Damage-associated Molecular Pattern Signaling Research Articles

Green leaf volatiles co-opt proteins involved in molecular pattern signalling in plant cells.

The green leaf volatiles (GLVs) Z-3-hexen-1-ol (Z3-HOL) and Z-3-hexenyl acetate (Z3-HAC) are airborne infochemicals released from damaged plant tissues that induce defenses and developmental responses in receiver plants, but little is known about their mechanism of action. We found that Z3-HOL and Z3-HAC induce similar but distinctive physiological and signaling responses in tomato seedlings and cell cultures. In seedlings, Z3-HAC showed a stronger root growth inhibition effect than Z3-HOL. In cell cultures, the two GLVs induced distinct changes in MAP kinase (MAPK) activity and proton fluxes as well as rapid and massive changes in the phosphorylation status of proteins within 5 min. Many of these phosphoproteins are involved in reprogramming the proteome from cellular homoeostasis to stress and include pattern recognition receptors, a receptor-like cytoplasmic kinase, MAPK cascade components, calcium signaling proteinsand transcriptional regulators. These are well-known components of damage-associated molecular pattern (DAMP) signaling pathways. These rapid changes in the phosphoproteome may underly the activation of defense and developmental responses to GLVs. Our data provide further evidence that GLVs function like DAMPs and indicate that GLVs coopt DAMP signaling pathways.

Elevated secretion of pro-collagen I-alpha and vascular endothelial growth factor as biomarkers of acetabular labrum degeneration and calcification in hip osteoarthritis: An explant study

BackgroundHip osteoarthritis (OA) involves structural degeneration of different joint compartments, including femoral head cartilage, periarticular ligaments and the acetabular labrum. However, the molecular mechanisms underlying labrum degeneration in hip OA remain poorly understood. AimTo assess secretion of putative biomarkers for OA from explanted human labrum tissues under basal and inflammatory conditions and to determine whether these could differentiate between OA and calcification status compared to fracture controls. MethodsIntact labrum specimens were collected from patients undergoing joint arthroplasty for primary hip OA (n ​= ​15, mean age 70) or non-OA femoral neck fracture (n ​= ​5, mean age 64). Tissues were dissected in equal-sized samples and explanted for one week. To mimic activation of inflammatory signaling by endogenous damage-associated molecular patterns (DAMP) tissue were stimulated with a toll-like receptor 4 (TLR4) agonist (1 ​μg/mL LPS). The involvement of transforming growth factor-beta (TGF-beta) signaling was evaluated by treatment with a TGF-beta type 1 receptor inhibitor (10 ​μM SB-505124). Secretion of aggrecan (ACAN), pro-collagen-I alpha (Pro-Col-Iα), cartilage oligomeric matrix protein (COMP), interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) was assessed by enzyme-linked immunosorbent assay (ELISA). Labrum calcification was evaluated by 3D whole mount fluorescent microscopy of ethyl cinnamate-based optically cleared tissues stained with Alcian blue/Alizarin red. ResultsWhole mount microscopy revealed non-OA fracture controls were non-calcified, whereas six OA labra (40%) were partially calcified or ossified. Basal secretion of Pro-Col-Iα and VEGF was increased four-fold in OA versus non-OA labra. Pro-Col-Iα levels were correlated with those of VEGF (r ​= ​0.65) and COMP (r ​= ​0.54). Stimulation of DAMP signaling through TLR4 affected secretion of IL-6, VEGF, COMP and Pro-Col-Iα, with distinct responses between non-OA and OA tissues. Inhibition of TGF-beta signaling specifically reduced elevated secretion of Pro-Col- Iα and VEGF in calcified OA labrum. ConclusionsSecretion of the putative OA biomarkers Pro-Col-Iα and VEGF is elevated in degenerated human acetabular labrum and may serve as indicators of OA and calcification status. Secretion of both factors was partially regulated by TGF-beta signaling in calcified OA labrum tissues.The Translational potential of this article:Our findings suggest that a biomarker panel consisting of Pro-Col-Iα/VEGF/COMP may be valuable for assessing subradiographic labrum degeneration and calcification in hip OA. Targeting TGF-beta signaling may offer a means to reduce vascular invasion and fibrosis in acetabular labrum tissue.

Apoptosis/Necroptosis Inducing Thiazole-Containing Artificial Polypeptide for Immunogenic Cell Death of Cancer.

Immunogenic cell death (ICD) has emerged as a promising approach to cancer immunotherapy. During ICD, cancer cell death and the release of damage-associated molecular pattern (DAMP) signals occur simultaneously. Increased production of reactive oxygen species (ROS) and severe endoplasmic reticulum stress are necessary for enhanced ICD. Furthermore, the levels of ROS and reduced glutathione (GSH) are involved in various cell death mechanisms. The thiazole ring structure has gained considerable interest as a functional moiety for anticancer agents. This study designed and synthesized a positively charged cell-penetrating polypeptide with a thiazole functional moiety (NS). The NS internalizes into the cancer cells through direct penetration and endo-lysosomal escape. The NS induces mitochondrial depolarization and ER stress in a concentration-dependent manner, leading to a significant ROS production and GSH depletion. Consequently, the ICD of cancer cells is activated, resulting in the release of DAMP signals. Furthermore, NS causes a shift in the cell death pathway from apoptosis to necroptosis as the concentration increases. In this study, we confirmed the possibility of NS as a promising ICD inducer that can be used while varying the concentration according to the cancer type.

Damage-Associated Molecular Patterns and Systemic Signaling.

Cellular damage inflicted by wounding, pathogen infection, and herbivory releases a variety of host-derived metabolites, degraded structural components, and peptides into the extracellular space that act as alarm signals when perceived by adjacent cells. These so-called damage-associated molecular patterns (DAMPs) function through plasma membrane localized pattern recognition receptors to regulate wound and immune responses. In plants, DAMPs act as elicitors themselves, often inducing immune outputs such as calcium influx, reactive oxygen species generation, defense gene expression, and phytohormone signaling. Consequently, DAMP perception results in a priming effect that enhances resistance against subsequent pathogen infections. Alongside their established function in local tissues, recent evidence supports a critical role of DAMP signaling in generation and/or amplification of mobile signals that induce systemic immune priming. Here, we summarize the identity, signaling, and synergy of proposed and established plant DAMPs, with a focus on those with published roles in systemic signaling.

Real-time assessment of kidney allografts during HOPE using flavin mononucleotide (FMN) - a preclinical study.

The gap between available donor grafts and patients on the waiting lists is constantly growing. This leads to an increased utilization of high-risk and therefore more vulnerable kidney grafts. The use of high-risk organs requires further optimization of machine preservation and assessment strategies before transplantation. Hypothermic machine perfusion (HMP) is the standard of care for kidneys originating from donation after circulatory death (DCD), whereas the evidence of HMP with additional oxygen (HOPE) is still very limited. Furthermore, an objective quality assessment of HMP-perfused kidneys is lacking. Recently, the release of mitochondria derived fragments, i.e., flavin mononucleotide (FMN) of complex I during machine liver perfusion was shown to be predictive for liver graft function before implantation. Therefore, the aim of this study was to evaluate, if FMN is useful also for assessment of kidney injury before use. A porcine perfusion model was used to investigate the feasibility of assessment of kidney grafts during hypothermic oxygenated perfusion (HOPE) with either 0, 30 or 60 minutes of warm ischemia. The model with warm ischemia times (WIT) of 30 min and 60 min, was used to mimic a clinically relevant scenario. A group with no warm ischemia time (0' WIT) served as control group. The groups underwent minimal static cold storage (SCS) of 2 h followed by 2 h of end-ischemic HOPE with repeated real-time FMN measurements. In a further step, these values were related to the release of damage-associated molecular patterns (DAMPs) and to the functionality of the respiratory chain, represented by the capacity of ATP production. We demonstrate, first, feasibility of perfusate FMN measurements in perfused kidneys, and secondly its correlation with donor warm ischemia time. Accordingly, FMN measurement showed significantly higher release in the 60-minute WIT group (n = 4) compared to the 30-minute WIT (n = 4) and the control group (n = 4). FMN release correlated also with DAMP signaling, such as the release of 8-OHdG and HMGB1. Finally, ATP replenishment proved to be best in control kidneys, followed by kidneys with 30 min and then by kidneys with 60 min of WIT. This study demonstrates the feasibility of FMN measurement in kidneys during HOPE. In addition, we show a correlation between FMN quantification and pre-existing kidney graft injury. Based on this, real-time FMN measurement during HOPE may be an objective assessment tool to accept high-risk kidneys for transplantation while minimizing post-transplant dysfunction, moving away from former "gut feeling" towards objective criteria in accepting marginal kidney grafts for transplantation. Graft evaluation based on these results may close the gap between available grafts and patients on the waiting lists by increasing utilization rates without significant impact for the recipients.

Toxicoproteomics of Mono(2-ethylhexyl) phthalate and Perfluorooctanesulfonic Acid in Models of Prostatic Diseases.

Benign and malignant prostatic diseases are common, costly, and burdensome; moreover, they share fundamental underlying molecular processes. Several ubiquitous contaminants may perturb these processes, possibly via peroxisome proliferator-activated receptor (PPAR) signaling, but the role of environmental exposures─particularly mixtures─in prostatic diseases is undefined. In the present study, nontumorigenic prostate stromal cells and metastatic prostate epithelial cells were exposed to ubiquitous exogenous PPAR ligands under different dosing paradigms, including a mixture, and effects were assessed via mass spectrometry-based global proteomics. In prostate stromal cells, environmentally relevant levels of mono(2-ethylhexyl) phthalate (MEHP), alone and in combination with perfluorooctanesulfonic acid, led to significant changes in proteins involved in key processes underlying prostatic diseases: oxidative stress defense, proteostasis, damage-associated molecular pattern signaling, and innate immune response signaling. A follow-up experiment in metastatic prostate epithelial cells showed that the occupationally relevant levels of MEHP perturbed similar processes, including lipid, cholesterol, steroid, and alcohol metabolism; apoptosis and coagulation regulation; wound response; and aging. This work shows that environmental exposures may contribute to prostatic diseases by perturbing key processes of a proposed adverse outcome pathway, including lipid metabolism, oxidative stress, and inflammation. Future in vivo research will investigate the role of contaminants in prostatic diseases and in preventative agents.

Damage signal induced by Bacillus thuringiensis infection triggers immune responses via a DAMP molecule in lepidopteran insect, Spodoptera exigua

Insect immunity defends the infection of an insect pathogenic bacterium, Bacillus thuringiensis (Bt). However, it was not clear on the recognition of Bt infection by the insect immune system. This study tested a physiological function of dorsal switch protein 1 (DSP1) in the Bt infection. DSP1 is classified into HMGB1-like damage-associated molecular pattern (DAMP) in insects. Upon Bt infection in a lepidopteran Spodoptera exigua, DSP1 was released from the nuclei of the midgut epithelium and activated immune responses. For this DSP1 release, a functional binding between Bt and its receptors on the midgut epithelium was required because any RNA interference (RNAi) treatments of Bt receptor (cadherin or ABCC) prevented the DSP1 release and became susceptible to the bacterial infection. The DSP1 release was required for the gene induction of Repat33, which is a member of response to pathogen gene family and its gene product mediated cellular and humoral immune responses against pathogen infection in S. exigua. The released DSP1 activated phospholipase A2 (PLA2) to produce eicosanoids, which induced the Repat33 expression because a hemocoelic injection of a recombinant DSP1 induced the Repat33 expression without Bt infection. However, any inhibition of PLA2 activity impaired the DAMP signaling between DSP1 and Repat33. DSP1 also up-regulated two other immune mediators, nitric oxide (NO) and a cytokine called plasmatocyte-spreading peptide (PSP). Either NO or PSP activated PLA2 to up-regulate Repat33 expression. These results suggest that Bt infection of the insect midgut generates a DAMP signal via DSP1 release, which turns on NO or the cytokine-PLA2-Repat33 immune signaling pathway.

Role of alarmins in poststroke inflammation and neuronal repair.

Severe loss of cerebral blood flow causes hypoxia and glucose deprivation in the brain tissue, resulting in necrotic cell death in the ischemic brain. Several endogenous molecules, called alarmins or damage-associated molecular patterns (DAMPs), are extracellularly released from the dead cells to activate pattern recognition receptors (PRRs) in immune cells that infiltrate into ischemic brain tissue following the disruption of the blood-brain barrier (BBB) after stroke onset. The activated immune cells produce various inflammatory cytokines and chemokines, triggering sterile cerebral inflammation in the ischemic brain that causes further neuronal cell death. Poststroke inflammation is resolved within several days after stroke onset, and neurological functions are restored to some extent as neural repair occurs around peri-infarct neurons. Clearance of DAMPs from the injured brain is necessary for the resolution of poststroke inflammation. Neurons and glial cells also express PRRs and receive DAMP signaling. Although the role of PRRs in neural cells in the ischemic brain has not yet been clarified, the signaling pathway is likely to be contribute to stroke pathology and neural repair after ischemic stroke. This review describes the molecular dynamics, signaling pathways, and functions of DAMPs in poststroke inflammation and its resolution.

Abstract P2052: Lowered Pro-fibrotic And Damage Associated Molecular Pattern Gene Expressions After Exercise Training In A Mouse Model Of Hypertrophic Cardiomyopathy

Background: Hypertrophic cardiomyopathy (HCM) is the most frequent inherited cardiac disease, most often caused by mutations in sarcomere genes. The disease is characterized by left ventricular (LV) hypertrophy, increased risk of arrhythmias and diastolic dysfunction, due to cardiomyocyte hypercontractility, myocyte disarray and fibrosis. Fibrosis is a marker of worse prognosis, adverse remodeling and an increased risk of sudden cardiac death. Hypothesis: We hypothesized that exercise training could attenuate fibrosis development in mice genetically predisposed to HCM. Methods: We allocated male α-Myh6 R403Q/+ to high intensity interval treadmill running or sedentary behavior (N=11 per group) for a total of 6 weeks. HCM was induced in all mice by cyclosporine A given via the feed for the last 3 of the 6 week exercise protocol. All mice underwent echocardiography and an exercise test at weeks 0, 3, and 6, before harvesting LVs for digital droplet PCR (ddPCR) and a qPCR immune array. Results: Exercise training lead to a 2-fold increase in exercise capacity as measured by total distance ran during exercise testing (p<0.0001). In exercised HCM mice, several key fibrosis-related genes were lowered compared to sedentary HCM mice as measured by ddPCR (N=11 per group): Collagen 1 by 51 % (p=0.03), collagen 3 by 54 % (p=0.02), fibronectin by 51 % (p=0.01) and lysyl oxidase by 42 % (p=0.05). An array of 87 selected immune-related genes showed that 39 genes were upregulated >1.5 fold in sedentary HCM mice compared to sedentary wild type controls (N=3 samples, pooled). Twelve upregulated genes belonged to damage associated-molecular patterns (DAMPs). Several of the DAMP genes were lower in exercised HCM mice compared to sedentary HCM mice (N=10-11 per group): Biglycan was lowered by 29 %, versican by 22 % and fibronectin by 55 %. Trends were also found for lowered expression of syndecan 1 (by 26 %), fibromodulin (by 55 %), and tenacin C (by 49 %) (0.07<p<0.08). There was no difference in maximal wall thickness or function measured by echocardiography. Conclusion: Exercise training led to a lower expression of key fibrosis-related genes in an inducible mouse model of HCM. Initial results indicate that attenuation of DAMP signaling might be involved in the mechanism for this effect.

Circulating extracellular histones exacerbate acute lung injury by augmenting pulmonary endothelial dysfunction via TLR4-dependent mechanism.

Extracellular histones released into the circulation following trauma, sepsis, and ARDS may act as potent damage-associated molecular pattern signals leading to multiple organ failure. Endothelial cell (EC) dysfunction caused by extracellular histones has been demonstrated in vitro and in vivo; however, precise mechanistic details of histone-induced EC dysfunction and exacerbation of ongoing inflammation remain poorly understood. This study investigated the role of extracellular histones in exacerbating preexisting endothelial dysfunction and acute lung injury. Histone subunits H3 and H4, but not H1, H2A, or H2B, induced permeability in human pulmonary EC. H3 and H4 at concentrations above 30 µg/mL caused EC inflammation reflected by activation of the NF-κB pathway, transcriptional activation, and release of cytokines and chemokines including IL-6 and IL-8, and increased mRNA and protein expression of EC adhesion molecules VCAM-1 and ICAM-1. Pharmacological inhibitors targeting Toll-like receptor TLR4 but not TLR2/6, blocked histone-induced EC dysfunction. H3 and H4 also strongly augmented EC permeability and inflammation caused by Gram-negative and Gram-positive bacterial particles, endotoxin, and TNFα. Heparin blocked histone-induced augmentation of EC inflammation caused by endotoxin and TNFα. Injection of histone in mouse models of lung injury caused by bacterial wall lipopolysaccharide (LPS) and heat-killed Staphylococcus aureus (HKSA) augmented ALI parameters: increased protein content, cell count, and inflammatory cytokine secretion in bronchoalveolar lavage fluid. Important clinical significance of these findings is in the demonstration that even a modest increase in extracellular histone levels can act as a severe exacerbating factor in conjunction with other EC barrier disruptive or proinflammatory agents.

IL-33 acts as a costimulatory signal to generate alloreactive Th1 cells in graft-versus-host disease.

Antigen-presenting cells (APCs) integrate signals emanating from local pathology and program appropriate T cell responses. In allogeneic hematopoietic stem cell transplantation (alloHCT), recipient conditioning releases damage-associated molecular patterns (DAMPs) that generate proinflammatory APCs that secrete IL-12, which is a driver of donor Th1 responses, causing graft-versus-host disease (GVHD). Nevertheless, other mechanisms exist to initiate alloreactive T cell responses, as recipients with disrupted DAMP signaling or lacking IL-12 develop GVHD. We established that tissue damage signals are perceived directly by donor CD4+ T cells and promoted T cell expansion and differentiation. Specifically, the fibroblastic reticular cell–derived DAMP IL-33 is increased by recipient conditioning and is critical for the initial activation, proliferation, and differentiation of alloreactive Th1 cells. IL-33 stimulation of CD4+ T cells was not required for lymphopenia-induced expansion, however. IL-33 promoted IL-12–independent expression of Tbet and generation of Th1 cells that infiltrated GVHD target tissues. Mechanistically, IL-33 augmented CD4+ T cell TCR-associated signaling pathways in response to alloantigen. This enhanced T cell expansion and Th1 polarization, but inhibited the expression of regulatory molecules such as IL-10 and Foxp3. These data establish an unappreciated role for IL-33 as a costimulatory signal for donor Th1 generation after alloHCT.

Toll-like receptors and damage-associated molecular patterns in the pathogenesis of heart transplant rejection.

Significant strides have been made in our understanding of the immune system and its role in cardiac transplant rejection. Despite the growing knowledge of immune responses, the mortality rate following cardiac transplantation remains grim. Related to procedural and pathological complications, toll-like receptor (TLR) and damage-associated molecular pattern (DAMP) signaling is the most direct and earliest interface between tissue integration and the innate immune response. This in turn can activate an adaptive immune response that further damages myocardial tissue. Furthermore, relevant literature on the status of DAMPs in the context of heart-transplantation remains limited, warranting further attention in clinical and translational research. This review aims to critically appraise the perspectives, advances, and challenges on DAMP-mediated innate immune response in the immune-mediated rejection of cardiac transplantation. Detailed analysis of the influence of TLR and DAMP signaling in mounting the immune response against the transplanted heart holds promise for improving outcomes through early detection and prevention of varied forms of organ rejection.

Damage Signaling by Extracellular Nucleotides: A Role for Cyclic Nucleotides in Elevating Cytosolic Free Calcium?

Extracellular ATP (eATP) is now held to be a constitutive damage-associated molecular pattern (DAMP) that is released by wounding, herbivory or pathogen attack. The concentration of eATP must be tightly regulated as either depletion or overload leads to cell death. In Arabidopsis thaliana, sensing of eATP is by two plasma membrane legume-like lectin serine–threonine receptor kinases (P2K1 and P2K2), although other receptors are postulated. The transcriptional response to eATP is dominated by wound- and defense-response genes. Wounding and pathogen attack can involve the cyclic nucleotides cyclic AMP (cAMP) and cyclic GMP (cGMP) which, in common with eATP, can increase cytosolic-free Ca2+ as a second messenger. This perspective on DAMP signaling by eATP considers the possibility that the eATP pathway involves production of cyclic nucleotides to promote opening of cyclic nucleotide-gated channels and so elevates cytosolic-free Ca2+. In silico analysis of P2K1 and P2K2 reveals putative adenylyl and guanylyl kinase sequences that are the hallmarks of “moonlighting” receptors capable of cAMP and cGMP production. Further, an Arabidopsis loss of function cngc mutant was found to have an impaired increase in cytosolic-free Ca2+ in response to eATP. A link between eATP, cyclic nucleotides, and Ca2+ signaling therefore appears credible.

A unique population of neutrophils generated by air pollutant–induced lung damage exacerbates airway inflammation

Diesel exhaust particles (DEPs) are the main component of traffic-related air pollution and have been implicated in the pathogenesis and exacerbation of asthma. However, the mechanism by which DEP exposure aggravates asthma symptoms remains unclear. This study aimed to identify a key cellular player of air pollutant-induced asthma exacerbation and development. We examined the distribution of innate immune cells in the murine models of asthma induced by house dust mite and DEP. Changes in immune cell profiles caused by DEP exposure were confirmed by flow cytometry and RNA-Seq analysis. The roles of sialic acid-binding, Ig-like lectin F (SiglecF)-positive neutrophils were further evaluated by adoptive transfer experiment and invitro functional studies. DEP exposure induced a unique population of lung granulocytes that coexpressed Ly6G and SiglecF. These cells differed phenotypically, morphologically, functionally, and transcriptionally from other SiglecF-expressing cells in the lungs. Our findings with murine models suggest that intratracheal challenge with DEPs induces the local release of adenosine triphosphate, which is a damage-associated molecular pattern signal. Adenosine triphosphate promotes the expression of SiglecF on neutrophils, and these SiglecF+ neutrophils worsen type 2 and 3 airway inflammation by producing high levels of cysteinyl leukotrienes and neutrophil extracellular traps. We also found Siglec8- (which corresponds to murine SiglecF) expressing neutrophils, and we found it in patients with asthma-chronic obstructive pulmonary disease overlap. The SiglecF+ neutrophil is a novel and critical player in airway inflammation and targeting this population could reverse or ameliorate asthma.

Abstract 104: Deletion Of Myd88 In T-Cells Worsens Pathology In A Mouse Model Of Non-Ischemic Heart Failure Through Enhanced T-Cell Survival And Effector Function:

Background: Heart failure (HF) is a leading cause of death worldwide, associated with cardiac and systemic inflammation. However, no anti-inflammatory therapies have shown success thus far. Damage associated molecular patterns (DAMPs) released in the heart can activate myeloid cells to promote antigen presentation to T-cells, which infiltrate the heart and participate in adverse cardiac remodeling. DAMP signaling converges onto the adaptor protein “Myeloid differentiation primary response 88” (MyD88). DAMP receptors and MyD88 are also expressed in T-cells, but their role in T-cell activation is unclear, and is unknown in the context of HF. We hypothesized that T-cell recognition of DAMPs through MyD88 causes “bystander activation” of T-cells and contributes to cardiac pathology in HF. Methods and Results: We reconstituted Tcra -/- mice, normally protected from HF, with WT or Myd88 -/- Type 1 helper T-cells (Th1) in the onset of transaortic constriction (TAC), a well-established model of HF. Surprisingly, we found that mice given Myd88 -/- Th1 cells exhibited significantly higher levels of cardiac T-cell infiltration, more severe fibrosis, and lower fractional shortening than mice given WT Th1 cells. We found that WT and Myd88 -/- Th1 cells had similar levels of IFNγ and Tbx21 by intracellular staining and RT-qPCR, indicating that MyD88 does not alter Th1 differentiation. However, Myd88 -/- Th1 cells secreted higher levels of IL-2 and TNFα, suggesting enhanced proliferative and pro-inflammatory effector function. We performed viability studies using live cell microscopy and measuring propidium iodide incorporation in real time, as well as by flow cytometry, and found that Myd88 -/- Th1 cells have a survival advantage compared to WT Th1 cells. Moreover, we found that Myd88 -/- Th1 cells exhibited higher levels of adhesion to ICAM-1 and VCAM-1, protein ligands involved in T-cell recruitment, compared to WT Th1 cells when perfused under conditions of shear flow. Conclusion: Together, these data demonstrate that T-cell MyD88 limits T-cell mediated pathology in HF by modulating Th1 effector function, survival, and adhesion ability. We identify novel role for T-cell MyD88 in cardiac inflammation that may be modulated in HF.

Dietary Polyphenol and Methylsulfonylmethane Supplementation Improves Immune, DAMP Signaling, and Inflammatory Responses During Recovery From All-Out Running Efforts.

Nutritional ingredients with defined mechanisms of action can be useful in the recovery of the body from the physical demands of a habitual training plan. The purpose of this study was to determine the effect of dietary supplementation with optimized curcumin, pomegranate ellagitannins, and MSM (R + MSM) on immune-associated mRNA during early recovery (i.e., up to 8 h post-exercise) following all-out running efforts (5-km, 10-km, and 21.1-km). Subjects (N = 14) were randomized to either a supplement (R + MSM) or a control group using an open label design. The study was completed over a period of 31-day prior to a scheduled half-marathon race. Venous blood samples were collected into PAXgene tubes at baseline, subsequent samples were collected at 2, 4, and 8 h after each running effort. A 574-plex mRNA Immunology Array (NanoString) was measured for each sample and ROSALIND® Advanced Analysis Software was used to examined changes in 31 annotated immune response pathways and specific mRNA changes. The greatest change in immune pathways occurred at 2 h (GSS > 3) followed by 4 h (GSS 2–3) and 8 h (GSS 1–2). R + MSM was associated with an increase in innate immunity (CAMP, LTF, TIRAP, CR1, IL1R1, CXCR1, PDCDILG2, and GNLY) and a blunted/smaller increase in damage-associated molecular pattern (DAMP) signaling/inflammation (TLR4, TLR5, S100A8, S100A9, and IFP35). We also found changes in immune-associated mRNA that have not been previously linked to exercise recovery (SOCS1, SOCS2, MME, CECAM6, MX1, IL-1R2, KLRD1, KLRK1, and LAMP3). Collectively these results demonstrate that supplementation with a combination of optimized curcumin, pomegranate ellagitannins, and methylsulfonylmethane resulted in changes that may improve biological recovery from all-out running efforts.

Mitochondrial fusion and fission: The fine-tune balance for cellular homeostasis.

Mitochondria are highly dynamic, maternally inherited cytoplasmic organelles, which fulfill cellular energy demand through the oxidative phosphorylation system. Besides, they play an active role in calcium and damage-associated molecular patterns signaling, amino acid, and lipid metabolism, and apoptosis. Thus, the maintenance of mitochondrial integrity and homeostasis is extremely critical, which is achieved through continual fusion and fission. Mitochondrial fusion allows the transfer of gene products between mitochondria for optimal functioning, especially under metabolic and environmental stress. On the other hand, fission is crucial for mitochondrial division and quality control. The imbalance between these two processes is associated with various ailments such as cancer, neurodegenerative and cardiovascular diseases. This review discusses the molecular mechanisms that control mitochondrial fusion and fission and how the disruption of mitochondrial dynamics manifests into various disease conditions.

Untangling Local Pro-Inflammatory, Reparative, and Regulatory Damage-Associated Molecular-Patterns (DAMPs) Pathways to Improve Transplant Outcomes.

Detrimental inflammatory responses after solid organ transplantation are initiated when immune cells sense pathogen-associated molecular patterns (PAMPs) and certain damage-associated molecular patterns (DAMPs) released or exposed during transplant-associated processes, such as ischemia/reperfusion injury (IRI), surgical trauma, and recipient conditioning. These inflammatory responses initiate and propagate anti-alloantigen (AlloAg) responses and targeting DAMPs and PAMPs, or the signaling cascades they activate, reduce alloimmunity, and contribute to improved outcomes after allogeneic solid organ transplantation in experimental studies. However, DAMPs have also been implicated in initiating essential anti-inflammatory and reparative functions of specific immune cells, particularly Treg and macrophages. Interestingly, DAMP signaling is also involved in local and systemic homeostasis. Herein, we describe the emerging literature defining how poor outcomes after transplantation may result, not from just an over-abundance of DAMP-driven inflammation, but instead an inadequate presence of a subset of DAMPs or related molecules needed to repair tissue successfully or re-establish tissue homeostasis. Adverse outcomes may also arise when these homeostatic or reparative signals become dysregulated or hijacked by alloreactive immune cells in transplant niches. A complete understanding of the critical pathways controlling tissue repair and homeostasis, and how alloimmune responses or transplant-related processes disrupt these will lead to new immunotherapeutics that can prevent or reverse the tissue pathology leading to lost grafts due to chronic rejection.

Exogenous ATP modulates PGE2 release in macrophages through sustained phosphorylation of CDK9 and p38 MAPK.

An important mediator of inflammation is prostaglandin E2 (PGE2 ), whose levels are determined by the activity of the enzyme cyclooxygenase (COX). Of the two isoforms of the enzyme, COX-2 has been shown to be induced in macrophages during inflammation. Although general COX inhibitors, belonging to the class of nonsteroidal anti-inflammatory drugs, or specific COX-2 inhibitors, called coxibs, are useful in the control of acute inflammation, adverse reactions were seen when used chronically in the treatment of rheumatoid arthritis or neurodegenerative diseases. Extracellular ATP (eATP) has been reported as a damage-associated molecular pattern signal. In this report, we show that eATP synergistically increases the levels of COX-2 enzyme and PGE2 in LPS-activated RAW264.7 macrophages and human monocytes. Activation of macrophages also occurred when cultured in media obtained from dying neurons that contained higher levels of ATP. We show that eATP increases the levels of COX-2 protein, which is sustained up to 36 h poststimulation. This is in turn due to sustained levels of phosphorylated, or activated, cyclin-dependent kinase 9 and p38 MAPK in ATP-treated cells compared to LPS-stimulated cells. The eATP-dependent increase in COX-2/PGE2 levels in LPS-activated RAW264.7 cells could be abolished using antagonists for purinergic P2X7-and P2Y6 receptors. Similarly, the increase in COX-2/PGE2 levels in the peritoneum of LPS-treated mice could be significantly abolished in mice that were preinjected with the nonspecific P2 receptor antagonist, suramin. P2 receptor antagonists, therefore, should be explored in our search for an ideal anti-inflammatory candidate.

Damage-associated molecular pattern (DAMP) activation in melanoma: investigation of the immunogenic activity of 15-deoxy, Δ12,14 prostamide J2.

Metastatic melanoma is the most deadly skin neoplasm in the United States. Outcomes for this lethal disease have improved dramatically due to the use of both targeted and immunostimulatory drugs. Immunogenic cell death (ICD) has emerged as another approach for initiating antitumor immunity. ICD is triggered by tumor cells that display damage-associated molecular patterns (DAMPs). These DAMP molecules recruit and activate dendritic cells (DCs) that present tumor-specific antigens to T cells which eliminate neoplastic cells. Interestingly, the expression of DAMP molecules occurs in an endoplasmic reticulum (ER) stress-dependent manner. We have previously shown that ER stress was required for the cytotoxic activity of the endocannabinoid metabolite, 15-deoxy, Δ12,14 prostamide J2 (15dPMJ2). As such, the current study investigates whether 15dPMJ2 induces DAMP signaling in melanoma. In B16F10 cells, 15dPMJ2 caused a significant increase in the cell surface expression of calreticulin (CRT), the release of ATP and the secretion of high-mobility group box 1 (HMGB1), three molecules that serve as surrogate markers of ICD. 15dPMJ2 also stimulated the cell surface expression of the DAMP molecules, heat shock protein 70 (Hsp70) and Hsp90. In addition, the display of CRT and ATP was increased by 15dPMJ2 to a greater extent in tumorigenic compared to non-tumorigenic melanocytes. Consistent with this finding, the activation of bone marrow-derived DCs was upregulated in co-cultures with 15dPMJ2-treated tumor compared to non-tumor melanocytes. Moreover, 15dPMJ2-mediated DAMP exposure and DC activation required the electrophilic cyclopentenone double bond within the structure of 15dPMJ2 and the ER stress pathway. These results demonstrate that 15dPMJ2 is a tumor-selective inducer of DAMP signaling in melanoma.