A novel in vitro model of long-lasting inflammation using primary human macrophages for osteoarthritis research

doi:10.21203/rs.3.rs-2620354/v1

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A novel in vitro model of long-lasting inflammation using primary human macrophages for osteoarthritis research

https://doi.org/10.21203/rs.3.rs-2620354/v1

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Background. Development of pre-clinical models of long-lasting inflammation is needed for the study of inflammatory diseases. Osteoarthritis (OA), the most common inflammatory joint pathology, is characterized by an abundance of M1-like macrophages producing several pro-inflammatory cytokines and proteolytic enzymes damaging the local tissues. Here we developed a novel in vitro model of long-lasting inflammation using primary human monocyte-derived macrophages continuously cultured for 15 days.

Results. We first screened synovial fluids from 11 OA patients using multiplex Ella^® and we found measurable levels of IL-6, CCL2, CCL5, CXCL8, CXCL10, TNFa, IL-1b, using them as read-outs of the following experiments. We then found an increased survival of macrophages differentiated with both M-CSF and GM-CSF (M-/GM-Mf) compared either with macrophages differentiated with M-CSF alone (M-Mf) or macrophages differentiated with GM-CSF alone (GM-Mf) and, consequently, decided to use them to set up the in vitro model of long-lasting inflammation. Consistently with the increased survival, the repeated stimulation of M-/GM-Mfs either with lipopolysaccharide + interferon-g (LPS+IFN-g) or Pam3CysSerLys4 (Pam3CSK4) led to the sustained production of IL-6, CCL2 and CXCL8 over time. Finally, to investigate the usefulness of our model for OA research, we repeatedly exposed stimulated macrophages to dexamethasone (DEX) and/or celecoxib (CEL), two anti-inflammatory drugs commonly used for OA therapy. We found an excellent anti-inflammatory activity but significant toxicity of DEX for concentrations over 10 nM, while CEL was not toxic and efficiently inhibited PGE₂ secretion. Of interest, the inflammatory profile of macrophages differentiated with M-CSF or GM-CSF and activated with the two pro-inflammatory stimuli (LPS+IFN-g vs Pam3CSK4) was not identical, overall showing production of more inflammatory mediators with GM-CSF-macrophages stimulated with Pam3CSK4.

Conclusions. We introduce here a novel easy-to-use in vitro culture model of long-lasting inflammation using primary human macrophages, that could be useful for the screening of new compounds and drug delivery systems, to improve the therapy of inflammatory disorders.

osteoarthritis

macrophages

inflammation

TLR ligands

in vitro model

immunotoxicity

ELISA.

Among numerous chronic inflammatory diseases, osteoarthritis (OA) is nowadays the most widely diffused age-related medical issue for which definitive therapies are not yet available (1). One third of the worldwide population over 65 years old is affected (2), with women more affected than men (3). OA, causing pain and disability, occurs mainly in the knee, although it can affect all the joints, limiting the mobility and healthy life of the patients. While physical damage of the joint can trigger this condition, unfavorable genetic background, unhealthy lifestyle, and lack of physical activity have been clearly correlated with the appearance and poor prognosis of the disease. OA secondary to a chronic physical damage is an occupational disease, affecting specific categories of workers (4,5), while acute physical damage results in post-traumatic OA, as observed for the knee or the ankle (6). Overweight and obesity are also well-established modifiable risk factors for OA (7) and people with a BMI > 30 kg/m² are more likely to require total knee arthroplasty (TKA) during their life (8). It has also been observed a predisposition for OA considering variants of genes related to muscle and skeletal development, neuronal function, immune response, adipogenesis and joint degeneration (9,10).

The pathophysiology of OA involves a complex network of altered cell types, changes in molecular pathways and also damaged mechanical structures (i.e. bone or extracellular matrix). Among the cellular components present in the microenvironment of the diseased-joint, macrophages play a key role in orchestrating the inflammatory response (11). It has been demonstrated that macrophages are extremely plastic immune cells (12), with the ability to acquire different immunomodulatory behavior depending on the surrounding microenvironment and to strongly influence other cell types in the joint (13). In healthy conditions, tissue-resident synovial macrophages protect the joint from inflammation. However, during OA, synovial macrophages acquire a M1-like pro-inflammatory phenotype and are detrimental for the joint’s structure (14,15). This knowledge is in line with additional reports showing how the continuous recruitment of monocytes (precursors of macrophages) towards the joint exacerbates inflammation and disease severity (16), overall demonstrating the central role played by myeloid cells in the pathogenesis, development and resolution of chronic inflammation.

OA-associated M1-like macrophages trigger inflammation (17) and tissue damage by producing a wide range of pro-inflammatory cytokines such as IL-1b, TNFa, IL-6, as well as chemokines able to recruit other immune cells, such as CCL2 (monocytes) or CXCL8 (neutrophils) (16,18–20). Extensive studies demonstrated the activation of specific molecular pathways in M1 macrophages, such as those downstream to the toll-like receptor 4 (TLR4) receptor (through MyD88 to activate NF-kB), as well as interferon regulatory factors (IRF) and transcription factors of the STAT family (STAT1 and STAT5 involved in M1 polarization, against STAT4 and STAT6 more observed in M2-like phenotypes) (21).

This state of chronic inflammation affects the synovia, bone osteoclasts (22) and chondrocytes, eventually resulting in bone reabsorption and remodeling (23), and, as a whole, worsening OA conditions.

A main limitation to the study of macrophages in OA is related to the availability of appropriate in vitro and in vivo models, reliably mimicking the inflamed joints observed in the clinic. While in vivo models in mice, rabbits or sheep, have been used to study the disease or for the pre-clinical testing of therapeutic strategies (13,24), in vitro models are also needed for the analysis of molecular signaling pathways and drug screenings (25). The literature reports numerous in vitro models of OA (26), some including macrophages (27–37) (Supplementary Table), but with some limitations. Firstly, some of them rely on the use of myeloid leukemia cell lines, such as THP-1 (27,29), J774 (28), RAW 264.7 (31,32) or U937 (33), which present mutations in molecular signaling pathways and a high rate of continuous proliferation, that might affect their immunotoxicity behavior, thus becoming a less reliable model. Secondly, when murine (30) or human (34–37) primary cells have been employed, the experimental time points were rather short (up to 2-7 days). Ideally an in vitro culture system should reproduce the chronic stimulation of macrophages, thus modeling the pathological environment of the OA joint. Here we have set up and optimized a new in vitro model of long-lasting inflammation using primary human monocyte-derived macrophages. The concomitant use of hrM-CSF and hrGM-CSF maximized macrophage survival up to 15 days of culture. As pro-inflammatory stimuli, either LPS+IFN-g or Pam3CysSerLys4 (Pam3CSK4) were used, as well as a cocktail of recombinant inflammatory cytokines (IL-1b, IFN-g, TNFa), and of synovial fluids from OA patients. Finally, as a validation of this model we tested the anti-inflammatory efficacy of dexamethasone and celecoxib, two compounds commonly used in the clinic for the treatment of OA (38).

2.1 Characterization of pro-inflammatory mediators in the synovial fluid from OA-patients with the novel Ella^® methodology

We first detected the presence of several inflammatory mediators in synovial fluid (SF) samples obtained from patients. To measure low cytokine concentrations commonly found in biological fluids, we used a novel multiplex ELISA method (Ella Automated Immunoassay System^®) with a high sensitivity. In parallel experiments, we compared the conventional ELISA and the Ella^® methodology, confirming the higher sensitivity of the latter, as quantifiable levels of the cytokine IL-6 were detected in the SF of 5/5 patients, while only in 1/5 patients by ELISA (Supplementary Figure 1). Quantification of a panel of 8 inflammatory mediators by Ella^® showed that IL-6 and the chemokines CXCL8, CCL2, CCL5 and CXCL10 were highly represented in the SFs of patients with OA, together with lower but detectable levels of TNFa, IL-1b and IFN-g (Figure 1A-H). These results indicated which inflammatory markers should be tested in the in vitro macrophage culture model.

2.2 Optimization of primary monocyte differentiation with hrM-CSF or hrGM-CSF and stimulation with different TLR agonists to obtain pro-inflammatory human macrophages

In preliminary experiments, we sought to determine the best combination of growth/differentiation factors and pro-inflammatory stimuli to obtain M1-like inflammatory macrophages in vitro. Human monocyte-derived macrophages (HMDMs) were differentiated for 5 days, either with hrM-CSF (M-Mfs), to obtain M0 non-polarized macrophages, or with hrGM-CSF (GM-Mfs) to pre-condition macrophages towards a pro-inflammatory phenotype (39–41). Fully differentiated macrophages were stimulated for 24 hours with LPS, engaging TLR4, in combination with IFN-g, to obtain classical M1 macrophages, or with Pam3CSK4 binding to TLR2. TLR2 is considered as the main receptor involved in the recognition of damage associated molecular patterns (DAMPs), such as fragments of matrix molecules degraded upon tissue damage, a condition found in joints affected by OA (42).

Exposure of macrophages to different CSFs plus different pro-inflammatory stimuli resulted in their polarization towards a heterogeneous phenotype, reflected by their ability to produce different amounts of cytokines (Figure 2A-H). In particular, though IL-6 production appears in response to all tested conditions, the stimulation by GM-Mfs with LPS+IFN-g led to a 5-fold increase in the production of IL-6 compared to the other conditions (Figure 2B). Macrophages differentiated with GM-CSF also secreted more IL-1b compared to M-CSF-differentiated cells, and less IL-10, confirming the notion that GM-CSF promotes pro-inflammatory macrophages (Figure 2C-D). In macrophages stimulated with the TLR2 agonist Pam3CSK4, CXCL8 levels were higher compared to macrophages stimulated with LPS+IFN-g (Figure 2E). Similarly, CCL2 was much higher after Pam3CSK4 stimulation but only in M-Mfs (Figure 2F). Conversely, the concentration of CXCL10, the production of which is known to be induced by IFN-g, was coherently increased with LPS+IFN-g but not with Pam3CSK4 in M-Mfs, although detectable levels were produced in GM-Mfs (Figure 2G). Finally, the primary pro-inflammatory cytokine TNFa presented higher levels after LPS+IFN-g stimulation (Figure 2H).

On the whole, GM-CSF induced a differentiation in macrophages that were more prone to inflammation, and TLR2 engagement by Pam3CSK4 stimulated higher secretion of inflammatory mediators, especially CXCL8 and CCL2.

In parallel, we exposed macrophages to a pool of synovial fluids from 7 patients (used as an endogenous source of pro-inflammatory stimuli) or a cocktail of primary inflammatory cytokines (TNFa + IL-1b + IFN-g). As shown in Figure S2A and B, the secreted levels of IL-6 and IL-10 were minimal, indicating that macrophages were not sufficiently stimulated. The results clearly demonstrated that to optimize an in vitro model of activated pro-inflammatory macrophages the use of TLR-agonists is necessary.

2.3 The concurrent use of M-CSF and GM-CSF increases the survival of macrophages in vitro in a long-term assay

The above experiments were performed with monocytes differentiated for a short period of 5 days and exposed to stimuli only once. To better mimic in vitro the conditions found in arthritic joints, we set up a longer assay (15 days) with repeated exposure to stimuli. We therefore set up a model using hrM-CSF in combination with repeated exposure to hrGM-CSF. Monocytes were first primed with M-CSF (from day 0 to day 5), next with hrGM-CSF (from day 2 to day 5) and then stimulated with hrGM-CSF every 2-3 days (Figure 3A, M-/GM-Mf) and we compared this model with the stimulation with single CSFs (Figure 3A, M-Mf or GM-Mf). We first checked macrophage viability, as M-CSF or GM-CSF-macrophages do not usually survive in vitro more than 8-9 days. Using the Alamar Blue viability assay (Figure 3B) and bright field microscopy examination (Figure 3C) we observed that most M-Mfs or GM-Mfs do not survive up to 15 days. Conversely, M-/GM-Mfs presented the highest viability at day 15, compared to cells differentiated with single CSFs, independently from the set of pro-inflammatory stimuli used (LPS+IFN-g or Pam3CSK4). Thus, the culture of M-/GM-Mfs up to 15 days was used for the subsequent long-lasting experiments.

2.4 M-/GM-Mfs retain the long-lasting ability to produce pro-inflammatory cytokines after exposure to TLR ligands

Next, we explored whether long-lasting cultured M-/GM-Mf macrophages maintained the ability to produce inflammatory cytokines after repeated exposure either to the cocktail of recombinant cytokines (IL-1b, IFN-g, TNFa) or to LPS+IFN-g or to Pam3CSK4, collecting supernatants at different timepoints (days 6, 8, 10, 13, 15). As for the short assay, we found that repeated stimulation with IL-1b, IFN-g, TNFa did not sufficiently activate macrophages to secrete inflammatory mediators (Figure S3), while the use of TLR ligands led to a prolonged inflammatory activity up to day 15.

In particular, M-/GM-Mfs continuously stimulated with Pam3CSK4 showed a sustained production overtime of IL-6 and CXCL8, with no significant decrease at any timepoint except at day 8, while the stimulation with LPS+IFNg led to a sustained production of CXCL8, but not IL-6 (Figure 4A-B). On the contrary, the chemokine CCL2 was the only one to remain stable over time in M-/GM-Mfs stimulated both with LPS+IFN-g and Pam3CSK4 (Figure 4C). The anti-inflammatory cytokine IL-10 gradually decreased in M-/GM-Mfs stimulated with LPS+IFN-g, while in those stimulated with Pam3CSK4 the production was partially restored after a significant drop on days 8 and 10 (Figure 4D). Surprisingly, with both stimuli TNFa was highly secreted at day 6, then not detectable anymore at days 8, but a recovery was registered at days 10 and 13 before a final drop at day 15 (Figure 4E). The decrease of some inflammatory mediators at day 8 is likely to be explained with the long-recognized phenomenon of “endotoxin tolerance”. It is known that pre-activated monocytes/macrophages show hyposensitivity to a secondary stimulation due to the inactivity of NF-kB (caused by high levels of inhibitory p50 homodimers) and lower production of pro-inflammatory cytokines (43,44). In addition, macrophages can induce tolerance also affecting other immune cell types, such as T cells (45).

In the subsequent stimulations, however, macrophages recovered their ability to produce inflammatory mediators, and Pam3CSK4 performed significantly better than LPS+IFN-g at most timepoints tested, leading us to choose this condition for the testing of the anti-inflammatory drugs in the long-term.

2.5 Dexamethasone and celecoxib show anti-inflammatory activity in the long-lasting in vitro model

For the validation of our long-lasting in vitro model using macrophages as an effective screening platform to test new therapeutic strategies, we studied the anti-inflammatory activity of dexamethasone (DEX) and celecoxib (CEL), two compounds widely used in OA therapy. Firstly, we examined the viability of monocytes and macrophages exposed for 24 hours to these drugs (Figures S4-S5), showing no significant toxicity for DEX up to 100 mM or for CEL up to 50 mM. Both drugs demonstrated good anti-inflammatory efficacy when treating monocytes or macrophages, inhibiting the secretion of IL-6, CXCL8 and IL-10 (Figures S4-S5). Similarly, following the long-lasting protocol, we studied the viability of macrophages exposed to DEX or CEL at different concentrations (5 doses), added 1 hour before the stimulation with Pam3CSK4 (every 2-3 days, as described in Figure 5A). Extensive toxicity was observed in macrophages exposed to DEX concentrations between 10 mM and 25 nM at days 10 and 15 (Figure 5B), leading us to select the lower dose of 10 nM for the next experiments. Instead, no toxicity was observed for any concentration of CEL from 10 nM till 25 mM (Figure 5C), nor for the combined exposure to DEX 10 nM + CEL 25 mM (Figure 5D).

Thus, macrophages were treated in the long-lasting assay with DEX 10 nM and/or CEL 25 mM, at each timepoint, 1 hour before stimulation with Pam3CSK4; levels of pro-inflammatory mediators were tested at days 6, 8, 10, 13 and 15. As shown in Figure 6A, a significant reduction in IL-6 levels was observed for DEX with or without CEL at each time point, while CEL alone only showed a trend to IL-6 inhibition (not significant versus control). For CXCL8, the best anti-inflammatory activity was observed with DEX either alone or in combination with CEL at days 8, 10 and 13, while no significant efficacy was found for CEL alone, except at day 6 (Figure 6B). In a similar manner, CCL2 was inhibited just in the conditions with DEX (alone or together with CEL), but not with CEL monotherapy (Figure 6C).

Finally, to better define the anti-inflammatory activity of CEL, we also investigated the secretion of PGE₂, as this is the final product of the COX-2 enzyme inhibited by CEL treatment (Figure 7). Differently from the previous mediators, PGE₂ was detectable in the controls just on days 6, 8 and 15; however, at these timepoints, a clear inhibition was observed in macrophages treated with CEL alone and/or in combination with DEX, showing a greater efficacy mediated by CEL.

As a whole, these data demonstrate that also in the long-lasting model using chronically stimulated inflammatory macrophages DEX and CEL maintain their anti-inflammatory activity.

Osteoarthritis (OA) represents a hot topic in biomedical research, as a growing number of people is affected worldwide, and its therapeutic management remains challenging and high-cost demanding for developed countries. The introduction of appropriate in vitro models of chronic inflammation could aid to dissect the pathophysiology of this disabling condition, and at the same time allow the testing of new treatments to ameliorate the therapeutic management of the disease, alleviate the symptoms and protect the joints from prolonged damage. To this scope, we have established a long-lasting in vitro model of chronic inflammation using human primary macrophages, which can mimic OA-conditions and response to treatment. Our novel approach differs from previously described in vitro models. Firstly, most of the investigations have so far relied on short timepoints (maximum 7 days) as detailed in Supplementary Table (27–37), while our long-term model of macrophage cultures shows satisfactory cell viability and continuous secretion of inflammatory mediators up to 15 days; this is a crucial aspect, as acute and chronic inflammation activate distinct molecular signaling pathways and result in the production of different pro-inflammatory mediators. Secondly, most in vitro models have used established myeloid leukemia cell lines which are not fully representative of human chronically inflamed macrophages (27–29,31–33,46,47). Indeed, the development of an in vitro long-term culture model mirroring chronic inflammation and using primary human macrophages is useful for OA, but also for research related to other autoimmune diseases, as they have been invoked in a wide range of conditions, such as rheumatoid arthritis (48,49) or systemic sclerosis (50).

The setup of a long-term in vitro model of inflammatory macrophages is challenging, as the joint microenvironment is composed of strongly interconnected immune and non-immune cells continuously exposed to a complex inflammatory milieu.

M-CSF and GM-CSF induce the differentiation of monocytes into mature macrophages, but their effects showed some distinct features. M-CSF is physiologically produced in steady-state conditions (51) and is mostly used in experimental protocols to obtain fully differentiated non-polarized M0-macrophages (52), while GM-CSF is released in pathologic conditions, and supports the differentiation of macrophages prone to an inflammatory phenotype (39–41,53). Our results are in line with the existing literature: GM-Mfs produced higher concentrations of IL-6 and IL-1b while reducing the levels of the anti-inflammatory cytokine IL-10. Plus, in the long-lasting model, the continuous addition of GM-CSF promoted the survival of macrophages and their pro-inflammatory activity, a condition that we could not achieve by replacing GM-CSF with M-CSF (data not shown).

Cultured macrophages were stimulated with two TLR agonists: LPS in combination with IFN-γ, to obtain classically activated M1-macrophages, and Pam3CSK4 engaging the TLR2, best mimicking the DAMPs present in the OA-inflammatory milieu (54). We also used synovial fluids from OA patients as a source of endogenous inflammatory mediators and a cocktail of cytokines (IL-1β + TNFα + IFN-γ), but the two latter approaches did not sufficiently activate macrophages in an “inflammatory mode”, even in the long-term assay with repeated stimulation. Both LPS+IFN-γ and Pam3CSK4 lead to a M1-like polarization of macrophages, but with different patterns of secreted cytokines. In brief, while the use of LPS+IFN-g induced the typical acute-like inflammatory response, with strong production of TNFa, IL-6 and CXCL10, the agonist Pam3CSK4 promoted higher release of chemokines such as CXCL8 and CCL2, involved in the continuous recruitment of leukocytes. This latter finding is in accordance with a study that used repeated stimulations of alveolar macrophages with either Pam3CSK4 or LPS (55). Of note, a study by Xue et al. showed that macrophages stimulated with Pam3CSK4 together with TNFa and PGE₂ (referred as M^TPP) acquired a phenotype that differed from the classical M1-like macrophages obtained with LPS, and resembling that of cells in chronic inflammatory conditions (such as granulomatous diseases) (56). Another investigation carried out with murine bone marrow-derived macrophages demonstrated that Pam3CSK4 activates primarily the MAPK pathway, while LPS is particularly involved in type I interferon responses (57). In the same study, repeated stimulation of TLR2 did not result in reduced expression of Il6 or Il10 genes, contrary to what was observed for LPS.

Our long-term in vitro model of chronic inflammation with repeated stimulation by Pam3CSK4 was used to validate the anti-inflammatory activity of DEX and CEL, two drugs commonly used in patients with OA. DEX, which is known to deeply alter the transcription profile of macrophages (58), showed a profound anti-inflammatory effect, but also severe toxicity at higher doses. In contrast, CEL, selectively inhibiting the COX-2 enzyme (59), presented a milder anti-inflammatory activity, but no toxicity at higher concentrations.

It is interesting to note that a single treatment of monocytes or macrophages showed different effects by the two drugs, both in terms of toxicity and anti-tumor efficacy. For example, CEL inhibited, at least partially, IL-6, CCL2 and IL-1b production in short-term cultured macrophages and monocytes, while being almost inefficient in long-term cultured macrophages. This apparently negative result has anyway relevance in the pathological context because new myeloid cells are continuously recruited in the inflamed joints from the bloodstream; from these data it is inferred that newly arrived monocytes should be more sensitive to the anti-inflammatory effect of CEL. Furthermore, it should be considered that CEL in the long-term assay maintained its ability to efficiently inhibit PGE₂, which is also an important mediator of inflammation.

Regarding DEX, while monocytes and macrophages tolerate a single dose of 100 mM, repeated exposure to concentrations higher than 10 nM, (i.e. 1000 times less) is toxic for these cells. Regarding the anti-inflammatory effect, even in the long-term culture DEX showed good efficacy and significantly inhibited IL-6, CXCL8 and CCL2.

In conclusion, this long-lasting culture model of chronically inflamed macrophages is simple and reproducible and may constitute a useful platform for the screening of new therapeutic strategies for OA and other inflammatory diseases. Taken together, our experimental results suggest that a chronologically distinct use of DEX and CEL could be implemented in a clinical situation. DEX could be used in the short-term at higher doses, inducing a deep reprogramming of macrophages towards an anti-inflammatory phenotype. While a combination of low doses of DEX with higher doses of CEL could result in a sustained amelioration of the inflammation, with no significant toxicities.

5.1 Isolation of primary human monocytes from healthy blood donors, differentiation in macrophages and short-term protocol

Human monocytes were isolated from buffy coats of blood donations of healthy donors using two sequential separation media, Ficoll and Percoll, as already described previously (52). Whole blood was washed with saline and centrifuged over the Lympholyte^® Human-Cell Separation Media (Euroclone, Cat#CL5020) to recover peripheral blood mononuclear cells (PBMCs). Subsequently, cells were centrifuged on top of Percoll (Cytiva, Cat#17-0891-01) adjusted for its osmolarity (285 mOsm/l) and diluted to a final concentration of 46% in “complete” RPMI (Euroclone, Cat#ECB2000), supplemented with 10% Fetal Bovine Serum (Euroclone, Cat#ECS0186L), 0.5% penicillin/streptomycin 10,000 U/mL (Gibco, Cat#15140122) and 0.5 % UltraGlutamine Supplement 200 mM (Lonza, Cat#BE17-605E/U1). For experiments employing monocytes, cells were directly challenged with pro-inflammatory stimuli and/or anti-inflammatory compounds (as detailed below). To obtain human monocyte derived macrophages (HMDMs), monocytes were resuspended in warm (37°C) RPMI without serum and plated (1.1*10⁶ cells/well in 24-well plates, 1.6*10⁶ cells/well in 12-well plates) to allow adhesion for 30 minutes at 37°C. Media was replaced with complete RPMI, containing the selected colony stimulating factor (CSF). To obtain M-CSF differentiated HMDMs (M-Mfs) media containing hrM-CSF 25 ng/ml (Peptrotech, Cat#300-25) was added, while GM-CSF differentiated macrophages (GM-Mfs) were obtained using hrGM-CSF (Peptrotech, Cat#300-03) at the same concentration. Monocytes, M-Mfs and GM-Mfs were then stimulated in the short-term (24 hours) with 3 different set of stimuli: i) LPS 100 ng/ml (Sigma, Cat#L4005) in combination with hrIFN-γ 50 ng/ml (Peprotech, Cat#300-02); ii) Pam3CSK4 100 ng/ml (InvivoGen, Cat#tlrl-pms); iii) a cocktail of pro-inflammatory recombinant cytokines, hrIL-1β (Peprotech, Cat# 200-01B), hrTNFα (Peprotech, Cat# 300-01A) and hrIFN-γ, all at the same concentration of 50 ng/ml.

The anti-inflammatory drugs used were dexamethasone (DEX, Acofarma, Cat#M0013570) and celecoxib (CEL, Merck, Cat# PHR1683) at indicated concentrations. The cells were exposed to these drugs for 1 hour before the addition of the pro-inflammatory stimuli and kept in the cell culture media until the end of the experiment.

5.2 Protocol for in vitro chronic stimulation of macrophages in the long-term

The long-lasting protocol was performed by replacing the culture media from day 5 to day 15 every 2-3 days (specifically on days 5, 6, 8, 10, 13). Plus, for this set of experiments, M/GM-Mfs were prepared by adding hrGM-CSF 25 ng/ml on day 2, on monocytes already plated with hrM-CSF 25 ng/ml on day 0, and by the supplement of hrGM-CSF every time the culture media was replaced. For the pro-inflammatory stimulation, the same 3 different set of stimuli introduced for the short-term protocol were used and the stimuli were added at each timepoint (described in each figure). Similarly, dexamethasone and celecoxib were added 1 hour before the pro-inflammatory stimuli and kept in the media until its replacement in the following timepoint.

5.3 Synovial fluid collection and processing

Synovial fluid (SF) samples were obtained from the knee of 11 patients (4 males, 7 females, 66.2±11.2, m±SD) undergoing TKA in the Knee Surgery unit at Humanitas Research Hospital. Informed consent was acquired from all the patients following the Declaration of Helsinki and the approval from the Ethical committee of our institute (study number: 2515; ID experimentation: 1341). All samples were collected under sterile conditions and centrifuged (430 rcf for 10 minutes at room temperature). The supernatant was then collected, and two sets of samples have been prepared: a small aliquot (0.4 ml) was stored at -80°C and used directly for the ELISA assays (see next section) while the remaining supernatants of all the samples were mixed to create a pool for direct stimulation of macrophages.

5.4 Toxicity and ELISA assays

All toxicity assays were performed using Alamar blue reagent (ThermoFisher, Cat#DAL1100) following the manufacturer’s protocol. ELISA assays were performed using commercial kits of the DuoSet series provided by Bio-Techne SRL: IL-6 (Cat#DY206), CXCL8 (Cat#DY208), IL-1β (Cat# DY201), TNFα (Cat#DY210), IL-10 (Cat#DY217B), CXCL10 (Cat#DY266), CCL2 (Cat#DY279). Synovial fluid aliquots were analyzed using the multiplex ELISA assay platform Ella (Bio-Techne SRL), using two different sets of cytokines: CCL2, CCL5, IL-6, IFN-γ on one plate, CXCL10, CXCL8, TNFα, IL-1β on the other one. These combinations were selected to match the standard curves for the different cytokines. The assays for PGE₂ were provided by Cayman (Cat#004CA514010-96).

5.5 Bright-field microscopy and staining

The images of macrophages stimulated with the different conditions were obtained by bright-field microscopy on samples stained with the Diff Quik® Staining Set (Medion Diagnostics AG, Cat# 130832) according to manufacturer’s protocol. The fixing agent and the two dyes provided by the kit were sequentially added for 1 minute on the cells, removing each one at the end of the incubation time. At the end, the plates were washed using tap water, dried overnight, and acquired in brightfield microscopy (Inverted Microscope IX53, Olympus).

5.6 Statistical analyses

All the statistical tests were performed with GraphPad Prism 9.4.1. Further details are provided in each corresponding figure legend.

CEL = celecoxib

DAMPs = damage associated molecular patterns

DEX = dexamethasone

IFN-g = interferon-g

IRF = interferon regulatory factors

LPS = lipopolysaccharide

OA = osteoarthritis

Pam3CSK4 = Pam3CysSerLys4

SF = synovial fluid

TKA = total knee arthroplasty

TLR4 = toll-like receptor 4

Ethics approval and consent to participate

Informed consent was acquired from all the patients following the Declaration of Helsinki and the approval from the Ethical committee of our institute (study number: 2515; ID experimentation: 1341)

Consent for publication

Not applicable

Availability of data and material

All data related to this paper can be requested from the corresponding authors

Competing interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 814444 (MEFISTO). AU is supported by a FIRC-AIRC fellowship for Italy. FTA is supported by a “Miguel Servet Grant” CP22/00106 by AES 2022, ISCIII, Spain.

Authors' contributions

PA, FTA, EK decided the scientific concept behind the designed experiments. AU and AP performed the in vitro experiments, under the scientific supervision of PA, FTA and CA. LAR, APL, RM, MC, NC, FMG helped in the isolation of HMDMs and in the quantification of the cytokines by ELISA. AU wrote a first draft of the manuscript, which was improved with the contributions from PA, FTA, EK, FR. All authors reviewed and agreed on the final version of the manuscript.

Acknowledgements

We wish to thank Sonia Valentino at Humanitas Research Hospital for the technical support.

Authors’ information

1. Department of Biomedical Sciences, Humanitas University, 20072 Pieve Emanuele (Milan) – Italy.

2. IRCCS Humanitas Research Hospital, 20089 Rozzano (Milan) – Italy.

3. Agorà Biomedical Sciences, Etromapmacs Pole, 71010 Lesina (Foggia) – Italy.

4. Center for Research in Molecular Medicine & Chronic Diseases (CiMUS), Universidade de Santiago de Compostela, 15706 Santiago de Compostela, Spain.

5. Instituto de Investigación Biomédica de A Coruña (INIBIC), Oncology Department, Complexo Hospitalario de A Coruña (CHUAC), 15006 A Coruña, Spain.

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No competing interests reported.

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A novel in vitro model of long-lasting inflammation using primary human macrophages for osteoarthritis research

Status:

Version 1

Abstract

Figures

1. Introduction

2. Results

2.1 Characterization of pro-inflammatory mediators in the synovial fluid from OA-patients with the novel Ella^® methodology

2.2 Optimization of primary monocyte differentiation with hrM-CSF or hrGM-CSF and stimulation with different TLR agonists to obtain pro-inflammatory human macrophages

2.3 The concurrent use of M-CSF and GM-CSF increases the survival of macrophages in vitro in a long-term assay

2.4 M-/GM-Mfs retain the long-lasting ability to produce pro-inflammatory cytokines after exposure to TLR ligands

2.5 Dexamethasone and celecoxib show anti-inflammatory activity in the long-lasting in vitro model

3. Discussion

4. Conclusions

5. Methods

5.1 Isolation of primary human monocytes from healthy blood donors, differentiation in macrophages and short-term protocol

5.2 Protocol for in vitro chronic stimulation of macrophages in the long-term

5.3 Synovial fluid collection and processing

5.4 Toxicity and ELISA assays

5.5 Bright-field microscopy and staining

5.6 Statistical analyses

6. List Of Abbreviations

7. Declarations

Ethics approval and consent to participate

Consent for publication

Availability of data and material

Competing interests

Funding

Authors' contributions

Acknowledgements

Authors’ information

8. References

Additional Declarations

Supplementary Files

Status:

Version 1

A novel in vitro model of long-lasting inflammation using primary human macrophages for osteoarthritis research

Status:

Version 1

Abstract

Figures

1. Introduction

2. Results

2.1 Characterization of pro-inflammatory mediators in the synovial fluid from OA-patients with the novel Ella® methodology

2.2 Optimization of primary monocyte differentiation with hrM-CSF or hrGM-CSF and stimulation with different TLR agonists to obtain pro-inflammatory human macrophages

2.3 The concurrent use of M-CSF and GM-CSF increases the survival of macrophages in vitro in a long-term assay

2.4 M-/GM-Mfs retain the long-lasting ability to produce pro-inflammatory cytokines after exposure to TLR ligands

2.5 Dexamethasone and celecoxib show anti-inflammatory activity in the long-lasting in vitro model

3. Discussion

4. Conclusions

5. Methods

5.1 Isolation of primary human monocytes from healthy blood donors, differentiation in macrophages and short-term protocol

5.2 Protocol for in vitro chronic stimulation of macrophages in the long-term

5.3 Synovial fluid collection and processing

5.4 Toxicity and ELISA assays

5.5 Bright-field microscopy and staining

5.6 Statistical analyses

6. List Of Abbreviations

7. Declarations

Ethics approval and consent to participate

Consent for publication

Availability of data and material

Competing interests

Funding

Authors' contributions

Acknowledgements

Authors’ information

8. References

Additional Declarations

Supplementary Files

Status:

Version 1

2.1 Characterization of pro-inflammatory mediators in the synovial fluid from OA-patients with the novel Ella^® methodology