TNF Drives Monocyte Dysfunction with Age and Results in Impaired Anti-pneumococcal Immunity.

Monocyte phenotype and output changes with age, but why this occurs and how it impacts anti-bacterial immunity are not clear. We found that, in both humans and mice, circulating monocyte phenotype and function was altered with age due to increasing levels of TNF in the circulation that occur as part of the aging process. Ly6C+ monocytes from old (18-22 mo) mice and CD14+CD16+ intermediate/inflammatory monocytes from older adults also contributed to this "age-associated inflammation" as they produced more of the inflammatory cytokines IL6 and TNF in the steady state and when stimulated with bacterial products. Using an aged mouse model of pneumococcal colonization we found that chronic exposure to TNF with age altered the maturity of circulating monocytes, as measured by F4/80 expression, and this decrease in monocyte maturation was directly linked to susceptibility to infection. Ly6C+ monocytes from old mice had higher levels of CCR2 expression, which promoted premature egress from the bone marrow when challenged with Streptococcus pneumoniae. Although Ly6C+ monocyte recruitment and TNF levels in the blood and nasopharnyx were higher in old mice during S. pneumoniae colonization, bacterial clearance was impaired. Counterintuitively, elevated TNF and excessive monocyte recruitment in old mice contributed to impaired anti-pneumococcal immunity since bacterial clearance was improved upon pharmacological reduction of TNF or Ly6C+ monocytes, which were the major producers of TNF. Thus, with age TNF impairs inflammatory monocyte development, function and promotes premature egress, which contribute to systemic inflammation and is ultimately detrimental to anti-pneumococcal immunity.

Streptococcus pneumoniae Colonization Disrupts the Microbial Community within the Upper Respiratory Tract of Aging Mice.

Nasopharyngeal colonization by the Gram-positive bacterium Streptococcus pneumonia is a prerequisite for pneumonia and invasive pneumococcal diseases. Colonization is asymptomatic, involving dynamic and complex interplay between commensals, the host immune system, and environmental factors. The elderly are at an increased risk of developing pneumonia, which might be due to changes in the respiratory microbiota that would impact bacterial colonization and persistence within this niche. We hypothesized that the composition of the upper respiratory tract (URT) microbiota changes with age and subsequently can contribute to sustained colonization and inefficient clearance of S. pneumoniae To test this, we used a mouse model of pneumococcal colonization to compare the composition of the URT microbiota in young, middle-aged, and old mice in the naive state and during the course of colonization using nasal pharyngeal washes. Sequencing of variable region 3 (V3) of the 16S rRNA gene was used to identify changes occurring with age and throughout the course of S. pneumonia colonization. We discovered that age affects the composition of the URT microbiota and that colonization with S. pneumoniae is more disruptive of preexisting communities in older mice. We have further shown that host-pathogen interactions followingS. pneumonia colonization can impact the populations of resident microbes, including Staphylococcus and Haemophilus. Together, our findings indicate alterations to the URT microbiota could be detrimental to the elderly, resulting in increased colonization of S. pneumonia and decreased efficiency in its clearance.

Age-Associated Microbial Dysbiosis Promotes Intestinal Permeability, Systemic Inflammation, and Macrophage Dysfunction.

Levels of inflammatory mediators in circulation are known to increase with age, but the underlying cause of this age-associated inflammation is debated. We find that, when maintained under germ-free conditions, mice do not display an age-related increase in circulating pro-inflammatory cytokine levels. A higher proportion of germ-free mice live to 600 days than their conventional counterparts, and macrophages derived from aged germ-free mice maintain anti-microbial activity. Co-housing germ-free mice with old, but not young, conventionally raised mice increases pro-inflammatory cytokines in the blood. In tumor necrosis factor (TNF)-deficient mice, which are protected from age-associated inflammation, age-related microbiota changes are not observed. Furthermore, age-associated microbiota changes can be reversed by reducing TNF using anti-TNF therapy. These data suggest that aging-associated microbiota promote inflammation and that reversing these age-related microbiota changes represents a potential strategy for reducing age-associated inflammation and the accompanying morbidity.

Tumor necrosis factor drives increased splenic monopoiesis in old mice.

Aging is accompanied by changes in hematopoiesis and consequently in leukocyte phenotype and function. Although age-related changes in bone marrow hematopoiesis are fairly well documented, changes in extramedullary hematopoiesis are less well described. We observed that 18-22-mo-old mice had larger spleens than young controls and found that the enlargement was caused by increased monopoiesis. Because extramedullary hematopoiesis is often driven by inflammation, we hypothesized that the chronic, low-level inflammation that occurs with age is a causal agent in splenomegaly. To test this theory, we compared the number of monocytes in 18-mo-old tumor necrosis factor-knockout mice, which are protected from age-associated inflammation, and found that they did not have increased extramedullary monopoiesis. To determine whether increased splenic monopoiesis is caused by intrinsic changes in the myeloid precursors that occur with age or by the aging microenvironment, we created heterochronic bone marrow chimeras. Increased splenic monopoiesis occurred in old recipient mice, regardless of the age of the donor mouse, but not in young recipient mice, demonstrating that these cells respond to signals from the microenvironment. These data suggest that decreasing the inflammatory microenvironment with age would be an effective strategy for reducing inflammatory diseases propagated by cells of myeloid lineage, which increase in number with age.

Circulating TNF and mitochondrial DNA are major determinants of neutrophil phenotype in the advanced-age, frail elderly.

Tumor necrosis factor (TNF), a potent inflammatory cytokine, and mitochondrial DNA (mtDNA), a product of inflammation-induced tissue damage, increase with age ("inflammaging") and many chronic diseases. Peripheral blood neutrophils, a critical component of innate immunity, have also been shown to be altered with age, and are exceptionally sensitive to external stimuli. Herein, we describe that the phenotype of neutrophils from the advanced-age, frail elderly (ELD) is determined by levels of circulating TNF and mtDNA. Neutrophils from ELD donors are morphologically immature, and have higher levels of intracellular reactive oxygen species (ROS) and expression of the activation markers CD11b and HLA-DR. The frequency of CD11b(++) neutrophils correlated with plasma TNF, and recombinant TNF elevated neutrophil CD11b ex vivo and in vivo. Furthermore, neutrophils from aged TNF-deficient mice expressed CD11b similar to young counterparts. The frequency of HLA-DR(+) neutrophils, on the other hand, positively correlated with circulating mtDNA, which increased neutrophil HLA-DR expression in a dose-dependent manner ex vivo. Cell-surface TLR-9 expression, however, was unaltered on neutrophils from ELD donors. In summary, we provide novel evidence that products of age-related inflammation modulate neutrophil phenotype in vivo. Given this, anti-inflammatory therapies may prove beneficial in improving neutrophil functionality in the elderly.

Characterization of inflammatory responses during intranasal colonization with Streptococcus pneumoniae.

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream(1). This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates(2), or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models. Our mouse model of intranasal colonization is adapted from human models(3) and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx(4-7). In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice(8), however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

The macrophage.

Macrophages are a diverse phenotype of professional phagocytic cells derived from bone-marrow precursors and parent monocytes in the peripheral blood. They are essential for the maintenance and defence of host tissues, doing so by sensing and engulfing particulate matter and, when necessary, initiating a pro-inflammatory response. Playing such a vast number of roles in both health and disease, the activation phenotype of macrophages can vary greatly and is largely dependent on the surrounding microenvironment. These phenotypes can be mimicked in experimental macrophage models derived from monocytes and in conjunction with stimulatory factors, although given the complexity of in vivo tissue spaces these model cells are inherently imperfect. Furthermore, experimental observations generated in mice are not necessarily conserved in humans, which can hamper translational research. The following chapter aims to provide an overview of how macrophages and their parent cell-type, monocytes, are classified, their development through the myeloid lineage, and finally, the general function of macrophages.