Dectin-1 stimulation promotes a distinct inflammatory signature in the setting of HIV-infection and aging.

Dectin-1 is an innate immune receptor that recognizes and binds ß-1, 3/1, 6 glucans on fungi. We evaluated Dectin-1 function in myeloid cells in a cohort of HIV-positive and HIV-negative young and older adults. Stimulation of monocytes with ß-D-glucans induced a pro-inflammatory phenotype in monocytes of HIV-infected individuals that was characterized by increased levels of IL-12, TNF-α, and IL-6, with some age-associated cytokine increases also noted. Dendritic cells showed a striking HIV-associated increase in IFN-α production. These increases in cytokine production paralleled increases in Dectin-1 surface expression in both monocytes and dendritic cells that were noted with both HIV and aging. Differential gene expression analysis showed that HIV-positive older adults had a distinct gene signature compared to other cohorts characterized by a robust TNF-α and coagulation response (increased at baseline), a persistent IFN-α and IFN-γ response, and an activated dendritic cell signature/M1 macrophage signature upon Dectin-1 stimulation. Dectin-1 stimulation induced a strong upregulation of MTORC1 signaling in all cohorts, although increased in the HIV-Older cohort (stimulation and baseline). Overall, our study demonstrates that the HIV Aging population has a distinct immune signature in response to Dectin-1 stimulation. This signature may contribute to the pro-inflammatory environment that is associated with HIV and aging.

PD-1highCXCR5-CD4+ peripheral helper T cells promote CXCR3+ plasmablasts in human acute viral infection.

T cell-B cell interaction is the key immune response to protect the host from severe viral infection. However, how T cells support B cells to exert protective humoral immunity in humans is not well understood. Here, we use COVID-19 as a model of acute viral infections and analyze CD4+ T cell subsets associated with plasmablast expansion and clinical outcome. Peripheral helper T cells (Tph cells; denoted as PD-1highCXCR5-CD4+ T cells) are significantly increased, as are plasmablasts. Tph cells exhibit "B cell help" signatures and induce plasmablast differentiation in vitro. Interestingly, expanded plasmablasts show increased CXCR3 expression, which is positively correlated with higher frequency of activated Tph cells and better clinical outcome. Mechanistically, Tph cells help B cell differentiation and produce more interferon γ (IFNγ), which induces CXCR3 expression on plasmablasts. These results elucidate a role for Tph cells in regulating protective B cell response during acute viral infection.

No evidence of fetal defects or anti-syncytin-1 antibody induction following COVID-19 mRNA vaccination.

The impact of Coronavirus Disease 2019 (COVID-19) mRNA vaccination on pregnancy and fertility has become a major topic of public interest. We investigated 2 of the most widely propagated claims to determine (1) whether COVID-19 mRNA vaccination of mice during early pregnancy is associated with an increased incidence of birth defects or growth abnormalities; and (2) whether COVID-19 mRNA-vaccinated human volunteers exhibit elevated levels of antibodies to the human placental protein syncytin-1. Using a mouse model, we found that intramuscular COVID-19 mRNA vaccination during early pregnancy at gestational age E7.5 did not lead to differences in fetal size by crown-rump length or weight at term, nor did we observe any gross birth defects. In contrast, injection of the TLR3 agonist and double-stranded RNA mimic polyinosinic-polycytidylic acid, or poly(I:C), impacted growth in utero leading to reduced fetal size. No overt maternal illness following either vaccination or poly(I:C) exposure was observed. We also found that term fetuses from these murine pregnancies vaccinated prior to the formation of the definitive placenta exhibit high circulating levels of anti-spike and anti-receptor-binding domain (anti-RBD) antibodies to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) consistent with maternal antibody status, indicating transplacental transfer in the later stages of pregnancy after early immunization. Finally, we did not detect increased levels of circulating anti-syncytin-1 antibodies in a cohort of COVID-19 vaccinated adults compared to unvaccinated adults by ELISA. Our findings contradict popular claims associating COVID-19 mRNA vaccination with infertility and adverse neonatal outcomes.

Impact of Aging and HIV Infection on the Function of the C-Type Lectin Receptor MINCLE in Monocytes.

Both aging and HIV infection are associated with an enhanced pro-inflammatory environment that contributes to impaired immune responses and is mediated in part by innate immune pattern-recognition receptors. MINCLE is a C-type lectin receptor that recognizes trehalose-6,6'-dimycolate or "cord factor," the most abundant glycolipid in Mycobacterium tuberculosis. Here, we evaluated MINCLE function in monocytes in a cohort of HIV-infected and uninfected young (21-35 years) and older adults (≥60 years) via stimulation of peripheral blood mononuclear cells with trehalose-6,6-dibehenate, a synthetic analog of trehalose-6,6'-dimycolate and measurement of cytokine production (interleukin [IL]-10, IL-12, IL-6, tumor necrosis factor-α) by multicolor flow cytometry. Our studies show an age- and HIV-associated increase in cytokine multifunctionality of monocytes both at the population and single cell level that was dominated by IL-12, IL-10, and IL-6. These findings provide insight into the host response to M. tuberculosis and possible sources for the pro-inflammatory environment seen in aging and HIV infection.

Aging impairs both primary and secondary RIG-I signaling for interferon induction in human monocytes.

Adults older than 65 account for most of the deaths caused by respiratory influenza A virus (IAV) infections, but the underlying mechanisms for this susceptibility are poorly understood. IAV RNA is detected by the cytosolic sensor retinoic acid-inducible gene I (RIG-I), which induces the production of type I interferons (IFNs) that curtail the spread of the virus and promote the elimination of infected cells. We have previously identified a marked defect in the IAV-inducible secretion of type I IFNs, but not proinflammatory cytokines, in monocytes from older (>65 years) healthy human donors. We found that monocytes from older adults exhibited decreased abundance of the adaptor protein TRAF3 (tumor necrosis factor receptor-associated factor 3) because of its increased proteasomal degradation with age, thereby impairing the primary RIG-I signaling pathway for the induction of type I IFNs. We determined that monocytes from older adults also failed to effectively stimulate the production of the IFN regulatory transcription factor IRF8, which compromised IFN induction through secondary RIG-I signaling. IRF8 played a central role in IFN induction in monocytes, because knocking down IRF8 in monocytes from younger adults was sufficient to replicate the IFN defects observed in monocytes from older adults, whereas restoring IRF8 expression in older adult monocytes was sufficient to restore RIG-I-induced IFN responses. Aging thus compromises both the primary and secondary RIG-I signaling pathways that govern expression of type I IFN genes, thereby impairing antiviral resistance to IAV.

ß-Hydroxybutyrate Deactivates Neutrophil NLRP3 Inflammasome to Relieve Gout Flares.

Aging and lipotoxicity are two major risk factors for gout that are linked by the activation of the NLRP3 inflammasome. Neutrophil-mediated production of interleukin-1ß (IL-1ß) drives gouty flares that cause joint destruction, intense pain, and fever. However, metabolites that impact neutrophil inflammasome remain unknown. Here, we identified that ketogenic diet (KD) increases ß-hydroxybutyrate (BHB) and alleviates urate crystal-induced gout without impairing immune defense against bacterial infection. BHB inhibited NLRP3 inflammasome in S100A9 fibril-primed and urate crystal-activated macrophages, which serve to recruit inflammatory neutrophils in joints. Consistent with reduced gouty flares in rats fed a ketogenic diet, BHB blocked IL-1ß in neutrophils in a NLRP3-dependent manner in mice and humans irrespective of age. Mechanistically, BHB inhibited the NLRP3 inflammasome in neutrophils by reducing priming and assembly steps. Collectively, our studies show that BHB, a known alternate metabolic fuel, is also an anti-inflammatory molecule that may serve as a treatment for gout.

DNA Methylation Regulates the Differential Expression of CX3CR1 on Human IL-7Rαlow and IL-7Rαhigh Effector Memory CD8+ T Cells with Distinct Migratory Capacities to the Fractalkine.

DNA methylation is an epigenetic mechanism that modulates gene expression in mammalian cells including T cells. Memory T cells are heterogeneous populations. Human effector memory (EM) CD8(+) T cells in peripheral blood contain two cell subsets with distinct traits that express low and high levels of the IL-7Rα. However, epigenetic mechanisms involved in defining such cellular traits are largely unknown. In this study, we use genome-wide DNA methylation and individual gene expression to show the possible role of DNA methylation in conferring distinct traits of chemotaxis and inflammatory responses in human IL-7Rα(low) and IL-7Rα(high) EM CD8(+) T cells. In particular, IL-7Rα(low) EM CD8(+) T cells had increased expression of CX3CR1 along with decreased DNA methylation in the CX3CR1 gene promoter compared with IL-7Rα(high) EM CD8(+) T cells. Altering the DNA methylation status of the CX3CR1 gene promoter changed its activity and gene expression. IL-7Rα(low) EM CD8(+) T cells had an increased migratory capacity to the CX3CR1 ligand fractalkine compared with IL-7Rα(high) EM CD8(+) T cells, suggesting an important biological outcome of the differential expression of CX3CR1. Moreover, IL-7Rα(low) EM CD8(+) T cells induced fractalkine expression on endothelial cells by producing IFN-γ and TNF-α, forming an autocrine amplification loop. Overall, our study shows the role of DNA methylation in generating unique cellular traits in human IL-7Rα(low) and IL-7Rα(high) EM CD8(+) T cells, including differential expression of CX3CR1, as well as potential biological implications of this differential expression.

Prolonged proinflammatory cytokine production in monocytes modulated by interleukin 10 after influenza vaccination in older adults.

We evaluated in vivo innate immune responses in monocyte populations from 67 young (aged 21-30 years) and older (aged ≥65 years) adults before and after influenza vaccination. CD14(+)CD16(+) inflammatory monocytes were induced after vaccination in both young and older adults. In classical CD14(+)CD16(-) and inflammatory monocytes, production of tumor necrosis factor α and interleukin 6, as measured by intracellular staining, was strongly induced after vaccination. Cytokine production was strongly associated with influenza vaccine antibody response; the highest levels were found as late as day 28 after vaccination in young subjects and were substantially diminished in older subjects. Notably, levels of the anti-inflammatory cytokine interleukin 10 (IL-10) were markedly elevated in monocytes from older subjects before and after vaccination. In purified monocytes, we found age-associated elevation in phosphorylated signal transducer and activator of transcription-3, and decreased serine 359 phosphorylation of the negative IL-10 regulator dual-specificity phosphatase 1. These findings for the first time implicate dysregulated IL-10 production in impaired vaccine responses in older adults.

Chitinase 3-like 1 suppresses injury and promotes fibroproliferative responses in Mammalian lung fibrosis.

Epithelial injury, alternative macrophage accumulation, and fibroproliferation coexist in the lungs of patients with idiopathic pulmonary fibrosis (IPF). Chitinase 3-like 1 (CHI3L1) is a prototypic chitinase-like protein that has been retained over species and evolutionary time. However, the regulation of CHI3L1 in IPF and its ability to regulate injury and/or fibroproliferative repair have not been fully defined. We demonstrated that CHI3L1 levels were elevated in patients with IPF. High levels of CHI3L1 are associated with progression--as defined by lung transplantation or death--and with scavenger receptor-expressing circulating monocytes in an ambulatory IPF population. In preterminal acute exacerbations of IPF, CHI3L1 levels were reduced and associated with increased levels of apoptosis. We also demonstrated that in bleomycin-treated mice, CHI3L1 expression was acutely and transiently decreased during the injury phase and returned toward and eventually exceeded baseline levels during the fibrotic phase. In this model, CHI3L1 played a protective role in injury by ameliorating inflammation and cell death, and a profibrotic role in the repair phase by augmenting alternative macrophage activation, fibroblast proliferation, and matrix deposition. Using three-dimensional culture system of a human fibroblast cell line, we found that CHI3L1 is sufficient to induce low grade myofibroblast transformation. In combination, these studies demonstrate that CHI3L1 is stimulated in IPF, where it represents an attempt to diminish injury and induce repair. They also demonstrate that high levels of CHI3L1 are associated with disease progression in ambulatory patients and that a failure of the CHI3L1 antiapoptotic response might contribute to preterminal disease exacerbations.

The role of Toll-like receptors in age-associated lung diseases.

The aging lung is faced with unique challenges. The lungs are the only internal organ with a direct interface with both the internal and the external environments and as a consequence are constantly sampling diverse, potentially injurious, elements. Therefore, the lungs have evolved a sophisticated, multilayered detection system to distinguish low-level, nonharmful signals from those that are toxic. A family of innate immune receptors, Toll-like receptors (TLRs), appears to serve such a function. Initially described as pattern-recognition receptors that recognize and protect against microbes, TLRs can also respond to diverse, nonmicrobial signals. The role of Toll-like receptors in noninfectious, age-related chronic lung disease is poorly understood. This review presents our current understanding of the biology of age-related lung diseases with a focus on the role of Toll-like receptors in idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, and late-onset asthma.

Age-associated elevation in TLR5 leads to increased inflammatory responses in the elderly.

Aging is accompanied by a progressive decline in immune function. Studies have shown age-related decreases in the expression and signaling efficiency of Toll-like receptors (TLRs) in monocytes and dendritic cells and dysregulation of macrophage TLR3. Using a multivariable mixed effect model, we report a highly significant increase in TLR5-induced production of IL-8 from monocytes of older individuals (P < 0.0001). Elevated IL-8 is accompanied by increased expression of TLR5, both protein and mRNA, and by increased levels of TLR5-mediated phosphorylation of MAPK p38 and ERK. We noted incomplete activation of NF-κB in response to TLR5 signaling in monocytes of elderly donors, as reflected by the absence of an associated increase in the production of TNF-α. Elevated TLR5 may provide a critical mechanism to enhance immune responsiveness in older individuals.

Dysregulation of human Toll-like receptor function in aging.

Studies addressing immunosenescence in the immune system have expanded to focus on the innate as well as the adaptive responses. In particular, aging results in alterations in the function of Toll-like receptors (TLRs), the first described pattern recognition receptor family of the innate immune system. Recent studies have begun to elucidate the consequences of aging on TLR function in human cohorts and add to existing findings performed in animal models. In general, these studies show that human TLR function is impaired in the context of aging, and in addition there is evidence for inappropriate persistence of TLR activation in specific systems. These findings are consistent with an overarching theme of age-associated dysregulation of TLR signaling that likely contributes to the increased morbidity and mortality from infectious diseases found in geriatric patients.

Age-associated decrease in TLR function in primary human dendritic cells predicts influenza vaccine response.

We evaluated TLR function in primary human dendritic cells (DCs) from 104 young (age 21-30 y) and older (> or =65 y) individuals. We used multicolor flow cytometry and intracellular cytokine staining of myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) and found substantial decreases in older compared with young individuals in TNF-alpha, IL-6, and/or IL-12 (p40) production in mDCs and in TNF-alpha and IFN-alpha production in pDCs in response to TLR1/2, TLR2/6, TLR3, TLR5, and TLR8 engagement in mDCs and TLR7 and TLR9 in pDCs. These differences were highly significant after adjustment for heterogeneity between young and older groups (e.g., gender, race, body mass index, number of comorbid medical conditions) using mixed-effect statistical modeling. Studies of surface and intracellular expression of TLR proteins and of TLR gene expression in purified mDCs and pDCs revealed potential contributions for both transcriptional and posttranscriptional mechanisms in these age-associated effects. Moreover, intracellular cytokine production in the absence of TLR ligand stimulation was elevated in cells from older compared with young individuals, suggesting a dysregulation of cytokine production that may limit further activation by TLR engagement. Our results provide evidence for immunosenescence in DCs; notably, defects in cytokine production were strongly associated with poor Ab response to influenza immunization, a functional consequence of impaired TLR function in the aging innate immune response.

Human innate immunosenescence: causes and consequences for immunity in old age.

The past decade has seen an explosion in research focusing on innate immunity. Through a wide range of mechanisms including phagocytosis, intracellular killing and activation of proinflammatory or antiviral cytokine production, the cells of the innate immune system initiate and support adaptive immunity. The effects of aging on innate immune responses remain incompletely understood, particularly in humans. Here we review advances in the study of human immunosenescence in the diverse cells of the innate immune system, including neutrophils, monocytes, macrophages, natural killer and natural killer T (NKT) cells and dendritic cells-with a focus on consequences for the response to infection or vaccination in old age.

Defective p53 engagement after the induction of DNA damage in cells deficient in topoisomerase 3beta.

The type IA topoisomerases have been implicated in the repair of dsDNA breaks by homologous recombination and in the resolution of stalled or damaged DNA replication forks; thus, these proteins play important roles in the maintenance of genomic stability. We studied the functions of one of the two mammalian type IA enzymes, Top3beta, using murine embryonic fibroblasts (MEFs) derived from top3beta(-/-) embryos. top3beta(-/-) MEFs proliferated more slowly than TOP3beta(+/+) control MEFs, demonstrated increased sensitivity to DNA-damaging agents such as ionizing and UV radiation, and had increased DNA double-strand breaks as manifested by increased gamma-H2-AX phosphorylation. However, incomplete enforcement of the G(1)-S cell cycle checkpoint was observed in top3beta(-/-) MEFs. Notably, ataxia-telangiectasia, mutated (ATM)/ATM and Rad3-related (ATR)-dependent substrate phosphorylation after UV-B and ionizing radiation was impaired in top3beta(-/-) versus TOP3beta(+/+) control MEFs, and impaired up-regulation of total and Ser-18-phosphorylated p53 was observed in top3beta(-/-) cells. Taken together, these results suggest an unanticipated role for Top3beta beyond DNA repair in the activation of cellular responses to DNA damage.

Toll-like receptors in older adults.

Toll-like receptors (TLRs) recognize a limited number of conserved elements in pathogens and, by activating antigen-presenting cells such as dendritic cells and monocytes and macrophages, play a crucial role in the immune response to infection and vaccination. Most data on TLR function in the context of human aging focus on responses to lipopolysaccharide, an integral component of gram-negative bacteria, which signals through TLR4. However, such studies have not led to a consensus conclusion and are limited by differences in epidemiological and laboratory methods. A recent comprehensive evaluation of TLR function in monocytes from older adults was conducted using a multivariable mixed statistical model to account for covariates. It was found that cytokine production after TLR1/2 engagement, which is essential for the recognition of triacylated lipopeptides found in a variety of bacteria, is substantially lower in monocytes from older adults. The upregulation of costimulatory proteins such as CD80, essential for optimal activation of T cells, on monocytes from older adults was less for all TLR ligands tested than for cells from young individuals, and the extent of CD80 upregulation predicted subsequent antibody response to influenza immunization. These and other consequences of aging on human TLR function may impair activation of the immune response and contribute to poorer vaccine responses and greater morbidity and mortality from infectious diseases in older adults. Such age-associated alterations have particular relevance in view of the interest in TLR agonists as therapeutic agents not only for infections, but also for allergic, autoimmune, and malignant disease.

Age-associated defect in human TLR-1/2 function.

The effects of aging on human TLR function remain incompletely understood. We assessed TLR function and expression in peripheral blood monocytes from 159 subjects in 2 age categories, 21-30 and >65 years of age, using a multivariable mixed effect model. Using flow cytometry to assess TLR-induced cytokine production, we observed a substantial, highly significant defect in TLR1/2-induced TNF-alpha (p = 0.0003) and IL-6 (p < 0.0001) production, in older adults compared with young controls. In contrast to findings in aged mice, other TLR (including TLR2/6)-induced cytokine production appeared largely intact. These differences were highly significant even after correcting for covariates including gender, race, medications, and comorbidities. This defect in TLR1/2 signaling may result from alterations in baseline TLR1 surface expression, which was decreased by 36% in older adults (p < 0.0001), whereas TLR2 surface expression was unaffected by aging. Production of IL-6 (p < 0.0001) and TNF-alpha (p = 0.003) after stimulation by N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-Cys-[S]-Ser1-[S]-Lys(4) trihydrochloride was strongly associated with TLR1 surface expression. Diminished TLR1/2 signaling may contribute to the increased infection-related morbidity and mortality and the impaired vaccine responses observed in aging humans.

Triggering TLR signaling in vaccination.

Toll-like receptors (TLRs) are a family of pattern-recognition receptors that are an important link between innate and adaptive immunity. Many established, as well as experimental, vaccines incorporate ligands for TLRs, not only to protect against infectious diseases but also in therapeutic immunization against noninfectious diseases, such as cancer. We review the underlying mechanisms by which engagement of TLR signaling pathways might trigger an adaptive immune response after immunization. Although the engagement of TLR signaling pathways is a promising mechanism for boosting vaccine responses, questions of efficacy, feasibility and safety remain the subject of active investigation.