Neutrophil-mediated innate immune resistance to bacterial pneumonia is dependent on Tet2 function.

Individuals with clonal hematopoiesis of indeterminate potential (CHIP) are at increased risk of aging related health conditions and all-cause mortality, but whether CHIP impacts risk of infection is much less clear. Using UK Biobank data, we revealed a positive association between CHIP and incident pneumonia in 438,421 individuals. We show that inflammation enhanced pneumonia risk, as CHIP carriers with a hypomorphic IL6 receptor polymorphism were protected. To better characterize the pathways of susceptibility, we challenged hematopoietic Tet Methylcytosine Dioxygenase 2 knockout (Tet2-/-) and floxed control mice (Tet2f/f) with Streptococcus pneumoniae. As with human CHIP carriers, Tet2-/- mice had hematopoietic abnormalities resulting in the expansion of inflammatory monocytes and neutrophils in peripheral blood. Yet, these cells were insufficient in defending against S. pneumoniae and resulted in increased pathology, impaired bacterial clearance, and higher mortality in Tet2-/- mice. We delineated the transcriptional landscape of Tet2-/- neutrophils and found that while inflammation-related pathways were upregulated in Tet2-/- neutrophils, migration and motility pathways were compromised. Using live-imaging techniques, we demonstrated impairments in motility, pathogen uptake and neutrophil extracellular trap (NET) formation by Tet2-/- neutrophils. Collectively, we show that CHIP is a risk factor for bacterial pneumonia related to innate immune impairments.

TNF is a critical cytokine in age-related dry eye disease.

Aging is a complex biological process that is characterized by low-grade inflammation, called inflammaging. Aging affects multiple organs including eye and lacrimal gland. Tumor necrosis factor (TNF) is a pleiotropic cytokine that participates in inflammation, activation of proteases such as cathepsin S, and formation of ectopic lymphoid organs. Using genetic and pharmacological approaches, we investigated the role of TNF in age-related dry eye disease, emphasizing the ocular surface and lacrimal gland inflammation. Our results show the increased protein and mRNA levels of TNF in aged lacrimal glands, accompanied by increased TNF, IL1ß, IL-18, CCL5, CXCL1, IL-2, IL-2 receptor alpha (CD25), IFN-γ, IL-12p40, IL-17, and IL-10 proteins in tears of aged mice. Moreover, genetic loss of the Tnf-/- in mice decreased goblet cell loss and the development of ectopic lymphoid structures in the lacrimal gland compared to wild-type mice. This was accompanied by a decrease in cytokine production. Treatment of mice at an early stage of aging (12-14-month-old) with TNF inhibitor tanfanercept eye drops for eight consecutive weeks decreased cytokine levels in tears, improved goblet cell density, and decreased the marginal zone B cell frequency in the lacrimal gland compared to vehicle-treated animals. Our studies indicate that modulation of TNF during aging could be a novel strategy for age-related dry eye disease.

Glucose Mediates Niche-Specific Repression of Staphylococcus aureus Toxic Shock Syndrome Toxin-1 through the Activity of CcpA in the Vaginal Environment.

Staphylococcus aureus chronically colonizes up to 30% of the human population on the skin or mucous membranes, including the nasal tract or vaginal canal. While colonization is often benign, this bacterium also has the capability to cause serious infections. Menstrual toxic shock syndrome (mTSS) is a serious toxinosis associated with improper use of tampons, which can induce an environment that is favorable to the production of the superantigen known as toxic shock syndrome toxin-1 (TSST-1). To better understand environmental signaling that influences TSST-1 production, we analyzed expression in the prototype mTSS strain S. aureus MN8. Using transcriptional and protein-based analysis in two niche-related media, we observed that TSST-1 expression was significantly higher in synthetic nasal medium (SNM) than in vaginally defined medium (VDM). One major divergence in medium composition was high glucose concentration in VDM. The glucose-dependent virulence regulator gene ccpA was deleted in MN8, and, compared with wild-type MN8, we observed increased TSST-1 expression in the ΔccpA mutant when grown in VDM, suggesting that TSST-1 is repressed by catabolite control protein A (CcpA) in the vaginal environment. We were able to relieve CcpA-mediated repression by modifying the glucose level in vaginal conditions, confirming that changes in nutritional conditions contribute to the overexpression of TSST-1 that can lead to mTSS. We also compared CcpA-mediated repression to other key regulators of tst, finding that CcpA regulation is dominant compared to other characterized regulatory mechanisms. This study underlines the importance of environmental signaling for S. aureus pathogenesis in the context of mTSS. IMPORTANCE Menstrual toxic shock syndrome (mTSS) is caused by strains of Staphylococcus aureus that overproduce a toxin known as toxic shock syndrome toxin-1 (TSST-1). This work studied how glucose levels in a model vaginal environment could influence the amount of TSST-1 that is produced by S. aureus. We found that high levels of glucose repress TSST-1 production, and this is done by a regulatory protein called catabolite control protein A (CcpA). The research also demonstrated that, compared with other regulatory proteins, the CcpA regulator appears to be the most important for maintaining low levels of TSST-1 in the vaginal environment, and this information helps to understand how changes in the vaginal environmental can lead to mTSS.

The Gut Microbiota and Unhealthy Aging: Disentangling Cause from Consequence.

The gut microbiota changes with age, but it is not clear to what degree these changes are due to physiologic changes, age-associated inflammation or immunosenescence, diet, medications, or chronic health conditions. Observational studies in humans find that there are profound differences between the microbiomes of long-lived and frail individuals, but the degree to which these differences promote or prevent late-life health is unclear. Studies in model organisms demonstrate that age-related microbial dysbiosis causes intestinal permeability, systemic inflammation, and premature mortality, but identifying causal relationships have been challenging. Herein, we review how physiological and immune changes contribute to microbial dysbiosis and the degree to which microbial dysbiosis contributes to late-life health conditions. We discuss the features of the aging microbiota that make it more amenable to diet and pre- and probiotic interventions. Health interventions that promote a diverse microbiome could influence the health of older adults.

Single cell and tissue-transcriptomic analysis of murine bladders reveals age- and TNFα-dependent but microbiota-independent tertiary lymphoid tissue formation.

Aging has multifaceted effects on the immune system, but how aging affects tissue-specific immunity is not well-defined. Bladder diseases characterized by chronic inflammation are highly prevalent in older women, but mechanisms by which aging promotes these pathologies remain unknown. Tissue transcriptomics of unperturbed, young and aged bladders identified a highly altered immune landscape as a fundamental feature of the aging female bladder. Detailed mapping of immune cells using single cell RNA-sequencing revealed novel subsets of macrophages and dendritic cells and unique changes to the immune repertoire in the aged bladder. B and T cells are highly enriched in aged bladders and spontaneously form organized bladder tertiary lymphoid tissues (bTLTs). Naïve, activated, and germinal center B cells and IgA+ plasma cells are found within bTLT and associated with increased urinary IgA concentrations. bTLTs form with increasing age and their formation is dependent on TNFα. Microbiota are not required to form bTLT, as aged germfree mice also harbor them. Thus, bTLTs require age-dependent TNFα but are independent of the microbiota. Our results indicate that chronic, age-associated inflammation underlies a fundamental alteration to the bladder and establishes a resource for further investigation of the bladder immune system in homeostasis, aging, and disease.

A penicillin-binding protein that can promote advanced-generation cephalosporin resistance and genome adaptation in the opportunistic pathogen Pseudomonas aeruginosa.

A previous soil metagenomics study recovered a novel cephalosporin resistance determinant, pbpTET A6, for which the exact resistance mechanism was unclear. This study used a three-dimensional structure-guided mutagenesis approach to demonstrate that PBPTET A6 is likely to be a class A penicillin-binding protein (PBP), and that its ability to confer cephalosporin resistance is directly linked to the functional integrity of its transpeptidase (TP) catalytic core. Screening of a library of PBPTET A6 variants carrying randomly introduced point mutations revealed additional residue modifications that compromised resistance, all of which were proximal to the TP active site except one which was found in a 29-amino-acid-long superstructure (α6-α7 loop) absent in other class A PBP homologues. Based on the site-specific mutagenesis results, it is hypothesized that residue arginine-400 plays an important role in limiting the access of certain cephalosporin compounds to the enzymatic core of the TP domain of PBPTET A6. Using a combination of adaptive evolution assays and whole-genome sequencing, the potential impact of PBPTET A6 on promoting the development of resistance in the clinically significant opportunistic pathogen Pseudomonas aeruginosa was investigated. Under the selective pressure of serial ceftazidime exposures, the pbpTET A6-expressing P. aeruginosa population readily evolved by excluding a ~400-kbp chromosomal element to acquire additional resistance against cephalosporins, suggesting that PBPTET A6 has a catalytic effect on facilitating antibiotic-resistance-associated genome adaptation. Overall, the soil environment contains genes conferring resistance to critically important antibiotics by cryptic mechanisms. Understanding what impact anthropogenic activities might have on the abundance and evolution of these genes should be a priority.

Regulation of toxic shock syndrome toxin-1 by the accessory gene regulator in Staphylococcus aureus is mediated by the repressor of toxins.

Toxic shock syndrome toxin-1 (TSST-1) is a superantigen (SAg) produced by Staphylococcus aureus thought to be responsible for essentially all cases of menstrual-associated toxic shock syndrome (TSS). As a potent exotoxin, it is not surprising that S. aureus has evolved multiple systems to control expression of TSST-1. Although the accessory gene regulator (Agr) system is recognized to enhance TSST-1 expression, how Agr regulates TSST-1 is unclear. Using an agr-null mutant, complementation experiments demonstrated that Agr controls TSST-1 expression through the activity of the RNAIII effector molecule. RNAIII can repress translation of the repressor of toxins (Rot) regulator, and deletion of rot increased expression of TSST-1 during the exponential phase of growth. Deletion of agr did not affect rot transcription, but did result in overexpression of the Rot protein, and Rot was also shown to bind and positively regulate the rot promoter. Overexpression of Rot dramatically repressed TSST-1, and Rot bound directly to the TSST-1 promoter. Deletion of both agr and rot in S. aureus returned TSST-1 expression to wild-type levels. This work demonstrates that Agr, although widely considered to be an inducer of TSST-1, has evolved in combination with Rot, to restrict the expression of this potent SAg.