The Mannose Receptor on Sinusoidal Lining Cells Mediates Two-Step Bacterial Clearance in the Human Spleen

The Mannose Receptor on Sinusoidal Lining Cells Mediates Two-Step Bacterial Clearance in the Human Spleen | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The Mannose Receptor on Sinusoidal Lining Cells Mediates Two-Step Bacterial Clearance in the Human Spleen Marco Rinaldo Oggioni, Alnabati Neama, Francesco Flandi, Tareq Al Saoudi, and 25 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7463569/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract The human spleen is the main organ in preventing invasive bacterial infection, yet the cellular mechanisms driving pathogen clearance remain poorly defined. This work shows that there is division of labour in the human spleen for eliminating bacteria from the circulation. Using a dual translational approach including ex vivo perfusion of human spleen and splenic primary cell cultures, we demonstrate that sinusoidal cells capture and retain bacteria via the CD206 receptor in the splenic red pulp to enable bactericidal activity by tissue resident macrophages. This activity was dependent on bacterial capsule, with unencapsulated bacteria being cleared irrespective of inhibition of the mannose receptor. This implies a specific two-step process to ensure efficient removal of encapsulated pathogens. These data change completely our understanding of pathogen clearance in the human spleen, with profound implications for the development of host-directed anti-infective strategies and for the evaluation of conjugate vaccine efficacy. Health sciences/Pathogenesis/Infection Biological sciences/Microbiology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The human spleen is the main secondary lymphoid organ involved in the control of systemic infection 1 , particularly through its critical role in the removal of encapsulated bacteria from the circulation 2 . Loss of the spleen by splenectomy, and other forms of functional asplenia, expose patients to the risk of overwhelming sepsis, most commonly caused by capsulated bacteria 3 , 4 , 5 . This has led to the inclusion of asplenia as a key indication for vaccination in many countries 6 . Despite this the pneumococcus remains the leading cause of post-splenectomy sepsis 7 . While the processes of bacterial clearance by the human spleen are critically understudied, the mechanisms by which the spleen removes Plasmodium falciparum -infected red blood cells are well documented due to exploitation of the translational ex vivo human spleen prefusion models 8 . In most human tissues, tissue resident macrophages express the mannose receptor (MRC1/CD206) 9 . However, in the human spleen neither the CD68 + CD163 + red pulp macrophages (RPMs) nor the CD68 + CD169 + perifollicular capillary sheath macrophages (PCSAMs) express the mannose receptor 9 . Instead, the CD206 receptor is expressed by the sinusoidal lining littoral cells, which make up the structure of the human spleen red pulp 9 , 11 . Splenic sinuses are the walled irregular structures draining the open circulation of the human splenic red pulp and are lined by sinus endothelia alternatively named sinusoidal lining cells (CD206 + , LYVE-1 + , CD141 + ) 11, 12 , 13 . The rationale behind this variation in CD206 expression in the human spleen is still unknown. The CD206 mannose receptor is a key molecule involved in the binding to bacteria, viruses and parasite-infected cells playing a key role in the interface between the human host and microbes. Binding of the mannose receptor to bacterial surface sugars, including capsular polysaccharides of Streptococcus pneumoniae and Klebsiella pneumoniae lipopolysaccharide (LPS) 10 , is one of the main mechanisms by which macrophages recognize these pathogens; and endocytosis of the receptor is associated to the uptake and the subsequent killing of the bacteria inside the macrophage compartments 14 . In addition, CD206 has been shown to mediate uptake of dengue virus and HBV into host cells 15 , 16 and recent work on the malaria parasite P. falciparum showed that infected red blood cells are efficiently captured via their surface polysaccharides by the CD206 + sinusoids, suggesting a crucial role of sinusoidal cells in the reduction of pathogen load 17 . Investigation of host-pathogen interactions in the spleen, using murine infection models, non-human primate infections and ex vivo porcine spleen perfusion, revealed that systemic S. pneumoniae infections were predominantly cleared by splenic red pulp macrophages 18 , 19 , 20 , 21 . While the spleen was the primary organ responsible for the clearance of bacteria, the rare occurrence of splenic replication in permissive macrophages facilitated re-seeding of bacteria to the bloodstream and initiation of systemic infection 18 , 19 . The translational ex vivo human spleen perfusion model confirmed detection of bacterial clusters in human splenic macrophages 21 . However, the mannose receptor expression on sinusoidal endothelial cells and not macrophages raised the question about pathogen clearance mechanisms. Using the unique translational platform of human organ ex vivo perfusion alongside primary splenic cell co-cultures of macrophages and sinusoids, we tested the hypothesis that CD206-mediated capture by sinusoidal endothelial cells underlies the human spleen’s distinctive ability to filter, retain, and clear encapsulated bacteria. This host-species-specific adaptation sheds new light on splenic innate immune surveillance and has important implications for vaccine efficiency and the development of host-targeted therapies. Results Cellular marker distribution in the human spleen. To determine the impact of CD206 expression on sinusoidal cells on the clearance of encapsulated bacteria in the human spleen, we applied a translational ex vivo human spleen perfusion model 21 using organs sourced through the TIMID trial (REC 18/EM/0057; ClinicalTrials.gov NCT04620824) and primary cells through the MOSIE trial (CE: 668/2023/Sper/AOUBo). Microscopy mapped the three red pulp markers, CD163, CD169, and CD206, to three distinct cell types: CD68 + CD163 + red pulp macrophages (RPMs), CD68 + CD169 + perifollicular capillary sheath–associated macrophages (PCSAMs), and sinusoid lining littoral CD206 + LYVE-1 + CD31 − cells 9, 22 (Fig. 1 A– 1 B; Fig. S1 A1-S1B). High-content scanning fluorescent microscopy revealed that RPMs and sinusoidal cells dominate the red pulp 11 (Fig. 1 A), while CD169 + macrophages clustered in sheaths around perifollicular capillaries, often forming ring-like structures adjacent to white pulp follicles 22 (Fig. 1 B). CD206 + sinusoids, CD163 + RPMs, and CD169 + macrophages occupied on average 36.5% (SD 4.13), 27.7% (SD 6.1), and 1.3% (SD 1.6) of the total splenic section area respectively, with ranges of 30.3–42.0%, 19.9–35.8%, and 0.07–4.6% (Fig. 1 C). Marker abundance varied between spleens, up to 1.2-fold for CD206 + , 2.7-fold for CD163 + , and intriguingly 12.1-fold for CD169 + macrophages (Fig. 1 D– 1 F), the latter reflecting differences in follicle density and microanatomy (Fig. S1 C1–S1C3). Importantly, serial biopsies from the same spleen at different infection stages showed no variation in CD163 or CD206 levels (Fig. S1 D1–S1D2), confirming the stability of these populations during perfusion and infection. Taken together, these observations reveal a compartmentalized immune architecture in human spleen, with CD206 + sinusoidal cells forming an extensive, blood-facing capture network; CD163 + macrophages distributed throughout the red pulp for removal of pathogens, senescent cells and debris; and rare CD169 + macrophages positioned around perifollicular regions, where they may regulate antigen entry into the white pulp 23 . This spatial arrangement underscores a division of labour in pathogen surveillance, placing CD206 + cells in an ideal position to intercept microbes during their first passage through the splenic open circulation. Ex vivo organ perfusion. Using the TIMID trial, human spleens were sourced for ex vivo normothermic organ perfusion to assess the splenic antibacterial clearance capacity. After cannulation and anticoagulant perfusion, spleens were transported on ice, connected to a normothermic circuit, and perfused with polymerised haemoglobin as an oxygen carrier. Systemic infection was simulated by introducing S. pneumoniae directly into the perfusion liquid mimicking hematogenous invasive human infections (Tab. S1), and serial biopsies and perfusate samples were taken over time to monitor the infection dynamics 21 . Across experiments, the spleen demonstrated a robust filtration capacity, removing over 90% of the inoculum within 60 minutes (Fig. 2A1-2B2, Fig. 2D1-2E2). Clearance kinetics were consistent whether challenged with individual strains or serotype mixtures (Tab. S1, Tab. S2). With limited organ availability, mixed infections guaranteed a more controlled experimental set-up allowing for both simultaneous testing of virulent and avirulent serotypes while minimising any potential impact of type-specific immunity of organ donors (Tab. S3). At a high-dose (1x10 8 cumulative CFU) of five equally counted serotypes (2, 4, 5, 6B, 19F), clearance was equally efficient across strains, regardless of invasive potential (Fig. 2A1–2B2) 24 . Similarly, the association of the bacteria with CD163 + RPMs and the less abundant CD169 + macrophages, based on image analysis, did not vary among serotypes (Fig. S2A1-S2A2). Neither the rapidity nor the extent of bacterial clearance in the human spleen was affected by the donor-derived level of antibacterial antibodies in the perfusion fluid (Tab. S3). At a lower dose (1x10 7 cumulative CFU), all serotypes were cleared from both perfusate and tissue within 30 minutes (Fig. 2D1–2E2). These kinetics underscore the spleen’s extraordinary capacity for rapid, non-discriminatory removal of encapsulated bacteria, even without high antibody titres. This observation aligns with the clinical reality of overwhelming post-splenectomy infection (OPSI), where the absence of splenic tissue dramatically impairs early pathogen removal 25 . The uniformity of clearance across serotypes highlights a fundamental property of splenic immunity: its filtering capacity operates independently of serotype-specific immune history, a fact critical to understanding infection risk in asplenic patients 25 . Contribution of the mannose receptor on sinusoids to bacterial clearance in the human spleen. The mannose receptor (CD206) is a C-type lectin that binds high-mannose structures found on many pathogens, including the capsules of S. pneumoniae and LPS of K. pneumoniae 10 , 26 , 27 . In most tissues, CD206 is expressed by macrophages and dendritic cells and mediates bacterial binding and uptake 10 , 28 . In the human spleen, however, macrophages lack CD206 entirely (Fig. 1 A– 1 B, Fig. S2B-S2C3), despite the spleen being the primary site for pneumococcal clearance. Instead, CD206 is confined to sinusoidal lining cells, a distribution that raises an important mechanistic question: does splenic clearance rely on CD206-mediated capture by these sinusoidal cells rather than by macrophages? To address this, we built on in vitro evidence that free mannose can block CD206–pathogen binding 10 . In the ex vivo perfusion model, spleens were pre-treated with 5 mM mannose alongside the 10–20 mM glucose required for host and bacterial metabolism. When challenged with a high pneumococcal dose (1x10 8 CFU), mannose-treated spleens failed to clear the bacteria, instead, counts instead accumulated progressively in both perfusate and tissue (Fig. 2C1-2C2). At a lower challenge dose (1x10 7 CFU), usually cleared within minutes, killing was completely abolished; bacteria instead proliferated rapidly (Fig. 2F1-2F2). These results identify the carbohydrate-binding activity of CD206 on sinusoidal lining cells as an essential initial step in pathogen clearance. Since the presence of glucose in the perfusion liquid and the cell culture medium blocks completely the pneumococcal mannose metabolism through carbon catabolite repression 29 , the observed escape from host-mediated clearance cannot be ascribed to an effect of mannose on the bacteria. Rather, it reflects direct interference with receptor-mediated capture. To complement the quantification of viable bacteria, we used high-content scanning fluorescence microscopy to examine the association of pneumococci with splenic macrophages and sinusoidal cells in tissue sections obtained during ex vivo perfusion. This analysis reinforced the clearance findings: across all time points, 16–21% of tissue-associated bacteria colocalized with CD206 (Fig. 3A1), a proportion that remained stable despite a 90% reduction in total bacterial load during the first hour (Fig. 2 ). Correspondingly, the area of sinusoidal tissue occupied by bacteria showed no decline (Fig. 3A2). Three-dimensional confocal reconstructions revealed discrete clusters of CD206 in direct contact with bacteria (Fig. 3B1-3B2). In mannose-treated spleens, bacterial association with CD206 fell sharply at early time points (Fig. 3A1), coinciding with higher viable counts and broader sinusoidal distribution later (Fig. 3A2). To determine the fate of these captured bacteria, we performed wheat germ agglutinin (WGA) staining, which confirmed that CD206 + sinusoidal cells do not internalise bacteria (Fig. 3C1-3C3). Instead, captured bacteria remained extracellular, suggesting that these cells act as stationary traps. Additional quantification showed that 20% of pneumococci colocalised with CD163 + RPMs at 30 min, 2 h, and 5 h post-infection (Fig. 3D1), and that mannose treatment significantly reduced this association (Fig. 3D1). Although CD169 + macrophages were less abundant, their bacterial association was also diminished by mannose (Fig. 3E1-3E2). To provide in vitro evidence for the phenotypes observed during ex vivo spleen perfusion, primary adherent cell cultures were established from human spleen homogenates. Primary spleen cultures contained 57% CD206⁺ sinusoidal cells and 35% CD163⁺ macrophages (Fig. 4A1), often linked by extensions (Fig. 4A2). Only CD14⁺ cells comprised only 1.4%, indicating rare monocytes, possibly overlapping with the CD206⁺ population 30 .Low-MOI infections showed that 2.1% of bacteria attached to CD206 + cells (Fig. 4B1) and 0.6% to CD163 + macrophages (Fig. 4B2). Mannose or anti-CD206 antibodies reduced bacterial binding to CD206 + cells (Fig. 4B1) but not to CD163 + macrophages (Fig. 4B2). The expression of CD206 on primary sinusoidal cells, and not macrophages, was confirmed by CD206 + co-expression with the sinusoidal marker LYVE-1 (Fig. 4 C). Three-dimensional reconstructions confirmed that pneumococci remained on the surface of CD206 + cells (Fig. 4D1–4D2), while bacteria appeared intracellular only in macrophages (Fig. 4E1–4E2). At MOI 10, bacterial counts dropped sharply within 30 min for both TIGR4 and D39 serotypes (Fig. 4F1, 4G1), but this bactericidal activity was lost when CD206 was blocked by mannose or antibodies (Fig. 4F1, 4G1). In contrast, when cultures were challenged with a non-encapsulated derivative of the TIGR4 strain, over 99% of bacteria were eliminated within 30 minutes (Fig. 4F2), and this killing was unaffected by mannose supplementation (Fig. 4F2). The same behaviour was observed for a rough non-encapsulated D39 strain (Fig. 4G2). These results strongly suggest that CD206-mediated bacterial attachment to sinusoidal cells is essential for enabling subsequent macrophage-mediated killing of encapsulated, sugar-coated pathogens. The control experiments with the non-encapsulated strains reinforce the conclusion that the CD206-dependent interaction between sinusoidal cells and macrophages is not required for the clearance of un-encapsulated bacteria. In addition to pneumococcal capsules, CD206 had been shown to bind also K. pneumoniae LPS 10 . Repeating the bactericidal assay with capsulated K2 capsule K. pneumoniae and K1 capsule Escherichia coli , confirmed CD206-dependence for any bactericidal activity of macrophages (Fig. 4H1-4H2). These in vitro results fully confirm the observations during organ perfusion, indicating that CD206-mediated bacterial attachment to sinusoidal cells is essential for enabling subsequent macrophage-mediated killing of encapsulated pathogens. These observations support a sequential “handoff” model: CD206 + sinusoidal cells capture encapsulated bacteria without internalisation, then present them to macrophages for phagocytosis and killing. Blocking CD206 prevents this initial capture and reduces downstream bacterial association with macrophages. Importantly, this arrangement mirrors receptor-mediated antigen transfer in other lymphoid organs 31 but it is specialised for bloodstream pathogen clearance. Unlike the traditional view that attributes CD206-mediated clearance to macrophages 9 , 27 , our data show that in the human spleen, these cells are non-phagocytic stromal elements. Functionally, they behave like biological flypaper, retaining pathogens at the sinusoidal lining surface until macrophages, particularly CD163 + and CD169 + subsets, engage them (Fig. S2B-S2C3). The mannose-inhibitable spectrum of this capture extends beyond pneumococcus to multiple encapsulated species, echoing mechanical retention of poorly deformable cells in splenic sinuses 32 , 33 but based on biochemical glycan recognition. The therapeutic implications are twofold: enhancing CD206-mediated capture could strengthen host defences, while systemic mannose administration, as in some urinary tract infection (UTIs) treatments 34 , could inadvertently impair splenic clearance. Our findings also clarify that in the spleen, CD206 is not a macrophage marker but a functional receptor on sinusoidal cells. This corrects earlier assumptions that CD206-mediated phagocytosis in spleen was macrophage-driven 9 , 35 repositioning these cells as dedicated antigen-capturing and presenting cells. By capturing encapsulated bacteria via mannose recognition, these sinusoidal cells transform a microbial evasion strategy, the anti-phagocytic capsule, into a vulnerability. The concept parallels earlier work showing that erythrocytes with exposed mannoses, including P. falciparum –infected cells, are preferentially retained and cleared 17 . Our results extend this to bacteria, demonstrating that the spleen uses mannose recognition to detect and trap both infected cells and encapsulated microbes. In summary, the human spleen is not just a passive filter but an active immune organ combining physical, biochemical, and receptor-mediated mechanisms to eliminate bloodstream threats. Redefining CD206 + sinusoidal cells as non-phagocytic, but indispensable for microbial capture, offers a new perspective on splenic immunity and opens the door to targeted interventions that reinforce this early line of defence. Mechanism of pathogen clearance by both CD163 + and of CD169 + macrophages. The two dominant splenic macrophage subsets, CD163 + RPMs and the less abundant CD169 + PCSAMs, operate in distinct but complementary niches 22 . Quantitative colocalization revealed that 15–30% of bacteria were associated with CD163 + macrophages, while 4–22% associated with CD169 + macrophages (Fig. 5A1-5A2). Notably, these proportions were consistent between early (30 min) and late (5 h) time points, despite > 90% clearance of bacteria from the perfusate within the first hour. In addition, microscopy analyses of tissue sections with high or low bacterial loads revealed a consistent ratio of bacteria-to-macrophage association across both CD163 + and CD169 + populations (Fig. 5B1-5B2), suggesting that even at high challenge doses, macrophage capacity for bacterial clearance was not saturated. The ratios remained consistent across different bacterial loads and serotypes (2, 4, 19F) (Fig S2A1-S2A2), indicating stable, load-independent pathogen engagement by these macrophage subsets. When fluorescent beads were perfused instead of live bacteria (Fig. S3A-S3C), macrophage uptake was slower, with association rates increasing progressively over time (Fig. 5A1–5A2). This slower uptake highlights the dynamic responsiveness of splenic macrophages to live microbes versus inert particles, reflecting an ability to accelerate engagement when genuine pathogens are detected. While these data do not allow quantification of the relative bactericidal contributions of each macrophage subset, they indicate that both CD163 + and CD169 + macrophages consistently retained similar bacterial loads over time, implying a comparable role in pneumococcal capture and clearance. To investigate the mechanisms of bacterial killing by human spleen tissue-resident macrophages, spleen sections, at different time points of infection (from 0 to 6 h) were stained for the phagosome maturation marker LAMP1. LAMP1 staining, marking lysosomal compartments, revealed a progressive increase in infected spleens, but not in uninfected controls (Fig. 5 C). This signal was localised mainly within macrophages (Fig. 5D1–5D2), consistent with active lysosomal engagement. Interestingly, minimal LAMP1–pneumococcus colocalization was observed, indicating that at early time points, bacteria-containing phagosomes may not yet have matured into fully degradative compartments (data not shown). Overall, the time-dependent increase in LAMP1 levels supports the conclusion that human spleen macrophages remain engaged in active phagocytosis during our ex vivo perfusion model and exhibit dynamic features of the phagocytic process. Apoptosis has long been recognized as a key mechanism by which human alveolar macrophages eliminate pneumococci 36 . To assess whether this mechanism is also active in human splenic macrophages, tissue sections were stained for cleaved caspase-3, the activated form of the caspase responsible for executing apoptotic proteolysis 37 , 38 . The infection triggered an early apoptotic response. Within 60 minutes, cleaved caspase-3 levels rose rapidly, over 10-fold in both CD163 + RPMs and in CD169 + macrophages (Fig. 5E1–5E2), with the latter showing 10x higher per-cell signal (Fig. S2D1–S2D2). In bead-perfused controls, this apoptotic rise was slower and less pronounced, suggesting that live bacterial infection accelerates cell death programmes. M30 staining, indicative of caspase-3–cleaved cytokeratin-18 39, 40 , increased steadily over 5–6 hours in both subsets (Fig. 5F1–5F2), with the M30-positive macrophage area expanding accordingly. Mannose-treated and untreated spleens showed similar trends and were pooled in the graphs (Fig. S2E1–S2E2). Considering that a human spleen contains approximately 2x10 10 macrophages 41 , we found that at the 1-hour mark, 0.65% of CD163 + macrophages were positive for cleaved caspase-3 (Fig. 5E1–5E2). This corresponds to roughly 7x10 7 to 3x10 8 apoptotic cells per spleen; numbers that closely match the bacterial challenge dose of 1x10 8 CFU. This correspondence suggests a direct link between the quantity of phagocytosed bacteria and the induction of macrophage apoptosis. The lysosomal activation observed after bacterial uptake, marked by increased LAMP1 expression, confirms that splenic macrophages engage in canonical phagolysosomal killing 42 . However, the concurrent detection of early cleaved caspase-3 within just 60 minutes, followed by M30 expression at 5–6 hours, indicates that bacterial clearance in the spleen is closely coupled to programmed cell death. This differs from the delayed apoptosis reported in other macrophage populations, such as alveolar macrophages, where cell death typically occurs in the resolution phase of infection 36 , 43 . The spleen’s context may explain this difference. As a blood-filtering organ, it is uniquely exposed to high concentrations of circulating pathogens. In such an environment, rapid apoptosis following phagocytosis could serve as both a terminal killing step and a containment measure, ensuring that intracellular pathogens cannot persist within surviving macrophages. This swift apoptotic turnover may also act as a chemotactic signal to recruit additional phagocytes, maintaining clearance efficiency even, as individual macrophages are lost. The timing of this response is likely critical for neutralising pathogens capable of manipulating macrophage survival. Neisseria meningitidis , for example, can delay apoptosis via nitric oxide detoxification 44 , prolonging its intracellular survival. The early initiation of apoptosis in splenic macrophages could therefore represent an evolved countermeasure against such strategies, an immunological “self-destruct” that denies bacteria a long-term niche. Our findings support a revised model of splenic antibacterial defence in which CD206⁺ sinusoidal cells and macrophages act in coordinated sequence. CD206⁺ lining cells first immobilise encapsulated bacteria via mannose-dependent glycan recognition without internalisation, preventing their return to circulation and enabling targeted clearance. CD163⁺ and CD169⁺ macrophages then receive these retained bacteria, phagocytose them, initiate lysosomal degradation, and undergo rapid apoptosis, ensuring efficient destruction while preventing intracellular persistence and systemic re-seeding. This architecture bears resemblance to antigen-handling systems in lymph nodes, where lymphatic endothelial cells and subcapsular sinus macrophages coordinate pathogen retention and transfer 31 . However, the splenic system is tuned for speed and broad-spectrum efficiency, particularly against encapsulated bacteria that evade complement-mediated lysis and antibody opsonisation. In effect, the capsule, a key virulence factor, becomes a vulnerability when confronted with CD206-mediated capture. The efficiency of this system underscores why loss of splenic function, whether through splenectomy, infarction, or disease-related atrophy 3 , 4 , 5 , carries such a high risk of overwhelming infection (OPSI), particularly from encapsulated organisms 25 . These patients lack both the macrophage-rich red pulp and the specialised sinusoidal cell network, severely compromising their capacity to trap and neutralise bloodstream bacteria. Functional hyposplenism, as seen in sickle cell disease or celiac disease, often involves reduced sinusoidal cell density, further increasing vulnerability to severe bacterial infections 45 , 46 . Spleen ex vivo perfusion, used for malaria⁸,¹⁷ and proposed for bacterial studies²¹, has limitations, some linked to reperfusion injury (e.g., high cytokines and soluble proteins) (Fig. S4)⁴⁷, whose impact we did not assess. Recognising the central role of CD206-mediated capture suggests new interventions, such as mannosylated nanoparticles to enhance vaccine delivery⁴⁸ or strategies to preserve/replace macrophages to boost killing capacity. Conversely, systemic free mannose, used to prevent UPEC adhesion³⁴, could transiently inhibit splenic CD206, impairing clearance of encapsulated pathogens. This risk may be important in patients with complicated UTI, concurrent bacteraemia, or reduced splenic reserve, warranting clinical caution. Declarations Funding: The work was funded by EU funding within the MUR PNRR Extended Partnership initiative on Emerging Infectious Diseases (Project no. PE00000007, INF-ACT) to MRO and GC, PRIN 2020 grant 202089LLEH and PRIN PNRR P2022M8KYE from the Italian Ministry of Education, BBSRC grant BB/V01465X/1 and the UK Biomedical Research Centre grant NIHR203327 to MRO and a BBSRC Impact Accelerator Account (IAA) award (RM32G0178B7) to KS NA is supported by a PhD fellowship from Umm AlQura University, Makkah, Saudi Arabia. Author contributions: NA acquired, analysed and interpreted the microbiological and microscopy data and contributed to the drafting of the manuscript, FF acquired, analysed and interpreted the microbiological and microscopy data, developed the primary cell cultures and drafted the first version of the manuscript, TS, TK, JI, WC and GGa. Managed of patient consent in UK and performed the surgical part of the experimental work, acquired, analysed and interpreted the data, GC, TR, DG, RH, ZJ, ST acquired, analysed and interpreted the microbiological and microscopy data and contributed to the drafting of the manuscript, GGe, GR, and FR managed patient consent and clinical data in Italy, EG, GC and KSt supervised microscopy work in Italy and UK respectively, S.B. contributed development of perfusion liquid, KSc, CDB, CEP, SF and CT supervised cell culture and microbiology work, MR and AD contribute to study design, experimental set-up, performed and oversaw surgical part of the project in Italy and UK respectively, LMP contributed to the study design, data analysis and oversaw the immunological part of the project and contributed to the drafting and finalising of the manuscript, MRO contributed to the study design, oversaw the microbiological and microscopy data analysis and contributed to the funding of the work. Acknowledgements: All authors thank the numerous patients in Leicester and Bologna consenting to participate in the clinical trials on which all the research work is based and the Research Governance Office of the University of Leicester and the respective office of the university of Bologna to sponsor the trials. Authors also thank Zaf Zafirelis from HbO2 Therapeutics (Souderton, PA, USA) for donating Hemopure, Kevin West for help with the H&E stains, and the University of Leicester Advanced Imaging Facility (RRID:SCR_020967) for use of microscope equipment, and the microscopy facility of the University of Bologna. References Morris DH, Bullock FD (1919) The importance of the spleen in resistance to infection. Ann Surg 70(5):513–531 Lammers AJ, De Porto AP, Florquin S, De Boer OJ, Bootsma HJ, Hermans PW, Van der Poll T (2011) Enhanced vulnerability for Streptococcus pneumoniae sepsis during asplenia is determined by the bacterial capsule. Immunobiology 216(8):863–870 Holdsworth RJ, Cuschieri A, Irving AD (1991) Postsplenectomy sepsis and its mortality rate: actual versus perceived risks. Br J Surg 78(9):1031–1038 Gea-Izquierdo E, Rodríguez-Caravaca G, Gil-Prieto R, Hernández-Barrera V, Gil-de-Miguel (2024) Á. Streptococcus pneumoniae infection in patients with asplenia: a Spanish perspective over a 25-Year period. Antibiotics , 13 (1), 104 Adriani KS, Brouwer MC, van der Ende A, van de Beek D (2013), June Bacterial meningitis in adults after splenectomy and hyposplenic states. In Mayo Clinic proceedings (Vol. 88, No. 6, pp. 571–578). Elsevier Pebody RG, Leino T, Nohynek H, Hellenbrand W, Salmaso S, Ruutu P (2005) Pneumococcal vaccination policy in Europe. Euro Surveill 10(9):174–178 Theilacker C, Ludewig K, Serr A, Schimpf J, Held J, Bögelein M, Kern WV (2016) Overwhelming postsplenectomy infection: a prospective multicenter cohort study. Clin Infect Dis 62(7):871–878 Buffet PA, Milon G, Brousse V, Correas JM, Dousset B, Couvelard A, David PH (2006) Ex vivo perfusion of human spleens maintains clearing and processing functions. Blood 107(9):3745–3752 Martinez-Pomares L, Hanitsch LG, Stillion R, Keshav S, Gordon S (2005) Expression of mannose receptor and ligands for its cysteine-rich domain in venous sinuses of human spleen. Lab Invest 85(10):1238–1249 Zamze S, Martinez-Pomares L, Jones H, Taylor PR, Stillion RJ, Gordon S, Wong SY (2002) Recognition of bacterial capsular polysaccharides and lipopolysaccharides by the macrophage mannose receptor. J Biol Chem 277(44):41613–41623 Qiu J, Salama ME, Hu CS, Li Y, Wang X, Hoffman R (2018) The characteristics of vessel lining cells in normal spleens and their role in the pathobiology of myelofibrosis. Blood Adv 2(10):1130–1145 Steiniger BS, Pfeffer H, Gaffling S, Lobachev O (2022) The human splenic microcirculation is entirely open as shown by 3D models in virtual reality. Sci Rep 12(1):16487 Steiniger BS (2015) Human spleen microanatomy: why mice do not suffice. Immunology 145(3):334–346 Stahl PD, Ezekowitz RAB (1998) The mannose receptor is a pattern recognition receptor involved in host defense. Curr Opin Immunol 10(1):50–55 Miller JL, deWet BJM, Martinez-Pomares L, Radcliffe CM, Dwek RA, Rudd PM, Gordon S (2008) The mannose receptor mediates dengue virus infection of macrophages. PLoS Pathog, 4(2), e17 den Brouw MLO, Binda RS, Geijtenbeek TB, Janssen HL, Woltman AM (2009) The mannose receptor acts as hepatitis B virus surface antigen receptor mediating interaction with intrahepatic dendritic cells. Virology 393(1):84–90 Cao H, Antonopoulos A, Henderson S, Wassall H, Brewin J, Masson A, Vickers MA (2021) Red blood cell mannoses as phagocytic ligands mediating both sickle cell anaemia and malaria resistance. Nat Commun 12(1):1792 Gerlini A, Colomba L, Furi L, Braccini T, Manso AS, Pammolli A, Oggioni MR (2014) The role of host and microbial factors in the pathogenesis of pneumococcal bacteraemia arising from a single bacterial cell bottleneck. PLoS Pathog, 10(3), e1004026 Ercoli G, Fernandes VE, Chung WY, Wanford JJ, Thomson S, Bayliss CD, Oggioni MR (2018) Intracellular replication of Streptococcus pneumoniae inside splenic macrophages serves as a reservoir for septicaemia. Nat Microbiol 3(5):600–610 Chung WY, Wanford JJ, Kumar R, Isherwood JD, Haigh RD, Oggioni MR, Ercoli G (2018) An ex vivo porcine spleen perfusion as a model of bacterial sepsis. ALTEX: Altern Anim Experimentation 36(1):29–38 Carreno D, Wanford JJ, Jasiunaite Z, Hames RG, Chung WY, Dennison AR, Oggioni MR (2021) Splenic macrophages as the source of bacteraemia during pneumococcal pneumonia. EBioMedicine , 72 Nagelkerke SQ, Bruggeman CW, Den Haan JM, Mul EP, Van Den Berg TK, Van Bruggen R, Kuijpers TW (2018) Red pulp macrophages in the human spleen are a distinct cell population with a unique expression of Fc-γ receptors. Blood Adv 2(8):941–953 Mebius RE, Kraal G (2005) Structure and function of the spleen. Nat Rev Immunol 5(8):606–616 Brueggemann AB, Griffiths DT, Meats E, Peto T, Crook DW, Spratt BG (2003) Clonal relationships between invasive and carriage Streptococcus pneumoniae and serotype-and clone-specific differences in invasive disease potential. J Infect Dis 187(9):1424–1432 Brigden ML (2001) Detection, education and management of the asplenic or hyposplenic patient. Am Family Phys 63(3):499–508 East L, Isacke CM (2002) The mannose receptor family. Biochim et Biophys Acta (BBA)-General Subj 1572(2–3):364–386 Martinez-Pomares L (2012) The mannose receptor. J Leukoc Biol 92(6):1177–1186 Subramanian K, Neill DR, Malak HA, Spelmink L, Khandaker S, Dalla Libera Marchiori G, Henriques-Normark B (2019) Pneumolysin binds to the mannose receptor C type 1 (MRC-1) leading to anti-inflammatory responses and enhanced pneumococcal survival. Nat Microbiol 4(1):62–70 Bidossi A, Mulas L, Decorosi F, Colomba L, Ricci S, Pozzi G, Oggioni MR (2012) A functional genomics approach to establish the complement of carbohydrate transporters in Streptococcus pneumoniae. PLoS ONE, 7(3), e33320 Gordon S, Plüddemann A, Mukhopadhyay S (2015) Sinusoidal immunity: macrophages at the lymphohematopoietic interface. Cold Spring Harb Perspect Biol 7(4):a016378 Phan TG, Green JA, Gray EE, Xu Y, Cyster JG (2009) Immune complex relay by subcapsular sinus macrophages and noncognate B cells drives antibody affinity maturation. Nat Immunol 10(7):786–793 Deplaine G, Safeukui I, Jeddi F, Lacoste F, Brousse V, Perrot S, Buffet PA (2011) The sensing of poorly deformable red blood cells by the human spleen can be mimicked in vitro. Blood J Am Soc Hematol 117(8):e88–e95 Safeukui I, Correas JM, Brousse V, Hirt D, Deplaine G, Mulé S, Buffet PA (2008) Retention of Plasmodium falciparum ring-infected erythrocytes in the slow, open microcirculation of the human spleen. Blood J Am Soc Hematol 112(6):2520–2528 Hayward G, Mort S, Hay AD, Moore M, Thomas NP, Cook J, Butler CC (2024) d-Mannose for prevention of recurrent urinary tract infection among women: a randomized clinical trial. JAMA Intern Med 184(6):619–628 Jackson DG, Prevo R, Clasper S, Banerji S (2001) LYVE-1, the lymphatic system and tumor lymphangiogenesis. Trends Immunol 22(6):317–321 Dockrell DH, Marriott HM, Prince LR, Ridger VC, Ince PG, Hellewell PG, Whyte MK (2003) Alveolar macrophage apoptosis contributes to pneumococcal clearance in a resolving model of pulmonary infection. J Immunol 171(10):5380–5388 Gao LY, Abu Kwaik Y (1999) Apoptosis in macrophages and alveolar epithelial cells during early stages of infection by Legionella pneumophila and its role in cytopathogenicity. Infection and immunity , 67 (2), 862–870. Häcker H, Fürmann C, Wagner H and Häcker G (2002) Caspase-9/-3 activation and apoptosis are induced in mouse macrophages upon ingestion and digestion of Escherichia coli bacteria. J Immunol 169(6):3172–3179 Leers MP, Kölgen W, Björklund V, Bergman T, Tribbick G, Persson B, Schutte B (1999) Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis. J Pathol 187(5):567–572 Ulukaya E, Karaagac E, Ari F, Oral AY, Adim SB, Tokullugil AH, Evrensel T (2011) Chemotherapy increases caspase-cleaved cytokeratin 18 in the serum of breast cancer patients. Radiol Oncol 45(2):116 Sender R, Weiss Y, Navon Y, Milo I, Azulay N, Keren L, Milo R (2023) The total mass, number, and distribution of immune cells in the human body. Proceedings of the National Academy of Sciences, 120(44), e2308511120 Flannagan RS, Cosío G, Grinstein S (2009) Antimicrobial mechanisms of phagocytes and bacterial evasion strategies. Nat Rev Microbiol 7(5):355–366 Preston JA, Bewley MA, Marriott HM, McGarry Houghton A, Mohasin M, Jubrail J, Dockrell DH (2019) Alveolar macrophage apoptosis–associated bacterial killing helps prevent murine pneumonia. Am J Respir Crit Care Med 200(1):84–97 Tunbridge AJ, Stevanin TM, Lee M, Marriott HM, Moir JW, Read RC, Dockrell DH (2006) Inhibition of macrophage apoptosis by Neisseria meningitidis requires nitric oxide detoxification mechanisms. Infect Immun 74(1):729–733 Di Sabatino A, Rosado MM, Cazzola P, Riboni R, Biagi F, Carsetti R, Corazza GR (2006) Splenic hypofunction and the spectrum of autoimmune and malignant complications in celiac disease. Clin Gastroenterol Hepatol 4(2):179–186 Stotesbury H, Kawadler JM, Saunders DE, Kirkham FJ (2021) MRI detection of brain abnormality in sickle cell disease. Expert Rev Hematol 14(5):473–491 Gravante G, Ong SL, Metcalfe MS, Sorge R, Sconocchia G, Orlando G, Dennison AR (2009), May Cytokine response to ischemia/reperfusion injury in an ex vivo perfused porcine liver model. In Transplantation proceedings (Vol. 41, No. 4, pp. 1107–1112). Elsevier Paurević M, Šrajer Gajdošik M, Ribić R (2024) Mannose ligands for mannose receptor targeting. Int J Mol Sci 25(3):1370 Additional Declarations There is NO Competing Interest. Supplementary Files Alnabatipapersupplementary.docx SUPPLEMENTARY INFORMATION Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7463569","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":518766300,"identity":"2e1cc701-1aad-4770-869e-22dc4c4396d1","order_by":0,"name":"Marco Rinaldo Oggioni","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFElEQVRIiWNgGAWjYPACCQYGdgYDMJMNiA98YGDgAXEP4NXCjKTl4AyYFjx6EFrAbB4gAeZi0aLbfvbhh497LPL4m5k3PmCoqLXrY+89eNi27Y6MOXsD4+EPmFrMzqQbS854JlEscZit2IDhzPHkNp5zCYdz257xWPYcwOowswNpbMw8ByQSGw7zmEkwth1LZpPIMQBqOcxjcCMBu5bzzyBa5qNoscSn5QbUlg0QLTV2YC2MeLU8Y5acAdSyEeSXhDMHEth4zhgc7Dl3GOiXgw0HzmBzWBrjhw8H6hLnHW/e+OBDRZ29fHuP8YcfZYftzdmbD3+owNSCChIYDic2ILiMDbgUIoM6e2JUjYJRMApGwcgCAJU8b6E3zDuTAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-4117-793X","institution":"University of Bologna","correspondingAuthor":true,"prefix":"","firstName":"Marco","middleName":"Rinaldo","lastName":"Oggioni","suffix":""},{"id":518766301,"identity":"a812b84b-397c-40c7-8115-dce840ca9d02","order_by":1,"name":"Alnabati Neama","email":"","orcid":"","institution":"University of Leicester","correspondingAuthor":false,"prefix":"","firstName":"Alnabati","middleName":"","lastName":"Neama","suffix":""},{"id":518766302,"identity":"303dfe28-4c0d-485f-bfcc-1252c9f0f304","order_by":2,"name":"Francesco Flandi","email":"","orcid":"","institution":"University of Bologna","correspondingAuthor":false,"prefix":"","firstName":"Francesco","middleName":"","lastName":"Flandi","suffix":""},{"id":518766303,"identity":"845b6db3-67fc-4e29-8f38-f5b7de9f449b","order_by":3,"name":"Tareq Al Saoudi","email":"","orcid":"","institution":"Leicester University Hospitals NHS Trust","correspondingAuthor":false,"prefix":"","firstName":"Tareq","middleName":"Al","lastName":"Saoudi","suffix":""},{"id":518766304,"identity":"5f58dc69-b88c-4ea9-90d2-55508c64169e","order_by":4,"name":"Giulia Cattabriga","email":"","orcid":"","institution":"University of Bologna","correspondingAuthor":false,"prefix":"","firstName":"Giulia","middleName":"","lastName":"Cattabriga","suffix":""},{"id":518766305,"identity":"03776d9a-6be0-489b-8963-a45f0749f6f3","order_by":5,"name":"Talia Richardson","email":"","orcid":"","institution":"University of Bologna","correspondingAuthor":false,"prefix":"","firstName":"Talia","middleName":"","lastName":"Richardson","suffix":""},{"id":518766306,"identity":"5c3b9981-302b-48ff-8f7f-14f830504e6a","order_by":6,"name":"Daniele Ghezzi","email":"","orcid":"","institution":"University of Bologna","correspondingAuthor":false,"prefix":"","firstName":"Daniele","middleName":"","lastName":"Ghezzi","suffix":""},{"id":518766307,"identity":"906816c5-6efb-4e40-ad5d-9805feab1489","order_by":7,"name":"Ryan Hames","email":"","orcid":"","institution":"University of Leicester","correspondingAuthor":false,"prefix":"","firstName":"Ryan","middleName":"","lastName":"Hames","suffix":""},{"id":518766308,"identity":"a8391e8e-71cb-4cdd-ba36-54252b96adc7","order_by":8,"name":"John Isherwood","email":"","orcid":"","institution":"Leicester University Hospitals NHS Trust","correspondingAuthor":false,"prefix":"","firstName":"John","middleName":"","lastName":"Isherwood","suffix":""},{"id":518766309,"identity":"f9d13110-83a2-4f1d-b648-cc7cd094bef7","order_by":9,"name":"Trisha Kanani","email":"","orcid":"","institution":"Leicester University Hospitals NHS Trust","correspondingAuthor":false,"prefix":"","firstName":"Trisha","middleName":"","lastName":"Kanani","suffix":""},{"id":518766310,"identity":"da66fe12-d390-4ddb-bd98-bd247d1621f3","order_by":10,"name":"Zydrune Jasiunaite","email":"","orcid":"","institution":"University of Leicester","correspondingAuthor":false,"prefix":"","firstName":"Zydrune","middleName":"","lastName":"Jasiunaite","suffix":""},{"id":518766311,"identity":"5ffcce6a-06cf-45ad-9065-ad02138e82d4","order_by":11,"name":"Shiying Tang","email":"","orcid":"","institution":"University of Leicester","correspondingAuthor":false,"prefix":"","firstName":"Shiying","middleName":"","lastName":"Tang","suffix":""},{"id":518766312,"identity":"c153ac57-13ab-429a-88ba-11af13110958","order_by":12,"name":"Giuliana Germinario","email":"","orcid":"","institution":"IRCCS Azienda Ospedaliero-Universitaria di Bologna","correspondingAuthor":false,"prefix":"","firstName":"Giuliana","middleName":"","lastName":"Germinario","suffix":""},{"id":518766313,"identity":"7ee20b77-955d-4b0a-a3cc-7f20177ede66","order_by":13,"name":"Giorgia Radi","email":"","orcid":"","institution":"University of Bologna","correspondingAuthor":false,"prefix":"","firstName":"Giorgia","middleName":"","lastName":"Radi","suffix":""},{"id":518766314,"identity":"136d7495-bc23-49e4-af2f-0d198c4c9828","order_by":14,"name":"Francesca Rizzo","email":"","orcid":"","institution":"IRCCS Azienda Ospedaliero-Universitaria di Bologna","correspondingAuthor":false,"prefix":"","firstName":"Francesca","middleName":"","lastName":"Rizzo","suffix":""},{"id":518766315,"identity":"77c3af04-1d5c-413b-a436-f0946502bb38","order_by":15,"name":"Katrin Schilcher","email":"","orcid":"","institution":"University of Leicester","correspondingAuthor":false,"prefix":"","firstName":"Katrin","middleName":"","lastName":"Schilcher","suffix":""},{"id":518766316,"identity":"ee8cf678-42a3-44bc-b10e-f1f0907feff6","order_by":16,"name":"Christopher Bayliss","email":"","orcid":"","institution":"University of Leicester","correspondingAuthor":false,"prefix":"","firstName":"Christopher","middleName":"","lastName":"Bayliss","suffix":""},{"id":518766317,"identity":"cf964817-357c-4a7f-a07d-818a52c59f75","order_by":17,"name":"Carola Parolin","email":"","orcid":"https://orcid.org/0000-0003-1536-9276","institution":"University of Bologna","correspondingAuthor":false,"prefix":"","firstName":"Carola","middleName":"","lastName":"Parolin","suffix":""},{"id":518766318,"identity":"81f1f86e-d623-408d-8bcf-6661ae7cdb81","order_by":18,"name":"Stefano Fedi","email":"","orcid":"","institution":"University of Bologna","correspondingAuthor":false,"prefix":"","firstName":"Stefano","middleName":"","lastName":"Fedi","suffix":""},{"id":518766319,"identity":"95270575-be53-4ce8-8bd5-cd36523a365d","order_by":19,"name":"Wen Yuan Chung","email":"","orcid":"https://orcid.org/0000-0003-0970-0639","institution":"University Hospitals of Leicester NHS Trust","correspondingAuthor":false,"prefix":"","firstName":"Wen","middleName":"Yuan","lastName":"Chung","suffix":""},{"id":518766320,"identity":"db788066-1b6e-4978-9e6e-04957539a990","order_by":20,"name":"Giuseppe Garcea","email":"","orcid":"","institution":"Leicester University Hospitals NHS Trust","correspondingAuthor":false,"prefix":"","firstName":"Giuseppe","middleName":"","lastName":"Garcea","suffix":""},{"id":518766321,"identity":"bf3526dc-956f-477c-b381-120d60a4ac3f","order_by":21,"name":"Enrico Giampieri","email":"","orcid":"https://orcid.org/0000-0003-2269-2338","institution":"University of Bologna","correspondingAuthor":false,"prefix":"","firstName":"Enrico","middleName":"","lastName":"Giampieri","suffix":""},{"id":518766322,"identity":"23274be1-4313-4603-adaf-a6faf339637e","order_by":22,"name":"Gastone Castellani","email":"","orcid":"https://orcid.org/0000-0003-4892-925X","institution":"University of Bologna","correspondingAuthor":false,"prefix":"","firstName":"Gastone","middleName":"","lastName":"Castellani","suffix":""},{"id":518766323,"identity":"ad2233f2-3933-408d-a68a-d9362180488b","order_by":23,"name":"Kees Straatman","email":"","orcid":"https://orcid.org/0000-0002-9812-492X","institution":"University of Leicester","correspondingAuthor":false,"prefix":"","firstName":"Kees","middleName":"","lastName":"Straatman","suffix":""},{"id":518766324,"identity":"dcba5077-f925-49fb-bc33-47779da08688","order_by":24,"name":"Stefano Bruno","email":"","orcid":"","institution":"University of Parma","correspondingAuthor":false,"prefix":"","firstName":"Stefano","middleName":"","lastName":"Bruno","suffix":""},{"id":518766325,"identity":"29dc6719-4d30-4491-8803-c50f7c74df01","order_by":25,"name":"Claudia Trappetti","email":"","orcid":"","institution":"University of Bologna","correspondingAuthor":false,"prefix":"","firstName":"Claudia","middleName":"","lastName":"Trappetti","suffix":""},{"id":518766326,"identity":"d2827edc-9c49-4d89-94b6-2eb94af5d37b","order_by":26,"name":"Matteo Ravaioli","email":"","orcid":"","institution":"University of Bologna","correspondingAuthor":false,"prefix":"","firstName":"Matteo","middleName":"","lastName":"Ravaioli","suffix":""},{"id":518766327,"identity":"3329852d-7726-48de-96e7-a14d0efc3659","order_by":27,"name":"Ashley Dennison","email":"","orcid":"","institution":"University Hospitals of Leicester, NHS Trust","correspondingAuthor":false,"prefix":"","firstName":"Ashley","middleName":"","lastName":"Dennison","suffix":""},{"id":518766328,"identity":"0a3621bc-4c91-4c65-94af-6821d26a79a5","order_by":28,"name":"Luisa Martinez-Pomares","email":"","orcid":"https://orcid.org/0000-0002-2331-127X","institution":"University of Nottingham","correspondingAuthor":false,"prefix":"","firstName":"Luisa","middleName":"","lastName":"Martinez-Pomares","suffix":""}],"badges":[],"createdAt":"2025-08-26 13:46:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7463569/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7463569/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91971499,"identity":"b61997c5-c04d-4a3a-863f-3ecd6575ec5e","added_by":"auto","created_at":"2025-09-23 09:17:20","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1531251,"visible":true,"origin":"","legend":"","description":"","filename":"Alnabatipaper.docx","url":"https://assets-eu.researchsquare.com/files/rs-7463569/v1/cbcbf3f5fbcd1afff0c769ca.docx"},{"id":91971075,"identity":"ab938095-0cac-4da3-a326-f3e4b637ca6c","added_by":"auto","created_at":"2025-09-23 09:09:20","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":24667,"visible":true,"origin":"","legend":"","description":"","filename":"NCOMMS2571126T.json","url":"https://assets-eu.researchsquare.com/files/rs-7463569/v1/a11b8d36e78efdb5d4b6c444.json"},{"id":91971099,"identity":"703ccced-376e-4373-a138-b51ce0235fe3","added_by":"auto","created_at":"2025-09-23 09:09:22","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2604974,"visible":true,"origin":"","legend":"","description":"","filename":"Alnabatipapersupplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-7463569/v1/9ad9f865f0cc9dcc02179f91.docx"},{"id":91971093,"identity":"b0067e5e-202c-4eff-90eb-07f4173f4d79","added_by":"auto","created_at":"2025-09-23 09:09:22","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":138207,"visible":true,"origin":"","legend":"","description":"","filename":"NCOMMS2571126T0enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7463569/v1/f3aa30a6df2b39514ca52a82.xml"},{"id":91971078,"identity":"0fc436e6-f747-4d9e-bbe4-a48781f85f09","added_by":"auto","created_at":"2025-09-23 09:09:20","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":441101,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7463569/v1/fce956af77022e1785c2dc8a.png"},{"id":91971094,"identity":"09a1e17d-54ed-4307-a0af-87da2d9859dc","added_by":"auto","created_at":"2025-09-23 09:09:22","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":27778,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7463569/v1/56d1aeb7f43da88088ec5606.png"},{"id":91971092,"identity":"cc442207-042d-4f9c-aeca-dd363b0933e8","added_by":"auto","created_at":"2025-09-23 09:09:22","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":997575,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7463569/v1/9f59272528e41420d5eb0edd.jpeg"},{"id":91971502,"identity":"6911306d-c2eb-4972-b18b-d8ee050747fa","added_by":"auto","created_at":"2025-09-23 09:17:21","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":280029,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7463569/v1/c4d1a60ece2054141ff66c9a.png"},{"id":91971096,"identity":"67e745d7-e316-4c24-8d2f-b1377f2e7904","added_by":"auto","created_at":"2025-09-23 09:09:22","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":99600,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7463569/v1/74509903d9cfb49a06d863e1.png"},{"id":91971085,"identity":"77ccdcfb-591e-4dde-9bc4-fcdde5bc1140","added_by":"auto","created_at":"2025-09-23 09:09:21","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":89897,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7463569/v1/e72b3eaead9a56172dcf3c5e.png"},{"id":91971083,"identity":"37a6df34-02df-4a96-9e77-62ddde2571e0","added_by":"auto","created_at":"2025-09-23 09:09:21","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":17018,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7463569/v1/3737df2bb786acb9c1989381.png"},{"id":91971087,"identity":"b6e95763-7407-4dd8-82eb-e40848214b69","added_by":"auto","created_at":"2025-09-23 09:09:21","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":303144,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7463569/v1/856e4904da61b752e89e39ab.png"},{"id":91971500,"identity":"befca256-66e0-4c2c-8500-a8a17d2dac06","added_by":"auto","created_at":"2025-09-23 09:17:21","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":49785,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7463569/v1/5db5f46b549e0201f98f5c5c.png"},{"id":91971501,"identity":"59210cbd-c1a6-4bdc-a356-84956d194c82","added_by":"auto","created_at":"2025-09-23 09:17:21","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":25731,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7463569/v1/eba886afe64f762d1e99786e.png"},{"id":91972806,"identity":"382f1b88-8ac9-4c95-90ad-37c71cc4109b","added_by":"auto","created_at":"2025-09-23 09:25:22","extension":"xml","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":135136,"visible":true,"origin":"","legend":"","description":"","filename":"NCOMMS2571126T0structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7463569/v1/ef0ec1af574703cd49e75dbc.xml"},{"id":91971091,"identity":"f74067e9-38ab-436a-ae64-36a19a33f959","added_by":"auto","created_at":"2025-09-23 09:09:22","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":147587,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7463569/v1/f35dabdfff6da36c327a8622.html"},{"id":91971100,"identity":"518adba2-22ca-4c42-bfcb-484d73bea587","added_by":"auto","created_at":"2025-09-23 09:09:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":267770,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQuantification of the major cell populations in the red pulp of the human spleen.\u003c/strong\u003e A-B) Representative high-content fluorescent microscopy images of spleen sections stained for CD206\u003csup\u003e+\u003c/sup\u003e sinusoidal cells (cyan), CD163\u003csup\u003e+\u003c/sup\u003e RPMs (magenta), and CD169\u003csup\u003e+\u003c/sup\u003e PCSAMs (red), with nuclear counterstaining using DAPI (grey). C) Quantification of marker-positive areas relative to the total DAPI area (μm²). D-F) Inter-individual variability in the abundance of each cell population across spleen samples. Spleens in figures are reported in Tab. S5.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7463569/v1/734c83ef6143398da7049a82.png"},{"id":91971090,"identity":"0b60e1ce-c3e9-4a19-851f-bb0cccf3311a","added_by":"auto","created_at":"2025-09-23 09:09:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":194860,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBacterial counts during \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eex vivo \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003espleen perfusion. \u003c/strong\u003eA1-F2)\u003cstrong\u003e \u003c/strong\u003eBacterial counts for six human spleens perfused with a mixture of pneumococcal isolates (Mix 1) (A-F). All organs were challenged with an equal mix of five \u003cem\u003eS. pneumoniae \u003c/em\u003eisolates (type 2: upward triangle; type 4: downward triangle; type 5: circle; type 6A: diamond; type 19F: square) at a cumulative dose of 1x10\u003csup\u003e8\u003c/sup\u003e CFU (high-dose; HD) (panels A-C) or 1x10\u003csup\u003e7\u003c/sup\u003e CFU (low-dose; LD) (panels D-F). Bacterial counts are reported for millilitres (mL) of perfusion liquid (A1-F1) and gram (g) of tissue biopsy (A2-F2). Panels C1–C2 and F1–F2 show bacterial counts in spleens perfused with 5 mM mannose in the perfusion liquid, added 30 minutes before bacterial challenge with the high-dose (C) and the low-dose (F) of pneumococci. No such treatment was given in spleens A, B, D and E. Bacterial counts are reported over six hours of infection. Dotted lines represent the limit of detection (2x10\u003csup\u003e2\u003c/sup\u003e CFU). Spleens perfused in these panels are listed in Tab. S5.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7463569/v1/5af712c5248809ab39725720.png"},{"id":91971079,"identity":"5295336c-dc46-492b-a89c-c833ce717e6e","added_by":"auto","created_at":"2025-09-23 09:09:20","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":997575,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssociation of pneumococci with splenic macrophages and sinusoidal cells during \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eex vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e perfusion, with and without mannose supplementation.\u003c/strong\u003e A1) Quantification of the percentage of tissue-associated pneumococci colocalising with CD206\u003csup\u003e+\u003c/sup\u003e sinusoidal cells at different time points post-infection (hour), under normal perfusion conditions (white bars) and with mannose supplementation (grey bars). A2) Analysis of the sinusoidal area occupied by bacteria over time of perfusion. Control samples are shown in white bars while mannose-supplemented tissues in grey bars. B1) 3D confocal reconstruction showing CD206 (red) expression in relation to pneumococci (green). B2) 3D reconstruction revealing the interaction between bacteria (green), CD206\u003csup\u003e+\u003c/sup\u003e sinusoidal cells (red) and CD163\u003csup\u003e+\u003c/sup\u003e macrophages (magenta). Blue shows the cell nuclei in DAPI staining. Images (panel B) are representative of ≥9 fields of view from n=2 independent human spleens and for two different (TIGR4 and 19F) pneumococcal serotype, with similar results obtained in all cases. C1-C3) WGA (C2-C3, yellow) staining of spleen section showing the localization of CD206 (C1-C3, red) and bacteria (C2-C3, green) on splenic sinusoidal cells. Cells nuclei are shown is DAPI staining (blue). Images (panel C) are representative of 2 fields of view from n=2 independent time point of infections (0 and 6 hours), with similar results obtained in all cases. D1-D2) Quantification of pneumococcal association with CD163⁺ RPMs at 30 minutes, 2 hours, and 5 hours post-infection under control conditions (white bars) and mannose supplementation (grey bars). E1-E2) Colocalization of pneumococci with CD169⁺ perifollicular macrophages during normal perfusion (white bars) and with mannose supplementation conditions (grey bars). Time is expressed in hour (h) of infection. Dotted lines indicate our limit of detections (D2, E2: 0.002 %). Marker associations (panels A, D and E) were calculated in Fiji (v1.53). Regions of interest (ROIs) were defined using the Image \u0026gt; Adjust \u0026gt; Threshold function for each cell population. These ROIs were then applied to the bacterial fluorescence channels to assess bacterial association to macrophages and sinusoidal cells through particles analysis. Statistical significance (panels A, D and E) was determined between the datasets for each time point by ordinary one way-ANOVA with Kruskal-Wallis post-hoc (ns: P\u0026gt;0.05; *: P≤0.05; **: P≤0.01; ***: P≤0.001; ****: P≤0.0001). Spleens analysed in these panels are listed in Tab. S5.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7463569/v1/904042161954f10f81d548b6.jpeg"},{"id":91971095,"identity":"b0771af2-7a68-4d95-8822-4b594591172a","added_by":"auto","created_at":"2025-09-23 09:09:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":221368,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCD206-dependent bacterial clearance by splenic adherent macrophages.\u003c/strong\u003e A1) Quantification of the primary splenic cell population after 24 hours of culture on collagen-coated wells. These data report the percentage of CD163\u003csup\u003e+\u003c/sup\u003e RPMs, CD206\u003csup\u003e+\u003c/sup\u003e sinusoidal cells and only negligible numbers of potential monocyte derived CD14\u003csup\u003e+\u003c/sup\u003e cells per field. A2) Characterization of the primary cell culture by immunofluorescence confocal microscopy. CD163\u003csup\u003e+\u003c/sup\u003e macrophages are shown in yellow, CD206\u003csup\u003e+\u003c/sup\u003e sinusoidal cells in red and CD163-CD206-CD14\u003csup\u003e+\u003c/sup\u003e cells in green. B1-B2) Analysis of pneumococcal association to CD206\u003csup\u003e+\u003c/sup\u003e sinusoidal cells (B1) and CD163\u003csup\u003e+\u003c/sup\u003e macrophages of three independent field of view for each condition (B2) (white bars: control condition; grey bars: inhibitory conditions D-mannose, anti-CD206 alone and in combination). C) Lyve-1 (yellow) expression by CD206\u003csup\u003e+\u003c/sup\u003e cells (red). Cells nuclei are shown in DAPI staining (blue). D1-E2) 3D confocal reconstruction of the interaction between bacteria (green), splenic sinusoids (red) and CD163\u003csup\u003e+\u003c/sup\u003e RPMs (yellow), showing intracellular bacteria only in macrophages. F1, G1) Quantification of bacterial survival 30 minutes post-infection for the TIGR4 (F1) and D39 strains (G1), during control conditions (white bars) and after the addition of 5mM mannose or anti-CD206 antibodies (grey bars) to the culture medium. F2-G2) Cultures infected with non-encapsulated derivatives of TIGR4 (F2) and D39 (G2). H1-H2) CD206-dependent killing of the \u003cem\u003eK. pneumoniae\u003c/em\u003e strain GMR15 (H1) and of the \u003cem\u003eE. coli\u003c/em\u003e strain UTI-89 (H2). F1-H2) experiments were performed in triplicates. Graphs report mean and standard deviation (SD) (F1: 43.7, SD 1.7; 94.8, SD 5.8; 86.3, SD 7.2; 90.8, SD 4.9. F2: 0.36, SD 0.01; 0.31, SD 0.04. G1: 31.7, SD 6.7; 79. 2, SD 9.1. G2: 16.3, SD 1.7; 19.4, SD 3.9; 19.4, SD 7.0. H1: 96.7, SD 4.0; 154, SD 10; 158, SD 13; 164, SD 7.3. H2: 38, SD 6.8; 55.4, SD 3.7). Dotted lines indicate our limit of detections (B1-B2: 0.03 %). Statistical significance was determined by ordinary one way-ANOVA (B; F1; G2; H1) and t-test (F2; G1; H2) (ns: P\u0026gt;0.05; *: P≤0.05; **: P≤0.01; ***: P≤0.001; ****: P≤0.0001). Spleens analysed in these panels are listed in Tab. S5.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7463569/v1/77fc573ec82b779a9041450b.png"},{"id":91971076,"identity":"9a3993b3-026e-4952-a5da-8bc604f6504e","added_by":"auto","created_at":"2025-09-23 09:09:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":302158,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBacterial uptake and bactericidal activity of human splenic macrophages during \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eex vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e perfusion.\u003c/strong\u003e A1-A2) Colocalization analysis of pneumococcal association with CD163\u003csup\u003e+\u003c/sup\u003e RPMs (A1, red) CD169\u003csup\u003e+\u003c/sup\u003e PCSAMs (A2, blue) over the course of the perfusion. Association of fluorescent micro-beads with CD163\u003csup\u003e+\u003c/sup\u003e (A1, light red) and CD169\u003csup\u003e+\u003c/sup\u003e macrophages (A2, light blue) are also reported. B1, B2) Linear association dynamics between bacterial burden and macrophage area occupation. Simple linear regression lines of best fit were determined for RPMs (B1, red) and PCSAMs (B2, blue). Each point represents a single analysed biopsy sample, and all spleens are included. Solid lines and filled circles represent D39, and dotted lines and open circles represent TIGR4. The R\u003csup\u003e2\u003c/sup\u003e value for each line is written adjacent to the appropriate line on each graph C) Quantification of LAMP1 over time, normalized to the total tissue area in different infected spleens (black bars) and in perfused control spleens (white bars). D1-D2) LAMP1 colocalization with CD163\u003csup\u003e+\u003c/sup\u003e (D1, red) and CD169\u003csup\u003e+\u003c/sup\u003e (D2, blue) macrophages in infected spleens (red and blue bars), in micro-beads perfused spleen (light-red and light-blue bars) and in control spleens (white bars) during the time of the perfusion. E1, E2) Quantification of cleaved caspase-3 signal in CD163\u003csup\u003e+\u003c/sup\u003e RPMs (E1) and CD169\u003csup\u003e+\u003c/sup\u003e PCSAMs (E2) over the time of perfusion. F1, F2) Analysis of the M30 levels in CD163\u003csup\u003e+\u003c/sup\u003e (F1, red bars) and CD169\u003csup\u003e+\u003c/sup\u003e (F2, blue bars) macrophages during infection. Panels A and C-F report means with standard deviations. Dotted lines represent our limit of detection (E1, E2, F1: 0.1%; F2: 0.01%). Time expressed in hours (h). Statistical significance was determined by ordinary one way-ANOVA with Kruskal-Wallis post-hoc (ns: P\u0026gt;0.05; *: P≤0.05; **: P≤0.01; ***: P≤0.001; ****: P≤0.0001). Spleens analysed in these panels are listed in Tab. S5.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7463569/v1/aa725ed945e66835dce11599.png"},{"id":91972807,"identity":"ea4ff866-a539-4a83-aebd-9127a6617d55","added_by":"auto","created_at":"2025-09-23 09:25:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2935015,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7463569/v1/53e116e4-203d-4638-90b3-b20ea2a20226.pdf"},{"id":91971504,"identity":"9f641e4c-e04b-4f01-a5a4-3a08c33d07d1","added_by":"auto","created_at":"2025-09-23 09:17:22","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2604974,"visible":true,"origin":"","legend":"SUPPLEMENTARY INFORMATION","description":"","filename":"Alnabatipapersupplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-7463569/v1/7e0838dc6081fee564d7b24c.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"The Mannose Receptor on Sinusoidal Lining Cells Mediates Two-Step Bacterial Clearance in the Human Spleen","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe human spleen is the main secondary lymphoid organ involved in the control of systemic infection\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, particularly through its critical role in the removal of encapsulated bacteria from the circulation\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Loss of the spleen by splenectomy, and other forms of functional asplenia, expose patients to the risk of overwhelming sepsis, most commonly caused by capsulated bacteria\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. This has led to the inclusion of asplenia as a key indication for vaccination in many countries\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Despite this the pneumococcus remains the leading cause of post-splenectomy sepsis\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. While the processes of bacterial clearance by the human spleen are critically understudied, the mechanisms by which the spleen removes \u003cem\u003ePlasmodium falciparum\u003c/em\u003e-infected red blood cells are well documented due to exploitation of the translational \u003cem\u003eex vivo\u003c/em\u003e human spleen prefusion models\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn most human tissues, tissue resident macrophages express the mannose receptor (MRC1/CD206)\u003csup\u003e9\u003c/sup\u003e. However, in the human spleen neither the CD68\u003csup\u003e+\u003c/sup\u003eCD163\u003csup\u003e+\u003c/sup\u003e red pulp macrophages (RPMs) nor the CD68\u003csup\u003e+\u003c/sup\u003eCD169\u003csup\u003e+\u003c/sup\u003e perifollicular capillary sheath macrophages (PCSAMs) express the mannose receptor\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Instead, the CD206 receptor is expressed by the sinusoidal lining littoral cells, which make up the structure of the human spleen red pulp\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Splenic sinuses are the walled irregular structures draining the open circulation of the human splenic red pulp and are lined by sinus endothelia alternatively named sinusoidal lining cells (CD206\u003csup\u003e+\u003c/sup\u003e, LYVE-1\u003csup\u003e+\u003c/sup\u003e, CD141\u003csup\u003e+\u003c/sup\u003e)\u003csup\u003e11, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The rationale behind this variation in CD206 expression in the human spleen is still unknown. The CD206 mannose receptor is a key molecule involved in the binding to bacteria, viruses and parasite-infected cells playing a key role in the interface between the human host and microbes. Binding of the mannose receptor to bacterial surface sugars, including capsular polysaccharides of \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e lipopolysaccharide (LPS)\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, is one of the main mechanisms by which macrophages recognize these pathogens; and endocytosis of the receptor is associated to the uptake and the subsequent killing of the bacteria inside the macrophage compartments\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. In addition, CD206 has been shown to mediate uptake of dengue virus and HBV into host cells\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e and recent work on the malaria parasite \u003cem\u003eP. falciparum\u003c/em\u003e showed that infected red blood cells are efficiently captured via their surface polysaccharides by the CD206\u003csup\u003e+\u003c/sup\u003e sinusoids, suggesting a crucial role of sinusoidal cells in the reduction of pathogen load\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eInvestigation of host-pathogen interactions in the spleen, using murine infection models, non-human primate infections and \u003cem\u003eex vivo\u003c/em\u003e porcine spleen perfusion, revealed that systemic \u003cem\u003eS. pneumoniae\u003c/em\u003e infections were predominantly cleared by splenic red pulp macrophages\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. While the spleen was the primary organ responsible for the clearance of bacteria, the rare occurrence of splenic replication in permissive macrophages facilitated re-seeding of bacteria to the bloodstream and initiation of systemic infection\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The translational \u003cem\u003eex vivo\u003c/em\u003e human spleen perfusion model confirmed detection of bacterial clusters in human splenic macrophages\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. However, the mannose receptor expression on sinusoidal endothelial cells and not macrophages raised the question about pathogen clearance mechanisms. Using the unique translational platform of human organ \u003cem\u003eex vivo\u003c/em\u003e perfusion alongside primary splenic cell co-cultures of macrophages and sinusoids, we tested the hypothesis that CD206-mediated capture by sinusoidal endothelial cells underlies the human spleen\u0026rsquo;s distinctive ability to filter, retain, and clear encapsulated bacteria. This host-species-specific adaptation sheds new light on splenic innate immune surveillance and has important implications for vaccine efficiency and the development of host-targeted therapies.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eCellular marker distribution in the human spleen.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo determine the impact of CD206 expression on sinusoidal cells on the clearance of encapsulated bacteria in the human spleen, we applied a translational \u003cem\u003eex vivo\u003c/em\u003e human spleen perfusion model\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e using organs sourced through the TIMID trial (REC 18/EM/0057; ClinicalTrials.gov NCT04620824) and primary cells through the MOSIE trial (CE: 668/2023/Sper/AOUBo). Microscopy mapped the three red pulp markers, CD163, CD169, and CD206, to three distinct cell types: CD68\u003csup\u003e+\u003c/sup\u003eCD163\u003csup\u003e+\u003c/sup\u003e red pulp macrophages (RPMs), CD68\u003csup\u003e+\u003c/sup\u003eCD169\u003csup\u003e+\u003c/sup\u003e perifollicular capillary sheath\u0026ndash;associated macrophages (PCSAMs), and sinusoid lining littoral CD206\u003csup\u003e+\u003c/sup\u003eLYVE-1\u003csup\u003e+\u003c/sup\u003eCD31\u003csup\u003e\u0026minus;\u003c/sup\u003e cells\u003csup\u003e9, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u0026ndash;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB; Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA1-S1B). High-content scanning fluorescent microscopy revealed that RPMs and sinusoidal cells dominate the red pulp\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), while CD169\u003csup\u003e+\u003c/sup\u003e macrophages clustered in sheaths around perifollicular capillaries, often forming ring-like structures adjacent to white pulp follicles\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). CD206\u003csup\u003e+\u003c/sup\u003e sinusoids, CD163\u003csup\u003e+\u003c/sup\u003e RPMs, and CD169\u003csup\u003e+\u003c/sup\u003e macrophages occupied on average 36.5% (SD 4.13), 27.7% (SD 6.1), and 1.3% (SD 1.6) of the total splenic section area respectively, with ranges of 30.3\u0026ndash;42.0%, 19.9\u0026ndash;35.8%, and 0.07\u0026ndash;4.6% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Marker abundance varied between spleens, up to 1.2-fold for CD206\u003csup\u003e+\u003c/sup\u003e, 2.7-fold for CD163\u003csup\u003e+\u003c/sup\u003e, and intriguingly 12.1-fold for CD169\u003csup\u003e+\u003c/sup\u003e macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u0026ndash;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), the latter reflecting differences in follicle density and microanatomy (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC1\u0026ndash;S1C3). Importantly, serial biopsies from the same spleen at different infection stages showed no variation in CD163 or CD206 levels (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD1\u0026ndash;S1D2), confirming the stability of these populations during perfusion and infection.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTaken together, these observations reveal a compartmentalized immune architecture in human spleen, with CD206\u003csup\u003e+\u003c/sup\u003e sinusoidal cells forming an extensive, blood-facing capture network; CD163\u003csup\u003e+\u003c/sup\u003e macrophages distributed throughout the red pulp for removal of pathogens, senescent cells and debris; and rare CD169\u003csup\u003e+\u003c/sup\u003e macrophages positioned around perifollicular regions, where they may regulate antigen entry into the white pulp\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. This spatial arrangement underscores a division of labour in pathogen surveillance, placing CD206\u003csup\u003e+\u003c/sup\u003e cells in an ideal position to intercept microbes during their first passage through the splenic open circulation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEx vivo\u003c/b\u003e \u003cb\u003eorgan perfusion.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eUsing the TIMID trial, human spleens were sourced for \u003cem\u003eex vivo\u003c/em\u003e normothermic organ perfusion to assess the splenic antibacterial clearance capacity. After cannulation and anticoagulant perfusion, spleens were transported on ice, connected to a normothermic circuit, and perfused with polymerised haemoglobin as an oxygen carrier. Systemic infection was simulated by introducing \u003cem\u003eS. pneumoniae\u003c/em\u003e directly into the perfusion liquid mimicking hematogenous invasive human infections (Tab. S1), and serial biopsies and perfusate samples were taken over time to monitor the infection dynamics\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Across experiments, the spleen demonstrated a robust filtration capacity, removing over 90% of the inoculum within 60 minutes (Fig.\u0026nbsp;2A1-2B2, Fig.\u0026nbsp;2D1-2E2). Clearance kinetics were consistent whether challenged with individual strains or serotype mixtures (Tab. S1, Tab. S2). With limited organ availability, mixed infections guaranteed a more controlled experimental set-up allowing for both simultaneous testing of virulent and avirulent serotypes while minimising any potential impact of type-specific immunity of organ donors (Tab. S3). At a high-dose (1x10\u003csup\u003e8\u003c/sup\u003e cumulative CFU) of five equally counted serotypes (2, 4, 5, 6B, 19F), clearance was equally efficient across strains, regardless of invasive potential (Fig.\u0026nbsp;2A1\u0026ndash;2B2)\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Similarly, the association of the bacteria with CD163\u003csup\u003e+\u003c/sup\u003e RPMs and the less abundant CD169\u003csup\u003e+\u003c/sup\u003e macrophages, based on image analysis, did not vary among serotypes (Fig. S2A1-S2A2). Neither the rapidity nor the extent of bacterial clearance in the human spleen was affected by the donor-derived level of antibacterial antibodies in the perfusion fluid (Tab. S3). At a lower dose (1x10\u003csup\u003e7\u003c/sup\u003e cumulative CFU), all serotypes were cleared from both perfusate and tissue within 30 minutes (Fig.\u0026nbsp;2D1\u0026ndash;2E2). These kinetics underscore the spleen\u0026rsquo;s extraordinary capacity for rapid, non-discriminatory removal of encapsulated bacteria, even without high antibody titres. This observation aligns with the clinical reality of overwhelming post-splenectomy infection (OPSI), where the absence of splenic tissue dramatically impairs early pathogen removal\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The uniformity of clearance across serotypes highlights a fundamental property of splenic immunity: its filtering capacity operates independently of serotype-specific immune history, a fact critical to understanding infection risk in asplenic patients\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eContribution of the mannose receptor on sinusoids to bacterial clearance in the human spleen.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe mannose receptor (CD206) is a C-type lectin that binds high-mannose structures found on many pathogens, including the capsules of \u003cem\u003eS. pneumoniae\u003c/em\u003e and LPS of \u003cem\u003eK. pneumoniae\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/em\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. In most tissues, CD206 is expressed by macrophages and dendritic cells and mediates bacterial binding and uptake\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In the human spleen, however, macrophages lack CD206 entirely (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u0026ndash;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, Fig. S2B-S2C3), despite the spleen being the primary site for pneumococcal clearance. Instead, CD206 is confined to sinusoidal lining cells, a distribution that raises an important mechanistic question: does splenic clearance rely on CD206-mediated capture by these sinusoidal cells rather than by macrophages?\u003c/p\u003e\u003cp\u003eTo address this, we built on \u003cem\u003ein vitro\u003c/em\u003e evidence that free mannose can block CD206\u0026ndash;pathogen binding\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In the \u003cem\u003eex vivo\u003c/em\u003e perfusion model, spleens were pre-treated with 5 mM mannose alongside the 10\u0026ndash;20 mM glucose required for host and bacterial metabolism. When challenged with a high pneumococcal dose (1x10\u003csup\u003e8\u003c/sup\u003e CFU), mannose-treated spleens failed to clear the bacteria, instead, counts instead accumulated progressively in both perfusate and tissue (Fig.\u0026nbsp;2C1-2C2). At a lower challenge dose (1x10\u003csup\u003e7\u003c/sup\u003e CFU), usually cleared within minutes, killing was completely abolished; bacteria instead proliferated rapidly (Fig.\u0026nbsp;2F1-2F2). These results identify the carbohydrate-binding activity of CD206 on sinusoidal lining cells as an essential initial step in pathogen clearance. Since the presence of glucose in the perfusion liquid and the cell culture medium blocks completely the pneumococcal mannose metabolism through carbon catabolite repression\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, the observed escape from host-mediated clearance cannot be ascribed to an effect of mannose on the bacteria. Rather, it reflects direct interference with receptor-mediated capture.\u003c/p\u003e\u003cp\u003eTo complement the quantification of viable bacteria, we used high-content scanning fluorescence microscopy to examine the association of pneumococci with splenic macrophages and sinusoidal cells in tissue sections obtained during \u003cem\u003eex vivo\u003c/em\u003e perfusion. This analysis reinforced the clearance findings: across all time points, 16\u0026ndash;21% of tissue-associated bacteria colocalized with CD206 (Fig.\u0026nbsp;3A1), a proportion that remained stable despite a 90% reduction in total bacterial load during the first hour (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Correspondingly, the area of sinusoidal tissue occupied by bacteria showed no decline (Fig.\u0026nbsp;3A2). Three-dimensional confocal reconstructions revealed discrete clusters of CD206 in direct contact with bacteria (Fig.\u0026nbsp;3B1-3B2). In mannose-treated spleens, bacterial association with CD206 fell sharply at early time points (Fig.\u0026nbsp;3A1), coinciding with higher viable counts and broader sinusoidal distribution later (Fig.\u0026nbsp;3A2).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo determine the fate of these captured bacteria, we performed wheat germ agglutinin (WGA) staining, which confirmed that CD206\u003csup\u003e+\u003c/sup\u003e sinusoidal cells do not internalise bacteria (Fig.\u0026nbsp;3C1-3C3). Instead, captured bacteria remained extracellular, suggesting that these cells act as stationary traps. Additional quantification showed that 20% of pneumococci colocalised with CD163\u003csup\u003e+\u003c/sup\u003e RPMs at 30 min, 2 h, and 5 h post-infection (Fig.\u0026nbsp;3D1), and that mannose treatment significantly reduced this association (Fig.\u0026nbsp;3D1). Although CD169\u003csup\u003e+\u003c/sup\u003e macrophages were less abundant, their bacterial association was also diminished by mannose (Fig.\u0026nbsp;3E1-3E2).\u003c/p\u003e\u003cp\u003eTo provide \u003cem\u003ein vitro\u003c/em\u003e evidence for the phenotypes observed during \u003cem\u003eex vivo\u003c/em\u003e spleen perfusion, primary adherent cell cultures were established from human spleen homogenates. Primary spleen cultures contained 57% CD206⁺ sinusoidal cells and 35% CD163⁺ macrophages (Fig.\u0026nbsp;4A1), often linked by extensions (Fig.\u0026nbsp;4A2). Only CD14⁺ cells comprised only 1.4%, indicating rare monocytes, possibly overlapping with the CD206⁺ population\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.Low-MOI infections showed that 2.1% of bacteria attached to CD206\u003csup\u003e+\u003c/sup\u003e cells (Fig.\u0026nbsp;4B1) and 0.6% to CD163\u003csup\u003e+\u003c/sup\u003e macrophages (Fig.\u0026nbsp;4B2). Mannose or anti-CD206 antibodies reduced bacterial binding to CD206\u003csup\u003e+\u003c/sup\u003e cells (Fig.\u0026nbsp;4B1) but not to CD163\u003csup\u003e+\u003c/sup\u003e macrophages (Fig.\u0026nbsp;4B2). The expression of CD206 on primary sinusoidal cells, and not macrophages, was confirmed by CD206\u003csup\u003e+\u003c/sup\u003e co-expression with the sinusoidal marker LYVE-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Three-dimensional reconstructions confirmed that pneumococci remained on the surface of CD206\u003csup\u003e+\u003c/sup\u003e cells (Fig.\u0026nbsp;4D1\u0026ndash;4D2), while bacteria appeared intracellular only in macrophages (Fig.\u0026nbsp;4E1\u0026ndash;4E2). At MOI 10, bacterial counts dropped sharply within 30 min for both TIGR4 and D39 serotypes (Fig.\u0026nbsp;4F1, 4G1), but this bactericidal activity was lost when CD206 was blocked by mannose or antibodies (Fig.\u0026nbsp;4F1, 4G1). In contrast, when cultures were challenged with a non-encapsulated derivative of the TIGR4 strain, over 99% of bacteria were eliminated within 30 minutes (Fig.\u0026nbsp;4F2), and this killing was unaffected by mannose supplementation (Fig.\u0026nbsp;4F2). The same behaviour was observed for a rough non-encapsulated D39 strain (Fig.\u0026nbsp;4G2). These results strongly suggest that CD206-mediated bacterial attachment to sinusoidal cells is essential for enabling subsequent macrophage-mediated killing of encapsulated, sugar-coated pathogens. The control experiments with the non-encapsulated strains reinforce the conclusion that the CD206-dependent interaction between sinusoidal cells and macrophages is not required for the clearance of un-encapsulated bacteria. In addition to pneumococcal capsules, CD206 had been shown to bind also \u003cem\u003eK. pneumoniae\u003c/em\u003e LPS \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Repeating the bactericidal assay with capsulated K2 capsule \u003cem\u003eK. pneumoniae\u003c/em\u003e and K1 capsule \u003cem\u003eEscherichia coli\u003c/em\u003e, confirmed CD206-dependence for any bactericidal activity of macrophages (Fig.\u0026nbsp;4H1-4H2). These \u003cem\u003ein vitro\u003c/em\u003e results fully confirm the observations during organ perfusion, indicating that CD206-mediated bacterial attachment to sinusoidal cells is essential for enabling subsequent macrophage-mediated killing of encapsulated pathogens.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThese observations support a sequential \u0026ldquo;handoff\u0026rdquo; model: CD206\u003csup\u003e+\u003c/sup\u003e sinusoidal cells capture encapsulated bacteria without internalisation, then present them to macrophages for phagocytosis and killing. Blocking CD206 prevents this initial capture and reduces downstream bacterial association with macrophages. Importantly, this arrangement mirrors receptor-mediated antigen transfer in other lymphoid organs\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e but it is specialised for bloodstream pathogen clearance.\u003c/p\u003e\u003cp\u003eUnlike the traditional view that attributes CD206-mediated clearance to macrophages\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, our data show that in the human spleen, these cells are non-phagocytic stromal elements. Functionally, they behave like biological flypaper, retaining pathogens at the sinusoidal lining surface until macrophages, particularly CD163\u003csup\u003e+\u003c/sup\u003e and CD169\u003csup\u003e+\u003c/sup\u003e subsets, engage them (Fig. S2B-S2C3). The mannose-inhibitable spectrum of this capture extends beyond pneumococcus to multiple encapsulated species, echoing mechanical retention of poorly deformable cells in splenic sinuses\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e but based on biochemical glycan recognition.\u003c/p\u003e\u003cp\u003eThe therapeutic implications are twofold: enhancing CD206-mediated capture could strengthen host defences, while systemic mannose administration, as in some urinary tract infection (UTIs) treatments\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, could inadvertently impair splenic clearance. Our findings also clarify that in the spleen, CD206 is not a macrophage marker but a functional receptor on sinusoidal cells. This corrects earlier assumptions that CD206-mediated phagocytosis in spleen was macrophage-driven\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e repositioning these cells as dedicated antigen-capturing and presenting cells. By capturing encapsulated bacteria via mannose recognition, these sinusoidal cells transform a microbial evasion strategy, the anti-phagocytic capsule, into a vulnerability. The concept parallels earlier work showing that erythrocytes with exposed mannoses, including \u003cem\u003eP. falciparum\u003c/em\u003e\u0026ndash;infected cells, are preferentially retained and cleared\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Our results extend this to bacteria, demonstrating that the spleen uses mannose recognition to detect and trap both infected cells and encapsulated microbes. In summary, the human spleen is not just a passive filter but an active immune organ combining physical, biochemical, and receptor-mediated mechanisms to eliminate bloodstream threats. Redefining CD206\u003csup\u003e+\u003c/sup\u003e sinusoidal cells as non-phagocytic, but indispensable for microbial capture, offers a new perspective on splenic immunity and opens the door to targeted interventions that reinforce this early line of defence.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMechanism of pathogen clearance by both CD163\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eand of CD169\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emacrophages.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe two dominant splenic macrophage subsets, CD163\u003csup\u003e+\u003c/sup\u003e RPMs and the less abundant CD169\u003csup\u003e+\u003c/sup\u003e PCSAMs, operate in distinct but complementary niches\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Quantitative colocalization revealed that 15\u0026ndash;30% of bacteria were associated with CD163\u003csup\u003e+\u003c/sup\u003e macrophages, while 4\u0026ndash;22% associated with CD169\u003csup\u003e+\u003c/sup\u003e macrophages (Fig.\u0026nbsp;5A1-5A2). Notably, these proportions were consistent between early (30 min) and late (5 h) time points, despite \u0026gt;\u0026thinsp;90% clearance of bacteria from the perfusate within the first hour. In addition, microscopy analyses of tissue sections with high or low bacterial loads revealed a consistent ratio of bacteria-to-macrophage association across both CD163\u003csup\u003e+\u003c/sup\u003e and CD169\u003csup\u003e+\u003c/sup\u003e populations (Fig.\u0026nbsp;5B1-5B2), suggesting that even at high challenge doses, macrophage capacity for bacterial clearance was not saturated. The ratios remained consistent across different bacterial loads and serotypes (2, 4, 19F) (Fig S2A1-S2A2), indicating stable, load-independent pathogen engagement by these macrophage subsets. When fluorescent beads were perfused instead of live bacteria (Fig. S3A-S3C), macrophage uptake was slower, with association rates increasing progressively over time (Fig.\u0026nbsp;5A1\u0026ndash;5A2). This slower uptake highlights the dynamic responsiveness of splenic macrophages to live microbes versus inert particles, reflecting an ability to accelerate engagement when genuine pathogens are detected. While these data do not allow quantification of the relative bactericidal contributions of each macrophage subset, they indicate that both CD163\u003csup\u003e+\u003c/sup\u003e and CD169\u003csup\u003e+\u003c/sup\u003e macrophages consistently retained similar bacterial loads over time, implying a comparable role in pneumococcal capture and clearance.\u003c/p\u003e\u003cp\u003eTo investigate the mechanisms of bacterial killing by human spleen tissue-resident macrophages, spleen sections, at different time points of infection (from 0 to 6 h) were stained for the phagosome maturation marker LAMP1. LAMP1 staining, marking lysosomal compartments, revealed a progressive increase in infected spleens, but not in uninfected controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). This signal was localised mainly within macrophages (Fig.\u0026nbsp;5D1\u0026ndash;5D2), consistent with active lysosomal engagement. Interestingly, minimal LAMP1\u0026ndash;pneumococcus colocalization was observed, indicating that at early time points, bacteria-containing phagosomes may not yet have matured into fully degradative compartments (data not shown). Overall, the time-dependent increase in LAMP1 levels supports the conclusion that human spleen macrophages remain engaged in active phagocytosis during our \u003cem\u003eex vivo\u003c/em\u003e perfusion model and exhibit dynamic features of the phagocytic process.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eApoptosis has long been recognized as a key mechanism by which human alveolar macrophages eliminate pneumococci\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. To assess whether this mechanism is also active in human splenic macrophages, tissue sections were stained for cleaved caspase-3, the activated form of the caspase responsible for executing apoptotic proteolysis\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. The infection triggered an early apoptotic response. Within 60 minutes, cleaved caspase-3 levels rose rapidly, over 10-fold in both CD163\u003csup\u003e+\u003c/sup\u003e RPMs and in CD169\u003csup\u003e+\u003c/sup\u003e macrophages (Fig.\u0026nbsp;5E1\u0026ndash;5E2), with the latter showing 10x higher per-cell signal (Fig. S2D1\u0026ndash;S2D2). In bead-perfused controls, this apoptotic rise was slower and less pronounced, suggesting that live bacterial infection accelerates cell death programmes. M30 staining, indicative of caspase-3\u0026ndash;cleaved cytokeratin-18\u003csup\u003e39, 40\u003c/sup\u003e, increased steadily over 5\u0026ndash;6 hours in both subsets (Fig.\u0026nbsp;5F1\u0026ndash;5F2), with the M30-positive macrophage area expanding accordingly. Mannose-treated and untreated spleens showed similar trends and were pooled in the graphs (Fig. S2E1\u0026ndash;S2E2).\u003c/p\u003e\u003cp\u003eConsidering that a human spleen contains approximately 2x10\u003csup\u003e10\u003c/sup\u003e macrophages\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, we found that at the 1-hour mark, 0.65% of CD163\u003csup\u003e+\u003c/sup\u003e macrophages were positive for cleaved caspase-3 (Fig.\u0026nbsp;5E1\u0026ndash;5E2). This corresponds to roughly 7x10\u003csup\u003e7\u003c/sup\u003e to 3x10\u003csup\u003e8\u003c/sup\u003e apoptotic cells per spleen; numbers that closely match the bacterial challenge dose of 1x10\u003csup\u003e8\u003c/sup\u003e CFU. This correspondence suggests a direct link between the quantity of phagocytosed bacteria and the induction of macrophage apoptosis.\u003c/p\u003e\u003cp\u003eThe lysosomal activation observed after bacterial uptake, marked by increased LAMP1 expression, confirms that splenic macrophages engage in canonical phagolysosomal killing\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. However, the concurrent detection of early cleaved caspase-3 within just 60 minutes, followed by M30 expression at 5\u0026ndash;6 hours, indicates that bacterial clearance in the spleen is closely coupled to programmed cell death. This differs from the delayed apoptosis reported in other macrophage populations, such as alveolar macrophages, where cell death typically occurs in the resolution phase of infection\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. The spleen\u0026rsquo;s context may explain this difference. As a blood-filtering organ, it is uniquely exposed to high concentrations of circulating pathogens. In such an environment, rapid apoptosis following phagocytosis could serve as both a terminal killing step and a containment measure, ensuring that intracellular pathogens cannot persist within surviving macrophages. This swift apoptotic turnover may also act as a chemotactic signal to recruit additional phagocytes, maintaining clearance efficiency even, as individual macrophages are lost. The timing of this response is likely critical for neutralising pathogens capable of manipulating macrophage survival. \u003cem\u003eNeisseria meningitidis\u003c/em\u003e, for example, can delay apoptosis via nitric oxide detoxification\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, prolonging its intracellular survival. The early initiation of apoptosis in splenic macrophages could therefore represent an evolved countermeasure against such strategies, an immunological \u0026ldquo;self-destruct\u0026rdquo; that denies bacteria a long-term niche.\u003c/p\u003e\u003cp\u003eOur findings support a revised model of splenic antibacterial defence in which CD206⁺ sinusoidal cells and macrophages act in coordinated sequence. CD206⁺ lining cells first immobilise encapsulated bacteria via mannose-dependent glycan recognition without internalisation, preventing their return to circulation and enabling targeted clearance. CD163⁺ and CD169⁺ macrophages then receive these retained bacteria, phagocytose them, initiate lysosomal degradation, and undergo rapid apoptosis, ensuring efficient destruction while preventing intracellular persistence and systemic re-seeding.\u003c/p\u003e\u003cp\u003eThis architecture bears resemblance to antigen-handling systems in lymph nodes, where lymphatic endothelial cells and subcapsular sinus macrophages coordinate pathogen retention and transfer\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. However, the splenic system is tuned for speed and broad-spectrum efficiency, particularly against encapsulated bacteria that evade complement-mediated lysis and antibody opsonisation. In effect, the capsule, a key virulence factor, becomes a vulnerability when confronted with CD206-mediated capture. The efficiency of this system underscores why loss of splenic function, whether through splenectomy, infarction, or disease-related atrophy \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, carries such a high risk of overwhelming infection (OPSI), particularly from encapsulated organisms\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. These patients lack both the macrophage-rich red pulp and the specialised sinusoidal cell network, severely compromising their capacity to trap and neutralise bloodstream bacteria. Functional hyposplenism, as seen in sickle cell disease or celiac disease, often involves reduced sinusoidal cell density, further increasing vulnerability to severe bacterial infections\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSpleen \u003cem\u003eex vivo\u003c/em\u003e perfusion, used for malaria⁸,\u0026sup1;⁷ and proposed for bacterial studies\u0026sup2;\u0026sup1;, has limitations, some linked to reperfusion injury (e.g., high cytokines and soluble proteins) (Fig. S4)⁴⁷, whose impact we did not assess. Recognising the central role of CD206-mediated capture suggests new interventions, such as mannosylated nanoparticles to enhance vaccine delivery⁴⁸ or strategies to preserve/replace macrophages to boost killing capacity. Conversely, systemic free mannose, used to prevent UPEC adhesion\u0026sup3;⁴, could transiently inhibit splenic CD206, impairing clearance of encapsulated pathogens. This risk may be important in patients with complicated UTI, concurrent bacteraemia, or reduced splenic reserve, warranting clinical caution.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eThe work was funded by EU funding within the MUR PNRR Extended Partnership initiative on Emerging Infectious Diseases (Project no. PE00000007, INF-ACT) to MRO and GC, PRIN 2020 grant 202089LLEH and PRIN PNRR P2022M8KYE from the Italian Ministry of Education, BBSRC grant BB/V01465X/1 and the UK Biomedical Research Centre grant NIHR203327 to MRO and a BBSRC Impact Accelerator Account (IAA) award (RM32G0178B7) to KS NA is supported by a PhD fellowship from Umm AlQura University, Makkah, Saudi Arabia.\u003c/p\u003e\u003ch2\u003eAuthor contributions:\u003c/h2\u003e\u003cp\u003eNA acquired, analysed and interpreted the microbiological and microscopy data and contributed to the drafting of the manuscript, FF acquired, analysed and interpreted the microbiological and microscopy data, developed the primary cell cultures and drafted the first version of the manuscript, TS, TK, JI, WC and GGa. Managed of patient consent in UK and performed the surgical part of the experimental work, acquired, analysed and interpreted the data, GC, TR, DG, RH, ZJ, ST acquired, analysed and interpreted the microbiological and microscopy data and contributed to the drafting of the manuscript, GGe, GR, and FR managed patient consent and clinical data in Italy, EG, GC and KSt supervised microscopy work in Italy and UK respectively, S.B. contributed development of perfusion liquid, KSc, CDB, CEP, SF and CT supervised cell culture and microbiology work, MR and AD contribute to study design, experimental set-up, performed and oversaw surgical part of the project in Italy and UK respectively, LMP contributed to the study design, data analysis and oversaw the immunological part of the project and contributed to the drafting and finalising of the manuscript, MRO contributed to the study design, oversaw the microbiological and microscopy data analysis and contributed to the funding of the work.\u003c/p\u003e\u003ch2\u003eAcknowledgements:\u003c/h2\u003e\u003cp\u003eAll authors thank the numerous patients in Leicester and Bologna consenting to participate in the clinical trials on which all the research work is based and the Research Governance Office of the University of Leicester and the respective office of the university of Bologna to sponsor the trials. Authors also thank Zaf Zafirelis from HbO2 Therapeutics (Souderton, PA, USA) for donating Hemopure, Kevin West for help with the H\u0026amp;E stains, and the University of Leicester Advanced Imaging Facility (RRID:SCR_020967) for use of microscope equipment, and the microscopy facility of the University of Bologna.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMorris DH, Bullock FD (1919) The importance of the spleen in resistance to infection. Ann Surg 70(5):513\u0026ndash;531\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLammers AJ, De Porto AP, Florquin S, De Boer OJ, Bootsma HJ, Hermans PW, Van der Poll T (2011) Enhanced vulnerability for Streptococcus pneumoniae sepsis during asplenia is determined by the bacterial capsule. Immunobiology 216(8):863\u0026ndash;870\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHoldsworth RJ, Cuschieri A, Irving AD (1991) Postsplenectomy sepsis and its mortality rate: actual versus perceived risks. Br J Surg 78(9):1031\u0026ndash;1038\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGea-Izquierdo E, Rodr\u0026iacute;guez-Caravaca G, Gil-Prieto R, Hern\u0026aacute;ndez-Barrera V, Gil-de-Miguel (2024) \u0026Aacute;. Streptococcus pneumoniae infection in patients with asplenia: a Spanish perspective over a 25-Year period. \u003cem\u003eAntibiotics\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e(1), 104\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAdriani KS, Brouwer MC, van der Ende A, van de Beek D (2013), June Bacterial meningitis in adults after splenectomy and hyposplenic states. In \u003cem\u003eMayo Clinic proceedings\u003c/em\u003e (Vol. 88, No. 6, pp. 571\u0026ndash;578). Elsevier\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePebody RG, Leino T, Nohynek H, Hellenbrand W, Salmaso S, Ruutu P (2005) Pneumococcal vaccination policy in Europe. Euro Surveill 10(9):174\u0026ndash;178\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTheilacker C, Ludewig K, Serr A, Schimpf J, Held J, B\u0026ouml;gelein M, Kern WV (2016) Overwhelming postsplenectomy infection: a prospective multicenter cohort study. Clin Infect Dis 62(7):871\u0026ndash;878\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBuffet PA, Milon G, Brousse V, Correas JM, Dousset B, Couvelard A, David PH (2006) Ex vivo perfusion of human spleens maintains clearing and processing functions. Blood 107(9):3745\u0026ndash;3752\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMartinez-Pomares L, Hanitsch LG, Stillion R, Keshav S, Gordon S (2005) Expression of mannose receptor and ligands for its cysteine-rich domain in venous sinuses of human spleen. Lab Invest 85(10):1238\u0026ndash;1249\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZamze S, Martinez-Pomares L, Jones H, Taylor PR, Stillion RJ, Gordon S, Wong SY (2002) Recognition of bacterial capsular polysaccharides and lipopolysaccharides by the macrophage mannose receptor. J Biol Chem 277(44):41613\u0026ndash;41623\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eQiu J, Salama ME, Hu CS, Li Y, Wang X, Hoffman R (2018) The characteristics of vessel lining cells in normal spleens and their role in the pathobiology of myelofibrosis. Blood Adv 2(10):1130\u0026ndash;1145\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSteiniger BS, Pfeffer H, Gaffling S, Lobachev O (2022) The human splenic microcirculation is entirely open as shown by 3D models in virtual reality. Sci Rep 12(1):16487\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSteiniger BS (2015) Human spleen microanatomy: why mice do not suffice. Immunology 145(3):334\u0026ndash;346\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStahl PD, Ezekowitz RAB (1998) The mannose receptor is a pattern recognition receptor involved in host defense. Curr Opin Immunol 10(1):50\u0026ndash;55\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMiller JL, deWet BJM, Martinez-Pomares L, Radcliffe CM, Dwek RA, Rudd PM, Gordon S (2008) The mannose receptor mediates dengue virus infection of macrophages. PLoS Pathog, 4(2), e17\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eden Brouw MLO, Binda RS, Geijtenbeek TB, Janssen HL, Woltman AM (2009) The mannose receptor acts as hepatitis B virus surface antigen receptor mediating interaction with intrahepatic dendritic cells. Virology 393(1):84\u0026ndash;90\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCao H, Antonopoulos A, Henderson S, Wassall H, Brewin J, Masson A, Vickers MA (2021) Red blood cell mannoses as phagocytic ligands mediating both sickle cell anaemia and malaria resistance. Nat Commun 12(1):1792\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGerlini A, Colomba L, Furi L, Braccini T, Manso AS, Pammolli A, Oggioni MR (2014) The role of host and microbial factors in the pathogenesis of pneumococcal bacteraemia arising from a single bacterial cell bottleneck. PLoS Pathog, 10(3), e1004026\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eErcoli G, Fernandes VE, Chung WY, Wanford JJ, Thomson S, Bayliss CD, Oggioni MR (2018) Intracellular replication of Streptococcus pneumoniae inside splenic macrophages serves as a reservoir for septicaemia. Nat Microbiol 3(5):600\u0026ndash;610\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChung WY, Wanford JJ, Kumar R, Isherwood JD, Haigh RD, Oggioni MR, Ercoli G (2018) An ex vivo porcine spleen perfusion as a model of bacterial sepsis. ALTEX: Altern Anim Experimentation 36(1):29\u0026ndash;38\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCarreno D, Wanford JJ, Jasiunaite Z, Hames RG, Chung WY, Dennison AR, Oggioni MR (2021) Splenic macrophages as the source of bacteraemia during pneumococcal pneumonia. \u003cem\u003eEBioMedicine\u003c/em\u003e, \u003cem\u003e72\u003c/em\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNagelkerke SQ, Bruggeman CW, Den Haan JM, Mul EP, Van Den Berg TK, Van Bruggen R, Kuijpers TW (2018) Red pulp macrophages in the human spleen are a distinct cell population with a unique expression of Fc-γ receptors. Blood Adv 2(8):941\u0026ndash;953\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMebius RE, Kraal G (2005) Structure and function of the spleen. Nat Rev Immunol 5(8):606\u0026ndash;616\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBrueggemann AB, Griffiths DT, Meats E, Peto T, Crook DW, Spratt BG (2003) Clonal relationships between invasive and carriage Streptococcus pneumoniae and serotype-and clone-specific differences in invasive disease potential. J Infect Dis 187(9):1424\u0026ndash;1432\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBrigden ML (2001) Detection, education and management of the asplenic or hyposplenic patient. Am Family Phys 63(3):499\u0026ndash;508\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEast L, Isacke CM (2002) The mannose receptor family. Biochim et Biophys Acta (BBA)-General Subj 1572(2\u0026ndash;3):364\u0026ndash;386\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMartinez-Pomares L (2012) The mannose receptor. J Leukoc Biol 92(6):1177\u0026ndash;1186\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSubramanian K, Neill DR, Malak HA, Spelmink L, Khandaker S, Dalla Libera Marchiori G, Henriques-Normark B (2019) Pneumolysin binds to the mannose receptor C type 1 (MRC-1) leading to anti-inflammatory responses and enhanced pneumococcal survival. Nat Microbiol 4(1):62\u0026ndash;70\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBidossi A, Mulas L, Decorosi F, Colomba L, Ricci S, Pozzi G, Oggioni MR (2012) A functional genomics approach to establish the complement of carbohydrate transporters in Streptococcus pneumoniae. PLoS ONE, 7(3), e33320\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGordon S, Pl\u0026uuml;ddemann A, Mukhopadhyay S (2015) Sinusoidal immunity: macrophages at the lymphohematopoietic interface. Cold Spring Harb Perspect Biol 7(4):a016378\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePhan TG, Green JA, Gray EE, Xu Y, Cyster JG (2009) Immune complex relay by subcapsular sinus macrophages and noncognate B cells drives antibody affinity maturation. Nat Immunol 10(7):786\u0026ndash;793\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDeplaine G, Safeukui I, Jeddi F, Lacoste F, Brousse V, Perrot S, Buffet PA (2011) The sensing of poorly deformable red blood cells by the human spleen can be mimicked in vitro. Blood J Am Soc Hematol 117(8):e88\u0026ndash;e95\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSafeukui I, Correas JM, Brousse V, Hirt D, Deplaine G, Mul\u0026eacute; S, Buffet PA (2008) Retention of Plasmodium falciparum ring-infected erythrocytes in the slow, open microcirculation of the human spleen. Blood J Am Soc Hematol 112(6):2520\u0026ndash;2528\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHayward G, Mort S, Hay AD, Moore M, Thomas NP, Cook J, Butler CC (2024) d-Mannose for prevention of recurrent urinary tract infection among women: a randomized clinical trial. JAMA Intern Med 184(6):619\u0026ndash;628\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJackson DG, Prevo R, Clasper S, Banerji S (2001) LYVE-1, the lymphatic system and tumor lymphangiogenesis. Trends Immunol 22(6):317\u0026ndash;321\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDockrell DH, Marriott HM, Prince LR, Ridger VC, Ince PG, Hellewell PG, Whyte MK (2003) Alveolar macrophage apoptosis contributes to pneumococcal clearance in a resolving model of pulmonary infection. J Immunol 171(10):5380\u0026ndash;5388\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGao LY, Abu Kwaik Y (1999) Apoptosis in macrophages and alveolar epithelial cells during early stages of infection by Legionella pneumophila and its role in cytopathogenicity. \u003cem\u003eInfection and immunity\u003c/em\u003e, \u003cem\u003e67\u003c/em\u003e(2), 862\u0026ndash;870. H\u0026auml;cker H, F\u0026uuml;rmann C, Wagner H and\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eH\u0026auml;cker G (2002) Caspase-9/-3 activation and apoptosis are induced in mouse macrophages upon ingestion and digestion of Escherichia coli bacteria. J Immunol 169(6):3172\u0026ndash;3179\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLeers MP, K\u0026ouml;lgen W, Bj\u0026ouml;rklund V, Bergman T, Tribbick G, Persson B, Schutte B (1999) Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis. J Pathol 187(5):567\u0026ndash;572\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eUlukaya E, Karaagac E, Ari F, Oral AY, Adim SB, Tokullugil AH, Evrensel T (2011) Chemotherapy increases caspase-cleaved cytokeratin 18 in the serum of breast cancer patients. Radiol Oncol 45(2):116\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSender R, Weiss Y, Navon Y, Milo I, Azulay N, Keren L, Milo R (2023) The total mass, number, and distribution of immune cells in the human body. Proceedings of the National Academy of Sciences, 120(44), e2308511120\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFlannagan RS, Cos\u0026iacute;o G, Grinstein S (2009) Antimicrobial mechanisms of phagocytes and bacterial evasion strategies. Nat Rev Microbiol 7(5):355\u0026ndash;366\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePreston JA, Bewley MA, Marriott HM, McGarry Houghton A, Mohasin M, Jubrail J, Dockrell DH (2019) Alveolar macrophage apoptosis\u0026ndash;associated bacterial killing helps prevent murine pneumonia. Am J Respir Crit Care Med 200(1):84\u0026ndash;97\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTunbridge AJ, Stevanin TM, Lee M, Marriott HM, Moir JW, Read RC, Dockrell DH (2006) Inhibition of macrophage apoptosis by Neisseria meningitidis requires nitric oxide detoxification mechanisms. Infect Immun 74(1):729\u0026ndash;733\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDi Sabatino A, Rosado MM, Cazzola P, Riboni R, Biagi F, Carsetti R, Corazza GR (2006) Splenic hypofunction and the spectrum of autoimmune and malignant complications in celiac disease. Clin Gastroenterol Hepatol 4(2):179\u0026ndash;186\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStotesbury H, Kawadler JM, Saunders DE, Kirkham FJ (2021) MRI detection of brain abnormality in sickle cell disease. Expert Rev Hematol 14(5):473\u0026ndash;491\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGravante G, Ong SL, Metcalfe MS, Sorge R, Sconocchia G, Orlando G, Dennison AR (2009), May Cytokine response to ischemia/reperfusion injury in an ex vivo perfused porcine liver model. In \u003cem\u003eTransplantation proceedings\u003c/em\u003e (Vol. 41, No. 4, pp. 1107\u0026ndash;1112). Elsevier\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePaurević M, Šrajer Gajdošik M, Ribić R (2024) Mannose ligands for mannose receptor targeting. Int J Mol Sci 25(3):1370\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7463569/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7463569/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe human spleen is the main organ in preventing invasive bacterial infection, yet the cellular mechanisms driving pathogen clearance remain poorly defined. This work shows that there is division of labour in the human spleen for eliminating bacteria from the circulation. Using a dual translational approach including \u003cem\u003eex vivo\u003c/em\u003e perfusion of human spleen and splenic primary cell cultures, we demonstrate that sinusoidal cells capture and retain bacteria via the CD206 receptor in the splenic red pulp to enable bactericidal activity by tissue resident macrophages. This activity was dependent on bacterial capsule, with unencapsulated bacteria being cleared irrespective of inhibition of the mannose receptor. This implies a specific two-step process to ensure efficient removal of encapsulated pathogens. These data change completely our understanding of pathogen clearance in the human spleen, with profound implications for the development of host-directed anti-infective strategies and for the evaluation of conjugate vaccine efficacy.\u003c/p\u003e","manuscriptTitle":"The Mannose Receptor on Sinusoidal Lining Cells Mediates Two-Step Bacterial Clearance in the Human Spleen","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-23 09:09:15","doi":"10.21203/rs.3.rs-7463569/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d566dc6e-eb0c-4137-bf0d-67c6f63474b1","owner":[],"postedDate":"September 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":55124908,"name":"Health sciences/Pathogenesis/Infection"},{"id":55124909,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-04-13T13:02:54+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-23 09:09:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7463569","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7463569","identity":"rs-7463569","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}