Progress 11/15/17 to 11/14/21
Outputs
Target Audience:Peers, academia and scientists. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?To learn more about diet and microbiome interactions, research staff attended the Diet & Optimum Health Conference/Linus Pauling Institute International Conference and Nutrition 2020 Live Online conference in June 2020. An undergraduate student has used results from her research project that was derived from our two animal studies to develop her senior honors thesis. How have the results been disseminated to communities of interest?Results were presented as 2 virtual posters at the Nutrition 2020 Live Online conference in June 2020. In addition, several publications have been published with 2 other publications in preparation. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Completion of feeding studies described in Objective 1 and 2 Due to the limitation in the number of aged mice we can acquire from National Institute of Aging, we have divided the proposed animal study described in Objective 1 into two separate mouse studies. The feeding study was completed in October 2018, and various tissues and fecal materials were collected for downstream analyses. Determined zinc status in young and old mice in zinc supplementation study Serum zinc levels were measured by ICP-OES in mice from the zinc supplementation study. Old mice had significant decrease in serum zinc despite being fed a zinc adequate diet. Zinc supplementation restored their serum zinc levels to those comparable to young mice. Measured systemic inflammatory response in young and old mice in zinc supplementation study The levels of six cytokines associated with inflammation (IL12, TNFa, IFNg, MCP1, IL10, and IL6) were determined in plasma samples from mice in the zin supplementation study. In agreement with published reports, we observed age-associated increase in plasma MCP1. Zinc supplementation had a significant effect in decreasing MCP1 levels in old mice Determined peripheral and mucosal Th1/Th2/Th17 cytokine response and changes in immune phenotypes in young and old mice in zinc supplementation study We collected mesenteric and inguinol lymph nodes from individual mouse at the time of necropsy from mice in zinc supplementation study. Lymphocytes were treated ex vivo with PMA/ionomycin or anti-CD3/CD28 to stimulate T cell proliferation and cytokine production. Differences in the quality and quantity of Th1/Th2/Th17 cytokines produced from lymphocytes derived from different age and treatment groups allow us to determine the effects of age and zinc status on two different immune sites (mesenteric and inguinol lymph nodes) that reflects alterations in mucosal and peripheral immune responses, respectively. The levels of seven cytokine (IL10, IL17, TNFa, IFNg, IL6, IL4, and IL2)l were measured from culture supernatants after immune cell stimulation. We observed significant effect of age in altering the immune profile, where age mice had increased IL17, TNFa, and IFNg production, and decreased IL2 production. Zinc supplementation had significant decrease or trend in reducing age-related increase in IL17, TNFa, and IFNg production, but had no effect on IL2 production. Using flow cytometry, we also examined the effects of age and zinc status on the naïve and memory CD4+ and CD8+ T cells in the lymph nodes using CD4, CD8, CD62L, and CD44 as markers to distinguish the different T cell subsets. With age, there was an expansion in memory T cell population, as defined by CD62LlowCD44high expression, and a decline in naïve T cell population, as defined by CD62LhighCD44low expression. Zinc supplementation had a significant effect in decreasing age-related increase in memory T cell population, with concomitant increase in naïve T cell populations. Old mice had reduced zinc and increased proinflammatory cytokines MCP1 and IL6 in the serum, increased Th1/Th17/inflammatory cytokines (IFNg, IL17, TNFa, respectively) and decreased naïve CD4 T-cells in the mesenteric lymph nodes (MLN). ZS significantly increased serum zinc levels, decreased TNFa, IFNg, IL17 in MLN, and increased naïve T-cell populations in aged mice. MZD further reduced serum zinc and increased serum IL6 levels in aged mice. Conclusion: ZS improved the immune function of aged mice and reduced inflammatory response, and MZD further increased age-related inflammation. Our data suggest that zinc status is an important contributing factor in age-related immune dysfunction and chronic inflammation. 16S microbiome analysis We purified fecal DNA samples from young and old mice from the zinc supplementation study. Purified fecal DNA (151 samples total) were prepared from fecal materials collected at the beginning of the study (wk 0 baseline), end of the study (wk 6), as well as colon and cecum contents (at necropsy). Using barcoded, degenerate PCR primers specific against 16S rRNA, we PCR amplified 16S amplicons from all 151 samples, and submitted them for amplicon sequencing using Illumina MiSEQ at the Center for Genome Research and Biocomputing core facility at Oregon State University. Identified differentially abundant sequence variants in zinc supplemented mice versus control mice We trimmed and filtered the 16S reads based on quality. We assigned taxonomic designations to each of the sequence variants, and determined the differentially abundant taxa based on diet (supplemented vs. adequate), age (young vs old), and timepoint (week 0 vs week 6). We identified 206 unique sequence variants in the filtered dataset. Beta diversity analysis using robust principle component analysis indicate that Age is the covariate associated with the first principle component axis. This result indicates that, in this study, age shows the biggest effect on the microbiome when taken as a whole. Over the timecourse series, few statistically significant differences were seen. Age shows a significant effect on overall microbial composition in the gut, according to a PERMANOVA test and a permutation test, regardless of zinc status. The primary microbial constituents, on average above 50% of the amplified sequences, were from either Bacteroides or Parabacteroides for young or old mice, respectively. Wilcoxon Rank Sum tests of each genus (ASVs agglomerated to the genus level) indicated that of the 124 genera in the study, 85 have a significant difference between the age groups. A follow-up Kruskal-Wallis test within each age group showed no significant results for the diet treatments. We also confirmed that, regardless of taxonomic level, we saw significant differences between the mice of different ages, including six of the seven phyla in the study. As a way to confirm the age effects, as well as explore diet and effects of host physiology associated with changes in the gut microbiome, we used a novel phylogenetic analysis approach called ClaaTU. In this approach, we apply a phylogenetic agglomeration method, independent of the assigned taxonomic ranks. This approach allows us to resolve relationships previously impossible, as we are otherwise restricted to the already defined ranks. We were able to confirm that age has the largest effect on the microbiome content, as well as identify several clades that have significant diet, zinc, and inflammation marker effects. Conclusion: Age effects overwhelm other effects on the mouse gut microbiome. Overall gut microbiome content is significantly different between the young and old mice, and individually, around 75% of the identified ASVs showed a significant difference between the age groups. A phylogenetic-based approach confirms the age effect, as well as allowed us to identify several previously uncovered diet effects. Changing a single micronutrient such as zinc shows smaller effects that, when paired with a split study design, are difficult to measure at the levels of the ASV or broad beta diversity. 7. Short chain fatty acid analysis Short chain fatty acid analyses (SCFA) were done using samples from both animal studies. We were able to detect and quantitate acetate, propionate, and butyrate from colon and cecum contents from our animals. Acetate levels in the cecum and colon showed changes associated with zinc restriction or supplementation in the diet. We are integrating these data with our microbiome data to explore correlation between changes in SCFA and microbiome (ASVs).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Gaulke CA, Rolshoven J, Wong CP, Hudson LG, Ho E, Sharpton TJ: Marginal Zinc Deficiency and Environmentally Relevant Concentrations of Arsenic Elicit Combined Effects on the Gut Microbiome. mSphere 2018, 3(6):e00521-18. doi: 10.1128/mSphere.00521-18
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Wong CP, Magnusson KR, Sharpton TJ, Ho E: Effects of zinc status on age-related T cell dysfunction and chronic inflammation. Biometals 2021, 34:291-301. doi: 10.1007/s10534-020-00279-5
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
Linus Pauling Institute International Conference 2019. Effects of zinc status and aging on age-related immune dysfunction and chronic inflammation.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
Linus Pauling Institute International Conference 2019. Zinc Status Elicits Age-Dependent Effects in the Gut Microbiome.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
American Society of Nutrition Conference 2020. Effects of Zinc Status and Aging on Age-Related Immune Dysfunction and Chronic Inflammation.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
American Society of Nutrition Conference 2020. Zinc Status Elicits Age-Dependent Effects in the Gut Microbiome.
|
Progress 11/15/19 to 11/14/20
Outputs
Target Audience:Peer scientists Changes/Problems:No cost extension was requested due to delay in research due to COVID What opportunities for training and professional development has the project provided?To learn more about diet and microbiome interactions, research staff attended the Nutrition 2020 Live Online conference in June 2020. An undergraduate student has used results from her research project that was derived from our two animal studies to develop her undergraduate research internship. How have the results been disseminated to communities of interest?Results were presented as 2 virtual posters at the Nutrition 2020 Live Online conference in June 2020. In addition, we have submitted a manuscript that described our findings related to zinc status and immune inflammatory response from our two animal studies to the journal Biometals. Manuscript has been reviewed, and is currently under revision, and will be resubmitted in November 2020 What do you plan to do during the next reporting period to accomplish the goals? We have plans to perform additional data integration to more closely examine interrelationships between microbiome, inflammatory parameters and other metadata. We will also focus on manuscript submissions for the coming year. We have nearly completed all analyses of the ASVs associated with age and diet. We are currently exploring significant differences correlating phylogenetic placement and ASV abundance, which cannot be determined using previously defined taxonomic designations as described above. After integrating these two synergistic methods of analysis of the microbiome data, a manuscript will be prepared and submitted.
Impacts
What was accomplished under these goals?
Completion of feeding studies described in Objective 1 and 2 Due to the limitation in the number of aged mice we can acquire from National Institute of Aging, we have divided the proposed animal study described in Objective 1 into two separate mouse studies. In the first mouse study (zinc supplementation study), groups of young (2mo) and old (24mo) mice were fed zinc adequate diet (30mg/kg zinc) or zinc supplemented diet (300mg/kg zinc) for 6wks. The feeding study was completed in June 2018, and various tissues and fecal materials were collected for downstream analyses. In the second mouse study (zinc deficiency study), groups of young (2mo) and old (24mo) mice were fed zinc adequate diet (30mg/kg zinc) or zinc deficient diet (6mg/kg zinc) for 6wks. The feeding study was completed in October 2018, and various tissues and fecal materials were collected for downstream analyses. Determined zinc status in young and old mice in zinc supplementation study Serum zinc levels were measured by ICP-OES in mice from the zinc supplementation study. Old mice had significant decrease in serum zinc despite being fed a zinc adequate diet. Zinc supplementation restored their serum zinc levels to those comparable to young mice. Measured systemic inflammatory response in young and old mice in zinc supplementation study The levels of six cytokines associated with inflammation (IL12, TNFa, IFNg, MCP1, IL10, and IL6) were determined in plasma samples from mice in the zin supplementation study. In agreement with published reports, we observed age-associated increase in plasma MCP1. Zinc supplementation had a significant effect in decreasing MCP1 levels in old mice Determined peripheral and mucosal Th1/Th2/Th17 cytokine response and changes in immune phenotypes in young and old mice in zinc supplementation study We collected mesenteric and inguinol lymph nodes from individual mouse at the time of necropsy from mice in zinc supplementation study. Lymphocytes were treated ex vivo with PMA/ionomycin or anti-CD3/CD28 to stimulate T cell proliferation and cytokine production. Differences in the quality and quantity of Th1/Th2/Th17 cytokines produced from lymphocytes derived from different age and treatment groups allow us to determine the effects of age and zinc status on two different immune sites (mesenteric and inguinol lymph nodes) that reflects alterations in mucosal and peripheral immune responses, respectively. The levels of seven cytokine (IL10, IL17, TNFa, IFNg, IL6, IL4, and IL2)l were measured from culture supernatants after immune cell stimulation. We observed significant effect of age in altering the immune profile, where age mice had increased IL17, TNFa, and IFNg production, and decreased IL2 production. Zinc supplementation had significant decrease or trend in reducing age-related increase in IL17, TNFa, and IFNg production, but had no effect on IL2 production. Using flow cytometry, we also examined the effects of age and zinc status on the naïve and memory CD4+ and CD8+ T cells in the lymph nodes using CD4, CD8, CD62L, and CD44 as markers to distinguish the different T cell subsets. With age, there was an expansion in memory T cell population, as defined by CD62LlowCD44high expression, and a decline in naïve T cell population, as defined by CD62LhighCD44low expression. Zinc supplementation had a significant effect in decreasing age-related increase in memory T cell population, with concomitant increase in naïve T cell populations. Old mice had reduced zinc and increased proinflammatory cytokines MCP1 and IL6 in the serum, increased Th1/Th17/inflammatory cytokines (IFNg, IL17, TNFa, respectively) and decreased naïve CD4 T-cells in the mesenteric lymph nodes (MLN). ZS significantly increased serum zinc levels, decreased TNFa, IFNg, IL17 in MLN, and increased naïve T-cell populations in aged mice. MZD further reduced serum zinc and increased serum IL6 levels in aged mice. Conclusion: ZS improved the immune function of aged mice and reduced inflammatory response, and MZD further increased age-related inflammation. Our data suggest that zinc status is an important contributing factor in age-related immune dysfunction and chronic inflammation. 16S microbiome analysis We purified fecal DNA samples from young and old mice from the zinc supplementation study. Purified fecal DNA (151 samples total) were prepared from fecal materials collected at the beginning of the study (wk 0 baseline), end of the study (wk 6), as well as colon and cecum contents (at necropsy). Using barcoded, degenerate PCR primers specific against 16S rRNA, we PCR amplified 16S amplicons from all 151 samples, and submitted them for amplicon sequencing using Illumina MiSEQ at the Center for Genome Research and Biocomputing core facility at Oregon State University. Identified differentially abundant sequence variants in zinc supplemented mice versus control mice We trimmed and filtered the 16S reads based on quality. We assigned taxonomic designations to each of the sequence variants, and determined the differentially abundant taxa based on diet (supplemented vs. adequate), age (young vs old), and timepoint (week 0 vs week 6). We identified 206 unique sequence variants in the filtered dataset. Beta diversity analysis using robust principle component analysis indicate that Age is the covariate associated with the first principle component axis. This result indicates that, in this study, age shows the biggest effect on the microbiome when taken as a whole. Over the timecourse series, few statistically significant differences were seen. Age shows a significant effect on overall microbial composition in the gut, according to a PERMANOVA test and a permutation test, regardless of zinc status. Generalized linear models (glms) developed for each amplicon sequence variant (ASV) were used to identify significant correlations for zinc status, age and specific taxa. The primary microbial constituents, on average above 50% of the amplified sequences, were from either Bacteroides or Parabacteroides for young or old mice, respectively. ASVs from the Muribaculaceae family were significantly associated with young mice, while those from the Lachnospiraceae were significantly associated with old mice. Of the 206 unique ASVs, the linear regressions identified 151 ASVs that had a significant age effect. Diet effects, and age by diet interactions, were rare. Conclusion: Age effects overwhelm other effects on the mouse gut microbiome. Overall gut microbiome content is signficantly different between the young and old mice, and individually, around 75% of the identified ASVs showed a significant difference between the age groups. Changing a single micronutrient such as zinc shows smaller effects that, when paired with a split study design, are difficult to measure. 7. Short chain fatty acid analysis Short chain fatty acid analyses (SCFA) were done using samples from both animal studies. We were able to detect and quantitate acetate, propionate, and butyrate from colon and cecum contents from our animals. Acetate levels in the cecum and colon showed changes associated with changes in zinc levels in the diet. We are integrating these data with our microbiome data to explore correlation between changes in SCFA and microbiome (ASVs).
Publications
|
Progress 11/15/18 to 11/14/19
Outputs
Target Audience:Peer and scientists Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Research staff attended the LPI International Conference 2019 to learn more about diet and microbiome interactions. A new undergraduate student obtained funding from OSU to gain experience with aging projects through the OSU Center for Healthy Aging. The student received additional 1:1 mentoring from other researchers on OSU campus doing aging research. How have the results been disseminated to communities of interest?Results presented as 2 posters at the Linus Pauling Institute International Conference in Aug 2019. What do you plan to do during the next reporting period to accomplish the goals?We have planned in the coming year to perform metabolomics analysis, with focus on short chain fatty acid products identified in stool samples from both studies. We have also completed sequencing of samples from stool, cecum and intestinal tissues. Additional comparisons of effects across these tissue types will be examined. Moreover, additional data integration will be perform to more closely examine interrelationships between microbiome, inflammatory parameters and other metadata. We will also focus on manuscript submissions for the coming year.
Impacts
What was accomplished under these goals?
1. Completion of feeding studies described in Objective 1 and 2 Due to the limitation in the number of aged mice we can acquire from National Institute of Aging, we have divided the proposed animal study described in Objective 1 into two separate mouse studies. In the first mouse study (zinc supplementation study), groups of young (2mo) and old (24mo) mice were fed zinc adequate diet (30mg/kg zinc) or zinc supplemented diet (300mg/kg zinc) for 6wks. The feeding study was completed in June 2018, and various tissues and fecal materials were collected for downstream analyses. In the second mouse study (zinc deficiency study), groups of young (2mo) and old (24mo) mice were fed zinc adequate diet (30mg/kg zinc) or zinc deficient diet (6mg/kg zinc) for 6wks. The feeding study was completed in October 2018, and various tissues and fecal materials were collected for downstream analyses. 2. Determined zinc status in young and old mice in zinc supplementation study Serum zinc levels were measured by ICP-OES in mice from the zinc supplementation study. Old mice had significant decrease in serum zinc despite being fed a zinc adequate diet. Zinc supplementation restored their serum zinc levels to those comparable to young mice. 3. Measured systemic inflammatory response in young and old mice in zinc supplementation study The levels of six cytokines associated with inflammation (IL12, TNFa, IFNg, MCP1, IL10, and IL6) were determined in plasma samples from mice in the zin supplementation study. In agreement with published reports, we observed age-associated increase in plasma MCP1. Zinc supplementation had a significant effect in decreasing MCP1 levels in old mice 4. Determined peripheral and mucosal Th1/Th2/Th17 cytokine response and changes in immune phenotypes in young and old mice in zinc supplementation study We collected mesenteric and inguinol lymph nodes from individual mouse at the time of necropsy from mice in zinc supplementation study. Lymphocytes were treated ex vivo with PMA/ionomycin or anti-CD3/CD28 to stimulate T cell proliferation and cytokine production. Differences in the quality and quantity of Th1/Th2/Th17 cytokines produced from lymphocytes derived from different age and treatment groups allow us to determine the effects of age and zinc status on two different immune sites (mesenteric and inguinol lymph nodes) that reflects alterations in mucosal and peripheral immune responses, respectively. The levels of seven cytokine (IL10, IL17, TNFa, IFNg, IL6, IL4, and IL2)l were measured from culture supernatants after immune cell stimulation. We observed significant effect of age in altering the immune profile, where age mice had increased IL17, TNFa, and IFNg production, and decreased IL2 production. Zinc supplementation had significant decrease or trend in reducing age-related increase in IL17, TNFa, and IFNg production, but had no effect on IL2 production. Using flow cytometry, we also examined the effects of age and zinc status on the naïve and memory CD4+ and CD8+ T cells in the lymph nodes using CD4, CD8, CD62L, and CD44 as markers to distinguish the different T cell subsets. With age, there was an expansion in memory T cell population, as defined by CD62LlowCD44high expression, and a decline in naïve T cell population, as defined by CD62LhighCD44low expression. Zinc supplementation had a significant effect in decreasing age-related increase in memory T cell population, with concomitant increase in naïve T cell populations. Old mice had reduced zinc and increased proinflammatory cytokines MCP1 and IL6 in the serum, increased Th1/Th17/inflammatory cytokines (IFNg, IL17, TNFa, respectively) and decreased naïve CD4 T-cells in the mesenteric lymph nodes (MLN). ZS significantly increased serum zinc levels, decreased TNFa, IFNg, IL17 in MLN, and increased naïve T-cell populations in aged mice. MZD further reduced serum zinc and increased serum IL6 levels in aged mice. Conclusion: ZS improved the immune function of aged mice and reduced inflammatory response, and MZD further increased age-related inflammation. Our data suggest that zinc status is an important contributing factor in age-related immune dysfunction and chronic inflammation. 5. 16S microbiome analysis We purified fecal DNA samples from young and old mice from the zinc supplementation study. Purified fecal DNA (151 samples total) were prepared from fecal materials collected at the beginning of the study (wk 0 baseline), end of the study (wk 6), as well as colon and cecum contents (at necropsy). Using barcoded, degenerate PCR primers specific against 16S rRNA, we PCR amplified 16S amplicons from all 151 samples, and submitted them for amplicon sequencing using Illumina MiSEQ at the Center for Genome Research and Biocomputing core facility at Oregon State University. 6. Identified differentially abundant sequence variants in zinc supplemented mice versus control mice We trimmed and filtered the 16S reads based on quality. We assigned taxonomic designations to each of the sequence variants, and determined the differentially abundant taxa based on diet (supplemented vs. adequate), age (young vs old), and timepoint (week 0 vs week 6). Using a statistical method that is aware of hierarchical relationships inherent to bacterial classification, we found no significant differences in the young mice in response to diet or timepoint. Conversely, we identified three significantly different taxa at week 6 in the old mice in three different genera: Acetatifactor, Romboutsia, and Clostridium sensu stricto 1. These taxa are commonly associated with the gut in various other studies. Age correlated with overall microbial composition in the gut, according to a PERMANOVA test and a permutation test, regardless of zinc status. Generalized linear models developed for each genus were used to identify significant correlations for zinc status, age and specific taxa. A significant interaction between age and MZD diets was found; no similar interactions were found with ZS diets. Conclusion: Zinc deficiency elicits a varied effect on the microbiome that is dependent upon host age. Conversely, zinc supplementation elicits smaller changes on the microbiome, and the changes identified are comparable regardless of age.
Publications
|
Progress 11/15/17 to 11/14/18
Outputs
Target Audience:peer scientists Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?For zinc supplementation study, we will continue with various tissue sample processing to examine the effects of age and zinc status on factors involved in regulating zinc metabolism, inflammatory response, and microbial population as described in Objective 1a and 1b. This includes examining changes in zinc transporters expression, inflammatory response in different tissue sites, as well as gut health as determined by alterations in endotoxin levels, short chain fatty acids, and markers of microbial translocation and enterocyte damage. The goal is to dissect how changes in zinc metabolism, inflammation, and gut health correlate with alterations in microbial communities. We will generate hypotheses as to the effect on the gut of the significantly different taxa identified. Furthermore, we will predict what functions might be resulting in these effects, based on previously determined functional analysis of enriched functions in these particular taxa. For zinc deficiency study, we just completed the mouse feeding study very recently. We will analyze the samples archived from this study following similar priority as described in zinc supplementation study. We will first confirm the serum zinc status in young and old mice fed zinc adequate or zinc deficient diet by ICP-OES. Changes in the systemic (plasma) inflammatory response will next be examined. Alterations in peripheral and mucosal Th1/Th2/Th17 immune response will also be measured. We will also begin to process fecal samples for microbiome analysis. Remaining archived tissues samples will be processed as described above for zinc supplementation study. The 16S amplicon data generated from the zinc deficient mice will be analyzed in a manner similar to the data generated from the zinc supplementation study. Significantly different taxa will be determined for each of the treatments and timepoints of the study.
Impacts
What was accomplished under these goals?
1. Completion of two mouse feeding studies described in Objective 1 Due to the limitation in the number of aged mice we can acquire from National Institute of Aging, we have divided the proposed animal study described in Objective 1 into two separate mouse studies. In the first mouse study (zinc supplementation study), groups of young (2mo) and old (24mo) mice were fed zinc adequate diet (30mg/kg zinc) or zinc supplemented diet (300mg/kg zinc) for 6wks. The feeding study was completed in June 2018, and various tissues and fecal materials were collected for downstream analyses. In the second mouse study (zinc deficiency study), groups of young (2mo) and old (24mo) mice were fed zinc adequate diet (30mg/kg zinc) or zinc deficient diet (6mg/kg zinc) for 6wks. The feeding study was completed in October 2018, and various tissues and fecal materials were collected for downstream analyses. 2. Determined zinc status in young and old mice in zinc supplementation study Serum zinc levels were measured by ICP-OES in mice from the zinc supplementation study. Old mice had significant decrease in serum zinc despite being fed a zinc adequate diet. Zinc supplementation restored their serum zinc levels to those comparable to young mice. 3. Measured systemic inflammatory response in young and old mice in zinc supplementation study The levels of six cytokines associated with inflammation (IL12, TNFa, IFNg, MCP1, IL10, and IL6) were determined in plasma samples from mice in the zin supplementation study. In agreement with published reports, we observed age-associated increase in plasma MCP1. Zinc supplementation had a significant effect in decreasing MCP1 levels in old mice 4. Determined peripheral and mucosal Th1/Th2/Th17 cytokine response and changes in immune phenotypes in young and old mice in zinc supplementation study We collected mesenteric and inguinol lymph nodes from individual mouse at the time of necropsy from mice in zinc supplementation study. Lymphocytes were treated ex vivo with PMA/ionomycin or anti-CD3/CD28 to stimulate T cell proliferation and cytokine production. Differences in the quality and quantity of Th1/Th2/Th17 cytokines produced from lymphocytes derived from different age and treatment groups allow us to determine the effects of age and zinc status on two different immune sites (mesenteric and inguinol lymph nodes) that reflects alterations in mucosal and peripheral immune responses, respectively. The levels of seven cytokine (IL10, IL17, TNFa, IFNg, IL6, IL4, and IL2)l were measured from culture supernatants after immune cell stimulation. We observed significant effect of age in altering the immune profile, where age mice had increased IL17, TNFa, and IFNg production, and decreased IL2 production. Zinc supplementation had significant decrease or trend in reducing age-related increase in IL17, TNFa, and IFNg production, but had no effect on IL2 production. Using flow cytometry, we also examined the effects of age and zinc status on the naïve and memory CD4+ and CD8+ T cells in the lymph nodes using CD4, CD8, CD62L, and CD44 as markers to distinguish the different T cell subsets. With age, there was an expansion in memory T cell population, as defined by CD62LlowCD44high expression, and a decline in naïve T cell population, as defined by CD62LhighCD44low expression. Zinc supplementation had a significant effect in decreasing age-related increase in memory T cell population, with concomitant increase in naïve T cell populations. 5. Prepared fecal DNA from young and old mice in zinc supplementation study for 16S RNA amplification and microbiome analysis We purified fecal DNA samples from young and old mice from the zinc supplementation study. Purified fecal DNA (151 samples total) were prepared from fecal materials collected at the beginning of the study (wk 0 baseline), end of the study (wk 6), as well as colon and cecum contents (at necropsy). Using barcoded, degenerate PCR primers specific against 16S rRNA, we PCR amplified 16S amplicons from all 151 samples, and submitted them for amplicon sequencing using Illumina MiSEQ at the Center for Genome Research and Biocomputing core facility at Oregon State University. 6. Identified differentially abundant sequence variants in zinc supplemented mice versus control mice We trimmed and filtered the 16S reads based on quality. We assigned taxonomic designations to each of the sequence variants, and determined the differentially abundant taxa based on diet (supplemented vs. adequate), age (young vs old), and timepoint (week 0 vs week 6). Using a statistical method that is aware of hierarchical relationships inherent to bacterial classification, we found no significant differences in the young mice in response to diet or timepoint. Conversely, we identified three significantly different taxa at week 6 in the old mice in three different genera: Acetatifactor, Romboutsia, and Clostridium sensu stricto 1. These taxa are commonly associated with the gut in various other studies. In addition, when the interaction effect of time is considered (week 0 vs week 6) in addition to the zinc supplementation level in the aged mice, five additional taxa are considered significant (Of the families Ruminococcaceae x 2, Lachnospiraceae, Streptococcaceae, and Eggerthellaceae). The significance of each of these taxa with respect to aging, zinc supplementation, and inflammation still needs to be investigated.
Publications
|
|