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In lung disease, persistence of KRT8-expressing aberrant basaloid cells in the alveolar epithelium is associated with impaired tissue regeneration and pathological tissue remodeling. We analyzed single cell RNA sequencing datasets of human interstitial lung disease and found the profibrotic Interleukin-11 (IL11) cytokine to be highly and specifically expressed in aberrant KRT8 + basaloid cells. IL11 is similarly expressed by KRT8 + alveolar epithelial cells lining fibrotic lesions in a mouse model of interstitial lung disease. Stimulation of alveolar epithelial cells with IL11 causes epithelial-to-mesenchymal transition and promotes a KRT8-high state, which stalls the beneficial differentiation of alveolar type 2 (AT2)-to-AT1 cells. Inhibition of IL11-signaling in AT2 cells in vivo prevents the accumulation of KRT8 + cells, enhances AT1 cell differentiation and blocks fibrogenesis, which is replicated by anti-IL11 therapy. These data show that IL11 inhibits reparative AT2-to-AT1 differentiation in the damaged lung to limit endogenous alveolar regeneration, resulting in fibrotic lung disease. In interstitial lung disease, aberrant KRT8+ basaloid cells impair alveolar repair mechanisms. Here they show that Interleukin-11 expression in KRT8+ cells potentiates their pathological properties and causes lung scarring, which can be prevented by anti-IL11 therapy.

The global shortage of donor corneas has garnered extensive interest in the development of graft alternatives suitable for endothelial keratoplasty using cultivated primary human corneal endothelial cells (CECs). We have recently described a dual media approach for the propagation of human CECs. In this work, we characterize the effects of a Rho-kinase inhibitor Y-27632 on the cultivation of CECs propagated using the dual media culture system. Seventy donor corneas deemed unsuitable for transplantation were procured for this study. We assessed the use of Y-27632 for its effect at each stage of the cell culture process, specifically for cell attachment, cell proliferation and during both regular passaging and cryopreservation. Lastly, comparison of donor-matched CEC-cultures expanded with or without Y-27632 was also performed. Our results showed that Y-27632 significantly improved the attachment and proliferation of primary CECs. A non-significant pro-survival effect was detected during regular cellular passage when CECs were pre-treated with Y-27632, an effect that became more evident during cryopreservation. Our study showed that the inclusion of Y-27632 was beneficial for the propagation of primary CECs expanded via the dual media approach and was able to increase overall cell yield by between 1.96 to 3.36 fold.

The advent of cell culture-based methods for the establishment and expansion of human corneal endothelial cells (CEnC) has provided a source of transplantable corneal endothelium, with a significant potential to challenge the one donor-one recipient paradigm. However, concerns over cell identity remain, and a comprehensive characterization of the cultured CEnC across serial passages has not been performed. To this end, we compared two established CEnC culture methods by assessing the transcriptomic changes that occur during in vitro expansion. In confluent monolayers, low mitogenic culture conditions preserved corneal endothelial cell state identity better than culture in high mitogenic conditions. Expansion by continuous passaging induced replicative cell senescence. Transcriptomic analysis of the senescent phenotype identified a cell senescence signature distinct for CEnC. We identified activation of both classic and new cell signaling pathways that may be targeted to prevent senescence, a significant barrier to realizing the potential clinical utility of in vitro expansion.

Corneal endothelial transplantation or endothelial keratoplasty has become the preferred choice of transplantation for patients with corneal blindness due to endothelial dysfunction. Currently, there is a worldwide shortage of transplantable tissue, and demand is expected to increase further with aging populations. Tissue-engineered alternatives are being developed, and are likely to be available soon. However, the cost of these constructs may impair their widespread use. A cost-minimization analysis comparing tissue-engineered constructs to donor tissue procured from eye banks for endothelial keratoplasty was performed. Both initial investment costs and recurring costs were considered in the analysis to arrive at a final tissue cost per transplant. The clinical outcomes of endothelial keratoplasty with tissue-engineered constructs and with donor tissue procured from eye banks were assumed to be equivalent. One-way and probabilistic sensitivity analyses were performed to simulate various possible scenarios, and to determine the robustness of the results. A tissue engineering strategy was cheaper in both investment cost and recurring cost. Tissue-engineered constructs for endothelial keratoplasty could be produced at a cost of US$880 per transplant. In contrast, utilizing donor tissue procured from eye banks for endothelial keratoplasty required US$3,710 per transplant. Sensitivity analyses performed further support the results of this cost-minimization analysis across a wide range of possible scenarios. The use of tissue-engineered constructs for endothelial keratoplasty could potentially increase the supply of transplantable tissue and bring the costs of corneal endothelial transplantation down, making this intervention accessible to a larger group of patients. Tissue-engineering strategies for corneal epithelial constructs or other tissue types, such as pancreatic islet cells, should also be subject to similar pharmacoeconomic analyses.

For healthspan and lifespan, ERK, AMPK and mTORC1 represent critical pathways and inflammation is a centrally important hallmark 1 – 7 . Here we examined whether IL-11, a pro-inflammatory cytokine of the IL-6 family, has a negative effect on age-associated disease and lifespan. As mice age, IL-11 is upregulated across cell types and tissues to regulate an ERK–AMPK–mTORC1 axis to modulate cellular, tissue- and organismal-level ageing pathologies. Deletion of Il11 or Il11ra1 protects against metabolic decline, multi-morbidity and frailty in old age. Administration of anti-IL-11 to 75-week-old mice for 25 weeks improves metabolism and muscle function, and reduces ageing biomarkers and frailty across sexes. In lifespan studies, genetic deletion of Il11 extended the lives of mice of both sexes, by 24.9% on average. Treatment with anti-IL-11 from 75 weeks of age until death extends the median lifespan of male mice by 22.5% and of female mice by 25%. Together, these results demonstrate a role for the pro-inflammatory factor IL-11 in mammalian healthspan and lifespan. We suggest that anti-IL-11 therapy, which is currently in early-stage clinical trials for fibrotic lung disease, may provide a translational opportunity to determine the effects of IL-11 inhibition on ageing pathologies in older people. IL-11 is identified as a key regulator of ERK–AMPK–mTORC1 signalling, metabolism, inflammation and age-related disease and lifespan in mouse and human.

Background Global shortage of donor corneas greatly restricts the numbers of corneal transplantations performed yearly. Limited ex vivo expansion of primary human corneal endothelial cells is possible, and a considerable clinical interest exists for development of tissue-engineered constructs using cultivated corneal endothelial cells. The objective of this study was to investigate the density-dependent growth of human corneal endothelial cells isolated from paired donor corneas and to elucidate an optimal seeding density for their extended expansion in vitro whilst maintaining their unique cellular morphology. Results Established primary human corneal endothelial cells were propagated to the second passage (P2) before they were utilized for this study. Confluent P2 cells were dissociated and seeded at four seeding densities: 2,500 cells per cm 2 (‘LOW’); 5,000 cells per cm 2 (‘MID’); 10,000 cells per cm 2 (‘HIGH’); and 20,000 cells per cm 2 (‘HIGH ×2 ’), and subsequently analyzed for their propensity to proliferate. They were also subjected to morphometric analyses comparing cell sizes, coefficient of variance, as well as cell circularity when each culture became confluent. At the two lower densities, proliferation rates were higher than cells seeded at higher densities, though not statistically significant. However, corneal endothelial cells seeded at lower densities were significantly larger in size, heterogeneous in shape and less circular (fibroblastic-like), and remained hypertrophic after one month in culture. Comparatively, cells seeded at higher densities were significantly homogeneous, compact and circular at confluence. Potentially, at an optimal seeding density of 10,000 cells per cm 2 , it is possible to obtain between 10 million to 25 million cells at the third passage. More importantly, these expanded human corneal endothelial cells retained their unique cellular morphology. Conclusions Our results demonstrated a density dependency in the culture of primary human corneal endothelial cells. Sub-optimal seeding density results in a decrease in cell saturation density, as well as a loss in their proliferative potential. As such, we propose a seeding density of not less than 10,000 cells per cm 2 for regular passage of primary human corneal endothelial cells.

Corneal transplantation is the only treatment available to restore vision for individuals with blindness due to corneal endothelial dysfunction. However, severe shortage of available donor corneas remains a global challenge. Functional regulatory compliant tissue-engineered corneal endothelial graft substitute can alleviate this reliance on cadaveric corneal graft material. Here, isolated primary human corneal endothelial cells (CEnCs) propagated using a dual media approach refined towards regulatory compliance showed expression of markers indicative of the human corneal endothelium, and can be tissue-engineered onto thin corneal stromal carriers. Both cellular function and clinical adaptability was demonstrated in a pre-clinical rabbit model of bullous keratopathy using a tissue-engineered endothelial keratoplasty (TE-EK) approach, adapted from routine endothelial keratoplasty procedure for corneal transplantation in human patients. Cornea thickness of rabbits receiving TE-EK graft gradually reduced over the first two weeks, and completely recovered to a thickness of approximately 400 µm by the third week of transplantation, whereas corneas of control rabbits remained significantly thicker over 1,000 µm ( p  < 0.05) throughout the course of the study. This study showed convincing evidence of the adaptability of the propagated CEnCs and their functionality via a TE-EK approach, which holds great promises in translating the use of cultured CEnCs into the clinic.

N-acetyl-p-aminophenol (APAP)-induced liver damage is associated with upregulation of Interleukin-11 (IL11), which is thought to stimulate IL6ST (gp130)-mediated STAT3 activity in hepatocytes, as a compensatory response. However, recent studies have found IL11/IL11RA/gp130 signaling to be hepatotoxic. To investigate further the role of IL11 and gp130 in APAP liver injury, we generated two new mouse strains with conditional knockout (CKO) of either Il11 (CKOIl11) or gp130 (CKOgp130) in adult hepatocytes. Following APAP, as compared to controls, CKOgp130 mice had lesser liver damage with lower serum Alanine Transaminase (ALT) and Aspartate Aminotransferase (AST), greatly reduced serum IL11 levels (90% lower), and lesser centrilobular necrosis. Livers from APAP-injured CKOgp130 mice had lesser ERK, JNK, NOX4 activation and increased markers of regeneration (PCNA, Cyclin D1, Ki67). Experiments were repeated in CKOIl11 mice that, as compared to wild-type mice, had lower APAP-induced ALT/AST, reduced centrilobular necrosis and undetectable IL11 in serum. As seen with CKOgp130 mice, APAP-treated CKOIl11 mice had lesser ERK/JNK/NOX4 activation and greater features of regeneration. Both CKOgp130 and CKOIl11 mice had normal APAP metabolism. After APAP, CKOgp130 and CKOIl11 mice had reduced Il6, Ccl2, Ccl5, Il1β, and Tnfα expression. These studies exclude IL11 upregulation as compensatory and establish autocrine, self-amplifying, gp130-dependent IL11 secretion from damaged hepatocytes as toxic and anti-regenerative.

Human ageing is associated with metabolic dysfunction, sarcopenia and frailty that taken together reduce healthspan. For age-associated diseases and lifespan, ERK, AMPK and mTORC1 represent critical pathways, across species1–7. Here we examined the hypothesis that IL11, recently shown to regulate ERK/mTORC1, is an inflammaging factor important for healthspan. As mice age, IL11 is progressively upregulated in liver, skeletal muscle, and fat to stimulate an ERK/AMPK/mTORC1 axis of cellular, tissue- and organismal-level ageing pathologies. In old mice, deletion of Il11 or Il11ra1 protects against metabolic multi-morbidity, sarcopenia, and frailty. Administration of anti-IL11 therapy to elderly mice for six months reactivates an age-repressed program of white fat beiging, reverses metabolic dysfunction, restores muscle function, and reduces frailty. Across studies, inhibition of IL11 lowers epigenetic age, reduces telomere attrition, and preserves mitochondrial function. Towards clinical translation, we generated, humanised, and engineered a neutralising, high-affinity IL11 antibody. These studies identify IL11 as a key inflammaging factor and therapeutic target for mammalian healthspan.