Single-cell landscapes reveal a conserved IL-6-hepcidin axis coordinates renal antibacterial defense and immunopathology in Larimichthys crocea

Abstract

The teleost kidney is a vital hematopoietic and secondary lymphoid organ, yet the cellular coordination and regulatory circuits driving its response to systemic infection remain poorly understood. Here, we present a high-resolution single-cell transcriptomic atlas of the large yellow croaker (Larimichthys crocea) kidney during challenge with the necrotizing pathogen Pseudomonas plecoglossicida. Our analysis reveals a profound remodeling of the renal immune microenvironment, characterized by a dramatic expansion of the myeloid compartment and the emergence of an integrated cell death signature, comprising apoptosis, ferroptosis, and necroptosis that underlies the formation of characteristic visceral white nodules. Through trajectory and RNA velocity analyses, we demonstrate that bacterial infection triggers “emergency granulopoiesis”, driving the rapid differentiation of neutrophils, macrophages and mast cells into hyper-inflammatory states. Crucially, we identify a non-canonical, multi-lineage antimicrobial response in which phagocytes serve as the primary sources of the antimicrobial peptide hepcidin, with selected activated B-cell and regulatory T-cell populations displaying additional hepcidin-associated signatures. We further establish the IL-6-JAK2/STAT3 signaling pathway as the master regulatory axis orchestrating this systemic defense. Pharmacological inhibition of JAK2 abolished hepcidin induction, while exogenous IL-6 administration significantly enhanced host survival and bacterial clearance. These findings delineate the evolutionary conservation of cytokine-mediated antimicrobial regulation and provide a comprehensive model of organ-level immune coordination in early vertebrates.

Keywords

Single-cell transcriptome sequencing / Teleost kidney / Larimichthys crocea / Immune cell landscape / Antibacterial immunity

References

[1] Álvarez CA, Santana PA, Salinas-Parra N, Beltrán D, Guzmán F, Vega B, Acosta F, Mercado L. Immune modulation ability of hepcidin from teleost fish. Animals (Basel), 2022, 12 1586

[2] Ao J, Mu Y, Xiang L-X, Fan D, Feng M, Zhang S, Shi Q, Zhu L-Y, Li T, Ding Y. Genome sequencing of the perciform fish Larimichthys crocea provides insights into molecular and genetic mechanisms of stress adaptation. PLoS Genet, 2015, 11 e1005118

[3] Audzevich T, Bashford-Rogers R, Mabbott NA, Frampton D, Freeman TC, Potocnik A, Kellam P, Gilroy DW. Pre/pro-B cells generate macrophage populations during homeostasis and inflammation. P Natl Acad Sci Usa, 2017, 114: E3954-E3963

[4] Bai Y, Wang J, Zhao J, Ke Q, Qu A, Deng Y, Zeng J, Gong J, Chen J, Pan Y. Genomic selection for visceral white-nodules diseases resistance in large yellow croaker. Aquaculture, 2022, 559 738421

[5] Baumgarth N. The double life of a B-1 cell: self-reactivity selects for protective effector functions. Nat Rev Immunol, 2011, 11: 34-46

[6] Boehm T, Iwanami N, Hess I. Evolution of the immune system in the lower vertebrates. Annu Rev Genom Hum G, 2012, 13: 127-149

[7] Bromage ES, Kaattari IM, Zwollo P, Kaattari SL. Plasmablast and plasma cell production and distribution in trout immune tissues. J Immunol, 2004, 173: 7317-7323

[8] Chan JT, Kadri S, Köllner B, Rebl A, Korytář T. RNA-seq of single fish cells–seeking out the leukocytes mediating immunity in teleost fishes. Front Immunol, 2022, 13 798712

[9] Cooper MD, Alder MN. The evolution of adaptive immune systems. Cell, 2006, 124: 815-822

[10] Dou J, Chen X, Zhang J, Yang L, Lin J, Zhu W, Huang D, Tan X. P. gingivalis induce macrophage polarization by regulating hepcidin expression in chronic apical periodontitis. Int Immunopharmacol, 2024, 142 113139

[11] Flajnik MF. A cold-blooded view of adaptive immunity. Nat Rev Immunol, 2018, 18: 438-453

[12] Flajnik MF, Kasahara M. Origin and evolution of the adaptive immune system: genetic events and selective pressures. Nat Rev Genet, 2010, 11: 47-59

[13] Ganz T. Hepcidin and iron regulation, 10 years later. Blood, 2011, 117: 4425-4433

[14] Geven EJ, Klaren PH. The teleost head kidney: integrating thyroid and immune signalling. Dev Comp Immunol, 2017, 66: 73-83

[15] Hentze MW, Muckenthaler MU, Galy B, Camaschella C. Two to tango: regulation of mammalian iron metabolism. Cell, 2010, 142: 24-38

[16] Hillegass JM, Villano CM, Cooper KR, White LA. Matrix metalloproteinase-13 is required for zebra fish (Danio rerio) development and is a target for glucocorticoids. Toxicol Sci, 2007, 100: 168-179

[17] Hu C-b, Huang C, Wang J, Hong Y, Fan D-d, Chen Y, Lin A-f, Xiang L-x, Shao J-z. PD-L1/BTLA checkpoint axis exploited for bacterial immune escape by restraining CD8⁺ T cell–initiated adaptive immunity in zebrafish. J Immunol, 2023, 211: 816-835

[18] Hu CB, Wang J, Hong Y, Li H, Fan DD, Lin AF, Xiang LX, Shao JZ. Single-cell transcriptome profiling reveals diverse immune cell populations and their responses to viral infection in the spleen of zebrafish. Faseb J, 2023, 37 e22951

[19] Hu C, Zhang N, Hong Y, Tie R, Fan D, Lin A, Chen Y, Xiang L-x, Shao J-z. Single-cell RNA sequencing unveils the hidden powers of zebrafish kidney for generating both hematopoiesis and adaptive antiviral immunity. Elife, 2024, 13 RP92424

[20] Hu C, Cui J, He R, Han M, Liu Y, Zhou J, Fan D, Ding X, Xiang L, Lou B. Single-cell profiling reveals immune cell diversity and infection-driven remodeling in the spleen of marine teleost Larimichthys crocea. Fund Res-China, 2025

[21] Hu C, Hong Y, Xiang L, Chen Y, Shao J, Zhang H. Single-cell sequencing: a sharp sword for deciphering biological innovations and evolutionary adaptations in lower vertebrates and invertebrates. Rev Aquacult, 2025, 17 e70073

[22] Jiang Y, Abernathy JW, Peatman E, Liu H, Wang S, Xu D-H, Kucuktas H, Klesius P, Liu Z. Identification and characterization of matrix metalloproteinase-13 sequence structure and expression during embryogenesis and infection in channel catfish (Ictalurus punctatus). Dev Comp Immunol, 2010, 34: 590-597

[23] Kaczmarek A, Vandenabeele P, Krysko DV. Necroptosis: the release of damage-associated molecular patterns and its physiological relevance. Immunity, 2013, 38: 209-223

[24] Kearney CJ, Martin SJ. An inflammatory perspective on necroptosis. Mol Cell, 2017, 65: 965-973

[25] Kwok AJ, Allcock A, Ferreira RC, Cano-Gamez E, Smee M, Burnham KL, Zurke Y-X, McKechnie S, Mentzer AJ. Neutrophils and emergency granulopoiesis drive immune suppression and an extreme response endotype during sepsis. Nat Immunol, 2023, 24: 767-779

[26] La Manno G, Soldatov R, Zeisel A, Braun E, Hochgerner H, Petukhov V, Lidschreiber K, Kastriti ME, Lönnerberg P, Furlan A. RNA velocity of single cells. Nature, 2018, 560: 494-498

[27] Lee P, Peng H, Gelbart T, Beutler E. The IL-6-and lipopolysaccharide-induced transcription of hepcidin in HFE-, transferrin receptor 2-, and β2-microglobulin-deficient hepatocytes. Proc Natl Acad Sci U S A, 2004, 101: 9263-9265

[28] Lee P, Peng H, Gelbart T, Wang L, Beutler E. Regulation of hepcidin transcription by interleukin-1 and interleukin-6. P Natl Acad Sci U S A, 2005, 102: 1906-1910

[29] Li J, Barreda DR, Zhang Y-A, Boshra H, Gelman AE, LaPatra S, Tort L, Sunyer JO. B lymphocytes from early vertebrates have potent phagocytic and microbicidal abilities. Nat Immunol, 2006, 7: 1116-1124

[30] Li Z, Fang M, Tang X, Zhang D, Wang Z. Disentangling genetic variation for endurance and resistance to visceral white-nodules disease in large yellow croaker (Larimichthys crocea) using genome information. Aquaculture, 2023, 564 739045

[31] Long GH, Graham AL. Consequences of immunopathology for pathogen virulence evolution and public health: malaria as a case study. Evol Appl, 2011, 4: 278-291

[32] Manz MG, Boettcher S. Emergency granulopoiesis. Nat Rev Immunol, 2014, 14: 302-314

[33] Matz H, Taylor RS, Redmond AK, Hill TM, Daniels RR, Beltran M, Henderson NC, Macqueen DJ, Dooley H. Organized B cell sites in cartilaginous fishes reveal the evolutionary foundation of germinal centers. Cell Rep, 2023

[34] McGinnis CS, Murrow LM, Gartner ZJ. DoubletFinder: doublet detection in single-cell RNA sequencing data using artificial nearest neighbors. Cell Syst, 2019, 8(329–337e324

[35] Mu Y, Huo J, Guan Y, Fan D, Xiao X, Wei J, Li Q, Mu P, Ao J, Chen X. An improved genome assembly for Larimichthys crocea reveals hepcidin gene expansion with diversified regulation and function. Commun Biol, 2018, 1: 195

[36] Neely HR, Flajnik MF. Emergence and evolution of secondary lymphoid organs. Annu Rev Cell Dev Bi, 2016, 32: 693-711

[37] Nemeth E, Tuttle MS, Powelson J, Vaughn MB, Donovan A, Ward DM, Ganz T, Kaplan J. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science, 2004, 306: 2090-2093

[38] Newton AH, Cardani A, Braciale TJ. The host immune response in respiratory virus infection: balancing virus clearance and immunopathology. Semin immunopathol, 2016Springer, 471-4824

[39] Pasparakis M, Vandenabeele P. Necroptosis and its role in inflammation. Nature, 2015, 517: 311-320

[40] Paudel S, Ghimire L, Jin L, Jeansonne D, Jeyaseelan S. Regulation of emergency granulopoiesis during infection. Front Immunol, 2022, 13 961601

[41] Pedersen ME, Vuong TT, Rønning SB, Kolset SO. Matrix metalloproteinases in fish biology and matrix turnover. Matrix Biol, 2015, 44: 86-93

[42] Peng J, Li W, Wang B, Zhang S, Xiao Y, Han F, Wang Z. UBE2G1 is a critical component of immune response to the infection of Pseudomonas plecoglossicida in large yellow croaker (Larimichthys crocea). Int J Mol Sci, 2022, 23 8298

[43] Richardson H, Emslie-Smith A, Senior B. Agar diffusion method for the assay of colicins. Appl Microbiol, 1968, 16: 1468-1474

[44] Shi W, Shao T, Li J-y, Fan D-d, Lin A-f, Xiang L-x, Shao J-z. BTLA–HVEM checkpoint axis regulates hepatic homeostasis and inflammation in a ConA-induced hepatitis model in zebrafish. J Immunol, 2019, 203: 2425-2442

[45] Shu FL, Jin LY, Liu H, Tao Z, Yin F, Xie JS, Zhou SM. The galU gene is required for in vivo survival of Pseudomonas plecoglossicida in large yellow croaker (Larimichthys crocea). J Fish Dis, 2023, 46: 229-238

[46] Sun Y-J, Peng X-Z, Tan H-J, He J-G, Yan Q-P (2025) Pseudomonas plecoglossicida hijacks host glycolysis to induce hypoglycemic shock in large yellow croakers (Larimichthys crocea). Zool Res 46

[47] Sunyer JO. Fishing for mammalian paradigms in the teleost immune system. Nat Immunol, 2013, 14: 320-326

[48] Tang Q, Iyer S, Lobbardi R, Moore JC, Chen H, Lareau C, Hebert C, Shaw ML, Neftel C, Suva ML. Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single-cell resolution using RNA sequencing. J Exp Med, 2017, 214: 2875-2887

[49] Tang Y, Xin G, Zhao L-M, Huang L-X, Qin Y-X, Su Y-Q, Zheng W-Q, Wu B, Lin N, Yan Q-P. Novel insights into host-pathogen interactions of large yellow croakers (Larimichthys crocea) and pathogenic bacterium Pseudomonas plecoglossicida using time-resolved dual RNA-seq of infected spleens. Zool Res, 2020, 41: 314

[50] Vanickova K, Milosevic M, Ribeiro Bas I, Burocziova M, Yokota A, Danek P, Grusanovic S, Chiliński M, Plewczynski D, Rohlena J. Hematopoietic stem cells undergo a lymphoid to myeloid switch in early stages of emergency granulopoiesis. Embo J, 2023, 42: EMBJ2023113527

[51] Wan F, Hu C-b, Ma J-x, Gao K, Xiang L-x, Shao J-z. Characterization of γδ T cells from zebrafish provides insights into their important role in adaptive humoral immunity. Front Immunol, 2017, 7: 675

[52] Wang LH, Kirken RA, Erwin RA, Yu C-R, Farrar WL. JAK3, STAT, and MAPK signaling pathways as novel molecular targets for the tyrphostin AG-490 regulation of IL-2-mediated T cell response. J Immunol, 1999, 162: 3897-3904

[53] Wrighting DM, Andrews NC. Interleukin-6 induces hepcidin expression through STAT3. Blood, 2006, 108: 3204-3209

[54] Wu L, Gao A, Li L, Chen J, Li J, Ye J. A single-cell transcriptome profiling of anterior kidney leukocytes from Nile tilapia (Oreochromis niloticus). Front Immunol, 2021, 12 783196

[55] Xie H, Ye M, Feng R, Graf T. Stepwise reprogramming of B cells into macrophages. Cell, 2004, 117: 663-676

[56] Yan L-t, Jiang Y, Xu Q, Ding G-m, Chen X-y, Liu M. Reproductive dynamics of the large yellow croaker Larimichthys crocea (Sciaenidae), a commercially important fishery species in China. Front Mar Sci, 2022, 9 868580

[57] Yin X, Chen N, Mu L, Bai H, Wu H, Qi W, Huang Y, Jian J, Wang A, Ye J. Identification and characterization of hepcidin from Nile Tilapia (Oreochromis niloticus) in response to bacterial infection and iron overload. Aquaculture, 2022, 546 737317

[58] You L-H, Yan C-Z, Zheng B-J, Ci Y-Z, Chang S-Y, Yu P, Gao G-F, Li H-Y, Dong T-Y, Chang Y-Z. Astrocyte hepcidin is a key factor in LPS-induced neuronal apoptosis. Cell Death Dis, 2017, 8: e2676

[59] Zhang S-P, Wang Z, Wang L-X, Liu S-J. AG490: an inhibitor of hepcidin expression in vivo. World J Gastroenterol, 2011, 17: 5032

[60] Zhang J, Zhou S, An S, Chen L, Wang G. Visceral granulomas in farmed large yellow croaker, Larimichthys crocea (Richardson), caused by a bacterial pathogen, Pseudomonas plecoglossicida. J Fish Dis, 2014, 37: 113-121

[61] Zhang W, Li B, Yu R, Xu W, Liu X, Su J, Yuan G. Hepcidin contributes to largemouth bass (Micropterus salmoides) against bacterial infections. Int J Biol Macromol, 2024, 266 131144

[62] Zhu L-y, Lin A-f, Shao T, Nie L, Dong W-r, Xiang L-x, Shao J-z. B cells in teleost fish act as pivotal initiating APCs in priming adaptive immunity: an evolutionary perspective on the origin of the B-1 cell subset and B7 molecules. J Immunol, 2014, 192: 2699-2714

[63] Zwollo P, Cole S, Bromage E, Kaattari S. B cell heterogeneity in the teleost kidney: evidence for a maturation gradient from anterior to posterior kidney. J Immunol, 2005, 174: 6608-6616

Rights & permissions

The Author(s)

Just Accepted

This article has successfully passed peer review and final editorial review, and will soon enter typesetting, proofreading and other publishing processes. The currently displayed version is the accepted final manuscript. The officially published version will be updated with format, DOI and citation information upon launch. We recommend that you pay attention to subsequent journal notifications and preferentially cite the officially published version. Thank you for your support and cooperation.