Currently, no human study has examined serum cytokine and chemokine profiles in patients with HME who present with either moderate or severe disease. Such studies are critical for enhancing our understanding of the pathophysiology of Ehrlichia-induced toxic shock. The development of profound shock, despite the paucity of infected cells in the blood in patients with severe infection with Ehrlichia, which lack LPS, has been taken as evidence for subtle immunologic dysfunction attributable to inflammatory mediators. In this study, we compared the levels of circulating pro-inflammatory cytokines and chemokines derived from or involved in the activation of immune cells in acute mild-to-moderate/nonfatal or severe/fatal HME with those seen in uninfected individuals from regions endemic for HME. We could not determine the statistical significance of the differences in the immune responses between the two patient groups due to small sample size. However, we believe that this difference is most likely to be biologically significant based on known levels of these immune molecules in healthy individuals. Our data here showed that fatal HME is associated with dysregulated cytokine and chemokine production with marked elevation of pro-and anti-inflammatory cytokines, and several chemokines that are chemo attractants to NK and T cells. The presence of uncontrolled elevation of inflammatory cytokines in patient with fatal HME could contribute to prolonged activation of Ehrlichia-target cells, which could possibly contribute to tissue injury and multi-organ failure.
It is possible that the majority of circulating cytokines and chemokines are produced within peripheral tissues that represent main sites of ehrlichial infection such as liver or lymphoid structures, from where they can leak into the bloodstream. It should be noted that these serum cytokine and chemokine expression levels were studied soon after admission, before progression of the disease or recovery in patients with fatal and nonfatal HME, respectively. Therefore, the immune profile could be considered to be predictive of subsequent adverse events or protection against Ehrlichia. In addition, serum cytokine and chemokine expression is measured more easily and is reproducible for rapid clinical diagnostic purposes.
A salient observation in this study was the high levels of IL-8 and G-CSF in patient with acute fatal HME. IL-8, a CXC chemokine, functions as a chemoattractant for neutrophils and T cells and plays an important role in the pathogenesis of septic shock associated with endotoxin [23–26]. G-CSF is shown to be produced as a compensatory mechanism during bacterial infection that are associated with bone marrow suppression [23–26]. G-CSF stimulates the survival, proliferation, differentiation and functions of neutrophils precursors and mature neutrophils [23–26]. However, despite the elevation of IL-8 and G-CSF, we observed low count of neutrophils in the blood of patient with fatal HME. This discordant result could be due to possible migration of neutrophils to the peripheral sites of infection or activation-induced cell death (AICD). In support of the first possibility, patient with fatal HME had a substantial elevation of several chemokines, suggesting that an active migration of inflammatory and immune cells to sites of infection are taking place. Although it is not yet clear whether neutrophils play a protective or pathogenic role during fatal ehrlichial infection in humans, our murine studies showed a correlation between expansion of neutrophils at peripheral sites of infection, ineffective bacterial elimination, and severe pathology in animal model of fatal ehrlichiosis,  suggesting that neutrophils may play a pathogenic role in fatal HME.
Activation of monocytes/macrophages killing mechanisms by IFN-γ (Th1 cytokine) is the pivotal step in controlling intracellular ehrlichial infection [7, 15, 28]. Our data showed that patient with fatal HME had weak Th1 cell-mediated immunity as marked by low serum level of IL-2 and IFN-γ. However, in the absence of kinetic data over the course of illness, it is not possible to determine whether low levels of these Th1 cytokines is due to defective induction of Th1 cells, suppression of T cell proliferation, or AICD. Our murine studies showed that lethal ehrlichial infection induces apoptosis of CD4 + T cells [7, 10–13, 18]. However, the exact mechanism by which CD4 + T cells undergo apoptosis in this model is not yet clear. TNFR and Fas mediated signals are known to be responsible for apoptosis of host cells during infections . Patient with fatal HME has higher expression of TNFR on the mononuclear cells than that detected in the patient with nonfatal HME, suggesting that signaling via TNFR, but not Fas/FasL signaling, may be relevant to observed weak Th1 response. Interestingly, our murine studies showed that lack of TNFR in lethally infected mice prolonged their survival and abrogated tissue injury .
The high levels of serum IL-10 and IL-13 in the patient with fatal HME compared to non fatal HME is consistent with our murine data where IL-10 peaks in the serum at later stages of lethal infection, which occurs before animals succumb to infection [7, 10, 12]. IL-10 is an immunosuppressive cytokine that inhibits effective elimination of intracellular bacteria and suppresses T cell proliferation . Thus, it is possible that IL-10 overproduction in the patient with fatal HME could be another potential mechanism that account for observed lymphopenia and week Th1 response.
Finally, our data demonstrated a differential TLRs expression during fatal and nonfatal HME with upregulation of TLR2 and TLR4 expression in patient with fatal HME. These data are consistent with TLR expression during sepsis caused by other lipopolysaccharide (LPS) positive Gram negative bacterial infection [21, 30]. However, unlike other Gram negative bacteria, Ehrlichia lack LPS, the natural ligand for TLR4. Therefore, upregulation of TLR4 in fatal disease could be induced by other microbial ligand or by endogenous TLR4 ligand such as the high-mobility group protein (HMGB1), which is secreted upon tissue damage .