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. 2022 Mar 17;90(3):e00558-21. doi: 10.1128/iai.00558-21

Borrelia burgdorferi Is a Poor Inducer of Gamma Interferon: Amplification Induced by Interleukin-12

Frederik R van de Schoor a,#, Hedwig D Vrijmoeth a,#, Michelle A E Brouwer a, Hadewych J M ter Hofstede a, Heidi L M Lemmers a, Helga Dijkstra a, Collins K Boahen a, Marije Oosting a, Bart-Jan Kullberg a, Joppe W Hovius b, Cees C van den Wijngaard c, Frank L van de Veerdonk a, Mihai G Netea a,d, Leo A B Joosten a,✉
Editor: De'Broski R Herberte
PMCID: PMC8929378  PMID: 35130450

ABSTRACT

Laboratory diagnosis of Lyme borreliosis (LB) is mainly based on serology, which has limitations, particularly in the early stages of the disease. In recent years there have been conflicting reports concerning a new diagnostic tool using the cytokine interferon-gamma (IFN-γ). Previous studies have generally found low concentrations of IFN-γ in early LB infection. The goal of this study is to investigate IFN-γ regulation during early LB and provide insights into the host response to B. burgdorferi. We performed in vitro experiments with whole blood assays and peripheral blood mononuclear cells (PBMCs) of LB patients and healthy volunteers exposed to B. burgdorferi and evaluated the IFN-γ response using ELISA and related interindividual variation in IFN-γ production to the presence of single nucleotide polymorphisms. IFN-γ production of B. burgdorferi-exposed PBMCs and whole blood was amplified by the addition of interleukin-12 (IL-12) to the stimulation system. This effect was observed after 24 h of B. burgdorferi stimulation in both healthy individuals and LB patients. The effect was highly variable between individuals, but was significantly higher in LB patients 6 weeks since the start of antibiotic treatment compared to healthy individuals. IL-12 p40 and IL-18 mRNA were upregulated upon exposure to B. burgdorferi, whereas IL-12 p35 and IFN-γ mRNA expression remained relatively unchanged. SNP Rs280520 in the downstream IL-12 pathway, Tyrosine Kinase 2, was associated with increased IFN-γ production. This study shows that IL-12 evokes an IFN-γ response in B. burgdorferi exposed cells, and that LB patients and healthy controls respond differently to this stimulation.

KEYWORDS: Lyme disease, borreliosis, erythema migrans, Borrelia, antibody responses, interferon-gamma

INTRODUCTION

Lyme borreliosis (LB) is caused by the tick-borne spirochete Borrelia burgdorferi sensu lato (B. burgdorferi s.l.) and has a broad clinical spectrum, ranging from early localized disease to severe disseminated manifestations. The most common early manifestation is erythema migrans (EM), and more rarely, B. burgdorferi s.l. infection can result in disseminated disease, including Lyme arthritis and Lyme neuroborreliosis (1).

Until today, the readily available and generally used method for diagnostic confirmation of LB is serology (2). The sensitivity of serology increases in weeks to months after infection, but is limited early in the disease (3). Also, serology cannot be used to monitor disease activity, as antibodies can remain detectable for years, even after antibiotic treatment and resolution of infection (2, 4). Furthermore, cross-reactivity and differences between commercial test kits may complicate clinical interpretation of serological results (5–7). Specifically, diagnostic parameters such as sensitivity and specificity can vary between tests. This can be due to the difference in antigens that are used. These include sonicated whole-cell, whole-cell combined with recombinant, or exclusively recombinant antigens.

Although the vast majority of LB patients recover completely after antibiotic treatment, signs and symptoms may persist and cause a significant decrease in quality of life (8–10). Persistent symptoms include fatigue, arthralgia, myalgia, and neurocognitive problems. After confirmed LB, these clinical manifestations are often referred to as post-treatment Lyme disease syndrome (PTLDS) (11). Similar symptoms are prevalent in up to 20% of the general population (10, 12–14), and proper identification of patients with LB is necessary to be able to initiate appropriate treatment. Therefore, additional understanding of immune responses in patients with active LB compared to those with past infection is warranted.

B. burgdorferi infection triggers both innate and adaptive immune responses, including a T helper 1 (Th1) response leading to IFN-γ release (15–19). Previous studies have demonstrated IFN-γ in the skin, cerebral spinal fluid, and synovial fluid of LB patients (19–28). Interestingly, in a cohort of 500 healthy subjects the IFN-γ production upon B. burgdorferi s.l. stimulation of human peripheral blood mononuclear cells (PBMCs) was limited (29). In contrast to IL-1β being rapidly produced, IFN-γ production was only detectable after 96 h of B. burgdorferi sensu stricto (s.s.) exposure of PBMCs in healthy volunteers (30). Therefore, in the present study, we aim to further unravel the mechanisms of IFN-γ production by human PBMCs upon B. burgdorferi s.l. exposure.

RESULTS

B. burgdorferi s.l. is a poor IFN-γ inducer.

Previously, we have shown that IFN-γ production by PBMCs obtained from healthy subjects and stimulated with B. burgdorferi for 24 h to 48 h was limited or even absent (29, 30). In the present study, IFN-γ production by B. burgdorferi-stimulated PBMCs was evaluated in a cohort of healthy individuals. After 24 or 48 h of incubation with live attenuated and viable B. burgdorferi s.s., minimal production of IFN-γ was detectable (Fig. 1A and B), whereas PBMCs exposed to heat-killed C. albicans (HKCA), a known inducer of memory T-cell responses, induced high production of IFN-γ. Seven days after initial B. burgdorferi s.s. exposure, IFN-γ production in healthy individuals was observed (Fig. 1C and 1F). Viable B. burgdorferi s.s. induces low amounts of IFN-γ by itself following 24 and 48 h of PBMC stimulation; however, HKCA is the more potent IFN-γ inducer (Fig. 1A and B). As a validation, these experiments were repeated in EM patients, using PBMCs thawed from liquid nitrogen (Fig. 1G), and compared to thawed cells from healthy individuals (Fig. 1D and E). PBMCs from EM patients also showed minimal IFN-γ production after B. burgdorferi exposure (Fig. 1G). In contrast to IFN-γ, innate proinflammatory cytokines were effectively produced by PBMCs of these EM patients upon stimulation with B. burgdorferi s.l. for 24 h (Fig. 1H). Accordingly, there was considerable IL-1β, IL-6, and IL-1Ra production following B. burgdorferi stimulation in the forester’s cohort, consisting of healthy subjects with high tick bite exposure. The production of pro-inflammatory cytokines, such as IL-1β and IL-6, was however modest compared to other pathogens used as stimuli (Figure. S1 in the supplemental material). Lastly, IFN-γ production was comparable in seropositive healthy individuals, defined by the presence of either B. burgdorferi IgM or IgG antibodies based on standard two-tiered testing, and seronegative individuals (Fig. 1I).

FIG 1.

FIG 1

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B. burgdorferi is a poor IFN-γ inducer. (A and B) IFN-γ production of B. burgdorferi-stimulated PBMCs of healthy controls (HCs) is minimal compared to stimulation with heat-killed Candida albicans (HKCA). PBMCs produce more IFN-γ when exposed to viable B. burgdorferi than to frozen/live-attenuated B. burgdorferi s.s. (C) Stimulation for 7 days with viable B. burgdorferi results in comparable IFN-γ levels as with HKCA stimulation. (D and E) PBMCs, thawed from liquid nitrogen, of a different cohort of healthy individuals were used as a validation experiment. (F) PBMCs of HCs produce more IFN-γ after B. burgdorferi s.s. exposure for 7 days in comparison to 24 or 48 h of stimulation; however, there are still many nonresponders. (G) IFN-γ production by PBMCs from 11 erythema migrans (EM) patients following B. burgdorferi s.l. stimulation is limited compared to HKCA stimulation. (H) Robust production of IL-1β, IL-6, IL-10, and IL-1Ra in contrast to IFN-γ by PBMCs of EM patients after 24 h stimulation. (I) The production of IFN-γ was not related to B. burgdorferi antibody status in healthy individuals. PBMCs, peripheral blood mononuclear cells; IFN-γ, interferon gamma; HCs, healthy controls; ACA, acrodermatitis chronic atrophicans; Bb ss, B. burgdorferi sensu stricto; Bb sl, B. burgdorferi s.l.; HKCA, heat-killed Candida albicans; EM, erythema migrans; LB, Lyme borreliosis; ns, not statistically significant; *, P < 0.05; **, P < 0.01; ***, P < 0.0001 tested with Wilcoxon signed-rank test for panels A–E and Mann-Whitney U test for panel I.

IL-12 p35 and IFN-γ gene expression is not upregulated upon B. burgdorferi infection.

Next, expression of IFN-γ and related cytokines upon B. burgdorferi recognition was evaluated at the transcriptional level. It is well-known that IL-12 and IL-18 synergize IFN-γ production (31, 32). Bioactive IL-12 p70, which can induce IFN-γ production, is a heterodimer formed by subunits p35 (IL-12α) and p40 (IL-12β) (33, 34). In a previously published study (35), blood samples of 29 LB patients, including 17 individuals with a single EM lesion and 12 with multiple EM, were collected before the start of treatment, and 3 weeks and 6 months later. Furthermore, 13 healthy controls, matched by age, sex, ethnicity, and comorbidity, were included. Transcriptome data of unstimulated PBMCs showed an upregulation of IL-12 p40 mRNA in patients at 3 weeks (Fig. 2B). However, no change in expression of the IL-12 p35 gene was observed (Fig. 2A). The IL-12 receptor subunits, IL-12Rβ1 and IL-12Rβ2, were not differentially expressed at any time point (Fig. 2C and D), suggesting there is no increased IL-12 signaling. Expression of IL-18 mRNA was significantly increased in EM patients compared to healthy controls (Fig. 2E) at baseline and after 3 weeks. IFN-γ expression was upregulated in some EM patients (Fig. 2E). We could validate these findings in transcriptome analysis of B. burgdorferi stimulated PBMCs isolated from 36 healthy individuals from another previously published data set (36) (Fig. 3). Similar to transcriptome data of EM patients, we observed upregulation of IL-12 p40 and IL-18 following 24 h of stimulation, but no change in IL-12 p35 and IFN-γ expression. This may indicate that, although IL-12 p40 and IL-18 mRNA are upregulated after B. burgdorferi infection, IFN-γ expression remains unchanged for most individuals.

FIG 2.

FIG 2

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Transcriptome PBMCs of LB patients and healthy volunteers. Transcriptome of unstimulated PBMCs of 29 physician-confirmed LB patients, including 17 with a single EM and 12 with multiple EM, compared to 13 matched healthy controls (HCs). The expression of cytokines is displayed as fragments per kilobase million (FPKM). (A) There was no difference in IL-12 p35 expression between HCs and LB patients. (B and E) IL-12 p40 and IL-18 are upregulated in PBMCs of LB patients at baseline and after 3 weeks compared to HCs. (C and D) IL-12Rβ1 and 2 expression was not different between the groups. (F) IFN-γ mRNA was upregulated in several LB patients at the time of diagnosis; however, most individuals showed no upregulation. (G) Expression of IL-7 was upregulated at the 3-week time point compared to HCs. (H) TYK2 mRNA was elevated in LB infection after 6 months compared to 3 weeks and baseline. (I) In the WBA condition, individuals with the Rs280520 SNP had higher IFN-γ production upon stimulation with B. burgdorferi in combination with IL-12. This SNP was present in 21 of 65 subjects investigated. FPKM, fragments per kilobase of exon model per million reads mapped; HCs, healthy controls; LB t0, Lyme borreliosis patients on baseline; LB t3wk, Lyme borreliosis patients 3 weeks after inclusion; LB t6m, Lyme borreliosis patients 6 months after inclusion; IL-12RB1 and 2, Interleukin 12 Receptor Subunit Beta 1 and 2; PBMC, peripheral blood mononuclear cell; TYK2, tyrosine kinase 2; SNP, single nucleotide polymorphism; WBA, whole blood assay; ns, not statistically significant; *, P < 0.05; **, P < 0.01; ***, P < 0.0001 calculated using independent-samples Mann-Whitney U test for comparing HC and LB patient cohort and Wilcoxon signed-ranked test for paired testing when comparing LB patient different time points in all panels.

FIG 3.

FIG 3

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Transcriptional response PBMCs of healthy volunteers to B. burgdorferi. Gene expression of relevant genes by microarray of 36 healthy individuals stimulated for 4 (A) and 24 h (B) with RPMI (medium control), heat-killed B. burgdorferi s.s. (Bb), Mycobacterium tuberculosis (MTB), and heat-killed Candida albicans (HKCA). The data are log2 transformed and in case of multiple transcripts per gene the mean is displayed, and scaling was performed per gene. IL-12 p35 expression after 4 and 24 h of B. burgdorferi stimulation is lacking compared to HKCA. IL-12 p40 is increased after 24 h of B. burgdorferi stimulation. These IL-12 p35 and p40 responses are in agreement with Fig. 2A and B. IFN-γ is not upregulated following B. burgdorferi stimulation, in contrast to IL-18 expression. After B. burgdorferi exposure, TYK2 and IL-7 expression are upregulated for both time points compared to the other stimuli. IL-12Rβ1 and IL-12Rβ2 expression increased after 4 h of B. burgdorferi stimulation; however, this could not be observed after 24 h. Bb, Borrelia burgdorferi sensu stricto; HCs, healthy controls; IL-12Rβ1 and 2, interleukin 12 receptor subunit beta 1 and 2; PBMC, peripheral blood mononuclear cell; TYK2, tyrosine kinase 2.

IL-12 is crucial for B. burgdorferi induced IFN-γ production by human PBMCs.

Next, we evaluated whether IL-12 and IL-18 are present following B. burgdorferi stimulation of PBMCs. In contrast to IL-1β, IL-12 was not detectable after 24 h or 48 h of B. burgdorferi s.s. stimulation in vitro, while IL-18 concentration was measurable in low concentrations (Fig. 4A). When PBMCs were exposed to B. burgdorferi s.s. and a low concentration of IL-12, a significant increase of IFN-γ production was observed compared with stimulation with either stimulus alone (Fig. 4B to H). IFN-γ production appeared to be dose-dependent of IL-12 concentrations, and could be observed in both EM patients and healthy controls (Fig. 4B, C, G, and H). IFN-γ was already detectable after 24 h of incubation (Fig. 4B, D, E, G, and H). This effect was inducible by stimulation with frozen/live attenuated and viable B. burgdorferi s.s. in combination with IL-12 (Fig. 4E and F). There was marked interindividual variability in IFN-γ induction upon IL-12 addition in both healthy individuals and EM patients.

FIG 4.

FIG 4

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IFN-γ production can be induced by addition of IL-12 to PBMCs of healthy individuals and EM patients. (A) PBMCs exposed to B. burgdorferi s.s. produce neither IL-12 nor IFN-γ, in contrast to IL-1β. IL-18 is produced in modest amounts upon stimulation with B. burgdorferi s.s. and is measurable after 48 h incubation. (B and C) Addition of IL-12 induces the production of IFN-γ by B. burgdorferi s.s.-stimulated PBMCs of 21 healthy volunteers in a dose-dependent manner after 24 and 48 h of stimulation. (D) IL-18bp partially reverses the IFN-γ production upon IL-12 and B. burgdorferi s.s. stimulation in a dose-dependent manner in 6 healthy donors. (E+F) Induction of IFN-γ was observed with both frozen/live-attenuated and viable B. burgdorferi in two different concentrations in combination with IL-12 (10 ng/mL). IFN-γ production was higher following 48 h compared to 24 h of stimulation. (G+H) PBMCs from patients with physician-confirmed EM produced IFN-γ upon stimulation with B. burgdorferi s.s. and IL-12. This could be observed at baseline (t0) and 6 weeks later (t6wk). (I) Six weeks after diagnosis IFN-γ concentrations upon PBMC stimulation were higher than at baseline (n = 16 at baseline and n = 9 after 6 weeks). HC, healthy control; EM, erythema migrans; B. burgdorferi s.s., B. burgdorferi sensu stricto; Bb mix, B. burgdorferi s.l.; IL-18bp, IL-18 binding protein; LB, Lyme borreliosis; PBMC, peripheral blood mononuclear cell; ns, not statistically significant; *, P < 0.05; **, P < 0.01; ***, P < 0.0001 calculated by Wilcoxon signed-ranked test for paired testing in panels B–H and Mann-Whitney U test for comparing groups in panel I.

The IL-12 induced IFN-γ response was observed in multiple settings, including in PBMCs of healthy controls isolated within 24 h and PBMCs isolated directly after blood collection (Fig. S2 in the supplemental material). Interestingly, IFN-γ production by B. burgdorferi s.s. and IL-12 stimulated PBMCs from LB patients was higher at 6 weeks after the diagnosis of EM compared to healthy individuals, but with considerable overlap between groups (Fig. 4I). In previous studies, IFN-γ production was observed this early in experiments with mitogens, heat-killed C. albicans, or anti-CD3/CD28 antibodies (Fig. 1A and B) (37–39). Altogether, our findings demonstrated that the addition of IL-12 induces early IFN-γ production upon B. burgdorferi exposure.

Genetic variations in TYK2 are associated with B. burgdorferi-induced IFN-γ production.

Further evaluation of downstream proteins of the IL-12 pathway showed that tyrosine kinase 2 (TYK2) mRNA expression was upregulated following B. burgdorferi exposure (Fig. 3A and B), whereas in PBMCs of EM patients, TYK2 mRNA was decreased at baseline compared to 3 weeks and 6 months later (Fig. 2H). TYK2 together with Janus Kinase 2 (JAK2) is tyrosine phosphorylated by induction of the IL-12 receptor and activates the signal transducer and activator of transcription (STAT) 1, 3, 4, and 5 (33). At the genomic level, single nucleotide polymorphisms (SNPs) in the IFN-γ signaling pathway were evaluated in data from 65 individuals from the 200 Functional Genomics Project (200FG) cohort (29). SNP rs280520 in the gene encoding TYK2 (Fig. 2I) was significantly associated with increased IFN-γ production in a whole blood assay (WBA). The association of this SNP with IFN-γ production upon IL-12 stimulation further argues for a role for the IL-12 pathway in the impaired IFN-γ induction upon B. burgdorferi exposure.

IL-18 production is important for the induction of IL-12 and B. burgdorferi IFN-γ production.

To assess the role of IL-18 in B. burgdorferi induced IFN-γ production, PBMCs were preincubated with IL-18 binding protein (IL-18bp) and IL-12 before B. burgdorferi stimulation. The induction of IFN-γ was partially reversed in 6 healthy donors (Fig. 4D). Thus, although IL-18 was not strongly enhanced by B. burgdorferi in vitro (Fig. 4A), the low amounts induced appeared to be bioactive. This shows that IL-12-induced IFN-γ production by B. burgdorferi is partially dependent on IL-18.

The addition of IL-12 and IL-7 enhances IFN-γ production.

To optimize IFN-γ production capacity of PBMCs after B. burgdorferi exposure in vitro, IL-7 in combination with IL-12 and B. burgdorferi was used to stimulate PBMCs thawed from liquid nitrogen. IL-7 is a well-known T-cell growth factor and acts synergistically with IL-12 (40–43). IL-7 mRNA of PBMCs was enhanced upon B. burgdorferi stimulation in healthy volunteers, and IL-7 mRNA was slightly upregulated in LB patients 3 weeks after infection (Fig. 2G and Fig. 3). We observed a cumulative effect of IL-7 to IL-12 and B. burgdorferi-stimulated PBMCs of both healthy individuals (Fig. 5A and B) and EM patients (Fig. 5D and E, Fig. S3), with higher IFN-γ concentrations in the EM group, compared to the healthy control group. However, similarly to stimulation with IL-12 alone (Fig. 4I), there was substantial overlap between groups (Fig. 5F).

FIG 5.

FIG 5

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Addition of IL-12 and IL-7 further enhances the production of IFN-γ in PBMCs. (A, B, D, and E) IL-7 and IL-12 enhance IFN-γ production compared to IL-12 alone in both HCs and EM patients. (C and F) IFN-γ levels were highest in EM patients 6 weeks after diagnosis compared to baseline and healthy controls. (G–I) Validation experiments in 200FG cohort. (G) 48 h of B. burgdorferi s.l. stimulation in the presence of IL-12 and IL-7 showed a significant upregulation of IFN-γ production compared to B. burgdorferi s.s. or B. burgdorferi s.l. alone. (H) Seven days of B. burgdorferi s.l. stimulation showed the same effect as 48 h of stimulation. (I) IFN-γ production was highest in individuals with positive Bb serology of whom PBMCs were stimulated with B. burgdorferi s.l. 5 × 106/mL, compared to individuals with negative serology (P = 0.0292), and compared to a lower concentration of B. burgdorferi s.l. Bb ss, Borrelia burgdorferi sensu stricto; Bb mix, Borrelia burgdorferi sensu lato; HKCA, heat-killed Candida albicans; Neg/Pos ser, negative/positive B. burgdorferi s.l. serology according to two-tier testing; PBMC, peripheral blood mononuclear cell. *, P < 0.05; **, P < 0.01; ***, P < 0.0001 by Wilcoxon signed-ranked test for paired testing for all panels, expect for panels C and F where the Mann-Whitney U test was used to compare healthy controls with LB patients.

Next, we explored the combination of IL-12, IL-7, and B. burgdorferi in the 200FG cohort, which consists of healthy foresters, most of whom report numerous tick bites each year (Fig. 5G and H). To maximize the effect on IFN-γ production, we stimulated PBMCs with a mix of B. burgdorferi s.s., B. garinii, and B. afzelii (Fig. 5G and H). We confirmed the added effect of IL-12 and IL-7, after 48 h and 7 days of B. burgdorferi s.l. stimulation compared to B. burgdorferi s.l. alone. Also, IL-22 and IL-10 production was significantly induced by the combination of IL-12 and IL-7, while IL-17 production was inhibited (Fig. S3). Moreover, IFN-γ production did not correlate with the reported number of tick bites in the last year (Fig. S3). In contrast, positive standard two-tiered B. burgdorferi s.l. serology, indicating previous exposure to the spirochete, was associated with a higher IFN-γ response (Fig. 5I). This could be observed when PBMCs were stimulated with the highest concentration of B. burgdorferi s.l. for either 48 h or 7 days of incubation, and exclusively in the presence of IL-12 and IL-7. Interestingly, in patients with active LB who had antibodies against Anaplasma phagocytophilum (Ap), the number of IL-12 secreting cells was reduced (44). As we could not measure IL-12 with an ELISA, we compared IFN-γ production of PBMCs stimulated with IL-12 and B. burgdorferi. We did not find a difference in IFN-γ production between IL-12/B. burgdorferi stimulated PBMCs of EM patients with or without Ap antibodies (Fig. S5). The reported seroprevalence was 8.1% in European foresters (45) and therefore we cannot rule out that previous Ap exposure influenced our results regarding IFN-γ production. Taken together, IL-7 in combination with IL-12 further enhances the ability of PBMCs to produce IFN-γ in response to B. burgdorferi s.l. in vitro, especially in individuals with previous B. burgdorferi exposure.

IFN-γ production in 24h- and 48h-stimulated whole blood.

Lastly, we assessed the effect of IL-12 on IFN-γ production in a whole blood assay (WBA) (Fig. 6). In PBMCs, the added effect of IL-12 on the induction of IFN-γ was B. burgdorferi s.s.-specific (Fig. S2A and B). However, in WBA, IFN-γ was also produced after stimulation with Pam3Cys, a TLR2 agonist, and HKCA in combination with recombinant IL-12 (Fig. 6A). Interestingly, the enhancing effect of IL-12 upon B. burgdorferi s.l. exposure was not exclusively observed for IFN-γ, but for other cytokines as well, including tumor necrosis factor-α (TNF-α), IL-1β (in whole blood stimulation), IL-10, and IL-22 (in PBMC stimulation) (Fig. S3, 4, and 5).

FIG 6.

FIG 6

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IFN-γ production in whole blood assays. (A) Addition of IL-12 to whole blood of healthy volunteers (n = 9) showed some induction of IFN-γ production in the presence of B. burgdorferi s.s., although it did not reach statistical significance compared to IL-12 alone (P = 0.1484). (B) In the forester cohort (n = 149) this effect was statistically significant compared to B. burgdorferi s.s. alone (P < 0.0001). (C and D) These experiments were performed with WB obtained from foresters. (C) Addition of IL-7 and IL-12 further enhances the production of IFN-γ in whole blood, especially when stimulated with a higher concentration of B. burgdorferi s.l. (D) After 48 h the effect is significantly greater in the higher B. burgdorferi s.l. concentration compared to 24 h. WBA, whole blood analysis; Bb ss, B. burgdorferi sensu stricto; Bb mix, B. burgdorferi sensu lato; LPS, lipopolysaccharide; *, P < 0.05; **, P < 0.01; ***, P < 0.0001 calculated by Wilcoxon signed-ranked test for paired testing for all panels.

Similar to PBMCs, the addition of IL-7 to IL-12 to WBA amplified the induction of IFN-γ. This effect depended on incubation time and the concentration of B. burgdorferi s.l. (Fig. 6C and D). In WBA, IL-1β and TNF-α production were also induced upon addition of IL-12 addition (Fig. S4B), as well as IL-12 combined with IL-7 to B. burgdorferi s.l. stimulation for 24 h (Fig. S3I). Overall, we demonstrate here that the addition of IL-12 and IL-7 to B. burgdorferi s.l. can elicit an IFN-γ response in a WBA.

DISCUSSION

This study demonstrates that B. burgdorferi alone is a poor early IFN-γ inducer in PBMCs isolated from healthy subjects, possibly due to the incapacity to induce bioactive IL-12, a crucial step for the activation of Th1 responses. Transcriptome analysis of B. burgdorferi-exposed PBMCs reveals that the IL-12 p35 mRNA was not upregulated. We show that the addition of recombinant IL-12 promotes B. burgdorferi-induced IFN-γ production in PBMCs, both in healthy individuals and EM patients. This effect is dose-dependent for both IL-12 and B. burgdorferi s.l. in PBMCs. Simultaneous addition of IL-7 and IL-12 to B. burgdorferi s.l. stimulation further enhances IFN-γ production. Of note, the IFN-γ response induced in patients at 6 weeks after the diagnosis of EM is higher than at baseline, and their IFN-γ production is much higher than in healthy individuals. Lastly, we demonstrate that the addition of recombinant IL-12 also amplifies IFN-γ production in whole blood exposed to B. burgdorferi s.l. for 24 h.

B. burgdorferi was unable to initiate IFN-γ production by PBMCs of healthy controls, EM patients, and even patients with disseminated LB following 24 or 48 h of stimulation. In an earlier study, IFN-γ production by PBMCs was slightly elevated in 36 LB patients compared to controls; however, there was a wide variety of disseminated LB manifestations, longer incubation times, and usage of sonicated B. burgdorferi (46). For the stimulation experiments, we used whole live B. burgdorferi spirochetes. Others have found that whole live B. burgdorferi elicited a comparable transcriptional profile as heat-killed B. burgdorferi (47) and that whole spirochetes led to enhanced immune responses compared to lysates (48). However, memory T cells recognize peptides, whereas we used the whole bacterium in the PBMC stimulation, and therefore we could have missed (part of) the specific T cell response in our model.

Another potential mechanism for the limited IFN-γ production by B. burgdorferi is the ability to interfere with mammalian immune responses by suppression of antigen presentation by host immune cells (49). Antigen presentation is vital for a sufficient adaptive immune response. Significant alterations in gene expression and protein production have previously been observed in human PBMCs by B. burgdorferi stimulation (49). Specifically, the antigen presentation pathway, and its proteins HLA-DM, MHC-II, and CD74 via TNF receptor I and RIP1 signaling, were severely downregulated, in monocyte subsets, monocyte-derived macrophages, and dendritic cells. Inhibition of antigen presentation was specific for B. burgdorferi, since the exposure of CD14+ monocytes to other pathogens resulted in a significantly different protein expression, and caused impaired T-cell recognition of B. burgdorferi. This might explain the delayed adaptive immune response in LB patients and ex vivo B. burgdorferi stimulations (22, 50–52). Interestingly, a decreased expression of IFN-γ in EM biopsies was associated with the presence of persisting symptoms, suggesting a relevant clinical consequence of this cytokine (22).

To produce the IFN-γ-inducing IL-12 p70 heterodimer, both IL-12 p35 and p40 genes need to be expressed (33, 53). We showed that while mRNA of both IL-18 and IL-12 p40 was upregulated, IL-12 p35 was unchanged and IFN-γ gene expression was only minimally induced in PBMCs from EM patients (35). Other studies have shown that in LPS-stimulated human monocytes, IL-12 p40 production was higher than IL-12 p70; therefore, lack of IL-12 p35 was thought to be the limiting factor for IL-12 production. mRNA expression confirmed that IL-12 p40 was produced in excess of IL-12 p35 (54). Moreover, IL-12 p40 can form homodimers, and IL-12 p80 has been shown to inhibit IFN-γ production by competitively binding to IL-12Rβ1 (34, 55–59). Additionally, IL-12 p40 can be paired with the p19 chain to form IL-23, which has previously been described to be induced in response to B. burgdorferi (18, 30). Thus, during B. burgdorferi s.l. infection, a lack of IL-12 p35 production combined with high IL-12 p40 levels produced by myeloid cells is likely to explain the poor IFN-γ induction.

The SNP rs280520 in the IL-12 downstream protein TYK2 was associated with higher concentrations of IFN-γ. This SNP is located in an intron region of the TYK2 gene and changes adenine to guanine (60). IL-12 signaling is disrupted in TYK2-deficient mice, rendering them more susceptible to viruses (61, 62). In addition, IL-12/IL-18 synergistic IFN-y induction was reduced in TYK2-deficient murine natural killer cells (NK) and T cells (63). Inherited TYK2 deficiency in humans impairs IL-12 dependent IFN-y signaling and may facilitate tuberculosis and viral infections (64–67). In contrast, TYK2 variants (I684S and P1104A) are associated with autoimmune diseases, including systemic lupus erythematosus, multiple sclerosis, Crohn’s disease, psoriasis, and type 1 diabetes (68–81). In our study, this SNP was associated with increased IFN-y production when stimulated with B. burgdorferi and IL-12. These studies show that TYK2 is involved in IL-12 mediated signaling of IFN-y and confirmed an important role for IL-12 signaling in B. burgdorferi induction of IFN-y.

Next, we observed that IL-12 was not produced following B. burgdorferi stimulation for 24 and 48 h and that the addition of IL-12 could induce the IFN-γ signal. Several studies have described that IL-12 stimulates T- and NK-cells to secrete IFN-γ (82–86). Interestingly, this includes one study of 17 seropositive LB patients, including 12 patients with chronic neuroborreliosis and 5 Lyme arthritis patients (87). They found that whole mononuclear cells from LB patients produced IL-12 in contrast to controls and that if the authors blocked IL-12 with a monoclonal antibody, the IFN-γ producing cell population was inhibited. In C3H mice, IL-12 induced the production of IFN-γ through activation of the p38 MAP kinase (88) and treatment with anti-IL-12 reduced arthritis but elevated B. burgdorferi load (89). Altogether, our findings together with these studies suggest that IFN-γ production in B. burgdorferi infection is partially under the control of IL-12. This is important as IFN-γ does not only reduce B. burgdorferi load but is also involved in inflammatory tissue damage (90).

When drafting a new diagnostic assay, a whole blood assay is preferred above PBMC-based experiments. We found that costimulation with B. burgdorferi and IL-12 also induced IFN-γ production in whole blood. For latent tuberculosis, the IGRA is widely accepted as a diagnostic tool (91), and studies have suggested similar techniques to discriminate active from past Q-fever using whole blood IFN-γ assays (92, 93). Two studies performed in Northern America reported a higher sensitivity for a whole blood IFN-γ assay in EM patients compared to serology (37, 94). However, we could not reproduce these findings in a European cohort (95), where B. afzelii is the most common cause of EM, in contrast to B. burgdorferi s.s. in Northern America. A validation study for other cellular tests for LB based on IFN-γ production among others is currently ongoing (96). Intriguingly, a recent study showed that stimulation of whole blood of 22 Lyme neuroborreliosis patients provided lower IL-12 and IFN-γ concentrations compared to healthy controls, which indicates a limited Th1 response (97). Although these authors did not stimulate with B. burgdorferi in their whole blood assay, they stimulated with various pathogens to study a broad function of different signaling pathways. This resulted in decreased IL-12 and IFN-γ production, which is in line with our results. Our findings were predominantly based on healthy volunteers and erythema migrans patients; however, this report expands on this group with a disseminated form of LB, Lyme neuroborreliosis.

Our study has several limitations. First, since erythema migrans is a clinical diagnosis, other (infectious) diseases presenting as a red skin lesion could have been misinterpreted as EM. Strict case definitions and physician confirmation of the diagnosis were used, to limit the chance of including patients with signs and symptoms that were not caused by B. burgdorferi s.l. infection (10, 98). Second, we observed a highly variable effect size of IL-12 on IFN-γ production between individuals, and overlapping IFN-γ concentrations between LB patients and healthy individuals. However, we were able to distinguish IFN-γ production by B. burgdorferi exposed PBMCs from unstimulated PBMCs by adding a combination of IL-12 and IL-7. Third, our experimental study was not powered beforehand. Fourth, it remains unknown whether IFN-γ induction after the addition of IL-12 (and IL-7) can be used to differentiate active B. burgdorferi infection from past infection. Also, the observed large interindividual differences in IFN-γ response, and their potential clinical consequences, are of interest and should be further investigated. IFN-γ producing cells, such as T cells and NK cells, could be studied in high responders compared to low responders to see if these cells respond differently to IL-12/IL-7. A validation study with large, well-defined groups of patients and healthy individuals is required to confirm the potential and clinical relevance of a diagnostic test.

In conclusion, we show that B. burgdorferi is a weak inducer of Th1 responses early during the infection and point toward an obstruction in the IL-12 pathway as a potential mechanism, supported by the association of an SNP in the IL-12 downstream protein TYK2 with increased IFN-γ production. The effects may be due to a lack of IL-12 p35 production and high IL-12 p40 levels. This study is not only relevant to unravel the pathogenesis of LB infection, but can also contribute to the search for a new diagnostic test for LB.

MATERIALS AND METHODS

Study participants and blood samples.

Blood samples from several groups of volunteers were obtained after written informed consent, in accordance with the principles of the Declaration of Helsinki. Ethical approval was obtained from the medical ethics committee (METC) Arnhem-Nijmegen (NL32357.091.10) and CMO Noord-Holland (NL50227.094.14).

First, peripheral blood mononuclear cells (PBMCs) from healthy volunteers were isolated from buffy coats (Sanquin Blood Bank, Nijmegen, the Netherlands) and freshly obtained blood samples. PBMCs were isolated immediately after blood collection, or the day after the collection in case of buffy coats. Data on experiments with freshly isolated PBMCs from six individuals were published previously (30). Second, whole blood and freshly isolated PBMCs were obtained from the 200FG cohort, a cohort of 200 foresters in The Netherlands (29). Blood samples for B. burgdorferi s.l. serology, PBMC isolation, and whole blood stimulation were acquired from 149 foresters in 2016 and 201 foresters in 2018 and 2019. Third, isolated PBMCs were available from 46 adult patients with physician-confirmed LB included in the LymeProspect study (10, 98). For this group, the baseline blood sample was drawn within 7 days after initiation of antibiotic therapy. Blood samples of EM patients were processed within 24 h after collection, while blood samples of patients with disseminated LB were isolated directly. Case definitions were based on the European Society of Clinical Microbiology and Infectious Diseases Study Group for Lyme borreliosis (ESGBOR) criteria (1). This group included patients with confirmed localized disease (EM) (n = 40), Lyme arthritis (n = 1), acrodermatitis chronica atrophicans (ACA) (n = 4), and Lyme neuroborreliosis (n = 1). From 35 patients (76%), a follow-up blood sample was acquired 6 weeks after the first.

Isolation of human peripheral blood mononuclear cells.

Blood was diluted 1:1 in phosphate-buffered saline (PBS), after which the PBMC fraction was obtained by density centrifugation over Ficoll-Paque (Pharmacia Biotech). PBMCs were isolated, washed three times in cold PBS, and resuspended in Roswell Park Memorial Institute (RPMI) 1640 (Dutch modification, including Phenol Red, HEPES (4-(2-HydroxyEthyl)-1-PiperazineEthaneSulfonic acid), sodium bicarbonate 1 g/L, Life Technologies, Nieuwekerk, The Netherlands) supplemented with 1% gentamicin, 2 mM l-glutamine, and 1 mM pyruvate. In experiments with an incubation time longer than 24 h, 20% serum, autologous if available, was used.

B. burgdorferi cultures.

B. burgdorferi s.s., ATCC strain 35210 (B31), B. afzelii PKO (low passage), and B. garinii, ATCC strain 51383 (20047) were cultured in MKP (modified Kelly-Pettenkofer)-II medium with 6% rabbit serum at 33°C. Spirochetes were grown to the mid-logarithmic phase and dark-field microscopy was used to check for motility. The number of spirochetes was determined using a Petroff-Hauser counting chamber. After harvesting, the bacteria were washed in PBS thrice, and divided to use directly in stimulation experiments as viable B. burgdorferi and stored at –80°C until use as live attenuated B. burgdorferi.

Stimulation experiments.

Whole blood (100 μL per well in a 48-wells plate) or PBMCs (5 × 105 cells per well in a round-bottom 96-wells plate) was stimulated with either RPMI medium as a negative control, B. burgdorferi spirochetes, or various other stimuli as positive controls. For the whole blood assay and the PBMC experiments, 400 μL and 100 μL of stimulus were used, respectively. Both a mix of Borrelia species indicated as B. burgdorferi s.l., and B. burgdorferi s.s. were used in the experiments. Borrelia s.l. mix was prepared by combining equal amounts of B. burgdorferi s.s., B. afzelii, and B. garinii. Various concentrations were used depending on the experiment. Multiplicity of infection (MOI) of 1 (5 × 106 spirochetes/mL), 0.2 (1 × 106 spirochetes/mL), 0.1 (5 × 105 spirochetes/mL) were used for PBMC stimulation. In previous preliminary experiments no clear differences between Borrelia s.l. mix and sensu stricto were observed, and these stimuli elicited the same type of responses. In the large cohort study, such as recruited for the LymeProspect study, the Borrelia s.l. mix was used for logistical reasons. Positive controls included heat-killed Candida albicans (HKCA) 1 × 106/mL, lipopolysaccharide (LPS) 100 ng/mL in case of experiments with cases included through the LymeProspect study (98) and 10 ng/mL otherwise, Pam3Cys (P3C) 10 μg/mL, and Mycobacterium tuberculosis lysate (MTB) 1 μg/mL. The acquisition and preparation of these positive controls have been described earlier (29, 99). The addition of stimuli was preceded by preincubation of cells for 1 h with recombinant human IL-12 (R&D Systems), recombinant human IL-7 (Fisher Scientific), and/or IL-18 binding protein (BP) (R&D Systems). After incubation for 24, 48, 72, or 7 days at 37°C with 5% CO2, plates were centrifuged and cell-free supernatants were collected and stored at –20°C until cytokine measurement.

Cytokine measurements.

Concentrations of human IFN-γ (Sanquin, Amsterdam), TNF-α, IL-1β, IL-1Ra, IL-6, IL-10, IL-17, IL-22, IL-18, and IL-12 (R&D Systems, Minneapolis) in the cell culture supernatants were measured using commercial ELISA kits, according to the manufacturer’s protocol.

Serological testing.

For the detection of B. burgdorferi s.l. antibodies in the 200FG cohort, ELISA (Serion/Virion GmbH, Wurzburg, Germany Borrelia IgM; ER-121-M and Borrelia IgG; ER-121-G for samples before 2017 and DiaSorin/LIAISON Saluggia, Italy Borrelia IgM 310010, Borrelia IgG 310880 for samples from 2017 on) were performed on all samples, and in case of equivocal or positive results, IgM and/or IgG immunoblot analysis was performed (EuroImmun, Lubeck, Germany; Borrelia IgG and IgM, DY-2131-3001-1G and DY-2131-3001-1M for samples before 2019 and Mikrogen GmbH, Neuried, Germany; recomLine Borrelia IgM; 4273 (4277); and Borrelia IgG; 4272 (4276) from 2019 on). All assays were performed according to the manufacturer’s instructions.

Single nucleotide polymorphism analysis.

DNA was isolated from whole blood of the 200FG cohort, as was described in a previous study (100). Single nucleotide polymorphisms (SNPs) were selected based on literature and gnomAD (https://gnomad.broadinstitute.org/) (Table S1).

Statistical analysis.

Statistical analysis was performed using GraphPad Prism (Version 5.03, San Diego, CA, USA). Data were analyzed using Mann-Whitney U-test for independent samples and Wilcoxon matched-pairs signed rank test for paired samples. Data are expressed as mean ± SEM, unless stated otherwise.

ACKNOWLEDGMENTS

The authors acknowledge the following colleagues for their valuable contribution: R. Ter Horst for his assistance with providing data of the SNPs, M.S.M. Willers for performing a part of the laboratory experiments, R. van Deuren for providing data of the transcriptome dataset, and F. Stelma for her assistance and the evaluation of the B. burgdorferi serology.

F.R.V.D.S., H.D.V., M.A.E.B., B.J.K., J.W.H., C.C.V.D.W., and L.A.B.J. are funded by the Netherlands Organization for Health Research and Development (ZonMw, project numbers 200330008, 522050001, 522001003, 522050002, 522050003), which has peer-reviewed the grant application. J.W.H. is supported by INTERREG as part of the NorthTick project. M.G.N. was supported by an ERC Advanced Grant (#833247) and a Spinoza grant from the Netherlands Organization for Scientific Research (NWO).

F.R.V.D.S. was primarily responsible for drafting this manuscript. F.R.V.D.S., H.D.V., and M.A.E.B. performed the laboratory experiments. M.O., H.J.M.T.H., H.L.M.L., and H.D. coordinated the 200FG project. H.L.M.L. and H.D. provided essential support for laboratory experiments. F.L.V.D.V. helped conceive novel concepts for laboratory experiments. J.W.H. kindly provided Borrelia strains. B.J.K., J.W.H., C.C.V.D.W., and L.A.B.J. are the principal investigators of the LymeProspect study. F.R.V.D.S., H.D.V., M.A.E.B., H.J.M.T.H., B.J.K., J.W.H., and C.C.V.D.W. reviewed and edited the manuscript. L.A.B.J. is the principal investigator for this project and had the supervision of the first authors. All authors contributed to the refinement of this manuscript and approved the final version.

B.J.K. and L.A.B.J. are co-inventors of the Spirofind, an experimental in-house assay for LD, which is owned by Radboudumc and was previously licensed for development to Boulder Diagnostics (Boulder, Colorado, USA) and subsequently Oxford Immunotec (Oxford, UK) until 2018. The other authors declare they have no conflicts of interest.

Footnotes

Supplemental material is available online only.

Supplemental file 1
Supplemental material. Download iai.00558-21-s0001.pdf, PDF file, 1.1 MB (1.1MB, pdf)

Contributor Information

Leo A. B. Joosten, Email: leo.joosten@radboudumc.nl.

De'Broski R. Herbert, University of Pennsylvania

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