Regulation of lymph node vascular growth by dendritic cells

Lymph nodes grow rapidly and robustly at the initiation of an immune response, and this growth is accompanied by growth of the blood vessels. Although the vessels are critical for supplying nutrients and for controlling cell trafficking, the regulation of lymph node vascular growth is not well understood. We show that lymph node endothelial cells begin to proliferate within 2 d of immunization and undergo a corresponding expansion in cell numbers. Endothelial cell proliferation is dependent on CD11c+ dendritic cells (DCs), and the subcutaneous injection of DCs is sufficient to trigger endothelial cell proliferation and growth. Lymph node endothelial cell proliferation is dependent on vascular endothelial growth factor (VEGF), and DCs are associated with increased lymph node VEGF levels. DC-induced endothelial cell proliferation and increased VEGF levels are mediated by DC-induced recruitment of blood-borne cells. Vascular growth in the draining lymph node includes the growth of high endothelial venule endothelial cells and is functionally associated with increased cell entry into the lymph node. Collectively, our results suggest a scenario whereby endothelial cell expansion in the draining lymph node is induced by DCs as part of a program that optimizes the microenvironment for the ensuing immune response

Chemokine Requirements for B Cell Entry to Lymph Nodes and Peyer's Patches

B cell entry to lymph nodes and Peyer's patches depends on chemokine receptor signaling, but the principal chemokine involved has not been defined. Here we show that the homing of CXCR4−/− B cells is suppressed in CCL19 (ELC)- and CCL21 (SLC)-deficient paucity of lymph node T cells mice, but not in wild-type mice. We also find that CXCR4 can contribute to T cell homing. Using intravital microscopy, we find that B cell adhesion to high endothelial venules (HEVs) is disrupted when CCR7 and CXCR4 are predesensitized. In Peyer's patches, B cell entry is dependent on CXCR5 in addition to CCR7/CXCR4. CXCL12 (SDF1) is displayed broadly on HEVs, whereas CXCL13 (BLC) is found selectively on Peyer's patch follicular HEVs. These findings establish the principal chemokine and chemokine receptor requirements for B cell entry to lymph nodes and Peyer's patches

Profiling the Essential Nature of Lipid Metabolism in Asexual Blood and Gametocyte Stages of Plasmodium falciparum

SummaryDuring its life cycle, Plasmodium falciparum undergoes rapid proliferation fueled by de novo synthesis and acquisition of host cell lipids. Consistent with this essential role, Plasmodium lipid synthesis enzymes are emerging as potential drug targets. To explore their broader potential for therapeutic interventions, we assayed the global lipid landscape during P. falciparum sexual and asexual blood stage (ABS) development. Using liquid chromatography-mass spectrometry, we analyzed 304 lipids constituting 24 classes in ABS parasites, infected red blood cell (RBC)-derived microvesicles, gametocytes, and uninfected RBCs. Ten lipid classes were previously uncharacterized in P. falciparum, and 70%–75% of the lipid classes exhibited changes in abundance during ABS and gametocyte development. Utilizing compounds that target lipid metabolism, we affirmed the essentiality of major classes, including triacylglycerols. These studies highlight the interplay between host and parasite lipid metabolism and provide a comprehensive analysis of P. falciparum lipids with candidate pathways for drug discovery efforts

Diversity-Oriented Synthesis Yields a Novel Lead for the Treatment of Malaria

Here, we describe the discovery of a novel antimalarial agent using phenotypic screening of Plasmodium falciparum asexual blood-stage parasites. Screening a novel compound collection created using diversity-oriented synthesis (DOS) led to the initial hit. Structure–activity relationships guided the synthesis of compounds having improved potency and water solubility, yielding a subnanomolar inhibitor of parasite asexual blood-stage growth. Optimized compound 27 has an excellent off-target activity profile in erythrocyte lysis and HepG2 assays and is stable in human plasma. This compound is available via the molecular libraries probe production centers network (MLPCN) and is designated ML238.Chemistry and Chemical Biolog

Normalization of the Lymph Node T Cell Stromal Microenvironment in lpr/lpr Mice Is Associated with SU5416-Induced Reduction in Autoantibodies

The vascular-stromal elements of lymph nodes can play important roles in regulating the activities of the lymphocytes within. During model immune responses, the vascular-stromal compartment has been shown to undergo proliferative expansion and functional alterations. The state of the vascular-stromal compartment and the potential importance of this compartment in a spontaneous, chronic model of autoimmunity have not been well studied. Here, we characterize the vascular expansion in MRL-lpr/lpr lymph nodes and attempt to ask whether inhibiting this expansion can interfere with autoantibody generation. We show that characteristics of vascular expansion in enlarging MRL-lpr/lpr lymph nodes resemble that of the VEGF-dependent expansion that occurs in wild-type mice after model immunization. Surprisingly, treatment with SU5416, an inhibitor of VEGF and other receptor tyrosine kinases, did not have sustained effects in inhibiting vascular growth, but attenuated the anti-dsDNA response and altered the phenotype of the double negative T cells that are expanded in these mice. In examining for anatomic correlates of these immunologic changes, we found that the double negative T cells are localized within ectopic follicles around a central B cell patch and that these T cell-rich areas lack the T zone stromal protein ER-TR7 as well as other elements of a normal T zone microenvironment. SU5416 treatment disrupted these follicles and normalized the association between T zone microenvironmental elements and T cell-rich areas. Recent studies have shown a regulatory role for T zone stromal elements. Thus, our findings of the association of anti-dsDNA responses, double negative T cell phenotype, and altered lymphocyte microenvironment suggest the possibility that lymphocyte localization in ectopic follicles protects them from regulation by T zone stromal elements and functions to maintain autoimmune responses. Potentially, altering the lymphocyte microenvironment that is set up by the vascular-stromal compartment can be a means by which to control undesired autoimmune responses

SU5416 reduces anti-dsDNA and alters T cell phenotype but without sustained effect on vascular growth.

<p>(A–D) MRL-lpr/lpr mice (JAX 006825) were treated for either 2 weeks (A) or 11.5 weeks (B–D) with indicated treatments starting at 8 weeks of age. (E–G) MRL-lpr/lpr (JAX 000485) were treated for 5–6 weeks with SU5416 starting at 10 weeks of age. (A) Endothelial cell numbers in brachial lymph nodes after 2 weeks of SU5416 treatment. n = 3 mice per condition. (B) Endothelial cell numbers in brachial lymph nodes after 11.5 weeks of SU5416 treatment. n = 4 DMSO mice and 3 SU5416 mice. Representative of 2 similar experiments. (C) Anti-dsDNA titers and (D) total IgG titers in serum over time. For (C–D), ELISA for serum from 0, 2, and 4 weeks was run independently from serum at 8, 10, and 11.5 weeks. n = 4 mice per treatment except at 11.5 weeks, when n = 3 for SU5416 (see text). Representative of 2 similar experiments. (E) Number of plasma cells per organ. n = 4 mice per treatment over 3 experiments. (F) Number of anti-dsDNA-secreting cells as determined by ELISPOT. n = 4 mice per treatment over 3 experiments. (G) Syndecan levels on indicated T cell populations from SU5416 or DMSO-treated mice. Representative of 4 mice over 3 experiments. For (A–F), * = p<.05 and * = p<.01 when SU5416 treatment compared to DMSO treatment using t-test.</p

SU5416 induces infiltration of ER-TR7 into the B cell areas within ectopic follicles.

<p>MRL-lpr-lpr mice were treated with SU5416 or DMSO vehicle for 11.5 weeks starting at 8 weeks of age and lymph nodes were examined. For comparison to a stimulated wild-type lymph node, 10 week old MRL+/+ mice were immunized with OVA/CFA and draining brachial lymph nodes were examined at day 8. (A, D) nearby sections from a single brachial lymph node from a DMSO-treated MRL-lpr/lpr mouse were stained as indicated. (B, E) Nearby sections from a single axillary lymph node from a DMSO-treated MRL-lpr/lpr mouse were stained as indicated. (C,F) Nearby sections from a single axillary lymph node from a SU5416-treated MRL-lpr/lpr mouse were stained as indicated. (G) Higher magnification of B cell follicle border indicated in (D, arrow). (H) Higher magnification of ectopic follicle border indicated in (E, arrow). (I, J) Higher magnification of border of B cell areas indicated in (F). (I) is magnification of area denoted by single arrow and (J) is magnification of area denoted by double arrows. Scale bars represent 500 um.</p

Follicles of double negative T cells exclude T zone constituents and SU5416 normalizes the microenvironment.

<p>MRL-lpr-lpr mice were treated with SU5416 or DMSO vehicle for 11.5 weeks starting at 8 weeks of age and lymph nodes were examined. For comparison to a stimulated wild-type lymph node, 10 week old MRL+/+ mice were immunized with OVA/CFA and draining brachial lymph nodes were examined at day 8. (Part i of A–F), nearby sections from a single day 8 MRL+/+ brachial lymph node were stained as indicated. (Part ii of A–F), nearby sections from a single brachial lymph node from a DMSO-treated MRL-lpr/lpr mouse were stained as indicated. (Part iii of A–F), higher magnification of boxed area in corresponding images in part ii. (Part iv of A–F), nearby sections from a single brachial lymph node from a SU5416 -treated MRL-lpr/lpr mouse were stained as indicated. (Part v of A–F), higher magnification of boxed area in corresponding images in part iv. (G–H) Higher magnification of (G) medullary and (H) T zone areas in (Fi). “F” denotes follicle. (I–J) Higher magnification of (I) medullary-like and (J) T zone-like areas in (Fiii). (K) Higher magnification of normalized T zone area in (Fv). Scale bars represent 500 um.</p