Monocyte NLRP3 inflammasome and interleukin-1ß activation modulated by alpha-1 antitrypsin therapy in deficient individuals.

INTRODUCTION: Altered complement component 3 (C3) activation in patients with alpha-1 antitrypsin (AAT) deficiency (AATD) has been reported. To understand the potential impact on course of inflammation, the aim of this study was to investigate whether C3d, a cleavage-product of C3, triggers interleukin (IL)-1ß secretion via activation of NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome. The objective was to explore the effect of AAT augmentation therapy in patients with AATD on the C3d/complement receptor 3 (CR3) signalling axis of monocytes and on circulating pro-inflammatory markers. METHODS: Inflammatory mediators were detected in blood from patients with AATD (n=28) and patients with AATD receiving augmentation therapy (n=19). Inflammasome activation and IL-1ß secretion were measured in monocytes of patients with AATD, and following C3d stimulation in the presence or absence of CR3 or NLRP3 inhibitors. RESULTS: C3d acting via CR3 induces NLRP3 and pro-IL-1ß production, and through induction of endoplasmic reticulum (ER) stress and calcium flux, triggers caspase-1 activation and IL-1ß secretion. Treatment of individuals with AATD with AAT therapy results in decreased plasma levels of C3d (3.0±1.2 µg/mL vs 1.3±0.5 µg/mL respectively, p<0.0001) and IL-1ß (115.4±30 pg/mL vs 73.3±20 pg/mL, respectively, p<0.0001), with a 2.0-fold decrease in monocyte NLRP3 protein expression (p=0.0303), despite continued ER stress activation. DISCUSSION: These results provide strong insight into the mechanism of complement-driven inflammation associated with AATD. Although the described variance in C3d and NLRP3 activation decreased post AAT augmentation therapy, results demonstrate persistent C3d and monocyte ER stress, with implications for new therapeutics and clinical practice.

Cigarette smoke exposed airway epithelial cell-derived EVs promote pro-inflammatory macrophage activation in alpha-1 antitrypsin deficiency.

BACKGROUND: Alpha-1 antitrypsin deficiency (AATD) is a genetic disorder most commonly secondary to a single mutation in the SERPINA1 gene (PI*Z) that causes misfolding and accumulation of alpha-1 antitrypsin (AAT) in hepatocytes and mononuclear phagocytes which reduces plasma AAT and creates a toxic gain of function. This toxic gain of function promotes a pro-inflammatory phenotype in macrophages that contributes to lung inflammation and early-onset COPD, especially in individuals who smoke cigarettes. The aim of this study is to determine the role of cigarette exposed AATD macrophages and bronchial epithelial cells in AATD-mediated lung inflammation. METHODS: Peripheral blood mononuclear cells from AATD and healthy individuals were differentiated into alveolar-like macrophages and exposed to air or cigarette smoke while in culture. Macrophage endoplasmic reticulum stress was quantified and secreted cytokines were measured using qPCR and cytokine ELISAs. To determine whether there is "cross talk" between epithelial cells and macrophages, macrophages were exposed to extracellular vesicles released by airway epithelial cells exposed to cigarette smoke and their inflammatory response was determined. RESULTS: AATD macrophages spontaneously produce several-fold more pro-inflammatory cytokines as compared to normal macrophages. AATD macrophages have an enhanced inflammatory response when exposed to cigarette smoke-induced extracellular vesicles (EVs) released from airway epithelial cells. Cigarette smoke-induced EVs induce expression of GM-CSF and IL-8 in AATD macrophages but have no effect on normal macrophages. Release of AAT polymers, potent neutrophil chemo attractants, were also increased from AATD macrophages after exposure to cigarette smoke-induced EVs. CONCLUSIONS: The expression of mutated AAT confers an inflammatory phenotype in AATD macrophages which disposes them to an exaggerated inflammatory response to cigarette smoke-induced EVs, and thus could contribute to progressive lung inflammation and damage in AATD individuals.

Sirtuin3 promotes the degradation of hepatic Z alpha-1 antitrypsin through lipophagy.

BACKGROUND: Alpha-1 antitrypsin deficiency (AATD) is a genetic disease caused by misfolding and accumulation of mutant alpha-1 antitrypsin (ZAAT) in the endoplasmic reticulum of hepatocytes. Hepatic ZAAT aggregates acquire a toxic gain-of-function that impacts the endoplasmic reticulum which is theorized to cause liver disease in individuals with AATD who present asymptomatic until late-stage cirrhosis. Currently, there is no treatment for AATD-mediated liver disease except liver transplantation. In our study of mitochondrial RNA, we identified that Sirtuin3 (SIRT3) plays a role in the hepatic phenotype of AATD. METHODS: Utilizing RNA and protein analysis in an in vitro AATD model, we investigated the role of SIRT3 in the pathophysiology of AATD-mediated liver disease while also characterizing our novel, transgenic AATD mouse model. RESULTS: We show lower expression of SIRT3 in ZAAT-expressing hepatocytes. In contrast, the overexpression of SIRT3 increases hepatic ZAAT degradation. ZAAT degradation mediated by SIRT3 appeared independent of proteasomal degradation and regular autophagy pathways. We observed that ZAAT-expressing hepatocytes have aberrant accumulation of lipid droplets, with ZAAT polymers localizing on the lipid droplet surface in a direct interaction with Perilipin2, which coats intracellular lipid droplets. SIRT3 overexpression also induced the degradation of lipid droplets in ZAAT-expressing hepatocytes. We observed that SIRT3 overexpression induces lipophagy by enhancing the interaction of Perilipin2 with HSC70. ZAAT polymers then degrade as a consequence of the mobilization of lipids through this process. CONCLUSIONS: In this context, SIRT3 activation may eliminate the hepatic toxic gain-of-function associated with the polymerization of ZAAT, providing a rationale for a potential novel therapeutic approach to the treatment of AATD-mediated liver disease.

Alpha 1 Antitrypsin-Deficient Macrophages Have Impaired Efferocytosis of Apoptotic Neutrophils.

Alpha 1 antitrypsin deficiency (AATD) is an autosomal co-dominant disorder characterized by a low level of circulating AAT, which significantly reduces protection for the lower airways against proteolytic burden caused by neutrophils. Neutrophils, which are terminally differentiated innate immune cells and play a critical role to clear pathogens, accumulate excessively in the lung of AATD individuals. The neutrophil burden in AATD individuals increases the risk for early-onset destructive lung diseases by producing neutrophil products such as reactive oxygen radicals and various proteases. The level of AAT in AATD individuals is not sufficient to inhibit the activity of neutrophil chemotactic factors such as CXCL-8 and LTB4, which could lead to alveolar neutrophil accumulation in AATD individuals. However, as neutrophils have a short lifespan, and apoptotic neutrophils are rapidly cleared by alveolar macrophages that outnumber the apoptotic neutrophils in the pulmonary alveolus, the increased chemotaxis activity does not fully explain the persistent neutrophil accumulation and the resulting chronic inflammation in AATD individuals. Here, we propose that the ability of alveolar macrophages to clear apoptotic neutrophils is impaired in AATD individuals and it could be the main driver to cause neutrophil accumulation in their lung. This study demonstrates that Z-AAT variant significantly increases the expression of pro-inflammatory cytokines including CXCL-8, CXCL1, LTB4, and TNFα in LPS-treated macrophages. These cytokines play a central role in neutrophil recruitment to the lung and in clearance of apoptotic neutrophils by macrophages. Our result shows that LPS treatment significantly reduces the efferocytosis ability of macrophages with the Z-AAT allele by inducing TNFα expression. We incubated monocyte-derived macrophages (MDMs) with apoptotic neutrophils and found that after 3 h of co-incubation, the expression level of CXCL-8 is reduced in M-MDMs but increased in Z-MDMs. This result shows that the expression of inflammatory cytokines could be increased by impaired efferocytosis. It indicates that the efferocytosis ability of macrophages plays an important role in regulating cytokine expression and resolving inflammation. Findings from this study would help us better understand the multifaceted effect of AAT on regulating neutrophil balance in the lung and the underlying mechanisms.

Augmentation of posterior wall acetabular fracture fixation using calcium-phosphate cement: a biomechanical analysis.

OBJECTIVE: This study investigates if the use of calcium phosphate cement as an adjunct to internal fixation for posterior wall acetabular fracture will result in acute restoration of joint loading parameters to the intact condition. METHODS: Ten fresh-frozen cadaveric pelves were used for this investigation. Simulated abductor mechanism was used to load the hip. Pressure-sensitive film was used to measure contact area and pressure within the anterior, superior, and posterior regions of the acetabulum for all experimental conditions. The hips were loaded under the following 4 conditions: 1) intact; 2) following posterior wall osteotomy; 3) following reduction and standard internal fixation; and 4) following reduction of the posterior wall using calcium phosphate cement, as a grout, in addition to internal fixation. A posterior wall fracture was created along an arc of 40-90 degrees about the acetabular rim. Extensometers were utilized to measure posterior wall fragment micromotion under conditions 3 and 4 above. Statistical analysis was performed using multivariate analysis of variance to assess the significance of the difference among and between conditions simultaneously for each region. Fragment motion data were analyzed using a 2-tailed t test. RESULTS: Fragment micromotion was reduced to 78 microm superiorly and 46 microm inferiorly with the use of calcium phosphate cement (P < 0.05). Creation of a posterior wall defect resulted in increased load in the superior acetabulum (1201N) as compared to the intact condition (902N, P = 0.024). Following reduction and internal fixation, the load distributed to the superior acetabulum (1132N) was not statistically different from the displaced condition. Following the addition of calcium phosphate cement, the load seen at the superior region of the acetabulum (883N) was less than fixation without calcium phosphate cement and was not different from the intact state (P = 0.85). CONCLUSION: The use of calcium-phosphate cement as a fracture grout with internal fixation resulted in a partial restoration of joint loading parameters toward the intact state. Further work will be needed to determine if similar types of augmented articular fixation may result in a clinical benefit.

Early motion after quadriceps and patellar tendon repairs: outcomes with single-suture augmentation.

BACKGROUND: Complications of immobilization after quadriceps and patellar tendon repairs include decreased patellar mobility, limited flexion, persistent pain, muscle weakness, and patella baja. In contrast, early motion limits muscle atrophy, accelerates tendon healing, and prevents joint stiffness. HYPOTHESIS: Quadriceps and patellar tendon repairs protected with a "relaxing suture" are strong enough to safely permit early motion, full weightbearing, and brace-free ambulation. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Twenty quadriceps and 30 patellar tendon ruptures were treated with a primary repair augmented with a single No. 5 Ethibond suture, a postoperative regimen of controlled motion and full weightbearing at 7 to 10 days, and brace-free ambulation at 6 weeks after surgery. At a minimum follow-up of 12 months, results of surgery were assessed with the Lysholm knee rating system. RESULTS: Six weeks after surgery, 120 degrees of flexion and brace-free ambulation were the goals and were achieved at a mean of 7.2 and 7.7 weeks, respectively. By 6 months, all patients reached their preinjury levels of activity (eg, basketball, softball, Rocky Mountain tour guide), 40 had full active extension, and 10 lacked 3 degrees to 10 degrees of active extension. There were no postoperative complications. At a mean follow-up of 4 years (range, 1-12 years), the Lysholm scores averaged 92 points (range, 84-100 points), and there were 35 excellent, 15 good, and no fair or poor results. CONCLUSION: Quadriceps and patellar tendon repairs protected by a relaxing suture were strong enough to safely permit early motion, weightbearing, and brace-free ambulation while producing good and excellent results.