William Housley

Natural but Not Inducible Regulatory T Cells Require TNF-α Signaling for In Vivo Function

TNF-α has a multifunctional role in autoimmune diseases as reflected in the variable responses of different human diseases to anti–TNF-α therapy. Recent studies have suggested that TNF-α modulates autoimmunity partially via effects on regulatory T cells (Tregs) and that these effects are mediated through the type II TNFR (TNFR2). We have investigated the requirement for TNFR2-expression on murine natural Tregs (nTregs) and induced Tregs (iTregs) in mediating suppression of colitis. Surprisingly, we find that TNFR2-expression is required for both spleen- and thymus-derived nTreg-mediated suppression, but is not required for iTreg-mediated suppression. Abnormal TNFR2−/− nTreg function was not associated with an in vivo decrease in accumulation, stability, or expression of markers known to be relevant in Treg function. Because iTregs are generated in the presence of TGF-β, we investigated whether activation in the presence of TGF-β could overcome the functional defect in TNFR2−/− nTregs. Although preactivation alone did not restore suppressive function of nTregs, preactivation in the presence of TGF-β did. These results identify potentially critical differences in activation requirements for nTregs versus iTregs. Furthermore, our findings are consistent with reports suggesting that nTregs are activated in sites of inflammation while iTregs are activated in lymph nodes. Finally, by demonstrating that nTregs require TNF-α for optimal function whereas iTregs do not, our results suggest that the enigma of variable responses of different human diseases to anti–TNF-α therapy may relate to whether nTregs or iTregs have the predominant regulatory role in a given disease.

Unique Lipids from a Common Human Bacterium Represent a New Class of Toll-Like Receptor 2 Ligands Capable of Enhancing Autoimmunity

Recent reports suggest that commensal bacteria may play a down-regulatory role in autoimmune disease. In the present studies, we demonstrate that phosphorylated dihydroceramides, uniquely structured lipids derived from the common human oral bacterium Porphyromonas gingivalis and from bacteria commonly found in the gastrointestinal tract and other organs, are capable of enhancing autoimmunity. We have previously reported that these lipids have proinflammatory effects on human fibroblasts in vitro and, in preliminary studies, have recovered these lipids from surgically removed human carotid atheroma, suggesting that they may play a role in human inflammatory disease. To investigate whether these lipids have functional effects on autoimmunity, we administered phosphorylated dihydroceramides to mice with the murine model of multiple sclerosis, experimental allergic encephalomyelitis (EAE). We find that these lipids, and particularly the phosphoethanolamine dihydroceramide (PE DHC) fraction, significantly enhanced EAE. Mechanistically, PE DHC enhances EAE in mice lacking natural killer T cells, fails to enhance EAE in Toll-like receptor 2 (TLR2)-deficient mice and, in vitro, induces dendritic cell interleukin-6 secretion in a TLR2-dependent manner. Finally, PE DHC-treated mice with EAE demonstrate a decreased percentage of spinal cord Foxp3+ T cells, suggesting that these lipids may affect regulatory aspects of adaptive immune responses. Overall, our results suggest that phosphorylated dihydroceramides derived from common human bacteria function as TLR2 ligands and may play a previously unrecognized role in human autoimmune diseases.

PDE8 Regulates Rapid Teff Cell Adhesion and Proliferation Independent of ICER

Background Abolishing the inhibitory signal of intracellular cAMP by phosphodiesterases (PDEs) is a prerequisite for effector T (Teff) cell function. While PDE4 plays a prominent role, its control of cAMP levels in Teff cells is not exclusive. T cell activation has been shown to induce PDE8, a PDE isoform with 40- to 100-fold greater affinity for cAMP than PDE4. Thus, we postulated that PDE8 is an important regulator of Teff cell functions. Methodology/Principal Findings We found that Teff cells express PDE8 in vivo. Inhibition of PDE8 by the PDE inhibitor dipyridamole (DP) activates cAMP signaling and suppresses two major integrins involved in Teff cell adhesion. Accordingly, DP as well as the novel PDE8-selective inhibitor PF-4957325-00 suppress firm attachment of Teff cells to endothelial cells. Analysis of downstream signaling shows that DP suppresses proliferation and cytokine expression of Teff cells from Crem −/− mice lacking the inducible cAMP early repressor (ICER). Importantly, endothelial cells also express PDE8. DP treatment decreases vascular adhesion molecule and chemokine expression, while upregulating the tight junction molecule claudin-5. In vivo, DP reduces CXCL12 gene expression as determined by in situ probing of the mouse microvasculature by cell-selective laser-capture microdissection. Conclusion/Significance Collectively, our data identify PDE8 as a novel target for suppression of Teff cell functions, including adhesion to endothelial cells.

PPARγ regulates retinoic acid‐mediated DC induction of Tregs

CD4+ CD25+ Foxp3+ Tregs are critical regulators of immune responses and autoimmune diseases. nTregs are thymically derived; iTregs are converted in the periphery from CD4+ CD25– Foxp3– Teffs. Recent studies reported that GALT CD103+ DCs mediated enhanced iTreg conversion via the secretion of RA. However, the factors regulating RA secretion and hence, the induction of iTregs by DCs are not yet clear. Activation of the nuclear hormone receptor PPARγ has been shown to induce RA expression in human DCs, and thus, we postulated that PPARγ activation in DCs may be an important regulator of RA secretion and iTreg generation. Using in vitro and in vivo approaches, we now demonstrate that PPARγ activation enhances iTreg generation through increased RA synthesis from murine splenic DCs. In addition, we demonstrate that inhibition of DC PPARγ decreases iTreg generation, suggesting a role for endogenous PPARγ ligands in this process. Overall, our findings suggest that PPARγ may be important as a factor that stimulates DCs to produce RA and as a potential mechanism by which PPARγ ligands ameliorate autoimmunity.

Cbl-b−/− T Cells Demonstrate In Vivo Resistance to Regulatory T Cells but a Context-Dependent Resistance to TGF-β

Cbl-b is an E3 ubiquitin ligase that negatively regulates T cell activation. Cbl-b−/− mice develop spontaneous autoimmunity, and Cbl-b dysregulation has been described in both murine and human autoimmune diseases. Although the mechanisms underlying the development of autoimmunity in Cbl-b−/− mice are not yet clear, we have reported that Cbl-b−/− CD4+CD25− effector T cells (Teffs) are resistant to CD4+CD25+ regulatory T cell (Treg)-mediated suppression in vitro and have suggested that this may be an important mechanism in the development of autoimmunity. To confirm the relevance of this resistance to autoimmune disease, we now show that Cbl-b−/− Teffs are resistant to suppression by Tregs in vivo and that this involves a resistance of truly naive Cbl-b−/− Teffs. Additionally, we show that Cbl-b−/− Tregs are fully functional in vivo, further suggesting that the regulatory abnormalities in Cbl-b−/− mice are related to defects in Teff, not Treg, function. To characterize the relevance of TGF-β sensitivity in Treg resistance, we examined in vivo Th17 generation and report that Cbl-b−/− mice are able to mount a normal Th17 response in vivo. As Cbl-b−/− Teffs have been shown to be insensitive to the suppressive effects of TGF-β in other in vivo models, the present results suggest that Cbl-b−/− Teffs demonstrate a context-dependent sensitivity to TGF-β in vivo. Overall, our results suggest that resistance to Tregs may be a bona fide mechanism underlying autoimmunity and that Cbl-b−/− mice offer unique approaches for studying the interrelationships between Treg function, TGF-β–mediated responses, and the development of autoimmunity.

Free Lipid A Isolated from Porphyromonas gingivalis Lipopolysaccharide Is Contaminated with Phosphorylated Dihydroceramide Lipids: Recovery in Diseased Dental Samples

ABSTRACT Recent reports indicate that Porphyromonas gingivalis mediates alveolar bone loss or osteoclast modulation through engagement of Toll-like receptor 2 (TLR2), though the factors responsible for TLR2 engagement have yet to be determined. Lipopolysaccharide (LPS) and lipid A, lipoprotein, fimbriae, and phosphorylated dihydroceramides of P. gingivalis have been reported to activate host cell responses through engagement of TLR2. LPS and lipid A are the most controversial in this regard because conflicting evidence has been reported concerning the capacity of P. gingivalis LPS or lipid A to engage TLR2 versus TLR4. In the present study, we first prepared P. gingivalis LPS by the Tri-Reagent method and evaluated this isolate for contamination with phosphorylated dihydroceramide lipids. Next, the lipid A prepared from this LPS was evaluated for the presence of phosphorylated dihydroceramide lipids. Finally, we characterized the lipid A by the matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and electrospray-MS methods in order to quantify recovery of lipid A in lipid extracts from diseased teeth or subgingival plaque samples. Our results demonstrate that both the LPS and lipid A derived from P. gingivalis are contaminated with phosphorylated dihydroceramide lipids. Furthermore, the lipid extracts derived from diseased teeth or subgingival plaque do not contain free lipid A constituents of P. gingivalis but contain substantial amounts of phosphorylated dihydroceramide lipids. Therefore, the free lipid A of P. gingivalis is not present in measurable levels at periodontal disease sites. Our results also suggest that the TLR2 activation of host tissues attributed to LPS and lipid A of P. gingivalis could actually be mediated by phosphorylated dihydroceramides.

Phosphorylated Dihydroceramides from Common Human Bacteria Are Recovered in Human Tissues

Novel phosphorylated dihydroceramide (PDHC) lipids produced by the periodontal pathogen Porphyromonas gingivalis include phosphoethanolamine (PE DHC) and phosphoglycerol dihydroceramides (PG DHC) lipids. These PDHC lipids mediate cellular effects through Toll-like receptor 2 (TLR2) including promotion of IL-6 secretion from dendritic cells and inhibition of osteoblast differentiation and function in vitro and in vivo. The PE DHC lipids also enhance (TLR2)-dependent murine experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. The unique non-mammalian structures of these lipids allows for their specific quantification in bacteria and human tissues using multiple reaction monitoring (MRM)-mass spectrometry (MS). Synthesis of these lipids by other common human bacteria and the presence of these lipids in human tissues have not yet been determined. We now report that synthesis of these lipids can be attributed to a small number of intestinal and oral organisms within the Bacteroides, Parabacteroides, Prevotella, Tannerella and Porphyromonas genera. Additionally, the PDHCs are not only present in gingival tissues, but are also present in human blood, vasculature tissues and brain. Finally, the distribution of these TLR2-activating lipids in human tissues varies with both the tissue site and disease status of the tissue suggesting a role for PDHCs in human disease.

Peroxisome proliferator-activated receptor gamma is required for CD4+ T cell-mediated lymphopenia-associated autoimmunity.

The nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) was shown to play an immunoregulatory role in many immune-related cell types, and activation of PPARγ was reported to be an effective therapeutic approach in murine and human autoimmune disease. However, despite an association between lymphopenia and autoimmunity, there has been no study on the role of T cell PPARγ in lymphopenia-associated autoimmunity. In the present studies, we examined the role of PPARγ in CD4+ T cells in two murine models of lymphopenia-associated autoimmunity. Surprisingly, we found that PPARγ expression in CD4+ CD25− T cells (T effector cells [Teffs]) is actually required for development of autoimmunity under lymphopenic conditions. Mechanistically, the inability of PPARγ-deficient (T-PPAR) Teffs to mediate lymphopenic autoimmunity is associated with a significant decrease in accumulation of Teffs in the spleen, lymph nodes, and tissues after adoptive transfer. This abnormal accumulation of T-PPAR Teffs was associated with defects in both in vivo proliferation and survival. Additionally, T-PPAR Teffs demonstrated decreased cytokine production in inflammatory sites and decreased expression of the homing receptor α4β7. Finally, these abnormalities in T-PPAR Teff function were not elicited by lymphopenia alone but also required the additional activation involved in the mediation of autoimmunity. Thus, in contrast to its documented immunosuppressive role, we identified an unexpected function for PPARγ in Teffs: a role in Teff proliferation and survival in lymphopenia-associated autoimmunity. These findings highlight both the multifunctional role of PPARγ in T cells and the complexity of PPARγ as a potential therapeutic target in autoimmunity.

Regulatory T-cells and cAMP suppress effector T-cells independently of PKA-CREM/ICER: a potential role for Epac.

cAMP signalling is both a major pathway as well as a key therapeutic target for inducing immune tolerance and is involved in Treg cell (regulatory T-cell) function. To achieve potent immunoregulation, cAMP can act through several downstream effectors. One proposed mechanism is that cAMP-mediated suppression, including immunosuppression by Treg cells, results from activation of PKA (protein kinase A) leading to the induction of the transcription factor ICER (inducible cAMP early repressor). In the present study, we examined CD4(+)CD25(-) Teff cell (effector T-cell) and CD4(+)CD25(+) Treg cell immune responses in Crem (cAMP-response-element modulator) gene-deficient mice which lack ICER (Crem(-/-)/ICER-deficient mice). ICER deficiency did not significantly alter the frequency or number of Treg cells and Teff cells. Treg cells or a pharmacological increase in cAMP suppressed Teff cells from Crem(+/+) and Crem(-/-)/ICER-deficient mice to an equivalent degree, demonstrating that ICER is dispensable in these functions. Additionally, activating the cAMP effector Epac (exchange protein directly activated by cAMP) suppressed Teff cells. Treg cells expressed low levels of all cyclic nucleotide Pde (phosphodiesterase) genes tested, but high levels of Epac. These data identify ICER as a redundant mediator of Treg cells and cAMP action on Teff cells and suggest that Epac may function as an alternative effector to promote cAMP-dependent Teff cell suppression.

Differential Expression and Function of PDE8 and PDE4 in Effector T cells: Implications for PDE8 as a Drug Target in Inflammation

Abolishing the inhibitory signal of intracellular cAMP is a prerequisite for effector T (Teff) cell function. The regulation of cAMP within leukocytes critically depends on its degradation by cyclic nucleotide phosphodiesterases (PDEs). We have previously shown that PDE8A, a PDE isoform with 40–100-fold greater affinity for cAMP than PDE4, is selectively expressed in Teff vs. regulatory T (Treg) cells and controls CD4+ Teff cell adhesion and chemotaxis. Here, we determined PDE8A expression and function in CD4+ Teff cell populations in vivo. Using magnetic bead separation to purify leukocyte populations from the lung draining hilar lymph node (HLN) in a mouse model of ovalbumin-induced allergic airway disease (AAD), we found by Western immunoblot and quantitative (q)RT-PCR that PDE8A protein and gene expression are enhanced in the CD4+ T cell fraction over the course of the acute inflammatory disease and recede at the late tolerant non-inflammatory stage. To evaluate PDE8A as a potential drug target, we compared the selective and combined effects of the recently characterized highly potent PDE8-selective inhibitor PF-04957325 with the PDE4-selective inhibitor piclamilast (PICL). As previously shown, PF-04957325 suppresses T cell adhesion to endothelial cells. In contrast, we found that PICL alone increased firm T cell adhesion to endothelial cells by ~20% and significantly abrogated the inhibitory effect of PF-04957325 on T cell adhesion by over 50% when cells were co-exposed to PICL and PF-04957325. Despite its robust effect on T cell adhesion, PF-04957325 was over two orders of magnitude less efficient than PICL in suppressing polyclonal Teff cell proliferation, and showed no effect on cytokine gene expression in these cells. More importantly, PDE8 inhibition did not suppress proliferation and cytokine production of myelin-antigen reactive proinflammatory Teff cells in vivo and in vitro. Thus, targeting PDE8 through PF-04957325 selectively regulates Teff cell interactions with endothelial cells without marked immunosuppression of proliferation, while PDE4 inhibition has partially opposing effects. Collectively, our data identify PF-04957325 as a novel function-specific tool for the suppression of Teff cell adhesion and indicate that PDE4 and PDE8 play unique and non-redundant roles in the control of Teff cell functions.