The Cytokine and Chemokine Profiles in Patients with Hand, Foot and Mouth Disease of Different Severities in Shanghai, China, 2010

<div><p>Background and purpose</p><p>Systemic upregulation of inflammatory cytokines is characteristic of critical severe hand, foot, and mouth disease (HFMD) with pulmonary edema. Thus, immunomodulatory medicines such as steroids, including methylprednisolone, have been proposed to treat patients with severe HFMD in China, because it is postulated that inflammatory cytokines play a role in the development of severe complications. This study is to further investigate the inflammatory response in the relatively mild HFMD patients, and whether steroid treatment has a beneficial effect on the suppression of inflammation in HFMD patients.</p><p>Method</p><p>We measured the levels of 50 kinds of chemokines, cytokines, growth factors and soluble receptors in serum samples from control patients without HFMD and the HFMD patients with or without prior treatment of intravenous methylprednisolone.</p><p>Results</p><p>Our present study found that even relatively mild HFMD patients without central nervous system (CNS) complications had elevated serum levels of inflammatory cytokines, including interleukin (IL)-3, IL-6, IL-12p40, and tumor necrosis factor (TNF)-α, which suggested systemic inflammation. In contrast, these patients also have decreased levels of other serum biomarkers, including IL-1Ra, IL-8, IL-16, soluble ICAM-1, CXCL-1, and CCL27. The dysregulation of cytokine and chemokine expression may be involved in CNS complications and unbalanced circulating leukocytes in HFMD patients. Surprisingly, patients treated with methylprednisolone had no difference in the expression levels of HFMD-associated biomarkers instead had slightly increased levels of IL-17A, which was not associated with the occurrence of HFMD.</p><p>Conclusion</p><p>Whether steroid treatment has any beneficial effect on the prognosis of HFMD patients requires to be further investigated.</p></div

The correlation between host biomarkers and disease prognosis.

<p>Levels of CCL27 (A), CXCL1 (B), and soluble VCAM-1 (C) in serum samples obtained from control patients (Ctrl, n = 20) and HFMD patients with or without CNS complications (n = 20, respectively). The line represents the average value. (D) Plots of soluble VCAM-1 concentrations in sera of HFMD patients against their maximal fever temperatures. Numbers above square brackets indicate P values for the corresponding comparisons.</p

Immune biomarkers whose levels in serum samples of HFMD patients were significantly different from those of control patients.

*<p>Significance was analyzed via Student's <i>t</i>-test and P values were further adjusted with the Benjamini-Hochberg procedure.</p

TLR3 Signaling in Macrophages Is Indispensable for the Protective Immunity of Invariant Natural Killer T Cells against Enterovirus 71 Infection

<div><p>Enterovirus 71 (EV71) is the most virulent pathogen among enteroviruses that cause hand, foot and mouth disease in children but rarely in adults. The mechanisms that determine the age-dependent susceptibility remain largely unclear. Here, we found that the paucity of invariant natural killer T (iNKT) cells together with immaturity of the immune system was related to the susceptibility of neonatal mice to EV71 infection. iNKT cells were crucial antiviral effector cells to protect young mice from EV71 infection before their adaptive immune systems were fully mature. EV71 infection led to activation of iNKT cells depending on signaling through TLR3 but not other TLRs. Surprisingly, iNKT cell activation during EV71 infection required TLR3 signaling in macrophages, but not in dendritic cells (DCs). Mechanistically, interleukin (IL)-12 and endogenous CD1d-restricted antigens were both required for full activation of iNKT cells. Furthermore, CD1d-deficiency led to dramatically increased viral loads in central nervous system and more severe disease in EV71-infected mice. Altogether, our results suggest that iNKT cells may be involved in controlling EV71 infection in children when their adaptive immune systems are not fully developed, and also imply that iNKT cells might be an intervention target for treating EV71-infected patients.</p></div

IL-12 and endogenous CD1d antigens are both required for full iNKT cell activation in EV71 infection.

<p>(A) EV71M-infected or-uninfected WT macrophages (Mock) were co-cultured with purified iNKT cells in the presence of neutralizing anti-CD1d (αCD1d), isotype control antibodies (Iso) or medium alone (M). (B) EV71M-infected or –uninfected WT or CD1d-deficient (CD1d^-/-) macrophages were co-cultured with purified iNKT cells. (C) EV71-infected WT macrophages were co-cultured with purified iNKT cells in the presence of the lipid synthesis inhibitor NB-DGJ or medium alone. (D) Seven-day-old WT or CD1d^-/- neonates (n = 3–5) were adoptively transferred with 5×10⁵ purified iNKT cells or saline control intraperitoneally and infected with 2×10⁵ PFU of EV71M. After 16 hours, splenocytes of saline (mock)-treated or EV71M-infected WT or CD1d^-/- mice were stained with TCRβ, CD1d tetramer, CD69 and DAPI. The CD69 MFI levels on iNKT cells are shown. (E) EV71M-infected WT macrophages were co-cultured with purified iNKT cells in the presence of neutralizing anti-IL-12, anti-IL-18 or isotype control antibodies. The IFN-γ concentrations in the 24-hour culture supernatants were quantified by ELISA. (F) The IL-12 (p70) concentrations in the supernatants of WT or TLR3^-/- macrophages infected with 10 MOIs of EV71M. All results represent the mean ± SD. NS, not significant; *, <i>P</i><0.05; **, <i>P</i><0.01. Data are representative of three (A, B, C, E, F) or two (D) independent experiments.</p

TLR3 is indispensable for iNKT cell activation in EV71 infection.

<p>(A) TLR3 signaling is required for IFN-γ production by EV71-infected splenocytes. Splenocytes from WT, TLR3^-/-, TLR7^-/- and MyD88^-/- mice (n = 3–5) were cultured with EV71M or mock for 24 hours, and IFN-γ production was quantified by ELISA. (B) TLR3 deficiency dramatically reduced the IFN-γ production of iNKT cells. Splenocytes from CD1d-deficient or WT C57BL/6 mice (n = 3) were infected with EV71M for 24 hours and then stained with TCRβ, CD1d tetramer and IFN-γ. Splenocytes were stimulated with PMA and ionomycin (PMA/I) or mock treated (mock), which served as positive and negative controls, respectively. IFN-γ-producing cells are shown among CD1d tetramer⁺ TCR⁺-gated cells. (C) iNKT cell activation in EV71 infection is dependent on TLR3 signaling. Macrophages from WT or TLR3^-/- mice were cultured in the presence of EV71M for 24 hours. After extensive washing, macrophages were co-cultured for 48 hours with purified iNKT cells from WT mice, and cytokine production was quantified by ELISA. (D) TLR3 signaling is required for <i>in vivo</i> activation of iNKT cells upon EV71M infection. Six-8 week-old WT or TLR3^-/- mice (n = 3–6) were injected with 1×10⁵ PFU of EV71M or saline intraperitoneally. After 16 hours, splenocytes of mock (PBS) or EV71M-infected WT or TLR3^-/- mice were stained with TCRβ, CD1d tetramer, CD69 and DAPI. The mean fluorescent intensity (MFI) of CD69 expression on iNKT cells is shown. NS, not significant; **, <i>P</i><0.01. (E) TLR3 triggering in macrophages activated iNKT cells. BMDCs or macrophages were cultured with medium alone or pI:C for 24 hours and then co-cultured with purified iNKT cells from WT mice for 48 hours. Cytokine production was quantified by ELISA. All results represent the mean values of cultures of 5 mice ± SD. *, <i>P</i><0.05. (F) The survival rates of 7-day-old wild-type (WT) mice and TLR3-deficient (TLR3^-/-) mice (n = 6, per group) after infection by 1×10⁶ PFU of EV71M. Data are representative of three (A, C, E, F) or two (B. D) independent experiments.</p

CD1d is essential for the protection of young mice from EV71 infection.

<p>Seven-day-old WT and CD1d^-/- mice were infected intraperitoneally with a lower dose (A, 2×10⁴ PFU) or higher dose (B, 2×10⁵ PFU) of EV71M. The clinical score (A) and survival (B, Kaplan-Meier curve) were monitored for the indicated period (n ≥ 10 per group). (C) Kaplan-Meier survival curves for 7-day-old WT and Jα18^-/- mice (n ≥ 5 per group) that were infected intraperitoneally with 2×10⁵ PFU of EV71M. The viral loads in each organ were examined by quantitative PCR on days 2 (D) and 4 (E) after the high dose of EV71M infection. Data are shown as the mean ± SD of three independent samples. NS, not significant; **, <i>P</i><0.01. Data are representative of two independent experiments.</p