2.2. Pro-Inflammatory Cytokines Stimulation of the Caco2/TC7 and HT29/MTX Co-Culture

Caco-2/TC7 (mono-culture) or Caco-2/TC7 and HT29-MTX (co-culture) were seeded on 6-well polycarbonate membrane cell culture inserts with HD 0.4 µm pores at a density of 4 × 10⁵ cells (Corning, Dutscher, Issy-les-Moulineaux, France). Culture medium was distributed on the insert (1 mL) and in the well (2 mL) supporting the insert. This set of experiments was realized in order to assess the modification of the response of the co-culture to the induction of an inflammatory response observed within the mono-culture. Indeed, Caco-2 cells are known to be able to produce inflammatory mediators in response to pro-inflammatory stimuli [24], and particularly the TC7 clone [25]. Cells were grown for 21 days to reach full differentiation. The culture medium was changed every other day. The day before the stimulation, cells were rinsed with phosphate buffered saline (PBS) and put into a serum-free medium. After 24 h in the depleted medium, cells were stimulated with a cocktail of pro-inflammatory cytokines—TNF-α (20 ng/mL), IL-1β (1 ng/mL) and IFN-ϒ (10 ng/mL) (BioTechne, Lille, France)—for 2, 6 or 24 h for the mono-culture and for 6 h for the co-culture, as this was the exposure timepoint that achieved the best stimulation of the mono-culture.

At the end of the stimulation, the insert medium (called below “apical medium”) and well medium (called below “basal medium”) were harvested and stored at −80 °C until further measurement of markers of intestinal inflammation and permeability. The cell layers were harvested in TriZOL and processed immediately.

To confirm the pro-inflammatory stimulation of the mono- and the co-culture by the cocktail of pro-inflammatory cytokines, we first measured the modification of the expression of genes involved: 1) in the pro-inflammatory signaling pathways—C-X-C motif chemokine ligand 8 (CXCL8) coding for IL-8, mitogen-activated protein kinase 14 (MAPK14) coding for p38-α, mitogen-activated protein kinase 8 (MAPK8) coding for JNK1, mitogen-activated protein kinase-kinase-kinase 1 (MAP3K1) coding for MEKK1, signal transducer and activator of transcription 3 (STAT3), nuclear factor-kappa B (NF-κB), tumor necrosis factors α (TNF-α), myeloid differentiation primary myeloid response 88 (MyD88); and 2) in the pro-apoptotic pathways—Caspase 3 and Caspase 9 (CASP3 and 9).

Total RNA was isolated from cells using TriZOL (Fisher Scientific SAS, Illkirch, France) and RNA concentration was measured spectrophotometrically (NanoDrop 2000, Thermo Scientific, Illkirch, France). cDNA was obtained from 1 µg RNA using the QuantiTect Reverse Transcription Kit following the manufacturer’s instructions (Qiagen, Courtaboeuf, France). Absorbance ratios at 260/280 nm and at 260/230 nm were measured by spectrophotometry to assess the purity of the DNA samples.

Primers and SYBR Green PCR master mix were respectively purchased from Eurofins Scientific France (Nantes, France) and Qiagen (Courtaboeuf, France) (Table 1). QPCRs were run on a StepOnePlus Real-Time PCR System (Applied Biosystem, Foster City, CA, USA) and the data were processed with the OneStepPlus software. Reactions were performed in duplicate. Levels of amplified cDNA were quantified using the 2^−ΔΔCT method (ΔΔCt = ΔCt_exposed − mean ΔCt_control) compared to Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) considered as the best housekeeping gene from two different genes tested (GAPDH vs. PPIA).

List of primers used in the study.

The levels of IL-8 secretion in both the basal and apical media of mono- and co-cultures were measured after stimulation with the cocktail of cytokines. Measurement of IL-8 in the media harvested at the end of stimulation were performed using an ELISA kit according to the manufacturer’s instructions (Human IL-8/CXCL8 DuoSet ELISA, #DY208, BioTechne, Lille, France) [26].