Tissue Inflammation Research Articles

Mitigative Effect and Mechanism of Caffeic Acid Combined with Umbilical Cord-Mesenchymal Stem Cells on LPS-Induced Mastitis.

Mastitis is an inflammation of the mammary gland tissue that can lead to decreased milk production and altered milk composition, carrying serious implications for the safety of dairy products. Although both caffeic acid (CA) and umbilical cord-mesenchymal stem cells (UC-MSCs) showed potential anti-inflammatory and immunomodulatory properties, little is known about their combined roles in treating mastitis. Here, we report the combined effects and mechanisms of CA and UC-MSCs on lipopolysaccharide (LPS)-induced mastitis. Based on the network pharmacological analysis, the potential relevant genes involved in the alleviating effects of CA on LPS-induced mastitis were inferred. In LPS-treated mammary epithelial cells, CA or/and UC-MSC conditioned medium (UC-MSC-CM) inhibited the phosphorylation of p65, p50, p38, IκB, and MKK3/6 proteins and the expression of downstream inflammatory factors TNF-α, IL-1β, IL-6, IL-8, and COX-2. Additionally, CA or/and hydrogel-loaded UC-MSCs also suppressed the activation of the above inflammatory pathway, leading to the alleviation of pathological damages in the LPS-induced mouse mastitis model. UC-MSCs exhibited more significant effects than CA, and the combined treatment of both was more effective. Our study sheds light on the synergistic and complementary effects of CA and UC-MSCs in alleviating mastitis, offering clues for understanding the regulation of the p38-MAPK/NF-κB↔TNF-α signal transduction loop in the tumor necrosis factor (TNF) pathway as a potential mechanism. This study provides a theoretical basis for developing a novel antibiotic alternative treatment of mastitis that may contribute to reducing economic losses in animal husbandry and protecting public health safety.

An abnormal increase in CD26(-)CD28(-) cytotoxic effector CD4 and CD8 T cell populations in patients with systemic lupus erythematosus.

CD26 is a human T cell costimulatory molecule as well as a T cell subset marker, and increase of CD26+ T cells in inflamed tissues and peripheral blood has been reported in diverse autoimmune diseases. In contrast, our group has previously shown that levels of circulating CD26+ T cells are decreased in patients with systemic lupus erythematosus (SLE), although the role of reduced CD26 T cell surface expression in SLE pathology remains to be elucidated. In the present study, we conducted CD26-based T cell subset analyses utilizing peripheral blood mononuclear cells from 57 SLE patients and 31 healthy adult volunteers. We show that the increase in CD26(-) T cell population reflects the abnormal expansion of CD26(-)CD28(-) cytotoxic subsets of both CD8 T cells and CD4 T cells in SLE patients. Single cell RNA sequencing analysis of the CD26(-)CD28(-) CD4 and CD8 T cell populations reveals unique characteristics with similarities to natural killer T cells. In addition, the level of CD26(-)CD28(-) T cells is increased in some active stage SLE patients with renal manifestation. Meanwhile, effect of prednisolone treatment on these populations varies from patient to patient, with levels of these cytotoxic effector populations still being elevated in some inactive stage SLE patients. Taken together, our data suggest that analysis of these populations in SLE may be a useful tool to classify this markedly heterogeneous condition.

The Role of Lymph-Adipose Crosstalk in Alcohol-Induced Perilymphatic Adipose Tissue Dysfunction.

Chronic alcohol use leads to metabolic dysfunction in adipose tissue. The underlying mechanisms and the contribution of alcohol-induced adipose tissue dysfunction to systemic metabolic dysregulation are not well understood. In our previous studies, we found that chronic alcohol feeding induces mesenteric lymphatic leakage, perilymphatic adipose tissue (PLAT) inflammation, and local insulin resistance in rats. The goal of this study was to further explore the link between alcohol-induced lymphatic leakage and PLAT immunometabolic dysregulation, locally and systemically, using in vivo and ex vivo approaches. Male rats received a Lieber-DeCarli liquid diet, of which 36% of the calories were from alcohol, for 10 weeks. Time-matched control animals were pair-fed. Adipokine levels were measured in PLAT, subcutaneous fat, plasma, and mesenteric lymph samples. Glucose tolerance was assessed after 10 weeks. Further, we used a novel ex vivo lymph-stimulated naïve PLAT explant approach to modeling lymph leakage to assess changes in adipokine secretion and expression of proinflammatory markers after stimulation with lymph from alcohol- or pair-fed animals. Our data show that chronic alcohol-fed rats presented PLAT-specific decreases in adiponectin and leptin levels, alterations in the expression of genes involved in lipid metabolic pathways, and associated impaired whole-body glucose homeostasis. Further, we found that direct naïve PLAT stimulation with lymph contents from alcohol-fed animals increased IL-6 expression in demonstrating the ability of lymph contents to differentially impact naïve adipose tissue. Overall, chronic alcohol feeding leads to depot-specific alterations in metabolic profile, impaired systemic glucose tolerance, and lymph-induced adipose tissue inflammation. The specific lymph components leading to PLAT immunometabolic dysregulation remain to be determined.

Inhibition of Lysosomal Cathepsin A and Neuraminidase 1 Interaction by Anti-Obesity Cyclic Peptide.

Chronic inflammation in adipose tissue is associated with metabolic disorders such as obesity and type 2 diabetes. Novel small molecules targeting adipocyte differentiation and fat accumulation offer potential for new anti-inflammatory and anti-obesity drugs. Here we show that the marine cyclic heptapeptide stylissatin A and its analogs (SAs) inhibit membranous neuraminidase 1 (Neu1) function by interacting with lysosomal protective protein cathepsin A (PPCA). Neu1 has been less explored as a therapeutic target due to the genetic defects leading to neurodegenerative disorders. However, unlike traditional neuraminidase inhibitors, SAs don't directly bind to Neu1 but modulate the molecular chaperone activity of PPCA. SAs caused degradation of perilipin 1 around lipid droplets and inhibited fat accumulation, along with decrease in membranous Neu1. Molecular docking and molecular dynamics simulations revealed that SAs interacted with activated PPCA at the Neu1 binding site. Focusing on this newfound protein-protein interaction inhibition mechanism could lead to the development of pharmaceuticals with fewer side effects.

Tanshinone IIA Exerts Cardioprotective Effects Through Improving Gut-Brain Axis Post-Myocardial Infarction.

Myocardial infarction (MI) is a lethal cardiovascular disease worldwide. Emerging evidence has revealed the critical role of gut dysbiosis and impaired gut-brain axis in the pathological progression of MI. Tanshinone IIA (Tan IIA), a traditional Chinese medicine, has been demonstrated to exert therapeutic effects for MI. However, the effects of Tan IIA on gut-brain communication and its potential mechanisms post-MI are still unclear. In this study, we initially found that Tan IIA significantly reduced myocardial inflammation, apoptosis and fibrosis, therefore alleviating hypertrophy and improving cardiac function following MI, suggesting the cardioprotective effect of Tan IIA against MI. Additionally, we observed that Tan IIA improved the gut microbiota as evidenced by changing the α-diversity and β-diversity, and reduced histopathological impairments by decreasing inflammation and permeability in the intestinal tissues, indicating the substantial improvement of Tan IIA in gut function post-MI. Lastly, Tan IIA notably reduced lipopolysaccharides (LPS) level in serum, inflammation responses in paraventricular nucleus (PVN) and sympathetic hyperexcitability following MI, suggesting that restoration of Tan IIA on MI-induced brain alterations. Collectively, these results indicated that the cardioprotective effects of Tan IIA against MI might be associated with improvement in gut-brain axis, and LPS might be the critical factor linking gut and brain. Mechanically, Tan IIA-induced decreased intestinal damage reduced LPS release into serum, and reduced serum LPS contributes to decreased neuroinflammation with PVN and sympathetic inactivation, therefore protecting the myocardium against MI-induced injury.

Probable IgG4-related Orbital Disease Masked by Exuberant Ocular Surface Pseudoepitheliomatous Hyperplasia

A 40-year-old woman presented with a mass in her OS for 2 years. Examination revealed a large conjunctival lesion on the nasal bulbar conjunctiva OS and a small upper tarsal conjunctival lesion in the OD. Biopsy OD revealed inflammatory granulation tissue, and OS revealed pseudoepitheliomatous hyperplasia with granulation tissue. The lesion responded well to steroids. She was lost to follow-up and returned with an enlarged lesion with orbital extension and corneal perforation. Orbital exenteration was performed. The entire conjunctiva showed extensive epithelial downgrowth and mononuclear round cell infiltrates, predominantly plasma cells admixed with neutrophils suggestive of aggressive pseudoepitheliomatous hyperplasia. Orbital tissue showed ill-defined granuloma formation with acute on chronic inflammation and plasma cells. The tissue IgG4/IgG ratio was 34%, suggestive of probable IgG4-related orbital disease. Based on this case, we conclude that IgG4-related orbital disease can rarely cause conjunctival and scleral inflammation with secondary pseudoepitheliomatous hyperplasia-like changes.

Targeting of oxidized Macrophage Migration Inhibitory Factor (oxMIF) with antibody ON104 attenuates the severity of glomerulonephritis.

The oxidized form of Macrophage Migration Inhibitory Factor (oxMIF) has been identified as the disease-related isoform of MIF, exerting pathological functions in inflamed tissue. In this study, we aimed to explore the in vivo effects of the neutralizing anti-oxMIF antibody ON104 in a rat model of crescentic glomerulonephritis (CGN), to better understand its disease modifying activities. WKY rats received a single intravenous injection of a rabbit nephrotoxic serum (NTS), targeting rat glomerular basement membrane to induce CGN. On day 4 and day 6, ON104 was given intraperitoneally (i.p.) and on day 8 urine, blood and kidney tissue were collected. ON104 substantially attenuated the severity of CGN demonstrated by reduced proteinuria, hematuria, as well as lower levels of kidney injury molecule (KIM)-1. ON104 treatment preserved the glomerular morphology and suppressed crescent formation, a hallmark of the disease. On the cellular level, oxMIF neutralization by ON104 strongly reduced the number of macrophages and neutrophils within the inflamed kidneys. In vitro, we identified human neutrophils, but not monocytes, as main producers of oxMIF among total peripheral cells. The present study demonstrates that oxMIF is a pertinent therapeutic target in a model of CGN which mechanistically resembles human immune mediated CGN. In this model, neutralization of oxMIF by ON104 leads to an improvement in both urinary abnormalities and histological pathological characteristics of the disease. ON104, thus has the potential to become a novel disease-modifying drug for the treatment of glomerulonephritis and other inflammatory kidney diseases.

The epithelial barrier theory and its associated diseases.

The prevalence of many chronic noncommunicable diseases has been steadily rising over the past six decades. During this time, over 350,000 new chemical substances have been introduced to the lives of humans. In recent years, the epithelial barrier theory came to light explaining the growing prevalence and exacerbations of these diseases worldwide. It attributes their onset to a functionally impaired epithelial barrier triggered by the toxicity of the exposed substances, associated with microbial dysbiosis, immune system activation, and inflammation. Diseases encompassed by the epithelial barrier theory share common features such as an increased prevalence after the 1960s or 2000s that cannot (solely) be accounted for by the emergence of improved diagnostic methods. Other common traits include epithelial barrier defects, microbial dysbiosis with loss of commensals and colonization of opportunistic pathogens, and circulating inflammatory cells and cytokines. In addition, practically unrelated diseases that fulfill these criteria have started to emerge as multimorbidities during the last decades. Here, we provide a comprehensive overview of diseases encompassed by the epithelial barrier theory and discuss evidence and similarities for their epidemiology, genetic susceptibility, epithelial barrier dysfunction, microbial dysbiosis, and tissue inflammation.

MICROBIOME: The trials and errors of developing an experimental model to study the impact of maternal gut microbiome disruption on perinatal asphyxia.

Maternal gut microbiome impairment has garnered attention for its potential role in influencing neurodevelopmental outcomes in offspring, especially in situations that increase brain vulnerability such as perinatal asphyxia (PA). Maternal microbiome and fetal brain interplay emerge as a critical link between maternal health and offspring neurodevelopment. This study aims to generate a model to assess the impact of maternal dysbiosis triggered by gestational antibiotic administration and PA on offspring neurodevelopment. Wistar rats were subjected to antibiotics in drinking water from the 11th gestational day until birth. On the 6th postnatal day, pups were subjected to PA/normoxia, resulting in four experimental groups: control-normoxia, antibiotics-normoxia, control-asphyxia, and antibiotics-asphyxia. Early-life behavioral tests were conducted between postnatal days 7 and 9. The initial antimicrobial cocktail (ampicillin, vancomycin, neomycin, clindamycin, amphotericin-B) led to an increased number of miscarriages, poor weight gain during pregnancy, reduced offspring weight, and changes in the maternal gut microbiome compared to control. Offspring presented impaired neurodevelopmental reflexes in both PA and antibiotic groups and increased hippocampal neuroinflammation. Due to these detrimental effects, a more pregnancy-safe antibiotic cocktail was used for a second experiment (ampicillin, vancomycin, neomycin, meropenem). This resulted in no miscarriages or pregnancy-weight loss but was still linked to gut microbiome disruption. PA impaired neurodevelopmental reflexes and increased neuroinflammation, effects amplified by antibiotic administration. These preliminary findings reveal the cumulative potential of maternal dysbiosis and PA on neurodevelopment impairment, emphasizing caution in gestational antimicrobial use. Further investigations should include offspring long-term follow-up and maternal behavior and integrate probiotics to counteract antibiotic effects. This study investigates the impact of maternal gut microbiome disruptions caused by gestational antibiotic treatment and low oxygen exposure shortly after birth on the development of the rats' babies. We found that both antibiotic exposure and reduced oxygen levels led to changes in early behavior and increased inflammation of the nervous tissue in the baby rats. Although using a different, potentially safer antibiotic combination reduced pregnancy complications, it still changed the bacteria in the mother's gut and worsened early behavior. These findings show that antibiotics during pregnancy can affect the developing brain of baby rats and careful consideration should be used before prescribing them. Future research will explore longer-term effects and potential medicines.

The association of systemic inflammatory biomarkers with metabolic dysfunction-associated steatotic liver disease and arterial hypertension

Objective — to explore the relationship between metabolic dysfunction‑associated steatotic liver disease (MASLD) on the background of arterial hypertension (AH) and blood inflammatory markers including C‑reactive protein (CRP), Interleukin‑6 (IL‑6), IL‑4, haptoglobin and pentraxin‑3 (PTX‑3). Materials and methods. We examined 102 patients with MASLD. They were divided into 3 groups: group A included 52 patients with isolated MASLD, group B — 23 patients with MASLD and AH stage I, and group C — 27 patients with MASLD and AH stage II. The control group (group D) comprised 20 apparently healthy people. Patients with viral hepatitis, liver cirrhosis, alcoholic liver disease, and AH stage III were excluded. Systemic inflammatory biomarkers analyses were performed using electrochemiluminescence, immunoenzymatic, and immunoturbidimetry techniques. Results. The systemic inflammatory biomarkers analyses revealed significantly higher levels of CRP (p=0.001), IL‑6 (p=0.01), haptoglobin (p<0.05) and PTX‑3 (p<0.01) and decreased levels of IL‑4 (p<0.05) in group B and group C in comparison with the group A and control group (p1 <0.01, p2=0.01). Also, there was a significant increase of CRP (p=0.01), IL‑6 (p=0.01), and PTX‑3 levels (p<0.05) in group B compared with group C. However, the relationship between the IL‑4 (p>0.05) and haptoglobin (p>0.05) levels and the progression of the AH stages have not been confirmed in our study. Conclusions. Our findings indicate the direct relationship between the systemic inflammatory biomarkers’ involvement in developing liver tissue inflammation and the further progression of MASLD. The obtained data indicate the relationship of AH and its stages with the development of chronic systemic inflammatory response in patients with MASLD and AH comorbid course.

Stiff-Soft Hybrid Biomimetic Nano-Emulsion for Targeted Liver Delivery and Treatment of Early Nonalcoholic Fatty Liver Disease

Background: Nonalcoholic fatty liver disease (NAFLD) poses a risk for numerous metabolic diseases. To date, the U.S. Food and Drug Administration has not yet approved any medications for the treatment of NAFLD, for which developing therapeutic drugs is urgent. Dihydromyricetin (DMY), the most abundant flavonoid in vine tea, has been shown to be hepatoprotective. Its application was limited by low bioavailability in vivo; Methods: In order to improve the bioavailability of DMY and achieve liver-targeted delivery, we designed a DMY-loaded stiff-soft hybrid biomimetic nano drug delivery system (DMY-hNE). The in vivo absorption, distribution, pharmacokinetic profiles, and anti-NAFLD efficacy of DMY-hNE were studied; Results: DMY-hNE was composed of a stiff core and soft shell, which led to enhanced uptake by gastrointestinal epithelial cells and increased penetration of the mucus barrier, thus improving the in vivo absorption, plasma DMY concentration, and liver distribution versus free DMY. In an early NAFLD mouse model, DMY-hNE effectively ameliorated fatty lesions accompanied with reduced lipid levels and liver tissue inflammation; Conclusions: These findings suggested that DMY-hNE is a promising platform for liver drug delivery and treatment of hepatopathy.

8640 Thyroid Orbitopathy in Pregnancy

Abstract Disclosure: Z. Maisonet -Feliciano: None. M. Alvarado: None. L.A. Gonzalez-Rodriguez: None. M. Ramirez: None. L. El Musa Penna: None. J. Segarra-Villafane: None. I.C. Arroyo: None. W. Medina-Torres: None. L.R. Sepulveda-Garcia: None. Thyroid orbitopathy, often associated with Graves' disease, is characterized by the inflammation and swelling of ocular tissues. Its occurrence or exacerbation during pregnancy is infrequent, primarily due to the immunosuppressive state inherent in pregnancy. In most instances, the immunosuppression associated with pregnancy leads to a reduction in TRAb levels, although they continue to be present in women who have an active form of the disease. The course and severity of ocular manifestation do not always correlate with thyroid hormone levels. Strict control of thyroid function is crucial in patients with thyroid eye disease. This report sheds light on the rare occurrence of thyroid orbitopathy during pregnancy, a condition that complicates treatment due to potential risks to both the mother and the child. In this case, a 35-year-old pregnant woman with no prior history of thyroid issues exhibited symptoms of hyperthyroidism in her first trimester, including unintended weight loss, anxiety, and tremors in the upper limbs. She also presented with ocular discomfort, diplopia, and eyelid lag. Thyroid tests confirmed Graves Disease, with a significant elevation in Thyroid Stimulating Immunoglobulin (TSI): 243 %, Free thyroxine (T4): 1.08 ng/dl and Triiodothyronine (T3): >1.89 units and decreased Thyroid Stimulation Hormone (TSH): 0.001 uIU/ml. Ophthalmic examination revealed mild Graves orbitopathy due to bilateral exophthalmos (measured by Hertel’s exophthalmometer) 21mm on both sides, Marginal reflex distance (MDR) 1: right eye 9 mm and left 7mm. MDR 2: right eye 6mm and left eye 7mm. Right upper eyelid retraction, lagophthalmos of the right eye, limitation in the upward movement of the left eye, and vertical diplopia on extreme gazes without signs of orbital compression. Considering her normal FT4 levels and the mild nature of her hyperthyroidism, the decision was made to prioritize the mother's treatment while minimizing fetal risk, thus opting not to initiate antithyroid medication but to closely monitor her thyroid function. Symptomatic management of her orbitopathy was provided including lubricating eye drops and an eye patch. Throughout the progression of her pregnancy, her TSI levels exhibited a decreasing trend while FT4 remained within normal limits. Following delivery, there was a notable improvement in her ocular symptoms, and her TSH started increasing up to normal values of 1.29 uIU/ml and FT4 remained in range. This report underscores the importance of recognizing uncommon presentations of autoimmune thyroid conditions, such as thyroid orbitopathy, during pregnancy. It emphasizes the complexities and necessary considerations in treating these cases. A collaborative approach involving endocrinology, ophthalmology, and obstetrics is essential to safeguard the health of both the mother and the fetus. Presentation: 6/1/2024

8301 A Rare Case of IgG4-Related Hypophysitis

Abstract Disclosure: P. Kesavan Chary: None. J.M. Silverstein: None. Background: IgG4-related disease (IgG4-RD) is an immune-mediated condition that presents as systemic inflammation and fibrosis of tissues, often affecting multiple organs. Isolated IgG4-related Hypophysitis is rare and characterized by lymphoplasmacytic infiltration of the pituitary gland by IgG4-positive plasma cells. A combination of clinical, serologic, radiologic, and histopathologic findings is used for diagnosis of IgG4-RD and approximately two-thirds of all patients have elevated serum IgG4 levels. We report a case of isolated IgG4-related Hypophysitis in a middle-aged woman serologically negative for IgG4-related disease. Case Report: A 52 year old female with history of steroid dependent severe persistent asthma, allergic rhinitis, and chronic sinusitis presented with one month of headache, polyuria, and polydipsia. MRI Brain demonstrated an enlarged, rounded pituitary gland 16 x 11 x 9 mm, with homogeneous enhancement, extension into the suprasellar cistern, and mild thickening of the infundibulum suspected to represent a pituitary adenoma. Lab evaluation revealed mild hyperprolactinemia (prolactin 83.2, normal 4.8-23.3 ng/mL) and secondary adrenal insufficiency based on an abnormal high-dose cosyntropin stimulation test in the setting of a low adrenocorticotrophic hormone (ACTH) level (<2.0 pg/mL). Remaining pituitary hormone levels were normal. Inpatient work-up for polyuria and polydipsia showed a low urine specific gravity of 1.003. An overnight water deprivation test was performed. Baseline urine osmolality (UOsm) was 95 mOsm/kg and serum sodium was 144 mmol/L; after 3 hours of water deprivation, serum Osm increased to 301 mOsm/kg (275-300 mOsm/kg) and UOsm only to 215 mOsm/kg, while her sodium increased to 145 mmol/L. Results were felt to be consistent with AVP deficiency (Central Diabetes Insipidus) and the patient was started on desmopressin. Because of the presence of AVP deficiency, further work-up was done to evaluate for an infiltrative process or malignancy. ANA, RPR, ESR, CRP, CBC, ACE level, T-SPOT, and IgG subclasses were unremarkable. Further imaging studies yielded a negative nuclear bone scan and PET/CT scan. For diagnostic purposes, the patient underwent biopsy via an endoscopic endonasal transsphenoidal approach. Pathology showed the presence of >10 IgG4+ plasma cells in a single high-power field, consistent with a diagnosis of IgG4-related Hypophysitis. Conclusion: Isolated IgG4-related Hypophysitis is the least common clinical presentation of IgG4-RD, which is itself a rare and relatively newly recognized condition. IgG4-related Hypophysitis should be suspected in patients presenting with AVP deficiency and pituitary stalk enlargement with an otherwise negative work up. Presentation: 6/2/2024

6822 Proptosis Improvement with Teprotumumab is Independent of Race, Ethnicity, Gender and Age from Three Global Randomized, Double-Masked, Placebo-Controlled Trials of Patients with Acute Thyroid Eye Disease (TED)

Abstract Disclosure: G.J. Kahaly: Consulting Fee; Self; Amgen Inc. Research Investigator; Self; Amgen Inc. S.T. Wester: Advisory Board Member; Self; Amgen Inc. Consulting Fee; Self; Immunovant, Lassen. Research Investigator; Self; Amgen Inc, Sling Therapeutics, Immunovant. C.Y. Liu: Other; Self; royalties from Wolters Kluwers Health. H. Kate: Employee; Self; Amgen Inc. Stock Owner; Self; Amgen Inc. E. Conrad: Employee; Self; Amgen Inc. Stock Owner; Self; Amgen Inc. R.J. Holt: Employee; Self; Amgen Inc. Stock Owner; Self; Amgen Inc. Y. Hiromatsu: Advisory Board Member; Self; Amgen Inc. Consulting Fee; Self; Amgen Inc. Research Investigator; Self; Amgen Inc. Introduction: Thyroid eye disease (TED) produces orbital tissue expansion, inflammation, proptosis (eye bulging) and diplopia (double vision), causing eye pain and appearance/visual changes. Teprotumumab, an insulin-like growth factor-1 receptor inhibitor, significantly improved signs and symptoms of TED in three placebo-controlled trials in patients with high inflammation. Here, we report pooled proptosis response in patient subgroups. Methods: Patients ≥18 years old from three placebo-controlled trials, two in the US and EU (NCT01868997, NCT03298867) and one in Japan (OPTIC-J; jRCT2031210453), with Graves’ disease and recent onset (≤9 month duration) active TED (Clinical activity score, CAS≥4, ≥3 for OPTIC-J) were included. Patients received eight infusions of teprotumumab or placebo over 21 weeks, with the final study visit at Week 24 (three weeks after final dose). Observed proptosis response, defined as ≥2 mm improvement from baseline in study eye without deterioration ≥2 mm in fellow eye at Week 24, versus placebo is reported by tobacco use (yes/no), race (White or Asian), and age (<65/≥65). Cochran-Mantel-Haenszel tests compared teprotumumab and placebo subgroups. Results: Of 111 teprotumumab and 114 placebo patients, 68.5% (76) and 76.3% (87) were female, respectively; mean (SD) age 50.3 (12.4) and 51.1 (13.1) years, respectively; mean (SD) TED duration 5.49 (2.32) and 5.98 (2.40) months, respectively; and 78.4% (87) and 73.7% (84) were never/former smokers, respectively. In the overall population, 80.2% (89/111) of teprotumumab patients and 14.0% (16/114) of placebo patients had a proptosis response (P<.0001). At Week 24, 70.8% (17/24) of teprotumumab and 23.3% (7/30) placebo smokers were proptosis responders; in non-smokers, 82.8% (72/87) of teprotumumab and 10.7% (9/84) placebo patients were proptosis responders (P<.0001 for both). In White patients, 78.9% (56/71) of teprotumumab and 15.8% (12/76) of placebo patients were proptosis responders; in Asian patients, 90.0% (27/30) of teprotumumab and 10.0% (3/30) of placebo patients were responders (P<.0001 for both). In patients <65 years, 79.2% (76/96) teprotumumab patients and 13.4% (13/97) placebo patients were responders; in patients ≥65 years, 86.7% (13/15) teprotumumab and 17.6% (3/17) placebo were responders (P<.0001 for both). Conclusions: Subgroup analysis of proptosis response by tobacco use, race, and age showed that teprotumumab treatment led to significantly higher proptosis response versus placebo in all groups. Public Presentation: 6/1/2024

8150 CD1a Lipid Presentation Contributes to T2D Phenotype in DIO Mouse Model

Abstract Disclosure: J.A. Ayala Angulo: None. K.D. Wiggins: None. M. Lopez: None. D. Nicholas: None. Type 2 Diabetes’s complex etiology, progression, and comorbidities are not fully explained by current models. Elevation of inflammatory markers and immune cell infiltration into tissues of T2D patients has been described in the literature extensively, but no clear answers as to what antigens are driving this response and how these observations contribute to disease progression exist. Recently, CD1a has been shown to present lipids to T cells and induce a memory response. Our previous work shows that T2D patients produce anti-lipid antibodies likely derived with T cell help, not present in healthy patients. Together, these data suggest a potential role for lipid-specific T cell responses to drive T2D progression through inflammatory stress, antibody production, and tissue infiltration. Current work with human CD1a transgenic B6 mice points to elevated liver steatosis and hepatitis along with higher glucose levels relative to low fat diet controls. CD1a mice show smaller islets suggesting pancreatic atrophy. This validates the CD1a-T cell mechanism as a potential contributor to T2D but also introduces a more physiologically relevant mouse model of T2D. Presentation: 6/1/2024

7990 Reduced Alpha-1 Antitrypsin Activity Is Associated with Higher Plasma Free Cortisol and Increased Tissue Glucocorticoid Exposure in Humans in vivo

Abstract Disclosure: L.D. Boyle: None. M. Nixon: None. C.M. Underhill: None. L.A. Hill: None. N.Z. Homer: None. M. Magennis: None. R. Andrew: None. G.L. Hammond: None. J.G. Lewis: None. R.H. Stimson: None. B.R. Walker: None. Background: Corticosteroid Binding Globulin (CBG) binds >85% of plasma cortisol, modulating its biological activity. Proteolytic cleavage by neutrophil elastase (NE) reduces CBG binding capacity, proposed to increase free cortisol availability to inflamed tissues. Genetic variation at the locus spanning SERPINA1 (encoding alpha-1 antitrypsin, AAT, the endogenous inhibitor of NE) and SERPINA6 (CBG) altered morning total plasma cortisol. We hypothesised that AAT deficiency increased CBG cleavage and hence free plasma cortisol, resulting in amplified tissue cortisol delivery and increased HPA axis negative feedback. We tested this in recall-by-genotype studies of people who are heterozygous for inactivating mutations in SERPINA1. Methods: 16 asymptomatic carriers of deleterious SERPINA1 single nucleotide polymorphisms (rs17580 & rs28929474) and 16 age-, gender- and BMI-matched controls were recruited from the Generation Scotland Biobank. To quantify in vivo whole body glucocorticoid appearance and clearance rates, and estimate tissue cortisol uptake, we performed arterio-venous plasma sampling across abdominal adipose and skeletal muscle during steady-state 9,11,12,12-[2H]4-cortisol tracer infusion. To assess endogenous negative feedback, participants underwent combined receptor antagonist stimulation of the HPA axis (‘CRASH’) testing using RU486 and spironolactone, or placebo in a double blind randomised crossover design. Total cortisol (LC-MS/MS), free cortisol (isotopic dilution & ultrafiltration), CBG binding capacity (radioligand displacement), CBG concentration and AAT (ELISA) were measured in plasma. Tissue cortisol (LC-MS/MS) and glucocorticoid-dependent transcripts (qPCR) were measured in adipose biopsy samples collected at end of study. Results: AAT was ∼30% lower in AAT+/- (p=0.0002 vs control). Plasma CBG concentration, binding capacity and total cortisol were similar between groups. However, plasma free cortisol fraction was higher in AAT+/- subjects (16.1 +/- 0.2 vs 13.9 +/- 0.04 %, p<0.0001). Adipose tissue from AAT+/- subjects displayed increased cortisol levels and increased glucocorticoid-responsive transcripts PER1 and LPL compared to controls. Consistent with release from CBG, total and free cortisol release across skeletal muscle was increased in AAT+/- subjects vs controls. The rate of appearance of 9,12,12-[2H]3-cortisol was reduced in AAT+/- (p=0.03 vs control), consistent with decreased whole body 11β-HSD1 activity. CRASH testing did not reveal additional differences between groups. Discussion: Subclinical AAT deficiency is associated with a higher free cortisol fraction and increased tissue glucocorticoid exposure. Although consistent with the hypothesis of local tissue-mediated control of glucocorticoid exposure via the NE/AAT/CBG axis, these findings were not accompanied by measurable changes in CBG. Presentation: 6/3/2024

9225 Reduced Alpha-1 Antitrypsin Activity Is Associated with Higher Plasma Free Cortisol and Increased Tissue Glucocorticoid Exposure in Humans in vivo

Abstract Disclosure: L.D. Boyle: None. M. Nixon: None. C.M. Underhill: None. L.A. Hill: None. N.Z. Homer: None. M. Magennis: None. R. Andrew: None. G.L. Hammond: None. J.G. Lewis: None. R.H. Stimson: None. B.R. Walker: None. Background Corticosteroid Binding Globulin (CBG) binds >85% of plasma cortisol, modulating its biological activity. Proteolytic cleavage by neutrophil elastase (NE) reduces CBG binding capacity, proposed to increase free cortisol availability to inflamed tissues. Genetic variation at the locus spanning SERPINA1 (encoding alpha-1 antitrypsin, AAT, the endogenous inhibitor of NE) and SERPINA6 (CBG) altered morning total plasma cortisol. We hypothesised that AAT deficiency increased CBG cleavage and hence free plasma cortisol, resulting in amplified tissue cortisol delivery and increased HPA axis negative feedback. We tested this in recall-by-genotype studies of people who are heterozygous for inactivating mutations in SERPINA1. Methods 16 asymptomatic carriers of deleterious SERPINA1 single nucleotide polymorphisms (rs17580 & rs28929474) and 16 age-, gender- and BMI-matched controls were recruited from the Generation Scotland Biobank. To quantify in vivo whole body glucocorticoid appearance and clearance rates, and estimate tissue cortisol uptake, we performed arterio-venous plasma sampling across abdominal adipose and skeletal muscle during steady-state 9,11,12,12-[2H]4-cortisol tracer infusion. To assess endogenous negative feedback, participants underwent combined receptor antagonist stimulation of the HPA axis (‘CRASH’) testing using RU486 and spironolactone, or placebo in a double blind randomised crossover design. Total cortisol (LC-MS/MS), free cortisol (isotopic dilution & ultrafiltration), CBG binding capacity (radioligand displacement), CBG concentration and AAT (ELISA) were measured in plasma. Tissue cortisol (LC-MS/MS) and glucocorticoid-dependent transcripts (qPCR) were measured in adipose biopsy samples collected at end of study. Results AAT was ∼30% lower in AAT+/- (p=0.0002 vs control). Plasma CBG concentration, binding capacity and total cortisol were similar between groups. However, plasma free cortisol fraction was higher in AAT+/- subjects (16.1 +/- 0.2 vs 13.9 +/- 0.04 %, p<0.0001). Adipose tissue from AAT+/- subjects displayed increased cortisol levels and increased glucocorticoid-responsive transcripts PER1 and LPL compared to controls. Consistent with release from CBG, total and free cortisol release across skeletal muscle was increased in AAT+/- subjects vs controls. The rate of appearance of 9,12,12-[2H]3-cortisol was reduced in AAT+/- (p=0.03 vs control), consistent with decreased whole body 11β-HSD1 activity. CRASH testing did not reveal additional differences between groups. Discussion Subclinical AAT deficiency is associated with a higher free cortisol fraction and increased tissue glucocorticoid exposure. Although consistent with the hypothesis of local tissue-mediated control of glucocorticoid exposure via the NE/AAT/CBG axis, these findings were not accompanied by measurable changes in CBG. Presentation: 6/3/2024

7975 TLR4 Signaling Modulates Reproductive and Metabolic Outcomes in a Mouse Model of Polycystic Ovary Syndrome

Abstract Disclosure: K.D. Wiggins: None. Z. Del Mundo: None. J.A. Ayala Angulo: None. M. Lopez: None. C. Garcia: None. C. Nguyen: None. J. De La Torre: None. A. Saba: None. D. Trinh: None. D. Nicholas: None. Polycystic Ovarian Syndrome (PCOS), characterized by disruptions in both reproductive and metabolic functions, is associated with abnormal immune system activity and chronic inflammation. Although the precise mechanistic drivers of chronic inflammation in PCOS are not fully elucidated, elevated serum levels of lipopolysaccharide (LPS) have been observed. We propose that Toll-like receptor 4 (TLR4) signaling, influenced by dysregulated LPS levels, is a central driver of chronic inflammation affecting both reproductive and metabolic pathways. To investigate this hypothesis, we subcutaneously implanted a nonsteroidal aromatase inhibitor, Letrozole (LET), in pubertal TLR4 knockout mice (TLR4KO) and C57BL/6J (wild type) mice. Our five-week LET treatment revealed that TLR4KO mice successfully entered all phases of the estrous cycle, with normalized Luteinizing Hormone (LH) and increased Follicle Stimulating Hormone (FSH) levels. These findings highlight TLR4's role in shaping cycling patterns and regulating fundamental hormonal pathways vital for reproductive health. Despite the genetic manipulation of the inflammatory pathway leading to a reduction in overall body weight gain, it had no discernible impact on the overall body composition of fat and lean mass. Additionally, our study found that dysregulation of blood glucose levels over time was independent of reduced body weight. TLR4KO mice demonstrated inefficient glucose clearance compared to wild-type mice, hinting at potential increases in insulin resistance, imbalances in adipose tissue inflammation, or possible alterations in the gut microbiota. Our study underscores the significance of disrupting the pro-inflammatory TLR4 pathway in shaping the reproductive and metabolic phenotypes in Letrozole-induced PCOS. The investigation into TLR4 becomes pivotal for comprehending the role of chronic inflammation in PCOS development or progression, offering valuable insights into potential therapeutic targets. Presentation: 6/2/2024

8729 Role of PARP1 in Macrophage-dependent Regulation of Adipose Tissue Inflammation, Adipogenesis, and Diet-induced Obesity

Abstract Disclosure: J. Zhu: None. R. Gupte: None. T.S. Nandu: None. W.L. Kraus: None. D. Huang: None. Adipose tissue macrophages play a significant role in maintaining adipose tissue homeostasis, as well as promoting chronic low-grade inflammation during the development of obesity and metabolic dysfunction. However, the crosstalk between macrophages and adipocytes, especially how macrophages affect adipogenesis in obese adipose tissue, are still poorly understood. We have investigated the function of PARP1 in the control of adipogenesis by using an adipocyte precursor-specific Parp1 knockout mouse model. Here, to investigate the role of macrophage PARP1 in regulating adipogenesis, we deleted Parp1 in the myeloid lineage by crossing Parp1loxp/loxp mice with LysMCre mice (Parp1 cKOLysMCre). Macrophages-specific knockout of Parp1 dramatically reduced LPS-induced pro-inflammatory gene expression in bone marrow-derived macrophages (BMDMs). To explore whether macrophage-specific Parp1 knockout affects adipocyte differentiation, we treated the primary preadipocytes from stromal vascular fraction (SVF) of white adipose tissue with the conditioned medium from LPS-stimulated control or Parp1 cKOLysMCre BMDMs throughout a 6-day period of differentiation. We observed that exposure of preadipocytes to the conditioned medium of Parp1 cKOLysMCre macrophages promoted adipogenesis by Oil Red O staining, as well as by monitoring the expression of key proadipogenic marker genes (e.g., Adipoq and Fabp4) by RT-qPCR. To further investigate the role of macrophage-specific deletion of Parp1 in metabolic outcomes, we fed the Parp1 cKOLysMCre mice with a high fat diet for 12 weeks. The knockout mice exhibited a significant increase in body weight compared to the control mice. Interestingly, we observed a reduction of the total number of macrophages resident in the adipose tissue from macrophage-specific Parp1 knockout mice. To explore the underlying mechanism, we performed single cell RNA-seq for adipose tissue macrophages using sorted SVF cells collected from HFD-fed mice. The results demonstrated dramatic reductions in macrophage M1 marker genes and pro-inflammatory transcription factors in Parp1 cKOLysMCre mice. Taken together, our study suggests that Parp1 knockout in macrophages selectively affects the recruitment and pro-inflammatory activation of macrophages, as well as adipocyte differentiation in adipose tissue. Importantly, despite the decreased adipose tissue inflammation, macrophage-specific Parp1 knockout enhanced adipogenesis, which leads to obesity in mice.This work was supported by a grant from the NIH/NIDDK (R01 DK069710) and funds from the Cecil H. and Ida Green Center for Reproductive Biology Sciences Endowment, to W.L.K., and a grant from NSFC (82270929) to D.H. Presentation: 6/1/2024

Immunometabolic cues recompose and reprogram the microenvironment around implanted biomaterials.

Circulating monocytes infiltrate and coordinate immune responses in tissues surrounding implanted biomaterials and in other inflamed tissues. Here we show that immunometabolic cues in the biomaterial microenvironment govern the trafficking of immune cells, including neutrophils and monocytes, in a manner dependent on the chemokine receptor 2 (CCR2) and the C-X3-C motif chemokine receptor 1 (CX3CR1). This affects the composition and activation states of macrophage and dendritic cell populations, ultimately orchestrating the relative composition of pro-inflammatory, transitory and anti-inflammatory CCR2+, CX3CR1+ and CCR2+ CX3CR1+ immune cell populations. In amorphous polylactide implants, modifying immunometabolism by glycolytic inhibition drives a pro-regenerative microenvironment principally by myeloid cells. In crystalline polylactide implants, together with arginase-1-expressing myeloid cells, T helper 2 cells and γδ+ T cells producing interleukin-4 substantially contribute to shaping the metabolically reprogrammed pro-regenerative microenvironment. Our findings inform the premise that local metabolic states regulate inflammatory processes in the biomaterial microenvironment.