Abstract
Black rhinoceros are critically endangered due to poaching in the wild (in situ). Globally, fewer than 200 animals are maintained as an ex situ insurance population. Unfortunately, the ex situ population faces major sustainability challenges from disease syndromes characterized by high inflammatory burdens and diverse manifestations of immunometabolic dysfunction, not known to be present among their wild counterparts. Overlapping ex situ disease phenotypes limit diagnostic specificity and highlight the need to define underlying disease mechanisms. In the present study, using a cohort of presumed clinically healthy and inflammatory black rhinoceros, we generated the first immunoproteomic profile of any endangered mammal species and identified 1,311 immune cell proteins. However, no significant differences were detected among clinical phenotypes. Therefore, we applied unsupervised machine learning approaches to detect molecular features suggestive of healthy versus inflammatory phenotypes. Forty-three proteins associated with inflammatory pathways were differentially expressed in a cohort of samples derived from both presumed healthy and inflammatory phenotypes. Results suggest subclinical disease may be relatively widespread ex situ, and that animals experience temporal fluctuations in inflammatory state over time. Findings implicate neutrophil degranulation and dysregulation of the oral-gut-liver axis as drivers of disease syndromes of ex situ black rhinoceros. The forty-three proteins associated with inflammatory pathways represent candidate inflammatory biomarkers to be assessed for clinical applications in future validation studies. Upon validation, these candidate biomarkers may guide management practices to strengthen long-term population sustainability.
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Data availability
Data and scripts used in this study are available at Smithsonian Institution’s Figshare site: https://figshare.com/s/b49afc55c401b6602358.
References
Emslie, R. The International Union for the Conservation of Nature Red List of Threatened Species: Diceros bicornis: e.T6557A152728945 (2020).
Sánchez-Barreiro, F. et al. Historic sampling of a vanishing beast: Population structure and diversity in the black rhinoceros. Mol. Biol. Evol. 40, msad180 (2023).
Smith, L., Ferrie, G. M. & Ferrie, G. M. Population analysis & breeding and transfer plan (American Association of Zoos and Aquariums, 2021).
Sullivan, K. E. et al. Safety and efficacy of a novel iron chelator (HBED; (N,N ′-Di(2‐hydroxybenzyl)ethylenediamine‐N,N′‐diacetic acid)) in equine (Equus caballus) as a model for black rhinoceros (Diceros bicornis). Anim. Physiol. Nutr. 106, 1107–1117 (2022).
Lagrot, I., Lagrot, J. F. & Bour, P. Probable extinction of the western black rhino, Diceros bicornis longipes: survey in northern Cameroon. (2007). (2006).
Dennis, P. M. et al. A review of some of the health issues of captive black rhinoceroses (Diceros bicornis). J. Zoo Wildl. Med. 38, 509–517 (2007).
Dennis, P. et al. IOD in rhinos - Epidemiology group report: Report from the epidemiology working group of the International Workshop on Iron Overload Disorder in browsing rhinoceros (February 2011). J. Zoo Wildl. Med. 43, S114–S116 (2012).
Radeke-Auer, K., Clauss, M., Stagegaard, J., Sonsbeek, L. G. R. B. V. & Lopez, J. Retrospective pathology review of captive black rhinoceros Diceros bicornis in the EAZA Ex-situ Programme (1995–2022). J. Zoo Aquarium Res. 11, 298–310 (2023).
Corder, M. L. et al. Metabolomic profiling implicates mitochondrial and immune dysfunction in disease syndromes of the critically endangered black rhinoceros (Diceros bicornis). Sci. Rep. 13(1), 15464 (2023).
Pouillevet, H., Soetart, N., Boucher, D., Wedlarski, R. & Jaillardon, L. Inflammatory and oxidative status in European captive black rhinoceroses: A link with iron overload disorder? PLoS ONE. 15, e0231514 (2020).
Schook, M. W., Wildt, D. E., Raghanti, M. A., Wolfe, B. A. & Dennis, P. M. Increased inflammation and decreased insulin sensitivity indicate metabolic disturbances in zoo-managed compared to free-ranging black rhinoceros (Diceros bicornis). Gen. Comp. Endocrinol. 217–218, 10–19 (2015).
Meyer, A. et al. Assessment of capillary zone electrophoresis and serum amyloid A quantitation in clinically normal and abnormal southern white rhinoceros (Ceratotherium simum simum) and southern black rhinoceros (Diceros bicornis minor). J. Zoo Wildl. Med. 53, 85 (2022).
Rispoli, L. A., Wojtusik, J. & Roth, T. L. Exploring serum ferritin’s connection to the acute phase response in zoo-managed African rhinoceroses. Zoo Biol. 44, 16–23 (2025).
Bruins-van Sonsbeek, L. G. R. et al. Rhinoceromics: A multi-amplicon study with clinical markers to transferrin saturation levels in ex-situ black rhinoceros (Diceros bicornis michaeli). Front. Microbiol. 16, 1515939 (2025).
Cray, C., Rodriguez, M., Dickey, M., Brewer, L. B. & Arheart, K. L. Assessment of serum amyloid A levels in the rehabilitation setting in the Florida manatee (Trichechus manatus latirostris). J. Zoo Wildl. Med. 44, 911–917 (2013).
Rhim, H., Kim, M., Gim, S. & Han, J.-I. Diagnostic value of serum amyloid A in differentiating the inflammatory disorders in wild birds. Front. Vet. Sci. https://doi.org/10.3389/fvets.2024.1284113 (2024).
Roth, T. L. & Vance, C. K. Corticosteroid-induced suppression of in vitro lymphocyte proliferation in four captive rhinoceros species. J. Zoo Wildl. Med. 38, 518–525 (2007).
Vance, C. K., Kennedy-Stoskopf, S., Obringer, A. R. & Roth, T. L. Comparative studies of mitogen- and antigen- induced lymphocyte proliferation in four captive rhinoceros species. J. Zoo Wildl. Med. 35, 435–446 (2004).
Citino, S. et al. IOD in rhinos - Veterinary group report: Report from the clinical medicine and pathology working group of the International Workshop on Iron Overload Disorder in browsing rhinoceros (February 2011). I. J. Zoo Wildl. Med. 43, S105–S107 (2012).
Wojtusik, J. & Roth, T. L. Investigation of factors potentially associated with serum ferritin concentrations in the black rhinoceros (Diceros bicornis) using a validated rhinoceros-specific assay. J. Zoo Wildl. Med. 49, 297–306 (2018).
Ameka, M. K. & Hasty, A. H. Paying the iron price: Liver iron homeostasis and metabolic disease. Comparative Physiology 12, 3641–3663 (2022).
Alexovič, M., Uličná, C., Sabo, J. & Davalieva, K. Human peripheral blood mononuclear cells as a valuable source of disease-related biomarkers: Evidence from comparative proteomics studies. Proteomics Clin. Appl. 18, 2300072 (2024).
Kleiveland, C. R. Peripheral Blood Mononuclear Cells In (ed. Verhoeckx, K.) (2015).
Končarević, S. et al. In-depth profiling of the peripheral blood mononuclear cells proteome for clinical blood proteomics. Int. J. Proteomics 2014, 1–9 (2014).
Pansarasa, O. et al. Biomarkers in human peripheral blood mononuclear cells: The state of the art in amyotrophic lateral sclerosis. Int. J. Mol. Sci. 23, 2580 (2022).
Sen, P., Kemppainen, E. & Orešič, M. Perspectives on systems modeling of human peripheral blood mononuclear cells. Front. Mol. Biosci. 4, 96 (2018).
Mhyre, T. R. et al. Proteomic analysis of peripheral leukocytes in Alzheimer’s disease patients treated with divalproex sodium. Neurobiol. Aging 29, 1631–1643 (2008).
He, P. et al. ITGA2 protein is associated with rheumatoid arthritis in Chinese and affects cellular function of T cells. Clin. Chim. Acta 523, 208–215 (2021).
Lepper, M. F. et al. Proteomic landscape of patient-derived CD4 + T cells in recent-onset type 1 diabetes. J. Proteome Res. 17, 618–634 (2018).
Rangel-Zúñiga, O. A. et al. Proteome from patients with metabolic syndrome is regulated by quantity and quality of dietary lipids. BMC Genomics 16, 509 (2015).
Leite, G. G. F. et al. Combined transcriptome and proteome leukocyte’s profiling reveals up-regulated module of genes/proteins related to low density neutrophils and impaired transcription and translation processes in clinical sepsis. Front. Immunol. https://doi.org/10.3389/fimmu.2021.744799 (2021).
Wright, C. et al. Ankylosing spondylitis monocytes show upregulation of proteins involved in inflammation and the ubiquitin proteasome pathway. Ann. Rheum. Dis. 68, 1626–1632 (2009).
Guito, J. C. et al. Asymptomatic infection of Marburg virus reservoir bats is explained by a strategy of immunoprotective disease tolerance. Curr. Biol. 31, 257-270.e5 (2021).
Irving, A. T. et al. Optimizing dissection, sample collection and cell isolation protocols for frugivorous bats. Methods. Ecol. Evol. 11, 150–158 (2020).
Roland, K. et al. Proteomic responses of peripheral blood mononuclear cells in the European eel (Anguilla anguilla) after perfluorooctane sulfonate exposure. Aquat. Toxicol. 128–129, 43–52 (2013).
Wilkerson, M. D. & Hayes, D. N. ConsensusClusterPlus: A class discovery tool with confidence assessments and item tracking. Bioinformatics 26, 1572–1573 (2010).
Hooijberg, E. H. et al. Assessment of the acute phase response in healthy and injured Southern White Rhinoceros (Ceratotherium simum simum). Front. Vet. Sci. 6, 475 (2020).
Yu, G. et al. clusterProfiler: An R package for comparing biological themes among gene clusters. OMICS 16(5), 284–287 (2012).
Liberzon, A. et al. Molecular signatures database (MSigDB) 3.0. Bioinformatics 27 (12), 1739–1740 (2011).
Maekawa, S. et al. RNA sequencing for ligature induced periodontitis in mice revealed important role of S100A8 and S100A9 for periodontal destruction. Sci. Rep. 9, 14663 (2019).
Dahlstrand Rudin, A. et al. The neutrophil subset defined by CD177 expression is preferentially recruited to gingival crevicular fluid in periodontitis. J. Leukoc. Biol. 109, 349–362 (2021).
Silbereisen, A. et al. Regulation of PGLYRP1 and TREM-1 during progression and resolution of gingival inflammation. JDR Clin. Transl. Res. 4, 352–359 (2019).
Yucel, Z. P. K. et al. Salivary biomarkers in the context of gingival inflammation in children with cystic fibrosis. J. Periodontol. 91, 1339–1347 (2020).
Lira-Junior, R. et al. S100A12 expression is modulated during monocyte differentiation and reflects periodontitis severity. Front. Immunol. 11, (2020).
Lundmark, A. et al. Gene expression profiling of periodontitis-affected gingival tissue by spatial transcriptomics. Sci. Rep. 8, 9370 (2018).
Franco, C., Patricia, H.-R., Timo, S., Claudia, B. & Marcela, H. Matrix metalloproteinases as regulators of periodontal inflammation. Int. J. Mol. Sci. 18, 440 (2017).
Baus-Domínguez, M. et al. Using genetics in periodontal disease to justify implant failure in Down syndrome patients. J. Clin. Med. 9, 2525 (2020).
Cavalla, F. et al. Proteomic profiling and differential messenger RNA expression correlate HSP27 and serpin family B member 1 to apical periodontitis outcomes. J. Endod. 43, 1486–1493 (2017).
Bose, A., Narayan, S. J. & Santosh, H. N. A randomized controlled crossover trial for reinforcement of epithelial barrier function by vitamin D induction of the antimicrobial peptide cathelicidin - A novel therapeutic approach in chronic periodontitis. RGUHS J. Dent. Sci. https://doi.org/10.26715/rjds.14_3_12 (2022).
Türkoğlu, O., Azarsız, E., Emingil, G., Kütükçüler, N. & Atilla, G. Are proteinase 3 and cathepsin C enzymes related to pathogenesis of periodontitis?. BioMed Res. Int. e420830 (2014).
Foratori-Junior, G. A. et al. Label-free quantitative proteomic analysis reveals inflammatory pattern associated with obesity and periodontitis in pregnant women. Metabolites 12, 1091 (2022).
Li, Q. et al. Proteomic analysis of human periodontal ligament cells under hypoxia. Proteome Sci. https://doi.org/10.1186/s12953-019-0151-2 (2019).
Devanoorkar, A., Kathariya, R., Guttiganur, N., Gopalakrishnan, D. & Bagchi, P. Resistin: A potential biomarker for periodontitis influenced diabetes mellitus and diabetes induced periodontitis. Dis. Markers e930206 (2014).
Velickovic, M. et al. Galectin-3, possible role in pathogenesis of periodontal diseases and potential therapeutic target. Front. Pharmacol. 12, (2021).
Xiong, Z., Fang, Y., Lu, S., Sun, Q. & Huang, J. Identification and validation of signature genes and potential therapy targets of inflammatory bowel disease and periodontitis. J. Inflamm. Res. 16, 4317–4330 (2023).
Gölz, L. et al. LPS from P. gingivalis and hypoxia increases oxidative stress in periodontal ligament fibroblasts and contributes to periodontitis. Mediat. Inflamm. e986264 (2014)
Berlutti, F., Pilloni, A., Pietropaoli, M., Polimeni, A. & Valenti, P. Lactoferrin and oral diseases: Current status and perspective in periodontitis. Annali di Stomatologia (Roma) 2, 10–18 (2012).
Liu, J. et al. Discovering genetic linkage between periodontitis and type 1 diabetes: A bioinformatics study. Front. Genet. 14, 75 (2023).
Bao, W., Wang, L., Liu, X. & Li, M. Predicting diagnostic biomarkers associated with immune infiltration in Crohn’s disease based on machine learning and bioinformatics. Eur. J. Med. Res. 28, 255 (2023).
Dheer, R. et al. Microbial signatures and innate immune gene expression in lamina propria phagocytes of inflammatory bowel disease patients. Cell. Mol. Gastroenterol. Hepatol. 9, 387–402 (2020).
Nowak, J. K. et al. Characterisation of the circulating transcriptomic landscape in inflammatory bowel disease provides evidence for dysregulation of multiple transcription factors including NFE2, SPI1, CEBPB, and IRF2. J. Crohns Colitis. 16, 1255–1268 (2022).
Iida, H. et al. Paraimmunobiotic bifidobacteria modulate the expression patterns of peptidoglycan recognition proteins in porcine intestinal epitheliocytes and antigen presenting cells. Cells 8, 891 (2019).
Guo, X. et al. Gut microbiota is a potential biomarker in inflammatory bowel disease. Front. Nutr. 8, 818902 (2022).
Zhang, X. et al. Widespread protein lysine acetylation in gut microbiome and its alterations in patients with Crohn’s disease. Nat. Commun. 11, 4120 (2020).
Rodrigues, D. M. et al. Matrix metalloproteinase 9 contributes to gut microbe homeostasis in a model of infectious colitis. BMC Microbiol. 12, 105 (2012).
Rahabi, M. et al. Divergent roles for macrophage C-type lectin receptors, dectin-1 and mannose receptors, in the intestinal inflammatory response. Cell Rep. 30, 4386-4398e.e5 (2020).
Kriaa, A. et al. Serine proteases at the cutting edge of IBD: Focus on gastrointestinal inflammation. FASEB J. 34, 7270–7282 (2020).
Yan, P. et al. Integrating the serum proteomic and fecal metaproteomic to analyze the impacts of overweight/obesity on IBD: a pilot investigation. Clin. Proteomics. 20, 6 (2023).
Su, T. et al. Myeloid-derived grancalcin instigates obesity-induced insulin resistance and metabolic inflammation in male mice. Nat. Commun. 15, 97 (2024).
Liang, W. et al. Intestinal cathelicidin antimicrobial peptide shapes a protective neonatal gut microbiota against pancreatic autoimmunity. Gastroenterology 162, 1288–1302e16 (2022).
Soussou, S. et al. Serine proteases and metalloproteases are highly increased in irritable bowel syndrome Tunisian patients. Sci. Rep. 13, 17571 (2023).
Melle, C. et al. Different expression of calgizzarin (S100A11) in normal colonic epithelium, adenoma and colorectal carcinoma. Int. J. Oncol. 28, 195–200 (2006).
Wang, C., Li, Y., Li, S., Chen, M. & Hu, Y. Proteomics combined with RNA sequencing to screen biomarkers of sepsis. Infect. Drug Resist. 15, 5575–5587 (2022).
Chen, L. et al. The landscape of isoform switches in sepsis: A multicenter cohort study. Sci. Rep. 12(1), 10276 (2022).
Volarevic, V. et al. Galectin-3 regulates indoleamine-2,3-dioxygenase-dependent cross-talk between colon-infiltrating dendritic cells and T regulatory cells and may represent a valuable biomarker for monitoring the progression of ulcerative colitis. Cells 8, 709 (2019).
Hinrichsen, F. et al. Microbial regulation of hexokinase 2 links mitochondrial metabolism and cell death in colitis. Cell Metab. 33, 2355-2366e.e8 (2021).
Martínez-Herrero, S. & Martínez, A. Adrenomedullin: Not just another gastrointestinal peptide. Biomolecules 12, 156 (2022).
Mui, L., Martin, C. M., Tschirhart, B. J. & Feng, Q. Therapeutic potential of annexins in sepsis and COVID-19. Front. Pharmacol. https://doi.org/10.3389/fphar.2021.735472 (2021).
Wang, W. et al. Dietary catalase supplementation alleviates deoxynivalenol-induced oxidative stress and gut microbiota dysbiosis in broiler chickens. Toxins 14, 830 (2022).
Deng, L. et al. Upregulation of soluble resistance-related calcium-binding protein (sorcin) in gastric cancer. Med. Oncol. 27, 1102–1108 (2010).
Kruzel, M. L., Zimecki, M. & Actor, J. K. Lactoferrin in a context of inflammation-induced pathology. Front. Immunol. https://doi.org/10.3389/fimmu.2017.01438 (2017).
Zhu, M., Dagah, O. M. A., Silaa, B. B. & Lu, J. Thioredoxin/glutaredoxin systems and gut microbiota in NAFLD: Interplay, mechanism, and therapeutical potential. Antioxidants 12, 1680 (2023).
Ryckman, C., Vandal, K., Rouleau, P., Talbot, M. & Tessier, P. A. Proinflammatory activities of S100: Proteins S100A8, S100A9, and S100A8/A9 induce neutrophil chemotaxis and adhesion 1. J. Immunol. 170, 3233–3242 (2003).
Henke, M. O. et al. Up-regulation of S100A8 and S100A9 protein in bronchial epithelial cells by lipopolysaccharide. Exp. Lung Res. 32, 331–347 (2006).
Saha, R. et al. Inflammatory signature in acute-on-chronic liver failure includes increased expression of granulocyte genes ELANE, MPO and CD177. Sci. Rep. 11, 18849 (2021).
Luo, Q. et al. Serum PGLYRP–1 is a highly discriminatory biomarker for the diagnosis of rheumatoid arthritis. Mol. Med. Rep. 19, 589–594 (2019).
Meijer, B., Gearry, R. B. & Day, A. S. The role of S100A12 as a systemic marker of inflammation. Int. J. Inflam. e907078 (2012).
Liu, W. & Rodgers, G. P. Olfactomedin 4 expression and functions in innate immunity, inflammation, and cancer. Cancer Metastasis Rev. 35, 201–212 (2016).
Snelgrove, R. J. et al. A critical role for LTA4H in limiting chronic pulmonary neutrophilic inflammation. Science 330, 90–94 (2010).
Choi, Y. J. et al. SERPINB1-mediated checkpoint of inflammatory caspase activation. Nat. Immunol. 20, 276–287 (2019).
Jo, M. et al. Astrocytic orosomucoid-2 modulates microglial activation and neuroinflammation. J. Neurosci. 37, 2878–2894 (2017).
Reinholz, M., Ruzicka, T. & Schauber, J. Cathelicidin LL-37: An antimicrobial peptide with a role in inflammatory skin disease. Ann. Dermatol. 24, 126–135 (2012).
Sawyer, A. J., Garand, M., Chaussabel, D. & Feng, C. G. Transcriptomic profiling identifies neutrophil-specific upregulation of cystatin F as a marker of acute inflammation in humans. Front. Immunol. https://doi.org/10.3389/fimmu.2021.634119 (2021).
Zhang, L., Zhu, T., Miao, H. & Liang, B. The calcium binding protein S100A11 and its roles in diseases. Front. Cell Dev. Biol. 9, 693262 (2021).
Bi, X. et al. GSTP1 inhibits LPS-induced inflammatory response through regulating autophagy in THP-1 cells. Inflammation 43(3), 1156–1169 (2020).
Nagaev, I., Bokarewa, M., Tarkowski, A. & Smith, U. Human resistin is a systemic immune-derived proinflammatory cytokine targeting both leukocytes and adipocytes. PLoS One 1, e31 (2006).
Liu, F.-T., Yang, R.-Y. & Hsu, D. K. Galectins in acute and chronic inflammation. Ann. N. Y. Acad. Sci. 1253, 80–91 (2012).
Steck, A. J., Kinter, J. & Renaud, S. Differential gene expression in nerve biopsies of inflammatory neuropathies. J. Peripher. Nerv. Syst. 16, 30–33 (2011).
Wyatt, E. et al. Regulation and cytoprotective role of hexokinase III. PLoS One 5, e13823 (2010).
Sun, J. et al. Adrenomedullin 2 attenuates LPS-induced inflammation in microglia cells by receptor-mediated cAMP-PKA pathway. Neuropeptides 85, 102109 (2021).
Han, P.-F. et al. Annexin A1 involved in the regulation of inflammation and cell signaling pathways. Chin. J. Traumatol. 23, 96–101 (2020).
Jang, B.-C. et al. Catalase induced expression of inflammatory mediators via activation of NF-κB, PI3K/AKT, p70S6K, and JNKs in BV2 microglia. Cell. Signal. 17, 625–633 (2005).
Wang, Y. et al. Soluble resistance-related calcium-binding protein participates in multiple diseases via protein-protein interactions. Biochimie 189, 76–86 (2021).
Cao, M.-Q. et al. Cross talk between oxidative stress and hypoxia via thioredoxin and HIF-2α drives metastasis of hepatocellular carcinoma. FASEB J. 34, 5892–5905 (2020).
Azarova, I. E., Klyosova, E. Y., Kolomoets, I. I. & Polonikov, A. V. Polymorphic variants of the neutrophil cytosolic factor 2 gene: Associations with susceptibility to type 2 diabetes mellitus and cardiovascular autonomic neuropathy. Russ. J. Genet. 58, 593–602 (2022).
Ma, J. et al. Glycogen metabolism regulates macrophage-mediated acute inflammatory responses. Nat. Commun. 11, 1769 (2020).
Miller, M. A. & Buss, P. E. Rhinoceridae (Rhinoceroses). in Fowler’s Zoo and Wild Animal Medicine, Volume 8 538–547 (Elsevier, 2015). https://doi.org/10.1016/B978-1-4557-7397-8.00055-4
Sprenkeler, E. G. G. et al. S100A8/A9 is a marker for the release of neutrophil extracellular traps and induces neutrophil activation. Cells 11, 236 (2022).
Burn, G. L., Foti, A., Marsman, G., Patel, D. F. & Zychlinsky, A. The neutrophil. Immunity 54, 1377–1391 (2021).
Eichelberger, K. R. & Goldman, W. E. Manipulating neutrophil degranulation as a bacterial virulence strategy. PLoS Pathog. 16, e1009054 (2020).
Lehman, H. K. & Segal, B. H. The role of neutrophils in host defense and disease. J. Allergy Clin. Immunol. 145, 1535–1544 (2020).
Taylor, L. A. et al. Tooth wear in captive rhinoceroses (Diceros, Rhinoceros, Ceratotherium: Perissodactyla) differs from that of free-ranging conspecifics. Contrib. Zool. 83, 107-S1 (2014).
Albuquerque-Souza, E. & Sahingur, S. E. Periodontitis, chronic liver diseases, and the emerging oral-gut-liver axis. Periodontol. 2000 89, 125–141 (2022).
Clauss, M. & Dierenfeld, E. S. The nutrition of browsers. in Zoo Wild Anim. Medicine 444–454 (Elsevier, 2008). https://doi.org/10.1016/B978-141604047-7.50058-0
Wei, L., Liu, M. & Xiong, H. Role of calprotectin as a biomarker in periodontal disease. Mediators Inflamm. 2019, 1–10 (2019).
Alder, M. N. et al. Olfactomedin 4 marks a subset of neutrophils in mice. Innate Immun. 25, 22–33 (2019).
Sansores-España, L. D. et al. Neutrophil N1 and N2 subsets and their possible association with periodontitis: A scoping review. Int. J. Mol. Sci. 23, 12068 (2022).
Silberman, M. S. & Fulton, R. B. Medical problems of captive and wild rhinoceros: A review of the literature and personal experiences. J. Zoo Anim. Med. 10, 6–16 (1979).
Gibson, K. M. et al. Gut microbiome differences between wild and captive black rhinoceros – Implications for rhino health. Sci. Rep. 9, 7570 (2019).
Planell, N. et al. Usefulness of transcriptional blood biomarkers as a non-invasive surrogate marker of mucosal healing and endoscopic response in ulcerative colitis. J. Crohns Colitis. 11, 1335–1346 (2017).
Brynjolfsson, S. F. et al. An antibody against triggering receptor expressed on myeloid cells 1 (TREM-1) dampens proinflammatory cytokine secretion by lamina propria cells from patients with IBD. Inflamm. Bowel Dis. 22, 1803–1811 (2016).
Dumitru, A. et al. Endotoxin inflammatory action on cells by dysregulated-immunological-barrier-linked ROS-apoptosis mechanisms in gut–liver axis. Int. J. Mol. Sci. 25, 2472 (2024).
Tilg, H., Moschen, A. R. & Szabo, G. Interleukin-1 and inflammasomes in alcoholic liver disease/acute alcoholic hepatitis and nonalcoholic fatty liver disease/nonalcoholic steatohepatitis. Hepatology 64, 955–965 (2016).
Xu, R., Huang, H., Zhang, Z. & Wang, F. S. The role of neutrophils in the development of liver diseases. Cell. Mol. Immunol. 11, 224–231 (2014).
Ganz, T., Goff, J., Klasing, K., Nemeth, E. & Roth, T. IOD in rhinos - Immunity Group Report: Report from the immunity, genetics and toxicology working group of the international workshop on iron overload disorder in browsing rhinoceros (February 2011). J. Zoo Wildl. Med. 43, S117–S119 (2012).
Sullivan, K. E., Mylniczenko, N. D., Nelson, S. E., Coffin, B. & Lavin, S. R. Practical management of iron overload disorder (IOD) in black rhinoceros (BR; Diceros bicornis). Animals 10, 1991 (2020).
Paglia, D. E. & Tsu, I.-H. Review of laboratory and necropsy evidence for iron storage disease acquired by browser rhinoceroses. J. Zoo Wildl. Med. https://doi.org/10.1638/2011-0177.1 (2012).
Ganz, T. & Nemeth, E. Iron homeostasis and its disorders in mice and men: Potential lessons for rhinos. J. Zoo Wildl. Med. 43, S19–S26 (2012).
Roth, T. L., Philpott, M. & Wojtusik, J. Rhinoceros serum labile plasma iron and associated redox potential: Interspecific variation, sex bias and iron overload disorder disconnect. Conserv. Physiol. 10, coac025 (2022).
Tang, J., Yan, Z., Feng, Q., Yu, L. & Wang, H. The roles of neutrophils in the pathogenesis of liver diseases. Front. Immunol. 12, 625472 (2021).
Obisesan, A. O., Zygiel, E. M. & Nolan, E. M. Bacterial responses to iron withholding by calprotectin. Biochemistry 60, 3337–3346 (2021).
Moles, A. et al. A TLR2/S100A9/CXCL-2 signaling network is necessary for neutrophil recruitment in acute and chronic liver injury in the mouse. J. Hepatol. 60, 782–791 (2014).
Oliveira, T. H. C. D., Marques, P. E., Proost, P. & Teixeira, M. M. M. Neutrophils: A cornerstone of liver ischemia and reperfusion injury. Lab. Invest. 98, 51–62 (2018).
Motiño, O. et al. Protective role of hepatocyte cyclooxygenase-2 expression against liver ischemia–reperfusion injury in mice. Hepatology 70, 650–665 (2019).
Jiang, W. & Banks, W. A. Viewpoint: Is lipopolysaccharide a hormone or a vitamin?. Brain Behav. Immun. 114, 1–2 (2023).
Opal, S. M. et al. Relationship between plasma levels of lipopolysaccharide (LPS) and LPS-binding protein in patients with severe sepsis and septic shock. J. Infect. Dis. 180, 1584–1589 (1999).
Senior, J. M., Proudman, C. J., Leuwer, M. & Carter, S. D. Plasma endotoxin in horses presented to an equine referral hospital: Correlation to selected clinical parameters and outcomes. Equine Vet. J. 43, 585–591 (2011).
Boribong, B. P., Lenzi, M. J., Li, L. & Jones, C. N. Super-low dose lipopolysaccharide dysregulates neutrophil migratory decision-making. Front. Immunol. 10, 359 (2019).
Liu, S. et al. Neutrophil extracellular traps are indirectly triggered by lipopolysaccharide and contribute to acute lung injury. Sci. Rep. 6, 37252 (2016).
Sandler, N. G. & Douek, D. C. Microbial translocation in HIV infection: Causes, consequences and treatment opportunities. Nat. Rev. Microbiol. 10, 655–666 (2012).
Fallon, J. P., Reeves, E. P. & Kavanagh, K. Inhibition of neutrophil function following exposure to the Aspergillus fumigatus toxin fumagillin. J. Med. Microbiol. 59, 625–633 (2010).
Danne, C. Neutrophils: Old cells in IBD, new actors in interactions with the gut microbiota. Clin. Transl. Med. 14, e1739 (2024).
Ficoll-Paque® PLUS and Ficoll-Paque® premium centrifugation media, Cytiva. VWR https://us.vwr.com/store/product/4779441/ficoll-paque-plus-and-ficoll-paque-premium-centrifugation-media-cytiva
Diceros bicornis minor (ID 8992) - Genome - NCBI. https://www.ncbi.nlm.nih.gov/genome/8992?genome_assembly_id=1737831
MetaMorpheus. Free, open-source PTM discovery. MetaMorpheus https://smith-chem-wisc.github.io/MetaMorpheus/
Solntsev, S. K., Shortreed, M. R., Frey, B. L. & Smith, L. M. Enhanced global post-translational modification discovery with MetaMorpheus. J. Proteome Res. 17, 1844–1851 (2018).
Wilmarth, P. PAW_BLAST. (2023).
Homo sapiens (Human) | Proteomes | UniProt. https://www.uniprot.org/proteomes/UP000005640
Stekhoven, D. stekhoven/missForest. (2024).
Stekhoven, D. J. & Bühlmann, P. MissForest—Non-parametric missing value imputation for mixed-type data. Bioinformatics 28, 112–118 (2012).
Ritchie, M. E. et al. Limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015).
Wilkinson, L. Ggplot2: Elegant graphics for data analysis by WICKHAM, H. Biometrics 67, 678–679 (2011).
Kolde, R. Pretty Heatmaps. R package. (2019).
Galili, T. Dendextend: An R package for visualizing, adjusting and comparing trees of hierarchical clustering. Bioinformatics 31, 3718–3720 (2015).
Acknowledgements
We acknowledge the contributions of the zoological institutions that made this work possible: Abilene Zoo, Blank Park Zoo, Lincoln Park Zoo, Cheyenne Mountain Zoo, Columbus Zoo and Aquarium, Denver Zoo, Disney’s Animal Kingdom®, El Coyote Ranch, Fort Worth Zoo, Fossil Rim Wildlife Center, Little Rock Zoo, Milwaukee County Zoo, Potter Park Zoo, Sedgwick County Zoo, White Oak Conservation , and South Africa National Parks (SANParks). We would also like to thank Sabrina Amann-Ross for graphic design support.
Funding
We would also like to acknowledge the International Rhino Foundation (B. Pukazhenthi), Morris Animal Foundation (B. Pukazhenthi and M. Corder), George Mason University (M. Corder and W. Zhou), Smithsonian Museum Conservation Institute (T. Cleland), and the Smithsonian’s National Zoo and Conservation Biology Institute (B. Pukazhenthi) for funding this research.
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MC, EP and BP conceived the project, analyzed the data, and wrote the main manuscript text.TB, JN, TPC, NWB, and AA assisted with data analysis and interpretation.TA, TC and WZ assisted with mass spectrometry data analysis and interpretation.MM, PB, LR, SC, JAG, PP, and HH assisted with sample collections and data interpretation.JLB and SP assisted with ELISA data generation, analysis, and interpretation.RD assisted with data analysis.JD assisted with statistical analysis and data interpretation.All authors reviewed the manuscript.
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Corder, M.L., Abulez, T., Cleland, T. et al. Immunoproteomic insights into inflammatory diseases of the critically endangered black rhinoceros (Diceros bicornis). Sci Rep (2026). https://doi.org/10.1038/s41598-026-43055-0
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DOI: https://doi.org/10.1038/s41598-026-43055-0
