Mitochondrial Fission Promotes the Continued Clearance of Apoptotic Cells by Macrophages.

Clearance of apoptotic cells (ACs) by phagocytes (efferocytosis) prevents post-apoptotic necrosis and dampens inflammation. Defective efferocytosis drives important diseases, including atherosclerosis. For efficient efferocytosis, phagocytes must be able to internalize multiple ACs. We show here that uptake of multiple ACs by macrophages requires dynamin-related protein 1 (Drp1)-mediated mitochondrial fission, which is triggered by AC uptake. When mitochondrial fission is disabled, AC-induced increase in cytosolic calcium is blunted owing to mitochondrial calcium sequestration, and calcium-dependent phagosome formation around secondarily encountered ACs is impaired. These defects can be corrected by silencing the mitochondrial calcium uniporter (MCU). Mice lacking myeloid Drp1 showed defective efferocytosis and its pathologic consequences in the thymus after dexamethasone treatment and in advanced atherosclerotic lesions in fat-fed Ldlr-/- mice. Thus, mitochondrial fission in response to AC uptake is a critical process that enables macrophages to clear multiple ACs and to avoid the pathologic consequences of defective efferocytosis in vivo.

Type I interferon and cancer.

Type I interferon (IFN) is a class of proinflammatory cytokines with a dual role on malignant transformation, tumor progression, and response to therapy. On the one hand, robust, acute, and resolving type I IFN responses have been shown to mediate prominent anticancer effects, reflecting not only their direct cytostatic/cytotoxic activity on (at least some) malignant cells, but also their pronounced immunostimulatory functions. In line with this notion, type I IFN signaling has been implicated in the antineoplastic effects of various immunogenic therapeutics, including (but not limited to) immunogenic cell death (ICD)-inducing agents and immune checkpoint inhibitors (ICIs). On the other hand, weak, indolent, and non-resolving type I IFN responses have been demonstrated to support tumor progression and resistance to therapy, reflecting the ability of suboptimal type I IFN signaling to mediate cytoprotective activity, promote stemness, favor tolerance to chromosomal instability, and facilitate the establishment of an immunologically exhausted tumor microenvironment. Here, we review fundamental aspects of type I IFN signaling and their context-dependent impact on malignant transformation, tumor progression, and response to therapy.

Transfer of Cell-Surface Antigens by Scavenger Receptor CD36 Promotes Thymic Regulatory T Cell Receptor Repertoire Development and Allo-tolerance.

The development of T cell tolerance in the thymus requires the presentation of host proteins by multiple antigen-presenting cell (APC) types. However, the importance of transferring host antigens from transcription factor AIRE-dependent medullary thymic epithelial cells (mTECs) to bone marrow (BM) APCs is unknown. We report that antigen was primarily transferred from mTECs to CD8α+ dendritic cells (DCs) and showed that CD36, a scavenger receptor selectively expressed on CD8α+ DCs, mediated the transfer of cell-surface, but not cytoplasmic, antigens. The absence of CD8α+ DCs or CD36 altered thymic T cell selection, as evidenced by TCR repertoire analysis and the loss of allo-tolerance in murine allogeneic BM transplantation (allo-BMT) studies. Decreases in these DCs and CD36 expression in peripheral blood of human allo-BMT patients correlated with graft-versus-host disease. Our findings suggest that CD36 facilitates transfer of mTEC-derived cell-surface antigen on CD8α+ DCs to promote tolerance to host antigens during homeostasis and allo-BMT.

Apoptotic Caspases Suppress Type I Interferon Production via the Cleavage of cGAS, MAVS, and IRF3.

Viral infection triggers host defenses through pattern-recognition receptor-mediated cytokine production, inflammasome activation, and apoptosis of the infected cells. Inflammasome-activated caspases are known to cleave cyclic GMP-AMP synthase (cGAS). Here, we found that apoptotic caspases are critically involved in regulating both DNA and RNA virus-triggered host defenses, in which activated caspase-3 cleaved cGAS, MAVS, and IRF3 to prevent cytokine overproduction. Caspase-3 was exclusively required in human cells, whereas caspase-7 was involved only in murine cells to inactivate cGAS, reflecting distinct regulatory mechanisms in different species. Caspase-mediated cGAS cleavage was enhanced in the presence of dsDNA. Alternative MAVS cleavage sites were used to ensure the inactivation of this critical protein. Elevated type I IFNs were detected in caspase-3-deficient cells without any infection. Casp3-/- mice consistently showed increased resistance to viral infection and experimental autoimmune encephalomyelitis. Our results demonstrate that apoptotic caspases control innate immunity and maintain immune homeostasis against viral infection.

Tissue-Resident Macrophages Are Locally Programmed for Silent Clearance of Apoptotic Cells.

Although apoptotic cells (ACs) contain nucleic acids that can be recognized by Toll-like receptors (TLRs), engulfment of ACs does not initiate inflammation in healthy organisms. Here we identified macrophage populations that continually engulf ACs in distinct tissues and found that these macrophages share characteristics compatible with immunologically silent clearance of ACs; such characteristics include high expression of AC recognition receptors, low expression of TLR9, and reduced TLR responsiveness to nucleic acids. Removal of the macrophages from tissues resulted in loss of many of these characteristics and the ability to generate inflammatory responses to AC-derived nucleic acids, suggesting that cues from the tissue microenvironment program macrophages for silent AC clearance. The transcription factors KLF2 and KLF4 control the expression of many genes within this AC clearance program. The coordinated expression of AC receptors with genes that limit responses to nucleic acids might ensure maintenance of homeostasis and thus represent a central feature of tissue macrophages.

Riding apoptotic bodies for cell-cell transmission by African swine fever virus.

African swine fever virus (ASFV), a devastating pathogen to the worldwide swine industry, mainly targets macrophage/monocyte lineage, but how the virus enters host cells has remained unclear. Here, we report that ASFV utilizes apoptotic bodies (ApoBDs) for infection and cell-cell transmission. We show that ASFV induces cell apoptosis of primary porcine alveolar macrophages (PAMs) at the late stage of infection to productively shed ApoBDs that are subsequently swallowed by neighboring PAMs to initiate a secondary infection as evidenced by electron microscopy and live-cell imaging. Interestingly, the virions loaded within ApoBDs are exclusively single-enveloped particles that are devoid of the outer layer of membrane and represent a predominant form produced during late infection. The in vitro purified ApoBD vesicles are capable of mediating virus infection of naive PAMs, but the transmission can be significantly inhibited by blocking the "eat-me" signal phosphatidyserine on the surface of ApoBDs via Annexin V or the efferocytosis receptor TIM4 on the recipient PAMs via anti-TIM4 antibody, whereas overexpression of TIM4 enhances virus infection. The same treatment however did not affect the infection by intracellular viruses. Importantly, the swine sera to ASFV exert no effect on the ApoBD-mediated transmission but can partially act on the virions lacking the outer layer of membrane. Thus, ASFV has evolved to hijack a normal cellular pathway for cell-cell spread to evade host responses.

The expanding family of neutrophil-derived extracellular vesicles.

Neutrophils are immune cells involved in several inflammatory and homeostatic processes. Their capacity to release cargo can be classified based on whether the cargo is released on its own, or in conjunction with plasma membrane structures. Examples of plasma membrane-free secretion modes are degranulation, neutrophil extracellular trap (NET) release, and cytokine release through inflammasome formation. The most studied membrane-covered neutrophil-derived structures are exosomes and ectosomes that are collectively called extracellular vesicles (EV). Apoptotic vesicles are another recognized EV subtype. Over the last decade, additional membrane-covered neutrophil-derived structures were characterized: migratory cytoplasts, migrasomes, and elongated neutrophil-derived structures (ENDS). All these structures are smaller than the neutrophils, cannot reproduce themselves, and thus meet the latest consensus definition of EVs. In this review, we focus on the less well-studied neutrophil EVs: apoptotic vesicles, cytoplasts, migrasomes, and ENDS.

Expanding roles of BCL-2 proteins in apoptosis execution and beyond.

The proteins of the BCL-2 family are known as key regulators of apoptosis, with interactions between family members determining permeabilisation of the mitochondrial outer membrane (MOM) and subsequent cell death. However, the exact mechanism through which they form the apoptotic pore responsible for MOM permeabilisation (MOMP), the structure and specific components of this pore, and what roles BCL-2 proteins play outside of directly regulating MOMP are incompletely understood. Owing to the link between apoptosis dysregulation and disease, the BCL-2 proteins are important targets for drug development. With the development and clinical use of drugs targeting BCL-2 proteins showing success in multiple haematological malignancies, enhancing the efficacy of these drugs, or indeed developing novel drugs targeting BCL-2 proteins is of great interest to treat cancer patients who have developed resistance or who suffer other disease types. Here, we review our current understanding of the molecular mechanism of MOMP, with a particular focus on recently discovered roles of BCL-2 proteins in apoptosis and beyond, and discuss what implications these functions might have in both healthy tissues and disease.

Non-apoptotic caspase activation ensures the homeostasis of ovarian somatic stem cells.

Current evidence has associated caspase activation with the regulation of basic cellular functions without causing apoptosis. Malfunction of non-apoptotic caspase activities may contribute to specific neurological disorders, metabolic diseases, autoimmune conditions and cancers. However, our understanding of non-apoptotic caspase functions remains limited. Here, we show that non-apoptotic caspase activation prevents the intracellular accumulation of the Patched receptor in autophagosomes and the subsequent Patched-dependent induction of autophagy in Drosophila follicular stem cells. These events ultimately sustain Hedgehog signalling and the physiological properties of ovarian somatic stem cells and their progeny under moderate thermal stress. Importantly, our key findings are partially conserved in ovarian somatic cells of human origin. These observations attribute to caspases a pro-survival role under certain cellular conditions.

Diosgenyl glucosamine conjugates increase pro-apoptotic and selective activities in cancer cell lines.

BACKGROUND INFORMATION: Antiproliferative and apoptotic activities have been attributed to the phytosteroid diosgenin ((25R)-spirost-5-en-3ß-ol; 1). It is known that combining glucose with two rhamnoses (the chacotrioside framework) linked to diosgenin increases its apoptotic activity. However, the effects of diosgenin glucosamine glycosides on different cancer cell types and cell death have not been entirely explored. RESULTS: This study reports the antiproliferative, cytotoxic, and apoptotic activities of diosgenin and its glycosylated derivative ((25R)-spirost-5-en-3ß-yl ß-D-glucopyranoside; 2). It also explores the effects of two diosgenin glucosamine derivates, diosgenin 2-acetamido-2-deoxy-ß-D-glucopyranoside (3), and diosgenin 2-amino-2-deoxy-ß-D-glucopyranoside hydrochloride (4), on different cancer cell lines. We found that all the compounds affected proliferative activity with minimal toxicity. In addition, all cancer cell lines showed morphological and biochemical characteristics corresponding to an apoptotic process. Apoptotic cell death was higher in all cell lines treated with compounds 2, 3 and 4 than in those treated with diosgenin. Moreover, compounds 3 and 4 induced apoptosis better than compounds 1 and 2. These results suggest that combining glucosamine with modified glucosamine attached to diosgenin has a greater apoptotic effect than diosgenin or its glycosylated derivative (compound 2). Furthermore, diosgenin and the abovementioned glycosides had a selective effect on tumour cells since the proliferative capacity of human lymphocytes, keratinocytes (HaCaT) and epithelial cells (CCD841) was not significantly affected. CONCLUSIONS: Altogether, these results demonstrate that diosgenin glucosamine compounds exert an antiproliferative effect on cancer cell lines and induce apoptotic effects more efficiently than diosgenin alone without affecting non-tumour cells. SIGNIFICANCE: This study evidences the pro-apoptotic and selective activities of diosgenyl glucosamine compounds in cancer cell lines.

The TRIM28/miR133a/CD47 axis acts as a potential therapeutic target in pancreatic necrosis by impairing efferocytosis.

Efferocytosis, the clearance of apoptotic cells by macrophages, plays a crucial role in inflammatory responses and effectively prevents secondary necrosis. However, the mechanisms underlying efferocytosis in acute pancreatitis (AP) remain unclear. In this study, we demonstrated the presence of efferocytosis in injured human and mouse pancreatic tissues. We also observed significant upregulation of CD47, an efferocytosis-related the "do not eat me" molecule in injured acinar cells. Subsequently, we used CRISPR-Cas9 gene editing, anti-adeno-associated virus (AAV) gene modification, and anti-CD47 antibody to investigate the potential therapeutic role of AP. CD47 expression was negatively regulated by upstream miR133a, which is controlled by the transcription factor TRIM28. To further investigate the regulation of efferocytosis and reduction of pancreatic necrosis in AP, we used miR-133a-agomir and pancreas-specific AAV-shTRIM28 to modulate CD47 expression. Our findings confirmed that CD47-mediated efferocytosis is critical for preventing pancreatic necrosis and suggest that targeting the TRIM28-miR133a-CD47 axis is clinically relevant for the treatment of AP.

Non-apoptotic regulated cell death mediates reprogramming of the tumour immune microenvironment by macrophages.

Tumour immune microenvironment (TIME) plays an indispensable role in tumour progression, and tumour-associated macrophages (TAMs) are the most abundant immune cells in TIME. Non-apoptotic regulated cell death (RCD) can avoid the influence of tumour apoptosis resistance on anti-tumour immune response. Specifically, autophagy, ferroptosis, pyroptosis and necroptosis mediate the crosstalk between TAMs and tumour cells in TIME, thus reprogram TIME and affect the progress of tumour. In addition, although some achievements have been made in immune checkpoint inhibitors (ICIs), there is still defect that ICIs are only effective for some people because non-apoptotic RCD can bypass the apoptosis resistance of tumour. As a result, ICIs combined with targeting non-apoptotic RCD may be a promising solution. In this paper, the basic molecular mechanism of non-apoptotic RCD, the way in which non-apoptotic RCD mediates crosstalk between TAMs and tumour cells to reprogram TIME, and the latest research progress in targeting non-apoptotic RCD and ICIs are reviewed.

Augmented mitochondrial apoptotic signaling impairs C2C12 myoblast differentiation following cellular aging through sequential passaging.

Aging is associated with the steady decline of several cellular processes. The loss of skeletal muscle mass, termed sarcopenia, is one of the major hallmarks of aging. Aged skeletal muscle exhibits a robust reduction in its regenerative capacity due to dysfunction (i.e., senescence, lack of self-renewal, and impaired differentiation) of resident muscle stem cells, called satellite cells. To replicate aging in vitro, immortalized skeletal muscle cells (myoblasts) can be treated with various agents to mimic age-related dysfunction; however, these come with their own set of limitations. In the present study, we used sequential passaging of mouse myoblasts to mimic impaired differentiation that is observed in aged skeletal muscle. Further, we investigated mitochondrial apoptotic mechanisms to better understand the impaired differentiation in these "aged" cells. Our data shows that sequential passaging (>20 passages) of myoblasts is accompanied with significant reductions in differentiation and elevated cell death. Furthermore, high-passage (HP) myoblasts exhibit greater mitochondrial-mediated apoptotic signaling through mitochondrial BAX translocation, CYCS and AIFM1 release, and caspase-9 activation. Finally, we show that inhibition of mitochondrial outer membrane permeability partly recovered differentiation in HP myoblasts. Together, our findings suggests that mitochondrial apoptotic signaling is a contributing factor to the diminished differentiation that is observed in aged myoblasts.

Inhibition of caspase-8 cascade restrains the osteoclastogenic fate of bone marrow cells.

Osteoclasts are multinucleated cells of hematopoietic origin, with a pivotal role in bone development and remodeling. Failure in osteoclast differentiation and activation leads to various bone disorders; thus, attention has focused on a search of molecules involved in osteoclast regulatory pathways. Caspase-8 appears to be an interesting candidate for further exploration, due to its potential function in bone development and homeostasis. Mouse bone marrow cells were differentiated into osteoclasts by RANKL stimulation. Increased activation of caspase-8 and its downstream executioner caspases (caspase-3 and caspase-6) was found during osteoclastogenesis. Subsequent inhibition of caspase-8, caspase-3, or caspase-6, respectively, during osteoclast differentiation showed distinct changes in the formation of TRAP-positive multinucleated cells and reduced expression of osteoclast markers including Acp5, Ctsk, Dcstamp, and Mmp9. Analysis of bone matrix resorption confirmed significantly reduced osteoclast function after caspase inhibition. The results clearly showed the role of caspases in the proper development of osteoclasts and contributed new knowledge about non-apoptotic function of caspases.

Different bacterial cargo in apoptotic cells drive distinct macrophage phenotypes.

The removal of dead cells (efferocytosis) contributes to the resolution of the infection and preservation of the tissue. Depending on the environment milieu, macrophages may show inflammatory (M1) or anti-inflammatory (M2) phenotypes. Inflammatory leukocytes are recruited during infection, followed by the accumulation of infected and non-infected apoptotic cells (AC). Efferocytosis of non-infected AC promotes TGF-ß, IL-10, and PGE2 production and the polarization of anti-inflammatory macrophages. These M2 macrophages acquire an efficient ability to remove apoptotic cells that are involved in tissue repair and resolution of inflammation. On the other hand, the impact of efferocytosis of infected apoptotic cells on macrophage activation profile remains unknown. Here, we are showing that the efferocytosis of gram-positive Streptococcus pneumoniae-AC (Sp-AC) or gram-negative Klebsiella pneumoniae-AC (Kp-AC) promotes distinct gene expression and cytokine signature in macrophages. Whereas the efferocytosis of Kp-AC triggered a predominant M1 phenotype in vitro and in vivo, the efferocytosis of Sp-AC promoted a mixed M1/M2 activation in vitro and in vivo in a model of allergic asthma. Together, these findings suggest that the nature of the pathogen and antigen load into AC may have different impacts on inducing macrophage polarization.

Exploring caspase functions in mouse models.

Caspases are enzymes with protease activity. Despite being known for more than three decades, caspase investigation still yields surprising and fascinating information. Initially associated with cell death and inflammation, their functions have gradually been revealed to extend beyond, targeting pathways such as cell proliferation, migration, and differentiation. These processes are also associated with disease mechanisms, positioning caspases as potential targets for numerous pathologies including inflammatory, neurological, metabolic, or oncological conditions. While in vitro studies play a crucial role in elucidating molecular pathways, they lack the context of the body's complexity. Therefore, laboratory animals are an indispensable part of successfully understanding and applying caspase networks. This paper aims to summarize and discuss recent knowledge, understanding, and challenges in caspase knock-out mice.

[6]-Shogaol Induces Apoptosis of Murine Bladder Cancer Cells.

BACKGROUND/AIMS: Bladder cancer is considered one of the most aggressive neoplasms due to its recurrence and progression profile, and even with the improvement in diagnosis and treatment methods, the mortality rate has not shown a declining trend in recent decades. From this perspective, the search and development of more effective and safer therapeutic alternatives are necessary. Phytochemicals are excellent sources of active principles with therapeutic potential. [6]-Shogaol is a phenolic compound extracted from the ginger rhizomes that has shown antitumor effects in a wide variety of cancer models. However, there is no record in the literature of studies reporting these effects in models of bladder cancer. Thus, this study aimed to investigate the in vitro cytotoxic and pro-apoptotic potential of [6]-Shogaol against murine bladder cancer urothelial cells (MB49). METHODS: The cytotoxic effects of [6]-Shogaol on cell viability (MTT method), cell morphology (light microscopy), alteration of proliferative processes (clonogenic assay), oxidative stress pathway (levels of reactive oxygen species) and the induction of apoptotic events (flow cytometry and high-resolution epifluorescence imaging) were evaluated in murine urothelial bladder cancer cell lines (MB49), relative to non-tumor murine fibroblasts (L929). RESULTS: The results showed that [6]-Shogaol was able to induce concentration-dependent cytotoxic effects, which compromised cell viability, exhibiting an inhibitory concentration of 50% of cells (IC50) of 146.8 µM for MB49 tumor cells and 236.0 µM for L929 non-tumor fibroblasts. In addition to inhibiting and altering the proliferative processes if colony formation, it presented pro-apoptotic activity identified through a quantitative analysis and the observation of apoptotic phenotypes, events apparently mediated by the induction of nuclear fragmentation. CONCLUSION: The data presented suggest that [6]-Shogaol has a higher concentration-dependent cytotoxic and apoptosis-inducing potential in MB49 cells than in L929 fibroblasts. These results may contribute to the development of therapeutic alternatives for bladder cancer.

Cucurbitacin I exerts its anticancer effects by inducing cell cycle arrest via the KAT2a-ube2C/E2F1 pathway and inhibiting HepG2-induced macrophage M2 polarization.

Hepatocellular carcinoma (HCC) is one of the most common malignancies globally, particularly prevalent in China, where it accounts for nearly half of the world's new cases and deaths each year, but has limited therapeutic options. This study systematically investigated the impact of cucurbitacin I on HCC cell lines including SK-Hep-1, Huh-7, and HepG2. The results revealed that cucurbitacin I not only inhibited cell proliferation, cell migration and colony formation, but also induced apoptosis in HCC cells. The apoptotic induction was accompanied by a decrease in the expression of the anti-apoptotic factor B-cell lymphoma 2 (Bcl2), and an elevation in the expression levels of pro-apoptotic factors, including tumor protein p53 (P53), bcl2 associated X-apoptosis regulator (Bax), and caspase3 (Cas3). Additionally, cucurbitacin I caused cell cycle arrest by modulating the lysine acetyltransferase 2A (KAT2A)-E2F transcription factor 1 (E2F1)/Ubiquitin-conjugating enzyme E2 C (UBE2C) signaling axis. In terms of regulation on tumor microenvironment, cucurbitacin I was demonstrated the ability to inhibit HCC cell-induced M2 polarization of macrophages. This comprehensive study unveils the multifaceted anti-cancer mechanisms of cucurbitacin I, providing robust support for its potential application in the treatment of HCC, offering new avenues for the future development of HCC treatment strategies.

Coreceptor AXL Facilitates African Swine Fever Virus Entry via Apoptotic Mimicry.

African swine fever (ASF) is an acute and hemorrhagic infectious disease caused by African swine fever virus (ASFV), which is listed as an animal epidemic disease that must be reported by The World Organization for Animal Health and that causes serious economic losses to China and even the whole world. Currently, the entry mechanism of ASFV is not fully understood. Especially in the early stages of virus entry, the host factors required for ASFV entry have not yet been identified and characterized. In this study, we demonstrated that ASFV externalized phosphatidylserine (PS) on the envelope functioned as viral apoptotic mimicry, which interacts with AXL, a tyrosine kinase receptor, to mediate ASFV entry into porcine alveolar macrophages (PAMs). We found that AXL was the most pronounced phosphatidylserine receptor (PSR) affecting ASFV entry in PAMs by RNA interference screening. Knockout AXL gene expression remarkably decreased ASFV internalization and replication in MA104 cells. Furthermore, the antibody against AXL extracellular domains effectively inhibited the ASFV entry. Consistent with these results, the deletion of the intracellular kinase domain of AXL and the treatment of the AXL inhibitor, R428, significantly inhibited the internalization of ASFV. Mechanistically, AXL facilitated the internalization of ASFV virions via macropinocytosis. Collectively, we provide evidence that AXL is a coreceptor for ASFV entry into PAMs, which expands our knowledge of ASFV entry and provides a theoretical basis for identifying new antiviral targets. IMPORTANCE African swine fever (ASF) is a highly contagious infectious disease caused by the ASF virus (ASFV), with a mortality rate of up to 100%. ASFV has caused huge economic losses to pig farming worldwide. Specific cellular surface receptors are considered crucial determinants of ASFV tropism. However, the host factors required for ASFV entry have not yet been identified, and the molecular mechanism of its entry remains unclear. Here, we found that ASFV utilized phosphatidylserine (PS) on the surface of virions to masquerade as apoptotic mimicry and facilitated virus entry by interacting with host factor AXL. We found that knockout of AXL remarkably decreased ASFV internalization and replication. The antibody against AXL extracellular domains and AXL inhibitor R428 significantly inhibited the internalization of ASFV via macropinocytosis. The current work deepens our understanding of ASFV entry and provides clues for the development of antiviral drugs to control ASFV infection.

Application of a pattern-based approach to histological diagnosis in very early onset IBD (VEO-IBD) in a multicentric cohort of children with emphasis on monogenic disease with IBD-like morphology.

AIMS: Very early-onset inflammatory bowel disease (VEO-IBD) is a clinical umbrella term referring to IBD-like symptoms arising in children before the age of 6 years, encompassing both 'pure' IBD, such as ulcerative colitis (UC) and Crohn's disease (CD) and monogenic diseases (MDs), the latter often involving genes associated with primary immunodeficiencies. Moreover, histological features in gastrointestinal (GI) biopsies in MD can also have IBD-like morphology, making differential diagnosis difficult. Correct diagnosis is fundamental, as MDs show a more severe clinical course and their inadequate/untimely recognition leads to inappropriate therapy. METHODS AND RESULTS: Biopsy samples from the lower and upper GI tract of 93 clinically diagnosed VEO-IBD children were retrospectively selected in a multicentre cohort and histologically re-evaluated by 10 pathologists blinded to clinical information. Each case was classified according to morphological patterns, including UC-like; CD-like; enterocolitis-like; apoptotic; eosinophil-rich; and IBD-unclassified (IBD-U). Nine (69%) MD children showed IBD-like morphology; only the IBD-U pattern correlated with MD diagnosis (P = 0.02) (available in 64 cases: 51 non-MD, true early-onset IBD/other; 13 MD cases). MD patients showed earlier GI symptom onset (18.7 versus 26.9 months) and were sent to endoscopy earlier (22 versus 37 months), these differences were statistically significant (P < 0.05). Upper GI histology was informative in 37 biopsies. CONCLUSIONS: The diagnosis of the underlying cause of VEO-IBD requires a multidisciplinary setting, and pathology, while being one of the fundamental puzzle pieces, is often difficult to interpret. A pattern-based histological approach is therefore suggested, thus aiding the pathologist in VEO-IBD reporting and multidisciplinary discussion.