Ureaplasma-Driven Neonatal Neuroinflammation: Novel Insights from an Ovine Model.

Ureaplasma species (spp.) are considered commensals of the adult genitourinary tract, but have been associated with chorioamnionitis, preterm birth, and invasive infections in neonates, including meningitis. Data on mechanisms involved in Ureaplasma-driven neuroinflammation are scarce. The present study addressed brain inflammatory responses in preterm lambs exposed to Ureaplasma parvum (UP) in utero. 7 days after intra-amniotic injection of UP (n = 10) or saline (n = 11), lambs were surgically delivered at gestational day 128-129. Expression of inflammatory markers was assessed in different brain regions using qRT-PCR and in cerebrospinal fluid (CSF) by multiplex immunoassay. CSF was analyzed for UP presence using ureB-based real-time PCR, and MRI scans documented cerebral white matter area and cortical folding. Cerebral tissue levels of atypical chemokine receptor (ACKR) 3, caspases 1-like, 2, 7, and C-X-C chemokine receptor (CXCR) 4 mRNA, as well as CSF interleukin-8 protein concentrations were significantly increased in UP-exposed lambs. UP presence in CSF was confirmed in one animal. Cortical folding and white matter area did not differ among groups. The present study confirms a role of caspases and the transmembrane receptors ACKR3 and CXCR4 in Ureaplasma-driven neuroinflammation. Enhanced caspase 1-like, 2, and 7 expression may reflect cell death. Increased ACKR3 and CXCR4 expression has been associated with inflammatory central nervous system (CNS) diseases and impaired blood-brain barrier function. According to these data and previous in vitro findings from our group, we speculate that Ureaplasma-induced caspase and receptor responses affect CNS barrier properties and thus facilitate neuroinflammation.

Erythrocyte transfusions are associated with retinopathy of prematurity in extremely low gestational age newborns.

AIM: Retinopathy of prematurity (ROP) is a major morbidity in preterm infants causing visual impairment including blindness. Prevention and timely treatment are critical. We investigated the potential role of red blood cell (RBC) transfusions as risk factor for ROP development. METHODS: Retrospective cohort study of data from 68 tertiary level neonatal intensive care units in Germany. Preterm infants born at 22 + 0 to 28 + 6 weeks of gestation between January 2009 and December 2021 were enrolled. RESULTS: We included n = 12 565 infants. Prevalence of any ROP was 49.2% with most infants being diagnosed with stage 1 (21.5%) and 2 disease (17.2%). ROP stage 3 was present in 10.2%, stage 4 in 0.3%, and ROP requiring treatment in 6.6%. Infants with ROP had significantly more frequently a history of RBC transfusions. Adjusting for confounders, RBC transfusions were associated with increased odds of ROP (OR 1.4, p < 0.001), ROP progression (OR 2.1, p < 0.01) and ROP requiring treatment (OR 3.6, p < 0.001). Restrictive transfusion approaches correlated with decreased (OR 0.7, p < 0.001), liberal regimes with increased odds (OR 1.2, p = 0.001). CONCLUSION: The present study confirmed an association of RBC transfusions and ROP. Our findings emphasise the need for anaemia prevention and critical re-evaluation of transfusion practices in preterm infants.

Imbalanced Inflammatory Responses in Preterm and Term Cord Blood Monocytes and Expansion of the CD14+CD16+ Subset upon Toll-like Receptor Stimulation.

Developmentally regulated features of innate immunity are thought to place preterm and term infants at risk of infection and inflammation-related morbidity. Underlying mechanisms are incompletely understood. Differences in monocyte function including toll-like receptor (TLR) expression and signaling have been discussed. Some studies point to generally impaired TLR signaling, others to differences in individual pathways. In the present study, we assessed mRNA and protein expression of pro- and anti-inflammatory cytokines in preterm and term cord blood (CB) monocytes compared with adult controls stimulated ex vivo with Pam3CSK4, zymosan, polyinosinic:polycytidylic acid, lipopolysaccharide, flagellin, and CpG oligonucleotide, which activate the TLR1/2, TLR2/6, TLR3, TLR4, TLR5, and TLR9 pathways, respectively. In parallel, frequencies of monocyte subsets, stimulus-driven TLR expression, and phosphorylation of TLR-associated signaling molecules were analyzed. Independent of stimulus, pro-inflammatory responses of term CB monocytes equaled adult controls. The same held true for preterm CB monocytes-except for lower IL-1ß levels. In contrast, CB monocytes released lower amounts of anti-inflammatory IL-10 and IL-1ra, resulting in higher ratios of pro-inflammatory to anti-inflammatory cytokines. Phosphorylation of p65, p38, and ERK1/2 correlated with adult controls. However, stimulated CB samples stood out with higher frequencies of intermediate monocytes (CD14+CD16+). Both pro-inflammatory net effect and expansion of the intermediate subset were most pronounced upon stimulation with Pam3CSK4 (TLR1/2), zymosan (TR2/6), and lipopolysaccharide (TLR4). Our data demonstrate robust pro-inflammatory and yet attenuated anti-inflammatory responses in preterm and term CB monocytes, along with imbalanced cytokine ratios. Intermediate monocytes, a subset ascribed pro-inflammatory features, might participate in this inflammatory state.

More than just inflammation: Ureaplasma species induce apoptosis in human brain microvascular endothelial cells.

BACKGROUND: Ureaplasma species (spp.) are commonly regarded as low-virulent commensals but may cause invasive diseases in immunocompromised adults and in neonates, including neonatal meningitis. The interactions of Ureaplasma spp. with host defense mechanisms are poorly understood. This study addressed Ureaplasma-driven cell death, concentrating on apoptosis as well as inflammatory cell death. METHODS: Human brain microvascular endothelial cells (HBMEC) were exposed to Ureaplasma (U.) urealyticum serovar 8 (Uu8) and U. parvum serovar 3 (Up3). Resulting numbers of dead cells as well as mRNA levels and enzyme activity of key agents in programmed cell death were assessed by flow cytometry, RNA sequencing, and qRT-PCR, respectively. xCELLigence data were used for real-time monitoring of changes in cell adhesion properties. RESULTS: Both Ureaplasma isolates induced cell death (p < 0.05, vs. broth). Furthermore, Ureaplasma spp. enhanced mRNA levels for genes in apoptosis, including caspase 3 (Up3 p < 0.05, vs. broth), caspase 7 (p < 0.01), and caspase 9 (Up3 p < 0.01). Caspase 3 activity was increased upon Uu8 exposure (p < 0.01). Vice versa, Ureaplasma isolates downregulated mRNA levels for proteins involved in inflammatory cell death, namely caspase 1 (Uu8 p < 0.01, Up3 p < 0.001), caspase 4 (Uu8 p < 0.05, Up3 p < 0.01), NOD-like receptor pyrin domain-containing 3 (Uu8 p < 0.05), and receptor-interacting protein kinase 3 (p < 0.05). CONCLUSIONS: By inducing apoptosis in HBMEC as main constituents of the blood-brain barrier, Ureaplasma spp. may provoke barrier breakdown. Simultaneous suppression of inflammatory cell death may additionally attenuate host defense strategies. Ultimate consequence could be invasive and long-term CNS infections by Ureaplasma spp.

An underestimated pathogen: Staphylococcus epidermidis induces pro-inflammatory responses in human alveolar epithelial cells.

OBJECTIVES: Conventionally regarded as a harmless skin commensal, Staphylococcus epidermidis accounts for the majority of neonatal late-onset sepsis and is shown to be associated with neonatal inflammatory morbidities, especially bronchopulmonary dysplasia. This study addressed the pro-inflammatory capacity of different S. epidermidis strains on human alveolar epithelial cells. METHODS: A549 cell monolayers were stimulated by live bacteria of S. epidermidis RP62A strain (biofilm-positive) and ATCC 12228 strain (biofilm-negative) at a multiplicity of infection ratio of 10 for 24 h. LPS (100 ng/ml) and Pam3CSK4 (1 µg/ml) were used for comparisons. Cell viability was measured by MTT method. The mRNA and protein expression of inflammatory mediators and toll-like receptor (TLR)-2 were assessed using RT-PCR, immunoassays and immunofluorescence. RESULTS: Both S. epidermidis strains induced expression of tumor necrosis factor (TNF)-α, IL-1ß, interleukin (IL)-6, IL-8, monocyte chemoattractant protein (MCP)-1, interferon γ-induced protein 10 (IP-10) and intercellular adhesion molecule (ICAM)-1, but not IL-10. The stimulatory effect of RP62A exceeded that of LPS (p < 0.05). RP62A strain showed a trend towards higher induction of pro-inflammatory mediators than ATCC 12228 strain. The co-stimulation with RP62A strain decreased cell viability compared to control and TLR agonists (p < 0.05). RP62A but not ATCC 12228 stimulated mRNA and protein expression of TLR2. CONCLUSIONS: S. epidermidis drives pro-inflammatory responses in lung epithelial cells in vitro. The pro-inflammatory capacity of S. epidermidis may differ between strains. Biofilm-positive S. epidermidis strain seems to induce more potent pulmonary pro-inflammation than biofilm-negative S. epidermidis strain.

Ureaplasma species modulate cell adhesion molecules and growth factors in human brain microvascular endothelial cells.

Ureaplasma species (spp.) are considered commensals of the adult urogenital tract, but may cause chorioamnionitis and preterm birth as well as sepsis and meningitis in neonates. Pathomechanisms in Ureaplasma-driven neuroinflammation are largely unknown. This study addressed mRNA and protein expression of intercellular and vascular cell adhesion molecules (ICAM-1, VCAM-1), granulocyte-colony stimulating factor (G-CSF), and vascular endothelial growth factor (VEGF) in native or lipopolysaccharide (LPS) co-stimulated human brain microvascular endothelial cells (HBMEC) exposed to Ureaplasma (U.) urealyticum or U. parvum. Ureaplasma spp. reduced G-CSF mRNA (p < 0.05) and protein expression (p < 0.01) and increased VEGF mRNA levels (p < 0.01) in native HBMEC. Upon co-stimulation, Ureaplasma isolates enhanced LPS-evoked VEGF and ICAM-1 mRNA expression (p < 0.05), but mitigated G-CSF and VCAM-1 mRNA responses (p < 0.05). In line with previous findings, our results indicate an ability of Ureaplasma spp. to compromise blood-brain barrier integrity, mitigate immune defense, and subdue neuroprotective mechanisms. This may facilitate intracerebral inflammation, allow chronic infections, and promote brain injury. More pronounced effects in co-stimulated cells may indicate an immunomodulatory capacity of Ureaplasma spp.

Ureaplasma Species Modulate Cytokine and Chemokine Responses in Human Brain Microvascular Endothelial Cells.

Ureaplasma species are common colonizers of the adult genitourinary tract and often considered as low-virulence commensals. Intraamniotic Ureaplasma infections, however, facilitate chorioamnionitis and preterm birth, and cases of Ureaplasma-induced neonatal sepsis, pneumonia, and meningitis raise a growing awareness of their clinical relevance. In vitro studies are scarce but demonstrate distinct Ureaplasma-driven impacts on immune mechanisms. The current study addressed cytokine and chemokine responses upon exposure of native or lipopolysaccharide (LPS) co-stimulated human brain microvascular endothelial cells (HBMEC) to Ureaplasma urealyticum or U. parvum, using qRT-PCR, RNA sequencing, multi-analyte immunoassay, and flow cytometry. Ureaplasma exposure in native HBMEC reduced monocyte chemoattractant protein (MCP)-3 mRNA expression (p < 0.01, vs. broth). In co-stimulated HBMEC, Ureaplasma spp. attenuated LPS-evoked mRNA responses for C-X-C chemokine ligand 5, MCP-1, and MCP-3 (p < 0.05, vs. LPS) and mitigated LPS-driven interleukin (IL)-1α protein secretion, as well as IL-8 mRNA and protein responses (p < 0.05). Furthermore, Ureaplasma isolates increased C-X-C chemokine receptor 4 mRNA levels in native and LPS co-stimulated HBMEC (p < 0.05). The presented results may imply immunomodulatory capacities of Ureaplasma spp. which may ultimately promote chronic colonization and long-term neuroinflammation.

Novel insights into neuroinflammation: bacterial lipopolysaccharide, tumor necrosis factor α, and Ureaplasma species differentially modulate atypical chemokine receptor 3 responses in human brain microvascular endothelial cells.

BACKGROUND: Atypical chemokine receptor 3 (ACKR3, synonym CXCR7) is increasingly considered relevant in neuroinflammatory conditions, in which its upregulation contributes to compromised endothelial barrier function and may ultimately allow inflammatory brain injury. While an impact of ACKR3 has been recognized in several neurological autoimmune diseases, neuroinflammation may also result from infectious agents, including Ureaplasma species (spp.). Although commonly regarded as commensals of the adult urogenital tract, Ureaplasma spp. may cause invasive infections in immunocompromised adults as well as in neonates and appear to be relevant pathogens in neonatal meningitis. Nonetheless, clinical and in vitro data on Ureaplasma-induced inflammation are scarce. METHODS: We established a cell culture model of Ureaplasma meningitis, aiming to analyze ACKR3 variances as a possible pathomechanism in Ureaplasma-associated neuroinflammation. Non-immortalized human brain microvascular endothelial cells (HBMEC) were exposed to bacterial lipopolysaccharide (LPS) or tumor necrosis factor-α (TNF-α), and native as well as LPS-primed HBMEC were cultured with Ureaplasma urealyticum serovar 8 (Uu8) and U. parvum serovar 3 (Up3). ACKR3 responses were assessed via qRT-PCR, RNA sequencing, flow cytometry, and immunocytochemistry. RESULTS: LPS, TNF-α, and Ureaplasma spp. influenced ACKR3 expression in HBMEC. LPS and TNF-α significantly induced ACKR3 mRNA expression (p < 0.001, vs. control), whereas Ureaplasma spp. enhanced ACKR3 protein expression in HBMEC (p < 0.01, vs. broth control). Co-stimulation with LPS and either Ureaplasma isolate intensified ACKR3 responses (p < 0.05, vs. LPS). Furthermore, stimulation wielded a differential influence on the receptor's ligands. CONCLUSIONS: We introduce an in vitro model of Ureaplasma meningitis. We are able to demonstrate a pro-inflammatory capacity of Ureaplasma spp. in native and, even more so, in LPS-primed HBMEC, underlining their clinical relevance particularly in a setting of co-infection. Furthermore, our data may indicate a novel role for ACKR3, with an impact not limited to auto-inflammatory diseases, but extending to infection-related neuroinflammation as well. AKCR3-induced blood-brain barrier breakdown might constitute a potential common pathomechanism.

Ureaplasma isolates differentially modulate growth factors and cell adhesion molecules in human neonatal and adult monocytes.

Generally regarded as commensal bacteria, the pathogenicity of Ureaplasma has often been considered low. Controversy remains concerning the clinical relevance of Ureaplasma infection in the pathogenesis of inflammation-related morbidities. Recently, we demonstrated Ureaplasma-driven pro-inflammatory cytokine responses in human monocytes in vitro. We hypothesized that Ureaplasma may induce further inflammatory mediators. Using qRT-PCR and multi-analyte immunoassay, we assessed the expression of granulocyte-colony stimulating factor (G-CSF), vascular endothelial growth factor (VEGF), intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in term neonatal and adult monocytes exposed to Ureaplasma urealyticum serovar 8 (Uu8) and U. parvum serovar 3 (Up3). Ureaplasma significantly induced VEGF mRNA in neonatal (Up3: p < 0.05, versus broth control) and adult monocytes (Uu8: p < 0.05) as well as ICAM-1 mRNA in neonatal cells (p < 0.05 each). As far as protein expression was concerned, Up3 stimulated VEGF release in both monocyte subsets (p < 0.01) and enhanced secretion of ICAM-1 protein in neonatal monocytes (p < 0.05). In adult cells, ICAM-1 protein release was increased upon exposure to both isolates (Uu8: p < 0.05, Up3: p < 0.01). Ureaplasma-induced responses did not significantly differ from corresponding levels mediated by E. coli lipopolysaccharide (LPS). The stimulatory effects were dose-dependent. Ureaplasma infection, on the contrary, did not affect G-CSF and VCAM-1 expression. Of note, co-infection of LPS-primed neonatal monocytes with Ureaplasma enhanced LPS-induced ICAM-1 release (Uu8: p < 0.05). Our results confirm Ureaplasma-driven pro-inflammatory activation of human monocytes in vitro, demonstrating a differential modulation of growth factors and cell adhesion molecules, that might promote unbalanced monocyte responses and adverse immunomodulation.

Caffeine modulates glucocorticoid-induced expression of CTGF in lung epithelial cells and fibroblasts.

BACKGROUND: Although caffeine and glucocorticoids are frequently used to treat chronic lung disease in preterm neonates, potential interactions are largely unknown. While anti-inflammatory effects of glucocorticoids are well defined, their impact on airway remodeling is less characterized. Caffeine has been ascribed to positive effects on airway inflammation as well as remodeling. Connective tissue growth factor (CTGF, CCN2) plays a key role in airway remodeling and has been implicated in the pathogenesis of chronic lung diseases such as bronchopulmonary dysplasia (BPD) in preterm infants. The current study addressed the impact of glucocorticoids on the regulation of CTGF in the presence of caffeine using human lung epithelial and fibroblast cells. METHODS: The human airway epithelial cell line H441 and the fetal lung fibroblast strain IMR-90 were exposed to different glucocorticoids (dexamethasone, budesonide, betamethasone, prednisolone, hydrocortisone) and caffeine. mRNA and protein expression of CTGF, TGF-ß1-3, and TNF-α were determined by means of quantitative real-time PCR and immunoblotting. H441 cells were additionally treated with cAMP, the adenylyl cyclase activator forskolin, and the selective phosphodiesterase (PDE)-4 inhibitor cilomilast to mimic caffeine-mediated PDE inhibition. RESULTS: Treatment with different glucocorticoids (1 µM) significantly increased CTGF mRNA levels in H441 (p < 0.0001) and IMR-90 cells (p < 0.01). Upon simultaneous exposure to caffeine (10 mM), both glucocorticoid-induced mRNA and protein expression were significantly reduced in IMR-90 cells (p < 0.0001). Of note, 24 h exposure to caffeine alone significantly suppressed basal expression of CTGF mRNA and protein in IMR-90 cells. Caffeine-induced reduction of CTGF mRNA expression seemed to be independent of cAMP levels, adenylyl cyclase activation, or PDE-4 inhibition. While dexamethasone or caffeine treatment did not affect TGF-ß1 mRNA in H441 cells, increased expression of TGF-ß2 and TGF-ß3 mRNA was detected upon exposure to dexamethasone or dexamethasone and caffeine, respectively. Moreover, caffeine increased TNF-α mRNA in H441 cells (6.5 ± 2.2-fold, p < 0.05) which has been described as potent inhibitor of CTGF expression. CONCLUSIONS: In addition to well-known anti-inflammatory features, glucocorticoids may have adverse effects on long-term remodeling by TGF-ß1-independent induction of CTGF in lung cells. Simultaneous treatment with caffeine may attenuate glucocorticoid-induced expression of CTGF, thereby promoting restoration of lung homeostasis.

The new generation synthetic reconstituted surfactant CHF5633 suppresses LPS-induced cytokine responses in human neonatal monocytes.

BACKGROUND: New generation synthetic surfactants represent a promising alternative in the treatment of respiratory distress syndrome in preterm infants. CHF5633, a new generation reconstituted agent, has demonstrated biophysical effectiveness in vitro and in vivo. In accordance to several well-known surfactant preparations, we recently demonstrated anti-inflammatory effects on LPS-induced cytokine responses in human adult monocytes. The present study addressed pro- and anti-inflammatory effects of CHF5633 in human cord blood monocytes. METHODS: Purified neonatal CD14(+) cells, either native or simultaneously stimulated with E. coli LPS, were exposed to CHF5633. TNF-α, IL-1ß, IL-8 and IL-10 as well as TLR2 and TLR4 expression were analyzed by means of real-time quantitative PCR and flow cytometry. RESULTS: CHF5633 did not induce pro-inflammation in native human neonatal monocytes and did not aggravate LPS-induced cytokine responses. Exposure to CHF5633 led to a significant decrease in LPS-induced intracellular TNF-α protein expression, and significantly suppressed LPS-induced mRNA and intracellular protein expression of IL-1ß. CHF5633 incubation did not affect cell viability, indicating that the suppressive activity was not due to toxic effects on neonatal monocytes. LPS-induced IL-8, IL-10, TLR2 and TLR4 expression were unaffected. CONCLUSION: Our data confirm that CHF5633 does not exert unintended pro-apoptotic and pro-inflammatory effects in human neonatal monocytes. CHF5633 rather suppressed LPS-induced TNF-α and IL-1ß cytokine responses. Our data add to previous work and may indicate anti-inflammatory features of CHF5633 on LPS-induced monocyte cytokine responses.