Umbilical cord-mesenchymal stem cells induce a memory phenotype in CD4+ T cells

Inflammation is a physiological state where immune cells evoke a response against detrimental insults. Finding a safe and effective treatment for inflammation associated diseases has been a challenge. In this regard, human mesenchymal stem cells (hMSC), exert immunomodulatory effects and have regenerative capacity making it a promising therapeutic option for resolution of acute and chronic inflammation. T cells play a critical role in inflammation and depending on their phenotype, they can stimulate or suppress inflammatory responses. However, the regulatory effects of hMSC on T cells and the underlying mechanisms are not fully elucidated. Most studies focused on activation, proliferation, and differentiation of T cells. Here, we further investigated memory formation and responsiveness of CD4+ T cells and their dynamics by immune-profiling and cytokine secretion analysis. Umbilical cord mesenchymal stem cells (UC-MSC) were co-cultured with either αCD3/CD28 beads, activated peripheral blood mononuclear cells (PBMC) or magnetically sorted CD4+ T cells. The mechanism of immune modulation of UC-MSC were investigated by comparing different modes of action; transwell, direct cell-cell contact, addition of UC-MSC conditioned medium or blockade of paracrine factor production by UC-MSC. We observed a differential effect of UC-MSC on CD4+ T cell activation and proliferation using PBMC or purified CD4+ T cell co-cultures. UC-MSC skewed the effector memory T cells into a central memory phenotype in both co-culture conditions. This effect on central memory formation was reversible, since UC-MSC primed central memory cells were still responsive after a second encounter with the same stimuli. The presence of both cell-cell contact and paracrine factors were necessary for the most pronounced immunomodulatory effect of UC-MSC on T cells. We found suggestive evidence for a partial role of IL-6 and TGFβ in the UC-MSC derived immunomodulatory function. Collectively, our data show that UC-MSCs clearly affect T cell activation, proliferation and maturation, depending on co-culture conditions for which both cell-cell contact and paracrine factors are needed

Screening of chorioamnionitis using volatile organic compound detection in exhaled breath: a pre-clinical proof of concept study

Chorioamnionitis is a major risk factor for preterm birth and an independent risk factor for postnatal morbidity for which currently successful therapies are lacking. Emerging evidence indicates that the timing and duration of intra-amniotic infections are crucial determinants for the stage of developmental injury at birth. Insight into the dynamical changes of organ injury after the onset of chorioamnionitis revealed novel therapeutic windows of opportunity. Importantly, successful development and implementation of therapies in clinical care is currently impeded by a lack of diagnostic tools for early (prenatal) detection and surveillance of intra-amniotic infections. In the current study we questioned whether an intra-amniotic infection could be accurately diagnosed by a specific volatile organic compound (VOC) profile in exhaled breath of pregnant sheep. For this purpose pregnant Texel ewes were inoculated intra-amniotically with Ureaplasma parvum and serial collections of exhaled breath were performed for 6 days. Ureaplasma parvum infection induced a distinct VOC-signature in expired breath of pregnant sheep that was significantly different between day 0 and 1 vs. day 5 and 6. Based on a profile of only 15 discriminatory volatiles, animals could correctly be classified as either infected (day 5 and 6) or not (day 0 and 1) with a sensitivity of 83% and a specificity of 71% and an area under the curve of 0.93. Chemical identification of these distinct VOCs revealed the presence of a lipid peroxidation marker nonanal and various hydrocarbons including n-undecane and n-dodecane. These data indicate that intra-amniotic infections can be detected by VOC analyses of exhaled breath and might provide insight into temporal dynamics of intra-amniotic infection and its underlying pathways. In particular, several of these volatiles are associated with enhanced oxidative stress and undecane and dodecane have been reported as predictive biomarker of spontaneous preterm birth in humans. Applying VOC analysis for the early detection of intra-amniotic infections will lead to appropriate surveillance of these high-risk pregnancies, thereby facilitating appropriate clinical course of action including early treatment of preventative measures for pre-maturity-associated morbidities

Neuroinflammation and structural injury of the fetal ovine brain following intra-amniotic Candida albicans exposure.

BackgroundIntra-amniotic Candida albicans (C. Albicans) infection is associated with preterm birth and high morbidity and mortality rates. Survivors are prone to adverse neurodevelopmental outcomes. The mechanisms leading to these adverse neonatal brain outcomes remain largely unknown. To better understand the mechanisms underlying C. albicans-induced fetal brain injury, we studied immunological responses and structural changes of the fetal brain in a well-established translational ovine model of intra-amniotic C. albicans infection. In addition, we tested whether these potential adverse outcomes of the fetal brain were improved in utero by antifungal treatment with fluconazole.MethodsPregnant ewes received an intra-amniotic injection of 10(7) colony-forming units C. albicans or saline (controls) at 3 or 5 days before preterm delivery at 0.8 of gestation (term ~ 150 days). Fetal intra-amniotic/intra-peritoneal injections of fluconazole or saline (controls) were administered 2 days after C. albicans exposure. Post mortem analyses for fungal burden, peripheral immune activation, neuroinflammation, and white matter/neuronal injury were performed to determine the effects of intra-amniotic C. albicans and fluconazole treatment.ResultsIntra-amniotic exposure to C. albicans caused a severe systemic inflammatory response, illustrated by a robust increase of plasma interleukin-6 concentrations. Cerebrospinal fluid cultures were positive for C. albicans in the majority of the 3-day C. albicans-exposed animals whereas no positive cultures were present in the 5-day C. albicans-exposed and fluconazole-treated animals. Although C. albicans was not detected in the brain parenchyma, a neuroinflammatory response in the hippocampus and white matter was seen which was characterized by increased microglial and astrocyte activation. These neuroinflammatory changes were accompanied by structural white matter injury. Intra-amniotic fluconazole reduced fetal mortality but did not attenuate neuroinflammation and white matter injury.ConclusionsIntra-amniotic C. albicans exposure provoked acute systemic and neuroinflammatory responses with concomitant white matter injury. Fluconazole treatment prevented systemic inflammation without attenuating cerebral inflammation and injury

Mesenchymal Stem Cells Induce T-Cell Tolerance and Protect the Preterm Brain after Global Hypoxia-Ischemia

Hypoxic-ischemic encephalopathy (HIE) in preterm infants is a severe disease for which no curative treatment is available. Cerebral inflammation and invasion of activated peripheral immune cells have been shown to play a pivotal role in the etiology of white matter injury, which is the clinical hallmark of HIE in preterm infants. The objective of this study was to assess the neuroprotective and anti-inflammatory effects of intravenously delivered mesenchymal stem cells (MSC) in an ovine model of HIE. In this translational animal model, global hypoxia-ischemia (HI) was induced in instrumented preterm sheep by transient umbilical cord occlusion, which closely mimics the clinical insult. Intravenous administration of 2 x 106MSC/kg reduced microglial proliferation, diminished loss of oligodendrocytes and reduced demyelination, as determined by histology and Diffusion Tensor Imaging (DTI), in the preterm brain after global HI. These anti-inflammatory and neuroprotective effects of MSC were paralleled by reduced electrographic seizure activity in the ischemic preterm brain. Furthermore, we showed that MSC induced persistent peripheral T-cell tolerance in vivo and reduced invasion of T-cells into the preterm brain following global HI. These findings show in a preclinical animal model that intravenously administered MSC reduced cerebral inflammation, protected against white matter injury and established functional improvement in the preterm brain following global HI. Moreover, we provide evidence that induction of T-cell tolerance by MSC might play an important role in the neuroprotective effects of MSC in HIE. This is the first study to describe a marked neuroprotective effect of MSC in a translational animal model of HIE

Comparison of ECG-based physiological markers for hypoxia in a preterm ovine model

BACKGROUND:\u3cbr/\u3eCurrent methods for assessing perinatal hypoxic conditions did not improve infant outcomes. Various waveform-based and interval-based ECG markers have been suggested, but not directly compared. We compare performance of ECG markers in a standardized ovine model for fetal hypoxia.\u3cbr/\u3eMETHODS:\u3cbr/\u3eSixty-nine fetal sheep of 0.7 gestation had ECG recorded 4 h before, during, and 4 h after a 25-min period of umbilical cord occlusion (UCO), leading to severe hypoxia. Various ECG markers were calculated, among which were heart rate (HR), HR-corrected ventricular depolarization/repolarization interval (QTc), and ST-segment analysis (STAN) episodic and baseline rise markers, analogue to clinical STAN device alarms. Performance of interval- and waveform-based ECG markers was assessed by correlating predicted and actual hypoxic/normoxic state.\u3cbr/\u3eRESULTS:\u3cbr/\u3eOf the markers studied, HR and QTc demonstrated high sensitivity (≥86%), specificity (≥96%), and positive predictive value (PPV) (≥86%) and detected hypoxia in ≥90% of fetuses at 4 min after UCO. In contrast, STAN episodic and baseline rise markers displayed low sensitivity (≤20%) and could not detect severe fetal hypoxia in 65 and 28% of the animals, respectively.\u3cbr/\u3eCONCLUSION:\u3cbr/\u3eInterval-based HR and QTc markers could assess the presence of severe hypoxia. Waveform-based STAN episodic and baseline rise markers were ineffective as markers for hypoxia

Global Hypoxia-Ischemia Induced Inflammation and Structural Changes in the Preterm Ovine Gut Which Were Not Ameliorated by Mesenchymal Stem Cell Treatment

Abstract Perinatal asphyxia, a condition of impaired gas exchange during birth, leads to fetal hypoxia-ischemia (HI) and is associated with postnatal adverse outcomes including intestinal dysmotility and necrotizing enterocolitis. Evidence from adult animal models of transient, locally induced intestinal HI has shown that inflammation is essential in HI-induced injury of the gut. Importantly, mesenchymal stem cell (MSC) treatment prevented this HI-induced intestinal damage. We therefore assessed whether fetal global HI induced inflammation, injury and developmental changes in the gut and whether intravenous MSC administration ameliorated these HI-induced adverse intestinal effects. In a preclinical ovine model, fetuses were subjected to umbilical cord occlusion (UCO), with or without MSC treatment, and euthanized 7 d after UCO. Global HI increased the number of myeloperoxidase-positive cells in the mucosa, upregulated messenger RNA (mRNA) levels of interleukin (IL)-1β and IL-17 in gut tissue and caused T-cell invasion in the intestinal muscle layer. Intestinal inflammation following global HI was associated with increased Ki67+ cells in the muscularis and subsequent muscle hyperplasia. Global HI caused distortion of glial fibrillary acidic protein immunoreactivity in the enteric glial cells and increased synaptophysin and serotonin expression in the myenteric ganglia. Intravenous MSC treatment did not ameliorate these HI-induced adverse intestinal events. Global HI resulted in intestinal inflammation and enteric nervous system abnormalities, which are clinically associated with postnatal complications, including feeding intolerance, altered gastrointestinal transit and necrotizing enterocolitis. The intestinal histopathological changes were not prevented by intravenous MSC treatment directly after HI, indicating that alternative treatment regimens for cell-based therapies should be explored

Prenatal administration of multipotent adult progenitor cells modulates the systemic and cerebral immune response in an ovine model of chorioamnionitis

Systemic and cerebral inflammation following antenatal infection (e.g. chorioamnionitis) and dysregulation of the blood brain barrier (BBB) are major risk factors for abnormal neonatal brain development. Administration of multipotent adult progenitor cells (MAPCs) represents an interesting pharmacological strategy as modulator of the peripheral and cerebral immune response and protector of BBB integrity. We studied the immunomodulatory and protective cerebrovascular potential of prenatally administered MAPCs in a preclinical ovine model for antenatal inflammation. Ovine fetuses were intra-amniotically (i.a.) exposed to lipopolysaccharide (LPS) or saline at gestational day 125, followed by the intravenous administration of 1*107 MAPCs or saline at gestational day 127. Circulating inflammation markers were measured. Fetal brains were examined immuno-histochemically post-mortem at gestational day 132. Fetal plasma IL-6 levels were elevated significantly 24 h after LPS administration. In utero systemic MAPC treatment after LPS exposure increased Annexin A1 (ANXA1) expression in the cerebrovascular endothelium, indicating enforcement of BBB integrity, and increased the number of leukocytes at brain barriers throughout the brain. Further characterisation of brain barrier-associated leukocytes showed that monocyte/choroid plexus macrophage (IBA-1+/CD206+) and neutrophil (MPO+) populations predominantly contributed to the LPS-MAPC-induced increase of CD45+cells. In the choroid plexus, the percentage of leukocytes expressing the proresolving mediator ANXA1 tended to be decreased after LPS-induced antenatal inflammation, an effect reversed by systemic MAPC treatment. Accordingly, expression levels of ANXA1 per leukocyte were decreased after LPS and restored after subsequent MAPC treatment. Increased expression of ANXA1 by the cerebrovasculature and immune cells at brain barriers following MAPC treatment in an infectious setting indicate a MAPC driven early defence mechanism to protect the neonatal brain against infection-driven inflammation and potential additional pro-inflammatory insults in the neonatal period