CD8+ T cells Are Preferentially Activated during Primary Low Dose Leishmania major Infection but Are Completely Dispensable during Secondary Anti-Leishmania Immunity

We previously showed that CD8⁺ T cells are required for optimal primary immunity to low dose Leishmania major infection. However, it is not known whether immunity induced by low dose infection is durable and whether CD8⁺ T cells contribute to secondary immunity following recovery from low dose infection. Here, we compared primary and secondary immunity to low and high dose L. major infections and assessed the influence of infectious dose on the quality and magnitude of secondary anti-Leishmania immunity. In addition, we investigated the contribution of CD8⁺ T cells in secondary anti-Leishmania immunity following recovery from low and high dose infections. We found that the early immune response to low and high dose infections were strikingly different: while low dose infection preferentially induced proliferation and effector cytokine production by CD8⁺ T cells, high dose infection predominantly induced proliferation and cytokine production by CD4⁺ T cells. This differential activation of CD4⁺ and CD8⁺ T cells by high and low dose infections respectively, was imprinted during in vitro and in vivo recall responses in healed mice. Both low and high dose-infected mice displayed strong infection-induced immunity and were protected against secondary L. major challenge. While depletion of CD4⁺ cells in mice that healed low and high dose infections abolished resistance to secondary challenge, depletion of CD8⁺ cells had no effect. Collectively, our results show that although CD8⁺ T cells are preferentially activated and may contribute to optimal primary anti-Leishmania immunity following low dose infection, they are completely dispensable during secondary immunity.     

Aging of Dry Desiccation-Tolerant Pollen Does Not Affect Protein Secondary Structure

Protein secondary structure and membrane phase behavior in aging Typha latifolia pollen were studied by means of Fourier transform infrared microspectroscopy (FTIR). Membranes isolated from fresh pollen occurred mainly in the liquid crystalline phase at room temperature, whereas the membrane fluidity of aged pollen was drastically decreased. This decrease did not result in large-scale irreversible protein aggregation, as was concluded from in situ FTIR assessment of the amide-1 bands. Curve-fitting on the infrared absorbance spectra enabled estimation of the proportion of different classes of protein secondary structure. Membrane proteins had a relatively large amount of [alpha]-helical structure (48%; band at 1658 cm-1), and turn-like structures (at 1637 and 1680 cm-1) were also detected. The secondary protein structure of isolated cytoplasmic proteins resembled that of proteins in whole pollen and was conserved upon drying in the absence of sucrose. The isolated cytoplasmic proteins had a large amount of [alpha]-helical structure (43%), and also [beta]-sheet (at 1637 and 1692 cm-1) and turn structures were detected. Heat-denaturing experiments with intact hydrated pollen showed low (1627 cm-1) and high (1692 cm-1) wave number bands indicating irreversible protein aggregates. The results presented in this paper show that FTIR is an extremely suitable technique to study protein secondary structure in intact plant cells of different hydration levels and developmental stages.     

IDO1 Deficiency Does Not Affect Disease in Mouse Models of Systemic Juvenile Idiopathic Arthritis and Secondary Hemophagocytic Lymphohistiocytosis

Objectives

Indoleamine 2,3-dioxygenase-1 (IDO1) is an immune-modulatory enzyme that catalyzes the degradation of tryptophan (Trp) to kynurenine (Kyn) and is strongly induced by interferon (IFN)-γ. We previously reported highly increased levels of IFN-γ and corresponding IDO activity in patients with hemophagocytic lymphohistiocytosis (HLH), a hyper-inflammatory syndrome. On the other hand, IFN-γ and IDO were low in patients with systemic juvenile idiopathic arthritis (sJIA), an autoinflammatory syndrome. As HLH can occur as a complication of sJIA, the opposing levels of both IFN-γ and IDO are remarkable. In animal models for sJIA and HLH, the role of IFN-γ differs from being protective to pathogenic. In this study, we aimed to unravel the role of IDO1 in the pathogenesis of sJIA and HLH.

Methods

Wild-type and IDO1-knockout (IDO1-KO) mice were used in 3 models of sJIA or HLH: complete Freund’s adjuvant (CFA)-injected mice developed an sJIA-like syndrome and secondary HLH (sHLH) was evoked by either repeated injection of unmethylated CpG oligonucleotide or by primary infection with mouse cytomegalovirus (MCMV). An anti-CD3-induced cytokine release syndrome was used as a non-sJIA/HLH control model.

Results

No differences were found in clinical, laboratory and hematological features of sJIA/HLH between wild-type and IDO1-KO mice. As IDO modulates the immune response via induction of regulatory T cells and inhibition of T cell proliferation, we investigated both features in a T cell-triggered cytokine release syndrome. Again, no differences were observed in serum cytokine levels, percentages of regulatory T cells, nor of proliferating or apoptotic thymocytes and lymph node cells.

Conclusions

Our data demonstrate that IDO1 deficiency does not affect inflammation in sJIA, sHLH and a T cell-triggered cytokine release model. We hypothesize that other tryptophan-catabolizing enzymes like IDO2 and tryptophan 2,3-dioxygenase (TDO) might compensate for the lack of IDO1.     

Acute Central Neuropeptide Y Administration Increases Food Intake but Does Not Affect Hepatic Very Low-Density Lipoprotein (Vldl) Production in Mice

Objective

Central neuropeptide Y (NPY) administration stimulates food intake in rodents. In addition, acute modulation of central NPY signaling increases hepatic production of very low-density lipoprotein (VLDL)-triglyceride (TG) in rats. As hypertriglyceridemia is an important risk factor for atherosclerosis, for which well-established mouse models are available, we set out to validate the effect of NPY on hepatic VLDL-TG production in mice, to ultimately investigate whether NPY, by increasing VLDL production, contributes to the development of atherosclerosis.

Research Design and Methods

Male C57Bl/6J mice received an intracerebroventricular (i.c.v.) cannula into the lateral (LV) or third (3V) ventricle of the brain. One week later, after a 4 h fast, the animals received an intravenous (i.v.) injection of Tran³⁵S (100 µCi) followed by tyloxapol (500 mg/kg body weight; BW), enabling the study of hepatic VLDL-apoB and VLDL-TG production, respectively. Immediately after the i.v. injection of tyloxapol, the animals received either an i.c.v. injection of NPY (0.2 mg/kg BW in artificial cerebrospinal fluid; aCSF), synthetic Y₁ receptor antagonist {"type":"entrez-nucleotide","attrs":{"text":"GR231118","term_id":"239536349","term_text":"GR231118"}}GR231118 (0.5 mg/kg BW in aCSF) or vehicle (aCSF), or an i.v. injection of PYY_3–36 (0.5 mg/kg BW in PBS) or vehicle (PBS).

Results

Administration of NPY into both the LV and 3V increased food intake within one hour after injection (+164%, p<0.001 and +367%, p<0.001, respectively). NPY administration neither in the LV nor in the 3V affected hepatic VLDL-TG or VLDL-apoB production. Likewise, antagonizing central NPY signaling by either PYY_3–36 or {"type":"entrez-nucleotide","attrs":{"text":"GR231118","term_id":"239536349","term_text":"GR231118"}}GR231118 administration did not affect hepatic VLDL production.

Conclusion

In mice, as opposed to rats, acute central administration of NPY increases food intake without affecting hepatic VLDL production. These results are of great significance when extrapolating findings on the central regulation of hepatic VLDL production between species.     

Chemokine-Like Receptor 1 Deficiency Does Not Affect the Development of Insulin Resistance and Nonalcoholic Fatty Liver Disease in Mice

The adipokine chemerin and its receptor, chemokine-like receptor 1 (Cmklr1), are associated with insulin resistance and nonalcoholic fatty liver disease (NAFLD), which covers a broad spectrum of liver diseases, ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). It is possible that chemerin and/or Cmklr1 exert their effects on these disorders through inflammation, but so far the data have been controversial. To gain further insight into this matter, we studied the effect of whole-body Cmklr1 deficiency on insulin resistance and NAFLD. In view of the primary role of macrophages in hepatic inflammation, we also transplanted bone marrow from Cmklr1 knock-out (Cmklr1^-/-) mice and wild type (WT) mice into low-density lipoprotein receptor knock-out (Ldlr^-/-) mice, a mouse model for NASH. All mice were fed a high fat, high cholesterol diet containing 21% fat from milk butter and 0.2% cholesterol for 12 weeks. Insulin resistance was assessed by an oral glucose tolerance test, an insulin tolerance test, and by measurement of plasma glucose and insulin levels. Liver pathology was determined by measuring hepatic inflammation, fibrosis, lipid accumulation and the NAFLD activity score (NAS). Whole-body Cmklr1 deficiency did not affect body weight gain or food intake. In addition, we observed no differences between WT and Cmklr1^-/- mice for hepatic inflammatory and fibrotic gene expression, immune cell infiltration, lipid accumulation or NAS. In line with this, we detected no differences in insulin resistance. In concordance with whole-body Cmklr1 deficiency, the absence of Cmklr1 in bone marrow-derived cells in Ldlr^-/- mice did not affect their insulin resistance or liver pathology. Our results indicate that Cmklr1 is not involved in the pathogenesis of insulin resistance or NAFLD. Thus, we recommend that the associations reported between Cmklr1 and insulin resistance or NAFLD should be interpreted with caution.     

Deficiency of Thrombospondin-4 in Mice Does Not Affect Skeletal Growth or Bone Mass Acquisition,but Causes a Transient Reduction of Articular Cartilage Thickness

Although articular cartilage degeneration represents a major public health problem, the underlying molecular mechanisms are still poorly characterized. We have previously utilized genome-wide expression analysis to identify specific markers of porcine articular cartilage, one of them being Thrombospondin-4 (Thbs4). In the present study we analyzed Thbs4 expression in mice, thereby confirming its predominant expression in articular cartilage, but also identifying expression in other tissues, including bone. To study the role of Thbs4 in skeletal development and integrity we took advantage of a Thbs4-deficient mouse model that was analyzed by undecalcified bone histology. We found that Thbs4-deficient mice do not display phenotypic differences towards wildtype littermates in terms of skeletal growth or bone mass acquisition. Since Thbs4 has previously been found over-expressed in bones of Phex-deficient Hyp mice, we additionally generated Thbs4-deficient Hyp mice, but failed to detect phenotypic differences towards Hyp littermates. With respect to articular cartilage we found that Thbs4-deficient mice display transient thinning of articular cartilage, suggesting a protective role of Thbs4 for joint integrity. Gene expression analysis using porcine primary cells revealed that Thbs4 is not expressed by synovial fibroblasts and that it represents the only member of the Thbs gene family with specific expression in articular, but not in growth plate chondrocytes. In an attempt to identify specific molecular effects of Thbs4 we treated porcine articular chondrocytes with human THBS4 in the absence or presence of conditioned medium from porcine synovial fibroblasts. Here we did not observe a significant influence of THBS4 on proliferation, metabolic activity, apoptosis or gene expression, suggesting that it does not act as a signaling molecule. Taken together, our data demonstrate that Thbs4 is highly expressed in articular chondrocytes, where its presence in the extracellular matrix is required for articular cartilage integrity.     

CD69 Does Not Affect the Extent of T Cell Priming

CD69 is rapidly upregulated on T cells upon activation. In this work we show that this is also the case for CD69 expression on dendritic cells (DC). Thus, the expression kinetics of CD69 on both cell types is reminiscent of the one of costimulatory molecules. Using mouse models of transgenic T cells, we aimed at evaluating the effect of monoclonal antibody (MAb)-based targeting and gene deficiency of CD69 expressed by either DC or T cells on the extent of antigen (Ag)-specific T cell priming, which could be the result of a putative role in costimulation as well as on DC maturation and Ag-processing and presentation. CD69 targeting or deficiency of DC did not affect their expression of costimulatory molecules nor their capacity to induce Ag-specific T cell proliferation in in vitro assays. Also, CD69 targeting or deficiency of transgenic T cells did not affect the minimal proliferative dose for different peptide agonists in vitro. In in vivo models of transgenic T cell transfer and local Ag injection, CD69 deficiency of transferred T cells did not affect the extent of the proliferative response in Ag-draining lymph nodes (LN). In agreement with these results, CD69 MAb targeting or gene deficiency of Vaccinia-virus (VACV) infected mice did not affect the endogenous formation of virus-specific CD8⁺ T cell populations at the peak of the primary immune response. Altogether our results argue against a possible role in costimulation or an effect on Ag processing and presentation for CD69.     

Distinct Macrophage Fates after in vitro Infection with Different Species of Leishmania: Induction of Apoptosis by Leishmania (Leishmania) amazonensis,but Not by Leishmania (Viannia) guyanensis

Leishmania is an intracellular parasite in vertebrate hosts, including man. During infection, amastigotes replicate inside macrophages and are transmitted to healthy cells, leading to amplification of the infection. Although transfer of amastigotes from infected to healthy cells is a crucial step that may shape the outcome of the infection, it is not fully understood. Here we compare L. amazonensis and L. guyanensis infection in C57BL/6 and BALB/c mice and investigate the fate of macrophages when infected with these species of Leishmania in vitro. As previously shown, infection of mice results in distinct outcomes: L. amazonensis causes a chronic infection in both strains of mice (although milder in C57BL/6), whereas L. guyanensis does not cause them disease. In vitro, infection is persistent in L. amazonensis-infected macrophages whereas L. guyanensis growth is controlled by host cells from both strains of mice. We demonstrate that, in vitro, L. amazonensis induces apoptosis of both C57BL/6 and BALB/c macrophages, characterized by PS exposure, DNA cleavage into nucleosomal size fragments, and consequent hypodiploidy. None of these signs were seen in macrophages infected with L. guyanensis, which seem to die through necrosis, as indicated by increased PI-, but not Annexin V-, positive cells. L. amazonensis-induced macrophage apoptosis was associated to activation of caspases-3, -8 and -9 in both strains of mice. Considering these two species of Leishmania and strains of mice, macrophage apoptosis, induced at the initial moments of infection, correlates with chronic infection, regardless of its severity. We present evidence suggestive that macrophages phagocytize L. amazonensis-infected cells, which has not been verified so far. The ingestion of apoptotic infected macrophages by healthy macrophages could be a way of amastigote spreading, leading to the establishment of infection.     

Low-Frequency Fluctuations of the Resting Brain: High Magnitude Does Not Equal High Reliability

The amplitude of low-frequency fluctuation (ALFF) measures low-frequency oscillations of the blood-oxygen-level-dependent signal, characterizing local spontaneous activity during the resting state. ALFF is a commonly used measure for resting-state functional magnetic resonance imaging (rs-fMRI) in numerous basic and clinical neuroscience studies. Using a test-retest rs-fMRI dataset consisting of 21 healthy subjects and three repetitive scans, we found that several key brain regions with high ALFF intensities (or magnitude) had poor reliability. Such regions included the posterior cingulate cortex, the medial prefrontal cortex in the default mode network, parts of the right and left thalami, and the primary visual and motor cortices. The above finding was robust with regard to different sample sizes (number of subjects), different scanning parameters (repetition time) and variations of test-retest intervals (i.e., intra-scan, intra-session, and inter-session reliability), as well as with different scanners. Moreover, the qualitative, map-wise results were validated further with a region-of-interest-based quantitative analysis using “canonical” coordinates as reported previously. Therefore, we suggest that the reliability assessments be incorporated in future ALFF studies, especially for the brain regions with a large ALFF magnitude as listed in our paper. Splitting single data into several segments and assessing within-scan “test-retest” reliability is an acceptable alternative if no “real” test-retest datasets are available. Such evaluations might become more necessary if the data are collected with clinical scanners whose performance is not as good as those that are used for scientific research purposes and are better maintained because the lower signal-to-noise ratio may further dampen ALFF reliability.     

Poly(Q) Expansions in ATXN7 Affect Solubility but Not Activity of the SAGA Deubiquitinating Module

Spinocerebellar ataxia type 7 (SCA7) is a debilitating neurodegenerative disease caused by expansion of a polyglutamine [poly(Q)] tract in ATXN7, a subunit of the deubiquitinase (DUB) module (DUBm) in the SAGA complex. The effects of ATXN7-poly(Q) on DUB activity are not known. To address this important question, we reconstituted the DUBm in vitro with either wild-type ATXN7 or a pathogenic form, ATXN7-92Q NT, with 92 Q residues at the N terminus (NT). We found that both forms of ATXN7 greatly enhance DUB activity but that ATXN7-92Q NT is largely insoluble unless it is incorporated into the DUBm. Cooverexpression of DUBm components in human astrocytes also promoted the solubility of ATXN7-92Q, inhibiting its aggregation into nuclear inclusions that sequester DUBm components, leading to global increases in ubiquitinated H2B (H2Bub) levels. Global H2Bub levels were also increased in the cerebellums of mice in a SCA7 mouse model. Our findings indicate that although ATXN7 poly(Q) expansions do not change the enzymatic activity of the DUBm, they likely contribute to SCA7 by initiating aggregates that sequester the DUBm away from its substrates.     

Genetic Characterization of pepP, Which Encodes an Aminopeptidase P Whose Deficiency Does Not Affect Lactococcus lactis Growth in Milk, Unlike Deficiency of the X-Prolyl Dipeptidyl Aminopeptidase

We sequenced the pepP gene of Lactococcus lactis, which encodes an aminopeptidase P (PepP), and demonstrated that the X-prolyl dipeptidyl aminopeptidase PepX plays a more important role than PepP in nitrogen nutrition. PepP shares homology with methionine aminopeptidases and could play a role in the maturation of nascent proteins.     

A Single Hot Event That Does Not Affect Survival but Decreases Reproduction in the Diamondback Moth,Plutella xylostella

Extremely hot events (usually involving a few hours at extreme high temperatures in summer) are expected to increase in frequency in temperate regions under global warming. The impact of these events is generally overlooked in insect population prediction, since they are unlikely to cause widespread mortality, however reproduction may be affected by them. In this study, we examined such stress effects in the diamondback moth, Plutella xylostella. We simulated a single extreme hot day (maximum of 40°C lasting for 3, 4 or 5 h) increasingly experienced under field conditions. This event had no detrimental effects on immediate mortality, copulation duration, mating success, longevity or lifetime fecundity, but stressed females produced 21% (after 3 or 4 h) fewer hatched eggs because of a decline in the number and hatching success of eggs laid on the first two days. These negative effects on reproduction were no longer evident in the following days. Male heat exposure led to a similar but smaller effect on fertile egg production, and exposure extended pre-mating period in both sexes. Our results indicate that a single hot day can have detrimental effects on reproduction, particularly through maternal effects on egg hatching, and thereby influence the population dynamics of diamondback moth.     

Kainic Acid Inhibits the Synaptosomal Plasma Membrane Glutamate Carrier and Allows Glutamate Leakage from the Cytoplasm but Does Not Affect Glutamate Exocytosis

Kainate inhibits the exchange of D-aspartate into guinea-pig cerebrocortical synaptosomes. Kainate inhibits the Ca2+-independent efflux of endogenous glutamate in the presence of a trapping system for the released amino acid but potentiates a Ca2+-independent net efflux of endogenous and labelled glutamate and aspartate in the absence of the trap. Dihydrokainate has a similar effect. No discrepancy is seen between the release of endogenous and exogenously accumulated amino acid. These results are consistent with the presence of a slow leak of glutamate or aspartate from the cytoplasm independent of the kainate-sensitive Na+-cotransport pathway. In the presence of the trap, glutamate effluxes by both pathways, whereas in the absence of the trap, the Na+-cotransport pathway opposes the leak. Neither in the presence or absence of the glutamate trap does kainate induce, inhibit, or otherwise affect the Ca2+-dependent release of endogenous glutamate. The results enable many of the apparent complexities in the presynaptic actions of kainate to be resolved.     

NOS2 Is Critical to the Development of Emphysema in Sftpd Deficient Mice but Does Not Affect Surfactant Homeostasis

Rationale

Surfactant protein D (SP-D) has important immuno-modulatory properties. The absence of SP-D results in an inducible NO synthase (iNOS, coded by NOS2 gene) related chronic inflammation, development of emphysema-like pathophysiology and alterations of surfactant homeostasis.

Objective

In order to test the hypothesis that SP-D deficiency related abnormalities in pulmonary structure and function are a consequence of iNOS induced inflammation, we generated SP-D and iNOS double knockout mice (DiNOS).

Methods

Structural data obtained by design-based stereology to quantify the emphysema-like phenotype and disturbances of the intracellular surfactant were correlated to invasive pulmonary function tests and inflammatory markers including activation markers of alveolar macrophages and compared to SP-D (Sftpd^−/−) and iNOS single knockout mice (NOS2^−/−) as well as wild type (WT) littermates.

Measurements and Results

DiNOS mice had reduced inflammatory cells in BAL and BAL-derived alveolar macrophages showed an increased expression of markers of an alternative activation as well as reduced inflammation. As evidenced by increased alveolar numbers and surface area, emphysematous changes were attenuated in DiNOS while disturbances of the surfactant system remained virtually unchanged. Sftpd^−/− demonstrated alterations of intrinsic mechanical properties of lung parenchyma as shown by reduced stiffness and resistance at its static limits, which could be corrected by additional ablation of NOS2 gene in DiNOS.

Conclusion

iNOS related inflammation in the absence of SP-D is involved in the emphysematous remodeling leading to a loss of alveoli and associated alterations of elastic properties of lung parenchyma while disturbances of surfactant homeostasis are mediated by different mechanisms.     

Superparamagnetic Iron Oxide Nanoparticles Labeling of Bone Marrow Stromal (Mesenchymal) Cells Does Not Affect Their “Stemness”

Superparamagnetic iron oxide nanoparticles (SPION) are increasingly used to label human bone marrow stromal cells (BMSCs, also called “mesenchymal stem cells”) to monitor their fate by in vivo MRI, and by histology after Prussian blue (PB) staining. SPION-labeling appears to be safe as assessed by in vitro differentiation of BMSCs, however, we chose to resolve the question of the effect of labeling on maintaining the “stemness” of cells within the BMSC population in vivo. Assays performed include colony forming efficiency, CD146 expression, gene expression profiling, and the “gold standard” of evaluating bone and myelosupportive stroma formation in vivo in immuncompromised recipients. SPION-labeling did not alter these assays. Comparable abundant bone with adjoining host hematopoietic cells were seen in cohorts of mice that were implanted with SPION-labeled or unlabeled BMSCs. PB+ adipocytes were noted, demonstrating their donor origin, as well as PB+ pericytes, indicative of self-renewal of the stem cell in the BMSC population. This study confirms that SPION labeling does not alter the differentiation potential of the subset of stem cells within BMSCs.