An Alternative Pathway of Imiquimod-Induced Psoriasis-Like Skin Inflammation in the Absence of Interleukin-17 Receptor A Signaling

Topical application of imiquimod (IMQ) on the skin of mice induces inflammation with common features found in psoriatic skin. Recently, it was postulated that IL-17 has an important role both in psoriasis and in the IMQ model. To further investigate the impact of IL-17RA signaling in psoriasis, we generated IL-17 receptor A (IL-17RA)–deficient mice (IL-17RAdel) and challenged these mice with IMQ. Interestingly, the disease was only partially reduced and delayed but not abolished when compared with controls. In the absence of IL-17RA, we found persisting signs of inflammation such as neutrophil and macrophage infiltration within the skin. Surprisingly, already in the naive state, the skin of IL-17RAdel mice contained significantly elevated numbers of Th17- and IL-17-producing γδ T cells, assuming that IL-17RA signaling regulates the population size of Th17 and γδ T cells. Upon IMQ treatment of IL-17RAdel mice, these cells secreted elevated amounts of tumor necrosis factor-α, IL-6, and IL-22, accompanied by increased levels of the chemokine CXCL2, suggesting an alternative pathway of neutrophil and macrophage skin infiltration. Hence, our findings have major implications in the potential long-term treatment of psoriasis by IL-17-targeting drugs

CRIS—A Novel cAMP-Binding Protein Controlling Spermiogenesis and the Development of Flagellar Bending

The second messengers cAMP and cGMP activate their target proteins by binding to a conserved cyclic nucleotide-binding domain (CNBD). Here, we identify and characterize an entirely novel CNBD-containing protein called CRIS (cyclic nucleotide receptor involved in sperm function) that is unrelated to any of the other members of this protein family. CRIS is exclusively expressed in sperm precursor cells. Cris-deficient male mice are either infertile due to a lack of sperm resulting from spermatogenic arrest, or subfertile due to impaired sperm motility. The motility defect is caused by altered Ca(2+) regulation of flagellar beat asymmetry, leading to a beating pattern that is reminiscent of sperm hyperactivation. Our results suggest that CRIS interacts during spermiogenesis with Ca(2+)-regulated proteins that--in mature sperm--are involved in flagellar bending

Instruction of haematopoietic lineage choices, evolution of transcriptional landscapes and cancer stem cell hierarchies derived from an AML1-ETO mouse model.

The t(8;21) chromosomal translocation activates aberrant expression of the AML1-ETO (AE) fusion protein and is commonly associated with core binding factor acute myeloid leukaemia (CBF AML). Combining a conditional mouse model that closely resembles the slow evolution and the mosaic AE expression pattern of human t(8;21) CBF AML with global transcriptome sequencing, we find that disease progression was characterized by two principal pathogenic mechanisms. Initially, AE expression modified the lineage potential of haematopoietic stem cells (HSCs), resulting in the selective expansion of the myeloid compartment at the expense of normal erythro- and lymphopoiesis. This lineage skewing was followed by a second substantial rewiring of transcriptional networks occurring in the trajectory to manifest leukaemia. We also find that both HSC and lineage-restricted granulocyte macrophage progenitors (GMPs) acquired leukaemic stem cell (LSC) potential being capable of initiating and maintaining the disease. Finally, our data demonstrate that long-term expression of AE induces an indolent myeloproliferative disease (MPD)-like myeloid leukaemia phenotype with complete penetrance and that acute inactivation of AE function is a potential novel therapeutic option

IL-6 regulates neutrophil microabscess formation in IL-17A-driven psoriasiform lesions

The lack of a generally accepted animal model for human psoriasis has hindered progress with respect to understanding the pathogenesis of the disease. Here we present a model in which transgenic IL-17A expression is targeted to the skin in mice, achievable after crossing our IL-17A(ind) allele to the K14-Cre strain. K14-IL-17A(ind/+) mice invariably develop an overt skin inflammation bearing many hallmark characteristics of human psoriasis including dermal infiltration of effector T cells, formation of neutrophil microabscesses, and hyperkeratosis. IL-17A expression in the skin results in upregulated granulopoiesis and migration of IL-6R-expressing neutrophils into the skin. Neutralization of IL-6 signaling efficiently reduces the observed pathogenesis in skin of IL-17A-overexpressing mice, with marked reductions in epidermal neutrophil abscess formation and epidermal thickening. Thus, IL-6 functions downstream of IL-17A to exacerbate neutrophil microabscess development in psoriasiform lesions

Genetic cell ablation reveals clusters of local self-renewing microglia in the mammalian central nervous system

During early embryogenesis, microglia arise from yolk sac progenitors that populate the developing central nervous system (CNS), but how the tissue-resident macrophages are maintained throughout the organism’s lifespan still remains unclear. Here, we describe a system that allows specific, conditional ablation of microglia in adult mice. We found that the microglial compartment was reconstituted within 1 week of depletion. Microglia repopulation relied on CNS-resident cells, independent from bone-marrow-derived precursors. During repopulation, microglia formed clusters of highly proliferative cells that migrated apart once steady state was achieved. Proliferating microglia expressed high amounts of the interleukin-1 receptor (IL-1R), and treatment with an IL-1R antagonist during the repopulation phase impaired microglia proliferation. Hence, microglia have the potential for efficient self-renewal without the contribution of peripheral myeloid cells, and IL-1R signaling participates in this restorative proliferation process

CRIS is a novel target for cAMP.

<p>(<b>A</b>) Sequence comparison of CNBDs from different proteins. Sequence alignment of CNBDs from mCRIS, cyclic nucleotide-gated channels (bCNGA1, rCNGA4), a hyperpolarization activated and cyclic nucleotide-gated channel (mHCN2), a regulatory subunit from PKA (bPKARI-B), the exchange protein directly-activated by cAMP (hEPAC1), the bacterial catabolite activator protein (CAP), and the ELK1 channel from zebrafish (zELK). Amino acids that have been shown to be essential for ligand binding <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003960#pgen.1003960-Cukkemane1" target="_blank">[1]</a> are highlighted with asterisks. The β strand that functions as an intrinsic ligand in the ELK channels is highlighted in grey. Secondary structure elements are indicated below (β sheets: β 1–8, black arrows; α helices: αA–C, PBC, grey boxes). (<b>B</b>) M4T model of the presumed CNBD of mCRIS in the presence of cAMP. (<b>C</b>) Close-up view of the CNBD model of mCRIS indicating important interactions of side chain and backbone atoms with cAMP. (<b>D–G</b>) Analysis of cAMP binding using FRET. (<b>D</b>) Model demonstrating that binding of cAMP changes the conformation of the CNBD resulting in a change in FRET. (<b>E</b>) Representative traces for the change in cerulean (blue) and citrine (yellow) emission during perfusion of cit-mCNBD-cer expressing CHO cells with 3 mM 8-Br-cAMP. Arrow indicates start of perfusion. (<b>F</b>) Average change in FRET (normalized emission ratio cerulean/FRET-citrine) during perfusion of cit-mCNBD-cer expressing cells with 3 mM 8-Br-cAMP (CNBD 8-Br-cAMP), 40 µM NKH477/100 µM IBMX (CNBD NKH/IBMX), 3 mM 8-Br-cGMP (CNBD 8-Br-cGMP), and cit-mCNBD-R288Q-cer expressing cells with 3 mM 8-Br-cAMP (CNBD-RQ 8-Br-cAMP), and 40 µM NKH477/100 µM IBMX (CNBD-RQ NKH/IBMX). Arrow indicates start of perfusion. (<b>G</b>) Average change in FRET after 10 min of perfusion (mean ± s.d., black; 95% confident interval, dotted, grey). N numbers and p values are indicated.</p

CRIS constitutes a new member of the CNBD-containing protein family.

<p>(<b>A</b>) Phylogenetic tree of CNBD-containing proteins. The different families have been labeled with different colors. Bootstrap values are shown as percentages. Scale bar shows amino acid substitution rate for the NJ (neighbor joining) tree. (<b>B</b>) Metazoan phylogeny describing the presence or absence of CRIS in metazoan genomes. The phylogenetic branching pattern was extracted from the Tree of Life project (<a href="http://www.toolweb.org/tree/" target="_blank">http://www.toolweb.org/tree/</a>) as of December 2012. The metazoan lineages known to contain CRIS are indicated by grey boxes, whereas those lineages that are believed to lack CRIS are indicated with white boxes.</p

Expression of putative CRIS interaction partners.

<p>(<b>A</b>) Analysis of protein expression in testis from wild-type (+/+) and <i>Cris</i>-deficient mice (−/−) by immunoblotting. Per lane, 50 µg total testis lysates have been loaded. Antibodies against KIF2A, IFT172, and ABCF2 are described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003960#pgen-1003960-t004" target="_blank">Table 4</a>. Loading control: β-tubulin. (<b>B</b>) Immunohistochemical analysis of IFT172 and KIF2A in mouse testis. Testis sections (+/+: wild-type, −/−: CRIS knockout) have been probed with IFT2- and KIF2A-specific antibodies and a fluorescent secondary antibody (green). DNA was stained with DAPI (blue). Scale bars are indicated. (<b>C</b>) Analysis of protein expression in sperm from wild-type (+/+) and <i>Cris</i>-deficient mice (−/−) by immunoblotting. Per lane, total protein from 5×10⁶ cells has been loaded. Loading control: β-tubulin. (<b>D</b>) Immunocytochemical analysis of IFT172 and KIF2A in mouse sperm. Sperm isolated from the cauda (+/+: wild-type, −/−: CRIS knockout) have been probed with IFT172- and KIF2A-specific antibodies and a fluorescent secondary antibody (green). DNA was stained with DAPI (blue). Scale bars are indicated.</p

CRIS is exclusively expressed in sperm precursor-cells.

<p>(<b>A</b>) Analysis of <i>Cris</i> mRNA expression by Northern blot. Left, mouse multi-tissue; right, mouse reproductive tissue. (<b>B</b>) Analysis of CRIS protein expression by immunoblotting using a CRIS-specific polyclonal antibody. Protein lysates: T, testis (50 µg); G, germ cells (50 µg); S, cauda sperm (100 µg); HA, mCRIS-HA expressing HEK293 cells (15 µg). Loading control: β-tubulin. (<b>C</b>) Identification of mCRIS in testis using mass spectrometry. Testis lysates were separated on a 1D SDS-PAGE, lanes were sliced, and analyzed by mass spectrometry. Unique peptides for mCRIS are indicated in blue. (<b>D</b>) Developmental expression pattern of mCRIS in testis. Proteins from mouse testis (30 µg/lane) have been probed with a CRIS-specific monoclonal antibody. The age of the mice (days after birth) is indicated. Control: mCRIS-HA expressing HEK cells (10 µg/lane); loading control: β-actin. (<b>E</b>) <i>In situ</i> hybridization. Testis sections (+/+: wild-type, −/−: CRIS knockout) have been labeled with a <i>Cris</i>-specific anti-sense probe. Dark staining indicates a positive signal. The corresponding sense probe showed no staining. Scale bars are indicated. (<b>F</b>) Immunohistochemical analysis of CRIS in mouse testis. Testis sections (+/+: wild-type, −/−: CRIS knockout) have been probed with a polyclonal CRIS-specific antibody and a fluorescent secondary antibody (red). DNA was stained with DAPI (blue). The secondary antibody unspecifically labels the interstitial cells in between the tubules (see −/−, right, secondary antibody only). Scale bars are indicated. (<b>G</b>) See (F) Higher magnification; dotted line: <i>lamina propria</i>. (<b>H</b>) Targeting strategy for the generation of <i>Cris</i>-deficient mice. Exons 5–7 (yellow boxes) have been replaced with a neomycin cassette (Neo) flanked by two lox-P elements (red arrow heads). Restriction sites, the corresponding fragment sizes, and the localization of probe 1 are indicated. (<b>I–J</b>) Verification of gene targeting. (<b>I</b>) Southern blot analysis using probe 1. The sizes of the fragments are indicated. (<b>J</b>) Analysis of CRIS expression in germ cells using a polyclonal CRIS-specific antibody (50 µg/lane). Loading control: calnexin.</p

Proteins identified by mass spectrometry.

<p>Proteins identified by mass spectrometry.</p