A neutralizing single-domain antibody that targets the trimer interface of the human metapneumovirus fusion protein.

IMPORTANCE: Human metapneumovirus (hMPV) is an important respiratory pathogen for which no licensed antivirals or vaccines exist. Single-domain antibodies represent promising antiviral biologics that can be easily produced and formatted. We describe the isolation and detailed characterization of two hMPV-neutralizing single-domain antibodies that are directed against the fusion protein F. One of these single-domain antibodies broadly neutralizes hMPV A and B strains, can prevent proteolytic maturation of F, and binds to an epitope in the F trimer interface. This suggests that hMPV pre-F undergoes trimer opening or "breathing" on infectious virions, exposing a vulnerable site for neutralizing antibodies. Finally, we show that this single-domain antibody, fused to a human IgG1 Fc, can protect cotton rats against hMPV replication, an important finding for potential future clinical applications.

Rethinking IL-1 Antagonism in Respiratory Viral Infections: A Role for IL-1 Signaling in the Development of Antiviral T Cell Immunity.

IL-1R integrates signals from IL-1α and IL-1ß, and it is widely expressed across tissues and immune cell types. While the expression pattern and function of IL-1R within the innate immune system is well studied, its role in adaptive immunity, particularly within the CD8 T cell compartment, remains underexplored. Here, we show that CD8 T cells dynamically upregulate IL-1R1 levels during priming by APCs, which correlates with their proliferation status and the acquisition of an effector phenotype. Notably, this IL-1 sensitivity persists in memory CD8 T cells of both mice and humans, influencing effector cytokine production upon TCR reactivation. Furthermore, our study highlights that antiviral effector and tissue-resident CD8 T cell responses against influenza A virus infection become impaired in the absence of IL-1 signaling. Altogether, these data support the exploitation of IL-1 activity in the context of T cell vaccination strategies and warrant consideration of the impact of clinical IL-1 inhibition on the rollout of T cell immunity.

Cleavage of cFLIP restrains cell death during viral infection and tissue injury and favors tissue repair.

Cell death coordinates repair programs following pathogen attack and tissue injury. However, aberrant cell death can interfere with such programs and cause organ failure. Cellular FLICE-like inhibitory protein (cFLIP) is a crucial regulator of cell death and a substrate of Caspase-8. However, the physiological role of cFLIP cleavage by Caspase-8 remains elusive. Here, we found an essential role for cFLIP cleavage in restraining cell death in different pathophysiological scenarios. Mice expressing a cleavage-resistant cFLIP mutant, CflipD377A, exhibited increased sensitivity to severe acute respiratory syndrome coronavirus (SARS-CoV)-induced lethality, impaired skin wound healing, and increased tissue damage caused by Sharpin deficiency. In vitro, abrogation of cFLIP cleavage sensitizes cells to tumor necrosis factor(TNF)-induced necroptosis and apoptosis by favoring complex-II formation. Mechanistically, the cell death-sensitizing effect of the D377A mutation depends on glutamine-469. These results reveal a crucial role for cFLIP cleavage in controlling the amplitude of cell death responses occurring upon tissue stress to ensure the execution of repair programs.

ADP-ribosylating adjuvant reveals plasticity in cDC1 cells that drive mucosal Th17 cell development and protection against influenza virus infection.

Migratory dendritic cells expressing CD103 are the targets for mucosal vaccines. These belong to either of two lineage-restricted subsets, cDC1 or cDC2 cells, which have been linked to priming of functionally distinct CD4 T cells. However, recent studies have identified plasticity in cDC2 cells with overlapping functions with cDC1 cells, while the converse has not been reported. We genetically engineered a vaccine adjuvant platform that targeted the cholera toxin A1 (CTA1) ADP-ribosylating enzyme to CD103+ cDC1 and cDC2 cells using a single-chain antibody (scFv) to CD103. Unexpectedly, intranasal immunization with the CTA1-svFcCD103 adjuvant modified cDC1 cells to effectively prime Th17 cells, a function previously limited to cDC2 cells. In fact, cDC2 cells were dispensible, while cDC1 cells, lacking in Batf3-/- mice, were critical. Following intranasal immunizations isolated cDC1 cells from mLN exclusively promoted Rorgt+ T cells and IL-17, IL-21, and IL-22 production. Strong CD8 T cell responses through antigen cross presentation by cDC1 cells were also observed. Single-cell RNAseq analysis revealed upregulation of Th17-promoting gene signatures in sorted cDC1 cells. Gene expression in isolated cDC2 cells was largely unaffected. Our finding represents a major shift of paradigm as we have documented functional plasticity in cDC1 cells.

Influenza Neuraminidase Characteristics and Potential as a Vaccine Target.

Neuraminidase of influenza A and B viruses plays a critical role in the virus life cycle and is an important target of the host immune system. Here, we highlight the current understanding of influenza neuraminidase structure, function, antigenicity, immunogenicity, and immune protective potential. Neuraminidase inhibiting antibodies have been recognized as correlates of protection against disease caused by natural or experimental influenza A virus infection in humans. In the past years, we have witnessed an increasing interest in the use of influenza neuraminidase to improve the protective potential of currently used influenza vaccines. A number of well-characterized influenza neuraminidase-specific monoclonal antibodies have been described recently, most of which can protect in experimental challenge models by inhibiting the neuraminidase activity or by Fc receptor-dependent mechanisms. The relative instability of the neuraminidase poses a challenge for protein-based antigen design. We critically review the different solutions that have been proposed to solve this problem, ranging from the inclusion of stabilizing heterologous tetramerizing zippers to the introduction of inter-protomer stabilizing mutations. Computationally engineered neuraminidase antigens have been generated that offer broad, within subtype protection in animal challenge models. We also provide an overview of modern vaccine technology platforms that are compatible with the induction of robust neuraminidase-specific immune responses. In the near future, we will likely see the implementation of influenza vaccines that confront the influenza virus with a double punch: targeting both the hemagglutinin and the neuraminidase.

In Vivo Therapy with M2e-Specific IgG Selects for an Influenza A Virus Mutant with Delayed Matrix Protein 2 Expression.

The ectodomain of matrix protein 2 (M2e) of influenza A viruses is a universal influenza A vaccine candidate. Here, we report potential evasion strategies of influenza A viruses under in vivo passive anti-M2e IgG immune selection pressure in severe combined immune-deficient (SCID) mice. A/Puerto Rico/8/34-infected SCID mice were treated with the M2e-specific mouse IgG monoclonal antibodies (MAbs) MAb 65 (IgG2a) or MAb 37 (IgG1), which recognize amino acids 5 to 15 in M2e, or with MAb 148 (IgG1), which binds to the invariant N terminus of M2e. Treatment of challenged SCID mice with any of these MAbs significantly prolonged survival compared to isotype control IgG treatment. Furthermore, M2e-specific IgG2a protected significantly better than IgG1, and even resulted in virus clearance in some of the SCID mice. Deep sequencing analysis of viral RNA isolated at different time points after treatment revealed that the sequence variation in M2e was limited to P10H/L and/or I11T in anti-M2e MAb-treated mice. Remarkably, in half of the samples isolated from moribund MAb 37-treated mice and in all MAb 148-treated mice, virus was isolated with a wild-type M2 sequence but with nonsynonymous mutations in the polymerases and/or the hemagglutinin genes. Some of these mutations were associated with delayed M2 and other viral gene expression and with increased resistance to anti-M2e MAb treatment of SCID mice. Treatment with M2e-specific MAbs thus selects for viruses with limited variation in M2e. Importantly, influenza A viruses may also undergo an alternative escape route by acquiring mutations that result in delayed wild-type M2 expression. IMPORTANCE Broadly protective influenza vaccine candidates may have a higher barrier to immune evasion compared to conventional influenza vaccines. We used Illumina MiSeq deep sequence analysis to study the mutational patterns in A/Puerto Rico/8/34 viruses that evolve in chronically infected SCID mice that were treated with different M2e-specific MAbs. We show that under these circumstances, viruses emerged in vivo with mutations in M2e that were limited to positions 10 and 11. Moreover, we discovered an alternative route for anti-M2e antibody immune escape, in which a virus is selected with wild-type M2e but with mutations in other gene segments that result in delayed M2 and other viral protein expression. Delayed expression of the viral antigen that is targeted by a protective antibody thus represents an influenza virus immune escape mechanism that does not involve epitope alterations.

A vulnerable, membrane-proximal site in human respiratory syncytial virus F revealed by a prefusion-specific single-domain antibody.

Human respiratory syncytial virus (RSV) is a major cause of lower respiratory tract disease, especially in young children and the elderly. The fusion protein (F) exists in a pre- and postfusion conformation and is the main target of RSV-neutralizing antibodies. Highly potent RSV-neutralizing antibodies typically bind sites that are unique to the prefusion conformation of F. In this study we screened a single-domain antibody (VHH) library derived from a llama immunized with prefusion-stabilized F and identified a prefusion F-specific VHH that can neutralize RSV A at subnanomolar concentrations. Structural analysis revealed that this VHH primarily binds to antigenic site I while also making contacts with residues in antigenic site III and IV. This new VHH reveals a previously underappreciated membrane-proximal region sensitive for neutralization.ImportanceRSV is an important respiratory pathogen. This study describes a prefusion F-specific VHH that primarily binds to antigenic site I of RSV F. This is the first time that a prefusion F-specific antibody that binds this site is reported. In general, antibodies that bind to site I are poorly neutralizing, whereas the VHH described here neutralizes RSV A at subnanomolar concentrations. Our findings contribute to insights into the RSV F antigenic map.

An Inside Job: Applications of Intracellular Single Domain Antibodies.

Sera of camelid species contain a special kind of antibody that consists only of heavy chains. The variable antigen binding domain of these heavy chain antibodies can be expressed as a separate entity, called a single domain antibody that is characterized by its small size, high solubility and oftentimes exceptional stability. Because of this, most single domain antibodies fold correctly when expressed in the reducing environment of the cytoplasm, and thereby retain their antigen binding specificity. Single domain antibodies can thus be used to target a broad range of intracellular proteins. Such intracellular single domain antibodies are also known as intrabodies, and have proven to be highly useful tools for basic research by allowing visualization, disruption and even targeted degradation of intracellular proteins. Furthermore, intrabodies can be used to uncover prospective new therapeutic targets and have the potential to be applied in therapeutic settings in the future. In this review we provide a brief overview of recent advances in the field of intracellular single domain antibodies, focusing on their use as research tools and potential therapeutic applications. Special attention is given to the available methods that allow delivery of single domain antibodies into cells.

The Opposing Effect of Type I IFN on the T Cell Response by Non-modified mRNA-Lipoplex Vaccines Is Determined by the Route of Administration.

mRNA-lipoplex vaccines are currently being explored in phase II clinical trials for the treatment of patients with advanced solid tumors. Mechanistically, these mRNA-lipoplex vaccines are characterized by the induction of type I interferon (IFN) centered innate responses. Earlier studies have identified type I IFNs as major regulators of the T cell response instigated by mRNA-lipoplex vaccines. However, stimulatory or, in contrast, profound inhibitory effects of type I IFNs were described depending on the study. In this mouse study, we demonstrated that the opposing roles of type I IFN signaling on the magnitude of the vaccine-evoked T cell responses is dependent on the route of mRNA-lipoplex administration and is regulated at the level of the T cells rather than indirectly through modulation of dendritic cell function. This study helps to understand the double-edged sword character of type I IFN induction upon mRNA-based vaccine treatment and may contribute to a more rational design of mRNA vaccination regimens.

Zinc inhibits lethal inflammatory shock by preventing microbe-induced interferon signature in intestinal epithelium.

The cytokine TNF drives inflammatory diseases, e.g., Crohn's disease. In a mouse model of TNF-induced systemic inflammatory response syndrome (SIRS), severe impact on intestinal epithelial cells (IECs) is observed. Zinc confers complete protection in this model. We found that zinc no longer protects in animals which lack glucocorticoids (GCs), or express mutant versions of their receptor GR in IECs, nor in mice which lack gut microbiota. RNA-seq studies in IECs showed that zinc caused reduction in expression of constitutive (STAT1-induced) interferon-stimulated response (ISRE) genes and interferon regulatory factor (IRF) genes. Since some of these genes are involved in TNF-induced cell death in intestinal crypt Paneth cells, and since zinc has direct effects on the composition of the gut microbiota (such as several Staphylococcus species) and on TNF-induced Paneth cell death, we postulate a new zinc-related anti-inflammatory mechanism. Zinc modulates the gut microbiota, causing less induction of ISRE/IRF genes in crypt cells, less TNF-induced necroptosis in Paneth cells, and less fatal evasion of gut bacteria into the system.

Cell-Mediated Responses to Human Metapneumovirus Infection.

Viruses are the most common cause of acute respiratory tract infections (ARTI). Human metapneumovirus (hMPV) frequently causes viral pneumonia which can become life-threatening if the virus spreads to the lungs. Even though hMPV was only isolated in 2001, this negative-stranded RNA virus has probably been circulating in the human population for many decades. Interestingly, almost all adults have serologic evidence of hMPV infection. A well-established host immune response is evoked when hMPV infection occurs. However, the virus has evolved to circumvent and even exploit the host immune response. Further, infection with hMPV induces a weak memory response, and re-infections during life are common. In this review, we provide a comprehensive overview of the different cell types involved in the immune response in order to better understand the immunopathology induced by hMPV. Such knowledge may contribute to the development of vaccines and therapeutics directed against hMPV.

Glucocorticoids limit lipopolysaccharide-induced lethal inflammation by a double control system.

Lipopolysaccharides (LPS) can lead to a lethal endotoxemia, which is a systemic inflammatory response syndrome (SIRS) characterized by a systemic release of cytokines, such as TNF. Endotoxemia is studied intensely, as a model system of Gram-negative infections. LPS- and TNF-induced SIRS involve a strong induction of interferon-stimulated genes (ISGs), some of which cause cell death in the intestinal epithelium cells (IECs). It is well known that glucocorticoids (GCs) protect against endotoxemia. By applying numerous mutant mouse lines, our data support a model whereby GCs, via their glucocorticoid receptor (GR), apply two key mechanisms to control endotoxemia, (i) at the level of suppression of TNF production in a GR monomer-dependent way in macrophages and (ii) at the level of inhibition of TNFR1-induced ISG gene expression and necroptotic cell death mediators in IECs in a GR dimer-dependent way. Our data add new important insights to the understanding of the role of TNF in endotoxemia and the two separate key roles of GCs in suppressing TNF production and activity.

TNF-α inhibits glucocorticoid receptor-induced gene expression by reshaping the GR nuclear cofactor profile.

Glucocorticoid resistance (GCR) is defined as an unresponsiveness to the therapeutic effects, including the antiinflammatory ones of glucocorticoids (GCs) and their receptor, the glucocorticoid receptor (GR). It is a problem in the management of inflammatory diseases and can be congenital as well as acquired. The strong proinflammatory cytokine TNF-alpha (TNF) induces an acute form of GCR, not only in mice, but also in several cell lines: e.g., in the hepatoma cell line BWTG3, as evidenced by impaired Dexamethasone (Dex)-stimulated direct GR-dependent gene up- and down-regulation. We report that TNF has a significant and broad impact on this transcriptional performance of GR, but no impact on nuclear translocation, dimerization, or DNA binding capacity of GR. Proteome-wide proximity-mapping (BioID), however, revealed that the GR interactome was strongly modulated by TNF. One GR cofactor that interacted significantly less with the receptor under GCR conditions is p300. NFκB activation and p300 knockdown both reduced direct transcriptional output of GR whereas p300 overexpression and NFκB inhibition reverted TNF-induced GCR, which is in support of a cofactor reshuffle model. This hypothesis was supported by FRET studies. This mechanism of GCR opens avenues for therapeutic interventions in GCR diseases.

Overexpression of Gilz Protects Mice Against Lethal Septic Peritonitis.

Sepsis in humans and experimental animals is characterized by an acute inflammatory response. glucocorticoids (GCs) are widely used for the treatment of many inflammatory disorders, yet their effectiveness in sepsis is debatable. One of the major anti-inflammatory proteins induced by GCs is glucocorticoid-induced leucine zipper (GILZ, coded by the TSC22D3 gene). We found that TSC22D3 mRNA expression is downregulated in white blood cells of human sepsis patients. Interestingly, transgenic GILZ-overexpressing mice (GILZ-tg) showed better survival rates in the cecal ligation and puncture (CLP) model of mouse sepsis. To our surprise, GILZ had only mild anti-inflammatory effects in this model, as the systemic proinflammatory response was not significantly reduced in GILZ-tg mice compared with control mice. During CLP, we observed reduced bacterial counts in blood of GILZ-tg mice compared with control mice. We found increased expression of Tsc22d3 mRNA specifically in peritoneal exudate cells in the CLP model, as well as increased capacity for bacterial phagocytosis of CD45 GILZ-tg cells compared with CD45 GILZ-wt cells. Hence, we believe that the protective effects of GILZ in the CLP model can be linked to a more efficient phagocytosis.

Selective Engagement of FcγRIV by a M2e-Specific Single Domain Antibody Construct Protects Against Influenza A Virus Infection.

Lower respiratory tract infections, such as infections caused by influenza A viruses, are a constant threat for public health. Antivirals are indispensable to control disease caused by epidemic as well as pandemic influenza A. We developed a novel anti-influenza A virus approach based on an engineered single-domain antibody (VHH) construct that can selectively recruit innate immune cells to the sites of virus replication. This protective construct comprises two VHHs. One VHH binds with nanomolar affinity to the conserved influenza A matrix protein 2 (M2) ectodomain (M2e). Co-crystal structure analysis revealed that the complementarity determining regions 2 and 3 of this VHH embrace M2e. The second selected VHH specifically binds to the mouse Fcγ Receptor IV (FcγRIV) and was genetically fused to the M2e-specific VHH, which resulted in a bi-specific VHH-based construct that could be efficiently expressed in Pichia pastoris. In the presence of M2 expressing or influenza A virus-infected target cells, this single domain antibody construct selectively activated the mouse FcγRIV. Moreover, intranasal delivery of this bispecific FcγRIV-engaging VHH construct protected wild type but not FcγRIV-/- mice against challenge with an H3N2 influenza virus. These results provide proof of concept that VHHs directed against a surface exposed viral antigen can be readily armed with effector functions that trigger protective antiviral activity beyond direct virus neutralization.

Glucocorticoid receptor dimers control intestinal STAT1 and TNF-induced inflammation in mice.

TNF is an important mediator in numerous inflammatory diseases, e.g., in inflammatory bowel diseases (IBDs). In IBD, acute increases in TNF production can lead to disease flares. Glucocorticoids (GCs), which are steroids that bind and activate the glucocorticoid receptor (GR), are able to protect animals and humans against acute TNF-induced inflammatory symptoms. Mice with a poor transcriptional response of GR dimer-dependent target genes were studied in a model of TNF-induced lethal inflammation. In contrast to the GRWT/WT mice, these GRdim/dim mice displayed a substantial increase in TNF sensitivity and a lack of protection by the GC dexamethasone (DEX). Unchallenged GRdim/dim mice had a strong IFN-stimulated gene (ISG) signature, along with STAT1 upregulation and phosphorylation. This ISG signature was gut specific and, based on our studies with antibiotics, depended on the gut microbiota. GR dimers directly bound to short DNA sequences in the STAT1 promoter known as inverted repeat negative GRE (IR-nGRE) elements. Poor control of STAT1 in GRdim/dim mice led to failure to repress ISG genes, resulting in excessive necroptosis induction by TNF. Our findings support a critical interplay among gut microbiota, IFNs, necroptosis, and GR in both the basal response to acute inflammatory challenges and pharmacological intervention by GCs.

Single-Domain Antibodies and Their Formatting to Combat Viral Infections.

Since their discovery in the 1990s, single-domain antibodies (VHHs), also known as Nanobodies®, have changed the landscape of affinity reagents. The outstanding solubility, stability, and specificity of VHHs, as well as their small size, ease of production and formatting flexibility favor VHHs over conventional antibody formats for many applications. The exceptional ease by which it is possible to fuse VHHs with different molecular modules has been particularly explored in the context of viral infections. In this review, we focus on VHH formats that have been developed to combat viruses including influenza viruses, human immunodeficiency virus-1 (HIV-1), and human respiratory syncytial virus (RSV). Such formats may significantly increase the affinity, half-life, breadth of protection of an antiviral VHH and reduce the risk of viral escape. In addition, VHHs can be equipped with effector functions, for example to guide components of the immune system with high precision to sites of viral infection.

Simultaneous Inhibition of Tumor Necrosis Factor Receptor 1 and Matrix Metalloproteinase 8 Completely Protects Against Acute Inflammation and Sepsis.

OBJECTIVES: Sepsis causes very high mortality and morbidity rates and remains one of the biggest medical challenges. This study investigates whether plasma levels of both matrix metalloproteinase 8 and tumor necrosis factor receptor 1 are associated with sepsis severity and also investigates the therapeutic applicability of simultaneous inhibition of the two molecules in sepsis. DESIGN: Observational human pilot study-prospective controlled animal study. SETTING: University hospital and research laboratory. SUBJECTS: Sepsis patients and C57BL/6 mice deficient for matrix metalloproteinase 8 and/or tumor necrosis factor receptor 1. INTERVENTION: Plasma and whole blood RNA were collected from 13 sepsis patients for 7 consecutive days and within 24 hours of admission to ICU. Matrix metalloproteinase 8 and tumor necrosis factor receptor 1 plasma and expression levels were determined in these patients. Mice deficient for both matrix metalloproteinase 8 and tumor necrosis factor receptor 1 were generated and subjected to endotoxemia and cecal ligation and puncture. Additionally, a bispecific Nanobody that simultaneously blocks matrix metalloproteinase 8 and tumor necrosis factor receptor 1 was created. MEASUREMENTS AND MAIN RESULTS: Plasma levels of matrix metalloproteinase 8 and tumor necrosis factor receptor 1 were positively correlated with the Sequential Organ Failure Assessment score (r, 0.51 and 0.58) and interleukin 6 levels (r, 0.59 and 0.52) in 13 sepsis patients. Combined elimination of tumor necrosis factor receptor 1 and matrix metalloproteinase 8 in double knockout mice resulted in superior survival in endotoxemia and CLP compared with single knockouts and wild-type mice. Cotreatment with our bispecific Nanobody in CLP resulted in improved survival rates (28% vs 19%) compared with untreated mice. CONCLUSIONS: Inhibition of matrix metalloproteinase 8 and tumor necrosis factor receptor 1 might have therapeutic potential to treat sepsis and proof-of-principle was provided as therapeutics that inhibit both tumor necrosis factor receptor 1 and matrix metalloproteinase 8 are effective in CLP.

Glucocorticoid Receptor-mediated transactivation is hampered by Striatin-3, a novel interaction partner of the receptor.

The transcriptional activity of the glucocorticoid receptor (GR) is co-determined by its ability to recruit a vast and varying number of cofactors. We here identify Striatin-3 (STRN3) as a novel interaction partner of GR that interferes with GR's ligand-dependent transactivation capacity. Remarkably, STRN3 selectively affects only GR-dependent transactivation and leaves GR-dependent transrepression mechanisms unhampered. We found that STRN3 down-regulates GR transactivation by an additional recruitment of the catalytic subunit of protein phosphatase 2A (PPP2CA) to GR. We hypothesize the existence of a functional trimeric complex in the nucleus, able to dephosphorylate GR at serine 211, a known marker for GR transactivation in a target gene-dependent manner. The presence of STRN3 appears an absolute prerequisite for PPP2CA to engage in a complex with GR. Herein, the C-terminal domain of GR is essential, reflecting ligand-dependency, yet other receptor parts are also needed to create additional contacts with STRN3.

Overexpression of Glucocorticoid-induced Leucine Zipper (GILZ) increases susceptibility to Imiquimod-induced psoriasis and involves cutaneous activation of TGF-ß1.

Psoriasis vulgaris is a chronic inflammatory skin disease affecting millions of people. Its pathophysiology is complex and involves a skin compartment with epidermal and immune cells which produce cytokines, e.g. belonging to the IL-23-Th17-cell axis. Glucocorticoids (GCs) are the most common therapeutics used in cutaneous inflammatory disorders and GC-induced leucine zipper (GILZ) has emerged as a mediator of GCs due to its anti-inflammatory actions, theoretically lacking GC side-effects. We evaluated whether GILZ may provide a better therapeutic index in comparison to GCs during the onset and progression of psoriasis by generating and characterizing a mouse model with generalized overexpression of this protein (GILZ-Tg mice) and the imiquimod (IMQ) psoriasis model. Unexpectedly, in GILZ-Tg mice, the severity of IMQ-induced psoriasis-like skin lesions as well as induction of cytokines commonly up-regulated in human psoriasis (Il-17, Il-22, Il-23, Il-6, S100a8/a9, and Stat3) was significantly more pronounced relative to GILZ-Wt mice. The increased susceptibility to IMQ-induced psoriasis of GILZ-Tg mice was significantly associated with skin-specific over-activation of TGF-ß1-mediated signaling via SMAD2/3. Our findings demonstrate that GILZ may behave as pro-inflammatory protein in certain tissues and that, similar to prolonged GC therapy, GILZ as an alternative treatment for psoriasis may also have adverse effects.