Richard M. Gallo

Regulation of the Actin Cytoskeleton by Rho Kinase Controls Antigen Presentation by CD1d

CD1d molecules are MHC class I-like molecules that present lipid Ags to NKT cells. Although we have previously shown that several different cell signaling molecules can play a role in the control of Ag presentation by CD1d, a defined mechanism by which a cell signaling pathway regulates CD1d function has been unclear. In the current study, we have found that the Rho kinases, Rho-associated, coiled-coil containing protein kinase (ROCK)1 and ROCK2, negatively regulate both human and mouse CD1d-mediated Ag presentation. Inhibition of ROCK pharmacologically, through specific ROCK1 and ROCK2 short hairpin RNA, or by using dendritic cells generated from ROCK1-deficient mice all resulted in enhanced CD1d-mediated Ag presentation compared with controls. ROCK regulates the actin cytoskeleton by phosphorylating LIM kinase, which, in turn, phosphorylates cofilin, prohibiting actin fiber depolymerization. Treatment of APCs with the actin filament depolymerizing agent, cytochalasin D, as well as knockdown of LIM kinase by short hairpin RNA, resulted in enhanced Ag presentation to NKT cells by CD1d, consistent with our ROCK inhibition data. Therefore, our overall results reveal a model whereby CD1d-mediated Ag presentation is negatively regulated by ROCK via its effects on the actin cytoskeleton.

Statins Impair CD1d-Mediated Antigen Presentation through the Inhibition of Prenylation

Statins are widely used as cholesterol-lowering agents that also decrease inflammation and target enzymes essential for prenylation, an important process in the activation and intracellular transport of proteins vital for a wide variety of cellular functions. Here, we report that statins impair a critical component of the innate immune response, CD1d-mediated Ag presentation. The addition of specific intermediates in the isoprenylation pathway reversed this effect, whereas specific targeting of enzymes responsible for prenylation mimicked the inhibitory effects of statins on Ag presentation by CD1d as well as MHC class II molecules. This study demonstrates the importance of isoprenylation in the regulation of Ag presentation and suggests a mechanism by which statins reduce inflammatory responses.

Anthrax lethal toxin impairs CD1d-mediated antigen presentation by targeting the extracellular signal-related kinase 1/2 mitogen-activated protein kinase pathway.

ABSTRACT Lethal toxin (LT) is a critical virulence factor of Bacillus anthracis and an important means by which this bacterium evades the hosts immune system. In this study, we demonstrate that CD1d-expressing cells treated with LT have reduced CD1d-mediated antigen presentation. We earlier showed an important role for the mitogen-activated protein kinase extracellular signal-regulated kinase 1/2 (ERK1/2) in the regulation of CD1d-mediated antigen presentation, and we report here that LT impairs antigen presentation by CD1d in an ERK1/2-dependent manner. Similarly, LT and the ERK1/2 pathway-specific inhibitor U0126 caused a decrease in major histocompatibility complex (MHC) class II-mediated antigen presentation. Confocal microscopy analyses revealed altered intracellular distribution of CD1d and LAMP-1 in LT-treated cells, similar to the case for ERK1/2-inhibited cells. These results suggest that Bacillus anthracis has the ability to evade the hosts innate immune system by reducing CD1d-mediated antigen presentation through targeting the ERK1/2 pathway.

STAT3 promotes CD1d-mediated lipid antigen presentation by regulating a critical gene in glycosphingolipid biosynthesis.

Cytokines that regulate the immune response signal through the Janus kinase / signal transducer and activation of transcription (JAK/STAT) pathway, but whether this pathway can regulate CD1d‐mediated lipid antigen presentation to natural killer T (NKT) cells is unknown. Here, we found that STAT3 promotes antigen presentation by CD1d. Antigen‐presenting cells (APCs) in which STAT3 expression was inhibited exhibited markedly reduced endogenous lipid antigen presentation to NKT cells without an impact on exogenous lipid antigen presentation by CD1d. Consistent with this observation, in APCs where STAT3 was knocked down, dramatically decreased levels of UDP glucose ceramide glucosyltransferase (UGCG), an enzyme involved in the first step of glycosphingolipid biosynthesis, were observed. Impaired lipid antigen presentation was reversed by ectopic expression of UGCG in STAT3‐silenced CD1d+ APCs. Hence, by controlling a fundamental step in CD1d‐mediated lipid antigen presentation, STAT3 signalling promotes innate immune responses driven by CD1d.

A VP22-Null HSV-1 Is Impaired in Inhibiting CD1d-Mediated Antigen Presentation

CD1d-restricted T (natural killer T [NKT]) cells are important for controlling a herpes simplex virus (HSV) infection. One of the mechanisms of immune evasion by HSV is to downregulate CD1d-mediated activation of NKT cells. VP22 is an HSV-1-encoded protein responsible for reorganizing the host cells cytoskeletal network and viral spreading. We have previously shown that modification of the cytoskeleton can alter CD1d-mediated antigen presentation. In this study, we found that an HSV-1 lacking VP22 (ΔUL49) was impaired in its ability to inhibit CD1d-mediated antigen presentation compared with the wild-type (WT) virus; this was reversed by a repair virus (UL49R) in CD1d-expressing cells. We further demonstrated that CD1d recycling was inhibited by infection with WT and UL49R, but not the ΔUL49 virus. Ectopic expression of VP22 in CD1d-expressing cells complemented the VP22-deficient virus in inhibiting antigen presentation. Moreover, inhibiting viral protein synthesis rescued VP22-dependent inhibition of CD1d antigen presentation. In conclusion, our findings suggest that VP22 is required (but not sufficient) for the inhibition of CD1d-mediated antigen presentation by an HSV-1 infection.

Forming a complex with MHC class I molecules interferes with mouse CD1d functional expression.

CD1d molecules are structurally similar to MHC class I, but present lipid antigens as opposed to peptides. Here, we show that MHC class I molecules physically associate with (and regulate the functional expression of) mouse CD1d on the surface of cells. Low pH (3.0) acid stripping of MHC class I molecules resulted in increased surface expression of murine CD1d on antigen presenting cells as well as augmented CD1d-mediated antigen presentation to NKT cells. Consistent with the above results, TAP1-/- mice were found to have a higher percentage of type I NKT cells as compared to wild type mice. Moreover, bone marrow-derived dendritic cells from TAP1-/- mice showed increased antigen presentation by CD1d compared to wild type mice. Together, these results suggest that MHC class I molecules can regulate NKT cell function, in part, by masking CD1d.