Transcriptional profiles of cytokines and chemokines reveal important pro-inflammatory response from endothelial cells during Orientia tsutsugamushi infection

Microbes and Infection 21 (2019) 313e320

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Microbes and Infection journal homepage: www.elsevier.com/locate/micinf

Original article

Transcriptional profiles of cytokines and chemokines reveal important pro-inflammatory response from endothelial cells during Orientia tsutsugamushi infection Hong Ge a, Christina M. Farris a, b, Min Tong a, Alice Maina a, Allen L. Richards a, * a b

Viral and Rickettsial Diseases Department, Infectious Diseases Directorate, Naval Medical Research Center, Silver Spring, MD, USA US Naval Medical Research Unit No. 2, Phnom Penh, Cambodia

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 August 2018 Accepted 14 January 2019 Available online 23 January 2019

Endothelial cells (EC) are key targets during Orientia tsutsugamushi infection. Knowledge of the proinflammatory response against O. tsutsugamushi by ECs is limited. The aim of the present study was to characterize the pro-inflammatory transcriptional response during the first 24 h of infection of the human dermal microvascular endothelial cell line with O. tsutsugamushi Karp by examining five-time points. The transcriptional profiles of 84 genes including cytokines, chemokines, growth factors, and TNF receptor superfamily genes were studied using a RT-PCR array. We identified 40 of the 84 genes that were up or down modulated during the early O. tsutsugamushi infection that differed remarkably from genes of non-infected cells. The modulated genes included: the interleukins (IL-1a/b, IL-4, IL-6, IL-7, IL10, IL-11, IL-18, and IL-24), chemokines (CXCL8, CCL2/MCP1, CCL5/RANTES, and CCL17), growth factors (NODAL, CNTF, and CSF2/GM-CSF), and TNFSF13B. IL-1b, IL-4, and IL-11 were highly induced at one hour post infection, whereas, CCL17 was profoundly up-regulated and IFNa2 was greatly down-regulated during the entire 24-hour time course. These results provide insight into the early pro-inflammatory response of endothelial cells to O. tsutsugamushi infection and indicate their potential role in the pathophysiology of the host's initial response to O. tsutsugamushi infection. © 2019 Published by Elsevier Masson SAS on behalf of Institut Pasteur.

Keywords: O. tsutsugamushi HMEC-1 Cytokines Chemokines

Orientia tsutsugamushi is an obligate intracellular Gramnegative bacterium that causes scrub typhus. This disease is characterized by fever, rash, eschar, myalgia, arthralgia, pneumonitis, and multi-organ infection that can lead to disseminated intravascular coagulation and organ failure without proper treatment [1]. O. tsutsugamushi can infect several types of host cells including endothelial cells (ECs) its major target, dendritic cells (DCs), macrophages, polymorphonuclear leukocytes (PMNs), and lymphocytes [2]. The mammalian innate immune response, including proinflammatory cytokines and chemokines, serves as the first line of defense against invading pathogens, such as O. tsutsugamushi. The cytokines and chemokines play important roles in recruiting leukocytes to inflammatory sites during bacterial infections. Upon O. tsutsugamushi infection, DCs are activated to produce and release cytokines and chemokines such as TNFa, IL-13b, IL-6, macrophage

* Corresponding author. Fax: þ1 301 319 7451. E-mail address: [email protected] (A.L. Richards). https://doi.org/10.1016/j.micinf.2019.01.002 1286-4579/© 2019 Published by Elsevier Masson SAS on behalf of Institut Pasteur.

inflammatory protein (MIP)-1a/b, MIP-2, MCP-1, CCL5 (RANTES) for leukocyte recruitment [2]. O. tsutsugamushi infection also induces IL-1b release in a time- and dose-dependent manner in LPS-primed macrophages through activation of caspase-1 in vitro [3]. In vivo, the O. tsutsugamushi induces the release of IL-1b and IL-18, together with IL-1a and MIF in serum and the spleen in C57BL/6 mice [3]. Pro-inflammatory cytokines such as IL-1b, IFNg, IL-10, IL-12p40, and TNFa, and chemokines such as CXCL9, CXCL10, and granzymes A and B have been shown to increase in patients with scrub typhus and may contribute to the essential innate immune response against O. tsutsugamushi [4]. The host innate immune response can be mediated by host pattern recognition receptors (PRRs) during initial infection, which serve as microbial sensors triggering signaling cascades resulting in the production of pro-inflammatory mediators [5]. The bestcharacterized of PRRs are Toll-like receptors (TLRs). TLR4 has been implicated in the response to LPS from Gram-negative bacteria [6], while TLR2 recognizes and responds to lipoprotein components from Gram-positive bacteria [7], through MyD88-NF-kB to activate the transcription of IL-1b. Another family of PRRs is

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comprised of NOD-like receptors (NLRs) that detect pathogenassociated molecular patterns (PAMPs) from bacteria and viruses in the cytoplasm to induce the secretion of IL-1b [8]. During O. tsutsugamushi infection, IL-1b production can be mediated by NLR inflammasomes, leading to the cleavage of IL-1b precursors into active IL-1b through caspase-1. O. tsutsugamushi is characterized by either lacking a peptidoglycan layer or a LPS component or by containing these components in unrecognizable forms [9]. It is also speculated that peptidoglycan-like structures are involved in the attachment of O. tsutsugamushi to DCs [10] and strong evidence of these peptidoglycan-like structures has been demonstrated recently [11]. ECs are important in the development of both innate and adaptive immune responses, and have been recently recognized as central and unfortunately undervalued in infectious diseases [12,13]. EC activation is necessary for leukocyte recruitment and plays a central role in control of Gram-positive and Gramenegative bacteria infections [12]. Treatment with LPS activates ECs, leading to the production of pro-inflammatory cytokines and chemokines, which in turn recruit immune cells to amplify the immune response. ECs also induce cytokine production by immune cells thus functioning as immune regulators through activation or suppression of immune cell function. In addition, ECs can serve as antigen presenters by expressing both MHC I and II antigens. Therefore, ECs have now been proposed as “conditional innate immune cells” [12e14]. In O. tsutsugamushi infection, studies on EC responses are very limited. ECs can be stimulated by proinflammatory cytokines including TNF-a and IL-1 released from cells of infected sites resulting the elevation of EC cell adhesion molecules (P-selectin, E-selectin, ICAM-1 and VCAM-1) and production of cytokines and chemokines such as IL-1a, IL-6, IL-8, TNFa, TNFb, CCL2, and CCL5 [2]. The transcriptions of the genes of the chemokines MCP-1, IL-8, and RANTES were also induced in human dermal microvascular endothelial cells (HMEC-1) upon O. tsutsugamushi infection [15]. In a recent study utilizing the human endothelial cell line (ECV304), it was shown that CCL5, CCL17, IL-1a, IL-6, IL-8, IL-10, IL-15, TNFa, and TNFb were induced in response to O. tsutsugamushi infection. This study also demonstrated that O. tsutsugamushi infection could activate the NOD1 pathway followed by IL-32 secretion, which in turn stimulated the production of IL1b, IL-6, IL-8, and ICAM-1 in ECV304 [10]. Conversely, cytokines may also cause tissue pathogenicity such as IL-33, which has been demonstrated to contribute to endothelial damage and renal injury in O. tsutsugamushi-infected mice [16]. However, the role of EC in the pro-inflammatory response against O. tsutsugamushi infection has been largely ignored compared to the studies on the roles of immune cells, and only limited reports on some cytokine genes produced by ECs in response to O. tsutsugamushi. Therefore, the overall characterizations of EC pro-inflammatory response to the infection process when encountering O. tsutsugamushi and the mechanisms underlying these responses are very much incomplete. To more completely investigate the ECs pro-inflammatory response to O. tsutsugamushi infection we characterized the transcriptional profiles for 84 genes from ECs using a cytokine/chemokine array, and identified 40 genes that were greatly modulated during O. tsutsugamushi infection. Our results demonstrated that ECs produced many common pro-inflammatory cytokines/chemokines as seen in other immune responsive cells, thus ECs must play an important role in the pro-inflammatory response against O. tsutsugamushi. ECs were also found to be unique in their cytokine responses to O. tsutsugamushi infection. Notably, CCL17 was identified as the gene that was not only profoundly up-regulated, but the upregulation remained throughout the entire 24-hour time course. These results suggest that ECs, as a major target for O. tsutsugamushi infection,

may contribute to, or play a central role in modulating the host human response to the infection. This study provides us additional information into understanding the innate immune and proinflammatory responses attributed to O. tsutsugamushi-infected human ECs. 1. Materials and methods 1.1. Endothelial cell culture and O. tsutsugamushi infection Human dermal microvascular endothelial cells (HMEC-1) [17] were purchased from ATCC (Manassas, VA). HMEC-1 is an immortalized cell line that retains the morphologic, phenotypic, and functional characteristics of normal human microvascular endothelial cells. The cells were cultured in MCDB131 (without Lglutamine) as base medium (Sigma, St. Louis, MO), supplemented with 10 ng/ml epidermal growth factor (EGF) (Sigma), 1 mg/ml hydrocortisone (Sigma), 10 mM glutamine (Sigma), and 10% FBS (Sigma) in 162 cm2flasks until confluence. The 6-well culture plates were seeded with 5  105 HMEC-1 and cultured over night at 37  C, 5% CO2. O. tsutsugamushi strain Karp seeds were then inoculated into HMEC-1 cells with pre-determined optimal multiplicity of infection (MOI) at 25:1. The cultures were rocked at room temperature for one hour, and then were returned to 37  C, 5% CO2 after replacement with fresh medium for 24 h. 1.2. Total RNA extraction and reverse transcription The cultures were harvested at 0, 1, 3, 6, 12, and 24 h (i.e. T0, T1, T3, T6, T12, and T24, respectively) post inoculation. The total RNA was extracted using QIAGEN RNeasy Mini kit according to the protocol provided by the manufacturer (Valencia, CA), with an additional step of on-column DNase I digestion to remove DNA completely. The yield, purity, and integrity of total RNA were determined by the O.D. 260 and 280 through spectrophotometer. The reverse transcription was done using the RT2 First Kit from QIAGEN according to the company's instruction. Approximately 0.5 mg of total RNA was incubated with genomic DNA elimination mix for 5 min at 42  C, followed by mixing with reversetranscription mix for 15 min at 42  C. The reaction was stopped by incubating mix at 95  C for 5 min. 1.3. Real time PCR array The Human Inflammatory Cytokines and Chemokines RT2 Profiler PCR Array (PAHS-150ZC) from QIAGEN was used to analyze cytokines/chemokines from HMEC-1 cells after inoculation with O. tsutsugamushi. This 96-well formatted plate array contains primers for amplification of 84 key genes central to the pro-inflammatory immune responses. These genes include not only a panel of cytokines and chemokines but also growth factors and hormones that exhibit cellular effects very similar to cytokine family response in a variety of cell types. The gene list in this array has been categorized into seven groups, i.e. twenty-six interleukins, thirty chemokines, two interferons, fifteen growth factors, nine TNF receptor superfamily members, four other cytokines, and sixteen antiinflammatory cytokines. Some genes are categorized into more than one group based on the function. For multiple control purposes, this array includes five house-keeping genes for normalization, one gene for genomic DNA contamination indicator, three genes for RT efficiency evaluation, and three genes for quantitative real-time PCR (qPCR) positive controls. Briefly, the synthesized cDNA was mixed with RT2 SYBR Green Mastermix, and then 25 ml of the PCR components mix was dispensed into each well of RT2 Profiler PCR Array that contained

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specific primers for the amplification of 84 genes. The qPCR was performed using the StepOnePlus (ThermoFisher Scientific, Waltham, MA) with the cycling conditions as follows: 1 cycle of 95  C for 10 min, followed by 40 cycles of 95  C for 15 s, and 60  C for 1 min.

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Before analysis, the original threshold values of qPCR were adjusted to the same value (0.1) across all RT2 Profiler PCR Arrays. All qPCR arrays were repeated once to obtain average CT for quantitation and comparison. The DDCT method was used to calculate the abundance of mRNA expression through www. SABiosciences.con/pcrarraydataanalysis.php for analysis. To ensure there was no significant genomic DNA contamination in the samples and good transcription as well as PCR efficiencies, all average CT values greater than 35 were considered as negative. The CT value(s) for the 84 genes were subtracted from average CT from 5 house-keeping genes (HKGs) to produce DCT, and then compared to corresponding DCT of genes in T0 array to yield DDCT. The differences in CT values were further evaluated to identify signature genes. We set the criteria that if the changes of transcriptional levels of genes were greater 2.0 or less than 0.5 fold difference compared to T0 at any time point, then these genes were selected as up-regulated or down-regulated in response to O. tsutsugamushi Karp infection.

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Fig. 1. The number of Orientia tsutsugamushi-infected HMEC-1 cell genes up-or downregulated over a 24-hour time course. A total of 84 genes were evaluated from O. tsutsugamushi-infected HMEC-1 cells. Forty genes were identified as up-regulated or down-regulated compared to the genes from uninfected cells. The selection criteria for up-regulated and down-regulated genes was 2 fold or 0.5 change in the level of mRNA (via cDNA quantitation by qPCR), respectively, compared to uninfected cells' mRNA. The number of genes up- or edown regulated shown is from the average of two qPCR assays.

2. Results 2.1. Overall cytokine responses to O. tsutsugamushi Karp infection and clustering of signature genes A total of 84 functional genes (including interleukins, chemokines, growth factors, TNF superfamily members, etc.) were evaluated using Human Cytokines and Chemokines Array from QIAGEN following infection as described in Materials and Methods. According to our selection criteria, 40 genes (47.6%) were identified involving either up- or down-regulated. There were 21, 15, 12, 16, and 16 genes that were transcriptionally modulated at T1, T3, T6, T12, and T24, respectively, with more genes modulated at T1 (Fig. 1). Among these, there was more up-regulation in the earlier time course (18, 8, 9, 7, and 8 from T1eT24), and more downregulation in the later time course (3, 7, 3, 9, 8 from T1eT24) (Fig. 1). Cluster analysis of the 40 genes according to the similarity of expression patterns reveals several expression patterns (Fig. 2). These genes clustered in the same group may functionally work together. There were 17 genes that were strongly induced at T1 (at the bottom of Fig. 2), including IL-1a, b, IL-4-7, IL16, CCL7, CCL17, and CCL19-20, CXCL8, CXCL13, NODAL, CNTF, TNFSF13B, and ADIPOQ. 2.2. Functional categories of signature genes Forty cytokine genes were considered as human endothelial cell signature genes and may represent important signaling molecules produced in the early response to O. tsutsugamushi infection in vitro. Functionally, they can be categorized as interleukins, chemokines, growth factors, TNF receptor superfamily members, and others. Among the 40 genes, 28 were up-regulated (Table 1A) and 14 down-regulated (Table 1B) at least at one time point for 2-fold changes during the time course. Moreover, 19 genes (46.3%) showed 2-fold changes for 2 time points. Notably, CCL17 was strongly up-regulated and IFNa2 down-regulated throughout entire time course after O. tsutsugamushi infection. Fourteen of the 40 genes were interleukins (35%, Fig. 3A). The most abundant induction of these interleukins was seen at T1. The

Fig. 2. The hierarchical cluster image of Orientia tsutsugamushi-infected HMEC-1 cell gene expression. Forty genes with changes of fluorescence ratios of 2.0 or 0.5 at any time point compared to uninfected control. Lane 1 is uninfected control; lane 2e6 indicates the time course from one hour (T1) to twenty-four hours (T24) after infection. The genes are grouped by the similarity in their expression patterns across the time course. The color intensity indicates relative magnitude of gene expression from high up-regulation (bright red) to high down-regulation (bright green).

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Table 1 Functional categories of 40 signature genes. A. The transcriptions of twenty-eight genes were up-regulated for 2.0 at least at one time point Chemokines (10): CCL1(I-309), CCL2 (MCP-1)***, CCL5 (RANTES)***, CCL7 (MCP-3), CCL17 (TARC)*****, CCL19, CCL20 (MIP-3a), IL-8 (CXCL8), CXCL10 (INP10)***, CXCL13 Interleukins (9): IL-1a**, IL-1b**, IL-4***, IL-6, IL-7***, IL-10, IL-11***, IL-18, IL-24** Growth factors (4): CNTF, CSF2 (GM-CSF), NODAL (TGFB family member), LIF TNF receptor superfamily members (3): LTA (TNFSF1), LTB (TNFSF3), TNFSF13B** Others (2): ADIPOQ (ACRP30), SPP1 B. The transcriptions of fourteen genes were down-regulated for 0.5 at least at one time point Chemokines (2): CX3CL1**, CXCL11 (I-TAC, IP-9)** Interleukins (6): IL-5, IL-13, IL-16, IL-17F***, IL-24, IL-27*** Growth factors (2): CSF3 (G-CSF), MSTN (GDF8, ligand of TGFB)** TNF receptor superfamily members (3): LTB (TNFSF3), TNFSF11***, TNFRSF11B** Others (1): IFNa2***** Note: a. **change in 2 time points; b. ***change in 3 time points; c. *****change in five time points.

IL-4 (10.87), IL-7 (8.90), IL-1b (7.56), IL-1a (2.65), IL-6 (2.75) are among top 5 highest inductions, while the transcription of IL-27 (0.32) was significantly decreased at T1. IL-10 was gradually induced from T1 to T6 with highest level of transcription seen at T6.

At T24, more genes that were down-regulated returned to baseline level, except, IL-1a/b and IL-18, which were the only three genes up-regulated at T24. IL-17F and IL-27 were significantly downregulated for 3 time points.

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Fig. 3. The 24-hour time course of HMEC-1 gene expression after Orientia tsutsugamushi infection. Graphical representation of the expression of interleukins (A), chemokines (B), growth factors (C), TNF super family (D), and others (E).

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2.3. The most abundantly expressed cytokine genes in endothelial cells By examining average DD CT values of all 84 genes, we found that fifteen genes were very active at the transcription levels with low DD CT values (between 1.1 and 10.5), while the average DD CT values for the rest of genes were near 17 over the time course (Fig. 4). In this category were included genes such as IL-6, IL-12A, TGFB2, CSF1, CCL20, CXCL1, CXCL2, CXCL12, MIF, GPI, VEGFA, and C5, indicating they were constitutively and highly expressed in ECs. Among these, IL-6 and CCL20 were not only constitutively expressed at high levels, but also were induced 2.0 fold changes during the time course (Fig. 3A,B). 3. Discussion 3.1. EC response pattern during O. tsutsugamushi infection The central role of ECs in response to other inflammatory diseases has been increasingly recognized [12]. In this study, we explored the transcriptional profiles of innate immunity from O. tsutsugamushi infected-ECs and provided more information on cytokines/chemokines and their possible roles in defense and survival from O. tsutsugamushi infection. The response course and outcome of bacterial infections of ECs represent a battle between host and pathogen. The host response can be blunted by pathogens through modulating the pro-inflammatory cytokine response by blocking cytokine production to escape killing [8,18]. Forty genes identified in this study may serve as signature genes upon early O. tsutsugamushi infection. These genes were either upor down-regulated for at least one point out of 5 time points during a 24-hour observation period. The top five induced interleukins at earlier time point T1 are IL-4, IL-7, IL-1b, IL-1a, and IL-6. Moreover, some genes were greatly modulated through three or five time points including up-regulation of CCL17, IL-4, IL-7, IL-11, CCL2, CCL5, and CXCL10, and down-regulation of IFNa2, IL17F, and IL27. The

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Twelve out of 40 modulated genes were categorized as chemokines in response to O. tsutsugamushi infection (30%, Fig. 3B). Similar to the interleukins, more chemokine gene responses were observed at T1 with nine genes up-regulated and one gene downregulated. The genes with elevated transcription included CCL2, CCL5, CCL7, CCL17, CCL19, CCL20, CXCL8, CXCL10, and CXCL13 at T1 with CCL17 at the top with 6.87 fold increases in transcription when compared to T0. Moreover, the induction of CCL17 was noticed in all five time points with peak at T1. CCL2 and CCL5, also called MCP-1 and RANTES, were induced over 3 time points. We also observed two down-regulated genes (CXCL11 and CX3CL) covered for 2 time points. The third group of genes selected was growth factors including 4 up-regulated genes of NODAL (Nodal homologs), CSF2 (GM-CSF), CNTF (ciliary neurotropic factor), and LIF (leukemia inhibitory factor) at earlier time points (T1-T3), as well as 1 down-regulated genes of CSF3 (G-CSF) at T3 and MSTN (myostatin) at T12-24 (Fig. 3C). The forth group of cytokines included the TNF super family members with five genes (TNFSF1, TNFSF3, TNFSF11, TNFRSF11B, and TNFSF13B (Fig. 3D). The most up-regulated gene among this family was TNFSF13B (5.08) at T1. The TNFSF11 and TNFRSF11B were down-regulated in 2 time points. The last group named “others” contained 3 genes: IFNa2, ADIPOQ (adiponectin, also called ACRP30), and SPP1 (secreted phosphoprotein 1) (Fig. 3E). Notably, IFNa2 was the only gene in this study that was found to be greatly down-regulated throughout the entire time course (0.13e0.16 fold reduction).

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Fig. 4. The constitutive expression of 12 dominant genes from Orientia tsutsugamushiinfected HMEC-1 cell over a 24-hour time course. These 12 genes were not obviously modulated upon O. tsutsugamushi infection, but their transcriptional levels remained constant at higher levels compared to other genes assessed. Their DCT values were below 10 after normalized to the CT values of house-keeping genes, while the average DCT values of the rest of genes were much higher, around 17.

roles and significance of these genes in immunity and pathogenicity against orientiae may be important through synergies or antagonism to decide the outcomes of ECs. Both Th1 (such as IL-1a/ b, IL-6, CSF2/GM-CSF2, CCL5/RANTES) and Th2 (such as IL-4, IL-7, IL8, IL-10, CCL2/MCP1) cytokines were involved. These suggested that ECs upon infection by O. tsutsugamushi produce Th1 response for immunity but also Th2 response for repairing and regulation, and possibly favoring O. tsutsugamushi growth leading to pathogenesis. 3.2. CCL17 In the present study, both CCL17 and CCL19 gene transcriptions were stimulated. Specifically, we observed a strong up-regulation of the chemokine CCL17 at all study points in ECs infected with O. tsutsugamushi Karp (Fig. 3B). This is the only gene in this study with up-regulation throughout the 24-hour infection period. CCL17 [also called thymus and activation-regulated chemokine (TARC)] plays an important role in T cell development in thymus as well as in trafficking and activation of mature T cells [19]. The CCL17 was originally implicated in the attraction of Th2 lymphocytes and thus considered as an M2 cytokine. Later, it was reported to be able to attract effector/memory Th1 lymphocytes [20]. CCL17 has been implicated in inflammatory diseases such as atopic dermatitis, intestinal infection, and asthma [21e23]. A recent report demonstrated that co-administration of CCL17 and CCL19 as a nasal adjuvant significantly enhanced the immunogenicity in an antidental caries DNA vaccine study in rodents [24]. It was found that CCL17 and CCL19 could induce a greater increase in the number of mature DCs in the spleen and DLNs (draining lymph nodes), and then stimulate antibody response as well [24]. Although CCL19 and its receptor CCR7 are considered essential molecules for facilitating the trafficking of mature DCs to secondary lymphoid organs and helping to establish a microenvironment to initiate primary immune response, CCL17 is required in this CCL19-CCR7-dependent migration of DCs [25]. Further, evidence indicated that GM-CSF could drive CCL17 production through an IFN regulatory factor-4 (IRF-4) pathway in human monocytes, murine macrophages, and mice [26]. In O. tsutsugamushi infection of ECV304 cells, CCL17 gene

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was reported to be up-regulated moderately in ECs along with other cytokines after infection with O. tsutsugamushi Boryong strain [10]. Our data demonstrate that CCL17 is strongly up-regulated during O. tsutsugamushi infection of HMEC-1 and further study on this gene is necessary. Given that CCL17 is activated in other infectious conditions, combined with its efficacy as an adjuvant in vaccine study, it is tempting to speculate that CCL17 may be one of critical players in the innate immune response against scrub typhus as well. The mechanisms as to how CCL17 and CCL19 regulate O. tsutsugamushi infection are currently unknown. 3.3. IL-1b, IL-4, IL-7, and IL-18 We demonstrated that IL-4, IL-7, and IL-1b were the top three cytokine genes among interleukins induced early in the infection of ECs by O. tsutsugamushi at T1 (Fig. 3A). IL-4 has many biological roles such as inducing the differentiation of naive helper T cells (Th0 cells) to Th2 cells and B cells into plasma cells. IL-4 has been shown to decrease the production of Th1 cells, macrophages, IFNg, and dendritic cells [27]. IL-7 is a cytokine important for B and T cell development [28]. IL-1b has been shown to play a key role in initiating and maintaining the inflammatory response against Gram-negative bacteria [29], and IL-1 receptor signaling is required for efficient host protection against O. tsutsugamushi infection [3]. In addition, IL-18 was induced at T1 and T24, a similar pattern to that of IL-1b. Both IL-1b and IL-18 belong to IL-1b family and their synergistic role has been implicated in many innate immune responses [30]. Besides, IL-1b together with TNF is commonly described as ECs activators after infection [31]. High levels of IL-1b, plus IFNg, IL-10, IL-12p40, TNFa have been detected in scrub typhus patients [32]. The O. tsutsugamushi induced secretion of IL-1b and corresponding activation of caspase-1 has been thought to be through ASC-and caspase-1-dependent pathway in macrophages [3]. 3.4. IL-10, IL-12 and IL-27 IL-10 is considered an important immunoregulatory cytokine that inhibits many cytokines including IL-1, IL-2, IL-3, IL-4, TNFa, IFNg, and GM-CSF to prevent immunopathological lesions due to overactive immune response during O. tsutsugamushi infection. But it can also cause persistence of pathogens by interfering with innate and adoptive immunity [33]. A significant positive relationship between IL-10 level and bacterial load has been reported in scrub typhus patients, (but this relationship with bacterial load has not been seen for IFNg, TNFa, and IL-1b) [34]. IL-10 could promote bacterial survival inside the hostile environment of activated murine macrophages by inhibiting TNFa during O. tsutsugamushi infection [35]. We noted an elevation of IL-10 at T3 (1.66 fold) and T6 (2.38 fold). This elevated IL-10 level may inhibit the proinflammatory responses and favor bacterial replication in ECs. In contrast to IL-10, IL-12 can stimulate the Th1 response and its family includes IL-12, IL-23, IL-27 and IL-35 [36]. We only noted slight induction of IL-12B gene at the early time point T1 (1.58 fold), while IL-12A was not up-regulated but constitutively expressed at higher levels over the time course (Fig. 4). The DD CT values were around 10 (Fig. 3A) and 18 (data not shown) in IL-12A and IL-12 B, respectively. In this study, IL-27, a member of IL-12 family, was down-regulated significantly at 3 time points (Fig. 3A). IL-27 plays an important function in regulating the activity of B- and T-lymphocytes by its interaction with a specific cell-surface receptor complex known as IL27R and gp130 to regulate both innate and adaptive immunity largely through Jak/Stat signaling pathway. It was initially linked with the development of Th1 responses, but it is now recognized as a possible antagonist of different classes of

inflammation through modifying CD4(þ) and CD8(þ) T cells, to induce IL-10, and to promote specialized T regulatory cell responses [37]. The significance of down-regulation of IL-27 is not clear in ECs. The balance of these cytokines at different levels may reflect a defense mechanism that ECs use to respond to O. tsutsugamushi infection. 3.5. IL-6 family We also observed several members from the IL-6 family (IL-6, IL11, LIF, CNTF, and IL-27) that were modulated. IL-6 has a broad effect on cells from both immune system and non-immune system. They are involved in both innate and adaptive immunities [38]. IL-6 has context-dependent pro- and anti-inflammatory properties and is now regarded as a keystone cytokine and a prominent target for clinical intervention [38]. IL-6 and IL-11 are two important molecules in this family. Their signaling includes the activation of the GP130-Janus kinase signaling cascade and transcription factor STAT3. In our study, IL-6 was not only stimulated at T1 but also constitutively expressed at very high levels throughout 24 h (Figs. 3A and 4), while IL-11 was up-regulated markedly at 3 time points. IL-11 has been thought to be associated with cell growth and differentiation that has been suggested a role for this cytokine in cancer and therapeutic targeting [39,40]. IL-11 has also been shown to improve platelet recovery after chemotherapy-induced thrombocytopenia and could stimulate the growth of certain lymphocytes [41]. It may be reasonable to assume that one of the roles of this elevated IL-11 in this study might reflect growth/repair characteristics in ECs via promoting the growth of platelets and ECs. 3.6. IL-17 In this transcriptional expression array, IL-17A was only upregulated slightly at T1 (data not shown) but IL-17F was downregulated profoundly from T6 to T24 (Fig. 3A). IL-17 family includes six members. Among them, IL-17A and IL-17F are best studied and both mediate pro-inflammatory responses [40]. Special T helper subset T cells (Th17 cells) are the major source of IL-17A and IL-17F. IL-17 can stimulate neutrophils to kill extracellular bacteria and fungi [42]. IL-17A and IL-17F play roles in the host defense against pathogens of epithelial and mucosal infections [40]. They can induce pro-inflammatory cytokines, anti-pathogenic peptides and chemokine secretion to recruit innate immune cells to eliminate pathogens [40]. IL-17 can also synergize with other cytokines such as TNFa, IL-1b, and IFNg to activate a large number of target genes [43]. Patients with the defective IL-17A/F suffer from high susceptibility to S. aureus, Streptococcus pneumonia and Candida albicans infections [44]. We found no obvious transcriptional elevation in IL17 A/F, which may suggest that IL-17A/F signaling may not be critical for ECs to release upon infection with O. tsutsugamushi or O. tsutsugamushi has a mechanism to inhibit the transcription of the IL-17 A/F genes. 3.7. IFNa2 IFNs are also key cytokines of the innate immune response stimulated by many pathogens and PAMPS from infected cells [45]. Type I IFNs (IFNa) can exert an antiviral action and are capable of decreasing the proliferation of dividing cells to perform immunomodulatory activities. Among them, IFNa2 is the first highly active human IFN for the use in research and clinical application for antiviral and antitumor medicine [46]. In humans, the IFNa2 gene is under constraints to prevent mutations, suggesting that it has an essential role in physiology [47]. It binds to its receptors (IFNaR1 and 2) to mainly activate JAK/STAT signaling pathway to trigger the

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transcription of a large family of responsive genes. In this study, we noted an extreme reduction of IFNa2 transcription over the whole time course in ECs during O. tsutsugamushi infection (Fig. 3E). Since we also observed relatively low induction on some of important Th1 response cytokines (such as IL-12), it might be speculated that, unlike immune cells, the inefficient expression of these antiinflammatory cytokines in ECs might be one of the reasons for ECs to become main target cells of O. tsutsugamushi infection. Since the IFNa2 has been successfully used in antiviral and antitumor clinics, it might also be worthy to find out if it is useful for clinical administration of IFNa2 in intervention for scrub typhus patients, especially for severe cases. Further investigation is needed. In conclusion we characterized 84 cytokine/chemokine genes at transcription level in order to explore important genes that are involved in innate immune response in O. tsutsugamushi infection. Forty out of 84 genes were identified as O. tsutsugamushi-infected ECs (HMEC-1) responsive genes. Moreover, the remarkable upregulation of CCL17 through entire time course may suggest its critical role in innate immunity against O. tsutsugamushi and needs to be further characterized. Furthermore, we observed a great down-regulation of IFNa2. Both Th1 and Th2 cytokines are involved but Th1 function appears dampened somehow that might reflect the nature of endothelial cells, differing from major immune cells. Our results indicated that ECs play an essential part in the proinflammatory response induced by O. tsutsugamushi infection. Data from this study provides new information and starting points for the further investigation on the mechanisms underlying the signaling cascades and production of pro-inflammatory cytokines that contribute to immunity and pathogenicity in scrub typhus. Conflict of interest The authors declare no conflict of interest. Acknowledgements This work was supported and funded by U.S. Military Infectious Diseases Research Program through work unit number WJ0057_14_NM. The views expressed in this article are those of the author and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the U.S. Government. Allen L. Richards is an employee of the U.S. Government. This work was prepared as part of his official duties. Title 17 U.S.C. x105 provides that ‘Copyright protection under this title is not available for any work of the United States Government.’ Title 17 U.S.C. x101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as part of that person's official duties. References [1] Kelly DJ, Richards AL, Temenak J, Strickman D, Dasch GA. The past and present threat of rickettsial diseases to military medicine and international public health. Clin Infect Dis 2002;34:S145e69. [2] Mansueto P, Vitale G, Cascio A, Seidita A, Pepe I, Carroccio A, et al. New insight into immunity and immunopathology of Rickettsial diseases. Clin Dev Immunol 2012;2012:967852. https://doi.org/10.1155/2012/967852. 26 pages. [3] Koo JE, Hong HJ, Dearth A, Kobayashi KS, Koh YS. Intracellular invasion of Orientia tsutsugamushi activates inflammasome in asc-dependent manner. PLoS One 2012;7:e39042. [4] Paris DH, Shelite TR, Day NP, Walker DH. Unresolved problems related to scrub typhus: a seriously neglected life-threatening disease. Am J Trop Med Hyg 2013;89:301e7. [5] Randow F, MacMicking JD, James LC. Cellular self-defense: how cellautonomous immunity protects against pathogens. Science 2013;340:701e6. [6] Poltorak A, Ricciardi-Castagnoli P, Citterio S, Beutler B. Physical contact between lipopolysaccharide and toll-like receptor 4 revealed by genetic complementation. Proc Natl Acad Sci U S A 2000;97:2163e7.

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