Skip to main content
NCBI home page
The Journal of Infectious Diseases logoLink to The Journal of Infectious Diseases
. 2025 Jan 8;232(1):162–169. doi: 10.1093/infdis/jiaf011

CCL17 Influences Borrelia burgdorferi Infection in the Heart

Xiaotian Tang 1,2,✉,3,#, Qian Yu 3,#, Yingjun Cui 4,#, Thomas M Hart 5, Freddie Rivas-Giorgi 6, Keith Calloway 7, Amrita Ray Mohapatra 8, Erol Fikrig 9
PMCID: PMC12308671  PMID: 39777514

Abstract

Lyme disease, caused by Borrelia burgdorferi, is transmitted to humans by Ixodes ticks. CCL17 is a potent chemokine that plays important roles in diverse illnesses, including autoimmune and infectious diseases. CCL17 knockout mice, infected with B. burgdorferi, had a reduced pathogen load in the heart compared to control animals. Mice lacking CCL17 also showed signs of immune alteration upon B. burgdorferi infection, including diverse serum levels of proinflammatory cytokines and less monocytes and macrophages infiltration. CCL17 also interacts directly with B. burgdorferi, the first demonstration that this chemokine has an affinity for a vector-borne pathogen.

Keywords: Lyme disease, Borrelia burgdorferi, CCL17, infection, heart

The chemokine CCL17 is highly associated with cardiac infection with B. burgdorferi in mice, which has implications for the treatment of Lyme carditis and/or related complications.

Arthropod-borne diseases are a leading cause of illness and death worldwide, and more than 80% of the global population live in areas at risk from at least one major vector-borne illness. Lyme disease is the most common vector-borne infection in North America and results in over 40 000 cases diagnosed annually in the United States. It is typically caused by the spirochete Borrelia burgdorferi and transmitted to humans primarily through the bite of black-legged ticks (Ixodes scapularis). Antibiotics are commonly used to treat infected individuals and are effective in most cases. Some patients, however, develop persistent symptoms. A human vaccine against B. burgdorferi was approved by the Food and Drug Administration but is not currently available [1].

Host factors directly or indirectly play crucial roles in the etiology of vector-borne disease. For example, human peptidoglycan recognition protein 1 (PGLYRP1) interacts with B. burgdorferi sensu lato and exhibits bactericidal activity [2]. Moreover, host secretoglobin family 1D member 2 inhibits growth of B. burgdorferi and affect susceptibility to Lyme disease [3]. Host cytokines and chemokines are secreted proteins with growth, differentiation, and activation functions that regulate the nature of immune responses. Of note, C-C chemokine ligand 17 (CCL17) is a thymus- and activation-regulated chemokine associated with diverse inflammatory and allergic diseases involving the lung, heart, and joints [4]. CCL17 plays a role in several infectious diseases, including Salmonella and encephalomyocarditis virus infections [5, 6]. CCL17 has been proposed as a therapeutic target in certain forms of cardiac hypertrophy, heart failure, and osteoarthritis [5, 7–9]. Lyme disease can cause serious complications, including arthritis, neurological illness, and carditis, which compelled us to investigate whether CCL17 is involved in infection caused by B. burgdorferi. Previous studies have shown that CCL17 levels in murine cells is induced upon stimulation with BmpA, a B. burgdorferi antigen [10]. Furthermore, CCL17 expression is also altered in mice infected with B. burgdorferi lacking bba57, which is required for early murine infection [11]. Collectively, these data suggest that CCL17 might play a role in B. burgdorferi infection in the vertebrate host. In this study, we investigated the importance of CCL17 during murine infection with B. burgdorferi. We found that CCL17 is highly associated with cardiac infection with B. burgdorferi, with changes of immune cell recruitment and production of inflammatory mediators in the heart. We also identified that CCL17 interacts directly with B. burgdorferi, representing the first demonstration that this chemokine has an affinity for a vector-borne pathogen.

METHODS

Ethics Statement

The animal experiments in this study were performed following the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health and Yale University with the protocol permit (number 2023–07941). In addition, the Yale Assurance Number is D16-00146 with an approval period of 4 May 2023 to 31 May 2027.

Animals

Breeding pairs of CCL17 heterozygous (CCL17+/−) mice in the C57/B6 background were generated by Dr Irmgard Förster at University of Bonn. The mice were kindly provided by Dr Kory J. Lavine's laboratory at Washington University in St Louis. The CCL17 knockout (KO) and wild-type mice littermates were generated and then genotyped by Transnetyx (www.transnetyx.com) as described in Feng et al [12].

Spirochetes

Thirteen strains of Lyme Borrelia were included in this study: B. burgdorferi isolates N40 [13], B31 [14], B31A [15], HP19 [16], CA8 [17], CT-1 [18], NT-1 [19]; Borrelia afzelii CB43 [20] and VS461 [21]; Borrelia bavariensis Pbi (National Center for Biotechnology Information [NCBI], txid290434), Pni [22], and PBN [22]; and Borrelia garinii G25 [23]. The N40 isolate was used for all experiments except for the one reported in Supplementary Figure 4. The spirochetes were grown in Barbour-Stoenner-Kelly H complete medium (Sigma-Aldrich, catalog No. B8291) in a 33°C setting incubator. The live cell density was determined by dark field microscopy and hemocytometer (INCYTO, catalog No. DHC-N01).

B. burgdorferi Infection in Mice

To infect mice with B. burgdorferi, the mice were injected subcutaneously with 100 μL of 1×105 cells/mL B. burgdorferi (strain N40). Three weeks postinfection, the mice were sacrificed, and ear skin, heart, spleen, and joints were aseptically collected and assessed for spirochete burden by quantitative real-time PCR (qPCR) as described in our previous study [24]. Briefly, qPCR was performed using iTaq Universal SYBR Green Supermix (Bio-Rad, catalog No. 1725124) with an initial denaturing step of 2 minutes at 95°C and 45 amplification cycles consisting of 20 seconds at 95°C followed by 15 seconds at 60°C, and 30 seconds at 72°C. The target genes and corresponding primer sequences are shown in Supplementary Table 1.

Analysis of Immune Cells in the Hearts of Wild Type and KO Mice

After 3 weeks’ B. burgdorferi infection, the hearts were dissected and washed with cold phosphate-buffered saline (PBS) to remove blood in 15-mL tubes. The hearts were then cut into pieces in 1 mL digestion buffer with Dulbecco's Modified Eagle Medium (DMEM; Gibco) with collagenase I (450 U/mL; Sigma, catalog No. 9001-12-1), hyaluronidase (60 U/mL, Sigma, catalog No. 37326-33-3), Dispase (5 U/mL), and DNase I (60 U/mL; Sigma, catalog No. 9003-98-9). To deactivate the enzymes, 500 μL Hanks’ Balanced Salt Solution (Gibco) was added to the samples. The samples were then filtered through 40-µm cell strainers to obtain single-cell suspensions. Red blood cell lysis was performed with 500 µL red blood lysis buffer for 5–10 minutes. Samples were washed with 500 µL DMEM and resuspended in 100 µL fluorescence-activated cell sorting (FACS) buffer (PBS with 2% FBS and 2 mM EDTA [Corning, catalog No. 46-034-CI]). The cells were stained using the LIVE/DEAD fixable violet stain kit (Invitrogen, catalog No. L34955). For testing neutrophils, monocytes, and macrophages, the cells were further incubated with fluorochrome-conjugated monoclonal antibodies against CD45 (peridinin chlorophyll [PerCP]; BD Pharmingen, catalog No. 561047), Ly6G (BV711; BioLegend, catalog No. 127643), CD11b (phycoerythrin [PE]; Biolegend, catalog No. 101208), CX3CR1 (allophycocyanin [APC]/Cyanine7; BioLegend, catalog No. 149047), Ly6C (fluorescein isothiocyanate [FITC]; BioLegend, catalog No. 128005), and CD64 (APC; BioLegend, catalog No. 139305) for 30 minutes at 4°C. For examining B cells, the cells were stained with CD45 (PerCP; BD Pharmingen, catalog No. 561047), CX3CR1 (APC/Cyanine7; BioLegend, catalog No. 149047), CD3 (APC; BioLegend, catalog No. 100235), and CD19 (FITC; BioLegend, catalog No. 115505) for 30 minutes at 4°C. For testing T cells, the cells were stained with CD45 (PerCP; BD Pharmingen, catalog No. 561047), CX3CR1 (APC/Cyanine7; BioLegend, catalog No. 149047), CD19 (Alexa Fluor 700; BioLegend, catalog No. 152413), CD3 (APC; BioLegend, catalog No. 100235), CD4 (PE-Cyanine5; BioLegend, catalog No. 100513), CD25 (FITC; BioLegend, catalog No. 101907), and CD8a (BV711; BioLegend, catalog No. 100747) for 30 minutes at 4°C and washed twice with PBS. The samples were run on a BD LSRII flow cytometer and analyzed using FlowJo software. The gating strategy followed the protocol in Feng et al [5].

Mouse Cytokine/Chemokine Arrays and qPCR

After 3 weeks’ B. burgdorferi infection, we quantified cytokines production in CCL17-KO and control mice by the Mouse Cytokine/Chemokine Array 32-plex (MD-32) performed by Eve Technologies. Serum collected from each group of mice was sent for cytokine/chemokine analyses. The cytokines/chemokines represented by this array were eotaxin, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), interleukin-1α (IL-1α), IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A, IFN-γ inducible protein-10 (IP-10), KC, leukemia inhibitory factor (LIF), lipopolysaccharide-induced CXC (LIX), monocyte chemoattractant protein-1 (MCP-1), macrophage colony-stimulating factor (M-CSF), monokine induced by interferon-γ (MIG), macrophage-inflammatory protein-1α (MIP-1α), MIP-1β, MIP-2, regulated upon activation normal T cell expressed and secreted (RANTES), and tumor necrosis factor-α (TNF-α).

We also evaluated the gene expression of cytokines/chemokines in hearts of CCL17-KO and control mice after 3 weeks’ B. burgdorferi infection. The mice were euthanized, and the hearts were dissected as described above. Total RNA of heart was extracted using RNeasy Fibrous Tissue Mini Kit according to the manufacturer's instructions (QIAGEN, catalog No. 74704). cDNA was synthesized, and qPCR was performed as described in our previous study [24]. Briefly, cDNA was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad, catalog No. 1708891). qPCR was performed using iTaq Universal SYBR Green Supermix (Bio-Rad, catalog No. 1725124) with an initial denaturing step of 2 minutes at 95°C and 45 amplification cycles consisting of 20 seconds at 95°C followed by 15 seconds at 60°C, and 30 seconds at 72°C. The target genes and corresponding primer sequences are shown in Supplementary Table 1.

Flow Cytometry to Validate B. burgdorferi-CCL17 Interaction

B. burgdorferi (N40) was cultured to a density of 106–107 cells/mL and harvested by centrifugation at 5000g for 15 minutes. Cells were washed twice with PBS and aliquoted to give 106 per sample. The cells were then blocked in 1% bovine serum albumin (BSA) for 1 hour at 4°C. After centrifugation, the pellet was suspended and incubated with human and mouse recombinant CCL17 (10 or 50 μg/mL) at 4°C for 2 hours. After coincubation, spirochetes were washed 3 times with PBS and fixed in 2% paraformaldehyde (PFA). After washing, the spirochetes were probed with anti 6X-His monoclonal antibody-conjugated to Alexa Fluor 488 (Invitrogen, catalog No. MA1-21315-488) and run through a BD LSRII flow cytometer (BD Bioscience) with 100 000 events for each sample. The data was then analyzed by FlowJo as described in our previous studies [24, 25].

Cryogenic Electron Microscopy to Validate B. burgdorferi-CCL17 Interaction

B. burgdorferi (N40) was cultured to a density of approximately 106–107 cells/mL, washed twice with PBS and aliquoted to give 106 per sample. The samples were then blocked in 1% BSA for 1 hour at 4°C. B. burgdorferi was then incubated with recombinant CCL17 (10 or 50 μg/mL) at 4°C for 1 hour. After washing and fixation in 2% PFA, the samples were probed with 12 nm colloidal gold AffiniPure goat anti-mouse immunoglobulin G (IgG H + L) (electron microscopy grade). The samples were then examined with a cryogenic electron microscopy (Thermo Scientific).

Immunofluorescence Assay to Validate B. burgdorferi-CCL17 Interaction

B. burgdorferi (N40) was cultured to a density of approximately 106–107 cells/mL, washed twice with PBS, and blocked in 1% BSA for 1 hour at 4°C. B. burgdorferi was then incubated with recombinant CCL17 with a His tag at 4°C for 2 hours. After washing and fixation in 2% PFA, the samples were probed for 1 hour with anti-6X-His monoclonal antibody conjugated to Alexa Fluor 488 (Thermo Fisher, catalog No. MA1-21315-488) to recognize CCL17-His. B. burgdorferi were then stained with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen, catalog No. P36935). After staining, the fluorescence signals were examined with a Leica SP5 fluorescence confocal microscope (Leica Microsystems).

ELISA to Validate B. burgdorferi-CCL17 Interaction

For the enzyme-linked immunosorbent assay (ELISA) assay, B. burgdorferi (N40) were washed twice with PBS, pelleted, and lysed using Bug-buster Protein Extraction Reagent (Novagen, catalog No. 70921-3). Protein concentration in the lysate was measured by absorbance at 280 nm. For the protease assay, the B. burgdorferi lysate was incubated in the presence or absence of proteinase K (QIAGEN, catalog No. 19131) for 30 minutes. In a 96-well plate, wells were coated with 200 ng of B. burgdorferi lysate. Samples were blocked with 1% BSA followed by incubation with recombinant CCL17 with a His tag at varying concentrations (10–300 ng) for 1 hour at 37°C. After washing and incubating with horseradish peroxidase (HRP) Anti-6X-His tag antibody to recognize CCL17-His (Abcam, catalog No. ab3553), KPL Sureblue TMB Microwell Peroxidase Substrate, 1-Component (Seracare, catalog No. 5120-0077) was added. The reaction was stopped with 2 M sulfuric acid, and absorbance was read at 450 nm.

Purification of Recombinant Proteins

BmpA and bba57 without a signal peptide were separately cloned into pET21 plasmid. Recombinant protein was expressed in Escherichia coli and was further purified by Ni-NTA agarose (QIAGEN, catalog No. 30230) as described by the manufacturer and in our previous study [24]. The eluted samples were filtered through a 0.22-mm filter and concentrated with a 10-kDa concentrator (MilliporeSigma, catalog No. Z740203) by centrifugation at 4°C. Recombinant protein purities were assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 4%–20% Mini-Protean TGX gels (Bio-Rad, catalog No. 4561094) and quantified using the bicinchoninic acid (BCA) Protein Estimation kit (Thermo Fisher Scientific, catalog No. 23225).

RESULTS

CCL17 Facilitates B. burgdorferi Infection in the Murine Heart

We assessed whether CCL17 plays a role in the pathogenesis of murine Lyme borreliosis by comparing the outcome of B. burgdorferi infection of CCL17-KO and wild-type mice. The mice were infected with 1 × 104 spirochetes, and the B. burgdorferi burden in the skin, spleen, joint, and heart tissues was assessed at 3 weeks postinfection. The B. burgdorferi burden in murine skin, joints, and spleen was similar in CCL17-KO (n = 9) and control mice (n = 9) (P > .05; Figure 1A). CCL17-KO mice, however, had a significantly lower spirochete burden in the heart compared to control mice at 3 weeks postinfection (P < .05; Figure 1A and Supplementary Figure 1).

Figure 1.

Figure 1.

Open in a new tab

CCL17 facilitates Borrelia burgdorferi infection in murine heart and affects murine heart immune responses. A, B. burgdorferi burden in the skin, spleen, joint, and heart of CCL17 WT and KO mice at 3 weeks postinfection. CCL17-KO mice (n = 9) had a significantly lower spirochete burden in the hearts compared to control mice (n = 9). A second experiment at 3–4 weeks yielded similar results (Supplementary Figure 1). B, More monocytes and macrophages were recruited to the hearts of control mice (n = 4) compared to CCL17-KO mice (n = 4) upon B. burgdorferi infection. Boxes indicate the median and the first (Q1) and third (Q3) quartiles, the bottom whisker indicates the lowest value no further than Q1 minus 1.5 times the IQR, and the top whisker indicates the largest value no further than Q3 plus 1.5 times IQR. C, Control mice (n = 9) had a higher production of multiple proinflammatory cytokines or chemokines including eotaxin, IFN-γ, IL-1α, IL-13, MCP-1/CCL2, and MIP-1β/CCL4 compared to KO mice (n = 9) after 3 weeks’ B. burgdorferi infection. The bars represent mean ± SD. D, The levels of IL-4 and IL-7 were lower produced in control mice after 3 weeks’ B. burgdorferi infection. The bars represent mean ± SD. E, CCL3 and IL-17A gene expression was significantly higher in the hearts of CCL17-KO mice (n = 8) compared to the control mice (n = 8) after 3 weeks’ B. burgdorferi infection. The bars represent mean ± SD. For all data, each dot represents 1 biological replicate. Statistical significance was assessed using a nonparametric Mann-Whitney test. *P < .05, **P < .01, ns P > .05. Abbreviations: APC, allophycocyanin; CCL17, C-C chemokine ligand 17; FITC, fluorescein isothiocyanate; IFN-γ, interferon-γ; IL, interleukin; KO, knockout; MCP-1, monocyte chemoattractant protein-1; MIP-1β, macrophage inflammatory protein-1β; ns, not significant; WT, wild type; IQR, interquartile range; SD, standard deviation.

CCL17 Affects the Murine Immune Response Upon B. burgdorferi Infection

Recent studies have shown that CCL17 positively correlates with myocardial injury and cardiac dysfunction by increasing the infiltration of CD4+ T cells, macrophages, dendritic cells, and neutrophils, and suppressing recruitment of regulatory T (Treg) cells [8, 12]. CCL17 also protects against viral myocarditis by suppressing the recruitment of Treg cells [5]. We therefore examined immune cell infiltration in the hearts of control and CCL17-KO mice upon B. burgdorferi infection. In addition to the reported Treg cell differences, we found that more monocytes (CX3CR1+LY6C+) and macrophages (CX3CR1+LY6C+CD64+) were recruited to the hearts of control mice (n = 4) compared to CCL17-KO mice (n = 4) (P < .05), while the other immune cells population were comparable (P > .05; Figure 1B and Supplementary Figure 2).

Cytokines and chemokines help orchestrate immune cell migration and maintain homeostasis. We therefore assessed the circulating serum cytokine profiles in both control and CCL17-KO mice at 3 weeks after infection, using a mouse cytokine/chemokine array panel. We found that control mice (n = 9) had a higher production of multiple proinflammatory cytokines or chemokines including eotaxin, IFN-γ, IL-1α, IL-13, MCP-1/CCL2, and MIP-1β/CCL4 (P < .05) (Figure 1C), than CCL17-KO mice (n = 9). In contrast, IL-4 and IL-7 levels were lower in control mice, compared with CCL17-KO mice upon B. burgdorferi infection (P < .05; Figure 1D). We then evaluated the expression of immune-related genes in the heart. We selected representative cytokine and chemokine genes, which were listed in our previous study [24]. We found the expression of 2 proinflammatory cytokines, CCL3 and IL-17A, was significantly higher in CCL17-KO mice (n = 8) compared to control animals (n = 8) (P < .05; Figure 1E and Supplementary Figure 3). These data indicate that mice lacking CCL17 showed signs of immune alteration upon B. burgdorferi infection.

CCL17 Interacts Directly With B. burgdorferi

As diverse pathogens can associate with host factors during infection [26], we examined whether CCL17 can directly interact with B. burgdorferi. We probed a recently developed BASEHIT (bacterial selection to elucidate host-microbe interactions in high throughput) library with B. burgdorferi [2, 27]. CCL17 is one of the exoproteins that passed the significant threshold, indicating B. burgdorferi-CCL17 binding. Flow cytometry further showed a strong binding of both mouse CCL17 and human CCL17 with B. burgdorferi in a dose-dependent manner (Figure 2A and 2B). We also observed that CCL17 has high affinity for B. burgdorferi, both using cryogenic electron microscopy (Figure 2C) and confocal microscopy (Figure 2D). We further tested whether CCL17 could bind to diverse Borrelia strains. Interestingly, CCL17 strongly binds to all the 13 Borrelia strains we tested except B31A, a high-passage and noninfectious strain, which showed moderate binding (Figure 2E and Supplementary Figure 4). To further examine whether CCL17 interacts with other bacteria, we evaluated the interaction between CCL17 and another gram-negative bacterium, E. coli. Flow cytometry assay showed that CCL17 does not bind to E. coli (Figure 2F), further suggesting that CCL17 binds to B. burgdorferi sensu lato. To investigate the potential B. burgdorferi ligand that interacts with CCL17, we performed ELISA-based binding and flow cytometry assays with protease-treated B. burgdorferi lysates. After treatment with proteinase K, B. burgdorferi had a significantly diminished ability to bind CCL17 (Figure 2G and 2H), revealing that CCL17 interacts with a B. burgdorferi protein ligand or ligands. Because CCL17 levels and expression were influenced by B. burgdorferi BmpA and bba57 [10, 11], we then examined whether these 2 lipoproteins are the potential ligands for CCL17. ELISA demonstrated that neither BmpA or bba57 had affinity for CCL17 (Supplementary Figure 5).

Figure 2.

Figure 2.

Open in a new tab

CCL17 binds to Borrelia burgdorferi. A, Binding of human CCL17 to B. burgdorferi in a dose-dependent manner (1 μg and 5 μg) as analyzed by flow cytometry. Human peptidoglycan recognition protein 1 (PGLYRP1) was used as the positive control. The background of Alexa Fluor 488-His antibody alone with B. burgdorferi is shown as Sec. B, Binding of mouse CCL17 to B. burgdorferi in a dose-dependent manner (1 μg and 5 μg) as analyzed by flow cytometry. C, Binding of CCL17 to B. burgdorferi as analyzed by cryogenic electron microscopy. The black dots are colloidal gold-coated CCL17. The control is colloidal gold AffiniPure goat anti-mouse IgG alone. D, Binding of CCL17 to B. burgdorferi as analyzed by immunofluorescence assay. B. burgdorferi bound to CCL17 was determined using an Alexa Fluor 488-conjugated 6X-His monoclonal antibody. E, CCL17 shows moderate binding to B. burgdorferi B31A strain. F, CCL17 did not bind to Escherichia coli as revealed by flow cytometry. B. burgdorferi was used as the positive control. G, After treatment with proteinase K, B. burgdorferi had significantly diminished ability to bind to CCL17 as revealed by ELISA. The bars represent mean ± SD. H, Flow cytometry showed reduced binding of B. burgdorferi to CCL17 after treatment with proteinase K. Abbreviations: Sec, secondary antibody; DAPI, 4′,6-diamidino-2-phenylindole; Bb, Borrelia burgdorferi; CCL17, C-C chemokine ligand 17; ELISA, enzyme-linked immunosorbent assay; SD, standard deviation.

DISCUSSION

Direct interactions between arthropod-borne pathogens and mammalian hosts are critical interfaces shaping the pathogenesis of vector-borne diseases. Specifically, host immune cell receptors (eg, Toll-like receptors) are stimulated by infectious agents to activate the immune system [28]. Host complement components and antimicrobial peptides (AMPs) can directly bind to pathogens and neutralize them [29]. In turn, pathogens have evolved diverse mechanisms to evade recognition by the receptors and killing by complement and AMPs [30–32]. Therefore, understanding the interaction nodes between microbes and host factors is crucial for a mechanistic understanding of pathogenesis.

Although binding of cytokines with bacteria has been characterized [26], whether a vector-borne pathogen has an affinity with cytokines/chemokines, and contributes to pathogenesis, has not been described. Here, our study provides an example of the role of CCL17 in a tick-borne infectious disease. We demonstrated that B. burgdorferi interact directly with CCL17. CCL17 strongly binds to all the 13 Borrelia strains we tested except B31A, a high-passage and noninfectious strain. Compared to B31, B31A lacks the cp32-6, cp32-7, lp25, lp28-1, lp28-4, lp36, and lp21 plasmids [15]. Therefore, the CCL17 binding partner may be the protein(s) encoded by one or more of these plasmids. In addition, a significantly decreased bacterial burden was observed in the hearts of CCL17-deficienct mice compared to wild-type mice. CCL17 has been associated with several infectious diseases that affect the intestine or heart. Erazo et al [6] found that Salmonella enterica serovar Typhimurium (STM) infection triggered upregulation of CCL17 expression in specific intestinal dendritic cell subsets in a tissue-specific manner; however, CCL17 only has a moderate influence on STM dissemination. In the heart, CCL17 protects against encephalomyocarditis virus by suppressing the recruitment of regulatory T cells [5]. Our data suggest that B. burgdorferi may utilize CCL17 for infection of the murine heart, and affects the infiltration of monocytes and macrophages into this organ. Our data suggest that CCL17 does not have a major role in murine Lyme arthritis, perhaps because the inflammatory infiltrate in the infected joints is primarily composed of neutrophils, while macrophages appear to predominate during Lyme carditis. Our murine heart data may also provide a clue to study human heart related to CCL17 as carditis is a less common manifestation of Lyme disease than Lyme arthritis.

Cytokine/chemokines are involved in complex and coordinated processes of the human immune system, and are implicated in the pathogenesis of numerous inflammatory and autoimmune diseases [33, 34]. They and their receptors have become candidates for drug development. Increasing numbers of studies have proposed that CCL17 may act as a therapeutic target for specific cardiac diseases, including cardiac hypertrophy and heart failure [7, 8, 12]. Inhibiting CCL17, in conjunction with antibiotics targeting the microbe itself, could potentially be important in the modulation of B. burgdorferi infection of the heart. Moreover, our recent studies suggest that CCL17 may interact with other pathogens such as Leptospira and Plasmodium [35], suggesting that this paradigm may be applicable to other infections. Overall, our studies show the importance of CCL17 in the pathogenesis of murine infection with the Lyme disease agent.

Supplementary Material

jiaf011_Supplementary_Data
jiaf011_supplementary_data.docx (6.1MB, docx)

Contributor Information

Xiaotian Tang, Zhejiang Key Laboratory of Biology and Ecological Regulation of Crop Pathogens and Insects, Ministry of Agriculture Key Lab of Molecular Biology of Crop Pathogens and Insect Pests, Institute of Insect Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China; Section of Infectious Diseases, Department of Internal Medicine, School of Medicine, Yale University, New Haven, Connecticut, USA.

Qian Yu, Section of Infectious Diseases, Department of Internal Medicine, School of Medicine, Yale University, New Haven, Connecticut, USA.

Yingjun Cui, Section of Infectious Diseases, Department of Internal Medicine, School of Medicine, Yale University, New Haven, Connecticut, USA.

Thomas M Hart, Section of Infectious Diseases, Department of Internal Medicine, School of Medicine, Yale University, New Haven, Connecticut, USA.

Freddie Rivas-Giorgi, Molecular Biochemistry and Biophysics Program, Yale College, New Haven, Connecticut, USA.

Keith Calloway, Section of Infectious Diseases, Department of Internal Medicine, School of Medicine, Yale University, New Haven, Connecticut, USA.

Amrita Ray Mohapatra, Section of Infectious Diseases, Department of Internal Medicine, School of Medicine, Yale University, New Haven, Connecticut, USA.

Erol Fikrig, Section of Infectious Diseases, Department of Internal Medicine, School of Medicine, Yale University, New Haven, Connecticut, USA.

Supplementary Data

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

Notes

Acknowledgments. We sincerely thank Dr Irmgard Förster at University of Bonn and Dr Kory J. Lavine at Washington University, St Louis for providing CCL17 knockout and wild-type mice. We also thank Dr Sukanya Narasimhan, Dr Chunyan Wang, Ms Kathleen DePonte, and Mr Ming-Jie Wu for their excellent technical assistance.

Disclaimer. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Financial support. This work was supported by the National Institutes of Health (grant numbers AI126033 and AI138949 to E. F.); the Steven and Alexandra Cohen Foundation (to E. F.); and the Howard Hughes Medical Institute Emerging Pathogens Initiative (to E. F.).

References

  • 1. Steere  AC, Sikand  VK, Meurice  F, et al.  Vaccination against Lyme disease with recombinant Borrelia burgdorferi outer-surface lipoprotein A with adjuvant. N Engl J Med  1998; 339:209–15. [DOI] [PubMed] [Google Scholar]
  • 2. Gupta  A, Arora  G, Rosen  CE, et al.  A human secretome library screen reveals a role for peptidoglycan recognition protein 1 in Lyme borreliosis. PLoS Pathog  2020; 16:e1009030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. Strausz  S, Abner  E, Blacker  G, et al.  SCGB1D2 inhibits growth of Borrelia burgdorferi and affects susceptibility to Lyme disease. Nat Commun  2024; 15:2041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Lupancu  TJ, Eivazitork  M, Hamilton  JA, Achuthan  AA, Lee  KM. CCL17/TARC in autoimmunity and inflammation—not just a T-cell chemokine. Immunol Cell Biol  2023; 101:600–9. [DOI] [PubMed] [Google Scholar]
  • 5. Feng  G, Zhu  C, Lin  CY, et al.  CCL17 protects against viral myocarditis by suppressing the recruitment of regulatory T cells. J Am Heart Assoc  2023; 12:e028442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Erazo  AB, Wang  N, Standke  L, et al.  CCL17-expressing dendritic cells in the intestine are preferentially infected by Salmonella but CCL17 plays a redundant role in systemic dissemination. Immun Inflamm Dis  2021; 9:891–904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. Zhang  Y, Tang  X, Wang  Z, et al.  The chemokine CCL17 is a novel therapeutic target for cardiovascular aging. Signal Transduct Target Ther  2023; 8:157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8. Zhang  Y, Ye  Y, Tang  X, et al.  CCL17 acts as a novel therapeutic target in pathological cardiac hypertrophy and heart failure. J Exp Med  2022; 219:e20200418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. Lee  MC, Saleh  R, Achuthan  A, et al.  CCL17 blockade as a therapy for osteoarthritis pain and disease. Arthritis Res Ther  2018; 20:62. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Zhao  H, Liu  A, Cui  Y, Liang  Z, Li  B, Bao  F. Borrelia burgdorferi basic membrane protein A could induce chemokine production in murine microglia cell line BV2. Microb Pathog  2017; 111:174–81. [DOI] [PubMed] [Google Scholar]
  • 11. Yang  X, Qin  J, Promnares  K, Kariu  T, Anderson  JF, Pal  U. Novel microbial virulence factor triggers murine Lyme arthritis. J Infect Dis  2013; 207:907–18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12. Feng  G, Bajpai  G, Ma  P, et al.  CCL17 aggravates myocardial injury by suppressing recruitment of regulatory T cells. Circulation  2022; 145:765–82. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. Barthold  SW, Moody  KD, Terwilliger  GA, Duray  PH, Jacoby  RO, Steere  AC. Experimental Lyme arthritis in rats infected with Borrelia burgdorferi. J Infect Dis  1988; 157:842–6. [DOI] [PubMed] [Google Scholar]
  • 14. Fraser  CM, Casjens  S, Huang  WM, et al.  Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi. Nature  1997; 390:580–6. [DOI] [PubMed] [Google Scholar]
  • 15. Caine  JA, Coburn  J. A short-term Borrelia burgdorferi infection model identifies tissue tropisms and bloodstream survival conferred by adhesion proteins. Infect Immun  2015; 83:3184–94. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Steere  AC, Grodzicki  RL, Kornblatt  AN, et al.  The spirochetal etiology of Lyme disease. N Engl J Med  1983; 308:733–40. [DOI] [PubMed] [Google Scholar]
  • 17. Pothineni  VR, Wagh  D, Babar  MM, et al.  Identification of new drug candidates against Borrelia burgdorferi using high-throughput screening. Drug Des Devel Ther  2016; 10:1307–22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. Hughes  CA, Kodner  CB, Johnson  RC. DNA analysis of Borrelia burgdorferi NCH-1, the first northcentral U.S. human Lyme disease isolate. J Clin Microbiol  1992; 30:698–703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. Foley  DM, Gayek  RJ, Skare  JT, et al.  Rabbit model of Lyme borreliosis: erythema migrans, infection-derived immunity, and identification of Borrelia burgdorferi proteins associated with virulence and protective immunity. J Clin Invest  1995; 96:965–75. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. Stepanova-Tresova  G, Kopecky  J, Kuthejlova  M. Identification of Borrelia burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii in Ixodes ricinus ticks from southern Bohemia using monoclonal antibodies. Zentralbl Bakteriol  2000; 289:797–806. [DOI] [PubMed] [Google Scholar]
  • 21. van Dam  AP, Kuiper  H, Vos  K, et al.  Different genospecies of Borrelia burgdorferi are associated with distinct clinical manifestations of Lyme borreliosis. Clin Infect Dis  1993; 17:708–17. [DOI] [PubMed] [Google Scholar]
  • 22. Rollins  RE, Wulbern  J, Rottgerding  F, et al.  Utilizing two Borrelia bavariensis isolates naturally lacking the PFam54 gene array to elucidate the roles of PFam54-encoded proteins. Appl Environ Microbiol  2022; 88:e0155521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. Godfroid  E, Ben Messaoud  A, Poliszczak  A, Lobet  Y, Bollen  A. Assignment of Borrelia burgdorferi strains G25 and VS461 to the Borrelia garinii and Borrelia afzelii genospecies, respectively: a comparison of OspA protein sequences. DNA Seq  1995; 5:251–4. [DOI] [PubMed] [Google Scholar]
  • 24. Tang  X, Arora  G, Matias  J, Hart  T, Cui  Y, Fikrig  E. A tick C1q protein alters infectivity of the Lyme disease agent by modulating interferon γ. Cell Rep  2022; 41:111673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. Tang  X, Cao  Y, Booth  CJ, et al.  Adiponectin in the mammalian host influences ticks’ acquisition of the Lyme disease pathogen Borrelia. PLoS Biol  2023; 21:e3002331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Hogbom  M, Ihalin  R. Functional and structural characteristics of bacterial proteins that bind host cytokines. Virulence  2017; 8:1592–601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. Sonnert  ND, Rosen  CE, Ghazi  AR, et al.  A host-microbiota interactome reveals extensive transkingdom connectivity. Nature  2024; 628:171–9. [DOI] [PubMed] [Google Scholar]
  • 28. Lani  R, Thariq  IM, Suhaimi  NS, Hassandarvish  P, Abu Bakar  S. From defense to offense: modulating toll-like receptors to combat arbovirus infections. Hum Vaccin Immunother  2024; 20:2306675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29. Kurokawa  C, Lynn  GE, Pedra  JH, Pal  U, Narasimhan  S, Fikrig  E. Interactions between Borrelia burgdorferi and ticks. Nat Rev Microbiol  2020; 18:587–600. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30. Hart  T, Nguyen  NTT, Nowak  NA, et al.  Polymorphic factor H-binding activity of CspA protects Lyme borreliae from the host complement in feeding ticks to facilitate tick-to-host transmission. PLoS Pathog  2018; 14:e1007106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31. Xie  J, Zhi  H, Garrigues  RJ, Keightley  A, Garcia  BL, Skare  JT. Structural determination of the complement inhibitory domain of Borrelia burgdorferi BBK32 provides insight into classical pathway complement evasion by Lyme disease spirochetes. PLoS Pathog  2019; 15:e1007659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Rana  VS, Kitsou  C, Dutta  S, et al.  Dome1–JAK–STAT signaling between parasite and host integrates vector immunity and development. Science  2023; 379:eabl3837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33. Siebert  S, Tsoukas  A, Robertson  J, McInnes  I. Cytokines as therapeutic targets in rheumatoid arthritis and other inflammatory diseases. Pharmacol Rev  2015; 67:280–309. [DOI] [PubMed] [Google Scholar]
  • 34. Takeuchi  T. Cytokines and cytokine receptors as targets of immune-mediated inflammatory diseases-RA as a role model. Inflamm Regen  2022; 42:35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Hart  TM, Sonnert  ND, Tang  X, et al.  An atlas of human vector-borne microbe interactions reveals pathogenicity mechanisms. Cell  2024; 187:4113–27.e13. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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

Supplementary Materials

jiaf011_Supplementary_Data
jiaf011_supplementary_data.docx (6.1MB, docx)

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

RESOURCES