodes following treatment with infliximab. In contrast, apoptosis of Ly6C+ macrophages in the ankles and popliteal lymph nodes, decreased migration of monocytes into the ankles, and a reduction of CCL2 were identified following the initiation of infliximab. These observations demonstrate that Ly6C+ macrophage apoptosis and decreased ingress of circulating monocytes into the joint are responsible for the initial reduction of macrophages following infliximab treatment in hTNF-Tg mice.

Association of Increased F4/80high Macrophages With Suppression of Serum-Transfer Arthritis in Mice With Reduced FLIP in Myeloid Cells.

OBJECTIVE: Macrophages are critical in the pathogenesis of rheumatoid arthritis (RA). We recently demonstrated that FLIP is necessary for the differentiation and/or survival of macrophages. We also showed that FLIP is highly expressed in RA synovial macrophages. This study was undertaken to determine if a reduction in FLIP in mouse macrophages reduces synovial tissue macrophages and ameliorates serum-transfer arthritis. METHODS: Mice with Flip deleted in myeloid cells (Flipf/f LysMc/+ mice) and littermate controls were used. Arthritis was induced by intraperitoneal injection of K/BxN serum. Disease severity was evaluated by clinical score and change in ankle thickness, and joints were examined by histology and immunohistochemistry. Cells were isolated from the ankles and bone marrow of the mice and examined by flow cytometry, real-time quantitative reverse transcriptase-polymerase chain reaction, or Western blotting. RESULTS: In contrast to expectations, Flipf/f LysMc/+ mice developed more severe arthritis early in the clinical course, but peak arthritis was attenuated and the resolution phase more complete than in control mice. Prior to the induction of serum-transfer arthritis, the number of tissue-resident macrophages was reduced. On day 9 after arthritis induction, the number of F4/80high macrophages in the joints of the Flipf/f LysMc/+ mice was not decreased, but increased. FLIP was reduced in the F4/80high macrophages in the ankles of the Flipf/f LysMc/+ mice, while F4/80high macrophages expressed an antiinflammatory phenotype in both the Flipf/f LysMc/+ and control mice. CONCLUSION: Our observations suggest that reducing FLIP in macrophages by increasing the number of antiinflammatory macrophages may be an effective therapeutic approach to suppress inflammation, depending on the disease stage.

SNAPIN is critical for lysosomal acidification and autophagosome maturation in macrophages.

We previously observed that SNAPIN, which is an adaptor protein in the SNARE core complex, was highly expressed in rheumatoid arthritis synovial tissue macrophages, but its role in macrophages and autoimmunity is unknown. To identify SNAPIN's role in these cells, we employed siRNA to silence the expression of SNAPIN in primary human macrophages. Silencing SNAPIN resulted in swollen lysosomes with impaired CTSD (cathepsin D) activation, although total CTSD was not reduced. Neither endosome cargo delivery nor lysosomal fusion with endosomes or autophagosomes was inhibited following the forced silencing of SNAPIN. The acidification of lysosomes and accumulation of autolysosomes in SNAPIN-silenced cells was inhibited, resulting in incomplete lysosomal hydrolysis and impaired macroautophagy/autophagy flux. Mechanistic studies employing ratiometric color fluorescence on living cells demonstrated that the reduction of SNAPIN resulted in a modest reduction of H+ pump activity; however, the more critical mechanism was a lysosomal proton leak. Overall, our results demonstrate that SNAPIN is critical in the maintenance of healthy lysosomes and autophagy through its role in lysosome acidification and autophagosome maturation in macrophages largely through preventing proton leak. These observations suggest an important role for SNAPIN and autophagy in the homeostasis of macrophages, particularly long-lived tissue resident macrophages.

CD11c-mediated deletion of Flip promotes autoreactivity and inflammatory arthritis.

Dendritic cells (DCs) are critical for immune homeostasis. To target DCs, we generated a mouse line with Flip deficiency in cells that express cre under the CD11c promoter (CD11c-Flip-KO). CD11c-Flip-KO mice spontaneously develop erosive, inflammatory arthritis, resembling rheumatoid arthritis, which is dramatically reduced when these mice are crossed with Rag(-/-) mice. The CD8α(+) DC subset is significantly reduced, along with alterations in NK cells and macrophages. Autoreactive CD4(+) T cells and autoantibodies specific for joint tissue are present, and arthritis severity correlates with the number of autoreactive CD4(+) T cells and plasmablasts in the joint-draining lymph nodes. Reduced T regulatory cells (Tregs) inversely correlate with arthritis severity, and the transfer of Tregs ameliorates arthritis. This KO line identifies a model that will permit in depth interrogation of the pathogenesis of rheumatoid arthritis, including the role of CD8α(+) DCs and other cells of the immune system.

Fas signaling in macrophages promotes chronicity in K/BxN serum-induced arthritis.

OBJECTIVE: A nonapoptotic role of Fas signaling has been implicated in the regulation of inflammation and innate immunity. This study was undertaken to elucidate the contribution of Fas signaling in macrophages to the development of arthritis. METHODS: K/BxN serum-transfer arthritis was induced in a mouse line in which Fas was conditionally deleted in the myeloid lineage (Cre(LysM) Fas(flox/flox) mice). The arthritis was assessed clinically and histologically. Expression of interleukin-1ß (IL-1ß), CXCL5, IL-10, IL-6, and gp96 was determined by enzyme-linked immunosorbent assay. Bone marrow-derived macrophages were activated with IL-1ß and gp96. Cell phenotype and apoptosis were analyzed by flow cytometry. RESULTS: Arthritis onset in Cre(LysM) Fas(flox/flox) mice was comparable with that observed in control mice; however, resolution was accelerated during the chronic phase. The attenuated arthritis was associated with reduced articular expression of the endogenous Toll-like receptor 2 (TLR-2) ligand gp96 and the neutrophil chemotactic chemokine CXCL5, and enhanced expression of IL-10. Activation with IL-1ß or gp96 induced increased IL-10 expression in Fas-deficient murine macrophages compared with control macrophages. IL-10 suppressed IL-6 and CXCL5 expression induced by IL-1ß plus gp96. IL-1ß-mediated activation of ERK, which regulates IL-10 expression, was increased in Fas-deficient mouse macrophages. CONCLUSION: Taken together, our findings indicate that impaired Fas signaling results in enhanced expression of antiinflammatory IL-10 and reduced expression of gp96, and these effects are associated with accelerated resolution of inflammation during the chronic phase of arthritis. These observations suggest that strategies to reduce endogenous TLR ligands and increase IL-10 may be beneficial in the treatment of rheumatoid arthritis.

TLR2 deletion promotes arthritis through reduction of IL-10.

RA is a chronic inflammatory disease characterized by the persistent expression of inflammatory cytokines from macrophages, which may be mediated, in part, through TLR2 signaling. Earlier studies demonstrate a role for TLR2 signaling in dampening the arthritis in IL-1Ra-/- mice, which was mediated through T cells. This study was performed to determine whether TLR2 signaling plays a role in the pathogenesis of T cell-independent arthritis triggered by transferring serum from K/BxN mice. We documented more severe arthritis in Tlr2-/- mice compared with WT controls. The Tlr2-/- mice also demonstrated increased inflammation, erosion, pannus formation, and osteoclastogenesis, as well as increased IL-1ß and decreased IL-10 within the joints. In vitro bone marrow-differentiated macrophages expressed comparable levels of activating and inhibitory FcγRs, however when stimulated with immune complexes, the Tlr2-/- macrophages expressed decreased IL-10 and reduced activation of Akt and ERK. Our findings indicate that Tlr2-/- promotes the effector phase of arthritis through decreased IL-10 by macrophages, which is important, not only as an anti-inflammatory cytokine but also in restraining the differentiation and activation of osteoclasts.

Glycoprotein 96 perpetuates the persistent inflammation of rheumatoid arthritis.

OBJECTIVE: The mechanisms that contribute to the persistent activation of macrophages in rheumatoid arthritis (RA) are incompletely understood. The aim of this study was to determine the contribution of endogenous gp96 in Toll-like receptor (TLR)-mediated macrophage activation in RA. METHODS: RA synovial fluid was used to activate macrophages and HEK-TLR-2 and HEK-TLR-4 cells. Neutralizing antibodies to TLR-2, TLR-4, and gp96 were used to inhibit activation. RA synovial fluid macrophages were isolated by CD14 negative selection. Cell activation was measured by the expression of tumor necrosis factor α (TNFα) or interleukin-8 messenger RNA. Arthritis was induced in mice by K/BxN serum transfer. The expression of gp96 was determined by immunoblot analysis, enzyme-linked immunosorbent assay, and immunohistochemistry. Arthritis was treated with neutralizing anti-gp96 antiserum or control serum. RESULTS: RA synovial fluid induced the activation of macrophages and HEK-TLR-2 and HEK-TLR-4 cells. RA synovial fluid-induced macrophage and HEK-TLR-2 activation was suppressed by neutralizing anti-gp96 antibodies only in the presence of high (>800 ng/ml) rather than low (<400 ng/ml) concentrations of gp96. Neutralization of RA synovial fluid macrophage cell surface gp96 inhibited the constitutive expression of TNFα. Supporting the role of gp96 in RA, joint tissue gp96 expression was induced in mice with the K/BxN serum-induced arthritis, and neutralizing antibodies to gp96 ameliorated joint inflammation, as determined by clinical and histologic examination. CONCLUSION: These observations support the notion that gp96 plays a role as an endogenous TLR-2 ligand in RA and identify the TLR-2 pathway as a therapeutic target.

FLIP: a novel regulator of macrophage differentiation and granulocyte homeostasis.

FLIP is a well-established suppressor of death receptor-mediated apoptosis. To define its essential in vivo role in myeloid cells, we generated and characterized mice with Flip conditionally deleted in the myeloid lineage. Myeloid specific Flip-deficient mice exhibited growth retardation, premature death, and splenomegaly with altered architecture and extramedullary hematopoiesis. They also displayed a dramatic increase of circulating neutrophils and multiorgan neutrophil infiltration. In contrast, although circulating inflammatory monocytes were also significantly increased, macrophages in the spleen, lymph nodes, and the peritoneal cavity were reduced. In ex vivo cultures, bone marrow progenitor cells failed to differentiate into macrophages when Flip was deleted. Mixed bone marrow chimera experiments using cells from Flip-deficient and wild-type mice did not demonstrate an inflammatory phenotype. These observations demonstrate that FLIP is necessary for macrophage differentiation and the homeostatic regulation of granulopoiesis.