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The gut microbiota is implicated in the adverse developmental outcomes of postnatal iron supplementation. To generate hypotheses on how changes to the gut microbiota by iron adversely affect development, and to determine whether the form of iron influences microbiota outcomes, we characterized gut microbiome and metabolome changes in Sprague-Dawley rat pups given oral supplements of ferrous sulfate (FS), ferrous bis-glycinate chelate (FC), or vehicle control (CON) on postnatal day (PD) 2−14. Iron supplementation reduced microbiome alpha-diversity (p < 0.0001) and altered short-chain fatty acids (SCFAs) and trimethylamine (TMA) in a form-dependent manner. To investigate the long-term effects of iron provision in early life, an additional cohort was supplemented with FS, FC, or CON until PD 21 and then weaned onto standard chow. At ~8 weeks of age, young adult (YA) rats that received FS exhibited more diverse microbiomes compared to CON (p < 0.05), whereas FC microbiomes were less diverse (p < 0.05). Iron provision resulted in 10,000-fold reduced abundance of Lactobacilli in pre-weanling and YA animals provided iron in early life (p < 0.0001). Our results suggest that in pre-weanling rats, supplemental iron form can generate differential effects on the gut microbiota and microbial metabolism that persist into adulthood.

Older kidney transplant recipients demonstrate increased rates of infection, and lower rates of rejection, compared with younger kidney transplant recipients. However, the mechanism behind this observation remains unknown. To develop a multifaceted view of age-associated immune dysfunction, we determined the function and phenotype of T cells predisposing to vulnerability to infection on a molecular level. Overlapping peptide pools representing the dominant CMV antigens were used to stimulate PBMC collected from 51 kidney transplant recipients, using cytokine secretion to determine specificity and intensity of response. Staphylococcal endotoxin B (SEB) was analyzed in parallel. To define immune cell subsets, we used single cell RNA sequencing (scRNAseq) to evaluate cellular surface markers and gene expression. We found increased frequency of SEB- and CMV-specific T cells was associated with freedom from infection, especially in older patients. Spatialized t-SNE analysis revealed decreased frequency of naïve T cells, increased frequency of TEMRA cells, and decreased frequency of IFNγ secreting T cells in patients with infection. Application of scRNAseq analysis revealed increased frequency of terminally differentiated T cells expressing NK-associated receptors and inhibitory markers. These findings offer unique insight into the mechanism behind vulnerability to infection in the kidney transplant recipient, revealing a specific T cell subtype of impaired antigen response and terminal effector phenotype as markers of T cell senescence.

After kidney transplantation, infection and death are important clinical complications, especially for the growing numbers of older patients with limited resilience to withstand adverse events. Evaluation of changes in gene expression in immune cells can reveal the underlying mechanisms behind vulnerability to infection. A cohort of 60 kidney transplant recipients was evaluated. Gene expression in peripheral blood mononuclear cells 3 months after kidney transplantation was analyzed to compare differences between patients with infection and those who were infection-free in the first-year post-transplant. Pro-inflammatory genes such as IL1B, CCL4, and TNF were found to be downregulated in post-transplant PBMC from patients who developed infection. In contrast, genes involved in metabolism, HLA genes, and transcripts involved in type I interferon innate antiviral responses were found to be upregulated. Promoter-based bioinformatic analyses implicated increased activity of interferon regulatory factors, erythroid nuclear factor (E2), and CCAAT-enhancer-binding protein (C/EBP) in patients who developed infections. Differential patterns of gene expression were observed in patients who developed infection after kidney transplantation, with patterns distinct from changes associated with patient age, suggesting possible mechanisms behind vulnerability to infection. Assessment of gene expression in blood may offer an approach for patient risk stratification and monitoring after transplantation.

Older kidney transplant recipients demonstrate increased rates of infection but decreased rates of rejection compared with younger recipients, suggesting that older transplant patients are functionally overimmunosuppressed. We hypothesized that this is a consequence of reduction in immunological activity due to biological aging and that an immune biological age, as determined by DNA methylation (DNAm), would be associated more strongly with incidence of infection than chronological age.

Methods

DNAm analysis was performed on peripheral blood mononuclear cell collected from 60 kidney transplant recipients representing older (≥age 60 y) and younger (aged 30-59 y) patients 3 months after transplantation. DNAm age was calculated based on methylation status of a panel of CpG sites, which have been previously identified as indicative of biological age.

Results

Correlation was seen between chronological and DNAm age; however, there were many patients with significant differences (either acceleration or slowing) between DNAm age and chronological age. A statistically significant association was seen between increased DNAm age and incidence of infection in the first year after kidney transplantation, whereas no significant association was seen between chronological age and infection.

Conclusions

Assessment of DNAm age holds promise as an approach for patient evaluation and individualization of immune suppression regimens. This analysis may provide insights into the immunological mechanism behind increased incidence of infection observed in older transplant patients. The ability to measure biological age would allow for patient risk stratification and individualization of immunosuppression, improving outcomes for the growing numbers of older patients undergoing kidney transplantation.

Background

The number of elderly patients with end-stage kidney disease requiring kidney transplantation continues to grow. Evaluation of healthy older adults has revealed proinflammatory changes in the immune system, which are posited to contribute to age-associated illnesses via "inflamm-aging." Immunologic dysfunction is also associated with impaired control of infections. Whether these immunologic changes are found in older kidney transplant recipients is not currently known, but may have important implications for risk for adverse clinical outcomes.

Methods

Three months after transplant, innate immune phenotype was evaluated by flow cytometry from 60 kidney transplant recipients (22 older [≥60 years] and 38 younger [<60 years old]). Multiplex cytokine testing was used to evaluate plasma cytokine levels. Younger patients were matched to older patients based on transplant type and induction immune suppression.

Results

Older kidney transplant recipients demonstrated decreased frequency of intermediate monocytes (CD14++CD16+) compared with younger patients (1.2% vs 3.3%, P = 0.007), and a trend toward increased frequency of proinflammatory classical monocytes (CD14++CD16-) (94.5% vs 92.1%) (P = 0.065). Increased levels of interferon-gamma (IFN-γ) were seen in older patients.

Conclusions

In this pilot study of kidney transplant recipients, we identified differences in the innate immune system in older as compared with younger patients, including increased levels of IFN-γ. This suggests that age-associated nonspecific inflammation persists despite immune suppression. The ability to apply noninvasive testing to transplant recipients will provide tools for patient risk stratification and individualization of immune suppression regimens to improve outcomes after transplantation.

Immunologic impairment may contribute to poor outcomes after implantation of mechanical circulatory support device (MCSD), with infection often as a terminal event. The study of immune dysfunction is of special relevance given the growing numbers of older patients with heart disease. The aim of the study was to define which immunologic characteristics are associated with development of adverse clinical outcomes after MCSD implantation. We isolated peripheral blood mononuclear cells (PBMC) from patients pre- and up to 20 days post-MCSD implantation and analyzed them by multiparameter flow cytometry for T cell dysfunction, including terminal differentiation, exhaustion, and senescence. We used MELD-XI and SOFA scores measured at each time point as surrogate markers of clinical outcome. Older patients demonstrated increased frequencies of terminally differentiated T cells as well as NKT cells. Increased frequency of terminally differentiated and immune senescent T cells were associated with worse clinical outcome as measured by MELD-XI and SOFA scores, and with progression to infection and death. In conclusion, our data suggest that T cell dysfunction, independently from age, is associated with poor outcomes after MCSD implantation, providing a potential immunologic mechanism behind patient vulnerability to multiorgan dysfunction and death. This noninvasive approach to PBMC evaluation holds promise for candidate evaluation and patient monitoring.

Noninvasive immunologic analysis of peripheral blood holds promise for explaining the mechanism of development of adverse clinical outcomes, and may also become a method for patient risk stratification before or after mechanical circulatory support device (MCSD) implantation. Dysregulation of the innate immune system is associated with increased patient age but has yet to be evaluated in the older patient with advanced heart failure undergoing MCSD surgery. Patients pre- and post-MCSD implantation had peripheral blood mononuclear cells (PBMC) and serum isolated. Multiparameter flow cytometry was used to analyze markers of innate cell function, including monocyte subtypes. Multiplex cytokine analysis was performed. MELD-XI and SOFA scores were utilized as surrogate markers of outcomes. Increased levels of pro-inflammatory cytokines including IL-15, TNF-α, and IL-10 were associated with increased MELD-XI and SOFA scores. IL-8, TNF- α, and IL-10 were associated with risk of death after MCSD implantation, even with correction for patient age. Increased frequency of 'classical' monocytes (CD14 + CD16-) were associated with increased MELD-XI and SOFA scores. This suggests that inflammation and innate immune system activation contribute to progression to multiorgan system failure and death after MCSD surgery. Development of noninvasive monitoring of peripheral blood holds promise for biomarker development for candidate selection and patient risk stratification.