Kazuki Nagashima

All Publications

• Mapping the T cell repertoire to a complex gut bacterial community. Nature Nagashima, K., Zhao, A., Atabakhsh, K., Bae, M., Blum, J. E., Weakley, A., Jain, S., Meng, X., Cheng, A. G., Wang, M., Higginbottom, S., Dimas, A., Murugkar, P., Sattely, E. S., Moon, J. J., Balskus, E. P., Fischbach, M. A. 2023

  Abstract

  Certain bacterial strains from the microbiome induce a potent, antigen-specific T cell response1-5. However, the specificity of microbiome-induced T cells has not been explored at the strain level across the gut community. Here, we colonize germ-free mice with complex defined communities (roughly 100 bacterial strains) and profile T cell responses to each strain. The pattern of responses suggests that many T cells in the gut repertoire recognize several bacterial strains from the community. We constructed T cell hybridomas from 92 T cell receptor (TCR) clonotypes; by screening every strain in the community against each hybridoma, we find that nearly all the bacteria-specific TCRs show a one-to-many TCR-to-strain relationship, including 13 abundant TCR clonotypes that each recognize 18 Firmicutes. By screening three pooled bacterial genomic libraries, we discover that these 13 clonotypes share a single target: a conserved substrate-binding protein from an ATP-binding cassette transport system. Peripheral regulatory T cells and T helper 17 cells specific for an epitope from this protein are abundant in community-colonized and specific pathogen-free mice. Our work reveals that T cell recognition of commensals is focused on widely conserved, highly expressed cell-surface antigens, opening the door to new therapeutic strategies in which colonist-specific immune responses are rationally altered or redirected.

  View details for DOI 10.1038/s41586-023-06431-8

  View details for PubMedID 37587342

  View details for PubMedCentralID 4128479

• Strain dropouts reveal interactions that govern the metabolic output of the gut microbiome. Cell Wang, M., Osborn, L. J., Jain, S., Meng, X., Weakley, A., Yan, J., Massey, W. J., Varadharajan, V., Horak, A., Banerjee, R., Allende, D. S., Chan, E. R., Hajjar, A. M., Wang, Z., Dimas, A., Zhao, A., Nagashima, K., Cheng, A. G., Higginbottom, S., Hazen, S. L., Brown, J. M., Fischbach, M. A. 2023; 186 (13): 2839-2852.e21

  Abstract

  The gut microbiome is complex, raising questions about the role of individual strains in the community. Here, we address this question by constructing variants of a complex defined community in which we eliminate strains that occupy the bile acid 7α-dehydroxylation niche. Omitting Clostridium scindens (Cs) and Clostridium hylemonae (Ch) eliminates secondary bile acid production and reshapes the community in a highly specific manner: eight strains change in relative abundance by >100-fold. In single-strain dropout communities, Cs and Ch reach the same relative abundance and dehydroxylate bile acids to a similar extent. However, Clostridium sporogenes increases >1,000-fold in the ΔCs but not ΔCh dropout, reshaping the pool of microbiome-derived phenylalanine metabolites. Thus, strains that are functionally redundant within a niche can have widely varying impacts outside the niche, and a strain swap can ripple through the community in an unpredictable manner, resulting in a large impact on an unrelated community-level phenotype.

  View details for DOI 10.1016/j.cell.2023.05.037

  View details for PubMedID 37352836

• Engineered skin bacteria induce antitumor T cell responses against melanoma. Science (New York, N.Y.) Chen, Y. E., Bousbaine, D., Veinbachs, A., Atabakhsh, K., Dimas, A., Yu, V. K., Zhao, A., Enright, N. J., Nagashima, K., Belkaid, Y., Fischbach, M. A. 2023; 380 (6641): 203-210

  Abstract

  Certain bacterial colonists induce a highly specific T cell response. A hallmark of this encounter is that adaptive immunity develops preemptively, in the absence of an infection. However, the functional properties of colonist-induced T cells are not well defined, limiting our ability to understand anticommensal immunity and harness it therapeutically. We addressed both challenges by engineering the skin bacterium Staphylococcus epidermidis to express tumor antigens anchored to secreted or cell-surface proteins. Upon colonization, engineered S. epidermidis elicits tumor-specific T cells that circulate, infiltrate local and metastatic lesions, and exert cytotoxic activity. Thus, the immune response to a skin colonist can promote cellular immunity at a distal site and can be redirected against a target of therapeutic interest by expressing a target-derived antigen in a commensal.

  View details for DOI 10.1126/science.abp9563

  View details for PubMedID 37053311

• Design, construction, and invivo augmentation of a complex gut microbiome. Cell Cheng, A. G., Ho, P., Aranda-Diaz, A., Jain, S., Yu, F. B., Meng, X., Wang, M., Iakiviak, M., Nagashima, K., Zhao, A., Murugkar, P., Patil, A., Atabakhsh, K., Weakley, A., Yan, J., Brumbaugh, A. R., Higginbottom, S., Dimas, A., Shiver, A. L., Deutschbauer, A., Neff, N., Sonnenburg, J. L., Huang, K. C., Fischbach, M. A. 2022

  Abstract

  Efforts to model the human gut microbiome in mice have led to important insights into the mechanisms of host-microbe interactions. However, the model communities studied to date have been defined or complex, but not both, limiting their utility. Here, we construct and characterize invitro a defined community of 104 bacterial species composed of the most common taxa from the human gut microbiota (hCom1). We then used an iterative experimental process to fill open niches: germ-free mice were colonized with hCom1 and then challenged with a human fecal sample. We identified new species that engrafted following fecal challenge and added them to hCom1, yielding hCom2. In gnotobiotic mice, hCom2 exhibited increased stability to fecal challenge and robust colonization resistance against pathogenic Escherichia coli. Mice colonized by either hCom2 or a human fecal community are phenotypically similar, suggesting that this consortium will enable a mechanistic interrogation of species and genes on microbiome-associated phenotypes.

  View details for DOI 10.1016/j.cell.2022.08.003

  View details for PubMedID 36070752

• Depletion of microbiome-derived molecules in the host using Clostridium genetics. Science (New York, N.Y.) Guo, C., Allen, B. M., Hiam, K. J., Dodd, D., Van Treuren, W., Higginbottom, S., Nagashima, K., Fischer, C. R., Sonnenburg, J. L., Spitzer, M. H., Fischbach, M. A. 2019; 366 (6471)

  Abstract

  The gut microbiota produce hundreds of molecules that are present at high concentrations in the host circulation. Unraveling the contribution of each molecule to host biology remains difficult. We developed a system for constructing clean deletions in Clostridium spp., the source of many molecules from the gut microbiome. By applying this method to the model commensal organism Clostridium sporogenes, we knocked out genes for 10 C. sporogenes-derived molecules that accumulate in host tissues. In mice colonized by a C. sporogenes for which the production of branched short-chain fatty acids was knocked out, we discovered that these microbial products have immunoglobulin A-modulatory activity.

  View details for DOI 10.1126/science.aav1282

  View details for PubMedID 31831639

• Host defense against oral microbiota by bone-damaging T cells NATURE COMMUNICATIONS Tsukasaki, M., Komatsu, N., Nagashima, K., Nitta, T., Pluemsakunthai, W., Shukunami, C., Iwakura, Y., Nakashima, T., Okamoto, K., Takayanagi, H. 2018; 9: 701

  Abstract

  The immune system evolved to efficiently eradicate invading bacteria and terminate inflammation through balancing inflammatory and regulatory T-cell responses. In autoimmune arthritis, pathogenic TH17 cells induce bone destruction and autoimmune inflammation. However, whether a beneficial function of T-cell-induced bone damage exists is unclear. Here, we show that bone-damaging T cells have a critical function in the eradication of bacteria in a mouse model of periodontitis, which is the most common infectious disease. Bacterial invasion leads to the generation of specialized TH17 cells that protect against bacteria by evoking mucosal immune responses as well as inducing bone damage, the latter of which also inhibits infection by removing the tooth. Thus, bone-damaging T cells, which may have developed to stop local infection by inducing tooth loss, function as a double-edged sword by protecting against pathogens while also inducing skeletal tissue degradation.

  View details for DOI 10.1038/s41467-018-03147-6

  View details for Web of Science ID 000425286700003

  View details for PubMedID 29453398

  View details for PubMedCentralID PMC5816021

• Targeted deletion of RANKL in M cell inducer cells by the Col6a1-Cre driver BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Nagashima, K., Sawa, S., Nitta, T., Prados, A., Koliaraki, V., Kollias, G., Nakashima, T., Takayanagi, H. 2017; 493 (1): 437-443

  Abstract

  The gut-associated lymphoid tissues (GALTs), including Peyer's patches (PPs), cryptopatches (CPs) and isolated lymphoid follicles (ILFs), establish a host-microbe symbiosis by the promotion of immune reactions against gut microbes. Microfold cell inducer (MCi) cells in GALTs are the recently identified mesenchymal cells that express the cytokine RANKL and initiate bacteria-specific immunoglobulin A (IgA) production via induction of microfold (M) cell differentiation. In the previous study, the Twist2-Cre driver was utilized for gene deletion in mesenchymal cells including MCi cells. In order to investigate MCi cells more extensively, it will be necessary to develop experimental tools in addition to the Twist2-Cre driver mice and characterize such drivers in specificity and efficiency. Here we show that M cell differentiation and IgA production are impaired in the targeted deletion of RANKL by the Col6a1-Cre driver. We compared Col6a1-Cre with Twist2-Cre in terms of the specificity for mesenchymal cells in GALTs. Col6a1-Cre CAG-CAT-EGFP mice exhibited EGFP expression in podoplanin+CD31- cells including MCi cells, while Twist2-Cre mice were shown to target endothelial cells and podoplanin+CD31- cells. Tnfsf11fl/ΔCol6a1-Cre mice exhibited the absence of M cells and severe IgA reduction together with an alteration in gut microbial composition. Moreover, we analyzed germ free mice to test whether changes in the microbiota are the cause of M cell deficiency. M cell differentiation was normal in the CPs/ILFs of germ free mice, indicating that MCi cells induce M cells independently of microbial colonization. This study demonstrates that Col6a1-Cre driver mice are as useful as Twist2-Cre driver mice for functional analyses of GALT-resident mesenchymal cells, including MCi cells.

  View details for DOI 10.1016/j.bbrc.2017.09.004

  View details for Web of Science ID 000413134200067

  View details for PubMedID 28882590

• Identification of subepithelial mesenchymal cells that induce IgA and diversify gut microbiota NATURE IMMUNOLOGY Nagashima, K., Sawa, S., Nitta, T., Tsutsumi, M., Okamura, T., Penninger, J. M., Nakashima, T., Takayanagi, H. 2017; 18 (6): 675-+

  Abstract

  Immunoglobulin A (IgA) maintains a symbiotic equilibrium with intestinal microbes. IgA induction in the gut-associated lymphoid tissues (GALTs) is dependent on microbial sampling and cellular interaction in the subepithelial dome (SED). However it is unclear how IgA induction is predominantly initiated in the SED. Here we show that previously unrecognized mesenchymal cells in the SED of GALTs regulate bacteria-specific IgA production and diversify the gut microbiota. Mesenchymal cells expressing the cytokine RANKL directly interact with the gut epithelium to control CCL20 expression and microfold (M) cell differentiation. The deletion of mesenchymal RANKL impairs M cell-dependent antigen sampling and B cell-dendritic cell interaction in the SED, which results in a reduction in IgA production and a decrease in microbial diversity. Thus, the subepithelial mesenchymal cells that serve as M cell inducers have a fundamental role in the maintenance of intestinal immune homeostasis.

  View details for DOI 10.1038/ni.3732

  View details for Web of Science ID 000401520900012

  View details for PubMedID 28436956

• LOX Fails to Substitute for RANKL in Osteoclastogenesis JOURNAL OF BONE AND MINERAL RESEARCH Tsukasaki, M., Hamada, K., Okamoto, K., Nagashima, K., Terashima, A., Komatsu, N., Win, S. J., Okamura, T., Nitta, T., Yasuda, H., Penninger, J. M., Takayanagi, H. 2017; 32 (3): 434-439

  Abstract

  Osteoclasts are the exclusive bone-resorbing cells that have a central role in bone homeostasis as well as bone destruction in cancer and autoimmune disease. Both mouse and human genetic studies have clearly proven that receptor activator of NF-κB ligand (RANKL; encoded by the Tnfsf11 gene) and its receptor RANK are essential for osteoclastogenesis. Although there have been several reports on RANKL-independent osteoclastogenesis, previous studies have never provided in vivo evidence showing RANKL can be substituted by other molecules using RANKL- or RANK-deficient genetic backgrounds. Thus, to date, there is no clear evidence of RANKL-independent osteoclastogenesis and no molecule has ever been proven capable of inducing osteoclast differentiation more efficiently than RANKL. Recently, lysyl oxidase (LOX), the enzyme that mediates collagen cross-linking, has been shown to induce human osteoclasts in the absence of RANKL and has a stronger osteoclastogenic activity than RANKL. Here, we investigated the effect of LOX on osteoclast differentiation using RANKL- and RANK-deficient cells to strictly explore RANKL-independent osteoclastogenesis. CD14+ human peripheral blood cells as well as osteoclast precursor cells derived from wild-type, RANKL- and RANK-deficient mice were treated with RANKL and/or LOX in short-term (3 days) or long-term (3 weeks) experimental settings. LOX treatment alone did not result in the formation of tartrate-resistant acid phosphatase (TRAP)+ cells or resorption pits in either short-term or long-term culture. In combination with RANKL, long-term treatment with LOX synergistically promoted osteoclastogenesis in cells derived from wild-type mice; however, this was abrogated in RANKL-deficient cells. Long-term treatment with LOX stimulated RANKL expression in mouse bone marrow stromal cells via the production of reactive oxygen species (ROS). Furthermore, LOX injection failed to rescue the phenotype of RANKL-deficient mice. These results suggest that LOX has the ability to induce RANKL expression on stromal cells; however, it fails to substitute for RANKL in osteoclastogenesis. © 2016 American Society for Bone and Mineral Research.

  View details for DOI 10.1002/jbmr.2990

  View details for Web of Science ID 000398055900004

  View details for PubMedID 27606829