Circulating mucosal-associated invariant T cells in subjects with recurrent urinary tract infections are functionally impaired

Abstract

Background: Urinary tract infection recurrence is common, particularly in women and immunocompromised patients, such as renal transplant recipients (RTRs). Mucosal-associated invariant T (MAIT) cells play a role in the antibacterial response by recognizing bacterial riboflavin metabolites produced by bacteria such as Escherichia coli. Here, we investigated whether MAIT cells are involved in the pathogenesis of recurrent urinary tract infections (RUTIs).

Methods: Using multichannel flow cytometry, we characterized the MAIT cell phenotype and function in blood from immunocompetent adults with (n = 13) and without RUTIs (n = 10) and in RTRs with (n = 9) and without RUTIs (n = 10).

Results: There were no differences in the numbers of MAIT cells between the study groups. MAIT cells in patients with RUTI expressed T-bet more often than those in controls. MAIT cells from immunocompetent RUTI participants required more antigen-presenting cells coincubated with E. coli to evoke a similar cytokine and degranulation response than those from controls. This effect was absent in the RTR with RUTI vs RTR control groups, where the overall percentage of MAIT cells that responded to stimulation was already reduced.

Conclusion: Circulating MAIT cells in immunocompetent individuals with RUTIs respond to bacterial stimuli with reduced efficacy, which suggests that they are involved in the pathogenesis of RUTIs.

Keywords: MAIT cells; recurrent urinary tract infection; renal transplantation.

Figure 1

Circulating MAIT cell numbers are similar in RUTI subjects and healthy controls. Comparison of PB MAIT cells between immunocompetent controls without RUTIs (CTRL) and immunocompetent participants with RUTIs (RUTI) and between RTRs without RUTIs (RTR CTRL) and RTRs with RUTIs (RTR RUTI) by flow cytometry. A, Scatterplots of the percentage of MAIT cells (MR1 BV421) within the CD3 population. B, Scatterplots of the percentage of MAIT cells expressing CD4 APC‐R700 and/or CD8 BV785. C, Scatterplots of the expression of CD161 PE on CD4− CD8+ (CD8+) and CD4− CD8− (DN) MAIT cells. A‐C, Statistical analysis: the Mann‐Whitney U test; the dash represents the median. No significant differences were found. D, Heatmap of the differentiation state of CD8+ and DN MAIT cells defined by CD45RA BV650/CCR7 BUV395/CD28 APC/CD27 APC‐FIRE750 expression. Almost all CD8+ and DN MAIT cells displayed a, not immediately cytotoxic, effector‐memory phenotype (CD45RA− CCR7− CD28+), mostly with the coexpression of CD27. The data shown are representative of six independent experiments with n = 6, 5, 7, 5, 10, and 9 donors per experiment. A total of 42 unique donors are shown (CTRL = 10; RUTI = 13, RTR CTRL = 9 and RTR RUTI = 9; 1 RTR RUTI had an RA‐downregulation deficiency* and was thus excluded from the analysis in D). APC, antigen‐presenting cell; CTRL, control; DN, double negative; MAIT, mucosal‐associated invariant T cell; RTR, renal transplant recipient; RUTI, recurrent urinary tract infection. *Tchilian, EZ, Beverley, PC. Altered CD45 expression and disease. Trends Immunol 2006;27: 146‐153

Figure 2

An increased number of circulating MAIT cells in RUTI participants express T‐bet comparison of the PB MAIT cell phenotype between immunocompetent controls without RUTIs (CTRL) and immunocompetent participants with RUTIs (RUTI) and between RTRs without RUTIs (RTR CTRL) and RTRs with RUTIs (RTR RUTI) by flow cytometry. A, Heatmap of the expression of IL‐7Rα PE‐Cy7, Eomes eFluor660, CXCR6 PE‐Cy7, granzyme K PerCPeFluor710, T‐bet PE‐Cy7, CXCR4 BUV395, CCR6 AF488, PD1 BB515, perforin BV510, CD69 APC‐FIRE750, granzyme B AF700, and Ki‐67 BV711 on CD4− CD8+ (CD8+) and CD4− CD8− (DN) MAIT cells. There were only a few differences between the groups. Markers expressed at significantly different levels between immunocompetent adults with and without RUTIs or RTRs with and without RUTIs are displayed in B and C. The expression of other markers was not significantly different between the compared groups (displayed in Figure S6). B, Scatterplots of the differentially expressed markers on CD8 MAIT cells. C, Scatterplots of the differentially expressed markers on DN MAIT cells. Statistical analysis: the Mann‐Whitney U test; the dash represents the median. Only significant P values are displayed: *P < .05, **P ≤ .01, ***P ≤ .001, ****P = .0001. The data shown are representative of six independent experiments with n = 6, 5, 7, 5, 10, and 9 donors per experiment. A total of 42 unique donors are shown (CTRL = 10; RUTI = 13, RTR CTRL = 9, and RTR RUTI = 9). APC, antigen‐presenting cell; CTRL, control; DN, double negative; MAIT, mucosal‐associated invariant T cell; RTR, renal transplant recipient; RUTI, recurrent urinary tract infection

Figure 3

Circulating MAIT cells in RUTI subjects have an increased activation threshold. Comparison of PB MAIT cell function between immunocompetent controls without RUTIs (CTRL) and immunocompetent participants with RUTIs (RUTI) and between RTRs without RUTIs (RTR CTRL) and RTRs with RUTIs (RTR RUTI). A, Schematic representation of MR1‐specific stimulation using Escherichia coli ‐loaded APCs. B, Scatterplots of the percentage of MAIT cells producing cytokines (TNF‐α [AF700], IFNү [BUV395], GM‐CSF [PE‐Dazzle594], IL‐2 [BV510], IL‐17A [BV650]), and degranulating (CD107A FITC) by flow cytometry after stimulation with either 104 E. coli‐loaded APCs, 105 E. coli‐loaded APCs, or PMA/ionomycin. Statistical analysis: the Mann‐Whitney U test; the dash represents the median. Only significant differences are displayed: *P < .05, **P ≤ .01, ***P ≤ .001, ****P = .0001. The data shown are representative of seven independent experiments with n = 2, 3, 3, 3, 10, 9, and 10 donors per experiment. Forty unique donors are shown (CTRL = 10; RUTI = 13, RTR CTRL = 9, and RTR RUTI = 7). APC, antigen‐presenting cell; CTRL, control; GM‐CSF, granulocyte‐macrophage colony‐stimulating factor; IFNү, interferon γ; IL‐2, interleukin‐2; MAIT, mucosal‐associated invariant T cell; PBMC, peripheral blood mononuclear cell; PMA, phorbol 12‐myristate 13‐acetate; RTR, renal transplant recipient; RUTI, recurrent urinary tract infection; TNF‐α, tumor necrosis factor α

Figure 4

The average number of MAIT cell functions after stimulation with 10⁴ Escherichia coli ‐loaded APCs is reduced in patients with RUTI. Comparison of the number of PB MAIT cell functions (TNF‐α [AF700]‐, IFNү [BUV395]‐, GM‐CSF [PE‐Dazzle594]‐, IL‐2 [BV510]‐, IL‐17A [BV650]‐production and/or expression of CD107A [FITC]) by flow cytometry following stimulation with 10⁴ E. coli‐loaded APCs (left panel) and 10⁵ E. coli‐loaded APCs (middle panel) and PMA‐ionomycin (right panel) between immunocompetent controls without RUTIs (CTRL) and immunocompetent participants with RUTIs (RUTI) and between RTRs without RUTIs (RTR CTRL) and RTRs with RUTIs (RTR RUTI). Scatterplots of the average number of MAIT cell functions. Statistical analysis: the Mann‐Whitney U test; the dash represents the median. Only significant differences are displayed: *P < .05, **P ≤ .01, ***P ≤ .001, ****P = .0001. The data shown are representative of seven independent experiments with n = 2, 3, 3, 3, 10, 9, and 10 donors per experiment. Forty unique donors are shown (CTRL = 10; RUTI = 13, RTR CTRL = 9, and RTR RUTI = 7). The heatmap of the percentage of MAIT cells with 0 to 6 of the evaluated functions is provided in Figure S8. APC, antigen‐presenting cell; CTRL, control; FITC, fluorescein isothiocyanate; GM‐CSF, granulocyte‐macrophage colony‐stimulating factor; IFNү, interferon γ; IL‐2, interleukin‐2; MAIT, mucosal‐associated invariant T cell; PMA, phorbol 12‐myristate 13‐acetate; RTR, renal transplant recipient; RUTI, recurrent urinary tract infection; TNF‐α, tumor necrosis factor α