Osteopontin drives KRAS-mutant lung adenocarcinoma.

Increased expression of osteopontin (secreted phosphoprotein 1, SPP1) is associated with aggressive human lung adenocarcinoma (LADC), but its function remains unknown. Our aim was to determine the role of SPP1 in smoking-induced LADC. We combined mouse models of tobacco carcinogen-induced LADC, of deficiency of endogenous Spp1 alleles, and of adoptive pulmonary macrophage reconstitution to map the expression of SPP1 and its receptors and determine its impact during carcinogenesis. Co-expression of Spp1 and mutant KrasG12C in benign cells was employed to investigate SPP1/KRAS interactions in oncogenesis. Finally, intratracheal adenovirus encoding Cre recombinase was delivered to LSL.KRASG12D mice lacking endogenous or overexpressing transgenic Spp1 alleles. SPP1 was overexpressed in experimental and human LADC and portended poor survival. In response to two different smoke carcinogens, Spp1-deficient mice developed fewer and smaller LADC with decreased cellular survival and angiogenesis. Both lung epithelial- and macrophage-secreted SPP1 drove tumor-associated inflammation, while epithelial SPP1 promoted early tumorigenesis by fostering the survival of KRAS-mutated cells. Finally, loss and overexpression of Spp1 was, respectively, protective and deleterious for mice harboring KRASG12D-driven LADC. Our data support that SPP1 is functionally involved in early stages of airway epithelial carcinogenesis driven by smoking and mutant KRAS and may present an important therapeutic target.

Osteopontin Promotes Protective Antigenic Tolerance against Experimental Allergic Airway Disease.

In the context of inflammation, osteopontin (Opn) is known to promote effector responses, facilitating a proinflammatory environment; however, its role during antigenic tolerance induction is unknown. Using a mouse model of asthma, we investigated the role of Opn during antigenic tolerance induction and its effects on associated regulatory cellular populations prior to disease initiation. Our experiments demonstrate that Opn drives protective antigenic tolerance by inducing accumulation of IFN-ß-producing plasmacytoid dendritic cells, as well as regulatory T cells, in mediastinal lymph nodes. We also show that, in the absence of TLR triggers, recombinant Opn, and particularly its SLAYGLR motif, directly induces IFN-ß expression in Ag-primed plasmacytoid dendritic cells, which renders them extra protective against induction of allergic airway disease upon transfer into recipient mice. Lastly, we show that blockade of type I IFNR prevents antigenic tolerance induction against experimental allergic asthma. Overall, we unveil a new role for Opn in setting up a tolerogenic milieu boosting antigenic tolerance induction, thus leading to prevention of allergic airway inflammation. Our results provide insight for the future design of immunotherapies against allergic asthma.

ERß in CD4+ T Cells Is Crucial for Ligand-Mediated Suppression of Central Nervous System Autoimmunity.

The development of therapies for multiple sclerosis targeting pathogenic T cell responses remains imperative. Previous studies have shown that estrogen receptor (ER) ß ligands could inhibit experimental autoimmune encephalomyelitis. However, the effects of ERß-specific ligands on human or murine pathogenic immune cells, such as Th17, were not investigated. In this article, we show that the synthetic ERß-specific ligand 4-(2-phenyl-5,7-bis[trifluoromethyl]pyrazolo[1,5-a]pyrimidin-3-yl)phenol (PHTPP) reversed established paralysis and CNS inflammation, characterized by a dramatic suppression of pathogenic Th responses as well as induction of IL-10-producing regulatory CD4(+) T cell subsets in vivo. Moreover, administration of PHTPP in symptomatic mice induced regulatory CD4(+) T cells that were suppressive in vivo. PHTPP-mediated experimental autoimmune encephalomyelitis amelioration was canceled in mice with ERß-deficient CD4(+) T cells only, indicating that expression of ERß by these cells is crucial for the observed therapeutic effect. Importantly, synthetic ERß-specific ligands acting directly on CD4(+) T cells suppressed human and mouse Th17 cells, downregulating Th17 cell signature gene expression and expanding IL-10-producing T cells among them. TGF-ß1 and aryl hydrocarbon receptor activation enhanced the ERß ligand-mediated expansion of IL-10-producing T cells among Th17 cells. In addition, these ERß-specific ligands promoted the induction and maintenance of Foxp3(+) T regulatory cells, as well as their in vitro suppressive function. Thus, ERß-specific ligands targeting pathogenic Th17 cells and inducing functional regulatory cells represent a promising subset of therapeutic agents for multiple sclerosis.

TIGIT Enhances Antigen-Specific Th2 Recall Responses and Allergic Disease.

T cell Ig and ITIM domain receptor (TIGIT), expressed on T, NK, and regulatory T cells, is known as an inhibitory molecule that limits autoimmunity, antiviral and antitumor immunity. In this report, we demonstrate that TIGIT enhances Th2 immunity. TIGIT expression was upregulated in activated Th2 cells from mice with experimental allergic disease and in Th2 polarization cultures. In addition, its high-affinity ligand CD155 was upregulated in mediastinal lymph node dendritic cells from allergic mice. In an in vitro setting, we observed that Tigit expression in Th2 cells and its interaction with CD155 expressed in dendritic cells were important during the development of Th2 responses. In addition, blockade of TIGIT inhibited Th2, but had no effect on either Th1 or Th17 polarization. In vivo blockade of TIGIT suppressed hallmarks of allergic airway disease, such as lung eosinophilia, goblet cell hyperplasia, Ag-specific Th2 responses, and IgE production, and reduced numbers of T follicular helper and effector Th2 cells. Thus, TIGIT is critical for Th2 immunity and can be used as a therapeutic target, especially in light of recent findings showing TIGIT locus hypomethylation in T cells from pediatric patients with allergic asthma.

ERß-Dependent Direct Suppression of Human and Murine Th17 Cells and Treatment of Established Central Nervous System Autoimmunity by a Neurosteroid.

Multiple sclerosis (MS), an autoimmune disease of the CNS, is mediated by autoreactive Th cells. A previous study showed that the neurosteroid dehydroepiandrosterone (DHEA), when administered preclinically, could suppress progression of relapsing-remitting experimental autoimmune encephalomyelitis (EAE). However, the effects of DHEA on human or murine pathogenic immune cells, such as Th17, were unknown. In addition, effects of this neurosteroid on symptomatic disease, as well as the receptors involved, had not been investigated. In this study, we show that DHEA suppressed peripheral responses from patients with MS and reversed established paralysis and CNS inflammation in four different EAE models, including the 2D2 TCR-transgenic mouse model. DHEA directly inhibited human and murine Th17 cells, inducing IL-10-producing regulatory T cells. Administration of DHEA in symptomatic mice induced regulatory CD4(+) T cells that were suppressive in an IL-10-dependent manner. Expression of the estrogen receptor ß by CD4(+) T cells was necessary for DHEA-mediated EAE amelioration, as well as for direct downregulation of Th17 responses. TGF-ß1 as well as aryl hydrocarbon receptor activation was necessary for the expansion of IL-10-producing T cells by DHEA. Thus, our studies demonstrate that compounds that inhibit pathogenic Th17 responses and expand functional regulatory cells could serve as therapeutic agents for autoimmune diseases, such as MS.

Osteopontin expression by CD103- dendritic cells drives intestinal inflammation.

Intestinal CD103(-) dendritic cells (DCs) are pathogenic for colitis. Unveiling molecular mechanisms that render these cells proinflammatory is important for the design of specific immunotherapies. In this report, we demonstrated that mesenteric lymph node CD103(-) DCs express, among other proinflammatory cytokines, high levels of osteopontin (Opn) during experimental colitis. Opn expression by CD103(-) DCs was crucial for their immune profile and pathogenicity, including induction of T helper (Th) 1 and Th17 cell responses. Adoptive transfer of Opn-deficient CD103(-) DCs resulted in attenuated colitis in comparison to transfer of WT CD103(-) DCs, whereas transgenic CD103(-) DCs that overexpress Opn were highly pathogenic in vivo. Neutralization of secreted Opn expressed exclusively by CD103(-) DCs restrained disease severity. Also, Opn deficiency resulted in milder disease, whereas systemic neutralization of secreted Opn was therapeutic. We determined a specific domain of the Opn protein responsible for its CD103(-) DC-mediated proinflammatory effect. We demonstrated that disrupting the interaction of this Opn domain with integrin α9, overexpressed on colitic CD103(-) DCs, suppressed the inflammatory potential of these cells in vitro and in vivo. These results add unique insight into the biology of CD103(-) DCs and their function during inflammatory bowel disease.

Osteopontin has a crucial role in allergic airway disease through regulation of dendritic cell subsets.

Osteopontin (Opn) is important for T helper type 1 (T(H)1) immunity and autoimmunity. However, the role of this cytokine in T(H)2-mediated allergic disease as well as its effects on primary versus secondary antigenic encounters remain unclear. Here we demonstrate that OPN is expressed in the lungs of asthmatic individuals and that Opn-s, the secreted form of Opn, exerts opposing effects on mouse T(H)2 effector responses and subsequent allergic airway disease: pro-inflammatory at primary systemic sensitization, and anti-inflammatory during secondary pulmonary antigenic challenge. These effects of Opn-s are mainly mediated by the regulation of T(H)2-suppressing plasmacytoid dendritic cells (DCs) during primary sensitization and T(H)2-promoting conventional DCs during secondary antigenic challenge. Therapeutic administration of recombinant Opn during pulmonary secondary antigenic challenge decreased established T(H)2 responses and protected mice from allergic disease. These effects on T(H)2 allergic responses suggest that Opn-s is an important therapeutic target and provide new insight into its role in immunity.

Regulation of adverse remodelling by osteopontin in a genetic heart failure model.

AIMS: Desmin, the muscle-specific intermediate filament protein, is a major target in dilated cardiomyopathy and heart failure in humans and mice. The hallmarks of desmin-deficient (des(-/-)) mice pathology include pronounced myocardial degeneration, extended fibrosis, and osteopontin (OPN) overexpression. We sought to identify the molecular and cellular events regulating adverse cardiac remodelling in des(-/-) mice and their potential link to OPN. METHODS AND RESULTS: In situ hybridization, histology, and immunostaining demonstrated that inflammatory cells and not cardiomyocytes were the source of OPN. RNA profile comparison revealed that activation of inflammatory pathways, sustained by innate immunity mechanisms, predominated among all changes occurring in degenerating des(-/-) myocardium. The expression of the most highly up-regulated genes (OPN: 226×, galectin-3: 26×, osteoactivin/Gpnmb/DC-HIL: 160× and metalloprotease-12: 98×) was associated with heart infiltrating macrophages. To evaluate the role of OPN, we generated des(-/-)OPN(-/-) mice and compared their cardiac function and remodelling indices with those of des(-/-). Osteopontin promoted cardiac dysfunction in this model since des(-/-)OPN(-/-) mice showed 53% improvement of left ventricular function, paralleled to an up to 44% reduction in fibrosis. The diminished fibrotic response in the absence of OPN could be partly mediated by a dramatic reduction in myocardial galectin-3 levels, associated with an impaired galectin-3 secretion by OPN-deficient infiltrating macrophages. CONCLUSION: Cardiomyocyte death due to desmin deficiency leads to inflammation and subsequent overexpression of a series of remodelling modulators. Among them, OPN seems to be a major regulator of des(-/-) adverse myocardial remodelling and it functions at least by potentiating galectin-3 up-regulation and secretion.

An integrin axis induces IFN-ß production in plasmacytoid dendritic cells.

Type I interferon (IFN) production by plasmacytoid dendritic cells (pDCs) has been mainly studied in the context of Toll-like receptor (TLR) activation. In the current report, we reveal that, in the absence of TLR activation, the integrin-binding SLAYGLR motif of secreted osteopontin (sOpn) induces IFN-ß production in murine pDCs. This process is mediated by α4ß1 integrin, indicating that integrin triggering may act as a subtle danger signal leading to IFN-ß induction. The SLAYGLR-mediated α4 integrin/IFN-ß axis is MyD88 independent and operates via a PI3K/mTOR/IRF3 pathway. Consequently, SLAYGLR-treated pDCs produce increased levels of type I IFNs following TLR stimulation. Intratumoral administration of SLAYGLR induces accumulation of IFN-ß-expressing pDCs and efficiently suppresses melanoma tumor growth. In this process, pDCs are crucial. Finally, SLAYGLR enhances pDC development from bone marrow progenitors. These findings open new questions on the roles of sOpn and integrin α4 during homeostasis and inflammation. The newly identified integrin/IFN-ß axis may be implicated in a wide array of immune responses.

T cell costimulation through CD28 depends on induction of the Bcl-xgamma isoform: analysis of Bcl-xgamma-deficient mice.

The molecular basis of CD28-dependent costimulation of T cells is poorly understood. Bcl-xgamma is a member of the Bcl-x family whose expression is restricted to activated T cells and requires CD28-dependent ligation for full expression. We report that Bcl-xgamma-deficient (Bcl-xgamma-/-) T cells display defective proliferative and cytokine responses to CD28-dependent costimulatory signals, impaired memory responses to proteolipid protein peptide (PLP), and do not develop PLP-induced experimental autoimmune encephalomyelitis (EAE). In contrast, enforced expression of Bcl-xgamma largely replaces the requirement for B7-dependent ligation of CD28. These findings identify the Bcl-xgamma cytosolic protein as an essential downstream link in the CD28-dependent signaling pathway that underlies T cell costimulation.

Osteopontin deficiency protects against airway remodeling and hyperresponsiveness in chronic asthma.

RATIONALE: Osteopontin (OPN) is a cytokine that is upregulated in epithelial cells and macrophages in the lungs of mice during chronic allergen challenge and airway remodeling and also in lungs of patients with asthma. However, it remains unclear whether OPN has an in vivo effect on lung remodeling in allergic asthma. Based on its ability to induce smooth muscle and fibroblast proliferation and migration we hypothesize that OPN regulates lung remodeling and also affects subsequent airway hyperresponsiveness (AHR). OBJECTIVES: Study the role of OPN in airway remodeling using OPN-knockout (KO) mice and a reversal approach administering recombinant mouse OPN (rOPN) in KO mice before challenge. METHODS: A chronic allergen-challenge model of airway remodeling with OPN KO mice, KO mice treated with rOPN, and human bronchial smooth muscle were used. MEASUREMENTS AND MAIN RESULTS: OPN deficiency protected mice against ova-induced AHR, which was associated with lower collagen and mucus production, gob-5 mRNA expression, submucosal cell area infiltration, and proliferation. Administration of rOPN to KO mice, just at the final five allergen challenges, exacerbated AHR and all the remodeling characteristics measured. In addition, rOPN increased the expression of IL-13 and pro-matrix metalloproteinase-9 in the lungs. Moreover, we demonstrated that rOPN induces proliferation of human BSM through binding to its alpha(v)beta3 integrin receptor and activation of PI3K/Akt downstream signaling pathway. CONCLUSIONS: We conclude that OPN deficiency protects against remodeling and AHR. Thus our data reveal OPN as a novel therapeutic target for airway remodeling and associated AHR in chronic asthma.

Suppression of autoimmune disease after vaccination with autoreactive T cells that express Qa-1 peptide complexes.

The ability of autoreactive T cells to provoke autoimmune disease is well documented. The finding that immunization with attenuated autoreactive T cells (T cell vaccination, or TCV) can induce T cell-dependent inhibition of autoimmune responses has opened the possibility that regulatory T cells may be harnessed to inhibit autoimmune disease. Progress in the clinical application of TCV, however, has been slow, in part because the underlying mechanism has remained clouded in uncertainty. We have investigated the molecular basis of TCV-induced disease resistance in two murine models of autoimmunity: herpes simplex virus-1 (KOS strain)-induced herpes stromal keratitis and murine autoimmune diabetes in non-obese diabetic (NOD) mice. We find that the therapeutic effects of TCV depend on activation of suppressive CD8 cells that specifically recognize Qa-1-bound peptides expressed by autoreactive CD4 cells. We clarify the molecular interaction between Qa-1 and self peptides that generates biologically active ligands capable of both inducing suppressive CD8 cells and targeting them to autoreactive CD4 cells. These studies suggest that vaccination with peptide-pulsed cells bearing the human equivalent of murine Qa-1 (HLA-E) may represent a convenient and effective clinical approach to cellular therapy of autoimmune disease.

Circulating conventional and plasmacytoid dendritic cell subsets display distinct kinetics during in vivo repeated allergen skin challenges in atopic subjects.

Upon allergen challenge, DC subsets are recruited to target sites under the influence of chemotactic agents; however, details pertinent to their trafficking remain largely unknown. We investigated the kinetic profiles of blood and skin-infiltrating DC subsets in twelve atopic subjects receiving six weekly intradermal allergen and diluent injections. The role of activin-A, a cytokine induced in allergic and tissue repair processes, on the chemotactic profiles of DC subsets was also examined. Plasmacytoid (pDCs) and conventional DCs (cDCs) were evaluated at various time-points in the blood and skin. In situ activin-A expression was assessed in the skin and its effects on chemokine receptor expression of isolated cDCs were investigated. Blood pDCs were reduced 1 h after challenge, while cDCs decreased gradually within 24 h. Skin cDCs increased significantly 24 h after the first challenge, inversely correlating with blood cDCs. Activin-A in the skin increased 24 h after the first allergen challenge and correlated with infiltrating cDCs. Activin-A increased the CCR10/CCR4 expression ratio in cultured human cDCs. DC subsets demonstrate distinct kinetic profiles in the blood and skin especially during acute allergic inflammation, pointing to disparate roles depending on each phase of the inflammatory response. The effects of activin-A on modulating the chemotactic profile of cDCs suggest it may be a plausible therapeutic target for allergic diseases.

Neurosteroids and microneurotrophins signal through NGF receptors to induce prosurvival signaling in neuronal cells.

The neurosteroid dehydroepiandrosterone (DHEA) exerts a portion of its neuroprotective effects by directly interacting with the nerve growth factor (NGF) receptors TrkA and p75(NTR) to induce prosurvival signaling. DHEA is an intermediate in the biosynthesis of estrogens and androgens that affects the endocrine system and potentially increases the risk for developing estrogen- and androgen-dependent tumors. We have synthesized 17-spiro analogs of DHEA that lack estrogenic or androgenic properties and bind to and activate NGF receptors, thus exerting potent neuroprotective effects without the tumor risk. These synthetic DHEA derivatives may serve as lead molecules to develop small agonists of NGF receptors that can penetrate the blood-brain barrier (microneurotrophins) with potential applications in the treatment of neurodegenerative diseases. The neuroprotective properties of microneurotrophins are now being tested in various animal models of neurodegenerative diseases.

Activin-A induces regulatory T cells that suppress T helper cell immune responses and protect from allergic airway disease.

Activin-A is a pleiotropic cytokine that participates in developmental, inflammatory, and tissue repair processes. Still, its effects on T helper (Th) cell-mediated immunity, critical for allergic and autoimmune diseases, are elusive. We provide evidence that endogenously produced activin-A suppresses antigen-specific Th2 responses and protects against airway hyperresponsiveness and allergic airway disease in mice. Importantly, we reveal that activin-A exerts suppressive function through induction of antigen-specific regulatory T cells that suppress Th2 responses in vitro and upon transfer in vivo. In fact, activin-A also suppresses Th1-driven responses, pointing to a broader immunoregulatory function. Blockade of interleukin 10 and transforming growth factor beta1 reverses activin-A-induced suppression. Remarkably, transfer of activin-A-induced antigen-specific regulatory T cells confers protection against allergic airway disease. This beneficial effect is associated with dramatically decreased maturation of draining lymph node dendritic cells. Therapeutic administration of recombinant activin-A during pulmonary allergen challenge suppresses Th2 responses and protects from allergic disease. Finally, we demonstrate that immune cells infiltrating the lungs from individuals with active allergic asthma, and thus nonregulated inflammatory response, exhibit significantly decreased expression of activin-A's responsive elements. Our results uncover activin-A as a novel suppressive factor for Th immunity and a critical controller of allergic airway disease.

T cell-dependent and -independent pathways to tissue destruction following herpes simplex virus-1 infection.

Viral infections can trigger tissue destruction through innate and/or adaptive immune mechanisms. Here we show that these pathways can be differentially activated after infection by different strains of the herpes simplex virus-1 (HSV-1) virus. Infection of murine corneal tissue by HSV-1 (KOS) triggers an autoreactive clone of CD4 cells that is cross-reactive with an HSV-1 epitope to initiate corneal destruction. In contrast, ocular infection by the HSV-1 (RE) strain induces murine corneal destruction through direct, T cell-independent, activation of the innate immune system. Although the relative role of these two pathways to blindness following clinical HSV-1 ocular infection is unknown, this analysis suggests a general experimental approach to evaluate the relative contribution of adaptive and innate immune mechanisms to virally induced host tissue destruction.

Cutting edge: Attenuated experimental autoimmune encephalomyelitis in eta-1/osteopontin-deficient mice.

Recent studies indicate that early T lymphocyte activation 1 (Eta-1), also known as osteopontin, is a cytokine contributing to the development of Th1 immunity. In the present report, the role of Eta-1 in experimental autoimmune encephalomyelitis (EAE), a disease associated with Th1 immunity, was examined by analysis of disease progression in Eta-1-deficient (Eta-1-/-) mice. Although incidence and onset of peptide-induced EAE were found to be similar in Eta-1-/- and Eta-1+/+ mice, Eta-1-/- mice displayed significantly lower mean maximal clinical score and faster recovery without spontaneous relapses. Accordingly, decreased inflammatory infiltration and demyelination were observed in the spinal cords of Eta-1-/- mice. Furthermore, in comparison to Eta-1+/+, Eta-1-/- CD4+ T cells had reduced expression of IFN-gamma and TNF-alpha upon ex vivo restimulation. Taken together, these results suggest that Eta-1 may sustain autoimmune responses by assisting in maintenance of Th1 immunity during EAE.