MicroRNA-19a and CD22 Comprise a Feedback Loop for B Cell Response in Sepsis

Abstract

Background: MicroRNA-19a (miR-19a), an oncogenic microRNA, has been recently reported to target CD22 in B cell lymphoma cell lines, but its role in inflammatory response is unclear. CD22 is a negative regulator for BCR signaling, and we hypothesize that miR-19a regulates B cell response by targeting CD22 in sepsis.

Material and methods: In order to determine whether miR-19a-CD22 pathway was involved in sepsis, and what role it played in the regulatory mechanisms, we detected the levels of miR-19a in B cells obtained from patients with sepsis, and measured the levels of miR-19a and CD22 expression in B cells activated by LPS in vitro. Additionally, we investigated the correlation between miR-19a and CD22, as well as the influence of this pathway on BCR signaling, in transfected B cells.

Results: We found that septic patients displayed up-regulated miR-19a in B cells. In vitro, miR-19a was increased in activated B cells, with CD22 expression initially enhanced but subsequently decreased. Moreover, overexpression of miR-19a resulted in an amplified BCR signaling, while overexpression of CD22 attenuated the effect of miR-19a and increased its expression.

Conclusions: Our study demonstrated that miR-19a and CD22 comprised a feedback loop for B cell response in sepsis, providing a potential therapeutic target to recover the immune homeostasis.

Figure 1

Expression of miR-19a and CD22 in B cells obtained from patients and controls. The relative expression levels of miR-19a in patients with sepsis (sepsis group) and in patients with non-infected SIRS (SIRS group) were significantly higher than that in healthy controls (control group), and the level in the sepsis group was elevated in comparison to that in the SIRS group (A). The levels of CD22 expression were displayed as geometrical mean fluorescence intensity (Geom. MFI) measured by flow cytometry, and were of no differences among different groups (B). Data are expressed as mean ±SD. * P<0.05, ** P<0.01.

Figure 2

Expressions of miR-19a and CD22 in activated B cells. PBMCs obtained from healthy volunteers were activated by LPS, and B cells were isolated after activation for the detection of miR-19a. The relative expression levels of miR-19a determined by qRT-PCR was increased by 2 days and 4 days in activated B cells compared to those in control B cells without stimulus (A). The mean fluorescence intensity (MFI) of CD22 on CD19 gated PBMCs determined by flow cytometry were increased by 2 days but decreased by 4 days (B). Data are expressed as mean ±SEM of 3 independent experiments. Histograms were obtained from 1 of 3 independent experiments that displayed similar results. * P<0.05.

Figure 3

Function of miR-19a in B cell activation. PBMCs transfected with miR-19a mimic or inhibitor (anti-miR19a), or corresponding controls (miR-NC, anti-miR-NC) were activated by LPS for 48 h. Activated B cells transfected with miR-19a mimic exhibited a higher level of p-BLNK, which was measured by Western blotting, while inhibition of miR-19a resulted in a suppressed level of p-BLNK (A). Overexpression of miR-19a induced decreases in CD22 mRNA while inhibition of miR-19a resulted in increased levels of CD22 mRNA (B). B cells transfected with miR-19a exhibited down-regulated CD22 expression, and inhibition of miR-19a enhanced CD22 expression (C). The effect of miR-19a mimic/inhibitor transfection was confirmed by the detection of miR-19a using qRT-PCR (D). Data are expressed as mean ±SEM of 3 independent experiments. Histograms and protein bands were obtained from 1 of 3 independent experiments that displayed similar results. * P<0.05, ** P<0.01.

Figure 4

Influence of CD22 overexpression on miR-19a. PBMCs were co-transfected with CD22 expression plasmid (pc-CD22) or control (pc-DNA), and with miR-19a mimic or control (miR-NC), and were activated by LPS for 48 h. Western blotting revealed that CD22 overexpression partially attenuated the up-regulation of p-BLNK induced by miR-19a transfection (A). qRT-PCR analysis demonstrated that overexpression of CD22 resulted in an increased level of miR-19a in activated B cells (B). The effect of CD22 plasmid was confirmed by the detection of CD22 mRNA using qRT-PCR (C). Data are expressed as mean ±SEM of 3 independent experiments. Protein bands were obtained from 1 of 3 independent experiments that displayed similar results. * P<0.05, ** P<0.01.