Nitrosothiols in the immune system: signaling and protection

Abstract

Significance: In the immune system, nitric oxide (NO) has been mainly associated with antibacterial defenses exerted through oxidative, nitrosative, and nitrative stress and signal transduction through cyclic GMP-dependent mechanisms. However, S-nitrosylation is emerging as a post-translational modification (PTM) involved in NO-mediated cell signaling.

Recent advances: Precise roles for S-nitrosylation in signaling pathways have been described both for innate and adaptive immunity. Denitrosylation may protect macrophages from their own S-nitrosylation, while maintaining nitrosative stress compartmentalized in the phagosomes. Nitrosothiols have also been shown to be beneficial in experimental models of autoimmune diseases, mainly through their role in modulating T-cell differentiation and function.

Critical issues: Relationship between S-nitrosylation, other thiol redox PTMs, and other NO-signaling pathways has not been always taken into account, particularly in the context of immune responses. Methods for assaying S-nitrosylation in individual proteins and proteomic approaches to study the S-nitrosoproteome are constantly being improved, which helps to move this field forward.

Future directions: Integrated studies of signaling pathways in the immune system should consider whether S-nitrosylation/denitrosylation processes are among the PTMs influencing the activity of key signaling and adaptor proteins. Studies in pathophysiological scenarios will also be of interest to put these mechanisms into broader contexts. Interventions modulating nitrosothiol levels in autoimmune disease could be investigated with a view to developing new therapies.

FIG. 1.

S-nitrosylation modulates the quaternary structure and function of surfactant protein D (SP-D). The SP-D dodecamer binds to signal inhibitory regulatory protein α (SIRP-α) and inhibits p38 mitogen-activated protein kinase (MAPK) activation via SHP-1, and it also binds toll-like receptor 4 (TLR4), thereby avoiding its dimerization and subsequent activation. Upon S-nitrosylation, SP-D dodecamers disassemble into trimers, which bind to CD-91/calreticulin, a process that activates p38 MAPK and triggers a proinflammatory response. Dodecamer disassembly might allow TLR4 dimerization and downstream activation of NF-κB (To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars).

FIG. 2.

Macrophages protect themselves from inducible nitric oxide synthase (iNOS)-induced S-nitrosylation. Proinflammatory stimuli, such as interferon-γ (IFN-γ) and lipopolysaccharide (LPS), trigger iNOS expression. This iNOS associates with vesicles through the Golgi, and it may be recruited to phagosomes where it produces large amounts of NO that can S-nitrosylate protein targets in a phagocyted cell, such as a bacterium. Thioredoxin (Trx) and glutathione (GSH)/S-nitrosoglutathione reductase (GSNOR) may denitrosylate macrophage proteins that become modified (To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars).

FIG. 3.

The biotin-switch technique (BST) is not sensitive enough to detect endogenous S-nitrosylation produced by iNOS activation in macrophages. (A) Murine macrophage cell line RAW 264.7 was treated with LPS and IFN-γ, and with auranofin, producing iNOS that was detected in western blots and NO measured as extracellular nitrite with the Griess reagent. (B) Cell extracts were subjected to the BST, blotted, and detected with avidin. Although there is a clear increase in iNOS-derived NO production after LPS+IFN-γ activation, differences in S-nitrosylation are only observed when the Trx pathway is inhibited with auranofin. Reprinted by permission from Tello et al. (174).

FIG. 4.

The NF-κB activation pathway is regulated by S-nitrosylation. S-nitrosylation of the p50 and p65 subunits of NF-κB causes the inhibition of its binding to DNA. Upstream inhibition of the IκB-kinase (IKK) complex's kinase activity is induced by S-nitrosylation of the IKKβ subunit, abrogating NF-κB activation. S-nitrosylation of CD40 inhibits binding and activation by CD40L. MyD88 S-nitrosylation disrupts binding to its upstream partner toll/interleukin-1 receptor adaptor protein (TIRAP), which might serve to delay the development of the immune response. On the other hand, S-nitrosylation of Src may activate NF-κB (To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars).

FIG. 5.

Model of endothelial nitric oxide synthase (eNOS) activation of N-Ras on the Golgi complex of antigen-stimulated T cells. The figure schematically depicts the possible compartmentalized signaling through which T-cell receptor (TCR) stimulation may lead to the selective S-nitrosylation and activation of N-Ras on the Golgi complex. Engagement of the TCR by antigens results in the phosphorylation of the CD3ζ chain and the recruitment of the adapter kinase ZAP-70. This kinase in turn phosphorylates LAT and PI3K, leading to activation of PLC-γ and Akt, respectively. PLC-γ activation produces diacylglycerol (DAG) in the plasma membrane and inositol 1,4,5-triphosphate (IP3) in the cytosol. IP3 release liberates Ca²⁺ from internal stores, which binds to calmodulin-associated eNOS and induces the translocation of RasGRP1 to the Golgi, where the levels of DAG are high. Simultaneously, active Akt can phosphorylate eNOS on Ser¹¹⁷⁷. As a result, eNOS might be fully activated to synthesize NO and S-nitrosylate inactive (GDP-bound) N-Ras on Cys¹¹⁸ (small red arrow). This would weaken its interaction with GDP, thereby facilitating RasGRP1-mediated GDP-GTP exchange on the Golgi complex (large red arrow) (To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars).

FIG. 6.

Hypothetical S-nitrosylation/denitrosylation mechanisms in T-cell development. (A) Local high extracellular levels of NO produced by iNOS from corticomedullar and medullar dendritic cells (DCs) in the thymus promote S-nitrosylation in thymocytes, fostering negative selection of CD4⁺CD8⁺ double-positive thymocytes. Protein S-nitrosylation in thymocytes may induce apoptosis by increasing p53 expression. The iNOS-derived NO may promote apoptosis through the S-nitrosylation of GAPDH. S-nitrosylated GAPDH can bind to Siah1 and translocate to the nucleus, where it increases the acetyltransferase activity of p300/CBP and the ensuing synthesis of p53, which can in turn activate caspase-1 through a still-to-be determined mechanism. (B) Through denitrosylation, upregulated GSNOR expression or activity in thymocytes ongoing CD4⁺ maturation may counteract the proapoptotic actions of protein S-nitrosylation mediated by iNOS-derived NO from DCs. (C) Low levels of NO generated in thymocytes by constitutive NOS (cNOS) during their cognate interactions with iNOS-nonexpressing DCs and macrophages (antigen-presenting cells [APC]) may protect thymocytes from cell death by S-nitrosylation of caspase-1 (To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars).