RAG1 Research Articles

Journey to the West: Trans-Pacific Historical Biogeography of Fringehead Blennies in the Genus Neoclinus (Teleostei: Blenniiformes).

Several temperate marine taxa of the northern hemisphere follow a trans-Pacific biogeographic track with representatives on either side of the intervening boreal waters. Shelter-dwelling blenniiform fishes of the genus Neoclinus exhibit this trans-Pacific distribution pattern with three species in the eastern North Pacific and eight species in the western North Pacific. We reconstructed the phylogeny of the Neocliniini (Neoclinus and the monotypic Mccoskerichthys) using six genetic markers: four mitochondrial genes (COI, cytochrome b, 12S and 16S), and two nuclear genes (RAG-1, TMO-4C4). Ancestral state reconstruction and molecular clock dating were used to explore hypothetical ancestral distributions and area relationships, and to estimate divergent times within this group. The monophyly of the genus Neoclinus, and the reciprocal monophyly of the eastern Pacific and western Pacific lineages were supported. Available evidence, including the eastern Pacific and western Atlantic occurrence of a New World clade of blennioid fishes that includes this lineage, supports the origin of the Neocliniini in the eastern Pacific with a single divergence event to the west across the North Pacific by the ancestor of the western Pacific clade. Estimated divergence time of the eastern and western Pacific clades of Neoclinus was 24.14 million year ago, which falls during the Oligocene epoch. Estimated times of divergence in other trans-Pacific lineages of marine fishes vary widely, from recent Pleistocene events to as early as 34 mya.

Neonatal thymectomy in children—accelerating the immunologic clock?

The thymus is critical for central tolerance and diverse T-lymphocyte repertoire development, to provide lifelong defense against pathogens while maintaining self-tolerance. Peak thymic output occurs in utero, during infancy, and in early childhood, diminishing throughout life. Infants with congenital heart disease requiring sternotomy often undergo thymectomy to clear the surgical field. The long-term effects of early thymectomy are just being appreciated. Many patients remain asymptomatic despite immunologic findings mirroring those of immunosenescence. Few develop increased infection or lymphoreticular malignancy risk. When considering the effects of infant thymectomy, patients with partial DiGeorge syndrome or hypomorphic recombination-activating gene (RAG) mutations may be instructive. These patients are lymphocytopenic, with increased early-onset infection and autoimmunity risk that is not seen in most patients who underwent thymectomy during infancy. The thymic structure of patients with partial DiGeorge syndrome or hypomorphic RAG is abnormal, with disrupted architecture inclining to perturbation of central tolerance. Similar findings may be seen in patients with myasthenia gravis, although disrupted peripheral tolerance may play a greater role in autoimmunity development. In conclusion, thymectomy during infancy may increase future risk of infection or autoimmunity, with premature immunosenescence mediated through disruption of central and peripheral tolerance mechanisms initiated by early cessation or diminution of thymic output. Ideally, some thymic tissue should be preserved at the time of surgery.

Cloning, characterization and ontogenetic expression profile of RAG-2 and IgM in angelfish (Pterophyllum scalare).

Early larval developmental stages of fish are highly susceptible to opportunistic pathogens until the complete maturation of the lymphoid organs. Knowledge of the expression pattern of important markers of adaptive immune system during the ontogenetic development is essential before vaccinating the fish. In the present study, Pterophyllum scalare (angelfish) was taken to explore the relative expression profile of developmental markers of adaptive immunity, recombination activating gene-2 (RAG-2) and immunoglobulin M (IgM). The fishes were bred and early developmental stages (0-45days post-hatched) were used to assess the expression profile. The genes, RAG-2 and IgM were cloned and sequenced with the base pair lengths of 1958bp and 225bp respectively. The mRNA expression of RAG-2 appeared at insignificant level at the first day of hatching, but the expression was significantly increased from 24 dph (days post-hatching) onwards and reached its peak at 27 dph. The results proved that the maturation of lymphoid organs was completed at 27 dph as the respective protein is involved in the V(D)J recombination, important for the maturation of lymphoid organs. A similar trend was also observed in the mRNA transcript levels of IgM gene and a significantly high expression was detected from 27 dph onwards. The present study suggested that the suitable time for vaccination in P. scalare could be taken at 27 dph, as the maturation and development of lymphoid organs is completed thus helps in stimulating the adaptive response of immunity against any pathogen.

C-C Motif Chemokine Receptor 7 Exacerbates Hypertension Through Effects on T Lymphocyte Trafficking.

Activated T lymphocytes that infiltrate blood pressure control organs make a critical contribution to the pathogenesis of hypertension. Dendritic cells act as potent antigen-presenting cells to stimulate prohypertensive T cells. However, the mechanisms that facilitate the recruitment of prohypertensive T cells and dendritic cells into the kidney's draining lymph node during hypertension require elucidation. As CCR7 (C-C motif chemokine receptor type 7) directs the homing of lymphocytes and dendritic cells into lymph nodes, we posited that dendritic cell-mediated T lymphocyte stimulation in the renal lymph node is CCR7 dependent and required for a full hypertensive response. We found that CCR7-deficient (CCR7 KO) mice had a blunted hypertensive response in our model of chronic Ang II (angiotensin II) infusion. Ang II-infused CCR7 KO animals had exaggerated accumulation of CD8+ T cells in the kidney but reduced numbers of CD4+ and CD8+ T cells in the kidney's draining lymph node. To understand whether CCR7-dependent homing of T lymphocytes or dendritic cells into the lymph node regulates the hypertensive response, we injected CCR7 KO or wild-type T cells or dendritic cells into CCR7 KO recipients, neither of which restored the full hypertensive response to Ang II infusion. However, adoptive transfer of wild-type but not CCR7 KO T lymphocytes into RAG1 (recombination-activating gene 1)-deficient mice that lack a lymphocyte niche restored full blood pressure elevation during Ang II infusion. Thus, CCR7-dependent interactions between T lymphocytes and dendritic cells are essential for T lymphocyte stimulation and hypertension accruing from inappropriate activation of the renin-angiotensin system.

Maternal immunity of tilapia broodstock vaccinated with polyvalent vaccine and resistance of their offspring againstStreptococcus agalactiae

This study aimed to examine the use of Streptococcus agalactiae polyvalent vaccine in tilapia broodstock and the effect of maternal immunity and resistance on their offspring against S. agalactiae strain. The broodstock was injected with polyvalent vaccine of S. agalactiae at a dose of 108 CFU per fish at 2nd gonadal maturity until spawning. Challenge test was carried out on the offspring at the 5, 10, 15 and 20 days after hatching using NK1, N17O, N14G, N3M, N4M strain respectively and combination of them. We observed immunological parameters in broodstock, eggs and larvae and relative per cent survival (RPS) of larvae after challenged with pathogenic S. agalactiae. The results showed that the leukocytes, phagocytic activity, respiratory burst, lysozyme activity and antibody levels of vaccinated broodstock had higher level compared with unvaccinated broodstock. The high level of the lysozyme activity, antibody levels and recombination activating gene 1 (RAG1) were also observed in eggs and larvae from vaccinated broodstock. Larvae produced from vaccinated broodstock when challenged with variety strain of pathogenic S. agalactiae had RPS value more than 50% until 20 days after hatching. In conclusion, polyvalent vaccine of S. agalactiae administrated in the broodstock could enhance immunity in the broodstock and protect their offspring from pathogenic S. agalactiae.

Biological significance of recombination-activating gene 1, RAG1 in optic nerve neuropathy

Biological significance of recombination-activating gene 1, RAG1 in optic nerve neuropathy

Mediator of DNA Damage Checkpoint Protein 1 Facilitates V(D)J Recombination in Cells Lacking DNA Repair Factor XLF.

DNA double-strand breaks (DSBs) trigger the Ataxia telangiectasia mutated (ATM)-dependent DNA damage response (DDR), which consists of histone H2AX, MDC1, RNF168, 53BP1, PTIP, RIF1, Rev7, and Shieldin. Early stages of B and T lymphocyte development are dependent on recombination activating gene (RAG)-induced DSBs that form the basis for further V(D)J recombination. Non-homologous end joining (NHEJ) pathway factors recognize, process, and ligate DSBs. Based on numerous loss-of-function studies, DDR factors were thought to be dispensable for the V(D)J recombination. In particular, mice lacking Mediator of DNA Damage Checkpoint Protein 1 (MDC1) possessed nearly wild-type levels of mature B and T lymphocytes in the spleen, thymus, and bone marrow. NHEJ factor XRCC4-like factor (XLF)/Cernunnos is functionally redundant with ATM, histone H2AX, and p53-binding protein 1 (53BP1) during the lymphocyte development in mice. Here, we genetically inactivated MDC1, XLF, or both MDC1 and XLF in murine vAbl pro-B cell lines and, using chromosomally integrated substrates, demonstrated that MDC1 stimulates the V(D)J recombination in cells lacking XLF. Moreover, combined inactivation of MDC1 and XLF in mice resulted in synthetic lethality. Together, these findings suggest that MDC1 and XLF are functionally redundant during the mouse development, in general, and the V(D)J recombination, in particular.

A Large Cohort of RAG1/2-Deficient SCID Patients-Clinical, Immunological, and Prognostic Analysis.

Severe combined immunodeficiency (SCID) is a fatal disorder resulting from various genetic defects. In the Middle East, where consanguineous marriage is prevalent, autosomal recessive mutations in recombination-activating genes (RAG) are a leading cause of SCID. We present a large cohort of SCID patients due to RAG1 or RAG2 mutations. Twenty-six patients with RAG1 or RAG2 deficiency, diagnosed at Sheba Medical Center, were retrospectively investigated. Clinical presentation, immunologic phenotype, genetic analysis, treatment, and outcome were analyzed. Majority of patients were referred from the Palestinian Authority. Most patients were males of Muslim Arab descent, 77% were born to consanguineous parents, and 65% had family history of immunodeficiency. Nearly all patients suffered from various infections before turning 2 months old, eight patients (31%) presented with Omenn and Omenn-like syndrome, and three patients (11%) had maternal engraftment. Notably, seven patients (27%) suffered from vaccine-derived infections, including a rare case of measles encephalitis. Nineteen patients underwent hematopoietic stem cell transplantation (HSCT) at a median age of 6 months, with a successful outcome for 72% of them. Genetic analysis revealed 11 different mutations (7 RAG2, 4 RAG1), two of them novel. Consanguineous marriages account for a genetic "founder effect." SCID is a pediatric emergency that dictates immediate precautions and curative treatment with HSCT. Due to lack of newborn screening for SCID within the Palestinian population, most patients in this cohort were diagnosed upon clinical symptoms, which led to a delayed diagnosis, harmful administration of contra-indicated live vaccines, delay in HSCT, and poor outcome.

Arsenic sulfide induces RAG1-dependent DNA damage for cell killing by inhibiting NFATc3 in gastric cancer cells

BackgroundArsenic sulfide was found to have potential anti-cancer activities, especially in gastric cancer. However, the underlying mechanism need to be further explored. This study was aimed to investigate the mechanism of arsenic compounds on gastric cancer.MethodsGastric cancer cell lines were infected with lentiviral vector carrying shNFATc3 and/or treated with arsenic sulfide. MTT assay were performed to assess cell growth. Flow cytometer assays were used to detect cell cycle and reactive oxygen species (ROS) level of gastric cancer cells. Western blot was carried out to detect nuclear factor of activated T-cells, cytoplasmic 3 (NFATc3), cell cycle markers, DNA damage pathway protein expression as well as other protein expression in gastric cancer cell lines. The expression of recombination activating gene 1 (RAG1) in gastric cancer cell lines was determined by RNA-sequencing analyses and Real-Time qPCR. The effect of NFATc3 on RAG1 were determined by CHIP-qPCR assay. The effect of arsenic sulfide on AGS cells was evaluated in vivo.ResultsWe show that arsenic sulfide as well as knockdown of NFATc3 resulted in increased double-strand DNA damage in gastric cancer cells by increasing the expression of RAG1, an endonuclease essential for immunoglobulin V(D) J recombination. Overexpression of NFATc3 blocked the expression of RAG1 expression and DNA damage induced by arsenic sulfide. Arsenic sulfide induced cellular oxidative stress to redistribute NFATc3, thereby inhibiting its transcriptional function, which can be reversed by N-acetyl-L-cysteine (NAC). We show that NFATc3 targets the promoter of RAG1 for transcriptional inhibition. We further showed that NFATc3 upregulation and RAG1 downregulation significantly associated with poor prognosis in patients with gastric cancer. Our in vivo experiments further confirmed that arsenic sulfide exerted cytotoxic activity against gastric cancer cells through inhibiting NFATc3 to activate RAG1 pathway.ConclusionThese results demonstrate that arsenic sulfide targets NFATc3 to induce double strand DNA break (DSB) for cell killing through activating RAG1 expression. Our results link arsenic compound to the regulation of DNA damage control and RAG1 expression as a mechanism for its cytotoxic effect.

Subclonal Variation of RAG-Mediated Diversification in B Cell Acute Lymphoblastic Leukemia

Background: Recombination-activating gene (RAG) mediates recombination of the immunoglobulin heavy chain variable gene (IGHV) in immature B lymphocytes. Aberrant targeting of RAG to non-IGH sites in B cell acute lymphoblastic leukemia (B-ALL) contributes to the development of driver mutations and clonal evolution (Papaemmanuil et al. Nat. Genet. 2014). This finding suggests that patients whose ALL involves increased RAG-mediated clonal diversification would exhibit more aggressive disease. As an initial step toward addressing this hypothesis, we asked whether the extent of RAG-mediated diversification in a patient's leukemia is consistent across all cells or variable between subclones. To assess variation in RAG-mediated subclone diversification, we interrogated rearranged IGH sequences from diagnostic B-ALL specimens to identify early subclones (SC) and to quantify RAG-derived sub-subclones (SSC) from each. We hypothesized that if RAG activity is a consistent feature of the leukemia, all SC within a single patient will have a comparable extent of SSC evolution. Methods: Amplicon-based IGHV sequencing identified the number of clonal IGH SC and their RAG-derived SSC in 22 pre-treatment adult and pediatric patients with newly-diagnosed B-ALL. Analysis was performed on peripheral blood (PB) for all patients in addition to diagnostic bone marrow (BM) for 16 of the 22 patients studied. Ultra-deep sequencing of IGH utilized 500 ng genomic DNA (representing approximately 80,000 cells/specimen) for the MiSeqDx platform, generating ~300 bp reads surrounding the VDJ junctional region. The NCBI IgBlast sequence analysis tool assigned IGH VDJ identities according to germline reference. Methods for determining subclones (SC) of shared clonal lineage involved classifying reads with a common Jh identity and 6 shared upstream Dh-Jh junctional nucleotides (termed "6N_Jx") according to defined methods (Gawad et al. Blood 2012). Further evolved sub-subclones (SCC) - as evidence of ongoing RAG activity - were defined by unique junctional sequences upstream of the common Dh-Jh junction (termed the "NDN" region). SSC were quantified according to their relative proportions within each SC family. Results: VDJ-rearranged SC families were detected for 20 of the 22 patients studied (median 2/patient; range 1-7); further analysis to assess variation in RAG-mediated diversification was limited to the 18 cases with ≥ 2 SC. In 9 of these 18, numerous evolved SSCs were identified from at least one SC in the specimen. In 4/9, there were starkly distinct levels of RAG-mediated diversity observed between intrapatient SC families, with some clonal precursors giving rise to numerous SSCs (up to 2,200 SSC per SC) while others showed minimal-to-no RAG-derived evolution (Table 1). Fifteen of 16 patients with matched BM and PB diagnostic specimens had detectable SCs. Of these, 73% (11/15) shared the dominant SC, while in 27% (4/15) the dominant SC differed between sites. However, regardless of which SC predominated, the extent of SSC diversification within each SC was preserved between the BM and the PB, with similar evolution patterns observed regardless of disease site. There was no relationship between SC read frequency and number of SSCs. Conclusions: Using deep sequencing of a single IGHV locus, wide variation in the extent of subclone diversification was observed in 4 of 20 patients with B-ALL. These findings indicate that the degree of RAG-mediated heterogeneity in B-ALL can range from minimal to extensive among distinct subclones in a single patient. The data underscore the relevance of single cell investigation of tumor characteristics to improving our understanding of the mechanisms of clonal evolution in lymphoid malignancies. Disclosures No relevant conflicts of interest to declare.

NK Cells from RAG- or DCLRE1C-Deficient Patients Inhibit HCMV.

The recombination-activating genes (RAGs) and the DNA cross-link repair 1C gene (DCLRE1C) encode the enzymes RAG1, RAG2 and Artemis. They are critical components of the V(D)J recombination machinery. V(D)J recombination is well known as a prerequisite for the development and antigen diversity of T and B cells. New findings suggested that RAG deficiency impacts the cellular fitness and function of murine NK cells. It is not known whether NK cells from severe combined immunodeficiency (SCID) patients with defective RAGs or DCLRE1C (RAGs−/DCLRE1C−-NK) are active against virus infections. Here, we evaluated the anti-HCMV activity of RAGs−/DCLRE1C−-NK cells. NK cells from six SCID patients were functional in inhibiting HCMV transmission between cells in vitro. We also investigated the expansion of HCMV-induced NK cell subset in the RAG- or DCLRE1C-deficient patients. A dynamic expansion of NKG2C+ NK cells in one RAG-2-deficient patient was observed post HCMV acute infection. Our study firstly reveals the antiviral activity of human RAGs−/ DCLRE1C−-NK cells.

Early exposure to environmental levels of sulfamethoxazole triggers immune and inflammatory response of healthy zebrafish larvae

Trace levels of antibiotics are increasingly being detected in aquatic environment and their potential toxicity to aquatic organisms is concerning. Sulfamethoxazole (SMX), a veterinary sulfonamide widely used across the globe, exists ubiquitously in aquatic environment with concentrations up to micrograms per liter. This study aims to investigate the effects of environmentally relevant levels (0.1, 1, 10, 100 μg/L) of SMX on the health of zebrafish during early development. Our results show that SMX delays the hatchment of embryos and reduces the body length. A dose-response relationship of oxidative stress indicators including total-antioxidant capacity (T-AOC), inducible nitric oxide synthase (iNOS) and total nitric oxide synthase (TNOS), catalase (CAT) has been observed. Additionally, SMX up-regulates the gene expression of several key proinflammatory cytokines and their corresponding proteins including interleukin-1β (IL-1β), interferon-γ (IFN-γ) and interleukin-11 (IL-11) and the expression of genes including interleukin-6 (il-6), tumor necrosis factor-α (tnf-α). This indicates that early exposure of SMX may evoke inflammation response in healthy fish. Inhibition of lysozyme and recombination-activating genes (rags) suggests that SMX suppresses the ability of zebrafish to resist pathogen. The reduction of the expression of Toll-like receptors (TLRs) related genes and significant correlations between TLRs and other immune-related genes reveal that TLRs might be an immunoregulator of SMX for zebrafish embryos and larvae. The novelty of this study lies in that early exposure to environmental levels of SMX not only affects the growth and development of zebrafish larvae, but also triggers oxidative stress and inflammation, resulting in a reduction in host immune defense via TLRs in healthy fish.

HIV‑1 integrase inhibitors targeting various DDE transposases: Retroviral integration versus RAG‑mediated recombination (Review).

Transposases are ubiquitous mobile genetic elements responsible for genome development, driving rearrangements, such as insertions, deletions and translocations. Across species evolution, some transposases are tamed by their host and are made part of complex cellular systems. The proliferation of retroviruses is also dependent on transposase related enzymes termed integrases. Recombination-activating gene protein (RAG)1 and metnase are just two examples of transposase domestication and together with retroviral integrases (INs), they belong to the DDE polynucleotidyl transferases superfamily. They share mechanistic and structural features linked to the RNase H-like fold, harboring a DDE(D) metal dependent catalytic motif. Recent antiretroviral compounds target the catalytic domain of integrase, but they also have the potential of inhibiting other related enzymes. In this review, we report the activity of different classes of integrase inhibitors on various DDE transposases. Computational simulations are useful to predict the extent of off-target activity and have been employed to study the interactions between RAG1 recombinase and compounds from three different pharmacologic classes. We demonstrate that strand-transfer inhibitors display a higher affinity towards the RAG1 RNase H domain, as suggested by experimental data compared to allosteric inhibitors. While interference with RAG1 and 2 recombination is associated with a negative impact on immune function, the inhibition of metnase or HTLV-1 integrase opens the way for the development of novel therapies for refractory cancers.

Kinks in the chain: examining recombination-activating gene scanning during V(D)J recombination.

Kinks in the chain: examining recombination-activating gene scanning during V(D)J recombination.

Genetic mutations and immunological features of severe combined immunodeficiency patients in Iran

BackgroundSevere combined immunodeficiency (SCID) is the most severe form of primary immunodeficiency disorders that is characterized by impaired early T lymphocyte differentiation and is variably associated with abnormal development of other lymphocyte lineages. SCID can be caused by mutations in more than 20 different genes. Molecular diagnosis in SCID patients contributes to genetic counseling, prenatal diagnosis, treatment modalities, and overall prognosis. In this cohort, the clinical, laboratory and genetic data related to Iranian SCID patients were comprehensively evaluated and efficiency of stepwise sequencing methods approach based on immunophenotype grouping was investigated MethodsClinical and laboratory data from 242 patients with SCID phenotype were evaluated. Molecular genetic analysis methods including Sanger sequencing, targeted gene panel and whole exome sequencing were performed on 62 patients. ResultsMortality rate was 78.9% in the cohort with a median follow-up of four months. The majority of the patients had a phenotype of T-NK-B+ (34.3%) and the most severe clinical manifestation and highest mortality rate were observed in T-NK-B- SCID cases. Genetic mutations were confirmed in 50 patients (80.6%), of which defects in recombination-activating genes (RAG1 and RAG2) were found in 16 patients (32.0%). The lowest level of CD4+ and CD8+ cells were observed in patients with ADA deficiency (p = 0.026) and IL2RG deficiency (p = 0.019), respectively. ConclusionCurrent findings suggest that candidate gene approach based on patient's immunophenotype might accelerate molecular diagnosis of SCID patients. Candidate gene selection should be done according to the frequency of disease-causing genes in different populations. Targeted gene panel, WES and WGS methods can be used for the cases which are not diagnosed using this method.

Mayaro Virus Replication Restriction and Induction of Muscular Inflammation in Mice Are Dependent on Age, Type-I Interferon Response, and Adaptive Immunity.

Mayaro virus (MAYV) is an emergent arbovirus first described in forest regions of the American continent, with recent and increasing notification of urban area circulation. Similar to Chikungunya (CHIKV) and other arthritogenic Alphavirus, MAYV-induced disease shows a high prevalence of persistent arthralgia, and myalgia. Despite this, knowledge regarding pathogenesis and characteristics of host immune response of MAYV infections are still limited. Here, using different ages of wild-type (WT), adult Type I Interferon receptor deficient (IFNAR–/–), and adult recombination activation gene-1 deficient (RAG–/–) mice, we have investigated the dependence of age, innate and adaptive immunity for the control of MAYV replication, tissue damage, and inflammation in mice. We have found that MAYV induces clinical signal and replicates in young WT mice, which gain the ability to restrict MAYV replication with aging. In addition, we observed that mice age and type I interferon response are related to restriction of MAYV infection and muscular inflammation in mice. Moreover, MAYV continues to replicate persistently in RAG–/– mice, being detected at blood and tissues 40 days post infection, indicating that adaptive immunity is essential to MAYV clearance. Despite chronic replication, infected adult RAG–/– mice did not develop an apparent signal of muscle damage in early and late infection. On the other hand, MAYV infection in young WT and adult IFNAR-/- mice triggers an increase in the expression of pro-inflammatory mediators, such as TNF, IL-6, KC, IL-1β, MCP-1, and RANTES, in muscle tissue, and decreases TGF-β expression, that were not significantly modulated in adult WT and RAG–/– mice. Taken together, our data demonstrated that age, innate and adaptive immunity are important to restrict MAYV replication and that adaptive immunity is also involved in MAYV-induced tissue damage. These results contribute to the comprehension of MAYV pathogenesis, and describe translational mice models for further studies of MAYV infection, vaccine tests, and therapeutic strategies against this virus.

Zika virus encephalitis in immunocompetent mice is dominated by innate immune cells and does not require T or B cells

BackgroundUntil the end of the twentieth century, Zika virus (ZIKV) was thought to cause a mostly mild, self-limiting disease in humans. However, as the geographic distribution of ZIKV has shifted, so too has its pathogenicity. Modern-day ZIKV infection is now known to cause encephalitis, acute disseminated encephalomyelitis, and Guillain-Barré syndrome in otherwise healthy adults. Nevertheless, the underlying pathogenetic mechanisms responsible for this shift in virulence remain unclear.MethodsHere, we investigated the contribution of the innate versus the adaptive immune response using a new mouse model involving intracranial infection of adult immunocompetent mice with a moderately low dose of ZIKV MR766. To determine the contribution of type I interferons (IFN-Is) and adaptive immune cells, we also studied mice deficient for the IFN-I receptor 1 (Ifnar1−/−) and recombination-activating gene 1 (Rag1−/−).ResultsWe show that intracranial infection with ZIKV resulted in lethal encephalitis. In wild-type mice, ZIKV remained restricted predominantly to the central nervous system (CNS) and infected neurons, whereas astrocytes and microglia were spared. Histological and molecular analysis revealed prominent activation of resident microglia and infiltrating monocytes that were accompanied by an expression of pro-inflammatory cytokines. The disease was independent of T and B cells. Importantly, unlike peripheral infection, IFN-Is modulated but did not protect from infection and lethal disease. Lack of IFN-I signaling resulted in spread of the virus, generalized inflammatory changes, and accelerated disease onset.ConclusionsUsing intracranial infection of immunocompetent wild-type mice with ZIKV, we demonstrate that in contrast to the peripheral immune system, the CNS is susceptible to infection and responds to ZIKV by initiating an antiviral immune response. This response is dominated by resident microglia and infiltrating monocytes and macrophages but does not require T or B cells. Unlike in the periphery, IFN-Is in the CNS cannot prevent the establishment of infection. Our findings show that ZIKV encephalitis in mice is dependent on the innate immune response, and adaptive immune cells play at most a minor role in disease pathogenesis.

Rag1 immunodeficiency-induced early aging and senescence in zebrafish are dependent on chronic inflammation and oxidative stress.

In mammals, recombination activating gene 1 (RAG1) plays a crucial role in adaptive immunity, generating a vast range of immunoglobulins. Rag1 −/− zebrafish (Danio rerio) are viable and reach adulthood without obvious signs of infectious disease in standard nonsterile conditions, suggesting that innate immunity could be enhanced to compensate for the lack of adaptive immunity. By using microarray analysis, we confirmed that the expression of immunity‐ and apoptosis‐related genes was increased in the rag1 −/− fish. This tool also allows us to notice alterations of the DNA repair and cell cycle mechanisms in rag1 −/− zebrafish. Several senescence and aging markers were analyzed. In addition to the lower lifespan of rag1 −/− zebrafish compared to their wild‐type (wt) siblings, rag1 −/− showed a higher incidence of cell cycle arrest and apoptosis, a greater amount of phosphorylated histone H2AX, oxidative stress and decline of the antioxidant mechanisms, an upregulated expression and activity of senescence‐related genes and senescence‐associated β‐galactosidase, respectively, diminished telomere length, and abnormal self‐renewal and repair capacities in the retina and liver. Metabolomic analysis also demonstrated clear differences between wt and rag1 −/− fish, as was the deficiency of the antioxidant metabolite l‐acetylcarnitine (ALCAR) in rag1 −/− fish. Therefore, Rag1 activity does not seem to be limited to V(D)J recombination but is also involved in senescence and aging. Furthermore, we confirmed the senolytic effect of ABT‐263, a known senolytic compound and, for the first time, the potential in vivo senolytic activity of the antioxidant agent ALCAR, suggesting that this metabolite is essential to avoid premature aging.

Up-regulation of syndecan-2 in proximal colon correlates with acute inflammation.

We previously reported that syndecan-2 expression is increased on the colonic epithelium during chronic inflammation. Here, we report that syndecan-2 exhibits a different pattern of site-specific colonic expression during acute inflammation. Syndecan-2 expression was up-regulated predominantly in the proximal colon of dextran sulfate sodium-induced colitis mice. The colitis-associated up-regulation of syndecan-2 was barely detected in Rag-1-/- (recombination activating gene 1 knockout) mice under colitis-inducing conditions. Increased syndecan-2 expression correlated with increased levels of infiltrated CD4+ IL-17A+ T cells in the proximal colon. Serum levels of IL-17A were increased during the acute inflammatory response in normal mice but not Rag-1-/- mice. IL-17A directly induced IL-17 receptor (IL-17RA) and syndecan-2 expression in ex vivo-cultured proximal colon tissues and adenoma cell lines from proximal colon. IL-17RA knockdown reduced the IL-17A-mediated syndecan-2 expression in SNU1235 cells. No elevation of syndecan-2 or IL-17RA was observed in colonic tissues from IL-17A-/- mice during colitis induction. Finally, increased expression of syndecan-2 and IL-17RA was observed in the proximal colons of cecal ligation and puncture-induced sepsis mice and infectious pan colitis patients. Together, these data suggest that acute inflammation induces syndecan-2 expression predominantly in the proximal colon via IL-17A-IL-17RA signaling during the early stage of the inflammatory response and that proximal colonic syndecan-2 might be a biomarker for acute inflammation.-Hong, H., Song, H.-K., Hwang, E. S., Lee, A. R., Han, D. S., Kim, S.-E., Oh, E.-S. Up-regulation of syndecan-2 in proximal colon correlates with acute inflammation.

DNA barcoding cannot discriminate between Sardinella tawilis and S. hualiensis (Clupeiformes: Clupeidae)

Sardinella tawilis, the only known freshwater sardinella in the world, is endemic to Taal Lake, Philippines. Previous studies found the Taiwan sardinella, S. hualiensis, to be morphologically very similar to S. tawilis and identified it as the marine sister species of S. tawilis. In this study, DNA barcoding using the mitochondrial cytochrome c oxidase I (COI) gene was carried out to analyze species demarcation in the Sardinella genus, focusing primarily on the relationship between S. tawilis and S. hualiensis. The neighbour-joining (NJ) tree that was constructed using Kimura 2-parameter (K2P) model showed a single clade for the two species with 100% bootstrap support. K2P interspecific genetic divergence ranged from 0% to 0.522%, which is clearly below the suggested 3–3.5% cutoff for species discrimination. Recombination activating gene 1 (RAG1), mitochondrial control region (CR), cytochrome b, 16S rRNA, and S7 markers were used to further validate the results. Sardinella tawilis and S. hualiensis clustered together with a bootstrap support of 99–100% in each of the NJ trees. Low interspecific genetic distances between S. tawilis and S. hualiensis for all the markers except CR could be attributed to incipient allopatric speciation.