Production Of Macrophage Inflammatory Protein-2 Research Articles

Oral carbohydrate solution cause an inflammatory response when aspirated into the lungs in mice.

Many studies have been published on the beneficial effects of oral carbohydrate solutions (OCS) administered prior to surgery. However, the risk of pulmonary aspiration cannot be excluded in all patients undergoing anesthesia. But, there are few studies on the safety of OCS at lung aspiration. Experiments were conducted with mice (Nine- to ten-week-old male BALB/c mice weighted 23-26g). Lung aspiration was performed by intratracheal administration of OCS and its major constituents, fructose and maltodextrin. Bronchoalveolar lavage fluid (BALF) was collected 3 and 24h after lung aspiration. The level of Tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and macrophage inflammatory protein-2 (MIP-2) were measured in BALF. The total white blood cell, neutrophil counts, wet to dry ratio and histological examination were performed in BALF and lung tissue, respectively, at 24h after aspiration. The OCS increased the level of TNF-α, IL-6 and MIP-2 at 3h and the neutrophil count at 24h in BALFs, compared to a phosphate-buffered saline (PBS) group. The increase in IL-6 level induced by OCS was maintained for 24h. The OCS also increased the number of white blood cells and the percentage of neutrophils in BALFs. Compared to fructose, maltodextrin significantly increased the production of MIP-2 in BALFs. OCS and maltodextrin also increased neutrophil recruitment in lung tissue. Aspiration of OCS may cause inflammation of the lungs. The preoperative use of OCS may require caution under specific clinical conditions, such as patients at risk of lung aspiration.

Taurine chloramine selectively regulates neutrophil degranulation through the inhibition of myeloperoxidase and upregulation of lactoferrin.

Taurine is a free amino acid rich in neutrophils, and neutrophils play an important role in the forefront defense against infection. Upon neutrophil activation, taurine reacts with hypochlorous acid (HOCl/OCl-) produced by the myeloperoxidase (MPO) system and gets converted to taurine chloramine (Tau-Cl). Neutrophils have three types of granules, of which the primary granule MPO, secondary granule lactoferrin, and tertiary granule matrix metalloproteinase (MMP)-9 are released into the extracellular space by a process called degranulation. MPO produces hypochlorous acid to kill microorganisms, and the released MPO forms neutrophil extracellular traps (NETs) with released chromatin. Excessive secretion of MPO causes oxidative damage to the surrounding tissues. Lactoferrin exerts antioxidant activity, prevents pro-inflammatory pathway activation, sepsis, and tissue damages, and delays neutrophil apoptosis. Our experimental results show that neutrophils released small amount of granules in an inactive state, and phorbol 12-myristate 13-acetate (PMA) and N-formyl-methionine-leucyl-phenylalanine induced neutrophil degranulation. Tau-Cl inhibited the PMA-induced degranulation of MPO and formation of NETs. While Tau-Cl increased the degranulation of lactoferrin, it had no effect on MMP-9 degranulation. MPO negatively regulated the production of macrophage inflammatory protein (MIP)-2, which stimulates the degranulation and migration of neutrophils. Tau-Cl abrogated MIP-2 expression, suggestive of its inhibitory effect on MPO release. The increase in the intracellular level of MPO may negatively regulates MIP-2 expression, thereby contributing to the further regulation of neutrophil degranulation and migration. Here, we suggest that Tau-Cl selectively inhibits MPO degranulation and stimulates lactoferrin degranulation from neutrophils, thereby protecting inflamed tissues from oxidative damage induced by excessively released MPO.

PEG-8 Laurate Fermentation of Staphylococcus epidermidis Reduces the Required Dose of Clindamycin Against Cutibacterium acnes.

The probiotic activity of skin Staphylococcus epidermidis (S. epidermidis) bacteria can elicit diverse biological functions via the fermentation of various carbon sources. Here, we found that polyethylene glycol (PEG)-8 Laurate, a carbon-rich molecule, can selectively induce the fermentation of S. epidermidis, not Cutibacterium acnes (C. acnes), a bacterium associated with acne vulgaris. The PEG-8 Laurate fermentation of S. epidermidis remarkably diminished the growth of C. acnes and the C. acnes-induced production of pro-inflammatory macrophage-inflammatory protein 2 (MIP-2) cytokines in mice. Fermentation media enhanced the anti-C. acnes activity of a low dose (0.1%) clindamycin, a prescription antibiotic commonly used to treat acne vulgaris, in terms of the suppression of C. acnes colonization and MIP-2 production. Furthermore, PEG-8 Laurate fermentation of S. epidermidis boosted the activity of 0.1% clindamycin to reduce the sizes of C. acnes colonies. Our results demonstrated, for the first time, that the PEG-8 Laurate fermentation of S. epidermidis displayed the adjuvant effect on promoting the efficacy of low-dose clindamycin against C. acnes. Targeting C. acnes by lowering the required doses of antibiotics may avoid the risk of creating drug-resistant C. acnes and maintain the bacterial homeostasis in the skin microbiome, leading to a novel modality for the antibiotic treatment of acne vulgaris.

1674-P: Stearate Potentiates MIP-1a Production with TNF-a via TLR4/TBK1/IKKe /IRF3 Signal Axis: Significance for Insulin Resistance

Increased levels of plasma free fatty acids (FAs) are an important contributor to chronic inflammation, insulin resistance and other obesity-related complications. However, the molecular mechanisms by which individual FAs contribute to obesity-associated inflammation and disorders remain incompletely understood. Here we report that dietary saturated FAs specifically stearate led to a profound production of macrophage inflammatory protein (MIP)-1α (CCL3) in the presence of TNF-α in monocytes, macrophages and adipose tissues. Using pharmacologic and genetic approaches, we identified the involvement of TLR4/TBK1/IKKε/IRF3 signal axis in this production of MIP-1α in response to stearate and TNF-α. Consistent with this, cultured adipose tissues from TLR4 knockout (KO) mice revealed that stearate and TNF-α treatment did not induce MIP-1α compared with TLR2 KO or wild type (C57BL/6) mice. Moreover, C57BL/6 mice fed a high fat diet (HFD) for 16 weeks showed elevated levels of plasma fatty acids, TNF-α and MIP-1α along with insulin resistance. MIP-1α was significantly correlated with TNF-α. Our human data shows that expression of TLR4/p-IRF3 was elevated in the PBMCs of obese individuals as compared to lean. Furthermore, elevated MIP-1α expression levels in obese fat tissues were significantly correlated with TNF-α and macrophage markers. In conclusion, these findings provide a mechanistic link between stearate and TNF-α for the overproduction of obesity associated MIP-1α, which could be used as a new target for the treatment of obesity-related chronic inflammation/insulin resistance. Disclosure S.P. Kochumon: None. F. Almulla: None. Funding Kuwait Foundation for the Advancement of Science (RA-AH-2016-007)

Distribution of dendritic cells in the septate uterus: An immunological perspective.

Septate uterus is associated with spontaneous abortion. Surgical intervention of the uterine septa (US) is frequently performed following spontaneous abortion; however, immunological mechanisms for spontaneous abortion in patients with septate uterus remain completely unknown. A total of 12 women with septate uterus who underwent hysteroscopic metroplasty and 10 women with uterine leiomyoma who underwent total hysterectomy were enrolled as the experimental and control groups, respectively. Immune cells, dendritic cells (DCs), macrophages, T cells, natural killer cells, invariant natural killer cells, and chemokine receptors in US and uterine myometrium tissue (UMT) were analyzed using flow cytometry and immunohistochemical staining. Additionally, the chemokine production of macrophage inflammatory protein 1 alpha (MIP-1α), regulated upon activation normal T-cell express sequence (RANTES), and macrophage inflammatory protein 3 beta (MIP-3β) from the viable cells obtained from the US and UMT samples was evaluated in an ex vivo study. The percentage of CD141+ DCs in US was significantly lower than that in UMT. Both US and UMT showed CCR1 and CCR5 expression on CD141+ DCs; however, the production of chemokines, MIP-1α, RANTES, and MIP-3β was abundant in UMT-obtained viable cells. The accumulation of CD141+ DCs was lower in US than that in UMT. This phenomenon may be caused by low chemokine productions in US. Our findings support the benefit of surgical intervention for septate uterus-that is, the elimination of inappropriate implantation sites.

Copy number variation of CCL3L1 among three major ethnic groups in Malaysia

BackgroundC-C motif Chemokine Ligand 3 Like 1 (CCL3L1) is a multiallelic copy number variable, which plays a crucial role in immunoregulatory and hosts defense through the production of macrophage inflammatory protein (MIP)-1α. Variable range of the CCL3L1 copies from 0 to 14 copies have been documented in several different populations. However, there is still lack of report on the range of CCL3L1 copy number exclusively among Malaysians who are a multi-ethnic population. Thus, this study aims to extensively examine the distribution of CCL3L1 copy number in the three major populations from Malaysia namely Malay, Chinese and Indian. A diploid copy number of CCL3L1 for 393 Malaysians (Malay = 178, Indian = 90, and Chinese = 125) was quantified using Paralogue Ratio Tests (PRTs) and then validated with microsatellites analysis.ResultsTo our knowledge, this is the first report on the CCL3L1 copy number that has been attempted among Malaysians and the Chinese ethnic group exhibits a diverse pattern of CCL3L1 distribution copy number from the Malay and Indian (p < 0.0001). The CCL3L1 ranged from 0 to 8 copies for both the Malay and Indian ethnic groups while 0 to 10 copies for the Chinese ethnic. Consequently, the CCL3L1 copy number among major ethnic groups in the Malaysian population is found to be significantly varied when compared to the European population (p < 0.0001). The mean/median reported for the Malay, Chinese, Indian, and European are 2.759/2.869, 3.453/3.290, 2.437/1.970 and 2.001/1.940 respectively.ConclusionThis study reveals the existence of genetic variation of CCL3L1 in the Malaysian population, and suggests by examining genetic diversity on the ethnicity, and specific geographical region could help in reconstructing human evolutionary history and for the prediction of disease risk related to the CCL3L1 copy number.

A Microtube Array Membrane (MTAM) Encapsulated Live Fermenting Staphylococcus epidermidis as a Skin Probiotic Patch against Cutibacterium acnes.

Antibiotics without selectivity for acne treatment may destroy the beneficial microbes in the human microbiome that helps to fight Cutibacterium acnes (C. acnes), a bacterium associated with inflammatory acne vulgaris. Probiotic treatment by direct application of live Staphylococcus epidermidis (S. epidermidis) onto the open acne lesions may run the risk of bloodstream infections. Here, we fabricated the polysulfone microtube array membranes (PSF MTAM) to encapsulate probiotic S. epidermidis. We demonstrate that the application of the encapsulation of S. epidermidis in PSF MTAM enhanced the glycerol fermentation activities of S. epidermidis. To mimic the granulomatous type of acne inflammatory acne vulgaris, the ears of mice were injected intradermally with C. acnes to induce the secretion of macrophage inflammatory protein-2 (MIP-2), a murine counterpart of human interleukin (IL)-8. The C. acnes-injected mouse ears were covered with a PST MTAM encapsulated with or without S. epidermidis in the presence of glycerol. The application of S. epidermidis-encapsulated PST MTAM plus glycerol onto the C. acnes-injected mouse ears considerably reduced the growth of C. acnes and the production of MIP-2. Furthermore, no S. epidermidis leaked from PSF MTAM into mouse skin. The S. epidermidis-encapsulated PST MTAM functions as a probiotic acne patch.

Triptolide mitigates radiation-induced pneumonitis via inhibition of alveolar macrophages and related inflammatory molecules.

Ionizing radiation-induced pulmonary injury is a major limitation of radiotherapy for thoracic tumors. We have demonstrated that triptolide (TPL) could alleviate IR-induced pneumonia and pulmonary fibrosis. In this study, we explored the underlying mechanism by which TPL mitigates the effects of radiotoxicity. The results showed that:(1) Alveolar macrophages (AMs) were the primary inflammatory cells infiltrating irradiated lung tissues and were maintained at a high level for at least 17 days, which TPL could reduce by inhibiting of the production of macrophage inflammatory protein-2 (MIP-2) and its receptor CXCR2.(2) Stimulated by the co-cultured irradiated lung epithelium, AMs produced a panel of inflammative molecules (IMs), such as cytokines (TNF-α, IL-6, IL-1α, IL-1β) and chemokines (MIP-2, MCP-1, LIX). TPL-treated AMs could reduce the production of these IMs. Meanwhile, AMs isolated from irradiated lung tissue secreted significantly high levels of IMs, which could be dramatically reduced by TPL.(3) TPL suppressed the phagocytosis of AMs as well as ROS production.Our results indicate that TPL mitigates radiation-induced pulmonary inflammation through the inhibition of the infiltration, IM secretion, and phagocytosis of AMs.

Suppression of colitis-associated carcinogenesis through modulation of IL-6/STAT3 pathway by balsalazide and VSL#3.

Recent studies suggest that the anti-inflammatory agent balsalazide (BSZ) and probiotic agent VSL#3 have potential therapeutic benefits for the treatment of patients with inflammatory bowel disease. However, their effectiveness in preventing colitis-associated carcinogenesis (CAC) remains uncertain. The aim of the present study was to determine the chemopreventive effects of BSZ and VSL#3 in the murine azoxymethane (AOM)/dextran sodium sulfate (DSS) model. C57B/L6J mice were randomly divided into four groups: CAC group, BSZ group, VSL#3 group, and BSZ + VSL#3 group. After 2 weeks, the AOM/DSS model was induced by AOM injection followed by two cycles of 2% DSS. During first and second cycles of DSS, the number of F4/80-positive macrophages was significantly lower in the drug-treated groups compared with the CAC group (P < 0.05). At the endpoint, the total numbers of tumors in the drug-treated groups were significantly low compared with the CAC group (P < 0.05), and the drug-treated groups had significantly lower F4/80-positive macrophages in the tumor stroma (P < 0.01). The protein production of macrophage inflammatory protein 1 beta, monocyte chemoattractant protein-1, interleukin (IL)-6, and IL-10 in the colon tissues decreased in concordance with the plasma concentrations of the cytokines (P < 0.05). The drug-treated groups revealed lower expression of p-STAT3 compared with the CAC group. In addition, BCL2 decreased, and BAX increased markedly in the BSZ + VSL#3 group. These results revealed that BSZ and VSL#3 have chemopreventive effects against CAC through IL-6/STAT3 suppression. BSZ and VSL#3 could be suitable options for chemoprevention of colorectal cancer.

Toll-Like Receptor 4 Agonistic Antibody Promotes Host Defense against Chronic Pseudomonas aeruginosa Lung Infection in Mice.

Chronic lower respiratory tract infection with Pseudomonas aeruginosa is difficult to treat due to enhanced antibiotic resistance and decreased efficacy of drug delivery to destroyed lung tissue. To determine the potential for restorative immunomodulation therapies, we evaluated the effect of Toll-like receptor 4 (TLR4) stimulation on the host immune response to Pseudomonas infection in mice. We implanted sterile plastic tubes precoated with P. aeruginosa in the bronchi of mice, administered the TLR4/MD2 agonistic monoclonal antibody UT12 intraperitoneally every week, and subsequently analyzed the numbers of viable bacteria and inflammatory cells and the levels of cytokines. We also performed flow cytometry-based phagocytosis and opsonophagocytic killing assays in vitro using UT12-treated murine peritoneal neutrophils. UT12-treated mice showed significantly enhanced bacterial clearance, increased numbers of Ly6G(+) neutrophils, and increased concentrations of macrophage inflammatory protein 2 (MIP-2) in the lungs (P < 0.05). Depletion of CD4(+) T cells eliminated the ability of the UT12 treatment to improve bacterial clearance and promote neutrophil recruitment and MIP-2 production. Additionally, UT12-pretreated peritoneal neutrophils exhibited increased opsonophagocytic killing activity via activation of the serine protease pathway, specifically neutrophil elastase activity, in a TLR4-dependent manner. These data indicated that UT12 administration significantly augmented the innate immune response against chronic bacterial infection, in part by promoting neutrophil recruitment and bactericidal function.

Cardiac RNA Induces Inflammatory Responses in Cardiomyocytes and Immune Cells via Toll-like Receptor 7 Signaling

We have recently reported that extracellular RNA (exRNA) released from necrotic cells induces cytokine production in cardiomyocytes and immune cells and contributes to myocardial ischemia/reperfusion injury. However, the signaling mechanism by which exRNA exhibits its pro-inflammatory effect is unknown. Here we hypothesize that exRNA directly induces inflammation through specific Toll-like receptors (TLRs). To test the hypothesis, we treated rat neonatal cardiomyocytes, mouse bone marrow-derived macrophages (BMDM), or mouse neutrophils with RNA (2.5-10 μg/ml) isolated from rat cardiomyocytes or the hearts from mouse, rat, and human. We found that cellular RNA induced production of several cytokines such as macrophage inflammatory protein-2 (MIP-2), ILs, TNFα, and the effect was completely diminished by RNase, but not DNase. The RNA-induced cytokine production was partially inhibited in cells treated with TLR7 antagonist or genetically deficient in TLR7. Deletion of myeloid differentiation primary response protein 88 (MyD88), a downstream adapter of TLRs including TLR7, abolished the RNA-induced MIP-2 production. Surprisingly, genetic deletion of TLR3 had no impact on the RNA-induced MIP-2 response. Importantly, extracellular RNA released from damaged cardiomyocytes also induced cytokine production. Finally, mice treated with 50 μg of RNA intraperitoneal injection exhibited acute peritonitis as evidenced by marked neutrophil and monocyte migration into the peritoneal space. Together, these data demonstrate that exRNA of cardiac origin exhibits a potent pro-inflammatory property in vitro and in vivo and that exRNA induces cytokine production through TLR7-MyD88 signaling.

Attenuated phagocytosis of secondary necrotic neutrophils by macrophages in aged and SMP30 knockout mice.

Secondary necrotic cells generated in vivo induce inflammatory responses; for example, the production of macrophage inflammatory protein-2 (MIP-2) and subsequent infiltration of neutrophils. The aim of the present study was to elucidate the effect of aging on the phagocytosis of secondary necrotic cells and the inflammatory responses by using either wild-type (WT) young mice, WT aged mice or senescence-accelerated mice (SMP30(-/-) mice). The phagocytosis of secondary necrotic neutrophils with resident macrophage from either WT young mice, WT aged mice or SMP30(-/-) mice was examined by coculturing macrophages with secondary necrotic neutrophils in vitro. To investigate the inflammatory response induced by secondary necrotic cells, time-dependent infiltration of neutrophils and production of MIP-2 were determined in the peritoneal cavity on the injection of secondary necrotic cells. The phagocytosis of secondary necrotic cells by macrophages from WT aged and SMP30(-/-) mice was significantly reduced as compared with that by macrophages from WT young mice. On peritoneal injection of secondary necrotic cells, the peak time of neutrophil infiltration was earlier in SMP30(-/-) mice than in WT young mice. The number of neutrophils in SMP30(-/-) mice at the peak time was also greater than that in WT young mice. Our findings showed that the phagocytosis of secondary necrotic cells was attenuated in aged mice and SMP30(-/-) mice, and that the MIP-2 production was enhanced and subsequently neutrophil infiltration was exaggerated on peritoneal injection of secondary necrotic cells into those mice.

β Common Receptor Mediates Erythropoietin-Conferred Protection on OxLDL-Induced Lipid Accumulation and Inflammation in Macrophages.

Erythropoietin (EPO), the key factor for erythropoiesis, also protects macrophage foam cells from lipid accumulation, yet the definitive mechanisms are not fully understood. β common receptor (βCR) plays a crucial role in the nonhematopoietic effects of EPO. In the current study, we investigated the role of βCR in EPO-mediated protection in macrophages against oxidized low-density lipoprotein- (oxLDL-) induced deregulation of lipid metabolism and inflammation. Here, we show that βCR expression was mainly in foamy macrophages of atherosclerotic aortas from apolipoprotein E-deficient mice. Results of confocal microscopy and immunoprecipitation analyses revealed that βCR was colocalized and interacted with EPO receptor (EPOR) in macrophages. Inhibition of βCR activation by neutralizing antibody or small interfering RNA (siRNA) abolished the EPO-conferred protection in oxLDL-induced lipid accumulation. Furthermore, EPO-promoted cholesterol efflux and upregulation of ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 were prevented by pretreatment with βCR neutralizing antibody or βCR siRNA. Additionally, blockage of βCR abrogated the EPO-conferred anti-inflammatory action on oxLDL-induced production of macrophage inflammatory protein-2. Collectively, our findings suggest that βCR may play an important role in the beneficial effects of EPO against oxLDL-elicited dysfunction of macrophage foam cells.

Neutrophil elastase inhibitor reduces ventilation-induced lung injury via nuclear factor-κB and NF-κB repressing factor in mice.

Mechanical ventilation used in patients with acute lung injury can damage pulmonary epithelial cells through production of inflammatory cytokines, oxygen radicals, and neutrophil infiltration, termed ventilator-induced lung injury. Neutrophil elastase, nuclear factor-κB (NF-κB), and NF-κB repressing factor (NRF) have previously been shown to participate in the regulation of macrophage inflammatory protein-2 (MIP-2) during airway inflammation. However, the mechanisms regulating interactions among mechanical ventilation, neutrophil influx, and NF-κB/NRF remain unclear. Thus, we hypothesized that neutrophil elastase inhibitor attenuated ventilation-induced neutrophil recruitment and MIP-2 production through inhibition of the NF-κB/NRF pathway. Male C57BL/6 mice were exposed to low-tidal-volume (6 mL/kg) or high-tidal-volume (30 mL/kg) mechanical ventilation using room air with or without 2 µg/g NF-κB inhibitor SN50 or 6 µg/g NRF short interfering RNA or 100 µg/g neutrophil elastase inhibitor administration. Nonventilated mice served as a control group. Evan blue dye, lung wet-to-dry weight ratio, free radicals, myeloperoxidase, histopathologic grading of lung tissue, inflammatory cytokines, Western blot of NF-κB and NRF, and gene expression of NRF were measured to establish the extent of lung injury. Neutrophil elastase inhibitor ameliorated high-tidal-volume ventilation-induced lung injury, neutrophil influx, production of MIP-2 and malondialdehyde, activation of NF-κB and NRF, apoptotic epithelial cell death, and disruption of bronchial microstructure in mice. Mechanical stretch-augmented acute lung injury was also attenuated through pharmacological inhibition of NF-κB activity by SN50 and NRF expression by NRF short interfering RNA. Our data suggest that neutrophil elastase inhibitor attenuates high-tidal-volume mechanical ventilation-induced neutrophil influx, oxidative stress, and production of MIP-2, at least partly, through inhibition of NF-κB/NRF pathway. Understanding the protective effects of neutrophil elastase inhibitor associated with the reduction of MIP-2 allow clarification of the pathophysiological mechanisms regulating severe lung inflammation and development of possible therapeutic strategies involved in acute lung injury.

마우스 대식세포를 이용한 일당귀 물추출물의 항염효능 연구

Objectives : The purpose of this study was to investigate the effects of Angelicae acutilobae Radix Water Extract (AA) on the production of cytokines in RAW 264.7 cell stimulated with lipopolysaccharide (LPS). Method : RAW 264.7 cells were cotreated with AA (50 and <TEX>$100{\mu}g/mL$</TEX>) and lipopolysaccharide (LPS; <TEX>$1{\mu}g/mL$</TEX>) for 24 hours. After 24 hours treatment, using bead-based multiplex cytokine assay, concentrations of various cytokines such as interleukin(IL)-6, tumor necrosis factor-alpha(TNF-<TEX>${\alpha}$</TEX>) granulocyte colony-stimulating factor(G-CSF), granulocyte macrophage colony-stimulating factor(GM-CSF), and macrophage inflammatory protein(MIP)-<TEX>$1{\alpha}$</TEX> were measured. Result : AA significantly inhibited LPS-induced production of IL-6 and MIP-<TEX>$1{\alpha}$</TEX> from LPS-stimulated RAW 264.7 cells at the concentration of <TEX>$50{\mu}g/mL$</TEX>. AA significantly inhibited LPS-induced production of TNF-<TEX>${\alpha}$</TEX> from LPS-stimulated RAW 264.7 cells at the concentration of <TEX>$100{\mu}g/mL$</TEX>. AA significantly inhibited LPS-induced production of G-CSF and GM-CSF in RAW 264.7 cells at the concentrations of 50 and <TEX>$100{\mu}g/mL$</TEX>. Conclusion : These results suggest that AA has anti-inflammatory effect related with its inhibition of proinflammatory cytokines such as IL-6, TNF-<TEX>${\alpha}$</TEX>, G-CSF, GM-CSF, and MIP-<TEX>$1{\alpha}$</TEX> in LPS-induced macrophages.

A Murine Model Of Hemorrhagic Shock/Resuscitation Demonstrates Transfusion Related Acute Lung Injury (TRALI) Induced By Stored Platelet Transfusions

Transfusion related acute lung injury (TRALI) is one of the leading causes of transfusion fatalities. There have been several animal models of TRALI developed including ex vivo lung models demonstrating the importance of human anti-neutrophil antibodies in TRALI and in vivo transfusion models showing how biological response modifiers can induce recipient lung damage. In addition, an in vivo antibody-mediated TRALI model has also been established and has shown close similarities with human TRALI. Most of the animal models have a two-hit paradigm where the first hit is delivered in the form of a noxious substance such as lipopolysaccharide and the second hit is a transfusion of either a blood product or an antibody. Here we describe a novel clinically relevant two-hit model where the first hit is hemorrhagic shock followed by a second hit in the form of a platelet transfusion. Severe combined immunodeficient (SCID) mice were anesthetized and had half of their total blood volume was removed via a carotid cannula to instigate hypovolemic shock. After one hour of shock, the mice were reperfused with either Ringer's lactate solution or fresh or three day aged platelets. 34-1-2s, a monoclonal anti-mouse MHC class I antibody known to induce TRALI was used as a positive reperfusion control. Two hours following reperfusion, the mice were sacrificed and blood was obtained via cardiac puncture in order to measure serum levels of the neutrophil chemoattractant macrophage inflammatory protein-2 (MIP-2). Lungs were removed to determine the percentage of accumulated neutrophils and to measure edema. Compared with control mice reperfused with Ringer's lactate solution or mice administered fresh platelets, serum MIP-2 levels in mice administered with three day aged platelets were significantly elevated and were comparable to those observed in mice infused with 34-1-2s. Pulmonary neutrophil accumulation in the mice transfused with 3 day stored platelets was also elevated to levels observed in 34-1-2s treated mice. Pulmonary edema as measured by lung Wet/Dry ratios was elevated in mice receiving the 3 day aged platelets. In vivo administration of a MIP-2 antagonist (SB225002, 4mg/kg) intraperitoneally immediately before reperfusion significantly reduced all TRALI symptoms. These results show that an animal model of hypovolemic shock exhibits TRALI upon reperfusion with stored platelets and is dependent on MIP-2 production. This clinically-relevant model will be valuable in studying potential therapeutic interventions to prevent TRALI following transfusions, Disclosures:No relevant conflicts of interest to declare.

Myeloperoxidase deficiency in mice exacerbates lung inflammation induced by nonviable Candida albicans

This study aimed to evaluate the effect of myeloperoxidase (MPO) deficiency on lung inflammation induced by nonviable Candida albicans (nCA). Mice were inoculated intranasally with nCA, and accumulation of neutrophils and macrophages in the bronchoalveolar lavage fluid was analyzed by flow cytometry. The levels of macrophage inflammatory protein 2 (MIP-2), keratinocyte-derived chemokine (KC), tumor necrosis factor (TNF)-α, and interleukin (IL)-1β in the lung were measured by ELISA. Production of MIP-2 and KC from neutrophils and macrophages was quantified in vitro. MIP-2 mRNA expression in the neutrophils was analyzed by real-time reverse transcription-PCR, and the extent of phosphorylation of ERK1/2 and Syk in the neutrophils was analyzed by Western blotting. The MPO(-/-) mice that received nCA showed more severe pneumonia than wild-type mice. Within 12 h of nCA administration, MPO(-/-) mice had significantly higher numbers of alveolar neutrophils and increased production of MIP-2 and KC relative to the responses seen in wild-type mice. Neutralization of MIP-2 and KC in vivo significantly reduced neutrophil infiltration. In vitro, production of MIP-2, but not that of KC, was enhanced in the nCA-stimulated neutrophils from MPO(-/-) mice, concomitant with up-regulation of Syk and ERK1/2. At 1 and 3 days after nCA administration, MPO(-/-) mice had significantly higher lung concentrations of TNF-α and IL-1β than wild-type mice. Pulmonary administration of nCA produced an altered inflammatory response in MPO(-/-) mice relative to wild-type mice. Enhanced MIP-2 production by MPO(-/-) neutrophils may at least partly contribute to exacerbated inflammation in mutant mice.

Suppressing NF-κB and NKRF Pathways by Induced Pluripotent Stem Cell Therapy in Mice with Ventilator-Induced Lung Injury

BackgroundHigh-tidal-volume mechanical ventilation used in patients with acute lung injury (ALI) can induce the release of inflammatory cytokines, as macrophage inflammatory protein-2 (MIP-2), recruitment of neutrophils, and disruption of alveolar epithelial and endothelial barriers. Induced pluripotent stem cells (iPSCs) have been shown to improve ALI in mice, but the mechanisms regulating the interactions between mechanical ventilation and iPSCs are not fully elucidated. Nuclear factor kappa B (NF-κB) and NF-κB repressing factor (NKRF) have been proposed to modulate the neutrophil activation involved in ALI. Thus, we hypothesized intravenous injection of iPSCs or iPSC-derived conditioned medium (iPSC-CM) would decrease high-tidal-volume ventilation-induced neutrophil infiltration, oxidative stress, and MIP-2 production through NF-κB/NKRF pathways.MethodsMale C57BL/6 mice, aged between 6 and 8 weeks, weighing between 20 and 25 g, were exposed to high-tidal-volume (30 ml/kg) mechanical ventilation with room air for 1 to 4 h after 5×107 cells/kg mouse iPSCs or iPSC-CM administration. Nonventilated mice were used as control groups.ResultsHigh-tidal-volume mechanical ventilation induced the increases of integration of iPSCs into the injured lungs of mice, microvascular permeability, neutrophil infiltration, malondialdehyde, MIP-2 production, and NF-κB and NKRF activation. Lung injury indices including inflammation, lung edema, ultrastructure pathologic changes and functional gas exchange impairment induced by mechanical ventilation were attenuated with administration of iPSCs or iPSC-CM, which was mimicked by pharmacological inhibition of NF-κB activity with SN50 or NKRF expression with NKRF short interfering RNA.ConclusionsOur data suggest that iPSC-based therapy attenuates high-tidal-volume mechanical ventilation-induced lung injury, at least partly, through inhibition of NF-κB/NKRF pathways. Notably, the conditioned medium of iPSCs revealed beneficial effects equal to those of iPSCs.

Myeloperoxidase deficiency induces MIP-2 production via ERK activation in zymosan-stimulated mouse neutrophils

Myeloperoxidase (MPO), a major constituent of neutrophils, catalyzes the production of hypochlorous acid (HOCl) from hydrogen peroxide (H2O2) and chloride anion. We have previously reported that MPO-deficient (MPO−/−) neutrophils produce greater amount of macrophage inflammatory protein-2 (MIP-2) in vitro than do wild type when stimulated with zymosan. In this study, we investigated the molecular mechanisms governing the up-regulation of MIP-2 production in the mutant neutrophils. Interestingly, we found that zymosan-induced production of MIP-2 was blocked by pre-treatment with U0126, an inhibitor of mitogen-activated protein kinase/extracellular-signal-regulated kinase (ERK), and with BAY11-7082, an inhibitor of nuclear factor (NF)-κB. Western blot analysis indicated that U0126 also inhibited the phosphorylation of p65 subunit of NF-κB (p65), indicating that MIP-2 was produced via the ERK/NF-κB pathway. Intriguingly, we found that ERK1/2, p65, and alpha subunit of inhibitor of κB (IκBα) in the MPO−/− neutrophils were phosphorylated more strongly than in the wild type when stimulated with zymosan. Exogenous H2O2 treatment in addition to zymosan stimulation enhanced the phosphorylation of ERK1/2 without affecting the zymosan-induced MIP-2 production. In contrast, exogenous HOCl inhibited the production of MIP-2 as well as IκBα phosphorylation without affecting ERK activity. The zymosan-induced production of MIP-2 in the wild-type neutrophils was enhanced by pre-treatment of the MPO inhibitor 4-aminobenzoic acid hydrazide. Collectively, these results strongly suggest that both lack of HOCl and accumulation of H2O2 due to MPO deficiency contribute to the up-regulation of MIP-2 production in mouse neutrophils stimulated with zymosan.

Blockade of Airway Inflammation by Kaempferol via Disturbing Tyk-STAT Signaling in Airway Epithelial Cells and in Asthmatic Mice

Asthma is characterized by bronchial inflammation causing increased airway hyperresponsiveness and eosinophilia. The interaction between airway epithelium and inflammatory mediators plays a key role in the asthmatic pathogenesis. The in vitro study elucidated inhibitory effects of kaempferol, a flavonoid found in apples and many berries, on inflammation in human airway epithelial BEAS-2B cells. Nontoxic kaempferol at ≤20 μM suppressed the LPS-induced IL-8 production through the TLR4 activation, inhibiting eotaxin-1 induction. The in vivo study explored the demoting effects of kaempferol on asthmatic inflammation in BALB/c mice sensitized with ovalbumin (OVA). Mouse macrophage inflammatory protein-2 production and CXCR2 expression were upregulated in OVA-challenged mice, which was attenuated by oral administration of ≥10 mg/kg kaempferol. Kaempferol allayed the airway tissue levels of eotaxin-1 and eotaxin receptor CCR3 enhanced by OVA challenge. This study further explored the blockade of Tyk-STAT signaling by kaempferol in both LPS-stimulated BEAS-2B cells and OVA-challenged mice. LPS activated Tyk2 responsible for eotaxin-1 induction, while kaempferol dose-dependently inhibited LPS- or IL-8-inflamed Tyk2 activation. Similar inhibition of Tyk2 activation by kaempferol was observed in OVA-induced mice. Additionally, LPS stimulated the activation of STAT1/3 signaling concomitant with downregulated expression of Tyk-inhibiting SOCS3. In contrast, kaempferol encumbered STAT1/3 signaling with restoration of SOCS3 expression. Consistently, oral administration of kaempferol blocked STAT3 transactivation elevated by OVA challenge. These results demonstrate that kaempferol alleviated airway inflammation through modulating Tyk2-STAT1/3 signaling responsive to IL-8 in endotoxin-exposed airway epithelium and in asthmatic mice. Therefore, kaempferol may be a therapeutic agent targeting asthmatic diseases.