Journal: Arthritis Research & Therapy

Article Title: Engineered self-regulating macrophages for targeted anti-inflammatory drug delivery

doi: 10.1186/s13075-024-03425-3

Figure Lengend Snippet: Overview of the therapeutic macrophages and inducible NF-κB lentiviral synthetic promoter. A Overview of the generation of engineered macrophages from the bone marrow of C57BL/6 mice following lentiviral transduction of the inducible NF-κB lentiviral circuit. B Response of IL-1 signaling in the therapeutic NF-κB-IL-1Ra circuit. (1) Following lentiviral transduction, (2) IL-1 signaling through the IL-1 receptor initiates a cascade leading to (3) nuclear translocation and increased transcriptional activity of NF-κB, activating an inflammatory transcriptional program and induces Il1rn expression, (4) driving production of IL-1Ra that acts as an IL-1 receptor antagonist (5) inhibiting activation of the NF-κB inflammatory cascade. An NRE was inserted upstream of the promoter to reduce background signal. To characterize activation dynamics, a separate inducible NF-κB circuit, denoted as ‘LUC’, was designed to drive the firefly luciferase gene downstream of the same promoter; IL-1Ra: interleukin-1 receptor antagonist; NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells; NRE: negative regulatory element

Article Snippet: IL-1Ra concentration was measured with DuoSet enzyme-linked immunosorbent assay (ELISA) specific to mouse IL-1Ra (R&D Systems).

Techniques: Transduction, Translocation Assay, Activity Assay, Expressing, Activation Assay, Luciferase

Journal: Arthritis Research & Therapy

Article Title: Engineered self-regulating macrophages for targeted anti-inflammatory drug delivery

doi: 10.1186/s13075-024-03425-3

Figure Lengend Snippet: NF-κB-IL-1Ra macrophages offer moderate chondroprotection against inflammatory mediators and inflammatory driven matrix degradation in response to IL-1β. A Timeline of co-culture experimental conditions. miPSC derived cartilage pellets were initially pre-treated with 0, 0.5 or 10 ng/mL of IL-1β prior to co-culture for 24 h. Pellets were placed into co-culture with transduced macrophages and re-treated with 0, 0.5, or 1 ng/mL of IL-1β for 48 h in a 24 well Transwell plate ( n = 3–6). B Total IL-1Ra in co-culture over 48 h, as measured by ELISA normalized to cell number, following treatment with IL-1β. C Concentration of nitric oxide secreted into the media normalized to cell number following treatment with IL-1β. D Gene expression of miPSC derived cartilage pellets in co-culture in reference to non-treated control when challenged with IL-1β for 48 h normalized to Gapdh and R18s ( n = 4–6). E Gene expression of engineered macrophages in co-culture in reference to non-treated control when challenged with IL-1β for 48 h normalized to Gapdh and R18s ( n = 4–6). F Amount of sGAG per pellet normalized to DNA content. G Total amount of sGAG secreted into the media. H and J Histological staining was quantified for the total area of positive tissue for all Safranin-O (red)/fast green (blue)/hematoxylin(purple)-stained tissue sections of miPSC derived cartilage co-cultured with NF-κB-LUC or NF-κB-IL-1Ra macrophages. I and K Histological staining was again quantified for the total area of positive tissue for all type II collagen tissue sections (brown stain) of miPSC derived cartilage co-cultured with NF-κB-LUC or NF-κB-IL-1Ra macrophages. Statistical significance were assessed using a two-way ANOVA with Tukey’s post hoc test; values represent mean with standard error of the mean (SEM) ( n = 4–6); histological samples ( n = 3–18)

Article Snippet: IL-1Ra concentration was measured with DuoSet enzyme-linked immunosorbent assay (ELISA) specific to mouse IL-1Ra (R&D Systems).

Techniques: Co-Culture Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Expressing, Control, Staining, Cell Culture

Journal: Arthritis Research & Therapy

Article Title: Engineered self-regulating macrophages for targeted anti-inflammatory drug delivery

doi: 10.1186/s13075-024-03425-3

Figure Lengend Snippet: Engineered macrophages with the NF-κB synthetic promoter dynamically autoregulate in response to inflammation in monolayer. A NF-κB-LUC macrophages demonstrate circuit activation by 4 h which is resolved by 72 h following treatment with IL-1β ( n = 5). B Relative luminescence normalized to a 24-hour baseline by NF-κB-LUC macrophages increases in response to higher concentrations of IL-1β over time ( n = 12); red arrow represents addition of stimulus. C NF-κB-IL-1Ra macrophage production of IL-1Ra, as measured by ELISA, following stimulation with 1 or 10 ng/ml of IL-1β at 0 and 48 h normalized to a non-treated control ( n = 5); red arrow represents addition of stimulus. D ELISA for IL-1Ra production from non-transduced and NF-κB-IL-1Ra macrophages in response to no treatment, 10 or 70 ng/mL IL-1β demonstrates significant production of IL-1Ra in NF-κB-IL-1Ra macrophages ( n = 4). E Gene expression of NF-κB-IL-1Ra BMDMs in monoculture in reference to non-transduced, non-treated BMDMs when challenged with 10 or 70 ng/mL IL-1β for 48 h normalized to Gapdh and R18s ( n = 4) demonstrated significant reduction in NF-κB responsive genes by NF-κB-IL-1Ra macrophages for both medium and high doses. Data was assessed using a one or two-way ANOVA with Tukey’s post hoc test; values represent mean with standard error of the mean (SEM) ( n = 4–12); RFU: relative luminescence unit; IL-1β: Interleukin-1 beta

Article Snippet: IL-1Ra concentration was measured with DuoSet enzyme-linked immunosorbent assay (ELISA) specific to mouse IL-1Ra (R&D Systems).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Control, Expressing

Journal: Arthritis Research & Therapy

Article Title: Engineered self-regulating macrophages for targeted anti-inflammatory drug delivery

doi: 10.1186/s13075-024-03425-3

Figure Lengend Snippet: NF-κB-IL-1Ra macrophages offer moderate chondroprotection against inflammatory mediators and inflammatory driven matrix degradation in response to IL-1β. A Timeline of co-culture experimental conditions. miPSC derived cartilage pellets were initially pre-treated with 0 or 10 ng/mL of IL-1β for 24 h. Pellets were then placed into new monoculture wells with 0 or 1 mg/mL of anakinra or in co-culture with transduced macrophages (NF-κB-LUC or NF-κB-IL-1Ra) and again treated with 0 or 10 ng/mL of IL-1β for an additional 48 h in a 24 well transwell plate ( n = 6). B IL-1Ra levels in the media following 48 h of monoculture or co-culture, as measured by ELISA. C Concentration of nitric oxide secreted into the media normalized to cell number following treatment with 0 or 10 ng/mL of IL-1β. D Gene expression of miPSC derived cartilage pellets in mono- and co-culture in reference to non-treated control pellets and E engineered macrophages in reference to a non-treated control when challenged with IL-1β for 48 h normalized to Gapdh and R18s ( n = 4–6). F Amount of sGAG per pellet normalized to DNA content. G and I Histological staining was quantified for the total area of positive tissue for all Safranin-O (red)/fast green(blue)/hematoxylin(purple)-stained tissue sections of miPSC derived cartilage; scale bar = 200 mm. H and J Histological staining was quantified for the total area of positive tissue for all type II collagen tissue sections (brown stain) of miPSC derived cartilage; scale bar = 200 mm. Statistical significance was assessed using a two-way ANOVA with Tukey’s post hoc test; values represent mean with standard error of the mean (SEM) ( n = 4–6)

Article Snippet: IL-1Ra concentration was measured with DuoSet enzyme-linked immunosorbent assay (ELISA) specific to mouse IL-1Ra (R&D Systems).

Techniques: Co-Culture Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Expressing, Control, Staining

Journal: Arthritis Research & Therapy

Article Title: Engineered self-regulating macrophages for targeted anti-inflammatory drug delivery

doi: 10.1186/s13075-024-03425-3

Figure Lengend Snippet: NF-κB-LUC and NF-κB-IL-1Ra macrophages differentially activate to co-culture with synovial fibroblasts following an inflammatory stimulus. A Timeline of co-culture experimental conditions. B NF-κB-LUC macrophages demonstrate circuit activation by 4 h following treatment with IL-1β that significantly increases in response to co-culture with synovial fibroblasts ( n = 6). C IL-1Ra production increased in response to IL-1β treatment with indirect and conditioned media demonstrating higher levels of production between control and therapeutic macrophages ( n = 3–5). D-F Gene expression of NF-κB-IL-1Ra and NF-κB-LUC macrophages with synovial fibroblasts when challenged with IL-1β for 48 h normalized to Gapdh and R18s ( n = 4) demonstrated significant reduction in NF-κB responsive genes when co-cultured with NF-κB- IL-1Ra macrophages. Data was assessed using a two-way ANOVA with Tukey’s post hoc test; values represent mean with standard error of the mean (SEM) ( n = 4–6)

Article Snippet: IL-1Ra concentration was measured with DuoSet enzyme-linked immunosorbent assay (ELISA) specific to mouse IL-1Ra (R&D Systems).

Techniques: Co-Culture Assay, Activation Assay, Control, Expressing, Cell Culture

Journal: Arthritis Research & Therapy

Article Title: Engineered self-regulating macrophages for targeted anti-inflammatory drug delivery

doi: 10.1186/s13075-024-03425-3

Figure Lengend Snippet: NF-κB-IL-1Ra engineered macrophages maintain presence for at least 7 days in the K/BxN serum transfer mouse model of RA. A Mice injected with NF-κB-IL-1Ra macrophages exhibited reduced ( B / F ) Bioluminescent imaging over time following injection of NF-κB-IL-1Ra and NF-κB-LUC macrophages in K/BxN mice demonstrated a reduction over time but as of day 7 there was still significant macrophage presence at the injection site ( C / G ) with ( D , H , I ) presence in the paws but ( E ) no presence in the clearance organs but. J Immunohistochemistry for GFP + staining confirmed injected macrophage presence in the paws of mice compared to non-treated and negative stained sections. Data was assessed using a two-way ANOVA with Tukey’s post hoc test; values represent mean with SEM ( n = 9–15); photons/second (p/s) with a baseline of 10 4 ; IP intraperitoneal; R, right; L, left; HR, hind right; HL, hind left

Article Snippet: IL-1Ra concentration was measured with DuoSet enzyme-linked immunosorbent assay (ELISA) specific to mouse IL-1Ra (R&D Systems).

Techniques: Injection, Imaging, Immunohistochemistry, Staining

Journal: Arthritis Research & Therapy

Article Title: Engineered self-regulating macrophages for targeted anti-inflammatory drug delivery

doi: 10.1186/s13075-024-03425-3

Figure Lengend Snippet: Engineered macrophages polarize following injection to a more inflammatory phenotype in the K/BxN treated mice. A and B Prior to injection macrophage efficiency was profiles using flow cytometry for positive CD11b and GFP labeling. C and D Macrophage activation was also examined for inflammatory marker CD80 and anti-inflammatory marker 206 ( n = 5–6). At 6 days following injection, peritoneal fluid at injection site was collected and examined to observe ( E-H ) presence of GFP positive macrophages still present at the injection site as well as ( I and J ) macrophage activation post injection using the same markers CD80 and CD206 ( n = 4–10). Data was assessed using a two-way ANOVA with Tukey’s post hoc test; values represent mean with SEM for NF-κB-IL-1Ra vs. NF-κB-LUC

Article Snippet: IL-1Ra concentration was measured with DuoSet enzyme-linked immunosorbent assay (ELISA) specific to mouse IL-1Ra (R&D Systems).

Techniques: Injection, Flow Cytometry, Labeling, Activation Assay, Marker

Journal: Arthritis Research & Therapy

Article Title: Engineered self-regulating macrophages for targeted anti-inflammatory drug delivery

doi: 10.1186/s13075-024-03425-3

Figure Lengend Snippet: NF-κB-IL-1Ra engineered macrophages mitigate disease in the K/BxN serum transfer mouse model of RA. Mice were examined daily in all four paws for ( A ) ankle thickness ( B ) clinical scores and ( C ) pain measurement by electric von Frey (EVF)* over time; *EVF was not taken at day 7. At terminal endpoint (day 7), despite the variability of animals in each treatment group, reduction was pronounced between all groups in terms of ( D ) ankle thickness and ( E ) clinical scores, but was not significantly different from baseline in terms of ( F ) pain measurement by SMALGO. G IL-1Ra present in the serum of mice was not significantly different by day 7 but mice that received NF-κB-IL-1Ra macrophages still showed less systemic inflammation as compared to mice with NF-κB-LUC macrophages or PBS controls. Data was assessed using a one or two-way ANOVA with Tukey’s post hoc test for * NF-κB-IL-1Ra vs. PBS and # NF-κB-LUC vs. PBS; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; values represent mean with SEM ( n = 9–15)

Article Snippet: IL-1Ra concentration was measured with DuoSet enzyme-linked immunosorbent assay (ELISA) specific to mouse IL-1Ra (R&D Systems).

Techniques:

Journal: Arthritis Research & Therapy

Article Title: Engineered self-regulating macrophages for targeted anti-inflammatory drug delivery

doi: 10.1186/s13075-024-03425-3

Figure Lengend Snippet: IL-1Ra engineered macrophages protect against bone damage. A Region of micro-CT analysis for all mice examining ( B ) Bone volume/tissue volume and ( C ) bone erosion as measured by bone surface/bone volume. At day 7, NF-κB-IL-1Ra macrophages somewhat reduced cartilage degradation ( D / E ) and inflammation ( F / G ) correlating to clinical score. Total animals per group in each range of clinical score is displayed as a percentage. Zoomed images are shown in the black boxes. Data was assessed using a one-way ANOVA with Tukey’s post hoc test; values represent mean with SEM ( n = 9–15)

Article Snippet: IL-1Ra concentration was measured with DuoSet enzyme-linked immunosorbent assay (ELISA) specific to mouse IL-1Ra (R&D Systems).

Techniques: Micro-CT