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Journal of Leukocyte Biology logoLink to Journal of Leukocyte Biology
. 2016 Apr 21;100(3):589–598. doi: 10.1189/jlb.4A0715-331R

Autophagy suppresses host adaptive immune responses toward Borrelia burgdorferi

Kathrin Buffen 1,2, Marije Oosting 1,2, Yang Li 3, Thirumala‐Devi Kanneganti 4, Mihai G Netea 1,2, Leo A B Joosten 1,2,✉
PMCID: PMC6608026  PMID: 27101991

Short abstract

Inhibition of autophagy increases the severity of murine Lyme arthritis and human adaptive immune responses against B. burgdorferi.

Keywords: IL‐17, IL‐23, Lyme disease

Abstract

We have previously demonstrated that inhibition of autophagy increased the Borrelia burgdorferi induced innate cytokine production in vitro, but little is known regarding the effect of autophagy on in vivo models of Borrelia infection. Here, we showed that ATG7‐deficient mice that were intra‐articular injected with Borrelia spirochetes displayed increased joint swelling, cell influx, and enhanced interleukin‐1β and interleukin‐6 production by inflamed synovial tissue. Because both interleukin‐1β and interleukin‐6 are linked to the development of adaptive immune responses, we examine the function of autophagy on Borrelia induced adaptive immunity. Human peripheral blood mononuclear cells treated with autophagy inhibitors showed an increase in interleukin‐17, interleukin‐22, and interferon‐γ production in response to exposure to Borrelia burgdorferi. Increased IL‐17 production was dependent on IL‐1β release but, interestingly, not on interleukin‐23 production. In addition, cytokine quantitative trait loci in ATG9B modulate the Borrelia induced interleukin‐17 production. Because high levels of IL‐17 have been found in patients with confirmed, severe, chronic borreliosis, we propose that the modulation of autophagy may be a potential target for anti‐inflammatory therapy in patients with persistent Lyme disease.

Abbreviations

ATG

= autophagy gene

CD

= cluster of differentiation

cQTL

= cytokine quantitative trait locus

EM

= erythema migrans

i.a.

= intra‐articular

KC

= keratinocyte chemoattractant

KO

= knockout

QTL

= quantitative trait locus

ra

= receptor antagonist

SNP

= single‐nucleotide polymorphism

SPF

= specific pathogen‐free

WT

= wild type

Introduction

Borrelia burgdorferi sensu lato, the causative agent of Lyme disease, stimulates a complex series of inflammatory events to eliminate the spirochete following infection [1]. The first most‐common sign of infection manifests as EM skin lesions, frequently accompanied by flu‐like symptoms [2]. If treated correctly, the prognosis for these patients is excellent; however, if untreated, hematogenous dissemination of spirochetes may give rise to a wide range of clinical manifestations, involving the central nervous system (mainly caused by Borrelia garinii), the skin (Borrelia afzelii) or the joints (B. burgdorferi) [3, 4].

Cytokines have an important role in the pathogenesis of Lyme disease by regulating the immune responses against Borrelia. Several in vitro studies have shown the importance of secreted IL‐1β in response to Borrelia [5, 6]. High amounts of this cytokine were found near the location of EM lesions after tick bites [7]. IL‐1 has a broad range of functions in mediating inflammation, providing protective immunity to infectious diseases, but it is also responsible for hyperinflammation in diseases associated with a dysregulated immune responses. In synergy with IL‐23, IL‐1β induces the production of IL‐17 and related cytokines from Th17 cells [8], which have been associated with increased joint damage in patients with rheumatoid arthritis or psoriasis [9, 10]. In addition, IL‐17 has been associated with the chronic stage of murine Lyme disease, and inhibition of IL‐17 by antibodies strongly reduced the development of Lyme arthritis, as shown by reduced joint swelling [11].

Previous studies have shown that autophagy, a highly conserved homeostatic mechanism that orchestrates the degradation of damaged cytosolic proteins, can modulate the IL‐1β response to pathogens, including Borrelia spirochetes [12]. Furthermore, inhibition of autophagy promotes the secretion of IL‐1β, which leads to elevated IL‐17 levels after LPS stimulation [13].

In this study, we investigated the role of autophagy in a KO mouse model of Lyme arthritis by injecting B. burgdorferi into the knee joints of the animals. ATG7 KO mice showed an increase in joint swelling, elevated cell influx into the joint cavity, as well as increased cytokine levels. In addition, we investigated the role of autophagy on Borrelia induced adaptive cytokines. The inhibition of autophagy by wortmannin increased the production of IL‐17, IL‐22, and IFN‐γ in response to Borrelia bacteria. The increase of IL‐17 was a specific response to elevated IL‐1β levels and independent of elevated IL‐23 levels. These findings underline the important role of autophagy in the pathogenesis of Lyme disease and suggest that modulation of autophagy could be a novel therapeutic strategy in that disease.

MATERIALS AND METHODS

Borrelia burgdorferi cultures

Borrelia burgdorferi, American Type Cell Culture strain 35210, was cultured at 33°C in Barbour‐Stoenner‐Kelly H medium (Sigma‐Aldrich, St. Louis, MO, USA), supplemented with 6% rabbit serum. Spirochetes were grown to late‐logarithmic phase and examined for motility by darkfield microscopy. Organisms were quantitated by fluorescence microscopy, after mixing 10 µl aliquots of culture material with 10 µl of an acridine orange solution, and were counted using a Petroff‐Hausser counting chamber (Hausser Scientific, Horsham, PA, USA). Bacteria were harvested by centrifugation of the culture at 7000 g for 15 min., washed twice with sterile PBS (pH 7.4), and diluted in the specified medium to required concentrations of 1 × 106 spirochetes/ml.

Animals

All mice were maintained at St. Jude Children's Research Hospital (kindly provided by Douglas R. Green, St. Jude Children's Research Hospital, Memphis, TN, USA) and have been described before [14]. Mice were housed in an SPF facility, and experiments were conducted under protocols approved by the St. Jude Children's Research Hospital's Committee on the Use and Care of Animals.

Isolation of human PBMCs and in vitro cytokine production

Venous blood was drawn from the cubital vein of healthy volunteers into 10‐ml EDTA tubes (Monoject, Medtronic, Dublin, Ireland). The mononuclear cell fraction was obtained by density centrifugation of blood diluted 1:1 in pyrogen‐free saline over Ficoll‐Paque reagent (GE Healthcare, Little Chalfont, United Kingdom). Cells were washed twice in saline and suspended in culture medium (RPMI 1640; Thermo Fisher Scientific Life Sciences, Waltham, MA, USA), supplemented with gentamicin 50 mg/ml, l‐glutamine 2 mM, and pyruvate 1 mM. Cells were counted in a Coulter counter (Beckman Coulter, Brea, California, USA), and the number was adjusted to 5 × 106 cells/ ml. A total of 5 × 105 mononuclear cells in a 100‐µl volume was added to round‐bottom 96‐well plates (Greiner Bio‐One, Monroe, NC, USA), which was incubated with either 100 µl of culture medium (negative control) or B. burgdorferi (106 spirochetes/ml). In some experiments, PBMCs were preincubated with culture medium or the autophagy inhibitor wortmannin (100 nM) for 60 min. At indicated time points, supernatants were collected and stored at −20°C until they were assayed.

CD4, CD8, CD56 depletion

To deplete cells from isolated PBMCs, cell subpopulations were labeled using magnetic beads coated with anti‐CD4, anti‐CD8, or anti‐CD56 (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany). Subsequently, cells were depleted over a depletion column according to the protocol supplied by the manufacturer. As the control for the isolation procedure, nondepleted PBMCs were also washed over the columns without coated beads.

Cytokine measurements

Concentrations of human IL‐1α, IL‐1β, IL‐1ra, IL‐23, TNF‐α, IL‐17A, IL17F, IL‐22, or IFN‐γ were determined in duplicates using specific, commercial ELISA kits (Sanquin, Amsterdam, The Netherlands; or R&D Systems, Minneapolis, MN, USA), in accordance with the manufacturers’ instructions.

Real‐time PCR

RNA from PBMCs was isolated using TRIzol reagent (Thermo Fisher Scientific Life Sciences) following the manufacturer's instructions. Isolated RNA was reversed‐transcribed into complementary DNA using iScript cDNA synthesis kit (Bio‐Rad Laboratories BV, Veenendaal, The Netherlands). Quantitative real‐time PCR was performed using Power SYBR Green PCR Master Mix (Thermo Fisher Scientific Life Sciences) using a 7300 Real‐Time PCR system (Thermo Fisher Scientific Life Sciences). In each PCR reaction, a melting curve analysis was included to control for a specific PCR amplification. Primers used for the experiments (final concentration 10 μM) are shown below. Real‐time qPCR data were corrected for expression of the housekeeping gene human B2M. Human IL‐1β: forward sequence, 5′–3′: GCC‐CTA‐AAC‐AGA‐TGA‐AGT‐GCT‐C, reversed sequence, 5′–3′: GAA‐CCA‐GCA‐TCT‐TCC‐TCA‐G; human IL‐23: forward sequence, 5′–3′: CAG‐CTT‐CAT‐GCC‐TCC‐CTA‐CT, reversed sequence, 5′–3′: GAC‐TGA‐GGC‐TTG‐GAA‐TCT‐GC; human TNF‐α: forward sequence, 5′–3′: TGG‐CCC‐AGG‐CAG‐TCA‐GA, reversed sequence, 5′–3′: GGT‐TTG‐CTA‐CAA‐CAT‐GGG‐CTA‐CA; human B2M: forward sequence, 5′–3′: ATG‐AGT‐ATG‐CCT‐GCC‐GTG‐TG, reversed sequence, 5′–3′: CCA‐AAT‐GCG‐GCA‐TCT‐TCA‐AAC; human IL‐17A: forward sequence, 5′–3′: CAA‐TCC‐CAA‐AAG‐GTC‐CTC‐AG, reversed sequence, 5′–3′: CAC‐TTT‐GCC‐TCC‐CAG‐ATC‐A; human IL‐17F: forward sequence, 5′–3′: GGC‐ATC‐ATC‐AAT‐GAA‐AAC‐CA, reversed sequence, 5′–3′: CTG‐TAC‐AAC‐TTC‐CGA‐GGG‐GTA; human IL‐22: forward sequence, 5′–3′: CAG‐CAG‐CCC‐TAT‐ATC‐ACC‐AA, reversed sequence, 5′‐3′: GGA‐ACA‐GTT‐TCT‐CCC‐CAA‐TG; and human IFN‐γ: forward sequence, 5′–3′: CGA‐GAT‐GAC‐TTC‐GAA‐AAG‐CTG, reversed sequence, 5′–3′: CAG‐TTC‐AGC‐CAT‐CAC‐TTG‐GA. Cycling conditions were 2 min at 50°C and 10 min at 95°C, followed by 40 cycles of 95°C for 15 s and 1 min at 60°C.

Intracellular IL‐17, IL‐22, and IFN‐γ flow cytometry

After 7 d of stimulation, PBMCs were stimulated for 4–6 h with PMA (50 ng/ml) (Sigma‐Aldrich), ionomycin (1 µg/ml) (Sigma‐Aldrich), and GolgiPlug (BD Biosciences, Franklin Lakes, NJ, USA), according to the protocols supplied by the manufacturers. Cells were stained extracellular using APC‐CD4, FITC‐CD4, PE‐CD56, PECy7‐CD56, ECD‐CD8, PE‐Cy7‐CD45, or APC‐CD45 antibodies (BD Biosciences or Beckman Coulter). Subsequently, cells were fixed and permeabilized with Cytofix/Cytoperm solution (eBioscience, San Diego, CA, USA) according to the protocols supplied by the manufacturer. Following permeabilization, cells were stained intracellularly with FITC‐conjugated anti‐IL‐17, FITC‐conjugated anti‐IFN‐γ or PE‐conjugated anti‐IL‐22 (BD Pharmingen or R&D) according to the protocols supplied by the manufacturers. The cells were measured on a FC500 flow cytometer (Beckman Coulter), and the data were analyzed using Kaluza 1.3 (Beckman Coulter).

Cytokine QTL mapping

Genotype and cytokine data could be generated for 391 healthy individuals. Gender information was coded as either 0 for women or 1 for men. The actual age and coded gender information were included as covariables in the linear regression model for cQTL mapping. Raw cytokine levels were log‐transformed then correlated with genotype data. A nominal P value ≤0.05 indicated a suggestive cytokine QTL.

Patella washouts and cytokine measurements

Protein levels of murine IL‐1β, IL‐6, or KC were measured in patellae washouts; 4 h after injection of 107 Borrelia spirochetes, patellae were isolated from inflamed knee joints and put in 0.05% Triton X‐100. After 2 freeze/thaw cycles, supernatant was harvested and centrifuged for 5 min at 10,000 rpm. Cytokines were determined by Luminex (Austin, TX, USA) technology; kits were obtained from Bio‐Rad Laboratories (Hercules, CA, USA).

Histologic analysis

Whole knee joints were removed and fixed in 4% formaldehyde for 7 d before decalcification in 5% formic acid and processing for paraffin embedding. Tissue sections (7 µm) were stained with H&E. Histopathologic changes in the knee joints were scored in the patellae/femur region.

Ethics statement

All human experiments were conducted according to the principles expressed in the Declaration of Helsinki. Before taking blood, informed written consent was obtained from each human subject. The study was approved by the review board of Radboud University Nijmegen Medical Centre.

Statistical analysis

Data are expressed as means ± sem unless otherwise indicated. Differences among experimental groups were tested using the 2‐sided Mann‐Whitney U test performed on GraphPad Prism 4.0 software (GraphPad Software, La Jolla, CA, USA). P values ≤0.05 were considered significant.

RESULTS

Borrelia induced joint inflammation is controlled by autophagy

As described before, autophagy modulates B. burgdorferi induced cytokine responses in vitro. To assess its role in vivo, we induced murine Lyme arthritis by injecting live spirochetes into knee joints of WT or ATG7‐deficient mice. Lyme arthritis, detected as joint swelling of the injected knee, could be seen in WT mice but was significantly increased in ATG7 KO mice ( Fig. 1A ). In addition to joint swelling, cytokine levels were measured in patella washouts. Significant differences in IL‐1β, IL‐6, and KC production could be detected when WT patellae were compared with ATG7‐deficient patellae (Fig. 1B–D). In addition, cell influx into the joint cavity at 4 h was assessed. In WT mice, only a few cells infiltrated into the joint cavity, in contrast to ATG7‐KO mice, in which more cells (mainly neutrophils) could be found (Fig. 1E and F).

Figure 1.

Figure 1

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Borrelia induced cytokine production and cell influx is modulated by autophagy. (A) Macroscopic score of the knees in either WT (white bars), or ATG7 KO mice (black bars) 24 h after i.a. injection of 1 × 107 live Borrelia species in 10 µl PBS. Data are means ± se from 5 animals in each group; **P < 0.01; 2‐tailed Mann‐Whitney U test. (B–D) At 4 h after i.a. injection of 1 × 107 live B. burgdorferi, patellae were cultured for 1 h, and IL‐1β, IL‐6, and KC protein levels were measured using Luminex. Data are means ± se; 5 animals in each group, *P < 0.05; **P < 0.01; 2‐sided Mann‐Whitney U test. (E) Murine Lyme arthritis in WT or ATG7 KO mice. Histology (H&E staining) after i.a. injection of B. burgdorferi in knee joints in WT mice (E) and ATG7 KO mice (F). JC, joint cavity; F, femur.

Time‐dependent cytokine production of PBMCs was stimulated with B. burgdorferi

To investigate the effect of autophagy on B. burgdorferi induced adaptive cytokines, we first measured the inflammatory response to B. burgdorferi itself, analyzing the levels of secreted cytokines by PBMCs stimulated with B. burgdorferi in a time‐dependent manner. After exposure to B. burgdorferi spirochetes, we observed an early increase of IL‐1β, IL‐23, and TNF‐α, which peaked after 24 h (Fig. 2A–C), as well as an increase in adaptive cytokines, such as IL‐17A, IL‐17F, IL‐22, and IFN‐γ ( Fig. 2D–G ).

Figure 2.

Figure 2

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Time‐dependent cytokine pattern associated with stimulation of live B. burgdorferi. Freshly isolated human PBMCs were stimulated with B. burgdorferi (multiplicity of infection, 0.2) for 2 h, 4 h, 24 h, 48 h, 4 d, or 7 d in IL‐1β (A), IL‐23 (B), TNF‐α (C), IL‐17F (D), IL‐17A (E), IL‐22 (F), and IFN‐γ (G). Cytokines were measured in the supernatants by specific ELISA.

Inhibition of autophagy enhances innate and adaptive cytokines production

Previously, the modulating effect of autophagy on B. burgdorferi induced IL‐1β has been shown ( Fig. 3A ) [12]. Because IL‐1Ra is a known natural antagonist of IL‐1β, we investigated the effect of autophagy on its production next to other innate cytokines, such as IL‐1α, TNF‐α, and IL‐23. The inhibition of autophagy decreased the B. burgdorferi induced IL‐1Ra production (Fig. 3B), shifting the IL‐1β:IL‐1ra ratio to a more prominent inflammatory response (Fig. 3C). Interestingly, the production of IL‐1α and TNF‐α were not affected by wortmannin (Fig. 3E and F). The increased amount of IL‐23 in B. burgdorferi stimulated autophagy‐incapable cells (Fig. 3D) led us to investigate the effect of autophagy on adaptive cytokines because IL‐23 and IL‐1β have been shown to be important in the production of IL‐17 [8, 15]. As shown in Fig. 3G–J, the production of IL‐17A, IL‐17F, IL‐22, as well as IFN‐γ was significantly increased in autophagy‐incapable cells stimulated with B. burgdorferi.

Figure 3.

Figure 3

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Modulation of B. burgdorferi induced inflammatory cytokine production by the inhibition of autophagy. Freshly isolated human PBMCs were preincubated for 1 h at 37°C in culture medium in the presence or absence of the autophagy‐inhibitor wortmannin (100 nM), followed by stimulation with B. burgdorferi (multiplicity of infection, 0.2) for 24 h (A–F) or 7 d (G–J). IL‐1α (E), IL‐1β (A), IL‐1ra (B), TNF‐α (F), IL‐23 (D), IL‐17A (G), IL‐17F (H), IL‐22 (J), and IFN‐γ (I) were measured in the supernatants by specific ELISA. Bars represent means ± se (error bars) of cells harvested from 10 volunteers. Bb, Borrelia burgdorferi. *P < 0.05; **P < 0.01; ***P < 0.0001, 2‐sided Mann‐Whitney U test.

Inhibition of autophagy alters cytokine expression at the transcriptional level

To examine whether inhibition of autophagy during exposure to Borrelia resulted in altered mRNA levels, we measured the transcription of several cytokines. After 24 h, IL‐1β and IL‐23 mRNA levels were strongly increased in human Borrelia stimulated PBMCs when autophagy was inhibited ( Fig. 4A and B ). As previously shown, the transcription of TNF‐α was not altered (Fig. 4C). The cytokine expression of IL‐17A, IL‐17F, IL‐22, and IFN‐γ was significantly increased after 4 d in autophagy‐blocked cells (Fig. 4D–G).

Figure 4.

Figure 4

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Modulated expression of B. burgdorferi induced inflammatory cytokines by the inhibition of autophagy. Freshly isolated human PBMCs were preincubated for 1 h at 37°C in culture medium in the presence or absence of the autophagy‐inhibitor wortmannin (100 nM), followed by stimulation with B. burgdorferi (multiplicity of infection, 0.2) for 24 h (A–C) or 4 d (D–G). IL‐1β (A), IL‐23 (B), TNF‐α (C), IL‐17A (D), IL‐17F (E), IFN‐γ (F), and IL‐22 (G) expression levels have been determined by RT‐PCR. Bars represent means ± se (error bars) of cells harvested from 10 volunteers. Bb, Borrelia burgdorferi. *P < 0.05; **P < 0.01; ***P < 0.0001, 2‐sided Mann‐Whitney U test.

The role of IL‐1 and IL‐23 in B. burgdorferi induced IL‐17, IL‐22, and IFN‐γ

In human PBMCs exposed to B. burgdorferi, IL‐17 production is dependent on IL‐1β and IL‐23. To determine whether the increased cytokine response in autophagy‐incapable cells was dependent on those cytokines, we blocked IL‐1 and IL‐23/IL‐12 by specific antibodies before we stimulated the cells with B. burgdorferi in the presence or absence of wortmannin. As expected, IL‐17 production was down‐regulated by inhibition of IL‐1 and IL‐23 in B. burgdorferi stimulated cells. Interestingly, the increase in the IL‐17 production induced by autophagy inhibition was reduced in the presence of anti‐IL‐1 ( Fig. 5A ), whereas the blockage of IL‐23 had no effect (Fig. 5B). The overall amount of IL‐22 and IFN‐γ was decreased in the presence of IL‐1 antibodies (Fig. 5C and E), but only IFN‐γ production was down‐regulated by anti IL‐23/IL‐12 (Fig. 5F); no effect on IL‐22 production could be detected (Fig. 5D).

Figure 5.

Figure 5

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Role of IL‐1β and IL‐23 on autophagy‐modulated B. burgdorferi–induced T cell responses. IL‐17 (A and B), IL‐22 (C and D), and IFN‐γ (E and F) were measured in culture supernatants of PBMCs stimulated with B. burgdorferi for 7 d, in the presence or absence of the autophagy‐inhibitor wortmannin. IL‐1 and IL‐23/IL12 were blocked by specific antibodies by 1 h of preincubation, as indicated. Bb, Borrelia burgdorferi. *P < 0.05; **P < 0.01; ***P < 0.0001.

Increase of Th17 cells is responsible for boosted B. burgdorferi induced IL‐17A in autophagy incapable cells

To further investigate cellular sources of IL‐17, IL‐22, and IFN‐γ in B. burgdorferi stimulated cells, PBMCs were incubated for 1 wk with the spirochete before flow cytometry was performed for intracellular cytokines in CD4+, CD8+, or CD56+ cells. CD4+ cells were the main producers of B. burgdorferi induced IL‐17, whereas IFN‐γ was produced in nearly equal amount of CD4+ and CD8+ cells; 12% of the overall IFN‐γ production was due to CD56+ cells ( Fig. 6A ). In addition, we wanted to assess whether the inhibition of autophagy increased a specific cell population that may be responsible for the increased cytokine response. A significant increase in CD4+IL‐17+ cells (almost 50%) could be found in autophagy‐incapable cells stimulated with B. burgdorferi, explaining the increase in IL‐17 production. Figure 6E demonstrates the increase of CD4+IL‐17+ autophagy‐incapable PBMCs stimulated with B. burgdorferi. The percentages of CD8+IL17+ and CD56+IL‐17+ cells did not differ between the stimulations (Fig. 6B). Furthermore, no difference could be seen between the subpopulations capability to produce IL‐22 or IFN‐γ (Fig. 6C and D).

Figure 6.

Figure 6

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Cellular source of autophagy‐modulated T cell responses induced by B. burgdorferi. (A) Assessment of surface markers CD4, CD56 and CD8 to elucidate the contribution of different cell types to the population of IL‐17A+, IL‐22+ and IFN‐γ+ cells. (B–D) Comparison of the capacity of CD4+, CD8+ and CD56+ cells stimulated by B. burgdorferi in the presence or absence of wortmannin to produce the inflammatory cytokines IL‐17, IFN‐γ or IL‐22. (E) Example of increased percentage of CD4+IL‐17+ cells stimulated with B. burgdorferi in the presence of wortmannin compared to cells stimulated with B. burgdorferi in the absence of autophagy modulators. B.b., Borrelia burgdorferi.

CD4+ cells are the main producers of adaptive cytokines in response to B. burgdorferi

To proof our previous findings of the cellular sources of IL‐17, IL‐22, and IFN‐γ, subpopulations known to be capable of producing these cytokines were depleted from the mixed‐cell population of PBMCs before exposure to B. burgdorferi in the presence or absence of wortmannin. For that purpose, cells expressing CD4, CD8, or CD56 were targeted. After depletion, equal numbers of regular PBMC or depleted cell populations were preincubated with RPMI‐1640 or wortmannin before stimulation with B. burgdorferi. Notably, CD4 depletion suppressed production of IL‐17, IL‐22, and IFN‐γ in response to spirochetes to background levels ( Fig. 7A–C ). CD8 depletion did only affect the amount of IFN‐γ production; IL‐17 and IL‐22 were independent of the presence of CD8+ cells (Fig. 7D–F). Depletion of CD56 had no effect on cytokine production at all (Fig. 7G–I).

Figure 7.

Figure 7

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T cell–dependent production of IL‐17A, IL‐22, and IFN‐γ by PBMCs exposed to live B. burgdorferi in the presence or absence of an autophagy inhibitor. After isolation, PBMCs were both depleted for CD4, CD8 or CD56 and stimulated with B. burgdorferi in the presence or absence of wortmannin. Release of IL‐17 (A, D, and G), IL‐22 (C, F, and I), or IFN‐γ (B, E, and H) was determined by ELISA after 7 d. Data are expressed as means ± se (error bars) of cells harvested from 10 volunteers. Bb, Borrelia burgdorferi. *P < 0.05, 2‐sided Mann‐Whitney U test.

cQTLs in ATG9B influence IL‐17 production

Genetic factors strongly influence clinical disease outcome by having a huge regulatory effect on cytokine production [16]. Therefore, we assessed genetic variability in autophagy genes that influence Borrelia induced cytokine production. cQTL analysis was performed as described previously [17]. Two SNPs in ATG9B (rs10266701 and rs13307588) showed suggestive cQTL (P < 0.05) for Borrelia induced IL‐17 ( Fig. 8A and B ) underlining our previous findings of the importance of autophagy for Borrelia induced cytokines.

Figure 8.

Figure 8

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SNPs in ATG9B affect IL‐17 levels induced by B. burgdorferi. Genetic variants in ATG9B (rs10266701 and rs13307588) modulate IL‐17 levels induced by B. burgdorferi. Box plots show the association of genotypes at SNP rs10266701 (P = 0.015) (A) and rs13307588 (P = 0.028) (B) with B. burgdorferi induced IL‐17 levels. The number of individuals per genotype is shown in parenthesis below each box plot.

DISCUSSION

The connection between autophagy and innate defense mechanisms has been made in several studies describing a regulatory role of autophagy on inflammasome activation and production of cytokines upon stimulation with microbial ligands [12, 18, 19–20]. Furthermore, it has been shown that autophagy modulates the production of T cell–derived cytokines, such as IL‐17, IL‐22, and IFN‐γ [13, 21], all cytokines known to also be produced after stimulation with B. burgdorferi [15, 22]. Because IL‐17 has been associated with increased joint damage in patients with rheumatoid arthritis [9] and elevated IL‐17 levels have been found in patients with confirmed neuroborreliosis [23], we examined the role of autophagy on the production of T cell–derived cytokines in response to B. burgdorferi.

In this study, we demonstrated that the secretion of adaptive inflammatory cytokines, such as IL‐17, IL‐22, and IFN‐γ, are highly elevated in autophagy‐incapable PBMCs in response to B. burgdorferi. IL‐1β and IL‐23 are needed to induce a Th17 responses [24, 25–26]; therefore, we investigated the role of autophagy in the modulation of their production. Elevated levels of IL‐23 were produced by PBMCs in response to B. burgdorferi when autophagy was inhibited. Previous studies have shown that IL‐23 promotes the development and expansion of Th17 cells. However, IL‐23 alone cannot drive differentiation of those cells from naïve CD4+ T cell precursors. IL‐1β signaling also has a critical role during the initial stages of Th17 cell differentiation; it enhances the metabolic fitness of rapidly dividing Th 17 cells during inflammation by the induction of phosphorylation of the mechanistic target of rapamycin [27]. In our experiments, both cytokines were present to induce the development of IL‐17. Apparently, the presence of IL‐23 is important for promoting the development of Th17 cells, but IL‐1β was the key driver of the amount of IL‐17 produced

A previous study by Strle et al. [28] showed elevated levels of IL‐23 in patients with post‐Lyme disease symptoms, and elevated levels of IL‐17 have been found in cerebrospinal fluid of patients with neuroborreliosis [23]. It has been suggested that Th17 cells and their associated cytokines are involved in the pathogenesis of Lyme arthritis [11, 29, 30]. Defective autophagy would explain an increased Th17 response, which might be associated with an increase in the severity of disease symptoms.

Using flow cytometric analysis of PBMCs and depletion of CD4, CD8, or CD56 T cell subsets, we determined that both cytokines IL‐17 and IL‐22 are primarily produced by CD4+ T cells in response to B. burgdorferi stimulation. Next to its production by CD4+ cells, we were able to demonstrate that a significant number of CD8+ and CD56+ cells express IFN‐γ in response to B. burgdorferi, which is in line with a previous report [31]. However, in contrast to that previous study, depletion of CD56+ cells did not alter B. burgdorferi induced IFN‐γ responses. Furthermore, the IFN+CD56+ cells did not expand upon stimulation with B. burgdorferi. These data suggest that NK cells do not have a major contribution to the IFN‐γ response to Borrelia in this setting.

With our in vitro data, we injected B. burgdorferi into the knee joints of ATG7‐deficient mice, showing increased joint swelling and more cell influx, compared with WT mice. It is known that the genetic background of mice influences susceptibility in several experimental disease models, including the induction and maintenance of experimental murine Lyme disease. C57Bl/6 mice clear the infection very fast, which explains the mild joint swelling and almost complete lack of cell influx from the bacteria. Long‐term effects on IL‐17 production could not be measured because the infection cleared soon after injection of the bacteria. To study the effect of autophagy on adaptive cytokines in mice, ATG‐KO needs to be developed in a mouse strain that is more susceptible to Borrelia infection, such as the C3H/H3N mice.

In summary, we have demonstrated a regulatory link between autophagy and T cell cytokine production in response to B. burgdorferi stimulation, which is dependent on IL‐1, but not on IL‐23, secretion. These findings further highlight autophagy as a potential target for anti‐inflammatory therapies in Lyme disease.

AUTHORSHIP

K.B. and M.O. contributed to the acquisition of data and analyzed and interpreted the data. Y.L., T.D.K., M.G.N., and L.A.B.J. contributed to study conception and design and revised the manuscript critically. All authors contributed substantially to the study's conception, design, and performance and approved this version of the article for publication.

DISCLOSURES

The authors declare no conflicts of interest.

ACKNOWLEDGMENTS

This work was supported by the Dutch Arthritis Association (NR 10‐1‐303), and a Vici grant of the Netherlands Organization for Scientific Research (NWO) supported M.G.N., and NWO VENI Grant 863.13.011 (to Y.L.).

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