Relevance of acquired T cell molecular defects in the immunopathogenesis of SLE.

Systemic lupus erythematosus (SLE) and other autoimmune diseases are thought to develop in genetically predisposed individuals when triggered by environmental factors. This paradigm does not fully explain disease development, as it fails to consider the delay between birth and disease expression. In this review, we discuss observations described in T cells from patients with SLE that are not related to hereditary factors and have therefore been considered secondary to the disease process itself. Here, we contextualize some of those observations and argue that they may represent a pathogenic layer between genetic factors and disease development. Acquired changes in T cell phenotype and function in the setting of SLE may affect the immune system, creating a predisposition towards a more inflammatory and pathogenic system that amplifies autoimmunity and facilitates disease development.

The helminth-derived peptide GK-1 induces an anti-tumoral CD8 T cell response associated with downregulation of the PD-1/PD-L1 pathway.

CD8 T cells can kill malignant cells in an antigen-specific manner. However, anti-tumoral responses are usually limited by suppressive factors that curb the effector responses of tumor-infiltrating CD8 T cells. Therapeutic strategies to overcome intra-tumoral T cell suppression, for example immune checkpoint inhibition, have been clinically effective in patients with cancer. Here, we provide data that demonstrates that GK-1, a peptide derived from the parasite Taenia crassiceps, promotes an anti-melanoma CD8 T cell response with heightened effector characteristics that leads to an increased amount of tumor-infiltrating CD44+ IFN-γ-producing CD8 T cells. The response induced by GK-1 was associated with a reduction in the expression of PD-1 and PD-L1 on tumor-infiltrating CD8 and dendritic cells, respectively, effects that led to a dramatic decrease in tumor burden. Our results suggest that the immunomodulatory properties of GK-1 may promote a CD8 T cell response that may be therapeutically useful in the setting of cancer.

Protein Tyrosine Kinase Fyn Regulates TLR4-Elicited Responses on Mast Cells Controlling the Function of a PP2A-PKCα/ß Signaling Node Leading to TNF Secretion.

Mast cells produce proinflammatory cytokines in response to TLR4 ligands, but the signaling pathways involved are not fully described. In this study, the participation of the Src family kinase Fyn in the production of TNF after stimulation with LPS was evaluated using bone marrow-derived mast cells from wild-type and Fyn-deficient mice. Fyn(-/-) cells showed higher LPS-induced secretion of preformed and de novo-synthesized TNF. In both cell types, TNF colocalized with vesicle-associated membrane protein (VAMP)3-positive compartments. Addition of LPS provoked coalescence of VAMP3 and its interaction with synaptosomal-associated protein 23; those events were increased in the absence of Fyn. Higher TNF mRNA levels were also observed in Fyn-deficient cells as a result of increased transcription and greater mRNA stability after LPS treatment. Fyn(-/-) cells also showed higher LPS-induced activation of TAK-1 and ERK1/2, whereas IκB kinase and IκB were phosphorylated, even in basal conditions. Increased responsiveness in Fyn(-/-) cells was associated with a lower activity of protein phosphatase 2A (PP2A) and augmented activity of protein kinase C (PKC)α/ß, which was dissociated from PP2A and increased its association with the adapter protein neuroblast differentiation-associated protein (AHNAK, desmoyokin). LPS-induced PKCα/ß activity was associated with VAMP3 coalescence in WT and Fyn-deficient cells. Reconstitution of MC-deficient Wsh mice with Fyn(-/-) MCs produced greater LPS-dependent production of TNF in the peritoneal cavity. Our data show that Fyn kinase is activated after TLR4 triggering and exerts an important negative control on LPS-dependent TNF production in MCs controlling the inactivation of PP2Ac and activation of PKCα/ß necessary for the secretion of TNF by VAMP3(+) carriers.

Morphine prevents lipopolysaccharide-induced TNF secretion in mast cells blocking IκB kinase activation and SNAP-23 phosphorylation: correlation with the formation of a ß-arrestin/TRAF6 complex.

We have previously shown that morphine pretreatment inhibits mast cell-dependent TNF production after LPS injection in the murine peritoneal cavity. In this study, we used bone marrow-derived mast cells (BMMCs) to investigate the molecular mechanisms of that inhibition. We found that morphine prevented LPS-induced TNF secretion in these cells. The observed inhibition was not due to morphine-induced TLR4 internalization and it was related to the blockage of preformed TNF secretion. LPS-induced TNF exocytosis in BMMCs was dependent on tetanus toxin-insensitive vesicle-associated membrane proteins and calcium mobilization, as well as PI3K, MAPK, and IκB kinase (IKK) activation. TNF secretion was also associated to the phosphorylation of synaptosomal-associated protein 23 (SNAP-23), which was found forming a complex with IKK in LPS-activated BMMCs. Morphine pretreatment prevented TLR4-dependent ERK and IKK phosphorylation. Analyzing the signaling events upstream of IKK activation, we found diminished TGF-ß-activated kinase 1 (TAK1) phosphorylation and TNFR-associated factor (TRAF) 6 ubiquitination in BMMCs pretreated with morphine and stimulated with LPS. Morphine pretreatment provoked a marked increase in the formation of a molecular complex composed of TRAF6 and ß-arrestin-2. Naloxone and a combination of µ and δ opioid receptor antagonists prevented morphine inhibitory actions. In conclusion, our results show that activation of µ and δ opioid receptors with morphine suppresses TLR4-induced TNF release in mast cells, preventing the IKK-dependent phosphorylation of SNAP-23, which is necessary for TNF exocytosis, and this inhibition correlates with the formation of a ß-arrestin-2/TRAF6 complex. To our knowledge, these findings constitute the first evidence of molecular crosstalk between opioid receptors and the TLR4 signal transduction system in mast cells.

Down-Regulation-Resistant STAT4 Risk Haplotype Contributes to Lupus Nephritis Through CD4+ T Cell Interferon-γ Production.

OBJECTIVE: Variants in STAT4 are associated with systemic lupus erythematosus (SLE) and other autoimmune diseases. We undertook this study to investigate how disease-associated variants affect STAT4 expression, in particular in CD4+ T cells where STAT4 plays an essential role. METHODS: We compared Th1 differentiation between naive CD4+ T cells from healthy donors homozygous for the risk (R/R) or nonrisk (NR/NR) alleles. We analyzed epigenetic marks in STAT4 and evaluated the relevance of its third intron, assessed the consequences of Stat4 overexpression in vivo in mice, and analyzed the effects of the STAT4 genotype in patients with lupus nephritis. RESULTS: Naive CD4+ T cells from NR/NR healthy donors down-regulated STAT4 in response to interleukin-12 (IL-12). In contrast, cells from R/R healthy donors maintained high levels. R/R cells exhibited a higher abundance of transcriptionally active STAT4 and increased interferon-γ production. Accordingly, R/R healthy donors exhibited a stronger induction of local active enhancer marks. Genetic editing confirmed the presence of a negative regulatory region in the STAT4 third intron, where most of the SLE-associated STAT4 single-nucleotide polymorphisms (SNPs) are located. In vivo forced expression demonstrated that increases in Stat4 levels in T cells enhanced glomerulonephritis in mice. Accordingly, the R/R genotype was associated with suboptimal response to treatment and with worse clinical outcomes in patients with proliferative lupus nephritis. CONCLUSION: The SLE-associated STAT4 haplotype correlates with an abnormal IL-12-mediated STAT4 transcriptional regulation. Carriers of the risk variant exhibit exaggerated CD4+ proinflammatory capacities that, in the context of SLE, contribute to more severe disease. R/R patients may benefit from blockade of the IL-12/STAT4 pathway.

Regulation of activated T cell survival in rheumatic autoimmune diseases.

Adaptive immune responses rely on the proliferation of T lymphocytes able to recognize and eliminate pathogens. The magnitude and duration of the expansion of activated T cell clones are finely regulated to minimize immunopathology and avoid autoimmunity. In patients with rheumatic autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis, activated lymphocytes survive and exert effector functions for prolonged periods, defying the mechanisms that normally curb their capacities during acute and chronic infections. Here, we review the molecular mechanisms that limit the duration of immune responses in health and discuss the factors that alter such regulation in the setting of systemic lupus erythematosus and rheumatoid arthritis. We highlight defects that could contribute to the development and progression of autoimmune disease and describe how chronic inflammation can alter the regulation of activated lymphocyte survival, promoting its perpetuation. These concepts might contribute to the understanding of the mechanisms that underlie the chronicity of inflammation in the context of autoimmunity.

Fas/FasL Signaling Regulates CD8 Expression During Exposure to Self-Antigens.

Activation of self-reactive CD8+ T cells induces a peripheral tolerance mechanism that involves loss of CD8 expression. Because genetic deficiency of Fas and Fasl causes the accumulation of double-negative (DN; CD3+ TCR-αß+ CD4- CD8-) T cells that have been proposed to derive from CD8+ cells, we decided to explore the role of Fas and FasL in self-antigen-induced CD8 downregulation. To this end, we quantified Fas and FasL induction by different stimuli and analyzed the effects of Fas/FasL deficiency during a protective immune response and after exposure to self-antigens. Our data describes how Fas and FasL upregulation differs depending on the setting of CD8 T cell activation and demonstrates that Fas/FasL signaling maintains CD8 expression during repetitive antigen stimulation and following self-antigen encounter. Together, our results reveal an unexpected role of Fas/FasL signaling and offer a new insight into the role of these molecules in the regulation of immune tolerance.

Protein phosphatase 2A B55ß limits CD8+ T cell lifespan following cytokine withdrawal.

How T cells integrate environmental cues into signals that limit the magnitude and length of immune responses is poorly understood. Here, we provide data that demonstrate that B55ß, a regulatory subunit of protein phosphatase 2A, represents a molecular link between cytokine concentration and apoptosis in activated CD8+ T cells. Through the modulation of AKT, B55ß induced the expression of the proapoptotic molecule Hrk in response to cytokine withdrawal. Accordingly, B55ß and Hrk were both required for in vivo and in vitro contraction of activated CD8+ lymphocytes. We show that this process plays a role during clonal contraction, establishment of immune memory, and preservation of peripheral tolerance. This regulatory pathway may represent an unexplored opportunity to end unwanted immune responses or to promote immune memory.

PPP2R2B hypermethylation causes acquired apoptosis deficiency in systemic autoimmune diseases.

Chronic inflammation causes target organ damage in patients with systemic autoimmune diseases. The factors that allow this protracted response are poorly understood. We analyzed the transcriptional regulation of PPP2R2B (B55ß), a molecule necessary for the termination of the immune response, in patients with autoimmune diseases. Altered expression of B55ß conditioned resistance to cytokine withdrawal-induced death (CWID) in patients with autoimmune diseases. The impaired upregulation of B55ß was caused by inflammation-driven hypermethylation of specific cytosines located within a regulatory element of PPP2R2B preventing CTCF binding. This phenotype could be induced in healthy T cells by exposure to TNF-α. Our results reveal a gene whose expression is affected by an acquired defect, through an epigenetic mechanism, in the setting of systemic autoimmunity. Because failure to remove activated T cells through CWID could contribute to autoimmune pathology, this mechanism illustrates a vicious cycle through which autoimmune inflammation contributes to its own perpetuation.

Tomosyn functions as a PKCδ-regulated fusion clamp in mast cell degranulation.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family proteins mediate membrane fusion critical for vesicular transport and cellular secretion. Mast cells rely on SNARE-mediated membrane fusion for degranulation stimulated by crosslinking of immunoglobulin E (IgE) bound to the Fcε receptor (FcεRI). We investigated the mechanisms downstream of receptor activation that control degranulation. We found that the SNARE binding protein tomosyn-1 (also known as STXBP5) inhibited FcεRI-stimulated degranulation of mast cells. After mast cell activation, tomosyn-1 was phosphorylated on serine and threonine residues, dissociated from the SNARE protein syntaxin 4 (STX4), and associated with STX3. We identified PKCδ as the major kinase required for tomosyn-1 threonine phosphorylation and for regulation of the interaction with STXs. Incubation with high IgE concentrations increased tomosyn-1 abundance in cultured mast cells. Similarly, in basophils from allergic patients with high amounts of serum IgE, the abundance of tomosyn-1 was increased as compared to that in patients with normal IgE concentrations. Our findings identified tomosyn-1 as an inhibitor of mast cell degranulation that required PKCδ to switch its interaction with STX partners during fusion. We suggest that the IgE-mediated increase in tomosyn-1 abundance in allergic patients may represent a counterregulatory mechanism to limit disease development.

Mast cells in renal inflammation and fibrosis: lessons learnt from animal studies.

Mast cells are hematopoietic cells involved in inflammation and immunity and have been recognized also as important effector cells in kidney inflammation. In humans, only a few mast cells reside in kidneys constitutively but in progressive renal diseases their numbers increase substantially representing an essential part of the interstitial infiltrate of inflammatory cells. Recent data obtained in experimental animal models have emphasized a complex role of these cells and the mediators they release as they have been shown both to promote, but also to protect from disease and fibrosis development. Sometimes conflicting results have been reported in similar models suggesting a very narrow window between these activities depending on the pathophysiological context. Interestingly in mice, mast cell or mast cell mediator specific actions became also apparent in the absence of significant mast cell kidney infiltration supporting systemic or regional actions via draining lymph nodes or kidney capsules. Many of their activities rely on the capacity of mast cells to release, in a timely controlled manner, a wide range of inflammatory mediators, which can promote anti-inflammatory actions and repair activities that contribute to healing, but in some circumstances or in case of inappropriate regulation may also promote kidney disease.

Morphine decreases early peritoneal innate immunity responses in Swiss-Webster and C57BL6/J mice through the inhibition of mast cell TNF-α release.

OBJECTIVE: To characterize immunosuppressive effects of morphine on the early innate immunity response of cytokine production in peritoneal cavity after LPS challenge. METHODS: The effects of a single i.p. administration of morphine (3.1 or 31 mg/kg) on LPS-induced tumor necrosis factor α (TNF-α) and monocyte chemoattractant protein-1 (CCL2) intraperitoneal release was tested in Swiss-Webster, C57BL/6J, mast cell deficient Kit(Wsh/Wsh) (W-sh) and mast cell reconstituted (W-sh-rec) mice. RESULTS: Morphine was found to inhibit LPS-induced TNF-α but not CCL2 release in the peritoneal cavity. Studies on mast cell deficient and reconstituted mice indicate that resident mast cells mediate selective morphine immunosuppression in the peritoneal cavity.