Rapid and Sensitive SERS-Based Lateral Flow Test for SARS-CoV2-Specific IgM/IgG Antibodies.

As an immune response to COVID-19 infection, patients develop SARS-CoV-2-specific IgM/IgG antibodies. Here, we compare the performance of a conventional lateral flow assay (LFA) with a surface-enhanced Raman scattering (SERS)-based LFA test for the detection of SARS-CoV-2-specific IgM/IgG in sera of COVID-19 patients. Sensitive detection of IgM might enable early serological diagnosis of acute infections. Rapid detection in serum using a custom-built SERS reader is at least an order of magnitude more sensitive than the conventional LFAs with naked-eye detection. For absolute quantification and the determination of the limit of detection (LOD), a set of reference measurements using purified (total) IgM in buffer was performed. In this purified system, the sensitivity of SERS detection is even 7 orders of magnitude higher: the LOD for SERS was ca. 100 fg/mL compared to ca. 1 µg/mL for the naked-eye detection. This outlines the high potential of SERS-based LFAs in point-of-care testing once the interference of serum components with the gold conjugates and the nitrocellulose membrane is minimized.

6-Color/1-Target Immuno-SERS Microscopy on the Same Single Cancer Cell.

There is an urgent clinical need for multicolor imaging of single cancer cells (no ensemble averaging) for identifying heterogenous expression of predictive biomarkers. Specifically, the comprehensive characterization of single disseminated tumor cells (sDTCs) responsible for metastatic relapse is the key to personalized therapy for patients. Current bioimaging methods lack the necessary multicolor capacity and suffer from background/autofluorescence. Both these central limitations can be overcome by immuno-SERS microscopy using SERS nanotags conjugated to antibodies. Here, we demonstrate the proof of concept for 6-color iSERS microscopy on the same single cancer cell. Human epidermal growth factor receptor 2 (HER2), the most prominent breast cancer marker, is localized on the membrane of single SkBr-3 cells, which overexpress HER2 and are an accepted model for sDTCs in breast cancer. This work paves the way for future multicolor/multitarget imaging for characterizing heterogeneous protein expression at the single-cell level.

Leishmania donovani inhibits macrophage apoptosis and pro-inflammatory response through AKT-mediated regulation of ß-catenin and FOXO-1.

In order to establish infection, intra-macrophage parasite Leishmania donovani needs to inhibit host defense parameters like inflammatory cytokine production and apoptosis. In the present study, we demonstrate that the parasite achieves both by exploiting a single host regulator AKT for modulating its downstream transcription factors, ß-catenin and FOXO-1. L. donovani-infected RAW264.7 and bone marrow-derived macrophages (BMDM) treated with AKT inhibitor or dominant negative AKT constructs showed decreased anti-inflammatory cytokine production and increased host cell apoptosis resulting in reduced parasite survival. Infection-induced activated AKT triggered phosphorylation-mediated deactivation of its downstream target, GSK-3ß. Inactivated GSK-3ß, in turn, could no longer sequester cytosolic ß-catenin, an anti-apoptotic transcriptional regulator, as evidenced from its nuclear translocation during infection. Constitutively active GSK-3ß-transfected L. donovani-infected cells mimicked the effects of AKT inhibition and siRNA-mediated silencing of ß-catenin led to disruption of mitochondrial potential along with increased caspase-3 activity and IL-12 production leading to decreased parasite survival. In addition to activating anti-apoptotic ß-catenin, phospho-AKT inhibits activation of FOXO-1, a pro-apoptotic transcriptional regulator. Nuclear retention of FOXO-1, inhibited during infection, was reversed when infected cells were transfected with dominant negative AKT constructs. Overexpression of FOXO-1 in infected macrophages not only documented increased apoptosis but promoted enhanced TLR4 expression and NF-κB activity along with an increase in IL-1ß and decrease in IL-10 secretion. In vivo administration of AKT inhibitor significantly decreased liver and spleen parasite burden and switched cytokine balance in favor of host. In contrast, GSK-3ß inhibitor did not result in any significant change in infectivity parameters. Collectively our findings revealed that L. donovani triggered AKT activation to regulate GSK-3ß/ß-catenin/FOXO-1 axis, thus ensuring inhibition of both host cell apoptosis and immune response essential for its intra-macrophage survival.

Leishmania donovani Exploits Myeloid Cell Leukemia 1 (MCL-1) Protein to Prevent Mitochondria-dependent Host Cell Apoptosis.

Apoptosis is one of the mechanisms used by host cells to remove unwanted intracellular organisms, and often found to be subverted by pathogens through use of host anti-apoptotic proteins. In the present study, with the help of in vitro and in vivo approaches, we documented that the macrophage anti-apoptotic protein myeloid cell leukemia 1 (MCL-1) is exploited by the intra-macrophage parasite Leishmania donovani to protect their "home" from actinomycin D-induced mitochondria-dependent apoptosis. Among all the anti-apoptotic BCL-2 family members, infection preferentially up-regulated expression of MCL-1 at both the mRNA and protein levels and compared with infected control, MCL-1-silenced infected macrophages documented enhanced caspase activity and increased apoptosis when subjected to actinomycin D treatment. Phosphorylation kinetics and ChIP assay demonstrated that infection-induced MCL-1 expression was regulated by transcription factor CREB (cAMP-response element-binding protein) and silencing of CREB resulted in reduced expression of MCL-1 and increased apoptosis. During infection, MCL-1 was found to be localized in mitochondria and this was significantly reduced in Tom70-silenced macrophages, suggesting the active role of TOM70 in MCL-1 transport. In the mitochondria, MCL-1 interacts with the major pro-apoptotic protein BAK and prevents BAK-BAK homo-oligomer formation thereby preventing cytochrome c release-mediated mitochondrial dysfunction. Silencing of MCL-1 in the spleen of infected mice showed decreased parasite burden and increased induction of splenocyte apoptosis. Collectively our results showed that L. donovani exploited the macrophage anti-apoptotic protein MCL-1 to prevent BAK-mediated mitochondria-dependent apoptosis thereby protecting its niche, which is essential for disease progression.

IRAK-M regulates the inhibition of TLR-mediated macrophage immune response during late in vitro Leishmania donovani infection.

Intramacrophage protozoan parasite Leishmania donovani, causative agent of visceral leishmaniasis, escapes Toll-like receptor (TLR) dependent early host immune response by inducing the deubiquitinating enzyme A20, which is sustained up to 6 h postinfection only. Therefore, Leishmania must apply other means to deactivate late host responses. Here, we elucidated the role of IL-1 receptor-associated kinase M (IRAK-M), a negative regulator of TLR signaling, in downregulating macrophage proinflammatory response during late hours of in vitro infection. Our data reveal a sharp decline in IRAK1 and IRAK4 phosphorylation at 24 h postinfection along with markedly reduced association of IRAK1-TNF receptor associated factor 6, which is mandatory for TLR activation. In contrast, IRAK-M was induced after A20 levels decreased and reached a maximum at 24 h postinfection. IRAK-M induction coincided with increased stimulation of TGF-ß, a hallmark cytokine of visceral infection. TGF-ß-dependent signaling-mediated induction of SMAD family of proteins, 2, 3, and 4 plays important roles in transcriptional upregulation of IRAK-M. In infected macrophages, siRNA-mediated silencing of IRAK-M displayed enhanced IRAK1 and IRAK4 phosphorylation with a concomitant increase in downstream NF-κB activity and reduced parasite survival. Taken together, the results suggest that IRAK-M may be targeted by L. donovani to inhibit TLR-mediated proinflammatory response late during in vitro infection.

Prostaglandin E2 negatively regulates the production of inflammatory cytokines/chemokines and IL-17 in visceral leishmaniasis.

Persistence of intracellular infection depends on the exploitation of factors that negatively regulate the host immune response. In this study, we elucidated the role of macrophage PGE2, an immunoregulatory lipid, in successful survival of Leishmania donovani, causative agent of the fatal visceral leishmaniasis. PGE2 production was induced during infection and resulted in increased cAMP level in peritoneal macrophages through G protein-coupled E-series prostanoid (EP) receptors. Among four different EPs (EP1-4), infection upregulated the expression of only EP2, and individual administration of either EP2-specific agonist, butaprost, or 8-Br-cAMP, a cell-permeable cAMP analog, promoted parasite survival. Inhibition of cAMP also induced generation of reactive oxygen species, an antileishmanial effector molecule. Negative modulation of PGE2 signaling reduced infection-induced anti-inflammatory cytokine polarization and enhanced inflammatory chemokines, CCL3 and CCL5. Effect of PGE2 on cytokine and chemokine production was found to be differentially modulated by cAMP-dependent protein kinase A (PKA) and exchange protein directly activated by cAMP (EPAC). PGE2-induced decreases in TNF-α and CCL5 were mediated specifically by PKA, whereas administration of brefeldin A, an EPAC inhibitor, could reverse decreased production of CCL3. Apart from modulating inflammatory/anti-inflammatory balance, PGE2 inhibited antileishmanial IL-17 cytokine production in splenocyte culture. Augmented PGE2 production was also found in splenocytes of infected mice, and administration of EP2 antagonist in mice resulted in reduced liver and spleen parasite burden along with host-favorable T cell response. These results suggest that Leishmania facilitates an immunosuppressive environment in macrophages by PGE2-driven, EP2-mediated cAMP signaling that is differentially regulated by PKA and EPAC.

Leishmania donovani activates uncoupling protein 2 transcription to suppress mitochondrial oxidative burst through differential modulation of SREBP2, Sp1 and USF1 transcription factors.

In order to reside and multiply successfully within the host macrophages, Leishmania parasites impair the generation of cellular as well as mitochondrial reactive oxygen species (ROS), which is a major host defense mechanism against any invading pathogen. Mitochondrial uncoupling protein 2 (UCP2) is strongly induced in Leishmania infection, both at mRNA and protein levels, to suppress the mitochondrial ROS generation. In the present study we have demonstrated that Leishmania donovani infection is associated with strong up-regulation of UCP2 at mRNA level which is the determining factor for its protein level upregulation. The transcriptional activation of UCP2 was mediated by increased nuclear translocation and DNA binding of sterol regulatory element binding protein 2 (SREBP2) and specificity protein 1 (Sp1) transcription factors with concomitant decrease of both the nuclear content and the promoter occupancy of upstream stimulatory factor 1 (USF1). siRNA-mediated silencing of SREBP2 or Sp1 was associated with decreased UCP2 expression in infected macrophages. In contrast, downregulation of USF1 resulted in activated transcription of UCP2. L. donovani infection resulted in degradation of USF1 thereby facilitating SREBP2 binding which in turn assisted in the association of Sp1 with the promoter ultimately culminating in elevated transcription of UCP2.

Leishmania donovani targets tumor necrosis factor receptor-associated factor (TRAF) 3 for impairing TLR4-mediated host response.

Intramacrophage pathogen Leishmania donovani escapes host immune response by subverting Toll-like receptor (TLR) signaling, which is critically regulated by protein ubiquitination. In the present study, we identified tumor necrosis factor receptor-associated factor (TRAF) 3, degradative ubiquitination of which is essential for TLR4 activation, as a target for Leishmania to deactivate LPS-mediated TLR4 signaling. We used LPS-treated RAW 264.7 cells and compared the TLR4-mediated immune response in these cells with L. donovani and L. donovani + LPS costimulated macrophages. TRAF3, which was ubiquitinated (2.1-fold over control) at lys 48 position and subsequently degraded following LPS treatment, persisted in L. donovani and L. donovani + LPS costimulated cells due to defective lys 48 ubiquitination. Lys 63-linked ubiquitination of upstream proteins in the cascade (cIAP1/2 and TRAF6), mandatory for TRAF3 degradation, was also reduced postinfection. This may be attributed to reduced association between ubiquitin-conjugating enzyme Ubc13 and TRAF6 during infection. Inhibition of TRAF3 before infection by shRNA in Balb/c mice showed enhanced IL-12 and TNF-α (10.8- and 8.1-fold over infected control) and decreased spleen parasite burden (61.3% suppression, P<0.001), thereby marking reduction in disease progression. Our findings identified TRAF3 as a novel molecular regulator exploited by Leishmania for successful infection.

Leishmania donovani prevents oxidative burst-mediated apoptosis of host macrophages through selective induction of suppressors of cytokine signaling (SOCS) proteins.

One of the mechanisms for establishment of infection employed by intra-macrophage pathogen-like Leishmania is inhibition of oxidative burst-mediated macrophage apoptosis to protect their niche for survival and replication. We tried to elucidate the underlying mechanism for this by using H2O2 for induction of apoptosis. Leishmania donovani-infected macrophages were much more resistant to H2O2-mediated apoptosis compared with control. Although infected cells were capable of comparable reactive oxygen species production, there was less activation of the downstream cascade consisting of caspase-3 and -7 and cleaved poly(ADP)-ribose polymerase. Suppressors of cytokine signaling (SOCS) 1 and 3 proteins and reactive oxygen species scavenging enzyme thioredoxin, known to be involved in stabilization of protein-tyrosine phosphatases, were found to be induced during infection. Induction of SOCS proteins may be mediated by Egr1, and silencing of Socs1 and -3 either alone or in combination resulted in reduced thioredoxin levels, enhanced activation of caspases, and increased apoptosis of infected macrophages. The induction of protein-tyrosine phosphatases, thioredoxin, SOCS, and Egr1 in L. donovani-infected macrophages was found to be unaffected by H2O2 treatment. SOCS knocked down cells also displayed decreased parasite survival thus marking reduction in disease progression. Taken together, these results suggest that L. donovani may exploit SOCS for subverting macrophage apoptotic machinery toward establishing its replicative niche inside the host.

Leishmania donovani exploits host deubiquitinating enzyme A20, a negative regulator of TLR signaling, to subvert host immune response.

TLRs, which form an interface between mammalian host and microbe, play a key role in pathogen recognition and initiation of proinflammatory response thus stimulating antimicrobial activity and host survival. However, certain intracellular pathogens such as Leishmania can successfully manipulate the TLR signaling, thus hijacking the defensive strategies of the host. Despite the presence of lipophosphoglycan, a TLR2 ligand capable of eliciting host-defensive cytokine response, on the surface of Leishmania, the strategies adopted by the parasite to silence the TLR2-mediated proinflammatory response is not understood. In this study, we showed that Leishmania donovani modulates the TLR2-mediated pathway in macrophages through inhibition of the IKK-NF-κB cascade and suppression of IL-12 and TNF-α production. This may be due to impairment of the association of TRAF6 with the TAK-TAB complex, thus inhibiting the recruitment of TRAF6 in TLR2 signaling. L. donovani infection drastically reduced Lys 63-linked ubiquitination of TRAF6, and the deubiquitinating enzyme A20 was found to be significantly upregulated in infected macrophages. Small interfering RNA-mediated silencing of A20 restored the Lys 63-linked ubiquitination of TRAF6 as well as IL-12 and TNF-α levels with a concomitant decrease in IL-10 and TGF-ß synthesis in infected macrophages. Knockdown of A20 led to lower parasite survival within macrophages. Moreover, in vivo silencing of A20 by short hairpin RNA in BALB/c mice led to increased NF-κB DNA binding and host-protective proinflammatory cytokine response resulting in effective parasite clearance. These results suggest that L. donovani might exploit host A20 to inhibit the TLR2-mediated proinflammatory gene expression, thus escaping the immune responses of the host.

Curative effect of 18ß-glycyrrhetinic acid in experimental visceral leishmaniasis depends on phosphatase-dependent modulation of cellular MAP kinases.

We earlier showed that 18ß-glycyrrhetinic acid (GRA), a pentacyclic triterpenoid from licorice root, could completely cure visceral leishmaniasis in BALB/c mouse model. This was associated with induction of nitric oxide and proinflammatory cytokine production through the up regulation of NF-κB. In the present study we tried to decipher the underlying cellular mechanisms of the curative effect of GRA. Analysis of MAP kinase pathways revealed that GRA caused strong activation of p38 and to a lesser extent, ERK in bone marrow-derived macrophages (BMDM). Almost complete abrogation of GRA-induced cytokine production in presence of specific inhibitors of p38 and ERK1/2 confirmed the involvement of these MAP kinases in GRA-mediated responses. GRA induced mitogen- and stress-activated protein kinase (MSK1) activity in a time-dependent manner suggested that GRA-mediated NF-κB transactivation is mediated by p38, ERK and MSK1 pathway. As kinase/phosphatase balance plays an important role in modulating infection, the effect of GRA on MAPK directed phosphatases (MKP) was studied. GRA markedly reduced the expression and activities of three phosphatases, MKP1, MKP3 and protein phosphatase 2A (PP2A) along with a substantial reduction of p38 and ERK dephosphorylation in infected BMDM. Similarly in the in vivo situation, GRA treatment of L. donovani-infected BALB/c mice caused marked reduction of spleen parasite burden associated with concomitant decrease of individual phosphatase levels. However, activation of kinases also played an important role as the protective effect of GRA was significantly abrogated by pharmacological inhibition of p38 and ERK pathway. Curative effect of GRA may, therefore, be associated with restoration of proper cellular kinase/phosphatase balance, rather than modulation of either kinases or phosphatases.