CD28-deficient Mice Research Articles

Antiviral CD8+ T Cells Restricted by Human Leukocyte Antigen Class II Exist during Natural HIV Infection and Exhibit Clonal Expansion

SummaryCD8+ T cell recognition of virus-infected cells is characteristically restricted by major histocompatibility complex (MHC) class I, although rare examples of MHC class II restriction have been reported in Cd4-deficient mice and a macaque SIV vaccine trial using a recombinant cytomegalovirus vector. Here, we demonstrate the presence of human leukocyte antigen (HLA) class II-restricted CD8+ T cell responses with antiviral properties in a small subset of HIV-infected individuals. In these individuals, T cell receptor β (TCRβ) analysis revealed that class II-restricted CD8+ T cells underwent clonal expansion and mediated killing of HIV-infected cells. In one case, these cells comprised 12% of circulating CD8+ T cells, and TCRα analysis revealed two distinct co-expressed TCRα chains, with only one contributing to binding of the class II HLA-peptide complex. These data indicate that class II-restricted CD8+ T cell responses can exist in a chronic human viral infection, and may contribute to immune control.

Proximal Tubule CD73 Is Critical in Renal Ischemia-Reperfusion Injury Protection.

CD73-derived adenosine plays an anti-inflammatory role in various organs. However, its role in renal ischemia-reperfusion injury (IRI) is controversial. We targeted CD73 mutant mice to determine the function of CD73 expressed by various renal cell types under mild IRI conditions. Mice with CD73 deletion in proximal tubules exhibited exacerbated IRI, comparable with that of CD73-/- mice compared with WT mice. Mice with CD73 deletions in other cell types, including cortical type 1 fibroblast-like cells, mesangial cells, macrophages, and dendritic cells, showed small or no increases in injury above control mice when subjected to threshold levels of ischemia. Results from adoptive transfer experiments between WT and CD73-/- mice and pharmacologic studies modulating enzymatic activity of CD73 and extracellular adenosine levels supported a critical role of adenosine generated by proximal tubule CD73 expression in abrogating IRI. Renal adenosine levels were lower before and after ischemia in CD73-deficient mice. However, reduction in total acid-extractable renal adenosine levels was inadequate to explain the marked difference in kidney injury in these CD73-deficient mice. Furthermore, CD73 inhibition and enzyme replacement studies showed no change in total kidney adenosine levels in treated mice compared with vehicle-treated controls. Protection from IRI in neutrophil-depleted WT recipients was sustained by repopulation with bone marrow neutrophils from WT mice but not by those lacking adenosine 2a receptors (from Adora2a-/- mice). These data support the thesis that local adenosine generated by cells at the injury site is critical for protection from IRI through bone marrow-derived adenosine 2a receptors.

279 Skin dendritic cells show increased migration to lymph nodes in CD73 deficient mice leading to exacerbated contact hypersensitivity reactions

279 Skin dendritic cells show increased migration to lymph nodes in CD73 deficient mice leading to exacerbated contact hypersensitivity reactions

Interleukin (IL)-39 [IL-23p19/Epstein-Barr virus-induced 3 (Ebi3)] induces differentiation/expansion of neutrophils in lupus-prone mice.

Interleukin (IL)-12 family cytokines play critical roles in autoimmune diseases. Our previous study has shown that IL-23p19 and Epstein-Barr virus-induced 3 (Ebi3) form a new IL-12 family heterodimer, IL-23p19/Ebi3, termed IL-39, and knock-down of p19 or Ebi3 reduced diseases by transferred GL7+ B cells in lupus-prone mice. In the present study, we explore further the possible effect of IL-39 on murine lupus. We found that IL-39 in vitro and in vivo induces differentiation and/or expansion of neutrophils. GL7+ B cells up-regulated neutrophils by secreting IL-39, whereas IL-39-deficient GL7+ B cells lost the capacity to up-regulate neutrophils in lupus-prone mice and homozygous CD19cre (CD19-deficient) mice. Finally, we found that IL-39-induced neutrophils had a positive feedback on IL-39 expression in activated B cells by secreting B cell activation factor (BAFF). Taken together, our results suggest that IL-39 induces differentiation and/or expansion of neutrophils in lupus-prone mice.

Beneficial Role of Erythrocyte Adenosine A2B Receptor-Mediated AMP-Activated Protein Kinase Activation in High-Altitude Hypoxia.

High altitude is a challenging condition caused by insufficient oxygen supply. Inability to adjust to hypoxia may lead to pulmonary edema, stroke, cardiovascular dysfunction, and even death. Thus, understanding the molecular basis of adaptation to high altitude may reveal novel therapeutics to counteract the detrimental consequences of hypoxia. Using high-throughput, unbiased metabolomic profiling, we report that the metabolic pathway responsible for production of erythrocyte 2,3-bisphosphoglycerate (2,3-BPG), a negative allosteric regulator of hemoglobin-O2 binding affinity, was significantly induced in 21 healthy humans within 2 hours of arrival at 5260 m and further increased after 16 days at 5260 m. This finding led us to discover that plasma adenosine concentrations and soluble CD73 activity rapidly increased at high altitude and were associated with elevated erythrocyte 2,3-BPG levels and O2 releasing capacity. Mouse genetic studies demonstrated that elevated CD73 contributed to hypoxia-induced adenosine accumulation and that elevated adenosine-mediated erythrocyte A2B adenosine receptor activation was beneficial by inducing 2,3-BPG production and triggering O2 release to prevent multiple tissue hypoxia, inflammation, and pulmonary vascular leakage. Mechanistically, we demonstrated that erythrocyte AMP-activated protein kinase was activated in humans at high altitude and that AMP-activated protein kinase is a key protein functioning downstream of the A2B adenosine receptor, phosphorylating and activating BPG mutase and thus inducing 2,3-BPG production and O2 release from erythrocytes. Significantly, preclinical studies demonstrated that activation of AMP-activated protein kinase enhanced BPG mutase activation, 2,3-BPG production, and O2 release capacity in CD73-deficient mice, in erythrocyte-specific A2B adenosine receptor knockouts, and in wild-type mice and in turn reduced tissue hypoxia and inflammation. Together, human and mouse studies reveal novel mechanisms of hypoxia adaptation and potential therapeutic approaches for counteracting hypoxia-induced tissue damage.

Role of CD69 in the pathogenesis of elastase-induced pulmonary inflammation and emphysema

Cluster of differentiation 69 (CD69), known as an early activation marker of lymphocytes, has been demonstrated to regulate inflammatory events in various disease models. Although the increased number of CD69-expressed T lymphocytes in the lungs of patients with chronic obstructive pulmonary disease (COPD) has been reported, a functional role of CD69 in the pathogenesis of COPD remains unknown. To address to this question, CD69-deficient (CD69KO) mice and wild-type (WT) mice were subjected to a mouse model of porcine pancreatic elastase (PPE)-induced pulmonary inflammation and emphysema. In the two genotypes, PPE increased counts of macrophages, neutrophils and lymphocytes in bronchoalveolar lavage fluid (BALF) and induced emphysematous changes in the lung, whereas those two pathological signs were significantly enhanced in CD69KO mice compared to WT mice. Moreover, the PPE-induced levels of IL-17 and IL-6 in BALF were significantly higher in CD69KO mice than in WT mice at the acute inflammatory phase. Immunofluorescent studies showed that IL-17 and IL-6 were predominantly expressed in CD4+ and γδ T cells and macrophages, respectively. Concomitant administration of IL-17- and IL-6-neutralizing antibodies significantly attenuated the PPE-induced emphysematous changes in the two genotypes. These findings suggest that CD69 negatively regulates the development of PPE-induced emphysema in part at least through modulating function of IL-17-producing T cells.

CD69 controls the uptake of L-tryptophan through LAT1-CD98 and AhR-dependent secretion of IL-22 in psoriasis.

The activation marker CD69 is expressed by skin γδ T cells. Here we found that CD69 controlled the aryl hydrocarbon receptor (AhR)-dependent secretion of interleukin 22 (IL-22) by γδ T cells, which contributed to the development of psoriasis induced by IL-23. CD69 associated with the aromatic-amino-acid-transporter complex LAT1-CD98 and regulated its surface expression and uptake of L-tryptophan (L-Trp) and the intracellular quantity of L-Trp-derived activators of AhR. In vivo administration of L-Trp, an inhibitor of AhR or IL-22 abrogated the differences between CD69-deficient mice and wild-type mice in skin inflammation. We also observed LAT1-mediated regulation of AhR activation and IL-22 secretion in circulating Vγ9(+) γδ T cells of psoriatic patients. Thus, CD69 serves as a key mediator of the pathogenesis of psoriasis by controlling LAT1-CD98-mediated metabolic cues.

A c-Myc/miR17-92/Pten Axis Controls PI3K-Mediated Positive and Negative Selection in B Cell Development and Reconstitutes CD19 Deficiency.

PI3K activity determines positive and negative selection of B cells, a key process for immune tolerance and B cell maturation. Activation of PI3K is balanced by phosphatase and tensin homolog (Pten), the PI3K's main antagonistic phosphatase. Yet, the extent of feedback regulation between PI3K activity and Ptenexpression during B cell development is unclear.Here, we show that PI3K control of this processis achieved post-transcriptionally by an axis composed of a transcription factor (c-Myc), a microRNA (miR17-92), and Pten. Enhancing activation of this axis through overexpression of miR17-92 reconstitutes the impaired PI3K activity for positive selection in CD19-deficient B cells and restores most of the B cell developmental impairments that are evident in CD19-deficient mice. Using a genetic approach of deletion and complementation, we show that the c-Myc/miR17-92/Pten axis critically controls PI3K activity and the sensitivity of immature B cells to negative selection imposed by activation-induced cell death.

Extracellular Adenosine Production by ecto-5'-Nucleotidase (CD73) Enhances Radiation-Induced Lung Fibrosis.

Radiation-induced pulmonary fibrosis is a severe side effect of thoracic irradiation, but its pathogenesis remains poorly understood and no effective treatment is available. In this study, we investigated the role of the extracellular adenosine as generated by the ecto-5'-nucleotidase CD73 in fibrosis development after thoracic irradiation. Exposure of wild-type C57BL/6 mice to a single dose (15 Gray) of whole thorax irradiation triggered a progressive increase in CD73 activity in the lung between 3 and 30 weeks postirradiation. In parallel, adenosine levels in bronchoalveolar lavage fluid (BALF) were increased by approximately 3-fold. Histologic evidence of lung fibrosis was observed by 25 weeks after irradiation. Conversely, CD73-deficient mice failed to accumulate adenosine in BALF and exhibited significantly less radiation-induced lung fibrosis (P < 0.010). Furthermore, treatment of wild-type mice with pegylated adenosine deaminase or CD73 antibodies also significantly reduced radiation-induced lung fibrosis. Taken together, our findings demonstrate that CD73 potentiates radiation-induced lung fibrosis, suggesting that existing pharmacologic strategies for modulating adenosine may be effective in limiting lung toxicities associated with the treatment of thoracic malignancies. Cancer Res; 76(10); 3045-56. ©2016 AACR.

CD69 Deficiency Enhances the Host Response to Vaccinia Virus Infection through Altered NK Cell Homeostasis.

During the host response to viral infection, the transmembrane CD69 protein is highly upregulated in all immune cells. We have studied the role of CD69 in the murine immune response to vaccinia virus (VACV) infection, and we report that the absence of CD69 enhances protection against VACV at both short and long times postinfection in immunocompetent and immunodeficient mice. Natural killer (NK) cells were implicated in the increased infection control, since the differences were greatly diminished when NK cells were depleted. This role of NK cells was not based on an altered NK cell reactivity, since CD69 did not affect the NK cell activation threshold in response to major histocompatibility complex class I NK cell targets or protein kinase C activation. Instead, NK cell numbers were increased in the spleen and peritoneum of CD69-deficient infected mice. That was not just secondary to better infection control in CD69-deficient mice, since NK cell numbers in the spleens and the blood of uninfected CD69(-/-) mice were already augmented. CD69-deficient NK cells from infected mice did not have an altered proliferation capacity. However, a lower spontaneous cell death rate was observed for CD69(-/-) lymphocytes. Thus, our results suggest that CD69 limits the innate immune response to VACV infection at least in part through cell homeostatic survival. We show that increased natural killer (NK) cell numbers augment the host response and survival after infection with vaccinia virus. This phenotype is found in the absence of CD69 in immunocompetent and immunodeficient hosts. As part of the innate immune system, NK lymphocytes are activated and participate in the defense against infection. Several studies have focused on the contribution of NK cells to protection against infection with vaccinia virus. In this study, it was demonstrated that the augmented early NK cell response in the absence of CD69 is responsible for the increased protection seen during infection with vaccinia virus even at late times of infection. This work indicates that the CD69 molecule may be a target of therapy to augment the response to poxvirus infection.

Exogenous induction of type I IFN protects mice from Cryptococcus neoformans and Cryptococcus gattii infections by distinct CD4+ T cell and CD8+ T cell-mediated mechanisms.

Abstract Rational Cryptococcus neoformans (Cn) causes deadly mycosis in AIDS patients whereas the Vancouver outbreak strain of Cryptococcus gattii (Cg) mostly infects otherwise healthy individuals but not AIDS patients even where the strains are equally prevalent in the environment. As HIV infection induces type I IFN (t1I) we hypothesized that exogenously induced t1I would differentially affect infections by Cn and Cg. Results Cn or Cg infected mice were dosed with poly(I:C)LC (PIC) to induce t1I. PIC-treated mice were protected from Cn and Cg infection-mediated death with reduced fungal burdens. Both Cn and Cg infection produced a non-protective immune response dominated by Th2 cell recruitment, M2/DC2 polarization and eosinophilia. PIC-treatment reversed the Th2 polarization toward increased Th1/17 cells, M1/DC1 polarization and reduced eosinophilia. PIC also induced activation of CD8 T cells in both Cg and Cn infected mice. Cg-infected CD8 cell-deficient mice were not protected by PIC treatment while PIC protection was intact in Cn-infected CD8 cell-deficient mice. In contrast, PIC protection was intact in Cg-infected CD4 cell-deficient mice while Cn-infected CD4 cell-deficient mice were not protected by PIC treatment. Conclusions These data show that t1I induction protects mice from Cn and Cg however protection was mediated by distinct immune mechanisms. PIC-mediated protection from Cn requires CD4 but not CD8 cells while PIC-mediated protection from Cg requires CD8 but not CD4 cells. Notably, Cn but not Cg was able to grow in CD4-deficient t1I-induced mice, which somewhat resemble the immunological condition of AIDS patients, suggesting a novel explanation for the observed AIDS patient tropism of these cryptococcal species.

High-Risk Corneal Graft Rejection in the Setting of Previous Corneal Herpes Simplex Virus (HSV)-1 Infection.

PurposeThe “high-risk phenotype” of corneal graft recipients is considered to be related to preexisting vascularization such as that associated with herpes simplex virus-1 (HSV-1) keratitis (HSK). The purpose of this study was to investigate the immunologic mechanisms underlying accelerated corneal graft rejection using a mouse model of HSK.MethodsHerpes simplex virus type 1 keratitis was induced in BALB/c mice. Syngeneic and allogeneic (C57BL/6 mice) corneal grafts were performed in mice with HSK at different times after infection. Some grafts were performed on HSV-infected CD4 T cell–deficient BALB/c mice. Clinical, histologic, immunologic, and virus detection studies were performed on samples of cornea, draining lymph node (LN), and trigeminal ganglion (TG) cells.ResultsCorneal grafts in mice with HSK rejected with higher frequency and more rapid tempo compared with grafts in uninfected mice. In corneas with HSK and vascularization at the time of grafting, both syngeneic and allogeneic corneal grafts failed with similar frequency and tempo. However, in the absence of preexisting inflammation and vascularization, syngeneic grafts were accepted when the grafts were performed at a late time point after HSV infection (42 days), whereas allografts were rejected at this time. In contrast, syngeneic grafts in nonvascularized HSV-infected recipients failed if they were performed within 10 days of HSV infection, an effect that was dependent on CD4 T cells, as demonstrated using CD4 deficient mice. Importantly, a variably sustained but strongly positive anti-HSV T-cell response was detected in allografted HSK recipients with a similar but lesser response in syngeneic hosts.ConclusionsA previous HSV-1 corneal infection predisposes donor grafts to a high risk of failure by both innate and adaptive immune mechanisms in which an anti-HSV CD4 T-cell response plays a prominent role.

CD73 Expressed on γδ T Cells Shapes Their Regulatory Effect in Experimental Autoimmune Uveitis.

γδ T cells can either enhance or inhibit an adaptive immune response, but the mechanisms involved are not fully understood. Given that CD73 is the main enzyme responsible for conversion of AMP into the immunosuppressive molecule adenosine, we investigated its role in the regulatory function of γδ T cells in experimental autoimmune uveitis (EAU). We found that γδ T cells expressed different amounts of CD73 during the different stages of EAU and that low CD73 expression on γδ T cells correlated with enhanced Th17 response-promoting activity. Functional comparison of CD73-deficient and wild-type B6 (CD73+/+) mice showed that failure to express CD73 decreased both the enhancing and suppressive effects of γδ T cells on EAU. We also demonstrated that γδ T cells expressed different amounts of CD73 when activated by different pathways, which enabled them to either enhance or inhibit an adaptive immune response. Our results demonstrate that targeting CD73 expression on γδ T cells may allow us to manipulate their pro- or anti-inflammatory effect on Th17 responses.

Early Prediction of Tumor Response to Treatment: Preclinical Validation of 99mTc-Duramycin.

Noninvasive imaging of cell death can provide an early indication of the efficacy of tumor treatment, aiding clinicians in distinguishing responding patients from nonresponding patients early on. (99m)Tc-duramycin is a SPECT tracer for cell death imaging. In this study, our aim was to validate the use of (99m)Tc-duramycin for imaging the early response of tumors to treatment. An in vitro binding assay was performed on COLO205 cells treated with 5-fluorouracil (3.1, 31, or 310 μM) and oxaliplatin (0.7 or 7 μM) or radiation (2 or 4.5 Gy). (99m)Tc-duramycin cell binding and the levels of cell death were evaluated after treatment. In vivo imaging was performed on 4 groups of CD1-deficient mice bearing COLO205 human colorectal cancer tumors. Each group included 6 tumors. The first group was given irinotecan (100 mg/kg), the second oxaliplatin (5 mg/kg), the third irinotecan (80 mg/kg) plus oxaliplatin (5 mg/kg), and the fourth vehicle (0.9% NaCl and 5% glucose). For radiotherapy studies, COLO205 tumors received 4.5 Gy, 2 fractions of 4.5 Gy in a 24-h interval, pretreatment with an 80 mg/kg dose of irinotecan combined with 2 fractions of 4.5 Gy in a 24-h interval, or no treatment (n = 5-6/group). Therapy response was evaluated by (99m)Tc-duramycin SPECT 24 h after the last dose of therapy. Blocking was used to confirm tracer specificity. Radiotracer uptake in the tumors was validated ex vivo using γ-counting, cleaved caspase-3, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) histology. Chemotherapy and radiotherapy increased (99m)Tc-duramycin binding to COLO205 cells in a concentration/dose- and time-dependent manner, which correlated well with cell death levels (P < 0.05) as analyzed by annexin V and caspase 3/7 activity. In vivo, (99m)Tc-duramycin uptake in COLO205 xenografts was increased 2.3- and 2.8-fold (P < 0.001) in mice treated with irinotecan and combination therapy, respectively. Blocking with unlabeled duramycin demonstrated specific binding of the radiotracer. After tumor irradiation with 4.5 Gy, (99m)Tc-duramycin uptake in tumors increased significantly (1.24 ± 0.07 vs. 0.57 ± 0.08 percentage injected dose per gram in the unirradiated tumors; P < 0.001). γ-counting of radioactivity in the tumors positively correlated with cleaved caspase-3 (r = 0.85, P < 0.001) and TUNEL (r = 0.81, P < 0.001) staining. We demonstrated that (99m)Tc-duramycin can be used to image induction of cell death early after chemotherapy and radiotherapy. It holds potential to be translated into clinical use for early assessment of treatment response.

Supporting data for the MS identification of distinct transferrin glycopeptide glycoforms and citrullinated peptides associated with inflammation or autoimmunity.

This data article presents the results of all the statistical analyses applied to the relative intensities of the detected 2D-DiGE protein spots for each of the 3 performed DiGE experiments. The data reveals specific subsets of protein spots with significant differences between WT and CD38-deficient mice with either Collagen-induced arthritis (CIA), or with chronic inflammation induced by CFA, or under steady-state conditions. This article also shows the MS data analyses that allowed the identification of the protein species which serve to discriminate the different experimental groups used in this study. Moreover, the article presents MS data on the citrullinated peptides linked to specific protein species that were generated in CIA+ or CFA-treated mice. Lastly, this data article provides MS data on the efficiency of the analyses of the transferrin (Tf) glycopeptide glycosylation pattern in spleen and serum from CIA+ mice and normal controls. The data supplied in this work is related to the research article entitled “identification of multiple transferrin species in spleen and serum from mice with collagen-induced arthritis which may reflect changes in transferrin glycosylation associated with disease activity: the role of CD38” [1]. All mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with identifiers PRIDE: PXD002644, PRIDE: PXD002643, PRIDE: PXD003183 and PRIDE: PXD003163.

CD38 Deficiency Protects the Heart from Ischemia/Reperfusion Injury through Activating SIRT1/FOXOs‐Mediated Antioxidative Stress Pathway

Ischemia/reperfusion (I/R) injury induces irreversible oxidative stress damage to the cardiac muscle. We previously observed that CD38 deficiency remarkably protects mouse embryonic fibroblasts (MEFs) from oxidative stress-induced injury. However, whether CD38 deficiency protects from I/R injury in the heart is not explored. Here, we showed that the hearts of CD38 deficient mice or wild type mice supplied with exogenous NAD were significantly protected from ischemia/reperfusion injury, seen as reduction of the myocardial infarct sizes when the mice were subjected to 30 min ischemia followed by 24 hours of reperfusion. Consistently, the protection of CD38 deficiency on hypoxia/reoxygenation (H/R) injury was confirmed with a CD38 knockdown H9c2 stable cell line. Furthermore, we observed that knockdown of CD38 remarkably inhibited ROS generation and intracellular Ca2+ overloading induced by H/R in H9c2 cells. The FOXO1 and FOXO3 expressions were significantly elevated by H/R injury in CD38 knockdown cells compared with normal H9c2 cells. The cell immunofluorescence assay showed that FOXO1 nuclear translocation was significantly increased in CD38 knockdown H9c2 cells. In addition, we demonstrated that the increase of FOXO1 nuclear translocation was associated with the increased expressions of antioxidant catalase and SOD2 and the attenuated expression of the ROS generation enzyme NOX4. In conclusion, our results provide new evidence that CD38 deficiency protects the heart from I/R injury through activating SIRT1/FOXOs-mediated antioxidative stress pathway.

Tetraspanin CD81, a modulator of immune suppression in cancer and metastasis

ABSTRACTCancer cells can escape the antitumor immune response by recruiting immune suppressor cells. However, although innate myeloid-derived suppressor cells (MDSCs) and T regulatory (Treg) cells accumulate normally in tumor-bearing CD81-deficient mice, both populations are impaired in their ability to suppress the antitumor immune response.

CD40 promotes MHC class II expression on adipose tissue macrophages and regulates adipose tissue CD4+ T cells with obesity.

Obesity activates both innate and adaptive immune responses in adipose tissue, but the mechanisms critical for regulating these responses remain unknown. CD40/CD40L signaling provides bidirectional costimulatory signals between antigen-presenting cells and CD4(+) T cells, and CD40L expression is increased in obese humans. Therefore, we examined the contribution of CD40 to the progression of obesity-induced inflammation in mice. CD40 was highly expressed on adipose tissue macrophages in mice, and CD40/CD40L signaling promoted the expression of antigen-presenting cell markers in adipose tissue macrophages. When fed a high fat diet, Cd40-deficient mice had reduced accumulation of conventional CD4(+) T cells (Tconv: CD3(+)CD4(+)Foxp3(-)) in visceral fat compared with wild-type mice. By contrast, the number of regulatory CD4(+) T cells (Treg: CD3(+)CD4(+)Foxp3(+)) in lean and obese fat was similar between wild-type and knockout mice. Adipose tissue macrophage content and inflammatory gene expression in fat did not differ between obese wild-type and knockout mice; however, major histocompatibility complex class II and CD86 expression on adipose tissue macrophages was reduced in visceral fat from knockout mice. Similar results were observed in chimeric mice with hematopoietic Cd40-deficiency. Nonetheless, neither whole body nor hematopoietic disruption of CD40 ameliorated obesity-induced insulin resistance in mice. In human adipose tissue, CD40 expression was positively correlated with CD80 and CD86 expression in obese patients with type 2 diabetes. These findings indicate that CD40 signaling in adipose tissue macrophages regulates major histocompatibility complex class II and CD86 expression to control the expansion of CD4(+) T cells; however, this is largely dispensable for the development of obesity-induced inflammation and insulin resistance in mice.

Time to Integrate to Nest Test Evaluation in a Mouse DSS-Colitis Model.

Severity assessment in laboratory animals is an important issue regarding the implementation of the 3R concept into biomedical research and pivotal in current EU regulations. In mouse models of inflammatory bowel disease severity assessment is usually undertaken by clinical scoring, especially by monitoring reduction of body weight. This requires daily observance and handling of each mouse, which is time consuming, stressful for the animal and necessitates an experienced observer. The time to integrate to nest test (TINT) is an easily applicable test detecting disturbed welfare by measuring the time interval mice need to integrate nesting material to an existing nest. Here, TINT was utilized to assess severity in a mouse DSS-colitis model. TINT results depended on the group size of mice maintained per cage with most consistent time intervals measured when co-housing 4 to 5 mice. Colitis was induced with 1% or 1.5% DSS in group-housed WT and Cd14-deficient mice. Higher clinical scores and loss of body weight were detected in 1.5% compared to 1% DSS treated mice. TINT time intervals showed no dose dependent differences. However, increased clinical scores, body weight reductions, and increased TINT time intervals were detected in Cd14 -/- compared to WT mice revealing mouse strain related differences. Therefore, TINT is an easily applicable method for severity assessment in a mouse colitis model detecting CD14 related differences, but not dose dependent differences. As TINT revealed most consistent results in group-housed mice, we recommend utilization as an additional method substituting clinical monitoring of the individual mouse.

Can CD44 Be a Mediator of Cell Destruction? The Challenge of Type 1 Diabetes.

CD44 is a multi-functional receptor with multiple of isoforms engaged in modulation of cell trafficking and transmission of apoptotic signals. We have previously shown that injection of anti-CD44 antibody into NOD mice induced resistance to type 1 diabetes (T1D). In this communication we describe our efforts to understand the mechanism underlying this effect. We found that CD44-deficient NOD mice develop stronger resistance to T1D than wild-type littermates. This effect is not explained by the involvement of CD44 in cell migration, because CD44-deficient inflammatory cells surprisingly had greater invasive potential than the corresponding wild type cells, probably owing to molecular redundancy. We have previously reported and we show here again that CD44 expression and hyaluronic acid (HA, the principal ligand for CD44) accumulation are detected in pancreatic islets of diabetic NOD mice, but not of non-diabetic DBA/1 mice. Expression of CD44 on insulin-secreting β cells renders them susceptible to the autoimmune attack, and is associated with a diminution in β-cells function (e.g., less insulin production and/or insulin secretion) and possibly also with an enhanced apoptosis rate. The diabetes-supportive effect of CD44 expression on β cells was assessed by the TUNEL assay and further strengthened by functional assays exhibiting increased nitric oxide release, reduced insulin secretion after glucose stimulation and decreased insulin content in β cells. All these parameters could not be detected in CD44-deficient islets. We further suggest that HA-binding to CD44-expressing β cells is implicated in β-cell demise. Altogether, these data agree with the concept that CD44 is a receptor capable of modulating cell fate. This finding is important for other pathologies (e.g., cancer, neurodegenerative diseases) in which CD44 and HA appear to be implicated.