Abstract
Purpose
The tick-borne bacterium Neoehrlichia mikurensis causes the infectious disease neoehrlichiosis in humans. Vascular endothelium is one of the target cells of the infection. Neoehrlichiosis patients with compromised B cell immunity present with more severe inflammation than immunocompetent patients. The aim of this study was to compare the cytokine profiles of immunocompetent and immunosuppressed patients with neoehrlichiosis.
Methods
Blood samples from Swedish and Norwegian immunosuppressed (N = 30) and immunocompetent (N = 16) patients with neoehrlichiosis were analyzed for the levels of 30 cytokines, using a multiplex cytokine assay and ELISA. A gender-matched healthy control group (N = 14) was analyzed in parallel. Data were analyzed using the multivariate method OPLS-DA.
Results
The multiplex cytokine analyses generated more cytokine results than did the uniplex ELISA analyses. Multivariate analysis of the multiplex cytokine results established that increased levels of FGF2, GM-CSF, CXCL10, and IFN-γ were associated with immunosuppressed patients, whereas increased levels of IL-15 and VEGF were associated with immunocompetent neoehrlichiosis patients. When multivariate analysis findings were confirmed with uniplex ELISA, it was found that both groups of patients had similarly elevated levels of VEGF, FGF2 and IFN-γ. In contrast, the immunosuppressed patients had clearly elevated levels of CXCL10, CXCL13 and BAFF, whereas the immunocompetent patients had the same levels as healthy controls.
Conclusion
Pro-angiogenic and type 1 cytokines were produced as part of the host response of neoehrlichiosis independent of immune status, whereas immunosuppressed neoehrlichiosis patients produced cytokines required for B cell-mediated defense.
Similar content being viewed by others
Introduction
Neoehrlichia mikurensis is an emerging tick-borne bacterium that can infect humans and cause neoehrlichiosis [1]. More than half of the published cases from Europe involve immunosuppressed patients who have presented with fever of uncertain cause, often in combination with thromboembolic and vascular events, such as repeated and severe thrombophlebitis, deep vein thrombosis, pulmonary embolism and transitory ischemic attacks [2]. In contrast, the clinical picture of neoehrlichiosis in immunocompetent individuals can vary from asymptomatic cases to febrile disease and even suspected fatal outcome [3,4,5]. Immunocompetent patients with neoehrlichiosis have presented with erythematous skin rashes in the absence of serological evidence to support a diagnosis of concomitant Borrelia-infection [6, 7]. We showed in a recent survey of a cohort of 40 Swedish neoehrlichiosis patients that while there was no difference in the incidence of vascular events between immunosuppressed and immunocompetent patients, the immunosuppressed ones tended to contract venous vascular events whereas the immunocompetent ones had involvement of the arterial side of the circulation [8]. N. mikurensis has been identified within circulating endothelial cells in the blood of patients with neoehrlichiosis, which implies that vascular endothelium is one of the targets of this infection [9].
Patients with compromised B cell immunity are susceptible to severe neoehrlichiosis. Patients at risk for grave disease are those with clonal B cell diseases, such as systemic rheumatic diseases, other autoimmune diseases and hematologic malignancies [1]. Biological agents directed against B cells, e.g., rituximab targeting CD20 on B cells, are important risk factors and are commonly used to treat multiple sclerosis patients, malignant B cell lymphomas and systemic rheumatic diseases [1, 10, 11]. Advanced age, recent chemotherapy, systemic corticosteroid treatment and splenectomy are additional risk factors for severe neoehrlichiosis [2].
N. mikurensis belongs to the family Anaplasmataceae, like the related human pathogenic bacterial species Anaplasma phagocytophilum and Ehrlichia chaffeensis [12]. However, unlike the latter two species, N. mikurensis has not yet been detected in North America, possibly because it has Ixodes ricinus as its main tick vector [2]. Due to its intracellular nature, N. mikurensis does not grow in blood cultures or any other cell-free media and it is consequently missed by routine microbiologic methods. At present, PCR is the sole diagnostic method available since no serological methods have been established [2].
To date, only two reports concerning cytokine responses in neoehrlichiosis patients have been published, comprising one immunosuppressed and two immunocompetent patients [7, 13]. The first case was a 77-year-old immunosuppressed individual with B cell chronic lymphocytic leukemia who exhibited increased levels of the cytokines, interleukin (IL)1β, 6, 8, 10, interferon gamma (IFN)-γ and tumor necrosis factor (TNF)-α [13]. The immunocompetent patients with neoehrlichiosis had increased levels of pro-inflammatory- and Th1 cytokines in serum, which correlated with concentrations of N. mikurensis DNA in serum [7]. The detected cytokines were increased levels of IFN-γ-induced protein 10 (CXCL10), IL-1β, IL-6, IL-12, IFN-γ, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1β, and TNF-α.
The objective of this study was to compare the cytokine responses in the blood of immunosuppressed and immunocompetent patients with neoehrlichiosis to increase the understanding of how immune defenses to this emerging pathogen are engaged depending on immune status.
Materials and methods
Study subjects
Blood samples derived from patients (N = 46) diagnosed by PCR with neoehrlichiosis were investigated together with samples from age- and gender-matched healthy controls (N = 14). Neoehrlichiosis patients were divided into two study groups, immunosuppressed (IS-Neo; N = 32) and immunocompetent (IC-Neo; N = 14). Patients were diagnosed at Sahlgrenska University Hospital, Gothenburg, Sweden (N = 36), Sørlandet Hospital, Kristiansand, Norway (N = 8) or in the Tick Borne Diseases STING study (N = 2) [14]. Clinical features of the study patients are listed in Table 1.
Ethics statements
All participants provided written informed consent for the study. The study was approved by the local Ethical Review Boards of Gothenburg (298-05 and 2018/658) and Uppsala (2015/249), Sweden and by the Norwegian Regional Committee for Medical and Health Research Ethics, the South-Eastern region (REK ref. 204409). The STING study [14] was approved by the Regional Ethical review board at Linköping University, Sweden (M132-06). All the participants provided written informed consent for the study.
Blood samples
Plasma and serum samples isolated from venous blood collected from the patients before the administration of antibiotics (doxycycline) were analyzed, together with reference plasma and serum samples from healthy gender-matched individuals (N = 14). The Swedish samples were collected between the years 2009 and 2019 and were stored at − 140 °C until analysis. Samples from Norwegian patients were serum samples, stored at − 70 °C until analysis.
Cytokine assays
The concentrations of 27 cytokines in diluted plasma and serum samples were analyzed using the fluorescence-based immunoassay Bioplex Pro™ human cytokine standard 27-plex panel (Bio-Rad Laboratory, Hercules, CA, USA) encompassing IL-1β, IL-1 receptor antagonist (IL-1RA), IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-17, eotaxin, fibroblast growth factor basic (FGF) 2, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), IFN-γ, CXCL10, MCP-1, MIP-1α, MIP-1β, platelet-derived growth factor-BB (PDGF-BB), RANTES, TNF-α; and vascular endothelial growth factor (VEGF). Cytokine data were analyzed using a Bio-Rad BioPlex 200 instrument equipped with BioPlex Manager software version 6.0 (Bio-Rad Laboratory). Data points that were measured as default “out of range” by the manufacturer’s software were manually determined by calculating the fluorescence intensity of each sample and comparing it with the fluorescence intensity of the standard curve, as described by Breen et al. [15]. The data sets derived from the 27-plex cytokine array are presented as the fold change of concentration for each cytokine level, relative to an average value of the healthy individuals. This was done to normalize assay-to-assay variation. Fold-change calculations were always based on data obtained in the same microtiter plate, to compensate for inter-assay variability.
Quantikine ELISA (R&D systems, Minneapolis, MN, USA) kits were used for uniplex concentration measurements of the cytokines CXCL10, chemokine (C-X-C motif) ligand 13 (CXCL13), IFN-γ, IL-15, IL-21, VEGF, GM-CSF, FGF2 and B cell-activating factor (BAFF). The serum and plasma samples were diluted 1:2 and analyzed in 96-well Half-Area Microplates (Corning, Tewksbury, MA, USA). Samples with high cytokine concentrations that were out of range were diluted and re-analyzed.
Statistics
The multivariate method, “orthogonal projection to latent structures by means of partial least squares-discriminant analysis” (OPLS-DA), was employed, using SIMCA-P software version 15.0.2 (MKS Data Analytics Solutions, Malmö, Sweden). Generated two-component models are given a value for explanatory power or robustness of fit, R2, which estimates the amount of variance in Y that is explained by the X-variables. A high value implies that the X-variables have generated a model capable of explaining differences between study groups. Models are also given a value for stability, Q2, which is determined with cross validation, whereby one study subject is removed from the model to test the capacity of the remaining subjects to separate the study groups. This was repeated for all subjects. A high value indicates that the model is stable no matter which subject is removed.
The Mann–Whitney test was used to compare groups of two and the Kruskal–Wallis test to compare groups of three, using GraphPad Prism 8 (GraphPad Software Inc., La Jolla CA, USA). P-values < 0.05 were considered statistically significant.
Results
Defining the cytokine patterns of immunosuppressed and immunocompetent patients with neoehrlichiosis using the multiplex cytokine assay
The levels of 27 cytokines were analyzed in the plasma of immunosuppressed patients with neoehrlichiosis (IS-Neo; N = 23), immunocompetent patients with neoehrlichiosis (IC-Neo; N = 7) and healthy controls (HC; N = 10). The IS-Neo group possessed higher levels of CXCL10, IFN-γ, FGF2, GM-CSF, IL-1RA, IL-5, IL-6, IL-10, IL-12, IL-17, MCP-1, MIP-1β and TNF-α, and lower levels of IL-15 compared with the IC-Neo group (Fig. 1). VEGF levels were also higher in the IS-Neo group but did not reach the statistical significance (p value = 0.0846).
The following cytokines were not detected or statistically significant in the fold change comparison between the samples from patients with neoehrlichiosis and the average levels of the healthy controls: Eotaxin, G-CSF, IL-1β, IL-2, IL-4, IL-7, IL-8, IL-9, IL-13 and MIP-1α. Two cytokines, PDGF-bb and RANTES, were not taken into consideration since they may leak from blood platelets if the sample is not immediately centrifuged and frozen, giving rise to potentially false-positive results [16, 17].
Using the multivariate OPLS-DA method, we constructed a model in which the study patients were set as Y-variables (Y1 for immunosuppressed and Y2 for immunocompetent) and cytokine levels (25 cytokines) were set as X-variables. The two study groups formed partly overlapping clusters and the generated two-component model (PC1 and PC2) had an explanatory power of 65% (a goodness of fit, R2Y = 0.65) and stability of 55% (Q2Y = 0.55) (Fig. 2A). Cytokines were grouped into four main categories: cell-mediated immunity, inflammation, growth factors and “other”. The cytokines that contributed to distinguishing the immunocompetent patients from the immunosuppressed patients with neoehrlichiosis are shown in a loading plot (Fig. 2B). Here, increased levels of FGF2, IFN-γ, GM-CSF and CXCL10 were associated with IS-Neo, whereas increased levels of VEGF and IL-15 were associated with IC-Neo. Therefore, these cytokines were chosen for further analyses, using the ELISA method.
Verification of multiplex cytokine data by uniplex ELISA
During the course of the study, additional patients were recruited. To confirm our multiplex cytokine assay results, we chose to verify our findings by re-testing patient samples for selected cytokines using uniplex ELISA kits. The levels of FGF2, IFN-γ, GM-CSF, CXCL10, IL-15, and VEGF were measured in plasma and serum samples previously analyzed with multiplex (IS-Neo; N = 23, IC-Neo; N = 7, HC; N = 10) and in the samples of newly recruited patients (IS-Neo; N = 9, IC-Neo; N = 7, HC; N = 4). CXCL10 was clearly elevated in the blood of IS-Neo, compared with the IC-Neo group and the healthy control group (Fig. 3A). In addition, the levels of VEGF (Fig. 3B), FGF2 (Fig. 3C), and IFN-γ (Fig. 3D) were similarly raised in the blood of both the IS-Neo and IC-Neo groups compared with the healthy controls. Regarding the IL-15 and GM-CSF levels, no significant differences were observed between the two study groups or healthy controls (Fig. 3E,F).
Additional cytokines
As most of immunosuppressed patients with neoehrlichiosis had suppressed B cell immunity (Table 1), cytokines of importance for B cell function were also analyzed, namely the levels of BAFF, CXCL13 and IL-21. A massive production of BAFF (Fig. 4A) was seen among the IS-Neo group with almost 17-fold higher levels, compared with the levels in the IC-Neo group and in healthy individuals. The IS-Neo patients also exhibited higher levels of CXCL13 than the IC-Neo patients and the healthy control group (Fig. 4B). No significant differences in IL-21 cytokine levels were observed between the study groups or healthy controls (Fig. 4C).
Discussion
The 27-plex cytokine assay yielded more findings than the ELISA assays. Multiplex cytokine assays make it possible to screen for many cytokines using limited volumes of patient sample but have lower reliability because of the large number of capture and detection antibodies employed. Heterophilic antibodies may be present in human sera and can bind to immunoglobulins of other species, giving rise to false-positive results by bridging capture and detection antibodies, or false-negative results by sterically blocking capture antibody binding sites, or both [18]. Such antibodies may also be present in animal sera used to manufacture the immunoassays. With this in mind, we chose to first screen the patient samples for cytokine patterns using multiplex assay in combination with the multivariate method for pattern recognition, and subsequently to verify these findings by (uniplex) ELISA.
The immunosuppressed patients and the immunocompetent patients with neoehrlichiosis had similarly elevated levels of IFN-γ, VEGF and FGF2. Interferon-gamma is the prototype cytokine for cell-mediated immunity, which facilitates the inactivation of intracellular microbes by various mechanisms, one of which is to boost the microbicidal capacity of macrophages and monocytes. Raised levels of IFN-γ were also seen in the immunosuppressed and immunocompetent patient cases published by our group, albeit those results were based on 6-plex and 27-plex immunoassays, respectively [7, 13]. Since N. mikurensis is an intracellular pathogen, cellular immunity is likely to be necessary for host control of infection. Many closely related bacteria of N. mikurensis, such as E. ruminantium and E. chaffeensis, induce type 1 cell-mediated immunity and IFN-γ production in infected hosts [19,20,21,22].
The finding of increased levels of the growth factors VEGF and FGF2 is novel. We showed in 2019 that the vascular endothelium is a target of neoehrlichial infection in humans [9]. Further, more than half of patients with neoehrlichial infection have evidence of inflamed and/or damaged blood vessels as we recently reported in a cohort study [8]. It is reasonable to assume that the increased levels of VEGF and FGF2 were produced to heal injured endothelium. VEGF and FGF2 stimulate migration and proliferation of endothelial cells to generate and stabilize new blood vessels [23]. Increased serum levels of VEGF and FGF2 have earlier been reported in patients with autoimmune vascular diseases such as polyarteritis nodosa [24] and Takayasu’s arteritis [25], two conditions with clinical pictures that can be confused with neoehrlichiosis [8].
The immunosuppressed neoehrlichiosis patients had clearly elevated levels of CXCL10, CXCL13 and BAFF, whereas the immunocompetent patients had the same levels as healthy controls. CXCL10, also known as interferon-gamma-induced protein 10, is secreted by several cell types such as monocytes, endothelial cells and fibroblasts in response to IFN-γ, which itself is mediated by the IL-12 cytokine family [26, 27]. CXCL10 is also involved in promotion of T cell adhesion to endothelial cells and angiogenesis [26]. CXCL10 is also an angiostatic cytokine that can counterbalance angiogenic activities such as FGF2-induced neovascularization [28].
BAFF and CXCL13 are important factors for B cell development and chemoattraction. BAFF is mainly expressed in monocytes and stimulates proliferation and differentiation of B cells [29]. CXCL13, also known as B cell-attracting chemokine 1, is expressed by both follicular dendritic cells and germinal center T follicular helper cells in the B cell follicles [30]. The elevated levels of these two cytokines in the immunosuppressed group of patients probably depends to a large extent on the fact that the majority of the patients had compromised B cell immunity due to clonal malignant or autoimmune diseases combined with anti-B cell therapy. A study by Rosengren et al. showed that serum CXCL13 is predictive of the rate of B cell repopulation following a course of the anti-CD20 monoclonal antibody, rituximab, which 67% of the immunosuppressed patients in this study were treated with [31]. Similarly, B cell depletion brought on by rituximab infusions leads to increased serum levels of BAFF [29].
To conclude, pro-angiogenic and type 1 cytokines (INF-γ) were produced as part of the host response of neoehrlichiosis, independent of immune status, whereas neoehrlichiosis patients with compromised B cell immunity had raised levels of cytokines needed to compensate for B cell depletion.
Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
References
Grankvist A, Andersson PO, Mattsson M et al (2014) Infections with the tick-borne bacterium “Candidatus Neoehrlichia mikurensis” mimic noninfectious conditions in patients with B cell malignancies or autoimmune diseases. Clin Infect Dis 58(12):1716–1722. https://doi.org/10.1093/cid/ciu189
Wenneras C (2015) Infections with the tick-borne bacterium Candidatus Neoehrlichia mikurensis. Clin Microbiol Infect 21(7):621–630. https://doi.org/10.1016/j.cmi.2015.02.030
von Loewenich FD, Geissdorfer W, Disque C et al (2010) Detection of “Candidatus Neoehrlichia mikurensis” in two patients with severe febrile illnesses: evidence for a European sequence variant. J Clin Microbiol 48(7):2630–2635. https://doi.org/10.1128/jcm.00588-10
Li H, Jiang J-F, Liu W et al (2012) Human infection with Candidatus neoehrlichia mikurensis, China. Emerg Infect Dis 18(10):1636–1639. https://doi.org/10.3201/eid1810.120594
Welc-Falęciak R, Siński E, Kowalec M et al (2014) Asymptomatic “Candidatus Neoehrlichia mikurensis” Infections in Immunocompetent Humans. J Clin Microbiol 52(8):3072. https://doi.org/10.1128/JCM.00741-14
Quarsten H, Grankvist A, Hoyvoll L et al (2017) Candidatus Neoehrlichia mikurensis and Borrelia burgdorferi sensu lato detected in the blood of Norwegian patients with erythema migrans. Ticks Tick Borne Dis 8(5):715–720. https://doi.org/10.1016/j.ttbdis.2017.05.004
Grankvist A, Sandelin LL, Andersson J et al (2015) Infections with Candidatus Neoehrlichia mikurensis and Cytokine Responses in 2 Persons Bitten by Ticks. Sweden Emerg Infect Dis 21(8):1462–1465. https://doi.org/10.3201/eid2108.150060
Höper L, Skoog E, Stenson M et al (2020) Vasculitis due to Candidatus Neoehrlichia mikurensis: a cohort study of 40 Swedish patients. Clin Infect Dis. https://doi.org/10.1093/cid/ciaa1217
Wass L, Grankvist A, Bell-Sakyi L et al (2019) Cultivation of the causative agent of human neoehrlichiosis from clinical isolates identifies vascular endothelium as a target of infection. Emerg Microbes Infect 8(1):413–425. https://doi.org/10.1080/22221751.2019.1584017
Andreasson K, Jonsson G, Lindell P et al (2015) Recurrent fever caused by Candidatus Neoehrlichia mikurensis in a rheumatoid arthritis patient treated with rituximab. Rheumatology (Oxford) 54(2):369–371. https://doi.org/10.1093/rheumatology/keu441
Dadgar A, Grankvist A, Wernbro L et al (2017) Fever of unknown origin in a multiple sclerosis patient on immunomodulatory therapy was due to neoehrlichiosis [Oklar feber hos patient med MS och rituximabbehandling var neoehrlichios - Ny fästingburen infektion som är svår att diagnostisera]. Lakartidningen. 114:6
Grankvist A, Jaén-Luchoro D, Wass L et al (2021) Comparative genomics of clinical isolates of the emerging tick-borne pathogen Neoehrlichia mikurensis. Microorganisms. https://doi.org/10.3390/microorganisms9071488
Welinder-Olsson C, Kjellin E, Vaht K et al (2010) First case of human “Candidatus Neoehrlichia mikurensis” infection in a febrile patient with chronic lymphocytic leukemia. J Clin Microbiol 48(5):1956–1959. https://doi.org/10.1128/jcm.02423-09
Cronhjort S, Wilhelmsson P, Karlsson L et al (2019) The tick-borne diseases STING study: Real-time PCR analysis of three emerging tick-borne pathogens in ticks that have bitten humans in different regions of Sweden and the Aland islands, Finland. Infect Ecol Epidemiol 9(1):1683935. https://doi.org/10.1080/20008686.2019.1683935
Breen EJ, Polaskova V, Khan A (2015) Bead-based multiplex immuno-assays for cytokines, chemokines, growth factors and other analytes: median fluorescence intensities versus their derived absolute concentration values for statistical analysis. Cytokine 71(2):188–198. https://doi.org/10.1016/j.cyto.2014.10.030
Boehlen F, Clemetson KJ (2001) Platelet chemokines and their receptors: what is their relevance to platelet storage and transfusion practice? Transfus Med 11(6):403–417. https://doi.org/10.1046/j.1365-3148.2001.00340.x
Bubel S, Wilhelm D, Entelmann M et al (1996) Chemokines in stored platelet concentrates. Transfusion 36(5):445–449. https://doi.org/10.1046/j.1537-2995.1996.36596282589.x
Phillips DJ, League SC, Weinstein P et al (2006) Interference in microsphere flow cytometric multiplexed immunoassays for human cytokine estimation. Cytokine 36(3–4):180–188. https://doi.org/10.1016/j.cyto.2006.12.002
Ismail N, Olano JP, Feng HM et al (2002) Current status of immune mechanisms of killing of intracellular microorganisms. FEMS Microbiol Lett 207(2):111–120. https://doi.org/10.1111/j.1574-6968.2002.tb11038.x
Grankvist A, Moore ER, Svensson Stadler L et al (2015) Multilocus sequence analysis of clinical “Candidatus Neoehrlichia mikurensis” strains from Europe. J Clin Microbiol 53(10):3126–3132. https://doi.org/10.1128/jcm.00880-15
Totté P, Bensaid A, Mahan SM et al (1999) Immune responses to Cowdria ruminantium infections. Parasitol Today (Personal ed) 15(7):286–290. https://doi.org/10.1016/s0169-4758(99)01467-2
Barnewall RE, Rikihisa Y (1994) Abrogation of gamma interferon-induced inhibition of Ehrlichia chaffeensis infection in human monocytes with iron-transferrin. Infect Immun 62(11):4804–4810
Laddha AP, Kulkarni YA (2019) VEGF and FGF-2: Promising targets for the treatment of respiratory disorders. Respir Med 156:33–46. https://doi.org/10.1016/j.rmed.2019.08.003
Kikuchi K, Hoashi T, Kanazawa S et al (2005) Angiogenic cytokines in serum and cutaneous lesions of patients with polyarteritis nodosa. J Am Acad Dermatol 53(1):57–61. https://doi.org/10.1016/j.jaad.2005.02.018
Pulsatelli L, Boiardi L, Assirelli E et al (2020) Imbalance between angiogenic and anti-angiogenic factors in sera from patients with large-vessel vasculitis. Clin Exp Rheumatol. 124(2):23–30
van den Borne P, Quax PH, Hoefer IE et al (2014) The multifaceted functions of CXCL10 in cardiovascular disease. Biomed Res Int 2014:893106. https://doi.org/10.1155/2014/893106
Antonelli A, Ferrari SM, Giuggioli D et al (2014) Chemokine (C-X-C motif) ligand (CXCL)10 in autoimmune diseases. Autoimmun Rev 13(3):272–280. https://doi.org/10.1016/j.autrev.2013.10.010
Yang J, Richmond A (2004) The angiostatic activity of interferon-inducible protein-10/CXCL10 in human melanoma depends on binding to CXCR3 but not to glycosaminoglycan. Mol Ther 9(6):846–855. https://doi.org/10.1016/j.ymthe.2004.01.010
Lavie F, Miceli-Richard C, Ittah M et al (2007) Increase of B cell-activating factor of the TNF family (BAFF) after rituximab treatment: insights into a new regulating system of BAFF production. Ann Rheum Dis 66(5):700–703. https://doi.org/10.1136/ard.2006.060772
Havenar-Daughton C, Lindqvist M, Heit A et al (2016) CXCL13 is a plasma biomarker of germinal center activity. Proc Natl Acad Sci 113(10):2702. https://doi.org/10.1073/pnas.1520112113
Rosengren S, Wei N, Kalunian KC et al (2011) CXCL13: a novel biomarker of B-cell return following rituximab treatment and synovitis in patients with rheumatoid arthritis. Rheumatology (Oxford) 50(3):603–610. https://doi.org/10.1093/rheumatology/keq337
Funding
Open access funding provided by University of Gothenburg. This study was supported by the European Union through the European Regional Development Fund and the Interreg North Sea Region Programme 2014–2020 as part of the NorthTick project (reference number J-No.: 38-2-7-19), The Swedish state under the agreement between the Swedish government and the county councils, the ALF-agreement ALF Research Fund (ALFGBG-827291), the Cancer and Allergy Foundation (2020-10154) and by the Swedish Research Council grants K2008-58X-14631-06-3 (PF) and 2020-01287 (CW).
Author information
Authors and Affiliations
Contributions
LW, CW and CL contributed to the study conception and design. Choice of methodology and supervision was given by CW. Material preparation, data collection and analysis were performed by LW and CL. HQ, P-EL, PF, ES and KN contributed with patient samples. The first draft of the manuscript was written by LW and all the authors commented and revised on previous versions of the manuscript. All the authors read and approved the final manuscript.
Corresponding author
Ethics declarations
Conflict of interest
PEL has been an external scientific expert to Valneva Austria GmbH, Vienna, Austria and Pfizer Inc, US. The other authors declare that they have no conflict of interest.
Ethics approval
All the procedures performed in the studies involving human participants were in accordance with the ethical standards of the regional research committees and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.
Consent to participate/publish
Informed consent was obtained from all the individual participants included in the study and the authors affirm that human research participants provided informed consent for publication.
Additional information
Edited by Volkhard A. J. Kempf.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Wass, L., Quarsten, H., Lindgren, PE. et al. Cytokine responses of immunosuppressed and immunocompetent patients with Neoehrlichia mikurensis infection. Med Microbiol Immunol 211, 133–141 (2022). https://doi.org/10.1007/s00430-022-00737-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00430-022-00737-6