Mycobacterial Phosphatidylinositol Mannosides Negatively Regulate Host Toll-like Receptor 4, MyD88-dependent Proinflammatory Cytokines, and TRIF-dependent Co-stimulatory Molecule Expression

Mycobacterium tuberculosis modulates host immune responses through proteins and complex glycolipids. Here, we report that the glycosylphosphatidylinositol anchor phosphatidyl-myo-inositol hexamannosides PIM₆ or PIM₂ exert potent anti-inflammatory activities. PIM strongly inhibited the Toll-like receptor (TLR4) and myeloid differentiation protein 88 (MyD88)-mediated release of NO, cytokines, and chemokines, including tumor necrosis factor (TNF), interleukin 12 (IL-12) p40, IL-6, keratinocyte-derived chemokine, and also IL-10 by lipopolysaccharide (LPS)-activated macrophages. This effect was independent of the presence of TLR2. PIM also reduced the LPS-induced MyD88-independent, TIR domain-containing adaptor protein inducing interferon β (TRIF)-mediated expression of co-stimulatory receptors. PIM inhibited LPS/TLR4-induced NFκB translocation. Synthetic PIM₁ and a PIM₂ mimetic recapitulated these in vitro activities and inhibited endotoxin-induced airway inflammation, TNF and keratinocyte-derived chemokine secretion, and neutrophil recruitment in vivo. Mannosyl, two acyl chains, and phosphatidyl residues are essential for PIM anti-inflammatory activity, whereas the inosityl moiety is dispensable. Therefore, PIM exert potent antiinflammatory effects both in vitro and in vivo that may contribute to the strategy developed by mycobacteria for repressing the host innate immunity, and synthetic PIM analogs represent powerful anti-inflammatory leads.Multifold interactions between Mycobacterium tuberculosis and host phagocytes determine immune responses to M. tuberculosis and tuberculosis pathogenesis (for review, see Refs. 1 and 2). Alveolar macrophages, the primary host cells for M. tuberculosis, and dendritic cells that carry mycobacterial antigens from the infection site to the draining lymph nodes to establish a T cell-mediated immune response contribute to modulate the innate immune response by secreting cytokines after recognition of microbial motives. Among them, TNF² is an essential mediator for granuloma formation and containment of M. tuberculosis infection. Similarly, IL-12, interferon γ, but also IL-1, IL-18, IL-23, and nitric oxide are required for host defense (1–6). Phagocytes are also a source of immuno-modulatory cytokines, such as IL-10 and transforming growth factor-β, which dampen the immune response and inflammation. Mycobacteria-derived molecules down-modulating the immune system have been described, including the protein ESAT-6, mannose-capped lipoarabinomannan (ManLAM), and lipomannans (LM) (7–12). Here, we report that phosphatidyl-myo-inositol mannosides (PIM), the glycosylphosphatidylinositol (GPI) anchor structure of LAM and LM, exert strong anti-inflammatory activities.Mycobacterial cell wall LAM, LM, and PIM are recognized by macrophages and dendritic cells through various pattern recognition receptors, including Toll-like receptors (TLRs) (13–16) and C-type lectins such as mannose receptor (MR/CD206) and dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN/CD209), central to M. tuberculosis binding and internalization by human dendritic cells (17–20). DC-SIGN and mannose receptor were proposed to mediate ManLAM inhibition of LPS-induced IL-12 production in dendritic cells, an activity ascribed to the mannosylated cap (8, 9). We showed recently that mycobacterial LM have a dual potential for pro-inflammatory and anti-inflammatory effects (11), tri- and tetra-acylated LM fractions exerting stimulatory effects through TLR2, TLR4, and MyD88 (21), whereas diacylated LM inhibit LPS-induced cytokine response independently of TLR2, SIGN-R1, and mannose receptor (12).PIM are biosynthetic precursors of LM and LAM (22–25). Dimannoside (PIM₂) and hexamannoside (PIM₆) PIM are the two most abundant classes of PIM found in M. tuberculosis H37Rv and Mycobacterium bovis BCG (see Fig. 1). PIM purification and molecular chemical characterization revealed four major acyl forms, mono- to tetra-acylated (lyso-PIM for one acyl, PIM for two acyl, Ac₁PIM for three acyl, and Ac₂PIM for four acyl, respectively; see Fig. 1) for both PIM₂ and PIM₆ (26–29). Higher order PIM with mannose cap-like structures were found to preferentially associate with human MR and to contribute to phagosome-lysosome fusion (20). The degree of acylation influenced higher order PIM association with the MR, whereas PIM₂ was recognized by DC-SIGN independently of its acylation degree. The complete synthesis of the different PIM has recently been reported (30–33).Open in a separate windowFIGURE 1.Natural PIM and synthetic PIM₁ and PIM₂ mimetics used in the study. Shown is a schematic representation of natural lyso-PIM₆, PIM₆, Ac₁PIM₆, Ac₂PIM₆, and PIM₂ (A) and synthetic PIM₁ (B) showing the C16 and C18 acyl groups on glycerol chain positions sn-2 and sn-1, the precursor PI, a synthetic mimetic of PIM₂ (PIM₂ mimetic) bearing C16 and C18 acyl chains, the de-acylated control molecule precursor of the PIM₂ mimetic (de-AcPIM₂ mimetic), and a PIM₂ mimetic with replacement of the phosphodiester moiety by a carbonate.Here, we analyzed isolated acyl forms of PIM and identified PIM₂ and PIM₆ but also synthetic PIM₁ and a mimetic of PIM₂ as strong inhibitors of endotoxin-induced proinflammatory responses in vitro and in vivo. Using macrophages from genetically modified mice, we characterized PIM inhibitory effects on MyD88, TRIF, and NFκB signaling pathways. Hence, not only complex mycobacterial lipoglycans like ManLAM and LM but also small molecular weight PIM analogues are potent inhibitors of host inflammatory responses.     

Mortality after Hospitalization for Pneumonia among Individuals with HIV, 1995–2008: A Danish Cohort Study

Background

HIV–infected persons are at increased risk of pneumonia, even with highly active antiretroviral treatment (HAART). We examined the impact of pneumonia on mortality and identified prognostic factors for death among HIV–infected.

Methodology/Principal Findings

In a nationwide, population-based cohort of individuals with HIV, we included persons hospitalized with pneumonia from the Danish National Hospital Registry and obtained mortality data from the Danish Civil Registration System. Comparing individuals with and without pneumonia, we used Poisson regression to estimate relative mortality and logistic regression to examine prognostic factors for death following pneumonia. From January 1, 1995, to July 1, 2008, we observed 699 episodes of first hospitalization for pneumonia among 4,352 HIV patients. Ninety-day mortality after pneumonia decreased from 22.4% (95% confidence interval [CI]: 16.5%–28.9%) in 1995–1996 to 8.4% (95% CI: 6.1%–11.6%) in 2000–2008. Mortality remained elevated for more than a year after hospitalization for pneumonia: adjusted mortality rate ratio 5.38 (95% CI: 4.27–6.78), 1.80 (95% CI: 1.36–2.37), and 1.62 (95% CI: 1.32–2.00) for days 0–90, 91–365, and 366+, respectively. The following variables predicted mortality within 90 days following hospitalization for pneumonia (adjusted Odds Ratios): male sex (3.77, 95% CI: 1.37–10.4), Charlson Comorbidity Index score ≥2 (3.86, 95% CI: 2.19–6.78); no current HAART (3.58, 95% CI: 1.83–6.99); history of AIDS (2.46, 95% CI: 1.40–4.32); age per 10 year increase (1.43, 95% CI: 1.11–1.85); and CD4+ cell count ≤200 (2.52, 95% CI: 1.37–4.65).

Conclusions/Significance

The first hospitalization for pneumonia among HIV–infected individuals was associated with elevated risk of death up to more than a year later. Use of HAART decreased the risk, independent of current CD4+ cell count. Prognosis following pneumonia improved over calendar time.     

Amino acid size, charge, hydropathy indices and matrices for protein structure analysis

Background  

Prediction of protein folding and specific interactions from only the sequence (ab initio) is a major challenge in bioinformatics. It is believed that such prediction will prove possible if Anfinsen's thermodynamic principle is correct for all kinds of proteins, and all the information necessary to form a concrete 3D structure is indeed present in the sequence.     

Molecular basis for triclosan activity involves a flipping loop in the active site

The crystal structure of the Escherichia coli enoyl reductase-NAD+-triclosan complex has been determined at 2.5 A resolution. The Ile192-Ser198 loop is either disordered or in an open conformation in the previously reported structures of the enzyme. This loop adopts a closed conformation in our structure, forming van der Waals interactions with the inhibitor and hydrogen bonds with the bound NAD+ cofactor. The opening and closing of this flipping loop is likely an important factor in substrate or ligand recognition. The closed conformation of the loop appears to be a critical feature for the enhanced binding potency of triclosan, and a key component in future structure-based inhibitor design.     

Phosphorylation of the mitochondrial protein Sab by stress-activated protein kinase 3

Mitogen-activated protein kinases (MAPKs) transduce extracellular signals into responses such as growth, differentiation, and death through their phosphorylation of specific substrate proteins. Early studies showed the consensus sequence (Pro/X)-X-(Ser/Thr)-Pro to be phosphorylated by MAPKs. Docking domains such as the "kinase interaction motif" (KIM) also appear to be crucial for efficient substrate phosphorylation. Here, we show that stress-activated protein kinase-3 (SAPK3), a p38 MAPK subfamily member, localizes to the mitochondria. Activated SAPK3 phosphorylates the mitochondrial protein Sab, an in vitro substrate of c-Jun N-terminal kinase (JNK). Sab phosphorylation by SAPK3 was dependent on the most N-terminal KIM (KIM1) of Sab and occurred primarily on Ser321. This appeared to be dependent on the position of Ser321 within Sab and the sequence immediately surrounding it. Our results suggest that SAPK3 and JNK may share a common target at the mitochondria and provide new insights into the substrate recognition by SAPK3.     

Neurons expressing the highest levels of gamma-synuclein are unaffected by targeted inactivation of the gene

Homologous recombination in ES cells was employed to generate mice with targeted deletion of the first three exons of the gamma-synuclein gene. Complete inactivation of gene expression in null mutant mice was confirmed on the mRNA and protein levels. Null mutant mice are viable, are fertile, and do not display evident phenotypical abnormalities. The effects of gamma-synuclein deficiency on motor and peripheral sensory neurons were studied by various methods in vivo and in vitro. These two types of neurons were selected because they both express high levels of gamma-synuclein from the early stages of mouse embryonic development but later in the development they display different patterns of intracellular compartmentalization of the protein. We found no difference in the number of neurons between wild-type and null mutant animals in several brain stem motor nuclei, in lumbar dorsal root ganglia, and in the trigeminal ganglion. The survival of gamma-synuclein-deficient trigeminal neurons in various culture conditions was not different from that of wild-type neurons. There was no difference in the numbers of myelinated and nonmyelinated fibers in the saphenous nerves of these animals, and sensory reflex thresholds were also intact in gamma-synuclein null mutant mice. Nerve injury led to similar changes in sensory function in wild-type and mutant mice. Taken together, our data suggest that like alpha-synuclein, gamma-synuclein is dispensable for the development and function of the nervous system.     

Anatomy of the nasal cartilages of the unilateral complete cleft lip nose

The purpose of this study was to disclose the relationship between the anomaly of the cartilaginous framework and the nasal deformity of cleft lip. The noses of six stillborn infants with unilateral complete cleft lip were carefully dissected. The size and weight of the lower lateral cartilages were measured to determine whether there was a significant difference between the normal and involved sides. The position of the nasal cartilages was observed, and the distance between them was measured to determine whether they were normal. The surgical dissection revealed that the lower lateral cartilages from both sides were asymmetrical in three dimensions, indicating the displacement of the lower lateral cartilage on the involved side. There was displacement of the cartilaginous septum and the upper lateral cartilage. The statistical evaluation did not demonstrate a significant difference between weight and size of the two sides. One of the major causative factors of nasal deformity is displacement of the nasal cartilages. There is no hypoplasia of nasal cartilage in newborn infants with cleft lip.