Extracellular Acidification Augments NLRP3-Mediated Inflammasome Signaling in Macrophages

Inflammation is a series of host defense processes in response to microbial infection and tissue injury. Inflammatory processes frequently cause extracellular acidification in the inflamed region through increased glycolysis and lactate secretion. Therefore, the immune cells infiltrating the inflamed region encounter an acidic microenvironment. Extracellular acidosis can modulate the innate immune response of macrophages; however, its role for inflammasome signaling still remains elusive. In the present study, we demonstrated that macrophages exposed to an acidic microenvironment exhibited enhanced caspase-1 processing and IL-1β secretion compared with those under physiological pH. Moreover, exposure to an acidic pH increased the ability of macrophages to assemble the NLR family pyrin domain containing 3 (NLRP3) inflammasome in response to an NLRP3 agonist.This acidosis-mediated augmentation of NLRP3 inflammasome activation occurred in bone marrow-derived macrophages but not in bone marrow-derived neutrophils. Notably, exposure to an acidic environment caused a reduction in the intracellular pH of macrophages but not neutrophils. Concordantly, macrophages, but not neutrophils, exhibited NLRP3 agonist-mediated translocation of chloride intracellular channel protein 1 (CLIC1) into their plasma membranes under an acidic microenvironment. Collectively, our results demonstrate that extracellular acidosis during inflammation can increase the sensitivity of NLRP3 inflammasome formation and activation in a CLIC1-dependent manner. Thus, CLIC1 may be a potential therapeutic target for NLRP3 inflammasome-mediated pathological conditions.

NLRP3 Exacerbate NETosis-Associated Neuroinflammation in an LPS-Induced Inflamed Brain

Neutrophil extracellular traps (NETs) exert a novel function of trapping pathogens. Released NETs can accumulate in inflamed tissues, be recognized by other immune cells for clearance, and lead to tissue toxicity. Therefore, the deleterious effect of NET is an etiological factor, causing several diseases directly or indirectly. NLR family pyrin domain containing 3 (NLRP3) in neutrophils is pivotal in signaling the innate immune response and is associated with several NET-related diseases. Despite these observations, the role of NLRP3 in NET formation in neuroinflammation remains elusive. Therefore, we aimed to explore NET formation promoted by NLRP3 in an LPS-induced inflamed brain. Wild-type and NLRP3 knockout mice were used to investigate the role of NLRP3 in NET formation. Brain inflammation was systemically induced by administering LPS. In such an environment, the NET formation was evaluated based on the expression of its characteristic indicators. DNA leakage and NET formation were analyzed in both mice through Western blot, flow cytometry, and in vitro live cell imaging as well as two-photon imaging. Our data revealed that NLRP3 promotes DNA leakage and facilitates NET formation accompanied by neutrophil death. Moreover, NLRP3 is not involved in neutrophil infiltration but is predisposed to boost NET formation, which is accompanied by neutrophil death in the LPS-induced inflamed brain. Furthermore, either NLRP3 deficiency or neutrophil depletion diminished pro-inflammatory cytokine, IL-1β, and alleviated blood-brain barrier damage. Overall, the results suggest that NLRP3 exacerbates NETosis in vitro and in the inflamed brain, aggravating neuroinflammation.These findings provide a clue that NLRP3 would be a potential therapeutic target to alleviate neuroinflammation.

Zika Virus Impairs Host NLRP3-mediated Inflammasome Activation in an NS3-dependent Manner

Zika virus (ZIKV) is a mosquito-borne flavivirus associated with severe neurological disorders including Guillain-Barré syndrome and microcephaly. The host innate immune responses against ZIKV infection are essential for protection; however, ZIKV has evolved strategies to evade and antagonize antiviral responses via its nonstructural (NS) proteins. Here, we demonstrated that ZIKV infection unexpectedly inhibits NLRP3-dependent inflammasome activation in bone marrow-derived macrophages and mixed glial cells from mouse brain. ZIKV infection led to increased transcript levels of proinflammatory cytokines such as IL-1β and IL-6 via activating NF-κB signaling. However, ZIKV infection failed to trigger the secretion of active caspase-1 and IL-1β from macrophages and glial cells even in the presence of LPS priming or ATP costimulation. Intriguingly, ZIKV infection significantly attenuated NLRP3-dependent, but not absent in melanoma 2-dependent caspase-1 activation and IL-1β secretion from both cells. ZIKV infection further blocked apoptosis-associated speck-like protein containing a caspase recruitment domain oligomerization in LPS/ATP-stimulated macrophages. Interestingly, expression of ZIKV NS3 protein reduced NLRP3-mediated caspase-1 activation and IL-1β secretion in macrophages, whereas NS1 and NS5 proteins showed no effects. Furthermore, NLRP3 was found to be degraded by the overexpression of ZIKV NS3 in 293T cells. Collectively, these results indicate that ZIKV evades host NLRP3 inflammasome-mediated innate immune responses in macrophages and glial cells; this may facilitate ZIKV's ability to enhance the replication and dissemination in these cells.

Cobalt Chloride-induced Hypoxia Ameliorates NLRP3-Mediated Caspase-1 Activation in Mixed Glial Cultures

Hypoxia has been shown to promote inflammation, including the release of proinflammatory cytokines, but it is poorly investigated how hypoxia directly affects inflammasome signaling pathways. To explore whether hypoxic stress modulates inflammasome activity, we examined the effect of cobalt chloride (CoCl2)-induced hypoxia on caspase-1 activation in primary mixed glial cultures of the neonatal mouse brain. Unexpectedly, hypoxia induced by oxygen-glucose deprivation or CoCl2 treatment failed to activate caspase-1 in microglial BV-2 cells and primary mixed glial cultures. Of particular interest, CoCl2-induced hypoxic condition considerably inhibited NLRP3-dependent caspase-1 activation in mixed glial cells, but not in bone marrow-derived macrophages. CoCl2-mediated inhibition of NLRP3 inflammasome activity was also observed in the isolated brain microglial cells, but CoCl2 did not affect poly dA:dT-triggered AIM2 inflammasome activity in mixed glial cells. Our results collectively demonstrate that CoCl2-induced hypoxia may negatively regulate NLRP3 inflammasome signaling in brain glial cells, but its physiological significance remains to be determined.

Salmonella Promotes ASC Oligomerization-dependent Caspase-1 Activation

Innate immune cells sense and respond to the cytoplasmic infection of bacterial pathogens through NLRP3, NLRC4 or AIM2 inflammasome depending on the unique molecular pattern of invading pathogens. The infection of flagellin- or type III secretion system (T3SS)-containing Gram-negative bacteria such as Salmonella enterica serovar Typhimurium (S. typhimurium) or Pseudomonas aeruginosa (P. aeruginosa) triggers NLRC4-dependent caspase-1 activation leading to the secretion of proinflammatory cytokines such as interleukin-1-beta (IL-1beta) and IL-18. Previous studies have shown that apoptosis-associated speck-like protein containing a CARD (ASC) is also required for Salmonella-induced caspase-1 activation, but it is still unclear how ASC contributes to the activation of NLRC4 inflammasome in response to S. typhimurium infection. In this study, we demonstrate that S. typhimurium triggers the formation of ASC oligomer in a potassium depletion-independent manner as determined by in vitro crosslinking and in situ fluorescence imaging. Remarkably, inhibition of potassium efflux failed to block Salmonella-promoted caspase-1 activation and macrophage cell death. These results collectively suggest that ASC is substantially oligomerized to facilitate the activation of caspase-1 in response to S. typhimurium infection. Contrary to NLRP3 inflammasome, intracellular potassium depletion is not critical for NLRC4 inflammasome signaling by S. typhimurium.

Pyrin Domain (PYD)-containing Inflammasome in Innate Immunity

Inflammasome is a cytosolic multiprotein complex to activate caspase-1 leading to the subsequent processing of inactive pro-interleukin-1-beta (Pro-IL-1beta) into its active interleukin-1 beta (IL-1beta) in response to pathogen- or danger-associated molecular pattern. In recent years, a huge progress has been made to identify inflammasome component as a molecular platform to recruit and activate caspase-1. Nucleotide-binding oligomerization domain-like receptor (NLR) family proteins such as NLRP1, NLRP3 or interleukin-1beta-converting enzyme (ICE)-protease activating factor (IPAF) have been first characterized to form inflammasome complex to induce caspase-1 activation. More recently, non-NLR type, pyrin-domain (PYD)-containing proteins such as pyrin or absent in melanoma2 (AIM2) were also proposed to form caspase-1-activating inflammasome machinery with apoptosis-associated speck-like protein containing a CARD (ASC), an essential adaptor molecule. Inflammasome pathways were shown to be crucial for protecting host organisms against diverse pathogen infections, but accumulating evidences also suggest that excessive activation of inflammasome/caspase-1 might be related to the pathogenesis of inflammation-related diseases. Indeed, mutations in NLRP3 or pyrin are closely associated with autoinflammatory diseases such as familial Mediterranean fever (FMF) syndrome or Muckle-Wells syndrome (MWS), indicating that the regulation of caspase-1 activity by inflammasome is a central process in these hereditary inflammatory disorders. Here, recent advances on the molecular mechanism of caspase-1 activation by PYD-containing inflammasomes are summarized and discussed.

Delayed Post-traumatic Vertebral Collapse: MR Categorization and MR-Pathology Correlation

STUDY DESIGN: A retrospective study. PURPOSE: To categorize the MR appearance of ischemic vertebral collapse and to correlate surgical and histologic findings. OVERVIEW OF LITERATURE: X-ray and MRI findings of delayed posttraumatic vertebral collapse shows several patterns. Histopathologic signs of osteonecrosis were present only in minor portion of cases sampled for biopsy of delayed post-traumatic vertebral collapse in the literature. METHODS: Twenty-one patients (22 vertebral bodies), with surgically and histopathologically proven ischemic vertebral collapse were included. The patients were examined with a 1.5 T MR imager. Spin echo T1- and T2-weighted images were obtained in axial and sagittal planes. Two experienced musculoskeletal radiologists, who reached consensus, evaluated the MR images. Then, MR-pathology correlations were made. RESULTS: Four different MR patterns were identified. Fluid patterns, were seen in 14% (3/22) of the affected vertebral bodies, and were characterized by hypo-intense signals on T1-weighted images, and hyper-intense signals, similar to water, on T2-weighted images. Extensive bone necrosis was predominant. Compression pattern, the most common pattern, found in 41% (9/22 vertebral bodies), was characterized by a marked decrease of anterior column height. Bone necrosis, granulation tissue, marrow fibrosis, and reactive new bone formation were found in relatively equal proportion. Granulation pattern, seen in 27% (6/22 vertebral bodies), was characterized by hypo-intense signals on T1-weighted images, and intermediate signals on T2-weighted images. Extensive granulation tissue was predominant. Mixed patterns were present in 18% (4/22), of the vertebral bodies. CONCLUSIONS: Awareness of histopathologic correlation of MR patterns in patients with delayed post-traumatic vertebral collapse may facilitate effective interpretation of clinical MR images of the spine.

Gout of the Hallucal Medial Sesamoid: A Case Report / 대한족부족관절학회지

Gout in the sesamoid of the great toe is very rare, such that to our best knowledge, there have been only four reports internationally. We present a case of hallucal medial sesamoid gout in the respect of the literature review, clinical, pathological features and surgical outcome.

Surgical Treatment of Displaced Intra-articular Calcaneal Fractures: Minimum of 2-year Follow-up

PURPOSE: To evaluate the overall clinical features and postoperative functional results of the intra-articular calcaneal fractures at more than 2 years follow-up, and also to compare the results at postoperative 1 year with the results at more than 2-year follow-up. MATERIALS AND METHODS: The study is based on 39 intra-articular calcaneal fractures (34 patients) that underwent surgical treatment from March 1997 to May 2002 with at least 2 years follow-up. The overall postoperative results were evaluated with Creighton-Nebraska functional scale. The comparison of results at postoperative 1 year was also performed with results at more than 2-year follow-up. RESULTS: By Sanders classifications, there were 13 type II fractures (33.3%), 20 type III (51.3%), and 6 type IV fractures (15.4%). Average follow-up period was 35 months (range: 24~87 months) and at final follow-up of more than 2 years, Creighton-Nebraska score was average 76.0 (range: 30~100) which significantly improved from postoperative 1-year results of 67.1 (range: 22~95) (p<0.05). CONCLUSION: The clinical outcome at more than 2 years after surgical treatment of intra-articular calcaneal fractures was quite promising, which significantly improved compared to 1-year results. Therefore, we concluded that functional results of calcaneal fractures should be evaluated at least 2 years after the treatment.

Tension Band Fixation for Type II Fracture of the Distal Clavicle

PURPOSE: To evaluate the efficacy of the tension band wire fixation for type II distal clavicle fractures. MATERIALS AND METHODS: Twenty one patients with type II distal clavicle fractures were evaluated, who were operated with tension band fixation technique with sparing AC joint, from May 2000 to December 2003, and could be followed-up for more than 1 year after operation. Average age at injury is 40.7 years old (14~73). 13 cases were males and 8 were females. And 16 cases were classified as type IIa and 5 cases as type IIb. Judgement of union was based on plain x-ray and clinical finding and postoperative assessment was evaluated on ASES and Constant scoring system. RESULTS: Outcomes in all patients showed more than good, average ASES score was 96.1 (88~98) and Constant score was 93.1 (82~100). Radiologic union was achieved at 11.7 (6~16) weeks postoperatively. One patient suffered from non union, and there was no other significant complications such as K-wire migrations, breakage, infection, and AC joint arthritis. CONCLUSION: Tension band fixation technique for type II distal clavicle fracture seems to be a useful and effective method, which is relatively simple and provides rigid fixation without violating the AC joint.

Subtalar Arthrodesis Using the Cannulated Compression Screw / 대한족부족관절학회지

PURPOSE: To analyze the overall clinical outcome, overall assessment, and patient's satisfaction rate of subtalar arthrodesis using the cannulated compression screw. MATERIALS AND METHODS: This study is based on 17 patients, 17 feet who underwent subtalar arthrodesis using the cannulated compression screw from March, 1997 to March, 2004 with at least 1 year follow-up. The average follow-up period was 33.0 months (12 to 72 months). Functional results were assessed using the American Orthopaedic Foot and Ankle Society Ankle-Hindfoot (AOFAS) score, and Visual Analysis Scale (VAS) pain score, patients' returning to previous occupation and patients' satisfaction rate were also evaluated. RESULTS: The mean AOFAS scores at final follow-up were 80.4 points (range 66~92). The satisfactory rates were as follow. Thirteen patients (76.4%) were at least satisfied with surgical result at final follow-up. Patients' VAS pain score was average 2.8 points (1~6). Fourteen (82.3%) patients returned to previous job at mean postoperative period of 11.3 months (range 3-18 months). Patients' work efficiency after returning to previous occupation was 68.7% (range 33~100%). There were 9 complications which were 3 cases of sural nerve injury, 1 case of valgus malunion, and 5 cases of the hindfoot residual pain. CONCLUSION: We obtained the satisfactory functional results with relatively high patient satisfaction rate of 76%. So we conclude that subtalar arthrodesis using the cannulated compression screw is a reliable method for addressing the painful end-stage subtalar osteoarthritis and unreconstructible comminuted calcaneal fractures. However we also found out that average 11 months were necessary for patients to return to their job.

Subtalar Arthrodesis Using the Cannulated Compression Screw / 대한족부족관절학회지

PURPOSE: To analyze the overall clinical outcome, overall assessment, and patient's satisfaction rate of subtalar arthrodesis using the cannulated compression screw. MATERIALS AND METHODS: This study is based on 17 patients, 17 feet who underwent subtalar arthrodesis using the cannulated compression screw from March, 1997 to March, 2004 with at least 1 year follow-up. The average follow-up period was 33.0 months (12 to 72 months). Functional results were assessed using the American Orthopaedic Foot and Ankle Society Ankle-Hindfoot (AOFAS) score, and Visual Analysis Scale (VAS) pain score, patients' returning to previous occupation and patients' satisfaction rate were also evaluated. RESULTS: The mean AOFAS scores at final follow-up were 80.4 points (range 66~92). The satisfactory rates were as follow. Thirteen patients (76.4%) were at least satisfied with surgical result at final follow-up. Patients' VAS pain score was average 2.8 points (1~6). Fourteen (82.3%) patients returned to previous job at mean postoperative period of 11.3 months (range 3-18 months). Patients' work efficiency after returning to previous occupation was 68.7% (range 33~100%). There were 9 complications which were 3 cases of sural nerve injury, 1 case of valgus malunion, and 5 cases of the hindfoot residual pain. CONCLUSION: We obtained the satisfactory functional results with relatively high patient satisfaction rate of 76%. So we conclude that subtalar arthrodesis using the cannulated compression screw is a reliable method for addressing the painful end-stage subtalar osteoarthritis and unreconstructible comminuted calcaneal fractures. However we also found out that average 11 months were necessary for patients to return to their job.