Pattern Molecules Research Articles

Correlation between circulating cell-free mitochondrial DNA content and severity of knee degeneration in patients with knee osteoarthritis: a cross-sectional study

BackgroundKnee osteoarthritis (KOA) is characterized by mitochondrial damage and increased inflammation. Circulating cell-free mitochondrial DNA (ccf-mtDNA), which originates from damaged mitochondria, is an endogenous damage-associated molecular pattern (DAMPs) molecule that may trigger inflammation and is recognized as a potential biomarker for various diseases. In this study, we investigated the potential association between plasma ccf-mtDNA content and its use as a diagnostic biomarker in patients with KOA.MethodsWe collected plasma samples from patients with KOA and healthy controls (HC). Subsequently, quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect ccf-mtDNA content in the plasma samples. We used the Kellgren–Lawrence (K-L) classification criteria to classify patients with KOA into four grades: I-IV. Disease severity in patients with KOA was assessed using the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). Next, Spearman analysis was performed to observe the correlation between ccf-mtDNA content and the K-L classification and WOMAC score. Logistic regression analysis was used to evaluate the relationship between ccf-mtDNA and KOA risk.ResultsIn total, we enrolled 60 patients with KOA and HC who were matched for age, sex, and body mass index (BMI). We found that plasma ccf-mtDNA contents were significantly higher in patients with KOA (median, 2.44; quartile range, 1.10–3.79) than in HC (median, 1.08; quartile range, 0.52–2.12) (P < 0.0001). Plasma ccf-mtDNA content sequentially increased following the KOA class I-IV group (P = 0.040) and positively correlated with the K-L classification (r = 0.369, P = 0.004) and WOMAC scores (r = 0.343, P = 0.007). The ccf-mtDNA content did not significantly differ between patients with bilateral and those with single KOA (P = 0.083). Patients with high levels of ccf-mtDNA had a significantly increased risk of KOA compared with those with low levels of ccf-mtDNA (odds ratio [OR], 4.15, 95% confidence interval [CI], 1.71–10.07; P = 0.002). Quartile analysis revealed a significant dose-dependent association (P trend < 0.001).ConclusionOur study’s findings showed that plasma ccf-mtDNA was highly expressed in patients with KOA compared with HC. Furthermore, ccf-mtDNA content is significantly associated with the severity and risk of KOA. Therefore, its detection may provide insight into the prevention and treatment of KOA.

Pyroptosis in asthma: inflammatory phenotypes, immune and non-immune cells, and novel treatment approaches.

Pyroptosis is a form of inflammatory programmed cell death, and is activated by pathogen infections or endogenous danger signals. The canonical pyroptosis process is characterized by the inflammasome (typically NLRP3)-mediated activation of caspase-1, which in turn cleaves and activates IL-1β and IL-18, as well as gasdermin D, which is a pore-forming executor protein, leading to cell membrane rupture, and the release of proinflammatory cytokines and damage-associated molecular pattern molecules. Pyroptosis is considered a part of the innate immune response. A certain level of pyroptosis can help eliminate pathogenic microorganisms, but excessive pyroptosis can lead to persistent inflammatory responses, and cause tissue damage. In recent years, pyroptosis has emerged as a crucial contributor to the development of chronic inflammatory respiratory diseases, such as asthma. The present study reviews the involvement of pyroptosis in the development of asthma, in terms of its role in different inflammatory phenotypes of the disease, and its influence on various immune and non-immune cells in the airway. In addition, the potential therapeutic value of targeting pyroptosis for the treatment of specific phenotypes of asthma is discussed.

Neutrophil extracellular traps formation is associated with postoperative complications in congenital cardiac surgery.

Pediatric patients with congenital heart disease (CHD) often require surgical repair using cardiopulmonary bypass. Despite advancements, mortality and complication rates remain significant. We prospectively examined 101 patients undergoing congenital cardiac surgery, identifying a mortality rate of 4.0% and a complication rate of 31.6%. Neonates and infants exhibitedmultiple complicationsmore frequently. Prolonged bypass time was significantly associated with complications, with each additional 30 min increasing the odds by 1.46 times (95% CI 1.01-2.10, p = 0.042). We further investigated the involvement of damage-associated molecular pattern (DAMP) molecules by proteomics and ELISA. Plasma levels of DAMPs, including histones and high mobility group box 1 (HMGB1), were significantly elevated in the complication group. As these molecules target Toll-like receptor (TLR)2 and TLR4, mRNA expression of TLR2 and TLR4 in neutrophils was upregulated in the complication group. In vitro and in vivo analyses demonstrated that histones and HMGB1 induced the formation of neutrophil extracellular traps (NETs). This finding aligned with greater NETs formation observed at the end of CPB and during the postoperative period in neonates and infants who developed postoperative complications. Targeting NETs and associated DAMPs may provide a novel therapeutic approach to mitigate complications in this patient population.

A myeloid differentiation–like protein in partnership with Toll5 from the pest insect Spodoptera litura senses baculovirus infection

Many types of viruses infect insects and other arthropods. In contrast, little is known about how arthropods sense viruses, although several innate immune pathways including Toll have antiviral functions. Large DNA viruses in the family Baculoviridae are used to control a number of pest insects. Here, we studied Spodoptera litura and Autographa californica multiple nucleopolyhedrovirus (AcMNPV) to test the hypothesis that one or more myeloid differentiation-like (ML) proteins and Toll family members sense baculoviruses. We identified 11 ML and 12 Toll genes in the S. litura genome. A series of experiments indicated that S. litura ML protein 11 (SlML-11) binds the budded form of AcMNPV and partners with S. litura Toll5 (SlToll5). SlML-11 also bound sphingomyelin (SPM), which is a component of the virion envelope. Disabling SlML-11 and SlToll5 increased susceptibility to infection, while priming larvae with SPM reduced susceptibility as measured by increased survival to the adult stage and clearance of AcMNPV from individuals that emerged as adults. We conclude that SPM is a pathogen-associated molecular pattern molecule while SlML-11 and SlToll5 interact to function as a pattern recognition receptor that senses AcMNPV.

Human iPSC-based disease modeling studies identify a common mechanistic defect and potential therapies for AMD and related macular dystrophies.

Age-related macular degeneration (AMD) and related macular dystrophies (MDs) primarily affect the retinal pigment epithelium (RPE) in the eye. A hallmark of AMD/MDs that drives later-stage pathologies is drusen. Drusen are sub-RPE lipid-protein-rich extracellular deposits, but how drusen forms and accumulates is not known. We utilized human induced pluripotent stem cell (iPSC)-derived RPE from patients with AMD and three distinct MDs to demonstrate that reduced activity of RPE-secreted matrix metalloproteinase 2 (MMP2) contributes to drusen in multiple maculopathies in a genotype-agnostic manner by instigating sterile inflammation and impaired lipid homeostasis via damage-associated molecular pattern molecule (DAMP)-mediated activation of receptor for advanced glycation end-products (RAGE) and increased secretory phospholipase 2-IIA (sPLA2-IIA) levels. Therapeutically, RPE-specific MMP2 supplementation, RAGE-antagonistic peptide, and a small molecule inhibitor of sPLA2-IIA ameliorated drusen accumulation in AMD/MD iPSC-RPE. Ultimately, this study defines a causal role of the MMP2-DAMP-RAGE-sPLA2-IIA axis in AMD/MDs.

LL37-DNA Complex Drives Vitiligo Progression Through TLR9-MyD88 Signaling Pathways.

Vitiligo is an autoimmune disorder characterized by chronic depigmentation and milk-white patches on the skin. Skin infiltration by autoreactive CD8+ T cells causes melanocyte destruction in vitiligo. Multiple risk factors, particularly immune-related inflammatory factors, are involved in the disappearance of melanocytes. LL37 is a classic damage-associated molecular pattern molecule that is involved in the development of various autoimmune diseases. An enhanced expression of LL37 in vitiligo is known; however, the exact role of LL37 in melanocyte loss has not yet been elucidated. In the present study, we detected increased LL37 expression in vitiligo serum and lesions. Furthermore, we confirmed that cultured keratinocytes released LL37 after treatment with H2O2. Moreover, the LL37-DNA complex enhanced the secretion of CXCL9, CXCL10, and CXCL16 from keratinocytes via the TLR9-MyD88 signaling pathway and facilitated the migration of CD8+ T cells. Altogether, our study demonstrates that LL37 released from keratinocytes binds to DNA and contributes to melanocyte destruction under oxidative stress-induced autoimmunity in vitiligo.

Evidence that extracellular HSPB1 contributes to inflammation in alcohol-associated hepatitis.

Alcohol-associated hepatitis (AH) is the most life-threatening form of alcohol-associated liver disease (ALD). AH is characterized by severe inflammation attributed to increased levels of ethanol, microbes or microbial components, and damage-associated molecular pattern (DAMP) molecules in the liver. HSPB1 (Heat Shock Protein Family B (Small) Member 1; also known as Hsp25/27) is a DAMP that is rapidly increased in and released from cells experiencing stress, including hepatocytes. The goal of this study was to define the role of HSPB1 in AH pathophysiology. Serum HSPB1 was measured in a retrospective study of 184 heathy controls (HC), heavy alcohol consumers (HA), patients with alcohol-associated cirrhosis (AC), and patients with AH recruited from major hospital centers. HSPB1 was also retrospectively evaluated in liver tissue from 10 HC and AH patients and an existing liver RNA-seq dataset. Finally, HSPB1 was investigated in a murine Lieber-DeCarli diet model of early ALD as well as cellular models of ethanol stress in hepatocytes and hepatocyte-macrophage communication during ethanol stress. Circulating HSPB1 was significantly increased in AH patients and levels positively correlated with disease-severity scores. Likewise, HSPB1 was increased in the liver of patients with severe AH and in the liver of ethanol-fed mice. In vitro , ethanol-stressed hepatocytes released HSPB1, which then triggered TNFα-mediated inflammation in macrophages. Anti-HSPB1 antibody prevented TNFα release from macrophages exposed to media conditioned by ethanol-stressed hepatocytes. Our findings support investigation of HSPB1 as both a biomarker and therapeutic target in ALD. Furthermore, this work demonstrates that anti-HSPB1 antibody is a rational approach to targeting HSPB1 with the potential to block inflammation and protect hepatocytes, without inactivating host defense. HSPB1 is significantly increased in serum and liver of patients with alcohol-associated hepatitis.Ethanol consumption leads to early increases in HSPB1 in the mouse liver.Hepatocytes subjected to ethanol stress release HSPB1 into the extracellular environment where it activates TNFα-mediated inflammation in macrophages.Anti-HSPB1 antibody blocks hepatocyte-triggered TNFα in a model of hepatocyte-macrophage communication during ethanol stress.

Sex-specific modulation of renal epigenetic and injury markers in aging kidney.

Sex differences in renal physiology and pathophysiology are now well established in rodent models and in humans. Epigenetic programming is known to be a critical component of renal injury, as studied mainly in male rodent models; however, not much is known about the impact of biological sex and age on the kidney epigenome. We sought to determine the influence of biological sex and age on renal epigenetic and injury markers, using male and female mice at 4 mo (4M; young), 12 mo (12M), and 24 mo (24M; aged) of age. Females had a significant increase in kidney and body weights and serum creatinine levels and a decrease in serum albumin levels from 4M to 24M of age, whereas minor changes were observed in male mice. Kidney injury molecule-1 levels in serum and renal tissue greatly enhanced from 12M to 24M in both males and females. Circulating histone 3 (H3; damage-associated molecular pattern molecules) levels extensively increased with age; however, males had higher levels than females. Overall, females had markedly high histone acetyltransferase (HAT) activity than age-matched males. Aged mice had decreased HAT activity and increased histone deacetylase activity than sex-matched 12M mice. Aged females had substantially decreased renal H3 methylation at lysine 9 and 27 and histone methyltransferase (HMT) activity than aged male mice. Antiaging protein Klotho levels were significantly higher in young males than age-matched females and decreased substantially with age in males, whereas epigenetic repressor of Klotho, trimethylated H3K27, and its HMT enzyme, enhancer of zeste homolog 2, increased consistently with age in both sexes. Moreover, nuclear translocation and activity of proinflammatory transcription factor nuclear factor-κB (p65) were significantly higher in aged mice. Taken together, our data suggest that renal aging lies in a range between normal and diseased kidneys but may differ between female and male mice, highlighting sex-related differences in the aging process.NEW & NOTEWORTHY Although there is evidence of sex-specific differences in kidney diseases, most preclinical studies have used male rodent models. The clinical data on renal injury have typically not been stratified by sex. Our findings provide convincing evidence of sex-specific differences in age-regulated epigenetic alterations and renal injury markers. This study highlights the importance of including both sexes for better realization of underlying sex differences in signaling mechanisms of aging-related renal pathophysiology.

Alternaria solani core effector Aex59 is a new member of the Alt a 1 protein family and is recognized as a PAMP

Early blight caused by Alternaria solani is a destructive disease in potato production. Here, through systematically screening of an effector protein pool consisting of 115 small cysteine-containing candidate Aex (Alternariaextracellular proteins) in A. solani, we identified a core effector protein named Aex59, a pathogen-associated molecular pattern (PAMP) molecule. Aex59 is uniquely present in the Ascomycota of fungi and can activate defense responses in multiple plants. Targeted gene disruption showed that Aex59 is a virulence factor and participates in spore development. Perception of Aex59 in Nicotiana benthamiana does not depend on the receptor-like kinases Brassinosteroid-associated kinase1 (BAK1) and Suppressor of BIR1-1 (SOBIR1), which are required for multiple pattern recognition receptors (PRR) pathways. Sequence analysis revealed that Aex59 is a new member of the Alt a 1 protein family and is a potential molecular marker capable of aiding in the classification of the fungi Alternaria spp.

Advanced Glycation End-Products Acting as Immunomodulators for Chronic Inflammation, Inflammaging and Carcinogenesis in Patients with Diabetes and Immune-Related Diseases.

Increased production of advanced glycation end products (AGEs) among reducing sugars (glucose, fructose, galactose, or ribose) and amino acids/proteins via non-enzymatic Maillard reaction can be found in lifestyle-related disease (LSRD), metabolic syndrome (MetS), and obesity and immune-related diseases. Increased serum levels of AGEs may induce aging, diabetic complications, cardiovascular diseases (CVD), neurodegenerative diseases (NDD), cancer, and inflamm-aging (inflammation with immunosenescence). The Maillard reaction can also occur among reducing sugars and lipoproteins or DNAs to alter their structure and induce immunogenicity/genotoxicity for carcinogenesis. AGEs, as danger-associated molecular pattern molecules (DAMPs), operate via binding to receptor for AGE (RAGE) or other scavenger receptors on cell surface to activate PI3K-Akt-, P38-MAPK-, ERK1/2-JNK-, and MyD88-induced NF-κB signaling pathways to mediate various pathological effects. Recently, the concept of "inflamm-aging" became more defined, and we have unveiled some interesting findings in relation to it. The purpose of the present review is to dissect the potential molecular basis of inflamm-aging in patients with diabetes and immune-mediated diseases caused by different AGEs.

Single Living Cell "Observation-Analysis" Integrated Platform Decodes Cell Migration Plasticity Orchestrated by Nucleocytoplasmic STAT3.

Cell migration requires the interplay among diverse migration patterns. The molecular basis of distinct migration programs is undoubtedly vital but not fully explored. Meanwhile, the lack of tools for investigating spontaneous migratory plasticity in a single living cell also adds to the hindrance. Here, we developed a micro/nanotechnology-enabled single-cell analytical platform to achieve coherent monitoring of spontaneous migratory pattern and signaling molecules. Via the platform, we unveiled a previously unappreciated STAT3 regionalization on the multifunctional regulations of migration. Specifically, nuclear STAT3 is associated with amoeboid migration, while cytoplasmic STAT3 promotes mesenchymal movement. Opposing effects of JAK2 multisite phosphorylation shape its response to STAT3 distribution in a dynamic and antagonistic manner, eventually triggering a reversible amoeboid-mesenchymal transition. Based on the above results, bioinformatics further revealed a possible downstream regulator of nucleocytoplasmic STAT3. Thus, our platform, as an exciting technological advance in single-cell migration research, can provide in-depth mechanism interpretations of tumor metastasis and progression.

Activation of dendritic cells by a synthesized small molecule for breast cancer vaccination.

e13149 Background: Dendritic cell-based vaccination is a promising immunotherapy for treating various types of cancer. However, tumor-loaded dendritic cells alone have limited treatment efficacy. We have designed a small molecule mimicking the action of damage-associated molecular pattern molecules as an adjuvant for activating dendritic cells in vitro, which demonstrated augmented treatment efficacy. Methods: We aimed at exploring the roles of the synthesized small molecule in augmenting the efficacy of dendritic cell-based cancer vaccination to target triple negative breast cancer. Breast cancer mouse model is constructed using a 4T1 tumor cell line. Data were analyzed using a two-tailed unpaired t-test. Results: Mouse dendritic cells could be activated in vitro by the small molecule as an adjuvant evidenced by increased expression of MHC-I, MHC-II and CD40. Augmented motility of dendritic cells was also observed after treatment with the adjuvant. The signaling of NFkB is remarkably activated in dendritic cells by the adjuvant, which facilitates the maturation of dendritic cells. Engraftment of adjuvant-primed dendritic cells loaded with 4T1 tumor antigen into 4T1 tumor-bearing mice efficiently reduced the tumor size and improved survival, compared with the group treated with dendritic cells only. After injection of adjuvant-activated dendritic cells, both CD4 T cell and CD8 T cell populations were expanded in the spleen and lymph nodes draining the tumor sites. Furthermore, the adjuvant could promote the production of IFN-γ and IL-2, cytokines required for T cell activation and expansion. Next, splenocytes were obtained from individual mice after dendritic cell-based treatment. Upon re-encounter with the tumor antigen in vitro, remarkable expansion of T cell populations with a memory cell feature was confirmed in splenocytes derived from mice that received adjuvant-treated dendritic cells. Consistently, production of TNF-α, IFN-γ and granzyme B, was profoundly increased in these T cells associated with the adjuvant treatment. Conclusions: Our work suggests a novel dendritic cell-based cancer immunotherapy by using a small molecule mimicking the action of damage-associated molecular pattern molecules, which have documented immune potentiation activities.

NLRP3 inflammasome activity and periodontal disease pathogenesis-A bidirectional relationship.

Periodontitis is an inflammatory oral disease that occurs as a result of the damaging effects of the immune response against the subgingival microflora. Among the mechanisms involved, the nucleotide-binding oligomerization domain, leucine-rich repeat-containing proteins family member NLRP3 (NLR family pyrin domain-containing 3), proposed as the key regulator of macrophage-induced inflammation, is strongly associated with periodontal disease due to the bacterial activators. This paper aimed to present key general concepts of NLRP3 inflammasome activation and regulation in periodontal disease. A narrative review was conducted in order to depict the current knowledge on the relationship between NLRP3 inflammasome activity and periodontal disease. Invitro and insitu studies were retrieved and commented based on their relevance in the field. The NLRP3 inflammasome activity stimulated by periodontal microbiota drive periodontal disease pathogenesis and progression. This occurs through the release of proinflammatory cytokines IL-1β, IL-18, and DAMPs (damage-associated molecular pattern molecules) following inflammasome activation. Moreover, the tissue expression of NLRP3 is dysregulated by oral microbiota, further exacerbating periodontal inflammation. The review provides new insights into the relationship between the NLRP3 inflammasome activity and periodontal disease pathogenesis, highlighting the roles and regulatory mechanism of inflammatory molecules involved in the disease process.

Tuning the diffusion constant to optimize the readout of positional information of spatial concentration patterns

Abstract Positional information encoded in spatial concentration patterns is crucial for the development of multicellular organisms. However, it is still unclear how such information is affected by the physically dissipative diffusion process. Here we study one-dimensional patterning systems with analytical derivation and numerical simulations. We find that the diffusion constant of the patterning molecules exhibits a nonmonotonic effect on the readout of the positional information from the concentration patterns. Specifically, there exists an optimal diffusion constant that maximizes the positional information. Moreover, we find that the energy dissipation due to the physical diffusion imposes a fundamental upper limit on the positional information.

Generation of 'semi-guided' cortical organoids with complex neural oscillations.

Temporal development of neural electrophysiology follows genetic programming, similar to cellular maturation and organization during development. The emergent properties of this electrophysiological development, namely neural oscillations, can be used to characterize brain development. Recently, we utilized the innate programming encoded in the human genome to generate functionally mature cortical organoids. In brief, stem cells are suspended in culture via continuous shaking and naturally aggregate into embryoid bodies before being exposed to media formulations for neural induction, differentiation and maturation. The specific culture format, media composition and duration of exposure to these media distinguish organoid protocols and determine whether a protocol is guided or unguided toward specific neural fate. The 'semi-guided' protocol presented here has shorter induction and differentiation steps with less-specific patterning molecules than most guided protocols but maintains the use of neurotrophic factors such as brain-derived growth factor and neurotrophin-3, unlike unguided approaches. This approach yields the cell type diversity of unguided approaches while maintaining reproducibility for disease modeling. Importantly, we characterized the electrophysiology of these organoids and found that they recapitulate the maturation of neural oscillations observed in the developing human brain, a feature not shown with other approaches. This protocol represents the potential first steps toward bridging molecular and cellular biology to human cognition, and it has already been used to discover underlying features of human brain development, evolution and neurological conditions. Experienced cell culture technicians can expect the protocol to take 1 month, with extended maturation, electrophysiology recording, and adeno-associated virus transduction procedure options.

Extracellular and intracellular inflammatory response of immune cells to radiation exposure

Background: Exposure to low doses of ionizing radiation is associated with increased risk of cardiovascular disease (CVD) in a dose-dependent manner. A person exposed to low doses of radiation have a 27-29% risk of developing and dying from CVD. Low doses of radiation initiate an inflammatory process that can increase the risk of developing CVD. Inflammation initiates atherosclerosis via immune cell infiltration subsequent to endothelial cell damage. We hypothesized that CD14+ monocytes will exhibit an amplified inflammatory response, as demonstrated by significant increases in the expression of inflammatory cytokines at 48 and 72 hours post radiation exposure compared to controls which would be magnified by the inflammatory stimulus LPS. Methods: Human venous peripheral blood was collected from 5 healthy young adults (24 ± 3 years, 22 ± 1 BMI, 5 Male) using an institutional review board (IRB)-approved protocol. Women were studied in days 1-5 of menstrual cycle. Peripheral blood mononuclear cells (PBMCs) were harvested by density gradient separation and re-suspended and incubated with anti-human CD14 microbeads. CD14+ immune cells were incubated in plates, exposed to 4 gray (Gy) of x-ray radiation, and then incubated for 48 and 72 hours. CD14+ immune cells were exposed to 1:500 dilution of 0.1 mg/mL of lipopolysaccharide (LPS) for the final 8 hours of incubation. The culture media was recovered, and assayed for secreted factors, specifically for IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, IFN-γ, and TNF-α. Additionally, flow cytometry analysis was performed to quantify intracellular content of IL-10, IL-8, IFN-g, IL-4, receptor for advanced glycation end products (RAGE), and damage-associated molecular pattern (DAMP) molecules (S100A8/9) involved in inflammatory activation. All data were analyzed with two-way repeated measures ANOVA. Results: Secreted Cytokines: Cytokine expression at 48 and 72 hours did not differ between the control, radiation (4Gy), and 4Gy+LPS groups. At 48 and 72 hours, there were no significant differences observed in the expression levels of IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNF-α in control, 4Gy, and 4Gy+LPS cells. Intracellular Content: Compared to control IL-4 content was increased at 48 and 72 hours, after exposure to 4Gy (48h: p=0.013 and 72h: p=0.008) and 4Gy+LPS (48h: p=0.022 and 72h: p=0.002). Furthermore, IL-4 was again increased at 72 hours after exposure to 4Gy+LPS compared to 48 hours (p=0.014). IL-10 content was also increased in after exposure to 4Gy and 4Gy+LPS at 72 hours compared to control (p=0.046 and p=0.007, respectively). S100A8/9 also showed a trending increase at 4Gy compared to control at 72 hours (p=0.11). No other content changed significantly. Conclusion: These data suggest that acute radiation exposure (4 Gy x-ray) has no effect on the secretome of CD14+ immune cells. However, the intracellular content of IL-4 was increased at both 48 and 72 hours, while IL-10 increased at 72 hours, indicating a shift from pro-inflammatory to anti-inflammatory intracellular content the further in time monocytes are from radiation exposure. Future work needs to test increased and repeated doses of radiation, as well as examine both x-ray and proton exposure. TRISH through NASA Cooperative Agreement NNX16AO69A and NIH HL150361. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sex and Age Differentially Regulate Epigenetic Modifications and Renal Injury Markers in Mice

Elucidation of epigenetic mechanism associated with aging renal physiology may reveal potential biomarkers and therapeutic targets for reversing aging renal pathologies. There is a sex difference in the age of onset of renal and cardiac disease, where females are protected against age related changes, including hypertension and fibrosis, until menopause. We hypothesized that sex and age would differentially regulate epigenetic modifications and renal physiology. To test this hypothesis, we examined kidney tissue and serum from 4-month (4M), 12-month (12M), and 24-month (24M)-old male and female C57BL/6JN mice (National Institute of Aging). Renal histone deacetylase (HDAC) and histone acetyltransferase (HAT) activities (ELISA) were significantly higher in all male mice groups compared with age-matched females. However, HAT activity decreased significantly in 24M males compared with 4M males and exhibited sex differences with 24M males having higher (p &lt; 0.05) activity that 24M females. Levels of histone 3 (H3) acetylation at lysine (K) 9 and 27 and methylation at K9 were comparable in all male and female groups. Nonetheless, age and sex differences were observed in H3K27 methylation levels with 24M males having significantly higher (p &lt; 0.05) levels as compared with 24M female mice. Serum creatinine (sCr) levels exhibited significant interaction between age and sex (p &lt; 0.0001). Aging significantly increased sCr levels in 24M mice compared with 4M mice in both sexes. However, sex-differences were evident in sCr levels with 24M female mice having higher levels compared with their age matched male counterparts. Of note, serum levels of kidney injury molecule-1 (KIM-1), a tubule injury biomarker were significantly higher in 24M mice compared with 4M and 12M sex-matched mice (male 24M, 410.1 ± 94.6 vs. 12M, 53.6 ± 17.2 and 4M, 27.9 ± 2.0; pg/ml, p &lt; 0.001; female 24M, 358.3 ± 56.1 vs. 12M, 63.8 ± 13.3 and 4M, 28.2 ± 5.0; pg/ml, p &lt; 0.001). Histones are released in the extracellular space during disease state, act as damage-associated molecular pattern molecules and activate inflammatory pathways. In the serum, circulating histone H3 levels showed significant interaction with age and sex (p &lt; 0.05) and increased significantly with age in both sexes. The data demonstrate that aged female mice exhibited higher levels of serum Cr and KIM-1 whereas aged males have higher KIM-1 levels as compared to sex matched young mice. Sex differences were observed in HDAC and HAT activity with males having higher enzymatic activity as compared to females. These findings will have important implications in age-related renal injury as aging is a primary risk factor for hypertension and related renal disease in men and women. Supported by the NIH grant R03AG075396. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Blocking CIRP protects against acute pancreatitis by improving mitochondrial function and suppressing pyroptosis in acinar cells

Acute pancreatitis (AP) continues to pose a major challenge as targeted therapeutic interventions are absent. Mitochondrial dysfunction and inflammasome-dependent pyroptosis are involved in the pathogenic mechanisms of AP. CIRP is a stress-response protein and a damage-associated molecular pattern (DAMP) molecule. In our previous studies, we discovered that excessive CIRP can directly damage pancreatic acinar cells. Nonetheless, the precise involvement of CIRP in AP is still unexplored. The primary aim of this study was to examine the potential involvement of CIRP in the development of pyroptosis and mitochondrial dysfunction in AP. To study this, an L-arginine-induced AP mouse model was used. Our results showed that Caspase-1-mediated pyroptosis and mitochondria-derived reactive oxygen species (ROS) were crucial factors in the occurrence of tissue damage and inflammation in AP. A substantial increase in the CIRP serum levels was observed in AP mice. Blocking CIRP by either CIRP gene knockout or systemic administration of C23, a competing inhibitor of CIRP, reduced ROS accumulation and pyroptosis in AP mice. These effects were associated with attenuated pancreatic injury and inflammation. In addition, CIRP-triggered mitochondrial dysfunction, autophagy impairment, and pyroptosis in pancreatic acinar cells were prevented by TAK242, an inhibitor of CIRP receptor TLR4. In conclusion, CIRP can induce mitochondrial dysfunction and pyroptosis in pancreatic acinar cells, and blocking CIRP may be a valuable approach to treating patients with AP.

Abstract 6674: Fungi utilizes exocytosis pathway to facilitate the release of DAMPs from pancreatic cancer cells

Abstract Recent studies overwhelmingly show that microbiome is pervasive to tumor and play key role in immunotherapy response and reprograming tumor immune microenvironment (TIME). However, it remains unknown how microbiome facilitates the host-immune cell crosstalk and promote tumorigenesis. Our recent study in pancreatic ductal adenocarcinoma (PDAC) show that intratumor fungal microbiome facilitate the release of a damage associated molecular pattern (DAMP) molecule IL-33 from cancer cells. The secreted IL33 from cancer cells in turns recruits ILC2 and TH2 cells which promotes PDAC tumorigenesis. However, the mechanism of fungal mediated IL-33 release from PDAC cells remains unknown. Our follow up studies using high throughput integrated proteomic profiling and immunoprecipitation revealed a group of exocyst complex proteins interacting with IL-33 in the presence of fungus. The exocyst complex consists of eight sub-unit proteins that tethers the secretory vesicles to the plasma membrane and implicated in cellular exocytosis. We found EXOC6B a component of exocyst complex, modulates IL-33 release via exocytosis pathway from the PDAC cells. Genetic depletion of EXOC6B resulted in shunted IL-33 release and significantly abrogated PDAC tumor progression. Mechanistically, we found that mycobiome activates a non-canonical TLR2-CARD9 pathway, thereby driving IL-33 nucleocytoplasmic transport and exocytosis. This study identifies a unique mechanism of EXOC6B mediated release of IL-33 that expands our knowledge on how host-mycobiome interaction shapes PDAC tumor microenvironment by intricate molecular mechanism. Our study identifies EXOC6B as a new therapeutics target involved in the mycobiome driven IL-33 secretion in pancreatic cancer. Citation Format: Aftab Alam, Shyamananda Singh Mayengbam, Sharon Senchanthisai, Bektas Irmak, Prasenjit Dey. Fungi utilizes exocytosis pathway to facilitate the release of DAMPs from pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6674.

Abstract 6742: PV 10 induces endoplasmic reticulum stress and autophagy, triggering immunogenic cell death and anti-tumor immunity in head and neck squamous cell carcinoma

Abstract Introduction: Head and neck squamous cell carcinoma (HNSCC) are complex, diverse cancers affecting mucosal linings of the upper aerodigestive tract. Globally, HNSCC is the sixth most common cancer, leading to over 890,000 new cases and more than 450,000 deaths annually. Primary risk factors include human papillomavirus (HPV) infection (HPV-positive HNSCC) and exposure to tobacco and excessive alcohol (HPV-negative HNSCC). Despite multimodal treatments, survival rates remain low due to recurrence and metastasis (R/M HNSCC), creating a need for safe, innovative therapies to reduce tumor burden with minimal adverse events. PV-10 (10% rose bengal sodium) is a novel intratumoral immunotherapeutic agent that has demonstrated promising anti-tumor activity in multiple solid tumor cancer types with few adverse effects in pre-clinical research and clinical trials. However, the role of PV-10 in HNSCCs and its role in enhancing immune response warrants further investigation. Methods: Cell viability was assessed using Alamar blue assay after PV-10 treatment of murine mEER and MTE-RAS HNSCC tumor cell lines. To evaluate the immunomodulatory properties of PV-10, we employed flow cytometry, confocal analysis, and luminescence-based assays. Additionally, we performed a DCFDA assay to measure the reactive oxygen species (ROS) and an Alexa Fluor 488 Annexin V/dead cell apoptosis assay to evaluate apoptosis. To gain insights into the mechanisms underlying PV-10's immunomodulatory effects in cancer cells and tumors from mice, western blotting and multiplex immunohistochemistry (mIHC) were used. Results: Our in vitro findings reveal that PV-10 induces cytotoxicity in both mEER and MTE-RAS cells. Notably, PV-10 promotes a significant increase in ROS, leading to an elevation in late apoptotic cells. Markers of immunogenic cell death (ICD), including a statistically significant increase in the release of damage-associated molecular pattern molecules HMGB1 and ATP, as well as enhanced surface expression of calreticulin, HSP-70, and HSP-90, were observed. At the molecular level, a remarkable activation of endoplasmic reticulum (ER) stress, pro-apoptotic protein, and autophagy markers were observed. Intratumoral PV-10 injection in vivo has shown significant tumor regression in both mEER and MTE-RAS tumors, and a complete response was noticed in some mice, indicating that PV-10 induces potent ICD in both mEER and MTE-RAS tumors. Conclusion: Our collated evidence strongly suggests that PV-10 induces ICD-associated immunogenicity in both mEER and MTE-RAS in vitro and in vivo, fostered by ROS-based ER stress and apoptosis. These findings hold promise for potential therapeutic avenues to manage HNSCC. Citation Format: Sowjanya Thatikonda, Ritu Chaudhary, Yeva Meshkovska, Maria I. Biernacki, Robbert Slebos, Shari P. Thomas, Amelio L. Antonio, Eric A. Wachter, Christine H. Chung. PV 10 induces endoplasmic reticulum stress and autophagy, triggering immunogenic cell death and anti-tumor immunity in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6742.