Zbigniew Mikulski

Bacterial colonization factors control specificity and stability of the gut microbiota

Mammals harbour a complex gut microbiome, comprising bacteria that confer immunological, metabolic and neurological benefits. Despite advances in sequence-based microbial profiling and myriad studies defining microbiome composition during health and disease, little is known about the molecular processes used by symbiotic bacteria to stably colonize the gastrointestinal tract. We sought to define how mammals assemble and maintain the Bacteroides, one of the most numerically prominent genera of the human microbiome. Here we find that, whereas the gut normally contains hundreds of bacterial species, germ-free mice mono-associated with a single Bacteroides species are resistant to colonization by the same, but not different, species. To identify bacterial mechanisms for species-specific saturable colonization, we devised an in vivo genetic screen and discovered a unique class of polysaccharide utilization loci that is conserved among intestinal Bacteroides. We named this genetic locus the commensal colonization factors (ccf). Deletion of the ccf genes in the model symbiont, Bacteroides fragilis, results in colonization defects in mice and reduced horizontal transmission. The ccf genes of B. fragilis are upregulated during gut colonization, preferentially at the colonic surface. When we visualize microbial biogeography within the colon, B. fragilis penetrates the colonic mucus and resides deep within crypt channels, whereas ccf mutants are defective in crypt association. Notably, the CCF system is required for B. fragilis colonization following microbiome disruption with Citrobacter rodentium infection or antibiotic treatment, suggesting that the niche within colonic crypts represents a reservoir for bacteria to maintain long-term colonization. These findings reveal that intestinal Bacteroides have evolved species-specific physical interactions with the host that mediate stable and resilient gut colonization, and the CCF system represents a novel molecular mechanism for symbiosis.

Lung-resident tissue macrophages generate Foxp3+ regulatory T cells and promote airway tolerance

Lung-resident antigen-presenting macrophages promote tolerance to inhaled antigens via the induction of regulatory T cells.

Activin A programs the differentiation of human TFH cells

SUMMARY Follicular helper T (TFH) cells are CD4+ T cells specialized in helping B cells and are associated both with protective antibody responses and autoimmune diseases. The promise of targeting TFH cells therapeutically has been limited by fragmentary understanding of extrinsic signals regulating human TFH cell differentiation. A screen of a human protein library identified activin A as new regulator of TFH cell differentiation. Activin A orchestrated expression of multiple TFH-associated genes, independently or in concert with additional signals. TFH programming by activin A was antagonized by the cytokine IL-2. Activin A’s capacity to drive TFH cell differentiation in vitro was conserved for non-human primates but not mice. Finally, activin A-induced TFH programming was dependent on SMAD2 and SMAD3 signaling and blocked by pharmacological inhibitors.Follicular helper T cells (TFH cells) are CD4+ T cells specialized in helping B cells and are associated both with protective antibody responses and autoimmune diseases. The promise of targeting TFH cells therapeutically has been limited by fragmentary understanding of extrinsic signals that regulate the differentiation of human TFH cells. A screen of a human protein library identified activin A as a potent regulator of TFH cell differentiation. Activin A orchestrated the expression of multiple genes associated with the TFH program, independently or in concert with additional signals. TFH cell programming by activin A was antagonized by the cytokine IL-2. Activin As ability to drive TFH cell differentiation in vitro was conserved in non-human primates but not in mice. Finally, activin-A-induced TFH programming was dependent on signaling via SMAD2 and SMAD3 and was blocked by pharmacological inhibitors.

IL-10-producing intestinal macrophages prevent excessive antibacterial innate immunity by limiting IL-23 synthesis.

Innate immune responses are regulated in the intestine to prevent excessive inflammation. Here we show that a subset of mouse colonic macrophages constitutively produce the anti-inflammatory cytokine IL-10. In mice infected with Citrobacter rodentium, a model for enteropathogenic Escherichia coli infection in humans, these macrophages are required to prevent intestinal pathology. IL-23 is significantly increased in infected mice with a myeloid cell-specific deletion of IL-10, and the addition of IL-10 reduces IL-23 production by intestinal macrophages. Furthermore, blockade of IL-23 leads to reduced mortality in the context of macrophage IL-10 deficiency. Transcriptome and other analyses indicate that IL-10-expressing macrophages receive an autocrine IL-10 signal. Interestingly, only transfer of the IL-10 positive macrophages could rescue IL-10-deficient infected mice. Therefore, these data indicate a pivotal role for intestinal macrophages that constitutively produce IL-10, in controlling excessive innate immune activation and preventing tissue damage after an acute bacterial infection.

Transcription factor Nr4a1 couples sympathetic and inflammatory cues in CNS-recruited macrophages to limit neuroinflammation

The molecular mechanisms that link the sympathetic stress response and inflammation remain obscure. Here we found that the transcription factor Nr4a1 regulated the production of norepinephrine (NE) in macrophages and thereby limited experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Lack of Nr4a1 in myeloid cells led to enhanced NE production, accelerated infiltration of leukocytes into the central nervous system (CNS) and disease exacerbation in vivo. In contrast, myeloid-specific deletion of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, protected mice against EAE. Furthermore, we found that Nr4a1 repressed autocrine NE production in macrophages by recruiting the corepressor CoREST to the Th promoter. Our data reveal a new role for macrophages in neuroinflammation and identify Nr4a1 as a key regulator of catecholamine production by macrophages.

Endothelial Protective Monocyte Patrolling in Large Arteries Intensified by Western Diet and AtherosclerosisNovelty and Significance

Rationale: Nonclassical mouse monocyte (CX3CR1high, Ly-6Clow) patrolling along the vessels of the microcirculation is critical for endothelial homeostasis and inflammation. Because of technical challenges, it is currently not established how patrolling occurs in large arteries. Objective: This study was undertaken to elucidate the molecular, migratory, and functional phenotypes of patrolling monocytes in the high shear and pulsatile environment of large arteries in healthy, hyperlipidemic, and atherosclerotic conditions. Methods and Results: Applying a new method for stable, long-term 2-photon intravital microscopy of unrestrained large arteries in live CX3CR1-GFP (green fluorescent protein) mice, we show that nonclassical monocytes patrol inside healthy carotid arteries at a velocity of 36 &mgr;m/min, 3× faster than in microvessels. The tracks are less straight but lead preferentially downstream. The number of patrolling monocytes is increased 9-fold by feeding wild-type mice a Western diet or by applying topical TLR7/8 (Toll-like receptor) agonists. A similar increase is seen in CX3CR1+/GFP/apoE−/− mice on chow diet, with a further 2- to 3-fold increase on Western diet (22-fold over healthy). In plaque conditions, monocytes are readily captured onto the endothelium from free flow. Stable patrolling is unaffected in CX3CR1-deficient mice and involves the contribution of LFA-1 (lymphocyte-associated antigen 1) and &agr;4 integrins. The endothelial damage in atherosclerotic carotid arteries was assessed by electron microscopy and correlates with the number of intraluminal patrollers. Abolishing patrolling monocytes in Nr4a1−/− apoE−/− mice leads to pronounced endothelial apoptosis. Conclusions: Arterial patrolling is a prominent new feature of nonclassical monocytes with unique molecular and kinetic properties. It is highly upregulated in hyperlipidemia and atherosclerosis in a CX3CR1-independent fashion and plays a potential role in endothelial protection.

Live cell imaging to understand monocyte, macrophage, and dendritic cell function in atherosclerosis

Ley et al. provide a review of the technology and accomplishments of dynamic imaging of myeloid cells in atherosclerosis.

Lymphatic Specific Disruption in the Fine Structure of Heparan Sulfate Inhibits Dendritic Cell Traffic and Functional T Cell Responses in the Lymph Node

Dendritic cells (DCs) are potent APCs essential for initiating adaptive immunity. Following pathogen exposure, trafficking of DCs to lymph nodes (LNs) through afferent lymphatic vessels constitutes a crucial step in the execution of their functions. The mechanisms regulating this process are poorly understood, although the involvement of certain chemokines in this process has recently been reported. In this study, we demonstrate that genetically altering the fine structure (N-sulfation) of heparan sulfate (HS) specifically in mouse lymphatic endothelium significantly reduces DC trafficking to regional LNs in vivo. Moreover, this alteration had the unique functional consequence of reducing CD8+ T cell proliferative responses in draining LNs in an ovalbumin immunization model. Mechanistic studies suggested that lymphatic endothelial HS regulates multiple steps during DC trafficking, including optimal presentation of chemokines on the surface of DCs, thus acting as a co-receptor that may function “in trans” to mediate chemokine receptor binding. This study not only identifies novel glycan-mediated mechanisms that regulate lymphatic DC trafficking, but it also validates the fine structure of lymphatic vascular-specific HS as a novel molecular target for strategies aiming to modulate DC behavior and/or alter pathologic T cell responses in lymph nodes.

Atheroprotective vaccination with MHC-II-restricted ApoB peptides induces peritoneal IL-10-producing CD4 T cells

Although immunization with major histocompatibility complex (MHC) class II-restricted apolipoprotein B (ApoB) peptides has been shown to be atheroprotective, the mechanism is unclear. Here, we investigated CD4+ T cell populations in immunized atherosclerotic mice. Peptides (16-mers) from mouse ApoB, the core protein of low-density lipoprotein (LDL), were screened for binding to I-Ab by computer prediction and confirmed by radiolabeled peptide competition. Three new peptides, P101 (FGKQGFFPDSVNKALY, 5.5 nM IC50), P102 (TLYALSHAVNSYFDVD, 6.8 nM), and P103 (LYYKEDKTSLSASAAS, 95 nM), were tested in an atherosclerosis model (Apoe-/- mice on Western diet). Immunization with each of the three peptides (1 time in complete Freunds adjuvant subcuntaneously and 4 time in incomplete Freunds adjuvant intraperitoneally) but not with adjuvant alone showed significantly reduced atherosclerotic plaques in the aortic root by serial sections and in the whole aorta by en face staining. There were no differences in body weight, LDL cholesterol, or triglycerides. Peritoneal leukocytes from ApoB peptide-immunized mice, but not control mice, secreted significant amounts of IL-10 (150 pg/ml). Flow cytometry showed that peptide immunization induced IL-10 in 10% of peritoneal CD4+ T cells, some of which also expressed chemokine (C-C motif) receptor 5 (CCR5). Vaccination with ApoB peptides expanded peritoneal FoxP3+ regulatory CD4+ T cells and more than tripled the number of CCR5+FoxP3+ cells. Similar trends were also seen in the draining mediastinal lymph nodes but not in the nondraining inguinal lymph nodes. We conclude that vaccination with MHC class II-restricted autologous ApoB peptides induces regulatory T cells (Tregs) and IL-10, suggesting a plausible mechanism for atheroprotection.NEW & NOTEWORTHY Vaccination against apolipoprotein B (ApoB), the protein of LDL, attracts attention as a novel approach to prevent atherosclerosis. We discovered major histocompatibility complex class II-restricted ApoB peptides, which reduce atherosclerosis and induce IL-10-producing CD4+ T cells and chemokine (C-C motif) receptor 5 expression on regulatory T cells, suggesting that immunization with ApoB peptides inhibits atherosclerosis by inducing anti-inflammatory cytokines.

The transcription factor NR4A3 controls CD103+ dendritic cell migration

The transcription factor NR4A3 (also known as NOR-1) is a member of the Nr4a family of nuclear receptors and is expressed in myeloid and lymphoid cells. Here, we have shown that Nr4a3 is essential for the migration of CD103+ dendritic cells (DCs) to lymph nodes (LNs). Nr4a3-deficient mice had very few CD103+ migratory DCs (mDCs) present in LNs, and mixed-chimera studies revealed that this migratory defect was cell intrinsic. We further found that CD103+ DCs from Nr4a3-deficient mice displayed a marked loss of surface expression of the chemokine CCR7. This defect in CCR7 expression was confined to CD103+ DCs, as CCR7 expression on T lymphocytes was unaffected. Moreover, CCR7 was not induced on CD103+ DCs from Nr4a3-deficient mice in response to either administration of the TLR7 agonist R848 or infection with Citrobacter rodentium in vivo. The transcription factor FOXO1 has been shown to regulate CCR7 expression. We found that FOXO1 protein was reduced in Nr4a3-deficient DCs through an AKT-dependent mechanism. Further, we found a requirement for NR4A3 in the maintenance of homeostatic mitochondrial function in CD103+ DCs, although this is likely independent of the NR4A3/FOXO1/CCR7 axis in the regulation of DC migration. Thus, NR4A3 plays an important role in the regulation of CD103+ mDCs by regulating CCR7-dependent cell migration.