Skip to main content
NCBI home page
The Journal of Infectious Diseases logoLink to The Journal of Infectious Diseases
. 2025 May 15;232(3):e403–e415. doi: 10.1093/infdis/jiaf221

Probiotic Lactobacillus Species Modulate Immune Responses During Vaginal Epithelial Cell Colonization

Marisa Valentine 1, Diletta Rosati 2, Axel Dietschmann 3, Tim B Schille 4,5, Mihai G Netea 6,7, Bernhard Hube 8,9,10,#,✉,c, Mark S Gresnigt 11,#,✉,c
PMCID: PMC12455308  PMID: 40372982

Abstract

Background

Vulvovaginal candidiasis (VVC), mainly caused by Candida albicans, affects approximately 75% of women at least once during their lifetime. VVC is characterized by fungal colonization, which leads to inappropriate local hyperinflammation and symptoms. Although the trigger of C. albicans pathogenicity is often unknown, antibiotic use and vaginal dysbiosis are associated with increased susceptibility to VVC. A healthy vaginal microbiota is normally dominated by Lactobacillus species, which are believed to keep C. albicans infections at bay. Probiotic lactobacilli are, therefore, explored to treat symptomatic VVC episodes. However, the influence of probiotic lactobacilli on immune responses in the context of VVC remains underexplored.

Methods

We investigated how probiotic lactobacilli influence vaginal epithelial and downstream inflammatory responses during C. albicans infection, using in vitro vaginal epithelial infection models and stimulating primary human immune cells with supernatants from these vaginal epithelial cells.

Results

Our study shows that although most of the tested probiotic lactobacilli reduced C. albicans-induced vaginal epithelial cell damage, some species, particularly Limosilactobacillus fermentum and Lacticaseibacillus paracasei, elicited proinflammatory responses even in the absence of C. albicans. Probiotic lactobacilli also differentially modulated the C. albicans killing efficiency and production of reactive oxygen species by neutrophils.

Conclusions

Overall, vaginal epithelial and downstream immune responses during co-cultivation with C. albicans and probiotic lactobacilli were mostly driven by specific bacterial species and their interactions with the vaginal epithelium. Therefore, the induction of “controlled” inflammation by probiotic lactobacilli may be beneficial to improve neutrophil function; however, whether this alleviates immunopathology warrants further investigation.

Keywords: vulvovaginal candidiasis, Candida albicans, immune response, probiotics, Lactobacillus

Vulvovaginal candidiasis, an infection mainly caused by the fungus Candida albicans, is associated with hyperinflammation. We found that different probiotic lactobacilli can differentially modulate vaginal epithelial cell and downstream immune responses during C. albicans infection.

Vulvovaginal candidiasis (VVC) is a female genital tract infection caused by the overgrowth of Candida species. Seventy-five percent of women experience VVC at least once, while 5%–8% report minimum four annual infections (recurrent VVC) [1]. VVC has a major socioeconomic impact by reducing quality of life [2].

Candida albicans (the predominant causative agent), unlike non-albicans species, causes inflammation-associated VVC [3]. Although normally commensal, C. albicans can overgrow and produce hyphae. Consequently, hyphae-associated virulence factors damage the vaginal mucosa, triggering interleukin-1α (IL-1α; alarmin) release, NLRP3 inflammasome activation, and neutrophil recruitment [4–6]. NLRP3 inflammasome-controlled IL-1β responses drive neutrophil-mediated inflammation [4, 7, 8]. However, recruited neutrophils fail to clear the infection, leading to immunopathology and persistent hyperinflammation that causes symptoms [1, 4, 9]. This dysfunctional response has been ascribed to host and fungal factors [10–12].

Factors and conditions, including antibiotic-induced dysbiosis, predispose to VVC [1]. A healthy vaginal microbiota is characterized by a low microbial diversity, typically dominated by a single Lactobacillus species of either Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, or Lactobacillus iners [1, 13]. Lactobacilli can antagonize C. albicans by secreting metabolites that lower pH, as well as impairing C. albicans adherence to host cells and hyphal formation [9]. For this reason, lactobacilli are explored as therapeutic microbes [14–16]. Probiotic lactobacilli can be as effective as fluconazole in clearing the fungal burden and alleviating VVC symptoms [14–16]. Lactobacilli can modulate immune responses, including defensin release by epithelial cells, making them promising candidates for VVC treatment [17–19]. Immunomodulation has been extensively studied in the context of the intestine [20, 21], but scarcely at the level of the vaginal epithelium.

It is crucial to consider vaginal epithelial cells in C. albicans-lactobacilli interaction studies, because the epithelium is the first line of defense and mounts initial responses against infection [5]. Anti-Candida activities of lactobacilli are mostly studied using models lacking host cells [22, 23], or focus solely on physical interactions with host cells [24]. Contrasting effects of lactobacilli on proinflammatory and anti-inflammatory cytokine responses of vaginal epithelial and cervical cells have been observed [19, 25]. Cross-talk between the vaginal epithelium and immune cells, however, has been mostly investigated in murine models in vivo, which do not completely recapitulate VVC in humans [26].

In our study, we evaluated how representative probiotic lactobacilli affect cytokine responses of human vaginal epithelial cells towards C. albicans infection and their subsequent effects on downstream immune responses.

METHODS

Human Vaginal Epithelial Cell Culture

A-431 vaginal epithelial cells (VECs; Deutsche Sammlung von Mikroorganismen und Zellkulturen, No. ACC91) were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco) with 10% heat-inactivated fetal bovine serum (Bio & Sell), according to the supplier's instructions. The cell line was authenticated by commercial short tandem repeat profiling (Eurofins Genomic) and monitored using polymerase chain reaction (PCR) mycoplasma test kits (PromoKine). Seeded VECs (2 × 104/well in 96-well and 4 × 105/well in 6-well plates) were incubated until confluency (37°C; 5% CO2; 2 days).

Microbial Culture and Maintenance

C. albicans SC5314 [27] was cultured on 1% yeast extract, 2% peptone, and 2% dextrose (YPD) medium (1.5% agar was added for solid medium). A single colony was inoculated into YPD medium and incubated overnight (30°C; 180 rpm). C. albicans cells were washed 3 times with phosphate buffered saline (PBS; pH 7.4), the cell number was enumerated with a Neubauer chamber, and adjusted.

Lactobacilli (Levilactobacillus brevis American Type Culture Collection [ATCC] 14869, Lacticaseibacillus casei ATCC 393, Limosilactobacillus fermentum ATCC 14931, Lacticaseibacillus paracasei ATCC 11578, Lacticaseibacillus rhamnosus ATCC 7469, and Ligilactobacillus salivarius ATCC 11741) were cultured on de Man, Rogosa, and Sharpe (MRS; Carl Roth) agar. MRS medium was inoculated and incubated 2–3 days (37°C; 5% CO2; 1% O2) without shaking. Bacterial cells were washed 3 times with PBS and the optical density (OD) adjusted in RPMI.

Heat-killed lactobacilli was prepared at 95°C for 30 minutes. Following washing, the cell concentration was adjusted, and cell suspensions (OD 0.2) stored at −20°C. Suspensions were plated on MRS agar to confirm heat-killing.

Generation of a mScarlet-Labeled C. albicans SC5314 Reporter Strain

The pNIM1R plasmid [28] was used to generate a mScarlet-labeled C. albicans SC5314 reporter strain. The C. albicans codon-optimized mScarlet open reading frame from pRB897 [29] was cloned into pNIM1R at SalI/BglII using the primers mSc-SalI-pNIM1R-F (5′-AATATAAATAGTCGACATGGTTTCTAAAGGTGAAGCAG-3′) and mSc-BglII-pNIM1-R (5′-TCCAGAATTTAGATCTCTAATATAATTCATCCATACCACCT-3′). The transformation cassette was excised with SacII and KpnI and transformed into SC5314 via polyethylene glycol/lithium acetate method [30]. Selection was performed on YPD containing 200 µg/mL nourseothricin. Successful integration into the ADH1 locus was verified by colony PCR.

Colonization and Infection

Cell culture medium was replaced with 50 µL (96-well) or 1 mL (6-well) serum-free RPMI and VECs were colonized with 50 µL (96-well) or 1 mL (6-well) bacteria (OD 0.2). After 18 hours, VECs were infected with C. albicans SC5314 at a multiplicity of infection (MOI) of 1, that is, 50 µL (96-well) or 1 mL (6-well) of cell suspension (4 × 105/mL). After 24 hours (37°C; 5% CO2), plates were centrifuged (200g; 10 minutes), and supernatant was collected. Supernatants for immune stimulations were filter sterilized (0.2 µm) before storage at −80°C.

Live-Cell Imaging of C. albicans

VECs were colonized with lactobacilli and infected with mScarlet-labeled C. albicans. The 96-well plate was imaged in an Incucyte SX5 live-cell analysis system (Sartorius; 37°C; 5% CO2). Phase contrast and fluorescence (excitation, 546–568 nm; emission, 576–639 nm) images were taken every 30 minutes for 5 hours at 20× magnification. Filamentation was quantified using the NeuroTrack tool (Incucyte software 2023A Rev2; Sartorius) [31].

Ethical Statement

Human peripheral blood was collected from healthy volunteers after receiving written informed consent. This study was performed following the principles outlined in the Declaration of Helsinki. The blood donation protocol and use of blood for this study were approved by the institutional ethics committee of Jena University Hospital (permission number 2207-01/08) and the local institutional review board in Nijmegen (CMO region Arnhem-Nijmegen, The Netherlands, no. 22992010/104).

Immune Cell Isolation and Stimulation

Peripheral blood mononuclear cells (PBMCs) and neutrophils were isolated as previously described using Ficoll-Paque density gradient centrifugation (GE Healthcare) [32]. Isolated cells were resuspended in RPMI and the concentrations adjusted.

PBMCs were seeded at a density of 5 × 105/well (100 µL of 5 × 106/mL) in round-bottom 96-well plates and stimulated with 50 µL of VEC supernatant or 5× diluted heat-killed bacterial stocks (OD 0.2). Fifty microliter RPMI was added to give a final volume of 200 µL. PBMCs were stimulated with supernatants from lactobacilli cultured on plastic alone in the same manner as on VECs. After 24 hours or 7 days (in the presence of 10% human serum; Bio & Sell), plates were centrifuged (200g; 10 minutes), and supernatants were collected and stored at −80°C.

Neutrophils were seeded in 96-well plates at a density of 1 × 105/well (100 µL of 1 × 106/mL) and first stimulated with 1 × 105/well C. albicans (50 µL of 2 × 106/mL) or L. rhamnosus (50 µL of OD 0.2) for 2 hours before the second stimulation. Neutrophils (5 × 104/well) were stimulated simultaneously with 5 × 105  C. albicans (MOI 10; 50 µL of 1 × 107/mL) and lactobacilli (50 µL of OD 1). After 3 hours, plates were centrifuged (200g; 10 minutes), and supernatants were collected and stored at −80°C.

C. albicans Survival in the Presence of Neutrophils

Neutrophils (1 × 105/well; 100 µL of 1 × 106/mL) were stimulated with C. albicans (MOI 1; 50 µL of 1 × 107/mL or 2 × 106/mL) in the presence of 4× diluted supernantant in RPMI (50 µL; total well volume 200 µL). Survival was also determined in the presence of neutrophils and lactobacilli incubated simultaneously or when first primed with 1 microorganism before stimulation with the other as described above.

After 3 hours of incubation (37°C; 5% CO2), plates were centrifuged (200g; 10 minutes). Samples were obtained by washing wells with H2O, and diluted using PBS. C. albicans (including inocula) was plated on YPD agar plates that were incubated (30°C; 2 days) and colony-forming units were enumerated.

Reactive Oxygen Species Detection

Neutrophils were seeded in white 96-well plates (5 × 104/well) and stimulated with C. albicans (25 µL of 4 × 105/mL) and/ or lactobacilli (25 µL of OD 0.2). Fifty microliters of a luminol-horseradish peroxidase (HRP) solution (200 µM luminol and 16 U HRP) was added. Chemiluminescence was measured every 2.5 minutes for 3 hours at 37°C in a microplate reader (Tecan). Unstimulated neutrophils served as negative controls.

Live-Cell Imaging of Neutrophil Extracellular Trap Formation

Neutrophils (2 × 104/well) were stimulated with mScarlet-labeled C. albicans (MOI 1; 50 µL of 2 × 105/well) in the absence and presence of live bacteria (50 µL of OD 0.2) in RPMI containing 0.1 µM SYTOX Green Nucleic Acid Stain (Invitrogen). The 96-well plate was imaged in an Incucyte SX5 live-cell analysis system (Sartorius; 37°C; 5% CO2). Phase contrast, orange (excitation, 546–568 nm; emission, 576–639 nm), and green fluorescence (excitation, 453–485 nm; emission, 494–533 nm) images were taken every 20 minutes for 12 hours at 20× magnification. Filamentation was quantified with the NeuroTrack tool (Incucyte software 2023A Rev2; Sartorius) [31] and the tool's nuclei parameter was used to identify SYTOX Green-positive NETosis events. The analysis definition can be shared upon request.

Cytokine Release and Epithelial Cell Damage

Cytokines were quantified using commercial enzyme-linked immunosorbent assay (ELISA) kits (DuoSet; R&D systems). Cell damage was measured as cytoplasmic enzyme lactate dehydrogenase activity with a cytotoxicity detection kit (Roche).

Statistical Analysis

GraphPad Prism version 10.4.0 was used to calculate significance using one-way or two-way ANOVA with a Holm-Šídák or Tukey multiple comparisons test. Significance is indicated as *P ≤ .05, **P ≤ .01, ***P ≤ .001, and ****P ≤ .0001.

RESULTS

Lactobacilli Dampen C. albicans-Induced Epithelial Damage But Not Immune Responses

The ability of different probiotic lactobacilli to reduce C. albicans-induced VEC damage was determined (Figure 1A). Bacteria alone did not damage VECs (Figure 1B). Except L. brevis and L. fermentum, lactobacilli reduced C. albicans-induced VEC damage (Figure 1B). While L. fermentum strongly reduced C. albicans hyphae length, C. albicans hyphae length was moderately increased in the presence of other species (Figure 1C and 1D).

Figure 1.

Alt text: Graphs and microscopy images labelled A to D. A is a graphical representation of the experimental procedure showing vaginal epithelial cells being colonized with lactobacilli and then infected with Candida albicans. B is a graph showing that different lactobacilli reduce C. albicans-induced vaginal epithelial cell damage. C is a graph showing the effect of different lactobacilli on C. albicans growth as hyphae over time. D is microscopy images showing C. albicans hyphal growth on vaginal epithelial cells in the presence of different lactobacilli.

Open in a new tab

The effect of different probiotic Lactobacillus species on the damage of Candida albicans-infected A-431 VECs and on C. albicans growth as hyphae. A, Schematic showing the experimental setup of A-431 VECs being colonized with lactobacilli alone for 18 h (top right), colonized for 18 h prior to C. albicans infection for an additional 24 h (bottom left), or infected with C. albicans alone for 24 h (bottom right). B, LDH released at 24 h post-infection by A-431 VECs that were colonized with different Lactobacillus species and subsequently infected with C. albicans. C, Growth of C. albicans as hyphae length over a time span of 5 h on A-431 VECs in the absence and presence of lactobacilli colonization. Significance is indicated for hyphae length at 5 h. D, Representative microscopy images showing the growth of mScarlet-labeled C. albicans 5 h post-infection on A-431 VECs in the absence and presence of the different Lactobacillus species. Scale bar = 200 µm. B and C, Bars and circles represent the mean ± standard error of the mean of n = 3 independent replicates. Means were compared for significance to their respective uninfected or infected controls using one-way (B) or two-way (C) ANOVA with a Holm-Šídák multiple comparisons test. **P ≤ .01, ***P ≤ .001, ****P ≤ .0001. Abbreviations: Lb, Levilactobacillus brevis; Lc, Lacticaseibacillus casei; LDH, lactate dehydrogenase; Lf, Limosilactobacillus fermentum; Lp, Lacticaseibacillus paracasei; Lr, Lacticaseibacillus rhamnosus; Ls, Ligilactobacillus salivarius; VEC, vaginal epithelial cell.

Subsequently, we studied whether the lactobacilli influenced VEC cytokine responses. Release of IL-1α and its endogenous inhibitor IL-1 receptor antagonist (IL-1Ra) by VECs seemed to be C. albicans driven (Figure 2A). IL-1α release tended to be lower and IL-1Ra release significantly reduced upon lactobacilli colonization (Figure 2A). VEC damage positively correlated with IL-1α and even more strongly with IL-1Ra (Figure 2B). C. albicans induced release of the neutrophil chemoattractant IL-8, which was not reduced by lactobacilli (Figure 2A). Instead, L. fermentum and L. paracasei induced IL-8 responses. Accordingly, VEC damage did not correlate with IL-8 release (Figure 2B). VECs did not release IL-1β nor tumor necrosis factor (TNF), while granulocyte-macrophage colony-stimulating factor (GM-CSF) release remained bacterial driven (Supplementary Figure 1).

Figure 2.

Alt text: Graphs showing how different lactobacilli affect cytokine release by vaginal epithelial cells and how certain cytokines, interleukin one alpha and interleukin one receptor antagonist, correlate with vaginal epithelial cell damage.

Open in a new tab

Inflammatory responses of Candida albicans-infected A-431 VECs in the absence and presence of different probiotic Lactobacillus species. A, IL-1α, IL-1Ra, and IL-8 released by VECs 24 h post-infection after being colonized with different Lactobacillus species and subsequently infected with C. albicans. B, Scatterplots showing correlations between released cytokines (IL-1α, IL-1Ra, and IL-8) and VEC damage (LDH in ng/ml); regression lines are shown in solid black and 95% confidence intervals as dotted lines. C, VEC-secreted ratio of IL-1α to IL-1Ra in the presence of different Lactobacillus species and C. albicans infection. A and C, Bars represent the mean ± standard error of the mean of n = 3 independent replicates. A and C, Means were compared for significance to their respective uninfected or infected controls using one-way ANOVA with a Holm-Šídák multiple comparisons test. *P ≤ .05, **P ≤ .01, ***P ≤ .001 , ****P ≤ .0001. Abbreviations: VEC, vaginal epithelial cell; IL, interleukin; IL-1Ra, IL-1 receptor antagonist; Lb, Levilactobacillus brevis; Lc, Lacticaseibacillus casei; Lf, Limosilactobacillus fermentum; Lp, Lacticaseibacillus paracasei; Lr, Lacticaseibacillus rhamnosus; Ls, Ligilactobacillus salivarius; LDH, lactate dehydrogenase; ns, not significant.

The balance between pro- and anti-inflammatory effects of lactobacilli on IL-1 cytokine responses by VECs was determined in terms of IL-1α/IL-1Ra ratios. Generally, low IL-1α/IL-1Ra ratios were observed in the presence of lactobacilli, highlighting a stronger tendency towards inhibition of IL-1R signaling by IL-1Ra (Figure 2C). L. rhamnosus and L. salivarius induced the strongest tendency towards anti-inflammatory IL-1Ra responses.

Downstream Immune Responses Are Driven by Probiotic Lactobacilli and Their Interaction With VECs

To investigate how lactobacilli, C. albicans, and VECs together influenced downstream immune responses, we stimulated PBMCs with VEC supernatants (Figure 3A). PBMC cytokine responses strongly depended on the lactobacilli species colonizing the VECs rather than the presence of C. albicans infection (Figure 3B). Supernatants from L. fermentum- or L. paracasei-colonized VECs elicited responses characterized by both anti- and proinflammatory cytokines (Figure 3B). Except for IL-8 and IL-1Ra, PBMC responses mirrored the probiotic species-specific pattern of IL-8 responses by VECs (Figure 4A). Downstream PBMC activation, therefore, is independent of VEC damage and driven by specific lactobacilli colonizing VECs.

Figure 3.

Alt text: Illustration and graphs showing how peripheral blood mononuclear cells were stimulated with vaginal epithelial cell supernatants for twenty-four hours and the effect on cytokine release by peripheral blood mononuclear cells.

Open in a new tab

Short-term cytokine response elicited upon 24-h stimulation of PBMCs with supernatants from A-431 VECs colonized with probiotic Lactobacillus species. A, Schematic showing how downstream PBMC responses were induced by stimulation with supernatants from A-431 VECs that were colonized and infected with lactobacilli and Candida albicans. B, Levels of secreted IL-10, IL-6, TNF, IL-8, IL-1β, and IL-1Ra after PBMCs were stimulated with filtered supernatants from A-431 VECs that were infected with C. albicans in the presence and absence of different Lactobacillus species. Bars represent the mean ± standard error of the mean of n = 6 independent replicates. Means were compared for significance to unstimulated PBMC controls using one-way ANOVA with a Holm-Šídák multiple comparisons test. *P ≤ .05, **P ≤ .01, ***P ≤ .001, P ≤ .0001. Abbreviations: IL, interleukin; IL-1Ra, IL-1 receptor antagonist; Lb, Levilactobacillus brevis; Lc, Lacticaseibacillus casei; Lf, Limosilactobacillus fermentum; Lp, Lacticaseibacillus paracasei; Lr, Lacticaseibacillus rhamnosus; Ls, Ligilactobacillus salivarius; PBMC, peripheral blood mononuclear cell; TNF, tumor necrosis factor, VEC, vaginal epithelial cell.

Figure 4.

Alt text: Graphs and heatmap labelled A to C. A is a heatmap showing the correlations between cytokines released by vaginal epithelial cells and peripheral blood mononuclear cells. B and C show the overall pro- and anti-inflammatory responses of peripheral blood mononuclear cells as tumor necrosis factor to interleukin ten and interleukin beta to interleukin one receptor antagonist ratios .

Open in a new tab

Overall pro- and anti-inflammatory effects of probiotic Lactobacillus species colonization on short-term stimulation of PBMCs. A, Heatmap showing the correlation between cytokine release from VECs and PBMCs stimulated with VEC supernatants for 24 h. Pearson correlation coefficients are indicated in the heatmap. Ratios of (B) TNF to IL-10 and (C) IL-1β to IL-1Ra in PBMCs stimulated with VEC supernatants colonized with lactobacilli and/ or infected with C. albicans. Bars represent the mean ± standard error of the mean of n = 6 independent replicates. Means were compared for significance to their respective unstimulated PBMC controls using one-way ANOVA with a Holm-Šídák multiple comparisons test. *P ≤ .05, **P ≤ .01, ****P ≤ .0001. Abbreviations: IL, interleukin; IL-1Ra, IL-1 receptor antagonist; Lb, Levilactobacillus brevis; Lc, Lacticaseibacillus casei; Lf, Limosilactobacillus fermentum; Lp, Lacticaseibacillus paracasei; Lr, Lacticaseibacillus rhamnosus; Ls, Ligilactobacillus salivarius; PBMC, peripheral blood mononuclear cell; TNF, tumor necrosis factor; VEC, vaginal epithelial cell.

The anti-inflammatory cytokine IL-10 was only detected in response to supernatants from lactobacilli-colonized VECs (Figure 3B). IL-1Ra showed a homogenously high response to supernatants of lactobacilli-colonized VECs. This suggests an overall potential of lactobacilli to induce an anti-inflammatory environment. Nevertheless, PBMCs stimulated with supernatants from L. fermentum-colonized VECs had higher TNF to IL-10 (Figure 4B) and IL-1β to IL-1Ra ratios (Figure 4C). When stimulating PBMCs for 7 days to investigate long-term responses (Figure 5A), we continued to observe that L. fermentum and L. paracasei elicited strong responses (Figure 5B). Stimulation with supernatants from VECs colonized by almost all bacterial species drove IL-6, IL-8, and IL-1Ra responses. Interestingly, while supernatants of C. albicans-infected VECs drove potent IL-8 responses, the colonization with probiotic species reduced IL-8 responses. Furthermore, in the context of C. albicans infection, most lactobacilli potentiated IL-6 and TNF responses. In terms of IL-1 signaling, most probiotic species except L. brevis increased IL-1β release, while the opposite pattern was observed for IL-1Ra. This may indicate that in the context of C. albicans infection, probiotic lactobacilli dampen release of IL-8 while polarizing towards proinflammatory IL-6, TNF, and IL-1β signaling. Independent of lactobacilli, long-term adaptive responses (IL-17 and IL-22) were, however, C. albicans-infection driven (Figure 5B).

Figure 5.

Alt text: Illustration and graphs showing how peripheral blood mononuclear cells were stimulated with vaginal epithelial cell supernatants for seven days and the effect on cytokine release by peripheral blood mononuclear cells.

Open in a new tab

Long-term cytokine response elicited upon 7-day stimulation of PBMCs with supernatants from A-431 VECs colonized with probiotic Lactobacillus species. A, Schematic showing how downstream PBMC responses were induced by stimulation with supernatants from A-431 VECs that were colonized and infected with lactobacilli and Candida albicans. B, Levels of secreted IL-10, IL-6, TNF, IL-8, IL-1β, IL-1Ra, IL-17, and IL-22 after PBMCs were stimulated with filtered supernatants from A-431 VECs that were infected with C. albicans in the presence and absence of different Lactobacillus species. Bars represent the mean ± standard error of the mean of n = at least 4 independent replicates. Means were compared for significance to unstimulated PBMC controls using one-way ANOVA with a Holm-Šídák multiple comparisons test. *P ≤ .05, **P ≤ .01, ***P ≤ .001, ****P ≤ .0001. Abbreviations: IL, interleukin; IL-1Ra, IL-1 receptor antagonist; Lb, Levilactobacillus brevis; Lc, Lacticaseibacillus casei; Lf, Limosilactobacillus fermentum; Lp, Lacticaseibacillus paracasei; Lr, Lacticaseibacillus rhamnosus; Ls, Ligilactobacillus salivarius; PBMC, peripheral blood mononuclear cell; TNF, tumor necrosis factor; VEC, vaginal epithelial cell.

Lactobacilli-Secreted Factors Directly Stimulate PBMCs

To distinguish between the role of VECs and lactobacilli in eliciting downstream PBMC responses, we stimulated PBMCs with supernatants of VECs colonized with heat-killed bacteria or supernatants from live bacterial cultures grown without VECs. L. fermentum, L. casei, and L. brevis were used to represent high, intermediate, and low stimulatory species, respectively (Figures 2–5). When stimulated with heat-killed bacteria, PBMCs were robustly activated (Supplementary Figure 2). We observed differences between supernatants from VECs colonized with dead lactobacilli and live lactobacilli grown on plastic (Figure 6A and 6B). Notably, supernatants from VECs colonized with heat-killed L. fermentum induced IL-10, IL-1β, IL-6, IL-8, and TNF responses in PBMCs (Figure 6A). Colonization of VECs with killed L. brevis and L. casei only induced IL-8 and IL-1Ra responses downstream (Figure 6A). Interestingly, when grown without VECs, L. brevis and L. fermentum supernatants induced IL-1β, IL-6, and TNF responses (Figure 6B), suggesting that bacteria themselves and their secreted components stimulate PBMCs responses.

Figure 6.

Alt text: Graphs labelled A to B with different panels showing cytokine release by peripheral blood mononuclear cells. In A is the response to supernatants from vaginal epithelial cells colonized with heat-killed lactobacilli. In B is the response to supernatants of live lactobacilli.

Open in a new tab

Cytokine response elicited upon 24-h stimulation of PBMCs with probiotic Lactobacillus species. A, Levels of secreted IL-10, IL-1β, IL-6, IL-8, TNF, and IL-1Ra after PBMCs were stimulated with filtered supernatants from A-431 VECs that were colonized with HK lactobacilli or (B) from live lactobacilli that were grown on plastic alone. Bars represent the mean ± standard error of the mean of n = 3 independent replicates. Means were compared for significance to their respective unstimulated PBMC controls using one-way ANOVA with a Holm-Šídák multiple comparisons test. *P ≤ .05, **P ≤ .01, ***P ≤ .001, ****P ≤ .0001. Abbreviations: HK, heat-killed; IL, interleukin; IL-1Ra, IL-1 receptor antagonist; Lb, Levilactobacillus brevis; Lc, Lacticaseibacillus casei; Lf, Limosilactobacillus fermentum; PBMC, peripheral blood mononuclear cell; TNF, tumor necrosis factor; VEC, vaginal epithelial cell.

Probiotic Lactobacillus Species Differently Modulate Neutrophil Responses

As dysfunctional neutrophils are a hallmark of VVC, we evaluated how probiotic lactobacilli affect neutrophil function. We used L. rhamnosus and L. brevis as a protective and non-protective species, respectively (Figure 1B). VEC supernatants did not differentially affect C. albicans killing by neutrophils (Supplementary Figure 3). When neutrophils were co-incubated with live L. brevis, C. albicans killing was compromised, whereas live or heat-killed L. rhamnosus had no effect (Figure 7A). L. brevis reduced IL-8 responses, suggesting that L. brevis can impair neutrophil activation. Neutrophils appeared activated in the presence of L. rhamnosus, reflected in increased reactive oxygen species production (Figure 7B). Both L. brevis and L. rhamnosus reduced neutrophil extracellular trap formation (NETosis), while L. rhamnosus made neutrophils more tolerant to C. albicans hyphae and inhibited spontaneous NETosis in uninfected neutrophils (Figure 7C). Prior exposure to L. rhamnosus also influenced neutrophil activity. Neutrophils were less effective at killing C. albicans and secreted more IL-8 (Figure 7D).

Figure 7.

Alt text: Graphs and microscopy images showing the interaction of different lactobacilli with neutrophils. A shows graphs of the effect of lactobacilli on Candida albicans survival in the presence of neutrophils and how this affects interleukin eight release. B shows that L. rhamnosus increases reactive oxygen production by neutrophils. C shows that lactobacilli reduced neutrophil extracellular trap formation by neutrophils in response to C. albicans and that L. rhamnosus makes neutrophils more resistant to induce neutrophil extracellular traps. C also has microscopy images of neutrophil extracellular traps in the presence of lactobacilli and C. albicans. D shows C. albicans survival and interleukin eight in the presence of neutrophils, first stimulated with L. rhamnosus and then C. albicans.

Open in a new tab

The effect of probiotic lactobacilli on neutrophil responses. A, Candida albicans survival (expressed as percentage CFUs of the inoculum) in the presence of live and HK lactobacilli after 3 h incubation with neutrophils, and IL-8 release after 3 h. B, ROS production over 3 h by neutrophils in the presence of C. albicans and live lactobacilli. C, NETosis (top graph) over 12 h by neutrophils in the presence of C. albicans and live lactobacilli as quantified using SYTOX Green nucleic acid staining. Additionally, the ratio of C. albicans filamentation (hyphae length) to NETosis (bottom graph) is shown with representative microscopy images showing mScarlet-labeled C. albicans and dead neutrophils in green. Scalebar = 200 µm. Significance is indicated for the end time points. D, C. albicans survival (expressed as percentage CFUs of the inoculum) after 3 h incubation with neutrophils that were stimulated with L. rhamnosus first before C. albicans stimulation, and IL-8 release after 3 h by neutrophils under these conditions. Bars represent the mean ± standard error of the mean of at least n = 3 independent replicates. Means were compared for significance using one-way (A and D) or two-way (C) ANOVA with a Holm-Šídák multiple comparisons test and one-way ANOVA with a Tukey multiple comparisons test (B). *P ≤ .05, **P ≤ .01, ***P ≤ .001, ****P ≤ .0001. Abbreviations: Ca, Candida albicans; CFU, colony-forming unit; HK, heat-killed; Lb, Levilactobacillus brevis; Lr, L. rhamnosus; IL, interleukin; ROS, reactive oxygen species; NETosis, neutrophil extracellular trap formation.

DISCUSSION

We show that probiotic lactobacilli modulate immune responses during crosstalk between VECs and primary human immune cells. While some probiotic lactobacilli dampened epithelial damage and IL-1Ra release, IL-8 responses were uncoupled from epithelial damage and strongly induced by specific species. PBMCs stimulated with VEC supernatants exhibited differential responses based on the specific probiotic species colonizing the VECs rather than epithelial damage.

In line with a previous study showing differential capacity of lactobacilli to antagonize C. albicans on the intestinal epithelium [33], we observed that lactobacilli protected against C. albicans-induced VEC damage to varying degrees. L. fermentum can reduce VEC damage via reduced C. albicans growth, while some lactobacilli increased C. albicans growth and likely reduce damage via other mechanisms such as metabolic adaptation [34]. L. fermentum was previously reported to reduce IL-8 release by HeLa cells [19]. Contrastingly, we found L. fermentum induced IL-8 release by A-431 VECs. This could be ascribed to bacterial strain or host cell differences. The peptidoglycan structure, a microbe-associated molecular pattern (MAMP) that can be recognized by VECs, can vary within the species, which likely contributes to differences in immunomodulation [35]. Additionally, metabolic differences influencing lactic acid, short-chain fatty acid (SCFA), and indole-3-aldehyde release could impact VEC responses [36–39]. PBMC responses are both lactobacilli species [40] and strain specific [41]. It would therefore be of interest to include the predominantly used probiotic strains in future studies to evaluate their specific immunomodulatory functions in depth.

We found that L. fermentum did not strongly protect against C. albicans-induced VEC damage and it elicited strong proinflammatory cytokine responses downstream in PBMCs. Contrastingly, L. brevis did not elicit proinflammatory responses, failed to prevent damage of VECs and associated IL-1Ra release, and poorly elicited downstream responses in PBMCs. This suggests that specific probiotics, rather than C. albicans-induced epithelial damage, drive downstream PBMC responses. This highlights the importance of studying lactobacilli in holistic models where host responses can be evaluated.

Importantly, probiotic lactobacilli differentially modulated VEC and PBMC responses. Immunoregulatory (not immunostimulatory) probiotics are recommended to treat inflammatory disease [42, 43]. However, it is unclear whether distinct immune stimulation can improve neutrophil functionality [4]. We found L. fermentum to be immunostimulatory, warranting further exploration for its potential to boost a protective immune response against C. albicans.

In the context of immunomodulation by lactobacilli, balances between pro- and anti-inflammatory responses are of interest. IL-10 to TNF and IL-10 to IL-12 ratios in vitro were shown to predict in vivo efficacy in treating colitis [44]. Further illustrating the importance of balance between pro- and anti-inflammatory responses, is the observation that TNF to IL-10 and IL-1β to IL-1Ra ratios were elevated in C. albicans-stimulated PBMCs from patients with recurrent VVC [45]. Apart from focusing on the acute effect of probiotic lactobacilli on PBMC responses, long-term responses were also considered. After 7-day stimulation, we observed that C. albicans infection elicited potent IL-8 responses that were dampened by epithelial colonization with lactobacilli. In addition, the presence of lactobacilli during infection led to pronounced proinflammatory IL-1β and IL-6 long-term responses. Interestingly, adaptive IL-17 and IL-22 responses were C. albicans driven. It would therefore be interesting to study the role of adaptive immune responses and trained immunity.

IL-1α is an alarmin released during epithelial cell damage [5]. Interestingly, its endogenous competitive antagonist IL-1Ra even more strongly correlated with VEC damage. This may be attributed to IL-1Ra not requiring processing, being intracellularly stored, and released upon damage [46]. The IL-1α and IL-1Ra responses suggest that colonization with some lactobacilli favors suppression of IL-1 signaling as IL-1Ra concentrations exceed IL-1α by two orders of magnitude. However, during C. albicans infection, colonization with probiotic lactobacilli prevented C. albicans-induced damage and associated IL-1Ra release, reducing the capacity to dampen IL-1 signaling.

While C. albicans infection induced IL-1α and IL-1Ra release by VECs, IL-8 responses tended to be driven by the probiotic that colonized VECs. When correlating downstream PBMC responses, most released cytokines (IL-10, IL-6, TNF, and IL-1β) mirrored the IL-8 responses from VECs. Responses to heat-killed lactobacilli suggest that MAMPs (such as cell wall components) [35] directly stimulate VECs and PBMCs. Additionally, stimulations with lactobacilli-conditioned supernatants suggest that secreted compounds mediate downstream responses. This corresponds to previous findings showing that supernatants of lactobacilli stimulate macrophages and Toll-like receptor 2 [36], potentially through SCFAs [37] and lactate [38].

We observed that L. rhamnosus and L. brevis differentially influenced neutrophil function. L. brevis reduced IL-8 release, NETosis, and compromised C. albicans killing, indicating that this species may impair neutrophil responses. L. rhamnosus increased neutrophil reactive oxygen species production, but did not affect C. albicans killing. Interestingly, L. rhamnosus made neutrophils more tolerant to C. albicans and less prone to death via NETosis. This suggests that neutrophils may be active for much longer, contributing to immunopathology, and may thus be more detrimental in VVC. IL-8 responses also increased in the presence of L. rhamnosus, which could have resulted from an additive or synergistic effect between bacterial MAMPs and C. albicans pathogen-associated molecular patterns (PAMPs), similar to previous reports on epithelial cells [25, 47]. L. rhamnosus priming of neutrophils before C. albicans stimulation reduced the ability to kill fungal cells. Potentially, L. rhamnosus elicits oxidative responses before neutrophils encounter C. albicans, thereby exhausting their capacity to mount sufficient oxidative response towards C. albicans. Probiotic lactobacilli may therefore influence neutrophil function during VVC and their immunomodulatory mechanisms warrant further detailed investigation in vivo.

In addition to cell wall differences, differences between L-lactate or D-lactate and quantity could lead to distinct neutrophil responses, because lactate regulates neutrophil function [48] and C. albicans’ resistance to neutrophil-mediated killing [49]. We studied normal-functioning neutrophils in vitro, whereas vaginal niche factors perturb neutrophil functionality during VVC [4, 11]. Future studies investigating neutrophils under dysfunctional conditions may reveal further influences of lactobacilli on neutrophil behavior.

In conclusion, we provide insight into immunomodulation by probiotic lactobacilli, showing these probiotics can reduce C. albicans-induced vaginal epithelial damage and elicit both pro- and anti-inflammatory responses. The overall impact of these immune responses elicited by probiotic lactobacilli in vivo remains unknown and warrants in-depth investigation.

Supplementary Material

jiaf221_Supplementary_Data
jiaf221_supplementary_data.zip (2.2MB, zip)

Notes

Acknowledgment . We thank our colleagues in the Microbial Pathogenicity Mechanisms, Afdeling Interne Geneeskunde, and Adaptive Pathogenicity Strategies laboratories where this work was conducted.

Author contributions . M. V. performed investigations, formal analysis, visualization, and wrote the original draft. D. R. and T. B. S. performed investigations. M. G. N. contributed supervision. A. D. performed formal analysis and visualization. M. S. G. contributed conceptualization, supervision, and funding acquisition, and wrote the original draft. B. H. contributed supervision and funding acquisition. All authors reviewed and edited the draft manuscript.

Financial support. This work was supported by the European Union Horizon 2020 Research and Innovation Program under the Marie Skłodowska-Curie Actions (grant number 812969; FunHoMic; M. V., D. R., B. H., and M. G. N.); the Deutsche Forschungsgemeinschaft (German Research Foundation) Emmy Noether grant (grant number 434385622/GR 5617/1-1; M. S. G. and A.D.) and Germany´s Excellence Strategy EXC 2051 (grant number 390713860 to T. B. S. and B. H.); the Research Foundation Flanders (Fonds Wetenschappelijk Onderzoek; FWO) StrategischBasisOnderzoek (SBO) project Defining Vulvovaginal Candidiasis Elements of Inferction and Remedy (DeVEnIR) (grant number S006424N to M. S. G.); the Free State of Thuringia and the European Union project A Live Broadcast of the Interactions Between Host and Fungal Pathogens (LiveFunPath; grant number 2023 FGI 0004 to M. S. G. and A. D.); and the European Society of Clinical Microbiology and Infectious Diseases (grant number 19059 to A. D.).

All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

Contributor Information

Marisa Valentine, Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute, Jena, Germany.

Diletta Rosati, Department of Internal Medicine and Radboud Centre for Infectious Diseases, Radboud University Medical Center, Nijmegen, The Netherlands.

Axel Dietschmann, Junior Research Group Adaptive Pathogenicity Strategies, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute, Jena, Germany.

Tim B Schille, Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute, Jena, Germany; Cluster of Excellence Balance of the Microverse, Friedrich Schiller University Jena, Jena, Germany.

Mihai G Netea, Department of Internal Medicine and Radboud Centre for Infectious Diseases, Radboud University Medical Center, Nijmegen, The Netherlands; Department for Immunology and Metabolism, Life and Medical Sciences Institute, University of Bonn, Bonn, Germany.

Bernhard Hube, Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute, Jena, Germany; Cluster of Excellence Balance of the Microverse, Friedrich Schiller University Jena, Jena, Germany; Institute of Microbiology, Friedrich Schiller University, Jena, Germany.

Mark S Gresnigt, Junior Research Group Adaptive Pathogenicity Strategies, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute, Jena, Germany.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

References

  • 1. Sobel  JD. Vulvovaginal candidosis. Lancet  2007; 369:1961–71. [DOI] [PubMed] [Google Scholar]
  • 2. Zhu  YX, Li  T, Fan  SR, Liu  XP, Liang  YH, Liu  P. Health-related quality of life as measured with the short-form 36 (SF-36) questionnaire in patients with recurrent vulvovaginal candidiasis. Health Qual Life Outcomes  2016; 14:65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. Willems  HME, Lowes  DJ, Barker  KS, Palmer  GE, Peters  BM. Comparative analysis of the capacity of the Candida species to elicit vaginal immunopathology. Infect Immun  2018; 86:e00527-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Yano  J, Peters  BM, Noverr  MC, Fidel  PL. Novel mechanism behind the immunopathogenesis of vulvovaginal candidiasis: “neutrophil anergy”. Infect Immun  2018; 86:e00684-17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5. Moyes  DL, Murciano  C, Runglall  M, Islam  A, Thavaraj  S, Naglik  JR. Candida albicans yeast and hyphae are discriminated by MAPK signaling in vaginal epithelial cells. PLoS One  2011; 6:e26580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Richardson  JP, Willems  HME, Moyes  DL, et al.  Candidalysin drives epithelial signaling, neutrophil recruitment, and immunopathology at the vaginal mucosa. Infect Immun  2018; 86:e00645-17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. Bruno  VM, Shetty  AC, Yano  J, Fidel  PL, Noverr  MC, Peters  BM. Transcriptomic analysis of vulvovaginal candidiasis identifies a role for the NLRP3 inflammasome. mBio  2015; 6:e00182-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8. Roselletti  E, Perito  S, Gabrielli  E, et al.  NLRP3 inflammasome is a key player in human vulvovaginal disease caused by Candida albicans. Sci Rep  2017; 7:17877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. Ardizzoni  A, Wheeler  RT, Pericolini  E. It takes two to tango: how a dysregulation of the innate immunity, coupled with Candida virulence, triggers VVC onset. Front Microbiol  2021; 12:692491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Yano  J, Noverr  MC, Fidel  PL. Vaginal heparan sulfate linked to neutrophil dysfunction in the acute inflammatory response associated with experimental vulvovaginal candidiasis. mBio  2017; 8:e00211-17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11. Ardizzoni  A, Sala  A, Colombari  B, et al.  Perinuclear anti-neutrophil cytoplasmic antibodies (pANCA) impair neutrophil candidacidal activity and are increased in the cellular fraction of vaginal samples from women with vulvovaginal candidiasis. J Fungi (Basel)  2020; 6:225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12. Roselletti  E, Pericolini  E, Nore  A, et al.  Zinc prevents vaginal candidiasis by inhibiting expression of an inflammatory fungal protein. Sci Transl Med  2023; 15:eadi3363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. Ravel  J, Gajer  P, Abdo  Z, et al.  Vaginal microbiome of reproductive-age women. Proc Natl Acad Sci U S A  2011; 108 (Suppl 1):4680–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Mandar  R, Soerunurk  G, Stsepetova  J, et al.  Impact of Lactobacillus crispatus-containing oral and vaginal probiotics on vaginal health: a randomised double-blind placebo controlled clinical trial. Benef Microbes  2023; 14:143–52. [DOI] [PubMed] [Google Scholar]
  • 15. Oerlemans  EFM, Bellen  G, Claes  I, et al.  Impact of a lactobacilli-containing gel on vulvovaginal candidosis and the vaginal microbiome. Sci Rep  2020; 10:7976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Mollazadeh-Narestan  Z, Yavarikia  P, Homayouni-Rad  A, et al.  Comparing the effect of probiotic and fluconazole on treatment and recurrence of vulvovaginal candidiasis: a triple-blinded randomized controlled trial. Probiotics Antimicrob Proteins  2023; 15:1436–46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Rizzo  A, Losacco  A, Carratelli  CR. Lactobacillus crispatus modulates epithelial cell defense against Candida albicans through Toll-like receptors 2 and 4, interleukin 8 and human beta-defensins 2 and 3. Immunol Lett  2013; 156:102–9. [DOI] [PubMed] [Google Scholar]
  • 18. Li  T, Liu  Z, Zhang  X, Chen  X, Wang  S. Local probiotic Lactobacillus crispatus and Lactobacillus delbrueckii exhibit strong antifungal effects against vulvovaginal candidiasis in a rat model. Front Microbiol  2019; 10:1033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. Santos  CMA, Pires  MCV, Leao  TL, et al.  Anti-inflammatory effect of two Lactobacillus strains during infection with Gardnerella vaginalis and Candida albicans in a HeLa cell culture model. Microbiology (Reading)  2018; 164:349–58. [DOI] [PubMed] [Google Scholar]
  • 20. Meijerink  M, Wells  JM. Probiotic modulation of dendritic cells and T cell responses in the intestine. Benef Microbes  2010; 1:317–26. [DOI] [PubMed] [Google Scholar]
  • 21. Qin  D, Ma  Y, Wang  Y, Hou  X, Yu  L. Contribution of lactobacilli on intestinal mucosal barrier and diseases: perspectives and challenges of Lactobacillus casei. Life (Basel)  2022; 12:1910. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22. Kohler  GA, Assefa  S, Reid  G. Probiotic interference of Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 with the opportunistic fungal pathogen Candida albicans. Infect Dis Obstet Gynecol  2012; 2012:636474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. De Gregorio  PR, Silva  JA, Marchesi  A, Nader-Macias  MEF. Anti-Candida activity of beneficial vaginal lactobacilli in in vitro assays and in a murine experimental model. FEMS Yeast Res  2019; 19:foz008. [DOI] [PubMed] [Google Scholar]
  • 24. Takano  T, Kudo  H, Eguchi  S, et al.  Inhibitory effects of vaginal lactobacilli on Candida albicans growth, hyphal formation, biofilm development, and epithelial cell adhesion. Front Cell Infect Microbiol  2023; 13:1113401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. Martinez  RC, Seney  SL, Summers  KL, Nomizo  A, De Martinis  EC, Reid  G. Effect of Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 on the ability of Candida albicans to infect cells and induce inflammation. Microbiol Immunol  2009; 53:487–95. [DOI] [PubMed] [Google Scholar]
  • 26. Cassone  A, Sobel  JD. Experimental models of vaginal candidiasis and their relevance to human candidiasis. Infect Immun  2016; 84:1255–61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. Gillum  AM, Tsay  EY, Kirsch  DR. Isolation of the Candida albicans gene for orotidine-5'-phosphate decarboxylase by complementation of S. cerevisiae ura3 and E. coli pyrF mutations. Mol Gen Genet  1984; 198:179–82. [DOI] [PubMed] [Google Scholar]
  • 28. Prieto  D, Roman  E, Correia  I, Pla  J. The HOG pathway is critical for the colonization of the mouse gastrointestinal tract by Candida albicans. PLoS One  2014; 9:e87128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29. Frazer  C, Hernday  AD, Bennett  RJ. Monitoring phenotypic switching in Candida albicans and the use of next-gen fluorescence reporters. Curr Protoc Microbiol  2019; 53:e76. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30. Walther  A, Wendland  J. An improved transformation protocol for the human fungal pathogen Candida albicans. Curr Genet  2003; 42:339–43. [DOI] [PubMed] [Google Scholar]
  • 31. Wurster  S, Kumaresan  PR, Albert  ND, Hauser  PJ, Lewis  RE, Kontoyiannis  DP. Live monitoring and analysis of fungal growth, viability, and mycelial morphology using the IncuCyte NeuroTrack processing module. mBio  2019; 10:e00673-19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Gresnigt  MS, Joosten  LA, Verschueren  I, et al.  Neutrophil-mediated inhibition of proinflammatory cytokine responses. J Immunol  2012; 189:4806–15. [DOI] [PubMed] [Google Scholar]
  • 33. Graf  K, Last  A, Gratz  R, et al.  Keeping Candida commensal: how lactobacilli antagonize pathogenicity of Candida albicans in an in vitro gut model. Dis Model Mech  2019; 12:dmm039719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34. Alonso-Roman  R, Last  A, Mirhakkak  MH, et al.  Lactobacillus rhamnosus colonisation antagonizes Candida albicans by forcing metabolic adaptations that compromise pathogenicity. Nat Commun  2022; 13:3192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Wells  JM. Immunomodulatory mechanisms of lactobacilli. Microb Cell Fact  2011; 10 (Suppl 1):S17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36. Ren  C, Cheng  L, Sun  Y. et al.  et al.  Lactic acid bacteria secrete toll like receptor 2 stimulating and macrophage immunomodulating bioactive factors. J Func Foods  2020; 66:103783. [Google Scholar]
  • 37. Ney  LM, Wipplinger  M, Grossmann  M, Engert  N, Wegner  VD, Mosig  AS. Short chain fatty acids: key regulators of the local and systemic immune response in inflammatory diseases and infections. Open Biol  2023; 13:230014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38. Manoharan  I, Prasad  PD, Thangaraju  M, Manicassamy  S. Lactate-dependent regulation of immune responses by dendritic cells and macrophages. Front Immunol  2021; 12:691134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39. Zelante  T, Iannitti  RG, Cunha  C, et al.  Tryptophan catabolites from microbiota engage aryl hydrocarbon receptor and balance mucosal reactivity via interleukin-22. Immunity  2013; 39:372–85. [DOI] [PubMed] [Google Scholar]
  • 40. Vissers  YM, Snel  J, Zuurendonk  PF, Smit  BA, Wichers  HJ, Savelkoul  HF. Differential effects of Lactobacillus acidophilus and Lactobacillus plantarum strains on cytokine induction in human peripheral blood mononuclear cells. FEMS Immunol Med Microbiol  2010; 59:60–70. [DOI] [PubMed] [Google Scholar]
  • 41. van Hemert  S, Meijerink  M, Molenaar  D, et al.  Identification of Lactobacillus plantarum genes modulating the cytokine response of human peripheral blood mononuclear cells. BMC Microbiol  2010; 10:293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42. Azad  MAK, Sarker  M, Wan  D. Immunomodulatory effects of probiotics on cytokine profiles. Biomed Res Int  2018; 2018:8063647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43. Mileti  E, Matteoli  G, Iliev  ID, Rescigno  M. Comparison of the immunomodulatory properties of three probiotic strains of lactobacilli using complex culture systems: prediction for in vivo efficacy. PLoS One  2009; 4:e7056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44. Foligne  B, Nutten  S, Grangette  C, et al.  Correlation between in vitro and in vivo immunomodulatory properties of lactic acid bacteria. World J Gastroenterol  2007; 13:236–43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45. Rosati  D, Bruno  M, Jaeger  M, et al.  An exaggerated monocyte-derived cytokine response to Candida hyphae in patients with recurrent vulvovaginal candidiasis. J Infect Dis  2022; 225:1796–806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46. Martin  P, Palmer  G, Rodriguez  E, et al.  Intracellular IL-1 receptor antagonist isoform 1 released from keratinocytes upon cell death acts as an inhibitor for the alarmin IL-1alpha. J Immunol  2020; 204:967–79. [DOI] [PubMed] [Google Scholar]
  • 47. Mailander-Sanchez  D, Braunsdorf  C, Grumaz  C, et al.  Antifungal defense of probiotic Lactobacillus rhamnosus GG is mediated by blocking adhesion and nutrient depletion. PLoS One  2017; 12:e0184438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48. Manosalva  C, Quiroga  J, Hidalgo  AI, et al.  Role of lactate in inflammatory processes: friend or foe. Front Immunol  2021; 12:808799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49. Rosati  D, Valentine  M, Bruno  M, et al.  Lactic acid in the vaginal milieu modulates the Candida-host interaction. Virulence  2025; 16:2451165. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

jiaf221_Supplementary_Data
jiaf221_supplementary_data.zip (2.2MB, zip)

Articles from The Journal of Infectious Diseases are provided here courtesy of Oxford University Press

RESOURCES