Alexander Visekruna

Heterogeneity in the Differentiation and Function of CD8+ T Cells

It is well established that CD8+ T cells constitute an important branch of adaptive immunity contributing to clearance of intracellular pathogens and providing long-term protection. These functions are mostly fulfilled by the best characterized subpopulation of CD8+ T cells, the cytotoxic T lymphocytes (also called Tc1 cells), owing to their ability to kill infected cells and to secrete cytokines such as interferon-γ and tumor necrosis factor-α. However, there is growing evidence for alternative CD8+ T cell fates influencing CD4+ T-cell-mediated responses in the context of allergy, autoimmunity and infections. Thus, like subpopulations of CD4+ T cells, also CD8+ T cells under particular conditions acquire the expression of interleukin (IL)-4, IL-5, IL-9, IL-13, IL-17 or suppressive activity and thereby influence immune responses. The process of CD8+ T-cell differentiation is dictated by antigen strength, co-stimulatory molecules and cytokines. These environmental cues induce transcription factors further specifying CD8+ T-cell decision into Tc1, Tc2, Tc9, Tc17 or CD8+ T regulatory fate. Here, we discuss our current understanding about functional diversity of effector CD8+ T cells and contribution of transcription factors to this process.

Tc9 cells, a new subset of CD8(+) T cells, support Th2-mediated airway inflammation.

Similar to T‐helper (Th) cells, CD8+ T cells also differentiate into distinct subpopulations. However, the existence of IL‐9‐producing CD8+ T (Tc9) cells has not been elucidated so far. We show that murine CD8+ T cells activated in the presence of IL‐4 plus TGF‐β develop into transient IL‐9 producers characterized by specific IFN‐γ and IL‐10 expression patterns as well as by low cytotoxic function along with diminished expression of the CTL‐associated transcription factors T‐bet and Eomesodermin. Similarly to the CD4+ counterpart, Tc9 cells required for their differentiation STAT6 and IRF4. Tc9 cells deficient for these master regulators displayed increased levels of Foxp3 that in turn suppressed IL‐9 production. In an allergic airway disease model, Tc9 cells promoted the onset of airway inflammation, mediated by subpathogenic numbers of Th2 cells. This support was specific for Tc9 cells because CTLs failed to exert this function. We detected increased Tc9 frequency in the periphery in mice and humans with atopic dermatitis, a Th2‐associated skin disease that often precedes asthma. Thus, our data point to the existence of Tc9 cells and to their supportive function in Th2‐dependent airway inflammation, suggesting that these cells might be a therapeutic target in allergic disorders.

rKLO8, a Novel Leishmania donovani – Derived Recombinant Immunodominant Protein for Sensitive Detection of Visceral Leishmaniasis in Sudan

Background For effective control of visceral leishmaniasis (VL) in East Africa, new rapid diagnostic tests are required to replace current tests with low sensitivity. The aim of this study is to improve diagnosis of VL in East Africa by testing a new antigen from an autochthonous L. donovani strain in Sudan. Methodology and Principle Findings We cloned, expressed and purified a novel recombinant protein antigen of L. donovani from Sudan, designated rKLO8, that contains putative conserved domains with significant similarity to the immunodominant kinesin proteins of Leishmania. rKLO8 exhibited 93% and 88% amino acid identity with cloned kinesin proteins of L. infantum (synonymous L. chagasi) (K39) and L. donovani (KE16), respectively. We evaluated the diagnostic efficiency of the recombinant protein in ELISA for specific detection of VL patients from Sudan. Data were compared with a rK39 ELISA and two commercial kits, the rK39 strip test and the direct agglutination test (DAT). Of 106 parasitologically confirmed VL sera, 104 (98.1%) were tested positive by rKLO8 as compared to 102 (96.2%) by rK39. Importantly, the patients sera showed increased reactivity with rKLO8 than rK39. Specificity was 96.1% and 94.8% for rKLO8- and rK39 ELISAs, respectively. DAT showed 100% specificity and 94.3% sensitivity while rK39 strip test performed with 81.1% sensitivity and 98.7% specificity. Conclusion The increased reactivity of Sudanese VL sera with the rKLO8 makes this antigen a potential candidate for diagnosis of visceral leishmaniasis in Sudan. However, the suitability at the field level will depend on its performance in a rapid test format.

The role of NF-κB activation during protection against Leishmania infection.

Members of the nuclear factor-κB (NF-κB) family of transcription factors regulate a variety of molecules involved in host defense against pathogens. A prominent role of NF-κB in innate and adoptive immunity is based on the regulation of inducible transcription of various genes whose products are essential components of the immune response such as cytokines, chemokines, and adhesion molecules. Since the discovery of the five members of the NF-κB transcription factor family, RelA, c-Rel, RelB, p50 and p52, considerable progress has been made toward better understanding how the different NF-κB homo- and heterodimers regulate such distinct subsets of target genes. All of the NF-κB molecules are activated by various infectious stimuli; however, there are still open questions related to the selective functions of individual NF-κB family members during a coordinated immune response to infection. Diverse parasites such as Toxoplasma gondii, Leishmania donovani, Leishmania major, and Trichuris muris have been reported to activate NF-κB signaling cascades, and a number of distinct parasite-derived molecules may actively interfere with the pathways that lead to NF-κB activation. In this review, we provide an overview on the role of NF-κB activation in leishmaniasis and discuss how individual NF-κB family members might perform their distinct and non-overlapping functions in the regulation of protective immunity to Leishmania infection.

Prevention of colitis-associated cancer by selective targeting of immunoproteasome subunit LMP7

Chronic inflammation is a well-known risk factor in development of intestinal tumorigenesis, although the exact mechanisms underlying development of colitis-associated cancer (CAC) still remain obscure. The activity and function of immunoproteasome has been extensively analyzed in the context of inflammation and infectious diseases. Here, we show that the proteasomal immunosubunit LMP7 plays an essential role in development of CAC. Mice devoid of LMP7 were resistant to chronic inflammation and formation of neoplasia, and developed virtually no tumors after AOM/DSS treatment. Our data reveal that LMP7 deficiency resulted in reduced expression of pro-tumorigenic chemokines CXCL1, CXCL2 and CXCL3 as well as adhesion molecule VCAM-1. As a consequence, an impaired recruitment and activity of tumor-infiltrating leukocytes resulting in decreased secretion of cytokines IL-6 and TNF-α was observed. Further, the deletion or pharmacological inhibition of LMP7 and consequent blockade of NF-κB abrogated the production of IL-17A, which possesses a strong carcinogenic activity in the gut. Moreover, in vivo administration of the selective LMP7 inhibitor ONX-0914 led to a marked reduction of tumor numbers in wild-type (WT) mice. Collectively, we identified the immunoproteasome as a crucial mediator of inflammation-driven neoplasia highlighting a novel potential therapeutic approach to limit colonic tumorigenesis.Chronic inflammation is a well-known risk factor in development of intestinal tumorigenesis, although the exact mechanisms underlying development of colitis-associated cancer (CAC) still remain obscure. The activity and function of immunoproteasome has been extensively analyzed in the context of inflammation and infectious diseases. Here, we show that the proteasomal immunosubunit LMP7 plays an essential role in development of CAC. Mice devoid of LMP7 were resistant to chronic inflammation and formation of neoplasia, and developed virtually no tumors after AOM/DSS treatment. Our data reveal that LMP7 deficiency resulted in reduced expression of pro-tumorigenic chemokines CXCL1, CXCL2 and CXCL3 as well as adhesion molecule VCAM-1. As a consequence, an impaired recruitment and activity of tumor-infiltrating leukocytes resulting in decreased secretion of cytokines IL-6 and TNF-α was observed. Further, the deletion or pharmacological inhibition of LMP7 and consequent blockade of NF-κB abrogated the production of IL-17A, which possesses a strong carcinogenic activity in the gut. Moreover, in vivo administration of the selective LMP7 inhibitor ONX-0914 led to a marked reduction of tumor numbers in wild-type (WT) mice. Collectively, we identified the immunoproteasome as a crucial mediator of inflammation-driven neoplasia highlighting a novel potential therapeutic approach to limit colonic tumorigenesis.

The Microbial Metabolite Butyrate Induces Expression of Th1-Associated Factors in CD4+ T Cells

Short-chain fatty acids (SCFAs), which are generated by the bacterial fermentation of dietary fibers, promote expansion of regulatory T cells (Tregs). Potential therapeutic value of SCFAs has been recently highlighted in the experimental models of T cell-mediated autoimmunity and allergic inflammation. These studies suggest that physiological intestinal concentrations of SCFAs within the millimolar range are crucial for dampening inflammation-mediated processes. Here, we describe opposing effects of SCFAs on T cell-mediated immune responses. In accordance with published data, lower butyrate concentrations facilitated differentiation of Tregs in vitro and in vivo under steady-state conditions. In contrast, higher concentrations of butyrate induced expression of the transcription factor T-bet in all investigated T cell subsets resulting in IFN-γ-producing Tregs or conventional T cells. This effect was mediated by the inhibition of histone deacetylase activity and was independent of SCFA-receptors FFA2 and FFA3 as well as of Na+-coupled SCFA transporter Slc5a8. Importantly, while butyrate was not able to induce the generation of Tregs in the absence of TGF-β1, the expression of T-bet and IFN-γ was triggered upon stimulation of CD4+ T cells with this SCFA alone. Moreover, the treatment of germ-free mice with butyrate enhanced the expression of T-bet and IFN-γ during acute colitis. Our data reveal that, depending on its concentration and immunological milieu, butyrate may exert either beneficial or detrimental effects on the mucosal immune system.

NALP expression in Paneth cells provides a novel track in IBD signaling.

PurposePaneth cells are part of the innate mucosal immunity of the gut with possible regulatory function. This study intends to identify the gene expression pattern of the orthotopic and metaplastic Paneth cells, searching for differences between metaplastic occurrence between Crohns disease and ulcerative colitis.MethodsPaneth cells were collected in RNAse-free conditions via micro dissection. RNA isolation and super amplification was followed by microarray analysis of whole genome expression activity of the orthotopic and metaplastic Paneth cells. Immunohistology of β-catenin and Frizzled-5 receptor was performed.ResultsHistological analysis showed no morphological or secretory change (Frizzled-5 receptor and β-catenin) in orthotopic and metaplastic Paneth cells. Microarray analysis indicated an increased, but not mutant activation of Wnt/β-catenin signaling and firstly showed expression of NALP 1, 7, 8 and 11 in metaplastic Paneth cells.ConclusionsPaneth cells might play a NALP-mediated role in the pathogenesis of IBD.

Transcription factor c-Rel is indispensable for generation of thymic but not of peripheral Foxp3+ regulatory T cells

The transcription factor c-Rel has been shown to be crucial for development of regulatory T cells (Tregs). Recent studies have reported that the expression of transcription factor Helios in Foxp3+ Tregs correlates with thymic origin of these cells (tTregs). Notably, we found that only the Helios+Foxp3+ Treg cell population was substantially reduced in c-Rel deficient mice. In contrast to a defective tTreg development, we observed an expansion of mucosal Tregs during the induction of acute colitis in rel−/− mice. Furthermore, we found a preferential accumulation of Helios−Foxp3+ Tregs in aged c-Rel deficient mice. This unexpected finding, together with the observation that naïve CD4+ T cells convert into Tregs in vitro in the absence of c-Rel and presence of IL-2, provide an evidence that extra-thymic generation of induced and peripheral Tregs (iTregs and pTregs) is independent of c-Rel. Moreover, the treatment with IL-2/anti-IL-2 mAb (JES6-1) resulted in a widespread increase of Helios+Foxp3+ Tregs in both wild-type (WT) and rel−/− mice. These data suggest that exogenous IL-2 administration compensates for defective IL-2 production and reduced tTreg numbers in c-Rel deficient mice. Our findings reveal that c-Rel is essential for the generation of tTregs but not for that of pTregs and iTregs.The transcription factor c-Rel has been shown to be crucial for development of regulatory T cells (Tregs). Recent studies have reported that the expression of transcription factor Helios in Foxp3+ Tregs correlates with thymic origin of these cells (tTregs). Notably, we found that only the Helios+Foxp3+ Treg cell population was substantially reduced in c-Rel deficient mice. In contrast to a defective tTreg development, we observed an expansion of mucosal Tregs during the induction of acute colitis in rel-/- mice. Furthermore, we found a preferential accumulation of Helios-Foxp3+ Tregs in aged c-Rel deficient mice. This unexpected finding, together with the observation that naïve CD4+ T cells convert into Tregs in vitro in the absence of c-Rel and presence of IL-2, provide an evidence that extra-thymic generation of induced and peripheral Tregs (iTregs and pTregs) is independent of c-Rel. Moreover, the treatment with IL-2/anti-IL-2 mAb (JES6-1) resulted in a widespread increase of Helios+Foxp3+ Tregs in both wild-type (WT) and rel-/- mice. These data suggest that exogenous IL-2 administration compensates for defective IL-2 production and reduced tTreg numbers in c-Rel deficient mice. Our findings reveal that c-Rel is essential for the generation of tTregs but not for that of pTregs and iTregs.

Chromatin Binding of c-REL and p65 Is Not Limiting for Macrophage IL12B Transcription During Immediate Suppression by Ovarian Carcinoma Ascites

Tumors frequently exploit homeostatic mechanisms that suppress expression of IL-12, a central mediator of inflammatory and anti-tumor responses. The p40 subunit of the IL-12 heterodimer, encoded by IL12B, is limiting for these functions. Ovarian carcinoma patients frequently produce ascites which exerts immunosuppression by means of soluble factors. The NFκB pathway is necessary for transcription of IL12B, which is not expressed in macrophages freshly isolated from ascites. This raises the possibility that ascites prevents IL12B expression by perturbing NFκB binding to chromatin. Here, we show that ascites-mediated suppression of IL12B induction by LPS plus IFNγ in primary human macrophages is rapid, and that suppression can be reversible after ascites withdrawal. Nuclear translocation of the NFκB transcription factors c-REL and p65 was strongly reduced by ascites. Surprisingly, however, their binding to the IL12B locus and to CXCL10, a second NFκB target gene, was unaltered, and the induction of CXCL10 transcription was not suppressed by ascites. These findings indicate that, despite its reduced nuclear translocation, NFκB function is not generally impaired by ascites, suggesting that ascites-borne signals target additional pathways to suppress IL12B induction. Consistent with these data, IL-10, a clinically relevant constituent of ascites and negative regulator of NFκB translocation, only partially recapitulated IL12B suppression by ascites. Finally, restoration of a defective IL-12 response by appropriate culture conditions was observed only in macrophages from a subset of donors, which may have important implications for the understanding of patient-specific immune responses.

IL-17 and TNF-α Are Key Mediators of Moraxella catarrhalis Triggered Exacerbation of Allergic Airway Inflammation

Alterations of the airway microbiome are often associated with pulmonary diseases. For example, detection of the bacterial pathogen Moraxella catarrhalis in the upper airways is linked with an increased risk to develop or exacerbate asthma. However, the mechanisms by which M. catarrhalis augments allergic airway inflammation (AAI) remain unclear. We here characterized the cellular and soluble mediators of M. catarrhalis triggered excacerbation of AAI in wt and IL-17 deficient as well as in animals treated with TNF-α and IL-6 neutralizing antibodies. We compared the type of inflammatory response in M. catarrhalis infected, house dust mite (HDM)-allergic and animals infected with M. catarrhalis at different time points of HDM sensitization. We found that airway infection of mice with M. catarrhalis triggers a strong inflammatory response with massive neutrophilic infiltrates, high amounts of IL-6 and TNF-α and moderate levels of CD4+ T-cell-derived IFN-γ and IL-17. If bacterial infection occurred during HDM allergen sensitization, the allergic airway response was exacerbated, particularly by the expansion of Th17u2009cells and increased TNF-α levels. Neutralization of IL-17 or TNF-α but not IL-6 resulted in accelerated clearance of M. catarrhalis and effectively prevented infection-induced exacerbation of AAI. Taken together, our data demonstrate an essential role for TNF-α and IL-17 in infection-triggered exacerbation of AAI.