Extracellular vesicles alter trophoblast function in pregnancies complicated by COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and resulting coronavirus disease (COVID-19) causes placental dysfunction, which increases the risk of adverse pregnancy outcomes. While abnormal placental pathology resulting from COVID-19 is common, direct infection of the placenta is rare. This suggests that pathophysiology associated with maternal COVID-19, rather than direct placental infection, is responsible for placental dysfunction and alteration of the placental transcriptome. We hypothesized that maternal circulating extracellular vesicles (EVs), altered by COVID-19 during pregnancy, contribute to placental dysfunction. To examine this hypothesis, we characterized maternal circulating EVs from pregnancies complicated by COVID-19 and tested their effects on trophoblast cell physiology in vitro . We found that the gestational timing of COVID-19 is a major determinant of circulating EV function and cargo. In vitro trophoblast exposure to EVs isolated from patients with an active infection at the time of delivery, but not EVs isolated from Controls, altered key trophoblast functions including hormone production and invasion. Thus, circulating EVs from participants with an active infection, both symptomatic and asymptomatic cases, can disrupt vital trophoblast functions. EV cargo differed between participants with COVID-19 and Controls, which may contribute to the disruption of the placental transcriptome and morphology. Our findings show that COVID-19 can have effects throughout pregnancy on circulating EVs and circulating EVs are likely to participate in placental dysfunction induced by COVID-19.

Impact of COVID-19 pandemic on research and careers of early career researchers: a DOHaD perspective.

The COVID-19 pandemic has exposed several inequalities worldwide, including the populations' access to healthcare systems and economic differences that impact the access to vaccination, medical resources, and health care services. Scientific research activities were not an exception, such that scientific research was profoundly impacted globally. Research trainees and early career researchers (ECRs) are the life force of scientific discovery around the world, and their work and progress in research was dramatically affected by the COVID-19 pandemic. ECRs are a particularly vulnerable group as they are in a formative stage of their scientific careers, any disruptions during which is going to likely impact their lifelong career trajectory. To understand how COVID-19 impacted lives, career development plans, and research of Developmental Origins of Health and Disease (DOHaD) ECRs, the International DOHaD ECR committee formed a special interest group comprising of ECR representatives of International DOHaD affiliated Societies/Chapters from around the world (Australia and New Zealand, Canada, French Speaking DOHaD, Japan, Latin America, Pakistan and USA). The anecdotal evidence summarized in this brief report, provide an overview of the findings of this special interest group, specifically on the impact of the evolving COVID-19 pandemic on daily research activities and its effects on career development plans of ECRs. We also discuss how our learnings from these shared experiences can strengthen collaborative work for the current and future generation of scientists.

Comprehensive dataset to assess morphological changes subsequent to bleomycin exposure.

Intratracheal bleomycin causes pulmonary injury, inflammation and fibrosis. The characteristic patchy nature of the injury makes analysis challenging. Histological assessment of lung injury is a useful tool to evaluate damage, however quantification is not standardized. We propose a multi-factorial approach to assess morphological changes subsequent to intratracheal bleomycin mediated lung injury. Lungs were inflation fixed with paraformaldehyde, sectioned and stained with hematoxylin and eosin. Whole slide images were scanned and ten 400x images were randomly chosen throughout the tissue for further analysis. Using ImageJ software, alveolar wall width was measured, nuclei were counted and airspace was quantified. Morphological changes were identified in mice instilled with bleomycin. This combination offers a robust measure of lung morphology especially in a heterogenous injury.

Neonatal IL-4 exposure decreases adipogenesis of male rats into adulthood.

The cytokine interleukin 4 (IL-4) can increase beige adipogenesis in adult rodents. However, neonatal animals use a distinct adipocyte precursor compartment for adipogenesis as compared with adults. In this study, we address whether IL-4 can induce persistent effects on adipose tissue when administered subcutaneously in the interscapular region during the neonatal period in Sprague-Dawley rats. We injected IL-4 into neonatal male rats during postnatal days 1-6, followed by analysis of adipose tissue and adipocyte precursors at 2 wk and 10 wk of age. Adipocyte precursors were cultured and subjected to differentiation in vitro. We found that a short and transient IL-4 exposure in neonates upregulated uncoupling protein 1 (Ucp1) mRNA expression and decreased fat cell size in subcutaneous white adipose tissue (WAT). Adipocyte precursors from mature rats that had been treated with IL-4 as neonates displayed a decrease in adiponectin (Adipoq) but no change in Ucp1 expression, as compared with controls. Thus, neonatal IL-4 induces acute beige adipogenesis and decreases adipogenic differentiation capacity long term. Overall, these findings indicate that the neonatal period is critical for adipocyte development and may influence the later onset of obesity.NEW & NOTEWORTHY We used neonatal injections in rat to show that IL-4 decreases adipogenesis and increases browning of white fat. In adulthood, adipocyte precursors show persistently decreased adipogenesis but not increased browning. These studies in the neonate are the first, to our knowledge, to show that IL-4 can have long-lasting effects.

Myeloid cell dynamics in bleomycin-induced pulmonary injury in mice; effects of anti-TNFα antibody.

Bleomycin is a cancer therapeutic known to cause lung injury which progresses to fibrosis. Evidence suggests that macrophages contribute to this pathological response. Tumor necrosis factor (TNF)α is a macrophage-derived pro-inflammatory cytokine implicated in lung injury. Herein, we investigated the role of TNFα in macrophage responses to bleomycin. Treatment of mice with bleomycin (3 U/kg, i.t.) caused histopathological changes in the lung within 3 d which culminated in fibrosis at 21 d. This was accompanied by an early (3-7 d) influx of CD11b+ and iNOS+ macrophages into the lung, and Arg-1+ macrophages at 21 d. At this time, epithelial cell dysfunction, defined by increases in total phospholipids and SP-B was evident. Treatment of mice with anti-TNFα antibody (7.5 mg/kg, i.v.) beginning 15-30 min after bleomycin, and every 5 d thereafter reduced the number and size of fibrotic foci and restored epithelial cell function. Flow cytometric analysis of F4/80+ alveolar macrophages (AM) isolated by bronchoalveolar lavage and interstitial macrophages (IM) by tissue digestion identified resident (CD11b-CD11c+) and immature infiltrating (CD11b+CD11c-) AM, and mature (CD11b+CD11c+) and immature (CD11b+CD11c-) IM subsets in bleomycin treated mice. Greater numbers of mature (CD11c+) infiltrating (CD11b+) AM expressing the anti-inflammatory marker, mannose receptor (CD206) were observed at 21 d when compared to 7 d post bleomycin. Mature proinflammatory (Ly6C+) IM were greater at 7 d relative to 21 d. These cells transitioned into mature anti-inflammatory/pro-fibrotic (CD206+) IM between 7 and 21 d. Anti-TNFα antibody heightened the number of CD11b+ AM in the lung without altering their activation state. Conversely, it reduced the abundance of mature proinflammatory (Ly6C+) IM in the tissue at 7 d and immature pro-fibrotic IM at 21 d. Taken together, these data suggest that TNFα inhibition has beneficial effects in bleomycin induced injury, restoring epithelial function and reducing numbers of profibrotic IM and the extent of pulmonary fibrosis.

Immune dysfunction in developmental programming of type 2 diabetes mellitus.

Intrauterine growth restriction (IUGR) is a common complication of pregnancy and increases the risk of the offspring developing type 2 diabetes mellitus (T2DM) later in life. Alterations in the immune system are implicated in the pathogenesis of IUGR-induced T2DM. The development of the fetal immune system is a delicate balance as it must remain tolerant of maternal antigens whilst also preparing for the post-birth environment. In addition, the fetal immune system is susceptible to an altered intrauterine milieu caused by maternal and placental inflammatory mediators or secondary to nutrient and oxygen deprivation. Pancreatic-resident macrophages populate the pancreas during fetal development, and their phenotype is dynamic through the neonatal period. Furthermore, macrophages in the islets are instrumental in islet development as they influence ß-cell proliferation and islet neogenesis. In addition, cytokines, derived from ß-cells and macrophages, are important to islet homeostasis in the fetus and adult and, when perturbed, can cause islet dysfunction. Several activated immune pathways have been identified in the islets of people who experienced IUGR, with alternations in the levels of IL-1ß and IL-4 as well as changes in TGFß signalling. Leptin levels are also altered. Immunomodulation has shown therapeutic benefit in T2DM and might be particularly useful in IUGR-induced T2DM.

Cell Origin and iNOS Function Are Critical to Macrophage Activation Following Acute Lung Injury.

In the intratracheal bleomycin (ITB) model of acute lung injury (ALI), macrophages are recruited to the lung and participate in the inflammation and resolution that follows injury. Macrophage origin is influential in determining activation; however, the specific phenotype of recruited and resident macrophages is not known. Inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of ALI; however, the effects of its inhibition are mixed. Here we examined how macrophage origin determines the phenotypic response to ALI. Further, we hypothesize cell specific iNOS is key to determining activation and recruitment. Using a chimeric mouse approach, we have identified recruited and resident macrophage populations. We also used the chimeric mouse approach to create either pulmonary or bone marrow NOS2-/- mice and systemically inhibited iNOS via 1400 W. We evaluated macrophage populations at the peak of inflammation (8 days) and the beginning of resolution (15 days) following ITB. These studies demonstrate tissue resident macrophages adopt a M2 phenotype specifically, but monocyte originated macrophages activate along a spectrum. Additionally, we demonstrated that monocyte originating macrophage derived iNOS is responsible for recruitment to the lung during the inflammatory phase. Further, we show that macrophage activation is dependent upon cellular origin. Finally, these studies suggest pulmonary derived iNOS is detrimental to the lung following ITB. In conclusion, macrophage origin is a key determinant in response to ALI and iNOS is central to recruitment and activation.

Maternal and neonatal response to COVID-19.

The risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to maternal and newborn health has yet to be determined. Several reports suggest pregnancy does not typically increase the severity of maternal disease; however, cases of preeclampsia and preterm birth have been infrequently reported. Reports of placental infection and vertical transmission are rare. Interestingly, despite lack of SARS-CoV-2 placenta infection, there are several reports of significant abnormalities in placenta morphology. Continued research on pregnant women infected with SARS-CoV-2 and their offspring is vitally important.

Reducing Th2 inflammation through neutralizing IL-4 antibody rescues myelination in IUGR rat brain.

BACKGROUND: Intrauterine growth restriction (IUGR) is a common complication of pregnancy and is associated with significant neurological deficits in infants, including white matter damage. Previous work using an animal model of IUGR has demonstrated that IUGR rats exhibit neurobehavioral deficits and developmental delays in oligodendrocyte maturation and myelination, but the mechanisms which cause this delay are unknown. Inflammation may be an important etiological factor in IUGR and has been recognized as playing a fundamental role in the pathogenesis of myelin disorders, including cerebral palsy. METHODS: To create the model, the uterine arteries of pregnant rats were ligated at embryonic day 15. Rats delivered spontaneously. Cytokine and chemokine expression was evaluated at one prenatal and three postnatal time points, and myelin protein expression and oligodendrocyte cell numbers were evaluated by several methods at postnatal day 14. IL-4 was identified as a potential inhibitor of myelination, and rat pups were injected with IL-4 function blocking antibody from postnatal days 1-5 and myelination was assessed. RESULTS: Here, we show a novel mechanism of white matter injury. IUGR induces an exaggerated Th2 response in the developing rat brain, including upregulation of several Th2 cytokines. Of these, IL-4 is significantly increased during the period corresponding to robust developmental myelination. We show that neutralizing IL-4 antibody therapy given in the newborn period ameliorates inflammation and restores myelin protein expression and oligodendrocyte cell number in the IUGR brain to control levels, demonstrating a novel role for Th2 responses and IL-4 in IUGR and white matter injury. In addition, IL-4 directly affects oligodendrocytes in vitro decreasing differentiation. CONCLUSIONS: In this study, we have identified inflammation as a factor in the decrease in myelin seen in an animal model of IUGR. IL-4, an inflammatory protein often thought to be protective in the adult, is specifically increased, and treatment of these animals to prevent this increase ameliorates white matter damage. Our results suggest that the immune system plays a role in IUGR that is different in the perinatal period than in the adult and preventing this exaggerated Th2 response may be a potential therapeutic target.

Framework for 3D histologic reconstruction and fusion with in vivo MRI: Preliminary results of characterizing pulmonary inflammation in a mouse model.

PURPOSE: Pulmonary inflammation is associated with a variety of diseases. Assessing pulmonary inflammation on in vivo imaging may facilitate the early detection and treatment of lung diseases. Although routinely used in thoracic imaging, computed tomography has thus far not been compellingly shown to characterize inflammation in vivo. Alternatively, magnetic resonance imaging (MRI) is a nonionizing radiation technique to better visualize and characterize pulmonary tissue. Prior to routine adoption of MRI for early characterization of inflammation in humans, a rigorous and quantitative characterization of the utility of MRI to identify inflammation is required. Such characterization may be achieved by considering ex vivo histology as the ground truth, since it enables the definitive spatial assessment of inflammation. In this study, the authors introduce a novel framework to integrate 2D histology, ex vivo and in vivo imaging to enable the mapping of the extent of disease from ex vivo histology onto in vivo imaging, with the goal of facilitating computerized feature analysis and interrogation of disease appearance on in vivo imaging. The authors' framework was evaluated in a preclinical preliminary study aimed to identify computer extracted features on in vivo MRI associated with chronic pulmonary inflammation. METHODS: The authors' image analytics framework first involves reconstructing the histologic volume in 3D from individual histology slices. Second, the authors map the disease ground truth onto in vivo MRI via coregistration with 3D histology using the ex vivo lung MRI as a conduit. Finally, computerized feature analysis of the disease extent is performed to identify candidate in vivo imaging signatures of disease presence and extent. RESULTS: The authors evaluated the framework by assessing the quality of the 3D histology reconstruction and the histology-MRI fusion, in the context of an initial use case involving characterization of chronic inflammation in a mouse model. The authors' evaluation considered three mice, two with an inflammation phenotype and one control. The authors' iterative 3D histology reconstruction yielded a 70.1% ± 2.7% overlap with the ex vivo MRI volume. Across a total of 17 anatomic landmarks manually delineated at the division of airways, the target registration error between the ex vivo MRI and 3D histology reconstruction was 0.85 ± 0.44 mm, suggesting that a good alignment of the ex vivo 3D histology and ex vivo MRI had been achieved. The 3D histology-in vivo MRI coregistered volumes resulted in an overlap of 73.7% ± 0.9%. Preliminary computerized feature analysis was performed on an additional four control mice, for a total of seven mice considered in this study. Gabor texture filters appeared to best capture differences between the inflamed and noninflamed regions on MRI. CONCLUSIONS: The authors' 3D histology reconstruction and multimodal registration framework were successfully employed to reconstruct the histology volume of the lung and fuse it with in vivo MRI to create a ground truth map for inflammation on in vivo MRI. The analytic platform presented here lays the framework for a rigorous validation of the identified imaging features for chronic lung inflammation on MRI in a large prospective cohort.

Surfactant dysfunction and lung inflammation in the female mouse model of lymphangioleiomyomatosis.

Pulmonary lymphangioleiomyomatosis (LAM) is a rare lung disease caused by mutations of the tumor suppressor genes, tuberous sclerosis complex (TSC) 1 or TSC2. LAM affects women almost exclusively, and it is characterized by neoplastic growth of atypical smooth muscle-like TSC2-null LAM cells in the pulmonary interstitium, cystic destruction of lung parenchyma, and progressive decline in lung function. In this study, we hypothesized that TSC2-null lesions promote a proinflammatory environment, which contributes to lung parenchyma destruction. Using a TSC2-null female murine LAM model, we demonstrate that TSC2-null lesions promote alveolar macrophage accumulation, recruitment of immature multinucleated cells, an increased induction of proinflammatory genes, nitric oxide (NO) synthase 2, IL-6, chemokine (C-C motif) ligand 2 (CCL2)/monocyte chemotactic protein 1 (MCP1), chemokine (C-X-C motif) ligand 1 (CXCL1)/keratinocyte chemoattractant (KC), and up-regulation of IL-6, KC, MCP-1, and transforming growth factor-ß1 levels in bronchoalveolar lavage fluid. Bronchoalveolar lavage fluid also contained an increased level of surfactant protein (SP)-D, but not SP-A, significant reduction of SP-B levels, and a resultant increase in alveolar surface tension. Consistent with the growth of TSC2-null lesions, NO levels were also increased and, in turn, modified SP-D through S-nitrosylation, forming S-nitrosylated SP-D, a known consequence of lung inflammation. Progressive growth of TSC2-null lesions was accompanied by elevated levels of matrix metalloproteinase-3 and -9. This report demonstrates a link between growth of TSC2-null lesions and inflammation-induced surfactant dysfunction that might contribute to lung destruction in LAM.

Tocopherol supplementation reduces NO production and pulmonary inflammatory response to bleomycin.

Bleomycin causes acute lung injury through production of reactive species and initiation of inflammation. Previous work has shown alteration to the production of reactive oxygen species results in attenuation of injury. Vitamin E, in particular, γ-tocopherol, isoform, has the potential to scavenge reactive oxygen and nitrogen species. This study examines the utility of dietary supplementation with tocopherols in reducing bleomycin-mediated acute lung injury. Male C57BL6/J mice were intratracheally instilled with PBS or 2 units/kg bleomycin. Animals were analyzed 3 and 8 days post instillation at the cellular, tissue, and organ levels. Results showed successful delivery of tocopherols to the lung via dietary supplementation. Also, increases in reactive oxygen and nitrogen species due to bleomycin are normalized in those mice fed tocopherol diet. Injury was not prevented but inflammation progression was altered, in particular macrophage activation and function. Inflammatory scores based on histology demonstrate limited progression of inflammation in those mice treated with bleomycin and fed tocopherol diet compared to control diet. Upregulation of enzymes and cytokines involved in pro-inflammation were limited by tocopherol supplementation. Day 3 functional changes in elastance in response to bleomycin are prevented, however, 8 days post injury the effect of the tocopherol diet is lost. The effect of tocopherol supplementation upon the inflammatory process is demonstrated by a shift in the phenotype of macrophage activation. The effect of these changes on resolution and the progression of pulmonary fibrosis has yet to be elucidated.