TLR4 signaling-induced heme oxygenase upregulation in the acute lung injury: role in hemorrhagic shock and two-hit induced lung inflammation

Resuscitated hemorrhagic shock is believed to promote the development of acute lung injury (ALI) by priming the immune system for an exaggerated inflammatory response to a second trivial stimulus. This work explored effects of TLR4 on hemorrhage-induced ALI and “second-hit” responses, and further explore the mechanisms involved in “second-hit” responses. Expression of HO-1, IL-10, lung W/D and MPO markedly increased at nearly all time-points examined in HSR/LPS group as compared with sham/LPS group in WT mice. In HSR/LPS mice, the induced amount of IL-10 and the expressions of HO-1 of WT mice were significantly higher compared with TLR-4d/d. This study provides in vivo evidence that pulmonary infections after LPS instillation contribute to local tissue release of pro-inflammatory mediators after HSR systemic. Activation of TLR4 might induce HO-1 expression and HO-1 modulates proinflammatory responses that are triggered via TLR4 signaling.     

An overview of cytokines and heat shock response in polytraumatized patients

Early after injury, local tissue damage induces a local and systemic inflammatory response that activates the immune system and leads to the development of systemic inflammatory response syndrome (SIRS). This post-traumatic response often results in uncontrolled release of inflammatory mediators and over-activation of the immune system, which occasionally results in multiple organ dysfunction syndrome (MODS). In parallel, a state of immunosuppression develops. This counter-regulating suppression of different cellular and humoral immune functions has been termed “compensatory anti-inflammatory response syndrome (CARS).” Both SIRS and CARS occur simultaneously even in the initial phase after injury. Pro- and anti-inflammatory cytokines have been suggested to play a major role in development of SIRS, although the degree of involvement of the different cytokines is quite disparate. While TNF-α and IL-1β are quite irrelevant for predicting organ dysfunction, IL-6 is the parameter that best predicts mortality. The hyperinflammatory state seems to be the cause of post-traumatic immunosuppression and heat shock proteins (HSPs), which have been proposed as one of the endogenous stimuli for the deterioration of the immune system acting as danger-associated molecular patterns (DAMPs). Extracellular HSPA1A released from injured tissues increase up to ten times immediately after trauma and even more in patients with MODS. It has powerful immune properties that could contribute to post-traumatic immunosuppression through several mechanisms that have been previously described, so HSPs could represent trauma-associated immunomodulatory mediators. For this reason, HSPA1A has been suggested to be a helpful early prognostic biomarker of trauma after severe injury: serial quantification of serum HSPA1A and anti-Hsp70 concentrations in the first hours after trauma is proposed to be used as a predictive biomarker of MODS and immunosuppression development in polytraumatized patients.     

Functions and cellular signaling by ribosomal extracellular RNA (rexRNA): Facts and hypotheses on a non-typical DAMP

Upon microbial infections with the subsequent host response of innate immunity, a variety of fragmented RNA- and DNA-based “Pathogen-associated molecular patterns” (PAMPs) are recognized mainly by endosomal or cytoplasmic host cell “Pattern recognition receptors” (PRRs), particularly “Toll-like receptors” (TLRs). Concomitantly, various self-extracellular RNA species (exRNAs) are present in extracellular body fluids where they contribute to diverse physiological and homeostatic processes. In principle, such exRNAs, including the most abundant one, ribosomal exRNA (rexRNA), are designated as “Danger-associated molecular patterns” (DAMPs) and are prevented by e.g. natural modifications from uncontrolled signaling via TLRs to avoid hyper-inflammatory responses or autoimmunity. Upon cellular stress or tissue damage/necrosis, the levels and composition of released self-exRNA species, either in free form, in complex with proteins or in association with extracellular vesicles (EVs), can change considerably. Among the self-exRNAs, rexRNA is considered as a non-typical DAMP, since it may induce inflammatory responses by cell membrane receptors, both in the absence or presence of PAMPs. Yet, its mode of receptor activation to mount inflammatory responses remains obscure. RexRNA also serves as a universal damaging factor in cardiovascular and other diseases independent of PRRs. In general, RNase1 provides a profound antagonist in these pathologies and in rexRNA-mediated inflammatory cell responses. Based on the extrapolation of the here described aspects of rexRNA-biology, further activities of this molecular entity are hypothesized that may stimulate additional research in this area.     

Intracellular signaling in neutrophil priming and activation

In order for neutrophils to function effectively in host defense, they have evolved specific attributes including the ability to migrate to the site of inflammation and release an array of toxic products including proteolytic enzymes, reactive oxygen species, and cationic proteins. While these compounds are intended for killing invading pathogens, if released inappropriately, they may also contribute to tissue damage. Such inflammatory tissue injury may be important in the pathogenesis of a variety of clinical disorders including arthritis, ischemia-reperfusion tissue injury, the systemic inflammatory response syndrome (SIRS), and the acute respiratory distress syndrome (ARDS). Despite the importance of neutrophil function in host defense and dysfunction in disease states, much remains unknown about the intracellular signaling pathways regulating neutrophil activity. This review will focus on the signaling molecules regulating leukocyte ‘effector’ functions including receptors, GTP-binding proteins, phospholipases, polyphosphoinositide metabolism, and protein kinases and phosphatases.     

The acute phase response of rainbow trout (Oncorhynchus mykiss) plasma proteins to viral, bacterial and fungal inflammatory agents

The innate arm of the immune system responds to inflammatory stimuli by the activation of phagocytes, and by altered levels of several plasma proteins. These changes in plasma proteins comprise a major component of the acute phase response, which is thought to be an adaptive response that contributes to regaining homeostasis after tissue injury or infection. In this study, rainbow trout (Oncorhynchus mykiss) were injected with a variety of potential inflammatory agents, and changes in the concentrations of plasma proteins were sought in polyacrylamide gels in which plasma proteins had been electrophoresed. Bacteria, viruses and yeast all induced changes in plasma protein profiles. Increases were first evident 2 days after injections, and most were evident within 1 week. The greatest number of changes occurred after injection with a Vibrio bacterin emulsified in Freund's incomplete adjuvant. While some proteins increased and others decreased following several treatments, other proteins changed only in response to injections of viruses or viral proteins, and others changed in response to bacterial components. Some proteins that increased after yeast injection decreased after injection of viral components. The partial amino acid sequence of one increased protein identified it as haptoglobin.     

Lipids,oxidized lipids,oxidation-specific epitopes,and Age-related Macular Degeneration

Age-related Macular Degeneration (AMD) is the leading cause of blindness among the elderly in western societies. While antioxidant micronutrient treatment is available for intermediate non-neovascular disease, and effective anti-vascular endothelial growth factor treatment is available for neovascular disease, treatment for early AMD is lacking due to an incomplete understanding of the early molecular events. The role of lipids, which accumulate in the macula, and their oxidation, has emerged as an important factor in disease development. These oxidized lipids can either directly contribute to tissue injury or react with amine on proteins to form oxidation-specific epitopes, which can induce an innate immune response. If inadequately neutralized, the inflammatory response from these epitopes can incite tissue injury during disease development. This review explores how the accumulation of lipids, their oxidation, and the ensuing inflammatory response might contribute to the pathogenesis of AMD. This article is part of a Special Issue entitled: Lipid modification and lipid peroxidation products in innate immunity and inflammation edited by Christoph J. Binder .     

Immunomodulatory activity of extracellular heat shock proteins and their autoantibodies

HSP are groups of stress‐inducible proteins which contribute to quality control by assisting the correct folding of both nascent and denatured proteins, and promoting the degradation of unrecoverable denatured proteins. HSP also help to maintain cellular homeostasis and protect from cell death through a mechanism called thermotolerance. Cells subjected to mild stress induce HSP which then protect them against subsequent stress. However, in cells subjected to severe stress, HSP promote apoptosis. Besides these intracellular events, HSP also exist in extracellular fluids, and have been shown to contribute to immunomodulation. In innate immunity extracellular HSP, like various microbial substances, induce various proinflammatory cytokines. In acquired immunity they interact with antigenic polypeptides and assist in antigen presentation. The extracellular HSP are so‐called adjuvant. Release of HSP from cells is triggered by stress and trauma, and is thus regarded as an immunological “danger signal”. In addition, anti‐HSP autoantibodies are frequently found in patients with autoimmune diseases and inflammatory disorders, and these autoantibodies can modulate the “danger signal” triggered by extracellular HSP.     

Acute multiple organ failure in adult mice deleted for the developmental regulator Wt1

There is much interest in the mechanisms that regulate adult tissue homeostasis and their relationship to processes governing foetal development. Mice deleted for the Wilms' tumour gene, Wt1, lack kidneys, gonads, and spleen and die at mid-gestation due to defective coronary vasculature. Wt1 is vital for maintaining the mesenchymal-epithelial balance in these tissues and is required for the epithelial-to-mesenchyme transition (EMT) that generates coronary vascular progenitors. Although Wt1 is only expressed in rare cell populations in adults including glomerular podocytes, 1% of bone marrow cells, and mesothelium, we hypothesised that this might be important for homeostasis of adult tissues; hence, we deleted the gene ubiquitously in young and adult mice. Within just a few days, the mice suffered glomerulosclerosis, atrophy of the exocrine pancreas and spleen, severe reduction in bone and fat, and failure of erythropoiesis. FACS and culture experiments showed that Wt1 has an intrinsic role in both haematopoietic and mesenchymal stem cell lineages and suggest that defects within these contribute to the phenotypes we observe. We propose that glomerulosclerosis arises in part through down regulation of nephrin, a known Wt1 target gene. Protein profiling in mutant serum showed that there was no systemic inflammatory or nutritional response in the mutant mice. However, there was a dramatic reduction in circulating IGF-1 levels, which is likely to contribute to the bone and fat phenotypes. The reduction of IGF-1 did not result from a decrease in circulating GH, and there is no apparent pathology of the pituitary and adrenal glands. These findings 1) suggest that Wt1 is a major regulator of the homeostasis of some adult tissues, through both local and systemic actions; 2) highlight the differences between foetal and adult tissue regulation; 3) point to the importance of adult mesenchyme in tissue turnover.     

The Mechano-Ubiquitinome of Articular Cartilage: Differential Ubiquitination and Activation of a Group of ER-Associated DUBs and ER Stress Regulators

Understanding how connective tissue cells respond to mechanical stimulation is important to human health and disease processes in musculoskeletal diseases. Injury to articular cartilage is a key risk factor in predisposition to tissue damage and degenerative osteoarthritis. Recently, we have discovered that mechanical injury to connective tissues including murine and porcine articular cartilage causes a significant increase in lysine-63 polyubiquitination. Here, we identified the ubiquitin signature that is unique to injured articular cartilage tissue upon mechanical injury (the “mechano-ubiquitinome”). A total of 463 ubiquitinated peptides were identified, with an enrichment of ubiquitinated peptides of proteins involved in protein processing in the endoplasmic reticulum (ER), also known as the ER-associated degradation response, including YOD1, BRCC3, ATXN3, and USP5 as well as the ER stress regulators, RAD23B, VCP/p97, and Ubiquilin 1. Enrichment of these proteins suggested an injury-induced ER stress response and, for instance, ER stress markers DDIT3/CHOP and BIP/GRP78 were upregulated following cartilage injury on the protein and gene expression levels. Similar ER stress induction was also observed in response to tail fin injury in zebrafish larvae, suggesting a generic response to tissue injury. Furthermore, a rapid increase in global DUB activity following injury and significant activity in human osteoarthritic cartilage was observed using DUB-specific activity probes. Combined, these results implicate the involvement of ubiquitination events and activation of a set of DUBs and ER stress regulators in cellular responses to cartilage tissue injury and in osteoarthritic cartilage tissues. This link through the ER-associated degradation pathway makes this protein set attractive for further investigation in in vivo models of tissue injury and for targeting in osteoarthritis and related musculoskeletal diseases.     

The effects of extracellular matrix proteins on neutrophil-endothelial interaction--a roadway to multiple therapeutic opportunities

Polymorphoneuclear leukocytes or neutrophils, a major component of white blood cells, contribute to the innate immune response in humans. Upon sensing changes in the microenvironment, neutrophils adhere to the vascular wall, migrate through the endothelial cell (EC)-pericyte bilayer, and subsequently through the extracellular matrix to reach the site of inflammation. These cells are capable of destroying microbes, cell debris, and foreign proteins by oxidative and non-oxidative processes. While primarily mediators of tissue homeostasis, there are an increasing number of studies indicating that neutrophil recruitment and transmigration can also lead to host-tissue injury and subsequently inflammation-related diseases. Neutrophil-induced tissue injury is highly regulated by the microenvironment of the infiltrated tissue, which includes cytokines, chemokines, and the provisional extracellular matrix, remodeled through increased vascular permeability and other cellular infiltrates. Thus, investigation of the effects of matrix proteins on neutrophil-EC interaction and neutrophil transmigration may help identify the proteins that induce pro- or anti-inflammatory responses. This area of research presents an opportunity to identify therapeutic targets in inflammation-related diseases. This review will summarize recent literature on the role of neutrophils and the effects of matrix proteins on neutrophil-EC interactions, with focus on three different disease models: 1) atherosclerosis, 2) COPD, and 3) tumor growth and progression. For each disease model, inflammatory molecules released by neutrophils, important regulatory matrix proteins, current anti-inflammatory treatments, and the scope for further research will be summarized.     

Passages in culture and stimulation conditions influence protein expression of primary fibroblasts

Fibroblasts (Fb) are key effector cells in systemic sclerosis (SSc). Fb stimulation with transforming growth factor beta 1 (TGF-β1) is considered as a positive control in studies assessing fibrogenesis. The lack of standardization of TGF-β1 stimulation might be responsible for discrepancies in experiments performed in different conditions. Using quantitative proteomics analysis, we evaluated the impact of changes in experimental conditions on proteomic profiles of primary Fb. Principal component analysis (PCA) identified several groups of differentially expressed proteins influenced by cell passage, culture medium, and both concentration and duration of exposure to TGF-β1 stimulation. Bioinformatics analysis revealed that late passages expressed proteins involved in senescence. TGF-β1 concentration and time of stimulation were correlated with the expression of proteins involved in the fibrogenesis and inflammatory processes. These data underline the need for standardization of culture conditions to allow inter-data comparisons in future in vitro studies, especially when using “omics” approaches.     

Periostin,a multifunctional matricellular protein in inflammatory and tumor microenvironments

The behavior and fate of cells in tissues largely rely upon their cross-talk with the tissue microenvironment including neighboring cells, the extracellular matrix (ECM), and soluble cues from the local and systemic environments. Dysregulation of tissue microenvironment can drive various inflammatory diseases and tumors. The ECM is a crucial component of tissue microenvironment. ECM proteins can not only modulate tissue microenvironment but also regulate the behavior of surrounding cells and the homeostasis of tissues. As a nonstructural ECM protein, periostin is generally present at low levels in most adult tissues; however, periostin is often highly expressed at sites of injury or inflammation and in tumors within adult organisms. Current evidence demonstrates that periostin actively contributes to tissue injury, inflammation, fibrosis and tumor progression. Here, we summarize the roles of periostin in inflammatory and tumor microenvironments.     

Free radical tissue damage: protective role of antioxidant nutrients

Highly reactive molecules called free radicals can cause tissue damage by reacting with polyunsaturated fatty acids in cellular membranes, nucleotides in DNA, and critical sulfhydryl bonds in proteins. Free radicals can originate endogenously from normal metabolic reactions or exogenously as components of tobacco smoke and air pollutants and indirectly through the metabolism of certain solvents, drugs, and pesticides as well as through exposure to radiation. There is some evidence that free radical damage contributes to the etiology of many chronic health problems such as emphysema, cardiovascular and inflammatory diseases, cataracts, and cancer. Defenses against free radical damage include tocopherol (vitamin E), ascorbic acid (vitamin C), beta-carotene, glutathione, uric acid, bilirubin, and several metalloenzymes including glutathione peroxidase (selenium), catalase (iron), and superoxide dismutase (copper, zinc, manganese) and proteins such as ceruloplasmin (copper). The extent of tissue damage is the result of the balance between the free radicals generated and the antioxidant protective defense system. Several dietary micronutrients contribute greatly to the protective system. Based on the growing interest in free radical biology and the lack of effective therapies for many of the chronic diseases, the usefulness of essential, safe nutrients in protecting against the adverse effects of oxidative injury warrants further study.     

Coordinated regulation of nutrient and inflammatory responses by STAMP2 is essential for metabolic homeostasis

Metabolic and inflammatory pathways crosstalk at many levels, and, while required for homeostasis, interaction between these pathways can also lead to metabolic dysregulation under conditions of chronic stress. Thus, we hypothesized that mechanisms might exist to prevent overt inflammatory responses during physiological fluctuations in nutrients or under nutrient-rich conditions, and we identified the six-transmembrane protein STAMP2 as a critical modulator of this integrated response system of inflammation and metabolism in adipocytes. Lack of STAMP2 in adipocytes results in aberrant inflammatory responses to both nutrients and acute inflammatory stimuli. Similarly, in whole animals, visceral adipose tissue of STAMP2(-/-) mice exhibits overt inflammation, and these mice develop spontaneous metabolic disease on a regular diet, manifesting insulin resistance, glucose intolerance, mild hyperglycemia, dyslipidemia, and fatty liver disease. We conclude that STAMP2 participates in integrating inflammatory and metabolic responses and thus plays a key role in systemic metabolic homeostasis.     

Two burgeoning families of platelet factor 4-related proteins: mediators of the inflammatory response

The inflammatory response involves the recruitment and activation of various types of cells from the systemic circulation and from local tissues. One important component of the inflammatory response is the activation of platelets at sites of tissue injury and inflammation. In particular, activated platelets release large amounts of two proteins, platelet factor 4 (PF4) and beta-thromboglobulin (beta TG), which mediate several inflammatory processes. Recently, many novel proteins that are structurally related to PF4 and beta TG have been identified. The PF4-related proteins are secreted by white blood cells, endothelial cells, and fibroblasts in response to various inflammatory and mitogenic stimuli. Like PF4, these proteins appear to be inflammatory response mediators; several of them are potent chemoattractants, activating agents, or mitogens for specific cell types that are involved in the inflammatory response. The study of PF4-related proteins provides new insight into the mechanisms of the immune response, and may result in the development of new therapeutic agents.     

Mobilization of copper among tissues in the estuarine crab Scylla serrata (Forskal) under imposed starvation

The quantitative changes in copper free and bound to proteins in haemolymph and different forms of copper in muscle and hepatopancreas under imposed starvation were studied in the estuarine mud crab Scylla serrata. During the course of starvation, both haemolymph copper free and bound to proteins significantly declined and the regression analyses of these data further revealed that the haemolymph copper-free proteins were more affected than copper-bound proteins. The multiple stress condition namely injury and exsanguination along with starvation resulted in an earlier release and/or degradation of both these proteins. Hepatopancreas periodically accumulates and releases copper during starvation. The copper levels in haemolymph and hepatopancreas during different days of starvation showed a close inverse relationship between these two tissues. These changes in hepatopancreas were predominantly reflected in the copper that exists in association with low molecular weight substances. It is found that the copper thus accumulated was partly released back into haemolymph and a fraction may be excreted. This study also indicates the major role played by the low molecular weight substances in accommodation, detoxification and mobilization of copper in the decapod hepatopancreas during imposed starvation.     

Oxidative Burst of Circulating Neutrophils Following Traumatic Brain Injury in Human

Besides secondary injury at the lesional site, Traumatic brain injury (TBI) can cause a systemic inflammatory response, which may cause damage to initially unaffected organs and potentially further exacerbate the original injury. Here we investigated plasma levels of important inflammatory mediators, oxidative activity of circulating leukocytes, particularly focusing on neutrophils, from TBI subjects and control subjects with general trauma from 6 hours to 2 weeks following injury, comparing with values from uninjured subjects. We observed increased plasma level of inflammatory cytokines/molecules TNF-α, IL-6 and CRP, dramatically increased circulating leukocyte counts and elevated expression of TNF-α and iNOS in circulating leukocytes from TBI patients, which suggests a systemic inflammatory response following TBI. Our data further showed increased free radical production in leukocyte homogenates and elevated expression of key oxidative enzymes iNOS, COX-2 and NADPH oxidase (gp91^phox) in circulating leukocytes, indicating an intense induction of oxidative burst following TBI, which is significantly greater than that in control subjects with general trauma. Furthermore, flow cytometry assay proved neutrophils as the largest population in circulation after TBI and showed significantly up-regulated oxidative activity and suppressed phagocytosis rate for circulating neutrophils following brain trauma. It suggests that the highly activated neutrophils might play an important role in the secondary damage, even outside the injured brain. Taken together, the potent systemic inflammatory response induced by TBI, especially the intensively increase oxidative activity of circulating leukocytes, mainly neutrophils, may lead to a systemic damage, dysfunction/damage of bystander tissues/organs and even further exacerbate secondary local damage. Controlling these pathophysiological processes may be a promising therapeutic strategy and will protect unaffected organs and the injured brain from the secondary damage.     

Syndecan-1 is an in vivo suppressor of Gram-positive toxic shock

Heparan sulfate proteoglycans bind to and regulate many inflammatory mediators in vitro, suggesting that they serve an important role in influencing inflammatory responses in vivo. Here we evaluated the role of syndecan-1, a major heparan sulfate proteoglycan, in modulating inflammatory responses in Gram-positive toxic shock, a systemic disease that is a significant cause of morbidity and mortality. Syndecan-1-null and wild-type mice were injected intraperitoneally with staphylococcal enterotoxin B, a pyrogenic superantigen, and their inflammatory responses were assessed. Syndecan-1-null mice showed significantly increased liver injury, vascular permeability, and death in response to staphylococcal enterotoxin B challenge compared with wild-type mice. Although serum levels of systemic IL-2 and IFNgamma were similar between the two backgrounds, those of TNFalpha and IL-6 were significantly increased in syndecan-1-null mice undergoing Gram-positive toxic shock. Furthermore, syndecan-1-null mice challenged with staphylococcal enterotoxin B showed enhanced T cell accumulation in tissues, whereas immunodepletion of T cells protected syndecan-1-null mice from the magnified systemic cytokine storm, inflammatory tissue injury, and death. Importantly, syndecan-1 shedding was induced in wild-type mice injected with staphylococcal enterotoxin B, and the administration of heparan sulfate, but not syndecan-1 core protein, rescued syndecan-1-null mice from lethal toxic shock by suppressing the production of TNFalpha and IL-6, and attenuating inflammatory tissue injury. Altogether, these data suggest that syndecan-1 shedding is a key endogenous mechanism that protects the host from Gram-positive toxic shock by inhibiting the dysregulation and amplification of the inflammatory response.