Parvulin 17-catalyzed Tubulin Polymerization Is Regulated by Calmodulin in a Calcium-dependent Manner.

Recently we have shown that the peptidyl-prolyl cis/trans isomerase parvulin 17 (Par17) interacts with tubulin in a GTP-dependent manner, thereby promoting the formation of microtubules. Microtubule assembly is regulated by Ca(2+)-loaded calmodulin (Ca(2+)/CaM) both in the intact cell and under in vitro conditions via direct interaction with microtubule-associated proteins. Here we provide the first evidence that Ca(2+)/CaM interacts also with Par17 in a physiologically relevant way, thus preventing Par17-promoted microtubule assembly. In contrast, parvulin 14 (Par14), which lacks only the first 25 N-terminal residues of the Par17 sequence, does not interact with Ca(2+)/CaM, indicating that this interaction is exclusive for Par17. Pulldown experiments and chemical shift perturbation analysis with (15)N-labeled Par17 furthermore confirmed that calmodulin (CaM) interacts in a Ca(2+)-dependent manner with the Par17 N terminus. The reverse experiment with (15)N-labeled Ca(2+)/CaM demonstrated that the N-terminal Par17 segment binds to both CaM lobes simultaneously, indicating that Ca(2+)/CaM undergoes a conformational change to form a binding channel between its two lobes, apparently similar to the structure of the CaM-smMLCK(796-815) complex. In vitro tubulin polymerization assays furthermore showed that Ca(2+)/CaM completely suppresses Par17-promoted microtubule assembly. The results imply that Ca(2+)/CaM binding to the N-terminal segment of Par17 causes steric hindrance of the Par17 active site, thus interfering with the Par17/tubulin interaction. This Ca(2+)/CaM-mediated control of Par17-assisted microtubule assembly may provide a mechanism that couples Ca(2+) signaling with microtubule function.

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.

CD4(+) FoxP3(+) regulatory T cells suppress fatal T helper 2 cell immunity during pulmonary fungal infection.

The opportunistic fungal pathogen Cryptococcus neoformans causes lung inflammation and fatal meningitis in immunocompromised patients. Regulatory T (Treg) cells play an important role in controlling immunity and homeostasis. However, their functional role during fungal infection is largely unknown. In this study, we investigated the role of Treg cells during experimental murine pulmonary C. neoformans infection. We show that the number of CD4(+) FoxP3(+) Treg cells in the lung increases significantly within the first 4 weeks after intranasal infection of BALB/c wild-type mice. To define the function of Treg cells we used DEREG mice allowing selective depletion of CD4(+) FoxP3(+) Treg cells by application of diphtheria toxin. In Treg cell-depleted mice, stronger pulmonary allergic inflammation with enhanced mucus production and pronounced eosinophilia, increased IgE production, and elevated fungal lung burden were found. This was accompanied by higher frequencies of GATA-3(+) T helper (Th) 2 cells with elevated capacity to produce interleukin (IL)-4, IL-5, and IL-13. In contrast, only a mild increase in the Th1-associated immune response unrelated to the fungal infection was observed. In conclusion, the data demonstrate that during fungal infection pulmonary Treg cells are induced and preferentially suppress Th2 cells thereby mediating enhanced fungal control.

Synergistic effects of IL-4 and TNFα on the induction of B7-H1 in renal cell carcinoma cells inhibiting allogeneic T cell proliferation.

BACKGROUND: The importance of B7-H molecules for the T cell/tumor communication and its impact on renal cell carcinoma (RCC) progression and prognosis has been recently described. Cytokine treatment of RCC has earlier been shown to be beneficial in preclinical settings, but its clinical implementation has not proven to be as effective. This might be partially explained by the yet incomplete picture of cellular alterations in tumor cells upon cytokine treatment investigated in detail in this study. METHODS: RCC tumor cell lines were treated with different cytokines alone or in combination. The constitutive and/or cytokine-induced expression of cytokine receptors signaling components and B7-H molecules in RCC cells were analysed by qPCR and flow cytometry. A mcherry reporter gene construct containing B7-H1 promoter was cloned and its activity was determined upon transfection in cytokine-stimulated cells. Cytokine pretreated tumor cells were co-cultured with allogeneic CD8+ T cells from healthy donors and T cell proliferation as well as cytokine secretion was determined. RESULTS: A heterogeneous, but constitutive B7-H1,-H2,-H3 and H4 expression was found on human RCC cell lines. IL-4 and TNFα treatment led to strong synergistic induction of B7-H1 in RCC cells, whereas B7-H2 was only increased by TNFα. In contrast, B7-H3 and B7-H4 expression were not altered by these cytokines. Treatment of RCC cells with TNFα and IL-4 was accompanied by an activation of signaling molecules like NF-κB, IκB and STAT6. The cytokine-mediated up-regulation of B7-H1 was due to transcriptional control as determined by an increased B7-H1 promoter activity in the presence of IL-4 and TNFα. Despite HLA class I and LFA-1 were also increased, the cytokine-mediated up-regulation of B7-H1 was more pronounced and caused an inhibition of allospecifc CD8+ T cell proliferation. CONCLUSION: Thus, IL-4 and TNFα, which could be released by immune cells of the tumor microenvironment, are able to control the B7-H1 expression in RCC thereby altering T cell responses. These data are of importance for understanding the complex interplay of tumor cells with immune cells orchestrated by a number of different soluble and membrane bound mediators and for the implementation of check point antibodies directed against B7-H1.

Identification of T helper (Th)1- and Th2-associated antigens of Cryptococcus neoformans in a murine model of pulmonary infection.

Cryptococcosis, caused by Cryptococcus neoformans, has been demonstrated to be controlled by T helper (Th)1 cells while Th2 cells are associated with fungal growth and dissemination. Although cryptococcal immunoreactive protein antigens were previously identified, their association with Th1 or Th2 immune responses was not provided. In mice, Th1-dependent IFN-γ induces the production of IgG2a, whereas the Th2 cytokine IL-4 stimulates the expression of IgG1 rendering each isotype an indicator of the underlying Th cell response. Therefore, we performed an immunoproteomic study that distinguishes Th1- and Th2-associated antigens by their reactivity with Th1-dependent IgG2a or Th2-dependent IgG1 antibodies in sera from C. neoformans-infected wild-type mice. We additionally analysed sera from Th2-prone IL-12-deficient and Th1-prone IL-4Rα-deficient mice extending the results found in wild-type mice. In total, ten, four, and three protein antigens associated with IgG1, IgG2a, or both isotypes, respectively, were identified. Th2-associated antigens represent promising candidates for development of immunotherapy regimens, whereas Th1-associated antigens may serve as candidates for vaccine development. In conclusion, this study points to intrinsic immunomodulatory effects of fungal antigens on the process of Th cell differentiation based on the identification of cryptococcal protein antigens specifically associated with Th1 or Th2 responses throughout mice of different genotypes.

Ketoacidosis alone does not predispose to mucormycosis by Lichtheimia in a murine pulmonary infection model.

Mucormycosis is a rare fungal infection; however, the number of cases increased during the last decades. The main risk factors are immunosuppression and uncontrolled diabetes mellitus. Although Lichtheimia species represent a common cause of mucormycosis in Europe, virulence and pathogenesis of this genus has not been investigated in detail yet. Using murine pulmonary infection models, we found that immunosuppression is essential for establishment of infection. The disease was characterized by necrosis, angioinvasion, thrombosis, and the lethal course of infection was associated with systemic activation of platelets. Furthermore, dissemination to internal organs was frequently observed. While the virulence potential of individual L. corymbifera and L. ramosa isolates differed, pathogenicity of both species was comparable. Although ketoacidosis promoted Rhizopus infection in mice, it did not predispose mice to infection with Lichtheimia in the absence of additional immunosuppression. This might partially explain the dominance of Rhizopus as cause of mucormycosis in countries with high prevalence of ketoacidotic patients.

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.

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.