Mucopolysaccharide diseases are a group of lysosomal storage disorders caused by deficiencies of hydrolase enzymes, leading to pathological glycosaminoglycan accumulation. A number of mucopolysaccharidosis (MPS) types are characterised by severe airway disease, the aetiology of which is poorly understood. There is ongoing evidence of significant clinical disease in the long-term despite disease modifying therapeutic strategies, including enzyme-replacement therapy (ERT). To provide a better understanding of this aspect of disease, we have characterised extracellular matrix (ECM) and inflammatory alterations in adenotonsillar tissue samples from 8 MPS patients.Adenotonsillar samples from MPS I, IVA and VI ERT treated patients and from a single enzyme naïve MPS IIIA individual were compared to non-affected control samples using quantitative immunohistochemistry, qPCR and biochemical analysis.Significantly increased lysosomal compartment size and total sulphated glycosaminoglycan  were identified in patient samples despite ERT. Heparan sulphate glycosaminoglycan was significantly elevated in MPS I and IIIA (p = 0.002), confirming incomplete reversal of disease. Collagen IV and laminin α-5  staining demonstrated increased ECM deposition within the reticular and capillary network of MPS samples. No significant change in the expression of the pro-inflammatory cytokines IL-1α, IL-6 or TNF-α was seen compared to control.This study suggests a role for ECM remodelling contributing to the obstructive phenotype of airway disease in MPS. Current therapeutic strategies with ERT fail to normalise these pathological alterations within adenotonsillar samples. Our findings lend novel insight into the pathological cascade of events, with primarily structural rather than inflammatory changes contributing to the continuing phenotype seen in patients despite current therapeutic regimes. Mucopolysaccharide diseases are a heterogeneous group of inherited metabolic lysosomal storage disorders with a combined incidence of 1 in 22,000 , 4.Airway involvement is a well-recognised feature of MPS I, II, IV and VI and significantly contributes to morbidity and premature mortality , 6. The The structural changes observed within the pharynx and laryngotracheal complex present as airway obstruction, most commonly secondary to adenotonsillar hypertrophy and tracheomalacia. A reactive environment has been described on endoscopic examination of the upper airways, with mucosal infiltration within the larynx and trachea and evidence of bronchitis noted within the distal airways , 18. AniThe airway in MPS provides a potential paradigm of both structural and inflammatory disease within a single organ system. The aetiology of airway deposits and of the inflammatory changes seen within the airway are yet to be elucidated. GAG induced perturbations in mucosal and extracellular matrix (ECM) function or induction of inflammatory cascades are presumed to play a role. However, this has not been demonstrated in MPS. We aim to identify the pathogenic events that continue to cause airway morbidity in the upper airway of MPS by determining and quantifying the structural and inflammatory phenotype of MPS patient tissue in comparison to unaffected controls.MPS patients undergoing adenotonsillectomy for the resolution of symptoms of upper airway obstruction and obstructive sleep apnoea (OSA) at the Royal Manchester Children’s Hospital, UK, were recruited to the study following informed written consent. Tissue samples deemed surplus to clinical requirements were collected from patients undergoing surgery for these clinically indicated procedures under ethical permission 13/NW/0029 (Greater Manchester North Research Ethics Committee) from April 2013 onwards.Clinical data on sample collection, patient demographics including phenotype, age at commencement of treatment and details of interventions for airway obstruction were documented.Overnight sleep oximetry studies were performed in recruited patients during unsedated natural sleep, using sleep oximetry equipment  using analysis criteria as previously described . StudiesHaematoxylin and eosin and periodic acid schiff (PAS) histological staining was performed according to standard protocols on identical 5μm sections from patient samples .Patient adenotonsillar samples were mounted in OCT  and cut as 5μm cryosections onto glass slides . Immunofluorescent staining against human lysosomal associated membrane protein 2  and heparan sulphate (HS) GAG  were performed using previously described protocols , 28]. Mohttp://rsb.info.nih.gov/ij). The mean staining intensity, based on relative grayscale pixel intensity (Arbitary units), from the 3 fields of view from 3 sections per organ in each mouse / individual were calculated [Images were acquired using the Pannoramic 250 Flash II automated digital scanning microscope  using a 20x/0.80 Plan Apo objective for brightfield and fluorescence scanning and a 40x/0.95 Plan Apo objective additionally for fluorescence and viewed using Pannoramic viewer (3D Histech Ltd) . Three slculated . Images -1 total protein.Total sulphated GAG quantification in patient samples was performed using the Blyscan Assay  as previously described . GAG levT method was used to determine relative mRNA expression [Patient samples of adenotonsillar tissue were placed in RNAlater solution  and stored at −20°C at the time of surgery. RNA was isolated for measurement by RT-PCR of IL-1α, IL-6, and TNF-α mRNA expression using TriZOL  and RNeasy Mini kit  and treated with Turbo DNase enzyme  to minimise DNA contamination. Quantification was performed using a Nanodrop spectrophotometer and 100 ng of total RNA reverse transcribed into cDNA . Taqman gene expression assays  containing sequence specific primers for human IL-1α (Assay ID:Hs00174092_m1), IL6 (Assay ID: Hs0098539_m1) and TNF-α (Assay ID: HS01113624_g1) were used to achieve target specific amplification. GAPDH was used as the endogenous comparator gene. RT-PCR was performed in triplicate on a StepOne Plus Real Time PCR system . Age matched non-affected patient controls were used as calibrator reference samples. The comparative ΔCpression .Descriptive statistics were calculated for demographics, sleep oximetry and biochemical data. Correlation coefficients were calculated with Pearson’s r and Spearman’s rho for normally and non-normally distributed data sets respectively. Student’s T-test, Mann-Whitney and one way and two-way ANOVA with post hoc analysis by Tukey’s multiple comparisons test were used to identify differences between the characteristics of subgroups. P-values <0.05 were considered significant. JMP v11.0 software (SAS) was used for this analysis. All raw data is provided as supporting information in The patients enrolled in this study were those identified as requiring adenotonsillectomy, adenoidectomy or tonsillectomy alone for clinical symptoms of upper airway obstruction. Twelve patients with a mean age of 5.5 years (range 2–12 years) were recruited to the study, composed of 8 MPS patients . All but the MPS IIIA patient were treated with ERT with a median age at commencement of 3 years. The median duration on ERT was 2.2 years (range 0.7–7.5years). Four individuals, without a diagnosis of metabolic disease, were recruited as controls and age and sex matched where possible. A specific history of tonsillitis was sought prior to enrolment, to exclude the potential confounding effect of infection on assessment of inflammatory profile within tissue. Subject demographic, treatment characteristics and sleep study outcomes are presented in All but 2 patients underwent overnight sleep oximetry studies, with the most recent pre-surgical study assessed . Six of the 8 (75%) MPS patients had evidence of severe SDB based on previously published criteria  .From the 12 recruited patients, 9 adenoid and 8 tonsil samples were processed and analysed. No significant differences were seen in staining intensity between adenoid and tonsillar samples from the same individual or in the group as a whole (p = 0.65). As a result adenotonsillar samples were pooled for analysis and grouped by MPS type, rather than tissue type.Collagen IV and laminin α-5 are known to localise to ECM reticular network and capillary basement membrane (BM). In order to determine architecture and ECM remodelling in adenotonsillar samples, H&E and immunostaining against these proteins was undertaken. The gross microscopic follicular architecture of adenotonsillar lymphoid tissue was preserved in MPS patients. High power magnification identified histiocytoid changes within the cells of the interfollicular zones and subepithelial stroma as shown in Collagen IV and laminin α-5 subunit demonstrated similar staining patterns within all ECM compartments of the adenoid and tonsil . In both2 = 0.64, p = 0.0017, In samples from the same individual, collagen IV staining correlated strongly with laminin reactivity, suggesting localisation to similar ECM components (R-1 vs MPS 8.9μg ml-1) and contributed to predominantly by MPS VI—demonstrating a 12-fold increase over control samples (p = 0.0007).To investigate GAG storage in patient tissue, HS accumulation was assessed with 10E4 antibody immunostaining and total tissue GAG was quantified using the Blyscan assay. HS localised to the basement membrane and vascular endothelial structures of the arteriole network in both control and MPS, in keeping with the structural role of PGs. Co-localisation with LAMP2, to within the lysosomal compartment of reticular matrix and capillary ECM components was more evident in HS storing MPS types I and III . Quantif2 = 0.44, p = 0.01; Lysosomal storage was demonstrated and quantified with LAMP2 and compared to control in adenotonsillar tissue of patients . LAMP2 rT method was used. As the control samples analysed were not paired to MPS samples, relative expression was conveyed as the mean 2-ΔCT and variability between MPS and control groups  demonstrated consistently raised expression of cytokines in tonsils relative to adenoids, although this failed to reach significance . As a result, adenoid and tonsil data are presented separately. A significant degree of variability in expression in our control samples was seen, raising the possibility of subclinical infection in some patients.Analysis demonstrated a reduction in expression of our pro-inflammatory cytokine panel in relation to control samples for all cytokines in adenoid tissue and IL-6 and TNF-α in tonsil. This reduction was significant for IL-6 in adenoid tissue .Our study performs the most detailed assessment to date of the structural and biochemical pathobiology within tissues contributing to upper airway obstruction in MPS. We find evidence of persistent lysosomal and GAG storage in adenotonsillar tissue despite ongoing treatment with ERT. We further observe significant alteration in ECM deposition, suggesting tissue remodelling occurring as a secondary process responsible for disease pathogenesis. We were unable to identify evidence of increased inflammation within these tissues.The characteristic architecture of adenotonsillar tissue, composed of a reticular fibre meshwork of various ECM proteins in close proximity to follicular lymphatic structures appears to be preserved in MPS. Previous studies of adenotonsillar tissue in MPS have focused solely on these histological appearances or crude GAG quantification –37. We hCollagen IV and laminin α-5 are the most widely expressed ECM components in the follicles, interfollicular zone and capillary network of lymphoid tissue  and our As has previously been determined in the CNS of MPS mice models , secondaExamination of serial sections demonstrates that pathological storage of HS appears to co-localise with collagen and laminin within the BM and reticular network of the tonsils and adenoids, thus making direct interaction more likely. This is in keeping with findings that PGs localise to the BM in tonsil  while stClinical and observational studies in the literature, including a systematic review of pre and post ERT polysomnography measures of obstruction, fail to demonstrate a significant improvement in OSA severity following commencement of ERT , 50, 51,Currently, there is no existing clinical or experimental evidence of altered immune function in the airway of patients with MPS. The interactions between pro and anti-inflammatory cytokines and GAGs and their PGs is complex . The cytQuantitative PCR was unable to show a significant upregulation in pro-inflammatory cytokine expression in MPS airway samples. In fact, relative expression of cytokines responsible for innate immunity was generally reduced in adenotonsillar tissue in comparison to control samples. This was exaggerated by a significant degree of variability amongst the control patient cohort, despite a history of tonsillitis being sought as an exclusion criterion. IL-6 plays both pro and anti-inflammatory roles, including in the acute response to microbial infection and is required for the transition from innate to acquired immunity . InteresAnalysis of patient samples alone has a number of drawbacks. The primary limitation of clinical studies of a rare and heterogeneous disease, such as MPS, is the small population available for recruitment. Infrequent surgery given the anaesthetic risks means that tissue availability is significantly limited. Indeed, no patients with MPS II underwent adenotonsillar surgery during the study recruitment period in our institution. Additionally, all individuals except MPS III and IVB patients are currently treated with either ERT or HSCT and hence patient samples were unavailable to provide comparative information on pre-therapeutic manifestations of disease. Determination of the untreated and lower respiratory tract phenotype maybe facilitated by use of a murine model of MPS, however it remains unclear whether mouse models of MPS provide an accurate reflection of airway manifestations seen in patients.We have performed a detailed analysis of airway pathobiology in patient samples, identifying novel findings that potentially explain the phenotype of airway disease in MPS. While ERT is known to reverse features such as hepatosplenomegaly, previous clinical and observational studies , 50, 51 As a consequence of these finding, we recommend that efforts be directed towards earlier diagnosis and initiation of ERT, for example with neonatal metabolic screening, to prevent the early and progressive substrate accumulation and that once established leads to irreversible and persistent upper airways obstruction. Secondly, once established and if significant, interventions, such as surgical adenotonsillectomy or non-invasive ventilatory support, in addition to ongoing disease modifying therapy, be targeted at relieving the obstructive manifestations noted within the airway of MPS.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.


Abuse, neglect, and other forms of early life stress (ELS) significantly increase risk for psychiatric disorders including depression. In this study, we show that ELS in a postnatal sensitive period increases sensitivity to adult stress in female mice, consistent with our earlier findings in male mice. We used RNA-sequencing in the ventral tegmental area, nucleus accumbens, and prefrontal cortex of male and female mice to show that adult stress is distinctly represented in the brain’s transcriptome depending on ELS history. We identify: 1) biological pathways disrupted after ELS and associated with increased behavioral stress sensitivity, 2) putative transcriptional regulators of the effect of ELS on adult stress response, and 3) subsets of primed genes specifically associated with latent behavioral changes. We also provide transcriptomic evidence that ELS increases sensitivity to future stress through enhancement of known programs of cortical plasticity. Early life stress alters behavioural response to adult stress in female mice. Here authors transcriptionally profile three brain regions involved in reward  in both male and female adult mice after early life and/or adult stress Nearly 1% of children in the US are victims of child abuse and neglect each year4, and many more children experience other forms of profound stress and trauma, including death of a caregiver, parental incarceration, or experience of a natural disaster. Studies in humans and animal models indicate that ELS increases risk for depression by sensitizing individuals to stress later in life, leading to a first appearance or synergistic worsening of depression-like symptoms9. Stress early in life can alter developmental brain trajectories, and not just steady-state processes, which has the potential to augment the long-term impact of stress. However, the ways in which ELS alters transcriptional development in the brain to increase risk for depression and depression-like behaviors are poorly understood.Early life stress (ELS) increases the lifetime risk of depression and is one of the strongest risk factors for developing mental illness8. Here, we first demonstrate that ELS in the same postnatal period likewise increases the susceptibility of female mice to adult stress. Using RNA-sequencing, we next asked whether ELS in male and female mice alters patterns of gene transcription in the brain’s reward circuitry, including ventral tegmental area (VTA), nucleus accumbens (NAc), and prefrontal cortex (PFC), all of which are implicated in stress responses and human depression. Our previous work found broad and long-lasting transcriptional changes in adult male VTA after ELS7. We hypothesized that similar biological pathways might be disrupted in male and female mice by ELS, even if specific genes within each pathway are distinct between the sexes. We also hypothesized that ELS-induced transcriptional changes in these brain regions would in turn lead to either unique transcriptional responses to additional stress in adulthood, or an exaggeration of earlier transcriptional alterations. We used a variety of analytical tools to determine biological processes, primed genes, and putative regulatory factors disrupted by ELS alone or in combination with adult stress to address these questions.We recently reported that ELS during a sensitive period from postnatal day P10–20, but not P2–12, increases the susceptibility to develop depression-related behavioral abnormalities after a second hit of stress in adulthood in male miceOverall, we present comprehensive genome-wide analysis of transcriptional changes in both male and female mice, across three brain reward regions, after ELS with or without subsequent adult stress. These transcriptional analyses provide a foundation for future research testing the causal role of molecular pathways implicated in ELS-induced stress sensitivity in both sexes.10. There were also main effects of both ELS and STVS on the ratio of novel/home cage latencies. Female mice that experienced two hits of stress had increased latency and ratios relative to ELS and STVS alone. Survival analysis indicated differences in latency to eat among ELS–STVS mice compared to ELS mice  and STVS mice . Main effects of both ELS and STVS on distance traveled . Adult sub-threshold variable stress (STVS) alone did not significantly alter behavior relative to standard-reared control female mice on any measure. There was an interaction between ELS and STVS on latency to eat Fig.  and on tled Fig.  in the N11 there was a main effect of STVS on latency to immobility, and female mice that experienced both ELS and STVS had shorter latency to immobility compared to ELS alone  also heightens later stress sensitivity. We did not find an effect of early–ELS on any individual measure , but did not find a significant relationship.We evaluated whether phase of estrus cycle impacted behavior in any test  was previously reported for male VTA only after either ELS or social defeat alone7. Here we extend this analysis and include transcriptional changes after ELS, adult stress (STVS or Defeat), and a combination of the two stresses (ELS + STVS/Defeat), in three brain reward regions from adult males and females. Significance of DEGs was set at uncorrected p < 0.05 and log2(fold-change (FC)) > |0.3785|  for broad pattern identification. There was 1–23% overlap in DEGs across the three stress conditions (ELS or STVS/Defeat or ELS + STVS/Defeat) in male and female VTA, male NAc, and male and female PFC  to identify patterns and strength of genome-wide overlap in a threshold-free manner. RRHO analyses confirmed similar directional regulation of genes by ELS and adult stresses  DAVID functional annotation for gene ontology of biological processes, (2) upstream regulator analysis with ingenuity pathway analysis, and (3) HOMER motif analysis to predict differential transcriptional regulators. These analyses were agnostic to direction of expression change. We hypothesized that, while distinct sets of genes may be regulated across sexes or across brain regions, similar biological processes or transcriptional regulators might be affected. Top gene ontology terms for male and female VTA, NAc, and PFC are shown in Fig. We next examined molecular pathways altered by a second hit of stress after ELS, compared to ELS alone. Multicellular organism development was again a top GO term in male VTA and male and female PFC Fig. . Interesp < 0.05) different from standard-control expression levels with LFC > |0.3875|, and also significantly (p < 0.05) different from both ELS-alone and adult stress-alone groups, in either direction without FC cutoff , and 1 primed gene in PFC .We hypothesized that ELS might increase sensitivity to adult stress through increases in developmentally defined plasticity programs. Knockout of 22. Here, we present systematic mapping of enduring ELS-induced transcriptomic changes in three reward-associated brain regions key to depression-like behaviors23, in both male and female mice, and we further assess transcriptomic responses to a second stress in adulthood.Stress experienced early in life can change circuit development and response to later stress and reward, which are likely mediated in part by transcriptional changes in the brain early in life and sustained across the lifespan25. We previously found that this same ELS procedure from P2 to 12 did not significantly alter depression-like behaviors in male mice, either before or after a second stress in adulthood, which may be due to immature stress-response circuitry or timing of molecular cascades that mediate adaptations to stressful stimuli26. In male mice, shifting the timing of ELS to P10–17 (or P10–20) also did not alter depression-like behaviors at baseline, but instead increased the risk that a second stress in adulthood would result in depression-like behaviors. These findings are in contrast to other rodent ELS paradigms that report baseline depression-like behaviors from early (<P14) postnatal stress in female mice27, and evidence that ELS prior to P9 may in fact blunt hippocampal plasticity and dampen responses to additional stress29. Studies of humans that experienced early life adversity have found earlier development of anxiety, depression, and other psychiatric illnesses, as well as latent vulnerability to these diseases32. For example, women abused as children are more likely to experience depression after additional adult abuse compared to women abused only as adults or only as children5. Key stress-responsive brain regions may mature faster in females than males33, and ELS may alter stress-related behaviors earlier in females than males34. Thus, we tested the hypothesis that stress in the earlier rather than later postnatal period might have greater impact on depression-like behavior in female mice. However, the greatest differences in individual and composite behavioral measures were found after the combination of ELS from P10–17 with STVS  induces susceptibility to subsequent adult stress. The mouse ELS paradigm used here was based on both rodent maternal separation studies and work demonstrating reduced nesting and/or bedding material induces disordered maternal care among rodent dams35 and later development of threat circuitry (see ref. 26). Inhibition of noradrenergic cells from P10–21, but not P2–9 or P56–67, increased anxiety-like and depression-like behavior in adult mice36. Dopamine signaling in the striatum matures rapidly from P10 through adolescence and is required from P18 to 28, but not from birth or in adulthood, for normal physiological development and related behaviors38. Rodent amygdala begins to support fear-learning at P10, plasticity and long-term potentiation develop in rodent hippocampus in the second postnatal week, and connectivity between human amygdala and hippocampus appears to develop later in children40. The impact of ELS on these and other brain regions important for stress and threat-learning would be interesting to transcriptionally profile in future studies. While there are mixed reports on whether timing of ELS experience in humans influences later depression42, it is also necessary to consider that mice are born premature relative to humans and for at least some brain systems rodent postnatal week-one is more similar to the second half of human gestation44.The later postnatal ELS that we find increases vulnerability to adult stress may be due to a later sensitive period of catecholamine circuitry development7. However, RNA-seq also revealed that adult stress after ELS does not simply amplify transcriptional differences observed after either stress alone, but instead results in unique transcriptional changes. Our results show that experience of stress in adulthood induces distinct transcriptional patterns depending on whether or not the mouse had previously experienced ELS, across multiple brain regions in both male and female mice. Similarly, chronic adolescent stress was found to induce distinct, latent patterns of gene expression in the hippocampus of male and female rats in response to an acute adult stress45. Together, these findings suggest that ELS alters transcriptional development of the brain- or, the lifelong potential (priming) of transcriptional regulation in response to subsequent stressful stimuli.The latent behavioral stress sensitivity induced by ELS is useful for disentangling the enduring transcriptional impact of ELS associated with vulnerability, from neurobiological changes associated with depression-like behaviors per se or with acute stress. Across three brain reward-associated regions, we find broadly similar transcriptional changes after ELS and adult stress. These similarities were visualized using both thresholded (heatmap) and threshold-free (RRHO) analyses Fig. . These f48. Nevertheless, it is still useful to consider here similarities in biological pathways affected by each stress or combination of stresses among male and female mice, even when distinct genes contribute to pathway enrichment. While the majority of predicted pathways and upstream regulators were unique, several GO terms related to brain and organism development were enriched in ELS DEG lists across sexes and regions. These predictions suggest that ELS alters brain development across reward regions in both sexes, through distinct genes, but potentially common pathways related to neuronal outgrowth and synapse formation and signaling. Moreover, several regulators predicted in females have been previously implicated in depression-like behavior in male mice. Neuronal PAS-domain protein 2 (NPAS2) is a predicted transcriptional regulator after two hits of stress in female NAc , and is also dysregulated in serum of depressed patients53 and hippocampus of rats after stress across the lifespan55. Ctnnb1 (which encodes ß-catenin) in male NAc D2 receptor subtype neurons was previously shown to mediate resilience to adult stress through DICER-mediated microRNA regulation56. DICER1 was also a top predicted upstream regulator in female PFC , experienced different adult stresses , and tissue was processed separately, we have deliberately avoided direct comparisons of potential sex differences in response to stress as sex and cohort variables cannot be distinguished. RNA-seq in both human and mouse brain reveals substantial sex differences at baseline, as well as in human depression and stressed miceNAc Fig. . SCNA  which included Kcnj13 (the voltage-gated inwardly rectifying potassium channel Kir7.1), Steap1 (six-transmembrane epithelian antigen of prostate 1), and Slc4a5 (electrogenic sodium bicarbonate cotransporter 4). Several of the primed genes identified by our analyses have been previously implicated in stress vulnerability and depression-like behavior in humans and rodents. Sgk1 (serum- and glucocorticoid-inducible kinase 1) was primed down in male VTA. While Sgk1 is increased in blood of depressed patients, it exerts a neuroprotective role under oxidative stress conditions which may be selectively suppressed in male VTA after ELS61. Npy has also been implicated in stress response (in different directions depending on region and acute vs. long-term sampling62) and is primed up in male VTA in our dataset and elevated in male and female hypothalamic regions after ELS from P2-963. Only one primed gene, Myrf (myelin regulatory factor) in female PFC, has been associated with major depression in human GWAS studies64. Empirical testing is needed to determine whether manipulation of primed genes, alone or in combination, is sufficient to alter sensitivity to adult stress.Our previous work focused on enduring transcriptional changes after ELS that may underlie vulnerability to stress in adulthood, while the current analyses extends to latent transcriptional changes revealed by the second hit of stress, which we call primed genes Fig. . DespiteLynx1, which binds nicotinic acetylcholine receptors17. Lynx1 knockout preserves experience-dependent plasticity past the critical period into adulthood17. Our hypothesis was supported in male NAc, and to a lesser degree in male VTA and female PFC  with ad libitum access to food and water. All experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee at Mount Sinai and of the Society for Neuroscience. All behavioral testing occurred during the animals’ light cycle. Experimenters were blind to experimental group, and order of testing was counterbalanced during behavioral experiments.All mating occurred in-house to prevent shipping stress during gestation. Two nulliparous C57BL/6J female mice (Jackson) were mated with one male in our animal facility. Males were removed after 5 days and females were separated into individual cages 1–3 days prior to parturition. Litters were weighed and counted and cages cleaned on the day of birth (PND0) but otherwise undisturbed. Cages were cleaned with minimal disruption to the litter once/week. Offspring were weaned at postnatal PND21 with males and females weaned separately into cages of 3–5 mice, keeping littermates together and only combining pups from different litters and of the same experimental condition to maintain three or more mice/cage. Male and female mice included here were from separate cohorts.71 from P10–17  or ELS groups. Pups were separated together as a litter to clean cages with distinct bedding for 3–4 h/day at random times each day during the light cycle to minimize predictability and habituation. EnviroDri nesting material was depleted to 1/3 of standard-reared cages during the days of separations and then restored on the final day. Early-ELS was identical but from P2 to 10, and separation cages were placed on warming pads to maintain constant low temperature (32–34 °C), since pups at that age cannot thermoregulate on their own.ELS consisted of a combination of maternal separation and limited nesting12. Adult stress for female mice consisted of 3 days of STVS13 , as previously reportedS13 Fig.  in order7. Female depression-like behaviors were tested in one cohort used for RNA-seq  and in an additional replication cohort which included early-ELS exposed mice . Beginning the day after the last day of adult stress, one behavioral test was conducted per day for 3 consecutive days, in the order described below, with the exception of sucrose preference which spanned behavioral testing days.Behavior of male mice was characterized and reported previously72. The test was performed under red light . Mice were placed in an empty cage and sprayed on the back with a 10% sucrose solution two times. Behavior was video recorded for 5 min The latency to begin grooming the back, and the total amount of time grooming thereafter  was scored by hand by an observer blind to experimental group. Increased latency and decreased total grooming time were considered indicators of depression-like or anxiety-like behavior13.Splash test was performed consistent with previous studies73. Upon single-housing, mice were acclimated overnight with two bottles of drinking water . The next day after conclusion of the splash test, water in one bottle was replaced with a 1% sucrose solution and both bottles weighed. Bottles were weighed again daily at the beginning of the light cycle for 2 days. Bottle locations were switched at each measurement to prevent location habituation. Percent sucrose preference was calculated as amount (g) sucrose solution consumed over total amount (g) of water and sucrose consumed.Sucrose preference, a measure of anhedonia-like behavior in mice, was assessed in a two-bottle choice test10. Mice were food restricted overnight before testing. On the day of testing, mice habituated to the testing room for at least 1 h. Under red light conditions, mice were then placed into a 40 × 40 × 20 cm arena with wood-chip bedding covering the floor and a single standard chow food pellet in the center of the arena. Mice were placed in the corner of the box, and the latency to eat was hand-scored for up to 5 min (first cohort) or 10 min (second cohort). A video-tracking system  measured locomotor activity. At the end of the test, mice were transferred to their home cage in standard lighting conditions, and the latency to eat was recorded by hand. A ratio of latency to eat in the novel arena/home cage was then calculated.Novelty suppressed feeding testing was adapted from previous workFor the forced swim test, mice were individually placed in beakers of 25 ± 1 °C water for 6 min with ambient lighting. Immobility was assessed by a video-tracking system as a measure of depression-like behavior .7. For the initial behavioral cohort  and SPSS  were used for all graphing and statistical analysis of behavior. Significance thresholds were set at n = 4–6 per group)1. Samples from female brain were from mice whose behavior is reported in the initial cohort here .Samples from male brain were from mice whose behavior was reported previously , and sequenced with 125-nt single-end reads at Beckman Coulter Genomics (currently Genewiz). Samples were multiplexed to produce >30M reads/sample. All RNA-seq files are available through Gene Expression Omnibus (GEO), accession GSE89692.Adult mice were cervically dislocated directly from the home cage 1 day after the final behavioral test. Brains were removed rapidly, placed into ice-cold PBS, and sliced into 1 mm-thick coronal sections in a slice matrix. Bilateral punches were made from VTA (16 gauge), NAc (14 gauge), and PFC (12 gauge) and flash-frozen in tubes on dry ice. Total RNA was isolated with TriZol reagent (Invitrogen) and purified with RNeasy Micro Kits (Qiagen). All RNA samples were determined to have A260/280 values ≥ 1.8 (Nanodrop); samples for RNA-seq had RIN values > 9 . 500 ng of purified RNA was used to prepare libraries for sequencing using the Truseq mRNA library prep kit (Illumina RS-122-2001/2). As previously reported, male VTA and NAc samples were pooled 3 animals/sample  prior to library preparation, and were sequenced on an Illumina Hi-seq machine with 50-nt single-end reads in the Mount Sinai Genomics Core Facilitiesn = 5–8 per group) and converted to cDNA using either SuperScript III (Invitrogen 18-080-400) or High-Capacity cDNA Reverse Transcription kits (Applied Biosystems 43-688-14). Primer efficiency and specificity were verified; sequences are given in Supplementary Data −∆∆Ct method with Hprt as a control gene. Genes were semi-randomly chosen across levels of significance, brain regions, and sexes. Only protein-coding genes with high-quality primer validation were included.RNA was extracted as above from VTA, NAc, or PFC tissue punches from independent cohorts of ELS and Std male and female mice  using Tophat2p < 0.05 for broad pattern identification  for each comparison. DEGs for each comparison represented in Venn Diagrams . Predicted upstream regulators included top predicted genes, cytokines, or endogenous chemicals, made from ≥3 up-regulated or down-regulated DEGs. HOMER motif analysis was used to predict differential transcriptional regulators including transcription factor-binding sites and transcriptional elements from enriched 8–10 base sequences located within −2000 to +1000 bases of the TSS of DEGs79.All genes included in differential gene expression analysis had a base mean expression >2. Significance was set at uncorrected p < 0.05) different from standard-control expression levels with LFC > |0.3875|, and also significantly (p < 0.05) different from both ELS-alone and adult stress-alone groups, in either direction without FC cutoff .Primed genes  multiplied by the sign of the fold change from the DESeq2 analysis, filtered to all genes with a mean base expression of >2 RPKM to avoid low-expression artifacts. RRHO was used to evaluate the overlap of differential expression lists between stress comparisons84. A two-sided version of this analysis was used to test for coincident and opposite enrichment15. RRHO difference maps were produced for pairs of RRHO maps  by calculating for each pixel the normal approximation of difference in log odds ratio and standard error of overlap between each matched comparison. This Z score was then converted to a P-value and corrected for multiple comparisons across pixels.Full threshold-free differential expression lists were ranked by the −log10 primary visual cortex tissue punch of male Lynx1−/− vs. WT mice (GEO: GSE8975718). Briefly, we used Limma85 to quantile normalized raw microrray probe-level data and RankProd86 to compute rank-based differential expression of mouse genes retaining genes with a false discovery rate < 0.0587, which we mapped to Entrez IDs to yield a 114 gene Lynx1−/− transcriptional signature.Genes differentially expressed across the entire transcriptome of early life and adult stress signatures were converted to Entrez IDs and unmapped and unexpressed genes were removed. A molecular matching score was calculated between the plasticity signature and a given stress signature by summing the logFC expression values in a stress signature that are shared with genes decreased in the plasticity signature and subtracting them from the sum of logFC expression values shared with genes increased in the plasticity signature to yield a summary measure of concordance between stress and plasticity signatures. High molecular match scores (>0) indicate a given stress signature mimics the plasticity signature whereas low molecular match scores (<0) indicate a given stress signature induces gene expression that opposes the plasticity signature.P value of molecular match scores (M) between a given stress and plasticity signature, we calculated an empirical P value for M given n = 10,000 permutations of M (Mperm), computed by shuffling the gene labels of the stress signature and recalculating M. The generalized Pareto distribution88 was used to improve accuracy of the P value estimation and multiple hypothesis tests were adjusted for using the false discovery rate87. To compare M for a given plasticity signature across the stress signatures, we normalized M with Mperm according to M (the effect size). M is computed similar to the approach by Zhang and Gant89. Analyses were completed in the R programming language (v 3.2.2).To estimate the Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Reporting SummarySource Data


The characterisation and clinical relevance of tumour-infiltrating lymphocytes (TILs) in leiomyosarcoma (LMS), a subtype of soft tissue sarcoma that exhibits histological heterogeneity, is not established. The use of tissue microarrays (TMA) in studies that profile TIL burden is attractive but given the potential for intra-tumoural heterogeneity to introduce sampling errors, the adequacy of this approach is undetermined. In this study, we assessed the histological inter- and intra-tumoural heterogeneity in TIL burden within a retrospective cohort of primary LMS specimens. Using a virtual TMA approach, we also analysed the optimal number of TMA cores required to provide an accurate representation of TIL burden in a full tissue section. We establish that LMS have generally low and spatially homogenous TIL burdens, although a small proportion exhibit higher levels and more heterogeneous distribution of TILs. We show that a conventional and practical number (e.g. ≤3) of TMA cores is adequate for correct ordinal categorisation of tumours with high or low TIL burden, but that many more cores (≥11) are required to accurately estimate absolute TIL numbers. Our findings provide a benchmark for the design of future studies aiming to define the clinical relevance of the immune microenvironments of LMS and other sarcoma subtypes. This approach offers several advantages over conventional examination of full tissue sections by minimising consumption of often limited tissue while providing various efficiencies in downstream sample processing and analysis. However, an inherent limitation to the use of TMAs is that, for each included specimen, only a small amount of tissue is sampled and arrayed, meaning that sampling error may lead to a distorted representation of the full tissue section. This limitation is of particular relevance in the study of tumour specimens, where intra-tumour spatial heterogeneity in terms of morphology and underlying molecular pathology is now well established in many cancer types2. Multiple studies have been undertaken to validate the TMA methodology in the assessment of various cancer biomarkers, with the aim of demonstrating that biomarker levels reported by TMAs are representative of results obtained when full sections are assessed. In this manner, it has been shown that expression levels of a diverse repertoire of tumour biomarkers are accurately reported through the assessment of TMAs, typically with the provision that between 1 and 3 replicate cores from each included tumour are assessed and aggregated6.Tissue microarrays (TMAs) are useful diagnostic and research tools that permit high-throughput histological and molecular studies of up to several hundred tissue specimens simultaneously by arraying them into a paraffin block10. TMAs continue to be frequently employed in studies that seek to investigate the potential role of TILs as putative biomarkers15. The success of such studies is dependent on the ability of the TMA approach to capture a sufficiently representative picture of the immune phenotypes present within the wider tumour. Observations of quantitative and qualitative spatial heterogeneity in the immune microenvironment of individual tumours of various cancer types call into question how well TMAs can provide such representation and whether the scope for sampling error renders them inappropriate for studies of tumour immunity20. There is currently little published evidence addressing this question23.Investigating the immune microenvironment as a potential source of cancer biomarkers is an area of renewed research interest. It is now known that the presence or absence of immune-related factors such as tumour-infiltrating lymphocytes (TIL) can serve as powerful prognostic and/or predictive biomarkers across a range of cancer types24. As with other STS subtypes, the immune microenvironment and its potential prognostic value is not well characterised in LMS. There is accumulating evidence that LMS is a disease that harbours extensive inter- and intra-tumoural genetic and morphological heterogeneity. For instance, recent genomic profiling analyses demonstrate that LMS is characterised by inter-tumour variability in somatic copy number alterations, a molecular characteristic found to have negative correlation with active anti-tumour immune response in a number of other cancers26. Furthermore, clinical evidence suggest that a small minority of LMS patients respond to immune checkpoint inhibitor therapy29. As such, inter-patient differences in the immune microenvironment of LMS may be useful for predicting response to therapy and prognosis. LMS often present with large primary tumours that exhibit intra-tumoural morphological heterogeneity of tumour cells and associated stroma, and consequently may also display intra-tumoural TIL heterogeneity30. To assess the suitability of TMAs for profiling TIL burden in LMS, we sought to address two questions in this study: 1) What is the extent of inter- and intra-tumour heterogeneity of TIL burden in LMS, by comparing related tumour blocks from spatially distinct areas of primary tumours and 2) how many TMA cores are required to provide sufficient representation of the TIL burden of the full tissue section?In this study, we assessed a cohort of leiomyosarcoma (LMS) tumour specimens to investigate the extent of inter- and intra-tumoural heterogeneity of TIL burden and how accurately TIL burden are represented by the TMA methodology, compared to full tumour sections. LMS are tumours of smooth muscle lineage that can arise from the uterus or from other anatomic sites including vessel walls. LMS are one of the more common soft tissue sarcoma (STS) subtypes, representing 10–20% of all STSSurgical resection specimens of primary LMS (n = 47) and accompanying annotation of baseline clinicopathological variables were identified and retrieved through retrospective review of departmental database and medical notes at a single specialist cancer centre. Histological diagnosis was confirmed by a specialist sarcoma histopathologist . Where available, 5 blocks containing formalin-fixed paraffin-embedded (FFPE) viable tumour from spatially distinct areas (at least 2 blocks each from tumour margins and core) of the same primary tumour were selected. Newly prepared haematoxylin and eosin (H&E) slides from each block were assessed to confirm presence of viable tumour material. Immunohistochemical (IHC) staining of T lymphocyte markers  was performed on consecutive 4 µm sections from each block . Human tonsillar tissue was used for positive control for expression of CD3, CD4 and CD8, with omission of primary antibody for negative control and use of Mouse IgG1  or Rabbit IgG  for CD4/CD8 or CD3 isotype controls respectively. IHC staining for B lymphocytes  was performed on all blocks from an initial set of 19 tumours – this was not expanded to all tumours due to uniformly low numbers of infiltrating B cells in this initial set.2) was manually counted by direct brightfield microscopy for each stained slide.The number of CD3, CD4, CD8 and CD20 IHC-positive lymphocytes in ten non-adjacent, tumour-containing high-power fields (HPF) . To generate a virtual TMA (vTMA), 1 mm diameter circular areas were selected from viable-tumour areas on each H&E image. For assessment of optimal number of cores, 20 × 1 mm areas were selected at random, while to assess for intra-tumoral variance between central and peripheral tumour regions, 20 × 1 mm areas were selected at both the tumour periphery (defined as within 3 mm of inked resection margin) and central (defined as ≥10 mm from nearest resection margin) regions. The corresponding areas were then selected on CD3 and CD8 digital slide images. Images of these areas at x10 magnification were exported as.tif files that were cropped to uniform 0.785 mmTo generate the physical TMA (pTMA), triplicate 1 mm diameter cores were sampled from areas of viable tumour within donor blocks from 44/47 LMS and re-embedded in an arrayed recipient paraffin block. Consecutive 4 µm sections from the arrayed block were stained for H&E, CD3 and CD8. After assessment of H&E slides to confirm viable tumour content, all CD3+ and CD8+ TILs were counted under direct brightfield microscopy. Average TIL number per 1 mm core (referred to herein as ‘TIL/core’) was calculated from triplicate cores for each tumour.To assess the extent of TIL burden in each of 47 LMS cases, an average number of infiltrating CD3+, CD4+ and CD8+ TIL per HPF (referred to herein as ‘TIL/HPF’) was calculated from 50 HPF per tumour (10 HPF from each of 5 related tumour blocks). Comparison of TIL burden of tumours from different anatomical sites of origin and grade was performed using 1-way ANOVA of Log2-transformed average TIL/HPF values with Prism v7.0 (GraphPad Software Inc).To assess the variability in TIL burden between different blocks from the same surgical specimen, we assessed the relative contribution of inter-block variation (block effect) and inter-tumour variation (tumour effect) on the total amount of variance in TIL numbers within the 47 LMS cohort by (i) Log2 transformation of all raw TIL/HPF count values (ii) calculation of average TIL/HPF with 95% confidence interval for each tumour block (average of 10 HPF), and across all 5 related blocks from each primary tumour (average of 50 HPF), and (iii) Ordinary 2 way ANOVA (Prism v7.0) to assess the percentage of total variability attributable to block effect, tumour effect, interaction between the two effects and residual variation. Correlation between Log2-transformed values for tumour average TIL/HPF and the standard deviation of average TIL/HPF of constituent blocks was assessed through calculation of Pearson correlation coefficients.n cores that fell within the following prescribed boundaries were then calculated: a) within +/− 20% of true TIL burden; b) within correct (i.e. same as true TIL burden) side of dichotomised ‘high/low’ boundary set at median of true TIL burdens from 47 LMS cohort; c) within correct quartile of 47 LMS cohort.Automated counts of infiltrating CD3+ and CD8+ TIL in all 20 × 1 mm vTMA cores for each tumour were used to calculate average TIL/core – this value was taken as representing the ‘true’ TIL burden of each tumour. Estimates of these true TIL burdens were then derived from the average TIL/core from all possible combinations of between 2 and 19 randomly-selected cores. The percentage of estimates generated from Automated counts of infiltrating CD3+ and CD8+ TIL in 20 × 1 mm vTMA cores were Log10-transformed and then used to derive sample frequency distributions, average TIL/core and standard deviation values from matched peripheral and central tumour areas in a subset of 6 tumours. Paired T tests were then performed to assess for differences between average TIL/core values from margin and core areas using Prism v7.0 (GraphPad Software Inc).The differences between Log2-transformed values of the estimated average TIL/core values derived from the pTMA and the true TIL burdens derived from the vTMA were calculated and plotted against the average of the two values in a Bland Altman plot along with 95% levels of agreement (Prism v7.0).Average of TIL/core values derived from triplicate cores within pTMA were used to identify each included LMS as having a ‘high’ or ‘low’ TIL burden, relative to the cohort median of true TIL burdens, as defined in the vTMA experiment. This high/low identification was then compared to a ‘gold standard’ high/low allocation, defined as the ‘true’ TIL burden of that tumour as derived from all 20 vTMA cores. Accuracy (%) of pTMA was defined as 100*(True Positive + True Negative)/Use of archival FFPE tumour samples and linked anonymised patient was approved by Institutional Review Board as part of the PROSPECTUS study, a Royal Marsden-sponsored non-interventional translational protocol .Adequate tumour material was identified for 47 patients with a confirmed diagnosis of LMS who had undergone radical resection of primary tumour  taken to represent the overall tumour TIL burden. Exemplar IHC images showing different degrees of CD3+ lymphocyte infiltration are shown in Fig. 36.For each case in the cohort, 5 tissue blocks that sampled spatially distinct tumour areas were assessed for TIL burden , indicated the near-absence of infiltrating B cells in a subset of 19 tumours. Across the different T lymphocyte markers, a dynamic range of 2–3 orders of magnitude (e.g. CD3 range 1–124 TIL/HPF) was seen in the extent of TIL burden between individual tumours Fig. . No signHaving established that overall TIL burdens can vary between individual LMS tumours, we assessed the extent of heterogeneity in average TIL/HPF between blocks taken from different regions from the same LMS specimen Fig. .Average TIL burden (stated as TIL/HPF) from each of 5 sampled blocks from the 47 cases are shown aligned with overall tumour average values in Fig. We performed 2-way ANOVA to objectively assess the relative extent that intra-tumoural heterogeneity (i.e. variation between blocks from the same tumour – ‘block effect’) and inter-tumoural heterogeneity  contributed to the overall amount of variation in TIL burden within the cohort Fig. . We founTaken together, these results indicate that while intra-tumoural heterogeneity was observed in a subset of LMS cases with higher overall TIL levels, intra-tumoural heterogeneity in TIL burden across the cohort was outweighed by the extent of inter-tumoural heterogeneity.in silico ‘virtual TMA’ (vTMA) that would allow for the iterative sampling of a number of cores that would be impractical for a physical TMA. We then assessed how many cores were required to produce an estimate of TIL burden that either (i) accurately recapitulated the true TIL burden of a tumour, or (ii) was sufficiently accurate to identify whether a tumour had high or low TIL burden, relative to the median or quartile TIL values of the entire cohort– this second approach was based on the observation that, in many published studies that have demonstrated clinical relevance of TIL numbers, similar rank-based categorisation was used, often based on dichotomisation around cohort median value36.To address the question of how many TMA cores must be sampled from a tumour to provide adequate representation of the overall TIL burden of a tumour, we devised an 2 - equivalent to approximately 50 HPF) were selected on H&E images, with the number of TILs within the corresponding areas (TIL/core) on CD3 and CD8 stained slides digitally counted  was required for >80% of estimated TIL burdens to fall within 20% of the ‘true’ CD3+ or CD8+ TIL burden for the corresponding tumour Fig. . However36. We found that sampling only 1 core was sufficient to correctly identify a majority of tumours as has having a ‘high’ or ‘low’ degree of infiltration, while 2–5 cores was adequate to correctly identify a majority of tumours as having ‘very low’, ‘low’, ‘high’ or ‘very high’ degree of TIL infiltration, based on categorical cut-offs at cohort quartiles. These data demonstrate that TMAs that include a practical number of replicate cores as used in previously reported studies (e.g. 3 or fewer) would be sufficiently representative when ordinal categorisation of TIL burden is planned. However, should precise quantification of the absolute value of true TIL burden be desired, a conventional TMA approach is unlikely to provide an adequate representation.Taken together, these data indicate that a large and likely impractical number of TMA cores (11 cores) must be sampled in order to accurately recapitulate the true burden of infiltrating T lymphocytes in LMS. However, many studies that have described an association between TILs and clinical outcome ultimately applied cut-off thresholds to assign TIL counts into ordinal categories (e.g. ‘high’ or ‘low’ infiltration) that reflect relative rather than absolute degree of infiltration33. In order to assess the degree of variability in TILs between the tumour periphery and central regions in our study, a subset of 6 LMS cases were subjected to a vTMA workflow  that included triplicate 1 mm cores from sampled tumours. Forty-four out of 47 LMS cases were included in this TMA. In 11/44 (25%) tumours, the same block was used for pTMA construction as was used for the vTMA model. In 33/44 (75%) tumours, due to insufficient tissue depth remaining in blocks used for the vTMA, a different tumour block from the same specimen was used for core sampling for the pTMA.In the vTMA model, a median of 11 cores were required to accurately estimate the absolute value for true TIL burden. We thus assessed if the triplicate cores used in pTMA were similarly inaccurate in estimating absolute true TIL burdens  was itself independently associated with worse prognosis. This suggests that spatial uniformity of TIL burden may vary between different cancer types and within different molecular and histological subtypes, and that spatial distribution itself may provide clinically relevant information, both findings that warrant caution when adopting TMA methods for assessing TIL burden. Immune microenvironment-based biomarkers that have been described in colorectal and breast cancers depend upon intra-tumoural spatial distinction of TIL, where comparison of infiltrates between the tumour core or invading margin, or between tumour nests and stromal components, have been shown to be important considerations8. In our study, we find that across the 6 cases analysed, there was no significant difference in TIL burden between tumour periphery and central regions. Comparative analysis indicates variation in TIL levels with some cases showing higher TIL levels in the periphery versus the core and others presenting the opposite observation. Cohen et al., have recently shown in a small cohort of 11 LMS cases that CD3+ TILs were present in both the central (11/11) and periphery (9/9) regions of the tumour. In their study, CD8+ TILs were found in a subset of cases in the central (10/11) and periphery (7/9) of the tumour41. Given the small number of cases assessed in both studies, further evidence in larger cohorts is required to determine if these observations are generally applicable to LMS and other STS subtypes.The applicability of our findings to other STS or epithelial cancers remains to be determined. Intra-tumoural heterogeneity in the immune microenvironment has been described in numerous epithelial cancer types, both within primary lesions and between different metastatic sites42. Our study does not provide any direct information on the use of TMAs to assess non-TIL immune factors and the extrapolation of our findings beyond TILs remains to be investigated.While TILs are accepted as central mediators of anti-tumour immune responses, the immune-tumour microenvironment constitutes a broad and complex range of cellular and protein factors that actively determine the nature and clinical consequence of any tumour-related immune responseOur data indicate that TMAs can provide a degree of representation of overall tumour TIL burden which is adequate for ordinal categorisation into high or low subgroups. The design of future studies of the immune microenvironment of tumours should acknowledge the inherent limitations of TMA methods and consider the incorporation of additional orthogonal approaches such as gene expression analysis and flow cytometry methodologies that are capable of providing complementary information regarding the composition of immune subsets leading to a more comprehensive and accurate representation of tumour immune microenvironment.Supplemental info


Despite the common use of lipid-lowering medications, cardiovascular diseases continue to be a significant health concern. Atherosclerosis, one of the most frequent causes of cardiovascular morbidity, involves extensive inflammatory activity and remodeling of the vascular endothelium. This relentless inflammatory condition can ultimately give rise to clinical manifestations, such as ischemic heart disease or stroke. Accumulating evidence over the past decades implicates cysteine protease cathepsins in cardiovascular disorders. In particular, Cathepsins B, L, and S are over-expressed during vascular inflammation, and their activity is associated with impaired clinical outcomes. Here we took advantage of these molecular events to introduce a non-invasive detection and treatment approach to modulate vascular inflammation using a Photosensitizing quenched Activity-Based Probed (PS-qABP) that targets these proteases.Methods: We tested the application of this approach in LDL receptor-deficient mice and used non-invasive imaging and heart cross-section staining to assess the theranostic efficacy of this probe. Moreover, we used fresh human endarterectomy tissues to analyze cathepsin signals on gel, and verified cathepsin identity by mass spectrometry.Results: We showed that our PS-qABP can rapidly accumulate in areas of inflammatory atheromas in vivo, and application of light therapy profoundly reduced lesional immune cell content without affecting smooth muscle cell and collagen contents. Lastly, using human tissue samples we provided proof-of-concept for future clinical applications of this technology.Conclusions: Photodynamic therapy guided by cysteine cathepsin activity is an effective approach to reduce vascular inflammation and attenuate atherosclerosis progression. This approach could potentially be applied in clinical settings. Despite the widespread usage of statins and other lipid-lowering medications, cardiovascular morbidity is still a major health issue Over the last decade, numerous efforts have been made to characterize clinically dangerous plaques (vulnerable plaques). In addition to reduced fibrous cap thickness and large necrotic areas An extension of this approach is to take advantage of these molecular cues and deliver a compound that could ameliorate the inflammatory activity of these vulnerable plaques. Such a therapeutic modality has been successfully demonstrated to alleviate cancer progression e6 on a polylysine polymer by Shon SM et al.,et al., significantly reduced the content of lesional macrophages but at a cost of a highly invasive procedure -/-Ldlr) mice using covalent probes. We show that YBN14 accumulates in atherosclerotic lesions of mice and attenuate disease burden by reducing the number of inflammatory cells and potentially increases plaque stability by increasing its collagen content. We also test the clinical application of such therapeutic modality in humans by showing that YBN14 binds to activated CTSs in patients derived tissue specimens.This rationale has prompted the design of chlorine et al., YBN14 theranostic probe was synthesized followed Ben-Nun -/-Ldlr) in the background of CL57BL/6 purchased from the Jackson Laboratory  were housed in specific pathogen-free conditions at the Hebrew University and were used with the approval of the animal ethics committee of the Hebrew University. Females, at the age of eight weeks and same weight, were used for experiments and challenged with a high-fat diet (HFD)  or normal chow (NC) for 12 weeks.LDL receptor-deficient mice  equipped with 710/760 nm excitation/emission filters. For photodynamic therapy, mice were fed HFD or normal chow for twelve weeks; then injected with YBN14 once a week for three weeks in combination with two light treatments  given 6 h and 12 h post each injection.Mice were fed high-fat diet (HFD) for 12 weeks and then were injected with YBN14  through the tail vein. For oC. Alternate cryosections of the aortic root (7 μm thick) were generated using microtome (Leica Biosystems) giving a total distance of approximately 100 μm from the annulus. Digital images were captured in Nikon-TL microscope.Mice were sacrificed, and the hearts were carefully dissected and embedded in optimal cutting temperature (OCT) tissue compound  and stored at -80 For lipid quantification, longitudinal sections were fixed in 4% paraformaldehyde  for 15 min at room temperature, washed in PBS x1 and covered with propylene glycol  for 5 min. Cryosections were then stained with Oil Red O  for 10 min at 37 °C and washed once with 60% 2-propanol and distilled water until the dye was completely removed from a negative control slide (containing no tissue). Lipid quantification was performed as described previously Collagen content was determined in methanol fixed tissue sections using the Picro Sirius Red Stain Kit . Quantitative determination of collagen content was performed on threshold images using adobe photoshop (Adobe). Collagen content was determined as described for lipid content analysis.All measurements and evaluations of lipids and collagen content in aortic sections were performed in a blinded fashion.® solution  and digital images were taken in Olympus IX83 microscope with UPLSAPO x60 oil lens. Image quantification was performed in ImageJ For immunofluorescence staining, serial cryostat sections (7 μm) of aortic roots were fixed in cold methanol (-20 °C for 5 min), air-dried, and blocked with CAS-Block™ solution . Primary antibodies used in this study were as follows: CD11b from eBioscience , alpha-smooth muscle actin from Novus (NB600-531). Slides were mounted with DAPI Fluoromount-Gg for 10 min) and stored at -20 oC until analyzed further. Cholesterol and triglycerides assessment were analyzed by an automated clinical diagnostic platform  using the following kits: CHOL2, HDLC3, and TRIGL kits from (Roche) according to the manufacturer's protocol.After 12 weeks of high-fat diet, blood samples were drawn from individual mice and allowed to clot at room temperature for 20 min. The serum was separated by centrifugation  at 37 oC. The reaction was stopped by the addition of Laemmli sample buffer  and heating at 95 oC for 5 min. Equal amount of proteins (50 μg) were separated on a 12.5% SDS-PAGE, and cathepsin activity was determined by the fluorescent signal as measured in an Odyssey scanner (LI-COR Biosciences) at 800 nm for YBN14, or in a Typhoon scanner for GB137.Cathepsin activity in human atherosclerotic tissue was determined as described previously 2). Intact tissues were immediately labeled with YBN14 (20 µM) for 4 h and then washed with acetate buffer to remove unbound probe. As a negative control, part of the samples was pre-incubated with GB111-NH2 (10 µM), a pan cathepsin inhibitor Fresh carotid plaques were obtained as described above, washed thrice with acetate buffer  for 5 min. Proteins were separated on 12.5% SDS-PAGE and scanned in an Odyssey scanner (LI-COR Biosciences) at 800 nm. Fluorescent gel printout was used as a template to cut small gel slices corresponding to labeled proteases for subsequent in-gel protein digestion 2 spectra data were searched using the MASCOT algorithm against human proteome. Mascot searches allowed for oxidation of methionine residues (16 Da), static modification of cysteine residues , tryptic peptides with one missed cleavage allowed, and a mass tolerance set to ± 0.25 Da for precursor mass and ±0.35 Da for product ion masses. The 'Decoy Database Search' was turned on. The resulting MS2 spectra matches were assembled and filtered according to MASCOT Protein scores.Identification of cathepsin proteases by SDS PAGE carried out according to Kaschani F P values lower than 0.05 were considered to be statistically significant.Statistical analyses were performed in GraphPad Prism 7. All data assumed to follow a normal distribution and statistical tests are described in details in the figure legends. in vivo in a diabetic murine model for atherosclerosis Ldlr-/-) Ldlr-/- mice were given high-fat diet (HFD) or normal chow (NC) as a control for twelve weeks. This time frame is sufficient to cause full-blown atherosclerosis in this animal model as evident in our experience and others in vivo, we injected YBN14 to the animals and visualized its fluorescent signal using the IVIS platform. Consistent with our previous observations Recently we demonstrated the application of ABP to detect cathepsin activity Ldlr-/- mice were given high-fat diet for twelve weeks and received YBN14 followed by two consecutive light treatments to induce photodynamic therapy (PDT). This treatment protocol was given once a week over a time interval of three weeks (Figure Since YBN14 can efficiently target lesional cathepsins, we next sought to determine its therapeutic efficacy. For this purpose, s Figure A. Atheros Figure B, C. Mors Figure D, E suggin vitro and analyzed cathepsin activity by complementary approaches as summarized in Figure 2 completely suppressed YBN14 fluorescent signal from cathepsin activity, indicating that YBN14 targets lesional cathepsins (Figure in situ characterization of proteases by mass spectrometry To check the potential application of this emerging technology in humans, we first examined whether cathepsins could bind YBN14 in human atheromas. For this purpose, we took fresh atherosclerotic plaques from patients, labeled them s Figure D. In a cCardiovascular diseases remain challenging despite the breadth of lipid-lowering medications. In fact, the inflammatory nature of atherogenesis is a significant contributor to vascular remodeling and its related medical complications In that sense, a concentrated effort to enhance the utility of molecular probes for clinical applications has been made for different types of inflammatory diseases including cancer in vivo using the conventional animal model of -/-Ldlrmice 18FDG in the clinical settings to capture inflammatory activity. More significantly, this study demonstrates the potential application of non- invasive phototherapy to treat vulnerable plaques that display a high degree of inflammatory activity. Specifically, the PS-qABP, YBN14 in combination with focal IR light therapy reduced lesional inflammatory cells, without showing any other adverse effect to SMC or the amount of plaque collagen content. Altogether, this data suggest that molecular phototherapy can overall improve plaque stability.First, we demonstrate that the PS-qABP, YBN14 can rapidly home in on atherosclerotic lesions In light of these encouraging results in the murine model, we sought to provide a proof of concept for the application of this technology in the clinical settings. For this purpose, we used fresh CP tissues from patients and labeled them with YBN14. Indeed, those plaques displayed high protease activity at the sites of injury. Moreover, we could validate that YBN14 targets cysteine cathepsins B and S by mass spectrometry, which is consistent with our previously reported observations on these proteases being highly reactive in symptomatic patients et al.,This study together with the data reported by Shon SM Supplementary figures and methods.Click here for additional data file.