Weisong Zhou

Prostaglandin I2 Analogs Inhibit Proinflammatory Cytokine Production and T Cell Stimulatory Function of Dendritic Cells

Signaling through the PGI2 receptor (IP) has been shown to inhibit inflammatory responses in mouse models of respiratory syncytial viral infection and OVA-induced allergic responses. However, little is known about the cell types that mediate the anti-inflammatory function of PGI2. In this study, we determined that PGI2 analogs modulate dendritic cell (DC) cytokine production, maturation, and function. We report that PGI2 analogs (iloprost, cicaprost, treprostinil) differentially modulate the response of murine bone marrow-derived DC (BMDC) to LPS in an IP-dependent manner. The PGI2 analogs decreased BMDC production of proinflammatory cytokines (IL-12, TNF-α, IL-1α, IL-6) and chemokines (MIP-1α, MCP-1) and increased the production of the anti-inflammatory cytokine IL-10 by BMDCs. The modulatory effect was associated with IP-dependent up-regulation of intracellular cAMP and down-regulation of NF-κB activity. Iloprost and cicaprost also suppressed LPS-induced expression of CD86, CD40, and MHC class II molecules by BMDCs and inhibited the ability of BMDCs to stimulate Ag-specific CD4 T cell proliferation and production of IL-5 and IL-13. These findings suggest that PGI2 signaling through the IP may exert anti-inflammatory effects by acting on DC.

A Functional IL-13 Receptor Is Expressed on Polarized Murine CD4+ Th17 Cells and IL-13 Signaling Attenuates Th17 Cytokine Production

IL-17A is produced from Th17 cells, and is involved in many autoimmune and inflammatory diseases. IL-13R has not previously been reported to be functionally expressed on T cells; however, we found that purified BALB/c CD4+ cells polarized to Th17 with TGF-β, IL-6, and IL-23 have increased mRNA and protein expression of IL-13Rα1 and mRNA expression of IL-4Rα compared with Th0, Th1, or Th2 polarized cells. The addition of IL-13 at Th17 polarization negatively regulated IL-17A and IL-21 expression, and reduced the number of CD4+ T cells producing IL-17A. Further, adding IL-13 at the time of Th17 cell restimulation attenuated IL-17A expression. CD4+ Th17 polarized cells from IL-4 knockout (KO) mice also had IL-13-induced inhibition of IL-17A production, but this was not observed in IL-4R KO and STAT6 KO mice. Addition of IL-13 at polarization increased IL-13R expression in wild-type Th17 cells. Further, IL-13 administration during Th17 polarization down-regulated retinoic acid-related-γT, the transcription required for Th17 development; increased STAT6 phosphorylation, and up-regulated GATA3, the transcription factor activated during the development of Th2 cells. This IL-13-mediated effect was specific to Th17 cells as IL-13 neither decreased IFN-γ expression by Th1 cells nor affected Th2 cell production of IL-4. Collectively, we have shown that Th17 cells express a functional IL-13R and that IL-13 negatively regulates IL-17A and IL-21 production by decreasing retinoic acid-related-γT expression and while increasing phosphorylation of STAT6 and GATA3 expression. Therefore, therapeutic intervention inhibiting IL-13 production could have adverse consequences by up-regulating Th17 inflammation in certain disease states.

Human TH17 cells express a functional IL-13 receptor and IL-13 attenuates IL-17A production.

BACKGROUND IL-13 is a central mediator of airway responsiveness and mucus expression in patients with allergic airway inflammation, and IL-13 is currently a therapeutic target for asthma. However, little is known about how IL-13 regulates human CD4(+) T-cell lineages because IL-13 receptor (IL-13R) α1, a subunit of IL-13R, has not previously been reported to exist on human T cells. OBJECTIVE We sought to determine whether human CD4(+) T(H)17 cells express IL-13Rα1 and whether IL-13 regulates T(H)17 cytokine production. METHODS Naive human CD4(+) cells were isolated from whole blood, activated with anti-CD3 and anti-CD28, and polarized to T(H)1, T(H)2, T(H)17, or induced regulatory T cells in the presence of IL-13 (0-10 ng/mL). Cell supernatants, total RNA, or total protein was examined 4 days after T(H)17 polarization. RESULTS T(H)17 cells, but not T(H)0, T(H)1, T(H)2, or induced regulatory T cells, expressed IL-13Rα1. IL-13 attenuated IL-17A production, as well as expression of retinoic acid-related orphan receptor, runt-related transcription factor-1, and interferon regulatory factor 4 in T(H)17-polarized cells. IL-13 neither inhibited IFN-γ production from T(H)1 cells nor inhibited IL-4 production from T(H)2 cells. Furthermore, attenuation of IL-17A production only occurred when IL-13 was present within 24 hours of T-cell activation or at the time of restimulation. CONCLUSIONS IL-13Rα1 is expressed on human CD4(+) T(H)17 cells, and IL-13 attenuates IL-17A production at polarization and restimulation. Although IL-13 is an attractive therapeutic target for decreasing symptoms associated with asthma, these results suggest that therapies inhibiting IL-13 production could have adverse side effects by increasing IL-17A production.

Prostaglandin I2 analogs inhibit Th1 and Th2 effector cytokine production by CD4 T cells.

An anti‐inflammatory effect of PGI2 has been suggested by increased inflammation in mice that are deficient in the PGI2 receptor (IP) or in respiratory syncytial viral‐ or OVA‐induced CD4 T cell‐associated responses. To determine the mechanism of the anti‐inflammatory effect, we hypothesized that PGI2 analogs inhibit CD4 T cell effector cytokine production. To test this hypothesis, we activated purified CD4 T cells with anti‐CD3 and anti‐CD28 antibodies under Th1 and Th2 polarizing conditions for 4 days and restimulated the T cells with anti‐CD3 in the presence of PGI2 analogs for 2 days. We found that PGI2 analogs (cicaprost and iloprost) inhibited the production of Th1 cytokines (IFN‐γ) and Th2 cytokines (IL‐4, IL‐10, and IL‐13) in a dose‐dependent pattern. The inhibitory effect was partially dependent on the IP receptor signaling and was correlated with elevated intracellular cAMP and down‐regulated NF‐κB activity. Pretreatment of the CD4 T cells with 8‐bromoadenosine‐3′,5′‐cyclic monophosphorothioate, Rp‐isomer, to inhibit a key signaling molecule in the cAMP pathway, protein kinase A (PKA), attenuated the suppressive effect of PGI2 analogs significantly, suggesting that PKA, in part, mediates the inhibition of the cytokine production. These data indicate that PGI2 analogs have an immune‐suppressive effect on previously activated and differentiated CD4 T cells in vitro and suggest that PGI2 may have a similar function in vivo.

Cutting Edge: Distal Regulatory Elements Are Required to Achieve Selective Expression of IFN-γ in Th1/Tc1 Effector Cells

Using a transgenic approach, we analyzed the contribution of introns located within the IFN-γ gene and distal regulatory regions to IFN-γ gene expression. Intron 1 and 3 from the IFN-γ gene displayed strong enhancer activity. This activity appeared to be dependent upon integration into the genome but resulted in a loss of Th1 selectivity. We also found that distal regulatory elements are not required for high level expression of the human IFN-γ gene, but rather for cell lineage-specific expression. An 8.6-kb human IFN-γ transgene was sufficient to yield high level expression but a 191-kb IFN-γ transgene with ∼90 kb of flanking 5′ and 3′ sequence was necessary to achieve both high level and Th1 selective expression of human IFN-γ.

Prostaglandin I2 Signaling and Inhibition of Group 2 Innate Lymphoid Cell Responses

RATIONALE Group 2 innate lymphoid cells (ILC2s) robustly produce IL-5 and IL-13, cytokines central to the asthma phenotype; however, the effect of prostaglandin (PG) I2 on ILC2 function is unknown. OBJECTIVES To determine the effect of PGI2 on mouse and human ILC2 cytokine expression in vitro and the effect of endogenous PGI2 and the PGI2 analog cicaprost on lung ILC2s in vivo. METHODS Flow-sorted bone marrow ILC2s of wild-type (WT) and PGI2 receptor-deficient (IP(-/-)) mice were cultured with IL-33 and treated with the PGI2 analog cicaprost. WT and IP(-/-) mice were challenged intranasally with Alternaria alternata extract for 4 consecutive days to induce ILC2 responses, and these were quantified. Prior to A. alternata extract, challenged WT mice were treated with cicaprost. Human flow-sorted peripheral blood ILC2s were cultured with IL-33 and IL-2 and treated with the PGI2 analog cicaprost. MEASUREMENT AND MAIN RESULTS We demonstrate that PGI2 inhibits IL-5 and IL-13 protein expression by IL-33-stimulated ILC2s purified from mouse bone marrow in a manner that was dependent on signaling through the PGI2 receptor IP. In a mouse model of 4 consecutive days of airway challenge with an extract of A. alternata, a fungal aeroallergen associated with severe asthma exacerbations, endogenous PGI2 signaling significantly inhibited lung IL-5 and IL-13 protein expression, and reduced the number of lung IL-5- and IL-13-expressing ILC2s, as well as the mean fluorescence intensity of IL-5 and IL-13 staining. In addition, exogenous administration of a PGI2 analog inhibited Alternaria extract-induced lung IL-5 and IL-13 protein expression, and reduced the number of lung IL-5- and IL-13-expressing ILC2s and the mean fluorescence intensity of IL-5 and IL-13 staining. Finally, a PGI2 analog inhibited IL-5 and IL-13 expression by human ILC2s that were stimulated with IL-2 and IL-33. CONCLUSIONS These results suggest that PGI2 may be a potential therapy to reduce the ILC2 response to protease-containing aeroallergens, such as Alternaria.

STAT1 Negatively Regulates Lung Basophil IL-4 Expression Induced by Respiratory Syncytial Virus Infection

IL-4 contributes to immunopathology induced in mice by primary respiratory syncytial virus (RSV) infection. However, the cellular source of IL-4 in RSV infection is unknown. We identified CD3−CD49b+ cells as the predominant source of IL-4 in the lungs of RSV-infected BALB/c mice. We ruled out T cells, NK cells, NKT cells, mast cells, and eosinophils as IL-4 expressors in RSV infection by flow cytometry. Using IL4 GFP reporter mice (4get) mice, we identified the IL-4-expressing cells in RSV infection as basophils (CD3−CD49b+FcεRI+c-kit−). Because STAT1−/− mice have an enhanced Th2-type response to RSV infection, we also sought to determine the cellular source and role of IL-4 in RSV-infected STAT1−/− mice. RSV infection resulted in significantly more IL-4-expressing CD3−CD49b+ cells in the lungs of STAT1−/− mice than in BALB/c mice. CD49b+IL-4+ cells sorted from the lungs of RSV-infected STAT1−/− mice and stained with Wright-Giemsa had basophil characteristics. As in wild-type BALB/c mice, IL-4 contributed to lung histopathology in RSV-infected STAT1−/− mice. Depletion of basophils in RSV-infected STAT1−/− mice reduced lung IL-4 expression. Thus, we show for the first time that a respiratory virus (RSV) induced basophil accumulation in vivo. Basophils were the primary source of IL-4 in the lung in RSV infection, and STAT1 was a negative regulator of virus-induced basophil IL-4 expression.

Signaling through the Prostaglandin I2 Receptor IP Protects against Respiratory Syncytial Virus-Induced Illness

ABSTRACT The role of prostanoids in modulating respiratory syncytial virus (RSV) infection is unknown. We found that RSV infection in mice increases production of prostaglandin I2 (PGI2). Mice that overexpress PGI2 synthase selectively in bronchial epithelium are protected against RSV-induced weight loss and have decreased peak viral replication and gamma interferon levels in the lung compared to nontransgenic littermates. In contrast, mice deficient in the PGI2 receptor IP have exacerbated RSV-induced weight loss with delayed viral clearance and increased levels of gamma interferon in the lung compared to wild-type mice. These results suggest that signaling through IP has antiviral effects while protecting against RSV-induced illness and that PGI2 is a potential therapeutic target in the treatment of RSV.

Cutting Edge: Oseltamivir Decreases T Cell GM1 Expression and Inhibits Clearance of Respiratory Syncytial Virus: Potential Role of Endogenous Sialidase in Antiviral Immunity

The sialoglycosphingolipid GM1 is important for lipid rafts and immune cell signaling. T cell activation in vitro increases GM1 expression and increases endogenous sialidase activity. GM1 expression has been hypothesized to be regulated by endogenous sialidase. We tested this hypothesis in vivo using a mouse model of respiratory syncytial virus (RSV) infection. RSV infection increased endogenous sialidase activity in lung mononuclear cells. RSV infection increased lung CD8+ T cell surface GM1 expression. Activated CD8+ T cells in the lungs of RSV-infected mice were GM1high. Treatment of RSV-infected mice with the sialidase/neuraminidase inhibitor oseltamivir decreased T cell surface GM1 levels. Oseltamivir treatment decreased RSV-induced weight loss and inhibited RSV clearance. Our data indicate a novel role for an endogenous sialidase in regulating T cell GM1 expression and antiviral immunity. Also, oseltamivir, an important anti-influenza drug, inhibits the clearance of a respiratory virus that lacks a neuraminidase gene, RSV.

Either IL-2 or IL-12 Is Sufficient to Direct Th1 Differentiation by Nonobese Diabetic T Cells

Th cell differentiation from naive precursors is a tightly controlled process; the most critical differentiation factor is the action of the driving cytokine: IL-12 for Th1 development, IL-4 for Th2 development. We found that CD4+ T cells from nonobese diabetic mice spontaneously differentiate into IFN-γ-producing Th1 cells in response to polyclonal TCR stimulation in the absence of IL-12 and IFN-γ. Instead, IL-2 was necessary and sufficient to direct T cell differentiation to the Th1 lineage by nonobese diabetic CD4+ T cells. Its ability to direct Th1 differentiation of both naive and memory CD4+ T cells was clearly uncoupled from its ability to stimulate cell division. Autocrine IL-2-driven Th1 differentiation of nonobese diabetic T cells may represent a genetic liability that favors development of IFN-γ-producing autoreactive T cells.