Innervation of nociceptor neurons in the spleen promotes germinal center responses and humoral immunity.

Peripheral sensory neurons widely innervate various tissues to continuously monitor and respond to environmental stimuli. Whether peripheral sensory neurons innervate the spleen and modulate splenic immune response remains poorly defined. Here, we demonstrate that nociceptive sensory nerve fibers extensively innervate the spleen along blood vessels and reach B cell zones. The spleen-innervating nociceptors predominantly originate from left T8-T13 dorsal root ganglia (DRGs), promoting the splenic germinal center (GC) response and humoral immunity. Nociceptors can be activated by antigen-induced accumulation of splenic prostaglandin E2 (PGE2) and then release calcitonin gene-related peptide (CGRP), which further promotes the splenic GC response at the early stage. Mechanistically, CGRP directly acts on B cells through its receptor CALCRL-RAMP1 via the cyclic AMP (cAMP) signaling pathway. Activating nociceptors by ingesting capsaicin enhances the splenic GC response and anti-influenza immunity. Collectively, our study establishes a specific DRG-spleen sensory neural connection that promotes humoral immunity, suggesting a promising approach for improving host defense by targeting the nociceptive nervous system.

Chemokines and Chemokine Receptors in Lymphoid Tissue Dynamics.

The continuous migration of immune cells between lymphoid and nonlymphoid organs is a key feature of the immune system, facilitating the distribution of effector cells within nearly all compartments of the body. Furthermore, reaching their correct position within primary, secondary, or tertiary lymphoid organs is a prerequisite to ensure immune cells' unimpaired differentiation, maturation, and selection, as well as their activation or functional silencing. The superfamilies of chemokines and chemokine receptors are of major importance in guiding immune cells to and within lymphoid and nonlymphoid tissues. In this review we focus on the role of the chemokine system in the migration dynamics of immune cells within lymphoid organs at the steady state and on how these dynamics are affected by infectious and inflammatory processes.

Physiological and Genetic Adaptations to Diving in Sea Nomads.

Understanding the physiology and genetics of human hypoxia tolerance has important medical implications, but this phenomenon has thus far only been investigated in high-altitude human populations. Another system, yet to be explored, is humans who engage in breath-hold diving. The indigenous Bajau people ("Sea Nomads") of Southeast Asia live a subsistence lifestyle based on breath-hold diving and are renowned for their extraordinary breath-holding abilities. However, it is unknown whether this has a genetic basis. Using a comparative genomic study, we show that natural selection on genetic variants in the PDE10A gene have increased spleen size in the Bajau, providing them with a larger reservoir of oxygenated red blood cells. We also find evidence of strong selection specific to the Bajau on BDKRB2, a gene affecting the human diving reflex. Thus, the Bajau, and possibly other diving populations, provide a new opportunity to study human adaptation to hypoxia tolerance. VIDEO ABSTRACT.

Emergence and Evolution of Secondary Lymphoid Organs.

For effective adaptive immunity to foreign antigens (Ag), secondary lymphoid organs (SLO) provide the confined environment in which Ag-restricted lymphocytes, with very low precursor frequencies, interact with Ag on Ag-presenting cells (APC). The spleen is the primordial SLO, arising in conjunction with adaptive immunity in early jawed vertebrates. The spleen, especially the spleen's lymphoid compartment, the white pulp (WP), has undergone numerous modifications over evolutionary time. We describe the progressive advancement of splenic WP complexity, which evolved in parallel with the increasing functionality of adaptive immunity. The Ag-presenting function of follicular dendritic cells (FDC) also likely emerged at the inception of adaptive immunity, and we propose that a single type of hematopoietically derived APC displayed Ag to both T and B cells. A dedicated FDC, derived from a vascular precursor, is a recent evolutionary innovation that likely permitted the robust affinity maturation found in mammals.

Reticular Fibroblasts Expressing the Transcription Factor WT1 Define a Stromal Niche that Maintains and Replenishes Splenic Red Pulp Macrophages.

Located within red pulp cords, splenic red pulp macrophages (RPMs) are constantly exposed to the blood flow, clearing senescent red blood cells (RBCs) and recycling iron from hemoglobin. Here, we studied the mechanisms underlying RPM homeostasis, focusing on the involvement of stromal cells as these cells perform anchoring and nurturing macrophage niche functions in lymph nodes and liver. Microscopy revealed that RPMs are embedded in a reticular meshwork of red pulp fibroblasts characterized by the expression of the transcription factor Wilms' Tumor 1 (WT1) and colony stimulating factor 1 (CSF1). Conditional deletion of Csf1 in WT1+ red pulp fibroblasts, but not white pulp fibroblasts, drastically altered the RPM network without altering circulating CSF1 levels. Upon RPM depletion, red pulp fibroblasts transiently produced the monocyte chemoattractants CCL2 and CCL7, thereby contributing to the replenishment of the RPM network. Thus, red pulp fibroblasts anchor and nurture RPM, a function likely conserved in humans.

Visualization of T Cell Migration in the Spleen Reveals a Network of Perivascular Pathways that Guide Entry into T Zones.

Lymphocyte homeostasis and immune surveillance require that T and B cells continuously recirculate between secondary lymphoid organs. Here, we used intravital microscopy to define lymphocyte trafficking routes within the spleen, an environment of open blood circulation and shear forces unlike other lymphoid organs. Upon release from arterioles into the red pulp sinuses, T cells latched onto perivascular stromal cells in a manner that was independent of the chemokine receptor CCR7 but sensitive to Gi protein-coupled receptor inhibitors. This latching sheltered T cells from blood flow and enabled unidirectional migration to the bridging channels and then to T zones, entry into which required CCR7. Inflammatory responses modified the chemotactic cues along the perivascular homing paths, leading to rapid block of entry. Our findings reveal a role for vascular structures in lymphocyte recirculation through the spleen, indicating the existence of separate entry and exit routes and that of a checkpoint located at the gate to the T zone.

IL-10-Dependent Crosstalk between Murine Marginal Zone B Cells, Macrophages, and CD8α+ Dendritic Cells Promotes Listeria monocytogenes Infection.

Type 1 CD8α+ conventional dendritic cells (cDC1s) are required for CD8+ T cell priming but, paradoxically, promote splenic Listeria monocytogenes infection. Using mice with impaired cDC2 function, we ruled out a role for cDC2s in this process and instead discovered an interleukin-10 (IL-10)-dependent cellular crosstalk in the marginal zone (MZ) that promoted bacterial infection. Mice lacking the guanine nucleotide exchange factor DOCK8 or CD19 lost IL-10-producing MZ B cells and were resistant to Listeria. IL-10 increased intracellular Listeria in cDC1s indirectly by reducing inducible nitric oxide synthase expression early after infection and increasing intracellular Listeria in MZ metallophilic macrophages (MMMs). These MMMs trans-infected cDC1s, which, in turn, transported Listeria into the white pulp to prime CD8+ T cells. However, this also facilitated bacterial expansion. Therefore, IL-10-mediated crosstalk between B cells, macrophages, and cDC1s in the MZ promotes both Listeria infection and CD8+ T cell activation.

Exposure to Bacterial CpG DNA Protects from Airway Allergic Inflammation by Expanding Regulatory Lung Interstitial Macrophages.

Living in a microbe-rich environment reduces the risk of developing asthma. Exposure of humans or mice to unmethylated CpG DNA (CpG) from bacteria reproduces these protective effects, suggesting a major contribution of CpG to microbe-induced asthma resistance. However, how CpG confers protection remains elusive. We found that exposure to CpG expanded regulatory lung interstitial macrophages (IMs) from monocytes infiltrating the lung or mobilized from the spleen. Trafficking of IM precursors to the lung was independent of CCR2, a chemokine receptor required for monocyte mobilization from the bone marrow. Using a mouse model of allergic airway inflammation, we found that adoptive transfer of IMs isolated from CpG-treated mice recapitulated the protective effects of CpG when administered before allergen sensitization or challenge. IM-mediated protection was dependent on IL-10, given that Il10-/- CpG-induced IMs lacked regulatory effects. Thus, the expansion of regulatory lung IMs upon exposure to CpG might underlie the reduced risk of asthma development associated with a microbe-rich environment.

Biomimetic noncationic lipid nanoparticles for mRNA delivery.

Although the tremendous progress has been made for mRNA delivery based on classical cationic carriers, the excess cationic charge density of lipids was necessary to compress mRNA through electrostatic interaction, and with it comes inevitably adverse events including the highly inflammatory and cytotoxic effects. How to develop the disruptive technologies to overcome cationic nature of lipids remains a major challenge for safe and efficient mRNA delivery. Here, we prepared noncationic thiourea lipids nanoparticles (NC-TNP) to compress mRNA by strong hydrogen bonds interaction between thiourea groups of NC-TNP and the phosphate groups of mRNA, abandoning the hidebound and traditional electrostatic force to construct mRNA-cationic lipids formulation. NC-TNP was a delivery system for mRNA with simple, convenient, and repeatable preparation technology and showed negligible inflammatory and cytotoxicity side effects. Furthermore, we found that NC-TNP could escape the recycling pathway to inhibit the egress of internalized nanoparticles from the intracellular compartment to the extracellular milieu which was a common fact in mRNA-LNP (lipid nanoparticles) formulation. Therefore, NC-TNP-encapsulated mRNA showed higher gene transfection efficiency in vitro and in vivo than mRNA-LNP formulation. Unexpectedly, NC-TNP showed spleen targeting delivery ability with higher accumulation ratio (spleen/liver), compared with traditional LNP. Spleen-targeting NC-TNP with mRNA exhibited high mRNA-encoded antigen expression in spleen and elicited robust immune responses. Overall, our work establishes a proof of concept for the construction of a noncationic system for mRNA delivery with good inflammatory safety profiles, high gene transfection efficiency, and spleen-targeting delivery to induce permanent and robust humoral and cell-mediated immunity for disease treatments.

Physical mechanisms of red blood cell splenic filtration.

The splenic interendothelial slits fulfill the essential function of continuously filtering red blood cells (RBCs) from the bloodstream to eliminate abnormal and aged cells. To date, the process by which 8 [Formula: see text]m RBCs pass through 0.3 [Formula: see text]m-wide slits remains enigmatic. Does the slit caliber increase during RBC passage as sometimes suggested? Here, we elucidated the mechanisms that govern the RBC retention or passage dynamics in slits by combining multiscale modeling, live imaging, and microfluidic experiments on an original device with submicron-wide physiologically calibrated slits. We observed that healthy RBCs pass through 0.28 [Formula: see text]m-wide rigid slits at 37 °C. To achieve this feat, they must meet two requirements. Geometrically, their surface area-to-volume ratio must be compatible with a shape in two tether-connected equal spheres. Mechanically, the cells with a low surface area-to-volume ratio (28% of RBCs in a 0.4 [Formula: see text]m-wide slit) must locally unfold their spectrin cytoskeleton inside the slit. In contrast, activation of the mechanosensitive PIEZO1 channel is not required. The RBC transit time through the slits follows a [Formula: see text]1 and [Formula: see text]3 power law with in-slit pressure drop and slip width, respectively. This law is similar to that of a Newtonian fluid in a two-dimensional Poiseuille flow, showing that the dynamics of RBCs is controlled by their cytoplasmic viscosity. Altogether, our results show that filtration through submicron-wide slits is possible without further slit opening. Furthermore, our approach addresses the critical need for in vitro evaluation of splenic clearance of diseased or engineered RBCs for transfusion and drug delivery.

Chemo- and mechanosensing by dendritic cells facilitate antigen surveillance in the spleen.

Spleen dendritic cells (DC) are critical for initiation of adaptive immune responses against blood-borne invaders. Key to DC function is their positioning at sites of pathogen entry, and their abilities to selectively capture foreign antigens and promptly engage T cells. Focusing on conventional DC2 (cDC2), we discuss the contribution of chemoattractant receptors (EBI2 or GPR183, S1PR1, and CCR7) and integrins to cDC2 positioning and function. We give particular attention to a newly identified role in cDC2 for adhesion G-protein coupled receptor E5 (Adgre5 or CD97) and its ligand CD55, detailing how this mechanosensing system contributes to splenic cDC2 positioning and homeostasis. Additional roles of CD97 in the immune system are reviewed. The ability of cDC2 to be activated by circulating missing self-CD47 cells and to integrate multiple red blood cell (RBC)-derived inputs is discussed. Finally, we describe the process of activated cDC2 migration to engage and prime helper T cells. Throughout the review, we consider the insights into cDC function in the spleen that have emerged from imaging studies.

Analysis of MRI-derived spleen iron in the UK Biobank identifies genetic variation linked to iron homeostasis and hemolysis.

The spleen plays a key role in iron homeostasis. It is the largest filter of the blood and performs iron reuptake from old or damaged erythrocytes. Despite this role, spleen iron concentration has not been measured in a large, population-based cohort. In this study, we quantify spleen iron in 41,764 participants of the UK Biobank by using magnetic resonance imaging and provide a reference range for spleen iron in an unselected population. Through genome-wide association study, we identify associations between spleen iron and regulatory variation at two hereditary spherocytosis genes, ANK1 and SPTA1. Spherocytosis-causing coding mutations in these genes are associated with lower reticulocyte volume and increased reticulocyte percentage, while these common alleles are associated with increased expression of ANK1 and SPTA1 in blood and with larger reticulocyte volume and reduced reticulocyte percentage. As genetic modifiers, these common alleles may explain mild spherocytosis phenotypes that have been observed clinically. Our genetic study also identifies a signal that co-localizes with a splicing quantitative trait locus for MS4A7, and we show this gene is abundantly expressed in the spleen and in macrophages. The combination of deep learning and efficient image processing enables non-invasive measurement of spleen iron and, in turn, characterization of genetic factors related to the lytic phase of the erythrocyte life cycle and iron reuptake in the spleen.

Lessons of Vascular Specialization From Secondary Lymphoid Organ Lymphatic Endothelial Cells.

Secondary lymphoid organs, such as lymph nodes, harbor highly specialized and compartmentalized niches. These niches are optimized to facilitate the encounter of naive lymphocytes with antigens and antigen-presenting cells, enabling optimal generation of adaptive immune responses. Lymphatic vessels of lymphoid organs are uniquely specialized to perform a staggering variety of tasks. These include antigen presentation, directing the trafficking of immune cells but also modulating immune cell activation and providing factors for their survival. Recent studies have provided insights into the molecular basis of such specialization, opening avenues for better understanding the mechanisms of immune-vascular interactions and their applications. Such knowledge is essential for designing better treatments for human diseases given the central role of the immune system in infection, aging, tissue regeneration and repair. In addition, principles established in studies of lymphoid organ lymphatic vessel functions and organization may be applied to guide our understanding of specialization of vascular beds in other organs.

Splenectomy ameliorates liver cirrhosis by restoring the gut microbiota balance.

BACKGROUND: Dysbiosis of gut microbiota is frequent in liver cirrhosis (LC) patients, and splenectomy (SP) has been reported to improve LC. Herein, we report the effects of SP on gut microbiota, especially on Veillonella parvula, a Gram-negative coccus of the gastrointestinal tract, in LC mice, and the underlying mechanism. METHODS: LC mice models were induced by tail vein injection of concanavalin A (ConA), followed by SP. 16 s rRNA sequencing was conducted to analyze the effects of ConA induction and SP on mouse gut microbiota and the gene expression affected by gut microbiota. LC mice receiving SP were gavaged with Veillonella parvula. Likewise, hepatic stellate cells (HSC) and hepatocytes (HC) were induced with conditioned medium (CM) of Veillonella parvula. RESULTS: SP alleviated LC in mice by restoring gut barrier function and maintaining gut microbiota balance, with Veillonella as the key genus. The Veillonella parvula gavage on LC mice reversed the ameliorative effect of SP. The CM of Veillonella parvula promoted the activation of HSC and the release of IL-6, IL-1ß, and TNF-α. Also, the CM of Veillonella parvula induced HC pyroptosis and the release of ALT and AST. Veillonella parvula represented an imbalance in the gut microbiota, thus enhancing gut-derived endotoxins in the liver with the main target being Tlr4/Nlrp3. Inhibition of Tlr4 blocked Veillonella parvula-induced HC damage, HSC activation, and subsequent LC progression. CONCLUSION: SP-mediated gut microbiota regulation ameliorates ConA-related LC progression by inhibiting Tlr4/Nlrp3 in the liver.

CD169+ macrophages in lymph node and spleen critically depend on dual RANK and LTbetaR signaling.

CD169+ macrophages reside in lymph node (LN) and spleen and play an important role in the immune defense against pathogens. As resident macrophages, they are responsive to environmental cues to shape their tissue-specific identity. We have previously shown that LN CD169+ macrophages require RANKL for formation of their niche and their differentiation. Here, we demonstrate that they are also dependent on direct lymphotoxin beta (LTß) receptor (R) signaling. In the absence or the reduced expression of either RANK or LTßR, their differentiation is perturbed, generating myeloid cells expressing SIGN-R1 in LNs. Conditions of combined haploinsufficiencies of RANK and LTßR revealed that both receptors contribute equally to LN CD169+ macrophage differentiation. In the spleen, the Cd169-directed ablation of either receptor results in a selective loss of marginal metallophilic macrophages (MMMs). Using a RANKL reporter mouse, we identify splenic marginal zone stromal cells as a source of RANKL and demonstrate that it participates in MMM differentiation. The loss of MMMs had no effect on the splenic B cell compartments but compromised viral capture and the expansion of virus-specific CD8+ T cells. Taken together, the data provide evidence that CD169+ macrophage differentiation in LN and spleen requires dual signals from LTßR and RANK with implications for the immune response.

Vago-splenic signal transduction of cardioprotection in humans.

BACKGROUND AND AIMS: The spleen serves as an important relay organ that releases cardioprotective factor(s) upon vagal activation during remote ischaemic conditioning (RIC) in rats and pigs. The translation of these findings to humans was attempted. METHODS: Remote ischaemic conditioning or electrical auricular tragus stimulation (ATS) were performed in 10 healthy young volunteers, 10 volunteers with splenectomy, and 20 matched controls. Venous blood samples were taken before and after RIC/ATS or placebo, and a plasma dialysate was infused into isolated perfused rat hearts subjected to global ischaemia/reperfusion. RESULTS: Neither left nor right RIC or ATS altered heart rate and heart rate variability in the study cohorts. With the plasma dialysate prepared before RIC or ATS, respectively, infarct size (% ventricular mass) in the recipient rat heart was 36 ± 6% (left RIC), 34 ± 3% (right RIC) or 31 ± 5% (left ATS), 35 ± 5% (right ATS), and decreased with the plasma dialysate from healthy volunteers after RIC or ATS to 20 ± 4% (left RIC), 23 ± 6% (right RIC) or to 19 ± 4% (left ATS), 26 ± 9% (right ATS); infarct size was still reduced with plasma dialysate 4 days after ATS and 9 days after RIC. In a subgroup of six healthy volunteers, such infarct size reduction was abrogated by intravenous atropine. Infarct size reduction by RIC or ATS was also abrogated in 10 volunteers with splenectomy, but not in their 20 matched controls. CONCLUSIONS: In humans, vagal innervation and the spleen as a relay organ are decisive for the cardioprotective signal transduction of RIC and ATS.

Macrophage-fibroblast circuits in the spleen.

Macrophages are an integral part of all organs in the body, where they contribute to immune surveillance, protection, and tissue-specific homeostatic functions. This is facilitated by so-called niches composed of macrophages and their surrounding stroma. These niches structurally anchor macrophages and provide them with survival factors and tissue-specific signals that imprint their functional identity. In turn, macrophages ensure appropriate functioning of the niches they reside in. Macrophages thus form reciprocal, mutually beneficial circuits with their cellular niches. In this review, we explore how this concept applies to the spleen, a large secondary lymphoid organ whose primary functions are to filter the blood and regulate immunity. We first outline the splenic micro-anatomy, the different populations of splenic fibroblasts and macrophages and their respective contribution to protection of and key physiological processes occurring in the spleen. We then discuss firmly established and potential cellular circuits formed by splenic macrophages and fibroblasts, with an emphasis on the molecular cues underlying their crosstalk and their relevance to splenic functionality. Lastly, we conclude by considering how these macrophage-fibroblast circuits might be impaired by aging, and how understanding these changes might help identify novel therapeutic avenues with the potential of restoring splenic functions in the elderly.

Exercise reduces pro-inflammatory lipids and preserves resolution mediators that calibrate macrophage-centric immune metabolism in spleen and heart following obesogenic diet in aging mice.

The study investigated the role of volunteer exercise and an obesogenic diet (OBD) in mice, focusing on the splenocardiac axis and inflammation-resolution signaling. Male C57BL/6J mice (2 months old) were assigned to control (CON) or OBD groups for ten months, then randomized into sedentary (Sed) or exercise (Exe) groups for two weeks. Leukocytes, heart function, structure, and spleen tissue examined for inflammation-resolution mediators and macrophage-centric gene transcripts. After two weeks of volunteer exercise, cardiac function shows limited changes, but structural changes were notable in the heart and spleen. Exercise induced cardiac nuclear hyperplasia observed in both CON and OBD groups. OBD-Sed mice showed splenic changes and increased neutrophils, whereas increased neutrophils were noted in the CON post exercise. OBD-Sed increased pro-inflammatory lipid mediators in the heart, reduced by exercise in OBD-Exe, while CON-Exe preserved resolution mediators. Chronic OBD-Sed depletes long chain fatty acids (DHA/EPA) in the heart and spleen, while exercise independently regulates lipid metabolism genes in both organs, affecting macrophage-centric lipid and lipoprotein pathways. Chronic obesity amplified cardiac inflammation, countered by exercise that lowered pro-inflammatory bioactive lipid mediators in the heart. OBD sustained inflammation in the heart and spleen, while exercise conserved resolution mediators in CON mice. In summary, these findings emphasize the interplay of diet with exercise and highlight the intricate connection of diet, exercise, inflammation-resolution signaling in splenocardiac axis and immune health.

Phosphorylation of spleen tyrosine kinase at Y346 negatively regulates ITAM-mediated signaling and function in platelets.

Spleen tyrosine kinase (Syk) is expressed in a variety of hemopoietic cells. Upon phosphorylation of the platelet immunoreceptor-based activation motif of the glycoprotein VI (GPVI)/Fc receptor gamma chain collagen receptor, both the tyrosine phosphorylation and activity of Syk are increased leading to downstream signaling events. Although it has been established that the activity of Syk is regulated by tyrosine phosphorylation, the specific roles of individual phosphorylation sites remain to be elucidated. We observed that Syk Y346 in mouse platelets was still phosphorylated when GPVI-induced Syk activity was inhibited. We then generated Syk Y346F mice and analyzed the effect this mutation exerts on platelet responses. Syk Y346F mice bred normally, and their blood cell count was unaltered. We did observe potentiation of GPVI-induced platelet aggregation and ATP secretion as well as increased phosphorylation of other tyrosines on Syk in the Syk Y346F mouse platelets when compared to WT littermates. This phenotype was specific for GPVI-dependent activation, since it was not seen when AYPGKF, a PAR4 agonist, or 2-MeSADP, a purinergic receptor agonist, was used to activate platelets. Despite a clear effect of Syk Y346F on GPVI-mediated signaling and cellular responses, there was no effect of this mutation on hemostasis as measured by tail-bleeding times, although the time to thrombus formation determined using the ferric chloride injury model was reduced. Thus, our results indicate a significant effect of Syk Y346F on platelet activation and responses in vitro and reveal its complex nature manifesting itself by the diversified translation of platelet activation into physiological responses.

Adenosine 2A receptor antagonists promote lymphocyte proliferation in sepsis by inhibiting Treg expression of PD-L1 in spleen.

The spleen is essential for lymphocyte proliferation, which is associated to sepsis prognosis. Adenosine 2A receptor (A2AR) blocking promotes lymphocyte proliferation in sepsis, however the mechanism is uncertain. Our sepsis cecum ligation perforation model showed that blocking A2AR increased survival and CD4+ cell numbers in a spleen-dependent mechanism. The sequencing of the transcriptome of the spleen indicated alterations in the expression of genes involved in the control of lymphocyte proliferation by inhibiting A2AR, including a reduction in the expression of PD-L1. Flow cytometry analysis of PD-L1 expression intensity in splenic cell subpopulations revealed that the Treg cell subpopulation was the strongest PD-L1-expressing cell population, and Treg PD-L1 expression decreased after blocking A2AR. In vitro activation of A2AR was able to upregulate PD-L1 expression of Treg and boost Treg capacity to limit lymphocyte proliferation, while blockage of PD-L1 partly reduced A2AR-activated Treg's ability to inhibit lymphocyte proliferation. In addition, blocking CREB phosphorylation significantly inhibited A2AR-induced PD-L1 expression. According to the findings of our research, inhibiting A2AR improves the prognosis of sepsis by lowering the level of PD-L1 expression by Treg in the spleen and reducing the inhibition of lymphocyte proliferation.