Zfp362 potentiates murine colonic inflammation by constraining Treg cell function rather than promoting Th17 cell differentiation.

Mucosal barrier integrity and pathogen clearance is a complex process influenced by both Th17 and Treg cells. Previously, we had described the DNA methylation profile of Th17 cells and identified Zinc finger protein (Zfp)362 to be uniquely demethylated. Here, we generated Zfp362-/- mice to unravel the role of Zfp362 for Th17 cell biology. Zfp362-/- mice appeared clinically normal, showed no phenotypic alterations in the T-cell compartment, and upon colonization with segmented filamentous bacteria, no effect of Zfp362 deficiency on Th17 cell differentiation was observed. By contrast, Zfp362 deletion resulted in increased frequencies of colonic Foxp3+ Treg cells and IL-10+ and RORγt+ Treg cell subsets in mesenteric lymph nodes. Adoptive transfer of naïve CD4+ T cells from Zfp362-/- mice into Rag2-/- mice resulted in a significantly lower weight loss when compared with controls receiving cells from Zfp362+/+ littermates. However, this attenuated weight loss did not correlate with alterations of Th17 cells but instead was associated with an increase of effector Treg cells in mesenteric lymph nodes. Together, these results suggest that Zfp362 plays an important role in promoting colonic inflammation; however, this function is derived from constraining the effector function of Treg cells rather than directly promoting Th17 cell differentiation.

Profiling of epigenetic marker regions in murine ILCs under homeostatic and inflammatory conditions.

Epigenetic modifications such as DNA methylation play an essential role in imprinting specific transcriptional patterns in cells. We performed genome-wide DNA methylation profiling of murine lymph node-derived ILCs, which led to the identification of differentially methylated regions (DMRs) and the definition of epigenetic marker regions in ILCs. Marker regions were located in genes with a described function for ILCs, such as Tbx21, Gata3, or Il23r, but also in genes that have not been related to ILC biology. Methylation levels of the marker regions and expression of the associated genes were strongly correlated, indicating their functional relevance. Comparison with T helper cell methylomes revealed clear lineage differences, despite partial similarities in the methylation of specific ILC marker regions. IL-33-mediated challenge affected methylation of ILC2 epigenetic marker regions in the liver, while remaining relatively stable in the lung. In our study, we identified a set of epigenetic markers that can serve as a tool to study phenotypic and functional properties of ILCs.

Acetyl-CoA-Carboxylase 1-mediated de novo fatty acid synthesis sustains Lgr5+ intestinal stem cell function.

Basic processes of the fatty acid metabolism have an important impact on the function of intestinal epithelial cells (IEC). However, while the role of cellular fatty acid oxidation is well appreciated, it is not clear how de novo fatty acid synthesis (FAS) influences the biology of IECs. We report here that interfering with de novo FAS by deletion of the enzyme Acetyl-CoA-Carboxylase (ACC)1 in IECs results in the loss of epithelial crypt structures and a specific decline in Lgr5+ intestinal epithelial stem cells (ISC). Mechanistically, ACC1-mediated de novo FAS supports the formation of intestinal organoids and the differentiation of complex crypt structures by sustaining the nuclear accumulation of PPARδ/ß-catenin in ISCs. The dependency of ISCs on cellular de novo FAS is tuned by the availability of environmental lipids, as an excess delivery of external fatty acids is sufficient to rescue the defect in crypt formation. Finally, inhibition of ACC1 reduces the formation of tumors in colitis-associated colon cancer, together highlighting the importance of cellular lipogenesis for sustaining ISC function and providing a potential perspective to colon cancer therapy.

Targeting cellular fatty acid synthesis limits T helper and innate lymphoid cell function during intestinal inflammation and infection.

CD4+ T cells contribute critically to a protective immune response during intestinal infections, but have also been implicated in the aggravation of intestinal inflammatory pathology. Previous studies suggested that T helper type (Th)1 and Th17 cells depend on de novo fatty acid (FA) synthesis for their development and effector function. Here, we report that T-cell-specific targeting of the enzyme acetyl-CoA carboxylase 1 (ACC1), a major checkpoint controlling FA synthesis, impaired intestinal Th1 and Th17 responses by limiting CD4+ T-cell expansion and infiltration into the lamina propria in murine models of colitis and infection-associated intestinal inflammation. Importantly, pharmacological inhibition of ACC1 by the natural compound soraphen A mirrored the anti-inflammatory effects of T-cell-specific targeting, but also enhanced susceptibility toward infection with C. rodentium. Further analysis revealed that deletion of ACC1 in RORγt+ innate lymphoid cells (ILC), but not dendritic cells or macrophages, decreased resistance to infection by interfering with IL-22 production and intestinal barrier function. Together, our study suggests pharmacological targeting of ACC1 as an effective approach for metabolic immune modulation of T-cell-driven intestinal inflammatory responses, but also reveals an important role of ACC1-mediated lipogenesis for the function of RORγt+ ILC.

Staphylococcus aureus Alpha-Toxin Limits Type 1 While Fostering Type 3 Immune Responses.

Staphylococcus aureus can cause life-threatening diseases, and hospital- as well as community-associated antibiotic-resistant strains are an emerging global public health problem. Therefore, prophylactic vaccines or immune-based therapies are considered as alternative treatment opportunities. To develop such novel treatment approaches, a better understanding of the bacterial virulence and immune evasion mechanisms and their potential effects on immune-based therapies is essential. One important staphylococcal virulence factor is alpha-toxin, which is able to disrupt the epithelial barrier in order to establish infection. In addition, alpha-toxin has been reported to modulate other cell types including immune cells. Since CD4+ T cell-mediated immunity is required for protection against S. aureus infection, we were interested in the ability of alpha-toxin to directly modulate CD4+ T cells. To address this, murine naïve CD4+ T cells were differentiated in vitro into effector T cell subsets in the presence of alpha-toxin. Interestingly, alpha-toxin induced death of Th1-polarized cells, while cells polarized under Th17 conditions showed a high resistance toward increasing concentrations of this toxin. These effects could neither be explained by differential expression of the cellular alpha-toxin receptor ADAM10 nor by differential activation of caspases, but might result from an increased susceptibility of Th1 cells toward Ca2+-mediated activation-induced cell death. In accordance with the in vitro findings, an alpha-toxin-dependent decrease of Th1 and concomitant increase of Th17 cells was observed in vivo during S. aureus bacteremia. Interestingly, corresponding subsets of innate lymphoid cells and γδ T cells were similarly affected, suggesting a more general effect of alpha-toxin on the modulation of type 1 and type 3 immune responses. In conclusion, we have identified a novel alpha-toxin-dependent immunomodulatory strategy of S. aureus, which can directly act on CD4+ T cells and might be exploited for the development of novel immune-based therapeutic approaches to treat infections with antibiotic-resistant S. aureus strains.

Epithelium-specific MyD88 signaling, but not DCs or macrophages, control acute intestinal infection with Clostridium difficile.

Infection with Clostridium difficile is one of the major causes of health care acquired diarrhea and colitis. Signaling though MyD88 downstream of TLRs is critical for initiating the early protective host response in mouse models of C. difficile infection (CDI). In the intestine, MyD88 is expressed in various tissues and cell types, such as the intestinal epithelium and mononuclear phagocytes (MNP), including DC or macrophages. Using a genetic gain-of-function system, we demonstrate here that restricting functional MyD88 signaling to the intestinal epithelium, but also to MNPs is sufficient to protect mice during acute CDI by upregulation of the intestinal barrier function and recruitment of neutrophils. Nevertheless, we also show that mice depleted for CD11c-expressing MNPs in the intestine display no major defects in mounting an effective inflammatory response, indicating that the absence of these cells is irrelevant for inducing host protection during acute infection. Together, our results highlight the importance of epithelial-specific MyD88 signaling and demonstrate that although functional MyD88 signaling in DC and macrophages alone is sufficient to correct the phenotype of MyD88-deficiency, these cells do not seem to be essential for host protection in MyD88-sufficient animals during acute infection with C. difficile.

MyD88 signaling in dendritic cells and the intestinal epithelium controls immunity against intestinal infection with C. rodentium.

MyD88-mediated signaling downstream of Toll-like receptors and the IL-1 receptor family is critically involved in the induction of protective host responses upon infections. Although it is known that MyD88-deficient mice are highly susceptible to a wide range of bacterial infections, the cell type-specific contribution of MyD88 in protecting the host against intestinal bacterial infection is only poorly understood. In order to investigate the importance of MyD88 in specific immune and nonimmune cell types during intestinal infection, we employed a novel murine knock-in model for MyD88 that enables the cell type-specific reactivation of functional MyD88 expression in otherwise MyD88-deficient mice. We report here that functional MyD88 signaling in CD11c+ cells was sufficient to activate intestinal dendritic cells (DC) and to induce the early group 3 innate lymphoid cell (ILC3) response as well as the development of colonic Th17/Th1 cells in response to infection with the intestinal pathogen C. rodentium. In contrast, restricting MyD88 signaling to several other cell types, including macrophages (MO), T cells or ILC3 did not induce efficient intestinal immune responses upon infection. However, we observed that the functional expression of MyD88 in intestinal epithelial cells (IEC) also partially protected the mice during intestinal infection, which was associated with enhanced epithelial barrier integrity and increased expression of the antimicrobial peptide RegIIIγ and the acute phase protein SAA1 by epithelial cells. Together, our data suggest that MyD88 signaling in DC and IEC is both essential and sufficient to induce a full spectrum of host responses upon intestinal infection with C. rodentium.

Rapid rebound of the Treg compartment in DEREG mice limits the impact of Treg depletion on mycobacterial burden, but prevents autoimmunity.

The development of an effective vaccine against tuberculosis (Tb) represents one of the major medical challenges of this century. Mycobacterium bovis Bacille Calmette-Guerin (BCG), the only vaccine available at present, is mostly effective at preventing disseminated Tb in children, but shows variable protection against pulmonary Tb, the most common form in adults. The reasons for this poor efficacy are not completely understood, but there is evidence that T regulatory cells (Tregs) might be involved. Similarly, Tregs have been associated with the immunosuppression observed in patients infected with Tb and are therefore believed to play a role in pathogen persistence. Thus, Treg depletion has been postulated as a novel strategy to potentiate M. bovis BCG vaccination on one side, while on the other, employed as a therapeutic approach during chronic Tb infection. Yet since Tregs are critically involved in controlling autoimmune inflammation, elimination of Tregs may therefore also incur the danger of an excessive inflammatory immune response. Thus, understanding the dynamics and function of Tregs during mycobacterial infection is crucial to evaluate the potential of Treg depletion as a medical option. To address this, we depleted Tregs after infection with M. bovis BCG or Mycobacterium tuberculosis (Mtb) using DEREG mice, which express the diphtheria toxin (DT) receptor under the control of the FoxP3 locus, thereby allowing the selective depletion of FoxP3+ Tregs. Our results show that after depletion, the Treg niche is rapidly refilled by a population of DT-insensitive Tregs (diTregs) and bacterial load remains unchanged. On the contrary, impaired rebound of Tregs in DEREG × FoxP3GFP mice improves pathogen burden, but is accompanied by detrimental autoimmune inflammation. Therefore, our study provides the proof-of-principle that, although a high degree of Treg depletion may contribute to the control of mycobacterial infection, it carries the risk of autoimmunity.

Dendritic cells coordinate innate immunity via MyD88 signaling to control Listeria monocytogenes infection.

Listeria monocytogenes (LM), a facultative intracellular Gram-positive pathogen, can cause life-threatening infections in humans. In mice, the signaling cascade downstream of the myeloid differentiation factor 88 (MyD88) is essential for proper innate immune activation against LM, as MyD88-deficient mice succumb early to infection. Here, we show that MyD88 signaling in dendritic cells (DCs) is sufficient to mediate the protective innate response, including the production of proinflammatory cytokines, neutrophil infiltration, bacterial clearance, and full protection from lethal infection. We also demonstrate that MyD88 signaling by DCs controls the infection rates of CD8α(+) cDCs and thus limits the spread of LM to the T cell areas. Furthermore, in mice expressing MyD88 in DCs, inflammatory monocytes, which are required for bacterial clearance, are activated independently of intrinsic MyD88 signaling. In conclusion, CD11c(+) conventional DCs critically integrate pathogen-derived signals via MyD88 signaling during early infection with LM in vivo.

MyD88 signalling in myeloid cells is sufficient to prevent chronic mycobacterial infection.

Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis that is responsible for almost 1.5 million deaths per year. Sensing of mycobacteria by the host's immune system relies on different families of receptors present on innate immune cells. Amongst them, several members of the TLR family are involved in the activation of immune cells by mycobacteria, yet the in vivo contribution of individual TLRs to the protective immune response remains controversial. On the contrary, MyD88, the adaptor molecule for most TLRs, plays a non-redundant role in the protection against tuberculosis and mice with a complete germline deletion of MyD88 succumb very early to infection. MyD88 is expressed in both immune and non-immune cells, but it is not clear whether control of mycobacteria requires ubiquitous or cell-type specific MyD88 expression. Therefore, using novel conditional switch-on mouse models, we aimed to investigate the importance of MyD88 signalling in DCs and macrophages for the induction of protective effector mechanisms against mycobacterial infection. We conclude that specific reactivation of MyD88 signalling in CD11c- or lysozyme M-expressing myeloid cells during Mycobacterium bovis Bacille Calmette-Guerin infection is sufficient to restore systemic and local inflammatory cytokine production and to control pathogen burden.

Protein kinase C-lambda knockout in embryonic stem cells and adipocytes impairs insulin-stimulated glucose transport.

Atypical protein kinase C (aPKC) isoforms have been suggested to mediate insulin effects on glucose transport in adipocytes and other cells. To more rigorously test this hypothesis, we generated mouse embryonic stem (ES) cells and ES-derived adipocytes in which both aPKC-lambda alleles were knocked out by recombinant methods. Insulin activated PKC-lambda and stimulated glucose transport in wild-type (WT) PKC-lambda(+/+), but not in knockout PKC-lambda(-/-), ES cells. However, insulin-stimulated glucose transport was rescued by expression of WT PKC-lambda in PKC-lambda(-/-) ES cells. Surprisingly, insulin-induced increases in both PKC-lambda activity and glucose transport were dependent on activation of proline-rich tyrosine protein kinase 2, the ERK pathway, and phospholipase D (PLD) but were independent of phosphatidylinositol 3-kinase (PI3K) in PKC-lambda(+/+) ES cells. Interestingly, this dependency was completely reversed after differentiation of ES cells to adipocytes, i.e. insulin effects on PKC-lambda and glucose transport were dependent on PI3K, rather than proline-rich tyrosine protein kinase 2/ERK/PLD. As in ES cells, insulin effects on glucose transport were absent in PKC-lambda(-/-) adipocytes but were rescued by expression of WT PKC-lambda in these adipocytes. Our findings suggest that insulin activates aPKCs and glucose transport in ES cells by a newly recognized PI3K-independent ERK/PLD-dependent pathway and provide a compelling line of evidence suggesting that aPKCs are required for insulin-stimulated glucose transport, regardless of whether aPKCs are activated by PI3K-dependent or PI3K-independent mechanisms.

Different regulation of Purkinje cell dendritic development in cerebellar slice cultures by protein kinase Calpha and -beta.

Activity of protein kinase C (PKC), and in particular the PKCgamma-isoform, has been shown to strongly affect and regulate Purkinje cell dendritic development, suggesting an important role for PKC in activity-dependent Purkinje cell maturation. In this study we have analyzed the role of two additional Ca(2+)-dependent PKC isoforms, PKCalpha and -beta, in Purkinje cell survival and dendritic morphology in slice cultures using mice deficient in the respective enzymes. Pharmacological PKC activation strongly reduced basal Purkinje cell dendritic growth in wild-type mice whereas PKC inhibition promoted branching. Purkinje cells from mice deficient in PKCbeta, which is expressed in two splice forms by granule but not Purkinje cells, did not yield measurable morphological differences compared to respective wild-type cells under either experimental condition. In contrast, Purkinje cell dendrites in cultures from PKCalpha-deficient mice were clearly protected from the negative effects on dendritic growth of pharmacological PKC activation and showed an increased branching response to PKC inhibition as compared to wild-type cells. Together with our previous work on the role of PKCgamma, these data support a model predicting that normal Purkinje cell dendritic growth is mainly regulated by the PKCgamma-isoform, which is highly activated by developmental processes. The PKCalpha isoform in this model forms a reserve pool, which only becomes activated upon strong stimulation and then contributes to the limitation of dendritic growth. The PKCbeta isoform appears to not be involved in the signaling cascades regulating Purkinje cell dendritic maturation in cerebellar slice cultures.

Robust circadian rhythmicity of Per1 and Per2 mutant mice in constant light, and dynamics of Per1 and Per2 gene expression under long and short photoperiods.

The Per1 and Per2 genes are components of the mammalian circadian clock. Mutations in these genes alter phase resetting in response to a nocturnal light pulse, and Per2 mutant mice are known to become arrhythmic in constant darkness. We show that under constant light conditions, Per2 mutant mice exhibit robust activity rhythms as well as body temperature rhythms with a period length that is less than 24 h. In Per1 mutants, the period length of both activity and body temperature rhythms is longer than 24 h in constant light. Per1 mutants prolong their period length (tao) when illuminance is increased, whereas Per2 mutants shorten their endogenous period. Additionally, the authors show that the circadian pattern of Per1 and Per2 gene expression in mice is modified under different photoperiods and that there is a mutual influence of these genes on their timing of expression. We propose that, in mice, the phase relationship between Per1 and Per2 gene expression might be critical for transducing day length information to the organism. Per1 could be part of a morning oscillator tracking dawn, and Per2 could be part of an evening oscillator tracking dusk.