Manolis Pasparakis

Age-associated hematopoietic skewing results in an increase in the neutrophil-to-lymphocyte ratio, which serves as a strong predictor of all-cause mortality in older adults, although its causes are incompletely understood. Here we show that cytotoxic CD4 ⁺ T lymphocytes accumulate in the bone marrow of mice during aging and induce myelopoiesis, increasing the neutrophil-to-lymphocyte ratio. T cell receptor-dependent induction of mitochondrial stress and activation of STING upregulates the chemokine CCL5 in CD4 ⁺ T lymphocytes. During aging, hematopoietic stem cells and downstream myeloid progenitors upregulate CCR5, the primary receptor for CCL5. Genetic ablation of Ccr5 in hematopoietic progenitors mitigates T cell-induced myeloid skewing and neutrophil expansion. Pharmacological blockade of CCR5 using the Food and Drug Administration-approved drug maraviroc normalizes myelopoiesis, reduces circulating and tissue-infiltrating neutrophils, and improves multiple aging-related biomarkers and functional outcomes in aged mice. Together, these findings demonstrate a T cell–bone marrow axis that exacerbates age-associated decline and highlight CCR5 inhibition as a potential geroprotective strategy.

Tumour necrosis factor (TNF) is a pleiotropic cytokine originally identified for its ability to kill cancer cells. However, a paradoxical tumour-promoting role for TNF emerged when early attempts to exploit its anti-tumour activity in cancer therapy produced conflicting outcomes, raising the question of whether TNF should be viewed as a therapeutic agent or a treatment target in cancer. Here, we demonstrate that expression of cFLIP, a catalytically inactive paralogue of caspase-8 (CASP8), determines the susceptibility of melanoma cells to TNF and thereby controls melanoma growth in a syngeneic, immune-competent mouse model of B16F10 cutaneous melanoma. B16F10 melanoma cells lacking cFLIP ( cFlip ^KO/KO cells) failed to grow in wild-type mice, whereas in TNF-deficient mice, cFlip ^KO/KO melanoma cells formed palpable tumours and exhibited robust subcutaneous growth. These findings indicate that TNF alone is sufficient to control melanoma growth in the absence of cFLIP. Importantly, the anti-tumour activity of TNF has predominantly been investigated through targeting cellular inhibitors of apoptosis proteins (cIAPs), which promotes RIPK1 activation and TNF-induced cytotoxicity. We show that genomic ablation of cIAPs or RIPK1, in contrast to cFLIP, neither triggered TNF-induced toxicity nor affected melanoma growth in vivo. Collectively, our data underscore the central role of cFLIP in regulating melanoma responses to TNF and suggest that endogenous immune surveillance as well as immunotherapies involving TNF could strongly benefit from cFLIP targeting strategies.

Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer-related death within this decade. Here, we show that its major driver oncogene KRAS activates the cGAS-STING-TBK1 axis, inducing a type I interferon (IFN) response that primes PDAC cells for necroptosis. Using genetically engineered mouse models, we find that cancer cell-specific deletion of caspase-8 is sufficient to trigger necroptotic cell death, eliminating most pancreatic precursor lesions. Mechanistically, KRAS-driven IFN signalling induces ISGF3-dependent expression of necroptosis-related interferon-stimulated genes, including MLKL. This renders PDAC cells selectively vulnerable to necroptosis upon caspase-8 inhibition. Therapeutically, pharmacologic caspase inhibition reduces tumour burden in aggressive PDAC models and human patient-derived organoids. A pan-cancer transcriptomic analysis links necroptosis gene expression with Ras pathway activity and IFN signatures across multiple tumour types. These findings reveal a KRAS-induced IFN program that sensitises tumour cells to necroptosis, highlighting a therapeutic vulnerability in PDAC with broader relevance across IFN-activated cancers.

Caspase-8 is a central regulator of death receptor signalling. It induces apoptosis and suppresses necroptosis through its catalytic activity, while it serves as a scaffold promoting inflammasome activation and NF-κB-mediated gene expression. The currently prevailing model is that homo-oligomerization promotes full activation of caspase-8 to induce apoptosis, while heterodimerization with its catalytically inactive homologue cFLIP mediates limited activation of caspase-8 to suppress necroptosis. Here, we show that caspase-8 homo-oligomerisation not only initiates apoptosis but also plays a critical role in inhibiting necroptosis in susceptible cell types in vivo and in vitro. Inhibition of caspase-8 homo-oligomerisation by knock-in mutation of critical residues in its second DED (F122G/L123G; Casp8 ^FGLG/FGLG ) prevented hepatocyte apoptosis and the development of hepatitis and hepatocellular carcinoma in mice lacking NEMO specifically in liver parenchymal cells. In this model, inhibition of caspase-8 homo-oligomerisation did not sensitize hepatocytes to necroptosis because these cells do not express RIPK3. In contrast, in the Sharpin ^cpdm/cpdm mouse model of dermatitis induced by caspase-8-dependent keratinocyte apoptosis, Casp8 ^{FGLG/FGLG /FGLG} mutation suppressed apoptosis but did not prevent skin inflammation. Combined inhibition of caspase-8 homo-oligomerisation and necroptosis fully prevented dermatitis in Sharpin ^cpdm/cpdm mice, demonstrating that inhibition of caspase-8 homo-oligomerization sensitized keratinocytes to MLKL-dependent necroptosis. Consistently, primary cells from Casp8 ^FGLG/FGLG mice were protected from apoptosis but became highly sensitized to necroptosis in response to TNF and FasL stimulation. Mechanistically, caspase-8(FGLG) expression altered TNFR1 complex II and Fas DISC assembly to promote RIPK3 recruitment and activation. Taken together, our results challenge the prevailing model of necroptosis inhibition by caspase-8 and suggest that distinct mechanisms of caspase-8 activation cooperate to regulate apoptotic and necroptotic signalling and tissue homeostasis.

Misregulation of tissue repair programs can severely compromise repair outcome. Timely clearance of inflammatory macrophages through regulated cell death is a prerequisite for resolution of inflammation and successful repair. How different modes of macrophage regulated cell death regulate repair and direct healing outcome remains unclear. Using inducible genetic models to trigger macrophage necroptosis (FADDiMKO) or enhance apoptosis of macrophages (cIAP1iMKOcIAP2-/-), we reveal opposing effects on the architecture of the wound tissue. Macrophage necroptosis profoundly disrupted tissue organization: FADDiMKO wounds exhibited reduced numbers of reparative (IL-4Rα⁺Ly6Clow) macrophages, failed inflammatory resolution, accompanied by a hemorrhagic granulation tissue and reduced myofibroblast differentiation in the mid-phase of repair. In striking contrast, excessive apoptosis preserved fundamental tissue architecture and vascular integrity, and reduced scar formation. Furthermore, single-cell transcriptomics demonstrated that macrophage necroptosis disrupts tissue-wide cellular communication networks essential for coordinated repair, in particular macrophage-fibroblast crosstalk. These findings establish that wound healing quality depends on both mode and rate of macrophage programmed death, providing a framework for therapeutic targeting of macrophages in wound healing disorders and fibrotic diseases.

DNA-protein cross-links (DPCs) are highly toxic DNA lesions that block replication and transcription, but their impact on organismal physiology is unclear. We identified a role for the metalloprotease SPRTN in preventing DPC-driven immunity and its pathological consequences. Loss of SPRTN activity during replication and mitosis lead to unresolved DNA damage, chromosome segregation errors, micronuclei formation, and cytosolic DNA release that activates the cyclic GMP-AMP synthase (cGAS)–stimulator of interferon genes (STING) pathway. In a Sprtn knock-in mouse model of Ruijs-Aalfs progeria syndrome, chronic cGas-Sting signaling caused embryonic lethality through inflammation and innate immune responses. Surviving mice displayed aging phenotypes beginning in embryogenesis, which persisted into adulthood. Genetic or pharmacological inhibition of cGas-Sting rescued embryonic lethality and alleviated progeroid phenotypes.

Most neuroendocrine cancers lack caspase 8 protein expression. While this feature was thought to facilitate escape from extrinsic apoptosis, its cancer-regulatory function has remained unexplored. Here, we devise a mouse model of small cell lung cancer (SCLC) recapitulating the lack of expression of caspase 8 seen in humans and uncover an unexpected role for necroptosis-fueled pre-tumoral inflammation resulting in reprogramming towards a neuronal progenitor cell-like state and increased metastatic disease. Notably, transcriptional signatures of this cellular state are enriched in relapsed and metastatic human SCLC. Mechanistically, caspase 8 loss within the pre-tumoral niche promotes inflammation marked by increased recruitment of regulatory T cells (Tregs) which are responsible for the promotion of metastatic disease. Importantly, inactivation of the necroptosis executioner MLKL reverses pre-tumoral inflammation, decreases metastasis as well as neuronal-like reprogramming. Taken together, our findings suggest that pre-tumoral inflammatory cell death contributes to neuronal progenitor mimicry, immunosuppression and increased metastasis in SCLC.

Aging is associated with a chronic, low-grade inflammatory state referred to as inflammaging, which contributes to impaired immune regulation and increased susceptibility to disease. While regulatory T (Treg) cells are key mediators of immune homeostasis, their role in the context of age-related inflammation remains poorly understood. Here we demonstrate that age-related changes in the microbiota promote impaired Treg cell function, resulting in the differentiation of inflammatory T cells. In agreement, we find that aged germ-free (GF) mice exhibited a more balanced immune profile, where the Treg cells are functional and pro-inflammatory mediators are reduced, suggesting that microbial exposure is essential for the establishment of inflammaging. Furthermore, we show that the use of old microbiota in young animals was sufficient to induce pro-inflammatory T cell responses and impaired mucosal Treg cell proliferation, while young microbiota restored Treg cell function in old animals. Mechanistically, we show that exposure to aged microbiota was associated with sustained TNF signaling, elevated oxidative stress, DNA damage, and increased expression of senescence markers such as γH2AX and p16 in Treg cells. These findings uncover a microbiota-TNF- dependent mechanism by which age-associated microbial dysbiosis drives Treg cell dysfunction and promotes immune aging, highlighting the therapeutic potential of microbiota-targeted strategies to restore immune homeostasis in the elderly.