Os comportamentos para formação da hierarquia surgem em cães domésticos (Canis familiaris) nas primeiras semanas de vida, na forma de posturas e brincadeiras. Estes comportamentos são a base para o aprendizado de atitudes que os adultos terão para afirmar sua posição no ranking hierárquico do grupo. Características individuais dos animais, como a personalidade, podem influenciar seu sistema imunológico, pela ação, em grande parte, de glicocorticóides. Outro hormônio bastante importante na relação entre Sistema Nervoso e Sistema Imune, é a testosterona, e por isso, o gênero também é uma importante peça neste conjunto. Pesquisas recentes mostram estes fatores no contexto neuroimune, utilizando para isso modelos animais, principalmente roedores. Porém raros estudos foram realizados utilizando cães domésticos. Para estudar os efeitos de características da personalidade e gênero sobre parâmetros imunológico de cães domésticos, utilizamos 30 cães beagle (15 machos e 15 fêmeas). O comportamento foi observado, por meio de análise animal-focal, e determinamos seu perfil em relação ao ranking hierárquico. Foram avaliados o cortisol, a testosterona, a contagem de leucócitos totais e diferenciais, a atividade de neutrófilos (burst oxidativo e fagocitose) e a fenotipagem de linfócitos, no momento basal, após o estímulo estressor de transporte, e 15 dias após uma alteração de agrupamento. Os resultados mostram que, sem estímulos estressores, o burst oxidativo de neutrófilos é maior em fêmeas. O estímulo estressor do transporte aumenta a atividade de neutrófilos, independente do gênero. Foi observado que, sem estímulos estressores, o perfil de dominância não influencia os parâmetros analisados, porém após o transporte e o reagrupamento, animais com atitudes dominantes tiveram maior porcentagem e intensidade de fagocitose dos neutrófilos. Portanto, nosso trabalho, sendo pioneiro no assunto, mostra que estímulos estressores, o gênero e a personalidade do animal podem influenciar parâmetros imunológicos. Novas pesquisas devem ser realizadas a fim de elucidar o (s) mecanismo (s) de ação que promove (m) estes resultados e demais aspectos envolvidos nesta relação. / Behaviors leading to the establishment of hierarchy appear in domestic dogs (Canis familiaris) in the first weeks of life, in the form of postures and games. These behaviors are the basis for learning attitudes displayed by adults that assert their position in the hierarchical ranking of the group. Individual characteristics of animals, such as personality may influence their immune system, mainly by the action of glucocorticoids. Another very important hormone in the relationship between nervous system and immune system, called neuroimmunomodulation, is testosterone, and therefore, the gender is also an important part in this set. Recent research shows these factors in the context neuroimmune, employing animal models, especially in rodents. Nonetheless, few studies have been conducted using domestic dogs. To study the effects of personality traits and gender on immune parameters of dogs, we used 30 beagles (15 males and 15 females). Behavior was observed, assessed and recorded, by animal-focal analyses, and the profile of each individual animal in its group, based on hierarchy ranking was described. We evaluated cortisol, testosterone, total leukocyte counts, and differential activity of neutrophils (oxidative burst and phagocytosis) and lymphocyte phenotyping. These parameters were assessed at baseline, after the stressor (stimulus of transportation), and 15 days after a change of the groups. Results show that without stressors, oxidative burst of neutrophils is higher in females. The stressor stimulus of transportation increases the activity of neutrophils regardless of gender. Without stressors, the profile of dominance does not influence the parameters measured, but after transport and reassembly, animals with dominant attitudes had the highest percentage and intensity of phagocytosis in neutrophils. Therefore, our work, being a pioneer in this subject, shows that depending on stressor stimuli, gender and personality of the animal influence immunological parameters. Further research should be conducted to elucidate the mechanism(s) of action that promotes these results and other aspects involved in this relationship.

Influenza, também conhecida como gripe, é uma doença infecciosa viral que acomete um grande número de indivíduos anualmente, sendo responsável por um elevado número de internações e óbitos. O agente etiológico é o Myxovirus influenzae, vírus envelopado, de RNA de fita simples e polaridade negativa. A vacinação é a forma mais eficaz de se prevenir a infecção pelo vírus, no entanto, a capacidade produtiva dessa vacina não é suficiente para a vacinação da totalidade da população mundial, principalmente em casos de pandemia. Esse projeto teve por objetivo desenvolver uma vacina influenza (fragmentada e inativada) adjuvada, visando aumentar a capacidade produtiva dessa vacina no Instituto Butantan, que hoje é estimada em aproximadamente 40 milhões de doses por campanha. A utilização de adjuvantes na formulação da vacina influenza é capaz de produzir a mesma resposta imunológica protetora contra esse vírus, utilizando uma quantidade menor dos antígenos vacinais, aumentando a capacidade de produção da vacina em até quatro vezes. Foram estudadas 23 formulações adjuvantes utilizando o esqualeno como referência (formulação similar ao MF59®, adjuvante desenvolvido pela Novartis), vitaminas lipossolúveis (vitaminas A, D e E), vitamina B2 (vitamina hidrossolúvel), MPLA (monofosforil lipídio A, produzido pelo Instituto Butantan como subproduto da vacina pertussis low) e gel de hidróxido de alumínio. Para tanto, foram avaliadas a resposta imune conferida a camundongos BALB/c após imunização com diferentes formulações de vacina influenza (fragmentada e inativada) adjuvada e a existência, ou não, de toxicidade induzida pelas formulações vacinais estudadas. As formulações vacinais mais promissoras farão parte das formulações candidatas para realizações de ensaios clínicos. Os animais foram imunizados por via intraperitoneal com as formulações vacinais e foram colhidas amostras de sangue para ensaios sorológicos (inibição de hemaglutinação e ELISA) e células esplênicas para avaliação celular (dosagem de citocinas por citometria de fluxo: IL-2, IL-4, IL-6, IL-10, IL-17 TNF-α e INF-γ). Além disso, em um dos experimentos avaliou-se a formação de memória imunológica contra influenza, parâmetro importante em se pensando em uma vacina. Os três primeiros experimentos foram uma triagem a partir da qual selecionaram-se as melhores formulações que foram testadas no último experimento. Nele foram avaliados além da indução de resposta imune a toxicidade e a memória imunológica. Todas as 23 formulações estudadas induziram resposta minimamente protetora nos animais, com exceção da formulação contendo apenas MPLA como adjuvante. As formulações que se mostraram mais promissoras continham além do gel de AI(OH)3 MPLA de B. pertussis ou vitamina B2. Isso sem considerar o tocoferol (vitamina E), que embora tenha apresentado bons resultados acabou preterido em decorrência de sua potencial relação com casos de narcolepsia descritos na literatura. O teste de memória foi capaz de demonstrar que essas formulações produzem resposta de memória imunológica duradoura. Assim, tem-se resultados promissores para novos estudos pré-clínicos e clínicos com a vacina influenza (fragmentada e inativada) sazonal (trivalente). / Influenza, also known as flu, is a viral infectious disease that infects a large number of people annually, being responsible by large morbidity and mortality rates. The etiologic agent is the Myxovirus influenzae, an enveloped virus with single-stranded RNA and negative polarity. Vaccination is the best way to prevent the virus infection; however, the production capacity of this vaccine is not sufficient to vaccinate the entire world population, especially in cases of pandemics. This project aimed to develop an adjuvanted influenza vaccine (split and inactivated), increasing the productive capacity of this vaccine in Instituto Butantan, which is estimated in approximately 40 million of doses by campaign. Influenza vaccines formulated with adjuvants can produce the same protective immunological response against the virus using less amount of antigen increasing the production capacity of this vaccine up to four times. Twenty-three adjuvants containing fat-soluble vitamins (vitamins A, D and E), vitamin B2 (water-soluble vitamin), MPLA (monophosphoryl lipid A, produced by Instituto Butantan as a byproduct of pertussis low vaccine production) and aluminum hydroxide gel were studied. An adjuvant similar to MF59® (Novartis adjuvant) containing squalene was used as control. The immune response elicited in BALB/c mice after immunization with the different formulations of the influenza vaccine and the existence or not of toxicity induced by the vaccines formulations were studied. The most promising formulation will be part of the candidate formulations of clinicai trials. The animais received the vaccine formulations intraperitoneally and at specific days blood samples were taken to serological tests (hemagglutination inhibition and ELISA). At the end, they were euthanized to collect the spleens and splenic cells were cultivated to evaluate cytokines by flow cytometry: IL-2, IL-4, IL-6, IL-10, IL-17 TNF-α and INF-γ. Furthermore, in one experiment the immunological memory against influenza was evaluated, an important parameter to vaccines. The most promising formulations contained besides to alum either B. pertussis MPLA or B2 vitamin. Tocopherol (vitamin E) presented good results too, however it has a potential relationship with reported cases of narcolepsy. The memory test was able to demonstrate that these formulations induced long lasting immune memory response. Thus, these are promising results for new pre-clinical and clinical trials with seasonal trivalent influenza vaccine (split and inactivated).

Drosophila is involved in a wide range of interactions with parasites and pathogens (parasitoid wasps, bacteria, fungi, viruses). Drosophila hosts vary greatly at the species, population and individual level, in their response against such organisms, and much of this variation has a genetic basis. In this thesis I explored three aspects of this variation. First, using recombination mapping based on SNPs and a variation of bulk segregant analysis, I identified a QTL region on the right arm of the third chromosome of D. melanogaster associated with resistance to at least some of the parasitoid species / strains used in the experiments. The location of the QTL was further explored with deficiency complementation mapping and was narrowed down to the 96D1-97B1 region. The success of the deficiency mapping suggests that the resistant allele is not completely dominant. Second, I investigated patterns of molecular evolution in a set of immunity-related genes, using sequences from a D. melanogaster and a D. simulans population and a set of genes without known involvement in immunity for comparison. I found evidence that several of these genes have evolved under different selection pressure in each species, possibly indicating interactions with different parasites. The immunity genes tested appear to be evolving faster compared to non-immunity genes, supporting the idea that the immune system is evolving under strong selective pressure from parasites. Finally, in a D. melanogaster – sigma virus system, I measured genetic variation in the transmission of different virus genotypes, in different environments. There was poor correlation between temperatures, suggesting that environmental heterogeneity could constraint evolution of resistance (to virus transmission). The correlation between viral genotypes was also low, although relatively stronger for more closely phylogenetically related viral strains. Such interactions between host genotypes, virus genotypes and environmental conditions can maintain genetic variation in virus transmission.

Virus-host interactions determine virus transmissibility and virulence, and underlie coevolution that shapes interesting biological phenomena such as the genetic architecture of host resistance and host range. Characterization of the virus factors that exert selective pressure on the host, and the host genes which underlie resistance and adaptation against viruses will help to define the mechanistic pathways embroiled in host-virus coevolution. In this thesis, I describe the viral causes and host consequences of host-virus coevolution. These include genomic signatures consistent with antagonistic coevolution in antiviral RNA interference pathway genes such as high rates of positive selection and polymorphism, loci that underlie genetic variation in resistance to virus infection, and apparent conflict between NF-κB signalling and DNA virus infection. The RNA interference (RNAi) pathway is the most general innate immune pathway in insects, underlined by the observation that many viruses encode suppressors of RNAi (VSRs). The relationship between RNAi and VSRs has garnered attention as a plausible battleground for host-virus antagonistic coevolution, and genomic patterns in Drosophila support this hypothesis. However, genomic patterns in the N-terminal domain of the key RNAi effector gene, Argonaute-2, have not been described. In Chapter 2, I sequence the Argonaute-2 N-terminal domain using PacBio long-read sequencing technology to describe variation within and across Drosophila species, and test whether this variation is associated with resistance to Drosophila C Virus. The RNAi pathway evolves adaptively in Drosophila, but this has not been formally extended across invertebrate species. In Chapter 3, I quantify rates of adaptive protein evolution and describe evidence for selective sweeps in RNAi pathway genes using population genomic data from 8 insect and nematode species. These analyses indicate that RNAi genes involved in suppression of transposable elements and defence against viruses evolve rapidly across invertebrates, and I identify genes with signatures of elevated adaptation in multiple insect species. Host genes that underlie host-virus interactions have been described in RNA virus infection of Drosophila, however substantially less attention has focussed on the host response to DNA viruses, primarily because no DNA viruses have been isolated from Drosophila. In Chapter 4, I describe the isolation of Kallithea virus, a Drosophila dsDNA nudivirus, and characterise the host response to infection and genetic variation in resistance. I find that Kallithea virus infection causes early male-specific lethality, a cessation of oogenesis, and induction of undescribed virus-responsive genes. Further, I describe genetic variation in resistance and tolerance to Kallithea virus infection, and identify a potential causal variant for virus-induced mortality in Cip4. Insect viruses commonly encode viral suppressors of RNAi, however there are a multitude of antiviral immune mechanisms besides RNAi which may select for viral-encoded inhibitors. In Chapter 5, I describe the requirement for RNAi and NF-κB in immunity against Kallithea virus, and map gp83 as a virus-encoded inhibitor of NF-κB signalling. I find that gp83 inhibits Toll signalling at the level of, or downstream of NF-κB transcription factors, and that this immunosuppressive function is conserved in other nudiviruses.

DNA damage response (DDR) pathways are essential in maintaining genomic integrity in cells, but many DDR proteins have other important functions such as in the innate immune sensing of cytoplasmic DNA. Some DDR proteins are known to be beneficial or restrictive to viral infection, but most remain uncharacterised in this respect. Non-homologous end joining (NHEJ) is a mechanism of double stranded DNA (dsDNA) repair that functions to rapidly mend broken DNA ends. The NHEJ machinery is well characterised in the context of DDR but recent studies have linked the same proteins to innate immune DNA sensing and, hence, anti-viral responses. The aim of this thesis is to further investigate the interplay between herpes simplex virus 1 (HSV-1), a dsDNA virus, and two NHEJ proteins, DNA protein kinase catalytic subunit (DNA-PKcs) and paralogue of XRCC4 and XLF (PAXX). PAXX was first described in the literature as a NHEJ protein in 2015, but whether it has any role in the regulation of virus infection has not been established. Here we show that PAXX acts as a restriction factor for HSV-1 because PAXX-/- (KO) cells produce a consistently higher titre of HSV-1 than the respective wild type (WT) cells. We hypothesised that this could be due to a role of PAXX binding viral DNA and directly inhibiting HSV-1 replication or activating an anti-viral innate immune response. We have been able to, at least partially, rule out both of these initial hypotheses by showing that there was a reduced number of viral genomes present in KO cells during active lytic infection, and that an identical level of type I interferons are produced from WT and KO cells during HSV-1 infection. Although further characterisation of HSV-1 infection in WT and KO cells has not defined the molecular mechanism of restriction of HSV-1 by PAXX, we have uncovered a potential role for PAXX in mitogen-activated protein kinase (MAPK) signalling. In addition, and consistent with its function in restriction of HSV-1 infection, we show that infection with this virus in WT cells induces a loss of nuclear PAXX protein. Preliminary data suggest that these changes in localisation may occur as a result of stimulation of the cells with DNA, but not the RNA analogue poly(I:C). The role of PAXX in the regulation of HSV-1 infection in vivo was investigated by studying KO mice. Despite previous observations that mice lacking NHEJ proteins have brain defects related to autoinflammatory pathology, there were no obvious defects in the development of Paxx-/- mice, and they had brains of normal weight. No significant difference in viral spread or viral protein expression was observed between WT and KO HSV-1 infected mice, and KO mice did not exhibit abnormal pathology. There were, however, small but significant differences in the cellular immune response to infection which might be explained by reduced MAPK signalling in KO cells. DNA-PKcs is another component of the NHEJ machinery that acts to assist in dsDNA break repair in the nucleus and as an innate sensor of cytoplasmic viral DNA, but the effect of DNA-PKcs on HSV-1 infection has not been fully explored. Murine skin fibroblasts (MSFs) derived from wild type and PRKDC-/- (DNA-PKcs deficient) mice were cultured ex vivo and used for innate immune studies. Although HSV-1 was able to infect and stimulate these cells, no differences in the stimulation of innate immune gene expression between the two genotypes was observed, suggesting that DNA-PKcs does not contribute to HSV-1 sensing in MSFs. It has previously been reported that the HSV-1 protein ICP0 targets DNA-PKcs for degradation, although the reason for this is unknown. We confirmed these data, although found it to be cell-type specific and explored this interaction further using PRKDC-/- RPE-1 cells created using CRISPR/Cas9. HSV-1 infection in these cells followed unusual dynamics, and the development of cytopathic effect was accelerated as compared to WT cells. Together these observations confirm that DNA-PKcs regulates HSV-1 infection, but more work is required to fully understand the mechanisms involved.

Rules of diplomatic immunity, which nowadays are enshrined in the Vienna Convention on Diplomatic Relations, play an important role in interstate diplomacy because they ensure the efficient performance of diplomatic functions. This thesis investigates a particular form of diplomatic immunity - diplomatic immunity ratione materiae. Unlike diplomatic immunity ratione personae, which pertains to the personal status of a diplomatic agent, diplomatic immunity ratione materiae depends in essence on the official nature of a particular act In practice, however, the determination of diplomatic immunity ratione materiae may meet with many conceptual and practical difficulties. For one, it is not always easy to distinguish the official acts of a diplomatic agent, who represents the sending State in the receiving State, from his or her private acts. In case of disagreement between the two States, questions may also arise as to who has the authority to make a final determination. The Vienna Convention does not offer much guidance on these issues; on the contrary, the Convention complicates them by employing, without adequate explanation, distinct formulas for different kinds of diplomatic immunity ratione materiae. This thesis examines these formulas in detail. On a general level, it is submitted that diplomatic immunity ratione materiae for certain types of activity constitutes not only a procedural bar to court proceedings but also a substantive exemption of individual responsibility. More specifically, it is argued that each formula must be understood in the light of the rationale behind immunity, the type of immunity concerned, and the specific functions or duties performed. In case of controversy, weight should be given to the opinion of the sending State, although the authority to make a decision lies ultimately with the court of the receiving State.

La Trypanosomose Humaine Africaine (THA) ou maladie du sommeil est une infection provoquée par un protozoaire du genre Trypanosoma. La recherche de nouvelles cibles thérapeutiques est nécessaire afin d’améliorer l’efficacité et la tolérance des traitements. Dans un premier travail, nous avons étudié l’importance de CYP51 (stérol 14α-déméthylase), une cible potentielle, par la technique d’interférence à ARN (RNAi). Nous avons démontré le caractère essentiel de cette enzyme pour le parasite, ainsi que les conséquences de sa déplétion sur la cytodiérèse. De plus, la survie de souris infectées par la souche CYP51RNAi induite était prolongée, montrant l’implication de CYP51 dans la virulence. La combinaison du posaconazole, un dérivé triazolé inhibant CYP51, à l’éflornithine a montré un effet similaire à la combinaison nifurtimox-éflornithine dans un modèle murin. Nos résultats soulignent l’intérêt potentiel d’un traitement ciblant CYP51 dans la trypanosomose. Du fait de l’importance de l’immunodépression dans la THA et de la capacité du trypanosome à échapper au système immunitaire de l’hôte, nous avons étudié, dans un deuxième travail, l’effet de T. gambiense et de son sécrétome (protéines excrétées/sécrétées) sur des cellules dendritiques humaines (DCs) in vitro. Nous avons ainsi montré une altération de la maturation des DCs induite par le LPS en présence du sécrétome. Nous avons également montré qu’une des protéines de ce sécrétome, TbKHC1, est exprimée par différentes espèces de trypanosomes. Elle est impliquée dans l’induction de l’arginase macrophagique chez la souris, un mécanisme d’échappement au système immunitaire. Ces travaux apportent des éléments pour une meilleure compréhension des phénomènes immunopathologiques rencontrés, dans la perspective de thérapeutiques ciblées et d’une approche vaccinale. / Trypanosoma brucei gambiense, an extracellular eukaryotic flagellate parasite, is the main causative agent of Human African Trypanosomiasis (HAT), also known as sleeping sickness. Trypanosomes have developped efficient mechanisms to escape the host immune response. New therapeutic options are needed for patients with HAT. Sterol 14α-demethylase (CYP51) is a potential drug target but its essentiality has not been studied in T. brucei. In a first study, we demonstrated its essentiality by RNA interference (CYP51RNAi) in vitro. CYP51RNAi induction caused morphological defects with multiflagellated cells, suggesting cytokinesis dysfunction. Additionally, the survival of CYP51RNAi infected-mice was improved, showing CYP51 RNAi effect on trypanosomal virulence. During infection with virulent strains, posaconazole-eflornithine and nifurtimox-eflornithine combinations showed similar improvement in mice survival. Thus, our results provide support for a CYP51 targeting based treatment in HAT. In a second work, we studied the innate host immune system characteristics in trypanosomiasis, as a severe immune dysregulation is present in HAT. To analyse the potential immunomodulatory activity of T. gambiense in human settings, we assess the effect of its secretome on dendritic cells (DCs) in vitro, using human monocyte-derived DCs. A significant inhibition of the LPS-induced maturation of DCs was observed with secretome. In line with this impairment, secretome down regulated cytokines production by LPS-activated DCs. TbKHC1, a kinesin heavy chain, is a component of the parasite secretome. We confirmed its role in parasitic escape to immune system by inducing arginase activity, in a murine model. Our results provide new information about the immune system characteristics during trypanosomiasis, which may help to uncover new therapeutic approachs in HAT.

O rompimento eritrocítico característico do ciclo de vida do Plasmodium resulta na liberação de ATP, o qual é reconhecido pelo receptor P2X7. Este estudo da malária experimental causada pelo P. chabaudi em camundongos P2X7-/- mostraram que estes animais são mais suscetíveis à infecção. Tanto in vitro, como ex vivo, as populações celulares fagocíticas mostraram-se sensíveis ao ATP extracelular, de uma forma P2X7 dependente, e a infecção pelo P. chabaudi mostrou ser um fator que aumenta esta sensibilidade. Estudos fenotípicos do baço e do fígado dos animais P2X7-/- indicaram que, com exceção da população CD11b+ Ly6G+, estes animais apresentam número inferior de células fagocíticas quando comparados aos animais C57BL/6. Além disso, há uma leve deficiência na ativação dos fagócitos, na produção de IFN-g e no número de células produtoras de IFN-g, TNF-a e IL-10 no baço dos animais P2X7-/- em relação aos C57BL/6. A maior suscetibilidade dos camundongos P2X7-/- à infecção pelo P. chabaudi deve-se, portanto, a uma resposta imunológica deficiente nestes animais. / The erythrocyte rupture is a step of the life cycle of Plasmodium in which ATP is released and is recognized by the P2X7. This study of experimental malaria caused by P. chabaudi in P2X7-/- mice showed that these mice are more susceptible to the infection. Either in vitro or ex vivo, the phagocyte cell populations were sensible to extracellular ATP in a P2X7-dependent way and the P. chabaudi infection acted as a component that increases this sensitivity. Phenotypic analysis of the spleen and liver from P2X7-/- mice showed that, excepted for the CD11b+ Ly6G+ population, these mice have a lower number of phagocyte cells when compared to C57BL/6 mice. Furthermore, there was a slight deficiency in phagocyte activation, IFN-g production and number of cells that produce important cytokines as IFN-g, IL-10 and TNF-a in P2X7-/- mice in comparison with C57BL/6 mice. The increased susceptibility of P2X7-/- mice to the P. chabaudi infection is due to a deficient immune response.

Control of retrotransposon expression in the mammalian germline is regulated by Argonaute family PIWI proteins and their associated small non-coding RNAs known as PIWI-interacting RNAs (piRNAs). To date, no study has demonstrated clear PIWI protein expression nor identified a cellular function(s) for PIWI proteins in the mammalian soma. In contrast to the germline-restricted expression of piRNA associated proteins, we observed that Miwi2 mRNA was induced specifically in epithelial cells during pneumococcal pneumonia. Further investigation showed that similar to its mRNA, MIWI2 protein was indeed expressed outside of the mammalian germline, and was localized to the cytoplasm of a discrete population of multiciliated lung epithelial cells. Immunoprecipitation of MIWI2 from whole lung lysates indicated that it was bound to a small RNA that was longer than a traditional piRNA. Microarray analysis revealed that depletion of MIWI2 in a murine epithelial cell line or in a whole animal model had no effect on retrotransposon expression, further suggesting that lung MIWI2 is independent of nuclear piRNA silencing pathways. Under basal conditions, MIWI2 was required for the normal maintenance of airway epithelial cell fate. In fact, Miwi2 deficiency resulted in an increase in club cells and decrease in ciliated cells indicating that MIWI2 could play a primary role in mucociliary homeostasis or clearance. Similarly, as MIWI2 is induced during lung infection we sought to determine if it participated in host innate immune responses to bacterial infection. Using a clinically relevant model of community acquired pneumonia, Miwi2 deficient mice exhibited an increased expression of inflammatory mediators and immune cell recruitment thus leading to enhanced bacterial clearance. Taken together, these data support the notion that MIWI2 exerts piRNA-independent functions outside of the germline in the ciliated lung epithelium to regulate innate immunity during pneumonia. More broadly, these studies shed light on new areas in PIWI protein and lung ciliated cell biology, and may have implications for multiple diseases including cancer, inflammatory disorders, and infectious diseases.

Despite the convergence of rapid technological advances in genomics and the maturing field of ecoimmunology, our understanding of the genes that regulate immunity in wild populations is still nascent. Previous work to assess immune function has relied upon relatively crude measures of immunocompetence. However, with next-generation RNA-sequencing, it is now possible to create a profile of gene expression in response to an immune challenge. In this study, captive zebra finch (Taeniopygia guttata; adult males) were challenged with bacterial lipopolysaccharide (2 mg/Kg BW; dissolved in 0.9% saline) or vehicle (0.9% saline) to stimulate the immune system. Two hours after injection, birds were euthanized and hypothalami, spleen, and red blood cells (RBCs) were collected. Taking advantage of the fully sequenced genome of zebra finch, total RNA was isolated, sequenced, and partially annotated in these tissue/cells. The data show 628 significantly upregulated transcripts in the hypothalamus, as well as 439 and 121 in the spleen and RBCs, respectively, relative to controls. Also, 134 transcripts in the hypothalamus, 517 in the spleen, and 61 in the RBCs were significantly downregulated. More specifically, a number of immunity-related transcripts (e.g., IL-1β, RSAD2, SOCS3) were upregulated among tissues/cells. Additionally, transcripts involved in metabolic processes (APOD, LRAT, RBP4) were downregulated, suggesting a potential trade-off in expression of genes that regulate immunity and metabolism. Unlike mammals, birds have nucleated RBCs, and these results suggest a novel transcriptomic response of RBCs to immune challenge. Lastly, molecular biomarkers could be developed to rapidly screen bird populations by simple blood sampling in the field.