Multifactorial Causes and Consequences of TLSP Production, Function, and Release in the Asthmatic Airway.

Disruption of the airway epithelium triggers a defensive immune response that begins with the production and release of alarmin cytokines. These epithelial-derived alarmin cytokines, including thymic stromal lymphopoietin (TSLP), are produced in response to aeroallergens, viruses, and toxic inhalants. An alarmin response disproportionate to the inhaled trigger can exacerbate airway diseases such as asthma. Allergens inhaled into previously sensitized airways are known to drive a T2 inflammatory response through the polarization of T cells by dendritic cells mediated by TSLP. Harmful compounds found within air pollution, microbes, and viruses are also triggers causing airway epithelial cell release of TSLP in asthmatic airways. The release of TSLP leads to the development of inflammation which, when unchecked, can result in asthma exacerbations. Genetic and inheritable factors can contribute to the variable expression of TSLP and the risk and severity of asthma. This paper will review the various triggers and consequences of TSLP release in asthmatic airways.

Th2 Cell Activation in Chronic Liver Disease Is Driven by Local IL33 and Contributes to IL13-Dependent Fibrogenesis.

BACKGROUND & AIMS: Type 2 immune responses contribute to liver fibrosis in parasite infections, but their role in other liver diseases is less well understood. Here, we aimed at unravelling mechanisms involved in T helper 2 (Th2) T-cell polarization, activation, and recruitment in human liver fibrosis and cirrhosis. METHODS: Tissues, cells, and serum from human livers were analyzed using quantitative reverse-transcription polymerase chain reaction, enzyme-linked immunosorbent assay, fluorescence in situ hybridization, immunostaining, flow cytometry, and various functional in vitro assays. Cellular interactions and soluble mediators involved in T-cell polarization and recruitment were studied, as well as their effect on hepatic stellate cell (HSC) activation, proliferation, and extracellular matrix synthesis. RESULTS: In human liver fibrosis, a stage-dependent increase in Th2-related transcription factors, Th2 cytokines, and trans-acting T-cell-specific transcription factor-expressing T cells was observed, and was highest in cirrhotic livers. The alarmin interleukin (IL)33 was found to be increased in livers and sera from patients with cirrhosis, to act as a chemotactic agent for Th2 cells, and to induce type 2 polarization of CD4+ T cells. Oval cells, liver sinusoidal endothelial cells, intrahepatic macrophages, and migrating monocytes were identified as sources of IL33. IL33-activated T cells, but not IL33 alone, induced HSC activation, as shown by Ki67 and α-smooth muscle actin staining, increased collagen type I alpha 1 chain messenger RNA expression, and wound healing assays. The profibrotic effect of IL33-activated T cells was contact-independent and could be antagonized using monoclonal antibodies against IL13. CONCLUSION: In patients with chronic liver disease, the alarmin IL33 promotes the recruitment and activation of CD4+ T cells with Th2-like properties, which activate paracrine HSC in an IL13-dependent manner and promotes fibrogenesis.

ATP/IL-33-Co-Sensing by Mast Cells (MCs) Requires Activated c-Kit to Ensure Effective Cytokine Responses.

Mast cells (MCs) are sentinel cells which represent an important part of the first line of defense of the immune system. MCs highly express receptors for danger-associated molecular patterns (DAMPs) such as the IL-33R and P2X7, making MCs to potentially effective sensors for IL-33 and adenosine-triphosphate (ATP), two alarmins which are released upon necrosis-induced cell damage in peripheral tissues. Besides receptors for alarmins, MCs also express the stem cell factor (SCF) receptor c-Kit, which typically mediates MC differentiation, proliferation and survival. By using bone marrow-derived MCs (BMMCs), ELISA and flow cytometry experiments, as well as p65/RelA and NFAT reporter MCs, we aimed to investigate the influence of SCF on alarmin-induced signaling pathways and the resulting cytokine production and degranulation. We found that the presence of SCF boosted the cytokine production but not degranulation in MCs which simultaneously sense ATP and IL-33 (ATP/IL-33 co-sensing). Therefore, we conclude that SCF maintains the functionality of MCs in peripheral tissues to ensure appropriate MC reactions upon cell damage, induced by pathogens or allergens.

Mitochondrial Impairment: A Link for Inflammatory Responses Activation in the Cardiorenal Syndrome Type 4.

Cardiorenal syndrome type 4 (CRS type 4) occurs when chronic kidney disease (CKD) leads to cardiovascular damage, resulting in high morbidity and mortality rates. Mitochondria, vital organelles responsible for essential cellular functions, can become dysfunctional in CKD. This dysfunction can trigger inflammatory responses in distant organs by releasing Damage-associated molecular patterns (DAMPs). These DAMPs are recognized by immune receptors within cells, including Toll-like receptors (TLR) like TLR2, TLR4, and TLR9, the nucleotide-binding domain, leucine-rich-containing family pyrin domain-containing-3 (NLRP3) inflammasome, and the cyclic guanosine monophosphate (cGMP)-adenosine monophosphate (AMP) synthase (cGAS)-stimulator of interferon genes (cGAS-STING) pathway. Activation of these immune receptors leads to the increased expression of cytokines and chemokines. Excessive chemokine stimulation results in the recruitment of inflammatory cells into tissues, causing chronic damage. Experimental studies have demonstrated that chemokines are upregulated in the heart during CKD, contributing to CRS type 4. Conversely, chemokine inhibitors have been shown to reduce chronic inflammation and prevent cardiorenal impairment. However, the molecular connection between mitochondrial DAMPs and inflammatory pathways responsible for chemokine overactivation in CRS type 4 has not been explored. In this review, we delve into mechanistic insights and discuss how various mitochondrial DAMPs released by the kidney during CKD can activate TLRs, NLRP3, and cGAS-STING immune pathways in the heart. This activation leads to the upregulation of chemokines, ultimately culminating in the establishment of CRS type 4. Furthermore, we propose using chemokine inhibitors as potential strategies for preventing CRS type 4.

ATP functions as a primary alarmin in allergen-induced type 2 immunity.

Environmental allergens that interact with the airway epithelium can activate cellular stress pathways that lead to the release of danger signals known as alarmins. The mechanisms of alarmin release are distinct from damage-associated molecular patterns (DAMPs), which typically escape from cells after loss of plasma membrane integrity. Oxidative stress represents a form of allergen-induced cellular stress that stimulates oxidant-sensing mechanisms coupled to pathways, which facilitate alarmin mobilization and efflux across the plasma membrane. In this review, we highlight examples of alarmin release and discuss their roles in the initiation of type 2 immunity and allergic airway inflammation. In addition, we discuss the concept of alarmin amplification, where "primary" alarmins, which are directly released in response to a specific cellular stress, stimulate additional signaling pathways that lead to secretion of "secondary" alarmins that include proinflammatory cytokines, such as IL-33, as well as genomic and mitochondrial DNA that coordinate or amplify type 2 immunity. Accordingly, allergen-evoked cellular stress can elicit a hierarchy of alarmin signaling responses from the airway epithelium that trigger local innate immune reactions, impact adaptive immunity, and exacerbate diseases including asthma and other chronic inflammatory conditions that affect airway function.

Mitochondrial Damage-Associated Molecular Patterns and Metabolism in the Regulation of Innate Immunity.

The innate immune system, as the host's first line of defense against intruders, plays a critical role in recognizing, identifying, and reacting to a wide range of microbial intruders. There is increasing evidence that mitochondrial stress is a major initiator of innate immune responses. When mitochondria's integrity is disrupted or dysfunction occurs, the mitochondria's contents are released into the cytosol. These contents, like reactive oxygen species, mitochondrial DNA, and double-stranded RNA, among others, act as damage-related molecular patterns (DAMPs) that can bind to multiple innate immune sensors, particularly pattern recognition receptors, thereby leading to inflammation. To avoid the production of DAMPs, in addition to safeguarding organelles integrity and functionality, mitochondria may activate mitophagy or apoptosis. Moreover, mitochondrial components and specific metabolic regulations modify properties of innate immune cells. These include macrophages, dendritic cells, innate lymphoid cells, and so on, in steady state or in stimulation that are involved in processes ranging from the tricarboxylic acid cycle to oxidative phosphorylation and fatty acid metabolism. Here we provide a brief summary of mitochondrial DAMPs' initiated and potentiated inflammatory response in the innate immune system. We also provide insights into how the state of activation, differentiation, and functional polarization of innate immune cells can be influenced by alteration to the metabolic pathways in mitochondria.

Brain alarm by self-extracellular nucleic acids: from neuroinflammation to neurodegeneration.

Neurological disorders such as stroke, multiple sclerosis, as well as the neurodegenerative diseases Parkinson's or Alzheimer's disease are accompanied or even powered by danger associated molecular patterns (DAMPs), defined as endogenous molecules released from stressed or damaged tissue. Besides protein-related DAMPs or "alarmins", numerous nucleic acid DAMPs exist in body fluids, such as cell-free nuclear and mitochondrial DNA as well as different species of extracellular RNA, collectively termed as self-extracellular nucleic acids (SENAs). Among these, microRNA, long non-coding RNAs, circular RNAs and extracellular ribosomal RNA constitute the majority of RNA-based DAMPs. Upon tissue injury, necrosis or apoptosis, such SENAs are released from neuronal, immune and other cells predominantly in association with extracellular vesicles and may be translocated to target cells where they can induce intracellular regulatory pathways in gene transcription and translation. The majority of SENA-induced signaling reactions in the brain appear to be related to neuroinflammatory processes, often causally associated with the onset or progression of the respective disease. In this review, the impact of the diverse types of SENAs on neuroinflammatory and neurodegenerative diseases will be discussed. Based on the accumulating knowledge in this field, several specific antagonistic approaches are presented that could serve as therapeutic interventions to lower the pathological outcome of the indicated brain disorders.

The Role of Alarmins in Osteoarthritis Pathogenesis: HMGB1, S100B and IL-33.

Osteoarthritis (OA) is a multifactorial disease in which genetics, aging, obesity, and trauma are well-known risk factors. It is the most prevalent joint disease and the largest disability problem worldwide. Recent findings have described the role of damage-associated molecular patterns (DAMPs) in the course of the disease. In particular, alarmins such as HMGB1, IL-33, and S100B, appear implicated in enhancing articular inflammation and favouring a catabolic switch in OA chondrocytes. The aims of this review are to clarify the molecular signalling of these three molecules in OA pathogenesis, to identify their possible use as staging biomarkers, and, most importantly, to find out whether they could be possible therapeutic targets. Osteoarthritic cartilage expresses increased levels of all three alarmins. HMGB1, in particular, is the most studied alarmin with increased levels in cartilage, synovium, and synovial fluid of OA patients. High levels of HMGB1 in synovial fluid of OA joints are positively correlated with radiological and clinical severity. Counteracting HMGB1 strategies have revealed improving results in articular cells from OA patients and in OA animal models. Therefore, drugs against this alarmin, such as anti-HMGB1 antibodies, could be new treatment possibilities that can modify the disease course since available medications only alleviate symptoms.

A unique maternal and placental galectin signature upon SARS-CoV-2 infection suggests galectin-1 as a key alarmin at the maternal-fetal interface.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic imposed a risk of infection and disease in pregnant women and neonates. Successful pregnancy requires a fine-tuned regulation of the maternal immune system to accommodate the growing fetus and to protect the mother from infection. Galectins, a family of ß-galactoside-binding proteins, modulate immune and inflammatory processes and have been recognized as critical factors in reproductive orchestration, including maternal immune adaptation in pregnancy. Pregnancy-specific glycoprotein 1 (PSG1) is a recently identified gal-1 ligand at the maternal-fetal interface, which may facilitate a successful pregnancy. Several studies suggest that galectins are involved in the immune response in SARS-CoV-2-infected patients. However, the galectins and PSG1 signature upon SARS-CoV-2 infection and vaccination during pregnancy remain unclear. In the present study, we examined the maternal circulating levels of galectins (gal-1, gal-3, gal-7, and gal-9) and PSG1 in pregnant women infected with SARS-CoV-2 before vaccination or uninfected women who were vaccinated against SARS-CoV-2 and correlated their expression with different pregnancy parameters. SARS-CoV-2 infection or vaccination during pregnancy provoked an increase in maternal gal-1 circulating levels. On the other hand, levels of PSG1 were only augmented upon SARS-CoV-2 infection. A healthy pregnancy is associated with a positive correlation between gal-1 concentrations and gal-3 or gal-9; however, no correlation was observed between these lectins during SARS-CoV-2 infection. Transcriptome analysis of the placenta showed that gal-1, gal-3, and several PSG and glycoenzymes responsible for the synthesis of gal-1-binding glycotopes (such as linkage-specific N-acetyl-glucosaminyltransferases (MGATs)) are upregulated in pregnant women infected with SARS-CoV-2. Collectively, our findings identify a dynamically regulated "galectin-specific signature" that accompanies the SARS-CoV-2 infection and vaccination in pregnancy, and they highlight a potentially significant role for gal-1 as a key pregnancy protective alarmin during virus infection.

Damage-mediated macrophage polarization in sterile inflammation.

Most of the leading causes of death, such as cardiovascular diseases, cancer, dementia, neurodegenerative diseases, and many more, are associated with sterile inflammation, either as a cause or a consequence of these conditions. The ability to control the progression of inflammation toward tissue resolution before it becomes chronic holds significant clinical potential. During sterile inflammation, the initiation of inflammation occurs through damage-associated molecular patterns (DAMPs) in the absence of pathogen-associated molecules. Macrophages, which are primarily localized in the tissue, play a pivotal role in sensing DAMPs. Furthermore, macrophages can also detect and respond to resolution-associated molecular patterns (RAMPs) and specific pro-resolving mediators (SPMs) during sterile inflammation. Macrophages, being highly adaptable cells, are particularly influenced by changes in the microenvironment. In response to the tissue environment, monocytes, pro-inflammatory macrophages, and pro-resolution macrophages can modulate their differentiation state. Ultimately, DAMP and RAMP-primed macrophages, depending on the predominant subpopulation, regulate the balance between inflammatory and resolving processes. While sterile injury and pathogen-induced reactions may have distinct effects on macrophages, most studies have focused on macrophage responses induced by pathogens. In this review, which emphasizes available human data, we illustrate how macrophages sense these mediators by examining the expression of receptors for DAMPs, RAMPs, and SPMs. We also delve into the signaling pathways induced by DAMPs, RAMPs, and SPMs, which primarily contribute to the regulation of macrophage differentiation from a pro-inflammatory to a pro-resolution phenotype. Understanding the regulatory mechanisms behind the transition between macrophage subtypes can offer insights into manipulating the transition from inflammation to resolution in sterile inflammatory diseases.

CA9 knockdown enhanced ionizing radiation-induced ferroptosis and radiosensitivity of hypoxic glioma cells.

PURPOSE: Ferroptosis is a type of regulatory cell death, caused by excessive lipid peroxidation This study aimed to explore whether ionizing radiation could induce ferroptosis in glioma cells and whether carbonic anhydrase 9 (CA9) knockdown could enhance the killing effect of ionizing radiation on hypoxic glioma cells through ferroptosis. MATERIALS AND METHODS: The protein levels of Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4) were detected by Western blot in glioma cells irradiated by different doses of X-ray. The relative mRNA levels of ferroptosis markers and intracellular iron-associated proteins were detected by Real-time qPCR. Lipid peroxidation of glioma cells was detected by oxidation-sensitive probe C11-BODIPY581/591 staining. CCK-8 Assay was used to detect cell viability after X-ray irradiation. Cloning formation assay was used to assess the radiosensitivity of glioma cells. The exposure of cell surface calreticulin was measured by immunofluorescence staining. RESULTS: X-ray induced lipid peroxidation and ferroptosis markers expression in U251 and GL261 glioma cells. Knockdown of CA9 in hypoxic glioma cells significantly altered the expression of iron regulation-related proteins and enhanced X-ray-induced ferroptosis and radiosensitivity. The ferroptosis inhibitor significantly improved the survival of cells irradiated by X-ray, while ferroptosis inducers (FINs) enhanced the lethal effect of X-ray on cells. Enhancing ferroptosis in glioma cells promoted the exposure and release of damage-associated molecular patterns (DAMPs). CONCLUSIONS: Ionizing radiation can induce ferroptosis in glioma cells. CA9 knockdown can enhance the radiosensitivity of hypoxic glioma cells and overcome the resistance of ferroptosis under hypoxia. Enhancing ferroptosis will become a new idea to improve the efficacy of radiotherapy for glioma.

Elevated interleukin-11 in systemic sclerosis and role in disease pathogenesis.

Systemic sclerosis (SSc) is an autoimmune connective tissue disease in which there is elevated inflammation, aberrant cytokine expression, and subsequent fibrosis. Interleukin-11 (IL-11) is a recently described profibrotic cytokine that can mediate fibrosis in the heart, lungs, and skin and is upregulated by transforming Growth Factor-ß (TGF-ß1). The objective of this study was to quantify the serum levels of IL-11 in early diffuse SSc patients. Also, if IL-11 could regulate the alarmin IL-33 in dermal fibroblasts was quantified. Early diffuse SSc patient sera was isolated and IL-11 was quantified by specific commercial ELISA compared to healthy control (n = 17). Healthy dermal fibroblasts were cultured in vitro and then serum starved and incubated with or without recombinant IL-11. At specific early and late time points the supernatant was quantified for the alarmin IL-33 by specific ELISA. In early diffuse SSc patients it was demonstrated that they have elevated IL-11 in their sera. In a subgroup of SSc patients with interstitial lung disease (ILD) this elevation was particularly pronounced compared to those devoid of fibrotic lung disease. In vitro incubation of healthy dermal fibroblasts led to a significant induction of IL-33 cytokine release into the cell media. IL-11 is a profibrotic cytokine that is elevated in early diffuse SSc and is particularly elevated in those with ILD. This suggests that IL-11 could be a possible biomarker of ILD in SSc. It was also found that IL-11 led to release of the cytokine alarmin IL-33 in fibroblasts at earlier time points but not late time points, suggesting early stimulation elicits an inflammatory response in the local microenvironment but prolonged stimulation leads to fibrosis.

Non-Vesicular Release of Alarmin Prothymosin α Complex Associated with Annexin-2 Flop-Out.

Nuclear protein prothymosin α (ProTα) is a unique member of damage-associated molecular patterns (DAMPs)/alarmins. ProTα prevents neuronal necrosis by causing a cell death mode switch in serum-starving or ischemic/reperfusion models in vitro and in vivo. Underlying receptor mechanisms include Toll-like receptor 4 (TLR4) and Gi-coupled receptor. Recent studies have revealed that the mode of the fatal stress-induced extracellular release of nuclear ProTα from cortical neurons in primary cultures, astrocytes and C6 glioma cells has two steps: ATP loss-induced nuclear release and the Ca2+-mediated formation of a multiple protein complex and its extracellular release. Under the serum-starving condition, ProTα is diffused from the nucleus throughout the cell due to the ATP loss-induced impairment of importin α-mediated nuclear transport. Subsequent mechanisms are all Ca2+-dependent. They include the formation of a protein complex with ProTα, S100A13, p40 Syt-1 and Annexin A2 (ANXA2); the fusion of the protein complex to the plasma membrane via p40 Syt-1-Stx-1 interaction; and TMEM16F scramblase-mediated ANXA2 flop-out. Subsequently, the protein complex is extracellularly released, leaving ANXA2 on the outer cell surface. The ANXA2 is then flipped in by a force of ATP8A2 activity, and the non-vesicular release of protein complex is repeated. Thus, the ANXA2 flop-out could play key roles in a new type of non-vesicular and non-classical release for DAMPs/alarmins, which is distinct from the modes conducted via gasdermin D or mixed-lineage kinase domain-like pseudokinase pores.

Induction of pro-inflammatory genes by fibronectin DAMPs in three fibroblast cell lines: Role of TAK1 and MAP kinases.

Changes in the organization and structure of the fibronectin matrix are believed to contribute to dysregulated wound healing and subsequent tissue inflammation and tissue fibrosis. These changes include an increase in the EDA isoform of fibronectin as well as the mechanical unfolding of fibronectin type III domains. In previous studies using embryonic foreskin fibroblasts, we have shown that fibronectin's EDA domain (FnEDA) and the partially unfolded first Type III domain (FnIII-1c) function as Damage Associated Molecular Pattern (DAMP) molecules to stimulate the induction of inflammatory cytokines by serving as agonists for Toll-Like Receptor-4 (TLR4). However, the role of signaling molecules downstream of TLR-4 such as TGF-ß Activated Kinase 1 (TAK1) and Mitogen activated protein kinases (MAPK) in regulating the expression of fibronectin DAMP induced inflammatory genes in specific cell types is not known. In the current study, we evaluate the molecular steps regulating the fibronectin driven induction of inflammatory genes in three human fibroblast cell lines: embryonic foreskin, adult dermal, and adult kidney. The fibronectin derived DAMPs each induce the phosphorylation and activation of TAK1 which results in the activation of two downstream signaling arms, IKK/NF-κB and MAPK. Using the specific inhibitor 5Z-(7)-Oxozeanol as well as siRNA, we show TAK1 to be a crucial signaling mediator in the release of cytokines in response to fibronectin DAMPs in all three cell types. Finally, we show that FnEDA and FnIII-1c induce several pro-inflammatory cytokines whose expression is dependent on both TAK1 and JNK MAPK and highlight cell-type specific differences in the gene-expression profiles of the fibroblast cell-lines.

Early activation and recruitment of invariant natural killer T cells during liver ischemia-reperfusion: the major role of the alarmin interleukin-33.

Over the past thirty years, the complexity of the αß-T cell compartment has been enriched by the identification of innate-like T cells (ITCs), which are composed mainly of invariant natural killer T (iNKT) cells and mucosal-associated invariant T (MAIT) cells. Based on animal studies using ischemia-reperfusion (IR) models, a key role has been attributed to iNKT cells in close connection with the alarmin/cytokine interleukin (IL)-33, as early sensors of cell-stress in the initiation of acute sterile inflammation. Here we have investigated whether the new concept of a biological axis of circulating iNKT cells and IL-33 applies to humans, and may be extended to other ITC subsets, namely MAIT and γδ-T cells, in the acute sterile inflammation sequence occurring during liver transplant (LT). From a prospective biological collection of recipients, we reported that LT was accompanied by an early and preferential activation of iNKT cells, as attested by almost 40% of cells having acquired the expression of CD69 at the end of LT (i.e. 1-3 hours after portal reperfusion), as opposed to only 3-4% of conventional T cells. Early activation of iNKT cells was positively correlated with the systemic release of the alarmin IL-33 at graft reperfusion. Moreover, in a mouse model of hepatic IR, iNKT cells were activated in the periphery (spleen), and recruited in the liver in WT mice, as early as the first hour after reperfusion, whereas this phenomenon was virtually missing in IL-33-deficient mice. Although to a lesser degree than iNKT cells, MAIT and γδ-T cells also seemed targeted during LT, as attested by 30% and 10% of them acquiring CD69 expression, respectively. Like iNKT cells, and in clear contrast to γδ-T cells, activation of MAIT cells during LT was closely associated with both release of IL-33 immediately after graft reperfusion and severity of liver dysfunction occurring during the first three post-operative days. All in all, this study identifies iNKT and MAIT cells in connection with IL-33 as new key cellular factors and mechanisms of acute sterile inflammation in humans. Further investigations are required to confirm the implication of MAIT and iNKT cell subsets, and to precisely assess their functions, in the clinical course of sterile inflammation accompanying LT.

Spinal alarmin HMGB1 and the activation of TLR4 lead to chronic stress-induced nociceptive hypersensitivity in rodents.

Chronic stress affects millions of people around the world, and it can trigger different behavioral disorders like nociceptive hypersensitivity and anxiety, among others. However, the mechanisms underlaying these chronic stress-induced behavioral disorders have not been yet elucidated. This study was designed to understand the role of high-mobility group box-1 (HMGB1) and toll-like receptor 4 (TLR4) in chronic stress-induced nociceptive hypersensitivity. Chronic restraint stress induced bilateral tactile allodynia, anxiety-like behaviors, phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38MAPK) and activation of spinal microglia. Moreover, chronic stress enhanced HMGB1 and TLR4 protein expression at the dorsal root ganglion, but not at the spinal cord. Intrathecal injection of HMGB1 or TLR4 antagonists reduced tactile allodynia and anxiety-like behaviors induced by chronic stress. Additionally, deletion of TLR4 diminished the establishment of chronic stress-induced tactile allodynia in male and female mice. Lastly, the antiallodynic effect of HMGB1 and TLR4 antagonists were similar in stressed male and female rats and mice. Our results suggest that chronic restraint stress induces nociceptive hypersensitivity, anxiety-like behaviors, and up-regulation of spinal HMGB1 and TLR4 expression. Blockade of HMGB1 and TLR4 reverses chronic restraint stress-induced nociceptive hypersensitivity and anxiety-like behaviors and restores altered HMGB1 and TLR4 expression. The antiallodynic effects of HMGB1 and TLR4 blockers in this model are sex independent. TLR4 could be a potential pharmacological target for the treatment of the nociceptive hypersensitivity associated with widespread chronic pain.

The roles of toll-like receptor 4, CD33, CD68, CD69, or CD147/EMMPRIN for monocyte activation by the DAMP S100A8/S100A9.

The S100A8/A9 heterocomplex is an abundant damage-associated molecular pattern and mainly expressed by monocytes, inflammatory activated keratinocytes and neutrophilic granulocytes. The heterocomplex as well as the heterotetramer are involved in a variety of diseases and tumorous processes. However, their detailed mode of action and especially which receptors are involved hereby remains to be fully revealed. Several cell surface receptors are reported to interact with S100A8 and/or S100A9, the best studied being the pattern recognition receptor TLR4. RAGE, CD33, CD68, CD69, and CD147, all of them are involved as receptors in various inflammatory processes, are also among these putative binding partners for S100A8 and S100A9. Interactions between S100 proteins and these receptors described so far come from a wide variety of cell culture systems but their biological relevance in vivo for the inflammatory response of myeloid immune cells is not yet clear. In this study, we compared the effect of CRISPR/Cas9 mediated targeted deletion of CD33, CD68, CD69, and CD147 in ER-Hoxb8 monocytes on S100A8 or S100A9 induced cytokine release with TLR4 knockout monocytes. Whereas deletion of TLR4 abolished the S100-induced inflammatory response in monocyte stimulation experiments with both S100A8 and S100A9, knockouts of CD33, CD68, CD69, or CD147 revealed no effect on the cytokine response in monocytes. Thus, TLR4 is the dominant receptor for S100-triggered inflammatory activation of monocytes.

Targeting alarmin release reverses Sjogren's syndrome phenotype by revitalizing Ca2+ signalling.

BACKGROUND: Primary Sjogren's syndrome (pSS) is a systemic autoimmune disease that is embodied by the loss of salivary gland function and immune cell infiltration, but the mechanism(s) are still unknown. The aim of this study was to understand the mechanisms and identify key factors that leads to the development and progression of pSS. METHODS: Immunohistochemistry staining, FACS analysis and cytokine levels were used to detect immune cells infiltration and activation in salivary glands. RNA sequencing was performed to identify the molecular mechanisms involved in the development of pSS. The function assays include in vivo saliva collection along with calcium imaging and electrophysiology on isolated salivary gland cells in mice models of pSS. Western blotting, real-time PCR, alarmin release, and immunohistochemistry was performed to identify the channels involved in salivary function in pSS. RESULTS: We provide evidence that loss of Ca2+ signaling precedes a decrease in saliva secretion and/or immune cell infiltration in IL14α, a mouse model for pSS. We also showed that Ca2+ homeostasis was mediated by transient receptor potential canonical-1 (TRPC1) channels and inhibition of TRPC1, resulting in the loss of salivary acinar cells, which promoted alarmin release essential for immune cell infiltration/release of pro-inflammatory cytokines. In addition, both IL14α and samples from human pSS patients showed a decrease in TRPC1 expression and increased acinar cell death. Finally, paquinimod treatment in IL14α restored Ca2+ homeostasis that inhibited alarmin release thereby reverting the pSS phenotype. CONCLUSIONS: These results indicate that loss of Ca2+ signaling is one of the initial factors, which induces loss of salivary gland function along with immune infiltration that exaggerates pSS. Importantly, restoration of Ca2+ signaling upon paquinimod treatment reversed the pSS phenotype thereby inhibiting the progressive development of pSS.

The NK cell receptor NKp46 recognizes ecto-calreticulin on ER-stressed cells.

Natural killer (NK) cell kill infected, transformed and stressed cells when an activating NK cell receptor is triggered1. Most NK cells and some innate lymphoid cells express the activating receptor NKp46, encoded by NCR1, the most evolutionarily ancient NK cell receptor2,3. Blockage of NKp46 inhibits NK killing of many cancer targets4. Although a few infectious NKp46 ligands have been identified, the endogenous NKp46 cell surface ligand is unknown. Here we show that NKp46 recognizes externalized calreticulin (ecto-CRT), which translocates from the endoplasmic reticulum (ER) to the cell membrane during ER stress. ER stress and ecto-CRT are hallmarks of chemotherapy-induced immunogenic cell death5,6, flavivirus infection and senescence. NKp46 recognition of the P domain of ecto-CRT triggers NK cell signalling and NKp46 caps with ecto-CRT in NK immune synapses. NKp46-mediated killing is inhibited by knockout or knockdown of CALR, the gene encoding CRT, or CRT antibodies, and is enhanced by ectopic expression of glycosylphosphatidylinositol-anchored CRT. NCR1)-deficient human (and Nrc1-deficient mouse) NK cells are impaired in the killing of ZIKV-infected, ER-stressed and senescent cells and ecto-CRT-expressing cancer cells. Importantly, NKp46 recognition of ecto-CRT controls mouse B16 melanoma and RAS-driven lung cancers and enhances tumour-infiltrating NK cell degranulation and cytokine secretion. Thus, NKp46 recognition of ecto-CRT as a danger-associated molecular pattern eliminates ER-stressed cells.