Faculty Bibliography

Rationale:Malignant pleural mesothelioma(MPM) has a poor prognosis with median survival of 12-24 months. We are not aware of prior studies examining the immune microenvironment in tumor draining lymph nodes (TDLN) in MPM. Our aim is to compare the tumor microenvironment(TME) and the microenvironment of TDLN. We hypothesize that the TME will display an immunosuppressive phenotype reflected in the TDLN.
Method(s):We performed digital spatial profiling(DSP) using the GeoMx (NanoString) platform on stored primary tumor and nodal biopsy specimens from 3 patients from our tumor bank. Samples from both primary tumor and lymph nodes were sectioned and labeled with pancytokeratin (CK). Tissue was then classified as "tumor" or "nontumor" using semi-automated segmentation based on pan-Cytokeratin (panK) labeling. The slides were then labeled with antibodies to 58 selected markers, with each unique antibody attached to a respective oligonucleotide. The tissue was exposed to UV light separately for tumor and non-tumor regions, cleaving the oligonucleotides from the attached antibodies. The oligonucleotides from the separate tumor and non-tumor regions were quantified using nCounter (NanoString).
Result(s):The non-neoplastic regions of the primary tumor contained higher expression of proteins associated with inflammatory cells including helper T-cells, cytotoxic T-cells, B-cells, macrophages, neutrophils, natural killer cells(Table 1). Furthermore, there was greater expression of immune checkpoint proteins, PD-L1 and CTLA-4, and CD163 and CD14, proteins associated with immunosuppressive macrophages, in the non-neoplastic region compared to the neoplastic region of the tumoe(Table 1). TDLNs contained similar levels of expression of lymphocyte markers, including those delineating cytotoxic T-cells and helper T-cells, as the primary tumor(Table 1). Despite this, TME expressed higher levels of T-cell exhaustion and immunsupression markers (FOXP3, LAG3, PD-1, CTLA-4) than TDLN(Table 1).
Conclusion(s):DSP is feasible in Formalin-fixed paraffin embedded (FFPE) mesothelioma specimens, providing a method for using quantitative immunopathology to study corresponding immune microenvironments. In our study, the non-tumor region of the primary tumor contained macrophages, lymphocytes, natural killer cells, and cancer-associated fibroblasts consistent with prior descriptions of the mesothelioma TME. Increased expression of immune checkpoint molecules in the non-tumor region suggests an immunosuppressive TME. TDLNs demonstrated similar lymphocyte markers, but without corresponding immune checkpoint expression of t suggesting the immunosuppressive phenotype of the TME may not be reflected in TDLNs. This pilot study is the first to use DSP to preliminarily characterize TDLNs in mesothelioma. We plan to apply this approach to stored additional MPM and NSCLC specimens to gain an in-depth understanding of the relationship between TME and TDLN

Isoprenylcysteine carboxyl methyltransferase (ICMT) is the third of three enzymes that sequentially modify the C-terminus of CaaX proteins, including RAS. Although all four RAS proteins are substrates for ICMT, each traffics to membranes differently by virtue of their hypervariable regions that are differentially palmitoylated. We found that among RAS proteins, NRAS was unique in requiring ICMT for delivery to the PM, a consequence of having only a single palmitoylation site as its secondary affinity module. Although not absolutely required for palmitoylation, acylation was diminished in the absence of ICMT. Photoactivation and FRAP of GFP-NRAS revealed increase flux at the Golgi, independent of palmitoylation, in the absence of ICMT. Association of NRAS with the prenyl-protein chaperone PDE6δ also required ICMT and promoted anterograde trafficking from the Golgi. We conclude that carboxyl methylation of NRAS is required for efficient palmitoylation, PDE6δ binding, and homeostatic flux through the Golgi, processes that direct delivery to the plasma membrane.

Hidradenitis suppurativa (HS) is a severe chronic inflammatory skin disease affecting human apocrine sweat gland-bearing skin regions. The overall prevalence of HS ranges from 0.05-4.1% with higher occurrence among females and African Americans, and strong associations with smoking and obesity. One unique feature of HS is the development of highly immunogenic keratinized sinus tracts that grow deeply in the dermis which further complicate HS pathogenesis and treatment. Using single cell transcriptomic analyses, we finely dissected different epidermal cell types in the HS lesional skin and revealed significant dysregulation of skin barrier function in the sinus tracts. We demonstrated that sinus tract keratinocytes exhibit dual cell fates of surface epidermis and skin appendages, and derived from progenitors in infundibulum of the apocrine-pilosebaceous unit. By analyzing ligand-receptor expressions between different skin appendages and immune cells, we highlighted Th17 and TNF responses at early and late stages during HS progression, respectively. Our work provides unprecedented understanding of pathological epidermal remodeling in human inflammatory diseases and important implications for therapeutics.
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A balanced intake of macronutrients-protein, carbohydrate and fat-is essential for the well-being of organisms. An adequate calorific intake but with insufficient protein consumption can lead to several ailments, including kwashiorkor¹. Taste receptors (T1R1-T1R3)² can detect amino acids in the environment, and cellular sensors (Gcn2 and Tor)³ monitor the levels of amino acids in the cell. When deprived of dietary protein, animals select a food source that contains a greater proportion of protein or essential amino acids (EAAs)⁴. This suggests that food selection is geared towards achieving the target amount of a particular macronutrient with assistance of the EAA-specific hunger-driven response, which is poorly understood. Here we show in Drosophila that a microbiome-gut-brain axis detects a deficit of EAAs and stimulates a compensatory appetite for EAAs. We found that the neuropeptide CNMamide (CNMa)⁵ was highly induced in enterocytes of the anterior midgut during protein deprivation. Silencing of the CNMa-CNMa receptor axis blocked the EAA-specific hunger-driven response in deprived flies. Furthermore, gnotobiotic flies bearing an EAA-producing symbiotic microbiome exhibited a reduced appetite for EAAs. By contrast, gnotobiotic flies with a mutant microbiome that did not produce leucine or other EAAs showed higher expression of CNMa and a greater compensatory appetite for EAAs. We propose that gut enterocytes sense the levels of diet- and microbiome-derived EAAs and communicate the EAA-deprived condition to the brain through CNMa.

OBJECTIVE:Endovascular abdominal aortic aneurysm repair (EVAR) is preferred to open surgical repair (OSR) for the treatment of abdominal aortic aneurysm (AAA) in high-risk patients. We sought to compare perioperative and long-term outcomes for EVAR in patients designated as unfit for OSR using a large national dataset. METHODS:The Vascular Quality Initiative database collected from 2013 to 2019 was queried for patients undergoing elective EVARs for AAA > 5cm. The patients were stratified into two cohorts based on the suitability for OSR (FIT vs. UNFIT). Primary outcomes included perioperative (in-hospital) major adverse events, perioperative mortality, and mortality at 1 and 5 years. Patient demographics and postoperative outcomes were analyzed to identify predictors of perioperative and long-term mortality. RESULTS:Of 16,183 EVARs, 1,782 patients were deemed unfit for OSR. The UNFIT cohort was more likely to be older and female, with higher proportions of HTN, CAD, CHF, COPD, and larger aneurysm diameter. Postoperatively, the UNFIT cohort was more likely to have cardiopulmonary complications (6.5% vs. 3%; P<.001), with higher perioperative mortality (1.7% vs. 0.6%; P<.001) and 1 and 5-year mortality (13% and 29% UNFIT vs. 5% and 14% FIT; P<.001). Subgroup analysis within the UNFIT cohort revealed those deemed unfit due to hostile abdomen had significantly lower 1 and 5-year mortality (6% and 20%) compared to those unfit due to cardiopulmonary compromise and frailty (14% and 30%; P=.451). Reintervention-free survival at 1 and 5-years was significantly higher in the FIT cohort (93% and 82%) as compared to the UNFIT cohort (85% and 68%; P<.001). Designation as unfit for OSR was an independent predictor of both perioperative (OR 1.59; 95% CI, 1.03-2.46; P=.038) and long-term mortality (HR 1.92; 95% CI, 1.69-2.17; P<.001). Advanced age (OR 2.91; 95% CI, 1.28-6.66; P=.011) was the strongest determinant of perioperative mortality while ESRD (HR 2.51; 95% CI, 1.78-3.55; P<.001) was the strongest predictor of long-term mortality. Statin (HR 0.77; 95% CI, 0.69-0.87; P<.001) and ACE inhibitor (HR 0.83; 95% CI, 0.75-0.93; P<.001) were protective of long-term mortality. CONCLUSION/CONCLUSIONS:Despite low perioperative mortality, long-term mortality of those designated by the operating surgeons as unfit for OSR was rather high in patients undergoing elective EVARs, likely due to the competing risk of death from their medical frailty. Unfit designation due to hostile abdomen did not confer any additional risks after EVAR. Judicious estimation of the patient's life expectancy is essential when considering treatment options in this subset of patients deemed unfit for OSR.

RATIONALE Coronavirus Disease-2019(COVID-19), caused by the severe acute respiratory syndrome coronavirus-2(SARS-CoV-2), causes multi-organ failure and death. Metabolic syndrome(MetSyn) characteristics are also risks for COVID-19. The Receptor for Advanced Glycation End-Products(RAGE) is a MetSyn mediator. SARS-CoV via its Spike protein binds ACE2 as its 1o-receptor, and may activate TLR2. Particulate matter(PM) similarly activates an innate immune response, partially via the RAGE receptor, and increases ACE2 expression. Excessive hyaluronan(HA) levels are found in lungs of COVID-19 patients. Reducing HA synthesis and stabilizing the HA shield surrounding cells may be therapeutic. A HA-binding peptide, P15-1, is anti-inflammatory and reduces HA. HA and its binding proteins may provide a link explaining synergistic ACE2 and RAGE signaling, reducing interaction of receptors with their ligands and ultimately inflammation-related changes in peripheral blood mononuclear cells(PBMCs), the severity of which correlate with patient outcome after SARS-CoV-2 exposure. Our focus is to develop novel therapeutic strategies for SARS-CoV-2 inflammation. To begin to explore our HYPOTHESIS that COVID-19 Spike protein and PM co-exposure synergistically induces an inflammatory phenotype and that phenotype can be mitigated by stabilizing the pericellular HA matrix and by inhibiting RAGE. METHODS. We performed in vitro exposure of PBMCs isolated from 9/11 World Trade Center(WTC) 1st- Responders to i. Media alone(MA); ii. WTC PM; iii. SARS-CoV-2 Spike RBD(C19); iv. C19 and PM; v. C19 and P15-1; vi. C19, PM and P15-1 vii. C19, PM and RAGE inhibitor(RAGEInh) FPS-ZM1; viii. LPS(positive control). Total mRNA levels for Cox-2, IL-1beta, IL-6 and MMP-13 24 hours after exposure were analyzed by real time PCR. Comparisons by Student's t- and Mann-Whitney U-tests. Correlations by Spearman's. Significance p<0.05. RESULTS COX-2, IL-1beta, IL-6 and MMP-13 mRNA levels were significantly increased 24-hrs after the administration of PM and C19. Co-exposure to PM and C19 yielded a synergistic increase in the mRNA of IL-beta, Figure 1B. P15-1 and RAGE inhibition significantly reduced mRNA levels of inflammatory markers in primary PBMCs exposed to C19, WTC PM, or a combination of the two, Figure 1. CONCLUSIONS Our work focuses on mitigating the COVID-19 inflammatory phenotype by stabilizing the pericellular HA matrix and by inhibiting RAGE. Preliminary data presented in this abstract will be further explored using PBMCs and cell lines in a multidisciplinary approach

Hidradenitis suppurativa (HS) is a prevalent inflammatory skin disease. HS causes deep, painful, recurrent abscesses. African Americans and females are at an increased risk. A lack of effective therapies and limited knowledge about HS pathogenesis contribute to unmet needs. Unlike other common inflammatory skin diseases, there has never been a genome-wide association study (GWAS) conducted for HS. Here, we performed a first GWAS for HS using data from the eMERGE network of electronic health record linked biorepositories (project NT227). We used HS diagnosis codes to identify cases and controls. We estimated ancestry with principal component analysis using a set of 40,156 SNPs. Our final cohort consisted of 600 HS cases and 82,611 controls with comparable multi-ethnic ancestry (lambda=1.005). Our cohort recapitulated HS race and gender predilections with genetically African female participants accounting for 35% of cases, but only 10% of controls. Genotype data for 6 million variants was tested for association, adjusting for five principal components. No locus exceeded our threshold for statistical significance. Importantly, there was no evidence for HLA association supporting classification of HS as inflammatory rather than autoimmune. Several loci approached the significance threshold, suggesting that an expansion in cohort size is needed to provide adequate power to detect associations. Interestingly, the lead SNP at one of the most significant loci (rs11075745; p=8x10^-7) is an eQTL for NFAT5, a mediator of NOTCH signaling whose expression is downregulated in HS lesional skin relative to patient-matched nonlesional skin. The risk allele influences expression in tissue specific manner. Our group is constructing multi-ethnic replication cohorts that will allow us to expand this study in the near future.
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Hidradenitis suppurativa (HS) is a severe chronic inflammatory skin disease lacking effective therapeutic options due to little understanding of the complex immune response within the lesional skin. Using single-cell transcriptomic analyses, we examined the signature changes in each immune cell types during HS progression, as well as in silico ligand-receptor predictions between different immune cell types to construct the interaction network that contribute to HS pathogenesis. Our results revealed a predominant Th17 response, as well as a distinct regulatory T cells existing in the lesional skin. We found that M1-polarized macrophages likely facilitate chemotaxis and IL1B responses in perilesional skin, while regulate lymphocyte activation and tissue remodeling in the lesional skin. In addition, we identified a significant increase of CCR7 expressing dendritic cells, as well as activated stromal fibroblasts expressing CCR7-ligand CCL19, which together support the organization of tertiary lymphoid organ (TLO)-like aggregates that contribute to persistent local inflammation. Importantly, we demonstrated a dense infiltration of plasma cells near sinus tracts, and that clonal expansion of the plasma cells frequently exists in HS patients. Together, our work provides a comprehensive understanding of immune responses and cytokine networks defining disease chronicity in HS, as well as significant implications for future therapeutics.
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Lysosomal trafficking and maturation in neurons remain poorly understood and are unstudied in vivo despite high disease relevance. We generated neuron-specific transgenic mice to track vesicular CTSD acquisition, acidification, and traffic within the autophagic-lysosomal pathway in vivo, revealing that mature lysosomes are restricted from axons. Moreover, TGN-derived transport carriers (TCs), not lysosomes, supply lysosomal components to axonal organelles. Ultrastructurally distinctive TCs containing TGN and lysosomal markers enter axons, engaging autophagic vacuoles and late endosomes. This process is markedly upregulated in dystrophic axons of Alzheimer models. In cultured neurons, most axonal LAMP1 vesicles are weakly acidic TCs that shuttle lysosomal components bidirectionally, conferring limited degradative capability to retrograde organelles before they mature fully to lysosomes within perikarya. The minor LAMP1 subpopulation attaining robust acidification are retrograde Rab7⁺ endosomes/amphisomes, not lysosomes. Restricted lysosome entry into axons explains the unique lysosome distribution in neurons and their vulnerability toward neuritic dystrophy in disease.