Inflammation

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) [1]. 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.

Asthma and chronic obstructive pulmonary disease (COPD) are two long-term inflammatory conditions of the lung airways, both related with exposure to environmental factors (like pollution and allergens) for asthma and smoking for COPD [2, 3]. The pharmaceutical approach for asthma and COPD is still relatively ineffective and based on the administration of drugs that will control the symptoms. These conditions may worsen over the years, requiring periodic hospitalization (asthma) or evolving by developing a cardiovascular-associated disease or lung cancer (COPD).

Atopic dermatitis (AD) and psoriasis are another two examples of long-term chronic diseases, characterised by a periodic flare of the symptoms which include red and itchy skin, swollen and cracked (in case of AD) or dry and scaly followed by abnormal skin growth (psoriasis) [4, 5]. As for asthma and COPD, the is no cure for AD and psoriasis and current treatments can just control the symptoms reducing the chances of disease progression.

Osteoarthritis (OA) is the most common type of arthritis affecting preferentially hands, knees, hips, lower back and neck [6]. 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.

Inflammatory bowel disease (IBD) 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 [7]. 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.

There has been extensive interest over many years in agents that could be used to reduce chronic inflammation, a process that may take years or even decades to produce detectable symptoms difficult to reverse. Therefore, there is a requirement for more definitive mechanistic studies and bioassays towards understanding the processes governing chronic inflammation that would facilitate the discovery of personalised therapeutic strategies. 

[1] Parnes O. The Lancet. 2008, Aug 23. 

[2] Papi A et al. The Lancet. 2018, Feb 24. 

[3] King TP. Clin Transl Med. 2015; 4: 26.

[4] Weidinger S. et al. The Lancet. 2015, Sep 13.

[5] Greb EJ et al. Nature Rev Dis Prim. 2016, Nov 24.

[6] Conaghan PG et al. Nature Rev Rheum. 2019, May 8.

[7] Guan Q. J Immunol Res. 2019, Dec 1.

1. Atopic Dermatitis

Primary human keratinocyte model

Primary healthy human keratinocytes were pre-treated with Compound – 004 at different concentrations for an hour before stimulating with a cocktail of Poly (I:C) + IL4 + IL13 and incubated at 37°C, 5% CO2 for another 48 hours. Analysis of inflammatory markers in the resulting supernatant at the end of the treatment were performed using Human Magnetic Luminex® Assay  Data is representative of 3 independent repeats (n=3).

Primary human keratinocyte model

2. Human follicle dermal papilla cells and Inflammation

3. Inflammatory Bowel Disease (IBD)

4. Osteoarthritis (OA)

Osteoarthritis (OA) Inflammation in vitro model – Healthy chondrocytes

Healthy chondrocytes were pre-treated with SM04690 and Pterosin B for 1 hour followed by stimulation with IL1β for 72 hours. Supernatants were analysed using Luminex and ELISA* for inflammation markers (A: TNFα, B: IL-6, C: MMP-1, D: LIF, E: IL-11, F: MMP-13, G: PGE2*). Lysates were analysed for COX-2 by ELISA (H). Each condition is representative of 3 technical repeats (n=3; mean±SEM). All treatment conditions were compared to the Stimulated conditions (***p<0.0005, **P<0.005, *p<0.05). Statistical analysis was performed using One-way ANOVA.

Osteoarthritis (OA) Inflammation in vitro model – OA chondrocytes

OA chondrocytes were pre-treated with SM04690 and Pterosin B for 1 hour followed by stimulation with IL1β for 72 hours. Supernatants were analysed using Luminex and ELISA* for inflammation markers (A: TNFα, B: IL-6, C: MMP-1, D: LIF, E: IL-11, F: MMP-13, G: PGE2*). Lysates were analysed for COX-2 by ELISA (H). Each condition is representative of 3 technical repeats (n=3; mean±SEM). All treatment conditions were compared to the Stimulated conditions (***p<0.0005, **P<0.005, *p<0.05). Statistical analysis was performed using One-way ANOVA.

Osteoarthritis Cartilage Degradation in vitro model – Healthy chondrocytes

Healthy chondrocytes were pre-treated with SM04690 and Pterosin B for 1 hour. Cells were stimulated with TNFα and Oncostatin M for 6 hours. Lysates were analysed using QuantiGene™ for gene expression of cartilage degradation markers (A: IHH, B: MMP-13, C: MMP-3, D: MMP-1, and E: COL10A1).  Results were normalised to the housekeeping genes GAPDH and HPRT1. Each condition is representative of 3 technical repeats (n=3; mean±SEM). All treatment conditions were compared to the Stimulated conditions (***p<0.0005, **P<0.005, *p<0.05). Statistical analysis was performed using One-way ANOVA.

Osteoarthritis Cartilage Degradation in vitro model – OA chondrocytes

OA chondrocytes were pre-treated with SM04690 and Pterosin B for 1 hour.  Cells were stimulated with IL1β for 6 hours. Lysates were analysed using QuantiGene™ for gene expression of cartilage degradation markers (A: IHH, B: MMP-13, C: MMP-3, D: MMP-1, and E: COL10A1).  Results were normalised to the housekeeping genes GAPDH and HPRT1. Each condition is representative of 3 technical repeats (n=3; mean±SEM). All treatment conditions were compared to the Stimulated conditions (***p<0.0005, **P<0.005, *p<0.05). Statistical analysis was performed using One-way ANOVA.

Osteoarthritis Hypertrophy in vitro model – Healthy chondrocytes

24 hrs

Healthy chondrocytes were pre-treated with SM04690 and Pterosin B for 1 hour, followed by stimulation with TGFβ1 for 24 hours. Lysates were analysed using QuantiGene™ for markers of hypertrophy (A: MMP-13, B: COL10A1, C: COL1A1, D: VEGFA, and E: RUNX2).  Results were normalised to the housekeeping genes GAPDH and HPRT1. Each condition is representative of 3 technical repeats (n=3; mean±SEM). All treatment conditions were compared to the Stimulated conditions (***p<0.0005, **P<0.005, *p<0.05). Statistical analysis was performed using One-way ANOVA

Osteoarthritis Hypertrophy in vitro model – Healthy chondrocytes

Healthy chondrocytes were pre-treated with SM04690 and Pterosin B for 1 hour, followed by stimulation with TGFβ1 for 48 hours. Lysates were analysed using QuantiGene™ for markers of hypertrophy (A: MMP-13, B: COL10A1, C: COL1A1, D: VEGFA, and E: RUNX2).  Results were normalised to the housekeeping genes GAPDH and HPRT1. Each condition is representative of 3 technical repeats (n=3; mean±SEM). All treatment conditions were compared to the Stimulated conditions (***p<0.0005, **P<0.005, *p<0.05). Statistical analysis was performed using One-way ANOVA.

48 hrs

Osteoarthritis Hypertrophy in vitro model – OA chondrocytes

OA chondrocytes were pre-treated with SM04690 and Pterosin B for 1 hour, followed by stimulation with TGFβ1 for 24 hours. Lysates were analysed using QuantiGene™ for markers of hypertrophy (A: MMP-13, B: COL10A1, C: COL1A1, D: VEGFA, and E: RUNX2).  Results were normalised to the housekeeping genes GAPDH and HPRT1. Each condition is representative of 3 technical repeats (n=3; mean±SEM). All treatment conditions were compared to the Stimulated conditions (***p<0.0005, **P<0.005, *p<0.05). Statistical analysis was performed using One-way ANOVA.

Osteoarthritis Hypertrophy in vitro model – OA chondrocytes

OA chondrocytes were pre-treated with SM04690 and Pterosin B for 1 hour, followed by stimulation with TGFβ1 for 48 hours. Lysates were analysed using QuantiGene™ for markers of hypertrophy (A: MMP-13, B: COL10A1, C: COL1A1, D: VEGFA, and E: RUNX2).  Results were normalised to the housekeeping genes GAPDH and HPRT1. Each condition is representative of 3 technical repeats (n=3; mean±SEM). All treatment conditions were compared to the Stimulated conditions (*p<0.05). Statistical analysis was performed using One-way ANOVA.

Osteoarthritis Cartilage Anabolism in vitro model – Healthy chondrocytes

Healthy chondrocytes were pre-treated with SM04690 and Pterosin B for 1 hour, followed by stimulation with TGFβ1 for 24 hours and 48 hours. Lysates were analysed using QuantiGene™ for markers of cartilage anabolism (A: COL2A1, B: SOX9, and C: ACAN).  Results were normalised to the housekeeping genes GAPDH and HPRT1. Each condition is representative of 3 technical repeats (n=3; mean ±SEM). All treatment conditions were compared to the Stimulated conditions (***p<0.0005, **P<0.005, *p<0.05). Statistical analysis was performed using One-way ANOVA.

Osteoarthritis Cartilage Anabolism in vitro model – OA chondrocytes

OA chondrocytes were pre-treated with SM04690 and Pterosin B for 1 hour, followed by stimulation with TGFβ1 for 24 hours and 48 hours. Lysates were analysed using QuantiGene™ for markers of cartilage anabolism (A: COL2A1, B: SOX9, and C: ACAN).  Results were normalised to the housekeeping genes GAPDH and HPRT1. Each condition is representative of 3 technical repeats (n=3; mean±SEM). All treatment conditions were compared to the Stimulated conditions (***p<0.0005, **P<0.005, *p<0.05). Statistical analysis was performed using One-way ANOVA.

5. Probiotics and its role on LPS-induced PBMC inflammation

PBMCs from 3 healthy volunteers were incubated for 24 hours with 5 probiotic strains (A, B, C, D, E) at 3 concentrations (Low, Medium, High) or with Vehicle (V) represented by cryoprotectant used to freeze the probiotics, Lipopolysaccharide (LPS) 100 ng/ml, or LPS 100ng/ml and Wortmannin 1000 nM (LPS+W). Bars represent the average of 3 donors and the bars represent Standard Error Mean (SEM).

6. Psoriasis

Psoriasis: Inflammation in vitro model

Effect of JAK inhibitor on LPS-induced IL8, TNFα and IL1α release from primary human epidermal keratinocytes after 48 hour stimulation

Psoriasis: Inflammation in vitro model

Primary human epidermal keratinocytes were pre-treated with JAK inhibitor or STAT3 inhibitor for 1 hour before stimulation with a cytokine cocktail of IL1α + TNFα +Oncostatin M for 48 hours. Cell culture supernatants were collected and expression levels of various inflammatory mediators measured by multiplex immunoassay. Results are expressed as pg/ml, mean ± SEM

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 for 5 days. BrdU incorporation assay was then performed and results are expressed as absorbance values. Data presented as Mean ± SEM

Analysis of keratinocyte differentiation markers:

Human epidermal keratinocytes were cutured in appropriate growth media and then 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.

Expression levels of key differentiation markers were analysed. Values are represented as Mean ± SEM Of the mRNA levels normalized to GAPDH mRNA levels.

7. Vascular Permeability

HUVEC cells were allowed to form a monolayer, prior to treatment with the VEGFR inhibitor axitinib (AX) and VEGF-A. A no treatment 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 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 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. This could account for the increase in fluorescence output observed with the  FITC-Dextran analysis as VEGF-A would have caused the HUVEC  monolayer to become more porous and permeable. Pre-treatment with AX maintained the integrity of the monolayer cell arrangement as that found in the untreated condition.

HUVEC transmigration
FITC-Dextran Vascular Permeability Assay

Confluent layer of Human umbilical vein endothelial cells (HUVECs) were treated with TNFα (100 ng/ml) in transwell inserts for 24 hours in the presence or absence of the test compounds. Disruptions of the barrier integrity or vascular permeability was assessed by FITC-Dextran permeability. Passage of fluorescent dextran molecules through the endothelial cell monolayer is proportional to the monolayer’s permeability. The extent of permeability was then obtained by measuring the fluorescence of the receiver plate well solution.

Signalling Pathway regulating vascular permeability in HUVECs

Test compound inhibits TNFα induced p38 phosphorylation in HUVEC cells. Confluent layer of Human umbilical vein endothelial cells (HUVECs) were treated with TNFα (100 ng/ml) in transwell inserts for 24 hours in the presence or absence of the test compounds.

Whole cell lysate was analysed using Luminex xMAP technology to measure the level of total and phosphorylated p38. Bars represent the ratio of phosphorylated versus total p38.

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