SIRPα on Mouse B1 Cells Restricts Lymphoid Tissue Migration and Natural Antibody Production.

The inhibitory immunoreceptor SIRPα is expressed on myeloid and neuronal cells and interacts with the broadly expressed CD47. CD47-SIRPα interactions form an innate immune checkpoint and its targeting has shown promising results in cancer patients. Here, we report expression of SIRPα on B1 lymphocytes, a subpopulation of murine B cells responsible for the production of natural antibodies. Mice defective in SIRPα signaling (SIRPαΔCYT mice) displayed an enhanced CD11b/CD18 integrin-dependent B1 cell migration from the peritoneal cavity to the spleen, local B1 cell accumulation, and enhanced circulating natural antibody levels, which was further amplified upon immunization with T-independent type 2 antigen. As natural antibodies are atheroprotective, we investigated the involvement of SIRPα signaling in atherosclerosis development. Bone marrow (SIRPαΔCYT>LDLR-/-) chimaeric mice developed reduced atherosclerosis accompanied by increased natural antibody production. Collectively, our data identify SIRPα as a unique B1 cell inhibitory receptor acting to control B1 cell migration, and imply SIRPα as a potential therapeutic target in atherosclerosis.

Celiac disease associated SNP rs17810546 is located in a gene silencing region.

GWAS studies have identified variant rs 17810546 in a non-coding region on chromosome 3 as a risk factor for several auto-immune diseases, including Celiac Disease. In silico analysis reveals that this variant is located in a transcription regulatory site. By means of reporter constructs we show that this region can override the expression rate of a gene as determined by its native promoter and that this modulation is influenced by the genetic composition of the haplotype which rs17810546 forms with a nearby other variant, rs761008. Secondly, we present data that this genetically imprinted modulation could be involved in Celiac Disease through the IL12A gene which is located 40 Kb downstream of this regulatory region. Based on our findings it is most likely that the IL12A gene does so as part of the cytokine IL-35.

Splenic volume differentiates complicated and non-complicated celiac disease.

BACKGROUND: Studies in small groups of patients indicated that splenic volume (SV) may be decreased in patients with celiac disease (CD), refractory CD (RCD) type II and enteropathy-associated T-cell lymphoma (EATL). OBJECTIVE: The objective of this article is to evaluate SV in a large cohort of uncomplicated CD, RCD II and EATL patients and healthy controls. METHODS: The retrospective cohort consisted of 77 uncomplicated CD (of whom 39 in remission), 29 RCD II, 24 EATL and 12 patients with both RCD II and EATL. The control group included 149 healthy living kidney donors. SV was determined on computed tomography. RESULTS: The median SV in the uncomplicated CD group was significantly larger than in controls (202 cm3 (interquartile range (IQR): 154-275) versus 183 cm3 (IQR: 140-232), p = 0.02). After correction for body surface area, age and gender, the ratio of SV in uncomplicated CD versus controls was 1.28 (95% confidence interval: 1.20-1.36; p < 0.001). The median SV in RCD II patients (118 cm3 (IQR 83-181)) was smaller than the median SV in the control group (p < 0.001). CONCLUSION: This study demonstrates large inter-individual variation in SV. SV is enlarged in uncomplicated CD. The small SV in RCD II may be of clinical relevance considering the immune-compromised status of these patients.

Intestinal epithelial cell transported TLR2 ligand stimulates Ly6C⁺ monocyte differentiation in a G-CSF dependent manner.

Microflora-induced TLR signaling in the intestinal epithelium is essential for a proper intestinal barrier function. Because of the close interactions of this epithelial layer with underlying mononuclear phagocytes, we hypothesized that epithelial TLR signaling may affect the differentiation of myeloid cell populations. In in vitro cultures we observed that colonic epithelial monolayers actively transported TLR2 ligands towards their basolateral side. The transported TLR2 ligands strongly stimulated the development of Ly6C(+) monocytes, while dendritic cell differentiation was inhibited. The TLR2 effect on monocyte and dendritic cell differentiation was mediated by the production of G-CSF. Mice lacking TLR signaling and mice that were treated with antibiotics showed decreased numbers of Ly6C(+) monocytes in bone marrow and spleen, which points to a role for microbial derived TLR-ligands in the homeostasis of Ly6C(+) monocytes. In conclusion, our results indicate that TLR ligands that are transported by intestinal epithelial cells stimulate Ly6C(+) monocyte development and suggest that this process may be involved in the maintenance of systemic Ly6C(+) monocyte numbers.

Dectin-1 deficiency does not affect atherosclerosis development in mice.

OBJECTIVE: Recent data suggest the involvement of dectin-1 in atherosclerosis through regulation of local reactive oxygen species production. The aim of the current study was to assess the effect of dectin-1 deficiency on atherosclerotic plaque development. METHODS: Using immunohistochemistry dectin-1 expression was observed on foamy macrophages in atherosclerotic lesions in mice. Following lethal irradiation LDLR(-/-) mice were reconstituted with bone marrow from either wild type or dectin-1(-/-) mice. After recovery, mice were fed a high fat diet for 9 weeks and atherosclerotic lesions were analyzed. RESULTS AND CONCLUSION: Overall, we found no significant differences in plaque size or severity between the groups. Also no differences were observed in granulocyte or macrophage composition of the plaques or in the ability to produce reactive oxygen species by macrophages from both groups. Dectin-1 is dispensable for the development of atherosclerotic lesions in mice.

Human buccal epithelium acquires microbial hyporesponsiveness at birth, a role for secretory leukocyte protease inhibitor.

OBJECTIVE: Repetitive interaction with microbial stimuli renders epithelial cells (ECs) hyporesponsive to microbial stimulation. Previously, we have reported that buccal ECs from a subset of paediatric patients with Crohn's disease are not hyporesponsive and spontaneously released chemokines. We now aimed to identify kinetics and mechanisms of acquisition of hyporesponsiveness to microbial stimulation using primary human buccal epithelium. DESIGN: Buccal ECs collected directly after birth and in later stages of life were investigated. Chemokine release and regulatory signalling pathways were studied using primary buccal ECs and the buccal EC line TR146. Findings were extended to the intestinal mucosa using murine model systems. RESULTS: Directly after birth, primary human buccal ECs spontaneously produced the chemokine CXCL-8 and were responsive to microbial stimuli. Within the first weeks of life, these ECs attained hyporesponsiveness, associated with inactivation of the NF-κB pathway and upregulation of the novel NF-κB inhibitor SLPI but no other known NF-κB inhibitors. SLPI protein was abundant in the cytoplasm and the nucleus of hyporesponsive buccal ECs. Knock-down of SLPI in TR146-buccal ECs induced loss of hyporesponsiveness with increased NF-κB activation and subsequent chemokine release. This regulatory mechanism extended to the intestine, as colonisation of germfree mice elicited SLPI expression in small intestine and colon. Moreover, SLPI-deficient mice had increased chemokine expression in small intestinal and colonic ECs. CONCLUSIONS: We identify SLPI as a new player in acquisition of microbial hyporesponsiveness by buccal and intestinal epithelium in the first weeks after microbial colonisation.

Antigen targeting reveals splenic CD169+ macrophages as promoters of germinal center B-cell responses.

Ag delivery to specific APCs is an attractive approach in developing strategies for vaccination. CD169(+) macrophages in the marginal zone of the spleen represent a suitable target for delivery of Ag because of their strategic location, which is optimal for the capture of blood-borne Ag and their close proximity to B cells and T cells in the white pulp. Here we show that Ag targeting to CD169(+) macrophages in mice resulted in strong, isotype-switched, high-affinity Ab production and the preferential induction and long-term persistence of Ag-specific GC B cells and follicular Th cells. In agreement with these observations, CD169(+) macrophages retained intact Ag, induced cognate activation of B cells, and increased expression of costimulatory molecules upon activation. In addition, macrophages were required for the production of cytokines that promote B-cell responses. Our results identify CD169(+) macrophages as promoters of high-affinity humoral immune responses and emphasize the value of CD169 as target for Ag delivery to improve vaccine responses.

PKCδ is dispensible for oxLDL uptake and foam cell formation by human and murine macrophages.

AIMS: Uptake of oxidized lipoprotein particles (oxLDL) and foam cell formation by macrophages is one of the first steps in the development of atherosclerosis. Recently, protein kinase C δ (PKCδ) has been implicated as a regulator of oxLDL uptake and foam cell formation via down-regulation of PKCß and scavenger receptors CD36 and SR-A expression. Here, we describe studies in which we have re-evaluated the role of PKCδ in oxLDL uptake and foam cell formation. METHODS AND RESULTS: PKCδ expression was silenced in the human monocytic cell lines and also in primary human monocytes to analyse oxLDL uptake and CD36 expression. Additionally, bone marrow-derived macrophages of PKCδ knockout mice and macrophages cultured from patients with rare null mutations in the PRKCD gene were tested for uptake of oxLDL and foam cell formation. Expression of scavenger receptor CD36 was determined and levels of PKCß isoforms were quantified. Neither a reduction in PKCδ levels nor its complete absence resulted in a detectable effect on the uptake of oxLDL and the formation of foam cells. CONCLUSION: PKCδ is dispensible for oxLDL uptake and foam cell formation by monocytes and macrophages.

The identification and developmental requirements of colonic CD169⁺ macrophages.

CD169-positive macrophages in the marginal zone of the spleen and subcapsular sinus of lymph nodes play an important role as gatekeepers, strategically located to capture pathogens. Here we identified a population of CD169-positive macrophages in the colon and investigated which factors influenced their development. Murine colonic CD115+ F4/80(lo) CD11c(lo) macrophages expressing CD169 were present in the lamina propria, mainly surrounding the crypts. In spite of the high levels of bacterial flora in the colon and the importance of Toll-like receptor signalling in mucosal homeostasis, the presence of CD169+ macrophages was not affected in mice that were deficient in MyD88-mediated Toll-like receptor signalling and in mice in which the bacterial flora was eradicated. Whereas the development of splenic CD169+ macrophages was dependent on lymphotoxin α, colonic CD169+ macrophages were present in normal numbers in lymphotoxin α-deficient mice. In contrast, reduced numbers of CD169+ macrophages were found in the colon of mice deficient in vitamin A, whereas CD169+ macrophages in the spleen were unaffected. In conclusion, we identified a new macrophage subset in the lamina propria of the colon characterized by the expression of CD169. Its differentiation, unlike CD169+ macrophages in lymphoid organs, is independent of lymphotoxin α signalling, but requires vitamin A.

Vitamin A and dendritic cell differentiation.

Vitamin A and its active metabolite retinoic acid are essential for the development and function of many tissues including the immune system. The induction of mucosal homing receptors on T and B cells by mucosal dendritic cells (DC) depends on the presence of vitamin A. Recent studies indicate that also the differentiation of CD11b+ DC subsets in the mucosa as well as the spleen depend on vitamin A signalling. As CD11b+ DC subsets exert non-redundant functions in anti-bacterial and anti-fungal immune responses, defects in CD11b+ DC differentiation will contribute to the clinical problems observed during vitamin A deficiency.

Genome-wide association study identifies variants associated with autoimmune hepatitis type 1.

BACKGROUND & AIMS: Autoimmune hepatitis (AIH) is an uncommon autoimmune liver disease of unknown etiology. We used a genome-wide approach to identify genetic variants that predispose individuals to AIH. METHODS: We performed a genome-wide association study of 649 adults in The Netherlands with AIH type 1 and 13,436 controls. Initial associations were further analyzed in an independent replication panel comprising 451 patients with AIH type 1 in Germany and 4103 controls. We also performed an association analysis in the discovery cohort using imputed genotypes of the major histocompatibility complex region. RESULTS: We associated AIH with a variant in the major histocompatibility complex region at rs2187668 (P = 1.5 × 10(-78)). Analysis of this variant in the discovery cohort identified HLA-DRB1*0301 (P = 5.3 × 10(-49)) as a primary susceptibility genotype and HLA-DRB1*0401 (P = 2.8 × 10(-18)) as a secondary susceptibility genotype. We also associated AIH with variants of SH2B3 (rs3184504, 12q24; P = 7.7 × 10(-8)) and CARD10 (rs6000782, 22q13.1; P = 3.0 × 10(-6)). In addition, strong inflation of association signal was found with single-nucleotide polymorphisms associated with other immune-mediated diseases, including primary sclerosing cholangitis and primary biliary cirrhosis, but not with single-nucleotide polymorphisms associated with other genetic traits. CONCLUSIONS: In a genome-wide association study, we associated AIH type 1 with variants in the major histocompatibility complex region, and identified variants of SH2B3and CARD10 as likely risk factors. These findings support a complex genetic basis for AIH pathogenesis and indicate that part of the genetic susceptibility overlaps with that for other immune-mediated liver diseases.

Reprogramming macrophages to an anti-inflammatory phenotype by helminth antigens reduces murine atherosclerosis.

Atherosclerosis is a lipid-driven inflammatory disease of the vessel wall, characterized by the chronic activation of macrophages. We investigated whether the helminth-derived antigens [soluble egg antigens (SEAs)] could modulate macrophage inflammatory responses and protect against atherosclerosis in mice. In bone marrow-derived macrophages, SEAs induce anti-inflammatory macrophages, typified by high levels of IL-10 and reduced secretion of proinflammatory mediators. In hyperlipidemic LDLR(-/-) mice, SEA treatment reduced plaque size by 44%, and plaques were less advanced compared with PBS-injected littermate controls. The atheroprotective effect of SEAs was found to be mainly independent of cholesterol lowering and T-lymphocyte responses but instead could be attributed to diminished myeloid cell activation. SEAs reduced circulating neutrophils and inflammatory Ly6C(high) monocytes, and macrophages showed high IL-10 production. In line with the observed systemic effects, atherosclerotic lesions of SEA-treated mice showed reduced intraplaque inflammation as inflammatory markers [TNF-α, monocyte chemotactic protein 1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and CD68], neutrophil content, and newly recruited macrophages were decreased. We show that SEA treatment protects against atherosclerosis development by dampening inflammatory responses. In the future, helminth-derived components may provide novel opportunities to treat chronic inflammatory diseases, as they diminish systemic inflammation and reduce the activation of immune cells.

A crucial role for retinoic acid in the development of Notch-dependent murine splenic CD8- CD4- and CD4+ dendritic cells.

The vitamin A metabolite retinoic acid is important for the function of the adaptive immune system, but the mechanism is not completely understood. Here we show that vitamin A is essential for the generation of Notch-dependent CD8(-) dendritic cells (DCs) in the spleen. We observed that CD8(-) CD4(-) (double negative (DN)) and CD4(+) DCs, but not CD8(+) DCs, express vitamin A regulated genes. To determine whether vitamin A levels influence splenic DC development, we generated mice that were fed a vitamin A-deficient diet. We detected a specific reduction of CD4(+) and DN DCs in the spleens of mice fed a vitamin A-deficient diet, while pre-DC numbers in both spleen and bone marrow were not affected. Vitamin A was specifically necessary for the development of RelB(high) , Notch-dependent CD4(+) , and DN DCs. In addition, DN DCs showed reduced proliferation during vitamin A deficiency. In contrast, mice that had received a diet with increased amounts of retinoic acid showed a significant expansion of Notch-dependent DN DCs. These data demonstrate that vitamin A stimulates the development of Notch-dependent splenic DCs and indicate that inefficient generation of DCs may contribute to the immune deficits observed during vitamin A deficiency.

Trichuris suis-induced modulation of human dendritic cell function is glycan-mediated.

Human monocyte-derived dendritic cells (DCs) show remarkable phenotypic changes upon direct contact with soluble products (SPs) of Trichuris suis, a pig whipworm that is experimentally used in therapies to ameliorate inflammation in patients with Crohn's disease and multiple sclerosis. These changes may contribute to the observed induction of a T helper 2 (Th2) response and the suppression of Toll-like receptor (TLR)-induced Th1 and Th17 responses by human DCs primed with T. suis SPs. Here it is demonstrated that glycans of T. suis SPs contribute significantly to the suppression of the lipopolysaccharide (LPS)-induced expression in DCs of a broad variety of cytokines and chemokines, including important pro-inflammatory mediators such as TNF-α, IL-6, IL-12, lymphotoxin α (LTA), C-C Motif Ligand (CCL)2, C-X-C Motif Ligands (CXCL)9 and CXCL10. In addition, the data show that human DCs strongly bind T. suis SP-glycans via the C-type lectin receptors (CLRs) mannose receptor (MR) and DC-specific ICAM-3-grabbing non-integrin (DC-SIGN). The interaction of DCs with T. suis glycans likely involves mannose-type glycans, rather than fucosylated glycans, which differs from DC binding to soluble egg antigens of the human worm parasite, Schistosoma mansoni. In addition, macrophage galactose-type lectin (MGL) recognises T. suis SPs, which may contribute to the interaction with immature DCs or other MGL-expressing immune cells such as macrophages. The interaction of T. suis glycans with CLRs of human DCs may be essential for the ability of T. suis to suppress a pro-inflammatory phenotype of human DCs. The finding that the T. suis-induced modulation of human DC function is glycan-mediated is novel and indicates that helminth glycans contribute to the dampening of inflammation in a wide range of human inflammatory diseases.

SIRPα controls the activity of the phagocyte NADPH oxidase by restricting the expression of gp91(phox).

The phagocyte NADPH oxidase mediates oxidative microbial killing in granulocytes and macrophages. However, because the reactive oxygen species produced by the NADPH oxidase can also be toxic to the host, it is essential to control its activity. Little is known about the endogenous mechanism(s) that limits NADPH oxidase activity. Here, we demonstrate that the myeloid-inhibitory receptor SIRPα acts as a negative regulator of the phagocyte NADPH oxidase. Phagocytes isolated from SIRPα mutant mice were shown to have an enhanced respiratory burst. Furthermore, overexpression of SIRPα in human myeloid cells prevented respiratory burst activation. The inhibitory effect required interactions between SIRPα and its natural ligand, CD47, as well as signaling through the SIRPα cytoplasmic immunoreceptor tyrosine-based inhibitory motifs. Suppression of the respiratory burst by SIRPα was caused by a selective repression of gp91(phox) expression, the catalytic component of the phagocyte NADPH oxidase complex. Thus, SIRPα can limit gp91(phox) expression during myeloid development, thereby controlling the magnitude of the respiratory burst in phagocytes.

Stromal cells of the mouse spleen.

The composition and function of stromal cells in the white pulp of the spleen resemble to a large extent the situation in other secondary lymphoid organs such as lymph nodes. The stromal cells play an important role in the support and guidance of lymphocytes and myeloid cells in the T and B cell zones of the spleen. Major differences of the spleen are found in the way cells enter the white pulp and the composition of stromal cells in the red pulp. In this review, the features of stromal cells of both white and red pulp will be described in light of the function of the spleen.

Nod2 improves barrier function of intestinal epithelial cells via enhancement of TLR responses.

Intestinal epithelial cells (IECs) form a physical barrier between the internal milieu and the intestinal microflora via the expression of tight junctions. TLR-mediated recognition of intestinal microflora by IECs is important for tight junction preservation, production of chemokines, and cell survival. Disturbance of the IEC barrier function results in bacterial invasion and contributes to the development of inflammatory bowel disease. We observed that muramyl dipeptide (MDP), a breakdown product of bacterial peptidoglycan, strongly enhances subsequent Toll-like receptor (TLR) responses in murine colonic epithelium cell lines. Prior exposure to MDP significantly increased the production of chemokines and cytokines and improved the barrier function induced by different TLR2 and TLR4 ligands. shRNA knock-down studies showed that MDP recognition by Nod2 mediated the enhancement of TLR responses. Our studies indicate that Nod2 stimulation by MDP significantly enhances TLR-mediated IEC barrier function and chemokine production. Failure of this protective mechanism may contribute to the increased risk of Crohn's disease in individuals with a loss-of-function mutation in NOD2.

Soluble helminth products suppress clinical signs in murine experimental autoimmune encephalomyelitis and differentially modulate human dendritic cell activation.

The increased incidence of auto-inflammatory and autoimmune diseases in the developed countries seems to be caused by an imbalance of the immune system due to the lack of proper regulation. Helminth parasites are well known modulators of the immune system and as such are of great interest for the treatment of these disorders. Clinical studies showed that administration of eggs of the pig nematode Trichuris suis to patients with inflammatory bowel disease reduces the disease severity. Here we demonstrate that treatment with soluble products from the nematodes T. suis and Trichinella spiralis induces significant suppression of symptoms in murine experimental autoimmune encephalomyelitis, a validated animal model for multiple sclerosis. These data show that infection with live nematodes is not a prerequisite for suppression of inflammation. To translate these results to the human system, the effects of soluble products of T. suis, T. spiralis and Schistosoma mansoni on the phenotype and function of human dendritic cells (DCs) were compared. Our data show that soluble products of T. suis, S. mansoni and T. spiralis suppress TNF-α and IL-12 secretion by TLR-activated human DCs, and that T. suis and S. mansoni, but not T. spiralis, strongly enhance expression of OX40L. Furthermore, helminth-primed human DCs differentially suppress the development of Th1 and/or Th17 cells. In conclusion, our data demonstrate that soluble helminth products have strong immunomodulatory capacities, but might exert their effects through different mechanisms. The suppressed secretion of pro-inflammatory cytokines together with an upregulation of OX40L expression on human DCs might contribute to achieve this modulation.