Matthew J. Turner

ST2 blockade reduces sST2-producing T cells while maintaining protective mST2-expressing T cells during graft-versus-host disease

Blocking sST2 before transplant with a neutralizing monoclonal antibody reduced GVHD severity and mortality. Blocking graft-versus-host disease Bone marrow transplantation replaces unhealthy bone marrow with bone marrow from a healthy donor. However, the donor-derived immune cells may recognize the transplant recipient as foreign and attack, resulting in graft-versus-host disease (GVHD). Now, Zhang et al. report that blocking soluble suppression of tumorigenicity 2 (sST2), a plasma marker for GVHD, with a neutralizing antibody can reduce GVHD severity and mortality. The blockade decreased the production of proinflammatory cytokines and increased the frequency of anti-inflammatory molecules and cells while maintaining graft-versus-leukemia activity. These data suggest that targeting sST2 may help decrease GVHD after bone marrow transplantation. Graft-versus-host disease (GVHD) remains a devastating complication after allogeneic hematopoietic cell transplantation (HCT). We previously identified high plasma soluble suppression of tumorigenicity 2 (sST2) as a biomarker of the development of GVHD and death. sST2 sequesters interleukin-33 (IL-33), limiting its availability to T cells expressing membrane-bound ST2 (mST2) [T helper 2 (TH2) cells and ST2+FoxP3+ regulatory T cells]. We report that blockade of sST2 in the peritransplant period with a neutralizing monoclonal antibody (anti-ST2 mAb) reduced GVHD severity and mortality. We identified intestinal stromal cells and T cells as major sources of sST2 during GVHD. ST2 blockade decreased systemic interferon-γ, IL-17, and IL-23 but increased IL-10 and IL-33 plasma levels. ST2 blockade also reduced sST2 production by IL-17–producing T cells while maintaining protective mST2-expressing T cells, increasing the frequency of intestinal myeloid–derived suppressor cells, and decreasing the frequency of intestinal CD103 dendritic cells. Finally, ST2 blockade preserved graft-versus-leukemia activity in a model of green fluorescent protein (GFP)–positive MLL-AF9 acute myeloid leukemia. Our findings suggest that ST2 is a therapeutic target for severe GVHD and that the ST2/IL-33 pathway could be investigated in other T cell–mediated immune disorders with loss of tolerance.

The environmental stressor ultraviolet B radiation inhibits murine antitumor immunity through its ability to generate platelet-activating factor agonists

Ubiquitous pro-oxidative stressor ultraviolet B radiation (UVB) to human or mouse skin generates platelet-activating factor (PAF) and novel oxidatively modified glycerophosphocholines (Ox-GPCs) with PAF-receptor (PAF-R) agonistic activity. These lipids mediate systemic immunosuppression in a process involving IL-10. The current studies sought to determine the functional significance of UVB-mediated systemic immunosuppression in an established model of murine melanoma. We show that UVB irradiation augments B16F10 tumor growth and is dependent on host, but not melanoma cell; PAF-R-expression as UVB or the PAF-R agonist, carbamoyl PAF (CPAF), both promote B16F10 tumor growth in wild-type (WT) mice, independent of whether B16F10 cells express PAF-Rs, but do not augment tumor growth in Pafr -/- mice. UVB-mediated augmentation of experimental murine tumor growth was inhibited with antioxidants, demonstrating the importance of Ox-GPC PAF-R agonists produced non-enzymatically. Host immune cells are required as CPAF-induced augmentation of tumor growth which is not seen in immunodeficient NOD SCID mice. Finally, depleting antibodies against IL-10 in WT mice or depletion of CD25-positive cells in FoxP3(EGFP) transgenic mice block UVB and/or CPAF-induced tumor growth supporting a requirement for IL-10 and Tregs in this process. These findings indicate that UVB-generated Ox-GPCs with PAF-R agonistic activity enhance experimental murine melanoma tumor growth through targeting host immune cells, most notably Tregs, to mediate systemic immunosuppression.

Cigarette Smoke Exposure Inhibits Contact Hypersensitivity via the Generation of Platelet-Activating Factor Agonists

Previous studies have established that pro-oxidative stressors suppress host immunity because of their ability to generate oxidized lipids with platelet-activating factor receptor (PAF-R) agonist activity. Although exposure to the pro-oxidative stressor cigarette smoke (CS) is known to exert immunomodulatory effects, little is known regarding the role of PAF in these events. The current studies sought to determine the role of PAF-R signaling in CS-mediated immunomodulatory effects. We demonstrate that CS exposure induces the generation of a transient PAF-R agonistic activity in the blood of mice. CS exposure inhibits contact hypersensitivity in a PAF-R–dependent manner as PAF-R–deficient mice were resistant to these effects. Blocking PAF-R agonist production either by systemic antioxidants or treatment with serum PAF-acetyl hydrolase enzyme blocked both the CS-mediated generation of PAF-R agonists and PAF-R–dependent inhibition of contact hypersensitivity (CHS) reactions, indicating a role for oxidized glycerophosphocholines with PAF-R agonistic activity in this process. In addition, cyclooxygenase-2 inhibition did not block PAF-R agonist production but prevented CS-induced inhibition of CHS. This suggests that cyclooxygenase-2 acts downstream of the PAF-R in mediating CS-induced systemic immunosuppression. Moreover, CS exposure induced a significant increase in the expression of the regulatory T cell reporter gene in Foxp3EGFP mice but not in Foxp3EGFP mice on a PAF-R–deficient background. Finally, regulatory T cell depletion via anti-CD25 Abs blocked CS-mediated inhibition of CHS, indicating the potential involvement of regulatory T cells in CS-mediated systemic immunosuppression. These studies provide the first evidence, to our knowledge, that the pro-oxidative stressor CS can modulate cutaneous immunity via the generation of PAF-R agonists produced through lipid oxidation.

A new itch to scratch for TSLP.

Atopic dermatitis (AD) is characterized by allergic inflammation and itch. Thymic stromal lymphopoietin (TSLP) is a pro-allergic cytokine implicated in AD. A paper in Cell transforms the understanding of the functional repertoire of TSLP in general and in AD in particular showing that TSLP can directly stimulate sensory neurons and provoke itch.

Topical Application of a Vitamin D Analogue Exacerbates Atopic Dermatitis and Induces the Atopic Dermatitis-like Phenotype in Stat6VT Mice

Calcipotriene is a topical vitamin D3 analogue approved for the treatment of plaque and scalp psoriasis. We report the case of a 2‐year‐old boy whose atopic dermatitis (AD) flared in response to application of calcipotriene 0.005% cream and solution for a mistaken diagnosis of plaque and scalp psoriasis. We investigated whether the patients eruption was secondary to an allergic contact dermatitis. In the Stat6VT mouse model of AD we tested whether calcipotriene could induce the otherwise‐spontaneous AD‐like phenotype. Closed patch testing was done on the patient with calcipotriene solution and cream, moisturizing cream, and 51% isopropanol. Stat6VT and wild‐type (WT) mice were treated for 7 days with calcipotriene solution or vehicle (isopropanol) applied to the right and left upper back skin, respectively, after which mice were followed longitudinally for 10 weeks. Biopsy specimens from prior treatment sites were then collected for histology and RNA isolation. RNA was analyzed for interleukin (IL‐4) expression using quantitative polymerase chain reaction. Patch testing was negative. Stat6VT mice, in contrast to WT mice, developed a persistent eczematous dermatitis at sites of calcipotriene application. Clinical and histologic features and high IL‐4 transcript levels were consistent with the spontaneous AD‐like phenotype seen in Stat6VT mice. At sites of active disease, calcipotriene can worsen a flare of AD. In Stat6VT mice, calcipotriene can induce the AD‐like phenotype.

Increased Th2 activity and diminished skin barrier function cooperate in allergic skin inflammation.

Atopic dermatitis (AD) is a chronic inflammatory skin disease induced by a complex interaction between susceptibility genes encoding skin barrier components and environmental allergen exposure that results in type 2 cytokine production. Although genetic lesions in either component can be risk factors for disease in patients, whether these pathways interact in the development of AD is not clear. To test this, we mated mice with T‐cell specific expression of constitutively active Stat6 (Stat6VT) that spontaneously develop allergic skin inflammation with Flaky tail (Ft) mice that have mutations in Flg and Tmem79 genes that each affect skin barrier function. Our results demonstrate that over 90% of the Stat6VT transgenic mice carrying the Ft alleles (Stat6VTxFt−/−) develop severe atopic dermatitis lesions by 3–5 months of age, compared with only 40% of Stat6VT mice that develop disease by 6–7 months of age. Further, histopathological analysis of skin tissues from Stat6VTxFt−/− mice revealed extensive thickening of the dermis with increased inflammatory infiltrates as compared with Stat6VT mice. Our study suggests that skin barrier defects and altered Th2 responses independently cooperate in the pathogenesis of allergic skin inflammation, similar to effects observed in patients with AD.

STAT6-Mediated Keratitis and Blepharitis: A Novel Murine Model of Ocular Atopic Dermatitis

PURPOSE Atopic dermatitis (AD) is a common inflammatory disease that can affect the eye, resulting in ocular pathologies, including blepharitis, keratitis, and uveitis; however, the pathogenic mechanisms underlying the ocular manifestations of AD are not well understood. METHODS In the present study, we characterized the ocular pathologies that develop in the Stat6VT mouse model of AD. We examined the cytokine profile of the eyelid lesions, measured the behavioral response, and documented the treatment response to topical steroids. RESULTS Our results show that Stat6VT mice spontaneously developed blepharitis, keratitis, and uveitis similar to that observed in patients with AD. Histologic findings of allergic inflammation in affected eyelids in this model include the presence of a lymphocyte-predominant infiltrate and tissue eosinophilia in the dermis. Gene expression analysis of affected eyelid tissue by quantitative PCR revealed increased amounts of mRNAs for the Th2 cytokines IL-4, IL-5, and IL-13. In addition, increased eyelid scratching was seen in Stat6VT mice with blepharitis. Topical treatment with the corticosteroid clobetasol reduced eyelid inflammation, tissue eosinophilia, and Th2 cytokine expression. CONCLUSIONS The development of AD-like ocular pathologies in this model supports the idea that in humans, AD-associated disease of the eye may be driven by Th2-mediated inflammation and demonstrates that the Stat6VT mouse may be a useful system in which to further investigate pathogenesis of and treatment strategies for blepharitis and other ocular diseases that develop in association with AD.

Poly-ADP ribose polymerase-14 limits severity of allergic skin disease

Poly‐ADP ribose polymerase‐14 (PARP14 or ARTD8) was initially identified as a transcriptional co‐activator for signal transducer and activator of transcription 6 (Stat6), where the presence of interleukin‐4 (IL‐4) and activated Stat6 induces the enzymatic activity of PARP14 that promotes T helper type 2 differentiation and allergic airway disease. To further our understanding of PARP14 in allergic disease, we studied the function of PARP14 in allergic inflammation of skin using mice that express constitutively active Stat6 in T cells (Stat6VT) and develop spontaneous inflammation of the skin. We mated Stat6VT mice to Parp14−/− mice and observed that approximately 75% of the Stat6VT × Parp14−/− mice develop severe atopic dermatitis (AD)‐like lesions, compared with about 50% of Stat6VT mice, and have increased morbidity compared with Stat6VT mice. Despite this, gene expression in the skin and the cellular infiltrates was only modestly altered by the absence of PARP14. In contrast, we saw significant changes in systemic T‐cell cytokine production. Moreover, adoptive transfer experiments demonstrated that decreases in IL‐4 production reflected a cell intrinsic role for PARP14 in Th2 cytokine control. Hence, our data suggest that although PARP14 has similar effects on T‐cell cytokine production in several allergic disease models, the outcome of those effects is distinct, depending on the target organ of disease.

Cytokines and Chemokines

Cytokines are small proteins used for intercellular communication in the immune system and beyond. They are subclassified based on membership in tumor necrosis factor, interleukin, interferon, transforming growth factor beta and chemokine superfamilies. Readily observed and easily accessible for manipulation and isolation, the skin is an excellent model system for studying the biology of cytokines. Important insights into mechanisms of cutaneous inflammation and immunity in health and disease have been and continue to be translated into the practice of dermatology and other areas of medicine. Cytokine-targeted therapies are now commonplace in the practice of dermatology, with many cytokine or cytokine receptor-target therapies in clinical trials for dermatologic diseases. As the list of available therapeutics grows, it is incumbent upon physicians to understand when and how to use these medications for safe and effective outcomes. To achieve this goal, a sound understanding of mechanisms of cutaneous inflammation and immunology mediated by cytokines is important.

ID: 91: ST2 BLOCKADE REDUCES SST2-PRODUCING T CELLS WHILE MAINTAINING PROTECTIVE MST2-EXPRESSING T CELLS DURING GRAFT-VERSUS-HOST DISEASE

Allogeneic hematopoietic stem cell transplantation is an important curative therapy for hematological malignancies and other blood disorders, but graft-versus-host disease (GVHD) remains a devastating complication. We previously identified high plasma soluble suppression of tumorigenicity 2 (ST2) as a biomarker of the development of GVHD and death. ST2, also known as the interleukin (IL)-33 receptor, is one of the newest members of the IL-1 receptor family, and its only known ligand is IL-33. Due to alternative splicing, ST2 has two main isoforms: a membrane-bound form (mST2), expressed on hematopoietic cells that promote type 2 helper T (Th2) cell immune responses, and a soluble form (sST2), secreted by non-hematopoietic cells. sST2 acts as a decoy receptor for IL-33, limiting its availability to mST2-expressing cells. Thus, we hypothesized that blockade of the sST2 will free IL-33 for signaling in mST2-expressing T cells [Th2 cells and ST2+FoxP3+ regulatory T cells (Tregs)], which will attenuate GVHD. First, we found the onset of GVHD symptoms was correlated with a marked increase of plasma ST2 in multiple clinically relevant GVHD murine models, and blockade of sST2 in the peri-transplant period with a neutralizing monoclonal antibody (anti-ST2 mAb) reduced GVHD clinical and histopathological severity and mortality. We identified intestinal stromal cells and T cells as major sources of sST2 during GVHD. ST2 blockade decreased systemic interferon-γ, IL-17, and IL-23 but increased IL-10 and IL-33 plasma levels. ST2 blockade also reduced sST2 production by IL-17–producing T cells while maintaining protective mST2-expressing Th2 cells and Tregs. In addition, ST2 blockade increased the frequency of intestinal myeloid-derived suppressor cells (MDSCs) and decreased the frequency of intestinal CD103 dendritic cells (DCs). Finally, whole transcriptome analysis of T cells comparing anti-ST2 mAb-treated mice versus IgG control-treated mice showed that anti-ST2 mAb administration up-regulated gene expression of cytokines and cytolytic molecules that have been implicated in antitumoral or graft-vs-leukemia (GVL) activity, such as IL-27, IL-18, IL-9, type I interferons, and granzyme A. We therefore postulated that ST2 blockade would not affect therapeutic GVL activity. To confirm this in a clinically relevant GVL model, we developed primary retrovirally induced MLL-AF9 eGFP+ leukemic cells on the C3H.SW background. The phenotype of the leukemic cells in this model is eGFP+, CD3−, B220− and Mac-1hiGr-1hi. Our results indicated that administration of anti-ST2 mAb or ST2−/− donor T cells preserved substantial GVL activity and resulted in significantly improved leukemia-free survival. In summary, our findings identify intestinal alloreactive T cells as an important source of the decoy receptor for IL-33 that can be blocked with two doses of anti-ST2 mAb in the peri-transplant period without inhibiting the beneficial mST2 expression on Th2 cells and Tregs or the GVL activity. In addition, ST2 blockade induced expansion of tolerogenic MDSCs while inhibiting immunogenic CD103 DCs during GVHD. This study offers new perspectives on the translation of drug-targetable biomarkers for selectively and safely treating GVHD and other T cell-mediated human disorders.