Human bladder uroepithelial cells synergize with monocytes to promote IL-10 synthesis and other cytokine responses to uropathogenic Escherichia coli

Abstract

Urinary tract infections are a major source of morbidity for women and the elderly, with Uropathogenic Escherichia coli (UPEC) being the most prevalent causative pathogen. Studies in recent years have defined a key anti-inflammatory role for Interleukin-10 (IL-10) in urinary tract infection mediated by UPEC and other uropathogens. We investigated the nature of the IL-10-producing interactions between UPEC and host cells by utilising a novel co-culture model that incorporated lymphocytes, mononuclear and uroepithelial cells in histotypic proportions. This co-culture model demonstrated synergistic IL-10 production effects between monocytes and uroepithelial cells following infection with UPEC. Membrane inserts were used to separate the monocyte and uroepithelial cell types during infection and revealed two synergistic IL-10 production effects based on contact-dependent and soluble interactions. Analysis of a comprehensive set of immunologically relevant biomarkers in monocyte-uroepithelial cell co-cultures highlighted that multiple cytokine, chemokine and signalling factors were also produced in a synergistic or antagonistic fashion. These results demonstrate that IL-10 responses to UPEC occur via multiple interactions between several cells types, implying a complex role for infection-related IL-10 during UTI. Development and application of the co-culture model described in this study is thus useful to define the degree of contact dependency of biomarker production to UPEC, and highlights the relevance of histotypic co-cultures in studying complex host-pathogen interactions.

Figure 1. Different cellular combinations reveal synergism between uroepithelial cells and monocytes for UPEC-induced IL-10 synthesis.

(A) Uroepithelial cells and monocytes in 96 well microtitre plate co-cultures produced peak levels of IL-10 protein (32-34 pg/mL) compared to monocultures that yielded 16-17 pg/mL following infection. T-cells did not increase IL-10 levels. (B) Substitution of monocytes with macrophages showed much higher levels of IL-10 with or without uroepithelial cells (> 200 pg/mL), but there was no significant difference between the two infected conditions (all infected groups significantly above controls, p ≤ 0.001; monocyte monocultures vs. monocyte co-cultures with uroepithelial cells, * p = 0.002; macrophage monocultures vs macrophage-uroepithelial dual co-cultures, ^‡ p = 0.003; SEM bars). (Mon = monocyte, Ep = uroepithelial cell, T = T-cell, MΦ = macrophage, Control = uninfected cells).

Figure 2. Use of membrane inserts demonstrates contact-dependent IL-10 synergism in UPEC-infected uroepithelial cell-monocyte dual co-cultures.

The level of IL-10 produced in co-cultures where uroepithelial cells and monocytes were in contact was double that of cultures where inserts were used to physically separate the two cell types. The schematics below graph show the cell type, infection and presence of inserts; blue = non-infected, red = infected (all control vs infected groups, p < 0.001; monocyte monocultures vs insert-containing dual co-cultures, * p ≤ 0.024; insert-containing dual co-cultures vs no insert dual co-cultures, ^† p ≤ 0.002; SEM bars).

Figure 3. IL-10 mRNA responses to UPEC in dual co-cultures that contain membrane inserts are principally monocyte-derived.

(A) Monocytes in dual co-cultures overall have > 10-fold abundance of IL-10 transcript compared to uroepithelial cells, as transcripts emerge 3.4 cycles (Ct = 25.0 vs Ct = 28.4) earlier in qPCR (monocyte vs uroepithelial cell Ct values, p = 0.007). (B) Monocytes from all insert-containing infected conditions showed a statistically significant increase in IL-10 mRNA (1.8-fold increase of averaged infected monocyte conditions over control non-infected monocytes; insert-containing infected monocytes from dual co-cultures vs uninfected control, * p = 0.009). Uroepithelial cells from insert-containing infected conditions did not show significant increases over non-infected (1.16-fold increase of averaged infected uroepithelial cell conditions over control non-infected uroepithelial cells). Mann–Whitney U-tests were used for comparisons and SEM bars are shown.

Figure 4. Contact between uroepithelial cells and monocytes drives synergistic and antagonistic biomarker production in UPEC-infected dual co-cultures.

(A) IL-10 production was synergistic in co-cultures that showed significant increases mediated by contact between uroepithelial cells and monocytes (compare Ic-Mon/Ep with Ni-Mon/Ep, p ≤ 0.0139). In contrast, the production of IL-6 (B), GM-CSF (C) and PDGF-BB (D) in insert-containing co-cultures was significantly higher than non-insert co-cultures (compare Ic-Mon/Ep with Ni-Mon/Ep), revealing antagonistic effects mediated by contact between monocytes and uroepithelial cells. Statistical comparisons are: ⁺ control vs corresponding infected; ^# insert-containing infected co-culture vs corresponding infected monoculture; ^┴ insert-containing infected co-culture vs no insert infected co-culture, notations are p < 0.05; Mann–Whitney U-test. (Ic-Ctrl = insert-containing dual co-culture uninfected, Ic-Ep = insert-containing uroepithelial infected, Ic-Mon = insert-containing monocyte infected, Ic-Mon/Ep = insert-containing monocyte and uroepithelial infected, Ni-Ctrl = no insert co-culture uninfected, Ni-Mon/Ep = no insert monocyte and uroepithelial infected, Mon-Ctrl = monocyte uninfected, Mon = monocyte infected, Ep-Ctrl = uroepithelial uninfected, Ep = uroepithelial infected).

Figure 5. UPEC-induced soluble-dependent biomarker responses in dual co-cultures grouped according to biomarker levels in uroepithelial monocultures.

Experimental Details are as per Figure 4. In this dataset, the most common type of response was a synergistic increase based on a soluble factor(s) conferred by presence of uroepithelial cells. Statistical comparisons are: ⁺ control vs corresponding infected; ^# insert-containing infected co-culture vs corresponding infected monoculture; ^┴ insert-containing infected co-culture vs no insert infected co-culture, notations are p < 0.05; Mann–Whitney U-test.

Figure 6. UPEC-induced soluble-dependent biomarker responses in dual co-cultures grouped according to biomarker levels in monocyte monocultures.

Experimental Details are as per Figure 4. In this dataset, the least common type of response was a synergistic increase based on a soluble factor(s) conferred by the presence of monocytes. Statistical comparisons are: ⁺ control vs corresponding infected; ^# insert-containing infected co-culture vs corresponding infected monoculture; ^┴ insert-containing infected co-culture vs no insert infected co-culture, notations are p < 0.05; Mann–Whitney U-test.

Figure 7. UPEC-induced soluble-dependent biomarker responses in dual co-cultures grouped according to low biomarker levels in monocultures.

Experimental Details are as per Figure 4. In this dataset, a common type of response was a synergistic increase based on a soluble factor(s) conferred by the presence of monocytes and uroepithelial cells, with low levels from monocultures absent in dual co-cultures. Statistical comparisons are: ⁺ control vs corresponding infected; ^# insert-containing infected co-culture vs corresponding infected monoculture; ^┴ insert-containing infected co-culture vs no insert infected co-culture, notations are p < 0.05; Mann–Whitney U-test.