Anti-Leishmania infantum Antibody-Producing Plasma Cells in the Spleen in Canine Visceral Leishmaniasis

The spleen is involved in visceral leishmaniasis immunopathogenesis, and presents alterations in white-pulp microenvironments that are associated with an increased susceptibility to coinfections and patient death. Plasmacytosis in splenic red pulp (RP) is one observed alteration, but the specificity of antibody-secreting cells and the distribution of them has not yet been evaluated. We biotinylated soluble L. infantum membrane antigens (bSLMA) used as probes in modified immunohistochemistry, and detected the presence of anti-L. infantum antibody-secreting cells. Were used spleens from eight dogs from the endemic area for canine visceral leishmaniasis (CanL), and three healthier controls. The spleen sections were cryopreserved, and we performed modified immunohistochemistry. The ratio of plasma cells which were reactive to bSLMA (Anti-Leish-PC) in the spleen RP and periarteriolar lymphatic sheath (PALS) were calculated. Dogs with CanL present hyperglobulinemia and more plasma cells in their RP than the controls. Furthermore, dogs with CanL presented a lower proportion of Anti-Leish-PC in their RP than in PALS. Likewise, dysproteinemia was related to RP and PALS plasmacytosis, and a more severe clinical profile.

The Multiple Facets of Iron Recycling

The production of around 2.5 million red blood cells (RBCs) per second in erythropoiesis is one of the most intense activities in the body. It continuously consumes large amounts of iron, approximately 80% of which is recycled from aged erythrocytes. Therefore, similar to the “making”, the “breaking” of red blood cells is also very rapid and represents one of the key processes in mammalian physiology. Under steady-state conditions, this important task is accomplished by specialized macrophages, mostly liver Kupffer cells (KCs) and splenic red pulp macrophages (RPMs). It relies to a large extent on the engulfment of red blood cells via so-called erythrophagocytosis. Surprisingly, we still understand little about the mechanistic details of the removal and processing of red blood cells by these specialized macrophages. We have only started to uncover the signaling pathways that imprint their identity, control their functions and enable their plasticity. Recent findings also identify other myeloid cell types capable of red blood cell removal and establish reciprocal cross-talk between the intensity of erythrophagocytosis and other cellular activities. Here, we aimed to review the multiple and emerging facets of iron recycling to illustrate how this exciting field of study is currently expanding.

PPARγ is essential for the development of bone marrow erythroblastic island macrophages and splenic red pulp macrophages

Tissue-resident macrophages play a crucial role in maintaining homeostasis. Macrophage progenitors migrate to tissues perinatally, where environmental cues shape their identity and unique functions. Here, we show that the absence of PPARγ affects neonatal development and VCAM-1 expression of splenic iron-recycling red pulp macrophages (RPMs) and bone marrow erythroblastic island macrophages (EIMs). Transcriptome analysis of the few remaining Pparg-deficient RPM-like and EIM-like cells suggests that PPARγ is required for RPM and EIM identity, cell cycling, migration, and localization, but not function in mature RPMs. Notably, Spi-C, another transcription factor implicated in RPM development, was not essential for neonatal expansion of RPMs, even though the transcriptome of Spic-deficient RPMs was strongly affected and indicated a loss of identity. Similarities shared by Pparg- and Spic-deficient RPM-like cells allowed us to identify pathways that rely on both factors. PPARγ and Spi-C collaborate in inducing transcriptional changes, including VCAM-1 and integrin αD expression, which could be required for progenitor retention in the tissue, allowing access to niche-related signals that finalize differentiation.

Memory CD8+ T cells mediate early pathogen-specific protection through localized delivery of chemokines and IFNγ to clusters of inflammatory monocytes

SummaryWhile cognate antigen drives clonal expansion of memory CD8+ T cells to achieve sterilizing immunity in immunized hosts, not much is known on how cognate antigen contributes to early mechanisms of protection before clonal expansion occurs. Herein, using distinct models of immunization, we establish that cognate antigen recognition by CD8+ TM cells on dendritic cells initiates their rapid and coordinated production of a burst of CCL3, CCL4 and XCL1 chemokines under the transcriptional control of IRF4. Using intravital microscopy imaging and in vivo monoclonal antibody labelling, we reveal that memory CD8+ T cells undergo antigen-mediated arrest in splenic red pulp clusters of CCR2+ monocytes where they locally deliver both IFNγ- and chemokine-potentiating microbicidal activities to achieve early protection. Thus, rapid and effective memory CD8+ T cell responses require a complex series of spatially and temporally coordinated stepwise molecular and cellular events that quickly restrict microbial pathogen growth and optimize the local delivery of effector molecules before clonal expansion occurs.

VLA-1 Binding to Collagen IV Controls Effector T Cell Suppression by Myeloid-Derived Suppressor Cells in the Splenic Red Pulp

Myeloid-derived suppressor cells (MDSCs) represent a major population controlling T cell immune responses. However, little is known about their molecular requirements for homing and T cell interaction to mediate suppression. Here, we investigated the functional role of the homing and collagen IV receptor VLA-1 (α1β1-integrin) on in vitro GM-CSF generated murine MDSCs from wild-type (WT) and CD49a/α1-integrin (Itga1−/−) gene-deficient mice. Here, we found that effector (Teff) but not naive (Tn) CD4+ T cells express VLA-1 and monocytes further up-regulated their expression after culture in GM-CSF when they differentiated into the monocytic subset of resting MDSCs (R-MDSCs). Subsequent activation of R-MDSCs by LPS+IFN-γ (A-MDSCs) showed increased in vitro suppressor potential, which was independent of VLA-1. Surprisingly, VLA-1 deficiency did not influence A-MDSC motility or migration on collagen IV in vitro. However, interaction times of Itga1−/− A-MDSCs with Teff were shorter than with WT A-MDSCs on collagen IV but not on fibronectin substrate in vitro. After injection, A-MDSCs homed to the splenic red pulp where they co-localized with Teff and showed immediate suppression already after 6 h as shown by inhibition of T cell proliferation and induction of apoptosis. Injection of A-MDSCs from Itga1−/− mice showed equivalent homing into the spleen but a reduced suppressive effect. Interaction studies of A-MDSCs with Teff in the subcapsular red pulp with intravital two-photon microscopy revealed also here that MDSC motility and migration parameters were not altered by VLA-1 deficiency, but the interaction times with Teff were reduced. Together, our data point to a new role of VLA-1 adhesion to collagen IV as a prerequisite for extended contact times with Teff required for suppression.

Exploring human splenic red pulp vasculature in virtual reality: details of sheathed capillaries and the open capillary network

We reconstructed serial sections of a representative adult human spleen to clarify the unknown arrangement of the splenic microvasculature, such as terminal arterioles, sheathed capillaries, the red pulp capillary network and venules. The resulting 3D model was evaluated in virtual reality (VR). Capillary sheaths often occurred after the second or third branching of a terminal arteriole and covered its capillary side or end branches. The sheaths started directly after the final smooth muscle cells of the arteriole and consisted of cuboidal CD271++ stromal sheath cells surrounded and infiltrated by B lymphocytes and macrophages. Some sheaths covered up to four sequential capillary bifurcations thus forming bizarre elongated structures. Each sheath had a unique form. Apart from symmetric dichotomous branchings inside the sheath, sheathed capillaries also gave off side branches, which crossed the sheath and freely ended at its surface. These side branches are likely to distribute materials from the incoming blood to sheath-associated B lymphocytes and macrophages and thus represent the first location for recognition of blood-borne antigens in the spleen. A few non-sheathed bypasses from terminal arterioles to the red pulp capillary network also exist. Red pulp venules are primarily supplied by sinuses, but they also exhibit a few connections to the capillary network. Thus, the human splenic red pulp harbors a primarily open microcirculation with a very minor closed part.

Multi-Focal Splenic Tumour in a Belgian Patient and a Brief Review of the Literature on Littoral Cell Angioma

We describe the case of a 66-year-old woman with littoral cell angioma (LCA) confirmed by histopathology and immunohistochemistry, to our knowledge the first case in Belgium. LCA is an extremely rare primary vascular tumour of the splenic red pulp, probably originating from littoral cells. If a splenic mass and nodules are incidentally identified on imaging and the patient has no associated signs or symptoms, LCA should be suspected. Histopathology and adjacent techniques are mandatory for definitive diagnosis. Splenectomy followed by adequate follow-up is necessary to exclude underlying pathology.

Towards understanding the cell surface phenotype, metabolic properties and immune functions of resident macrophages of the peritoneal cavity and splenic red pulp using high resolution quantitative proteomics

Background: Resident macrophages (Mϕs) are distributed throughout the body and are important for maintaining tissue homeostasis and for defence against infections. Tissue Mϕs are highly adapted to their microenvironment and thought to mediate tissue-specific functions involving metabolism and immune defence that are not fully elucidated. Methods: We have used high resolution quantitative proteomics to gain insights into the functions of two types of resident tissue Mϕs: peritoneal cavity Mϕs and splenic red pulp Mϕs. The cellular expression levels of many proteins were validated by flow cytometry and were consistently in agreement with the proteomics data. Results: Peritoneal and splenic red pulp macrophages displayed major differences in cell surface phenotype reflecting their adaptation to different tissue microenvironments and tissue-specific functions. Peritoneal Mϕs were shown to be enriched in a number of key enzymes and metabolic pathways normally associated with the liver, such as metabolism of fructose, detoxification, nitrogen homeostasis and the urea cycle. Supporting these observations, we show that peritoneal Mϕs are able to utilise glutamine and glutamate which are rich in peritoneum for urea generation. In comparison, splenic red pulp Mϕs were enriched in proteins important for adaptive immunity such as antigen presenting MHC molecules, in addition to proteins required for erythrocyte homeostasis and iron turnover. We also show that these tissue Mϕs may utilise carbon and nitrogen substrates for different metabolic fates to support distinct tissue-specific roles. Conclusions: This study provides new insights into the functions of tissue Mϕs in immunity and homeostasis. The comprehensive proteomics data sets are a valuable resource for biologists and immunologists.