Two biochemically distinct lipophosphoglycans from Leishmania braziliensis and Leishmania infantum trigger different innate immune responses in murine macrophages

BACKGROUND: The dominant, cell surface lipophosphoglycan (LPG) of Leishmania is a multifunctional molecule involved in the interaction with vertebrate and invertebrate hosts. Although the role of LPG on infection has been extensively studied, it is not known if LPG interspecies variations contribute to the different immunopathologies of leishmaniases. To investigate the issue of interspecies polymorphisms, two Leishmania species from the New World that express structural variations of side chains of LPG repeat units were examined. In this context, the procyclic form of L. braziliensis LPG (strain M2903), is devoid of side chains, while the L. infantum LPG (strain BH46) has up to three glucoses residues in the repeat units. METHODS: Mice peritoneal macrophages from Balb/c, C57BL/6 and knock-out (TLR2 -/-, TLR4 -/-) were primed with IFN-γ and stimulated with purified LPG from both species. Nitric oxide and cytokine production, MAPKs (ERK, p38 and JNK) and NF-kB activation were evaluated. RESULTS: Macrophages stimulated with L. braziliensis LPG, had a higher TNF-α, IL-1β, IL-6 and NO production than those stimulated with that of L. infantum. Furthermore, the LPGs from the two species resulted in differential kinetics of signaling via MAPK activation. L. infantum LPG exhibited a gradual activation profile, whereas L. braziliensis LPG showed a sharp but transient activation. L. braziliensis LPG was able to activate NF-kB. CONCLUSION: These data suggest that two biochemically distinct LPGs were able to differentially modulate macrophage functions

A Subset of Liver NK T Cells is Activated During \u3cem\u3eLeishmania donovani\u3c/em\u3e Infection by CD1d-Bound Lipophosphoglycan

Natural killer (NK) T cells are activated by synthetic or self-glycolipids and implicated in innate host resistance to a range of viral, bacterial, and protozoan pathogens. Despite the immunogenicity of microbial lipoglycans and their promiscuous binding to CD1d, no pathogen-derived glycolipid antigen presented by this pathway has been identified to date. In the current work, we show increased susceptibility of NK T cell–deficient CD1d−/− mice to Leishmania donovani infection and Leishmania-induced CD1d-dependent activation of NK T cells in wild-type animals. The elicited response was Th1 polarized, occurred as early as 2 h after infection, and was independent from IL-12. The Leishmania surface glycoconjugate lipophosphoglycan, as well as related glycoinositol phospholipids, bound with high affinity to CD1d and induced a CD1d-dependent IFNγ response in naive intrahepatic lymphocytes. Together, these data identify Leishmania surface glycoconjugates as potential glycolipid antigens and suggest an important role for the CD1d–NK T cell immune axis in the early response to visceral Leishmania infection

A Subset of Liver NK T Cells Is Activated during Leishmania donovani Infection by CD1d-bound Lipophosphoglycan

Natural killer (NK) T cells are activated by synthetic or self-glycolipids and implicated in innate host resistance to a range of viral, bacterial, and protozoan pathogens. Despite the immunogenicity of microbial lipoglycans and their promiscuous binding to CD1d, no pathogen-derived glycolipid antigen presented by this pathway has been identified to date. In the current work, we show increased susceptibility of NK T cell–deficient CD1d−/− mice to Leishmania donovani infection and Leishmania-induced CD1d-dependent activation of NK T cells in wild-type animals. The elicited response was Th1 polarized, occurred as early as 2 h after infection, and was independent from IL-12. The Leishmania surface glycoconjugate lipophosphoglycan, as well as related glycoinositol phospholipids, bound with high affinity to CD1d and induced a CD1d-dependent IFNγ response in naive intrahepatic lymphocytes. Together, these data identify Leishmania surface glycoconjugates as potential glycolipid antigens and suggest an important role for the CD1d–NK T cell immune axis in the early response to visceral Leishmania infection

Glycoinositolphospholipids from Leishmania braziliensis and L. infantum: Modulation of Innate Immune System and Variations in Carbohydrate Structure

The essential role of the lipophosphoglycan (LPG) of Leishmania in innate immune response has been extensively reported. However, information about the role of the LPG-related glycoinositolphospholipids (GIPLs) is limited, especially with respect to the New World species of Leishmania. GIPLs are low molecular weight molecules covering the parasite surface and are similar to LPG in sharing a common lipid backbone and a glycan motif containing up to 7 sugars. Critical aspects of their structure and functions are still obscure in the interaction with the vertebrate host. In this study, we evaluated the role of those molecules in two medically important South American species Leishmania infantum and L. braziliensis, causative agents of visceral (VL) and cutaneous Leishmaniasis (CL), respectively. GIPLs derived from both species did not induce NO or TNF-α production by non-primed murine macrophages. Additionally, primed macrophages from mice (BALB/c, C57BL/6, TLR2−/− and TLR4−/−) exposed to GIPLs from both species, with exception to TNF-α, did not produce any of the cytokines analyzed (IL1-β, IL-2, IL-4, IL-5, IL-10, IL-12p40, IFN-γ) or p38 activation. GIPLs induced the production of TNF-α and NO by C57BL/6 mice, primarily via TLR4. Pre incubation of macrophages with GIPLs reduced significantly the amount of NO and IL-12 in the presence of IFN-γ or lipopolysaccharide (LPS), which was more pronounced with L. braziliensis GIPLs. This inhibition was reversed after PI-specific phospholipase C treatment. A structural analysis of the GIPLs showed that L. infantum has manose rich GIPLs, suggestive of type I and Hybrid GIPLs while L. braziliensis has galactose rich GIPLs, suggestive of Type II GIPLs. In conclusion, there are major differences in the structure and composition of GIPLs from L. braziliensis and L. infantum. Also, GIPLs are important inhibitory molecules during the interaction with macrophages

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Types of GIPLs.

<p>For information on M2, M3, iM2, GIPL-A, isoM3 and isoM4, see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001543#s1" target="_blank">introduction</a>. Fatty acid chains vary in different GIPL species: The predominant type fatty acid in Type-1 and Hybrid GIPLs is C_18∶0, in type-2 GIPLs the predominant lipids are C_18∶0, C_22∶0 C_24∶0 and C_26∶0. “R” in Type 1 GIPLs represent a protein linked to the GIPL structure by a ethanolamine phosphate residue (e.g. gp63 surface metalloprotease) <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001543#pntd.0001543-McConville1" target="_blank">[31]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001543#pntd.0001543-Ralton1" target="_blank">[78]</a>.</p

Thin layer chromatography (TLC) of <i>Leishmania</i> glycoinositolphospholipids (GIPLs).

<p>(A) Purified intact GIPLs: Lane 1, <i>L. braziliensis</i> GIPLs (Gb); lane 2, <i>L. infantum</i> GIPLs (Gi) and lane 3, <i>L. donovani</i> GIPLs (Gd). The assignments for <i>L. donovani</i> structures are: isoM2 as Manα1-3Manα1-4GlcN-PI; isoM3 as Manα1-6(Manα1-3)Manα1-4GlcN-PI and isoM4 as Manα1-2Manα1-6(Manα1-3)Manα1-4GlcN-PI <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001543#pntd.0001543-McConville2" target="_blank">[32]</a>. LPG, lipophosphoglycan; GPI, glicosyl phosphatidylinositol. (B) Deaminated GIPLs. Lane 1, <i>L. braziliensis</i> untreated GIPLs (Gb); lane 2, deaminated <i>L. braziliensis</i> GIPLs (Gb deam.); lane 3, <i>L. infantum</i> untreated GIPLs (Gi) and Lane 4, deaminated <i>L. infantum</i> GIPLs (Gi deam.).</p

Fluorophore-assisted carbohydrate electrophoresis (FACE) of <i>Leishmania</i> GIPLs.

<p>Lane 1, oligoglucose ladder represented by G2-G7; lane 2, <i>L. braziliensis</i> GIPLs (Gb) and lane 3, <i>L. infantum</i> GIPLs (Gi) and lane 4, <i>L. donovani</i> GIPLs (Gd).</p

Monosaccharide profile of <i>Leishmania</i> glycoinositolphospholipids (GIPLs).

<p>(A) Fluorophore-assisted carbohydrate electrophoresis (FACE). Lane 1, standards represented by galactose, glucose and mannose (100 µg/ml); lane 2, <i>L. braziliensis</i> GIPLs (Gb); lane 3, <i>L. infantum</i> GIPLs (Gi) and Lane 4, <i>L. donovani</i> GIPLs (Gd). (B) High performance liquid chromatography (HPLC). Gal, galactose; Glc, glucose and Man, mannose.</p

Modulation of nitrite, TNF-α and IL-12 production by <i>Leishmania</i> GIPLs in BALB/c macrophages.

<p>Cells were incubated with GIPLs (25 µg/ml) from <i>L. braziliensis</i> (Gb) <i>and L. infantum</i> (Gi) for 15 min prior to stimulation with IFN-γ (100 IU/ml) (A) or LPS (100 ng/mL) (B). Nitrite content was measured by Griess reaction; TNF-α and IL-12 concentrations were measured by ELISA. P<0.05 was considered significant. Results are the representation of three experiments.</p