Faculty Bibliography

Ulcerative colitis is an idiopathic, chronic inflammatory bowel disease. Its pathogenesis is multifactorial involving inflammation and immune dysregulation. Proinflammatory TNFα/NFκB signaling is believed to play a cardinal role in ulcerative colitis. Growing evidence indicates the molecular interactions between the cellular metabolites and different phases of inflammation. This study aims to identify the metabolites that can inhibit TNFα/NFκB signaling and are potentially therapeutic against various TNFα-associated inflammatory diseases, particularly inflammatory bowel diseases. We performed in vitro and in vivo screening of cellular metabolites to inhibit TNFα/NFκB signaling. Multiple confirmation assays, including NFκB translocation, quantitative real-time PCR, ELISA, immunofluorescence staining, and RNA sequencing analysis were executed. Drug affinity-responsive target stability assay with proteomics was utilized for target identification. cPLA2 ablated mice with dextran sodium sulfate-induced colitis were employed to assess pyruvate's dependence on its molecular target in attenuating ulcerative colitis pathogenesis. Metabolite screening and subsequent validation with multiple approaches led to the isolation of pyruvate, a glycolytic metabolite, and a critical node in several metabolic pathways, as a novel inhibitor of TNFα/NFκB signaling. Importantly, pyruvate suppressed inflammation, preserved colonic histology, maintained tight junction proteins, and regulated permeability in the ulcerative colitis model. Additionally, cPLA2 was identified as a previously unknown target of pyruvate and pyruvate largely lost its therapeutic effects against ulcerative colitis in cPLA2-deficient mice. Conclusively, this study not only unveils pyruvate as an antagonist of TNFα/NFκB signaling and therapeutic intervention against colitis but also provides mechanistic insight into the mode of action of pyruvate.

Allergic contact dermatitis (ACD) is a delayed-type IV hypersensitivity response driven by innate and adaptive immune cells. While specific immune regulations of these cell types are amply elucidated, their regulations by extracellular matrix (ECM) components and T cell mediated adaptive immunity in ACD remains unclear. Lumican and biglycan are ECM proteoglycans abundant in the dermis and lymph node, known to regulate innate immune myeloid cells, but have not been investigated in lymphoid cell regulations in ACD. By immunohistology we localized lumican and biglycan in skin biopsies of psoriatic patients. Using wild type (WT), lumican and biglycan knockout mice, we investigated CD4⁺T cell infiltration, activation and proliferation in the skin and draining lymph node (dLN) of CHS-challenged mice by immunohistochemistry and flow cytometry. We used the OT-II adoptive transfer model to test antigen specific CD4⁺T cell activation. We assessed interactions of the proteoglycans with LFA-1 on T cells by confocal microscopy. Compared to WTs, the knockouts showed severe ear inflammation, with increased CD4⁺T cells infiltration in the dermis. CHS-challenged knockout mice dLN showed increased T-bet, STAT1 and -STAT4 signaling, indicating enhanced Th1 commitment and proliferation. We found that WT lymph node fibroblastic reticular cells (FRCs) secrete lumican, biglycan and decorin, a related proteoglycan, while none are expressed by naive or activated T cells. Lumican and biglycan interact with LFA-1 on T cell surfaces, and in vitro all three proteoglycans suppress CD4⁺T cell activation. Secreted by dLN FRCs, lumican, biglycan, and possibly decorin interact with LFA-1 on CD4⁺T cells to restrict its activation and reduce dermatitis severity.

PURPOSE/OBJECTIVE:The purpose of this study is to examine the effect of age on outcomes following repair of acute displaced patella fractures Methods: 248 patients who sustained a displaced patella fracture and underwent open reduction and internal fixation were identified. Patients included underwent a similar operative protocol, were prescribed a standard post-operative protocol of therapy, and were seen at standard follow-up intervals. Patients were divided into groups of < 65 years old (young) and ≥ 65 years old (older). Statistical analysis was run to determine if there was a significant difference in range of knee motion and rate of major complications. RESULTS:Of the 248 patients, 149 were young and 99 were older. The mean age of the older group was 74.5 ± 6.7 and the mean age of the young group was 50 ± 12. Fracture pattern and BMI were similar the groups, however the older group had a higher average CCI (p<0.001). Additionally, the groups had similar length of follow up (p=0.693) and similar mean time to radiographic healing (p=0.533). Older patients had limited knee extension at 6 months (compared young patients (p=0.031). Finally, older patients had a higher rate of all complications compared to young patients. Two percent of older patients developed a fracture related infection (FRI), 4% developed a symptomatic nonunion and 11% were underwent re-operation including removal of hardware, total knee replacement, irrigation and debridement and manipulation under anesthesia. CONCLUSION/CONCLUSIONS:Complication rates following patella fracture fixation in older patients were higher than young patients, despite having similar injury patterns, surgical treatment and follow up. These findings can better inform treating physicians during surgical intervention of older patients with patella fractures.

Pathology in large vessels frequently develops at specific locations, implying that local stressors and spatially restricted gene expression are likely contributors to disease susceptibility. Here we perform single-cell transcriptomics in the carotids, the aortic arch and the thoracic and abdominal aorta to identify site- and sex-specific differences that could inform about vulnerability. Our findings revealed (1) regionally defined transcriptional profiles, (2) signatures associated with embryonic origins and (3) differential contributions of sex-specific effectors. Furthermore, cross-referencing regional-specific signatures with available genome-wide association study and expression quantitative trait loci databases identified 339 disease candidates associated with aorta distensibility, stiffness index and blood pressure. CPNE8 and SORBS2 were further evaluated and highlighted as strong causal candidates. Sex differences were predominantly observed in the thoracic and abdominal aorta. MCAM (CD146), a transcript with sex-skewed expression and lower in male mice and men, had significantly reduced expression in human aortic aneurysms. The findings reveal underlying diversity within vascular smooth muscle cell populations relevant to understanding site-specific and sex-specific variation of vascular pathologies.

Decreased brain levels of coenzyme Q₁₀ (CoQ₁₀), an endogenously synthesized lipophilic antioxidant^1,2, underpin encephalopathy in primary CoQ₁₀ deficiencies^3,4 and are associated with common neurodegenerative diseases and the ageing process^5,6. CoQ₁₀ supplementation does not increase CoQ₁₀ pools in the brain or in other tissues. The recent discovery of the mammalian CoQ₁₀ headgroup synthesis pathway, in which 4-hydroxyphenylpyruvate dioxygenase-like protein (HPDL) makes 4-hydroxymandelate (4-HMA) to synthesize the CoQ₁₀ headgroup precursor 4-hydroxybenzoate (4-HB)⁷, offers an opportunity to pharmacologically restore CoQ₁₀ synthesis and mechanistically treat CoQ₁₀ deficiencies. To test whether 4-HMA or 4-HB supplementation promotes CoQ₁₀ headgroup synthesis in vivo, here we administered 4-HMA and 4-HB to Hpdl^-/- mice, which model an ultra-rare, lethal mitochondrial encephalopathy in humans. Both 4-HMA and 4-HB were incorporated into CoQ₉ and CoQ₁₀ in the brains of Hpdl^-/- mice. Oral treatment of Hpdl^-/- pups with 4-HMA or 4-HB enabled 90-100% of Hpdl^-/- mice to live to adulthood. Furthermore, 4-HB treatment stabilized and improved the neurological symptoms of a patient with progressive spasticity due to biallelic HPDL variants. Our work shows that 4-HMA and 4-HB can modify the course of mitochondrial encephalopathy driven by HPDL variants and demonstrates that CoQ₁₀ headgroup intermediates can restore CoQ₁₀ synthesis in vivo.

BACKGROUND/UNASSIGNED:Electronic cigarettes (e-cigarettes) are the most used tobacco product among youth, and adults who smoke combustible cigarettes favor e-cigarettes over approved cessation aids. Despite the lower perceived harm of vaping compared with smoking, acute inhalation of e-cigarette aerosol elicits cardiovascular responses that may lead to persistent damage when repeated over time. METHODS/UNASSIGNED:We exposed female hypercholesterolemic mice to either pod-mod e-cigarette aerosol or filtered air daily for 24 weeks. We assessed the long-term effects of vaping on aortic stiffness and vasoreactivity while investigating the underlying cellular and molecular mechanisms of injury. RESULTS/UNASSIGNED:Chronic inhalation of e-cigarette aerosol triggered the accumulation of inflammatory signals systemically and within aortic tissues, as well as T-lymphocyte accrual in the aortic wall. Limited eNOS (endothelial nitric oxide synthase) expression and enhanced superoxide radical production curbed NO bioavailability in the aorta of mice exposed to e-cigarette aerosol despite iNOS (inducible nitric oxide synthase) induction, impairing the endothelium-dependent vasodilation that regulates blood flow distribution. Inhalation of e-cigarette aerosol thickened and stiffened aortic tissues via collagen deposition and remodeling, hindering the storage of elastic energy and limiting the cyclic distensibility that enables the aorta to function as a pressure reservoir. These effects combined contributed to raising systolic and pulse pressure above control levels. CONCLUSIONS/UNASSIGNED:Chronic inhalation of aerosol from pod-mod e-cigarettes promotes oxidative stress, inflammation, and fibrosis within aortic tissues, significantly impairing passive and vasoactive aortic functions. This evidence provides new insights into the biological processes that increase the risk of adverse cardiovascular events as a result of pod-mod e-cigarette vaping.

Age-related inflammation or 'inflammaging' increases disease burden and controls lifespan. Adipose tissue macrophages (ATMs) are critical regulators of inflammaging; however, the mechanisms involved are not well understood in part because the molecular identities of niche-specific ATMs are unknown. Using intravascular labeling to exclude circulating myeloid cells followed by single-cell sequencing with orthogonal validation via multiparametric flow cytometry, we define sex-specific changes and diverse populations of resident ATMs through lifespan in mice. Aging led to depletion of vessel-associated macrophages, expansion of lipid-associated macrophages and emergence of a unique subset of CD38⁺ age-associated macrophages in visceral adipose tissue with inflammatory phenotype. Notably, CD169⁺CD11c^- ATMs are enriched in a subpopulation of nerve-associated macrophages (NAMs) that declines with age. Depletion of CD169⁺ NAMs in aged mice increases inflammaging and impairs lipolysis suggesting catecholamine resistance in visceral adipose tissue. Our findings reveal NAMs are a specialized ATM subset that control adipose homeostasis and link inflammation to tissue dysfunction during aging.

Chemotherapy-induced alopecia and radiation-induced alopecia, the thinning or loss of hair due to cytotoxic chemotherapy and radiation therapy, respectively, are distressing adverse effects of cancer treatment. Chemotherapy, targeted therapies, and radiation therapy used in pediatric oncology often lead to alopecia by damaging hair follicles, with varying degrees of severity depending on the specific treatment type, mechanism of action, and damage-response pathway involved. Pediatric chemotherapy-induced alopecia, radiation-induced alopecia, and permanent alopecia, defined as hair regrowth that remains incomplete 6 months or more after treatment, have significant negative impacts on mental health, self-esteem, and social interactions, highlighting the need for further research into supportive care strategies. There are currently no standard interventions for chemotherapy-induced alopecia or radiation-induced alopecia in children, with most recommendations limited to gentle hair care and camouflaging techniques during treatment. Scalp cooling has demonstrated safety and efficacy in reducing chemotherapy-induced alopecia in adults and is currently under investigation in children and adolescents. Topical and low-dose oral minoxidil have been studied in children for other hair loss disorders and may improve hair regrowth after chemotherapy or radiation. Increased awareness and continued research into management strategies for pediatric chemotherapy-induced alopecia and radiation-induced alopecia are necessary to help mitigate its significant negative impact on quality of life.

The advent of large-scale sequencing in both development and disease has identified large numbers of candidate genes that may be linked to important phenotypes. We have developed a rapid, scalable system for assessing the role of candidate genes using zebrafish. We generated transgenic zebrafish in which Cas9 was knocked in to the endogenous mitfa locus, a master transcription factor of the melanocyte lineage. The main advantage of this system compared to existing techniques is maintenance of endogenous regulatory elements. We used this system to identify both cell-autonomous and non-cell-autonomous regulators of normal melanocyte development. We then applied this to the melanoma setting to demonstrate that loss of genes required for melanocyte survival can paradoxically promote more aggressive phenotypes, highlighting that in vitro screens can mask in vivo phenotypes. Our genetic approach offers a versatile tool for exploring developmental processes and disease mechanisms that can readily be applied to other cell lineages.

Animals need daily intakes of three macronutrients: sugar, protein, and fat. Under fasted conditions, however, animals prioritize sugar as a primary source of energy. They must detect ingested sugar-specifically D-glucose-and quickly report its presence to the brain. Hypothalamic neurons that can respond to the caloric content in the gut regardless of the identity of macronutrient have been identified, but until now, the existence of neurons that can encode the specific macronutrients remained unknown. We found that a subset of corticotropin-releasing factor (CRF)-expressing neurons in the hypothalamic paraventricular nucleus (CRF^PVN) respond specifically to D-glucose in the gut, separately from other macronutrients or sugars. CRF^PVN neuronal activity is essential for fasted mice to develop a preference for D-glucose. These responses of CRF^PVN neurons to intestinal D-glucose require a specific spinal gut-brain pathway including the dorsal lateral parabrachial nuclei. These findings reveal the neural circuit that encodes the identity of D-glucose.