Immune Mediators of protective and pathogenic immune responses in patients with mild and fatal human monocytotropic ehrlichiosis

A 7 year old patient with acute lymphocytic leukemia (ALL), who has been in complete remission, was admitted to the hospital after 4 days of persistent fever for which he had been treated as an outpatient with ceftriaxone. Upon admission, his laboratory studies revealed marked leukopenia (WBC count, 500/μl), anemia (Hgb, 11.5 g/dl), thrombocytopenia (platelet count, 164,000/μl), neutropenia (absolute neutrophil count of 420/μl), and increased liver enzymes (ALT 636 U/L and AST 1726 U/L). Blood culture was negative for bacterial and fungal infections. However, Ehrlichia chaffeensis DNA was detected in the blood by PCR, but ehrlichiae culture was negative. The acute serum contained no antibodies to Ehrlichia. There had been no known tick bite; however, the patient had recently returned from camp in a wooded area known to harbor ticks. He was placed on broad spectrum antibiotics: cefepime, vancomycin, levofloxacin and doxycycline. CT scan of the chest demonstrated right lower lobe pneumonia with moderate right pleural effusion. Cerebrospinal fluid contained a normal concentration of protein and glucose. Shortly after admission, he developed increasing respiratory distress and hypoxia, which necessitated his transfer to the pediatric intensive care unit (PICU). Later, the patient had increasing respiratory distress and began having seizure-like activity, developed significant hyperkalemia, and respiratory and metabolic acidosis. Despite prolonged resuscitation, the patient’s condition deteriorated with multiorgan failure syndrome, and septic shock. He died on day 6 after his admission to the hospital. The cause of death was attributed to septic shock secondary to ehrlichiosis. Autopsy examination showed; 1) cardiomegaly with diffuse focal calcifications in both ventricles; 2) diffuse alveolar damage; 3) hepatic periportal inflammation and focal fibrosis; and 4) lymphocytic depletion in the spleen and lymph nodes. The latter was consistent with presence of leukopenia.

Blood and serum samples were collected from a patient with fatal HME, a patient with mild HME as well as gender- and age-matched healthy volunteers. The serum and blood samples from patient were collected upon hospital admission and thus represent the acute or early phase of illness, a time when ongoing inflammatory responses were likely to be at a maximum. Patient specimens and medical informations that are reported in this study have been collected with the approval of the office of research, Institutional Review Board (IRB) at Meharry Medical College. For patient samples, no consent form is obtained. This study was considered by the IRB as an exempted study, i.e. research that involve the collection of records and diagnostic specimens that are submitted to the clinical laboratory at Vanderbilt Medical Center for diagnostic purposes. Residual samples that remain after performing the required laboratory diagnostic tests are considered as discarded samples and thus used in this study. Patient samples and medical data were collected and recorded in a manner that subjects cannot be identified, directly or through any identifiers linked to the subjects. Blood and sera from healthy controls were collected under the approved IRB protocol and the informed consent was obtained from healthy individuals.

Total mRNA was extracted from peripheral mononuclear cells, and samples were stored at −80°C until processed. Serum and blood samples were collected from healthy individuals as controls.

A two-step colorimetric microtiter plate PCR assay capable of detecting and discriminating medically important Ehrlichia species was used as described before [19, 20]. Isolation of Ehrlichia from patients' blood and strain identification were performed as described previously [3‐5].

Total RNA was extracted from human blood using PureLink RNA Mini-extraction Kit (Invitrogen). The quality of RNA was checked before cDNA preparation. Reverse transcription of 1μg of RNA was carried out with the iScript cDNA Synthesis Kit (Invitrogen, Wisc., USA), following the manufacturer’s protocols. The relative expression levels of genes of interest were studied by real-time PCR using My iQ and SYBR green detection system (Bio-Rad TM, USA). The primer sequences are summarized in Table 1. At the end of each reaction, a melting-curve analysis was performed to confirm the absence of primer-dimers. The data were analyzed using the 2 – ΔΔ Ct method with Ct denoting the threshold cycle of PCR amplification at which product is first detected by fluorescence. Expression levels of every gene were normalized to the expression level of GAPDH.

In the serum samples obtained at admission or shortly afterwards, the pro- and anti-inflammatory cytokines and chemokines that we measured fell into five groups including: pro-inflammatory cytokines (IL-1α, IL-1β, IL-6, IL-17, TNF-α), Th1 cytokines (IL-2, IFN-γ, IL-12p70), Th2 cytokines (IL-4, IL-5, IL-9), immunosuppressive or anti-inflammatory cytokines ( IL-10, IL-13), neutrophil chemoattractant and growth factors (IL-8, G-CSF), and T cell, NK cell, and monocyte ckemokines (eotaxin, IP-10, MCP-1, MIP-1α, MIP-1β, RANTES). Our data show that fatal HME is associated with substantially increased levels of pro-inflammatory cytokines such as IL-1α, IL-1β, TNF-α and IL-6 compared to nonfatal HME, and healthy controls (Figure 1A). The levels of anti-inflammatory cytokines such as IL-10 and IL-13 were substantially increased in fatal disease compared to non-fatal HME and controls (Figure 1B). On the other hand, Th1 response marked by the production of Th1 cytokines; IL-2 and IFN-γ was lower in fatal HME compared to non-fatal HME (Figure 1C). Weak Th1 response in the patient with fatal HME was associated with a slight increase in the levels of Th2 cytokines (IL-4 and IL-9) (Figure 1B) and Th17-cytokine (IL-17) (Figure 1C) when compared to patient with non-fatal HME and healthy controls. Interestingly, the levels of IL-8 (Figure 1D) and G-CSF (Figure 1D), a neutrophil chemoattractant and neutrophil growth factor, were greatly increased in fatal HME compared to nonfatal HME and controls, while the level of GM-CSF was slightly lower in fatal HME than that detected in nonfatal HME (Figure 1D).

Macrophage/monocyte and NK cell chemoattractants such as MCP-1α, MIP-1α, and MIP-1β were highest in fatal HME compared to non-fatal HME and healthy controls (Figure 1E). In contrast, the concentrations of the T cell chemokine RANTES and IFN-γ dependent chemokine (IP-10) were lower in fatal HME than in non-fatal HME, but higher than in healthy controls (Figure 1E). The strong clustering of high levels of pro-inflammatory cytokines and monocyte/NK cell chemokines, but the lower levels of Th1 cytokines, the T cell chemokine RANTES, and anti-inflammatory cytokines suggests that fatal disease is associated with excessive pro-inflammatory, but weak Th1, response at least at the time the patients are hospitalized.

One of the main clinical manifestations of HME is marked leukopenia, mainly affecting the lymphocyte population. Similarly, in an animal model of fatal ehrlichiosis, death is accompanied by a substantial decrease in the T cell population, mainly CD4 + T cells. As expected, these cases of fatal and nonfatal HME were associated with a decline in the absolute number of lymphocytes, with severe lymphopenia in the fatal case (absolute lymphocyte count: fatal HME = 124/μl and nonfatal HME = 1720/μl). To examine whether the difference in lymphocyte levels in these HME patients was due to apoptosis, we examined the expression of apoptotic markers on peripheral blood leukocytes. Our data show that leukocytes from the patient with milder infection expressed a higher level of FAS mRNA compared to the patient with fatal ehrlichial infection and healthy controls. In contrast, leukocytes from the patient with fatal Ehrlichia infection expressed a higher level of TNFR mRNA than that expressed in leukocytes from the patient with milder Ehrlichia infection or healthy controls (Figure 2A). There was no difference in mRNA expression of FASL between all groups.

Recognition of specific microbial ligands by toll like receptors (TLRs) has been shown to play a major role in induction of the innate and acquired immune responses against bacteria, viruses, and protozoa [21, 22]. We examined whether the above quantitative differences in serum levels of cytokines and chemokines during fatal and nonfatal HME are due to altered signals through TLRs. Our data show that leukocytes from the patient with fatal HME expressed ~ 2-fold higher levels of TLR2 and TLR4 compared to leukocytes from the patient with nonfatal HME or healthy controls (Figure 2B). There was no difference in expression of TLR9 mRNA between all groups (Figure 2B).