Inflammation

Inflammation is a natural process initiated when we need to defend ourselves from intruders like foreign bodies, irritant molecules or pathogens (like bacteria and viruses).

In response to this aggression, our body unleashes the neutrophils, a type of white blood cell which move toward the affected area through the enlargement of the small blood vessels, releasing specific molecules (like cytokines, reactive oxygen species and proteases) defending our body. This process is called acute inflammation appearing with key signs: pain (continuous or just on touching the area affected), redness and heat (increased blood supply and flow), loss of functionality (difficult to move the limb, breathing or loss of smell) and swelling, due to the development of an edema following fluids build up. Sometimes these symptoms may not occur, but the person may experience tiredness or fever. Acute inflammation may last for few days or persist for few weeks; in this case is called sub-acute inflammation.

Inflammation is a controlled process and should disappear as soon as the harmful substance or the pathogen is removed. Persistence of this defence mechanism for months or even years, called chronic inflammation, could result in tissue damage producing severe conditions like asthma, chronic obstructive pulmonary disease, atopic dermatitis, psoriasis, osteoarthritis and inflammatory bowel disease.

Cellomatics Biosciences provides physiologically relevant in vitro inflammatory disease models to investigate the efficacy and/or toxicity of test compounds. These assays can be customised to suit the client’s requirements with options for bespoke assays with multiple end-point measurements.

Atopic Dermatitis

Atopic dermatitis (AD) is a chronic, complex allergic inflammatory skin disease that affects 10-20% of the population, characterised by a periodic flare of the symptoms which include red and itchy skin, swollen and cracked. Initial immune response begins with the T helper (Th)2 response and is then shifted to Th1 in the chronic phase.

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Atopic Dermatitis - Feasibility study

Primary healthy human keratinocytes were treated with different concentrations of IL-13 along with Poly I:C for 48 hours. Inflammatory markers in the resulting supernatants were analysed using a Human Magnetic Luminex® Assay (n=3, mean±SEM).

Atopic Dermatitis

Primary healthy human keratinocytes were treated with different concentrations of drug along with Poly I:C+ IL-13 (50 ng/ml) for 48 hours. Inflammatory markers in the resulting supernatants were analysed using a Human Magnetic Luminex® Assay (n=3, mean±SEM).

Psoriasis

An inflammatory skin disorder, triggered or exacerbated by a number of genetic, environmental, or immunological factors. It is characterised by: Hyperproliferation of keratinocytes, Abnormal epidermal differentiation and Infiltration of inflammatory cells.

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Primary human keratinocyte differentiation

Keratinocyte differentiation was assessed following treatment with calcium + serum, treatment with a cytokine cocktail, or as a consequence of reaching high confluence (untreated). The majority of untreated control cells differentiated by day 6. Differentiated cells could be readily identified by their large size and cobblestone morphology (white arrows). Treatment with a combination of calcium and serum caused the keratinocytes to rapidly differentiate (>24 hours), resulting in flattened, fibrous, skin-like morphology which was sustained throughout the 6 days of treatment. The cytokine-treated cells (exposed to TNF-α, Oncostatin-M and IL1-α) seemed to exbibit delayed, aberrant differentiation, which is one of the hallmarks of psoriasis. Black arrows highlight irregular filament formation visible only in cytokine treated cells.

Keratinocyte Inflammatory Mediators

Human epidermal keratinocytes were differentiated in media supplemented with FBS and Calcium. The cells were then pre-treated with JAK3/STAT3 inhibitors followed by stimulation with a cytokine cocktail of IL1α + TNFα + Oncostatin M for 48 hours. Supernatants were analysed for various inflammatory mediators using Luminex Multiplex Assay (n=3±SEM;*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001)

Keratinocyte Differentiation Markers

Human epidermal keratinocytes were allowed to differentiate in media supplemented with FBS and Calcium. The cells were then pre-treated with JAK3/STAT3 inhibitors followed by stimulation with a cytokine cocktail of IL1α + TNFα + Oncostatin M for 48 hours. Gene expression levels of key differentiation markers were analysed using Luminex Quantigene™ Assay. The mRNA expressions are normalised to GAPDH and fold change calculated relative to the unstimulated control (n=3±SEM; *p<0.05;**p<0.01;***p<0.001; ****p<0.0001).

Psoriasis: Proliferation in vitro model

Healthy Human epidermal keratinocytes were stimulated with recombinant IL17A (100 ng/ml) and cultured in the presence or absence of STAT3 Inhibitor (Cryptotanshinone) for 5 days. A BrdU incorporation assay was then performed and results are expressed as absorbance values (n=3, mean±SEM; **p<0.01; ***p<0.001).

Psoriasis: Inflammation in vitro model

Effect of JAK Inhibitor on LPS-induced IL8, TNFα and IL1α release from primary human epidermal keratinocytes after 48 hours of treatment. The analytes were measured in cell supernatants using a Luminex Multiplex Assay (n=3, mean±SEM; ****p<0.0001).

IBD

It is a term used to describe used to describe disorders that involve chronic inflammation of the digestive tract. Ulcerative colitis is mainly localised into the colon (large intestine), while Crohn’s disease can affect any part of the digestive system. In both cases, symptoms may range from mild to severe, with periods of active illness followed by temporary remission. There is currently no cure for IBD, and medications tend to reduce the symptoms preventing severe complications, which include colon cancer.

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Caco-2 Model for IBD: Tight Junction Proteins

Caco-2 cells were differentiated for 21 days and stained for tight junction proteins such as ZO-1 (purple) and Occludin (orange) along with DAPI (blue– nucleus). The images were captured at 20X magnification using high content imager and the intensities of each fluorescence analysed.

Caco-2 Model for IBD: TEER Reported

Caco-2 cells were seeded onto trans-well inserts and allowed to differentiate for 21 days and TEER was measured regularly. On day 21 (Basal), the monolayer was treated Dexamethasone and stimulated with LPS+ IL1beta cocktail for 48 hours. TEER measurements were continued at 24 and 48 hours post treatment (pt) and reported as Ω.cm2 (n=3±SEM). 

Caco-2 Model for IBD: Tight Junction Proteins

Caco-2 cells differentiated for 21 days were treated with Dexamethasone followed by stimulation with LPS+ IL1beta cocktail for 48 hours. The monolayers were stained for tight junction proteins such as ZO-1 (green) with DAPI (blue– nucleus). The images were captured using ImageXpress Pico Imager and the fluorescent intensities analysed to obtain Percentage (%) of Positive Cells.

Caco-2 Model for IBD: Permeability

Caco-2 cells were differentiated for 21 days and treated with Dexamethasone followed by stimulation with LPS+ IL1beta cocktail for 48 hours. FITC-Dextran was added to the apical chamber along with the stimulants. Breaks in the monolayer were detected by the permeability of FITC-Dextran into the basal chamber. A significant reduction in FITC dextran was observed in Dexamethasone when compared to stimulated condition (****p<0.0001; n=3±SEM). The RFU of all the conditions were normalised to the untreated control (100). A red dotted line indicates the level of the untreated.

Caco-2 Model for IBD: Inflammatory Mediators

Caco-2 cells were differentiated for 21 days and subsequently treated with Dexamethasone, followed by stimulation with LPS+ IL1beta cocktail for 8 hours. mRNA expression in the cell lysates was quantified using the Luminex Quantigene™ Assay. A second set of differentiated Caco-2 cells were maintained for 48 hours post treatment. The supernatants were analysed after 48h for inflammatory mediators using a Luminex Multiplex Assay (**p<0.01; ***p<0.001; ****p<0.0001; n=3 mean±SEM).  

Osteoarthritis (OA)

It is the most common type of arthritis affecting preferentially hands, knees, hips, lower back and neck. It occurs when the smooth cushion between bones, called cartilage, become damaged after a sustained inflammatory response and thus joints become painful, swollen and hard to move. Pain, reduced mobility and adverse effects from medications could substantially affect the quality of life.

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Osteoarthritis Inflammation in vitro model

Primary healthy human chondrocytes were pre-treated with SM04690 and Pterosin B, followed by stimulation with IL1β for 72 hours. Supernatants were analysed for Inflammatory mediators using Luminex Multiplex Assay (n=3±SEM; ).

Osteoarthritis Cartilage Degradation in vitro model

Primary healthy human chondrocytes were pre-treated with SM04690 and Pterosin B, followed by stimulation with TNFα and Oncostatin M for 6 hours. Cell lysates were analysed for cartilage degradation markers using Luminex Quantigene™ Assay. Gene expressions were normalised to house-keeping genes (HKG- GAPDH and HPRT1) and fold changes calculated relative to the untreated (vehicle control) (n=3±SEM).

Osteoarthritis Cartilage Anabolism in vitro model

Primary healthy human chondrocytes were pre-treated with SM04690 and Pterosin B, followed by stimulation with TGFβ1 for 24 and 48 hours. Cell lysates were analysed for markers of cartilage anabolism (COL2A1; SOX9 and ACAN) using Luminex Quantigene™ Assay. Gene expressions were normalised to house-keeping genes (HKG- GAPDH and HPRT1) and fold changes calculated relative to the untreated (vehicle control) (n=3±SEM).

Osteoarthritis Hypertrophy in vitro model

Primary healthy human chondrocytes were pre-treated with SM04690 and Pterosin B, followed by stimulation with TGFβ1 for 48 hours. Cell lysates were analysed for hypertrophy markers using Luminex Quantigene™ Assay. Gene expressions were normalised to house-keeping genes (HKG- GAPDH and HPRT1) and fold changes calculated relative to the untreated (vehicle control) (n=3±SEM).

Other Models

Our team has developed other models including Vascular permeability assay and a human follicle dermal papilla cell based inflammatory model.

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Vascular Permeability

HUVECs were allowed to form a monolayer, prior to treatment with the VEGFR inhibitor Axitinib (AX) and VEGF-A. Untreated control was also included as part of the analysis. (A) FITC-Dextran mediated measure of permeability. VEGF-A treatment resulted in an increase in monolayer permeability as indicated by the increase in fluorescence output (RFU) relative to the untreated condition. Pre-treatment with AX reduced the fluorescence output despite the addition of VEGF-A, showing that AX reverses the permeabilisation effect of VEGF-A. (B) Calcein-AM staining of HUVEC cells after drug treatment. A tight HUVEC monolayer was formed in the untreated condition but upon treatment with VEGF-A, the cellular arrangement has changed indicating movement and motility. Pre-treatment with AX maintained the integrity of the monolayer cell arrangement as that found in the untreated condition. 

Human Dermal Papilla Cells and Inflammation

Human Follicle Dermal Papilla Cells were treated with Minoxidil and Emodin for 48 hours. Cell lysates were then analysed for TGFβ1, TGFβ2, IGF1 and VEGFA gene expression using Quantigene™ Multiplex Assay. Changes in expression levels for each treatment condition were normalised to the house keeping gene and compared to the vehicle control (***p<0.001, **p<0.01, *<p0.05; n=3±SEM). 

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To request a consultation where we can discuss your exact requirements, please contact Cellomatics Biosciences.