Life science research focuses on deciphering the biochemical mechanisms that regulate cell proliferation and function and largely depends on the use of tissue culture methods in which cells are grown on two-dimensional hard plastic or glass surfaces. However, the flat surface of the tissue culture plate represents a poor topological approximation of the complex three-dimensional (3D) architecture of a tissue or organ composed of various cell types, extracellular matrix (ECM) and interstitial fluids. Moreover, if we consider a cell as a perfectly defined volume, flattened cells have full access to the environment and limited cell-to-cell contact. However if the cell is a cube in a simple cuboidal epithelium, then its access to the lumen is limited to one face, with the opposite face facing the basal membrane and the remaining four faces lying in close contact with neighbouring cells. This is of great importance when considering the access of viruses and bacteria to the cell surface, the excretion of soluble factors or proteins or the signalling within or between cells. This short review discusses various cell culture approaches to improve the simulation of the 3D environment of cells.

To analyse the effects of season and reproductive status on seasonal changes in body composition of female muskoxen (Ovibos moschatus), we examined 196 muskox cows fromVictoria Island, Northwest Territories, Canada, during 16 collections carried out by Inuit hunters in April, May, July, August, September, and November from 1989 to 1993. During this time, the density of muskoxen on the island increased slowly and they numbered about45,000 in 1993. Hunting and predation by wolves (Canis lupus) were limited, and there was little evidence of muskox mortality due to malnutrition. Adult muskox cows ( 4 years old) were leanest in May, when the ingesta‐free body weight (IFBW) and estimated fat weight of newly‐lactating cows were 116 ± 2 kg (adjusted mean ± S.E.) and 13.7 ± 0.8 kg, respectively, and those of non‐lactating cows were 112 ± and 12.2 ± 1.6 kg. These weights peaked in September at 166 ± 4 kg and 37.8± 1.8 kg in lactating cows and 176 ± 7 and 45.1 ± 3.4 kg in non‐lactating cows. The most rapid losses of fat (242g/day) and protein (55g/day) occurred i lactating cows in late winter. The weight of ingesta, primarily rumen contents, increase during the year from April (20.9% of body weight) to November (27.8%) and was exceptionally high for ruminants. Seasonal patterns in 2‐ and 3‐year‐old females were generally similar to those in older cows, although the younger cows were leaner and weighed less. Pregnancy rates increased from 0 in females 18–23 months old to 25% in cows 30–35 months old and 63% in females 42 months old. Mammary gland weight in lactating females peaked in July and August, suggesting that peak milk production did not occur until at least two months after calving, and lactation was sometimes extended to a year or more. Lactation in November did not preclude pregnancy, but females which were pregnant at this time were fatter and heavier than non‐pregnant cows. These results indicate a high‐amplitude annual cycle of body reserves in female muskoxen, and suggest a considerable sensitivity of pregnancy rates to nutritional influences.

Rewiring of host cytokine networks is a key feature of inflammatory bowel diseases (IBD) such as Crohn’s disease (CD). Th1-type cytokines—IFN-γ and TNF-α—occupy critical nodes within these networks and both are associated with disruption of gut epithelial barrier function. This may be due to their ability to synergistically trigger the death of intestinal epithelial cells (IECs) via largely unknown mechanisms. In this study, through unbiased kinome RNAi and drug repurposing screens we identified JAK1/2 kinases as the principal and nonredundant drivers of the synergistic killing of human IECs by IFN-γ/TNF-α. Sensitivity to IFN-γ/TNF-α-mediated synergistic IEC death was retained in primary patient-derived intestinal organoids. Dependence on JAK1/2 was confirmed using genetic loss-of-function studies and JAK inhibitors (JAKinibs). Despite the presence of biochemical features consistent with canonical TNFR1-mediated apoptosis and necroptosis, IFN-γ/TNF-α-induced IEC death was independent of RIPK1/3, ZBP1, MLKL or caspase activity. Instead, it involved sustained activation of JAK1/2-STAT1 signalling, which required a nonenzymatic scaffold function of caspase-8 (CASP8). Further modelling in gut mucosal biopsies revealed an intercorrelated induction of the lethal CASP8-JAK1/2-STAT1 module during ex vivo stimulation of T cells. Functional studies in CD-derived organoids using inhibitors of apoptosis, necroptosis and JAKinibs confirmed the causative role of JAK1/2-STAT1 in cytokine-induced death of primary IECs. Collectively, we demonstrate that TNF-α synergises with IFN-γ to kill IECs via the CASP8-JAK1/2-STAT1 module independently of canonical TNFR1 and cell death signalling. This non-canonical cell death pathway may underpin immunopathology driven by IFN-γ/TNF-α in diverse autoinflammatory diseases such as IBD, and its inhibition may contribute to the therapeutic efficacy of anti-TNFs and JAKinibs.

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