Project description:Toll like receptor 4 (TLR4), an innate immunity gene, is involved in responses to several pulmonary agonists including endotoxin (LPS; Poltorak et al.,1998), ozone (O3 ,Kleeberger et. al., 2001), Pseudomonas aeruginosa (Faure et al, 2004), and hyperoxia (Zhang et al, 2005). TLR4 appears to partially mediate the response to LPS- and O3-induced lung injury, however, TLR4 is protective for prevention of injury in Pseudomonas aeruginosa infection and against acute lung injury (hyperoxia). The mechanism behind this protection is unclear. We previously demonstrated that TLR4 was also protective against BHT-induced chronic inflammation and tumor promotion (Bauer et al, 2005). C.C3H-Tlr4Lps-d (BALBLps-d) mice, congenic for a 10 cM region of C3H/HeJ chromosome 4 that contains Tlr4 (Vogel et al, 1994), have a missence mutation that renders TLR4 dysfunctional. The Tlr4 mutation likely abrogates signaling by disrupting a direct point of contact with other signaling molecules (Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol 2004;4(7):499-511.). Bronchoalveolar lavage fluid (BALF) alveolar macrophages, lymphocytes, and total protein content were significantly elevated in BALBLps-d mice compared to BALB/c (BALB; Tlr4 sufficient) mice following chronic BHT (Bauer et al., 2005). BALBLps-d mice also had a significant increase in tumor multiplicity (60%) over that of BALB mice in response to an MCA/BHT tumor promotion protocol. While this was the first model to demonstrate a protective role for TLR4 in chronic lung inflammation and tumorigenesis, the downstream mechanism regulating this protective response remains unknown. Using Affymetrix microarray analysis followed by GeneSpring and Ingenuity pathway analyses, we herein identified known and novel downstream pathways and their interactions that may be involved in the protective mechanism elicited by TLR4. We therefore hypothesize that these pathways and interactions amongst the genes identified during the tumor promotion/chronic inflammation stage are in part influencing the differential strain response observed during tumorigenesis. Keywords: time course, tumor study Protocol 1 - 3 biological replicates after chronic dosing in each mouse strain Protocol 2 - multiple replicates after MCA/BHT tumor progression model used

Project description:Toll like receptor 4 (TLR4), an innate immunity gene, is involved in responses to several pulmonary agonists including endotoxin (LPS; Poltorak et al.,1998), ozone (O3 ,Kleeberger et. al., 2001), Pseudomonas aeruginosa (Faure et al, 2004), and hyperoxia (Zhang et al, 2005). TLR4 appears to partially mediate the response to LPS- and O3-induced lung injury, however, TLR4 is protective for prevention of injury in Pseudomonas aeruginosa infection and against acute lung injury (hyperoxia). The mechanism behind this protection is unclear. We previously demonstrated that TLR4 was also protective against BHT-induced chronic inflammation and tumor promotion (Bauer et al, 2005). C.C3H-Tlr4Lps-d (BALBLps-d) mice, congenic for a 10 cM region of C3H/HeJ chromosome 4 that contains Tlr4 (Vogel et al, 1994), have a missence mutation that renders TLR4 dysfunctional. The Tlr4 mutation likely abrogates signaling by disrupting a direct point of contact with other signaling molecules (Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol 2004;4(7):499-511.). Bronchoalveolar lavage fluid (BALF) alveolar macrophages, lymphocytes, and total protein content were significantly elevated in BALBLps-d mice compared to BALB/c (BALB; Tlr4 sufficient) mice following chronic BHT (Bauer et al., 2005). BALBLps-d mice also had a significant increase in tumor multiplicity (60%) over that of BALB mice in response to an MCA/BHT tumor promotion protocol. While this was the first model to demonstrate a protective role for TLR4 in chronic lung inflammation and tumorigenesis, the downstream mechanism regulating this protective response remains unknown. Using Affymetrix microarray analysis followed by GeneSpring and Ingenuity pathway analyses, we herein identified known and novel downstream pathways and their interactions that may be involved in the protective mechanism elicited by TLR4. We therefore hypothesize that these pathways and interactions amongst the genes identified during the tumor promotion/chronic inflammation stage are in part influencing the differential strain response observed during tumorigenesis. Keywords: time course, tumor study

Project description:Anti-tick vaccines have proved to be an effective and sustainable method for the control of tick infestations and tick-borne diseases with clear advantages over the application of chemical acaricides (Šmit and Postma, 2016; de la Fuente, 2018; Ndawula and Tabor, 2020). Moreover, the efficacy of acaricide application against Ornithodoros ticks is seriously limited owing to their endophilic/nidicolous life style, which make these ticks less accessible to the chemical acaricides (Astigaraga et al., 1995). Success in tick vaccine development is largely dependent on identification of new and highly protective tick antigens. Searching of new candidate protective antigens is currently being approached among tick molecules that play important biological functions at the tick-host interface, and more precisely among the salivary and intestinal proteins involved in biological processes specifically evolved by ticks to adapt to haematophagy (de la Fuente et al., 2016; Oleaga et al., 2021; Pérez-Sánchez et al., 2021). Accordingly, next-generation sequencing (NGS) and high-throughput proteomics technologies are been used to explore the transcriptome and proteome of the salivary glands/saliva and midguts of an increasing number of tick species and obtain the corresponding sialomes and mialomes (Chmelař et al., 2016; Almeida-Martins et al., 2020; Mans et al., 2020; Oleaga et al., 2021). These studies have identified a wealth of tick molecules related to tick haematophagy, tick-host interplay and pathogen transmission, which can then be scrutinized and filtered in vaccinomics pipelines for selecting candidate protective antigens (Chmelař et al., 2016; Maruyama et al., 2017; Antunes et al., 2018; de la Fuente et al., 2018; Ren et al., 2019; Couto et al., 2021). Similarly, we were also interested in characterizing the O. erraticus sialome. As far as O. erraticus saliva must contain all the bioactive molecules that the tick need to successfully feed, decoding its composition will lead to the discovery of new antigen targets for developing vaccines for the control and prevention of O. erraticus infestations and the diseases it transmits. Accordingly, the objective of the present work was to obtain the proteome of the saliva of O. erraticus adult ticks. For this, we have used a proteomics informed by transcriptomics approach to analyse female and male saliva separately using two different mass spectrometry approaches: liquid chromatography-tandem mass spectrometry (LC-MS/MS) in data-dependent acquisition (DDA) mode, and Sequential Window Acquisition of all Theoretical fragment ion spectra Mass Spectrometry (SWATH MS). SWATH MS is a specific variant of data-independent acquisition (DIA) methods that combines deep proteome coverage capabilities with quantitative consistency and accuracy (Ludwig et al., 2018). Here we reported the identification of 387 non-redundant proteins in the saliva of O. erraticus adult ticks as well as a qualitative and quantitative comparison of the saliva protein composition between both sexes. The integration of O. erraticus sialoproteomic and sialotranscriptomic datasets facilitate a better understanding of the physiology of feeding in O. erraticus and will drive the discovery of new and more effective antigen targets for development of anti-tick vaccines.

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