Feeding cessation and antibiotics improve clinical symptoms and alleviate gut and systemic inflammation in preterm pigs sensitive to necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a microbiota- and feeding-related gut inflammatory disease in preterm infants. The standard of care (SOC) treatment for suspected NEC is antibiotic treatment and reduced enteral feeding, but how SOC treatment mitigates NEC remains unclear. We explored whether SOC treatment alone or combined with an anti-inflammatory protein (inter-alpha inhibitor protein, IAIP) supplementation improves outcomes in a preterm piglet model of formula-induced NEC. Seventy-one cesarean-delivered preterm piglets were initially fed formula, developing NEC symptoms by day 3, and then randomized into CON (continued feeding) or SOC groups (feeding cessation and antibiotics), each with or without human IAIP (2×2 factorial design). By day 5, IAIP treatment did not significantly influence outcomes, whereas SOC treatment effectively reduced NEC lesions, diarrhea, and bloody stools. Notably, SOC treatment improved gut morphology and function, dampened gut inflammatory responses, altered the colonic microbiota composition, and modulated systemic immune responses. Plasma proteomic analysis revealed the effects of SOC treatment on organ development and systemic inflammatory responses. Collectively, these findings suggest that SOC treatment significantly prevents NEC progression in preterm piglets via effects on gut structure, function, and microbiota, as well as systemic immune and inflammatory responses. Timely feeding cessation and antibiotics are critical factors in preventing NEC progression in preterm infants, while the benefits of additional human IAIP treatment remain to be established.

Association between decreased cord blood inter-alpha inhibitor levels and neonatal encephalopathy at birth.

BACKGROUND: Inter-alpha inhibitor proteins (IAIPs) are structurally related proteins found in the systemic circulation with immunomodulatory anti-inflammatory properties. Reduced levels are found in inflammatory related conditions including sepsis and necrotizing enterocolitis, and in neonatal rodents after exposure to hypoxia ischemia. In the current study, cord blood IAIP levels were measured in neonates with and without exposure to hypoxic-ischemic encephalopathy (HIE). METHODS: This is a prospective cohort study including infants born ≥36 weeks over a one-year period. Term pregnancies were divided into two groups: a "reference control" (uncomplicated term deliveries), and "moderate to severe HIE" (qualifying for therapeutic hypothermia). IAIPs were quantified using a sensitive ELISA on the cord blood samples. RESULTS: The study included 57 newborns: Reference control group (n = 13) and moderate/severe HIE group (n = 44). Measurement of IAIP cord blood concentrations in moderate to severe HIE group [278.2 (138.0, 366.0) µg/ml] revealed significantly lower IAIP concentrations compared with the control group [418.6 (384.5, 445.0) µg/ml] (p = 0.002). CONCLUSIONS: These findings suggest a potential role for IAIPs as indicators of neonates at risk for HIE. IAIP levels could have diagnostic implications in the management of HIE. Future research is required to explore the relationship between HIE and IAIPs as biomarkers for disease severity. CATEGORY OF STUDY: Translational.

Neuroprotective efficacy of hypothermia and Inter-alpha Inhibitor Proteins after hypoxic ischemic brain injury in neonatal rats.

Therapeutic hypothermia is the standard of care for hypoxic-ischemic (HI) encephalopathy. Inter-alpha Inhibitor Proteins (IAIPs) attenuate brain injury after HI in neonatal rats. Human (h) IAIPs (60 â€‹mg/kg) or placebo (PL) were given 15 â€‹min, 24 and 48 â€‹h to postnatal (P) day-7 rats after carotid ligation and 8% oxygen for 90 â€‹min with (30 â€‹°C) and without (36 â€‹°C) exposure to hypothermia 1.5 â€‹h after HI for 3 â€‹h. Hemispheric volume atrophy (P14) and neurobehavioral tests including righting reflex (P8-P10), small open field (P13-P14), and negative geotaxis (P14) were determined. Hemispheric volume atrophy in males was reduced (P â€‹< â€‹0.05) by 41.9% in the normothermic-IAIP and 28.1% in the hypothermic-IAIP compared with the normothermic-PL group, and in females reduced (P â€‹< â€‹0.05) by 30.3% in the normothermic-IAIP, 45.7% in hypothermic-PL, and 55.2% in hypothermic-IAIP compared with the normothermic-PL group after HI. Hypothermia improved (P â€‹< â€‹0.05) the neuroprotective effects of hIAIPs in females. The neuroprotective efficacy of hIAIPs was comparable to hypothermia in female rats (P â€‹= â€‹0.183). Treatment with hIAIPs, hypothermia, and hIAIPs with hypothermia decreased (P â€‹< â€‹0.05) the latency to enter the peripheral zone in the small open field test in males. We conclude that hIAIPs provide neuroprotection from HI brain injury that is comparable to the protection by hypothermia, hypothermia increases the effects of hIAIPs in females, and hIAIPs and hypothermia exhibit some sex-related differential effects.

Inter-Alpha Inhibitor Proteins Modify the Microvasculature after Exposure to Hypoxia-Ischemia and Hypoxia in Neonatal Rats.

Microvasculature develops during early brain development. Hypoxia-ischemia (HI) and hypoxia (H) predispose to brain injury in neonates. Inter-alpha inhibitor proteins (IAIPs) attenuate injury to the neonatal brain after exposure to HI. However, the effects of IAIPs on the brain microvasculature after exposure to HI have not been examined in neonates. Postnatal day-7 rats were exposed to sham treatment or right carotid artery ligation and 8% oxygen for 90 min. HI comprises hypoxia (H) and ischemia to the right hemisphere (HI-right) and hypoxia to the whole body, including the left hemisphere (H-left). Human IAIPs (hIAIPs, 30 mg/kg) or placebo were injected immediately, 24 and 48 h after HI/H. The brains were analyzed 72 h after HI/H to determine the effects of hIAIPs on the microvasculature by laminin immunohistochemistry and calculation of (1) the percentage area stained by laminin, (2) cumulative microvessel length, and (3) density of tunneling nanotubes (TNTs), which are sensitive indicators of the earliest phases of neo-vascularization/collateralization. hIAIPs mainly affected the percent of the laminin-stained area after HI/H, cumulative vessel length after H but not HI, and TNT density in females but not males. hIAIPs modify the effects of HI/H on the microvasculature after brain injury in neonatal rats and exhibit sex-related differential effects. Our findings suggest that treatment with hIAIPs after exposure to H and HI in neonatal rats affects the laminin content of the vessel basal lamina and angiogenic responses in a sex-related fashion.

Inter-alpha Inhibitor Proteins Ameliorate Brain Injury and Improve Behavioral Outcomes in a Sex-Dependent Manner After Exposure to Neonatal Hypoxia Ischemia in Newborn and Young Adult Rats.

Hypoxic-ischemic (HI) brain injury is a major contributor to neurodevelopmental morbidities. Inter-alpha inhibitor proteins (IAIPs) have neuroprotective effects on HI-related brain injury in neonatal rats. However, the effects of treatment with IAIPs on sequential behavioral, MRI, and histopathological abnormalities in the young adult brain after treatment with IAIPs in neonates remain to be determined. The objective of this study was to examine the neuroprotective effects of IAIPs at different neurodevelopmental stages from newborn to young adults after exposure of neonates to HI injury. IAIPs were given as 11-sequential 30-mg/kg doses to postnatal (P) day 7-21 rats after right common carotid artery ligation and exposure to 90 min of 8% oxygen. The resulting brain edema and injury were examined by T2-weighted magnetic resonance imaging (MRI) and cresyl violet staining, respectively. The mean T2 values of the ipsilateral hemisphere from MRI slices 6 to 10 were reduced in IAIP-treated HI males + females on P8, P9, and P10 and females on P8, P9, P10, and P14. IAIP treatment reduced hemispheric volume atrophy by 44.5 ± 29.7% in adult male + female P42 rats and improved general locomotor abilities measured by the righting reflex over time at P7.5, P8, and P9 in males + females and males and muscle strength/endurance measured by wire hang on P16 in males + females and females. IAIPs provided beneficial effects during the learning phase of the Morris water maze with females exhibiting beneficial effects. IAIPs confer neuroprotection from HI-related brain injury in neonates and even in adult rats and beneficial MRI and behavioral benefits in a sex-dependent manner.

Changes in Cellular Localization of Inter-Alpha Inhibitor Proteins after Cerebral Ischemia in the Near-Term Ovine Fetus.

Inter-alpha Inhibitor Proteins (IAIPs) are key immunomodulatory molecules. Endogenous IAIPs are present in human, rodent, and sheep brains, and are variably localized to the cytoplasm and nuclei at multiple developmental stages. We have previously reported that ischemia-reperfusion (I/R) reduces IAIP concentrations in the fetal sheep brain. In this study, we examined the effect of I/R on total, cytoplasmic, and nuclear expression of IAIPs in neurons (NeuN+), microglia (Iba1+), oligodendrocytes (Olig2+) and proliferating cells (Ki67+), and their co-localization with histones and the endoplasmic reticulum in fetal brain cells. At 128 days of gestation, fetal sheep were exposed to Sham (n = 6) or I/R induced by cerebral ischemia for 30 min with reperfusion for 7 days (n = 5). Although I/R did not change the total number of IAIP+ cells in the cerebral cortex or white matter, cells with IAIP+ cytoplasm decreased, whereas cells with IAIP+ nuclei increased in the cortex. I/R reduced total neuronal number but did not change the IAIP+ neuronal number. The proportion of cytoplasmic IAIP+ neurons was reduced, but there was no change in the number of nuclear IAIP+ neurons. I/R increased the number of microglia and decreased the total numbers of IAIP+ microglia and nuclear IAIP+ microglia, but not the number of cytoplasmic IAIP+ microglia. I/R was associated with reduced numbers of oligodendrocytes and increased proliferating cells, without changes in the subcellular IAIP localization. IAIPs co-localized with the endoplasmic reticulum and histones. In conclusion, I/R alters the subcellular localization of IAIPs in cortical neurons and microglia but not in oligodendrocytes or proliferating cells. Taken together with the known neuroprotective effects of exogenous IAIPs, we speculate that endogenous IAIPs may play a role during recovery from I/R.

High-mobility group box-1 and inter-alpha inhibitor proteins: In vitro binding and co-localization in cerebral cortex after hypoxic-ischemic injury.

The high-mobility group box-1 (HMGB1) protein is a transcription-regulating protein located in the nucleus. However, it serves as a damage-associated molecular pattern protein that activates immune cells and stimulates inflammatory cytokines to accentuate neuroinflammation after release from damaged cells. In contrast, Inter-alpha Inhibitor Proteins (IAIPs) are proteins with immunomodulatory effects including inhibition of pro-inflammatory cytokines. We have demonstrated that IAIPs exhibit neuroprotective properties in neonatal rats exposed to hypoxic-ischemic (HI) brain injury. In addition, previous studies have suggested that the light chain of IAIPs, bikunin, may exert its anti-inflammatory effects by inhibiting HMGB1 in a variety of different injury models in adult subjects. The objectives of the current study were to confirm whether HMGB1 is a target of IAIPs by investigating the potential binding characteristics of HMGB1 and IAIPs in vitro, and co-localization in vivo in cerebral cortices after exposure to HI injury. Solid-phase binding assays and surface plasmon resonance (SPR) were used to determine the physical binding characteristics between IAIPs and HMGB1. Cellular localizations of IAIPs-HMGB1 in neonatal rat cortex were visualized by double labeling with anti-IAIPs and anti-HMGB1 antibodies. Solid-phase binding and SPR demonstrated specific binding between IAIPs and HMGB1 in vitro. Cortical cytoplasmic and nuclear co-localization of IAIPs and HMGB1 were detected by immunofluorescent staining in control and rats immediately and 3 hours after HI. In conclusion, HMGB1 and IAIPs exhibit direct binding in vitro and co-localization in vivo in neonatal rats exposed to HI brain injury suggesting HMGB1 could be a target of IAIPs.

Effects of Juvenile or Adolescent Working Memory Experience and Inter-Alpha Inhibitor Protein Treatment after Neonatal Hypoxia-Ischemia.

Hypoxic-Ischemic (HI) brain injury in the neonate contributes to life-long cognitive impairment. Early diagnosis and therapeutic interventions are critical but limited. We previously reported in a rat model of HI two interventional approaches that improve cognitive and sensory function: administration of Inter-alpha Inhibitor Proteins (IAIPs) and early experience in an eight-arm radial water maze (RWM) task. Here, we expanded these studies to examine the combined effects of IAIPs and multiple weeks of RWM assessment beginning with juvenile or adolescent rats to evaluate optimal age windows for behavioral interventions. Subjects were divided into treatment groups; HI with vehicle, sham surgery with vehicle, and HI with IAIPs, and received either juvenile (P31 initiation) or adolescent (P52 initiation) RWM testing, followed by adult retesting. Error rates on the RWM decreased across weeks for all conditions. Whereas, HI injury impaired global performance as compared to shams. IAIP-treated HI subjects tested as juveniles made fewer errors as compared to their untreated HI counterparts. The juvenile group made significantly fewer errors on moderate demand trials and showed improved retention as compared to the adolescent group during the first week of adult retesting. Together, results support and extend our previous findings that combining behavioral and anti-inflammatory interventions in the presence of HI improves subsequent learning performance. Results further indicate sensitive periods for behavioral interventions to improve cognitive outcomes. Specifically, early life cognitive experience can improve long-term learning performance even in the presence of HI injury. Results from this study provide insight into typical brain development and the impact of developmentally targeted therapeutics and task-specific experience on subsequent cognitive processing.

Novel Neuroprotective Agents to Treat Neonatal Hypoxic-Ischemic Encephalopathy: Inter-Alpha Inhibitor Proteins.

Perinatal hypoxia-ischemia (HI) is a major cause of brain injury and mortality in neonates. Hypoxic-ischemic encephalopathy (HIE) predisposes infants to long-term cognitive deficits that influence their quality of life and place a large burden on society. The only approved treatment to protect the brain after HI is therapeutic hypothermia, which has limited effectiveness, a narrow therapeutic time window, and is not considered safe for treatment of premature infants. Alternative or adjunctive therapies are needed to improve outcomes of full-term and premature infants after exposure to HI. Inter-alpha inhibitor proteins (IAIPs) are immunomodulatory molecules that are proposed to limit the progression of neonatal inflammatory conditions, such as sepsis. Inflammation exacerbates neonatal HIE and suggests that IAIPs could attenuate HI-related brain injury and improve cognitive outcomes associated with HIE. Recent studies have shown that intraperitoneal treatment with IAIPs can decrease neuronal and non-neuronal cell death, attenuate glial responses and leukocyte invasion, and provide long-term behavioral benefits in neonatal rat models of HI-related brain injury. The present review summarizes these findings and outlines the remaining experimental analyses necessary to determine the clinical applicability of this promising neuroprotective treatment for neonatal HI-related brain injury.

Effects of inter-alpha inhibitor proteins on brain injury after exposure of neonatal rats to severe hypoxia-ischemia.

Hypoxic-ischemic (HI) brain injury is one of the most common neurological problems occurring in premature and full-term infants after perinatal complications. Hypothermia is the only treatment approved for HI encephalopathy in newborns. However, this treatment is only partially protective, cannot be used to treat premature infants, and has limited efficacy to treat severe HI encephalopathy. Inflammation contributes to the evolution of HI brain injury in neonates. Inter-alpha Inhibitor Proteins (IAIPs) are immunomodulatory proteins that have neuroprotective properties after exposure to moderate HI in neonatal rats. The objective of the current study was to determine the neuroprotective efficacy of treatment with IAIPs starting immediately after or with a delay of one hour after exposure to severe HI of 120 min duration. One hundred and forty-six 7-day-old rat pups were randomized to sham control, HI and immediate treatment with IAIPs (60 mg/kg) or placebo (PL), and sham, HI and delayed treatment with IAIPs or PL. IAIPs or PL were given at zero, 24, and 48 h after HI or 1, 24 and 48 h after HI. Total brain infarct volume was determined 72 h after exposure to HI. Treatment with IAIPs immediately after HI decreased (P < 0.05) infarct volumes by 58.0% and 44.5% in male and female neonatal rats, respectively. Delayed treatment with IAIPs after HI decreased (P < 0.05) infarct volumes by 23.7% in male, but not in female rats. We conclude that IAIPs exert neuroprotective effects even after exposure to severe HI in neonatal rats and appear to exhibit some sex-related differential effects.

Pharmacokinetics of Inter-Alpha Inhibitor Proteins and Effects on Hemostasis After Hypoxic-Ischemic Brain Injury in Neonatal Rats.

BACKGROUND: Hypoxic-ischemic (HI) brain injury is a leading cause of long-term neurodevelopmental morbidities in neonates. Human plasma-derived Inter-Alpha Inhibitor Proteins (hIAIPs) are neuroprotective after HI brain injury in neonatal rats. The light chain (bikunin) of hIAIPs inhibits proteases involved in the coagulation of blood. Newborns exposed to HI can be at risk for significant bleeding in the brain and other organs. OBJECTIVE: The objectives of the present study were to assess the pharmacokinetics (PK) and the duration of bleeding after intraperitoneal (IP) administration of hIAIPs in HI-exposed male and female neonatal rats. METHODS: HI was induced with the Rice-Vannucci method in postnatal (P) day-7 rats. After the right common carotid artery ligation, rats were exposed to 90 min of 8% oxygen. hIAIPs (30 mg/kg, IP) were given immediately after Sham or HI exposure in the PK study and serum was collected 1, 6, 12, 24, or 36 h after the injections. Serum hIAIP concentrations were measured with a competitive ELISA. ADAPT5 software was used to fit the pooled PK data considering first-order absorption and disposition. hIAIPs (60 mg/kg, IP) were given in the bleeding time studies at 0, 24 and 48 h after HI with tail bleeding times measured 72 h after HI. RESULTS: IP administration yielded significant systemic exposure to hIAIPs with PK being affected markedly including primarily faster absorption and reduced elimination as a result of HI and modestly of sex-related differences. hIAIP administration did not affect bleeding times after HI. CONCLUSION: These results will help to inform hIAIP dosing regimen schedules in studies of neuroprotection in neonates exposed to HI.

Inter-alpha inhibitor proteins attenuate lipopolysaccharide-induced blood-brain barrier disruption and downregulate circulating interleukin 6 in mice.

Circulating levels of inter-alpha inhibitor proteins change dramatically in acute inflammatory disorders, which suggest an important contribution to the immunomodulatory system. Human blood-derived inter-alpha inhibitor proteins are neuroprotective and improve survival of neonatal mice exposed to lipopolysaccharide. Lipopolysaccharide augments inflammatory conditions and disrupts the blood-brain barrier. There is a paucity of therapeutic strategies to treat blood-brain barrier dysfunction, and the neuroprotective effects of human blood-derived inter-alpha inhibitor proteins are not fully understood. To examine the therapeutic potential of inter-alpha inhibitor proteins, we administered human blood-derived inter-alpha inhibitor proteins to male and female CD-1 mice after lipopolysaccharide exposure and quantified blood-brain barrier permeability of intravenously injected 14C-sucrose and 99mTc-albumin. We hypothesized that human blood-derived inter-alpha inhibitor protein treatment would attenuate lipopolysaccharide-induced blood-brain barrier disruption and associated inflammation. Lipopolysaccharide increased blood-brain barrier permeability to both 14C-sucrose and 99mTc-albumin, but human blood-derived inter-alpha inhibitor protein treatment only attenuated increases in 14C-sucrose blood-brain barrier permeability in male mice. Lipopolysaccharide stimulated a more robust elevation of male serum inter-alpha inhibitor protein concentration compared to the elevation measured in female serum. Lipopolysaccharide administration also increased multiple inflammatory factors in serum and brain tissue, including interleukin 6. Human blood-derived inter-alpha inhibitor protein treatment downregulated serum interleukin 6 levels, which were inversely correlated with serum inter-alpha inhibitor protein concentration. We conclude that inter-alpha inhibitor proteins may be neuroprotective through mechanisms of blood-brain barrier disruption associated with systemic inflammation.

Inter-alpha Inhibitor Proteins Modulate Neuroinflammatory Biomarkers After Hypoxia-Ischemia in Neonatal Rats.

Neuroinflammation contributes to hypoxic-ischemic (HI) brain injury. Inter-alpha inhibitor proteins (IAIPs) have important immunomodulatory properties. Human (h) plasma-derived IAIPs reduce brain injury and improve neurobehavioral outcomes after HI. However, the effects of hIAIPs on neuroinflammatory biomarkers after HI have not been examined. We determined whether hIAIPs attenuated HI-related neuroinflammation. Postnatal day-7 rats exposed to sham-placebo, or right carotid ligation and 8% oxygen for 90 minutes with placebo, and hIAIP treatment were studied. hIAIPs (30 mg/kg) or PL was injected intraperitoneally immediately, 24, and 48 hours after HI. Rat complete blood counts and sex were determined. Brain tissue and peripheral blood were prepared for analysis 72 hours after HI. The effects of hIAIPs on HI-induced neuroinflammation were quantified by image analysis of positively stained astrocytic (glial fibrillary acid protein [GFAP]), microglial (ionized calcium binding adaptor molecule-1 [Iba-1]), neutrophilic (myeloperoxidase [MPO]), matrix metalloproteinase-9 (MMP9), and MMP9-MPO cellular markers in brain regions. hIAIPs reduced quantities of cortical GFAP, hippocampal Iba-1-positive microglia, corpus callosum MPO, and cortical MMP9-MPO cells and the percent of neutrophils in peripheral blood after HI in male, but not female rats. hIAIPs modulate neuroinflammatory biomarkers in the neonatal brain after HI and may exhibit sex-related differential effects.

Inter-α-inhibitor Ameliorates Endothelial Inflammation in Sepsis.

PURPOSE: Vascular endothelial cells demonstrate severe injury in sepsis, and a reduction in endothelial inflammation would be beneficial. Inter-α-Inhibitor (IαI) is a family of abundant plasma proteins with anti-inflammatory properties and has been investigated in human and animal sepsis with encouraging results. We hypothesized that IαI may protect endothelia from sepsis-related inflammation. METHODS: IαI-deficient or sufficient mice were treated with endotoxin or underwent complement-induced lung injury. VCAM-1 and ICAM-1 expression was measured in blood and lung as marker of endothelial activation. Human endothelia were exposed to activated complement C5a with or without IαI. Blood from human sepsis patients was examined for VCAM-1 and ICAM-1 and levels were correlated with blood levels of IαI. RESULTS: IαI-deficient mice showed increased endothelial activation in endotoxin/sepsis- and complement-induced lung injury models. In vitro, levels of endothelial pro-inflammatory cytokines and cell growth factors induced by activated complement C5a were significantly decreased in the presence of IαI. This effect was associated with decreased ERK and NFκB activation. IαI levels were inversely associated with VCAM-1 and ICAM-1 levels in a human sepsis cohort. CONCLUSIONS: IαI ameliorates endothelial inflammation and may be beneficial as a treatment of sepsis.

Neuroprotective effects of inter-alpha inhibitor proteins after hypoxic-ischemic brain injury in neonatal rats.

Hypoxic-ischemic (HI) brain injury is one of the most common neurological problems occurring in the perinatal period. Hypothermia is the only approved intervention for neonatal HI encephalopathy. However, this treatment is only partially protective, has a narrow therapeutic time window after birth and only can be used to treat full-term infants. Consequently, additional therapies are critically needed. Inflammation is an important contributing factor to the evolution of HI brain injury in neonates. Inter-alpha Inhibitor Proteins (IAIPs) are immunomodulatory proteins with anti-inflammatory properties. We have previously shown that IAIPs reduce neuronal cell death and improve behavioral outcomes when given after carotid artery ligation, but before hypoxia in male neonatal rats. The objective of the current study was to investigate the neuroprotective effects of treatment with IAIPs given immediately or 6 h after HI in both male and female neonatal rats. HI was induced with the Rice-Vannucci method in postnatal (P) day 7 rats. After ligation of the right common carotid artery, P7 rats were exposed to 90 min of hypoxia (8% oxygen). Human plasma-derived IAIPs or placebo (phosphate buffered saline) was given at zero, 24, and 48 h after HI. Brains were perfused, weighed and fixed 72 h after HI at P10. In a second, delayed treatment group, the same procedure was followed except that IAIPs or placebo were given at 6, 24 and 48 h after HI. Separate sham-operated, placebo-treated groups were exposed to identical protocols but were not exposed to carotid artery ligation and remained in room air. Rat sex was recorded. The effects of IAIPs on HI brain injury were examined using histopathological scoring and immunohistochemical analyses of the brain and by using infarct volume measurements on frozen tissue of the entire brain hemispheres ipsilateral and contralateral to HI injury. IAIPs given immediately after HI improved (P < 0.050) histopathological brain injury across and within the cingulate, caudate/putamen, thalamus, hippocampus and parietal cortex in males, but not in females. In contrast, IAIPs given immediately after HI reduced (P < 0.050) infarct volumes of the hemispheres ipsilateral to HI injury in similarly both the males and females. Treatment with IAIPs also resulted in higher (P < 0.050) brain weights compared with the placebo-treated HI group, reduced (P < 0.050) neuronal and non-neuronal cell death in the cortex and total hemisphere, and also increased the total area of oligodendrocytes determined by CNPase in the ipsilateral hemisphere and corpus callosum (P < 0.050) of male, but not female subjects exposed to HI. Delayed treatment with IAIPs 6 h after HI did not improve histopathological brain injury in males or females, but resulted in higher (P < 0.050) brain weights compared with the placebo-treated HI males. Therefore, treatment with IAIPs immediately after HI improved brain weights and reduced neuropathological brain injury and cell death in male rats, and reduced infarct volume in both male and female neonatal rats. We conclude that IAIPs exert neuroprotective effects after exposure to HI in neonatal rats and may exhibit some sex-related differential effects.

Intravascular heavy chain-modification of hyaluronan during endotoxic shock.

During inflammation, the covalent linking of the ubiquitous extracellular polysaccharide hyaluronan (HA) with the heavy chains (HC) of the serum protein inter alpha inhibitor (IαI) is exclusively mediated by the enzyme tumor necrosis factor α (TNFα)-stimulated-gene-6 (TSG-6). While significant advances have been made regarding how HC-modified HA (HC-HA) is an important regulator of inflammation, it remains unclear why HC-HA plays a critical role in promoting survival in intraperitoneal lipopolysaccharide (LPS)-induced endotoxemia while exerting only a modest role in the outcomes following intratracheal exposure to LPS. To address this gap, the two models of intraperitoneal LPS-induced endotoxic shock and intratracheal LPS-induced acute lung injury were directly compared in TSG-6 knockout mice and littermate controls. HC-HA formation, endogenous TSG-6 activity, and inflammatory markers were assessed in plasma and lung tissue. TSG-6 knockout mice exhibited accelerated mortality during endotoxic shock. While both intraperitoneal and intratracheal LPS induced HC-HA formation in lung parenchyma, only systemically-induced endotoxemia increased plasma TSG-6 levels and intravascular HC-HA formation. Cultured human lung microvascular endothelial cells secreted TSG-6 in response to both TNFα and IL1ß stimulation, indicating that, in addition to inflammatory cells, the endothelium may secrete TSG-6 into circulation during systemic inflammation. These data show for the first time that LPS-induced systemic inflammation is uniquely characterized by significant vascular induction of TSG-6 and HC-HA, which may contribute to improved outcomes of endotoxemia.

Rapid clearance of heavy chain-modified hyaluronan during resolving acute lung injury.

BACKGROUND: Several inflammatory lung diseases display abundant presence of hyaluronic acid (HA) bound to heavy chains (HC) of serum protein inter-alpha-inhibitor (IαI) in the extracellular matrix. The HC-HA modification is critical to neutrophil sequestration in liver sinusoids and to survival during experimental lipopolysaccharide (LPS)-induced sepsis. Therefore, the covalent HC-HA binding, which is exclusively mediated by tumor necrosis factor α (TNFα)-stimulated-gene-6 (TSG-6), may play an important role in the onset or the resolution of lung inflammation in acute lung injury (ALI) induced by respiratory infection. METHODS: Reversible ALI was induced by a single intratracheal instillation of LPS or Pseudomonas aeruginosa in mice and outcomes were studied for up to six days. We measured in the lung or the bronchoalveolar fluid HC-HA formation, HA immunostaining localization and roughness, HA fragment abundance, and markers of lung inflammation and lung injury. We also assessed TSG-6 secretion by TNFα- or LPS-stimulated human alveolar macrophages, lung fibroblast Wi38, and bronchial epithelial BEAS-2B cells. RESULTS: Extensive HC-modification of lung HA, localized predominantly in the peri-broncho-vascular extracellular matrix, was notable early during the onset of inflammation and was markedly decreased during its resolution. Whereas human alveolar macrophages secreted functional TSG-6 following both TNFα and LPS stimulation, fibroblasts and bronchial epithelial cells responded to only TNFα. Compared to wild type, TSG-6-KO mice, which lacked HC-modified HA, exhibited modest increases in inflammatory cells in the lung, but no significant differences in markers of lung inflammation or injury, including histopathological lung injury scores. CONCLUSIONS: Respiratory infection induces rapid HC modification of HA followed by fragmentation and clearance, with kinetics that parallel the onset and resolution phase of ALI, respectively. Alveolar macrophages may be an important source of pulmonary TSG-6 required for HA remodeling. The formation of HC-modified HA had a minor role in the onset, severity, or resolution of experimental reversible ALI induced by respiratory infection with gram-negative bacteria.

Alterations in inter-alpha inhibitor protein expression after hypoxic-ischemic brain injury in neonatal rats.

Hypoxic-ischemic (HI) brain injury is frequently associated with premature and/or full-term birth-related complications that reflect widespread damage to cerebral cortical structures. Inflammation has been implicated in the long-term evolution and severity of HI brain injury. Inter-Alpha Inhibitor Proteins (IAIPs) are immune modulator proteins that are reduced in systemic neonatal inflammatory states. We have shown that endogenous IAIPs are present in neurons, astrocytes and microglia and that exogenous treatment with human plasma purified IAIPs decreases neuronal injury and improves behavioral outcomes in neonatal rats with HI brain injury. In addition, we have shown that endogenous IAIPs are reduced in the brain of the ovine fetus shortly after ischemic injury. However, the effect of HI on changes in circulating and endogenous brain IAIPs has not been examined in neonatal rats. In the current study, we examined changes in endogenous IAIPs in the systemic circulation and brain of neonatal rats after exposure to HI brain injury. Postnatal day 7 rats were exposed to right carotid artery ligation and 8% oxygen for 2h. Sera were obtained immediately, 3, 12, 24, and 48h and brains 3 and 24h after HI. IAIPs levels were determined by a competitive enzyme-linked immunosorbent assay (ELISA) in sera and by Western immunoblots in cerebral cortices. Serum IAIPs were decreased 3h after HI and remained lower than in non-ischemic rats up to 7days after HI. IAIP expression increased in the ipsilateral cerebral cortices 24h after HI brain injury and in the hypoxic contralateral cortices. However, 3h after hypoxia alone the 250kDa IAIP moiety was reduced in the contralateral cortices. We speculate that changes in endogenous IAIPs levels in blood and brain represent constituents of endogenous anti-inflammatory neuroprotective mechanism(s) after HI in neonatal rats.

TNF-stimulated gene 6 promotes formation of hyaluronan-inter-α-inhibitor heavy chain complexes necessary for ozone-induced airway hyperresponsiveness.

Exposure to pollutants, such as ozone, exacerbates airway inflammation and hyperresponsiveness (AHR). TNF-stimulated gene 6 (TSG-6) is required to transfer inter-α-inhibitor heavy chains (HC) to hyaluronan (HA), facilitating HA receptor binding. TSG-6 is necessary for AHR in allergic asthma, because it facilitates the development of a pathological HA-HC matrix. However, the role of TSG-6 in acute airway inflammation is not well understood. Here, we hypothesized that TSG-6 is essential for the development of HA- and ozone-induced AHR. TSG-6-/- and TSG-6+/+ mice were exposed to ozone or short-fragment HA (sHA), and AHR was assayed via flexiVent. The AHR response to sHA was evaluated in the isolated tracheal ring assay in tracheal rings from TSG-6-/- or TSG-6+/+, with or without the addition of exogenous TSG-6, and with or without inhibitors of Rho-associated, coiled-coil-containing protein kinase (ROCK), ERK, or PI3K. Smooth-muscle cells from mouse tracheas were assayed in vitro for signaling pathways. We found that TSG-6 deficiency protects against AHR after ozone (in vivo) or sHA (in vitro and in vivo) exposure. Moreover, TSG-6-/- tracheal ring non-responsiveness to sHA was reversed by exogenous TSG-6 addition. sHA rapidly activated RhoA, ERK, and Akt in airway smooth-muscle cells, but only in the presence of TSG-6. Inhibition of ROCK, ERK, or PI3K/Akt blocked sHA/TSG-6-mediated AHR. In conclusion, TSG-6 is necessary for AHR in response to ozone or sHA, in part because it facilitates rapid formation of HA-HC complexes. The sHA/TSG-6 effect is mediated by RhoA, ERK, and PI3K/Akt signaling.