Number Of Splenic Macrophages Research Articles

M1 cholinergic signaling in the brain modulates cytokine levels and splenic cell sub-phenotypes following cecal ligation and puncture.

The contribution of the central nervous system to sepsis pathobiology is incompletely understood. In previous studies, administration of endotoxin to mice decreased activity of the vagus anti-inflammatory reflex. Treatment with the centrally-acting M1 muscarinic acetylcholine (ACh) receptor (M1AChR) attenuated this endotoxin-mediated change. We hypothesize that decreased M1AChR-mediated activity contributes to inflammation following cecal ligation and puncture (CLP), a mouse model of sepsis. In male C57Bl/6 mice, we quantified basal forebrain cholinergic activity (immunostaining), hippocampal neuronal activity, serum cytokine/chemokine levels (ELISA) and splenic cell subtypes (flow cytometry) at baseline, following CLP and following CLP in mice also treated with the M1AChR agonist xanomeline. At 48h. post-CLP, activity in basal forebrain cells expressing choline acetyltransferase (ChAT) was half of that observed at baseline. Lower activity was also noted in the hippocampus, which contains projections from ChAT-expressing basal forebrain neurons. Serum levels of TNFα, IL-1β, MIP-1α, IL-6, KC and G-CSF were higher post-CLP than at baseline. Post-CLP numbers of splenic macrophages and inflammatory monocytes, TNFα+ and ILβ+ neutrophils and ILβ+ monocytes were higher than baseline while numbers of central Dendritic Cells (cDCs), CD4+ and CD8+ T cells were lower. When, following CLP, mice were treated with xanomeline activity in basal forebrain ChAT-expressing neurons and in the hippocampus was significantly higher than in untreated animals. Post-CLP serum concentrations of TNFα, IL-1β, and MIP-1α, but not of IL-6, KC and G-CSF, were significantly lower in xanomeline-treated mice than in untreated mice. Post-CLP numbers of splenic neutrophils, macrophages, inflammatory monocytes and TNFα+ neutrophils also were lower in xanomeline-treated mice than in untreated animals. Percentages of IL-1β+ neutrophils, IL-1β+ monocytes, cDCs, CD4+ T cells and CD8+ T cells were similar in xanomeline-treated and untreated post-CLP mice. Our findings indicate that M1AChR-mediated responses modulate CLP-induced alterations in serum levels of some, but not all, cytokines/chemokines and affected splenic immune response phenotypes.

Repeated social defeat stress leads to immunometabolic shifts in innate immune cells of the spleen

Psychosocial stress has been shown to prime peripheral innate immune cells, which take on hyper-inflammatory phenotypes and are implicated in depressive-like behavior in mouse models. However, the impact of stress on cellular metabolic states that are thought to fuel inflammatory phenotypes in immune cells are unknown. Using single cell RNA-sequencing, we investigated mRNA enrichment of immunometabolic pathways in innate immune cells of the spleen in mice subjected to repeated social defeat stress (RSDS) or no stress (NS). RSDS mice displayed a significant increase in the number of splenic macrophages and granulocytes (p < 0.05) compared to NS littermates. RSDS-upregulated genes in macrophages, monocytes, and granulocytes significantly enriched immunometabolic pathways thought to play a role in myeloid-driven inflammation (glycolysis, HIF-1 signaling, MTORC1 signaling) as well as pathways related to oxidative phosphorylation (OXPHOS) and oxidative stress (p < 0.05 and FDR<0.1). These results suggest that the metabolic enhancement reflected by upregulation of glycolytic and OXPHOS pathways may be important for cellular proliferation of splenic macrophages and granulocytes following repeated stress exposure. A better understanding of these intracellular metabolic mechanisms may ultimately help develop novel strategies to reverse the impact of stress and associated peripheral immune changes on the brain and behavior.

Repeated dose treatment of Tinospora cordifolia polysaccharide rich extract activates splenic antigen presenting cells with no associated organ toxicity

Tinospora cordifolia is a medically valuable plant widely used in Ayurveda and has been attributed with immunomodulatory properties. We compared the immunomodulation of repeated doses of the purified polysaccharide G1-4A and polysaccharide-rich extract (PRE) of T. cordifolia in BALB/c mice. Since both had comparable effects, we further compared the repeated dose effects of 50 and 500 mg/kg b.w PRE per dose with particular emphasis on histological changes in different organs, splenic cell death and DNA damage and immunomodulation. In mice treated with both concentrations of PRE, no gross changes were observed in any organ by histopathological analyses along with normal biochemical parameters similar to control. There was no increase in splenic lymphocyte cell death or DNA damage. A significant increase in the number of splenic macrophages with no increase in phagocytosis was observed. Increased expression of phenotypic maturation markers was observed in splenic dendritic cells and not in bone marrow-derived dendritic cells of PRE-treated mice. Increased activation of B cells in response to mitogen with no change in such a response by T cells was also observed. These results from repeated dose experiments indicate that PRE treatment has no associated organ toxicity and activates antigen-presenting cells and thus can be used instead of G1-4A as a promising immunotherapeutic adjuvant.

Effects of low dose radiation on immune cells subsets and cytokines in mice.

Whole-body exposure to low-dose radiation due to diagnostic imaging procedures, occupational hazards and radiation accidents is a source of concern. In this study, we analyzed the effects of single and long-term low-dose irradiation on the immune system. Male Balb/c mice received a single whole-body dose of irradiation (0.01, 0.05, 0.2, 0.5 or 1Gy). For long-term irradiation, mice were irradiated 10 times (total dose of 0.2, 0.5 or 1Gy) over a period of 6weeks. Two days after single or long-term irradiation, the numbers of splenic macrophages, natural killer cells and dendritic cells were reduced, and the spleen organ coefficient was decreased. At 2Days after long-term low-dose irradiation, the number of white blood cells in the peripheral blood of the mice decreased. Between 7 and 14Days after long-term low-dose irradiation, the number of immune cells in the thymus and spleen began to increase and then stabilized. Th1/Th2 cytokines and reactive oxygen species-related proteins first decreased and then increased to a plateau. Our results show a significant difference in the effects of single and long-term low-dose irradiation on the immune system.

Receptor tyrosine kinase MerTK suppresses an allogenic type I IFN response to promote transplant tolerance.

Recipient infusion of donor apoptotic cells is an emerging strategy for inducing robust transplantation tolerance. Daily clearance of billions of self-apoptotic cells relies on homeostatic engagement of phagocytic receptors, in particular, receptors of the tyrosine kinase family TAM (Tyro3, Axl, and MerTK), to maintain self-tolerance. However, an outstanding question is if allogeneic apoptotic cells trigger the same receptor system for inducing allogeneic tolerance. Here, we employed allogeneic apoptotic splenocytes and discovered that the efferocytic receptor MerTK on recipient phagocytes is a critical mediator for transplantation tolerance induced by this strategy. Our findings indicate that the tolerogenic properties of allogeneic apoptotic splenocytes require MerTK transmission of intracellular signaling to suppress the production of inflammatory cytokine interferon α (IFN-α). We further demonstrate that MerTK is crucial for subsequent expansion of myeloid-derived suppressor cells and for promoting their immunomodulatory function, including maintaining graft-infiltrating CD4+ CD25+ Foxp3+ regulatory T cells. Consequently, recipient MerTK deficiency resulted in failure of tolerance by donor apoptotic cells, and this failure could be effectively rescued by IFN-α receptor blockade. These findings underscore the importance of the efferocytic receptor MerTK in mediating transplantation tolerance by donor apoptotic cells and implicate MerTK agonism as a promising target for promoting transplantation tolerance.

Paquinimod prevents development of diabetes in the non-obese diabetic (NOD) mouse.

Quinoline-3-carboxamides (Q compounds) are immunomodulatory compounds that have shown efficacy both in autoimmune disease and cancer. We have in here investigated the impact of one such compound, paquinimod, on the development of diabetes in the NOD mouse model for type I diabetes (T1D). In cohorts of NOD mice treated with paquinimod between weeks 10 to 20 of age and followed up until 40 weeks of age, we observed dose-dependent reduction in incidence of disease as well as delayed onset of disease. Further, in contrast to untreated controls, the majority of NOD mice treated from 15 weeks of age did not develop diabetes at 30 weeks of age. Importantly, these mice displayed significantly less insulitis, which correlated with selectively reduced number of splenic macrophages and splenic Ly6Chi inflammatory monocytes at end point as compared to untreated controls. Collectively, these results demonstrate that paquinimod treatment can significantly inhibit progression of insulitis to T1D in the NOD mouse. We propose that the effect of paquinimod on disease progression may be related to the reduced number of these myeloid cell populations. Our finding also indicates that this compound could be a candidate for clinical development towards diabetes therapy in humans.

Praziquantel Targets M1 Macrophages and Ameliorates Splenomegaly in Chronic Schistosomiasis.

Splenomegaly is a common feature of many infectious diseases, including schistosomiasis japonica. However, the immunopathogenesis and the treatment of splenomegaly due to schistosomiasis have been largely neglected. Praziquantel (PZQ), a classical schistosomicide, has been demonstrated by us and others to have antifibrotic and anti-inflammatory activities against schistosomiasis. In this study, we investigated the effect of PZQ on alleviating the splenomegaly caused by Schistosoma japonicum infection in mice. The results showed that the number of macrophages, especially the number of M1 macrophages, was significantly increased in the enlarged spleens of infected mice (P < 0.001). After PZQ treatment for 4 weeks, the number of splenic macrophages, especially the number of M1 macrophages, was significantly reduced (P < 0.001) by the way of apoptosis, and another schistosomicide, mefloquine, had no effect either on the splenomegaly or on reducing the number of macrophages. Furthermore, by using the murine macrophage line RAW 264.7, we found that PZQ could inhibit the formation of the NLRP3 inflammasome and attenuate phagocytic activity in M1 macrophages. Thus, our studies suggest that PZQ plays a powerful role in ameliorating the splenomegaly caused by S. japonicum infection, which presents a new strategy for the therapy of splenomegaly resulting from other pathological conditions.

The absence of TNF permits myeloid Arginase 1 expression in experimental L. monocytogenes infection

During an immune response inflammatory macrophages with their wide variety of effector mechanisms including the expression of inducible nitric oxide synthase play an important part in the defense against invading pathogens. The inflammatory phenotype requires the presence of TNF which suppresses alternative activation. In the bacterial Listeria monocytogenes infection model inflammatory macrophages are crucial for protection. After infection, TNF-deficient hosts have a similar number of splenic macrophages but die rapidly. A more detailed analysis of these cells showed that while inducible nitric oxide synthase is expressed at a comparable level TNF-deficient macrophages show an increased expression of Arginase 1.

Deficiency in Interleukin-1 receptor-associated kinase (IRAK) 4 activity attenuates manifestations of murine Lupus

Abstract IL-1R-associated kinase (IRAK) 4 mediates host defense against infections. As an active kinase, IRAK4 elicits full spectra of MyD88-dependent responses, while kinase-inactive IRAK4 induces a subset of cytokines and negative regulators whose expression is not regulated by mRNA stability. IRAK4 kinase activity is critical for resistance against Streptococcus pneumonia, but its role in autoimmunity is incompletely understood. In this study, we determined the role of IRAK4 kinase activity in murine lupus. Lupus development in BXSB male mice expressing the Y chromosome autoimmunity accelerator (Yaa) increased basal and Toll-like receptor (TLR) 4/7-induced phosphorylation of IRAK1, mitogen-activated protein kinases, p65 nuclear factor-κB (NF-κB), enhanced TNF-α and CCL5 gene expression in splenic macrophages, but decreased levels of Toll-interacting protein (Tollip) and IRAK-M, without affecting IRAK4 or IRAK1 expression. Mice harboring kinase-inactive IRAK4 on the lupus-prone Yaa background had reduced glomerulonephritis, splenomegaly, serum anti-nuclear antibodies, decreased numbers of splenic macrophages, total and TNF-a+ dendritic cells, activated T- and B-lymphocytes, and lower TNF-α expression in macrophages compared to lupus-prone mice with functional IRAK4. Furthermore, mice expressing kinase-inactive IRAK4 had decreased proteinuria and increased survival in the chemically-induced (pristine) lupus model. Thus, IRAK4 kinase activity significantly contributes to murine lupus and could represent a new target for therapeutic interventions.

Deficiency in IRAK4 activity attenuates manifestations of murine Lupus.

Interleukin-1 receptor-associated kinase (IRAK) 4 mediates host defense against infections. As an active kinase, IRAK4 elicits full spectra of myeloid differentiation primary response protein (MyD) 88-dependent responses, while kinase-inactive IRAK4 induces a subset of cytokines and negative regulators whose expression is not regulated by mRNA stability. IRAK4 kinase activity is critical for resistance against Streptococcus pneumoniae, but its involvement in autoimmunity is incompletely understood. In this study, we determined the role of IRAK4 kinase activity in murine lupus. Lupus development in BXSB mice expressing the Y chromosome autoimmunity accelerator (Yaa) increased basal and Toll-like receptor (TLR) 4/7-induced phosphorylation of mitogen-activated protein kinases, p65 nuclear factor-κB (NF-κB), enhanced tumor necrosis factor (TNF)-α and C-C motif chemokine ligand (CCL) 5 gene expression in splenic macrophages, but decreased levels of Toll-interacting protein and IRAK-M, without affecting IRAK4 or IRAK1 expression. Mice harboring kinase-inactive IRAK4 on the lupus-prone Yaa background manifested blunted TLR signaling in macrophages and reduced glomerulonephritis, splenomegaly, serum anti-nuclear antibodies, numbers of splenic macrophages, total and TNF-α+ dendritic cells, activated T- and B-lymphocytes, and lower TNF-α expression in macrophages compared with lupus-prone mice with functional IRAK4. Thus, IRAK4 kinase activity contributes to murine lupus and could represent a new therapeutic target.

Dexamethasone Regulates Macrophage and Cd4+Cd25+ Cell Numbers in the Chicken Spleen

Dexamethasone (DEX) is a corticoid hormone that is experimentally used to mimic the effects of increased levels of endogenous corticosterone observed during the stress response. Currently, stress is considered one of the major predisposing factors for diseases in the poultry industry. The aim of this study was to analyze the effects of DEX and/or of a 20-fold coccidial vaccine dose on leukocyte phenotypes in the spleen and cecal tonsils of chickens. Twenty specific-pathogen-free (SPF) Leghorn chickens were divided into four groups: a non-treated group (NT), a DEX-treated group (Dex), a vaccinated group (V) and a DEX-treated+vaccinated group (Dex+V). On experimental day (ED) 42, each bird in the vaccinated groups received a anti-coccidial vaccine. DEX was injected in the birds of the Dex and Dex+V groups (0.9 mg/kg) onED42 and ED45. The immunophenotyping was performed by flow cytometry analysis of splenocytes and cecal tonsils cells onED48. DEX treatment per se was unable to change CD4+CD8+, CD4+CD8+ and CD4-CD8+ populations with TCRgd or CD28 in the spleen, or macrophages and T lymphocytes in the cecal tonsils. V group birds presented higher numbers of splenic macrophages compared with those measured in the Dex+V group. The number of CD4+CD25+ cells in the spleen of birds of the V group was higher than those measured in the other experimental groups. Our data suggest that CD4+CD25+ cells and macrophages might be influenced by DEX treatment in spleen, but not in the cecal tonsils of chickens inoculated with Eimeria.

Osteoclasts Are Required for Hematopoietic Stem and Progenitor Cell Mobilization but Not for Stress Erythropoiesis in Plasmodium chabaudi adami Murine Malaria.

The anemia and inflammation concurrent with blood stage malaria trigger stress haematopoiesis and erythropoiesis. The activity of osteoclasts seems required for the mobilization of hematopoietic stem and progenitor cells (HSPC) from the bone marrow to the periphery. Knowing that BALB/c mice with acute Plasmodium chabaudi adami malaria have profound alterations in bone remodelling cells, we evaluated the extent to which osteoclasts influence their hematopoietic response to infection. For this, mice were treated with osteoclast inhibiting hormone calcitonin prior to parasite inoculation, and infection as well as hematological parameters was studied. In agreement with osteoclast-dependent HSPC mobilization, administration of calcitonin led to milder splenomegaly, reduced numbers of HSPC in the spleen, and their retention in the bone marrow. Although C-terminal telopeptide (CTX) levels, indicative of bone resorption, were lower in calcitonin-treated infected mice, they remained comparable in naive and control infected mice. Calcitonin-treated infected mice conveniently responded to anemia but generated less numbers of splenic macrophages and suffered from exacerbated infection; interestingly, calcitonin also decreased the number of macrophages generated in vitro. Globally, our results indicate that although osteoclast-dependent HSC mobilization from bone marrow to spleen is triggered in murine blood stage malaria, this activity is not essential for stress erythropoiesis.

Targeting splenic hematopoietic stem cells in cardiovascular disease

Inflammation plays a crucial role in cardiovascular disease. For example, inflammatory monocytes, after being recruited to atherosclerotic plaques, secrete proteinases that erode the fibrous cap. This may lead to plaque rupture and subsequent myocardial infarction (MI). Patients with acute myocardial infarction have elevated levels of blood leukocytes [1]. Peripheral blood leukocyte count after MI correlates with in-hospital mortality, underscoring role of inflammatory cells in post-MI myocardial remodeling. The spleen provides steady flow of leukocytes to the infarct and atherosclerotic plaques. Since monocyte turnover at the sites of inflammation is very high [2], hematopoietic stem and progenitor cells (HSPC) differentiate into myeloid cells to keep up with the increased demand. MI drives splenic HSPC into the cell cycle [3] and induces their lineage differentiation. Subsequent imaging studies [4, 5] suggested splenocyte proliferation after acute myocardial infarction in patients. Although splenic myelopoiesis exaggerates inflammation in cardiovascular disease, the components of splenic hematopoietic niche are not well understood. Since macrophages play a major role in bone marrow HSC maintenance, we investigated if macrophages regulate hematopoiesis in the spleen [6]. Toward this end, we developed lipidoid nanoparticles containing siRNA against CD115, the receptor for MCS-F, which is involved in macrophage differentiation, proliferation and survival. CD115 knockdown significantly reduced splenic macrophage numbers in mice injected with the Toll like receptor ligand lipopolysaccharide. Concomitantly, splenic HSC, HSPC and granulocyte macrophage progenitor numbers were diminished after knocking down CD115. Levels of VCAM-1, an HSC retention factor, were reduced in the spleen; however, HSC proliferation and apoptosis were unaltered, indicating impaired retention of splenic HSC after CD115 knockdown. Reduced splenic retention was in line with elevated HSPC levels in the blood after siCD115 treatment. Additionally, we found similar diminished splenic HSC numbers after depletion of splenic macrophages in CD169-iDTR mice with diphtheria toxin, bolstering the hypothesis that macrophages maintain splenic HSC. CD115 knockdown curtailed myeloid cell supply to the infarct in mice on day 4 after coronary ligation. Furthermore, the treatment also abated inflammation in atherosclerotic lesions in ApoE−/− mice fed with high fat diet. This reduction of inflammation was likely a consequence of mitigated extramedullary myelopoiesis due to impaired HSC maintenance after CD115 knockdown. In agreement with reduced inflammation, the treatment reduced plaque size and necrotic core area, and thickened fibrous cap. These are features of stable atherosclerotic plaques. Since VCAM-1 was the only HSC retention factor that was downregulated in splenocytes after CD115 knockdown, we investigated which hematopoietic cells express VCAM-1. Out of all hematopoietic cells tested, only macrophages expressed VCMA-1 at high levels. An adoptive transfer experiment revealed that splenic HSPC reside in close proximity with VCAM-1+ macrophages. In fact, more than 90% of HSPC reside within 10 μm distance from this macrophage subset. These data indicate that VCAM-1+ macrophages maintain splenic HSC. To investigate this further, we formulated siRNA against VCAM-1 in lipidoid nanoparticles which enrich in macrophages but not endothelial cells. VCAM-1 knockdown in macrophages significantly reduced myelopoiesis and HSC numbers in the spleen. Of note, the treatment did not change HSC numbers in the bone marrow, indicating involvement of other cell types and redundant mechanisms of retention for bone marrow HSC maintenance. Further, macrophage-specific VCAM-1 knockdown reduced inflammation in atherosclerosis and improved characteristics of stable atherosclerotic plaques. In summary, the study suggests that VCAM-1+ macrophages are important components of splenic HSC niche. In absence of VCAM-1 or splenic macrophages, HSC depart from the spleen, resulting in decreased myelopoiesis after MI and in mice with atherosclerosis. We previously showed that MI-induced splenic HSPC proliferation is stem cell factor-dependent [3]. Endothelial cells are major source of stem cell factor, which maintain HSC in the bone marrow [7]. However, it is not known if SCF secreted by splenic endothelial cells after MI triggers myelopoiesis. The bone marrow HSC niche is a complex microenvironment and orchestrated by different cells such as nestin+ mesenchymal stem cell, endothelial cells and regulatory T cells. It remains to be investigated which splenic cells besides macrophages maintain HSC.

12. GM-CSF But Not M-CSF is Critical to the Therapeutic Mechanism of Action and Efficacy of Pulmonary Macrophage Transplantation (PMT) Therapy of Hereditary Pulmonary Alveolar Proteinosis (hPAP)

Rationale: Hereditary pulmonary alveolar proteinosis (hPAP) is a disorder caused by recessive mutations in the GM-CSF receptor α or β gene (CSF2RA or CSF2RB), which is characterized by surfactant accumulation and hypoxemic respiratory failure, and for which no pharmacologic therapy exits. Using a murine hPAP model (Csf2rb–/–) phenotypically identical to children with hPAP, we recently reported a new gene/cell transplantation therapy approach – pulmonary macrophage transplantation (PMT) (Nature. 2014, 514: 450), with spontaneous engraftment – without preparative myeloablation, extraordinary therapeutic efficacy, a durable treatment effect, and no safety signal. Interestingly, both GM-CSF and M-CSF are elevated in the lungs of hPAP and provide a possible explanation for the remarkable survival advantage observed for transplanted macrophages compared to endogenous cells. The present study was undertaken to determine the potential relative contribution of these factors to the therapeutic mechanism of action of PMT therapy in preclinical studies in Csf2rb–/– mice.Methods: Csf2rb–/– mice received a single PMT ‘treatment’ – intrapulmonary instillation of wild type (WT) or (as a control) Csfr2b–/– bone marrow derived macrophages (BMDM) (2×106 per mouse) in the absence or presence of a specific M-CSF receptor kinase inhibitor (GW2580) or vehicle. Two months later, therapeutic efficacy was evaluated by measuring both disease severity (bronchoalveolar lavage (BAL) turbidity, BAL surfactant protein D (SP-D) levels) and pulmonary biomarkers of hPAP (BAL GM-CSF, M-CSF levels).Results: We first demonstrated the activity of GW2580 in vivo by measuring splenic macrophage numbers in Csf2rb–/– mice, which were reduced in GW2580-treated compared to untreated mice (2.8±0.2% vs 6.5±0.5%, n=13, 10 /group; p 0.05; all comparisons).Conclusions: Results demonstrate that GM-CSF but not M-CSF contributes to the efficacy of PMT therapy that restores surfactant homeostasis in mice with hPAP.Funding: ATS, CCHMC, NIH UL1T77 (TS); NIH R01HL085453, R21HL106134, R01HL118342 (BCT)

Immunomodulatory activity of orphan drug Elmiron® in female B6C3F1/N mice

Interstitial cystitis (IC) is a chronic disorder characterized by bladder discomfort and urinary urgency in the absence of identifiable infection. Despite the expanding use in IC treatment and other chronic conditions, the effects of Elmiron® treatment on immune system remain unknown. Therefore, female B6C3F1/N mice were orally administered Elmiron® daily for 28-days at doses of 63, 125, 250, 500 or 1000mg/kg to evaluate its immunomodulatory effects. Mice treated with Elmiron® had a significant increase in absolute numbers of splenic macrophages (63, 500 and 1000mg/kg) and natural killer (NK) cells (250 and 1000mg/kg). Elmiron® treatment did not affect the humoral immune response or T cell proliferative response. However, innate immune responses such as phagocytosis by liver macrophages (1000mg/kg) and NK cell activity were enhanced (500 and 1000mg/kg). Further analysis using a disease resistance model showed that Elmiron®-treated mice demonstrated significantly increased anti-tumor activity against B16F10 melanoma cells at the 500 and 1000mg/kg doses. Collectively, we conclude that Elmiron® administration stimulates the immune system, increasing numbers of specific cell populations and enhancing macrophage phagocytosis and NK cell activity in female B6C3F1/N mice. This augmentation may have largely contributed to the reduced number of B16F10 melanoma tumors.

Trypanosomiasis-Induced Th17-Like Immune Responses in Carp

BackgroundIn mammalian vertebrates, the cytokine interleukin (IL)-12 consists of a heterodimer between p35 and p40 subunits whereas interleukin-23 is formed by a heterodimer between p19 and p40 subunits. During an immune response, the balance between IL-12 and IL-23 can depend on the nature of the pathogen associated molecular pattern (PAMP) recognized by, for example TLR2, leading to a preferential production of IL-23. IL-23 production promotes a Th17-mediated immune response characterized by the production of IL-17A/F and several chemokines, important for neutrophil recruitment and activation. For the cold blooded vertebrate common carp, only the IL-12 subunits have been described so far.Methodology/Principal FindingsCommon carp is the natural host of two protozoan parasites: Trypanoplasma borreli and Trypanosoma carassii. We found that these parasites negatively affect p35 and p40a gene expression in carp. Transfection studies of HEK293 and carp macrophages show that T. carassii-derived PAMPs are agonists of carp TLR2, promoting p19 and p40c gene expression. The two protozoan parasites induce different immune responses as assessed by gene expression and histological studies. During T. carassii infections, in particular, we observed a propensity to induce p19 and p40c gene expression, suggestive of the formation of IL-23. Infections with T. borreli and T. carassii lead to an increase of IFN-γ2 gene expression whereas IL-17A/F2 gene expression was only observed during T. carasssii infections. The moderate increase in the number of splenic macrophages during T. borreli infection contrasts the marked increase in the number of splenic neutrophilic granulocytes during T. carassii infection, along with an increased gene expression of metalloproteinase-9 and chemokines.Conclusion/SignificanceThis is the first study that provides evidence for a Th17-like immune response in fish in response to infection with a protozoan parasite.

Macrophages as novel target cells for erythropoietin

Our original demonstration of immunomodulatory effects of erythropoietin in multiple myeloma led us to the search for the cells in the immune system that are direct targets for erythropoietin. The finding that lymphocytes do not express erythropoietin receptors led to the hypothesis that other cells act as direct targets and thus mediate the effects of erythropoietin. The finding that erythropoietin has effects on dendritic cells thus led to the question of whether macrophages act as target cells for erythropoietin. The effects of erythropoietin on macrophages were investigated both in-vivo and in-vitro. The in-vivo studies were performed on splenic macrophages and inflammatory peritoneal macrophages, comparing recombinant human erythropoietin-treated and untreated mice, as well as transgenic mice over-expressing human erythropoietin (tg6) and their control wild-type counterparts. The in-vitro effects of erythropoietin on macrophage surface markers and function were investigated in murine bone marrow-derived macrophages treated with recombinant human erythropoietin. Erythropoietin was found to have effects on macrophages in both the in-vivo and in-vitro experiments. In-vivo treatment led to increased numbers of splenic macrophages, and of the splenic macrophages expressing CD11b, CD80 and major histocompatibility complex class II. The peritoneal inflammatory macrophages obtained from erythropoietin-treated mice displayed increased expression of F4/80, CD11b, CD80 and major histocompatibility complex class II, and augmented phagocytic activity. The macrophages derived in-vitro from bone marrow cells expressed erythropoietin receptor transcripts, and in-vitro stimulation with erythropoietin activated multiple signaling pathways, including signal transducer and activator of transcription (STAT)1 and 5, mitogen-activated protein kinase, phosphatidylinositol 3-kinase and nuclear factor kappa B. In-vitro erythropoietin treatment of these cells up-regulated their surface expression of CD11b, F4/80 and CD80, enhanced their phagocytic activity and nitric oxide secretion, and also led to augmented interleukin 12 secretion and decreased interleukin 10 secretion in response to lipopolysaccharide. Our results show that macrophages are direct targets of erythropoietin and that erythropoietin treatment enhances the pro-inflammatory activity and function of these cells. These findings point to a multifunctional role of erythropoietin and its potential clinical applications as an immunomodulating agent.

Oral administration of a soluble 1–3, 1–6 β-glucan during prophylactic survivin peptide vaccination diminishes growth of a B cell lymphoma in mice

β-glucans are biological response modifiers with activatory effects on macrophages, dendritic cells (DC), granulocytes and NK cells. In this study, we investigated the effect of a soluble yeast-derived β-(1–3), (1–6)- d-glucan on prophylactic peptide vaccination against the B cell lymphoma A20 in syngeneic Balb/c mice. We found that repeated immunizations with two MHC class-I restricted peptides derived from the tumor antigen survivin combined with oral co-administration of β-glucan could significantly diminish intradermal tumor growth, whereas peptide vaccination alone failed to control tumor growth. β-glucan as single agent induced only a weak but non-significant growth inhibitory effect. To determine whether the tumor inhibitory effect of the combined treatment was associated with the induction of a tumor-specific immune response we quantified splenic DC and macrophages, analyzed the maturation of DC and measured the frequency of peptide-specific CD8 + and CD4 + T cells. Treated mice showed significantly increased numbers of splenic macrophages and mature DC compared to untreated tumor-bearing mice. After restimulation with both peptides in vitro elevated levels of interferon (IFN)-γ-secreting CD8 + T cells were found in two of four tested mice following treatment and one of four mice showed a strong increase of interleukin (IL)-4-secreting CD4 + T cells. Our data reveal a beneficial effect of β-(1–3), (1–6)- d-glucan in tumor growth inhibition by tumor-specific peptide vaccination which may rely on a function of the polymeric sugar as immunological adjuvant.

Dietary Fish Oil Increases the Number of Splenic Macrophages Secreting TNF-α and IL-10 But Decreases the Secretion of These Cytokines by Splenic T Cells from Mice ,2

Dietary fish oil has immunomodulatory effects that are partly mediated by its effects on cytokine secretion. In this paper, we examine whether dietary fish oil has different effects on cytokine secretion by T cells and macrophages. Female BalbC mice were fed diets supplemented with 18% fish oil + 2% corn oil or 20% corn oil. Concanavalin A (ConA)- and LPS-induced TNF-α and IL-10 secretion by splenocytes was examined using ELISA. Dietary fish oil decreased ConA induced-, but increased LPS-induced, TNF-α and IL-10 secretion by total murine splenocytes. Dietary fish oil increased the number of splenocytes secreting TNF-α and IL-10, following stimulation with LPS, by 123 and 38%, respectively, but did not affect cytokine secretion by each cell, as determined using enzyme-linked immunospot. Spleens from mice fed the fish oil diet had over 2-fold higher proportion of macrophages with high expression of CD11b than spleens from mice fed the corn oil diet. In addition, fish oil increased the proportion of total and CD11b+ splenocytes that expressed the LPS receptor complex molecules, CD14 and toll-like receptor (TLR)4/myeloid differentiation factor-2 (MD-2), by 85 and 28%, respectively. The increased proportion of macrophages expressing the LPS receptor complex molecules, CD14 and TLR4/MD-2, in spleens from mice fed the fish oil diet may explain the increased number of cells that secreted the cytokines after LPS stimulation.

IL-10-deficiency unmasks unique immune system defects and reveals differential regulation of organ-specific autoimmunity in non-obese diabetic mice

Interleukin (IL)-10 is a potent anti-inflammatory cytokine and ablation of IL-10 exacerbates Th1-type autoimmune diseases. Even though type 1 diabetes (T1D) in NOD mice is believed to be Th1-mediated, the incidence and severity of T1D is unaltered in IL-10-deficient NOD mice raised under pathogen-free conditions. We describe for the first time, the outcome of IL-10 deficiency on islet and other organ-specific autoimmunity in NOD mice raised in a conventional facility. IL-10-deficient NOD mice under such conditions were protected from spontaneous as well as cyclophosphamide-induced diabetes, but were susceptible to diabetes induced by adoptive transfer of splenocytes from spontaneously diabetic NOD mice. Whereas the incidence of rectal prolapse was very high in this NOD.IL-10 −/− mouse colony, IL-10-deficient C57Bl/6 mice raised under similar conditions seldom developed rectal prolapse. While injection of complete Freund’s adjuvant (CFA) significantly reduced insulitis, it did not ameliorate colitis in IL-10-deficient NOD mice indicating differential regulation of organ-specific autoimmunity by CFA. Phenotypic characterization of IL-10 −/− mice revealed a significant increase in splenic macrophage numbers in NOD but not on the B6 background. This was accompanied by a heightened systemic inflammatory cytokine response and mortality following in vivo challenge with a toll-like receptor 9 agonist, CpG-containing DNA.