Tissue S100/calgranulin expression and blood neutrophil-to-lymphocyte ratio (NLR) in prostatic disorders in dogs.

BACKGROUND: Prostatic carcinoma (PCA) is a rare but severe condition in dogs that is similar to the androgen-independent form of PCA in men. In contrast to humans, PCA is difficult to diagnose in dogs as reliable biomarkers, available for PCA screening in human medicine, are currently lacking in small animal oncology. Calprotectin (S100A8/A9) and S100A12 are Ca2+-binding proteins of the innate immune system with promising potential to distinguish malignant from benign urogenital tract conditions, similar to the blood neutrophil-to-lymphocyte-ratio (NLR). However, both have not yet been extensively investigated in dogs with PCA. Thus, this study aimed to evaluate the expression of the S100/calgranulins (calprotectin, S100A12, and their ratio [Cal-ratio]) in prostatic biopsies from nine dogs with PCA and compare them to those in dogs with benign prostatic lesions (eight dogs with prostatitis and ten dogs with benign prostatic hyperplasia [BPH]) as well as five healthy controls. In addition, blood NLRs were investigated in twelve dogs with PCA and 22 dogs with benign prostatic conditions. RESULTS: Tissue S100A8/A9+ cell counts did not differ significantly between tissue from PCA and prostatitis cases (P = 0.0659) but were significantly higher in dogs with prostatitis than BPH (P = 0.0013) or controls (P = 0.0033). S100A12+ cell counts were significantly lower in PCA tissues than in prostatitis tissue (P = 0.0458) but did not differ compared to BPH tissue (P = 0.6499) or tissue from controls (P = 0.0622). Cal-ratios did not differ significantly among the groups but were highest in prostatitis tissues and significantly higher in those dogs with poor prostatitis outcomes than in patients that were still alive at the end of the study (P = 0.0455). Blood NLR strongly correlated with prostatic tissue S100A8/A9+ cell counts in dogs with PCA (ρ = 0.81, P = 0.0499) but did not differ among the disease groups of dogs. CONCLUSIONS: This study suggests that the S100/calgranulins play a role in malignant (PCA) and benign (prostatic inflammation) prostatic conditions and supports previous results in lower urinary tract conditions in dogs. These molecules might be linked to the inflammatory environment with potential effects on the inflammasome. The blood NLR does not appear to aid in distinguishing prostatic conditions in dogs. Further investigation of the S100/calgranulin pathways and their role in modulation of tumor development, progression, and metastasis in PCA is warranted.

Tissue S100/calgranulin expression and blood neutrophil-to-lymphocyte ratio (NLR) in dogs with lower urinary tract urothelial carcinoma.

BACKGROUND: Urothelial carcinoma (UC) is the most common neoplasm of the canine lower urinary tract, affecting approximately 2% of dogs. Elderly female patients of certain breeds are predisposed, and clinical signs of UC can easily be confused with urinary tract infection or urolithiasis. Diagnosis and treatment are challenging given the lack of disease-specific markers and treatments. The S100A8/A9 complex and S100A12 protein are Ca2+-binding proteins expressed by cells of the innate immune system and have shown promise as urinary screening markers for UC. The neutrophil-to-lymphocyte ratio (NLR) can also aid in distinguishing certain neoplastic from inflammatory conditions. Our study aimed to evaluate the tissue expression of S100/calgranulins and the blood NLR in dogs with UC. Urinary bladder and/or urethral tissue samples from dogs with UC (n = 10), non-neoplastic inflammatory lesions (NNUTD; n = 6), and no histologic changes (n = 11) were evaluated using immunohistochemistry. Blood NLRs were analyzed in dogs with UC (n = 22) or NNUTD (n = 26). RESULTS: Tissue S100A12-positive cell counts were significantly higher in dogs with lower urinary tract disease than healthy controls (P = 0.0267 for UC, P = 0.0049 for NNUTD), with no significant difference between UC and NNUTD patients. Tissue S100A8/A9-positivity appeared to be higher with NNUTD than UC, but this difference did not reach statistical significance. The S100A8/A9+-to-S100A12+ ratio was significantly decreased in neoplastic and inflamed lower urinary tract tissue compared to histologically normal specimens (P = 0.0062 for UC, P = 0.0030 for NNUTD). NLRs were significantly higher in dogs with UC than in dogs with NNUTD, and a cut-off NLR of ≤ 2.83 distinguished UC from NNUTD with 41% sensitivity and 100% specificity. Higher NLRs were also associated with a poor overall survival time (P = 0.0417). CONCLUSIONS: These results confirm that the S100/calgranulins play a role in the immune response to inflammatory and neoplastic lower urinary tract diseases in dogs, but the tissue expression of these proteins appears to differ from their concentrations reported in urine samples. Further investigations of the S100/calgranulin pathways in UC and their potential as diagnostic or prognostic tools and potential therapeutic targets are warranted. The NLR as a routinely available marker might be a useful surrogate to distinguish UC from inflammatory conditions.

Intestinal S100/Calgranulin Expression in Cats with Chronic Inflammatory Enteropathy and Intestinal Lymphoma.

Diagnosing chronic inflammatory enteropathies (CIE) in cats and differentiation from intestinal lymphoma (IL) using currently available diagnostics is challenging. Intestinally expressed S100/calgranulins, measured in fecal samples, appear to be useful non-invasive biomarkers for canine CIE but have not been evaluated in cats. We hypothesized S100/calgranulins to play a role in the pathogenesis of feline chronic enteropathies (FCE) and to correlate with clinical and/or histologic disease severity. This retrospective case-control study included patient data and gastrointestinal (GI) tissues from 16 cats with CIE, 8 cats with IL, and 16 controls with no clinical signs of GI disease. GI tissue biopsies were immunohistochemically stained using polyclonal α-S100A8/A9 and α-S100A12 antibodies. S100A8/A9+ and S100A12+ cells were detected in all GI segments, with few significant differences between CIE, IL, and controls and no difference between diseased groups. Segmental inflammatory lesions were moderately to strongly correlated with increased S100/calgranulin-positive cell counts. Clinical disease severity correlated with S100A12+ cell counts in cats with IL (ρ = 0.69, p = 0.042) and more severe diarrhea with colonic lamina propria S100A12+ cells with CIE (ρ = 0.78, p = 0.021) and duodenal S100A8/A9+ cells with IL (ρ = 0.71, p = 0.032). These findings suggest a role of the S100/calgranulins in the pathogenesis of the spectrum of FCE, including CIE and IL.

Canine tissue-associated CD4+CD8α+ double-positive T cells are an activated T cell subpopulation with heterogeneous functional potential.

Canine CD4+CD8α+ double-positive (dp) T cells of peripheral blood are a unique effector memory T cell subpopulation characterized by an increased expression of activation markers in comparison with conventional CD4+ or CD8α+ single-positive (sp) T cells. In this study, we investigated CD4+CD8α+ dp T cells in secondary lymphatic organs (i.e. mesenteric and tracheobronchial lymph nodes, spleen, Peyer's patches) and non-lymphatic tissues (i.e. lung and epithelium of the small intestine) within a homogeneous group of healthy Beagle dogs by multi-color flow cytometry. The aim of this systematic analysis was to identify the tissue-specific localization and characteristics of this distinct T cell subpopulation. Our results revealed a mature extrathymic CD1a-CD4+CD8α+ dp T cell population in all analyzed organs, with highest frequencies within Peyer's patches. Constitutive expression of the activation marker CD25 is a feature of many CD4+CD8α+ dp T cells independent of their localization and points to an effector phenotype. A proportion of lymph node CD4+CD8α+ dp T cells is FoxP3+ indicating regulatory potential. Within the intestinal environment, the cytotoxic marker granzyme B is expressed by CD4+CD8α+ dp intraepithelial lymphocytes. In addition, a fraction of CD4+CD8α+ dp intraepithelial lymphocytes and of mesenteric lymph node CD4+CD8α+ dp T cells is TCRγδ+. However, the main T cell receptor of all tissue-associated CD4+CD8α+ dp T cells could be identified as TCRαß. Interestingly, the majority of the CD4+CD8α+ dp T cell subpopulation expresses the unconventional CD8αα homodimer, in contrast to CD8α+ sp T cells, and CD4+CD8α+ dp thymocytes which are mainly CD8αß+. The presented data provide the basis for a functional analysis of tissue-specific CD4+CD8α+ dp T cells to elucidate their role in health and disease of dogs.

Orf virus (ORFV) infection in a three-dimensional human skin model: Characteristic cellular alterations and interference with keratinocyte differentiation.

ORF virus (ORFV) is the causative agent of contagious ecthyma, a pustular dermatitis of small ruminants and humans. Even though the development of lesions caused by ORFV was extensively studied in animals, only limited knowledge exists about the lesion development in human skin. The aim of the present study was to evaluate a three-dimensional (3D) organotypic culture (OTC) as a human skin model for ORFV infection considering lesion development, replication of the virus, viral gene transcription and modulation of differentiation of human keratinocytes by ORFV. ORFV infection of OTC was performed using the ORFV isolate B029 derived from a human patient. The OTC sections showed a similar structure of stratified epidermal keratinocytes as human foreskin and a similar expression profile of the differentiation markers keratin 1 (K1), K10, and loricrin. Upon ORFV infection, OTCs exhibited histological cytopathic changes including hyperkeratosis and ballooning degeneration of the keratinocytes. ORFV persisted for 10 days and was located in keratinocytes of the outer epidermal layers. ORFV-specific early, intermediate and late genes were transcribed, but limited viral spread and restricted cell infection were noticed. ORFV infection resulted in downregulation of K1, K10, and loricrin at the transcriptional level without affecting proliferation as shown by PCNA or Ki-67 expression. In conclusion, OTC provides a suitable model to study the interaction of virus with human keratinocytes in a similar structural setting as human skin and reveals that ORFV infection downregulates several differentiation markers in the epidermis of the human skin, a hitherto unknown feature of dermal ORFV infection in man.

Orf virus infection of human keratinocytes and dermal fibroblasts: Limited virus detection and interference with intercellular adhesion molecule-1 up-regulation.

Orf virus (Parapoxvirus ovis, ORFV) is a dermatotropic virus causing pustular dermatitis in small ruminants and humans. We analysed isolated human primary keratinocytes (KC) and dermal fibroblasts (FB) for cell death and virus replication by infection with a patient-derived ORFV isolate. ORFV infection was associated with rapid induction of cell death in KC allowing for considerable virus removal. Upon infection with ORFV, KC and FB harboured intracytoplasmic ORFV and showed viral protein presence; however, missing virus spread indicated an abortive infection. Upon ORFV exposure, KC but not FB secreted the pro-inflammatory cytokine interleukin (IL)-6. ORFV infection enhanced the frequency of KC expressing intercellular adhesion molecule (ICAM)-1 which was independent of IL-6. Interestingly, ORFV inhibited ICAM-1 up-regulation on infected but not on non-infected KC. Even interferon-γ, a potent inducer of ICAM-1, up-regulated ICAM-1 only on non-infected KC. Transfer of ORFV-free supernatant from infected to non-infected KC induced ICAM-1 on non-infected KC pointing to the involvement of soluble mediator(s). Similarly as in KC, in FB interference with ICAM-1 up-regulation by ORFV infection was also observed. In conclusion, we shed light on epidermal and dermal defense mechanisms to ORFV infection and point to a novel ICAM-1-related immune evasion mechanism of ORFV in human skin.

Lung epithelium is the major source of IL-33 and is regulated by IL-33-dependent and IL-33-independent mechanisms in pulmonary cryptococcosis.

Inhalation of the fungus Cryptococcus neoformans (C. neoformans) results in pulmonary cryptococcosis associated with IL-33-dependent type 2 immunity. Lung epithelium represents the initial contact site of infection. The role of IL-33 in type 2 immunity has been analyzed, but the source of this cytokine and its effect on lung epithelial cell function in pulmonary cryptococcosis remained unclear. In mice infected with C. neoformans, we identified alveolar type 2 epithelial cells as major source of IL-33. On both, IL-33-positive and IL-33-negative epithelial cells, IL-33 receptor expression was detectable. Therefore, we studied the role of IL-33 receptor expression for IL-33 synthesis during fungal infection on lung epithelial cells and found no auto-/paracrine IL-33 induction. Next, the effect of IL-33 on epithelial E-cadherin expression, a cell-to-cell adhesion molecule, was analyzed. Fungal infection resulted in E-cadherin downregulation in an IL-33-dependent manner on pulmonary epithelial cells both at the single-cell and at the population level. On the other hand, epithelial cells from infected mice upregulated surfactant protein C (SP-C) and CXCL15 mRNA production together with but independently of IL-33. In conclusion, lung epithelium represents a significant source of IL-33 in pulmonary cryptococcosis and is regulated in an IL-33-dependent but also IL-33-independent manner.

Therapeutic expansion of CD4+FoxP3+ regulatory T cells limits allergic airway inflammation during pulmonary fungal infection.

Allergic asthma can be frequently caused and exacerbated by sensitization to ubiquitous fungal allergens associated with pulmonary mucus production, airway hyperresponsiveness and bronchial constriction, resulting in a complex disease that is often difficult to treat. Fungal infections are frequently complicated by the development of a type 2 immune response that prevents successful elimination of the fungal pathogen. Furthermore, production of type 2 cytokines triggers allergic airway inflammation. Following intranasal infection of BALB/c mice with the fungusCryptococcus neoformans, we recently described a more pronounced type 2 immune response in the absence of regulatory T (Treg) cells. To determine whether Treg cell expansion is able to suppress type 2-related fungal allergic inflammation, we increased Treg cell numbers during pulmonaryC. neoformansinfection by administration of an interleukin (IL)-2/anti-IL-2 complex. Expansion of Treg cells resulted in reduced immunoglobulin E production and decreased allergic airway inflammation including reduced production of pulmonary mucus and type 2 cytokines as well as production of immunosuppressive cytokines such as IL-10 and transforming growth factor-ß1. From our data we conclude that Treg cells and/or their suppressive mediators represent potential targets for therapeutic intervention during allergic fungal airway disease.

A novel experimental model of Cryptococcus neoformans-related immune reconstitution inflammatory syndrome (IRIS) provides insights into pathogenesis.

Antiretroviral therapy (ART) has yielded major advances in fighting the HIV pandemic by restoring protective immunity. However, a significant proportion of HIV patients co-infected with the opportunistic fungal pathogen Cryptococcus neoformans paradoxically develops a life-threatening immune reconstitution inflammatory syndrome (IRIS) during antiretroviral therapy. Despite several clinical studies, the underlying pathomecha-nisms are poorly understood. Here, we present the first mouse model of cryptococcal IRIS that allows for a detailed analysis of disease development. Lymphocyte-deficient RAG-1(-/-) mice are infected with C. neoformans and 4 weeks later adoptively transferred with purified CD4(+) T cells. Reconstitution of CD4(+) T cells is sufficient to induce a severe inflammatory disease similar to clinical IRIS in C. neoformans-infected RAG-1(-/-) mice of different genetic backgrounds and immunological phenotypes (i.e. C57BL/6 and BALB/c). Multiorgan inflammation is accompanied by a systemic release of distinct proinflammatory cytokines, i.e. IFN-γ, IL-6, and TNF-α. IRIS development is characterized by infection-dependent activation of donor CD4(+) T cells, which are the source of IFN-γ. Interestingly, IFN-γ-mediated effects are not required for disease induction. Taken together, this novel mouse model of cryptococcal IRIS provides a useful tool to verify potential mechanisms of pathogenesis, revealing targets for diagnosis and therapeutic interventions.

IL-4 receptor-alpha-dependent control of Cryptococcus neoformans in the early phase of pulmonary infection.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes lung inflammation and meningoencephalitis in immunocompromised people. Previously we showed that mice succumb to intranasal infection by induction of pulmonary interleukin (IL)-4Rα-dependent type 2 immune responses, whereas IL-12-dependent type 1 responses confer resistance. In the experiments presented here, IL-4Rα⁻/⁻ mice unexpectedly show decreased fungal control early upon infection with C. neoformans, whereas wild-type mice are able to control fungal growth accompanied by enhanced macrophage and dendritic cell recruitment to the site of infection. Lower pulmonary recruitment of macrophages and dendritic cells in IL-4Rα⁻/⁻ mice is associated with reduced pulmonary expression of CCL2 and CCL20 chemokines. Moreover, IFN-γ and nitric oxide production are diminished in IL-4Rα⁻/⁻ mice compared to wild-type mice. To directly study the potential mechanism(s) responsible for reduced production of IFN-γ, conventional dendritic cells were stimulated with C. neoformans in the presence of IL-4 which results in increased IL-12 production and reduced IL-10 production. Together, a beneficial role of early IL-4Rα signaling is demonstrated in pulmonary cryptococcosis, which contrasts with the well-known IL-4Rα-mediated detrimental effects in the late phase.

Abrogation of IL-4 receptor-α-dependent alternatively activated macrophages is sufficient to confer resistance against pulmonary cryptococcosis despite an ongoing T(h)2 response.

In the murine model of pulmonary infection with Cryptococcus neoformans, IL-4 receptor α (IL-4Rα)-dependent polyfunctional T(h)2 cells induce disease progression associated with alternative activation of lung macrophages. To characterize the effector role of IL-4Rα-dependent alternatively activated macrophages (aaMph), we intra-nasally infected mice with genetically ablated IL-4Rα expression on macrophages (LysM(Cre)IL-4Rα(-/lox) mice) and IL-4Rα(-/lox) littermates. LysM(Cre)IL-4Rα(-/lox) mice were significantly more resistant to pulmonary cryptococcosis with higher survival rates and lower lung burden than non-deficient heterozygous littermates. Infected LysM(Cre)IL-4Rα(-/lox) mice had reduced but detectable numbers of aaMph expressing arginase-1, chitinase-like enzyme (YM1) and CD206. Similar pulmonary expression of inducible nitric oxide synthase was found in LysM(Cre)IL-4Rα(-/lox) and IL-4Rα(-/lox) control mice, but macrophages from LysM(Cre)IL-4Rα(-/lox) mice showed a higher potential to produce nitric oxide. In contrast to the differences in the macrophage phenotype, pulmonary T(h)2 responses were similar in infected LysM(Cre)IL-4Rα(-/lox) and IL-4Rα(-/lox) mice with each mouse strain harboring polyfunctional T(h)2 cells. Consistently, type 2 pulmonary allergic inflammation associated with eosinophil recruitment and epithelial mucus production was present in lungs of both LysM(Cre)IL-4Rα(-/lox) and IL-4Rα(-/lox) mice. Our results demonstrate that, despite residual IL-4Rα-independent alternative macrophage activation and ongoing T(h)2-dependent allergic inflammation, abrogation of IL-4Rα-dependent aaMph is sufficient to confer resistance in pulmonary cryptococcosis. This is even evident on a relatively resistant heterozygous IL-4Rα(+/-) background indicating a key contribution of macrophage IL-4Rα expression to susceptibility in allergic bronchopulmonary mycosis.

Overexpression of TGF-ß1 in macrophages reduces and stabilizes atherosclerotic plaques in ApoE-deficient mice.

Although macrophages represent the hallmark of both human and murine atherosclerotic lesions and have been shown to express TGF-ß1 (transforming growth factor ß1) and its receptors, it has so far not been experimentally addressed whether the pleiotropic cytokine TGF-ß1 may influence atherogenesis by a macrophage specific mechanism. We developed transgenic mice with macrophage specific TGF-ß1 overexpression, crossed the transgenics to the atherosclerotic ApoE (apolipoprotein E) knock-out strain and quantitatively analyzed both atherosclerotic lesion development and composition of the resulting double mutants. Compared with control ApoE(-/-) mice, animals with macrophage specific TGF-ß1 overexpression developed significantly less atherosclerosis after 24 weeks on the WTD (Western type diet) as indicated by aortic plaque area en face (p<0.05). Reduced atherosclerotic lesion development was associated with significantly less macrophages (p<0.05 after both 8 and 24 weeks on the WTD), significantly more smooth muscle cells (SMCs; p<0.01 after 24 weeks on the WTD), significantly more collagen (p<0.01 and p<0.05 after 16 and 24 weeks on the WTD, respectively) without significant differences of inner aortic arch intima thickness or the number of total macrophages in the mice pointing to a plaque stabilizing effect of macrophage-specific TGF-ß1 overexpression. Our data shows that macrophage specific TGF-ß1 overexpression reduces and stabilizes atherosclerotic plaques in ApoE-deficient mice.

Cutting edge: TGF-beta signaling is required for the in vivo expansion and immunosuppressive capacity of regulatory CD4+CD25+ T cells.

Data regarding the role of TGF-beta for the in vivo function of regulatory CD4(+)CD25(+) T cells (Treg) are controversial. A transgenic mouse model with impaired TGF-beta signaling specifically in T cells was used to assess the role of endogenous TGF-beta for the in vivo function of CD4(+)CD25(+) Treg in a murine model of colitis induced by dextran sulfate. Transfer of wild-type, but not transgenic CD4(+)CD25(+) Treg was found to suppress colitis in wild-type mice. In addition, by transferring CFSE-labeled CD4(+)CD25(+) Treg we could demonstrate that endogenous TGF-beta promotes the expansion of CD4(+)CD25(+) Treg in vivo. Transgenic mice themselves developed reduced numbers of peripheral CD4(+)CD25(+) Treg and were more susceptible to the induction of colitis, which could be prevented by the transfer of wild-type Treg. These data indicate that TGF-beta signaling in CD4(+)CD25(+) Treg is required for their in vivo expansion and suppressive capacity.

TGFbeta regulates the CD4+CD25+ T-cell pool and the expression of Foxp3 in vivo.

Factors influencing the development of CD4+CD25+ T-cells in vivo are poorly understood. In order to investigate the contribution of TGFbeta1 to the development and function of CD4+CD25+ T-cells, we generated a gain of function mutation resulting in the overexpression of an active form of TGFbeta1 in T-cells under control of the human CD2 promoter. In peripheral lymphoid organs and in the thymus, the frequency of CD4+CD25+ T-cells was increased in transgenic mice. This appeared to be due to an autocrine effect of TGFbeta on T-cells, since concomitant impairment of TGFbeta-signaling in double transgenic mice resulted in a phenotype similar to wild type. In contrast, in single transgenic mice with impaired TGFbeta-signaling in T-cells, CD4+CD25+ T-cell numbers were reduced in peripheral lymphoid organs but not in the thymus. In addition, TGFbeta was found to regulate the expression of Foxp3 in vivo, a transcription factor essential for the generation and function of regulatory T-cells. In CD4+CD25+ T-cells, TGFbeta1 increased the expression of Foxp3, whereas a decreased expression was seen in CD4+CD25+ T-cells with impaired TGFbeta-signaling. TGFbeta1 induced the expression of IL-10 in transgenic T-cells, but the increased in vitro suppressive capacity observed in transgenic CD4+CD25+ T-cells was due to the secretion of TGFbeta and not IL-10. Therefore, our study provides in vivo evidence for a role of TGFbeta in the homeostasis of CD4+CD25+ T-cells.

Susceptibility to collagen-induced arthritis is modulated by TGFbeta responsiveness of T cells.

The objective of our study was to determine the regulatory effects that endogenous transforming growth factor beta (TGFbeta) exerts on T cells in the pathogenesis of collagen-induced arthritis (CIA). CIA was induced in transgenic mice expressing a dominant negative TGFbeta type II receptor in T cells under the control of the human CD2 promoter. Clinical and histological arthritis scores were determined and experiments on disease induction and the healing phase of disease were performed. The proliferation and cytokine production of draining lymph node cells in vitro were analyzed. Transgenic mice were more susceptible to induction of CIA. The overall incidence was higher in transgenic mice than in wild-type mice (57% vs 35%, P < 0.05). Affected transgenic animals displayed a significantly higher clinical (4.5 +/- 0.6 vs 1.67 +/- 0.19, P = 0.001) and histological arthritis score (8.01 +/- 0.9 vs 4.06 +/- 1.1, P < 0.05). Draining lymph node cells of transgenic mice secreted more tumor necrosis factor alpha and IFNgamma and proliferated more vigorously in response to collagen type II and upon CD3/CD28 costimulation in vitro. Therefore, the regulation of T cells by endogenous TGFbeta is important for the maintenance of joint integrity after arthritis induction. Defects in TGFbeta-signalling as a susceptibility factor for rheumatoid arthritis may warrant further investigation.

A key pathogenic role for the STAT1/T-bet signaling pathway in T-cell-mediated liver inflammation.

TH1 cytokines have been suggested to contribute to the pathogenesis of T-cell-mediated liver injury and inflammation. However, the molecular signaling pathways involved in such injury are still poorly understood. In the present study, we investigated the role of the STAT1/T-bet signaling pathway in a murine model of T-cell-mediated liver inflammation induced by the application of concanavalin A (Con A) using newly created STAT1 transgenic mice as well as STAT1- and T-bet-deficient mice. Liver injury induced by Con A was associated with an increase of both pSTAT1 and T-bet levels in the liver. Furthermore, functional studies suggested a pathogenic role for STAT1 in Con A-induced liver injury, because transgenic mice overexpressing STAT1 under the control of the CD2 promoter/enhancer construct showed elevated interferon gamma (IFN-gamma) and IRF-1 levels as well as significantly augmented liver injury following administration of Con A. Consistently, we observed that both STAT1-deficient and T-bet-deficient mice were protected from such T-cell-dependent liver injury. In conclusion, these findings suggest a key pathogenic role for the STAT1/T-bet signaling pathway for T-cell activation in the Con A model of T-cell-mediated liver pathology.

TGF-beta regulates airway responses via T cells.

Allergic asthma is characterized by airway hyperreactivity, inflammation, and a Th2-type cytokine profile favoring IgE production. Beneficial effects of TGF-beta and conflicting results regarding the role of Th1 cytokines have been reported from murine asthma models. In this study, we examined the T cell as a target cell of TGF-beta-mediated immune regulation in a mouse model of asthma. We demonstrate that impairment of TGF-beta signaling in T cells of transgenic mice expressing a dominant-negative TGF-beta type II receptor leads to a decrease in airway reactivity in a non-Ag-dependent model. Increased serum levels of IFN-gamma can be detected in these animals. In contrast, after injection of OVA adsorbed to alum and challenge with OVA aerosol, transgenic animals show an increased airway reactivity and inflammation compared with those of wild-type animals. IL-13 levels in bronchoalveolar lavage fluid and serum as well as the number of inducible NO synthase-expressing cells in lung infiltrates were increased in transgenic animals. These results demonstrate an important role for TGF-beta signaling in T cells in the regulation of airway responses and suggest that the beneficial effects observed for TGF-beta in airway hyperreactivity and inflammation may be due to its regulatory effects on T cells.

Impairment of TGF-beta signaling in T cells increases susceptibility to experimental autoimmune hepatitis in mice.

In autoimmune hepatitis, strong TGF-beta1 expression is found in the inflamed liver. TGF-beta overexpression may be part of a regulatory immune response attempting to suppress autoreactive T cells. To test this hypothesis, we determined whether impairment of TGF-beta signaling in T cells leads to increased susceptibility to experimental autoimmune hepatitis (EAH). Transgenic mice of strain FVB/N were generated expressing a dominant-negative TGF-beta type II receptor in T cells under the control of the human CD2 promoter/locus control region. On induction of EAH, transgenic mice showed markedly increased portal and periportal leukocytic infiltrations with hepatocellular necroses compared with wild-type mice (median histological score = 1.8 +/- 0.26 vs. 0.75 +/- 0.09 in wild-type mice; P < 0.01). Increased IFN-gamma production (118 vs. 45 ng/ml) and less IL-4 production (341 vs. 1,256 pg/ml) by mononuclear cells isolated from transgenic livers was seen. Impairment of TGF-beta signaling in T cells therefore leads to increased susceptibility to EAH in mice. This suggests an important role for TGF-beta in immune homeostasis in the liver and may teleologically explain TGF-beta upregulation in response to T cell-mediated liver injury.