Induction Of Proinflammatory Cytokines Research Articles

Autonomous circadian rhythms in the human hepatocyte regulate hepatic drug metabolism and inflammatory responses.

Critical aspects of physiology and cell function exhibit self-sustained ~24-hour variations termed circadian rhythms. In the liver, circadian rhythms play fundamental roles in maintaining organ homeostasis. Here, we established and characterized an in vitro liver experimental system in which primary human hepatocytes display self-sustained oscillations. By generating gene expression profiles of these hepatocytes over time, we demonstrated that their transcriptional state is dynamic across 24 hours and identified a set of cycling genes with functions related to inflammation, drug metabolism, and energy homeostasis. We designed and tested a treatment protocol to minimize atorvastatin- and acetaminophen-induced hepatotoxicity. Last, we documented circadian-dependent induction of pro-inflammatory cytokines when triggered by LPS, IFN-β, or Plasmodium infection in human hepatocytes. Collectively, our findings emphasize that the phase of the circadian cycle has a robust impact on the efficacy and toxicity of drugs, and we provide a test bed to study the timing and magnitude of inflammatory responses over the course of infection in human liver.

Sarm1 Controls the MYD88-Mediated Inflammatory Responses in Inflammatory Bowel Disease via the Regulation of TRAF3 Recruitment

ABSTRACT Background Sterile alpha and TIR motif-containing 1 (Sarm1) is known as a negative regulator of inflammatory responses. However, its role in inflammatory bowel disease (IBD) is still unclear. Objective This study aimed to explore the function of Sarm1 in IBD and its underlying mechanisms. Sarm1 and tumor necrosis factor (TNF) receptor associated factor 3 (TRAF3) knockout (KO) micewere established. Methods The colitis was induced using dextran sulfate sodium (DSS). Bone marrow-derived macrophages (BMDMs) were isolated and stimulated with lipopolysaccharides (LPS) or cytidine phosphate guanosine(CpG). Inflammatory cytokines were measured viaELISA. qPCR and Western blotting were used to determine the levels of the mRNA and protein expression, respectively. Results It was demonstrated that reduced expression of Sarm1 was correlated with the severity of IBD in ulcerative colitis patients, and also with the reduction of pro-inflammatory cytokines in the mouse model induced by DSS. It was further observed that Sarm1 KO enhanced the induction of pro-inflammatory cytokines in both animal and in vitro cell models. Sarm1 deficiency in macrophages increased the severity of colitis in the mouse model induced by DSS. Moreover, Sarm1 regulatedTRAF3 recruitment to myeloid differentiation primary response protein 88 (MyD88), which in turn controlled the MYD88-mediated inflammatory responses. Conclusions In summary, our data suggest that Sarm1 controls the MYD88-mediated inflammatory responses in IBD via its regulation of TRAF3 recruitment.

Serum Mac-2 binding protein glycosylation isomer and galectin-3 levels in adult-onset Still's disease and their association with cytokines.

Autoinflammation with cytokine dysregulation may be implicated in the pathophysiology of adult-onset Still's disease (AOSD); however, the relationship between galectins and cytokines in patients with active AOSD remains unknown. We aimed to examine the relationship between circulating cytokines/chemokines and galectin-3 (Gal-3) or its ligand, Mac-2 binding protein glycosylation isomer (M2BPGi), in Japanese patients with AOSD. We recruited 44 consecutive patients diagnosed with AOSD according to the Yamaguchi criteria, 50 patients with rheumatoid arthritis (RA) as disease controls, and 27 healthy participants. Serum M2BPGi levels were directly measured using a HISCL M2BPGi reagent kit and an automatic immunoanalyzer (HISCL-5000). Serum Gal-3 concentrations were measured by enzyme-linked immunosorbent assay. The serum levels of 69 cytokines were analyzed in patients with AOSD using a multi-suspension cytokine array. We performed a cluster analysis of each cytokine expressed in patients with AOSD to identify specific molecular networks. Significant increases in the serum concentrations of Gal-3 and M2BPGi were found in the serum of patients with AOSD compared with patients with RA and healthy participants (both p <0.001). There were significant positive correlations between serum Gal-3 levels and AOSD disease activity score (Pouchot score, r=0.66, p <0.001) and serum ferritin levels. However, no significant correlations were observed between serum M2BPGi levels and AOSD disease activity scores (Pouchot score, r = 0.32, p = 0.06) or serum ferritin levels. Furthermore, significant correlations were observed between the serum levels of Gal-3 and various inflammatory cytokines, including interleukin-18, in patients with AOSD. Immunosuppressive treatment in patients with AOSD significantly reduced serum Gal-3 and M2BPGi levels (p = 0.03 and 0.004, respectively). Although both Gal-3 and M2BPGi were elevated in patients with AOSD, only Gal-3 was a useful biomarker for predicting disease activity in AOSD. Our findings suggest that circulating Gal-3 reflects the inflammatory component of AOSD, which corresponds to proinflammatory cytokine induction through inflammasome activation cascades.

Abstract CT068: Interim results of the ongoing phase 1-2 clinical trial of KVA12123, an engineered IgG1 targeting VISTA, as monotherapy and in combination with pembrolizumab in patients with advanced solid tumors

Abstract V-domain Ig Suppressor of T-cell Activation (VISTA), a promising immuno-oncology target, is primarily expressed by immunosuppressive myeloid cells that infiltrate solid tumors. VISTA may contribute to resistance to anti-PD-(L)1 and anti-CTLA-4 therapies and correlates with a poor prognosis. KVA12123 is a human IgG1 monoclonal antibody that specifically binds to VISTA at neutral and acidic pHs. It was designed to improve pharmacokinetic (PK) characteristics as well as reduce the risk of cytokine release syndrome (CRS). KVA12123 exhibited strong anti-tumor activity alone and in combination with anti-PD1 in preclinical studies. It demonstrated an excellent safety profile in non-human primates. The VISTA-101 clinical trial is a first-in-human, Phase 1/2, multicenter, open-label, safety, PK and pharmacodynamic (PD) evaluation of KVA12123, both as monotherapy and in combination with pembrolizumab, in adult patients with advanced solid tumors (NCT05708950). METHODS: The phase 1 of VISTA-101 is an accelerated Bayesian Optimal Interval Design consisting of two arms: Part A) KVA12123 monotherapy dose escalation from 3 mg to 1000 mg including 6 cohorts and Part B) KVA12123 dose escalation from 30 mg to 1000 mg in combination with 400 mg fixed dosing of pembrolizumab including 4 cohorts. The primary objectives are safety, tolerability and to define a recommended phase 2 dose. Safety, PK and receptor occupancy data were considered during dose escalation. VISTA-101 is also assessing PD and predictive biomarkers of response. Patient tumor response is evaluated by iRECIST. RESULTS: Forty-two patients have been enrolled in the dose escalation monotherapy arm of the study. KVA12123 was administered at doses ranging from 3 to 1000 mg every 2 weeks over a 6-week cycle. KVA12123 was well tolerated at all dose levels and no DLTs were observed. The most frequently observed adverse events were grade 1 or 2 transient infusion-related reactions. No evidence of CRS-associated cytokines were detected. PK analysis demonstrated greater than dose-proportional exposure consistent with target-mediated metabolism. A VISTA receptor occupancy assay demonstrated full target engagement at doses of 30 mg and above. PD analysis indicated a dose-dependent induction of pro-inflammatory myeloid derived cytokines and chemokines involved in immune cell activation and recruitment to the tumor microenvironment including CCL2, CCL3, CCL4 and CXCL10. Increases in nonclassical monocytes, NK cells, CD4+ and CD8+ T cells were also demonstrated, consistent with observations made in preclinical models and indicative of VISTA target engagement. Patient tumor response evaluations in the monotherapy arm will be reported. The study continues to evaluate KVA12123 both as a monotherapy and in combination with pembrolizumab to define the recommended phase 2 dose. Citation Format: Evan Y. Yu, Jason Henry, Manish R. Patel, Paul Swiecicki, Ida Micaily, Benjamin Garmezy, Kalyan Banda, Vinny Hayreh, Kurt Lustig, Yulia Ovechkina, Shawn P. Iadonato, Thierry Guillaudeux, Lee Rosen. Interim results of the ongoing phase 1-2 clinical trial of KVA12123, an engineered IgG1 targeting VISTA, as monotherapy and in combination with pembrolizumab in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT068.

Functional Analysis of cag Pathogenicity Island Diversity in Helicobacter pylori Isolated from Multi-Ethnic Patients in Malaysia

cagPAI is an important pathogenic marker contributing to disease severity caused by H. pylori infection, encoding proteins for type four secretion systems (T4SS) and the pro-inflammatory cytokines (IL-8) induction in infected host. The heterogeneity of cagPAI genes results in different clinical outcomes in H. pylori infection. The aim of this study was to investigate the functional diversity of cagPAI genes in clinical H. pylori strains isolated from patients with gastroduodenal diseases. Among the isolates investigated, 6 out of 27 (22.2%) harbor intact cagPAI, 3 strains (11.1%) have complete absence of cagPAI, and 18 strains (66.7%) contain a partially deleted island. IL-8 concentration is observed significantly higher among cagPAI-positive isolates (P = 0.045). Of note, IL-8 secretion in the gastric cells infected with H. pylori strains isolated from Malay patients was significantly higher than the gastric cells infected with the strains from non-Malay patients (P = 0.046). The secretion of IL-8 appears in similarly high among intact and partially deleted cagPAI isolates. East Asian cagA isolates induce a notably higher level of IL-8 than that of Western cagA. Inflammatory activities are observed and IL-8 concentration in the milder stage was found in significantly higher quantities than in the severe stage (P = 0.027). The ‘hummingbird’ phenotype was observed to cause severe effects in infected cells with cagPAI-intact H. pylori. The genetic variability of cagPAI isolates from multi-ethnicity patients may have unique implications for disease outcomes with respect to proinflammatory secretion.

Transcriptional and phenotypical alterations associated with a gradual benzo[a]pyrene-induced transition of human bronchial epithelial cells into mesenchymal-like cells

The role of benzo[a]pyrene (BaP), a prominent genotoxic carcinogen and aryl hydrocarbon receptor (AhR) ligand, in tumor progression remains poorly characterized. We investigated the impact of BaP on the process of epithelial-mesenchymal transition (EMT) in normal human bronchial epithelial HBEC-12KT cells. Early morphological changes after 2-week exposure were accompanied with induction of SERPINB2, IL1, CDKN1A/p21 (linked with cell cycle delay) and chemokine CXCL5. After 8-week exposure, induction of cell migration and EMT-related pattern of markers/regulators led to induction of further pro-inflammatory cytokines or non-canonical Wnt pathway ligand WNT5A. This trend of up-regulation of pro-inflammatory genes and non-canonical Wnt pathway constituents was observed also in the BaP-transformed HBEC-12KT-B1 cells. In general, transcriptional effects of BaP differed from those of TGFβ1, a prototypical EMT inducer, or a model non-genotoxic AhR ligand, TCDD. Carcinogenic polycyclic aromatic hydrocarbons could thus induce a unique set of molecular changes linked with EMT and cancer progression.

Crosstalk between keratinocytes and neutrophils shapes skin immunity against S. aureus infection.

Staphylococcus aureus (S. aureus) infection of the skin leads to a rapid initial innate immune response with keratinocytes in the epidermis as the initial sensors. Polymorphonuclear neutrophils (PMNs) are the first innate immune cells to infiltrate infection sites where they provide an effective first-line of defense. Previous work of our group showed that in inflamed skin a crosstalk between PMNs and keratinocytes results in enhanced S. aureus skin colonization. In this work, we used an in vitro co-culture model to studied the crosstalk between primary human keratinocytes (PHKs) and PMNs in a sterile environment and upon S. aureus infection. We investigated the influence of PHKs on PMN activation by analyzing PMN lifespan, expression of degranulation markers and induction of proinflammatory cytokines. Furthermore, we analyzed the influence of PMNs on the inflammatory response of PHKs. Finally, we investigated the influence of the skin microbiome on PMN-mediated skin inflammation. We show that co-culture of PMNs with PHKs induces activation and degranulation of PMNs and significantly enhances their lifespan compared to PMN cultivation alone by an IL-8 mediated mechanism and, furthermore, primes PMNs for enhanced activity after S. aureus infection. The prolonged incubation with PMNs also induces inflammatory responses in PHKs which are further exacerbated in the presence of S. aureus and induces further PMN recruitment thus fueling skin inflammation. Interestingly, infection of PHKs with the skin commensal S. epidermidis reduces the inflammatory effects of PMNs in the skin and exhibits an anti-inflammatory effect. Our data indicate that skin infiltrating PMNs and PHKs influence each other in such a way to enhance skin inflammation and that commensal bacteria are able to reduce the inflammatory effect.

Calcitriol promotes M2 polarization of tumor-associated macrophages in 4T1 mouse mammary gland cancer via the induction of proinflammatory cytokines

Our research found that vitamin D3 (VD3) treatment increased lung metastasis in mice with 4T1 murine breast cancer (BC). This study aims to investigate the impact of VD3 on the activation of tumor-associated macrophages (TAMs) in BC. Mice bearing 4T1, E0771, 67NR BC cells, and healthy mice, were fed diets with varying VD3 contents (100—deficient, 1000—normal, and 5000 IU/kg—elevated). Some mice in the 1000 and 100 IU/kg groups received calcitriol. We studied bone metastasis and characterized TAMs and bone marrow-derived macrophages (BMDMs). 4T1 cells had higher bone metastasis potential in the 5000 IU/kg and calcitriol groups. In the same mice, an elevated tumor osteopontin level and M2 polarization of TAMs (MHCIIlow CD44high phenotype) were observed. Gene expression analysis confirmed M2 polarization of 4T1 (but not 67NR) TAMs and BMDMs, particularly in the 100 IU + cal group (increased Mrc1, Il23, and Il6). This polarization was likely due to COX-2/PGE2 induction in 4T1 calcitriol-treated cells, leading to increased proinflammatory cytokines like IL-6 and IL-23. Future studies will explore COX-2/PGE2 as a primary mediator of calcitriol-stimulated inflammation in the BC microenvironment, especially relevant for BC patients with VD3 deficiency and supplementation.

Comparative analysis of the interactions of different Streptococcus suis strains with monocytes, granulocytes and the complement system in porcine blood

Streptococcus suis (S. suis) is an important porcine pathogen causing meningitis, arthritis, and septicemia. Serotypes 2 and 14 are the most common zoonotic ones worldwide, whereas serotypes 2, 9, and 7 are very important in pigs in Europe. To cause invasive infections S. suis needs to enter the bloodstream. Consequently, the immune response in blood represents an important line of defense and bacteremia plays a key role in the pathogenesis of invasive S. suis infections. We investigated the working hypothesis that S. suis strains of the same serotype but different clonal complex (CC) might exhibit substantial differences in the interaction with components of the immune system in porcine blood. The experimental design of this study includes comparative analysis of 8 virulent strains belonging to 4 serotypes with strains of the same serotype being genetically not closely related. Significant differences between two strains of the same serotype but different clonal complex were recorded in the flow cytometric analysis of association with different leukocytes for serotype 9 and 14. Our results demonstrate that the serotype 9 strain of CC94 shows significantly increased association with monocytes and survival in porcine blood of conventional piglets as well as a tendency towards decreased composition of C3 in plasma of these piglets in comparison to the serotype 9 strain of CC16. Correlation analysis of C3 deposition on the bacterial surface and survival in respective blood samples of 8-week-old piglets demonstrated a negative correlation indicating that C3 deposition is a crucial step to limit bacterial survival and proliferation of different S. suis pathotypes in the blood of these piglets. In summary, our results indicate that the capsule composition of a S. suis strain is not alone sufficient to determine association with leukocytes, activation of complement, induction of proinflammatory cytokines, oxidative burst, and bacterial survival in porcine blood. In this study, substantial differences in these host–pathogen interactions were observed between strains of the same serotype. Therefore, a more comprehensive characterization of the field isolates, including at least MLST analysis to determine the sequence type/clonal complex, is recommended.

JAK2-V617F Promotes up-Regulation of Pro-Inflammatory Cytokines Including IL-1 upon Adhesion-Induced Activation of β1/β2 Integrins

Introduction In JAK2-V617F (JAK2-VF) mutated cells, over-activation of β1/β2 integrin inside-out signaling has been described (Edelmann-Stephan et al., 2017, JCI). Integrins play a fundamental role in cell interaction and cell invasion during inflammatory processes. In patients suffering from Myeloproliferative Neoplasms (MPNs), highly elevated pro-inflammatory cytokine levels including IL-1β play an important role in pathophysiology. Importantly, Rai et al. (2023, BioRχ) have shown that IL-1β promotes initiation of MPN. In this study, we investigated a potential link between integrin over-activation and elevated pro-inflammatory cytokines including IL-1α and IL-1β in JAK2-VF induced disease. Methods We employed (1) murine myeloid progenitor 32D cells co-transfected with EPO-R and JAK2-WT or JAK2-VF, (2) granulocytes and hematopoietic progenitor cell populations from total bone marrow of Vav1-Cre x JAK2 +/+ and Vav1-Cre x JAK2 VF/+ mice (described by Mullally et al. 2010), and (3) human granulocytes isolated from PB of JAK2-VF positive patients and healthy volunteers. Cells were allowed to adhere on immobilized VCAM-1/ICAM-1 or on BSA (control) and subsequently subjected to further analysis including (1) qPCR analysis and ELISA of a panel of inflammatory cytokines (IL-1β, IL-1α, TNF-α, IL-6, CXCL10), (2) Western-blotting monitoring integrin outside-in signaling pathways, (3) FLICA assays assessing adhesion-induced active caspase-1, (4) RNAseq of mouse and human granulocytes (3 JAK2-VF patients, 2 controls). Results In the 32D cell model, adhesion on mVCAM-1/mICAM-1 strongly induced mRNA expression of IL-1α, IL1-β, IL-6, TNF-α and CXCL10. Interestingly, cytokine RNA induction was significantly higher in 32D JAK2-VF cells as compared to the 32D JAK2-WT cells (Figure 1). By applying a panel of integrin signaling inhibitors we could show an essential involvement of pivotal signaling nodes such as PI3K, Syk, FAK and NFκB, respectively. Next, cytokine proteins were investigated by ELISA. Adhesion on mVCAM-1/mICAM-1 increased intracellular IL-1α and IL-1β cytokine levels. Again, this effect was strongly amplified by the JAK2-VF mutation (Figure 2). Importantly, both cytokines were found in their mature form, indicating intact protein processing. RNAseq of bone marrow (BM) derived mouse granulocytes (including immature pro-and pre-neutrophils) also showed significant upregulation of a number of pro-inflammatory genes including Nlrp3, Il1a, Il1b, Tnf, Cxcl2 and Cxcl10 upon adhesion on mVCAM-1/mICAM-1. However, no significant differences between WT and VF cells were observed. In contrast, RNAseq of VCAM-1/ICAM-1 stimulated human JAK2-VF positive PB granulocytes (which represent terminally differentiated cells) revealed no cytokine up-regulation. Based on this finding, we hypothesize that the adhesion-induced cytokine phenotype is restricted to more immature cell populations as observed in the 32D cell model. Therefore, we are currently investigating (by qPCR and intracellular IL-1 immunophenotyping) hematopoietic progenitor cell populations from Vav1-Cre x JAK2 +/+ and Vav1-Cre x JAK2 VF/+ mice. Since active IL-1β requires processing by the inflammasome, FLICA assays for active caspase-1 were performed using mouse hematopoietic progenitor cells. Importantly, CD41+ cells and megakaryocyte progenitors (MKP) showed an increased FLICA signal upon adhesion on mVCAM-1/mICAM-1. This indicates that caspase 1 is activated upon adhesion in distinct progenitor cell populations. Finally, to support our hypothesis that JAK2-VF augments induction of pro-inflammatory cytokines upon adhesion to VCAM-1/ICAM-1, Vav1-Cre x JAK2 VF/+ mice (n=8) were treated in vivo with anti-β1/β2-integrin antibodies for 1 week. As expected we found a strong reduction of IL-1α concentration in serum of antibody treated mice (528.7 ± 82.2 pg/ml) as compared to PBS-treated control mice (1302.3 ± 370.4 pg/ml, p = 0.0541) Conclusion Here, we show a functional link between elevated cytokine levels and integrin activation in JAK2-VF induced disease. Adhesion to VCAM-1/ICAM-1 promoted the expression of a number of pro-inflammatory cytokines which are known to play important roles in initiation, clinical symptoms and prognosis of MPNs. Further studies aimed at precisely defining the primary cell populations involved in the mouse MPN disease models as well as in humans are currently ongoing.

Middle East respiratory coronavirus (MERS-CoV) internalized by llama alveolar macrophages does not result in virus replication or induction of pro-inflammatory cytokines

Severe Middle East respiratory syndrome (MERS) is characterized by massive infiltration of immune cells in lungs. MERS-coronavirus (MERS-CoV) replicates in vitro in human macrophages, inducing high pro-inflammatory responses. In contrast, camelids, the main reservoir for MERS-CoV, are asymptomatic carriers. Although limited infiltration of leukocytes has been observed in the lower respiratory tract of camelids, their role during infection remains unknown. Here we studied whether llama alveolar macrophages (LAMs) are susceptible to MERS-CoV infection and can elicit pro-inflammatory responses. MERS-CoV did not replicate in LAMs; however, they effectively capture and degrade viral particles. Moreover, transcriptomic analyses showed that LAMs do not induce pro-inflammatory cytokines upon MERS-CoV sensing.

Anti-inflammatory effects of yeast-derived vacuoles on LPS-induced murine macrophage activation.

Saccharomyces cerevisiae is a single-celled fungal microorganism. S. cerevisiae-derived vacuoles are closely related to mammalian lysosomes, which play a role in the degradation of macromolecules by various hydrolytic enzymes. This study evaluated the anti-inflammatory efficacy of S. cerevisiae-vacuoles by inhibiting inflammatory mediators induced by lipopolysaccharide (LPS). The results showed that treatment with 5, 10, and 20 µg/mL of S. cerevisiae-derived vacuoles almost completely inhibited the LPS-induced expression of iNOS protein and mRNA. Moreover, vacuoles significantly reduced the mRNA expression of tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), and interleukin 6 (IL-6) in LPS-stimulated macrophages compared to the control cells. The immunofluorescence analysis confirmed that S. cerevisiae-derived vacuoles inhibited the translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in LPS-stimulated cells. Taken together, the treatment with S. cerevisiae-derived vacuoles alone activated macrophages, but LPS-activated macrophages modulated pro-inflammatory mediators by downregulating the NF-κB pathway. These results suggest that S. cerevisiae-derived vacuoles may have therapeutic potential in the treatment of inflammatory diseases. In conclusion, our study provides new insights into the immunomodulatory effects of S. cerevisiae-derived vacuoles and their potential as a novel anti-inflammatory agent. IMPORTANCE This study investigates the potential of using vacuoles derived from the yeast Saccharomyces cerevisiae as a new anti-inflammatory therapy. Inflammation is a natural response of the immune system to invading pathogens, but when it is dysregulated, it can lead to chronic diseases. The researchers found that treating macrophages with vacuoles significantly reduced the production of pro-inflammatory cytokines and iNOS, markers of inflammation when they were stimulated with lipopolysaccharide. The study also showed that vacuoles inhibited the NF-κB signaling pathway, which is involved in the induction of pro-inflammatory cytokines in macrophages. These findings suggest that S. cerevisiae-derived vacuoles may have potential as a new therapeutic agent for regulating the inflammatory response in various diseases. Further studies are needed to evaluate the efficacy and safety of vacuoles in vivo and to elucidate the underlying mechanisms of their anti-inflammatory effects.

SARS-CoV-2 replicates in the human testis with slow kinetics and has no major deleterious effects ex vivo.

SARS-CoV-2 is a new virus responsible for the Covid-19 pandemic. Although SARS-CoV-2 primarily affects the lungs, other organs are infected. Alterations of testosteronemia and spermatozoa motility in infected men have raised questions about testicular infection, along with high level in the testis of ACE2, the main receptor used by SARS-CoV-2 to enter host cells. Using an organotypic culture of human testis, we found that SARS-CoV-2 replicated with slow kinetics in the testis. The virus first targeted testosterone-producing Leydig cells and then germ-cell nursing Sertoli cells. After a peak followed by the upregulation of antiviral effectors, viral replication in the testis decreased and did not induce any major damage to the tissue. Altogether, our data show that SARS-CoV-2 replicates in the human testis to a limited extent and suggest that testicular damages in infected patients are more likely to result from systemic infection and inflammation than from viral replication in the testis.

FRI657 Case Series On GLP-1 Receptor Agonist Related Acute Pancreatitis

Abstract Disclosure: A.U. Kulkarni: None. V. Mehta: None. M. Renzu: None. K. Pereira: None. M.A. Rather: None. Introduction: Acute pancreatitis (AP) has now become one of the leading causes of GI related hospital admissions in the United States. With increasing use of GLP-1 Receptor Agonists (GLP-1 RA) in T2DM and obesity, they are now being identified as one such class of drugs which can cause AP. We present this case series to discuss these adverse effects. Case 1: A 50 yo AA male with a history of T2DM and hypertension presented with acute severe epigastric pain, nausea and vomiting for 5 days. Vitals were stable. On physical exam, tenderness in the RUQ was noted. Labs revealed lipase of 313 U/l (11-82 U/l), BG of 213 mg/dL, HbA1c 11.3, normal LFT and lipid panel. Blood EtOH was negative. He denied tobacco, alcohol, and illicit drug use. CT abdomen showed acute interstitial edematous pancreatitis without cholelithiasis or choledocholithiasis. Liver ultrasound showed hepatic steatosis. He noted recent initiation of Semaglutide 6 months ago which was stopped 3 weeks prior to this visit due to abdominal discomfort. Previous Dulaglutide therapy was also discontinued due to GI intolerance. Home medication for DM otherwise included metformin and basal insulin. Case 2: A 68 yo AA woman with a history of T2DM, hypothyroidism presented after a week of severe epigastric pain, radiating to her back, with nausea and vomiting. Labs showed lipase 593 U/l (11-82 U/l), BG of 427 mg/dl, HbA1c of 10. Social history was non-contributory. CT Abdomen showed AP and no gallstones or bile duct dilatation. LFTs and lipid panel were unremarkable. Chart review noted prior admission for AP a month ago. Her home medications were basal Insulin and Dulaglutide (4 yrs). Drug induced AP from Dulaglutide was suspected. Lack of clinical improvement after 3 days of management prompted RUQ ultrasound, which was prematurely terminated due to pain during the scan. Subsequent CCK-HIDA was negative for acute cholecystitis but noted low gallbladder EF. Thus dulaglutide was identified as a potential trigger along with gallbladder dysmotility as a possible etiology for her recurrent AP. Both patients were managed with IV fluids, NPO diet and morphine. They were instructed to stop GLP-1 RA and discharged with PCP follow-up. Discussion: GLP-1 RA are now being widely used due to low risk of hypoglycemia and weight loss. Clinical trials with GLP-1 RA have shown mild, asymptomatic increase in lipase, amylase levels as well as rare incidences of AP. This has been attributed to the stimulation of pancreatic beta cells and exocrine duct cells leading to acinar cell hypertrophy, proinflammatory cytokine induction, causing pancreatic injury. Though drug-induced AP is uncommon and is a diagnosis of exclusion, detailed history taking, medication review and investigating for all other causes is of utmost importance. A good understanding of the drugs associated with AP should alert the physician to identify these agents and promptly discontinue them to prevent further complications. Presentation: Friday, June 16, 2023

Ethanol Induces Neuroinflammation in a Chronic Plus Binge Mouse Model of Alcohol Use Disorder via TLR4 and MyD88-Dependent Signaling.

Ethanol induces neuroinflammation, which is believed to contribute to the pathogenesis of alcohol use disorder (AUD). Toll-like receptors (TLRs) are a group of pattern recognition receptors (PRRs) expressed on both immune cells, including microglia and astrocytes, and non-immune cells in the central nervous system (CNS). Studies have shown that alcohol activates TLR4 signaling, resulting in the induction of pro-inflammatory cytokines and chemokines in the CNS. However, the effect of alcohol on signaling pathways downstream of TLR4, such as MyD88 and TRIF (TICAM) signaling, has not been evaluated extensively. In the current study, we treated male wild-type, TLR4-, MyD88-, and TRIF-deficient mice using a chronic plus binge mouse model of AUD. Evaluation of mRNA expression by qRT-PCR revealed that ethanol increased IL-1β, TNF-α, CCL2, COX2, FosB, and JunB in the cerebellum in wild-type and TRIF-deficient mice, while ethanol generally did not increase the expression of these molecules in TLR4- and MyD88-deficient mice. Furthermore, IRF3, IRF7, and IFN-β1, which are associated with the TRIF-dependent signaling cascade, were largely unaffected by alcohol. Collectively, these results suggest that the TLR4 and downstream MyD88-dependent signaling pathways are essential in ethanol-induced neuroinflammation in this mouse model of AUD.

Ex vivo infection model for Francisella using human lung tissue.

Tularemia is mainly caused by Francisella tularensis (Ft) subsp. tularensis (Ftt) and Ft subsp. holarctica (Ftt) in humans and in more than 200 animal species including rabbits and hares. Human clinical manifestations depend on the route of infection and range from flu-like symptoms to severe pneumonia with a mortality rate up to 60% without treatment. So far, only 2D cell culture and animal models are used to study Francisella virulence, but the gained results are transferable to human infections only to a certain extent. In this study, we firstly established an ex vivo human lung tissue infection model using different Francisella strains: Ftt Life Vaccine Strain (LVS), Ftt LVS ΔiglC, Ftt human clinical isolate A-660 and a German environmental Francisella species strain W12-1067 (F-W12). Human lung tissue was used to determine the colony forming units and to detect infected cell types by using spectral immunofluorescence and electron microscopy. Chemokine and cytokine levels were measured in culture supernatants. Only LVS and A-660 were able to grow within the human lung explants, whereas LVS ΔiglC and F-W12 did not replicate. Using human lung tissue, we observed a greater increase of bacterial load per explant for patient isolate A-660 compared to LVS, whereas a similar replication of both strains was observed in cell culture models with human macrophages. Alveolar macrophages were mainly infected in human lung tissue, but Ftt was also sporadically detected within white blood cells. Although Ftt replicated within lung tissue, an overall low induction of pro-inflammatory cytokines and chemokines was observed. A-660-infected lung explants secreted slightly less of IL-1β, MCP-1, IP-10 and IL-6 compared to Ftt LVS-infected explants, suggesting a more repressed immune response for patient isolate A-660. When LVS and A-660 were used for simultaneous co-infections, only the ex vivo model reflected the less virulent phenotype of LVS, as it was outcompeted by A-660. We successfully implemented an ex vivo infection model using human lung tissue for Francisella. The model delivers considerable advantages and is able to discriminate virulent Francisella from less- or non-virulent strains and can be used to investigate the role of specific virulence factors.

Identification and elucidation of cross talk between SLAM Family Member 7 (SLAMF7) and Toll-like receptor (TLR) pathways in monocytes and macrophages

To further elucidate the expression, regulation and function of Signaling Lymphocytic Activation Molecule Family (SLAMF) protein members in human monocytes and macrophages. Un-differentiated monocytic THP-1 cell (u-THP-1) and differentiated THP-1 macrophage (d-THP-1) were used as culture models in the study. Responses of cells to the differentiation agents phorbol ester (25 ng/ml) and TLR (Toll-like receptor) ligands were assessed. RT-PCR and Western blot analysis were used to determine mRNA and protein level. Pro-inflammatory cytokine mRNA expression levels and phagocytosis were used as functional markers. Data analyzed using t-test, one-way or two-way ANOVA followed by post hoc test. SLAMFs were differentially expressed in THP-1 cells. Differentiation of u-THP-1 to d-THP-1 led to significantly higher SLAMF7 mRNA and protein levels than other SLAMF. In addition, TLR stimuli increased SLAMF7 mRNA expression but not protein expression. Importantly, SLAMF7 agonist antibody and TLR ligands synergistically increased the mRNA expression levels of IL-1β, IL-6 and TNF-α, but had no effect on phagocytosis. SLAMF7 knocked-down in d-THP-1 significantly lowered TLR-induced mRNA expressions of pro-inflammatory markers. SLAM family proteins are differentially regulated by differentiation and TLRs. SLAMF7 enhanced TLR-mediated induction of pro-inflammatory cytokines in monocytes and macrophages but not phagocytosis.

Aging exaggerates pulpal pain sensation by increasing the expression levels of nociceptive neuropeptides and inflammatory cytokines

BackgroundDental pain is a main clinical problem in the elderly population and its assessment and treatment make special challenges for health care services. However, the age-induced alteration in dental pain perception and the underlying molecular mechanism(s) has not yet been fully clarified. MethodsHere, the effect of aging on nociceptive behaviors following inflammatory dental pulp pain was evaluated. Since prostaglandins, nociceptive neuropeptides, and inflammatory cytokines have critical roles in the development of aging as well as pain signaling, the expression levels of COX-2, CGRP, IL-1β, IL-6, TNF-α and its converting enzyme TACE were assessed in the trigeminal ganglion of young and aged rats with dental pain. Dental pulp pain was induced by intradental application of capsaicin (100 μg). The immunofluorescence (COX-2 and CGRP) and western blot techniques were used. ResultsThe data showed that aged animals have different pattern of pain. So that, the mean of nociceptive scores was significantly greater in aged rats at 10 and 15 min after capsaicin injection. In aged rats, dental pain was persisting over 7 h, while it was disappeared at 300 min in young rats. Molecular data showed that dental pain significantly increased the expression of COX-2, CGRP, IL-1β, IL-6, TNF-α and TACE in the trigeminal ganglion of the young and aged rats. In addition, the amount of those parameters, except TACE, in capsaicin-treated aged animals were significantly (p < 0.05) greater than those in capsaicin-treated young rats. ConclusionIt seems that the induction of pro-inflammatory cytokines in an acute inflammatory pulpal pain model may contribute, at least in part to the increased nociceptive behaviors and pain perception in aged rats.

In-Silico CLEC5A mRNA expression analysis to predict Dengue susceptibility in cancer patients

Dengue fever is the fastest-growing infectious disease in the world. It is the leading vector-borne viral neglected tropical disease. The most acute immune response to dengue virus infection is dengue shock syndrome and hemorrhagic fever, which is due to the activation of CLEC5A C-type lectin domain family 5, member A (CLEC5A). It is a cell surface receptor, and its well-known ligand is the dengue virus. It gets activated by the attachment of dengue virion, which, as a result, phosphorylates its adaptor protein DAP12 leading to the induction of various pro-inflammatory cytokines. Clinical data suggested that the kidneys and lungs are among the major hit organs in the case of severe dengue infection. Here we predict kidney and lung cancer patients are vulnerable to dengue virus infection as CLEC5A mRNA expression in tumor samples using publicly available software such as TIMER and GEPIA database. We also identified the immunomodulatory role CLEC5A gene therefore targeting it could be a vital tool to cure dengue.

Effects of Moringa oleifera Seed Oil on Cultured Human Sebocytes In Vitro and Comparison with Other Oil Types.

The seeds of Moringa oleifera (horseradish tree) contain about 40% of one of the most stable vegetable oils (Moringa seed oil). Therefore, the effects of Moringa seed oil on human SZ95 sebocytes were investigated and were compared with other vegetable oils. Immortalized human SZ95 sebocytes were treated with Moringa seed oil, olive oil, sunflower oil, linoleic acid and oleic acid. Lipid droplets were visualized by Nile Red fluorescence, cytokine secretion via cytokine antibody array, cell viability with calcein-AM fluorescence, cell proliferation by real-time cell analysis, and fatty acids were determined by gas chromatography. Statistical analysis was performed by the Wilcoxon matched-pairs signed-rank test, the Kruskal-Wallis test and Dunn's multiple comparison test. The vegetable oils tested stimulated sebaceous lipogenesis in a concentration-dependent manner. The pattern of lipogenesis induced by Moringa seed oil and olive oil was comparable to lipogenesis stimulated by oleic acid with also similar fatty acid secretion and cell proliferation patterns. Sunflower oil induced the strongest lipogenesis among the tested oils and fatty acids. There were also differences in cytokine secretion, induced by treatment with different oils. Moringa seed oil and olive oil, but not sunflower oil, reduced the secretion of pro-inflammatory cytokines, in comparison to untreated cells, and exhibited a low n-6/n-3 index. The anti-inflammatory oleic acid detected in Moringa seed oil probably contributed to its low levels of pro-inflammatory cytokine secretion and induction of cell death. In conclusion, Moringa seed oil seems to concentrate several desired oil properties on sebocytes, such as high content level of the anti-inflammatory fatty acid oleic acid, induction of similar cell proliferation and lipogenesis patterns compared with oleic acid, lipogenesis with a low n-6/n-3 index and inhibition of secretion of pro-inflammatory cytokines. These properties characterize Moringa seed oil as an interesting nutrient and a promising ingredient in skin care products.