Melissa A. Hausburg

Systemic inflammatory response syndrome, driven by trauma or infection, involves excessive innate immune activation. Dexamethasone (DEX), a synthetic glucocorticoid, suppresses inflammation, although adverse side effects limit its clinical use. The inflammasome, a multiprotein complex that assembles in response to inflammatory stimuli like ATP after lipopolysaccharide (LPS) priming, facilitates caspase-1 cleavage and release of the proinflammatory cytokine, interleukin (IL)-1β. We investigated whether DEX and decanoic acid (DA), a saturated medium-chain fatty acid, inhibited caspase-1 activity and IL-1β release in an LPS/ATP-stimulated THP-1 macrophage model and whether co-treatment would allow for pharmacologic DEX dose-sparing in vitro. PMA-differentiated THP-1macrophages were primed with LPS overnight and stimulated with ATP for 3 h in the presence of DA, DEX, or both. Viability and cytotoxicity were assessed. Caspase-1 activity and IL-1β were quantified, and EC ₅₀ values were calculated by nonlinear regression. In control conditions, LPS/ATP stimulation induced 5.56 (q1: 3.51 – q3: 6.38) ng/mL of IL-1β release. DA and DEX inhibited IL-1β with full dose-response EC ₅₀ values of 131.7 μM and 24.29 nM, respectively. DA concentrations exceeding 800 μM decreased cell viability. An 8 x 9-point DA/DEX dose matrix (0–500 μM DA; 0 to 1e4 nM DEX) was used to interrogate drug interaction for IL-1β inhibition and caspase-1 activity. DEX EC ₅₀ values remained within a narrow 95% confidence interval of 17.5–26.6 nM, regardless of DA background concentration. DA reduced caspase-1 enzymatic activity with a calculated EC ₅₀ value of ~140 μM; whereas, DEX did not. These data show that DA and DEX act independently. Furthermore, compared to DEX alone, the presence of DA allowed for equivalent in vitro IL-1β suppression at lower DEX concentrations, supporting pharmacologic DEX dose-sparing. Determination of whether this pharmacologic effect translates to clinically meaningful corticosteroid dose reduction requires further study.

Background/Objectives: This study examined whether dexamethasone (Dex) and decanoic acid (DA) exert complementary effects on inflammatory signaling, catabolic gene expression, and peroxisome proliferator-activated receptor gamma (PPARγ) activation in human chondrocytes. Methods: Two donor-specific primary human chondrocyte models, each derived from a single knee donor, were used, in which cells were treated with Dex (≤0.1 µM), DA (≤500 µM), or combinations thereof. Acute Interleukin-1β(IL-1β)-induced PGE₂ release was evaluated in normal donor cells, whereas prolonged transcriptional responses and PPARγ DNA-binding activity were evaluated in OA donor cells. IL-1β-induced prostaglandin E₂ (PGE₂) release was measured by competitive ELISA after 24 h. Glyceraldehyde-3-phosphate dehydrogenase-normalized transcription of Collagen type II alpha 1, Collagen type I alpha 1, Aggrecan, SRY-Box Transcription Factor 9, Runt-Related Transcription Factor 2, and Matrix Metalloproteinase 13 (MMP13) was assessed by qRT-PCR at 7, 10–14, and 20–28 days. In addition, PPARγ DNA-binding activity was measured after 1 week. Interactions were evaluated by Loewe additivity and highest single agent (HSA) analyses. Results: Dex and DA each reduced PGE₂ release, whereas co-treatment increased potency and maximal inhibition, with a Loewe combination index of 0.4 at 1 nM Dex plus 19 µM DA. In the temporal analysis, Dex plus DA showed a positive MMP13 ΔCt difference versus Dex at 10–14 days (2.42 ± 2.14; p = 0.065) and a more consistent difference at 20–28 days (1.11 ± 0.25; p = 0.016). This agreed with fixed-dose HSA findings at 14 and 28 days (ΔHSA ≈ −75 for 0.1 µM Dex plus 100 µM DA versus Dex). Co-treatment was also associated with increased PPARγ activation beyond the HSA reference (ΔHSA ≈ 0.09 for 0.1 µM Dex plus 100–250 µM DA versus Dex). Conclusions: Dex plus DA co-treatment produced complementary effects on acute PGE₂ inhibition, as well as late-onset MMP13 suppression and PPARγ activity in the donor-specific chondrocyte models studied. These hypothesis-generating findings provide an in vitro rationale for future evaluation of this combination as a corticosteroid-sparing strategy.

Previous abdominal surgery (PAS) increases risk of small bowel obstruction (SBO) due to adhesions, and appendectomy (appy) is an independent risk factor for abdominal adhesion-related complications. Peritoneal inflammation, e.g., acute appendicitis (AA), causes formation of reactive ascitic fluid (rA) that activates peritoneum surface mesothelial cells (MCs) to form adhesions. Pathologic adhesions may arise if restoration of MC-regulated fibrinolysis and secretion of glycocalyx (GCX) are disrupted. Proteins affecting these processes may originate from peritoneal rA. This is a prospective observational IRB-approved study at three Level 1 trauma centers where rA is collected prior to surgical intervention for non-perforated AA or adhesiolysis for SBO. Samples from 48 appy and 15 SBO patients were used to treat human MCs and subjected to quantification of 85 inflammatory mediators. Results were compared between patients with surgically naïve abdomens (naïve) and patients with >1 PAS. Select rA caused MCs to form clusters of fibroblastic cells, extracellular matrix fibers (FIB), and secretion of GCX. PAS and naïve patient rA fluids were clustered into “fiber-GCX” (FIB-GCX) groups: highFIB-highGCX, highFIB-lowGCX, noFIB-highGCX, noFIB-lowGCX, and noFIB-noGCX. Between groups, 26 analytes were differentially abundant including innate immune response, wound healing, and mucosal defense proteins. Factors that contributed to the differences between groups were rA-induced highFIB and history of PAS. Overall, PAS patient rA showed a muted immune response compared to rA from naïve patients. Our data suggest that abdominal surgery may negatively impact future immune responses in the abdomen. Further, quantifying immunomodulators in peritoneal rA may lead to the development a personalized approach to post-surgical adhesion treatment and prevention.

Inflammatory responses to the novel coronavirus SARS-CoV-2, which causes COVID-19, range from asymptomatic to severe. Here we present a follow-up analysis of a longitudinal study characterizing COVID-19 immune responses from a father and son with distinctly different clinical courses. The father required a lengthy hospital stay for severe symptoms, whereas his son had mild symptoms and no fever yet tested positive for SARS-CoV-2 for 29 days. Father and son, as well as another unrelated COVID-19 patient, displayed a robust increase of SERPING1, the transcript encoding C1 esterase inhibitor (C1-INH). We further bolstered this finding by incorporating a serum proteomics dataset and found that serum C1-INH was consistently increased in COVID-19 patients. C1-INH is a central regulator of the contact and complement systems, potentially linking COVID-19 to complement hyperactivation, fibrin clot formation, and immune depression. Furthermore, despite distinct clinical cases, significant parallels were observed in transcripts involved interferon and B cell signaling. As symptoms were resolving, widespread decreases were seen in immune-related transcripts to levels below those of healthy controls. Our study provides insight into the immune responses of likely millions of people with extremely mild symptoms who may not be aware of their infection with SARS-CoV-2 and implies a potential for long-lasting consequences that could contribute to reinfection risk.