TRPML Cation Channels in Inflammation and Immunity

Background: In 1883, Ilya Mechnikov discovered phagocytes and established the concept of phagocytosis by macrophages. In 1908, he was awarded the Nobel Prize in Physiology/Medicine for his findings, which laid the foundations for today's understanding of the innate immune response. Only in the 1960s, Max Cooper and Robert Good significantly advanced our understanding of the immune system by demonstrating that B- and T-cells cooperate to regulate the adaptive immune response. Both, innate and adaptive immune response are essential to effectively protect the individual against infectious agents, such as viruses, bacterial or insect toxins, or allergens. Innate immune responses occur rapidly upon exposure to noxious or infectious agents or organisms, in contrast to the adaptive immune system that needs days rather than hours to develop and acts primarily on the basis of antigen-specific receptors expressed on the surface of B- and T-lymphocytes. In recent years, it has become evident that endosomes and lysosomes are involved in many aspects of immune cell function, such as phagocytosis, antigen presentation and processing by antigen-presenting cells, release of proinflammatory mediators, e.g., by mast cells, or secretion of the pore-forming protein perforin by cytotoxic T lymphocytes. Several lysosomal storage disorders (LSDs) have been associated with defects in immune system function or immune system hyperactivity, such as Gaucher, Fabry, or Niemann-Pick type C1 disease, mucopolysaccharidoses (MPS), gangliosidosis, or juvenile neuronal ceroid lipofuscinosis (JNCL). Beside accumulating evidence on the importance of endolysosomes in immune cell function, recent results suggest direct roles of endolysosomal ion channels, such as the TRPML channels (mucolipins), which are members of the transient receptor potential (TRP) superfamily of non-selective cation channels, for different aspects of immune cell function. The aim of this review is to discuss the current knowledge about the roles of TRPML channels in inflammation and immunity, and to assess their potential as drug targets to influence immune cell functions. Advances: Examples of recently established roles of TRPML channels in immune system function and immune response include the TRPML1-mediated modulation of secretory lysosomes, granzyme B content, and tuning of effector function in NK cells, TRPML1-dependent directional dendritic cell (DC) migration and DC chemotaxis, and the role of TRPML2 in chemokine release from LPS-stimulated macrophages. Outlook: Although our understanding of the functional roles of TRPML channels in inflammation and immunity is still in its infancy, a few interesting findings have been made in the past years, encouraging further and more detailed work on the role of TRPMLs, e.g., in intracellular trafficking and release of chemokines, cytokines, or granzyme B, or in phagocytosis and bacterial toxin and virus trafficking through the endolysosomal machinery

Lung emphysema and impaired macrophage elastase clearance in mucolipin 3 deficient mice

Lung emphysema and chronic bronchitis are the two most common causes of chronic obstructive pulmonary disease. Excess macrophage elastase MMP-12, which is predominantly secreted from alveolar macrophages, is known to mediate the development of lung injury and emphysema. Here, we discovered the endolysosomal cation channel mucolipin 3 (TRPML3) as a regulator of MMP-12 reuptake from broncho-alveolar fluid, driving in two independently generated Trpml3-/- mouse models enlarged lung injury, which is further exacerbated after elastase or tobacco smoke treatment. Mechanistically, using a Trpml3IRES-Cre/eR26-τGFP reporter mouse model, transcriptomics, and endolysosomal patch-clamp experiments, we show that in the lung TRPML3 is almost exclusively expressed in alveolar macrophages, where its loss leads to defects in early endosomal trafficking and endocytosis of MMP-12. Our findings suggest that TRPML3 represents a key regulator of MMP-12 clearance by alveolar macrophages and may serve as therapeutic target for emphysema and chronic obstructive pulmonary disease

Endolysosomal cation channels and toxic chronic lung disease

Chronic obstructive pulmonary disease (COPD) is a prevalent disease that affects million people worldwide. Classified as the third most common cause of death globally, it accounts for more than 3 million deaths per year. Smoking constitutes the main risk factor for developing COPD, while other factors include inhalation of environmental pollutants such as exhaust fumes from the industry or vehicles, occupational dusts and fumes, or genetic predispositions. Characteristic for COPD is a chronically inflamed lung and permanent pathological changes of the airways and lung tissue. Consequently, this leads to common symptoms like chronic cough, sputum, and shortness of breath, all of which significantly affect the quality of life. Two common forms of COPD can be described: chronic bronchitis associated with mucus hyper-production, and emphysema resulting from destruction of alveolar tissue, e.g. due to an excess of the macrophage elastase MMP-12. However, treatment options are poor, as no drugs are currently available that can slow down COPD progression or reduce the disease mortality. Thus, it is important and necessary to uncover novel treatment strategies and drug tarets. In this dissertation, the endolysosomal cation channel TRPML3 was investigated in the context of COPD and emphysema development with the aim to evaluate its potential as therapeutic target. Being located on membranes of intracellular organelles such as early endosomes and lysosomes, TRPML3 is involved in membrane fusion and fission events including endocytosis, trafficking, exocytosis, and autophagy. Here, by the use of the following novel reporter mouse line, Trpml3-IRES-Cre/eR26-τGFP, TRPML3 was found to be mainly expressed in alveolar macrophages (AMΦ) in the lungs. Further studies on lung function, and examination of lung tissues revealed that Trpml3 deficient (Trpml3-/-) mice show a pulmonary emphysema phenotype. This was demonstrated under both basal conditions and toxic conditions, meaning the exposure to cigarette smoke to induce COPD, and the application of a porcine pancreatic elastase to induce emphysema in the lungs. Analysis of bronchoalveolar lavage fluid (BAL-F) obtained from Trpml3-/- mice unveiled increased levels of the AMΦ-specific protease MMP-12, whereas other inflammatory cytokines, proteases and antiproteases, i.e. tissue inhibitor of metalloproteinases (TIMPs), were unchanged. Furthermore, loss of TRPML3 in AMΦ did not affect lysosomal exocytosis, but resulted in impairments in endolysosomal trafficking and endocytosis, being causative for the increased levels of MMP-12 also found in AMΦ cell culture supernatants and likely in BAL-F. Overall, this work discovered a significant role of TRPML3 in COPD and emphysema development. Mice lacking TRPML3 were unveiled to be particularly susceptible for developing pulmonary emphysema, attributed to the reduced capacity of Trpml3-/- AMΦ to balance the MMP-12 concentration in the lungs properly (Figure 1). Functioning as such a key regulator for MMP-12, TRPML3 may be considered as novel therapeutic target structure for the treatment of COPD

Correction factors for self-selection when evaluating screening programmes

Objective: In screening programmes there is recognized bias introduced through participant self-selection (the healthy screenee bias). Methods used to evaluate screening programmes include Intention-to-screen, per-protocol, and the post hoc approach in which, after introducing screening for everyone, the only evaluation option is participants versus non-participants. All methods are prone to bias through self-selection. We present an overview of approaches to correct for this bias. Methods: We considered four methods to quantify and correct for self-selection bias. Simple calculations revealed that these corrections are actually all identical, and can be converted into each other. Based on this, correction factors for further situations and measures were derived. The application of these correction factors requires a number of assumptions. Results: Using as an example the German Neuroblastoma Screening Study, no relevant reduction in mortality or stage 4 incidence due to screening was observed. The largest bias (in favour of screening) was observed when comparing participants with non-participants. Conclusions: Correcting for bias is particularly necessary when using the post hoc evaluation approach, however, in this situation not all required data are available. External data or further assumptions may be required for estimation

Neuroblastoma Screening at 1 Year of Age: The Final Results of a Controlled Trial

Background: Neuroblastoma screening aims to reduce neuroblastoma-related mortality. A controlled trial showed no reduction in stage 4 disease incidence and preliminary mortality data. This article presents epidemiologic and clinical data 20 years after cessation of the screening program. Methods: The patients with detected disease in the screening area were compared with the clinically diagnosed patients in the control area and in the prestudy and poststudy cohorts. All statistical tests were 2-sided. Results: The cumulative incidence for children aged 1 to 6 years in the birth study cohorts (1994-1999) in the screening arm was 13.4 cases per 100 000 births (95% confidence interval [CI] = 12.2 to 14.6) based on 61.2% of screening participants and 38.8% of nonparticipants. Screening participants had a cumulative incidence of 15.7 (95% CI = 14.0 to 17.4) per 100 000 births. The cumulative incidence in the contemporary control cohort was 9.3 (95% CI = 8.2 to 10.3) per 100 000 births, 7.6 (95% CI = 6.8 to 8.4) in the prestudy cohort, and 8.1 (95% CI = 7.4 to 8.9) in the poststudy cohort from 2000 to 2004 (P<.001 each). The increased incidence in the screening cohort was restricted to stages 1 through 3, while stage 4 incidence was not reduced. The cumulative mortality for deaths within 10 years from diagnosis and per 100 000 births remained unchanged. Patients with stage 4 disease detected by screening had better biological characteristics and an improved outcome compared with those stage 4 cases not detected by screening. Conclusions: Neuroblastoma screening at 1 year of age reduced neither stage 4 incidence nor neuroblastoma mortality and was affected by overdiagnosis, leading to unnecessary treatment. A few screening-detected stage 4 cases represent a biologically interesting subgroup but do not change the recommendation to close the catecholamine-based neuroblastoma screening book

Cardiovascular Health Status And Genetic Risk In Survivors of Childhood Neuroblastoma and Nephroblastoma Treated With Doxorubicin: Protocol of the Pharmacogenetic Part of the LESS-Anthra Cross-Sectional Cohort Study

Background: In childhood cancer survivors (survival of 5 years or more after diagnosis), cardiac toxicity is the most common nonmalignant cause of death attributed to treatment-related consequences. Identifying patients at risk of developing late cardiac toxicity is therefore crucial to improving treatment outcomes. The use of genetic markers has been proposed, together with clinical risk factors, to predict individual risk of cardiac toxicity from cancer therapies, such as doxorubicin. Objective: The primary aim of this study is to evaluate the value of multimarker genetic testing for RARG rs2229774, UGT1A6 rs17863783, and SLC28A3 rs7853758 for predicting doxorubicin-induced cardiotoxicity. The secondary aim is to replicate previously described associations of candidate genetic markers with doxorubicin-induced cardiotoxicity. Moreover, we will evaluate the prevalence of cardiovascular dysfunction in childhood cancer survivors after neuroblastoma or nephroblastoma. Methods: This is the pharmacogenetic substudy of the research project Structural Optimization for Children With Cancer After Anthracycline Therapy (LESS-Anthra). We invited 2158 survivors of childhood neuroblastoma or nephroblastoma treated with doxorubicin according to the trial protocols of SIOP 9/GPOH, SIOP 93-01/GPOH, SIOP 2001/GPOH, NB 90, NB 97, or NB 2004 to participate in this prospective cross-sectional cohort study. The study participants underwent a cardiological examination and were asked to provide a blood or saliva sample for genotyping. The study participants' health statuses and cardiovascular diagnoses were recorded using a questionnaire completed by the cardiologist. Digital echocardiographic data were centrally evaluated to determine the contractile function parameters. Medical data on the tumor diagnosis and treatment protocol were taken from the study documentation. Survivors were screened for variants of several candidate genes by TaqMan genotyping. Results: This study includes 657 survivors treated with doxorubicin for childhood cancer, the largest German cohort assembled to date to investigate cardiovascular late effects. Data analyses are yet to be completed. Conclusions: This study will define the genetic risk related to 3 marker genes proposed in a pharmacogenetic guideline for risk assessment. Moreover, the results of this study will show the prevalence of cardiovascular dysfunction in survivors of pediatric neuroblastoma or nephroblastoma who were treated with doxorubicin. The results will help to improve primary treatment and follow-up care, thus reducing cardiovascular late effects in the growing population of childhood cancer survivors

TRPML3 distribution in salivary gland, adrenal gland, and pituitary gland.

(A) Overview image of the salivary gland with its lobules. Two submandibular lymph nodes (LN) are located at the sites. (B) Zoomed image of the upper lmph node (LN). (C) Zoomed image of the salivary gland tissue reveals the typical structure of secretory acini (arrowheads), consisting of several secretory cells. Some of these acini show τGFP expression. (D-F) Immunostainings show CgA expression in secretory cells. Some of the secretory cells building up one acinus reveal higher CgA expression than others. This higher CgA expression is colocalizing with τGFP (merged/yellow). (G) Immunostaining of the kidney with the adrenal gland (AG) sitting on top using CgA to label neuroendocrine cells. (H-K) Magnified images of the adrenal gland from (G). DAPI staining reveals the adrenal medulla (AM) and the adrenal cortex (AC). CgA antibody stains the catecholamine secreting chromaffin cells in the adrenal medulla. τGFP+ cells are nearly absent from the adrenal medulla, but seem to be more prominent in outer regions of the adrenal cortex. (L) Overview image of the pituitary gland. A, adenohypophyis. N, neurohypophysis. I, intermediate lobe. τGFP expression was mainly found throughout the adenohypophysis. (M-O) Zoomed image and immunostaining of the adenohypophysis using an antibody against CgA reveals several endocrine cells (CgA+ cells) overlapping with τGFP (arrowheads).</p

TRPML3 distribution in olfactory bulb and nose.

(A, B) Overview images of coronal sections of the olfactory bulb. GL, glomerular layer. MCL, mitral cell layer. GCL, granule cell layer. τGFP expression is mainly detected in the GL. (C, D) Magnified images of the glomerular layer (GL) showing prominent τGFP expression in the glomeruli. (E) Coronal section of the nose. NC, nasal cavity. PD, pharyngeal duct (nasopharynx). S, nasal septum. (F-H) Zoomed images of the nasal septum (S) and olfactory epithelium (OE). τGPF signal is found in olfactory receptor neurons (ORNs) of the olfactory epithelium (OE) and in the bundles of axons (BA) that are projecting to the glomeruli of the olfactory bulb.</p

Alveolar macrophages of neonatal and adult express TRPML3.

Immunofluorescence images using F4/80 antibody as macrophage marker (A) in 10 μM lung cryosections or (B) on cells isolated by bronchoalveolar lavage. Images clearly show co-localization of τGFP and F4/80 confirming TRPML3 expression in lung macrophages. (C) Trpml3 levels in lung increase from birth to adult. Trpml3 detected by RT-qPCR and normalized as in Fig 1. Levels are displayed relative to normalized Trpml3 in E18 lung. (D, E) In situ hybridization on sections of neonatal (P2) lungs from Trpml3+/+ mice shows a pattern of scattered positive cells (arrowheads) detected by both 5’Trpml3 (B) and 3’Trpml3 (C) probes. (F-K): Immunohistochemistry using nonfluorescent detection (ABC+DAB) shows NT and CT1 antibodies specifically label scattered cells (arrowheads) in sections of adult (P48) Trpml3+/+ (F-I) but not Trpml3-/- lungs (J, K). (L, M): Co-immunohistochemistry on sections of adult (P48) Trpml3+/+ lungs indicates that NT colabels F4/80 positive macrophages. Scale bars indicate 50 μm in D, E, F, G, J, K and 10 μm in H, I, L, M.</p

Percent AQP2+ and AQP1+ tubes as a function of NT+ tubes.

Percent AQP2+ and AQP1+ tubes as a function of NT+ tubes.</p