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Crohn’s disease [CD] is one of the two most common forms of inflammatory bowel disease, affecting over half a million Americans. Like many other diseases with chronic inflammation, some patients with CD develop tertiary lymphoid organs [TLO] in areas of the gastrointestinal tract with active disease. TLOs are organized clusters of lymphocytes, similar in structure to secondary lymphoid organs, though they develop after birth and their contribution to pathogenesis in CD, or other diseases, is unclear. We, and others, have also found B cell rich lymphoid aggregates in the mesenteric fat of CD patients along dramatically remodeled lymphatic vessels. TNFΔARE/+ is a murine model of ileal inflammation that recapitulates several key features of ileal CD, including development of mesenteric tertiary lymphoid organs. Use of this model revealed that mesenteric TLOs block cellular and molecular export from the gut, leading us to wonder if mechanisms that interfere with their development might reduce ileitis. TNFΔARE/+ mice that lack B cells revealed that B cells are required for tertiary lymphoid organ formation in this model. Without these TLOs, lymphatic outflow from the intestine was restored. Nonetheless, histological and flow cytometric approaches reveal no difference in local inflammation in the ileum. However, systemic inflammation, as assessed by metabolic cages and changes in body weight over time, increased. This suggests that TLOs may act to trap inflammatory signals locally, preventing systemic dissemination of inflammatory cells or mediators. Work supported by NIH grants DP1-DK109668-04 and T32-DK077653-27 and the Kenneth Rainin Foundation

Immunoglobulin A (IgA) secretion by plasma cells (PCs), terminally differentiated B cells residing in the intestinal lamina propria, assures microbiome homeostasis and protects the host against enteric infections. However, whether exposure to diet-derived and commensal-derived signals instruct tissue resident PCs effector function and dynamically shape IgA immune responses at the mucosal barrier remain largely uninvestigated. Here, we demonstrated that intestinal epithelial cells (IECs) integrate luminal input to produce 7α,25-dihydroxycholesterol (7α,25-HC), a cholesterol metabolite (oxysterol) with immunomodulatory functions. In IECs, both cholesterol uptake via NPC1L1 and commensal recognition via MyD88 controlled oxysterol production. Mice lacking the oxysterol enzyme CH25H specifically in IECs abolished 7α,25-HC production and allowed to study oxysterol generation and activity in the small intestine. Inability of IECs to generate 7α,25-HC enhanced IgA secretion by PCs in the gut, suggesting that oxysterol negatively regulate humoral response at the mucosal barriers. Mechanistically, we showed that intestinal PCs sensed 7α,25-HC via the chemoattractant receptor GPR183 to position in the lamina propria tissue. This IEC-PC axis was rapidly modulated by cholesterol dietary content and tuned Salmonella-specific IgA response. Our finding revealed a new mechanism linking dietary cholesterol and humoral immune responses centered around PC localization for efficient mucosal protection. Supported by grants from Kenneth Rainin Foundation, Innovator Award, Charles H. Hood Foundation Child Health Research Awards Program, The Leukemia and Lymphoma Society New Idea Award and the Multiple Myeloma Research Fellowship, NIH ( AI40098 ) and The American Association of Immunologists Careers in Immunology Fellowship Program.

Crohn’s disease [CD] is one of the two common forms of inflammatory bowel disease and it affects over half a million Americans. Like many other diseases marked by chronic inflammation, some patients with CD develop tertiary lymphoid organs [TLO] in regions of the gut with active disease. TLOs are organized clusters of adaptive immune cells, similar in function and structure to secondary lymphoid organs. Recently, we found B cell rich lymphoid aggregates in the mesenteric fat of CD patients positioned along dramatically remodeled lymphatic vessels, but their function, specifically of the B cells within these aggregates, is completely unknown. Using complementary imaging, flow cytometry, and single cell RNA sequencing approaches, we analyzed B cells present in the mesenteric fat from CD patients. Whereas B cells were essentially not present in the mesentery under homeostatic conditions, they were significantly increased in the inflamed mesenteric fat of CD patients. Single cell RNA sequencing data revealed differences in B cell populations in the mesenteric fat that potentially implicate B cells as contributing to lymphangiogenesis associated with TLOs. Simultaneously, we used a murine model of ileal inflammation to probe mechanism. Imaging and flow cytometry showed that the B cells in TLOs from TNFΔARE/+mice were comparable to the B cells from CD patients. Additionally, TNFΔARE/+/μMT mice, which lack B cells, further revealed that B cells may be required for the lymphatic remodeling seen in the mesentery.

The concept that impaired lymphatic flow during the progression of Crohn’s disease, a major form of inflammatory bowel disease, emerged decades ago, but remains unresolved. In Crohn’s disease, tertiary lymphoid organs (TLOs) are associated with collecting lymphatic vessels (CLVs) of the mesentery that govern lymph outflow from the intestine. Whether TLOs affect lymph transport is unknown. In the TNFDARE mouse model of Crohn’s-like ileitis, TLOs formed at valve regions which supported lymphangiogenic outgrowths to the TLOs. Photoconversion approaches to study cell trafficking indicated that TLOs received immune cells traveling from the intestine but scarcely allowed their egress, effectively trapping DCs, B, and T cells. Using adoptive transfer of microbiota-dependent TCR we found reduced overall Tregs differentiation and their presence shifted from the lymph nodes to TLOs, altering intestinal-induced responses. Strikingly, although anti-TNF therapy dampens gut inflammation, TLO are refractory the therapy. Moreover, passage of soluble fluorescent tracer through TLO-rich CLVs was also impeded, indicating transport defects were intrinsic to the CLVs. Indeed, lymph flow diverted at TLO to neighboring CLV branches, where dysfunctional valves allowed lymph return to the gut wall, accompanied by lymph leakage at the TLOs. Culturing lymphatic endothelial cells with TNF demonstrate that the cytokine alone can reduce key genes found in lymphatic valves. These data indicate that TNF disrupts lymphatic valve maintenance pathways, promoting formation of mesenteric TLOs that broadly impair lymph transit of molecules and cells from the intestine, impairing immune surveillance of the mucosal barrier, altering homeostasis.

Metabolic regulation has been recognized as a powerful principle guiding immune responses. Inflammatory macrophages undergo extensive metabolic rewiring marked by the production of substantial amounts of itaconate, which has recently been described as an immunoregulatory metabolite. Itaconate and its membrane-permeable derivative dimethyl itaconate (DI) selectively inhibit a subset of cytokines, including IL-6 and IL-12 but not TNF. The major effects of itaconate on cellular metabolism during macrophage activation have been attributed to the inhibition of succinate dehydrogenase, yet this inhibition alone is not sufficient to account for the pronounced immunoregulatory effects observed in the case of DI. Furthermore, the regulatory pathway responsible for such selective effects of itaconate and DI on the inflammatory program has not been defined. Here we show that itaconate and DI induce electrophilic stress, react with glutathione and subsequently induce both Nrf2 (also known as NFE2L2)-dependent and -independent responses. We find that electrophilic stress can selectively regulate secondary, but not primary, transcriptional responses to toll-like receptor stimulation via inhibition of IκBζ protein induction. The regulation of IκBζ is independent of Nrf2, and we identify ATF3 as its key mediator. The inhibitory effect is conserved across species and cell types, and the in vivo administration of DI can ameliorate IL-17-IκBζ-driven skin pathology in a mouse model of psoriasis, highlighting the therapeutic potential of this regulatory pathway. Our results demonstrate that targeting the DI-IκBζ regulatory axis could be an important new strategy for the treatment of IL-17-IκBζ-mediated autoimmune diseases.

Current understanding of Crohn’s disease (CD) is that hereditary factors render the mucosal surface susceptible by either deregulated interactions between the bacterial flora and surface epithelial cells or an overwhelming host immune response. Given that lymph flow is essential for leukocyte trafficking and lipid absorption, understanding the effects of chronic inflammation in the intestinal-draining lymphatics is crucial for IBD treatment. Proper immune responses in the gut require immune cell migration towards secondary lymphoid organs via mesenteric collecting vessels. Our group described newly developed tertiary lymphoid organs (TLO) in mesenteric fat of CD biopsies, that are formed in association with lymphatic collecting vessels. However, mouse models that resemble lymphatic impairment and TLO formation in IBD are missing. Using a unique IBD model of Crohn’s disease-like ileitis (TNFΔARE mice) we demonstrate that these mice develop mesenteric TLO. Under TNFΔARE background, we generated a lymphatic reporter and a transgenic photoconvertible lines. Our results show that mesenteric TLO are integrated with collecting lymphatic vessels, obstructing leukocyte and lymph traffic from the intestine. Using adoptive transfer of microbiota-dependent TCR Tregs we found that TLO have the capacity to differentiate naïve T cells, yet they harbor tissue-resident T cells, demonstrating their duality. Strikingly, although anti-TNF therapy reduce ileum inflammation, TLO are resistant to the treatment, contributing to TNF therapy relapse or inefficacy. Our results demonstrate that chronic intestinal inflammation extend to the mesentery, impairing immune surveillance of the gut mucosal barrier, altering homeostasis.

Humoral immunity requires cross-talk between T follicular helper (Tfh) cells and B cells. Nevertheless, a detailed understanding of this intercellular interaction during secondary immune responses is lacking. We examined this by focusing on the response to a soluble, unadjuvanted, pathogen-derived Ag (soluble extract of Schistosoma mansoni egg [SEA]) that induces type 2 immunity. We found that activated Tfh cells persisted for long periods within germinal centers following primary immunization. However, the magnitude of the secondary response did not appear to depend on pre-existing Tfh cells. Instead, Tfh cell populations expanded through a process that was dependent on memory T cells recruited into the reactive LN, as well as the participation of B cells. We found that, during the secondary response, IL-4 was critical for the expansion of a population of plasmablasts that correlated with increased SEA-specific IgG1 titers. Additionally, following immunization with SEA (but not with an Ag that induced type 1 immunity), IL-4 and IL-21 were coproduced by individual Tfh cells, revealing a potential mechanism through which appropriate class-switching can be coupled to plasmablast proliferation to enforce type 2 immunity. Our findings demonstrate a pivotal role for IL-4 in the interplay between T and B cells during a secondary Th2 response and have significant implications for vaccine design.

Lipoproteins trapped in arteries drive atherosclerosis. Extravascular low-density lipoprotein undergoes receptor uptake, whereas high-density lipoprotein (HDL) interacts with cells to acquire cholesterol and then recirculates to plasma. We developed photoactivatable apoA-I to understand how HDL passage through tissue is regulated. We focused on skin and arteries of healthy mice versus those with psoriasis, which carries cardiovascular risk in man. Our findings suggest that psoriasis-affected skin lesions program interleukin-17-producing T cells in draining lymph nodes to home to distal skin and later to arteries. There, these cells mediate thickening of the collagenous matrix, such that larger molecules including lipoproteins become entrapped. HDL transit was rescued by depleting CD4 + T cells, neutralizing interleukin-17, or inhibiting lysyl oxidase that crosslinks collagen. Experimental psoriasis also increased vascular stiffness and atherosclerosis via this common pathway. Thus, interleukin-17 can reduce lipoprotein trafficking and increase vascular stiffness by, at least in part, remodeling collagen.

Lymphatic vessels transport interstitial fluid, soluble Ag, and immune cells from peripheral tissues to lymph nodes (LNs), yet the contribution of peripheral lymphatic drainage to adaptive immunity remains poorly understood. We examined immune responses to dermal vaccination and contact hypersensitivity (CHS) challenge in K14-VEGFR-3-Ig mice, which lack dermal lymphatic capillaries and experience markedly depressed transport of solutes and dendritic cells from the skin to draining LNs. In response to dermal immunization, K14-VEGFR-3-Ig mice produced lower Ab titers. In contrast, although delayed, T cell responses were robust after 21 d, including high levels of Ag-specific CD8+ T cells and production of IFN-γ, IL-4, and IL-10 upon restimulation. T cell-mediated CHS responses were strong in K14-VEGFR-3-Ig mice, but importantly, their ability to induce CHS tolerance in the skin was impaired. In addition, 1-y-old mice displayed multiple signs of autoimmunity. These data suggest that lymphatic drainage plays more important roles in regulating humoral immunity and peripheral tolerance than in effector T cell immunity.

C-C motif chemokine receptor 2 (CCR2) axis is a major chemokine axis that recruits myeloid cells including monocytes and macrophages. Thus far, CCR2−/− mice have not been found to be more susceptible to infection with Euro-American lineage 4 strains of Mycobacterium tuberculosis (Mtb). Here, using a prototype W-Beijing family lineage 2 clinical Mtb strain, HN878, we show that CCR2−/− mice exhibit increased susceptibility to tuberculosis (TB). Following exposure to Mtb HN878, alveolar macrophages (AMs) are amongst the earliest cells infected. Using a novel labeling technique, we show that AMs accumulate early in the airways following Mtb HN878 infection and express CCR2. During disease progression, CCR2-expressing AMs exit the airways and localize within the TB granulomas to mediate protective immunity. RNA-sequencing of sorted airway and non-airway AMs show distinct gene expression profiles, suggesting that upon exit from airways, AMs become classically activated. Furthermore, absence of CCR2+ cells specifically at the time of AM egress from the airways resulted in enhanced susceptibility to Mtb infection, increased accumulation of neutrophils, and loss of Mtb control. Interestingly, we provide new evidence that infection with an Mtb strain HN878 mutant lacking phenolic glycolipid (PGL) expression still resulted in increased susceptibility in CCR2−/− mice. Together, our data provide novel evidence for a critical protective role for CCR2 in AM localization within the TB granulomas to mediate protective immunity against clinically relevant and emerging Mtb infections.

Macrophages reside in the vascular intima of the aorta in plaque prone regions. Intima macrophages were found to be ‘tissue resident’, possessing the ability to self-renew independent of circulating monocytes and having a unique dependence on Csf1 and Flt3 pathways for development. Lineage tracing and depletion experiments showed these cells were regulators of early plaque development in models of atherosclerosis. Using bulk RNAseq on intima resident macrophages and single cell sequencing data from total leukocytes from C57Bl/6 mouse aorta, we identified a core gene expression profile associated with intima resident macrophages. Comparing this gene signatures to single-cell gene expression data obtained from total leukocytes from atherosclerotic aortas identified overlapping gene expression with foamy macrophages, despite foamy macrophages developing exclusively from recruited monocytes at this stage of disease. The intima resident gene signature was expressed in foamy cells in addition to cholesterol handling genes, but not in newly recruited monocytes. Importantly, the intima gene signature was only associated with cells lacking expression of inflammatory genes. Together this suggests there is a niche-driven gene expression program within the aortic intima influencing macrophage differentiation and function. Targeting this newly identified gene set may be important for regulation of the inflammatory pathways and foamy macrophage differentiation in atherosclerosis.

Tissue-resident macrophages occupy key roles in immunity and physiology within organ microenvironments. Many tissue macrophages derive from embryonic progenitors and self-maintain locally as unique tissue-specific populations. Resident macrophages can also expand in response to type 2 stimuli including parasites and contribute to control of infection via the alternative activation program. However, the transcriptional basis for this capacity to proliferate in situ is poorly understood. We have observed that the transcription factor basic helix-loop-helix, member e40 (Bhlhe40) is highly expressed in a subset of hematopoietic cell types, including large peritoneal macrophages (LPMs). Based on this data, we hypothesized that Bhlhe40 is a key part of the LPM transcriptional network. We have found that peritoneal macrophages are selectively reduced in Bhlhe40−/− mice, in contrast to other resident macrophages. Mixed bone marrow chimeras, conditional knockout mice, and other approaches demonstrated a specific, intrinsic defect in mature, Bhlhe40-deficient LPMs. Bhlhe40−/− LPMs exhibited an alternative activation-like profile and impaired self-renewal. These functional perturbations were correlated with altered expression of gene sets pertaining to the endoplasmic reticulum (ER) and protein homeostasis, as well as ER morphology changes in Bhlhe40−/− LPMs. Using models of peritoneal type 2 immunity, we observed near-total loss of expansion of Bhlhe40-deficient LPMs, correlated with impaired control of an intestinal helminth. Our findings demonstrate critical roles for Bhlhe40 as a tissue-specific regulator of resident macrophage self-renewal and expansion during type 2 immunity.

Resident macrophages in peritoenal and pleural cavities exist as two distinct populations: F4/80+ICAM2+macrophages and MHCII+ macrophages. F4/80+ ICAM2+macrophages are derived from CX3CR1+embryonic precursors, and maitained by the transcription factor Gata6. However, the origin and regulating factors that maintain MHCII+ macrophages remain unknown. Here, we show that the MHCII+ macrophages arise postnatally from monocytes, which continuously replenish this subset through adulthood. Gene expression analysis identified distinct surface markers such as CD226 and revealed that the transcription factor IRF4 was selectively expressed in these macrophages relative to other organs. Monocytes first enter peritoneal or pleural cavities to become MHC II+ cells that up-regulate CD226 later as they continue to mature. In the absence of IRF4 or after administration of oral antibiotics, MHCII+CD226−CD11c− monocyte-derived cells accumulated in peritoneal and pleural cavities, but CD11c+ CD226+ macrophages were lost. Thus, MHC II+ resident peritoneal and pleural macrophages are continuously replenished by blood monocytes recruited to the peritoneal and pleural cavities constitutively, starting after birth, where they require IRF4 and signals likely derived from the microbiome to fully differentiate.

The lymphatic system is essential for the generation of immune responses by facilitating immune cell trafficking to lymph nodes. Dendritic cells (DCs), the most potent APCs, exit tissues via lymphatic vessels, but the mechanisms of interaction between DCs and the lymphatic endothelium and the potential implications of these interactions for immune responses are poorly understood. In this study, we demonstrate that lymphatic endothelial cells (LECs) modulate the maturation and function of DCs. Direct contact of human monocyte-derived DCs with an inflamed, TNF-α-stimulated lymphatic endothelium reduced expression of the costimulatory molecule CD86 by DCs and suppressed the ability of DCs to induce T cell proliferation. These effects were dependent on adhesive interactions between DCs and LECs that were mediated by the binding of Mac-1 on DCs to ICAM-1 on LECs. Importantly, the suppressive effects of the lymphatic endothelium on DCs were observed only in the absence of pathogen-derived signals. In vivo, DCs that migrated to the draining lymph nodes upon inflammatory stimuli, but in the absence of a pathogen, showed increased levels of CD86 expression in ICAM-1-deficient mice. Together, these data demonstrate a direct role of LECs in the modulation of immune response and suggest a function of the lymphatic endothelium in preventing undesired immune reactions in inflammatory conditions.

In vivo data suggest that monocytes participate critically in cross-presentation, but other data suggest that lymph node resident dendritic cells (DCs) mainly cross-present. Here, we utilized a three-dimensional model of a blood vessel wall that endogenously supports DC development from human monocytes, and we incorporated dying autologous cells in the subendothelial matrix of the model. Flu-infected dying cells promoted monocytes to become mature DCs and cross-present cell-associated Ags for the activation of CTLs. Similar responses were induced by loading the dying cells with the TLR7/8 ligand ssRNA, whereas dying cells loaded with TLR3 ligand were less efficient. Monocyte-derived DCs that developed in this model cross-presented Ag to T cells efficiently regardless of whether they engulfed detectable amounts of labeled dying cells. Unexpectedly, the monocyte-derived cells that directly engulfed dying cells in vitro were not the major APCs stimulating CD8+ lymphocytes. Instead, bystander DCs acquired more robust capacity to cross-prime through receipt of MHC class I/peptide from the phagocytic, monocyte-derived cells. In mice, lymph node-homing monocyte-derived DCs processed Ags from engulfed cells and then transferred MHC class I/peptide complexes to confer cross-priming capacity to MHC class I-deficient lymph node resident CD8α+ DCs. Thus, natural or synthetic TLR7/8 agonists contained within dying cells promote the conversion of monocytes to DCs with capacity for cross-presentation and for “cross-dressing” other DCs. These data reveal a way in which migratory monocyte-derived DCs and other DCs, like lymph node resident DCs, both mediate cross-presentation.

There are two major myeloid pulmonary dendritic cell (DC) populations: CD103+ DCs and CD11bhigh DCs. In this study, we investigated in detail the origins of both myeloid DC pools using multiple experimental approaches. We show that, in resting lung, Ly-6ChighCCR2high monocytes repopulated CD103+ DCs using a CCR2-dependent mechanism, and these DCs preferentially retained residual CCR2 in the lung, whereas, conversely, Ly-6ClowCCR2low monocytes repopulated CD11bhigh DCs. CX3CR1 was required to generate normal numbers of pulmonary CD11bhigh DCs, possibly because Ly-6Clow monocytes in the circulation, which normally express high levels of CX3CR1, failed to express bcl-2 and may have diminished survival in the circulation in the absence of CX3CR1. Overall, these data demonstrate that the two circulating subsets of monocytes give rise to distinct tissue DC populations.

FTY720 stimulates CCR7-driven T cell homing to peripheral lymph nodes (LN) by direct activation of sphingosine 1-phosphate receptors, along with the participation of multidrug transporters, 5-lipoxygenase, and G protein-coupled receptors for chemokines. In this study, we demonstrate that FTY720 also directly stimulates in vitro T cell chemotaxis to CCR2-CCL2, but not to a variety of other chemokines, including CCR5-CCL3/4/5 and CXCR4-CXCL12. FTY720 influences CCR2-CCL2-driven migration through activation of the multidrug transporters, Abcb1 and Abcc1, and through 5-lipoxygenase activity. In vivo administration of FTY720 induces chemokine-dependent migration of T cells in the thymus, peripheral blood, LN, and spleen. The CCR7 and CCR2 chemokine ligands are required for both T cell sequestration in LN and thymic T cell egress following FTY720 administration. Furthermore, FTY720 administration uncovers a requirement for CXCR4 ligands for LN homing, but not for thymic egress, and CCR5 for thymic egress, but not LN homing. FTY720-driven splenic and peripheral blood T cell egress are both independent of CCR2, CCR5, CCR7, or CXCR4. These results indicate that FTY720- and sphingosine 1-phosphate receptor-stimulated T cell migration are dependent on the restricted usage of chemokine receptor-ligand pairs within discrete anatomic compartments.

Purified monocytes infected with influenza A virus do not become mature dendritic cells (DCs) and they present viral peptides poorly to autologous memory T cells. In this study, we investigated whether influenza A-infected monocytes matured to DCs with a high capacity to stimulate T cells when they were infected with influenza A virus in a model tissue setting wherein they were cocultured with endothelium grown on a type I collagen matrix. Intercellular interactions with endothelium strongly promoted the Ag-presenting capacity of monocyte-derived cells infected with influenza A virus, and the heterologous coculture system also enhanced production of IFN-α by monocytes in the absence of plasmacytoid cells. Production of IFN-α in the presence of endothelium correlated with monocyte differentiation to mature DCs and their ability to stimulate proliferation and IFN-γ production by autologous T cells. Monocyte-derived cells that developed into migratory DCs promoted proliferation of influenza A virus-specific CD4+ and CD8+ cells, whereas those that developed into macrophages promoted proliferation of CD8+ T cells only. This onset of APC activity could be partially blocked with Ab to the IFN-αβ receptor when monocytes were infected with UV-treated virus, but neutralizing this pathway was inconsequential when monocytes were infected with live virus. Thus, type I IFN and direct contact with endothelium promote development of influenza A virus-presenting activity in monocyte-derived cells in a setting in which this differentiation does not depend on plasmacytoid cells. However, when infected with live influenza virus, the role of type I IFN in mediating differentiation and Ag-presenting capacity is expendable, apparently due to other mechanisms of virus-mediated activation.

Tissue-resident macrophages occupy key roles in immunity and physiology, both as sensors of and first responders to homeostatic perturbations. The majority of resident macrophages derive from embryonic progenitors and self-maintain locally as unique tissue-specific populations. The transcriptional basis for tissue-resident macrophage identity is poorly understood. We have observed that the transcription factor basic helix loop helix, member e40 (Bhlhe40) is highly expressed in a subset of hematopoietic cell types, including peritoneal macrophages. Based on these data, we hypothesized that Bhlhe40 is a key part of the peritoneal macrophage transcriptional network and adapts nascent macrophages to the functional demands of the peritoneal niche. We have found that peritoneal macrophages are selectively reduced in Bhlhe40−/− mice, in contrast to other resident macrophages. Furthermore, in the absence of Bhlhe40 the remaining large peritoneal macrophages (LPMs) are replaced by monocyte-derived cells to a greater extent than in wildtype mice. Bhlhe40-deficient peritoneal macrophages exhibit altered polarization and self-renewal. Mixed bone marrow chimeras and other approaches demonstrated a specific, intrinsic defect in mature Bhlhe40−/− LPMs. Using models of peritoneal immunity, we observed impaired responses of Bhlhe40-deficient peritoneal macrophages. Our preliminary findings demonstrate a role for Bhlhe40 in peritoneal macrophage biology and lead us to propose that Bhlhe40 transcriptionally controls a tissue-specific program in macrophages tailored to the peritoneal environment.

Atherosclerosis is the leading cause of heart disease, which is the largest contributor to deaths across the world. This disease involves the development of inflammation and plaque formation within the lumen of mid- and large-sized arteries following cholesterol-enriched diet. Our current study uses intravital two photon imaging to examine the initiating and propagating factors of this disease. Atherogenesis occurs at sites of endothelial damage most commonly near branching points and areas of high sheering stress in vasculature, including the aortic arch and carotid bifurcation. A minimally invasive technique was developed allowing us to monitor the movement of cells inside the vasculature. This technique involves isolating the right common carotid artery near the bifurcation and positioning it to monitor monocyte dynamics on the endothelium without disrupting the flow. Using a variety of fluorescent proteins on myeloid backgrounds crossed with LDLR−/− mice, we measure the monocyte dynamics on both normal diet and cholesterol diet at multiple different time points. We can trace the movement of monocytes in the blood and measure the interaction with resident cells in the luminal wall, finding that monocyte rolling is influenced by localization to plaque-areas and treatment with high cholesterol diet. Defining these differences in interactions and determining mechanisms of monocyte recruitment are key to understanding the initiation and growth of atherosclerotic plaque over time.

Disruption in the movement of blood or lymph leads to potentially fatal diseases, most commonly atherosclerosis in the blood and lymphedema/lymphangitis in the lymph. There have been an enormous amount of studies done on blood vasculature in the past few decades, but our understanding of lymphatics in health and disease remains poor. Lymphatics interact systemically and locally with the blood vasculature and we have recently proven that dendritic cells are an important regulator in lymphatic function. Some of these processes can only be proven in a living animal as the interactions and impact of immune cells on vascular flow dynamics and vice versa cannot been mimicked in vitro. Our most recent intravital imaging studies allow the visualization of resident macrophage dynamics within a plaque. Due to the fast movement of cells in the vasculature and the sensitivity of cells to phototoxicity it was challenging to generate a palette of dyes which are low in cytotoxicity able to label the structures and cell populations of interest. It is possible by using fluorophores (Fluorescent proteins and conventional dyes) efficient enough to be excited with low laser power and an emission sufficient enough for being detected. Recently, a femtosecond pulsed Titanium Sapphire (Ti:sap) laser with averaged power ≥ 1W above 1200 nm became available. We have coupled a laser of this type (InSight DS+) together with an conventional Ti:sap-laser (Mai Tai DeepSee) to an customized ultra fast and sensitive scanning Microscope. Using these settings we can image up to five fluorophores in intravital settings. This approach enables us to visualize lymphatics and blood vessels together with myeloid cells and lymphocytes in vivo.

Recently, we identified that Batf3-dependent DCs were solely responsible for the rejection of minor antigen-mismatched grafts, and demonstrated in Batf3−/− female mice, lacking Batf3-dependent DCs, the complete acceptance of minor antigen-mismatched male cells, which are normally rejected in WT female mice. Intriguingly, this rejection occurs in the absence of PAMPs, leading to considerable uncertainty of how endogenous APC subtypes become activated and licensed to induce an adaptive immune response against male cells in female mice. Investigating this gap in knowledge is the main goal of this study. Rejection of minor antigen-mismatched grafts involves all branches of the immune system: adaptive immunity (T cells), humoral immunity (B cells) and innate immunity (DCs and antigen-presenting monocytes). Although we showed that depletion of one cell type dampens or omits the inflammatory outcome of minor antigen-mismatched graft rejection, here we show how lymphoid and myeloid cell subtypes act sequentially and in concert for the induction of minor antigen-mismatched graft rejection without the presence of PAMPs. In conclusion, we believe that understanding the mechanisms of action against minor antigen-mismatched graft rejection will also apply more broadly to other diseases such as autoimmunity and cancer, in which an immune response is elicited against self-associated minor antigens.