Peeing Pentraxins

Antimicrobial agents secreted into urine potentially play a powerful role in the defense of the urinary tract. In this issue of Immunity, Jaillon et al. (2014) describe a role for pentraxin 3molecules in complementing the host’s cellular innate immune responses to uropathogens.

The urinary tract, which comprises the kidneys, ureters, bladder and urethra, serves asthe body’s drainage system for removing wastes and extra water. Despite its close proximity to the gastrointestinal tract, where trillions of microbesare harbored, the urinary tract, for the most part, appears sterile. The resistance to infection of the urinary tract is partially ascribable to its unique anatomy and to the flushing action of urine and urinary mucins which rapidly eliminate any contaminating bacteria (Chromek and Brauner, 2008). In addition, the urinary tract constitutively produces antimicrobial agents such as cathelcidins that directly kill pathogens (Chromek et al., 2006). Tamm-Horsfall protein (THP), also known as uromodulin, is another constituent of urine secreted by the kidneys which serve to aggregate various bacteria facilitating their early removal by urine (Saemann et al., 2005).

Prospective uropathogens that resist these attempts at clearance are then challenged by the innate immune system of the urinary tract. The bladder epithelium is richly endowed with immunosensory molecules, such as toll-lie receptor 4 (TLR4), which upon activation, promptly initiates a broad spectrum of antibacterial responses (Schilling et al., 2003). These responses include local secretion of several proinflammatory cytokines and chemokines, which is accompanied by a rapid neutrophil response. In this issue of Immunity, Jaillon et al. (2014) report that complementing these cellular innate responses in the urinary tract is ahumoral arm of the innate immune responses comprising of secretion into the urine of a soluble pattern recognition molecule pentraxin 3 (PTX3). These authors report that PTX3 secretions in urine was markedly enhanced in humans and mice following UTIs and that mice deficient in PTX3 were found to be highly impaired in their capacity to clear uropathogenic Escherichia. Coli following vesicular challenge. PTX3 appears to be the first humoral pattern recognition molecule Implicated in innate immune defense against UTI.

Pentraxins are a superfamily of evolutionarily conserved secreted proteins, which contain an 8 amino acid-long pentraxin signature in the C-terminus pentraxin domain (Bottazzi et al., 2010). Different pentraxins are able to bind distinct microbial pattern molecules, such as lipopolysacchrides (LPS), lipoproteins, and outer membrane proteins (Omp). Binding of soluble pentraxins to their corresponding ligands on microbes triggers complement-mediated killing, direct pathogen neutralization, or promotes the opsonizationand uptake of the pathogen by phagocytic cells (Bottazzi et al., 2010). PTX3 is a prominent member of the pentraxin family which has been reported to play a protective role against several bacterial and fungal pathogens (Garlanda et al., 2002), but their contribution to the clearance of uropathogens have not previously been examined.

Jaillon et al. (2014) observed that PTX3 levels locally, in the urine, as well as in the blood were markedly enhanced following intravesicular challenge of wild type mice with uropathogenic E.coli(UPEC). The functional significance of PTX3 production in the urinary tract was deduced by comparing bacterial burden in the bladder and kidneys of wild type and the Ptx3-deficient mice following UPEC challenge. The bacterial burden was significantly higher in Ptx3^−/− mice compared to wild type mice, and this difference paralleled the number of macroscopic abscesses in the kidneys of the two groups of mice. Compared to infected wild type mice, infected Ptx3^−/− mice experienced early and persistent exacerbated inflammation, accompanied by markedly more severe loss in the body weight. Interestingly, the primary sources of PTX3 in the urinary tract were the epithelial cells lining the kidneys and the bladder as well as the large population of leukocytes recruited into the urinary tract following infection. Studies undertaken in vitro using uroepithelial cell lines indicated that the TLR4-Myd88 signaling axis on these cells was responsible for promoting the PTX3 response to bacterial infection. The authors next investigated how PTX3 proteins promoted bacterial clearance in the urinary tract. They observed that PTX3 served as opsonins, directly binding UPEC and promoting uptake by neutrophils. To explain the increased clearance of bacteria in the urinary tract of wild type mice the authors showed that the phagosomes of wild type neutrophils containing PTX3-coated UPEC matured at faster rates than in phagocytes bearing uncoated UPEC suggesting that PTX3 promoted increased intracellular killing of UPEC by neutrophils. Clinical evidence supporting a role for PTX3 in immunity in the urinary tract comes from the findings that PTX3 levels in the urine were markedly higher in UTI patients than in the urines of healthy donors or patients with other types of infections. There even appeared to be significant differences in PTX3 levels in the urine of UTI patients with varying severity as PTX3 levels in patients with acute pyelonephritis (APN), were significantly higher than in cystitis patients or individuals with asymptomatic bacteriuria. Consistent with recent reports linking genetic polymorphisms in the PTX3 locus to both the production of PTX3 in different individuals and their corresponding susceptibility to fungal infections (Cunha et al., 2014), Jaillon et al. (2014) found that PTX3 polymorphisms were associated with susceptibility to acute pyelonephritis. Thus, there appears to be multiple lines of evidence pointing to PTX3 as being an essential component of the innate immune network of the urinary tract.

That urine is a rich growth medium for bacterial growth has been widely recognized since 1863 when Louis Pasteur was credited with making this observation (Asscher et al., 1966). As urine is usually retained in the bladder for hours, the bladder represents a powerful incubator for bacterial growth. Bacterial numbers in the bladder can actually reach levels in excess of 10⁸ per ml. Indeed, 1×10⁵ per ml of urine is the critical number traditionally employed in clinical laboratory to differentiate between infection and contamination. Controlling the high numbers of bacteria attained in the urine represents a significant challenge to the urinary tract. One counter measure employed by the host’s immune defenses is secretion into the urine of fluid-phase pattern recognitions molecules (PRMs). These molecules can directly bind and aggregate bacteria in the urine so that they are either unable to attach to bladder walls or are internalized and killed in greater numbers by immune cells. Until now, most of the fluid phase PRMs in urine were thought to be constitutively produced (e.g. THP). While these readymade components could be highly effective in eliminating the occasional intruder into the lower urinary tract, their amounts are not sufficient to eliminate bacteria that have already begun to multiply in great numbers in the urine. The current study by Jaillon et al. (2014) reveals that the urinary tract have in reserve another family of fluid-phase PRMs that are actively induced and secreted in direct proportion to the size of the microbial challenge and which is triggered by the LPS-sensing immune surveillance molecule TLR4.

Although compelling and informative, this study also raises many questions. For example, it is not known how phagosomes encasing PTX3-opsonized UPEC are induced to rapidly mature leading to the demise of its microbial cargo. The impact of PTX3 on bacterial numbers in the infected mouse bladder and kidneys is very striking. Although the authors have shown with in vitro supporting data that PTX3 promotes bacterial clearance by acting as an opsonin, it may not be the whole story. First, little difference in bacterial clearance between wild type and Ptx3^−/− mice was observed on day 1 in the bladder when neutrophil numbers in the urinary tract was possibly at its highest level. Indeed, significant differences in bacterial numbers in the bladders and kidneys in the two groups of mice were mostly seen on day 5. Second, although neutrophils are highly effective in bacterial clearance it is unclear how effective they will be in the urinary tract as pH, osmolality and toxins in the urine may negatively impact their phagocytic and bactericidal activities. Thus, this study may represent just the tip of the iceberg with regard to PTX3’s protective role in the urinary tract. There is emerging data suggesting pentraxins play a key role in modulating various arms of the inflammatory response and that PTX3 may have several cellular targets (Soares et al., 2006). With the finding that most of the protective actions of PTX3 are evident only by the 5^th day of infection, there is also a possibility that PTX3 may be achieving its effects via the adaptive immune system. Thus, while PTX3 has a significant protective role in the urinary tract, its mode of action might be multifaceted.

Urine volume and composition has often been employed as a barometer of the physiological state of the host. Jaillon et al. (2014) have shown the value of interrogating urine samples in revealing novel components of the innate immune response. Interestingly, they have also reported that there appears to be a direct correlation between PTX3 levels in the urine and the magnitude of infection, raising the possibility of utilizing PTX3 as a biomarker for UTIs which could be a valuable adjunct to current diagnostic approaches.

Figure 1. PTX3 functions as an opsonin promoting UPEC uptake by neutrophils.

Invading uropathogenic E.coliare sensed by TLR4 molecules lining the surface of the superficial epithelium of the urinary tract. The activation of TLR4 leads to the production and secretion of PTX3 via the MyD88 signaling cascade, probably by translocation of the downstream NF-κB transcription factor into the nucleus. Secreted PTX3 bindsbacteria in the urine, promoting their uptake into neutrophil phagosomes that rapidly mature because of the presence of PTX3 and culminating in early clearance of the bacteria.