An Exaggerated Monocyte-Derived Cytokine Response to Candida Hyphae in Patients With Recurrent Vulvovaginal Candidiasis
Abstract
Background
Recurrent vulvovaginal candidiasis (RVVC) affects up to 8% of women. The immunopathogenesis is poorly understood but it has been suggested that RVVC might be due to dysregulated innate immune response. The aim of this study was to compare cytokine profiles in stimulated primary mononuclear cells (PBMCs) from RVVC and healthy individuals.
Methods
PBMCs isolated from RVVC patients (n = 24) and healthy volunteers (n = 30) were stimulated with unspecific and pathogen-specific antigens. Cytokine production was assessed after 24 hours, 48 hours, and 7 days using ELISA.
Results
No significant differences in cytokine production were found in T helper 1 (Th1), Th2, and Th17 immunity in response to both unspecific and pathogen-specific stimulations. Tumor necrosis factor-α (TNF-α) production in response to C. albicans hyphae was significantly higher in patients than controls and within the patient group, a significant positive correlation was found between interleukin-1β (IL-1β) and both TNF-α and IL-6. Both IL-1β/IL-1Ra and TNF-α/IL-10 ratios in Candida hyphae-stimulated PBMCs were significantly higher in patients than controls.
Conclusions
Women affected by RVVC showed increased monocytes-derived cytokine production, which might contribute to an exaggerated vaginal immune response to Candida hyphae. RVVC patients show no defective Th-dependent adaptive immune response upon Candida stimulation.
Keywords: recurrent vulvovaginal candidiasis, RVVC, Candida albicans, immune response, innate immunity, cytokines
———
Peripheral blood mononuclear cell stimulations show an increased monocytes-derived cytokine production, but a normal T helper cell function, in women with recurrent vulvovaginal candidiasis (RVVC). This suggest autoinflammation rather than immunodeficiency to be central in the pathogenesis of RVVC.
———
Vulvovaginal candidiasis (VVC) is a debilitating condition, affecting 75% of women of childbearing age globally every year [
]. Although Candida infections at the vaginal mucosa are not life-threatening, they strongly affect quality of life [
]. Women experiencing more than 3 episodes annually are classified as having recurrent VVC (RVVC) [
]. Because no vaccines are currently available, antifungal formulations are the only therapeutic options, although short courses do not prevent recurrences [
]. Host-related risk factors, such as diabetes mellitus, pregnancy, oral contraceptives usage, and immunosuppressive and antibiotic therapy, are associated with RVVC [
,
]. However, the aforementioned predisposing factors are not present in the majority of the patients and, so far, no sole causative agent has been found to be responsible for (R)VVC onset. To date, the immunopathogenesis of (R)VVC is poorly understood. As a higher incidence of mucosal Candida infections has been found in patients with reduced cell-mediated immunity (CMI) [
], a dysregulated adaptive immunity in response to Candida has been hypothesized to be involved in RVVC susceptibility. However, studies from both human and mouse models failed to identify a role for either systemic or local CMI deficiency (reviewed in [
]). An exacerbated innate immune response, particularly due to IL-1β hyperproduction and polymorphonuclear neutrophils (PMNs) dysfunction, has been proposed to be responsible for RVVC immunopathogenesis [
,
].
The purpose of the current study was to investigate the immune response in RVVC patients and healthy individuals. Thus, we assessed both adaptive and innate cytokine production capacity of peripheral blood mononuclear cells (PBMCs) challenged with both unspecific and pathogen-specific antigens.
METHODS
Patient Selection and Healthy Volunteers’ Recruitment
Patients and healthy volunteers were recruited at Radboud University Medical Center (Nijmegen, the Netherlands). We included 24 consecutive women with RVVC. RVVC was defined as an episode with typical symptoms and vulvovaginal signs, with at least 3 reported episodes in the previous or following year and at least 1 episode in which microbiological culture was confirmed. Immunocompromised women were excluded. Inclusion of healthy controls was approved by the local institutional review board (region Arnhem-Nijmegen, number 2299 2010/104). According to the Dutch law, the patient study was exempt from approval by an ethics committee because the blood samples were part of regular care.
Isolation and Stimulation of Peripheral Blood Mononuclear Cells
For the purpose of the study, we isolated PBMCs, which are a heterogeneous mixed population consisting of lymphoid cells (T cells, B cells, and natural killer [NK] cells) and myeloid (monocytes and dendritic cells) cells. PBMCs are a useful and easily accessible material to investigate both the early cytokine production by monocytes and the later adaptive response by T-helper cells (Th-1, Th-2, and Th-17) against intruding pathogens, such as Candida albicans. PBMCs were isolated from EDTA blood by Ficoll-Paque density gradient centrifugation as described previously [
]. The PBMCs of at least 1 control was included in the same experiment. PBMCs were stimulated with RPMI (negative control), Escherichia coli lipopolysaccharide (LPS; 10 ng/mL), β-glucan (10 µg/mL), LPS plus muramyl dipeptide (5 µg/mL), phytoemoagglutinin (10 µg/mL), C. albicans UC820 (American Type Culture Collection [ATCC] MYA-3573) yeast heat-killed (HK) (1×105 cells/mL), C. albicans UC820 (ATCC MYA-3573) hyphae HK (1×106 cells/mL), and C. albicans UC820 (ATCC MYA-3573) yeast live (1×105 cells/mL) at 37°C for 24 hours, 48 hours, and 7 days (in the presence of 10% serum with the culture medium). Cell culture supernatants were collected and stored at −20°C until used for enzyme-linked immunosorbent assay (ELISA).
In particular, to investigate the Th17 immune response, interleukin (IL)-17 and IL-22 were measured after 7 days’ stimulation. To elicit an effective protection against Candida infection, a balance in Th1 and Th2 response is crucial [
]. To test whether the Th1/Th2 function was impaired in RVVC patients, PBMCs were incubated for 48 hours with both unspecific and pathogen-specific stimuli, followed by measurements of interferon-γ (IFN-γ) and IL-10 levels. The innate immune response was investigated by the capacity of C. albicans yeast and hyphae to induce tumor necrosis factor-α (TNF-α), IL-6, IL-1β, and IL-1Ra in PBMCs. Cytokine concentrations were determined using commercial ELISA kits for TNF-α, IL-6, IL-1β, IL-1Ra, IL-17, IL-22 (R&D systems), IL-10, and IFN-γ (Sanquin Research) following the manufacturer’s instructions.
Statistical Analysis
Data are expressed as mean ± standard error of the mean (SEM). The Mann-Whitney U test was used for the comparison of cytokine concentrations between the 2 independent groups. The healthy control group included both female and male subjects. No differences in cytokine production were found between men and women (
). In addition, we did not find a relevant influence of control volunteers’ age on cytokine production in line with previous findings [
] (
). For the assessment of correlations Spearman rank correlation coefficient was calculated. A P value of < .05 was considered statistically significant. Generation of graphs and statistical analyses were performed using GraphPad Prism 5.
RESULTS
Patient Characteristics
Twenty-four RVVC patients and 30 healthy volunteers were included in the study and their clinical characteristics are presented in
. Briefly, 19 patients (79.1%) were using systemic hormonal contraception and 2 (8.3%) were taking other medication at the time of diagnosis. Fourteen patients were diagnosed with atopy (58.8%) and 7 patients (29.1%) reported other previous vaginal infections.
Table 1.
Clinical Characteristics of RVVC Patients and Healthy Volunteers Included in the Study
RVVC (n = 24)Control (n = 30)CharacteristicValue95% CIValue95% CISex (M/F)0/2413/17Age, y, mean (± SD)30.00 (± 8.827)26.5–33.542.41 (±17.36)36.1–48.7Atopy, No. (%)14 (58.8)NASystemic hormonal
contraception, No. (%)19 (79.1)NAOther medication usage, No. (%)2 (8.3)NAPrevious vaginal infections, No. (%)7 (29.1)NACandida species, No. (%)C. albicans19 (79.1)NAUnspecified2 (8.3)NAC. dubliniensis1 (4.1)NAC. glabrata, C. lusitaniae1 (4.1)NAC. famata, C. parapsilosis1 (4.1)NABlood count Leukocytes (×10 6/mL)7.126 (± 1.44)6.50–7.75… Neutrophils (×106/mL)4.314 (± 1.21)3.78–4.85… Lymphocytes (×103/mL)2.097 (± 0.43)1.91–2.283… Monocytes (×106/mL)0.5123 (± 0.10).47–.56… Eosinophils (×106/mL)0.1427 (± 0.11).093–.19… Basophils (×106/mL)0.035 (± 0.02).03–.04…Mannose binding lectin, mg/L, reference 1.0–5.52.0641.325–2.803…Abbreviations: CI, confidence interval; RVVC, recurrent vulvovaginal candidiasis.
Normal Dectin-1 Signaling
Because the β-glucan receptor dectin-1, together with Toll-like receptor-2 (TLR-2) and TLR-4, plays a central role in Candida recognition and cytokine production [
], we checked whether dectin-1 signaling was defective. To do this, we compared IL-6 production of PBMCs after exposure to LPS (TLR4 ligand), β-glucan (dectin-1 ligand), and the combination of both. As illustrated in
, overall, we did not find any significant alteration in dectin-1 signaling in the RVVC group; however, PBMCs from 3 of the 24 RVVC patients showed no synergism between β-glucan and LPS stimulation (as was present in all controls), which may indicate a partial defect in dectin-1 pathway.
Th1, Th2, and Th17 Function in RVVC
Neither unspecific nor pathogen-specific stimulations showed significant differences in IL-17 (
) and IL-22 (
) production levels, between PBMCs from patients and controls, except for a significant increase in IL-22 production in patients upon LPS plus β-glucan stimulation (
; P = .0109). Focusing on the patient group, we next investigated the interrelationship between IL-17 and IL-22 levels in RVVC patients and found a significant positive correlation between IL-17 and IL-22 levels in both yeast-stimulated (
; r = 0.6337, P = .0004) and hyphae-stimulated (
; r = 0.7161, P < .0001) PBMCs.
Figure 1.
Th-17 response in PBMCs from RVVC patients and controls.
PBMCs from healthy controls and RVVC patients were stimulated with RPMI, Escherichia coli LPS (10 ng/mL), β-glucan (10 µg/mL), LPS plus β-glucan, LPS plus MDP (5µg/mL), PHA (10 µg/mL), Candida albicans UC820 yeast HK (1 × 105 cells/mL), or C. albicans UC820 hyphae HK (1 × 106 cells/mL) for 7 days in the presence of 10% serum at 37 °C. IL-17 (A) and IL-22 (B) levels in culture supernatants were measured by ELISA. Results are means ± SEM. Significance was determined with Mann-Whitney U test. * P value < .05 was considered statistically significant. Correlation within patients of IL-17 and IL-22 levels upon C. albicans yeast (UC820, HK) (C) and hyphae (UC820, HK) (D) stimulation of PBMCs. Correlation were analyzed using Spearman rank correlation coefficient test. Abbreviations: ELISA, enzyme-linked immunosorbent assay; HK, heat killed; IL, interleukin; LPS, lipopolysaccharide; MDP, muramyl dipeptide; PBMC, peripheral blood mononuclear cell; PHA, phytoemoagglutinin; RVVC, recurrent vulvovaginal candidiasis; SEM, standard error of the mean.
Overall, PBMCs from RVVC patients displayed a similar or lower trend for IL-10 production when compared to controls (
). Similarly, no significant differences in IFN-γ release (
) were observed between the 2 groups. Taken together, these findings suggest a normal Th1/Th2 function in RVVC patients.
Figure 2.
Th-1 and Th-2 response in PBMCs from RVVC patients and controls. PBMCs from healthy controls and RVVC patients were stimulated with RPMI, Escherichia coli LPS (10 ng/mL), β-glucan (10 µg/mL), LPS plus β-glucan, LPS plus MDP (5 µg/mL), PHA (10 µg/mL), Candida albicans UC820 yeast HK (1 × 105 cells/mL), C. albicans UC820 hyphae HK (1 × 106 cells/mL), or C. albicans yeast UC820 live (1 × 105 cells/mL) for 48 hours at 37°C. IL-10 (A) and IFN-γ (B) levels were measured by ELISA in culture supernatants. Results are means ± SEM. Significance was determined with Mann-Whitney U test. Abbreviations: ELISA, enzyme-linked immunosorbent assay; HK, heat killed; IL, interleukin; IFN-γ, interferon-γ; LPS, lipopolysaccharide; MDP, muramyl dipeptide; PBMC, peripheral blood mononuclear cell; PHA, phytoemoagglutinin; RVVC, recurrent vulvovaginal candidiasis; SEM, standard error of the mean; Th, T helper cell.
Innate Immunity: Exaggerated Cytokine Response to Candida Hyphae
TNF-α levels in PBMCs from RVVC patients in response to Candida hyphae were statistically significantly higher than controls (P = .0013). IL-6 and IL-1β production by PBMC of RVVC patients did not differ from that in controls, whereas IL-1Ra concentrations tended to be lower in patients than in controls (
). Conversely, monocytes-derived cytokine production in response to C. albicans yeast stimulation was similar in patients compared to controls (
). C. albicans hyphae-induced IL-1β levels in PBMCs from RVVC patients were positively correlated with both TNF-α (r = 0.6045, P = .0009) and IL-6 (r = 0.5414, P = .0031), as shown in
. Similar results were found in yeast-stimulated PBMCs from RVVC patients (
; TNF-α, r = 0.7104, P < .0001; IL-6, r = 0.5671, P = .0019).
Figure 3.
Innate response to Candida albicans hyphae. Cytokine induction in PBMCs after 24 hours stimulation with C. albicans hyphae HK (UC820, 1 × 106 cells/mL). A, TNF-α, IL-6, IL-1β, and IL-1Ra levels were quantified in culture supernatants by ELISA. Results are means ± SEM. Significance was determined with Mann-Whitney U test. B, Correlation of cytokines in hyphae-stimulated PBMCs within RVVC patients. Correlations were analyzed using Spearman rank correlation coefficient test. * P value < .05 was considered statistically significant. Abbreviations: ELISA, enzyme-linked immunosorbent assay; HK, heat killed; IL, interleukin; IL-1Ra, interleukin-1 receptor a; PBMC, peripheral blood mononuclear cell; RVVC, recurrent vulvovaginal candidiasis; SEM, standard error of the mean; TNF-α, tumor necrosis factor-α.
Unbalanced Proinflammatory Response
After Candida yeast stimulation, we observed a positive correlation between IL-1β and IL-1Ra in PBMCs from patients (
; r = 0.5322, P = .0037), indicating that the IL-1β production was adequately counterbalanced by IL-1Ra levels; accordingly, the IL-1β/IL-1Ra ratio, reflecting IL-1 bioactivity, was not significantly different between controls and patients (
, right). On the contrary, IL-1β and IL-1Ra levels in hyphae-stimulated PBMCs from RVVC patients were not significantly correlated (
left) and the IL-1β/IL-1Ra ratio was significantly higher in patients compared to controls (
right; P = .0169). Moreover, the TNF-α/IL-10 ratio between controls and patients resulted in significantly higher TNF-α production in PBMCs from RVVC patients only upon stimulation with Candida hyphae (
right; P = .0015).
Figure 4.
Increased IL-1β bioactivity. A, Left, correlation between IL-1β and IL-1Ra in PBMCs from RVVC patients stimulated with Candida albicans yeast HK (UC820, 1 × 105 cells/mL). Right, difference in ratio of IL-1β and IL-1Ra levels between controls and patients. B, Left, correlation between IL-1β and IL-1Ra in PBMCs from RVVC patients stimulated with C. albicans hyphae HK (UC820, 1 × 106 cells/mL). Right, difference in ratio of IL-1β and IL-1Ra levels between controls and patients. C, Difference in ratio of IL-1β and IL-1Ra levels between controls and patients in PBMCs exposed to C. albicans yeast (left) and hyphae (right). Correlations were analyzed using Spearman rank correlation coefficient test. * P value < .05 was considered statistically significant. Abbreviations: HK, heat killed; IL-1Ra, interleukin-1 receptor a; IL-1β, interleukin-1β; NS, nonsignificant; PBMC, peripheral blood mononuclear cell; RVVC, recurrent vulvovaginal candidiasis.
To investigate a possible a role for atopy in RVVC pathogenesis, as previously suggested [
], we stratified the RVVC group into 2 clinical subgroups based on atopic constitution and compared cytokine production capacity between the 2 subgroups and the healthy controls. We observed a trend towards higher production of proinflammatory cytokines upon challenge with Candida yeast and hyphae, with significantly higher TNF-α levels in Candida hyphae-stimulated PBMCs (
; P = .0066). Furthermore, IL-1β/IL-1Ra and TNF-α/IL-10 ratios were higher in atopic individuals when compared with the control and nonatopic groups upon Candida hyphae stimulation (
; P = .0014 and P = .0345, respectively).
Figure 5.
The influence of atopy on immune response. RVVC patients were divided into 2 clinical subgroups based on atopic constitution. A, Cytokine production capacity (TNF-α, IL-1β, and IL-6) in the 2 subgroups and healthy controls in PBMCs culture supernatants after 24 hours stimulation with Candida albicans yeast (above) and hyphae (below). Results are mean ± SEM. Significance was determined with Mann-Whitney U test. B, Above, difference in IL-1β/IL-1Ra ratio between controls and patients in PBMCs exposed to C. albicans yeast (left) or hyphae (right). Below, difference in TNF-α/IL-10 ratio between controls and patients in PBMCs exposed to C. albicans yeast (left) or hyphae (right). Correlations were analyzed using Spearman rank correlation coefficient test. * P value < .05 was considered statistically significant. Abbreviations: IL, interleukin; IL-1Ra, interleukin-1 receptor a; PBMC, peripheral blood mononuclear cell; RVVC, recurrent vulvovaginal candidiasis; SEM, standard error of the mean; TNF-α, tumor necrosis factor-α.
DISCUSSION
Our cohort of women with idiopathic RVVC was characterized by an inappropriately high proinflammatory innate cytokine response to Candida hyphae. Firstly, PBMCs from women with RVVC displayed an increased TNF-α production. Secondly, PBMCs from women with RVVC also showed a substantial increase of IL-1β bioactivity compared to controls, upon stimulation with Candida hyphae, especially due to relatively low IL-1Ra concentrations. In contrast, we found no evidence of defective or altered Th1, Th2, or Th17 function. Cumulatively, these findings support the hypothesis that RVVC is likely the result of a vaginal immune exuberant response to Candida infection, similar to localized autoinflammatory diseases such as gout or hidradenitis suppurativa.
Our main findings are in line with mouse and clinical studies suggesting RVVC is the result of an acute local innate immune response driven by a positive feedback loop mechanism characterized by an exaggerated IL-1β production via NLRP3 inflammasome and failure of fungal clearance by infiltrated, but ineffective, PMNs [
]. Current pharmacologic treatment of RVVC consists solely of antifungal prophylaxis. Long-term weekly fluconazole effectively reduces recurrences, although approximately 10% of women have breakthrough infections [
]. More importantly, half of women relapse after cessation of fluconazole prophylaxis. Our findings offer a scientific base for a completely new therapeutic approach for RVVC, targeting of the underlying autoinflammatory state. IL-1β is a pivotal cytokine in many autoinflammatory diseases and blocking its effect, for example by the IL-1 receptor antagonist anakinra, has been reported to be very effective [
]. Hence, similar approaches should be tested in RVVC.
In addition to dysregulated IL-1 activity, we investigated whether RVVC individuals display a defective T helper function. Indeed, defective Th17 has been reported to be one of the main causes of other mucosal Candida infections (eg, oral and esophageal). However, we did not observe a defective Th1/Th2/Th17 response in RVVC patients, strengthening the hypothesis that deficiencies in adaptive immunity are not involved in RVVC pathophysiology. In line with our results, prior studies revealed that RVVC does not occur in patients with genetic defects in adaptive immunity leading to mucosal fungal infections: for example, deficiencies of STAT3, a signal mediator activated also in response to type I [
] and type II [
] interferon, or mutations in IL-12/IL-12R signaling pathway, lead to more frequent oral mucosal candidiasis or different intracellular pathogen infections (ie, mycobacterial infections [
]) rather than to RVVC [
]. In contrast to Carvalho et al [
], we found only a slight decrease of IFN-γ in RVVC patients in response to C. albicans antigens and normal IL-10 concentrations between the 2 groups, suggestive for very little dysregulation of Th1/Th2-related immunity. In addition, we did not find diminished IL-17 production, similarly to previous findings reporting a normal systemic CMI [
,
]. Interestingly, the decrease in absolute CD4+ T-lymphocyte count characteristic of HIV patients, inborn errors in the IL-17 pathway (eg, IL-17RA, IL-17F), or mutations in STAT1, STAT3, and AIRE [
,
], resulting in chronic mucocutaneous candidiasis, hyper IgE syndrome, and autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, are commonly associated with fungal infections such as cutaneous and oropharyngeal candidiasis [
] but not RVVC. The role of IL-22 in VVC is still not clear: while in vitro studies reported that the absence of IL-22 dampens IL-1Ra production, resulting in unrestrained inflammasome function and symptomatic VVC [
], we found normal IL-22 production capacity in RVVC patients. Moreover, individuals bearing a polymorphism in the novel candidate susceptibility factor SIGLEC15 have been reported recently to have high IL-22 concentrations as well as IL-17A and IFN-γ [
]. Taken together, these observations suggest that defects in IL-22 signaling pathway are not the primary cause of RVVC, at least not in most patients.
C. albicans is a dimorphic fungus and its switch from yeast to hyphae morphology mirrors the shift from a commensal to an opportunistic relationship with the host. The ability to form hyphae is required for the exuberant inflammatory response [
,
], as supported by the induction of less immunopathology by Candida strains defective in the yeast to hyphae transition, like C. glabrata [
]. The lower rate of infection of C. glabrata and other non-albicans species, excluding C. tropicalis, as opposed to C. albicans (present in around 90% of cases) at the vaginal mucosa, is likely attributable to their inability to form hyphae. Nevertheless, C. glabrata is the second leading cause of VVC as well as severe candidiasis, meaning there are other morphology-independent virulence factors, such as candidalysin expression, biofilm formation, and phospholipase secretion, responsible vaginitis immunopathology [
].
Lastly, we did not find any significative alterations in dectin-1 function overall in our cohort. However, 3 of 24 patients did not display a synergistic effect when PBMCs were costimulation occurred with β-glucan and LPS: we cannot exclude therefore a partial dectin-1 defect in a minority of RVVC patients. It is important to note that, in particular, a complete loss-of-function mutation of dectin-1 is associated with increased susceptibility to mucocutaneous Candida infection [
,
]. Homozygous individuals dectin-1 Tyr238X deficient are rare in the western population [
], explaining why dectin-1 deficiency is a risk factor at the individual level but it is not responsible for RVVC susceptibility in the overall RVVC population [
]. Presumably, due to the small number of patients in our cohort and the rarity of the loss-of-function phenotype in the population, we did not include any homozygous individuals. This suggests that other individuals with the clinical phenotype are most likely characterized by an autoinflammatory condition rather than a genetic deficiency.
Interestingly, in our cohort more than half of the patients recruited for this study were diagnosed with atopic disease and increased IL-1β and TNF-α bioactivity were measured in comparison to healthy individuals. These results further strengthen the hypothesis that RVVC is a hypersensitivity response and that atopy might play a role in the pathogenesis of RVVC rather than being a confounder, as also suggested in other studies [
,
]. The biological mechanisms that link atopy and RVVC, however, still need to be elucidated.
The main limitation of this study is that we measured cytokine production in supernatant from PBMCs and not in vaginal fluids. Further studies are warranted to investigate the crucial role of the microbiome, metabolome, and inflammatory mediators (ie, PMNs) in vaginal anti-Candida immunity.
In conclusion, we provide evidence that women with RVVC are characterized by an inappropriately strong cytokine production profile that is reminiscent of an autoinflammatory disease, rather than a defect in T-cell mediated immunity. A hallmark is the increased bioactivity of IL-1β in response to invasive C. albicans infection, which is inadequately counterbalanced by IL-1Ra production, thus, resulting in recurrent Candida vaginitis. This potentially offers the opportunity for new treatment options aimed at reducing the excessive inflammation.
Supplementary Data
Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.
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Notes
Acknowledgment. The authors thank Trees Jansen for the help with the cytokine assessment.
Financial support. This work was supported by the European Union Horizon 2020 Research and Innovation Programme under the Marie Skłodowska‐Curie grant agreement (grant number 812969). M. G. N. was supported by a Spinoza Grant from the Netherlands Organization for Scientific Research and a European Research Council Advanced Grant (grant number 833247).
Potential conflicts of interest. All authors: No reported conflicts of interest. 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.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
jiaa444_suppl_Supplementary_Figure_S1A_Part1
jiaa444_suppl_Supplementary_Figure_S1A_Part2
jiaa444_suppl_Supplementary_Figure_S1B_Part1
jiaa444_suppl_Supplementary_Figure_S1B_Part2
jiaa444_suppl_Supplementary_Figure_S2
jiaa444_suppl_Supplementary_Figure_S3
jiaa444_suppl_Supplementary_Figure_S4