Dendritic cells (DCs) play a critical role in the development of acute lung injury (ALI) / acute respiratory distress syndrome (ARDS), but the underlying mechanisms remain poorly understood, due to their heterogeneous phenotype and function. In this study, a novel DC subset is defined in mice, Ly6C⁺ cDC2, which corresponds to CD14⁺ cDC2 in humans. These subsets highly express C-X-C motif chemokine receptor 1 (Cxcr1) and exhibit pro-inflammatory effects during ALI. Ex vivo, Ly6C⁺ cDC2s release higher levels of Il-6 and Il-1β, thereby promoting naïve T cells to differentiate into Th17 cells. Notably, Cxcr1 deficiency reduced the release of Il-6 and Il-1β from Ly6C⁺ cDC2s and shifted naïve T cells toward Treg differentiation, resulting in a decreased Th17/Treg ratio. In vivo, adoptive transfer of Ly6C⁺ cDC2s increased the Th17/Treg ratio in the lungs and spleens of LPS-treated mice, exacerbating lung injury. Specific depletion of Cxcr1 in DCs significantly reduced the severity of ALI and mortality. Mechanistically, it is found that Cxcr1 regulates the expression of Il-6 and Il-1β in Ly6C⁺ cDC2s through the MEK1/ERK/NF-κB pathway. Collectively, pro-inflammatory Ly6C⁺ cDC2s are identified as key effector cells mediating the role of Cxcr1 signaling in modulating T cell differentiation, driving the progression of ALI.

Adaptation to hypoxia is a major challenge for the survival of Mycobacterium tuberculosis (Mtb) in vivo. Interferon (IFN)-γ-producing CD8+ T cells contribute to control of Mtb infection, in part by promoting antimicrobial activities of macrophages. Whether Mtb counters these responses, particularly during hypoxic conditions, remains unknown. Using metabolomic, proteomic and genetic approaches, here we show that Mtb induced Rv0884c (SerC), an Mtb phosphoserine aminotransferase, to produce d-serine. This activity increased Mtb pathogenesis in mice but did not directly affect intramacrophage Mtb survival. Instead, d-serine inhibited IFN-γ production by CD8+ T cells, which indirectly reduced the ability of macrophages to restrict Mtb upon co-culture. Mechanistically, d-serine interacted with WDR24 and inhibited mTORC1 activation in CD8+ T cells. This decreased T-bet expression and reduced IFN-γ production by CD8+ T cells. Our findings suggest an Mtb evasion mechanism where pathogen metabolic adaptation to hypoxia leads to amino acid-dependent suppression of adaptive anti-TB immunity.

OBJECTIVES This study intends to appraise the feasibility of double sleeve lobectomy after neoadjuvant chemotherapy in central non-small-cell lung cancer with bronchovascular aggression. METHODS This retrospective study included non-small-cell lung cancer patients who received double sleeve lobectomy from January 2014 to June 2020. Patients were divided into 2 groups: the neoadjuvant chemotherapy group and the non-neoadjuvant chemotherapy group. Demographic data and perioperative outcomes were compared between these 2 groups. RESULTS Of the 110 patients who received double sleeve lobectomy during this period, 35 patients (31.8%) received neoadjuvant chemotherapy. Compared with the non-neoadjuvant chemotherapy group, patients who received neoadjuvant chemotherapy were associated with younger age (P = 0.026), smaller pathologic tumour size (P = 0.005), higher forced expiratory volume in 1 s (P = 0.007), higher forced expiratory volume in 1 s of predicted value (P = 0.005) and higher clinical stage (P < 0.001). In the neoadjuvant chemotherapy group, 18 patients (51.4%) attained a partial response and 17 patients (48.6%) achieved stable disease. The postoperative hospital stays (P = 0.042) and chest tube drainage duration (P = 0.030) were longer in the neoadjuvant chemotherapy group and other perioperative performances were similar between these 2 groups. No statistically significant difference was reported in postoperative complications and mortality between these 2 groups. CONCLUSIONS The intraoperative performance and postoperative outcomes of double sleeve lobectomy following neoadjuvant chemotherapy were similar to direct surgery, indicating that double sleeve lobectomy after neoadjuvant chemotherapy is feasible and safe in central lung cancer involving both the pulmonary artery and bronchus.

BACKGROUND This study evaluated the efficacy of bronchial sleeve lobectomy with pulmonary arterioplasty compared with pneumonectomy in centrally located non-small cell lung cancer with bronchovascular invasion.METHODS The cohort consisted of 212 patients receiving pneumonectomy and 156 patients undergoing bronchial sleeve lobectomy with pulmonary arterioplasty. Propensity score matching was used to create a fully balanced cohort, after which, baseline characteristics, perioperative performance, and oncologic results were compared between the 2 groups.RESULTS A total of 139 pneumonectomy patients were matched with 139 sleeve lobectomy patients. In the matched cohort, bronchial sleeve lobectomy with pulmonary arterioplasty was associated with longer operative time (P < .001), decreased perioperative transfusion rate (P = .002), shorter postoperative hospital stays (P < .001), shorter intensive care unit stays (P = .040), and lower Clavien-Dindo Classification (P = .016). In respect to survival outcomes, a log-rank test revealed no significant difference in overall survival (P = .381) and recurrence-free survival (P = .619) between the 2 surgical procedures.CONCLUSIONS Bronchial sleeve lobectomy with pulmonary arterioplasty could achieve superior perioperative outcomes and equivalent oncologic efficacy compared with pneumonectomy, indicating that this complex procedure is safe and reliable for centrally located non-small cell lung cancer concurrently involving the pulmonary artery and bronchus.(Ann Thorac Surg 2022;113:934-41)(c) 2022 by The Society of Thoracic Surgeons

Objectives:In this study, we apply a clustering method to proteomic data sets from bovine and human models of post-traumatic osteoarthritis (PTOA) to distinguish clusters of proteins based on their kinetics of release from cartilage and examined these groups for PTOA biomarker candidates. We then quantified the effects of dexamethasone (Dex) on the kinetics of release of the cartilage media proteome.Design:Mass spectrometry was performed on sample medium collected from two separate experiments using juvenile bovine and human cartilage explants (3 samples/treatment condition) during 20- or 21-day treatment with inflammatory cytokines (TNF-α, IL-6, sIL-6R) with or without a single compressive mechanical injury. All samples were incubated with or without 100 nM Dex. Clustering was performed on the correlation between normalized averaged release vectors for each protein.Results:Our proteomic method identified the presence of distinct clusters of proteins based on the kinetics of their release over three weeks of culture. Clusters of proteins with peak release after one to two weeks had biomarker candidates with increased release compared to control. Dex rescued some of the changes in protein release kinetics the level of control, and in all conditions except control, there was late release of immune-related proteins.Conclusions:We demonstrate a clustering method applied to proteomic data sets to identify and validate biomarkers of early PTOA progression and explore the relationships between the release of spatially related matrix components. Dex restored the kinetics of release to many matrix components, but not all factors that contribute to cartilage homeostasis.

Background: The detection value of different types of specimens for programmed death ligand-1 (PD-L1) expression remains controversial. As such, the purpose of this meta-analysis was to compare the detection value of biopsy specimens and surgical resection specimens for PD-L1 expression in patients with non-small cell lung cancer (NSCLC). Methods: PubMed and Web of Science were searched prior to December 2020 to identify studies that compared the detection value of biopsy specimens and surgical resection specimens for PD-L1 expression in NSCLC. Quality Assessment of Diagnostic Accuracy Studies (QUADAS)-2 scale was used to evaluate the quality of the literature included. The detection value of different types of specimens for PD-L1 expression was then assessed. Besides, the relative risk (RR) with 95% CI were pooled using Review Manager 5.3 software and Stata 14.0 software. Results: The meta-analysis involved 12 articles and included 877 patients. There was no significant difference in the detection rate of PD-L1 at the 1% cutoff between biopsy specimens and surgical resection specimens (RR =0.89, 95% CI: 0.70-1.12, P=0.33). However, there was a significant difference between two groups when the cutoff is 50% (RR =0.69, 95% CI: 0.58-0.83, P<0.01). In addition, a subgroup analysis of the type of biopsy specimens and the PD-L1 qualitative immunohistochemistry (IHC) assays showed that the detection rate of PD-L1 in small biopsies and using the SP142 antibody were lower than in surgical specimens and using other antibodies for both the 1% and 50% cut-offs (P<0.01). Conclusions: Current evidence suggests that caution must be taken when using biopsy specimens from patients with advanced NSCLC to evaluate PD-L1 status eligible for immunotherapy, additional biopsy specimens sampling may be needed to minimize the risk of tumor misclassification. In addition, PD-L1 qualitative IHC assays and the type of biopsy specimens related to PD-L1 expression detection.

Purpose: Recent progress in the field of post-traumatic osteoarthritis (PTOA) has suggested that low doses of steroid drugs such as dexamethasone (Dex) may be used in early post-injury interventions to prevent disease progression. There are a number of potential molecular biomarkers for patients at risk of PTOA that have been identified in synovial fluid aspirates from joint-injured patients, but it is critical to understand the kinetics of cartilage matrix breakdown to identify the windows of time at which such biomarkers might be released. Proteomics is a powerful tool that can be applied to explore the proteome throughout a time course of matrix breakdown, as well as to understand the effects of Dex on the processes affecting cartilage catabolism. In this study, we use an ex vivo explant model of human PTOA to model disease progression with or without Dex treatment and characterize the kinetics of the cartilage release proteome. Methods: Human cartilage explants (3 mm x 1 mm) were harvested from the tibial plateau (Collins Grade 1) of a 74-year old male donor obtained postmortem through the Gift of Hope Organ and Tissue Donor Network (Itasca, IL). All procedures were approved by both Rush University and MIT. Explants were treated with or without inflammatory cytokines (100 ng/mL TNFa, 50 ng/mL IL-6, 250 ng/mL sIL-6R) and/or a single compressive mechanical injury, and cultured in serum-free DMEM with 1% ITS for 21 days. Cytokine and injury+cytokine-treated samples were incubated with or without 100 nM Dex. Medium changes were carried out every three days and spent medium was collected and stored at -20°C for analysis. Proteomics analyses using in-solution trypsin digestion followed by LC/MS/MS (Q-ExactiveTM) were performed for medium samples at all time points for the identification and quantification of released proteins using Proteome Discoverer 2.3 (Thermo). Statistical analysis was performed using Matlab (Mathworks) and the R package limma. The amount of each protein released at each timepoint was normalized to the total amount released, then averaged across three biological replicates. These release vectors were clustered based on correlation (Fig. 1A). Cluster enrichment for proteins with increased or decreased release from control was determined by selecting the number of proteins in each cluster from the total population and generating 10,000 bootstrapped distributions. Results: Spent media was filtered for proteins identified in at least 70% of samples, with missing values imputed via the k-nearest neighbor method (k=5), resulting in 416 proteins for analysis. The media proteomes were then clustered based on the fraction of total protein release per day. In the untreated controls, the three major clusters were a cluster with peak release at the first timepoint followed by a decreasing release (Fig. 1B), which contained nearly all the identified matrix proteins, and two additional clusters with an increased release at day 12 (Fig. 1C), which had many intracellular proteins, assumed to be markers of a low level of cell death two weeks into culture. For both the cytokine (Fig. 2A,i) and injury+cytokine (Fig. 2B,i) treated conditions, a large cluster was present with peak release at day 3. These clusters both were enriched for matrix proteins with decreased release versus control, such as collagens VI, IX, and XI. The cytokine-treated samples had two clusters enriched for intracellular proteins on day 12 (Fig. 2A,ii-iii) not present with the addition of mechanical injury. The increased release of proteases and matrix proteins, including aggrecan, metalloproteinases, and collagen I, was apparent in a cluster with a day 12-18 peak (Fig. 2A,iv), and in two clusters that had an earlier release for injury+cytokines (Fig. 2B,ii-iii). Both models of disease had late release of cathespins and immune proteins peaking at day 18 (Fig. 2A,v-vi and Fig. 2B,iv), with no change in the total release of these proteins versus control. There were many proteins that freely diffused from cartilage (Fig. 1B) under control conditions but experienced peak release at a later time in the two disease conditions, and the addition of Dex to both disease models restored most of these proteins to the same behavior as in control (Fig. 3A,i and Fig. 3B,i), with the notable exceptions of many proteases, some collagens, and immune factors. Both Dex treatment regimens caused a group of proteins including MMPs to experience peak release on day 12 or day 8 (Fig. 3A,ii and Fig. 3B,ii), and that had increased release versus control. These proteins had decreased fold change total release from their non-Dex treated counterparts. The Dex-treated conditions had clusters of proteins with peak release on day 18 that were highly enriched for immune proteins, proteases, and protease inhibitors with increased release versus control (Fig. 3A,iv and Fig. 3B,iii-v).Fig. 3Clusters with more than 15 proteins for cytokine+Dex (A) and injury+cytokine+Dex (B) treatment conditions. Grey: no change in total amount released from control. Red: increased total release from control. Blue: decreased total release from control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Conclusions: Our analysis identified the presence of distinct clusters of proteins based on the kinetics of their release over three weeks of culture. In both models of PTOA progression, our data suggest that many biomarkers for cartilage matrix breakdown are released at their peak nine to fifteen days after the initial injury. Notably, the addition of a single mechanical impact injury caused matrix breakdown to begin several days sooner than cytokines alone. This approach allows us to not only observe changes in matrix breakdown, but also effects on the immune response of cartilage: in all treatment conditions except control, there was a late peak in the release of complement factors. Dex did not restore the kinetics of cartilage matrix breakdown entirely to control behavior and had a significant effect on the release of immune factors late into the experiment. This highlights the need to better understand the effect of Dex not only on the matrix constituents, but other pathways involved in cartilage homeostasis. The authors acknowledge Gift of Hope Organ & Tissue Donor Network (Itasca, IL), Rush Klaus Kuettner Endowed Chair (SC), donors' families , and NIH/NCATS grant UH3TR002186.

Pathogenic mycobacteria induce the formation of hypoxic granulomas during latent tuberculosis (TB) infection, in which the immune system contains, but fails to eliminate the mycobacteria. Fatty acid metabolism-related genes are relatively overrepresented in the mycobacterial genome and mycobacteria favor host-derived fatty acids as nutrient sources. However, whether and how mycobacteria modulate host fatty acid metabolism to drive granuloma progression remains unknown. Here, we report that mycobacteria under hypoxia markedly secrete the protein Rv0859/MMAR_4677 (Fatty-acid degradation A, FadA), which is also enriched in tuberculous granulomas. FadA acts as an acetyltransferase that converts host acetyl-CoA to acetoacetyl-CoA. The reduced acetyl-CoA level suppresses H3K9Ac-mediated expression of the host proinflammatory cytokine Il6, thus promoting granuloma progression. Moreover, supplementation of acetate increases the level of acetyl-CoA and inhibits the formation of granulomas. Our findings suggest an unexpected mechanism of a hypoxia-induced mycobacterial protein suppressing host immunity via modulation of host fatty acid metabolism and raise the possibility of a novel therapeutic strategy for TB infection.

Mycobacterial arabinogalactan (AG) is an essential cell wall component of mycobacteria and a frequent structural and bio-synthetical target for anti-tuberculosis (TB) drug development. Here, we report that mycobacterial AG is recognized by galectin-9 and exacerbates mycobacterial infection. Administration of AG-specific aptamers inhibits cellular infiltration caused by Mycobacterium tuberculosis (Mtb) or Mycobacterium bovis BCG, and moderately increases survival of Mtb-infected mice or Mycobacterium marinum-infected zebrafish. AG interacts with carbohydrate recognition domain (CRD) 2 of galectin-9 with high affinity, and galectin-9 associates with transforming growth factor β-activated kinase 1 (TAK1) via CRD2 to trigger subsequent activation of extracellular signal-regulated kinase (ERK) as well as induction of the expression of matrix metalloproteinases (MMPs). Moreover, deletion of galectin-9 or inhibition of MMPs blocks AG-induced pathological impairments in the lung, and the AG-galectin-9 axis aggravates the process of Mtb infection in mice. These results demonstrate that AG is an important virulence factor of mycobacteria and galectin-9 is a novel receptor for Mtb and other mycobacteria, paving the way for the development of novel effective TB immune modulators.

Purpose: Post-traumatic osteoarthritis (PTOA) affects millions of patients around the world each year, but currently has no prognostic biomarker. ‘Omics-level analyses allow for comprehensive observations of cartilage responses in models of PTOA. In this study, we utilize data on the secreted proteome from human cartilage under inflammatory cytokine stress to discover novel biomarker candidates released in response to disease stress and characterize the kinetics of release of proteins from the cartilage matrix. We also examine the contributions of proteolytic degradation versus changes in chondrocyte biosynthesis. Methods: Human cartilage explants (3 mm x 1 mm) were harvested from the tibial plateau (Collins Grade 1) of a 74-year old male donor obtained postmortem through the Gift of Hope Organ and Tissue Donor Network (Itasca, IL). All procedures were approved by both Rush University and MIT. Explants were treated with or without inflammatory cytokines (100 ng/mL TNFa, 50 ng/mL IL-6, 250 ng/mL sIL-6R) and cultured in serum-free DMEM with 1% ITS for 21 days. Treated samples were incubated with or without 100 nM Dex. Medium changes were carried out every three days and used medium was collected and stored at -80°C for analysis. Proteomics analyses using in-solution trypsin digestion followed by LC/MS/MS (Q-ExactiveTM) were performed for medium samples at all time points for the identification and quantification of released proteins using Proteome Discoverer 2.3 (Thermo). Statistical analysis was performed using Matlab (Mathworks) and p-values calculated using Kruskal-Wallis tests and Benjamini-Hochberg corrections (p < 0.05, FDR = 0.25). Proteins were clustered based on vectors of protein release per timepoint normalized to the total amount of protein released over the full 21 days and averaged between three biological replicates. Enrichment analysis was performed using the STRING database (String Consortium). Results: Spent media from human cartilage explant culture was analyzed (LC/MS/MS) and filtered for proteins identified in at least 70% of samples, with missing values imputed via a normal distribution centered around the lowest abundance found for each protein. Proteins with significantly increased release to the media versus control were enriched for those involved in ECM organization (FDR = 2.04E-18) and immune responses (FDR = 2.56E-5), including previously identified putative PTOA biomarkers such as collagen II, MMP3, IL8, ACAN, and CCL2. Clustering of proteins released from cytokine-treated cartilage based on the correlation between their normalized average time release profiles resulted in eight distinct clusters (Figure 1). The proteins having increased release over control after cytokine treatment nearly all fall into one cluster, Cluster V, demonstrating a peak release on day 18 of culture. This finding is further supported by comparing the log2 fold change of each protein in Cluster V to its control, which yields a statistically significant average increase from control values (Figure 2). Clusters I and III (Figure 2) both contain proteins with a statistically significant decreased release from control (Figure 2). Cluster I is highly enriched for ECM-related proteins, including collagens IX, XI, and XIV, that follow the profile of proteins freely diffusing out of cartilage, not appearing to be affected by the progression of matrix breakdown like Cluster V. Cluster I proteins have significantly decreased release from cartilage in the cytokine-treated condition, suggesting that the more dominant effect on these proteins after cytokine exposure is not due to matrix breakdown, but another biological process such as the rate of synthesis of these proteins or the regulation of fibrillogenesis, which collagen XI, IX, and XIV all contribute to.Fig. 2: Violin plot of log2 fold change from control for each protein in clusters I, III, and V.[]View Large Image Figure ViewerDownload Hi-res image Download (PPT) Conclusions: This analysis revealed the increased loss of matrix proteins 18 days into culture, validating several known PTOA biomarker candidates and suggesting several more for further clinical investigation (CCL20 & 5, serum amyloid A, and MMPs 10 and 13.) However, not all matrix proteins are affected the same by the increase in proteases, but may see a greater effect on their release due to changes in their rate of synthesis instead of their breakdown and subsequent release from diseased cartilage, such as collagens IX and XI, though this must be further validated.

OBJECTIVES:In this exploratory study, we used discovery proteomics to follow the release of proteins from bovine knee articular cartilage in response to mechanical injury and cytokine treatment. We also studied the effect of the glucocorticoid Dexamethasone (Dex) on these responses.DESIGN:Bovine cartilage explants were treated with either cytokines alone (10 ng/ml TNFα, 20 ng/ml IL-6, 100 ng/ml sIL-6R), a single compressive mechanical injury, cytokines and injury, or no treatment, and cultured in serum-free DMEM supplemented with 1% ITS for 22 days. All samples were incubated with or without addition of 100 nM Dex. Mass spectrometry and western blot analyses were performed on medium samples for the identification and quantification of released proteins.RESULTS:We identified 500 unique proteins present in all three biological replicates. Many proteins involved in the catabolic response of cartilage degradation had increased release after inflammatory stress. Dex rescued many of these catabolic effects. The release of some proteins involved in anabolic and chondroprotective processes was inconsistent, indicating differential effects on processes that may protect cartilage from injury. Dex restored only a small fraction of these to the control state, while others had their effects exacerbated by Dex exposure.CONCLUSIONS:We identified proteins that were released upon cytokine treatment which could be potential biomarkers of the inflammatory contribution to cartilage degradation. We also demonstrated the imperfect rescue of Dex on the effects of cartilage degradation, with many catabolic factors being reduced, while other anabolic or chondroprotective processes were not.

BACKGROUND:Operative safety and oncologic adequacy of thoracoscopic sleeve lobectomy remain controversial. As such, the purpose of this meta-analysis was to evaluate evidence comparing thoracoscopy and thoracotomy in sleeve lobectomy for centrally located non-small cell lung cancer (NSCLC). METHODS:Electronic searches of PubMed and Web of Science databases were undertaken from inception to March 2020. Comparative studies about thoracoscopic and thoracotomy sleeve lobectomy, with evaluation for perioperative outcomes and oncological results were identified. The following outcomes were measured in this meta-analysis: operating time, blood loss, numbers of lymph node, postoperative hospital stay, chest drainage time, postoperative complication rate, mortality, overall survival (OS). The standardized difference (SMD), relative risk (RR) and hazard ratio (HR) with 95% confidence intervals (CI) were pooled using Stata software. RESULTS:Six studies generating 281 thoracoscopy and 369 thoracotomy cases were finally included. There was no significant difference in intraoperative blood loss, number of resected lymph nodes, chest drainage time, postoperative complication rate and mortality between two groups. However, thoracoscopic sleeve lobectomy was associated with longer operation time (SMD 0.59, 95% CI: 0.14 to 1.03, P=0.010). And shorter postoperative hospital stays (SMD -0.24, 95% CI: -0.51 to 0.03, P=0.078) were observed in the thoracoscopy group with marginal significance. Furthermore, sleeve lobectomy via thoracoscopy could achieve comparable OS compared to that via thoracotomy (HR 0.69, 95% CI: 0.38 to 1.00; P<0.001). In addition, there were no evident publication bias in all observational outcomes. CONCLUSIONS:Current evidence suggests that thoracoscopic sleeve lobectomy is a safe and efficient surgical procedure for centrally located NSCLC, with comparable perioperative outcomes and equivalent oncological results compared to thoracotomy sleeve lobectomy.

Active matrix metalloproteases (MMPs) play a significant role in the pathogenesis of many diseases including osteoarthritis (OA), which involves progressive proteolytic degradation of cartilage. Clinical success of OA interventions that target MMPs has been limited by a lack of information about the presence and activity of specific disease-related proteases. We therefore developed a chemoproteomics approach based on MS to characterize the release and activity of MMPs in an in vitro model of the early inflammatory phase of posttraumatic OA (PTOA). We designed and synthesized chemical activity-based probes (ABPs) to identify active MMPs in bovine cartilage explants cultured for 30 days with the proinflammatory cytokine, interleukin-1α. Using these probes in an activity-based protein profiling-multidimensional identification technology (ABPP-MudPIT) approach, we identified active MMP-1, -2, -3, -7, -9, -12, and -13 in the medium after 10 days of culture, the time at which irreversible proteolysis of the collagen network in the explant was detected using proteolytic activation of FRET-quenched MMP substrates. Total MMP levels were quantified by shotgun proteomics, which, taken with ABPP-MudPIT data, indicated the presence of predominantly inactive MMPs in the culture medium. The selectivity of the ABPP-MudPIT approach was further validated by detection of specific endogenous MMPs activated de novo with 4-aminophenylmurcuric acetate. The utility of the new ABPP-MudPIT approach for detecting molecular biomarkers of PTOA disease initiation and potential targets for therapeutics motivates possible application in other diseases involving MMP activity.

Purpose: Traumatic joint injury leads to mechanical damage of cartilage as well as elevated levels of pro-inflammatory cytokines in the synovial fluid. Taken together, these insults often progress to post-traumatic osteoarthritis. In this exploratory study, we used discovery proteomics to follow the release of extracellular matrix proteins caused by mechanical damage and cytokine treatment of human knee cartilage explants. We then studied the effects of dexamethasone in this model system to elucidate the mechanism(s) by which this glucocorticoid was found to markedly alter the response to inflammation. Methods: Human cartilage explants (3 mm x 1 mm) were harvested from the tibial plateau (Collins Grade 1) of a 74-year old male donor obtained postmortem through the Gift of Hope Organ and Tissue Donor Network (Itasca, IL). All procedures were approved by both Rush University and MIT. Groups of 7 explants were treated (individually) with either cytokines alone (cyt: 100 ng/mL TNFα, 50 ng/mL IL-6, 250 ng/mL sIL-6R), a single compressive mechanical impact injury (inj: radially unconfined; 60% strain; 300%/s strain rate), cytokines+injury (cyt+inj) or not treated (control), and cultured in serum-free DMEM (supplemented with 1% ITS) for 21 days. Additionally, treated samples were incubated with or without addition of 100 nM dexamethasone (Dex). Medium changes were carried out every 3 days; used medium was collected and stored at −80°C for analysis. Cartilage explants were flash-frozen at the end of the 21 day culture. LC/MS/MS (Q-ExactiveTM) was performed for medium samples at all the time points for the identification and quantification of proteins. sGAG release was quantified via the DMMB assay. Proteome Discoverer 2.1 software was used for the analysis and the quantification was made at the MS1 level. Results: GAG release was greatly elevated by cyt and cyt+inj, but this release was markedly decreased by treatment with Dex. LC-MS experiments from 49 different samples showed release of 487 proteins to the medium, identified by 4403 peptides. Each protein was identified with at least 2 unique peptides. 199 of these released proteins were common to all experimental treatment groups. In total, 260 extracellular proteins representing both high and low abundant proteins were identified. Together, the human explant system showed a dramatic increase in release of proteins and protein fragments upon treatments incorporating cytokines. Of approximately 40 ECM proteins that showed increased response to cytokines, the release of the majority of these proteins was inhibited by addition of Dex including interleukin-8, ICAM-1 and VCAM-1, proteases (e.g., MMP-1 (Fig 1) and MMP-13) as well as matrix proteins such as aggrecan and COMP. Dex treatment also reduced the release of protein fragments measured as neopetitopes, including the COMP (S77) neoepitope (Fig 2), and that from collagen III (G948). Conclusions: This proteomics approach allowed us to unambiguously identify and quantify multiple proteins in the medium of human cartilage explant cultures upon treatments that mimic the earliest events in post-traumatic-OA, including injurious compression and inflammation. We followed the quantitative release of more than 400 proteins including cytokines, proteases as well as other extracellular proteins throughout the 21 day culture period. Treatment with dexamethasone had a marked effect by reducing critical cytokine-mediated explant responses.Figure 2COMP S77 neoepitope release into the explant culture media.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Background A hallmark of osteoarthritis is increased proteolytic cleavage of aggrecan. Cross talk between cartilage and the synovium + joint capsule (SJC) can drive cartilage degradation by activating proteases in both tissues. We investigated aggrecan proteolysis patterns in cartilage explants using a physiologically relevant explant model of joint injury combining cartilage mechanical compression and coincubation with SJC. Methods Bovine cartilage explants were untreated; coincubated with SJC; or subjected to mechanical injury and coincubated with SJC, mechanical injury alone, or mechanical injury and incubated with tumor necrosis factor-α (TNF-α). To compare the patterns of aggrecan proteolysis between 6 h and 16 days, release of sulfated glycosaminoglycans and specific proteolytic aggrecan fragments into medium or remaining in cartilage explants was measured by dimethylmethylene blue and Western blot analysis. Results Aggrecanase activity toward aggrecan was observed in all conditions, but it was directed toward the TEGE↓ARGS interglobular domain (IGD) site only when cartilage was coincubated with SJC or TNF-α. Matrix metalloproteinase (MMP) activity at the aggrecan IGD site (IPES↓FFGV) was not detected when cartilage was exposed to TNF-α (up to 6 days), but it was in all other conditions. Compared with when bovine cartilage was left untreated or subjected to mechanical injury alone, additional aggrecan fragment types were released into medium and proteolysis of aggrecan started at an earlier time when SJC was present. Conclusions Indicative of different proteolytic pathways for aggrecan degradation, the SJC increases both aggrecanase and MMP activity toward aggrecan, whereas TNF-α inhibits MMP activity against the IGD of aggrecan.

Purpose: The combination of insulin-like growth factor 1 (IGF-1) and dexamethasone (Dex) treatment has been shown to reduce aggrecan and collagen degradation and promote sGAG synthesis in human and bovine cartilage explants treated with IL-1 (one of the pro-inflammatory cytokines reported to be upregulated in the synovial fluid after traumatic joint injuries). However, the underlying cellular signaling mechanisms are not well understood. Since phosphorylation is an important post-translational modification in mediating cellular response to stimuli, we employed an untargeted LC/MS/MS approach to study the phosphorylation changes in chondrocytes following treatment with IL-1, IGF-1, and Dex to achieve a better understanding of how human chondrocytes respond to these stimuli. Methods: Chondrocytes were isolated postmortem from the ankle and knee cartilage (Collins Grade 1) from the same donor (56-year--old female), equilibrated for 3 days, and subjected to 8 treatment conditions: untreated control, IL-1 (10ng/mL), Dex (100nM), IGF-1 (300ng/mL), IL-1+Dex, IL-1+IGF-1, IL-1+Dex+IGF-1, and Dex+IGF. After 30 minutes of treatment, cells were lysed in 8M urea and cell lysates were reduced, alkylated and trypsin digested into peptides. Each peptide sample was labelled with isobaric tags using the 8-plex iTRAQ reagent protocol (Sciex), and the labelled peptides from the different conditions were combined. Two immunoprecipitation (IP) steps were carried out to enrich for phosphopeptides (one IP using phosphotyrosine (pY) antibodies and a second IP using an antibody specific to the pSer/pThr-Pro motif). The products from each IP were further enriched for phosphopeptides using an IMAC (immobilized metal ion affinity chromatography) column. Phosphopeptides were resolved by reverse phase HPLC coupled to tandem mass spectrometry. The identification and relative quantification of the proteins was carried out with Discoverer software. In separate tests, cartilage explants (3mmx1mm) harvested from the femoropatellar groove of 1–2 weeks old calves were used for treatments with kinase inhibitors. Results: In the phosphoproteomics analysis of pY peptides, 229 and 167 peptides were identified and quantified in ankle and knee chondrocyte samples, respectively. Among these pY peptides, 74 peptides were found in both ankle and knee chondrocyte samples. Interestingly, we found an upregulation in the phosphorylation levels (more than 2x relative to untreated control) of key components of the MAPK pathway (including ERK1, ERK2, ERK5, JNK1, JNK2, P38α, and P38γ) following IL-1 treatment (Fig 1). Treatment with IGF and Dex did not affect the IL-1 stimulated phosphorylation changes of these proteins, except for JNK1 and JNK2, which had slight downregulation in phosphorylation with the addition of Dex. In addition to MAPK protein peptides, IL-1 treatment also upregulated the phosphorylation (more than 2x) of 68 peptides with the pS/pT-P motif, indicating that a large number of substrates of the MAPK pathways are being modulated in response to IL-1 treatment. Inhibition of the MAPK pathways using small molecule inhibitors revealed that the JNK inhibitor, SP600125, reduced GAG loss, rescued sGAG synthesis, and improved viability of IL-1 treated bovine cartilage explants (Fig 2). Conclusions: We harnessed an innovative phosphoproteomics approach to understand the changes in phosphorylation caused by treatment of human chondrocytes with IL-1, Dex and IGF-1 (individually and in combinations) that result in reprogramming of these cells. Using this approach, we found key regulatory kinases, including p38, JNK1/2, ERK1/2, ERK5, and substrates of the MAPK pathways that were highly upregulated in phosphorylation as a result of cytokine treatment. We further found that inhibition of JNK1/2 pathway has the anti-catabolic effect of reducing GAG loss and anabolic effect of promoting sGAG synthesis in cartilage explants treated with IL-1.Fig 2APercent sGAG loss, from immature bovine explants treated with IL-1 +/- SP600125 (8 days experiment). 47 disks/condition from 8 animals were used, values are mean +/- SEM. B. Normalized sulfate incorporation rate measured during Day 6–8 of the same disks used in A. C. Bovine chondrocyte viability in cartilage disks in response to 8 day treatments. Cells were fluorescently labeled with fluorescein diacetate (green, viable) and propidium iodide (red, non-viable).View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Injury to the joint provokes a number of local pathophysiological changes, including synthesis of inflammatory cytokines, death of chondrocytes, breakdown of the extra‐cellular matrix of cartilage, and reduced synthesis of matrix macromolecules. These processes combine to engender the subsequent development of post‐traumatic osteoarthritis (PTOA). To prevent this from happening, it is necessary to inhibit these disparate responses to injury; given their heterogeneity, this is challenging. However, dexamethasone has the necessary pleiotropic properties required of a drug for this purpose. Using in vitro models, we have shown that low doses of dexamethasone sustain the synthesis of cartilage proteoglycans while inhibiting their breakdown after injurious compression in the presence or absence of inflammatory cytokines. Under these conditions, dexamethasone is non‐toxic and maintains the viability of chondrocytes exposed chronically to such cytokines as interleukin (IL) ‐1, IL‐6, and tumor necrosis factor‐α. Moreover, the anti‐inflammatory properties of dexamethasone have been appreciated for decades. In view of this information, we have initiated a pilot clinical study to determine whether a single, intra‐articular injection of dexamethasone into the wrist shows promise in preventing PTOA after intra‐articular fracture of the distal radius.