Antonio Santos

All Publications

• FZD5 controls intestinal crypt homeostasis and colonic Wnt surrogate agonist response. Developmental cell Mu, Q., Ha, A., Santos, A. J., Lo, Y. H., van Unen, V., Miao, Y., Tomaske, M., Guzman, V. K., Alwahabi, S., Yuan, J. J., Deng, L., Li, L., Garcia, K. C., Kuo, C. J. 2024

  Abstract

  The rapidly regenerating intestinal epithelium requires crypt intestinal stem cells (ISCs). Wnt/β-catenin signaling maintains crypt homeostasis and Lgr5+ ISCs, and WNT ligands bind Frizzled receptors (FZD1-10). Identifying specific FZD(s) essential for intestinal homeostasis has been elusive; however, bioengineered antagonists blocking Wnt binding to FZD5 and FZD8 deplete the gut epithelium in vivo, highlighting potential roles. Here, an epithelial-specific Fzd5 knockout (KO) elicited lethal pan-intestinal crypt and villus loss, whereas an Lgr5+ ISC-specific Fzd5 KO depleted Lgr5+ ISCs via premature differentiation and repressed Wnt target genes. Fzd5-null phenotypes were rescued by constitutive β-catenin activation in vivo and in both mouse and human enteroids. KO of Fzd5, not Fzd8, in enteroids ablated responsiveness to dual-specificity FZD5/FZD8-selective Wnt surrogate agonists, which ameliorated DSS-induced colitis in wild-type and Fzd8 KO mice. Overall, FZD5 is a dominant and essential regulator of crypt homeostasis, Lgr5+ ISCs, and intestinal response to Wnt surrogate agonists, with implications for therapeutic mucosal repair.

  View details for DOI 10.1016/j.devcel.2024.10.022

  View details for PubMedID 39579768

• A human autoimmune organoid model reveals IL-7 function in coeliac disease. Nature Santos, A. J., van Unen, V., Lin, Z., Chirieleison, S. M., Ha, N., Batish, A., Chan, J. E., Cedano, J., Zhang, E. T., Mu, Q., Guh-Siesel, A., Tomaske, M., Colburg, D., Varma, S., Choi, S. S., Christophersen, A., Baghdasaryan, A., Yost, K. E., Karlsson, K., Ha, A., Li, J., Dai, H., Sellers, Z. M., Chang, H. Y., Dunn, J. C., Zhang, B. M., Mellins, E. D., Sollid, L. M., Fernandez-Becker, N. Q., Davis, M. M., Kuo, C. J. 2024

  Abstract

  In vitro models of autoimmunity are constrained by an inability to culture affected epithelium alongside the complex tissue-resident immune microenvironment. Coeliac disease (CeD) is an autoimmune disease in which dietary gluten-derived peptides bind to the major histocompatibility complex (MHC) class II human leukocyte antigen molecules (HLA)-DQ2 or HLA-DQ8 to initiate immune-mediated duodenal mucosal injury1-4. Here, we generated air-liquid interface (ALI) duodenal organoids from intact fragments of endoscopic biopsies that preserve epithelium alongside native mesenchyme and tissue-resident immune cells as a unit without requiring reconstitution. The immune diversity of ALI organoids spanned T cells, B and plasma cells, natural killer (NK) cells and myeloid cells, with extensive T-cell and B-cell receptor repertoires. HLA-DQ2.5-restricted gluten peptides selectively instigated epithelial destruction in HLA-DQ2.5-expressing organoids derived from CeD patients, and this was antagonized by blocking MHC-II or NKG2C/D. Gluten epitopes stimulated a CeD organoid immune network response in lymphoid and myeloid subsets alongside anti-transglutaminase 2 (TG2) autoantibody production. Functional studies in CeD organoids revealed that interleukin-7 (IL-7) is a gluten-inducible pathogenic modulator that regulates CD8+ T-cell NKG2C/D expression and is necessary and sufficient for epithelial destruction. Furthermore, endogenous IL-7 was markedly upregulated in patient biopsies from active CeD compared with remission disease from gluten-free diets, predominantly in lamina propria mesenchyme. By preserving the epithelium alongside diverse immune populations, this human in vitro CeD model recapitulates gluten-dependent pathology, enables mechanistic investigation and establishes a proof of principle for the organoid modelling of autoimmunity.

  View details for DOI 10.1038/s41586-024-07716-2

  View details for PubMedID 39048815

• Organoid modeling of lung-resident immune responses to SARS-CoV-2 infection. Research square Choi, S. S., van Unen, V., Zhang, H., Rustagi, A., Alwahabi, S. A., Santos, A. J., Chan, J. E., Lam, B., Solis, D., Mah, J., Röltgen, K., Trope, W., Guh-Siesel, A., Lin, Z., Beck, A., Edwards, C., Mallajosyula, V., Martin, B. A., Dunn, J. C., Shrager, J., Baric, R. A., Pinsky, B., Boyd, S. D., Blish, C. A., Davis, M. M., Kuo, C. J. 2023

  Abstract

  Tissue-resident immunity underlies essential host defenses against pathogens, but analysis in humans has lacked in vitro model systems where epithelial infection and accompanying resident immune cell responses can be observed en bloc. Indeed, human primary epithelial organoid cultures typically omit immune cells, and human tissue resident-memory lymphocytes are conventionally assayed without an epithelial infection component, for instance from peripheral blood, or after extraction from organs. Further, the study of resident immunity in animals can be complicated by interchange between tissue and peripheral immune compartments. To study human tissue-resident infectious immune responses in isolation from secondary lymphoid organs, we generated adult human lung three-dimensional air-liquid interface (ALI) lung organoids from intact tissue fragments that co-preserve epithelial and stromal architecture alongside endogenous lung-resident immune subsets. These included T, B, NK and myeloid cells, with CD69+CD103+ tissue-resident and CCR7- and/or CD45RA- TRM and conservation of T cell receptor repertoires, all corresponding to matched fresh tissue. SARS-CoV-2 vigorously infected organoid lung epithelium, alongside secondary induction of innate cytokine production that was inhibited by antiviral agents. Notably, SARS-CoV-2-infected organoids manifested adaptive virus-specific T cell activation that was specific for seropositive and/or previously infected donor individuals. This holistic non-reconstitutive organoid system demonstrates the sufficiency of lung to autonomously mount adaptive T cell memory responses without a peripheral lymphoid component, and represents an enabling method for the study of human tissue-resident immunity.

  View details for DOI 10.21203/rs.3.rs-2870695/v1

  View details for PubMedID 37205380

  View details for PubMedCentralID PMC10187413

• Progenitor identification and SARS-CoV-2 infection in human distal lung organoids. Nature Salahudeen, A. A., Choi, S. S., Rustagi, A., Zhu, J., van Unen, V., de la O, S. M., Flynn, R. A., Margalef-Catala, M., Santos, A. J., Ju, J., Batish, A., Usui, T., Zheng, G. X., Edwards, C. E., Wagar, L. E., Luca, V., Anchang, B., Nagendran, M., Nguyen, K., Hart, D. J., Terry, J. M., Belgrader, P., Ziraldo, S. B., Mikkelsen, T. S., Harbury, P. B., Glenn, J. S., Garcia, K. C., Davis, M. M., Baric, R. S., Sabatti, C., Amieva, M. R., Blish, C. A., Desai, T. J., Kuo, C. J. 2020

  Abstract

  The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange. Three-dimensional in vitro human distal lung culture systems would strongly facilitate investigation of pathologies including interstitial lung disease, cancer, and SARS-CoV-2-associated COVID-19 pneumonia. We generated long-term feeder-free, chemically defined culture of distal lung progenitors as organoids derived from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids exhibited AT1 transdifferentiation potential while basal cell organoids developed lumens lined by differentiated club and ciliated cells. Single cell analysis of basal organoid KRT5+ cells revealed a distinct ITGA6+ITGB4+ mitotic population whose proliferation further segregated to a TNFRSF12Ahi subfraction comprising ~10% of KRT5+ basal cells, residing in clusters within terminal bronchioles and exhibiting enriched clonogenic organoid growth activity. Distal lung organoids were created with apical-out polarity to display ACE2 on the exposed external surface, facilitating SARS-CoV-2 infection of AT2 and basal cultures and identifying club cells as a novel target population. This long-term, feeder-free organoid culture of human distal lung, coupled with single cell analysis, identifies unsuspected basal cell functional heterogeneity and establishes a facile in vitro organoid model for human distal lung infections including COVID-19-associated pneumonia.

  View details for DOI 10.1038/s41586-020-3014-1

  View details for PubMedID 33238290

• Next-Generation Surrogate Wnts Support Organoid Growth and Deconvolute Frizzled Pleiotropy In Vivo. Cell stem cell Miao, Y. n., Ha, A. n., de Lau, W. n., Yuki, K. n., Santos, A. J., You, C. n., Geurts, M. H., Puschhof, J. n., Pleguezuelos-Manzano, C. n., Peng, W. C., Senlice, R. n., Piani, C. n., Buikema, J. W., Gbenedio, O. M., Vallon, M. n., Yuan, J. n., de Haan, S. n., Hemrika, W. n., Rösch, K. n., Dang, L. T., Baker, D. n., Ott, M. n., Depeille, P. n., Wu, S. M., Drost, J. n., Nusse, R. n., Roose, J. P., Piehler, J. n., Boj, S. F., Janda, C. Y., Clevers, H. n., Kuo, C. J., Garcia, K. C. 2020

  Abstract

  Modulation of Wnt signaling has untapped potential in regenerative medicine due to its essential functions in stem cell homeostasis. However, Wnt lipidation and Wnt-Frizzled (Fzd) cross-reactivity have hindered translational Wnt applications. Here, we designed and engineered water-soluble, Fzd subtype-specific "next-generation surrogate" (NGS) Wnts that hetero-dimerize Fzd and Lrp6. NGS Wnt supports long-term expansion of multiple different types of organoids, including kidney, colon, hepatocyte, ovarian, and breast. NGS Wnts are superior to Wnt3a conditioned media in organoid expansion and single-cell organoid outgrowth. Administration of Fzd subtype-specific NGS Wnt in vivo reveals that adult intestinal crypt proliferation can be promoted by agonism of Fzd5 and/or Fzd8 receptors, while a broad spectrum of Fzd receptors can induce liver zonation. Thus, NGS Wnts offer a unified organoid expansion protocol and a laboratory "tool kit" for dissecting the functions of Fzd subtypes in stem cell biology.

  View details for DOI 10.1016/j.stem.2020.07.020

  View details for PubMedID 32818433

• The Intestinal Stem Cell Niche: Homeostasis and Adaptations TRENDS IN CELL BIOLOGY Santos, A. M., Lo, Y., Mah, A. T., Kuo, C. J. 2018; 28 (12): 1062–78

  View details for DOI 10.1016/j.tcb.2018.08.001

  View details for Web of Science ID 000450302500009

• The Intestinal Stem Cell Niche: Homeostasis and Adaptations. Trends in cell biology Santos, A. J., Lo, Y., Mah, A. T., Kuo, C. J. 2018

  Abstract

  The intestinal epithelium is a rapidly renewing cellular compartment. This constant regeneration is a hallmark of intestinal homeostasis and requires a tightly regulated balance between intestinal stem cell (ISC) proliferation and differentiation. Since intestinal epithelial cells directly contact pathogenic environmental factors that continuously challenge their integrity, ISCs must also actively divide to facilitate regeneration and repair. Understanding niche adaptations that maintain ISC activity during homeostatic renewal and injury-induced intestinal regeneration is therefore a major and ongoing focus for stem cell biology. Here, we review recent concepts and propose an active interconversion of the ISC niche between homeostasis and injury-adaptive states that is superimposed upon an equally dynamic equilibrium between active and reserve ISC populations.

  View details for PubMedID 30195922

• TANGO1 builds a machine for collagen export by recruiting and spatially organizing COPII, tethers and membranes ELIFE Raote, I., Ortega-Bellido, M., Santos, A. M., Foresti, O., Zhang, C., Garcia-Parajo, M. F., Campelo, F., Malhotra, V. 2018; 7

  Abstract

  Collagen export from the endoplasmic reticulum (ER) requires TANGO1, COPII coats, and retrograde fusion of ERGIC membranes. How do these components come together to produce a transport carrier commensurate with the bulky cargo collagen? TANGO1 is known to form a ring that corrals COPII coats, and we show here how this ring or fence is assembled. Our data reveal that a TANGO1 ring is organized by its radial interaction with COPII, and lateral interactions with cTAGE5, TANGO1-short or itself. Of particular interest is the finding that TANGO1 recruits ERGIC membranes for collagen export via the NRZ (NBAS/RINT1/ZW10) tether complex. Therefore, TANGO1 couples retrograde membrane flow to anterograde cargo transport. Without the NRZ complex, the TANGO1 ring does not assemble, suggesting its role in nucleating or stabilising this process. Thus, coordinated capture of COPII coats, cTAGE5, TANGO1-short, and tethers by TANGO1 assembles a collagen export machine at the ER.

  View details for DOI 10.7554/eLife.32723

  View details for Web of Science ID 000427504300001

  View details for PubMedID 29513218

  View details for PubMedCentralID PMC5851698