Fibrin-rich environment drives catheter-associated UTIs by suppressing macrophage defenses

Armando M Marrufo; Christopher Gager; Jonathan J Molina; Marissa J Andersen; Alyssa A La Bella; Jessica J Kean; Katelyn C Fealy; Elizabeth R Lucas; Ellsa Wongso; Tamanna Urmi; Kassandra Arias-Parbul; Railyn Webster; Peter V Stuckey; Kurt N Kohler; Deborah Donahue; Victoria A Ploplis; Matthew J Flick; Francis J Castellino; Felipe H Santiago-Tirado; Ana L Flores-Mireles

doi:10.1038/s41467-026-76660-8

. 2026 Aug 25;17:10183. doi: 10.1038/s41467-026-76660-8

Fibrin-rich environment drives catheter-associated UTIs by suppressing macrophage defenses

Armando M Marrufo ^1,^#, Christopher Gager ^1,^#, Jonathan J Molina ^1,², Marissa J Andersen ¹, Alyssa A La Bella ¹, Jessica J Kean ¹, Katelyn C Fealy ¹, Elizabeth R Lucas ¹, Ellsa Wongso ¹, Tamanna Urmi ¹, Kassandra Arias-Parbul ¹, Railyn Webster ^1,³, Peter V Stuckey ¹, Kurt N Kohler ¹, Deborah Donahue ⁴, Victoria A Ploplis ^4,⁵, Matthew J Flick ^6,⁷, Francis J Castellino ^4,⁵, Felipe H Santiago-Tirado ¹, Ana L Flores-Mireles ^1,^2,^✉

PMCID: PMC13612831 PMID: 42786163

Abstract

Catheter-associated urinary tract infections (CAUTIs) pose severe clinical challenges, often leading to urosepsis and multidrug resistance. The wound-healing process triggered by catheter-induced bladder damage deposits fibrinogen and its polymerized form, fibrin. However, uropathogens exploit the fibrinogen and fibrin matrix to form a protective biofilm. While catheter-induced inflammation recruits innate immune cells, particularly macrophages, to the site of infection, the persistence of uropathogens suggests that the local bladder environment alters their antimicrobial function. Our research pinpoints the coagulation cascade as a key driver of this dysfunction, finding a differential macrophage polarization influenced by fibrinogen and fibrin. We showed that urinary catheterization creates a fibrin-rich matrix that was correlated with polarizing macrophages into an anti-inflammatory M2-like state, suppressing their bactericidal response. In contrast, using mice expressing fibrinogen locked in the form of a monomer, we demonstrated that monomeric fibrinogen promotes a pro-inflammatory M1-like polarization state. Notably, skewing a M1-like macrophage state with GM-CSF failed to clear the infection and systemic dissemination, suggesting a dominant role for fibrin matrix in suppressing M1-like macrophage-mediated antimicrobial activity. Together, these findings show that the catheterized bladder provides signals that significantly alter macrophage function, creating an ideal niche for pathogen persistence.

Subject terms: Antimicrobial responses, Pathogens

Marrufo et al. show that fibrinolytic deficiency resulting in extravascular fibrin formation in the bladder predisposes hosts to CAUTIs and systemic dissemination by modulating macrophages’ bactericidal response.

Introduction

Macrophages (Mφs) are plastic cells of the innate immune system that tailor their function in response to microenvironmental signals, like cytokines, chemokines, and pathogen/damage-associated molecular patterns (PAMPs/DAMPs)^1–3. Mφs exhibit this plasticity by polarizing into a continuum from M1-like (pro-inflammatory) to M2-like (anti-inflammatory) states^1–3. On each end of the continuum, M1-like Mφs drive microbicidal responses^1,2 while M2-like Mφs resolve inflammation by dampening its antimicrobial response and promoting tissue repair and remodeling^1,4. Because these functions are opposing, the M1 to M2 ratio balance must be precisely regulated to maintain tissue health^1–3,5. Chronic M1-Mφ activation contributes to inflammatory diseases like IBD and atherosclerosis^3,6–9, whereas excessive M2-Mφ activity is linked to cancer progression and impaired pathogen clearance^3,10,11. In response to complex microenvironmental cues, Mφs can receive mixed signals that lead them to co-express both M1-like and M2-like characteristics. These cells, referred to as hybrid M1/M2-like Mφs, could drive either beneficial or maladaptive host outcomes, such as regulating extracellular matrix remodeling, impeding pathogen clearance, or promoting atherosclerotic lesions^3,12–14.

Beyond cytokines and pathogens, components of the coagulation cascade are potent regulators of Mφ polarization. Soluble fibrinogen (Fg) and its polymerized form, fibrin, have been suggested to differentially program Mφ polarization¹⁵. An ex vivo study found that soluble Fg and fibrin exert distinct effects on the polarization of bone-marrow-derived Mφs (BMDMφs)¹⁵. The mechanism of fibrin(ogen)-dependent polarization is linked to Mφ receptors, including Mac-1 (α_Mβ₂ or CD11b/CD18), CD11c/CD18 (α_Xβ₂), and toll-like receptor-4 (TLR-4)^16–19. Moreover, dysfunction of Mφ polarization has been implicated in coagulopathy-related diseases, such as hemophilia and thrombotic disorders^3,20,21.

Previously, we found that the local levels of Fg and fibrin play a critical role in the outcome of catheter-associated urinary tract infections (CAUTI)²², which are one of the leading causes of nosocomial infections. In humans and mice, urinary catheterization induces bladder tissue damage, leading to the accumulation of Fg and fibrin, collectively termed fibrin(ogen)^23–25. As a response to injury, fibrin(ogen) is necessary for restoring hemostasis and promoting wound healing, but in CAUTI it also has detrimental consequences due to uropathogens (e.g., Escherichia coli and Enterococcus faecalis) exploiting fibrin(ogen) as a scaffold for biofilm formation^{22,23,25–29}. This process creates a central paradox. The catheterized bladder is a complex inflammatory environment with robust infiltration of immune cells, including Mφs, that should clear the infection^28–33. Despite the immune presence, pathogen clearance fails, leading to persistent CAUTI^22,33. This strongly suggests an underlying state of immune dysregulation. Our own work highlights the critical role of fibrin in this failure; we previously demonstrated that mice with coagulopathies resulting in excess fibrin accumulation are highly susceptible to persistent CAUTI and systemic dissemination by diverse uropathogens²². While the role of fibrin(ogen) providing a physical scaffold for uropathogen colonization is known, its direct impact on the responding immune cells is not. Therefore, it is critical to understand how Fg and fibrin specifically modulate the bactericidal function of Mφs within this unique microenvironment.

This study reveals that urinary catheterization creates a fibrin-rich environment that programs Mφs into an anti-inflammatory M2-like Mφ state, suppressing their ability to kill the two most prevalent uropathogens, E. coli and E. faecalis. Using knock-in mice, we demonstrate that the effect of fibrin formation correlates with an M2-like Mφ phenotype associated with suppressed bacterial clearance, whereas its soluble Fg promotes an M1-like Mφ phenotype characterized by enhanced bactericidal response. We then tested whether therapeutically forcing an M1-like Mφ phenotype with GM-CSF could resolve the infection. While therapeutic administration of GM-CSF successfully induces an M1-like Mφ phenotype, this surprisingly failed to promote bacterial clearance. This critical disconnect was linked to shared phagocytosis activity among all Mφ subpopulations, including those with poor bactericidal capacity. This shared phagocytosis likely undermines effective bacterial clearance, contributing to dissemination. Our research identifies the fibrin-driven environment of the catheterized bladder as a dominant, immunosuppressive force in CAUTI. Ultimately, this research demonstrates that mitigating CAUTI requires a paradigm shift beyond traditional antimicrobial therapies to actively target the pathological host microenvironment created by urinary catheterization.

Results

Urinary catheterization-induced inflammation promotes robust fibrin(ogen) and macrophage recruitment

Urinary catheterization induces a robust innate immune response marked by a significant influx of Mφs (Supplementary Fig. 1), compared to uncomplicated UTIs (uUTIs, absence of catheter) (Fig. 1)^31,32. Despite this strong immune presence, uropathogens such as E. coli and E. faecalis, are still able to colonize and persist in the catheterized bladder^{22,25,26,31–36}. At 24 h post infection (hpi), we investigated how catheterization alters Mφ response by utilizing established mouse models of uncomplicated UTI (uUTI, absence of catheter), catheterization alone, and CAUTI using E. coli and E. faecalis^37,38.

We then assessed Mφ recruitment, localization, and whether Mφs interact with fibrin(ogen) and the pathogen in the bladder. To quantify Mφ abundance, we gated for Mφ populations (Fig. 1a, b, Supplementary Fig. 1). Naive mouse bladders showed low abundance of Mφs (Fig. 1b), residing in the lamina propria (LP) (Fig. 1c and Supplementary Fig. 2). Catheterized-only wild-type mice displayed a significant increase of Mφs when compared to naive bladders (Fig. 1b). Importantly, we found that Mφs in the LP were colocalizing with fibrin(ogen), which is recruited to the bladder due to the catheter-induced damage (Fig. 1c and Supplementary Fig. 3). In uUTI, Fg was not detected in the bladder, regardless of the pathogen (Fig. 1c, Supplementary Figs. 4 and 5). This Fg-deficient environment had profoundly influenced pathogen fitness. While uropathogenic E. coli (which accounts for up to 95% of uUTI cases) successfully established a persistent infection, E. faecalis failed to do so (Fig. 1c, Supplementary Figs. 4 and 5). This disparity is consistent with prior research indicating that fibrin(ogen) is required for E. faecalis biofilm formation and pathogenicity^{22–24,28,29}. This difference in colonization outcome (persistent E. coli vs. transient E. faecalis) correlated directly with the level of host defense, specifically leading to greater Mφ recruitment during E. coli infection (Fig. 1b, c, Supplementary Figs. 4 and 5).

A robust Mφ response was observed during CAUTI, comparable to the effect of catheterization alone (Fig. 1b). Interestingly, Mφs also continued to colocalize with fibrin(ogen) in the LP instead of moving to the lumen, where both fibrin(ogen) and uropathogens were found (Fig. 1c, Supplementary Figs. 6 and 7). Fibrin(ogen) that accumulated in the bladder lumen and urothelium instead promoted robust colonization by both uropathogens, providing E. coli an additional niche through the formation of biofilms (Fig. 1c, Supplementary Figs. 6 and 7). Our data showed that catheter-induced bladder damage is a more potent stimulus for Mφ recruitment than the infection itself. Interestingly, Mφs interact with Fg/fibrin, highlighting a potentially important role for Fg/fibrin in modulating the immune response.

Macrophage polarization in the catheterized bladder

Based on our data, we assessed how the catheterized bladder environment modulated Mφ polarization across the M1-like to M2-like spectrum. By flow cytometry, we gated for Mφs (Live cells CD45⁺CD11b⁺F4/80⁺) and analyzed the expression of inducible nitric oxide synthase (iNOS) and Arginase-1 (Arg1) as markers for M1- and M2-like Mφ phenotypes, respectively^1,2,15,39 (Supplementary Fig. 8). We identified and quantified four distinct populations: M0- (non-polarized), M1- (pro-inflammatory/antimicrobial), hybrid M1/M2- (both M1-like and M2-like characteristics), and M2-like Mφs (anti-inflammatory/wound healing) (Fig. 2).

We observed distinct Mφ polarization populations across different bladder conditions (Fig. 2). Catheterization alone significantly increased M2-like Mφs compared to naive bladders. Compared to catheterization alone, infection of catheterized bladders with either E. coli or E. faecalis showed an increased trend in M0-, M1-, and M1/M2-like Mφ subpopulations (Fig. 2a–c). During uUTI, Mφ polarization was pathogen-dependent compared to naive bladders. Specifically, E. coli infection increased the proportion of M1/M2-like Mφs phenotypes. In contrast, E. faecalis infection led to an increase in M1- and M1/M2-like Mφ polarization (Fig. 2b, c). The catheterized environment drives robust Mφ recruitment, establishing a large cell population biased towards M0- and M2-like Mφ polarization that is then uniquely modulated by pathogens during CAUTI (Fig. 2a, d).

Fibrin suppresses fibrinogen-induced M1-like response

The catheterized bladder contains both soluble Fg and its polymer, fibrin, which have been suggested to differentially polarize Mφ by inducing pro-inflammatory M1-like (iNOS +) and pro-reparative M2-like Mφ (Arg1 +) polarization, respectively^15,39. Thus, we investigated their differential effects on Mφ polarization (Fig. 3). We utilized RAW 264.7 mouse Mφs to determine if Fg and/or fibrin directly influence Mφ polarization, measuring the abundance of M1-like Mφ (iNOS⁺Arg1^-) and M2-like Mφ (iNOS^-Arg1⁺). Due to the significant in vivo prevalence of a hybrid phenotype expressing both markers (iNOS⁺ and Arg1⁺), hybrid population was quantified in all subsequent analyses (Supplementary Fig. 9). These Fg and/or fibrin effects were benchmarked against cells treated with standard polarizing cytokines for M1-like (e.g., IFN-γ + LPS^40–42 or GM-CSF^43–47), M2-like (IL-4^48,49), and mixed M1/M2-like Mφ (1: IFN-γ + LPS + IL-4 or 2: GM-CSF + IL-4) phenotypes.

From the selected concentrations and conditions from Supplementary Fig. 9, we next treated BMDMφs and RAW 264.7 cells with cytokines, soluble Fg, or fibrin for 24 h (Fig. 3, Supplementary Fig. 10). Immunofluorescence analysis of BMDMφs and RAW 264.7 cells for iNOS (M1-like Mφ marker) and Arg1 (M2-like Mφ marker) consistently revealed a hybrid M1/M2-like Mφ subpopulation co-expressing both markers in all conditions, at different levels (Fig. 3 and Supplementary Fig. 10).

Soluble Fg and fibrin modulated differently BMDMφ polarization (Fig. 3a), with soluble Fg induced a significantly higher presence of M1-like Mφs (Fig. 3c). To confirm that this M1-like polarization was not an artifact of endotoxin contamination, we quantified endotoxin levels in our Fg preparations, verifying that any traces were negligible and remained strictly below acceptable thresholds (Supplementary Fig. 11). As an additional control, a mutant fibrinogen locked in its monomeric soluble form (Fg^AEK) was used, finding that it also drove M1-like Mφ polarization, mirroring the effect of wild-type soluble Fg (Supplementary Fig. 12). Conversely, fibrin significantly promoted M2-like Mφ polarization (Fig. 3e). This effect predominated even during co-treatment with soluble Fg (or Fg^AEK), indicating that fibrin signaling overrides Fg-induced M1-Mφ polarization (Fig. 3e, Supplementary Figs. 10 and 12b). This outcome contrasted with mixed-cytokine controls; for example, GM-CSF + IL-4 treatment resulted in M1-like Mφ predominance in BMDMφs, while IFN-γ + LPS + IL-4 led to a majority hybrid M1/M2-like Mφ population (Fig. 3c, d). These findings were largely confirmed in RAW 264.7 cells, although in this cell line, the GM-CSF + IL-4 treatment also resulted in a predominant hybrid M1/M2-like Mφ population (Supplementary Fig. 10). Our findings demonstrate that soluble Fg and fibrin exerted differential effects on Mφ polarization: soluble Fg favored an M1-like Mφ phenotype while fibrin skewed toward M2-like Mφ polarization that is pronounced when soluble Fg is present (Fig. 3e).

Catheterized bladder environment with fibrin accumulation enhances M2-like Mφ response

Building on our prior work showing that fibrin accumulation exacerbates CAUTI²², we investigated whether fibrin is a key driver of the catheter-induced inflammation seen in CAUTI. To do this, we catheterized wild-type (Fg^+/+) mice and Fg^AEK mice (cannot form fibrin) and mock-infected them for 24 h. We then assessed bladder edema by weight and analyzed fibrin(ogen) accumulation and key fibrinolysis markers (urokinase plasminogen activator [uPA], D-dimer) by western blot (Supplementary Fig. 13). As hypothesized, bladders from Fg^AEK mice showed a significant reduction in both bladder weight and fibrin(ogen) accumulation. (Supplementary Fig. 13a, b). Furthermore, D-dimer, a product of fibrin degradation, was detected only in wild-type (Fg^+/+) mice, whereas uPA, the main plasmin activator during CAUTI²², was present in the bladders of both Fg^+/+ and Fg^AEK mice (Supplementary Fig. 13c). Together, these results show that catheterization triggers fibrin accumulation, outpacing the active fibrinolytic system, directly contributing to bladder inflammation.

To understand the immunogenic effect of the complex catheterized bladder environment and fibrin(ogen) on Mφ polarization, we analyzed the Mφ subpopulations by flow cytometry (Fig. 4, Supplementary Figs. 14 and 15). We used a comprehensive set of models, including wild-type (Fg^+/+) mice and mice with distinct coagulopathies: those unable to form fibrin (Fg^AEK)^22,50,51, those with excess fibrin accumulation (Pg^−/− and uPA^−/−)^22,52,53, and those with excessive fibrinolysis (PAI-1^−/−)^22,54 (Fig. 4a, Table 1). These mice were studied in the context of uUTI and CAUTI caused by either E. coli or E. faecalis. A Principal Component Analysis (PCA) revealed the primary factors that correlate with Mφ polarization (Fig. 4b, c). The analysis identified two main clusters of variables: (1) M1-like Mφ polarization was enhanced by conditions with low or no fibrin, such as uUTI and coagulopathies that cause bleeding or excessive fibrinolysis (Fg^AEK and PAI-1^−/− mice). (2) M2-like Mφ polarization was strongly associated with conditions promoting fibrin accumulation, namely urinary catheterization and fibrinolytic deficiencies (WT, Pg^−/−, and uPA^−/− mice). While the fibrin environment influenced the primary polarization state, the specific uropathogen provided a secondary layer of modulation (Fig. 4b, Supplementary Fig. 15). During uUTI, E. coli infection correlated with a strong M1-like Mφ response, while E. faecalis shifted it toward a hybrid M1/M2-like phenotype (Fig. 4b–e). However, in host strains with severe coagulation defects, genotype was the dominant factor determining polarization. Specifically, Fg^AEK mice consistently exhibited an M1-like Mφ phenotype, while uPA^−/− mice favored an M2-like Mφ state, regardless of the pathogen (Fig. 4b, c).

Fig. 4 — a Coagulation cascade diagram. C57BL/6 female WT and coagulation-transgenic mice bladders were uninfected or infected with ~10⁷ CFUs of *E. coli* UTI89 or *E. faecalis* OG1RF in absence (uUTI) or presence of a catheter (CAUTI) for 24 h. Coagulation-transgenic mice used are: Fg^AEK (soluble Fg, no fibrin formation), Pg^−/− and uPA^−/− (elevated fibrin accumulation, no plasmin activation), and PAI-1^−/− (continuous fibrin clot degradation). Naive mice were neither catheterized nor infected. Flow cytometry analysis was performed in single cells to identify differential Mφs populations (Supplementary Fig. 8, gating strategy). b PCA analysis from data described (c–e) were analyzed to identify variance of Mφ polarization in relation to: (1) uropathogen; (2) infection model (uUTI or CAUTI); and (3) host coagulopathies. c–e Average percentages of M0- (gray), M1- (red), M1/M2- (orange), and M2-like Mφs (yellow) out of total Mφ population after flow cytometry analysis were plotted on heatmaps by Mφs polarization phenotype and infection type; c uninfected catheterized-only (Cath); d *E. coli* UTI89 infected; or e *E. faecalis* OG1RF infected. Naive bladders were used as control, infection only was included as a reference. Heatmaps are paired with a legend. Total n counts over 5 independent experiments are: (1) n = 22 for WT naive; (2) catheterized only: n = 9 for WT; n = 7 for Fg^AEK; n = 4 for Pg^−/−; n = 6 for uPA^−/−; n = 7 for PAI-I^−/−; (3) *E. coli* infection: n = 7 for uUTI WT; n = 7 for CAUTI WT; n = 7 for Fg^AEK; n = 4 for Pg^−/−; n = 6 for uPA^−/−; n = 8 for PAI-I^−/−; (4) *E. faecalis* infection: n = 6 for uUTI WT; n = 7 for CAUTI WT; n = 6 for Fg^AEK; n = 4 for Pg^−/−; n = 6 for uPA^−/−; n = 8 for PAI-I^−/−. Individual data points, n values and statistical analyzes are described in Supplementary Fig. 15.

Table 1.

Table of mouse strains used in this study

Mouse strain	Genotype	Phenotype	References
Wild-type (WT) mice	Fg^+/+	Presence of both Fg and fibrin
Fg AEK mutant form mice	Fg^AEK	Fibrinopeptide A cannot be cleaved by thrombin resulting in absence of fibrin polymer formation	Prasad et al.⁵⁰
Plasminogen (Pg)-deficient mice	Pg^−/−	Impaired fibrinolysis resulting in elevated fibrin accumulation	Ploplis et al.⁵³
Urokinase plasminogen activator (uPA)-deficient mice	uPA^−/−	Impaired activation of PG resulting in fibrinolysis deficiency leading to elevated fibrin accumulation	Carmeliet et al.⁵²
Plasminogen activator inhibitor-1 (PAI-1)-deficient mice	PAI-1^−/−	Impaired inhibition of UPA activity resulting in continuous degradation of fibrin clots leading to elevated fibrin degradation products	Carmeliet et al.⁵⁴

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Diagram displayed in Fig. 4a.

In summary, the balance between fibrin accumulation and clearance in the catheterized bladder in part modulates Mφ polarization, shaping the outcome of CAUTI. Our findings show that conditions promoting fibrin accumulation, such as impaired fibrinolysis, associated with an M2-like Mφ phenotype, potentially worsening the infection. Conversely, conditions that reduce fibrin levels, either by preventing its formation (Fg^AEK) or enhancing its degradation (PAI-1^−/−), promote an M1-like Mφ polarization associated with a more effective antimicrobial response.

Fibrin-associated M2-like Mφs exhibited altered antimicrobial response against CAUTI pathogens

To determine how soluble Fg and fibrin directly modulate the antimicrobial functions of Mφs, we performed gentamicin protection assays. We stimulated mouse BMDMφs with various cytokines, soluble Fg, fibrin, or a combination of both, and evaluated their capacity to phagocytose and subsequently kill E. coli or E. faecalis at 3 h post-phagocytosis (Fig. 5, Supplementary Fig. 16c, d). To control for initial bacterial loading, the bacterial burden in the supernatant was quantified (Supplementary Fig. 16a, b). Our results revealed that soluble Fg and fibrin exert opposing effects on Mφ antimicrobial capacity. Uninduced Mφs readily phagocytosed E. coli or LPS-coated beads but were more effective at killing the internalized E. faecalis (Fig. 5a, h, Supplementary Fig. 16c–e). As expected, GM-CSF-induced M1-like Mφ polarization enhanced bactericidal activity against both pathogens. In contrast, M2-like Mφs induced by IL-4 reduced bactericidal activity, and the outcome was pathogen-dependent. These M2-like Mφs reduced clearance of intracellular E. faecalis and showed increased killing against E. coli (Fig. 5b, c, i, j). Interestingly, interaction with soluble Fg enhanced both phagocytosis and bacterial killing (Fig. 5e, l, Supplementary Fig. 16c–f). Conversely, engagement with fibrin significantly reduced these two functions, even in presence of soluble Fg (Fig. 5f, g, m, n). To determine whether these responses translate to human Mφs, we also stimulated human THP-1-derived Mφs with the same conditions to evaluate the phagocytosis of LPS-coated beads in parallel with mouse BMDMφs (Supplementary Fig. 16e, f). Consistent with our mouse BMDMφs findings, human Mφs exhibited analogous phagocytosis trends for each stimulus. This suggests that the divergent immunomodulatory effects of Fg and fibrin are conserved in humans, though further translational studies are warranted. Together, these results demonstrate that soluble Fg promotes a potent, M1-like antimicrobial response, while fibrin favors a M2-like response that suppresses their phagocytic and bactericidal functions.

Fig. 5 — BMDMφs were either a, h uninduced (M0-Mφs) or stimulated with 100 ng/mL of b, i GM-CSF (M1-Mφs), c, j IL-4 (M2-Mφs), or d, k cotreated with GM-CSF and IL-4 (M1/M2-Mφs), e, l 1.5 mg/mL of Fg or f, m) fibrin or g, n both simultaneously for 24 h. Mφs were then infected with either a–g opsonized *E. coli* UTI89 or h–n *E. faecalis* OG1RF at a multiplicity-of-infection ratio of 2 (100,000 bacteria to 50,000 Mφs) for initial uptake (45 min). Supernatant was removed after initial uptake and BMDMφs were washed three times with 1X DPBS then treated with gentamicin and penicillin/streptomycin for 15 min (served as t = 0 post-treatment) or 3 h (t = 3) to remove non-internalized extracellular bacteria before being exposed to pure sterile distilled water for intracellular bacteria retrieval and CFU counts. Assays were performed over 11 independent experiments. The horizontal line represents median value. Each data point is one replicate. The horizontal broken line represents the limit of detection (LOD) of viable bacteria. The two-tailed Mann–Whitney U test was used to determine significant difference between time points; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Exact p-values and individual n values are provided in the source data.

GM-CSF-Induced M1-like Mφ polarization is insufficient to overcome the pro-pathogen catheterized environment

The catheterized bladder favors an M2-like Mφ polarization (Fig. 4) and is deficient in the M1-like inducing cytokine GM-CSF²². We therefore hypothesized that therapeutically administering GM-CSF, a clinically established immunostimulant^43–47, could repolarize Mφs to an antimicrobial M1-like Mφ state and improve CAUTI outcomes. To test this, we pre-treated mice with GM-CSF, IL-4 (as an M2-like polarization control), both, or a vehicle (phosphate-buffered saline, PBS), and then catheterized for 12 h, with or without infection by E. coli or E. faecalis (Fig. 6a). We assessed treatment efficacy on Mφ polarization in both catheter-only and CAUTI conditions (Fig. 6b, Supplementary Fig. 17).

First, we used principal component analysis (PCA) to determine the primary drivers of Mφ polarization (Fig. 6b). The analysis confirmed that the two main factors influencing the Mφ phenotype were the cytokine treatment administered and the presence of a pathogen (Fig. 6b). In vehicle-treated control mice, catheterization alone prompted a significant shift toward M2-like Mφ polarization (Fig. 6b, c; Supplementary Fig. 17a, m). As hypothesized, therapeutic cytokine treatments effectively directed this polarization. IL-4 treatment amplified the M2-like Mφ shift compared to the control, while any treatment combination containing GM-CSF robustly promoted M1-like Mφ polarization (Fig. 6b, c; Supplementary Fig. 17g, j, m). Specifically, only catheterized bladders treated with GM-CSF (both with and without IL-4) reduced the populations of M0-, M2-, and M1/M2-like Mφs, strongly skewing the environment toward an antimicrobial M1-Mφ state (Fig. 6b, c; Supplementary Fig. 17d, g, j, m).

Crucially, these cytokine-driven effects were dominant even during an active infection (Fig. 6b, d, e). The M1-like skewing pattern persisted during CAUTI with either E. coli or E. faecalis, indicating that the Mφ phenotype was primarily dictated by the therapeutic cytokine environment rather than the specific pathogen involved (Fig. 6b, d, e; Supplementary Fig. 17e, f, h, i, k, l, n, o). In summary, these results show that we can successfully manipulate Mφ polarization in the bladder using GM-CSF to drive a potent M1-like Mφ response.

M1-like polarization unable to improve bacterial clearance

Although we successfully repolarized Mφs toward a M1-like Mφ phenotype, this did not translate into enhanced antimicrobial activity or better CAUTI outcomes. We compared the specific treatments with the vehicle control, finding that GM-CSF treatment, either alone or with IL-4, failed to reduce the bacterial burden in the bladder or on the catheter for either pathogen. In fact, the combination of GM-CSF and IL-4 significantly increased bladder colonization by E. coli (Fig. 6f). Furthermore, no cytokine treatment reduced bladder inflammation or edema, as measured by bladder weight (Supplementary Fig. 18a, b).

We next evaluated the effect of our treatments on systemic bacterial dissemination during E. coli infection. While most treatments did not significantly alter bacterial spread, the co-administration of GM-CSF and IL-4 resulted in a significant reduction of the bacterial burden specifically in the heart (Supplementary Fig. 19). We also observed non-significant trends, with GM-CSF alone trending to decrease dissemination and IL-4 alone trending to increase it (Supplementary Fig. 19a–c). During E. faecalis CAUTI, the cytokine treatments had opposite effects on bacterial dissemination. GM-CSF treatment alone decreased bacterial dissemination, an effect that was statistically significant in the spleen (Supplementary Fig. 19e). Conversely, treatments involving IL-4 (both alone and with GM-CSF) had an increased dissemination for both pathogens, which reached statistical significance in the heart (Supplementary Fig. 19).

In conclusion, despite successfully repolarizing Mφs to a pro-inflammatory M1-like Mφ state, this strategy failed to reduce local bacterial burden or inflammation and, in some cases, worsened the bladder infection. However, the treatments exerted complex, pathogen-specific effects on bacterial dissemination, where GM-CSF alone reduced systemic spread while interventions involving IL-4 exhibited the opposite effect.

Shared uropathogen phagocytosis by Mφ subpopulations

The failure to improve overall bacterial clearance prompted us to ask whether Mφs was phagocytizing pathogens and if their killing ability was polarization dependent. We assessed initial pathogen uptake by infecting mice with GFP-expressing E. coli or E. faecalis, following the experimental model in Fig. 6a. Subsequent flow cytometry analysis allowed us to quantify the phagocytic (GFP⁺) Mφs and determine which subpopulations were involved (Fig. 7; Supplementary Fig. 20).

Fig. 7 — C57BL/6 WT female mice were i.p. injected with two doses of either sterile 1X PBS (vehicle) or cytokines at 12 and 4 h prior to catheterization and/or infection as described in Fig. 6a. Mice were catheterized and infected (a–e) with either ~10⁸ CFUs of *E. coli* UTI89-GFP strain or e–j *E. faecalis* OG1RF-GFP strain. Twelve hours post-catheterization and/or infection, bladders were harvested and digested to isolate single cells for flow cytometry analysis and staining as described in Fig. 6a. a, f Percent of Mφs that phagocytized the pathogen (GFP⁺-Mφs) and did not phagocytize the pathogen (GFP^--Mφs). Two-tailed Mann–Whitney U test was used to determine significance. Polarized Mφs populations containing b–e *E. coli*-GFP or g–j *E. faecalis*-GFP in the bladders of cytokine-treated mice. Total n counts over 4 independent experiments are: (1) n = 12 for WT naive; (2) catheterized only: n = 5 for Vehicle; n = 6 for GM-CSF; n = 5 for IL-4; n = 6 for GM-CSF + IL-4; (3) *E. coli* infection: n = 8 for Vehicle; n = 8 for GM-CSF; n = 9 for IL-4; n = 6 for GM-CSF + IL-4; (4) *E. faecalis* infection: n = 8 for Vehicle; n = 6 for GM-CSF; n = 10 for IL-4; n = 6 for GM-CSF + IL-4. For (b–e), the two-tailed Kruskal–Wallis test with Dunn’s multiple comparisons was used to test for significance between groups. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Exact p-values are provided in source data. Full gating strategy in Supplementary Fig. 20.

While vehicle and GMCSF + IL-4 cotreatment showed an increased trend in phagocytosis during E. coli infection, IL-4 was the only treatment that showed significant effect when compared to the GFP^- Mφ population (Fig. 7a, f). During E. faecalis infection, vehicle and GM-CSF treatment (with and without IL-4) displayed an increase in Mφ phagocytosis. To identify the responsible cell types, we analyzed the phagocytic (GFP ⁺) Mφ and found that in the control group, all subpopulations (M0-, M1-, M1/M2-, and M2-like Mφs) contributed to pathogen uptake for both pathogens (Fig. 7b, g). Similarly, phagocytosis was performed by all Mφ subpopulations across all cytokine treatments for both pathogens (Fig. 7). Within the GM-CSF treatment group, M1-like Mφ showed significantly enhanced phagocytic activity compared to their non-polarized M0- and hybrid M1/M2-like Mφ for E. coli (Fig. 7c). Likewise, GM-CSF-treatment significantly increased phagocytosis of E. faecalis in M1- compared to M0-like Mφs. In IL-4-treated mice, phagocytosis by both M1- and M2-like Mφs was significantly enhanced relative to that of the M0- and M1-like Mφ populations, regardless of the pathogen (Fig. 7d, i). GM-CSF and IL-4 cotreatment significantly enhanced phagocytosis of E. coli by M1- and M2-like Mφs and E. faecalis by M1-like Mφs, respectively (Fig. 5e, j). Although GM-CSF treatment increased M1-like Mφs phagocytosis, these subpopulations exhibit differential pathogen killing capacity (Fig. 5), likely undermining effective bacterial clearance and explains the pathogen persistence during CAUTI (Fig. 6) and systemic dissemination (Supplementary Fig. 19).

Discussion

The use of urinary catheters is on the rise, driven by an aging population and an increase in chronic and lifestyle-related diseases. However, their increasing use is also driving a higher incidence of CAUTI^{30,37,38,55–57}. CAUTIs are a leading cause of healthcare-associated infections and frequently result in deadly urosepsis³⁷, with a 25% of sepsis cases coming from urinary isolates⁵⁸. A major paradox in the field is how these uropathogens are able to persist during CAUTI and urosepsis^22,33, despite the robust immune cell recruitment into the catheterized bladder, especially Mφs (Fig. 1)^30–33. Here, we showed that the Mφs’ ability to clear pathogens is hindered by the complexity of the catheterized bladder environment, which modulates Mφ polarization (Fig. 2). Our study revealed that fibrin effects on Mφ response overcame soluble Fg-induced M1-like Mφ polarization, resulting in a sustained M2-like Mφ polarization (Fig. 3). Consistently, mice with coagulopathies that resulted in fibrin clot accumulation correlated with robust M2-like Mφ polarization during urinary catheterization (Fig. 4). Importantly, we observed that Mφ interaction with soluble Fg is associated with a pro-phagocytosis response against both E. coli and E. faecalis, while interaction with fibrin displayed reduced pathogen clearance in vitro and ex vivo (Fig. 5 and Supplementary Fig. 16). Since phagocytosis and intracellular bacterial clearance are rapid processes that can occur within minutes, our phagocytosis assessment is limited to our post-45-min infection. Our in vitro and ex vivo studies suggest that fibrin may alter bactericidal response; however, bacterial effector mechanisms such as phagosome acidification, rapid phagocytosis, reactive oxygen species, and nitric oxide production were not directly assessed, which warrants further study. Together, this suggested that reprogramming of the Mφs with an immunostimulant GM-CSF could help control the infection. However, M1-like Mφ reprogramming with GM-CSF treatment did not contribute to reducing infection in vivo (Fig. 6). Our study indicates that the catheter-induced bladder inflammation and fibrin(ogen) accumulation produces mixed signals that contributes to dysfunctional Mφ polarization, potentially resulting in impaired antimicrobial response against infection.

Our findings indicate that soluble Fg and solid fibrin exert opposing effects on Mφ polarization. Prior studies have demonstrated that the distinct structural conformations of Fg and fibrin fundamentally alter how they engage with Mφs receptors^16,59,60. Specifically, fibrin(ogen) has been reported to interact with Mφs’ receptors, CD11b/CD18 (α_Mβ₂, Mac-1, CR3), CD11c/CD18 (α_Xβ₂, CR4), and toll-like receptor-4 (TLR-4), modulating inflammatory response^{15,16,18,19,61}. Furthermore, integrin-mediated interactions with dense, thick fibrin matrices have been shown to promote reparative M2-like polarization. This occurs via the induction of transglutaminase 2 (Tgm2), a downstream regulator of the FAK signaling pathway, which drives mitochondrial biogenesis and subsequently reduces the production of M1-associated reactive oxygen species^62,63. Conversely, interactions with sparse, thin fibrin matrices yield the opposite polarization effect^62,63. These conformational differences between Fg and varied fibrin architectures likely dictate their distinct signaling outcomes. We acknowledge, however, that without targeted receptor-blocking or pathway-perturbation experiments, the specific receptors driving this M2-like phenotype remain inferential. Given that the catheterized bladder environment features both soluble Fg and varied fibrin architectures, future studies examining the interplay between specific fibrin(ogen) architectures, integrin signaling (such as Mac-1 or TLR4), and canonical Mφ polarization pathways are critically needed to elucidate the intracellular mechanisms governing host antimicrobial responses.

Interestingly, Hsieh et al. showed that BMDMφs stimulated by soluble Fg activates proinflammatory M1-like cytokine secretion of TNF-α, IL-6, MCP-1, MIG, MIP-1α, MIP-1β, and CCL5 similar to IFN-γ + LPS cotreatment, while Mφs interacting with fibrin matrices expressed IL-10, G-CSF and TGF-β1, similar to IL-4 + IL-13 cotreatment¹⁵. Importantly, the same study demonstrated that fibrin protected Mφs from M1-like activation (IFN-γ and LPS), maintaining high IL-10 and low M1-like TNF-α levels¹⁵. TNF-α levels were also abrogated even when Mφs were first cocultured with fibrin and Fg before IFN-γ and LPS co-stimulation, suggesting that fibrin was still able to outcompete these M1-like signals, including soluble Fg¹⁵. In contrast, co-culturing Mφs with Fg and M2-like signals (IL-4 and IL-13) yielded elevated levels of both IL-10 and TNF-α, suggesting a hybrid M1/M2-like phenotype¹⁵. However, future investigations are need to elucidate the complex interplay between Fg, fibrin, and canonical polarization signals in regulating Mφ behavior and function during CAUTIs and other biomaterial-associated inflammation. Furthermore, it is unclear whether soluble Fg and fibrin use the same collection of Mφ receptors to differentially modulate polarization in the catheterized bladder and how this interaction affects specific signaling pathways and transcriptional profiles.

Mφ polarization is a spectrum of M1-like to M2-like phenotype with plasticity to switch between polarized states. Our findings strongly support this model; specifically, we identified a distinct population of hybrid M1/M2-like Mφs within the catheterized bladder that co-express both iNOS and Arg1 markers, demonstrating their intermediate state (Fig. 4). A limitation of the present study is the reliance on iNOS and Arg-1 to evaluate Mφ polarization. While these remain the gold-standard markers for characterizing M1- and M2-like phenotypes in mouse models, defining discrete populations, particularly hybrid M1/M2-like states, is complicated by the mixed inflammatory signals inherent to the catheterized bladder microenvironment. Because Mφ polarization occurs along a dynamic phenotypic continuum, capturing the full spectrum of intermediate subpopulations will require the use of expanded marker panels in future investigations. Finally, while our data clearly demonstrate the impact of these M1/M2-like shifts in mice, further research is required to elucidate whether these specific Mφ polarization dynamics fully reflect the pathology of human CAUTIs.

Our findings revealed that modulation of hybrid Mφ populations was impacted when combination signals were present, including GM-CSF + IL-4 or fibrin + soluble Fg ex vivo and in vivo (Figs. 2–6 and Supplementary Fig. 17). Typically, uropathogens present surface molecules (PAMPs) that drive M1-like polarization, such as LPS on E. coli and lipoteichoic acid (LTA) on E. faecalis^64–66. However, we observed different outcomes for E. coli and E. faecalis in the catheterized bladder. In fact, E. coli promoted a hybrid M1/M2-like Mφ population, whereas E. faecalis drove M2-like Mφ polarization (Fig. 4). This could be related to its active manipulation of the fibrin(ogen)-rich catheterized bladder environment; we have shown that E. faecalis uses its SprE protease to target the fibrinolytic system, resulting in an increase of fibrin accumulation in the bladder²². This results in a two-for-one survival strategy as the fibrin not only provides a scaffold for its biofilm²², but could also subvert the immune response by promoting a detrimental M2-like Mφ state (Fig. 4). In doing so, E. faecalis engineers a pro-microbial niche that benefits itself and other potential uropathogens by enhancing biofilm platforms while suppressing immune surveillance.

Our in vivo and in vitro results show that while all Mφ subpopulations can phagocytize pathogens, their killing ability varies significantly, depending on both the Mφ polarization state and the specific pathogen (Figs. 5 and 7). We identified M1-like Mφs, particularly those induced by GM-CSF or soluble Fg (Figs. 3, 4b–e, and 6b–e), as the most potent bactericidal cells (Fig. 5). GM-CSF has been shown to critically regulate M1-like Mφ antimicrobial activation in mouse models of intestinal Citrobacter rodentium infection and colitis while suppressing M2-like Mφ wound-healing response associated with intestinal fibrosis⁶⁷. This led us to test GM-CSF as a therapy, which yielded surprising, divergent outcomes. The treatment successfully induced M1-like polarization in the catheterized bladder (Fig. 6) with this subpopulation having exhibited higher pathogen phagocytosis in vivo (Fig. 7c, h). While the therapy failed to reduce the local bacterial burden in the bladder (Fig. 6f, h), it was the most effective in decreasing systemic dissemination (Supplementary Fig. 19). This suggests that while these M1-like Mφs may struggle to eradicate the massive bacterial biofilm communities in the bladder lumen, they are efficient in controlling the bacteria invading deeper bladder tissue to prevent systemic dissemination. It is possible that these M1-like Mφs may be contributing to inflammation, tissue damage, and pathology associated with catheterization rather than its role in bacterial clearance seen in uncomplicated UTIs. This warrants further investigation.

Interestingly, the combination GM-CSF and IL-4 treatment exacerbated the bladder infection. This treatment led to a significant increase in colonization by E. coli and a similar, near-significant trend for E. faecalis (Fig. 6f, h). Notably, this treatment had differential effects in systemic dissemination dependent on the pathogen. While E. coli dissemination was similar to GM-CSF treatment alone, the E. faecalis dissemination profile instead mirrored IL-4 treatment alone (Supplementary Fig. 19). This difference in systemic dissemination was mirrored in our ex vivo killing assays, where BMDMφ treated with GM-CSF + IL-4 increased killing E. coli but not E. faecalis (Fig. 5d, k). This is likely explained by their distinct surface molecules. The LPS on E. coli is a potent activator of Mφ bactericidal pathways through the TLR-4 receptor which enhances phagocytosis, phagolysosome maturation, and pathogen degradation using reactive oxygen species and nitric oxide production^68–70. On the other hand, LTA on E. faecalis is known to actively hinder the Mφ‘s ability to eliminate the bacteria^71,72. This indicates that the catheterized bladder environment provides mixed polarization signals, coming from the host response and the pathogen, creating a dysfunctional immune response that may drive persistent CAUTI and systemic dissemination.

A key paradox in CAUTI research is how systemic dissemination occurs since patients with uUTIs have a low risk of bloodstream infections whereas CAUTIs often lead to urosepsis²⁸. For uUTI, motile pathogens like E. coli can use their flagella to swim up the urinary tract to the kidneys. However, their spread into the bloodstream is typically blocked by a combination of anatomical barriers and a robust immune response³⁸. Thus, a key question is how non-motile pathogens, such as E. faecalis, cause CAUTI-related urosepsis. Our findings point to the idea that Mφs could be acting as a “Trojan Horse.” Since our in vivo reprograming experiments revealed that while all Mφ subpopulations can phagocytize pathogens (Fig. 7), M2-like Mφs consistently exhibited high phagocytosis in all treatment conditions, often reaching statistical significance (Fig. 7). This was especially clear in the IL-4 treatment group, where high M2-like Mφ phagocytosis alarmingly correlated with systemic bacterial dissemination of the bacteria as compared to the vehicle control (Fig. 7d, i and Supplementary Fig. 19). This is consistent with our ex vivo findings, which shows that these M2-Mφs are avid eaters with impaired bactericidal activity (Fig. 5c, j), ultimately contributing to pathogen persistence. Our finding that a fibrin-rich environment correlates with enhanced M2-like Mφ polarization providing a direct mechanism for our previous observation that fibrinolytic deficiencies led to increased systemic dissemination during mono- and polymicrobial CAUTI²². Because GM-CSF treatment reduced dissemination without decreasing the bladder burden, our findings suggest that Mφs primarily function to contain the infection locally in the bladder rather than directly clearing the bacteria during short-term catheterization. Future studies utilizing prolonged catheterization models will address whether sustained GM-CSF treatment eventually reduces both bladder infection and systemic dissemination.

Pathogens, including bacteria, fungi, and viruses, can hijack Mφs to spread while evading the immune system^73–76. However, there is a critical need to determine how fibrin directly alters Mφ intracellular mechanisms to potentially create a protective niche for uropathogens to survive and colonize. For example, a recent study demonstrated that perivascular Mφs protect the bladder-blood barrier by releasing Mφ extracellular traps (METs) to contain E. coli from disseminating from the urinary tract⁷⁷. However, it remains unknown whether this containment mechanism is pathogen-specific, or whether fibrin-induced M2-like Mφs exhibit a diminished capacity to produce these extracellular traps. Furthermore, future investigations should assess whether GM-CSF treatment promotes MET formation, which could provide a mechanistic explanation for the reduced bacterial dissemination observed for both pathogens in our model. Together, elucidating these intracellular mechanisms can provide a powerful explanation for promoting higher risk of urosepsis during CAUTI. This model explains why this life-threatening complication is common in catheterized patients but rare in simple UTIs²⁸, as it provides a transport vehicle for both motile and non-motile pathogens to escape the bladder.

Dysregulated wound healing often drives Mφ dysfunction, leading to chronic inflammatory pathologies^{22,31,32,78,79}. We previously demonstrated that continuous, catheter-induced bladder damage disrupts the temporal sequence of wound healing and compromises the host’s capacity to resolve inflammation^{23,25,27–29,37,38}. Specifically, IL-6 emerges as the primary inflammatory cytokine elevated as early as 1-h post-catheterization across the bladder, bloodstream, and liver^22,31,32, with its levels increased further upon infection^22,31. IL-6 is known to upregulate hepatic Fg expression⁸⁰, which subsequently increases Fg levels in the bloodstream and drives its recruitment to the damaged tissue⁸¹. Notably, increased of catheter-induced inflammation and IL-6 levels correlates temporally with increased Fg concentrations in the catheterized bladders of both mice and patients^22,25. This Fg deposition creates a physical platform that accelerates microbial colonization and further fuels bladder inflammation^{22–24,26,27,35,36}. Because Mφ recruitment begins at 1 h but does not peak until 24 h³², this kinetic delay indicates that mechanical trauma prompts an immediate spike in proinflammatory mediators to drive Fg deposition first, establishing an infectious niche before a robust Mφ recruitment.

Though the temporal cascade of immune cell recruitment to the catheterized bladder is well-established^31,32, how this microenvironment subsequently alters Mφ polarization has remained unknown. This study bridges this gap, revealing that the catheterized bladder creates a fibrin-rich microenvironment that correlates with enhanced M2- and hybrid M1/M2-like Mφ polarization. Although evaluated within a short-term (up to 24-h) in female mice, this early M2-like skew represents a key mechanism driving the host’s inability to clear the infection, ultimately fostering persistent CAUTIs and urosepsis.

While M2-like Mφs typically mediate anti-inflammatory responses and tissue remodeling, dysregulated or excessive M2-like polarization can drive detrimental pathologies, including oncogenesis, asthma, and chronic bacterial and fungal infections^82–85. Crucially, our findings shift the paradigm of CAUTI pathogenesis by demonstrating that catheter-induced inflammation establishes a specialized microenvironment that dictates Mφ polarization, shaping it through a dynamic ‘tug-of-war’ of opposing signals released concurrently by the injured bladder and the invading pathogen. For instance, host-derived proinflammatory mediators and soluble Fg release favor M1-like bactericidal response to active infection, typical of acute inflammation. Conversely, pathogens such as E. faecalis and SARS-CoV-2, can actively subvert M1-like response by directly or indirectly skewing Mφs towards M2-like response^22,86. Compounding this, biomaterial-induced mechanical trauma triggers extracellular matrix remodeling marked by fibrin clotting and collagen deposition to promote healing⁸⁷. All these signals could modulate Mφs to transition from hybrid M1/M2-like phenotype towards M2-like^12,87,88. This competing interplay within the CAUTI microenvironment highlights the complexity of isolating the exact mechanisms that regulate Mφ behavior along this phenotypic continuum, which includes these uncharacterized hybrid populations. Thus, further mechanistic studies focusing specifically on the role of hybrid M1/M2-like Mφs are needed. Finally, because this study evaluated the effects of short-term catheterization, investigating Mφ polarization dynamics following catheter removal will be critical to determine the durability of this immune reprogramming, long-term urothelial remodeling, and subsequent patient susceptibility to subsequent uUTIs.

Ultimately, our findings demonstrate that the catheterized bladder is subjected to a complex microenvironment of mixed signals that dysregulates Mφ polarization and compromise pathogen clearance. By distinctly differentiating the independent effects of soluble Fg and fibrin on Mφ antimicrobial responses, this work opens new avenues for understanding immune evasion in the urinary tract. While this study was restricted to acute (24-h) catheterization in female mice due to strict male anatomical barriers for indwelling catheterization, where surgical alternatives would confound readouts via trauma-induced Fg recruitment and exacerbate inflammation, investigating long-term dynamics and sex-dependent variations remains crucial for future studies. This is emphasized by the narrow 2:1 female-to-male ratio in CAUTIs compared to the 4:1 ratio in uUTIs^28,29,37. Investigating these sex differences is critical, as Fg levels are naturally higher in women than in men and progressively increase with age, an elevation heavily tied to post-menopausal hormonal shifts^89–93. These baseline variations likely influence Mφ recruitment and polarization. Because our previous work established that levels of Fg and fibrin dynamics dictate susceptibility to severe CAUTI and urosepsis in fibrinolytic-deficient populations⁹⁴, characterizing this unique catheterized bladder environment is essential to developing new preventative therapies against CAUTI and its associated urosepsis.

Methods

Ethics statements

All mouse studies including infections and procedures are approved by The University of Notre Dame Institutional Animal Care and Use Committee as part of protocol number 25-01-9009. All animal handling, use, and care followed approved protocol and Guide for the Care and Use of Laboratory Animals from the National Research Council⁹⁵. This study utilized 6- to 8-week-old female wild-type C57BL/6 mice purchased from Jackson Laboratory, Charles River, and transgenic mice (Table 1) bred in W.M. Keck Center for Transgenic Research at the University of Notre Dame’s Harper Cancer Research Institute. All mice were maintained and housed under a 14-h light/10-h dark cycle with ambient temperature (~70 °C) and humidity (~50%) in the University of Notre Dame Freimann Life Science Center.

Study design

This study was performed using a preclinical CAUTI mouse model and transgenic mouse strains in accordance with approved protocols to investigate how urinary catheterization affects Mφs’ antimicrobial response in CAUTIs. Our objective was to understand how coagulopathies that results in impaired fibrin clot dissolution modulates Mφs’ polarization and impairs its function to control bacterial infections. Having found M1-, hybrid M1/M2-, and M2-like Mφ subpopulations in our studies, we investigated whether soluble Fg without clot formation and fibrin accumulation that differentially polarize these subpopulations also modulates its phagocytic and bactericidal activity against both E. coli and E. faecalis. Six- to eight-week-old female mice were randomly assigned to groups based on its genotype (WT, Fg^AEK, Pg^−/−, uPA^−/−, and PAI-1^−/−, Table 1) and then randomly partitioned into one of these two groups first: (1) catheterized or (2) non-catheterized before being sub-grouped further into either uninfected or infected with either pathogen. Furthermore, for in vivo Mφ polarization studies, mice were further subdivided into either receiving an i.p. injection of vehicle (1X PBS) or cytokines (GM-CSF, IL-4, or GM-CSF + IL4). In all experiments, a minimum of 3 and maximum of 16 mice comprise an experimental group and 3 to 4 independent experiments were performed. Sample size was determined based on previous CAUTI studies and was unchanged in this study^23,27,95. Data collection is detailed in the following subsections. All data including outliers are included in statistical analysis. For quantifying Mφ populations in flow cytometry analysis, bladders from multiple mice were not pooled together. Mice who lost catheters before time of sacrifice were excluded from all analyses. End points for mouse studies were determined before start of experiments and researchers were not blinded to experimental groups. Statistical analysis is detailed at the end of this section.

Bacterial strains and growth conditions

Uropathogenic E. coli (UPEC) UTI89 HK::GFP, E. coli UTI89 (no GFP), E. faecalis OG1RF, and E. faecalis OG1RF pAOJ20 (GFP) strains were used in mouse, in vitro, and ex vivo studies. UPEC UTI89 was first inoculated in 10 mL of Luria Broth (LB, MP Biomedicals). For UPEC UTI89 HK::GFP strain, LB was supplemented with the final concentration of 50 µg/mL kanamycin (Santa Cruz Biotechnology) shaking for 4 h at 37 °C. After shaking, the inoculum was diluted in LB with kanamycin at 1:1000 and then grown for 24 h in static conditions overnight at 37 °C before being grown again for the next 24 h in the same static conditions. E. faecalis OG1RF was grown in static conditions overnight at 37 °C in 10 mL of brain heart infusion (BHI, Hardy Diagnostics) supplemented with final concentrations of 25 µg/mL rifampicin and 25 µg/mL fusidic acid (Chem-Implex). For E. faecalis OG1RF pAOJ20 (GFP), the inoculum was grown in static conditions overnight at 37 °C in 10 mL of brain heart infusion supplemented with final concentrations of 20 µg/mL rifampicin, 20 µg/mL fusidic acid, and 10 µg/mL chloramphenicol.

In vivo experimental model

Female mice were first anesthetized by inhaling isoflurane and then were transurethrally implanted through the urethra with a 5-mm-long silicone tubing (Braintree Scientific SIL 025) on a 30-gauge needle for 24 h as previously described⁹⁵. For CAUTIs, mice were then immediately transurethrally injected with 50 µl of approximately 10⁷ colony-forming units (CFUs) of either E. coli UTI89 or E. faecalis OG1RF in 1X PBS. For uncomplicated UTIs, mice were infected as described without implantation. Naive mice were neither implanted nor infected. After 24 h, mice were anesthetized and then sacrificed by cervical dislocation for collection of bladders, kidneys, spleen, heart, and catheters (where applicable) for downstream applications. For bacterial enumeration, organs were homogenized, and catheters were sonicated for CFU counts.

In vivo Mφ polarization model

Female mice were intraperitoneally injected with two doses of either sterile 1X PBS as vehicle control, 200 ng/mouse of GM-CSF (IrvineScientific)⁹⁶, 200 ng/mouse of IL-4 (PeproTech)⁹⁷, or 200 ng/mouse of both GM-CSF + IL-4 simultaneously 12 and 4 h prior to catheterization and/or infection with 10⁸ CFUs of either E. coli UTI89 or E. faecalis OG1RF in 1X PBS. Twelve hours post-catheterization and/or infection, mice were sacrificed for collection of bladders, kidneys, spleen, heart, and catheters for either single-cell isolation for flow cytometry analysis or bacterial CFU enumeration.

Cell culturing

The RAW 264.7 Mφ-like cell line (ATCC TIB-71) was cultured in Dulbecco modified Eagle medium (DMEM, Corning) with 4.5 g/L glucose, 2 mM L-glutamine (VWR), 1 mM sodium pyruvate (Corning), 10% fetal bovine serum (FBS, ThermoFisher Scientific), 100 units/mL penicillin, and 100 µg/mL streptomycin (Corning). THP-1 human monocytes (ATCC TIB-202) were cultured using complete growth media composed of Roswell Park Memorial Institute (RPMI) 1640 1x media supplemented with 2 mM L-glutamine, 4.5 g/L glucose, 2 mM L-glutamine, 1 mM sodium pyruvate, 10% FBS, 100 units/mL penicillin, and 100 µg/mL streptomycin. THP-1s were differentiated into Mφs by stimulation with 16 nM phorbol 12-myristate 13-acetate (PMA) for 48 h, followed by a 24 h recovery period in PMA-free, complete media. Cells were passaged no more than ten times for both cell lines. Upon isolation and separation of monocytes from bone marrow, bone marrow-derived monocytes were cultured in DMEM media supplemented with 4.5 g/L glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate, 10% FBS, 100 units/mL penicillin and 100 µg/mL streptomycin, and 25 ng/mL recombinant mouse Macrophage Colony Stimulating Factor (M-CSF, IrvineScientific) for 5–7 days before use. Cells were maintained in 37 °C 5% CO₂ incubator before use.

Isolation of bone-marrow derived monocytes

Bone marrow from femurs of 6- to 8-week-old female C57BL/6 mice were flushed with Roswell Park Memorial Institute (RPMI) 1640 media with 1X L-glutamine supplemented with 5.95 g/L HEPES (VWR), and 1 mM sodium pyruvate to retrieve all cells. Peripheral blood mononuclear cells (PBMCs) were separated from red blood cells and neutrophils using centrifugation separation with density gradients Histopaque-1077 (Sigma-Aldrich) and Histopaque-1119 (Sigma-Aldrich). After centrifugation, PBMCs were then isolated, washed with 1X DPBS, and treated in DMEM media supplemented with 4.5 g/L glucose, 2 mM L-glutamine, 1 mM sodium pyruvate, 10% FBS, 100 units/mL penicillin and 100 µg/mL streptomycin, and 25 ng/mL recombinant mouse M-CSF for 5–7 days for differentiation into Mφs.

Isolation of soluble fibrinogen from Fg^AEK mice

Blood was collected from the submandibular vein of anesthetized Fg^AEK mice following puncture by lancet. The Eppendorf tubes containing whole blood were centrifuged at 4 °C, 8000 × g for 5 min and the supernatant blood plasma collected via transfer to non-stick Eppendorf tubes. Plasma was then frozen at −80 °C prior to Fg^AEK extraction. In summary, cryo-preserved Fg^AEK plasma was slowly thawed on ice before adjustment to 10% Ethanol for overnight incubation at 4 °C. The samples were then centrifuged at 4 °C, 8000 × g for at least 5 min to pellet precipitated Fg^AEK. The resulting pellets were resuspended in 20 mM Sodium Citrate (pH 7.4) and quantified via BCA assay.

Fibrin gel formation

For forming fibrin gels, soluble human Fg (Enzyme Research Laboratories) or isolated Fg from Fg^AEK mutant mice was treated with 2 U/mL of thrombin (Sigma-Aldrich) at 37 °C for 1 h. After 1 h, fibrin gels were washed with 1X Dulbecco’s PBS (DPBS) three times to remove residual thrombin from the gel.

Endotoxin quantification for fibrinogen stock

Endotoxin levels of stock Fg solution (14 mg/ml) and subsequent 1:10 serial dilutions (1.0 mg/ml, 0.1 mg/ml, and 0.01 mg/ml) was analyzed using the Pierce Chromogenic Endotoxin Quant Kit (ThermoFisher Scientific) according to the manufacturer’s instruction. Endotoxin-free water and 1.0 mg/ml LPS served as positive and negative controls, respectively. Absorbance was immediately measured at 405 nm using a microplate reader. Mean blank-corrected absorbance values were used to generate a linear regression standard curve to determine the endotoxin concentration (EU/mL) of each sample.

In vitro and ex vivo polarization of Mφs

Uninduced (M0-like) RAW 264.7, THP-1, and BMDMφs were cultured in its respective media without cytokine treatment. For M1-like polarization, cells were treated with 100 ng/mL GM-CSF (IrvineScientific) or IFN-γ (R&D Systems) and LPS for 24 h. For M2-like polarization, cells were treated with 100 ng/mL IL-4 (PeproTech) for 24 h. To obtain a hybrid M1/M2-like phenotype, cells were induced with 100 ng/mL of GM-CSF and IL-4 or 100 ng/mL of each LPS, IFN-γ, and IL-4 for 24 h.

Immunofluorescent staining of RAW 264.7 and BMDMφs

For IF analysis, RAW 264.7 or BMDMφs were stimulated with the listed concentrations for 24 h as described in the preceding section. For each condition, approximately 500,000 Mφs were seeded on glass-bottom petri dishes for 24 h before staining. For soluble Fg, Mφs were resuspended in 1.5 mg/mL of soluble Fg^AEK conjugated with an Alexa Fluor 488 antibody for detection. Additionally, another set of Mφs were resuspended in 1.5 mg/mL of soluble Alexa Fluor 650-conjugated Fg (Fg⁶⁵⁰, Invitrogen). To form fibrin gels on dishes, the glass bottom was first coated with 1.5 mg/mL of unconjugated Fg before polymerization with 2 U/mL of thrombin as described previously. Fibrin gels were then washed with 1X DPBS three times to remove residual thrombin. For Mφs simultaneously interacting with both fibrin and Fg, the glass bottom was first coated with the 1.5 mg/mL fibrin gel, then washed with 1X DPBS three times. Mφs were resuspended in cell culture media containing 1.5 mg/mL of soluble Fg^AEK or Alexa Fluor 650-conjugated Fg. After a 24-h incubation, supernatants were removed before fixing Mφs with 10% formalin in 1X DPBS for 20 min at room temperature. Cells were then washed three times in 1X DPBS before blocking at room temperature in a moist chamber. After blocking, cells were then permeabilized with 0.3% Triton X-100 in 1X DPBS for 20 min to allow intracellular staining. After washing, cells were then blocked with 1X DPBS with 1% BSA and 0.3% Triton X-100 for 1 h before incubation with rabbit anti-iNOS (1:100) and goat anti-Arg1 (1:100) primary antibodies at 4 °C overnight. After incubation, cells were washed in 1X PBS three times then incubated with DyLight 488 donkey anti-goat and DyLight 550 donkey anti-rabbit secondary antibodies (1:500) at room temperature for 2 h. Sections were then washed with 1X DPBS before staining with Hoechst dye (1:10,000) for imaging.

Immunofluorescent staining and imaging of bladders

Bladders were harvested and fixed in 10% formalin overnight at 4 °C, before being processed and sectioned for staining as previously described³⁵. Sections were deparaffinized with xylene, rehydrated with isopropanol, and washed with water. Antigen retrieval was done by boiling the sections in 10 mM sodium citrate, rinsed in water, and then washed in 1X PBS. Sections were then blocked with 1X PBS with 1% BSA and 0.3% Triton X-100 for 1 h before incubation with corresponding primary antibodies at 4 °C overnight. One set of bladders were stained with goat anti-fibrin(ogen) (1:100), rabbit anti-E. coli (1:100) or anti-Group D Streptococcus (1:100), and rat anti-F4/80 (1:100) antibodies. Another set of bladders were stained with rabbit anti-iNOS (1:100), goat anti-Arg1 (1:100), and rat anti-F4/80 (1:100) antibodies. After incubation, sections were washed in 1X PBS three times then incubated with corresponding secondary antibodies (1:500) at room temperature for 2 h. Next, sections were washed with 1X PBS before staining with Hoechst dye (1:10,000). Imaging was performed using the Zeiss Axio Observer and analyzed using Zeiss Zen Pro, Zeiss Apotome, and ImageJ software.

Flow cytometry analysis

Mouse bladders were harvested and treated with digestion solution (0.34 U/mL Liberase^TM (Roche) and 100 µg/mL DNaseI (ThermoFisher) in 1X DPBS). Bladders were incubated on a heat block shaker for 1 h at 37 °C and 250 rpm. Bladders were vortexed every 15 min at a high speed for 30 seconds. After 1 h, digestion was arrested with a 1X DPBS supplemented with 2% FBS (FACS Buffer) and 0.2 µM EDTA before passing through 40-µm cell strainers. Following a 20-min incubation with anti-mouse CD16/32 monoclonal antibodies (1:500) in FACS Buffer, cells were washed three times with FACS Buffer before staining with far red viability dye (Invitrogen) for 20 min. After washing excess viability dye, cells were then stained with conjugated antibodies to detect cell surface markers for 1 h: PE-Cy5 anti-CD45, Brilliant Violet (BV) 650 anti-CD11b, and Super Bright (SB) 780 anti-F4/80. Cells were then washed three times with FACS buffer before fixed and permeabilized with 0.1% Triton X-100 in FACS Buffer for 20 min at 4 °C. Cells were then directly incubated with conjugated antibodies to detect intracellular markers for 45 min: eFluor 450 anti-Arginase-1 and PE anti-iNOS monoclonal antibodies. Cells were then washed before final resuspension in FACS Buffer. Data was acquired using BD Fortessa and analyzed by FlowJo v10. Data was presented as percentage Mφs, calculated by the number of events gated for M0- (Arg1^-iNOS^-), M1- (Arg1^-iNOS⁺), hybrid M1/M2- (Arg1⁺iNOS⁺), or M2-like (Arg1⁺iNOS^-) over the total number of events gated for Mφs (Live cells CD45⁺CD11b⁺F4/80⁺). Gating strategy in Supplementary Fig. 8. Manufacturers of listed antibodies and reagents are provided in Supplementary Data 1.

Gentamicin protection assay

Approximately 50,000 BMDMφs were seeded per well in a 96-well flat-bottom culture plate. Mφs were stimulated with the listed concentrations as described in the “In vitro and ex vivo polarization of Mφs” section. Furthermore, Mφs were also seeded with either 1.5 mg/mL of soluble Fg (Fg or Fg^AEK), fibrin gel, or both simultaneously. During the 24-h stimulation at 37 °C 5% CO₂, Mφs were cultured in DMEM media supplemented with 10% FBS and 1X penicillin/streptomycin. After 24 h, supernatant of cell culture media was removed, cells were washed three times with 1X DPBS, and replaced with fresh cell culture media free of penicillin/streptomycin. Cells were then infected with either E. coli UTI89 or E. faecalis OG1RF strains at a multiplicity-of-infection ratio of 2 for a period of 45 min to allow uptake of the pathogen in a 37 °C 5% CO₂ incubator. After 45 min, supernatant was removed and plated to measure CFUs. Mφs were then washed three times with 1X DPBS before being replaced with DMEM media supplemented with 100 µg/mL gentamicin and 1X penicillin/streptomycin to remove non-internalized bacteria. Mφs were then incubated in a 37 °C 5% CO₂ incubator for 30 min (serving as t = 0) and 3-h post-uptake. After each time point, media was removed, and cells were washed three times with 1X DPBS before treatment with distilled water for 15 min to lyse the Mφs. Water was then plated on either LB or BHI media plates and incubated overnight at 37 °C for CFU counts.

LPS-coated beads-based phagocytosis assay

After Mφ harvest, approximately 50,000 cells were seeded per well into a 96-well optic culture plate to assess phagocytosis following stimulation by these varying treatments: the listed concentrations documented within “In vitro and ex vivo polarization of Mφs”, 1.5 mg/mL of soluble Fg (Fg or Fg^AEK), 1.5 mg/mL fibrin gel, or both concurrently. Stimulation took place over a 24 h period at 37 °C 5% CO₂ in DMEM media (BMDMφs) supplemented with 10% FBS and 1X penicillin/streptomycin or complete THP-1 growth media. Mφ phagocytic capacity was quantified using the Vybrant™ Phagocytosis Assay Kit (ThermoFisher Scientific) with the addition of a prepared fluorescein-labeled E. coli BioParticle suspension to each well (100 µg/well). The plates were then incubated for 2 h at 37 °C 5% CO₂ to allow for phagocytosis, per manufacturer’s instruction. Following incubation, the extracellular fluorescence was quenched by the addition of Trypan Blue for 1 min at room temperature. Fluorescence was immediately measured at an excitation wavelength of ~480 nm and an emission wavelength of ~520 nm. The measured fluorescence directly reflected the quantity of E. coli BioParticles successfully phagocytosed by the Mφs. All measurements were normalized to untreated Mφs.

Western blot

Approximately 10⁶ RAW 264.7 were seeded per well in a 6-well flat-bottom culture plate. Mφs were stimulated as described in the “In vitro and ex vivo polarization of Mφs” section, but at concentrations of 12.5, 25, 50, and 100 ng/mL of inducers. Additionally, Mφs were also seeded with soluble Fg, fibrin gel, or both simultaneously at concentrations of 0.1875, 0.375, 0.75, and 1.5 mg/mL. After 24 h, supernatant was removed, cells were harvested and centrifuged into a pellet and resuspended in 5X SDS buffer. The cell lysate was then boiled at 95 °C for 5 min before 15 µL of the sample was loaded in an SDS-PAGE gel. For probing Arginase-1, samples were run on 14% acrylamide gels for 4 and half hours at 120 V. The gel was then transferred to polyvinylidene difluoride membrane (PVDF, Millipore Sigma) using a semi-dry transfer. After transfer, membranes were blocked in 5% non-fat milk in 1X PBS for 1 h at room temperature before incubation with rabbit anti-beta actin (1:7500) and goat anti-Arginase-1 (1:100) primary antibodies diluted in 2% non-fat milk in 1X PBS-Tween (0.1%, PBS-T) overnight at 4 °C. Membranes were washed with 1X PBS-T before being probed with IRDye680RD donkey anti-rabbit and IRDye800CW donkey anti-goat secondary antibodies (LI-COR Bioscience) diluted in 2% non-fat milk in 1X PBS-T-SDS (0.01% SDS) for 1 h at room temperature. For probing iNOS, samples were run on 7% acrylamide gels for 2 h at 120 V. Then, the gel was transferred to PVDF membrane using a wet tank transfer method running at 4 °C for 2 and half hours. After transfer, membranes were blocked before incubation with rat anti-beta actin (1:7500) and rabbit anti-iNOS (1:100) primary antibodies as previously described. Immunoblots for fibrin(ogen), D-dimer, and uPA, rabbit anti-Fg (Abcam ab34269; 1:1000), rabbit anti-D-Dimer (Bioss bs-3514R; 1:1000), and rabbit anti-uPA (Proteintech 17968-1-AP; 1:1000) primary antibodies were used. As a loading control, goat anti-β-actin (Abcam ab8229; 1:10,000) was used. After overnight, membranes were probed with secondary antibodies IRDye680RD donkey anti-rat and IRDye800CW donkey anti-rabbit as previously described. Membranes were then visualized on an Odyssey CxL Reader. Median fluorescent intensity (MFI) was taken using image studio to determine densitometry of Fg and normalized to β-actin. Measurements were graphed into Graphpad Prism 9 for statistical analysis.

Statistical analysis

Data derived from these studies were entered into GraphPad Prism 8 software to generate statistical results and graphs. At least more than three independent experiments with replicates were performed for all studies. For data that was parametric, unpaired student two-tailed t-tests was used to determine significance between samples. When data was nonparametric, medians was used to represent the distribution and either the two-tailed Mann–Whitney U tests or Kruskal–Wallis with Dunn’s multiple comparisons test was performed, where appropriate. For all tests performed, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Statistical test listed in each figure legend. For principal component analysis (PCA), averages of each treatment/condition and Mφ phenotype counts were placed into GraphPad Prism for PCA and generated principal components. Principal components that account for 90% of the data variation were plotted and labeled appropriately according to catheterization type and infection pathogen.

List of all materials, reagents, antibodies, and dyes used in this study

List of all materials, reagents, antibodies, and dyes can be found in Supplementary Data 1.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Supplementary information

Supplementary information^{(15.9MB, pdf)}

41467_2026_76660_MOESM2_ESM.pdf^{(27.6KB, pdf)}

Description of Additional Supplementary Files

Supp Data 1^{(15.2KB, xlsx)}

Reporting Summary^{(117KB, pdf)}

Transparent Peer Review file^{(1.7MB, pdf)}

Source data

Source Data^{(13.4MB, xlsx)}

Acknowledgements

We thank the members of both A.L.F.M. and F.H.S.T. laboratories for their guidance on experimental design, materials, and comments. We also thank M.J.F. for providing us the Fg^AEK transgenic mice. We also thank the Freimann Life Science Center for mouse breeding and husbandry. Additionally, we thank Dr. Sara Cole from the Notre Dame Integrated Imaging Facility for tissue processing.

Author contributions

A.L.F.M. conceived and supervised the research. Conceptualization: A.M.M., C.G., and A.L.F.M. Formal analysis: A.M.M., C.G., J.J.M., J.J.K., and A.L.F.M. Methodology: A.M.M., C.G., J.J.M., J.J.K., A.L.F.M., D.D., F.H.S.T., V.A.P., M.J.F., and F.J.C. Investigation: A.M.M., A.L.F.M., C.G., J.J.M., J.J.K., M.J.A., K.C.F., A.A.L., E.R.L., E.W., T.U., K.A.P., R.W., P.V.S., and K.N.K. Visualization: A.M.M., C.G., and A.L.F.M. Supervision: A.L.F.M. Writing—original draft: A.M.M., C.G., and A.L.F.M. Writing—review and editing: A.M.M., C.G., J.J.M., J.J.K., M.J.A., A.A.L., E.R.L., E.W., T.U., K.A.P., R.W., P.V.S., K.N.K., V.A.P., M.J.F., D.D., F.J.C., F.H.S.T., and A.L.F.M.

Peer review

Peer review information

Nature Communications thanks Juan de Dios Ruizrosado, Laura Schwartz, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Funding

This work was done in the Flores-Mireles Laboratory and funded by institutional funds from the University of Notre Dame (to A.L.F.M.), the National Institute of Health’s National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) grants R01DK128805 (to A.L.F.M. and A.M.M.), the Diversity supplement R01DK12880501-A1S1 (to A.M.M.); R01-HL013423 (to D.D., V.A.P., and F.J.C.); R01-HL160046 and U01-HL143403 (to M.J.F.); R01AI177875 and R21AI71742 (to F.H.S.-T.); and from Good Venture Foundation (Open Philanthropy) grant (to A.L.F.M., A.M.M., A.A.L, J.J.M., M.J.A., C.G., K.N.K., and E.W.).

Data availability

Source data is provided in the Source data file and in the Supplementary Information. Source data are provided with this paper.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Armando M. Marrufo, Christopher Gager.

Supplementary information

The online version contains supplementary material available at https://doi.org/10.1038/s41467-026-76660-8.

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PERMALINK

Fibrin-rich environment drives catheter-associated UTIs by suppressing macrophage defenses

Armando M Marrufo

Christopher Gager

Jonathan J Molina

Marissa J Andersen

Alyssa A La Bella

Jessica J Kean

Katelyn C Fealy

Elizabeth R Lucas

Ellsa Wongso

Tamanna Urmi

Kassandra Arias-Parbul

Railyn Webster

Peter V Stuckey

Kurt N Kohler

Deborah Donahue

Victoria A Ploplis

Matthew J Flick

Francis J Castellino

Felipe H Santiago-Tirado

Ana L Flores-Mireles

Abstract

Introduction

Results

Urinary catheterization-induced inflammation promotes robust fibrin(ogen) and macrophage recruitment

Fig. 1. Urinary catheterization induces robust fibrin(ogen) accumulation and Mφ response.

Macrophage polarization in the catheterized bladder

Fig. 2. Urinary catheterizations induce diverse Mφ polarization populations.

Fibrin suppresses fibrinogen-induced M1-like response

Fig. 3. Fibrin induced M2-like Mφ polarization predominates even in the presence of soluble Fg.

Catheterized bladder environment with fibrin accumulation enhances M2-like Mφ response

Fig. 4. Soluble fibrinogen and fibrin differentially polarize Mφs during catheterization.

Table 1.

Fibrin-associated M2-like Mφs exhibited altered antimicrobial response against CAUTI pathogens

Fig. 5. Fibrin-induced Mφs exhibit altered antimicrobial response against pathogens.

GM-CSF-Induced M1-like Mφ polarization is insufficient to overcome the pro-pathogen catheterized environment

Fig. 6. Mφ reprogramming is not sufficient to promote bacterial clearance during CAUTI.

M1-like polarization unable to improve bacterial clearance

Shared uropathogen phagocytosis by Mφ subpopulations

Fig. 7. Differential phagocytosis of bacteria by polarized Mφ during CAUTI.

Discussion

Methods

Ethics statements

Study design

Bacterial strains and growth conditions

In vivo experimental model

In vivo Mφ polarization model

Cell culturing

Isolation of bone-marrow derived monocytes

Isolation of soluble fibrinogen from FgAEK mice

Fibrin gel formation

Endotoxin quantification for fibrinogen stock

In vitro and ex vivo polarization of Mφs

Immunofluorescent staining of RAW 264.7 and BMDMφs

Immunofluorescent staining and imaging of bladders

Flow cytometry analysis

Gentamicin protection assay

LPS-coated beads-based phagocytosis assay

Western blot

Statistical analysis

List of all materials, reagents, antibodies, and dyes used in this study

Reporting summary

Supplementary information

Source data

Acknowledgements

Author contributions

Peer review

Peer review information

Funding

Data availability

Competing interests

Footnotes

Supplementary information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

Isolation of soluble fibrinogen from Fg^AEK mice