Bioengineered Nanoparticles Loaded-Hydrogels to Target TNF Alpha in Inflammatory Diseases

Rheumatoid Arthritis (RA) is an incurable autoimmune disease that promotes the chronic impairment of patients’ mobility. For this reason, it is vital to develop therapies that target early inflammatory symptoms and act before permanent articular damage. The present study offers two novel therapies based in advanced drug delivery systems for RA treatment: encapsulated chondroitin sulfate modified poly(amidoamine) dendrimer nanoparticles (NPs) covalently bonded to monoclonal anti-TNF α antibody in both Tyramine-Gellan Gum and Tyramine-Gellan Gum/Silk Fibroin hydrogels. Using pro-inflammatory THP-1 (i.e., human monocytic cell line), the therapy was tested in an inflammation in vitro model under both static and dynamic conditions. Firstly, we demonstrated effective NP-antibody functionalization and TNF-α capture. Upon encapsulation, the NPs were released steadily over 21 days. Moreover, in static conditions, the approaches presented good anti-inflammatory activity over time, enabling the retainment of a high percentage of TNF α. To mimic the physiological conditions of the human body, the hydrogels were evaluated in a dual-chamber bioreactor. Dynamic in vitro studies showed absent cytotoxicity in THP-1 cells and a significant reduction of TNF-α in suspension over 14 days for both hydrogels. Thus, the developed approach showed potential for use as personalized medicine to obtain better therapeutic outcomes and decreased adverse effects.

Increased LPS levels coexist with systemic inflammation and result in monocyte activation in severe COVID-19 patients.

This study aimed to evaluate the link between microbial translocation markers and systemic inflammation at the earliest time-point after hospitalization and at the last 72 h of hospitalization in survivors and non-survivors COVID-19 patients. Sixty-six SARS-CoV-2 RT-PCR+ infected patients and nine non-COVID-19 pneumonia controls were admitted in this study. Blood samples were collected at hospital admission (T1) (Controls and COVID-19+ patients) and 0-72 h before hospital discharge (T2, alive or dead) to analyze systemic cytokines and chemokines, LPS concentrations, and soluble CD14 (sCD14) levels. THP-1 human monocytic cell line was incubated with plasma from survivors and non-survivors COVID-19 patients and their phenotype, activation status, TLR4, and chemokine receptors were analyzed by flow cytometry. COVID-19 patients presented higher IL-6, IFN-γ, TNF-α, TGF-β1, CCL2/MCP-1, CCL4/MIP-1β, and CCL5/RANTES levels than controls. Moreover, LPS and sCD14 were higher at hospital admission in SARS-CoV-2-infected patients. Non-survivors COVID-19 patients had increased LPS levels concomitant with higher IL-6, TNF-α, CCL2/MCP-1, and CCL5/RANTES levels at T2. Increased expression of CD16 and CCR5 were identified in THP-1 cells incubated with the plasma of survivor patients obtained at T2. The incubation of THP-1 with T2 plasma of non-survivors COVID-19 leads to higher TLR4, CCR2, CCR5, CCR7, and CD69 expression. In conclusion, increased microbial translocation during hospitalization coexist with the inflammatory condition of SARS-CoV-2 infection and could lead to higher monocyte activation in non-survivors COVID-19 patients.

Comparative Proteomic Analysis Reveals Varying Impact on Immune Responses in Phorbol 12-Myristate-13-Acetate-Mediated THP-1 Monocyte-to-Macrophage Differentiation

Macrophages are sentinels of the innate immune system, and the human monocytic cell line THP-1 is one of the widely used in vitro models to study inflammatory processes and immune responses. Several monocyte-to-macrophage differentiation protocols exist, with phorbol 12-myristate-13-acetate (PMA) being the most commonly used and accepted method. However, the concentrations and duration of PMA treatment vary widely in the published literature and could affect the probed phenotype, however their effect on protein expression is not fully deciphered. In this study, we employed a dimethyl labeling-based quantitative proteomics approach to determine the changes in the protein repertoire of macrophage-like cells differentiated from THP-1 monocytes by three commonly used PMA-based differentiation protocols. Employing an integrated network analysis, we show that variations in PMA concentration and duration of rest post-stimulation result in downstream differences in the protein expression and cellular signaling processes. We demonstrate that these differences result in altered inflammatory responses, including variation in the expression of cytokines upon stimulation with various Toll-like receptor (TLR) agonists. Together, these findings provide a valuable resource that significantly expands the knowledge of protein expression dynamics with one of the most common in vitro models for macrophages, which in turn has a profound impact on the immune as well as inflammatory responses being studied.

DENGUE VIRUS ALTERS SIALIC ACID RESIDUES CONFIGURATION IN MACROPHAGES

The activation of the innate immune response requires sialic acid residues removal. Nevertheless, it is unknown the role of these changes during the Dengue virus infection. We determine if during Dengue virus infection, the sialic acid residues alter on the macrophages. The human monocytic cell line THP-1 was differentiated into macrophages and were infected with Dengue virus. The changes in sialic acid were evaluated by lectin blot in the cellular lysate. The activity of neuraminidase was defined by RT-PCR and fluorescence assays. Macrophages infection with DENV-2 reduces α-2,6 sialic acid residues at 24 h, and α-2,3 sialic acid residues lower at 48 h in some proteins. Transcriptional profile and enzymatic activities of Neu-1 showed a narrow decrease. Sialic acid residues oscillation in varied conformations and times suggest a role of a selective mechanism to remove these residues. The lesser participation of Neu-1 in this process could be concomitant to other similar enzymes such as sialyl-transferases, or the phenomenon requires minimal activity to have a relevant biological function.

Engineered IgG1-Fc Molecules Define Valency Control of Cell Surface Fcγ Receptor Inhibition and Activation in Endosomes

The inhibition of Fcγ receptors (FcγR) is an attractive strategy for treating diseases driven by IgG immune complexes (IC). Previously, we demonstrated that an engineered tri-valent arrangement of IgG1 Fc domains (SIF1) potently inhibited FcγR activation by IC, whereas a penta-valent Fc molecule (PentX) activated FcγR, potentially mimicking ICs and leading to Syk phosphorylation. Thus, a precise balance exists between the number of engaged FcγRs for inhibition versus activation. Here, we demonstrate that Fc valency differentially controls FcγR activation and inhibition within distinct subcellular compartments. Large Fc multimer clusters consisting of 5-50 Fc domains predominately recruited Syk-mScarlet to patches on the plasma membrane, whereas PentX exclusively recruited Syk-mScarlet to endosomes in human monocytic cell line (THP-1 cells). In contrast, SIF1, similar to monomeric Fc, spent longer periods docked to FcγRs on the plasma membrane and did not accumulate and recruit Syk-mScarlet within large endosomes. Single particle tracking (SPT) of fluorescent engineered Fc molecules and Syk-mScarlet at the plasma membrane imaged by total internal reflection fluorescence microscopy (SPT-TIRF), revealed that Syk-mScarlet sampled the plasma membrane was not recruited to FcγR docked with any of the engineered Fc molecules at the plasma membrane. Furthermore, the motions of FcγRs docked with recombinant Fc (rFc), SIF1 or PentX, displayed similar motions with D ~ 0.15 μm2/s, indicating that SIF1 and PentX did not induce reorganization or microclustering of FcγRs beyond the ligating valency. Multicolor SPT-TIRF and brightness analysis of docked rFc, SIF1 and PentX also indicated that FcγRs were not pre-assembled into clusters. Taken together, activation on the plasma membrane requires assembly of more than 5 FcγRs. Unlike rFc or SIF1, PentX accumulated Syk-mScarlet on endosomes indicating that the threshold for FcγR activation on endosomes is lower than on the plasma membrane. We conclude that the inhibitory effects of SIF1 are mediated by stabilizing a ligated and inactive FcγR on the plasma membrane. Thus, FcγR inhibition can be achieved by low valency ligation with SIF1 that behaves similarly to FcγR docked with monomeric IgG.

Discovery of a highly potent novel rifampicin analog by preparing a hybrid of the precursors of the antibiotic drugs rifampicin and clofazimine

Tuberculosis (TB) is an infectious disease caused by the bacillus Mycobacterium tuberculosis (Mtb). The present work reports the design and synthesis of a hybrid of the precursors of rifampicin and clofazimine, which led to the discovery of a novel Rifaphenazine (RPZ) molecule with potent anti-TB activity. In addition, the efficacy of RPZ was evaluated in-vitro using the reference strain Mtb H37Rv. Herein, 2,3 diamino phenazine, a precursor of an anti-TB drug clofazimine, was tethered to the rifampicin core. This 2,3 diamino phenazine did not have an inherent anti-TB activity even at a concentration of up to 2 µg/mL, while rifampicin did not exhibit any activity against Mtb at a concentration of 0.1 µg/mL. However, the synthesized novel Rifaphenzine (RPZ) inhibited 78% of the Mtb colonies at a drug concentration of 0.1 µg/mL, while 93% of the bacterial colonies were killed at 0.5 µg/mL of the drug. Furthermore, the Minimum Inhibitory Concentration (MIC) value for RPZ was 1 µg/mL. Time-kill studies revealed that all bacterial colonies were killed within a period of 24 h. The synthesized novel molecule was characterized using high-resolution mass spectroscopy and NMR spectroscopy. Cytotoxicity studies (IC50) were performed on human monocytic cell line THP-1, and the determined IC50 value was 96 µg/mL, which is non-cytotoxic.

Dose-dependent phorbol 12-myristate-13-acetate-mediated monocyte-to-macrophage differentiation induces unique proteomic signatures in THP-1 cells

Macrophages are sentinels of the innate immune system, and the human monocytic cell line THP-1 is one of the widely used in vitro models to study immune responses. Several monocyte-to-macrophage differentiation protocols exist, with phorbol 12-myristate-13-acetate (PMA) being the widely used and accepted method. However, the concentrations and duration of PMA treatment vary widely in the published literature and their effect on protein expression is not fully deciphered. In this study, we employed a dimethyl labeling-based quantitative proteomics approach to determine the changes in the protein repertoire of macrophage-like cells differentiated from THP-1 monocytes by three commonly used PMA-based differentiation protocols. Our analysis shows that variations in PMA concentration and duration of rest post-stimulation result in downstream differences in the protein expression and cellular processes. We demonstrate that these differences result in altered gene expression of cytokines upon stimulation with various TLR agonists. Together, these findings provide a valuable resource that significantly expands the knowledge of protein expression dynamics with one of the most common in vitro models for macrophages, which in turn has a profound impact on the immune responses being studied.

Macrophage-derived Netrin-1 participates in endometriosis-associated pain

Background: Endometriosis is a common disease in reproductive-age women and usually causes pelvic pain. Endometriosis pain is considered as a kind of neuropathic pain and infiltrating nerve fiber in endometriotic lesions may play an important role. Netrin-1 is widely reported as an axon guidance cue that regulates axonal attraction or rejection in neural injury and regeneration. In this study, we aim to determine the role of Netrin-1 in endometriosis-related pain. Methods: Peripheral blood, peritoneal fluid, and endometrial tissues were sampled from women with (n=37) and without (n=23) endometriosis. Serum Netrin-1 concentrations, endometrial expression levels of Netrin-1 and its receptors including DCC, A2BAR, UNC5B, UNC5C and DSCAM were assessed. The polarization phenotypes of the peritoneal macrophages were identified by detecting the marker expression of M1 (CD86+) /M2 (CD163+) macrophages via flow cytometry. Lipopolysaccharide (LPS) and interferon gamma (IFN-γ) stimulated human monocytic cell line (THP-1) and rat alveolar macrophage-derived cell line (NR8383) cells to induce M1 phenotype macrophages. The expression levels of M1 markers and Netrin-1 in THP-1/NR8383 cells were determined. Results: The expression levels of Netrin-1 in serum and endometriotic lesions were significantly higher in women with endometriosis when compared with those in women without endometriosis (P<.05), and both were correlated with pain symptoms (P<.05). Netrin-1 was co-expressed with CD 68 (a macrophage marker) in endometriotic lesions, and was synthesized and secreted by THP-1 and NR8383 cells in process of M1 polarization. In women with endometriosis, peritoneal macrophages were polarized towards M1 phenotype. In addition, increased expression of DCC and A2BAR, and decreased expression of UNC5B, UNC5C and DSCAM in endometriotic lesions were found. Conclusions: These results suggest that Netrin-1 production by macrophages in endometriotic lesions may play an important role in endometriosis pain.