Corrigendum: Proteinase-Mediated Macrophage Signaling in Psoriatic Arthritis.

[This corrects the article DOI: 10.3389/fimmu.2020.629726.].

Proteinase-Mediated Macrophage Signaling in Psoriatic Arthritis.

Objective: Multiple proteinases are present in the synovial fluid (SF) of an arthritic joint. We aimed to identify inflammatory cell populations present in psoriatic arthritis (PsA) SF compared to osteoarthritis (OA) and rheumatoid arthritis (RA), identify their proteinase-activated receptor 2 (PAR2) signaling function and characterize potentially active SF serine proteinases that may be PAR2 activators. Methods: Flow cytometry was used to characterize SF cells from PsA, RA, OA patients; PsA SF cells were further characterized by single cell 3'-RNA-sequencing. Active serine proteinases were identified through cleavage of fluorogenic trypsin- and chymotrypsin-like substrates, activity-based probe analysis and proteomics. Fluo-4 AM was used to monitor intracellular calcium cell signaling. Cytokine expression was evaluated using a multiplex Luminex panel. Results: PsA SF cells were dominated by monocytes/macrophages, which consisted of three populations representing classical, non-classical and intermediate cells. The classical monocytes/macrophages were reduced in PsA compared to OA/RA, whilst the intermediate population was increased. PAR2 was elevated in OA vs. PsA/RA SF monocytes/macrophages, particularly in the intermediate population. PAR2 expression and signaling in primary PsA monocytes/macrophages significantly impacted the production of monocyte chemoattractant protein-1 (MCP-1). Trypsin-like serine proteinase activity was elevated in PsA and RA SF compared to OA, while chymotrypsin-like activity was elevated in RA compared to PsA. Tryptase-6 was identified as an active serine proteinase in SF that could trigger calcium signaling partially via PAR2. Conclusion: PAR2 and its activating proteinases, including tryptase-6, can be important mediators of inflammation in PsA. Components within this proteinase-receptor axis may represent novel therapeutic targets.

Iron nanoparticle-labeled murine mesenchymal stromal cells in an osteoarthritic model persists and suggests anti-inflammatory mechanism of action.

Osteoarthritis (OA) is characterized by cartilage degradation and chronic joint inflammation. Mesenchymal stem cells (MSCs) have shown promising results in OA, but their mechanism of action is not fully understood. We hypothesize that MSCs polarize macrophages, which are strongly associated with joint inflammation to more homeostatic sub-types. We tracked ferumoxytol (Feraheme™, iron oxide nanoparticle)-labeled murine MSCs (Fe-MSCs) in murine OA joints, and quantified changes to joint inflammation and fibrosis. 10-week-old C57BL/6 male mice (n = 5/group) were induced to undergo osteoarthritis by destabilization of medical meniscus (DMM) or sham surgery. 3 weeks post-surgery, mice were injected intra-articularly with either fluorescent dye-(DiR) labeled or DiR-Fe-MSC or saline to yield 4 groups (n = 5 per group for each timepoint [1, 2 and 4weeks]). 4 weeks after injection, mice were imaged by MRI, and scored for i) OARSI (Osteoarthritis Research Society International) to determine cartilage damage; ii) immunohistochemical changes in iNOS, CD206, F4/80 and Prussian Blue/Sca-1 to detect pro-inflammatory, homeostatic and total macrophages and ferumoxytol -labeled MSCs respectively, and iii) Masson's Trichrome to detect changes in fibrosis. Ferumoxytol-labeled MSCs persisted at greater levels in DMM vs. SHAM-knee joints. We observed no difference in OARSI scores between MSC and vehicle groups. Sca-1 and Prussian Blue co-staining confirmed the ferumoxytol label resides in MSCs, although some ferumoxytol label was detected in proximity to MSCs in macrophages, likely due to phagocytosis of apoptotic MSCs, increasing functionality of these macrophages through MSC efferocytosis. MRI hypertintensity scores related to fluid edema decreased in MSC-treated vs. control animals. For the first time, we show that MSC-treated mice had increased ratios of %CD206+: %F4/80+ (homeostatic macrophages) (p<0.05), and decreased ratios of %iNOS+: %F4/80+ macrophages (p<0.01), supporting our hypothesis that MSCs may modulate synovial inflammation.

Bone Marrow Mesenchymal Stromal Cell Treatment in Patients with Osteoarthritis Results in Overall Improvement in Pain and Symptoms and Reduces Synovial Inflammation.

Patients with late-stage Kellgren-Lawrence knee osteoarthritis received a single intra-articular injection of 1, 10, or 50 million bone marrow mesenchymal stromal cells (BM-MSCs) in a phase I/IIa trial to assess safety and efficacy using a broad toolset of analytical methods. Besides safety, outcomes included patient-reported outcome measures (PROMs): Knee Injury and Osteoarthritis Outcome Score (KOOS) and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC); contrast-enhanced magnetic resonance imaging (MRI) for cartilage morphology (Whole Organ MRI Scores [WORMS]), collagen content (T2 scores), and synovitis; and inflammation and cartilage turnover biomarkers, all over 12 months. BM-MSCs were characterized by a panel of anti-inflammatory markers to predict clinical efficacy. There were no serious adverse events, although four patients had minor, transient adverse events. There were significant overall improvements in KOOS pain, symptoms, quality of life, and WOMAC stiffness relative to baseline; the 50 million dose achieved clinically relevant improvements across most PROMs. WORMS and T2 scores did not change relative to baseline. However, cartilage catabolic biomarkers and MRI synovitis were significantly lower at higher doses. Pro-inflammatory monocytes/macrophages and interleukin 12 levels decreased in the synovial fluid after MSC injection. The panel of BM-MSC anti-inflammatory markers was strongly predictive of PROMs over 12 months. Autologous BM-MSCs are safe and result in significant improvements in PROMs at 12 months. Our analytical tools provide important insights into BM-MSC dosing and BM-MSC reduction of synovial inflammation and cartilage degradation and provide a highly predictive donor selection criterion that will be critical in translating MSC therapy for osteoarthritis. Stem Cells Translational Medicine 2019;8:746&757.

Synovial fluid monocyte/macrophage subsets and their correlation to patient-reported outcomes in osteoarthritic patients: a cohort study.

BACKGROUND: Chronic, low-grade inflammation of the synovium (synovitis) is a hallmark of osteoarthritis (OA), thus understanding of OA immunobiology, mediated by immune effectors, is of importance. Specifically, monocytes/macrophages (MΦs) are known to be abundantly present in OA joints and involved in OA progression. However, different subsets of OA MΦs have not been investigated in detail, especially in terms of their relationship with patient-reported outcome measures (PROMs). We hypothesized that levels of synovial fluid (SF) MΦ subsets are indicative of joint function and quality of life in patients with OA, and can therefore serve as biomarkers and therapeutic targets for OA. METHODS: In this cohort study, synovial fluid leukocytes (SFLs, N = 86) and peripheral blood mononuclear cells (n = 53) from patients with knee OA were characterized. Soluble MΦ receptors and chemokine (sCD14, sCD163, CCL2, CX3CL1) levels were detected in SF using immunoassays. Linear models, adjusted for sex, age and body mass index, were used to determine associations between SF MΦs and soluble factors with PROMs (N = 83). Pearson correlation was calculated to determine correlation between MΦ subsets, T cells and soluble factors. RESULTS: SF MΦs were the most abundant SFLs. Within these, the double-positive CD14+CD16+-MΦ subset is enriched in knee OA SF compared to the circulation. Importantly, MΦ subset ratios correlated with PROMs, specially stiffness, function and quality of life. Interestingly, the SF CD14+CD16+-MΦ subset ratio correlated with SF chemokine (C-C motif) ligand 2 (CCL2) levels but not with levels of sCD163 or sCD14; we found no association between PROMs and either SF CCL2, sCD163, sCD14 or CX3CL1 (which was below detection levels). All SF MΦs displayed high levels of HLA-DR, suggesting an activated phenotype. Correlation between OA SF MΦ subsets and activated CD4+ T cell subsets suggests modulation of CD4+ T cell activation by MΦs. CONCLUSION: SF MΦ subsets are associated with knee OA PROMs and display an activated phenotype, which may lead to modulation of CD4+ T cell activation. Knee OA SF MΦ subsets could serve as knee OA function biomarkers and as targets of novel therapeutics.

TLR3 or TLR4 Activation Enhances Mesenchymal Stromal Cell-Mediated Treg Induction via Notch Signaling.

Mesenchymal stromal cells (MSCs) are the subject of numerous clinical trials, largely due to their immunomodulatory and tissue regenerative properties. Toll-like receptors (TLRs), especially TLR3 and TLR4, are highly expressed on MSCs and their activation can significantly modulate the immunosuppressive and anti-inflammatory functions of MSCs. While MSCs can recruit and promote the generation of regulatory T cells (Tregs), the effect of TLR activation on MSC-mediated Treg induction is unknown. In this study, we investigated the effect of ligand-mediated activation of TLR3 and TLR4 on Treg induction by human MSCs. We found that generation of Tregs in human CD4(+) lymphocyte and MSC cocultures was enhanced by either TLR3 or TLR4 activation of MSCs and that the increase was abolished by TLR3 and TLR4 gene-silencing. Augmented Treg induction by TLR-activated MSCs was cell contact-dependent and associated with increased gene expression of the Notch ligand, Delta-like 1. Moreover, inhibition of Notch signaling abrogated the augmented Treg levels in the MSC cocultures. Our data show that TLR3 or TLR4 activation of MSCs increases Treg induction via the Notch pathway and suggest new means to enhance the potency of MSCs for treating disorders with an underlying immune dysfunction, including steroid resistant acute graft-versus-host disease. Stem Cells 2017;35:265-275.

A Systematic Study of the Effect of Different Molecular Weights of Hyaluronic Acid on Mesenchymal Stromal Cell-Mediated Immunomodulation.

INTRODUCTION: Osteoarthritis (OA) is associated with chronic inflammation, and mesenchymal stromal cells (MSCs) have been shown to provide pain relief and reparative effects in clinical investigations. MSCs are often delivered with hyaluronic acid (HA), although the combined mechanism of action is not fully understood; we thus investigated the immunomodulatory effects of combining MSCs with different molecular weights (MW) of HA. METHODS: HAs with MWs of 1.6 MDa (hHA), 150 kDa or 7.5 kDa, were added to MSCs alone or MSC-immune cell co-cultures. Gene expression analyses, flow cytometry and cytokine measurements were assessed to determine the effect of HAs on the MSC interactions with immune cells. RESULTS: MSCs in the presence of HAs, in both normal and lymphocyte-conditioned medium, showed negligible changes in gene expression. While addition of hHA resulted in increased proliferation of activated lymphocytes, both in the presence and absence of MSCs, the overall combined effect was a more regulated, homeostatic one; this was supported by higher ratios of secreted IL10/IFNγ and IL10/IL2, in lymphocyte cultures, than with lower MW HAs or no HA, both in the presence and absence of MSCs. In addition, examination of monocyte-derived macrophages showed an increased M2 macrophage frequency (CD14+CD163+CD206+) in the presence of hHA, both with and without MSCs. CONCLUSIONS: hHA produces a less pro-inflammatory environment than lower MW HAs. Moreover, combining hHA with MSCs has an additive effect on the MSC-mediated immunomodulation, suggestive of a more potent combination treatment modality for OA.

The effects of human umbilical cord perivascular cells on rat hepatocyte structure and functional polarity.

Hepatocyte culture is a useful tool for the study of their biology and the development of bioartificial livers. However, many challenges have to be overcome since hepatocytes rapidly lose their normal phenotype in vitro. We have recently demonstrated that human umbilical cord perivascular cells (HUCPVCs) are able to provide support to hepatocytes. In the present study we go further into exploring the effects that HUCPVCs have in the functional polarization, and both the internal and external organization, of hepatocytes. Also, we investigate HUCPVC-hepatocyte crosstalk by tracking both the effects of HUCPVCs on hepatocyte transcription factors and those of hepatocytes on the expression of hepatotrophic factors in HUCPVCs. Our results show that HUCPVCs maintain the functional polarity of hepatocytes ex vivo, as judged by the secretion of fluorescein into bile canaliculi, for at least 40 days. Transmission electron microscopy revealed that hepatocytes in coculture organize in an organoid-like structure embedded in extracellular matrix surrounded by HUCPVCs. In coculture, hepatocytes displayed a higher expression of C/EBPα, implicated in maintenance of the mature hepatocyte phenotype, and HUCPVCs upregulated hepatocyte growth factor and Jagged1 indicating that these genes may play important roles in HUCPVC-hepatocyte interactions.

Effects of two mesenchymal cell populations on hepatocytes and lymphocytes.

The inflammatory response to liver injury plays an important role in the onset of liver fibrosis, which may ultimately lead to liver failure. The attenuation of inflammation and hepatocyte rescue are, therefore, of the utmost importance for recovery. Mesenchymal stromal cells (MSCs) from adult bone marrow have been shown to rescue hepatocyte function. Here we explore a more plentiful source of neonatal MSCs: human umbilical cord perivascular cells (HUCPVCs). We cocultured HUCPVCs or bone marrow-derived mesenchymal stromal cells (BM-MSCs) with rat hepatocytes or human peripheral blood mononuclear cells in order to identify their effects on hepatocyte functionality and the proliferation of phytohemagglutinin-stimulated peripheral blood mononuclear cells (phaPBMCs). The expression of hepatotrophic factors by both types of MSCs in the presence of hepatocytes and the functional implications of blocking putative MSC anti-inflammatory factors were compared. Both types of MSCs improved albumin secretion, ureagenesis, hepatospecific gene expression, cytochrome P450 (CYP) activity, and functional hepatocyte mass maintenance. However, although HUCPVCs had an improved effect on the maintenance of ureagenesis, BM-MSCs had a strong effect on hepatocyte CYP activity. Additionally, each MSC type differentially expressed putative hepatotrophic factors, whereas phaPBMC proliferation was significantly decreased. Indoleamine 2,3-dioxygenase (IDO) was the main immunosuppressive mechanism used by both types of MSCs, but HUCPVCs exhibited higher expression of programmed death 1 ligands. However, the functional significance of the difference in anti-inflammatory factor expression still remains to be elucidated. Thus, both MSC types can serve as hepatocyte stromal cells and mitigate inflammation with IDO, but they present differences in the manner in which they affect hepatocytes and in the expression of both hepatotrophic and anti-inflammatory factors.

Human umbilical cord perivascular cells improve rat hepatocyte function ex vivo.

Hepatocyte functionality and survival decrease rapidly in culture, and both can be improved using bone marrow-derived mesenchymal stromal cells (MSCs). We have previously described an alternative, more plentiful source of MSCs coming from the perivascular area of the umbilical cord, human umbilical cord perivascular cells (HUCPVCs). Our objective was therefore to ascertain whether HUCPVCs could serve as hepatocyte stromal cells ex vivo. For this purpose, rat hepatocytes were cocultured in contact with HUCPVCs (contact coculture). Also, HUCPVCs were cocultured separated from hepatocytes with a semipermeable membrane (noncontact coculture) to assess soluble factor interactions. Next, an HUCPVC-conditioned medium (CM) was used to investigate the possibility of HUCPVC-free support, while flash-frozen HUCPVCs were employed to investigate the effects of nonsoluble interactions. In all experiments, medium samples were taken daily to assess the production of albumin. Also, at certain days, the levels of cytochrome P450 (CYP) activity and urea secretion were tested. RNA extraction was performed at the end of experiments. Our results show that HUCPVCs in contact and noncontact cocultures with hepatocytes improve albumin gene expression and secretion compared to monoculture. Flash-frozen HUCPVCs had a late improvement in albumin secretion, while CM improved it for a short period. Ureagenesis maintenance was improved by contact coculture and flash-frozen HUCPVCs. CYP activity was significantly increased in the presence of flash-frozen HUCPVCs and in noncontact cocultures. We conclude that HUCPVCs can act as stromal cells for rat hepatocytes, and that soluble and nonsoluble factors induce differential effects on hepatocytes.

Mesenchymal stromal cells as supportive cells for hepatocytes.

Hepatocytes and hematopoietic stem cells (HSCs) appear to share many of the same requirements for their survival, functionality, and proliferation. This may be due to a shared location during fetal development. Moreover, hepatocytes and HSCs are unable to function, or even survive, without stromal cell support. Bone marrow-derived mesenchymal stromal cells (MSCs) support the proliferation and functionality, not only of HSCs, but also of hepatocytes. Although knowledge of the mechanisms underlying HSCs' support is far more advanced than for hepatocytes, data suggest that many agents important for HSCs also maintain the normal hepatocyte phenotype in vitro. Thus, it is possible that new techniques for the maintenance and expansion of HSCs may also be useful for hepatocytes. Bone marrow-derived MSCs are easily cultured and expanded in vitro, and some data suggest that they are immunoregulatory as well as relatively nonimmunogenic. These observations suggest that allogeneic MSCs may be useful not only in supporting hepatocyte growth and proliferation but also in modulating immune responses such as stellate cell activation.

La expresión gráfica en la fabricación de prototipos. Descripción del proceso de un prototipo de mano derecha / The proficiency in dreafting oriented to the fabrication of prototipe. Description of prototype fabrication process

El avance vertiginoso de los sistemas en programas de 3-D y el maquinado de control numérico, han revolucionado el expresión gráfica como herramienta de excepción para la expresión del pensamiento abstracto ingenieril y guía de procesos de manufacturado. Mediante la aplicación del dibujo técnico: ensamblado y en explosión, y posteriormente pasado por dibujo asistido por computador (CAD) se diseña el prototipo de una mano derecha que incluye el equivalente a la articulación del cuerpo, para luego proceder a maquinado manual y evaluación mecánica comparada del modelo, el cual cumple con el 92 por ciento de los movimientos de la mano...