Sukhwinder Singh

Plasmacytoid dendritic cells and type I IFN: 50 years of convergent history.

It has been 50 years since the initial descriptions of what are now known as plasmacytoid dendritic cells (pDC) and type I IFN. pDC, which are infrequent cells found in the peripheral blood and lymphoid organs, are the most potent producers of type I and type III IFNs in the body. pDC produce IFN-alpha in response to both DNA and RNA enveloped viruses by virtue of their ribonucleic acids signaling in the endosome through TLR9 and TLR7, respectively. This stimulation, which also occurs with DNA or RNA-containing immune complexes and synthetic TLR7 and -9 agonists, is dependent upon the transcription factor IRF-7, which is expressed at high constitutive levels in pDC. In addition to releasing as much as 3-10pg of IFN-alpha/cell, pDC are also potent modulators of the immune response. In this review, we discuss the signaling pathways in pDC, their roles in linking innate and adaptive immunity, and their roles in infectious disease and autoimmunity.

C-terminal SH3 domain of CrkII regulates the assembly and function of the DOCK180/ELMO Rac-GEF.

Genetic studies in Caenorhabiditis elegans identified an evolutionarily conserved CED‐2 (CrkII), CED‐5 (DOCK180), CED‐12 (ELMO), CED‐10 (Rac1) module important for cell migration and phagocytosis of apoptotic cells. Previous studies have shown that DOCK180 and ELMO comprise an unconventional bipartite Dbl homology domain‐independent Rac guanine nucleotide exchange factor (Rac‐GEF); but it is still unclear how CrkII functions in Rac‐GEF activity. In this study, we have characterized a unique function of CrkII in phagocytosis and Rac activation mediated by the C‐terminal SH3 domain, a region of CrkII that has no clear cellular or biochemical function. We found that mutations that disrupt the C‐terminal SH3 domain of CrkII (CrkII‐SH3‐C) abrogate engulfment of apoptotic cells and impair cell spreading on extracellular matrix. Surprisingly, despite the effects on engulfment, W276K CrkII strongly potentiated Rac‐GTP loading when ectopically expressed in HEK 293T cells. Contrary to the effects of the true dominant negative SH2 domain mutants (R38K CrkII) and SH3‐N domain mutants (W170K CrkII) that prevent macromolecular assembly of signaling proteins, W276K CrkII increases association between DOCK180 and CrkII as well as constitutive tethering of the Crk/DOCK180/ELMO protein complex that interacted with RhoG. Our results indicate that while N‐terminal SH3 of CrkII promotes assembly between CrkII and DOCK180, the C‐terminal SH3 of CrkII regulates the stability and turnover of the DOCK180/ELMO complex. Studies with W276K CrkII may offer a unique opportunity to study the structure and function of the DOCK180/ELMO Rac‐GEF.

Drug-Induced Reactivation of Apoptosis Abrogates HIV-1 Infection

HIV-1 blocks apoptosis, programmed cell death, an innate defense of cells against viral invasion. However, apoptosis can be selectively reactivated in HIV-infected cells by chemical agents that interfere with HIV-1 gene expression. We studied two globally used medicines, the topical antifungal ciclopirox and the iron chelator deferiprone, for their effect on apoptosis in HIV-infected H9 cells and in peripheral blood mononuclear cells infected with clinical HIV-1 isolates. Both medicines activated apoptosis preferentially in HIV-infected cells, suggesting that the drugs mediate escape from the viral suppression of defensive apoptosis. In infected H9 cells, ciclopirox and deferiprone enhanced mitochondrial membrane depolarization, initiating the intrinsic pathway of apoptosis to execution, as evidenced by caspase-3 activation, poly(ADP-ribose) polymerase proteolysis, DNA degradation, and apoptotic cell morphology. In isolate-infected peripheral blood mononuclear cells, ciclopirox collapsed HIV-1 production to the limit of viral protein and RNA detection. Despite prolonged monotherapy, ciclopirox did not elicit breakthrough. No viral re-emergence was observed even 12 weeks after drug cessation, suggesting elimination of the proviral reservoir. Tests in mice predictive for cytotoxicity to human epithelia did not detect tissue damage or activation of apoptosis at a ciclopirox concentration that exceeded by orders of magnitude the concentration causing death of infected cells. We infer that ciclopirox and deferiprone act via therapeutic reclamation of apoptotic proficiency (TRAP) in HIV-infected cells and trigger their preferential elimination. Perturbations in viral protein expression suggest that the antiretroviral activity of both drugs stems from their ability to inhibit hydroxylation of cellular proteins essential for apoptosis and for viral infection, exemplified by eIF5A. Our findings identify ciclopirox and deferiprone as prototypes of selectively cytocidal antivirals that eliminate viral infection by destroying infected cells. A drug-based drug discovery program, based on these compounds, is warranted to determine the potential of such agents in clinical trials of HIV-infected patients.

Storage lipid studies in tuberculosis reveal that foam cell biogenesis is disease-specific

Foam cells are lipid-laden macrophages that contribute to the inflammation and tissue damage associated with many chronic inflammatory disorders. Although foam cell biogenesis has been extensively studied in atherosclerosis, how these cells form during a chronic infectious disease such as tuberculosis is unknown. Here we report that, unlike the cholesterol-laden cells of atherosclerosis, foam cells in tuberculous lung lesions accumulate triglycerides. Consequently, the biogenesis of foam cells varies with the underlying disease. In vitro mechanistic studies showed that triglyceride accumulation in human macrophages infected with Mycobacterium tuberculosis is mediated by TNF receptor signaling through downstream activation of the caspase cascade and the mammalian target of rapamycin complex 1 (mTORC1). These features are distinct from the known biogenesis of atherogenic foam cells and establish a new paradigm for non-atherogenic foam cell formation. Moreover, they reveal novel targets for disease-specific pharmacological interventions against maladaptive macrophage responses.

Toxoplasma gondii Inactivates Human Plasmacytoid Dendritic Cells by Functional Mimicry of IL-10

Plasmacytoid dendritic cells (pDCs) are the major producers of IFN-α, an antiviral cytokine involved in immunomodulation and control of HIV type 1 replication, whereas Toxoplasma gondii is a life-threatening opportunistic infection in AIDS patients. During infection with HIV type 1, human pDCs decrease in circulation and remaining pDC produce lower amounts of IFN-α in response to viral stimulation. In this study, we investigated the impact of coinfection with T. gondii on the innate virus-directed responses of human pDCs. Using intracellular flow cytometry and fluorescence microscopy, we determined that T. gondii invaded but did not induce IFN-α or TNF-α in human pDC. However, T. gondii inhibited IFN-α and TNF-α produced in response to HSV and HIV, thus functionally inactivating pDC. IFN-α production was inhibited only in cells infected by T. gondii, which inhibited neither uptake of GFP-HSV nor localization of TLR9 in CD71+ endosomes, directing us to investigate downstream events. Using imaging flow cytometry, we found that both T. gondii and IL-10 inhibited virus-induced nuclear translocation, but not phosphorylation, of IFN response factor 7. Blockade of IFN response factor 7 nuclear translocation and inhibition of the IFN-α response was partially reversed by a deficiency in the T. gondii–derived ROP16 kinase, known to directly phosphorylate STAT3, a critical mediator of IL-10’s anti-inflammatory effects. Taken together, our results indicate that T. gondii suppresses pDC activation by mimicking IL-10’s regulatory effects through an ROP16 kinase-dependent mechanism. Our findings further imply a convergent mechanism of inhibition of TLR signaling by T. gondii and IL-10 and suggest potential negative consequences of HIV/T. gondii coinfection.