LKB1 isoform expression modulates T cell plasticity downstream of PKCθ and IL-6.

Following activation, CD4 T cells undergo metabolic and transcriptional changes as they respond to external cues and differentiate into T helper (Th) cells. T cells exhibit plasticity between Th phenotypes in highly inflammatory environments, such as colitis, in which high levels of IL-6 promote plasticity between regulatory T (Treg) cells and Th17 cells. Protein Kinase C theta (PKCθ) is a T cell-specific serine/threonine kinase that promotes Th17 differentiation while negatively regulating Treg differentiation. Liver kinase B1 (LKB1), also a serine/threonine kinase and encoded by Stk11, is necessary for Treg survival and function. Stk11 can be alternatively spliced to produce a short variant (Stk11S) by transcribing a cryptic exon. However, the contribution of Stk11 splice variants to Th cell differentiation has not been previously explored. Here we show that in Th17 cells, the heterogeneous ribonucleoprotein, hnRNPLL, mediates Stk11 splicing into its short splice variant, and that Stk11S expression is diminished when Hnrnpll is depleted using siRNA knock-down approaches. We further show that PKCθ regulates hnRNPLL and, thus, Stk11S expression in Th17 cells. We provide additional evidence that exposing induced (i)Tregs to IL-6 culminates in Stk11 splicing downstream of PKCθAltogether our data reveal a yet undescribed outside-in signaling pathway initiated by IL-6, that acts through PKCθ and hnRNPLL to regulate Stk11 splice variants and facilitate Th17 cell differentiation. Furthermore, we show for the first time, that this pathway can also be initiated in developing iTregs exposed to IL-6, providing mechanistic insight into iTreg phenotypic stability and iTreg to Th17 cell plasticity.

Effects of Linoleic Acid-Rich Diet on Plasma Profiles of Eicosanoids and Development of Colitis in Il-10-/- Mice.

Dietary intake of linoleic acid (LA, 18:2ω-6) has risen dramatically in recent decades. Previous studies have suggested a high intake of LA could increase tissue concentrations of proinflammatory and protumorigenic ω-6-series eicosanoid metabolites, increasing risks of inflammation and associated diseases. However, the effects of a LA-rich diet on in vivo profiles of eicosanoids and development of inflammatory diseases are understudied. Here, we treated spontaneous colitis-prone (Il-10-/-) mice with a control diet (∼3 Cal% LA) or a LA-rich diet (∼9 Cal% LA) for 18 weeks and analyzed the effects of the LA-rich diet on profiles of eicosanoids and development of colitis. We found that treatment with the LA-rich diet increased the tissue level of LA: the liver levels of LA were 5.8 ± 0.6% in the control diet-treated mice versus 11.7 ± 0.7% in the LA-rich diet-treated mice (P < 0.01). The plasma concentrations of a series of LA-derived metabolites, including 9-hydroxyoctadecadienoic acid (HODE), 9,10-dihydroxyoctadecenoic acid (DiHOME), 12,13-DiHOME, and 13-HODE were significantly increased by treatment with the LA-rich diet (P < 0.05). However, the LA-rich diet had little effect on the severity of colitis in the treated Il-10-/- mice. These results suggest a limited role of increased consumption of dietary LA on promoting colitis in the Il-10-/- model.

Cell-Penetrating Anti-Protein Kinase C Theta Antibodies Act Intracellularly to Generate Stable, Highly Suppressive Regulatory T Cells.

Regulatory T cells maintain immunological tolerance and dampen inflammatory responses. Administering regulatory T cells can prevent the immune-mediated tissue destruction of graft-versus-host disease, which frequently accompanies hematopoietic stem cell transfer. Neutralizing the T cell-specific kinase, protein kinase C theta, which promotes T cell effector functions and represses regulatory T cell differentiation, augments regulatory T cell immunosuppression and stability. We used a synthetic, cell-penetrating peptide mimic to deliver antibodies recognizing protein kinase C theta into primary human CD4 T cells. When differentiated ex vivo into induced regulatory T cells, treated cells expressed elevated levels of the regulatory T cell transcriptional regulator forkhead box P3, the surface-bound immune checkpoint receptor programmed death receptor-1, and pro-inflammatory interferon gamma, previously ascribed to a specific population of stable, highly suppressive human induced regulatory T cells. The in vitro suppressive capacity of these induced regulatory T cells was 10-fold greater than that of T cells differentiated without antibody delivery. When administered at the time of graft-versus-host disease induction, using a humanized mouse model, antibody-treated regulatory T cells were superior to non-treated T cells in attenuating lethal outcomes. This antibody delivery approach may overcome obstacles currently encountered using patient-derived regulatory T cells as a cell-based therapy for immune modulation.

CD28 Signaling Drives Notch Ligand Expression on CD4 T Cells.

Notch signaling provides an important cue in the mammalian developmental process. It is a key player in T cell development and function. Notch ligands such as Delta-like ligands (DLL) 1, 3, 4, and JAG1, 2 can impact Notch signaling positively or negatively, by trans-activation or cis-inhibition. Trans and cis interactions are receptor-ligand interaction on two adjacent cells and interaction on the same cell, respectively. The former sends an activation signal and the later, a signal for inhibition of Notch. However, earlier reports suggested that Notch is activated in the absence of Notch ligand-expressing APCs in a purified population of CD4 T cells. Thus, the role of ligands in Notch activation, in a purified population of CD4 T cells, remains obscure. In this study, we demonstrate that mature CD4 T cells are capable of expressing Notch ligands on their surface very early upon activation with soluble antibodies against CD3 and CD28. Moreover, signaling solely through CD28 induces Notch ligand expression and CD3 signaling inhibits ligand expression, in contrast to Notch which is induced by CD3 signaling. Additionally, by using decoys, mimicking the Notch extracellular domain, we demonstrated that DLL1, DLL4, and JAG1, expressed on the T cells, can cis-interact with the Notch receptor and inhibit activation of Notch. Thus, our data indicate a novel mechanism of the regulation of Notch ligand expression on CD4 T cells and its impact on activated Notch.

Triclocarban Exposure Exaggerates Spontaneous Colonic Inflammation in Il-10-/- Mice.

Triclocarban (3,4,4'-trichlorocarbanilide, TCC) is a high-volume chemical used as an antimicrobial ingredient in many consumer and personal care products. In 2016, the Food and Drug Administration removed TCC from over-the-counter hand washing products. However, TCC remains approved to use in many other products and is a ubiquitous contaminant in the environment; furthermore, many common food crops can efficiently accumulate environmental TCC, resulting in potential human exposure through oral ingestion of contaminated food products. Therefore, human exposure to TCC could be a long-lasting and serious problem. A better understanding of its impact on human health could lead to important impact for public health and regulatory policy. Using a spontaneous colonic inflammation model in Il-10-/- mice, here we demonstrate that exposure to TCC, at doses relevant to human exposure, exaggerates spontaneous colonic inflammation in Il-10-/- mice, with reduced colon length, increase fecal concentration of lipocalin 2, enhanced gene expression of Il-6 and Ifn-γ in the colon, and exaggerated crypt damage in the colon. Collectively, these results support that TCC could be a potential environmental risk factor of colitis and associated gut diseases.

Cymerus™ iPSC-MSCs significantly prolong survival in a pre-clinical, humanized mouse model of Graft-vs-host disease.

The immune-mediated tissue destruction of graft-vs-host disease (GvHD) remains a major barrier to greater use of hematopoietic stem cell transplantation (HSCT). Mesenchymal stem cells (MSCs) have intrinsic immunosuppressive qualities and are being actively investigated as a therapeutic strategy for treating GvHD. We characterized Cymerus™ MSCs, which are derived from adult, induced pluripotent stem cells (iPSCs), and show they display surface markers and tri-lineage differentiation consistent with MSCs isolated from bone marrow (BM). Administering iPSC-MSCs altered phosphorylation and cellular localization of the T cell-specific kinase, Protein Kinase C theta (PKCθ), attenuated disease severity, and prolonged survival in a humanized mouse model of GvHD. Finally, we evaluated a constellation of pro-inflammatory molecules on circulating PBMCs that correlated closely with disease progression and which may serve as biomarkers to monitor therapeutic response. Altogether, our data suggest Cymerus iPSC-MSCs offer the potential for an off-the-shelf, cell-based therapy to treat GvHD.

Rotenone Treatment Reveals a Role for Electron Transport Complex I in the Subcellular Localization of Key Transcriptional Regulators During T Helper Cell Differentiation.

Recent advances in our understanding of tumor cell mitochondrial metabolism suggest it may be an attractive therapeutic target. Mitochondria are central hubs of metabolism that provide energy during the differentiation and maintenance of immune cell phenotypes. Mitochondrial membranes harbor several enzyme complexes that are involved in the process of oxidative phosphorylation, which takes place during energy production. Data suggest that, among these enzyme complexes, deficiencies in electron transport complex I may differentially affect immune responses and may contribute to the pathophysiology of several immunological conditions. Once activated by T cell receptor signaling, along with co-stimulation through CD28, CD4 T cells utilize mitochondrial energy to differentiate into distinct T helper (Th) subsets. T cell signaling activates Notch1, which is cleaved from the plasma membrane to generate its intracellular form (N1ICD). In the presence of specific cytokines, Notch1 regulates gene transcription related to cell fate to modulate CD4 Th type 1, Th2, Th17, and induced regulatory T cell (iTreg) differentiation. The process of differentiating into any of these subsets requires metabolic energy, provided by the mitochondria. We hypothesized that the requirement for mitochondrial metabolism varies between different Th subsets and may intersect with Notch1 signaling. We used the organic pesticide rotenone, a well-described complex I inhibitor, to assess how compromised mitochondrial integrity impacts CD4 T cell differentiation into Th1, Th2, Th17, and iTreg cells. We also investigated how Notch1 localization and downstream transcriptional capabilities regulation may be altered in each subset following rotenone treatment. Our data suggest that mitochondrial integrity impacts each of these Th subsets differently, through its influence on Notch1 subcellular localization. Our work further supports the notion that altered immune responses can result from complex I inhibition. Therefore, understanding how mitochondrial inhibitors affect immune responses may help to inform therapeutic approaches to cancer treatment.

Intracellular Delivery of Anti-pPKCθ (Thr538) via Protein Transduction Domain Mimics for Immunomodulation.

Targeting cellular proteins with antibodies, to better understand cellular signaling pathways in the context of disease modulation, is a fast-growing area of investigation. Humanized antibodies are increasingly gaining attention for their therapeutic potential, but the collection of cellular targets is limited to those secreted from cells or expressed on the cell surface. This approach leaves a wealth of intracellular proteins unexplored as putative targets for antibody binding. Protein kinase Cθ (PKCθ) is essential to T cell activation, proliferation, and differentiation, and its phosphorylation at specific residues is required for its activity. Here we report on the design, synthesis, and characterization of a protein transduction domain mimic capable of efficiently delivering an antibody against phosphorylated PKCθ (Thr538) into human peripheral mononuclear blood cells and altering expression of downstream indicators of T cell activation and differentiation. We used a humanized, lymphocyte transfer model of graft-versus-host disease, to evaluate the durability of protein transduction domain mimic:Anti-pPKCθ modulation, when delivered into human peripheral mononuclear blood cells ex vivo. We demonstrate that protein transduction domain mimic:Antibody complexes can be readily introduced with high efficacy into hard-to-transfect human peripheral mononuclear blood cells, eliciting a biological response sufficient to alter disease progression. Thus, protein transduction domain mimic:Antibody delivery may represent an efficient ex vivo approach to manipulating cellular responses by targeting intracellular proteins.