Different inflammatory responses are associated with Ureaplasma parvum-induced UTI and urolith formation

Abstract

Background: Epidemiologic studies show a strong association between Ureaplasmas and urogenital tract disease in humans. Since healthy humans can be colonized with Ureaplasmas, its role as a pathogen remains controversial. In order to begin to define the role of the host in disease, we developed a rodent model of urinary tract infection (UTI) using Fischer 344 (F344) rats. Animals were inoculated with sterile broth, 10(1), 10(3), 10(5), 10(7), or 10(9) log CFU of a rat-adapted strain of Ureaplasma parvum.

Results: Infected animals exhibited two distinct profiles, asymptomatic UTI and UTI complicated with struvite urolithiasis. Inoculum dose of U. parvum affected the incidence of UTI, and 50% to 57% of animals inoculated with >or= 10(7) CFU of U. parvum remained infected (p < 0.04). However, inoculum dose did not influence immune response to U. parvum. Asymptomatic UTI was characterized by a minimal immune response that was predominantly monocytic and lymphocytic, with limited lesions, and elevated urinary levels of IFN-gamma, IL-18 and MCP-1 (P <or= 0.02). UTI complicated with struvite formation was characterized by an exaggerated immune response that was mostly neutrophilic (P <or= 0.0001), with lesions that showed extensive uroepithelial hyperplasia (P <or= 0.0001), and a predominance of IL-1 alpha, IL-1 beta, and GRO/KC in the urine (P <or= 0.02). Animals with asymptomatic UTI also had a significantly high rate of kidney infection (P <or= 0.0005).

Conclusion: Complications associated with U. parvum infection are primarily dependent upon host-specific factors rather than Ureaplasma microbial load. The immune response in F344 rats is similar to that which occurs in humans with ureaplasmal associated disease. Therefore, this model of infection is a useful tool for elucidating U. parvum-host interactions that confer UTI and disease.

Figure 1

Summary of the bladder lesions found in F344 rats experimentally infected with U. parvum. Panel A is a 200× magnification of bladder tissue from a Control rat. This sample represents a lesion score of 0 for degree of inflammation, degree of epithelial change and inflammatory cell type. Panels B, C, and D are tissue sections from animals inoculated with U. parvum that had a lesion score of 3 for degree of inflammation, degree of epithelial change and inflammatory cell type. Panel B is a 200× magnification of bladder tissue from an animal within the struvite group. The asterisk demarcates the extensive edema and fibrinous exudate infiltrating the submucosa. The black arrow points to uroepithelial effacement. Panel C is a 400× magnification of extensive uroepithelial hyperplasia. Panel D is a magnified inset of Panel B that highlights the array of white blood cells that comprised in the inflammatory cellular infiltrate in the tissues of infected animals. The blue arrow is pointing to a neutrophil. The green arrow is pointing to a tissue macrophage. The yellow arrow is pointing to a plasma cell. The red arrow is pointing to a lymphocyte.

Figure 2

Lesion scores of bladder tissue from F344 rats inoculated with U. parvum. Lesion score analysis was done prior to grouping each sample into a clinical profile (Panels A, B, and C) or inoculum dose groups (Panel D). Data is a summation of 5 separate experiments. In panels A, B, and C, nonparametric lesion scores were grouped according to the clinical profile and analyzed by Kruskall- Wallis test (Control, n = 6; Negative, n = 36; UTI, n = 11; Struvite, n = 10). Panel D are the results of an inflammatory cell type lesion score analysis performed on bladder tissues from animals in the Negative group. Scoring for cell types that comprised the inflammatory infiltrate was: 1 for primarily mononuclear cells (lymphocytes, plasma cells and macrophages), 2 for mononuclear cells and neutrophils, and 3 for mononuclear cells, neutrophils and fibrous infiltrates. Data is a summation of 5 separate experiments. Nonparametric lesion scores were grouped according to the inoculum dose of U. parvum. Raw lesion scores were analyzed by Kruskall- Wallis test (n = 10 for log 1 CFU, n = 11 for log 3 CFU, n = 8 for log 5 CFU, n = 5 for log 7 CFU, and n = 3 for log 9 CFU). Values in all graphs represent raw lesion scores for each biological replicate. Horizontal bars demarcate the median value for each clinical profile group.

Figure 3

Summary of the kidney lesions found in F344 rats experimentally infected with U. parvum. Panel A is a 200× magnification of kidney tissue from a Control rat and demonstrates the lack of inflammatory lesions that are characteristic in animals inoculated with U. parvum. Panels B, C, and D are tissue sections from animals inoculated with U. parvum that had a lesion score of 4 for total area affected. Panel B is a 400× magnification demonstrating the inflammatory infiltrate extending from the renal pelvic space into the interstitium with uroepithelium largely intact. The black arrow is pointing to uroepithelial hyperplasia. Panel C is a 400× magnification of renal tubules. The black arrow is pointing to the extensive inflammatory infiltrate throughout the renal tubular interstitium. The yellow arrow is pointing to a glomerulus. Panel D is a 600× magnification of renal uroepithelium at the edge of the pelvic space. The black arrow is pointing to extensive hemorrhage and disruption of the uroepithelial barrier by a fibrinous inflammatory infiltrate.

Figure 4

Urine chemokines detected in F344 rats inoculated with sterile 10B or U. parvum. Data represent the mean ± SD of a combination of 5 separate experiments. Urine chemokine concentrations were grouped according to clinical profile, control (n = 6), negative (n = 36), UTI (n = 16), and Struvite (n = 13). P values within each graph were obtained by one-way ANOVA. Fisher's PLSD test revealed that GRO/KC concentrations in the struvite group were significantly greater than the control, negative and UTI groups. Fisher's PLSD test revealed that MCP-1 concentrations in the control group was significantly greater than the negative and control groups.

Figure 5

Urine cytokines detected in F344 rats inoculated with sterile 10B or U. parvum. Data represent the mean ± SD of a combination of 5 separate experiments. Urine cytokine concentrations were grouped according to clinical profile, control (n = 6), negative (n = 36), UTI (n = 16), and Struvite (n = 13). P values within each graph were obtained by one-way ANOVA. Fisher's PLSD test revealed that IL-1α, IL-1β, IL-6, and TNF-α concentrations in the struvite group were significantly greater than control, negative, and UTI groups. Fisher's PLSD test revealed IL-10 concentrations in the struvite group were significantly greater than negative and UTI groups.

Figure 6

Global profiling of urine cytokines detected in control and U. parvum infected F344 rats. The clustered heat map represents the standardized LS means for each cytokine that had a significantly different pattern of expression among clinical groups (P ≤ 0.02). Values were obtained by one-way ANOVA using a row by row modeling with Fischer C correction for multiple comparisons. Two main cytokine cluster patterns were identified in the analysis and are demarcated by the green and red cluster tree. The number of biological replicates were n = 6 for control, n = 16 for UTI group, n = 13 for the Struvite group.

Figure 7

Profiling the inflammatory response to different doses of U. parvum in culture negative F344 rats. Panel A is a clustered heat map representing the standardized LS means for each cytokine with a significantly different pattern of expression among infection dose groups (P 0.05). Values were obtained by one-way ANOVA using a row by row modeling with Fischer C correction for multiple comparisons. Two main cytokine cluster patterns were identified in the analysis and are demarcated by the green and red cluster tree. The number of biological replicates were n = 6 for control, n = 10 for log 1 CFU, n = 11 for log 3 CFU, n = 8 for log 5 CFU, n = 5 for log 7 CFU, and n = 3 for log 9 CFU. The red arrow is highlighting the pattern of cytokines present in the urine of culture negative rats that were inoculated with log 5 CFU. Animals within this dose group were the only animals to exhibit an obvious inflammatory cell infiltrate comprising a mixture of mononuclear cells with neutrophils (P 0.006, Figure 2, Panel D).