Possible role of Toxoplasma gondii in brain cancer through modulation of host microRNAs

Background: The obligate intracellular protozoan parasite Toxoplasma gondii infects humans and other warm-blooded animals and establishes a chronic infection in the central nervous system after invasion. Studies showing a positive correlation between anti-Toxoplasma antibodies and incidences of brain cancer have led to the notion that Toxoplasma infections increase the risk of brain cancer. However, molecular events involved in Toxoplasma induced brain cancers are not well understood. Presentation of the hypothesis Toxoplasma gains control of host cell functions including proliferation and apoptosis by channelizing parasite proteins into the cell cytoplasm and some of the proteins are targeted to the host nucleus. Recent studies have shown that Toxoplasma is capable of manipulating host micro RNAs (miRNAs), which play a central role in post-transcriptional regulation of gene expression. Therefore, we hypothesize that Toxoplasma promotes brain carcinogenesis by altering the host miRNAome using parasitic proteins and/or miRNAs. Testing the hypothesis The miRNA expression profiles of brain cancer specimens obtained from patients infected with Toxoplasma could be analyzed and compared with that of normal tissues as well as brain cancer tissues from Toxoplasma uninfected individuals to identify dysregulated miRNAs in Toxoplasma-driven brain cancer cells. Identified miRNAs will be further confirmed by studying cancer related miRNA profiles of the different types of brain cells before and after Toxoplasma infection using cell lines and experimental animals. Expected outcome The miRNAs specifically associated with brain cancers that are caused by Toxoplasma infection will be identified. Implications of the hypothesis Toxoplasma infection may promote initiation and progression of cancer by modifying the miRNAome in brain cells. If this hypothesis is true, the outcome of this research would lead to the development of novel biomarkers and therapeutic tools against Toxoplasma driven brain cancers

Impact of SIV infection on mycobacterial lipid-reactive T cell responses in Bacillus Calmette-Guérin (BCG) inoculated macaques

BackgroundAlthough BCG vaccine protects infants from tuberculosis (TB), it has limited efficacy in adults against pulmonary TB. Further, HIV coinfection significantly increases the risk of developing active TB. In the lack of defined correlates of protection in TB disease, it is essential to explore immune responses beyond conventional CD4 T cells to gain a better understanding of the mechanisms of TB immunity.MethodsHere, we evaluated unconventional lipid-reactive T cell responses in cynomolgus macaques following aerosol BCG inoculation and examined the impact of subsequent SIV infection on these responses. Immune responses to cellular lipids of M. bovis and M. tuberculosis were examined ex vivo in peripheral blood and bronchioalveolar lavage (BAL).ResultsPrior to BCG inoculation, innate-like IFN-γ responses to mycobacterial lipids were observed in T cells. Aerosol BCG exposure induced an early increase in frequencies of BAL γδT cells, a dominant subset of lipid-reactive T cells, along with enhanced IL-7R and CXCR3 expression. Further, BCG exposure stimulated greater IFN-γ responses to mycobacterial lipids in peripheral blood and BAL, suggesting the induction of systemic and local Th1-type response in lipid-reactive T cells. Subsequent SIV infection resulted in a significant loss of IL-7R expression on blood and BAL γδT cells. Additionally, IFN-γ responses of mycobacterial lipid-reactive T cells in BAL fluid were significantly lower in SIV-infected macaques, while perforin production was maintained through chronic SIV infection.ConclusionsOverall, these data suggest that despite SIV-induced decline in IL-7R expression and IFN-γ production by mycobacterial lipid-reactive T cells, their cytolytic potential is maintained. A deeper understanding of anti-mycobacterial lipid-reactive T cell functions may inform novel approaches to enhance TB control in individuals with or without HIV infection

Paucity of CD4+ Natural Killer T (NKT) Lymphocytes in Sooty Mangabeys Is Associated with Lack of NKT Cell Depletion after SIV Infection

Lack of chronic immune activation in the presence of persistent viremia is a key feature that distinguishes nonpathogenic simian immunodeficiency virus (SIV) infection in natural hosts from pathogenic SIV and HIV infection. To elucidate novel mechanisms downmodulating immune activation in natural hosts of SIV infection, we investigated natural killer T (NKT) lymphocytes in sooty mangabeys. NKT lymphocytes are a potent immunoregulatory arm of the innate immune system that recognize glycolipid antigens presented on the nonpolymorphic MHC-class I-like CD1d molecules. In a cross-sectional analysis of 50 SIV-negative and 50 naturally SIV-infected sooty mangabeys, ligand α-galactosylceramide loaded CD1d tetramers co-staining with Vα24-positive invariant NKT lymphocytes were detected at frequencies ≥0.002% of circulating T lymphocytes in approximately half of the animals. In contrast to published reports in Asian macaques, sooty mangabey NKT lymphocytes consisted of CD8+ and CD4/CD8 double-negative T lymphocytes that were CXCR3-positive and CCR5-negative suggesting that they trafficked to sites of inflammation without being susceptible to SIV infection. Consistent with these findings, there was no difference in the frequency or phenotype of NKT lymphocytes between SIV-negative and SIV-infected sooty mangabeys. On stimulation with α-galactosylceramide loaded on human CD1d molecules, sooty mangabey NKT lymphocytes underwent degranulation and secreted IFN-γ, TNF-α, IL-2, IL-13, and IL-10, indicating the presence of both effector and immunoregulatory functional capabilities. The unique absence of CD4+ NKT lymphocytes in sooty mangabeys, combined with their IL-10 cytokine-secreting ability and preservation following SIV infection, raises the possibility that NKT lymphocytes might play a role in downmodulating immune activation in SIV-infected sooty mangabeys

Loss of Effector and Anti-Inflammatory Natural Killer T Lymphocyte Function in Pathogenic Simian Immunodeficiency Virus Infection

Chronic immune activation is a key determinant of AIDS progression in HIV-infected humans and simian immunodeficiency virus (SIV)-infected macaques but is singularly absent in SIV-infected natural hosts. To investigate whether natural killer T (NKT) lymphocytes contribute to the differential modulation of immune activation in AIDS-susceptible and AIDS-resistant hosts, we compared NKT function in macaques and sooty mangabeys in the absence and presence of SIV infection. Cynomolgus macaques had significantly higher frequencies of circulating invariant NKT lymphocytes compared to both rhesus macaques and AIDS-resistant sooty mangabeys. Despite this difference, mangabey NKT lymphocytes were functionally distinct from both macaque species in their ability to secrete significantly more IFN-γ, IL-13, and IL-17 in response to CD1d/α-galactosylceramide stimulation. While NKT number and function remained intact in SIV-infected mangabeys, there was a profound reduction in NKT activation-induced, but not mitogen-induced, secretion of IFN-γ, IL-2, IL-10, and TGF-β in SIV-infected macaques. SIV-infected macaques also showed a selective decline in CD4+ NKT lymphocytes which correlated significantly with an increase in circulating activated memory CD4+ T lymphocytes. Macaques with lower pre-infection NKT frequencies showed a significantly greater CD4+ T lymphocyte decline post SIV infection. The disparate effect of SIV infection on NKT function in mangabeys and macaques could be a manifestation of their differential susceptibility to AIDS. Alternately, these data also raise the possibility that loss of anti-inflammatory NKT function promotes chronic immune activation in pathogenic SIV infection, while intact NKT function helps to protect natural hosts from developing immunodeficiency and aberrant immune activation

Enhanced Th1/Th17 Functions of CD161+ CD8+ T Cells in Mucosal Tissues of Rhesus Macaques.

Expression of the C-type lectin-like receptor CD161 by human T cells is associated with type-17 responses, which play critical regulatory roles in immunity and inflammation at mucosal sites. However, the functions of CD161-expressing T cells in macaques, the pre-clinical model of several human diseases, remain unknown. This study examined the phenotypic and functional characteristics of CD161+ T cells in peripheral blood, mucosal tissues and lymph nodes of rhesus macaques. Majority of CD161-expressing T cells in peripheral blood and lung/intestinal mucosal tissues of rhesus macaques were found to be CD8+CD4- in phenotype. There was a significant enrichment of CD161+CD8+ T cells in the lungs and colonic mucosa (16.1%±6.6 and 16.8%±5.7) in comparison to peripheral blood (4.2%±1.2) and mesenteric lymph nodes (1.3%±0.8). Regardless of the tissue compartment, CD161+CD8+ T cells mainly comprised of γδ T cells and TCR Vα7.2+ MAIT cells (up to 80%), and displayed Th1 and Th17 cytokine responses to mitogen stimulation. Mucosal CD161+CD8+ T cells were characterized by very high expression of CD69, a recent activation marker that is preferentially expressed on tissue resident cells. Furthermore, lung and colonic mucosal CD161+CD8+ T cells showed enhanced IFN-γ, IL-17, and Perforin production in comparison to those in blood. Thus, macaque CD161+CD8+ T cells represent mucosal tissue-homing innate-like CD8+ T-cell populations with Th1/Th17 type cytokine and cytotoxic effector functions that can potentially enhance the recruitment of adaptive immune cells and control initial pathogen burden/dissemination in tissues. Analysis of their role in early immune responses to mucosal pathogens will be valuable in the design of vaccines and therapeutics

Innate CD161⁺ T cell cytokine secretion and cytotoxic response in blood and mucosal tissues.

<p>HMBPP-specific and fixed <i>E</i>.<i>coli</i>-induced secretion of IFN-γ (A) and Perforin (B) was analyzed in lymhocytes from PBMC, lung and colonic mucosa using enzyme-linked immunosorbent spot (ELISpot) assay. Enzyme-linked immunosorbent assay (ELISA) for TNF-α, IFN-γ, IL-17 and IL-13 in culture supernatants collected 16 hours following HMBPP (C) and <i>E</i>.<i>coli</i> (D) stimulation of lymhocytes from PBMC, lung and colonic mucosa. Data obtained from 6 rhesus macaques. Asterisks denote significant differences (p<0.05) calculated by the Wilcoxon matched-pairs signed rank test.</p

Trafficking marker expression profile of peripheral blood CD161⁺ CD8⁺ T cells in rhesus macaques.

<p>(A) Frequencies of expression of the trafficking markers α4β7, CD69, CXCR3, CXCR5, CCR5, CCR6, and IL-18Rα by <i>ex vivo</i> CD161⁺CD8⁺ T cells in the circulation of healthy rhesus macaques (Mean and SEM shown for data obtained from six animals). (B) Heat map showing relative expression of the chemokine receptors CCR5, CCR6, CXCR3, and CXCR5 generated using Prism 6 software. (C) Pie charts and arcs showing the proportion of different combinations of the chemokine receptors on CD161⁺CD8⁺ T cells as analyzed by Pestle and SPICE 5.3. (D) Boolean analysis showing the range and mean frequencies of CCR5, CCR6, CXCR3, and CXCR5 by CD161⁺CD8⁺ T cells in the 6 rhesus macaques.</p

Frequency and composition of CD161⁺ CD8⁺ T cells in tissues.

<p>(A) CD161⁺CD8⁺ T cell frequencies in matched samples from PBMC, mesenteric lymph nodes (mes LN), colon and lung tissue of rhesus macaques (n = 6). (B) CD69 expression on CD161⁺CD8⁺ T cells in PBMC, mes LN, colon and lung. (C) Frequencies of MAIT cells (CD161⁺ Vα7.2⁺ T cells), γδ T cells (pan TCR γδ⁺), and iNKT (PBS-57-loaded CD1d TM⁺) cells respectively in CD161⁺CD8⁺ T cells of un-infected rhesus macaques. Asterisks denote significant differences (p = 0.03) calculated by the Wilcoxon matched-pairs signed rank test.</p

CD161⁺ CD8⁺ T cell cytokine production in mucosal tissues.

<p>(A) Intracellular levels of TNF-α and IL-17 cytokines by mitogen-stimulated CD161⁺CD8⁺ T cells in PBMC, colon and lung of uninfected rhesus macaques (n = 5–6). Intracellular cytokine staining was performed on freshly isolated cells stimulated for 16h with PMA/Ionomycin. Data shows mean and SEM after subtraction of background in unstimulated cells incubated with medium alone. (B) Ratios of IL-17 to TNF-α in CD161⁺CD8⁺ T cells isolated from PBMC, colon and lung showing increase in Th17-type responses in mucosal tissues. Asterisks denote significant differences (p<0.05) calculated by the Wilcoxon matched-pairs signed rank test.</p