Marginal Zone Macrophages Research Articles

Systemic lupus erythematosus favors the generation of IL-17 producing double negative T cells

Mature double negative (DN) T cells are a population of αβ T cells that lack CD4 and CD8 coreceptors and contribute to systemic lupus erythematosus (SLE). The splenic marginal zone macrophages (MZMs) are important for establishing immune tolerance, and loss of their number or function contributes to the progression of SLE. Here we show that loss of MZMs impairs the tolerogenic clearance of apoptotic cells and alters the serum cytokine profile, which in turn provokes the generation of DN T cells from self-reactive CD8+ T cells. Increased Ki67 expression, narrowed TCR V-beta repertoire usage and diluted T-cell receptor excision circles confirm that DN T cells from lupus-prone mice and patients with SLE undergo clonal proliferation and expansion in a self-antigen dependent manner, which supports the shared mechanisms for their generation. Collectively, our results provide a link between the loss of MZMs and the expansion of DN T cells, and indicate possible strategies to prevent the development of SLE.

Marginal zone SIGN-R1+ macrophages are essential for the maturation of germinal center B cells in the spleen

The mechanisms that regulate germinal center (GC) B cell responses in the spleen are not fully understood. Here we use a combination of pharmacologic and genetic approaches to delete SIGN-R1+ marginal zone (MZ) macrophages and reveal their specific contribution to the regulation of humoral immunity in the spleen. We find that while SIGN-R1+ macrophages were not essential for initial activation of B cells, they were required for maturation of the response and development of GC B cells. These defects could be corrected when follicular helper T (Tfh) cells were induced before macrophage ablation or when Tfh responses were enhanced. Moreover, we show that in the absence of SIGN-R1+ macrophages, DCIR2+ dendritic cells (DCs), which play a key role in priming Tfh responses, were unable to cluster to the interfollicular regions of the spleen and were instead displaced to the MZ. Restoring SIGN-R1+ macrophages to the spleen corrected positioning of DCIR2+ DCs and rescued the GC B cell response. Our study reveals a previously unappreciated role for SIGN-R1+ macrophages in regulation of the GC reaction and highlights the functional specification of macrophage subsets in the MZ compartment.

The spatiotemporal dynamics of type I IFN response determine viral infection outcome

Abstract Type I interferons (IFN-I) play a critical role in shaping the antiviral immune response early after an infection. However, the dynamics by which different immune cell subsets regulate the IFN-I response during the early stages of acute and chronic infections is not completely understood. Here we used the Lymphocytic Choriomeningitis Virus (LCMV)-infection mouse model system to characterize the dynamics of the IFN-I response in acute and chronic infections. Time-resolved spleen-transcriptomes revealed that during an acute infection, IFN-I showed two peaks of expression at days 2 and 5 post-infection. In contrast, in chronically infected mice a single peak of IFN-I genes appeared at day 1. Further analysis revealed that IFN-I genes in acute infection were co-expressed with genes related to inflammatory macrophages, suggesting an important role of these cells determining infection fate. Indeed, we identified metallophilic macrophages as an important source of Ifnb only during acute infection and demonstrated that the subsequent IFN-I receptor (IFNAR) signaling is necessary to induce pro-inflammatory macrophages. In contrast, during chronic infection, early depletion of marginal zone macrophages results in a lack of IFN-I production and the inflammatory response is not induced. Importantly, blockage of the second peak of IFN-I response by IFNAR blockage during an acute infection also resulted in exhaustion of virus-specific CD8 T cells and prevention of lymphoid tissue fibrosis. Further studies are ongoing to decipher the regulatory mechanisms underlying the characterized events, thus revealing universal concepts related to infection fate decisions that are also relevant for persistent human infections such as HIV or HCV.

Dominant role of splenic marginal zone lipid rafts in the classical complement pathway against S. pneumoniae

Lipid rafts (LRs) play crucial roles in complex physiological processes, modulating innate and acquired immune responses to pathogens. The transmembrane C-type lectins human dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) and its mouse homolog SIGN-R1 are distributed in LRs and expressed on splenic marginal zone (MZ) macrophages. The DC-SIGN-C1q or SIGN-R1-C1q complex could mediate the immunoglobulin (Ig)-independent classical complement pathway against Streptococcus pneumoniae. Precise roles of LRs during this complement pathway are unknown. Here we show that LRs are indispensable for accelerating the DC-SIGN- or SIGN-R1-mediated classical complement pathway against S. pneumoniae, thus facilitating rapid clearance of the pathogen. The trimolecular complex of SIGN-R1-C1q-C4 was exclusively enriched in LRs of splenic MZ macrophages and their localization was essential for activating C3 catabolism and enhancing pneumococcal clearance, which were abolished in SIGN-R1-knockout mice. However, DC-SIGN replacement on splenic MZ macrophage’s LRs of SIGN-R1-depleted mice reversed these defects. Disruption of LRs dramatically reduced pneumococcal uptake and decomposition. Additionally, DC- SIGN, C1q, C4, and C3 were obviously distributed in splenic LRs of cadavers. Therefore, LRs on splenic SIGN-R1+ or DC-SIGN+ macrophages could provide spatially confined and optimal bidirectional platforms, not only for usual intracellular events, for example recognition and phagocytosis of pathogens, but also an unusual extracellular event such as the complement system. These findings improve our understanding of the orchestrated roles of the spleen, unraveling a new innate immune system initiated from splenic MZ LRs, and yielding answers to several long-standing problems, including the need to understand the profound role of LRs in innate immunity, the need to identify how such a small portion of splenic SIGN-R1+ macrophages (<0.05% of splenic macrophages) effectively resist S. pneumoniae, and the need to understand how LRs can promote the protective function of DC-SIGN against S. pneumoniae in the human spleen.

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+ Tcell 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 insteaddiscovered 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+ Tcells. However, thisalso facilitated bacterial expansion. Therefore, IL-10-mediated crosstalk between B cells, macrophages, and cDC1s in the MZ promotes both Listeria infection and CD8+ Tcell activation.

S904 THE FUNCTIONAL RESPONSE OF IRON‐RECYCLING AND INFECTION CONTROL‐SPECIALIZED MACROPHAGES IS IMPAIRED BY TRANSFUSIONS AND AMELIORATED BY IRON CHELATION

Background:Iron overload is common in anemic patients who require regular transfusions. Transfusional iron overload is associated with enhanced susceptibility to infections, a common cause of morbidity/mortality in these patient populations.Aims:Here we studied how iron overload, besides supporting bacterial growth, may alter macrophage response to infectious stimuli.Methods:We applied a mouse model of repeated allogeneic RBC transfusions and investigated the impact of transfusions on the phenotypic plasticity of different macrophage populations as well as their responses to infectious cues.Results:Using this model, we have recently shown that repeated transfusions promote macrophage switching to a M2‐like anti‐inflammatory phenotype, hallmarked by reduced inflammatory marker expression and inflammatory cytokine release. Consistently, in transfused animals, macrophages develop a blunted M1‐like inflammatory phenotype in response to LPS, leading to reduced circulating inflammatory cytokines. These observations indicate that transfusion‐induced erythrophagocytosis dampens the inflammatory response to infectious cues by impairing macrophage functions. Transfusion‐blunted M1 polarization occurs in both iron‐recycling hepatic Kupffer cells and splenic red pulp macrophages (RPM). We now have extended our analysis to additional macrophage sub‐populations of the spleen which play a critical role in the phagocytosis of blood‐borne pathogens and control of innate/adaptive immune response to infections and found that also these cells, marginal zone (MZM) and marginal metallophilic (MMM) macrophages, are affected by RBC transfusions and show a suppression of M1 markers. Since MZMs and MMMs are not directly involved in erythrophagocytosis, we speculate that the blunted M1 polarization of iron‐recycling macrophages attenuates the pro‐inflammatory activation of other macrophages, including MZMs and MMMs, with potential impact on the susceptibility and response to infections. We now have administered an iron chelator, deferasirox, during the transfusion period, and show that iron chelation therapy partially restores the phenotype of Kupffer cells and RPMs by rescuing marker expression and shifting the transfusion‐triggered M2‐like macrophage population to a M1‐like (both with and without LPS). Interestingly, chelation therapy significantly improves the expression of M1 markers also in MZMs and MMMs, suggesting that chelation would help to control infections by improving the response of different macrophage sub‐populations. Importantly, we have confirmed our findings in a mouse model of MDS receiving repeated transfusions. Combined with marked cytopenias and iron‐boosted microorganism growth, the weak pro‐inflammatory activation of macrophages might contribute to the increased propensity of transfused MDS patients to develop infections.Summary/Conclusion:Our data indicate that transfusions blunt macrophage inflammatory response by affecting macrophage plasticity towards an anti‐inflammatory phenotype with reduced potential to counteract infections. By restoring cell polarization, iron chelation likely improves the functional response of macrophages to invading microorganisms. Transfusion practice might therefore increase the risk of infections not solely through increased iron availability to microorganisms, but also through the impaired activation of a broad plethora of macrophages, involved in iron recycling and infection control. Transfused patients might benefit from chelation by both restricting iron from pathogens and improving macrophage‐induced immune response.

Lipid membranes accelerate amyloid formation in the mouse model of AA amyloidosis

Introduction: AA amyloidosis develops as a result of prolonged inflammation and is characterized by deposits of N-terminal proteolytic fragments of the acute phase reactant serum amyloid A (SAA). Macrophages are usually found adjacent to amyloid, suggesting their involvement in the formation and/or degradation of the amyloid fibrils. Furthermore, accumulating evidence suggests that lipid membranes accelerate the fibrillation of different amyloid proteins.Methods: Using an experimental mouse model of AA amyloidosis, we compared the amyloidogenic effect of liposomes and/or amyloid-enhancing factor (AEF). Inflammation was induced by subcutaneous injection of silver nitrate followed by intravenous injection of liposomes and/or AEF to accelerate amyloid formation.Results: We showed that liposomes accelerate amyloid formation in inflamed mice, but the amyloidogenic effect of liposomes was weaker compared with AEF. Regardless of the induction method, amyloid deposits were mainly found in the marginal zones of the spleen and coincided with the depletion of marginal zone macrophages, while red pulp macrophages and metallophilic marginal zone macrophages proved insensitive to amyloid deposition.Conclusions: We conclude that increased intracellular lipid content facilitates AA amyloid fibril formation and show that the mouse model of AA amyloidosis is a suitable system for further mechanistic studies.

Pleiotropic Impacts of Macrophage and Microglial Deficiency on Development in Rats with Targeted Mutation of the Csf1r Locus.

We have produced Csf1r-deficient rats by homologous recombination in embryonic stem cells. Consistent with the role of Csf1r in macrophage differentiation, there was a loss of peripheral blood monocytes, microglia in the brain, epidermal Langerhans cells, splenic marginal zone macrophages, bone-associated macrophages and osteoclasts, and peritoneal macrophages. Macrophages of splenic red pulp, liver, lung, and gut were less affected. The pleiotropic impacts of the loss of macrophages on development of multiple organ systems in rats were distinct from those reported in mice. Csf1r−/− rats survived well into adulthood with postnatal growth retardation, distinct skeletal and bone marrow abnormalities, infertility, and loss of visceral adipose tissue. Gene expression analysis in spleen revealed selective loss of transcripts associated with the marginal zone and, in brain regions, the loss of known and candidate novel microglia-associated transcripts. Despite the complete absence of microglia, there was little overt phenotype in brain, aside from reduced myelination and increased expression of dopamine receptor-associated transcripts in striatum. The results highlight the redundant and nonredundant functions of CSF1R signaling and of macrophages in development, organogenesis, and homeostasis.

Identification and isolation of splenic tissue-resident macrophage sub-populations by flow cytometry.

Tissue-resident macrophages in the spleen, including red pulp and white pulp macrophages, marginal zone macrophages (MZMs) and marginal zone metallophilic macrophages (MMMs), are highly heterogeneous as a consequence of adaptation to tissue-specific environments. Each macrophage sub-population in the spleen is usually identified based on the localization, morphology and membrane antigen expression by immunohistochemistry. However, their phenotypical and functional characteristics remain incompletely understood due to the difficulty of identification and isolation by flow cytometry. We used a cocktail of three enzymes (Collagenase D, Dispase I and DNase I), rather than traditional mechanical grinding, for isolation of each sub-population, which resulted in significant improvement of isolation of these macrophage sub-populations, particularly MZMs and MMMs, as determined by CD11bhiF4/80medTim4hi and CD11bhiF4/80medTim4med, respectively. This method should be helpful for molecular and functional characterization of each splenic resident macrophage sub-population.

Experimental Stroke Differentially Affects Discrete Subpopulations of Splenic Macrophages.

Changes to the immune system after stroke are complex and can result in both pro-inflammatory and immunosuppressive consequences. Following ischemic stroke, brain resident microglia are activated and circulating monocytes are recruited to the injury site. In contrast, there is a systemic deactivation of monocytes/macrophages that may contribute to immunosuppression and the high incidence of bacterial infection experienced by stroke patients. The manipulation of macrophage subsets may be a useful therapeutic strategy to reduce infection and improve outcome in patients after stroke. Recent research has enhanced our understanding of the heterogeneity of macrophages even within the same tissue. The spleen is the largest natural reservoir of immune cells, many of which are mobilized to the site of injury after ischemic stroke and is notable for the diversity of its functionally distinct macrophage subpopulations associated with specific micro-anatomical locations. Here, we describe the effects of experimental stroke in mice on these distinct splenic macrophage subpopulations. Red pulp (RP) and marginal zone macrophages (MZM) specifically showed increases in density and alterations in micro-anatomical location. These changes were not due to increased recruitment from the bone marrow but may be associated with increases in local proliferation. Genes associated with phagocytosis and proteolytic processing were upregulated in the spleen after stroke with increased expression of the lysosome-associated protein lysosomal-associated membrane proteins specifically increased in RP and MZM subsets. In contrast, MHC class II expression was reduced specifically in these populations. Furthermore, genes associated with macrophage ability to communicate with other immune cells, such as co-stimulatory molecules and inflammatory cytokine production, were also downregulated in the spleen after stroke. These findings suggest that selective splenic macrophage functions could be impaired after stroke and the contribution of macrophages to stroke-associated pathology and infectious complications should be considered at a subset-specific level. Therefore, optimal therapeutic manipulation of macrophages to improve stroke outcome is likely to require selective targeting of functionally and spatially distinct subpopulations.

Early cellular interactions and immune transcriptome profiles in human factor VIII‐exposed hemophilia A mice

Background Developing factor VIII (FVIII) inhibitory antibodies is the most serious complication in hemophilia A treatment, representing a significant health and economic burden. A better understanding of the early events in an immune response leading to this outcome may provide insight into inhibitor development. Objective To identify early mediators of FVIII immunity and to detail immune expression profiles in the spleen and liver. Methods C57Bl/6F8 E16 knockout mice were infused with 5-20μg (2000-8000IUkg-1 ) of recombinant FVIII. Spleens were frozen at various time-points post-infusion and stained for FVIII and cellular markers. Splenic and liver RNA expression analysis was performed 3h post-infusion of 0.6μg (240IUkg-1 ) FVIII by nCounter technology using a 561-gene immunology panel. Results FVIII localization in the spleen did not change over 2.5h. We observed significantly higher co-localization of FVIII with MARCO+ cells compared with SIGLEC1+ and SIGNR1+ in the splenic marginal zone. FVIII exhibited little co-localization with CD11c+ dendritic cells and the macrophage mannose receptor, CD206. Following FVIII infusion, the splenic mRNA profiling identified genes such as Tnfaip6 and Il23r, which are implicated in chemotaxis and a proinflammatory Th17 response, respectively. In contrast, an upregulation of Gfi1 in the liver suggests an anti-inflammatory environment. Conclusions FVIII co-localizes predominantly with marginal zone macrophages (MARCO+ ) in the murine spleen following intravenous infusion. Targeting pathways that are implicated in the early FVIII innate immune response in the spleen may lead to therapeutic interventions to prevent inhibitor formation.

Crucial role of Mer tyrosine kinase in the maintenance of SIGN-R1+ marginal zone macrophages.

Mer Tyrosine Kinase receptor (Mer) is involved in anti-inflammatory efferocytosis. Here we report elevated spontaneous germinal center (Spt-GC) responses in Mer-deficient mice (Mer-/- ) that are associated with the loss of SIGN-R1+ marginal zone macrophages (MZMs). The dissipation of MZMs in Mer-/- mice occurs independently of reduced cellularity or delocalization of marginal zone B cells, sinusoidal cells or of CD169+ metallophillic macrophages. We find that MZM dissipation in Mer-/- mice contributes to apoptotic cell (AC) accumulation in Spt-GCs and dysregulation of the GC checkpoint, allowing an expansion of DNA-reactive B cells in GCs. We further observe that bone marrow derived macrophages from Mer-/- mice produce more TNFα, and are susceptible to cell death upon exposure to ACs compared to WT macrophages. Anti-TNFα Ab treatment of Mer-/- mice is, however, unable to reverse MZM loss, but results in reduced Spt-GC responses, indicating that TNFα promotes Spt-GC responses in Mer-/- mice. Contrary to an anti-TNFα Ab treatment, treatment of Mer-/- mice with a synthetic agonist for the transcription factor LXRα rescues a significant number of MZMs invivo. Our data suggest that Mer-LXRα signaling plays an important role in the differentiation and maintenance of MZMs, which in turn regulate Spt-GC responses and tolerance.

Type I Interferon Signaling to Dendritic Cells Limits Murid Herpesvirus 4 Spread from the Olfactory Epithelium.

Murid herpesvirus 4 (MuHV-4) is a B cell-tropic gammaherpesvirus that can be studied in vivo Despite viral evasion, type I interferons (IFN-I) limit its spread. After MuHV-4 inoculation into footpads, IFN-I protect lymph node subcapsular sinus macrophages (SSM) against productive infection; after peritoneal inoculation, they protect splenic marginal zone macrophages, and they limit MuHV-4 replication in the lungs. While invasive infections can be used to test specific aspects of host colonization, it is also important to understand natural infection. MuHV-4 taken up spontaneously by alert mice enters them via olfactory neurons. We determined how IFN-I act in this context. Blocking IFN-I signaling did not increase neuronal infection but allowed the virus to spread to the adjacent respiratory epithelium. In lymph nodes, a complete IFN-I signaling block increased MuHV-4 lytic infection in SSM and increased the number of dendritic cells (DC) expressing viral green fluorescent protein (GFP) independently of lytic infection. A CD11c+ cell-directed signaling block increased infection of DC only. However, this was sufficient to increase downstream infection, consistent with DC providing the main viral route to B cells. The capacity of IFN-I to limit DC infection indicated that viral IFN-I evasion was only partly effective. Therefore, DC are a possible target for IFN-I-based interventions to reduce host colonization.IMPORTANCE Human gammaherpesviruses infect B cells and cause B cell cancers. Interventions to block virus binding to B cells have not stopped their infection. Therefore, we must identify other control points that are relevant to natural infection. Human infections are difficult to analyze. However, gammaherpesviruses colonize all mammals. A related gammaherpesvirus of mice reaches B cells not directly but via infected dendritic cells. We show that type I interferons, an important general antiviral defense, limit gammaherpesvirus B cell infection by acting on dendritic cells. Therefore, dendritic cell infection is a potential point of interferon-based therapeutic intervention.

Bim suppresses the development of SLE by limiting myeloid inflammatory responses.

The Bcl-2 family is considered the guardian of the mitochondrial apoptotic pathway. We demonstrate that Bim acts as a molecular rheostat by controlling macrophage function not only in lymphoid organs but also in end organs, thereby preventing the break in tolerance. Mice lacking Bim in myeloid cells (LysMCreBimfl/fl) develop a systemic lupus erythematosus (SLE)-like disease that mirrors aged Bim-/- mice, including loss of marginal zone macrophages, splenomegaly, lymphadenopathy, autoantibodies (including anti-DNA IgG), and a type I interferon signature. LysMCreBimfl/fl mice exhibit increased mortality attributed to glomerulonephritis (GN). Moreover, the toll-like receptor signaling adaptor protein TRIF (TIR-domain-containing adapter-inducing interferon-β) is essential for GN, but not systemic autoimmunity in LysMCreBimfl/fl mice. Bim-deleted kidney macrophages exhibit a novel transcriptional lupus signature that is conserved within the gene expression profiles from whole kidney biopsies of patients with SLE. Collectively, these data suggest that the Bim may be a novel therapeutic target in the treatment of SLE.

Receptor Activator of NF-κB Orchestrates Activation of Antiviral Memory CD8 T Cells in the Spleen Marginal Zone

SummaryThe spleen plays an important role in protective immunity to bloodborne pathogens. Macrophages and dendritic cells (DCs) in the spleen marginal zone capture microbial antigens to trigger adaptive immune responses. Marginal zone macrophages (MZMs) can also act as a replicative niche for intracellular pathogens, providing a platform for mounting the immune response. Here, we describe a role for RANK in the coordinated function of antigen-presenting cells in the spleen marginal zone and triggering anti-viral immunity. Targeted deletion of RANK results in the selective loss of CD169+ MZMs, which provide a niche for viral replication, while RANK signaling in DCs promotes the recruitment and activation of anti-viral memory CD8 T cells. These studies reveal a role for the RANKL/RANK signaling axis in the orchestration of protective immune responses in the spleen marginal zone that has important implications for the host response to viral infection and induction of acquired immunity.

Chronic Brucella Infection Induces Selective and Persistent Interferon Gamma-Dependent Alterations of Marginal Zone Macrophages in the Spleen.

The spleen is known as an important filter for blood-borne pathogens that are trapped by specialized macrophages in the marginal zone (MZ): the CD209+ MZ macrophages (MZMs) and the CD169+ marginal metallophilic macrophages (MMMs). Acute systemic infection strongly impacts MZ populations and the location of T and B lymphocytes. This phenomenon has been linked to reduced chemokine secretion by stromal cells. Brucella spp. are the causative agent of brucellosis, a widespread zoonotic disease. Here, we used Brucella melitensis infection as a model to investigate the impact of chronic stealth infection on splenic MZ macrophage populations. During the late phase of Brucella infection, we observed a loss of both MZMs and MMMs, with a durable disappearance of MZMs, leading to a reduction of the ability of the spleen to take up soluble antigens, beads, and unrelated bacteria. This effect appears to be selective as every other lymphoid and myeloid population analyzed increased during infection, which was also observed following Brucella abortus and Brucella suis infection. Comparison of wild-type and deficient mice suggested that MZ macrophage population loss is dependent on interferon gamma (IFN-γ) receptor but independent of T cells or tumor necrosis factor alpha receptor 1 (TNF-αR1) signaling pathways and is not correlated to an alteration of CCL19, CCL21, and CXCL13 chemokine mRNA expression. Our results suggest that MZ macrophage populations are particularly sensitive to persistent low-level IFN-γ-mediated inflammation and that Brucella infection could reduce the ability of the spleen to perform certain MZM- and MMM-dependent tasks, such as antigen delivery to lymphocytes and control of systemic infection.

Abstract 002: Neuropilin1: New Splenic Mechanisms Mediating the Guidance of Neural Cues in Immune Cells Relevant for Hypertension

Investigation of immune mechanisms involved in hypertension (HTN) is one of the most emerging research area in the field. On this note, we found that hypertensive hits activate nervous system in splenic marginal zone (MZ) that represents the boundary between lymphoid white pulp and innate red pulp. The fact that both innate and adaptive immunity contribute to HTN strongly suggests that neuroimmune mechanisms couple myeloid and lymphoid cells in transducing hypertensive hits into increased blood pressure (BP). We previously showed that Placental Growth Factor (PlGF), a VEGF homolog, released in the splenic MZ upon Angiotensin II (AngII), mediates this interaction. Here we dissect the downstream pathway conveying nervous outflow into immune activation. PlGF is expected to act through its major receptor VEGFR1, its only known tyrosine kinase receptor. First we found that it is mainly expressed in splenic innate immune cells and vasculature, both in basal conditions and upon AngII. To test VEGFR1 in HTN, we challenged transgenic mice with defective VEGFR1 signaling (Flt1-TK) with AngII. To our surprise, they raised BP upon 28-days AngII as WT mice did (SBP: Flt1-TK 139±2; WT 138±2; ***p&lt; 0.001), thus suggesting the existence of alternative pathways. PlGF may also act on a co-receptor, Neuropilin1 (Nrp1), known to enhance responses to ligands. In addition, Nrp1 has unique functions in neurons’ guidance during development and in the maturation of dendritic cells from monocytes. We found Nrp1 basally expressed and strongly increased upon AngII, especially in MZ macrophages. To test the hypothesis that PlGF/Nrp1 pathway could mediate a neuroimmune activation of dendritic cells relevant for HTN, we selectively deleted the receptor in myeloid lineage, generating Nrp1 fl;fl ;LysM-Cre transgenic mice. After assessing Nrp1deletion in splenic monocyte/macrophages by FACS, we infused AngII and found a protection from T cells costimulation and egression from the spleen. More important, they were protected from HTN (SBP: Nrp1 fl;fl ;LysM-Cre +/- 140±3; Nrp1 fl;fl ;LysM-Cre -/- 105±2; ***p&lt; 0.001). Our data indicate a crucial role of Nrp1 in HTN, mediating non-canonical PlGF signaling in innate immunity that serves as a guidance of nerves priming adaptive immune responses.

Mer tyrosine kinase (MerTK) deficiency drives the loss of marginal zone macrophages with subsequent accumulation of apoptotic cells and dysregulation of germinal centers

Abstract MerTK is a scavenger receptor involved in anti-inflammatory efferocytosis. We previously reported that MerTK deficiency on a mixed B6 and 129 background (B6-129.MerKO) led to dysregulated germinal center (GC) responses and accumulation of apoptotic cells (ACs), primarily in the GCs. Here we report that the dysregulation of GCs in B6-129.MerKO mice strongly correlates with the loss of marginal zone macrophages (MZMs) starting at two months of age. In addition to MerTK, MZMs also express other AC-scavenger receptors like SIGNR1, MARCO and Tim-4, which are also lost from the splenic MZ of B6-129.MerKO mice. We do not observe a loss or translocation of marginal zone B cells (MZBs) or marginal zone metallophillic macrophages (MMMs) from the marginal zone, indicating the exclusive role of MZMs in deregulated GC responses observed in this model. Preliminary results using splenic and bone marrow derived macrophages show reduced expression of a pro-survival heat shock protein Hsp27 and its phosphorylation through p38 and MAPKAPK-2 in the absence of MerTK. We also observe increased cell death in MerTK deficient macrophages vs. WT macrophages, when challenged with ACs. Our data indicate that reduced Hsp27 expression due to MerTK deficiency may lead to cell death of MZMs. We are further investigating if the increased availability of self-antigens through AC accumulation, due to the loss of MZMs can lead to positive selection of self-reactive B cells in the GCs, causing autoimmunity. Our data indicate that MerTK deficiency and ACs enhance antigen presentation by macrophages, DCs and B cells. In accordance, we observe an increase in activated T cells in MerTK deficient mice, which may promote dysregulation of GC responses observed in this model.

Splenic Ly6Ghigh mature and Ly6Gint immature neutrophils contribute to eradication of S. pneumoniae.

The spleen plays an integral protective role against encapsulated bacterial infections. Our understanding of the associated mechanisms is limited to thymus-independent (TI) antibody production by the marginal zone (MZ) B cells, leaving the contribution of other splenic compartments such as the red pulp (RP) largely unexplored despite asplenic patients succumbing to the infection in the first 24 h, suggesting important antibody-independent mechanisms. In this study, using time-lapse intravital imaging of the spleen, we identify a tropism for Streptococcus pneumoniae in this organ mediated by tissue-resident MZ and RP macrophages and a protective role for two distinct splenic neutrophil populations (Ly6Ghi and Ly6Gintermediate) residing in the splenic RP. Splenic mature neutrophils mediated pneumococcal clearance in the spleen by plucking bacteria off the surface of RP macrophages that caught the majority of bacteria in a complement-dependent manner. This neutrophil phagocytic capacity was further enhanced after TI antibody production. Resident immature neutrophils (Ly6Gintermediate) in the spleen undergo emergency proliferation and mobilization from their splenic niche after pneumococcal stimulation to increase the effector mature neutrophil pool. We demonstrate that splenic neutrophils together with two macrophage populations and MZ B cells regulate systemic S. pneumoniae clearance through complementary mechanisms.

Cutting Edge: Marginal Zone Macrophages Regulate Antigen Transport by B Cells to the Follicle in the Spleen via CD21.

Marginal zone macrophages (MZM) are strategically located in the spleen, lining the marginal sinus where they sense inflammation and capture Ag from the circulation. One of the receptors expressed by MZM is scavenger receptor macrophage receptor with collagenous structure (MARCO), which has affinity for modified self-antigens. In this article, we show that engagement of MARCO on murine macrophages induces extracellular ATP and loss of CD21 and CD62L on marginal zone B cells. Engagement of MARCO also leads to reduction of Ag transport by marginal zone B cells and affects the subsequent immune response. This study highlights a novel function for MZM in regulating Ag transport and activation, and we suggest that MARCO-dependent ATP release regulates this through shedding of CD21 and CD62L. Because systemic lupus erythematosus patients were shown to acquire autoantibodies against MARCO, this highlights a mechanism that could affect a patient's ability to combat infections.