Identification and characterization of endo-α-, exo-α-, and exo-ß-D-arabinofuranosidases degrading lipoarabinomannan and arabinogalactan of mycobacteria.

The cell walls of pathogenic and acidophilic bacteria, such as Mycobacterium tuberculosis and Mycobacterium leprae, contain lipoarabinomannan and arabinogalactan. These components are composed of D-arabinose, the enantiomer of the typical L-arabinose found in plants. The unique glycan structures of mycobacteria contribute to their ability to evade mammalian immune responses. In this study, we identified four enzymes (two GH183 endo-D-arabinanases, GH172 exo-α-D-arabinofuranosidase, and GH116 exo-ß-D-arabinofuranosidase) from Microbacterium arabinogalactanolyticum. These enzymes completely degraded the complex D-arabinan core structure of lipoarabinomannan and arabinogalactan in a concerted manner. Furthermore, through biochemical characterization using synthetic substrates and X-ray crystallography, we elucidated the mechanisms of substrate recognition and anomer-retaining hydrolysis for the α- and ß-D-arabinofuranosidic bonds in both endo- and exo-mode reactions. The discovery of these D-arabinan-degrading enzymes, along with the understanding of their structural basis for substrate specificity, provides valuable resources for investigating the intricate glycan architecture of mycobacterial cell wall polysaccharides and their contribution to pathogenicity.

LRRK2 is required for CD38-mediated NAADP-Ca2+ signaling and the downstream activation of TFEB (transcription factor EB) in immune cells.

CD38 is a cell surface receptor capable of generating calcium-mobilizing second messengers. It has been implicated in host defense and cancer biology, but signaling mechanisms downstream of CD38 remain unclear. Mutations in LRRK2 (leucine-rich repeat kinase 2) are the most common genetic cause of Parkinson disease; it is also a risk factor for Crohn disease, leprosy, and certain types of cancers. The pathogenesis of these diseases involves inflammation and macroautophagy/autophagy, processes both CD38 and LRRK2 are implicated in. Here, we mechanistically and functionally link CD38 and LRRK2 as upstream activators of TFEB (transcription factor EB), a host defense transcription factor and the master transcriptional regulator of the autophagy/lysosome machinery. In B-lymphocytes and macrophages, we show that CD38 and LRRK2 exist in a complex on the plasma membrane. Ligation of CD38 with the monoclonal antibody clone 90 results in internalization of the CD38-LRRK2 complex and its targeting to the endolysosomal system. This generates an NAADP-dependent calcium signal, which requires LRRK2 kinase activity, and results in the downstream activation of TFEB. lrrk2 KO macrophages accordingly have TFEB activation defects following CD38 or LPS stimulation and fail to switch to glycolytic metabolism after LPS treatment. In overexpression models, the pathogenic LRRK2G2019S mutant promotes hyperactivation of TFEB even in the absence of CD38, both by stabilizing TFEB and promoting its nuclear translocation via aberrant calcium signaling. In sum, we have identified a physiological CD38-LRRK2-TFEB signaling axis in immune cells. The common pathogenic mutant, LRRK2G2019S, appears to hijack this pathway.Abbreviations:ADPR: ADP-ribose; AMPK: AMP-activated protein kinase; BMDM: bone marrow-derived macrophage; cADPR: cyclic-ADP-ribose; COR: C-terminal of ROC; CTSD: cathepsin D; ECAR: extracellular acidification rate; EDTA: ethylenediaminetetraacetic acid; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GPN: Gly-Phe ß-naphthylamide; GSK3B/GSK3ß: glycogen synthase kinase 3 beta; GTP: guanosine triphosphate; KD: knockdown; LAMP1: lysosomal-associated membrane protein 1; LRR: leucine rich repeat; LRRK2: leucine rich repeat kinase 2; mAb: monoclonal antibody; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MAPK/ERK: mitogen-activated protein kinase; MCOLN1: mucolipin 1; MFI: mean fluorescence intensity; mRNA: messenger RNA; MTOR: mechanistic target of rapamycin kinase; NAADP: nicotinic acid adenine dinucleotide phosphate; NAD: nicotinamide adenine dinucleotide; NADP: nicotinamide adenine dinucleotide phosphate; PD: Parkinson disease; PPP3CB: protein phosphatase 3, catalytic subunit, beta isoform; q-RT-PCR: quantitative reverse transcription polymerase chain reaction; ROC: Ras of complex; siRNA: small interfering RNA; SQSTM1/p62: sequestome 1; TFEB: transcription factor EB; TPCN: two pore channel; TRPM2: transient receptor potential cation channel, subfamily M, member 2; ZKSCAN3: zinc finger with KRAB and SCAN domains 3.

A LRRK2 GTP Binding Inhibitor, 68, Reduces LPS-Induced Signaling Events and TNF-α Release in Human Lymphoblasts.

Mutations in the leucine-rich repeat kinase-2 (LRRK2) gene cause autosomal-dominant Parkinson's disease (PD) and contribute to sporadic PD. Common genetic variation in LRRK2 modifies susceptibility to immunological disorders including Crohn's disease and leprosy. Previous studies have reported that LRRK2 is expressed in B lymphocytes and macrophages, suggesting a role for LRRK2 in immunological functions. In this study, we characterized the LRRK2 protein expression and phosphorylation using human lymphoblasts. Lipopolysaccharide (LPS), a proinflammatory agent, induced the increase of LRRK2 expression and kinase activities in human lymphoblasts in a time-dependent manner. Moreover, LPS activated the Toll-like receptor (TLR) signaling pathway, increased TRAF6/LRRK2 interaction, and elevated the phosphorylation levels of MAPK (JNK1/2, p38, and ERK1/2) and IkBα. Treatment with LRRK2 inhibitor 68 reduced LPS-induced TRAF6/LRRK2 interaction and MAPK and IkBα phosphorylation, thereby reducing TNF-α secretion. These results indicate that LRRK2 is actively involved in proinflammatory responses in human lymphoblasts, and inhibition of GTP binding by 68 results in an anti-inflammation effect against proinflammatory stimuli. These findings not only provide novel insights into the mechanisms of LRRK2-linked immune and inflammatory responses in B-cell-like lymphoblasts, but also suggest that 68 may also have potential therapeutic value for LRRK2-linked immunological disorders.

Photoperiod Affects Leptin Action on the Choroid Plexus in Ewes Challenged with Lipopolysaccharide-Study on the mRNA Level.

The ovine choroid plexus (ChP) expresses the long isoform of the leptin receptor, which makes this structure a potential target for leptin action. In sheep, leptin concentration in plasma is higher during long days (LD) than short days (SD). This study evaluates the influence a of photoperiod on leptin impact on the gene expression of Toll-like receptor 4 (TLR4), proinflammatory cytokines (IL1B, IL6), their receptors (IL1R1, IL1R2, ILRN, IL6R, IL6ST) and inflammasome components necessary for pro-IL-1ß activation (NLRP3, PYCARD, CASP1), chemokine (CCL2), leptin receptor isoforms (LEPRa, LEPRb) and a suppressor of cytokine signalling (SOCS3) in the ChP of ewes treated or not with lipopolysaccharide (LPS). Studies were conducted on adult female sheep divided into four groups (n = 6 in each): control, leptin (20 µg/kg), LPS (400 ng/kg), and LPS and leptin injected under SD and LD photoperiods. The leptin alone did not affect the gene expression but in co-treatment with LPS increased (p < 0.05) IL1B but only during SD, and SOCS3, IL1R2, IL1RN, IL6ST and CCL2 only during LD, and decreased (p < 0.05) the IL1R1 expression only during SD photoperiod. This indicates that the immunomodulatory action of leptin on the ChP is manifested only under the LPS challenge and is photoperiodically dependent.

[The use of synthetic glycoconjugates as components of the immunochromatographic test for rapid serological diagnosis of leprosy.]

The glycoconjugates with BSA (bovine serum albumin) were synthesized using a next saccharide: disaccharide derivative M.leprae PGL-1 (phenolic glycolipid-1); a complex of the disaccharide fragment and the branched hexasaccharide fragment LAM (lipoarabinomannan); diarabinofuranose fragment LAM. These glycoconjugates were used as antigenic components for leprosy rapid serotest construction in immunochromatographic format (leprosy LF serotest). The data obtained with sera of leprosy patients, patients who have been in contact with leprosy, and healthy donors indicate that the most promising antigenic component is a BSA conjugate with two synthetic epitopes - a disaccharide derivative of PGL-1 and a branched hexasaccharide fragment of LAM. The leprosy LF serotest with such glycoconjugate demonstrated the greatest diagnostic sensitivity for main forms of leprosy - paucibacillary (PB) and multibacillary (MB).

Oral administration of Debaryomyces hansenii CBS8339-ß-glucan induces trained immunity in newborn goats.

Beta-glucans from yeast can induce trained immunity in in vitro and in vivo models. Intraperitoneal doses of ß-glucans in mammals have shown to induce trained immunity, but the training effects of orally administering ß-glucans are unknown. Newborn goats are susceptible to infections in the neonatal stage, so the induction of trained immunity could improve animal survival. This study aimed to describe the in vitro effects of immunological training by ß-glucan from Debaryomyces hansenii (ß-Dh) on caprine monocytes, as well as its in vivo effects using oral doses on newborn goats upon challenge with lipopolysaccharide (LPS). Hence in vitro, goat monocytes trained with ß-Dh up-regulated the gene expression of macrophage surface markers (CD11b and F4/80) whereas enhanced cell survival and high phagocytic ability was found upon LPS challenge. In the in vivo experiment, newborn goats stimulated with two doses (day -7 and - 4) of ß-Dh (50 mg/kg) and challenged (day 0) with LPS showed an increase in respiratory burst activity, IL-1ß, IL-6, and TNFα production in plasma, and transcription of the macrophage surface markers. This study has demonstrated for the first time that trained immunity was induced with oral doses of ß-glucan upon LPS challenge in mammals using newborn goats.

Lipoarabinomannan from Mycobacterium indicus pranii shows immunostimulatory activity and induces autophagy in macrophages.

Mycobacterium indicus pranii (MIP) known for its immunotherapeutic potential against leprosy and tuberculosis is undergoing various clinical trials and also simultaneously being studied in animal models to get insight into the mechanistic details contributing to its protective efficacy as a vaccine candidate. Studies have shown potential immunomodulatory properties of MIP, the most significant being the ability to induce strong Th1 type of response, enhanced expression of pro-inflammatory cytokines, activation of APCs and lymphocytes, elicitation of M.tb specific poly-functional T cells. All of these form crucial components of host-immune response during M.tb infection. Also, MIP was found to be potent inducer of autophagy in macrophages which resulted in enhanced clearance of M.tb from MIP and M.tb co-infected cells. Hence, we further examined the component/s of MIP responsible for autophagy induction. Interestingly, we found that MIP lipids and DNA were able to induce autophagy but not the protein fraction. LAM being one of the crucial components of mycobacterial cell-wall lipids and possessing the ability of immunomodulation; we isolated LAM from MIP and did a comparative study with M.tb-LAM. Stimulation with MIP-LAM resulted in significantly high secretion of pro-inflammatory cytokines and displayed high autophagy inducing potential in macrophages as compared to M.tb-LAM. Treatment with MIP-LAM enhanced the co-localization of M.tb within the phago-lysosomes and increased the clearance of M.tb from the infected macrophages. This study describes LAM to be a crucial component of MIP which has significant contribution to its immunotherapeutic efficacy against TB.

Dapsone modulates lipopolysaccharide-activated bone marrow cells by inducing cell death and down-regulating tumor necrosis factor-α production.

Dapsone, an antibiotic, has been used to cure leprosy. It has been reported that dapsone has anti-inflammatory activity in hosts; however, the anti-inflammatory mechanism of dapsone has not been fully elucidated. The present study investigated the anti-inflammatory effects of dapsone on bone marrow cells (BMs), especially upon exposure to lipopolysaccharide (LPS). We treated BMs with LPS and dapsone, and the treated cells underwent cellular activity assay, flow cytometry analysis, cytokine production assessment, and reactive oxygen species assay. LPS distinctly activated BMs with several characteristics including high cellular activity, granulocyte changes, and tumor necrosis factor alpha (TNF-α) production increases. Interestingly, dapsone modulated the inflammatory cells, including granulocytes in LPS-treated BMs, by inducing cell death. While the percentage of Gr-1 positive cells was 57% in control cells, LPS increased that to 75%, and LPS plus dapsone decreased it to 64%. Furthermore, dapsone decreased the mitochondrial membrane potential of LPS-treated BMs. At a low concentration (25 µg/mL), dapsone significantly decreased the production of TNF-α in LPS-treated BMs by 54%. This study confirmed that dapsone has anti-inflammatory effects on LPS-mediated inflammation via modulation of the number and function of inflammatory cells, providing new and useful information for clinicians and researchers.

Anti-Lipoarabinomannan-Specific Salivary IgA as Prognostic Marker for Leprosy Reactions in Patients and Cellular Immunity in Contacts.

Leprosy causes the most common peripheral neuropathy of infectious etiology, posing an important public health problem worldwide. Understanding the molecular and immunological mechanisms of nerve damage induced by M. leprae is mandatory to develop tools for early diagnosis and preventive measures. The phenolic glycolipid 1 (PGL-1) and lipoarabinomannan (LAM) antigens are major components of the bacterial surface and are implicated on leprosy immunopathogenesis and neural damage. Although the anti-PGL-1 serum IgM is highly used for operational classification of patients, the anti-LAM salivary IgA (sIgA) has not been investigated as diagnostic or prognostic marker in leprosy. Our aim was to assess the presence of anti-LAM sIgA in leprosy patients and their contacts in order to demonstrate whether such expression was associated with leprosy reactions. Distinct patterns of anti-LAM slgA were observed among groups, which were stratified into treatment-naïve patients (116), patients who completed multidrug therapy-MDT (39), household contacts (111), and endemic controls (11). Both anti-LAM sIgA and anti-PGL-I serum IgM presented similar prognostic odds toward leprosy reactions [(odds ratio) OR = 2.33 and 2.78, respectively]. Furthermore, the anti-LAM sIgA was highly correlated with multibacillary (MB) forms (OR = 4.15). Contrarily, among contacts the positive anti-LAM sIgA was highly correlated with those with positive Mitsuda test, suggesting that the presence of anti-LAM slgA may act as an indicator of cellular immunity conferred to contacts. Our data suggest that anti-LAM slgA may be used as a tool to monitor patients undergoing treatment to predict reactional episodes and may also be used in contacts to evaluate their cellular immunity without the need of Mitsuda tests.

In Situ complement activation and T-cell immunity in leprosy spectrum: An immunohistological study on leprosy lesional skin.

Mycobacterium leprae (M. leprae) infection causes nerve damage and the condition worsens often during and long after treatment. Clearance of bacterial antigens including lipoarabinomannan (LAM) during and after treatment in leprosy patients is slow. We previously demonstrated that M. leprae LAM damages peripheral nerves by in situ generation of the membrane attack complex (MAC). Investigating the role of complement activation in skin lesions of leprosy patients might provide insight into the dynamics of in situ immune reactivity and the destructive pathology of M. leprae. In this study, we analyzed in skin lesions of leprosy patients, whether M. leprae antigen LAM deposition correlates with the deposition of complement activation products MAC and C3d on nerves and cells in the surrounding tissue. Skin biopsies of paucibacillary (n = 7), multibacillary leprosy patients (n = 7), and patients with erythema nodosum leprosum (ENL) (n = 6) or reversal reaction (RR) (n = 4) and controls (n = 5) were analyzed. The percentage of C3d, MAC and LAM deposition was significantly higher in the skin biopsies of multibacillary compared to paucibacillary patients (p = <0.05, p = <0.001 and p = <0.001 respectively), with a significant association between LAM and C3d or MAC in the skin biopsies of leprosy patients (r = 0.9578, p< 0.0001 and r = 0.8585, p<0.0001 respectively). In skin lesions of multibacillary patients, MAC deposition was found on axons and co-localizing with LAM. In skin lesions of paucibacillary patients, we found C3d positive T-cells in and surrounding granulomas, but hardly any MAC deposition. In addition, MAC immunoreactivity was increased in both ENL and RR skin lesions compared to non-reactional leprosy patients (p = <0.01 and p = <0.01 respectively). The present findings demonstrate that complement is deposited in skin lesions of leprosy patients, suggesting that inflammation driven by complement activation might contribute to nerve damage in the lesions of these patients. This should be regarded as an important factor in M. leprae nerve damage pathology.

Clofazimine Biocrystal Accumulation in Macrophages Upregulates Interleukin 1 Receptor Antagonist Production To Induce a Systemic Anti-Inflammatory State.

Clofazimine (CFZ) is a poorly soluble antibiotic and anti-inflammatory drug indicated for the treatment of leprosy. In spite of its therapeutic value, CFZ therapy is accompanied by the formation of drug biocrystals that accumulate within resident tissue macrophages, without obvious toxicological manifestations. Therefore, to specifically elucidate the off-target consequences of drug bioaccumulation in macrophages, we compared the level of inflammasome activation in CFZ-accumulating organs (spleen, liver and lung) in mice after 2 and 8 weeks of CFZ treatment when the drug exists in soluble and insoluble (biocrystalline) forms, respectively. Surprisingly, the results showed a drastic reduction in caspase 1 and interleukin-1ß (IL-1ß) cleavage in the livers of mice treated with CFZ for 8 weeks (8-week-CFZ-treated mice) compared to 2-week-CFZ-treated and control mice, which was accompanied by a 3-fold increase in hepatic IL-1 receptor antagonist (IL-1RA) production and a 21-fold increase in serum IL-1RA levels. In the lung and spleen, IL-1ß cleavage and tumor necrosis factor alpha expression were unaffected by soluble or biocrystal CFZ forms. Functionally, there was a drastic reduction of carrageenan- and lipopolysaccharide-induced inflammation in the footpads and lungs, respectively, of 8-week-CFZ-treated mice. This immunomodulatory activity of CFZ biocrystal accumulation was attributable to the upregulation of IL-1RA, since CFZ accumulation had minimal effect in IL-1RA knockout mice or 2-week-CFZ-treated mice. In conclusion, CFZ accumulation and biocrystal formation in resident tissue macrophages profoundly altered the host's immune system and prompted an IL-1RA-dependent, systemic anti-inflammatory response.

M. leprae components induce nerve damage by complement activation: identification of lipoarabinomannan as the dominant complement activator.

Peripheral nerve damage is the hallmark of leprosy pathology but its etiology is unclear. We previously identified the membrane attack complex (MAC) of the complement system as a key determinant of post-traumatic nerve damage and demonstrated that its inhibition is neuroprotective. Here, we determined the contribution of the MAC to nerve damage caused by Mycobacterium leprae and its components in mouse. Furthermore, we studied the association between MAC and the key M. leprae component lipoarabinomannan (LAM) in nerve biopsies of leprosy patients. Intraneural injections of M. leprae sonicate induced MAC deposition and pathological changes in the mouse nerve, whereas MAC inhibition preserved myelin and axons. Complement activation occurred mainly via the lectin pathway and the principal activator was LAM. In leprosy nerves, the extent of LAM and MAC immunoreactivity was robust and significantly higher in multibacillary compared to paucibacillary donors (p = 0.01 and p = 0.001, respectively), with a highly significant association between LAM and MAC in the diseased samples (r = 0.9601, p = 0.0001). Further, MAC co-localized with LAM on axons, pointing to a role for this M. leprae antigen in complement activation and nerve damage in leprosy. Our findings demonstrate that MAC contributes to nerve damage in a model of M. leprae-induced nerve injury and its inhibition is neuroprotective. In addition, our data identified LAM as the key pathogen associated molecule that activates complement and causes nerve damage. Taken together our data imply an important role of complement in nerve damage in leprosy and may inform the development of novel therapeutics for patients.

Serological responses to prednisolone treatment in leprosy reactions: study of TNF-α, antibodies to phenolic glycolipid-1, lipoarabinomanan, ceramide and S100-B.

BACKGROUND: Corticosteroids have been extensively used in the treatment of immunological reactions and neuritis in leprosy. The present study evaluates the serological response to steroid treatment in leprosy reactions and neuritis. METHODS: Seven serological markers [TNF-α, antibodies to Phenolic glycolipid-1 (PGL-1 IgM and IgG), Lipoarabinomannan (LAM IgG1 and IgG3), C2-Ceramide and S100 B] were analyzed longitudinally in 72 leprosy patients before, during and after the reaction. At the onset of reaction these patients received a standard course of prednisolone. The levels of the above markers were measured by Enzyme linked immunosorbent assay (ELISA) and compared with the individuals own value in the month prior to the reaction and presented as percentage increase. RESULTS: One month before the reaction individuals showed a varying increase in the level of different markers such as TNF-α (53%) and antibodies to Ceramide (53%), followed by to PGL-1 (51%), S100B (50%) and LAM (26%). The increase was significantly associated with clinical finding of nerve pain, tenderness and new nerve function impairment. After one month prednisolone therapy, there was a fall in the levels [TNF-α (60%), C2-Ceramide (54%), S100B (67%), PGL-1(47%) and LAM (52%)] with each marker responding differently to steroid. CONCLUSION: Reactions in leprosy are inflammatory processes wherein a rise in set of serological markers can be detected a month before the clinical onset of reaction, some of which remain elevated during their action and steroid treatment induces a variable fall in the levels, and this forms the basis for a variable individual response to steroid therapy.

An attempt to improve pure neural leprosy diagnosis using immunohistochemistry tests in peripheral nerve biopsy specimens.

The diagnosis of pure neural leprosy (PNL) is based on clinical and laboratory data, including the histopathology of nerve biopsy specimens and detection of Mycobacterium leprae DNA by polymerase chain reaction (PCR). Given that histopathologic examination and PCR methods may not be sufficient to confirm the diagnosis, immunolabeling of lipoarabinomanan (LAM) and/or phenolic glycolipid 1 (PGL-1) M. leprae wall components was utilized in the present investigation in an attempt to detect any vestigial presence of M. leprae in acid-fast bacilli (AFB) nerve samples. Twenty-three PNL nerve samples (6 AFB and 17 AFBPCR) were cryosectioned and subjected to LAM and PGL-1 immunohistochemical staining by immunoperoxidase. Five nonleprosy nerve samples were used as controls. The 6 AFB samples showed LAM/PGL-1 immunoreactivity. Among the 17 AFB samples, 8 revealed LAM and/or PGL-1 immunoreactivity. In 17 AFBPCR patients, just 7 yielded LAM and/or PGL-1 nerve results. In the PNL cases, the detection of immunolabeled LAM and PGL-1 in the nerve samples would have contributed to an enhanced diagnostic efficiency in the absence of molecular diagnostic facilities.

IL-10 production from dendritic cells is associated with DC SIGN in human leprosy.

The defective antigen presenting ability of antigen presenting cells (APCs) modulates host cytokines and co-stimulatory signals that may lead to severity of leprosy. In the present study, we sought to evaluate the phenotypic features of APCs along with whether DC SIGN (DC-specific intercellular adhesion molecule-grabbing nonintegrin) influences IL-10 production while moving from tuberculoid (BT/TT) to lepromatous (BL/LL) pole in leprosy pathogenesis. The study revealed an increased expression of DC SIGN on CD11c⁺ cells from BL/LL patients and an impaired form of CD83 (∼50 kDa). However, the cells after treatment with GM-CSF+IL-4+ManLAM showed an increased expression of similar form of CD83 on DCs. Upon treatment with ManLAM, DCs were found to show increased nuclear presence of NF-κB, thus leading to higher IL-10 production. High IL-10 production from ManLAM treated PBMCs further suggested the role of DC SIGN in subverting the DCs function towards BL/LL pole of leprosy. Anti-DC SIGN treatment resulting in restricted nuclear ingression of NF-κB as well as its acetylation along with enhanced T cell proliferation validated our findings. In conclusion, Mycobacterium leprae component triggers DC SIGN on DCs to induce production of IL-10 by modulating intracellular signalling pathway at the level of transcription factor NF-κB towards BL/LL pole of disease.

Identification of active compounds from Caesalpinia sappan L. extracts suppressing IL-6 production in RAW 264.7 cells by PLS.

ETHNOPHARMACOLOGICAL RELEVANCE: Caesalpinia sappan L. is distributed in Southeast Asia and also used as herbal medicine for the treatment of various diseases such as burning sensations, leprosy, dysentery, osteoarthritis and rheumatoid arthritis (RA). The overproduction of IL-6 plays an important role in the prognosis of RA, but the active compounds from the extracts of Caesalpinia sappan L. suppressing IL-6 production remain unknown. AIMS OF THE STUDY: Identifying the main active compounds of Caesalpinia sappan L. extracts inhibiting the IL-6 production in LPS-stimulated RAW 264.7 cells by partial least squares (PLS). MATERIALS AND METHODS: Sixty-four samples with different proportions of compounds were prepared from Caesalpinia sappan L. by supercritical CO2 fluid extraction (SCFE) and refluxing. Each of 64 samples was applied to RAW 264.7 cells with LPS to evaluate whether IL-6 production by LPS is affected by addition of each sample. The IL-6 production in medium was determined by ELISA and the inhibitory activity of each sample was analyzed. In addition, the fingerprints of these 64 samples were also established by ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC-MS). We used the PLS, a simplified method, to evaluate the results from IL-6 production and fingerprints. RESULTS: Each of 64 samples markedly suppressed LPS-induced IL-6 production in RAW cells. The fingerprints by UPLC-MS clearly revealed variations among 64 samples produced in different extract conditions. The PLS analysis with IL-6 production and fingerprints by UPLC-MS suggested that the peaks 71, 93, 150, 157, 168 have more influence on the inhibitory activity of Caesalpinia sappan L. extracts. The peaks 71, 93, 150 are likely representing sappanone A, protosappanin E and neoprotosappanin, respectively. The peaks 157 and 168 are still at large. CONCLUSION: This is the first report that sappanone A, protosappanin E, neoprotosappanin and two unidentified compounds can be considered as possible active compounds that might inhibit IL-6 production. Further studies are needed to confirm the effectiveness of these five compounds on IL-6 production and possible mechanism.

PARK2 mediates interleukin 6 and monocyte chemoattractant protein 1 production by human macrophages.

Leprosy is a persistent infectious disease caused by Mycobacterium leprae that still affects over 200,000 new patients annually. The host genetic background is an important risk factor for leprosy susceptibility and the PARK2 gene is a replicated leprosy susceptibility candidate gene. The protein product of PARK2, Parkin, is an E3 ubiquitin ligase that is involved in the development of various forms of Parkinsonism. The human macrophage is both a natural host cell of M. leprae as well as a primary mediator of natural immune defenses, in part by secreting important pro-inflammatory cytokines and chemokines. Here, we report that down-regulation of Parkin in THP-1 macrophages, human monocyte-derived macrophages and human Schwann cells resulted in a consistent and specific decrease in interleukin-6 (IL-6) and monocyte chemoattractant protein 1 (MCP-1/CCL2) production in response to mycobacteria or LPS. Interestingly, production of IL-6 at 6 hours by THP-1 cells stimulated with live M. leprae and M. bovis BCG was dependent on pretreatment with 1,25-dihydroxyvitamin D(3) (VD). Parkin knockdown in VD-treated cells blocked IL-6 induction by mycobacteria. However, IκB-α phosphorylation and levels of IκB-ξ, a nuclear protein required for IL-6 expression, were not affected by Parkin silencing. Phosphorylation of MAPK ERK1/2 and p38 was unaffected by Parkin silencing while JNK activation was promoted but did not explain the altered cytokine production. In a final set of experiments we found that genetic risk factors of leprosy located in the PARK2 promoter region were significantly correlated with M. leprae sonicate triggered CCL2 and IL6 transcript levels in whole blood assays. These results associated genetically controlled changes in the production of MCP-1/CCL2 and IL-6 with known leprosy susceptibility factors.

Identification of serological biomarkers of infection, disease progression and treatment efficacy for leprosy

Although leprosy is curable with drug treatment, the identification of biomarkers of infection, disease progression and treatment efficacy would greatly help to reduce the overall prevalence of the disease. Reliable biomarkers would also reduce the incidence of grade-2 disability by ensuring that those who are most at risk are diagnosed and treated early or offered repeated treatments in the case of relapse. In this study, we examined the reactivity of sera from lepromatous and tuberculoid leprosy patients (LPs) against a panel of 12 recombinant Mycobacterium leprae proteins and found that six proteins were strongly recognised by multibacillary (MB) patients, while only three were consistently recognised by paucibacillary patients. To better understand the dynamics of patient antibody responses during and after drug therapy, we measured antibody titres to four recombinant proteins, phenolic glycolipid-I and lipoarabinomannan at baseline and up to two years after diagnosis to investigate the temporal changes in the antibody titres. Reactivity patterns to individual antigens and decreases in antibody titres were patient-specific. Antibody titres to proteins declined more rapidly vs. those to carbohydrate and glycolipid antigens. Compared to baseline values, increases in antibody titres were observed during reactional episodes in one individual. Additionally, antibody responses against a subset of antigens that provided a good prognostic indicator of disease progression were analysed in 51 household contacts of MB index cases for up to two years. Although the majority of these contacts showed no change or exhibited decreases in antibody titres, seven individuals developed higher titres towards one or more of these antigens and one individual with progressively higher titres was diagnosed with borderline lepromatous leprosy 19 months after enrolment. The results of this study indicate that antibody titres to specific M. leprae antigens can be used to monitor treatment efficacy in LPs and assess disease progression in those most at risk for developing this disease.

Phenolic-glycolipid-1 and lipoarabinomannan preferentially modulate TCR- and CD28-triggered proximal biochemical events, leading to T-cell unresponsiveness in mycobacterial diseases.

BACKGROUND: Advanced stages of leprosy show T cell unresponsiveness and lipids of mycobacterial origin are speculated to modulate immune responses in these patients. Present study elucidates the role of phenolicglycolipid (PGL-1) and Mannose-capped lipoarabinomannan (Man-LAM) on TCR- and TCR/CD28- mediated signalling. RESULTS: We observed that lipid antigens significantly inhibit proximal early signalling events like Zap-70 phosphorylation and calcium mobilization. Interestingly, these antigens preferentially curtailed TCR-triggered early downstream signalling events like p38 phosphorylation whereas potentiated that of Erk1/2. Further, at later stages inhibition of NFAT binding, IL-2 message, CD25 expression and T-cell blastogenesis by PGL-1 and Man-LAM was noted. CONCLUSION: Altogether, we report that Man-LAM and PGL-1 preferentially interfere with TCR/CD28-triggered upstream cell signalling events, leading to reduced IL-2 secretion and T-cell blastogenesis which potentially could lead to immunosupression and thus, disease exacerbation, as noted in disease spectrum.