Ccr5 deficiency regulates the proliferation and trafficking of natural killer cells under physiological conditions

Chemokines were shown to govern the trafficking of immune cells and may also play important roles in the survival and activation of these cells. We report here that under physiological conditions, the bone marrow (BM), spleen, blood and liver of Ccr5, but not of Ccr1-deficient mice, contain reduced numbers of NK cells. NK cells in the BM of Ccr5-deficient mice proliferate to a lesser extent compared to WT mice. Furthermore, spleen NK cells derived from Ccr5-deficient mice that were transplanted into irradiated recipients failed to proliferate in the host. Ccr5, but not Ccr1-deficient NK cells, failed to migrate in vitro in response to RANTES and MIP-1β but not MIP-1β or SDF-1 and had reduced activation, lower expression levels of NK cell markers and a slightly reduced capacity to adhere to target cells and stimulate their killing. Using the polyI:C mouse model for NK trafficking, we found that in the absence of Ccr5, but not Ccr1, NK cells failed to accumulate in the liver. In contrast, using the influenza viral infection as a model to evaluate NK cell proliferation, we found that Ccr5-deficient NK cells in the BM had a higher proliferation rate than WT NK cells. These results suggest a role for Ccr5 in NK cell proliferation and circulation under physiological conditions and a complex role for Ccr5 in determining the fate of NK cells under pathological conditions.     

Activation-induced cytidine deaminase induces reproducible DNA breaks at many non-Ig Loci in activated B cells

After immunization or infection, activation-induced cytidine deaminase (AID) initiates diversification of immunoglobulin (Ig) genes in B cells, introducing mutations within the antigen-binding V regions (somatic hypermutation, SHM) and double-strand DNA breaks (DSBs) into switch (S) regions, leading to antibody class switch recombination (CSR). We asked if, during B cell activation, AID also induces DNA breaks at genes other than IgH genes. Using a nonbiased genome-wide approach, we have identified hundreds of reproducible, AID-dependent DSBs in mouse splenic B cells shortly after induction of CSR in culture. Most interestingly, AID induces DSBs at sites syntenic with sites of translocations, deletions, and amplifications found in human B cell lymphomas, including within the oncogene B cell lymphoma11a (bcl11a)/evi9. Unlike AID-induced DSBs in Ig genes, genome-wide AID-dependent DSBs are not restricted to transcribed regions and frequently occur within repeated sequence elements, including CA repeats, non-CA tandem repeats, and SINEs.     

Targeted maximum likelihood estimation of effect modification parameters in survival analysis

The Cox proportional hazards model or its discrete time analogue, the logistic failure time model, posit highly restrictive parametric models and attempt to estimate parameters which are specific to the model proposed. These methods are typically implemented when assessing effect modification in survival analyses despite their flaws. The targeted maximum likelihood estimation (TMLE) methodology is more robust than the methods typically implemented and allows practitioners to estimate parameters that directly answer the question of interest. TMLE will be used in this paper to estimate two newly proposed parameters of interest that quantify effect modification in the time to event setting. These methods are then applied to the Tshepo study to assess if either gender or baseline CD4 level modify the effect of two cART therapies of interest, efavirenz (EFV) and nevirapine (NVP), on the progression of HIV. The results show that women tend to have more favorable outcomes using EFV while males tend to have more favorable outcomes with NVP. Furthermore, EFV tends to be favorable compared to NVP for individuals at high CD4 levels.     

Functional characterization of aquaporin-4 specific T cells: towards a model for neuromyelitis optica

Background

Antibodies to the water channel protein aquaporin-4 (AQP4), which is expressed in astrocytic endfeet at the blood brain barrier, have been identified in the serum of Neuromyelitis optica (NMO) patients and are believed to induce damage to astrocytes. However, AQP4 specific T helper cell responses that are required for the generation of anti-AQP4 antibodies and most likely also for the formation of intraparenchymal CNS lesions have not been characterized.

Methodology/Principal Findings

Using overlapping 15-meric peptides of AQP4, we identified the immunogenic T cell epitopes of AQP4 that are restricted to murine major histocompatibility complex (MHC) I-A^b. The N-terminal region of AQP4 was highly immunogenic. More precisely, the intracellular epitope AQP4_22–36 was detected as major immunogenic determinant. AQP4_82–108 (located in the second transmembrane domain), AQP4_139–153 (located in the second extracellular loop), AQP4_211–225 (located in the fifth transmembrane domain), AQP4_235–249 (located in the sixth transmembrane domain), as well as AQP4_289–306 in the intracellular C-terminal region were also immunogenic epitopes. AQP4_22–36 and AQP4_289–303 specific T cells were present in the natural T cell repertoire of wild type C57BL/6 mice and T cell lines were raised. However, active immunization with these AQP4 peptides did not induce signs of spinal cord disease. Rather, sensitization with AQP4 peptides resulted in production of IFN-γ, but also IL-5 and IL-10 by antigen-specific T cells. Consistent with this cytokine profile, the AQP4 specific antibody response upon immunization with full length AQP4 included IgG1 and IgG2, which are associated with a mixed Th2/Th1 T cell response.

Conclusions and Significance

AQP4 is able to induce an autoreactive T cell response. The identification of I-A^b restricted AQP4 specific T cell epitopes will allow us to investigate how AQP4 specific autoimmune reactions are regulated and to establish faithful mouse models of NMO that include both cellular and humoral responses against AQP4.     

Structural and mechanical analysis of tectorial membrane Tecta mutants

The tectorial membrane (TM) is an extracellular matrix of the cochlea whose prominent role in hearing has been demonstrated through mutation studies. The C1509G mutation of the Tecta gene, which encodes for the α-tectorin protein, leads to hearing loss. The heterozygote TM only attaches to the first row of outer hair cells (OHCs), and the homozygote TM does not attach to any OHCs. Here we measured the morphology and mechanical properties of wild-type, heterozygous, and homozygous Tecta TMs. Morphological analyses conducted with second- and third-harmonic imaging, scanning electron microscopy, and immunolabeling revealed marked changes in the collagen architecture and stereocilin-labeling patterns of the mutant TMs. The mechanical properties of the mutant TM were measured by force spectroscopy. Whereas the axial Young's modulus of the low-frequency (apical) region of Tecta mutant TM samples was similar to that of wild-type TMs, it significantly decreased in the basal region to a value approaching that found at the apex. Modeling simulations suggest that a reduced TM Young's modulus is likely to reduce OHC stereociliary deflection. These findings argue that the heterozygote C1509G mutation results in a lack of attachment of the TM to the OHCs, which in turn reduces both the overall number of OHCs that are involved in mechanotransduction and the degree of mechanotransduction exhibited by the OHCs that remain attached to the TM.     

ndufa7 plays a critical role in cardiac hypertrophy

Cardiac hypertrophy is a common pathological change in patients with progressive cardiac function failure, which can be caused by hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM) or arterial hypertension. Despite years of study, there is still limited knowledge about the underlying molecular mechanisms for cardiac hypertrophy. NDUFA7, a subunit of NADH:ubiquinone oxidoreductase (complex I), has been reported to be a novel HCM associated gene. However, the biological role of NDUFA7 in heart remains unknown. In this study, we found that NDUFA7 exhibited high expression in the heart, and its level was significantly decreased in mice model of cardiac hypertrophy. Moreover, we demonstrated that ndufa7 knockdown in developing zebrafish embryos resulted in cardiac development and functional defects, associated with increased expression of pathological hypertrophy biomarkers nppa (ANP) and nppb (BNP). Mechanistic study demonstrated that ndufa7 depletion promoted ROS production and calcineurin signalling activation. Moreover, NDUFA7 depletion contributed to cardiac cell hypertrophy. Together, these results report for the first time that ndufa7 is implicated in pathological cardiac hypertrophy.     

CD4+NKG2D+ T Cells Exhibit Enhanced Migratory and Encephalitogenic Properties in Neuroinflammation

Migration of encephalitogenic CD4⁺ T lymphocytes across the blood-brain barrier is an essential step in the pathogenesis of multiple sclerosis (MS). We here demonstrate that expression of the co-stimulatory receptor NKG2D defines a subpopulation of CD4⁺ T cells with elevated levels of markers for migration, activation, and cytolytic capacity especially when derived from MS patients. Furthermore, CD4⁺NKG2D⁺ cells produce high levels of proinflammatory IFN-γ and IL-17 upon stimulation. NKG2D promotes the capacity of CD4⁺NKG2D⁺ cells to migrate across endothelial cells in an in vitro model of the blood-brain barrier. CD4⁺NKG2D⁺ T cells are enriched in the cerebrospinal fluid of MS patients, and a significant number of CD4⁺ T cells in MS lesions coexpress NKG2D. We further elucidated the role of CD4⁺NKG2D⁺ T cells in the mouse system. NKG2D blockade restricted central nervous system migration of T lymphocytes in vivo, leading to a significant decrease in the clinical and pathologic severity of experimental autoimmune encephalomyelitis, an animal model of MS. Blockade of NKG2D reduced killing of cultivated mouse oligodendrocytes by activated CD4⁺ T cells. Taken together, we identify CD4⁺NKG2D⁺ cells as a subpopulation of T helper cells with enhanced migratory, encephalitogenic and cytotoxic properties involved in inflammatory CNS lesion development.     

Oxidative Stress-Induced DNA Damage and Repair in Human Peripheral Blood Mononuclear Cells: Protective Role of Hemoglobin

Background

DNA repair is a cellular defence mechanism responding to DNA damage caused in large part by oxidative stress. There is a controversy with regard to the effect of red blood cells on DNA damage and cellular response.

Aim

To investigate the effect of red blood cells on H₂O₂-induced DNA damage and repair in human peripheral blood mononuclear cells.

Methods

DNA breaks were induced in peripheral blood mononuclear cells by H₂O₂ in the absence or presence of red blood cells, red blood cells hemolysate or hemoglobin. DNA repair was measured by ³H-thymidine uptake, % double-stranded DNA was measured by fluorometric assay of DNA unwinding. DNA damage was measured by the comet assay and by the detection of histone H2AX phosphorylation.

Results

Red blood cells and red blood cells hemolysate reduced DNA repair in a dose-dependent manner. Red blood cells hemolysate reduced % double-stranded DNA, DNA damage and phosphorylation of histone H2AX. Hemoglobin had the same effect as red blood cells hemolysate on % double-stranded DNA.

Conclusion

Red blood cells, via red blood cells hemolysate and hemoglobin, reduced the effect of oxidative stress on peripheral blood mononuclear cell DNA damage and phosphorylation of histone H2AX. Consequently, recruitment of DNA repair proteins diminished with reduction of DNA repair. This suggests that anemia predisposes to increased oxidative stress induced DNA damage, while a higher hemoglobin level provides protection against oxidative-stress-induced DNA damage.