Mycobacterium tuberculosis (Mtb) is able to evade the immune defenses and may persist for years, decades and even lifelong in the infected host. Mtb cell wall components may contribute to such persistence by modulating several pivotal types of immune cells. Dendritic cells (DCs) are the most potent antigen-presenting cells and hence play a crucial role in the initial immune response to infections by connecting the innate with the adaptive immune system.
We investigated the effects of two of the major mycobacterial cell wall-associated types of glycolipids, mannose-capped lipoarabinomannan (ManLAM) and phosphatidylinositol mannosides (PIMs) purified from the Mtb strains H37Rv and Mycobacterium bovis, on the maturation and cytokine profiles of immature human monocyte-derived DCs. ManLAM from Mtb H37Rv stimulated the release of pro-inflammatory cytokines TNF, IL-12, and IL-6 and expression of co-stimulatory (CD80, CD86) and antigen-presenting molecules (MHC class II). ManLAM from M. bovis also induced TNF, IL-12 and IL-6 but at significantly lower levels. Importantly, while ManLAM was found to augment LPS-induced DC maturation and pro-inflammatory cytokine production, addition of PIMs from both Mtb H37Rv and M. bovis strongly reduced this stimulatory effect.
These results indicate that the mycobacterial cell wall contains macromolecules of glycolipid nature which are able to induce strong and divergent effects on human DCs; i.e while ManLAM is immune-stimulatory, PIMs act as powerful inhibitors of DC cytokine responses. Thus PIMs may be important Mtb-associated virulence factors contributing to the pathogenesis of tuberculosis disease. These findings may also aid in the understanding of some earlier conflicting reports on the immunomodulatory effects exerted by different ManLAM preparations.
... Research on the host response to Mtb has so far mainly focused on antigens of protein/peptide nature. However, the immune response to Mtb is initiated mainly through the interaction of Mtb cell envelope components, mostly glycolipids, with cells of the innate immune system (3), which trigger activating or repressive responses in terms of cytokine production (4,5). Glycolipids are abundantly expressed in all mycobacterial species. ...
... Tuberculin PPD (RT 50) was obtained from Statens Serum Institute, and PHA from Invivogen. LAM and PIM were prepared in-house (5). PIM contains both PIM2 and PIM6 isoforms, differing in number of fatty acyl constituents (5). ...
... LAM and PIM were prepared in-house (5). PIM contains both PIM2 and PIM6 isoforms, differing in number of fatty acyl constituents (5). ...
Upon infection with Mycobacterium tuberculosis (Mtb) the host immune response might clear the bacteria, control its growth leading to latent tuberculosis (LTB), or fail to control its growth resulting in active TB (ATB). There is however no clear understanding of the features underlying a more or less effective response. Mtb glycolipids are abundant in the bacterial cell envelope and modulate the immune response to Mtb, but the patterns of response to glycolipids are still underexplored. To identify the CD45+ leukocyte activation landscape induced by Mtb glycolipids in peripheral blood of ATB and LTB, we performed a detailed assessment of the immune response of PBMCs to the Mtb glycolipids lipoarabinomannan (LAM) and its biosynthetic precursor phosphatidyl-inositol mannoside (PIM), and PPD. At 24 h and 5 days of stimulation, cell profiling and secretome analysis was done using mass cytometry and high-multiplex immunoassay. PIM mainly affected antigen-presenting cells to produce both proinflammatory (IL-2, IL-6, IL-17A, TNF-α and GM-CSF), and IL-4 and IL-10 cytokines, but not IFN-γ. LAM triggered a similar, albeit weaker, response. By contrast, PPD induced an increase in IFN-γ-producing cells. Moreover, PPD also led to increased numbers of IL-2, IL-6, IL-10, IL-17A, TNF-α and GrzB-producing cells. Treatment with an anti-TLR2 antibody led to partial inhibition of PIM-induced IL-6 production in myeloid cells, suggesting that PIM induces IL-6 production through TLR2. Expansion of monocyte subsets in response to PIM or LAM was reduced in both ATB and LTB as compared to healthy controls, suggesting a hyporesponsive/tolerance pattern in Mtb-infected individuals.
... Research on the host response to Mtb has so far mainly focused on protein-based antigens. However, the immune response to Mtb is initiated mainly through the interaction of Mtb cell envelope components, mostly glycolipids, with distinct cells of the innate immune system (3), which trigger activating or repressive responses in terms of cytokine production (4,5). The ability of Mtb lipids to traffic outside infected cells (6)(7)(8) renders the direct contact of Mtb cell envelope glycolipids with distinct immune cells an important aspect of the immune response (9). ...
... Tuberculin PPD (RT 50) was obtained from Statens Serum Institute, and PHA from In vivogen. LAM and PIMs were prepared as previously described in detail (5). Briefly, heatkilled bacteria were frozen and thawed several times, sonicated and extracted in 40% hot phenol for 1 h at 70°C. ...
Upon infection with Mycobacterium tuberculosis (Mtb) the host immune response might clear the bacteria, control its growth leading to latent tuberculosis (LTB), or fail to control its growth resulting in active TB (ATB). There is however no clear understanding of the features underlying a more or less effective response. Mtb glycolipids are abundant in the bacterial cell envelope and modulate the immune response to Mtb, but the patterns of response to glycolipids are still underexplored. To identify the CD45+ leukocyte activation landscape induced by Mtb glycolipids in peripheral blood of ATB and LTB, we performed a detailed assessment of the immune response of PBMCs to the Mtb glycolipids lipoarabinomannan (LAM) and its biosynthetic precursor phosphatidyl-inositol mannoside (PIM), and purified-protein derivate (PPD). At 24 h of stimulation, cell profiling and secretome analysis was done using mass cytometry and high-multiplex immunoassay. PIM induced a diverse cytokine response, mainly affecting antigen-presenting cells to produce both pro-inflammatory and anti-inflammatory cytokines, but not IFN-γ, contrasting with PPD that was a strong inducer of IFN-γ. The effect of PIM on the antigen-presenting cells was partly TLR2-dependent. Expansion of monocyte subsets in response to PIM or LAM was reduced primarily in LTB as compared to healthy controls, suggesting a hyporesponsive/tolerance pattern derived from Mtb infection.
... Furthermore, the difference in immune cells and experimental conditions used also lead to the discrepancies between different immunological assays. [33,39,40] More critically, the SL-I used in most of these assays does not represent the major SGL component of the mycobacterial outer membranes. ...
Mycobacterium tuberculosis (Mtb) causes tuberculosis as one major threat to human health, which has been deteriorated owing to the emerging multidrug resistance. Mtb contains a complex lipophilic cell wall structure that is important for bacterial persistence. Among the lipid components, sulfoglycolipids (SGLs), known to induce immune cell responses, are composed of a trehalose core attached with a conserved sulfate group and 1–4 fatty acyl chains in an asymmetric pattern. At least one of these acyl chains is polymethylated with 3–12 methyl branches. Although Mtb SGL can be isolated from bacterial culture, resulting SGL is still a homologous mixture, impeding accurate research studies. This up‐to‐date review covers the chemical synthesis and immunological studies of Mtb SGLs and structural analogues, with an emphasis on the development of new glycosylation methods and the asymmetric synthesis of polymethylated scaffolds. Both are critical to advance further research on biological functions of these complicated SGLs.
... ManLAM from virulent Mtb H37Rv induced the maturation of DCs and secretion of IL-6, IL-12 and TNF-a in human DCs (Mazurek et al., 2012), and promotes increased Ag presentation (Yonekawa et al., 2014). Subsequently, it has been reported that ManLAM-induced DC activation occurs via Dectin-2 (Yonekawa et al., 2014). ...
Dendritic cells (DCs) are principal defense components that play multifactorial roles in translating innate immune responses to adaptive immunity in Mycobacterium tuberculosis (Mtb) infections. The heterogeneous nature of DC subsets follows their altered functions by interacting with other immune cells, Mtb, and its products, enhancing host defense mechanisms or facilitating pathogen evasion. Thus, a better understanding of the immune responses initiated, promoted, and amplified or inhibited by DCs in Mtb infection is an essential step in developing anti-tuberculosis (TB) control measures, such as host-directed adjunctive therapy and anti-TB vaccines. This review summarizes the recent advances in salient DC subsets, including their phenotypic classification, cytokine profiles, functional alterations according to disease stages and environments, and consequent TB outcomes. A comprehensive overview of the role of DCs from various perspectives enables a deeper understanding of TB pathogenesis and could be useful in developing DC-based vaccines and immunotherapies.
... , mutations influencing human IL-17F and IL-17-receptor A (IL-17RA) function lead to the development of chronic fungal infection because of a compromised IL-17-mediated immunity(Puel et al., 2011).Dectin-2 recognises MAN-LAM from M. tuberculosis; MAN-LAM has modulatory effects on host immunity as mentioned above(Briken et al., 2004, Mazurek et al., 2012.Yonekawa et al. (2014) reported that bone marrow-derived dendritic cells (BMDCs) stimulated with Man-LAM produce both pro-(TNF-α, IL-6) and anti-(IL-10) inflammatory cytokines, which was completely abrogated in Clec4n-/-BMDCs. The author suggested that this production of pro-and anti-inflammatory cytokines are beneficial for host organisms to maintain balanced immune responses(Yonekawa et al., 2014). ...
Colonisation and chronic lung infection by the opportunistic pathogen Pseudomonas aeruginosa (PA) is the leading cause of morbidity and mortality in cystic fibrosis patients. A critical key determinant of PA pathogenicity is the switch from planktonic to the biofilm mode of growth, which facilitates chronic infections and makes PA eradication extremely difficult. Biofilms are aggregates of microorganisms in which the cells are enclosed in an extracellular polymeric substance (EPS) that contains proteins, extracellular DNA (eDNA) and exopolysaccharides. Psl and Pel are two important exopolysaccharides utilised by PA to construct biofilms. While Psl is composed of D-mannose, D-glucose, and L-rhamnose, Pel contains N-acetylgalactosamine (GalNAc) and N-acetylglucosamine (GlcNAc). C-type lectins receptors (CLRs) are pattern recognition receptors (PRRs) that recognise carbohydrate motifs and are expressed by innate immune cells, including dendritic cells (DCs), macrophages and neutrophils. The overarching aim of this thesis is to investigate the interplay between PA and innate immune cells and how it is affected by biofilm formation. We hypothesise that engagement of CLRs through recognition of carbohydrate structures within PA biofilms could modulate the immune response and interfere with innate immune cells function, hence facilitating the persistence of infection. Previously, our lab demonstrated that PA biofilms and biofilm-associated carbohydrates purified from the Pel-deficient mutant ΔwspF Δpel, engage two CLRs, Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN, CD209) and Mannose receptor (MR, CD206). In this study, we show that Dectin-2 (CLEC6A) is another CLRs that binds PA biofilms and purified biofilm carbohydrates; Dectin-2 binds to biofilms formed by PA strains with different EPS composition as well as biofilms formed by PA wound isolates. Similarly to MR and DC-SIGN, Dectin-2 also recognised purified biofilm-associated carbohydrates. High and low molecular fractions of purified biofilm carbohydrates were tested in this study and Dectin-2, together with DC-SIGN and MR preferentially bound to the HMW preparations. Biofilm-associated HMW carbohydrates did not induce Dectin-2 signalling on huDectin-2 reporter cells or HEK-Blue™ mDectin-2 cells but showed antagonistic activity blocking the response of muDectin-2 reporter cells to the Dectin-2 ligand zymosan. To microscopically examine the distribution of CLRs ligands within the biofilms and to study the interaction of immune cells with biofilm, we optimised biofilm generation in substrates suitable for microscopic examination. Confocal analysis shows DC-SIGN, Dectin-2 and, weakly, MR ligands within PAO1 biofilms concentrated into discrete clusters with additional DC-SIGN ligands dispersed among bacteria aggregates. Monocyte-derived DCs (moDCs) (DC-SIGN+ and MR+) cultured with biofilms formed by strains that overexpress Psl (ΔwspF and ΔwspF Δpel) tend to display round morphology and similar findings were noted when moDCs were incubated with purified biofilm carbohydrates. In a different set of experiments cytokine production by human moDCs in response to biofilms was tested and no differences were seen among biofilms with different carbohydrate compositions or after addition of purified biofilm carbohydrates. Taken together, these data indicate that DC-SIGN, MR and Dectin-2 are receptors for PA biofilms and biofilm-associated carbohydrates. The results of this research support the idea that CLRs engagements upon biofilm recognition by innate immune cells might modulate immune cells function. As the first responder cells of the innate immune system, neutrophils possess a unique set of migration features. Several studies found that neutrophils were not able to eradicate infection caused by biofilm-forming PA, which might indicate that biofilm formation was hindering neutrophil’s activity. Here, we describe an in vitro assay that allows us to examine neutrophil behaviour, using live and confocal microscopy, following their interaction with PA, as a planktonic or biofilm cultures. Using live microscopy analysis, we observed that (1) biofilms reduced neutrophil velocity, net distance covered and the mean square displacement. Using confocal microscopy analysis, we observed that (2) neutrophils appeared unable to release granules when incubated with biofilms. Finally, (3) preliminary observations indicated that cells exhibit higher circularity when incubated with biofilm formed by strains expressing Psl or Psl-deficient biofilms in the presence of purified biofilm carbohydrates. The findings from this study have gone some way towards enhancing our understanding of the potential impact of biofilm formation on innate immune cells which might open new avenues for therapeutic approaches.
Background
The pathogen responsible for tuberculosis is called Mycobacterium tuberculosis. Its interaction with macrophages has a significant impact on the onset and progression of the disease.
Methods
The respiratory pathway allows Mycobacterium tuberculosis to enter the body's lungs where it battles immune cells before being infected latently or actively. In the progress of tuberculosis, Mycobacterium tuberculosis activates the body's immune system and creates inflammatory factors, which cause tissue inflammation to infiltrate and the creation of granulomas, which seriously harms the body. Toll-like receptors of macrophage can mediate host recognition of Mycobacterium tuberculosis, initiate immune responses, and participate in macrophage autophagy. New host-directed therapeutic approaches targeting autophagy for drug-resistant Mycobacterium tuberculosis have emerged, providing new ideas for the effective treatment of tuberculosis.
Conclusions
In-depth understanding of the mechanisms by which macrophage autophagy interacts with intracellular Mycobacterium tuberculosis, as well as the study of potent and specific autophagy-regulating molecules, will lead to much-needed advances in drug discovery and vaccine design, which will improve the prevention and treatment of human tuberculosis.
Glycolipids constitute a major part of the cell envelope of Mycobacterium tuberculosis (Mtb). They are potent immunomodulatory molecules recognized by several immune receptors like pattern recognition receptors such as TLR2, DC-SIGN and Dectin-2 on antigen-presenting cells and by T cell receptors on T lymphocytes. The Mtb glycolipids lipoarabinomannan (LAM) and its biosynthetic relatives, phosphatidylinositol mannosides (PIMs) and lipomannan (LM), as well as other Mtb glycolipids, such as phenolic glycolipids and sulfoglycolipids have the ability to modulate the immune response, stimulating or inhibiting a pro-inflammatory response. We explore here the downmodulating effect of Mtb glycolipids. A great proportion of the studies used in vitro approaches although in vivo infection with Mtb might also lead to a dampening of myeloid cell and T cell responses to Mtb glycolipids. This dampened response has been explored ex vivo with immune cells from peripheral blood from Mtb-infected individuals and in mouse models of infection. In addition to the dampening of the immune response caused by Mtb glycolipids, we discuss the hyporesponse to Mtb glycolipids caused by prolonged Mtb infection and/or exposure to Mtb antigens. Hyporesponse to LAM has been observed in myeloid cells from individuals with active and latent tuberculosis (TB). For some myeloid subsets, this effect is stronger in latent versus active TB. Since the immune response in individuals with latent TB represents a more protective profile compared to the one in patients with active TB, this suggests that downmodulation of myeloid cell functions by Mtb glycolipids may be beneficial for the host and protect against active TB disease. The mechanisms of this downmodulation, including tolerance through epigenetic modifications, are only partly explored.
Lipids represent a less explored class of virulence-associated pathogenic molecules in infectious diseases. Lipids are amphipathic molecules that as part of the cell membranes, orchestrate various cellular processes by modulating membrane biophysical properties such as fluidity, stiffness, packing, curvature, and organization. Changes in the aforementioned membrane properties, in turn, alter lipid/protein diffusion, localization, lipid–protein interactions, and finally their activity. Hence, lipids play critical roles in infectious diseases by intervening in cellular signaling, protein trafficking, membrane fusion, and protein functions. Mycobacterium tuberculosis (Mtb)—the single leading cause of tuberculosis—serves as an epitome of pathogens that use their glycolipids to fine-tune various lipid-dependent interactions with the host cell. The Mtb cell membrane is composed of three layers: inner membrane, arabino-peptidoglycan layer, and outer membrane, and it is the noncovalently attached glycolipids within the outer membrane that are primarily involved in pathogenesis. In this chapter, we give an up-to-date account of recent findings on the biophysical consequences of Mtb lipid-host cell membrane contact, while elucidating the complex cell membrane structure of Mtb, and the specific immunomodulatory properties of distinct Mtb lipids. The presented work showcases how Mtb uses its structurally diverse glycolipids to impact specific host processes including immune modulation during host contact, bestowing the pathogen with a lipid-centric virulence strategy to spatiotemporally regulate host cell interactions by modulating host membrane structure and function.
One of the major defining features of Mycobacterium tuberculosis (Mtb), the bacterium causing tuberculosis, is its characteristic hydrophobic cell wall. The Mtb cell wall is sometimes described as a “miracle of nature” due to its intricate and complex architecture and the abundance of several lipids not found in other genera. These factors, together with their location at the direct interface of the interaction with host, render Mtb cell wall lipids directly responsible for diverse facets of infection such as recognition, uptake, trafficking, and survival. In this chapter, we outline the diverse chemical nature of the Mtb cell envelope components and posit them in the context of their known interaction with host receptors, pathogenesis mechanisms, and outcomes. How the diverse Mtb lipids collectively contribute to distinct infection outcomes remains to be discovered. New research using emerging tools could reveal some of these secrets and provide new armaments against this ancient disease.
In the present study we demonstrate that human monocytes activated by lipopolysaccharides (LPS) were able to produce high levels of interleukin 10 (IL-10), previously designated cytokine synthesis inhibitory factor (CSIF), in a dose dependent fashion. IL-10 was detectable 7 h after activation of the monocytes and maximal levels of IL-10 production were observed after 24-48 h. These kinetics indicated that the production of IL-10 by human monocytes was relatively late as compared to the production of IL-1α, IL-1β, IL-6, IL-8, tumor necrosis factor Oi(TNFα), and granulocyte colony-stimulating factor (G-CSF), which were all secreted at high levels 4-8 h after activation. The production of IL-10 by LPS activated monocytes was, similar to that of IL-1α, IL-1β, IL-6, IL-8, TNFα, granulocyte-macrophage colony-stimulating factor (GM-CSF), and G-CSF, inhibited by IL-4. Furthermore we demonstrate here that IL-10, added to monocytes, activated by interferon γ(IFN-γ), LPS, or combinations of LPS and IFN-γ at the onset of the cultures, strongly inhibited the production of IL-1α, IL-1β, IL-6, IL-8, TNFα, GM-CSF, and G-CSF at the transcriptional level. Viral-IL-10, which has similar biological activities on human cells, also inhibited the production of TNFα and GM-CSF by monocytes following LPS activation. Activation of monocytes by LPS in the presence of neutralizing anti-IL-10 monoclonal antibodies resulted in the production of higher amounts of cytokines relative to LPS treatment alone, indicating that endogenously produced IL-10 inhibited the production of IL-1α, IL-1β, IL-6, IL-8, TNFα, GM-CSF, and G-CSF. In addition, IL-10 had autoregulatory effects since it strongly inhibited IL-10 mRNA synthesis in LPS activated monocytes. Furthermore, endogenously produced IL-10 was found to be responsible for the reduction in class II major histocompatibility complex (MHC) expression following activation of monocytes with LPS. Taken together our results indicate that IL-10 has important regulatory effects on immunological and inflammatory responses because of its capacity to downregulate class II MHC expression and to inhibit the production of proinflammatory cytokines by monocytes.
Mannosylated molecules on the Mycobacterium tuberculosis surface are important determinants in the immunopathogenesis of tuberculosis. To date, much attention has been paid to mannose-capped
lipoarabinomannan, which mediates phagocytosis and intracellular trafficking of M. tuberculosis by engaging the macrophage mannose receptor and subsequently binds to intracellular CD1b molecules for presentation to T
cells. Another important mannosylated lipoglycan on the M. tuberculosis surface is lipomannan (LM). Comparative structural detail of the LMs from virulent and avirulent strains is limited as is
knowledge regarding their differential capacity to be recognized by the adaptive immune response. Here, we purified LM from
the avirulent M. smegmatis and the virulent M. tuberculosis H37Rv, performed a comparative structural biochemical analysis, and addressed their ability to stimulate CD1b-restricted T cell
clones. We found that M. tuberculosis H37Rv produces a large neutral LM (TB-LM); in contrast, M. smegmatis produces a smaller linear acidic LM (SmegLM) with a high succinate content. Correspondingly, TB-LM was not as efficiently
presented to CD1b-restricted T cells as SmegLM. Thus, here we correlate the structure-function relationships for LMs with
CD1b-restricted T cell responses and provide evidence that the structural features of TB-LM contribute to its diminished T
cell responsiveness.
Mannosylated lipoarabinomannans are multifaceted molecules. They have been shown to exert an immunosuppressive role in the
immunopathogenesis of tuberculosis. They are also described as antigens of host double negative αβ T-cells. Delimitation of
ManLAMs epitopes require knowledge of the precise structure of these molecules. The two major functional domains (the cap
motifs and the phosphatidylinositol anchor) of the parietal and cellular ManLAMs ofMycobacterium tuberculosis H37Rv were investigated here. Using capillary electrophoresis, we established that parietal and cellular ManLAMs share the
same capping motifs, mono-, di-, and trimannosyl units with the same relative abundance. By31P NMR analysis of the native LAMs in Me2SO-d
6, the major acyl-form of both parietal and cellular H37Rv ManLAM anchors, typified by the P3 phosphorus resonance, comprised
a diacylglycerol unit. Three other acyl-forms were characterized in the cellular ManLAMs. Comparative analysis of the cellular
Mycobacterium bovis BCG andM. tuberculosis ManLAM acyl-forms revealed the presence of the same populations, but with different relative abundance. The biological importance
of the H37Rv ManLAM acyl-form characterization is discussed, particularly concerning the molecular mechanisms of binding of
ManLAMs to the CD1 proteins involved in the presentation of ManLAMs to T-cell receptors.
Lipoarabinomannan (LAM) is a major and structurally important outer cell wall component of all mycobacteria. LAM is also generally regarded as an important immunomodulating substance affecting several immunologic networks and hence important in the pathogenesis of mycobacterial infections. We here describe a new method for large-scale purification of mycobacterial LAM. A crude cell wall preparation was prepared from batch-grown Mycobacterium tuberculosis H37Rv. From this cell wall preparation LAM was purified by sequential extractions and chromatographic steps. From 20 g dry weight cell wall preparation 313 mg of highly purified (>98%) LAM was obtained in only 3 days. The LAM content of the final purification step was quantified by ELISA using reference LAM as standard. The identity and purity of the LAM preparation was further confirmed by comparison with reference LAM preparation from M. tuberculosis strain Erdman in polyacrylamide gel electrophoresis and Western blots, using reference anti-LAM monoclonals CS-35 and CS-40.
Synergistic interplay between Mycobacterium tuberculosis (Mtb) and HIV in coinfected individuals leads to the acceleration of both tuberculosis and HIV disease. Mtb, as well as HIV, may modulate the function of many immune cells, including DCs. To dissect the bystander impact of Mφs infected with Mtb on DC functionality, we here investigated changes in DC phenotype, cytokine profiles, and HIV-1 transinfecting ability. An in vitro system was used in which human monocyte-derived DCs were exposed to soluble factors released by Mφs infected with mycobacteria, including virulent clinical Mtb isolates and nonvirulent BCG. Soluble factors secreted from Mtb-infected Mφs, and to a lesser extent BCG-infected Mφs, resulted in the production of proinflammatory cytokines and partial upregulation of DC maturation markers. Interestingly, the HIV-1 transinfecting ability of DCs was enhanced upon exposure to soluble factors released by Mtb-infected Mφs. In summary, our study shows that DCs exposed to soluble factors released by mycobacteria-infected Mφs undergo maturation and display an augmented ability to transmit HIV-1 in trans. These findings highlight the important role of bystander effects during the course of Mtb-HIV coinfection and suggest that Mtb-infected Mφs may contribute to an environment that supports DC-mediated spread and amplification of HIV in coinfected individuals.
Simple sugars, oligosaccharides, polysaccharides, and their derivatives, including the methyl ethers with free or potentially free reducing groups, give an orange-yellow color when treated with phenol and concentrated sulfuric acid. The reaction is sensitive and the color is stable. By use of this phenol-sulfuric acid reaction, a method has been developed to determine submicro amounts of sugars and related substances. In conjunction with paper partition chromatography the method is useful for the determination of the composition of polysaccharides and their methyl derivatives.
The aim of the study was to investigate the interaction between manLAM and DC-SIGN influencing DCs maturation and downstream immune response using small interfering RNA-expressing lentiviral vectors to specifically knockdown DC-SIGN. Our data indicated that DC-SIGN knockdown alone in DCs did not affect the maturation or the immunological function of lipopolysacharide (LPS)-activated DCs. Surface molecules were dramatically down-regulated in DCs primed with manLAM but not in mock control DCs (P<0.05). Meanwhile, manLAM enhanced the production of the immunosuppressive cytokine IL-10 in DCs (P<0.05). The level of IFN-γ was significantly down-regulated in the supernatants of naive T cells after co-cultured with DCs primed with manLAM (P<0.05). We demonstrated that DCs primed with manLAM may partially impair maturation phenotypes and immune response in LPS-activated DCs. However, the alterations of DCs function and downstream immune response caused by manLAM were reversed by the knockdown of DC-SIGN.
