MAP Kinase Phosphatase-2 Plays a Critical Role in Response to Infection by Leishmania mexicana

In this study we generated a novel dual specific phosphatase 4 (DUSP4) deletion mouse using a targeted deletion strategy in order to examine the role of MAP kinase phosphatase-2 (MKP-2) in immune responses. Lipopolysaccharide (LPS) induced a rapid, time and concentration-dependent increase in MKP-2 protein expression in bone marrow-derived macrophages from MKP-2+/+ but not from MKP-2−/− mice. LPS-induced JNK and p38 MAP kinase phosphorylation was significantly increased and prolonged in MKP-2−/− macrophages whilst ERK phosphorylation was unaffected. MKP-2 deletion also potentiated LPS-stimulated induction of the inflammatory cytokines, IL-6, IL-12p40, TNF-α, and also COX-2 derived PGE2 production. However surprisingly, in MKP-2−/− macrophages, there was a marked reduction in LPS or IFNγ-induced iNOS and nitric oxide release and enhanced basal expression of arginase-1, suggesting that MKP-2 may have an additional regulatory function significant in pathogen-mediated immunity. Indeed, following infection with the intracellular parasite Leishmania mexicana, MKP-2−/− mice displayed increased lesion size and parasite burden, and a significantly modified Th1/Th2 bias compared with wild-type counterparts. However, there was no intrinsic defect in MKP-2−/− T cell function as measured by anti-CD3 induced IFN-γ production. Rather, MKP-2−/− bone marrow-derived macrophages were found to be inherently more susceptible to infection with Leishmania mexicana, an effect reversed following treatment with the arginase inhibitor nor-NOHA. These findings show for the first time a role for MKP-2 in vivo and demonstrate that MKP-2 may be essential in orchestrating protection against intracellular infection at the level of the macrophage

TLR1/2 Activation during Heterologous Prime-Boost Vaccination (DNA-MVA) Enhances CD8+ T Cell Responses Providing Protection against Leishmania (Viannia)

Leishmania (Viannia) are the predominant agents of leishmaniasis in Latin America. Given the fact that leishmaniasis is a zoonosis, eradication is unlikely; a vaccine could provide effective prevention of disease. However, these parasites present a challenge and we do not fully understand what elements of the host immune defense prevent disease. We examined the ability of vaccination to protect against L. (Viannia) infection using the highly immunogenic heterologous prime-boost (DNA-modified vaccinia virus) modality and a single Leishmania antigen (TRYP). Although this mode of vaccination can induce protection against other leishmaniases (cutaneous, visceral), no protection was observed against L. (V.) panamensis. However, we found that if the vaccination was modified and the innate immune response was activated through Toll-like receptor1/2(TLR1/2) during the DNA priming, vaccinated mice were protected. Protection was dependent on CD8 T cells. Vaccinated mice had higher CD8 T cell responses and decreased levels of cytokines known to promote infection. Given the long-term persistence of CD8 T cell memory, these findings are encouraging for vaccine development. Further, these results suggest that modulation of TLR1/2 signaling could improve the efficacy of DNA-based vaccines, especially where CD8 T cell activation is critical, thereby contributing to effective and affordable anti parasitic vaccines

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

IgG recognizes a single GIPL spot on TLC immunoblot.

<p><b><i>A</i></b>. Purified GIPLs [2×10⁸ cell equivalents (c.e.)] from <i>L. mexicana</i> amastigotes (lanes 1 and 4), <i>L. mexicana</i> stationary-phase promastigotes (lanes 2 and 5), and <i>L. major</i> stationary-phase promastigotes (lanes 3 and 6) were separated by TLC. Lanes 1–3 were stained by immunoblot with serum from <i>L. mexicana</i>-infected mice (from 29 wks). Lanes 4–6 were stained with orcinol to visualize GIPLs. O, origin; F, front; GIPL, the GIPL recognized by anti-serum. <b><i>B</i></b>. Immunoblot as in <i>A</i>: <i>L. mexicana</i> lesion amastigote GIPL (lane 1, 2×10⁸ c.e.), <i>L. mexicana</i> axenic amastigote GIPL (lane 2, 10⁸ c.e.). Data represent at least 12 experiments using 4 different sera, with similar results.</p

Phospholipase A₂ abolishes IgG binding to the dominant GIPL.

<p><i>L. mexicana</i> amastigote GIPLs (2×10⁸ cell equivalents) were digested with bee venom phospholipase A₂ (PLA₂) and then partitioned into water-saturated <i>n</i>-butanol and water. The aqueous phase was purified by Sep-pak C18 column as described in the <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002224#s2" target="_blank">Materials and Methods</a>. Samples were separated by TLC, and an immunoblot was performed as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002224#pntd-0002224-g005" target="_blank">Fig. 5</a>. Lane 1, untreated <i>L. mexicana</i> amastigote GIPLs; 2, aqueous phase of mock digest; 3, <i>n</i>-butanol phase of mock digest; 4, aqueous phase of PLA₂ digest; 5, <i>n</i>-butanol phase of PLA₂ digest. The sample in lanes 4 and 5 was not digested to completion. Data represent 4 experiments with similar results.</p

Antibodies from <i>L. mexicana</i>-infected patients induce IL-10 from human monocytes.

<p>LPS-stimulated human blood monocytes were infected with <i>L. mexicana</i> axenic amastigotes pre-opsonized with uninfected Mexican control sera (nl), or sera from patients with localized cutaneous leishmaniasis (LCL), or sera from patients with diffuse cutaneous leishmaniasis (DCL). Supernatants were harvested at 20 hours and assayed for IL-10 by ELISA. Sera from individual people are shown in different colors on the left. To allow comparisons, subjects A–D in each group have the same colors as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002224#pntd-0002224-g009" target="_blank">Fig. 9</a>. All LCL and DCL patient sera induced significantly more IL-10 than the uninfected control mean value (<i>P</i><0.05). Unstimulated monocytes without LPS (media) produced no detectable IL-10. LCL patient D serum-opsonized amastigotes induced no IL-10 in the absence of LPS [LCL-D-aa (no LPS)]. All parasite-infected cells stimulated with LPS made more IL-10 than LPS alone (LPS). An uninfected Pennsylvania control (PA nl-aa+LPS), the means for Mexican uninfected controls (Mex nl-aa+LPS), amastigotes opsonized with serum from LCL patients (LCL-aa+LPS), and DCL patients (DCL-aa+LPS) are also shown. Mex nl-aa+LPS, LCL-aa+LPS, and DCL-aa+LPS show the mean and SE for four patients; other samples show mean and SE for quadruplicate samples of a single condition. *, <i>P</i><0.05 for DCL-aa+LPS vs. Mex nl-aa+LPS. N.D., none detected. Data represent 2 experiments with similar results.</p

The immunodominant glycolipid recognized by <i>L. mexicana</i>-infected mouse serum is a GPI and has a branched mannose chain.

<p><i>L. mexicana</i> amastigote GIPLs (lanes 1–4, 6, 8, 9) were digested with <i>T. brucei</i> GPI-PLC, human GPI-PLD, or jack bean α-mannosidase (JBAM), extracted with <i>n</i>-butanol, and separated by TLC. Then serum from <i>L. mexicana</i>-infected mice was used for immunoblot. In addition, <i>Trypanosoma brucei</i> GPI glycolipids were also analyzed by TLC immunoblot in comparison with <i>L. mexicana</i> amastigote GIPLs. Lane 1, 8×10⁷ c.e. GIPL, mock GPI-PLC; lane 2, 8×10⁷ c.e., GPI-PLC; lane 3, 8×10⁷ c.e., mock GPI-PLD; lane 4, 8×10⁷ c.e., GPI-PLD; lane 5, GPI-PLD without <i>L. mex</i> GIPL; lane 6, 2×10⁸ c.e. GIPL; lane 7, 2×10⁸ c.e. <i>T. brucei</i> GPIs; lane 8, 8×10⁸ c.e. GIPL, mock JBAM; lane 9, 8×10⁸ c.e., JBAM. O, origin; F, front; A, immunodominant GIPL; B, de-branched mannose product of immunodominant GIPL. Data represent at least 3 experiments with similar results.</p

<i>L. mexicana</i>-infected B6 mice have an IgG1 response to GIPLs that is parasite species-specific and predicts parasite load.

<p><b><i>A</i></b>. GIPLs from <i>L. mexicana</i> amastigotes were purified and dried onto a polyvinyl chloride 96-well plate, and sera from <i>L. mexicana</i>-infected (<i>L. mex</i>), <i>L. major</i>-infected (<i>L. major</i>), and uninfected (uninf.) B6 mice were assayed for IgG1 binding by ELISA. <b><i>B</i></b>. Serum from B6 mice infected with <i>L. mexicana</i> for 28 wks (1/15 dil) was assayed for anti-GIPL IgG1 as in <i>A</i> and correlated with log parasite loads (also at 28 wks) determined by limiting dilution. <b><i>C</i></b>. Anti-GIPL IgG1 responses at 16 wks post-infection were correlated with parasite loads at 29 wks post-infection in the same mice. <b><i>D</i></b>. Anti-GIPL IgG1 responses at 16 wks of infection were plotted against lesion sizes at 16 wks. Individual dots represent individual mice. The data represent two experiments with similar results. R² for correlations are shown.</p

mAb binds GIPLs and the surface of amastigotes, and induces IL-10 from macrophages.

<p><b><i>A</i></b>. A newly developed mAb (26H3T-B4) was bound to <i>L. mexicana</i> axenic amastigotes at varying amounts per 10⁶ axenic amastigotes and detected using PE-goat anti-mouse IgG by flow cytometry, with geometric mean fluorescence intensity (GMFI) calculated for samples in triplicate; a mouse IgG1 isotype control was used at the same concentrations (isotype ctrl). Triplicate samples with no antibody (no Ab) are shown with a line for comparison. At all concentrations, GMFI were different for 26H3T-B4 vs. isotype control; GMFI for 26H3T-B4 were also different from no antibody controls. <i>P</i><0.02 for all comparisons. <b><i>B</i></b>. Biotinylated 26H3T-B4 was tested for GIPL binding by ELISA at varying concentrations. <b><i>C</i></b>. Bone marrow-derived macrophages were incubated in media alone, or stimulated with lipopolysaccharide (LPS), <i>L. mexicana</i> axenic amastigotes+LPS (a.a.+LPS), or amastigotes opsonized with 26H3T-B4+LPS (26H3T-B4 ops a.a.+LPS), and then supernatants were assayed for IL-10 by ELISA. *, <i>P</i><0.004 for all comparisons. Data represent at least 2 experiments with similar results.</p

Opsonization with early and late <i>L. mexicana</i> anti-sera.

<p>Sera from mice infected with <i>L. mexicana</i> for 9 wks (n = 5) and 27 wks (n = 6) were used to opsonize <i>L. mexicana</i> axenic amastigotes. <b><i>A</i></b>. Parasite surface mouse-IgG1 and -IgG2a/c were detected by flow cytometry. Unopsonized amastigotes (thin black), uninfected serum (thick black), and serum from infected mice (colored) are shown. <b><i>B</i></b>. Data from <i>A</i> with Geometric Mean Fluorescence Intensity (GMFI) are shown for each time point, with mean and SE. <i>P</i> = 6×10⁻⁵ for the difference between 9- and 27-wk samples. Data are representative of 2 similar experiments.</p