TLR10: An Intriguing Toll-Like Receptor with Many Unanswered Questions.

BACKGROUND: Toll-like receptors (TLRs) are one of the first pattern recognition receptors found in the innate immune system. The TLR family has 12 members (TLR1-TLR9, TLR11-TLR13) in mice and 10 members (TLR1-TLR10) in humans, with TLR10 being the latest identified. SUMMARY: Considerable research has been performed on TLRs; however, TLR10 is known as an orphan receptor for the lack of information on its signalling, role, and ligands. Even though there are recent studies pointing towards the potential TLR10 ligands, their function and signalling pathway are yet to be determined. KEY MESSAGES: This review gives an insight into recent findings on TLR10's pro- and anti-inflammatory properties, with the goal of outlining existing results and indicating future research topics on this receptor.

The Development and Validation of Radiopharmaceuticals Targeting Bacterial Infection.

The International Atomic Energy Agency organized a technical meeting at its headquarters in Vienna, Austria, in 2022 that included 17 experts representing 12 countries, whose research spanned the development and use of radiolabeled agents for imaging infection. The meeting focused largely on bacterial pathogens. The group discussed and evaluated the advantages and disadvantages of several radiopharmaceuticals, as well as the science driving various imaging approaches. The main objective was to understand why few infection-targeted radiotracers are used in clinical practice despite the urgent need to better characterize bacterial infections. This article summarizes the resulting consensus, at least among the included scientists and countries, on the current status of radiopharmaceutical development for infection imaging. Also included are opinions and recommendations regarding current research standards in this area. This and future International Atomic Energy Agency-sponsored collaborations will advance the goal of providing the medical community with innovative, practical tools for the specific image-based diagnosis of infection.

CD34 Protein: Its expression and function in inflammation.

CD34 has spear-headed the field of basic research and clinical transplantation since the first reports of its expression on hematopoietic stem cells (HSCs). Expressed in mice, humans, rats and other species, CD34 has been used for more than 40 years as a hematopoietic stem and progenitor cell marker. It was later found that muscle satellite cells and epidermal precursors can also be identified with the aid of CD34. Despite the usefulness of CD34 as a marker of HSCs, its overall purpose in animal physiology has remained unclear. This review recaptures CD34 structure, evolutionary conservation, proposed functions, and role in lung inflammation, to describe current research findings and to provide guidance for future studies on CD34.

Modulation of low-dose ozone and LPS exposed acute mouse lung inflammation by IF1 mediated ATP hydrolysis inhibitor, BTB06584.

Ozone and bacterial lipopolysaccharide (LPS) are common air pollutants that are related to high hospital admissions due to airway hyperreactivity and increased susceptibility to infections, especially in children, older population and individuals with underlying conditions. We modeled acute lung inflammation (ALI) by exposing 6-8 week old male mice to 0.005 ppm ozone for 2 h followed by 50 µg of intranasal LPS. We compared the immunomodulatory effects of single dose pre-treatment with CD61 blocking antibody (clone 2C9.G2), ATPase inhibitor BTB06584 against propranolol as the immune-stimulant and dexamethasone as the immune-suppressant in the ALI model. Ozone and LPS exposure induced lung neutrophil and eosinophil recruitment as measured by respective peroxidase (MPO and EPX) assays, systemic leukopenia, increased levels of lung vascular neutrophil regulatory chemokines such as CXCL5, SDF-1, CXCL13 and a decrease in immune-regulatory chemokines such as BAL IL-10 and CCL27. While CD61 blocking antibody and BTB06584 produced maximum increase in BAL leukocyte counts, protein content and BAL chemokines, these treatments induced moderate increase in lung MPO and EPX content. CD61 blocking antibody induced maximal BAL cell death, a markedly punctate distribution of NK1.1, CX3CR1, CD61. BTB06584 preserved BAL cell viability with cytosolic and membrane distribution of Gr1 and CX3CR1. Propranolol attenuated BAL protein, protected against BAL cell death, induced polarized distribution of NK1.1, CX3CR1 and CD61 but presented with high lung EPX. Dexamethasone induced sparse cell membrane distribution of CX3CR1 and CD61 on BAL cells and displayed very low lung MPO and EPX levels despite highest levels of BAL chemokines. Our study unravels ATPase inhibitor IF1 as a novel drug target for lung injury.

Regulation of TLR10 Expression and Its Role in Chemotaxis of Human Neutrophils.

Toll-like receptors are innate immune receptors that play a critical role in pathogen-associated molecular pattern recognition. TLR10 was recently identified and very limited data are available on its expression, mechanisms that regulate its expression, and its role in primary immune cells. To study the expression pattern of TLR10 in primary immune cells, we examined TLR10 protein expression in naive and Escherichia coli lipopolysaccharide (LPS)-activated human neutrophils. Human neutrophils challenged with LPS showed a decrease in total and surface TLR10 expression at 90 min. TLR10 in LPS-activated neutrophils colocalized with flotallin-1, a lipid raft marker, and EEA-1, an early endosomal marker, to suggest its endocytosis. There was increased colocalization of TLR10 with TLR4 at LPS 60 min followed by decrease at later LPS treatment times. Treatment with TLR4 neutralizing antibody decreased cytoplasmic localization of TLR10 in LPS-treated neutrophils. Reactive oxygen species (ROS) depletion and neutralization of p65 subunit of NF-κB in LPS-treated neutrophils decreased TLR10 expression. Live cell imaging of LPS-activated neutrophils showed TLR10 translocation in the leading edge and TLR10 knockdown in neutrophils reduced their fMLP-induced chemotaxis and the number of neutrophils with pseudopodia but without affecting the expression of key proteins of actin nucleation process, ARP-3 and Diap1. Taken together, our findings show that neutrophil activation alters TLR10 expression through ROS production and NF-κB regulation, and TLR10 knockdown reduced neutrophil chemotaxis.

Research article expression of surfactant protein-A and D, and CD9 in lungs of 1 and 30 day old foals.

BACKGROUND: Respiratory diseases are a major cause of morbidity and mortality in the horses of all ages including foals. There is limited understanding of the expression of immune molecules such as tetraspanins and surfactant proteins (SP) and the regulation of the immune responses in the lungs of the foals. Therefore, the expression of CD9, SP-A and SP-D in foal lungs was examined. RESULTS: Lungs from one day old (n = 6) and 30 days old (n = 5) foals were examined for the expression of CD9, SP-A, and SP-D with immunohistology and Western blots. Western blot data showed significant increase in the amount of CD9 protein (p = 0.0397) but not of SP-A and SP-D at 30 days of age compared to one day. Immunohistology detected CD9 in the alveolar septa and vascular endothelium but not the bronchiolar epithelium in the lungs of the foals in both age groups. SP-A and SP-D expression was localized throughout the alveolar septa including type II alveolar epithelial cells and the vascular endothelium of the lungs in all the foals. Compared to one day old foals, the expression of SP-A and SP-D appeared to be increased in the bronchiolar epithelium of 30 day old foals. Pulmonary intravascular macrophages were also positive for SP-A and SP-D in 30 days old foals and these cells are not developed in the day old foals. CONCLUSIONS: This is the first data on the expression of CD9, SP-A and SP-D in the lungs of foals.

Loss of Nucleobindin-2/Nesfatin-1 increases lipopolysaccharide-induced murine acute lung inflammation.

NUCB2/nesfatin-1 is expressed in variety of tissues. Treatment with nesfatin-1 reduces inflammation in rat models of subarachnoid hemorrhage-induced oxidative brain damage and traumatic brain injury as well as myocardial injury. There is only one study showing anti-inflammatory actions of nesfatin-1 on acute lung inflammation. To more precisely determine the role of NUCB2/nesfatin-1 in acute lung inflammation, we conducted a study using NUCB2/nesfatin-1 knockout (NKO) mice as well as neutrophils isolated from the bone marrows of WT and NKO mice. Our findings suggest that the absence of NUCB2/nesfatin-1 significantly increases the accumulation of adherent neutrophils by approximately 3 times compared with WT within LPS-treated lungs. Integrating this with observations from both BALF and neutrophil cytokine expression, we propose that although neutrophils lacking NUCB2/nesfatin-1 individually secrete less pro-inflammatory cytokines compared with stimulated WT cells, the result of knocking out NUCB2/nesfatin-1 is net pro-inflammatory. No change was found in NUCB2/nesfatin-1 mRNA or protein expression comparing WT LPS and PBS-treated samples. Taken together, our results show that NUCB2/nesfatin-1 is constitutively expressed in mouse lungs and neutrophils and demonstrates anti-inflammatory properties in mouse lungs during acute lung injury, by inhibiting adherent neutrophil accumulation and inflammatory cytokine expression.

Lack of CD34 produces defects in platelets, microparticles, and lung inflammation.

Lung innate immune activation results in acute lung inflammation, which is characterized by alveolar barrier disruption and accumulation of cellular lung aggregates comprising neutrophils, platelets, mononuclear cells, and microparticles. CD34 is a sialomucin, with pan-selectin affinity and recently shown to protect the endothelial barrier in a bleomycin-induced lung injury model. However, there is very little information about the fundamental role of CD34 in regulation of the lung innate immune response. We hypothesized that CD34 regulates leukocyte recruitment by promoting optimal platelet activation (aggregation and spread) during bacterial lipopolysaccharide (LPS)-induced acute lung injury. Therefore, we utilized CD34 knock-out (KO) and wild-type (WT) mice to analyze and compare the morphology and expression of leukocyte subsets from the pulmonary and systemic compartments. We utilized the chemotactic N-formylated tri-peptide, fMLP, to understand platelet aggregation in vitro, and the fundamental immune stimulant, LPS, to induce lung injury and understand platelet activation ex vivo. Our data reveal that under steady-state conditions, KO mice possess large aggregates of integrin ß3 (CD61)-positive microparticles in peripheral blood. Moreover, the KO mice recruit a large number of neutrophils to lungs, which are not cleared even at 36-h post-LPS exposure. The KO mice display an increased platelet CD61 expression, which aggregates, but does not spread normally in response to in vitro fMLP treatment. The KO platelets display similar deficits in their spreading ability even after ex vivo LPS exposure. Thus, our data demonstrate that CD34 modulates platelet biology, microparticle aggregation, and neutrophil recruitment during murine lung inflammation.

Characterization of low-dose ozone-induced murine acute lung injury.

Ozone is a toxic and highly reactive gaseous oxidizing chemical with well-documented adverse health effects in humans. On the basis of animal and human data, environmental guidelines and air quality standards recommend a threshold for exposure of no more than 0.063 ppm of ozone (daily concentrations). This research describes a standardized sensitive model of sterile murine lung inflammation induced by exposing mice to acute (0, 4 or 24 hr), yet low, levels of ozone (0.005, 0.05 or 0.5 ppm), one that are below the current recommendations for what is considered a safe or "ambient" ozone concentration for humans. Ozone led to concentration and time-dependent phlogistic cell death in the bronchoalveolar lavage, lung epithelial damage and hemorrhage. Interestingly, we observed distinct large bright CD11b positive cells in the bronchoalveolar lavage, upregulation of lung vascular and alveolar ATP synthase as well as plasminogen and bronchiolar angiostatin expression in ozone-exposed mice, platelet and neutrophil accumulation in the lung vasculature and an eotaxin-2, IL-16, CXCL5, CXCL12, and CXCL13 dominant inflammatory response leading to lung injury. Using a fluorescent intravital microscopy set up, we quantified ozone-induced extensive alveolar cellular damage. We observed ozone-induced actin filament disorganization, perturbed respiratory mechanics, acute suppression of the alveolar reactive oxygen species (ROS) production and mitochondrial potential in ventilated lungs. We present evidence of systemic, as well as pulmonary toxicity, at 40-fold lower ozone concentrations than previously reported in mice. The findings are important in establishing a sensitive means of quantifying structural and functional lung disorganization following exposure to an aerosolized pollutant, even at levels of ozone exposure previously thought to be safe in humans.

Neutrophils in the lung: "the first responders".

Neutrophils, constituting the first line of defense, perform vital functions during immune surveillance. A key feature that assists in their prompt response to an inflammatory signal is rapid migration to the affected site. They are normally short-lived but can be activated to live longer under the influence of an inflammatory stimulus. They can, thereby, release their toxic granule contents that are differentially housed inside the cytoplasm. Although these events are well characterized in the peripheral circulation, we are still far from fully understanding their recruitment in lungs. Lungs are a reservoir of neutrophils under steady-state. In the event of an infection or injury, they promptly activate and recruit to the alveolar compartment as well as the airways. Lung intravital microscopy has revealed that neutrophils display novel features during steady- and activated-state highlighting key differences in the lung vasculature compared to peripheral sites. This review will discuss neutrophil biology in lung inflammation and will highlight the role of angiostatin, an anti-angiogenic molecule, as well as vitronectin, an acute phase secreted plasma protein, in lung neutrophil recruitment.

RGD-tagged helical rosette nanotubes aggravate acute lipopolysaccharide-induced lung inflammation.

Rosette nanotubes (RNT) are a novel class of self-assembled biocompatible nanotubes that offer a built-in strategy for engineering structure and function through covalent tagging of synthetic self-assembling modules (G∧C motif). In this report, the G∧C motif was tagged with peptide Arg-Gly-Asp-Ser-Lys (RGDSK-G∧C) and amino acid Lys (K-G∧C) which, upon co-assembly, generate RNTs featuring RGDSK and K on their surface in predefined molar ratios. These hybrid RNTs, referred to as K(x)/RGDSK(y)-RNT, where x and y refer to the molar ratios of K-G∧C and RGDSK-G∧C, were designed to target neutrophil integrins. A mouse model was used to investigate the effects of intravenous K(x)/RGDSK(y)-RNT on acute lipopolysaccharide (LPS)-induced lung inflammation. Healthy male C57BL/6 mice were treated intranasally with Escherichia coli LPS 80 µg and/or intravenously with K9°/RGDSK¹°-RNT. Here we provide the first evidence that intravenous administration of K9°/RGDSK¹°-RNT aggravates the proinflammatory effect of LPS in the mouse. LPS and K9°/RGDSK¹°-RNT treatment groups showed significantly increased infiltration of polymorphonuclear cells in bronchoalveolar lavage fluid at all time points compared with the saline control. The combined effect of LPS and K9°/RGDSK¹°-RNT was more pronounced than LPS alone, as shown by a significant increase in the expression of interleukin-1ß, MCP-1, MIP-1, and KC-1 in the bronchoalveolar lavage fluid and myeloperoxidase activity in the lung tissues. We conclude that K9°/RGDSK¹°-RNT promotes acute lung inflammation, and when used along with LPS, leads to exaggerated immune response in the lung.