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Interleukin-34 (IL-34) is a recently defined cytokine, showing a functional overlap with macrophage colony stimulating factor (M-CSF). Little is known about IL-34 production by exogenous pathogens infection in human individuals. In this study, we found that IL-34 level was increased in the peripheral blood mononuclear cells (PBMCs) and serum samples from a cohort of 155 patients infected by influenza A virus (IAV) comparing to that of 145 healthy individuals. Expression of IL-34 in IAV infected PBMCs was blocked by IL-22 speciï¬c siRNA, indicating IL-34 was induced through IL-22 in the inflammatory cascade. Interestingly, we found that IL-22 mRNA expression and protein secretion activated by IAV infection was significantly suppressed by over-expression of IL-34 but increased by IL-34-specific siRNA, suggesting there was a feedback mechanism between IL-34 and IL-22. In conclusion, IL-34 is induced by IAV infection via IL-22 in the inflammatory cascade. Our results provide that IL-34 is a potential target for anti-inflammatory medicine screening.
Keywords: Influenza A virus; Interleukin-34; Interleukin-22; Inflammatory
Inï¬‚uenza viruses (IV) are highly contagious single-stranded RNA viruses in the Orthomyxoviridae family and its genome consists of eight distinct RNA segments which encode 11-12 proteins (Neumann et al. 2004; Medina et al. 2011). Seasonal strains (e.g. Inï¬‚uenza A viruses, H3N2) that cause annual epidemics can cause severe disease in immunocompromised individuals, including the young and the elderly (Hurt et al. 2012; Rappupli et al. 2012). The host cytokine immune response provides the first line of defense against Inï¬‚uenza A viruses (IAV) infection (Teijaro et al. 2011). Studies have also shown that IAV infection induces excessive cytokine production (Cytokine storm) and robust recruitment of leukocytes which are hypothesized to be major contributors to severe disease in humans (Jewell et al. 2010; Iwasaki et al. 2011). However, the mechanisms underlying the increased induction of cytokines during IAV infection have to date been largely unclear.
Interleukin-22 (IL-22) is a member of the IL-10 family and produced by CD4+ T helper 17 (Th17) cells, natural killer (NK) cells, CD11c+ myeloid cells, and lymphoid tissue inducer-like (LTi) cells (Rutz et al. 2013; Cella et al. 2009). The IL-22 receptor is composed of the IL-22R1 and IL-10R2 subunits and is found on cells of nonhematopoietic origin in the skin, kidney, liver, lung, and gut (Wolk et al. 2004; Ouyang et al. 2008). IL-22 plays a dual role in autoimmune and inï¬‚ammatory diseases. In experimental noninfectious systems, IL-22 exerts a potent protective effect on hepatocytes and epithelial cells at barrier surfaces (Aujia et al. 2008; Zheng et al. 2008). On the other hand, uncontrolled IL-22 involved in the inflammatory disorders such as psoriasis, lupus erythematosus, and rheumatoid arthritis (Van et al. 2012; Zhang et al. 2007).
Interleukin-34 (IL-34) is a recently discovered cytokine and has been identified by functional screening of a library of secreted proteins (Lin et al. 2008; Melanie et al. 2012). IL-34 is speciï¬cally expressed in splenic tissues, predominantly in the red pulp region, and supported human monocyte survival and to promote the formation of the colony-forming unit-macrophage (CFU-M) in human bone marrow cell cultures (Chemel et al. 2012; Hwang et al. 2012). IL-34 is a dimeric glycoprotein which presents different amino acid sequence with colony-stimulating factor 1 (CSF-1) (Baud’huin et al. 2010). Murine IL-34 also promotes peritoneal macrophages survival and bone marrow progenitor cell proliferation (Wang et al. 2012; Dai et al. 2002). Although several functions of IL-34 have been described, a clear role for IL-34 in inflammatory network has not been established.
A number of investigations demonstrate that viral infections stimulate IL-22 expression (Stoyan et al. 2013; Christophe et al. 2012). However, the role of IAV infection in the regulation of the newly described pro-inflammatory factor IL-34 expression is still unclear. Furthermore, since IL-22 and IL-34 are obligatory mediators of inflammation, the question arises as to whether there is an interaction between the two proteins or they act as independent effectors of host inflammatory response to viral infection. The aim of this study was to investigate the role of IAV infection in the regulation of IL-34 expression and to determine the molecular mechanisms responsible. Our results showed that IAV infection activates IL-22 and IL-32 expression by a heretofore unrecognized mechanism, in which IAV stimulates IL-34 expression through IL-22, and IL-34 feedback inhibits IL-22 expression.
Materials and Methods
Participants included 155 IAV patients . Samples from healthy individuals were randomly selected as controls from the local blood donation center. As shown in Table 1 and Table 2, the main clinical and demographic characteristics of the population were presented in this study. Written informed consents were obtained from all patients. This study was approved by the Ethics Committee and was performed according to the Declaration of Helsinki Principles.
Virus and Constructs
The influenza virus strain A/chicken/Hubei/327/2004 (H5N1) used in this study was provided by China Center for Type Culture Collection (CCTCC). Stock virus was propagated in 10-day-old embryonated chicken eggs for 36 to 48 h at 37 °C. The allantoic fluid was then harvested, and aliquots were stored at -80°C before being used. Mammalian expression plasmids for IL-22 and IL-34 were constructed by standard molecular biology techniques.
Peripheral Blood Mononuclear Cell (PBMC) Isolation and Transfection
PBMCs were isolated from venous blood from healthy individuals. PBMC fractions were obtained by density centrifugation using Histopaque as per the manufacturer’s instructions (Sigma, St. Louis, MO). PBMCs were transfected with plasmid DNA by electroporation with an Amaxa Nucleofector II Device according to the manufacturers’ protocols.
IL-22 and IL-34 small interfering RNA (IL-22-RNAi and IL-34-RNAi) and their negative control were synthesized by RiBo Biotech (GuangZhou RiBo Biotech). siRNA was used according to the manufacturers’ protocols.
Quantitative RT-PCR (qRT-PCR) analysis
Total RNA was isolated using TRIzol (Invitrogen, Basel, Switzerland). Cellular RNA samples were reverse-transcribed using random primers. qRT-PCR was performed using a LightCycler 480 (Roche) and the SYBR Green system (Applied Biosystems). GAPDH was amplified as an internal control. For IL-22, the primer was 5’-GAAGAAGTGCTGTTCCCTCAATC-3’ and 3’-ATTCCTCTGGATATGCAGGTCAT-5’. For IL-10, the primer was 5’-GTGGAGACGCTGCTGCTGAATGT-3’ and 3’-CTCTGGGCTGCAAGACTGAGGAC-5’. For GAPDH, the primer was 5’-GGAAGGTGAAGGTCGGAGTCAACGG-3’ and 3’-CTCGCTCCTGGAAGATGGTGATGGG-5’.
For the quantiï¬cation of IL-22 and IL-34 released in culture supernatants, Human IL-22 ELISA Kit (eBioscience) and Human IL-34 Elisa Kit (eBioscience) were used according to the manufacturer’s protocol.
Measurement data are presented as the mean ± SD or mean ± SEM. Data that satisfied the normal distribution criterion were used in the group design of the t-test for the statistical analysis. The differences among the groups were tested by a one-way ANOVA followed by a post hoc test (LSD). A value of p < 0.05 was considered significant. All statistical analyses were performed using professional statistical software (SPSS 15.0 for Windows, SPSS Inc., Chicago). All experiments were repeated at least three times with similar results.
To investigate IL-22 and IL-34 expressions during IAV infection, PBMCs were isolated from IAV patients (n = 51) and healthy individuals (n = 47). As determined by qRT-PCR, the mRNA levels of IL-22 and IL-34 were increased by 8.4-fold and 9.9-fold in the IAV patients compared to the healthy individuals (Fig. 1A, B; Table 1). Signiï¬cant differences in serum IL-22 and IL-34 levels were observed between IAV patients (n = 104) and healthy individuals (n = 98) as determined by ELISA (IL-22: 148.49 ± 45.02 pg/ml versus 61.75 ± 23 pg/ml; IL-34:172.53 ± 39.33 pg/ml versus 66.85 ± 27.53 pg/ml) (Fig. 1C, D; Table 2). These results indicated that IL-22 and IL-34 expressions were elevated in the patients infected with IAV.
IL-22 and IL-34 activation in response to IAV infection in a time- and dose- dependent manner
It has been reported that IV can activate the expression of IL-22. Initially, we investigated whether IAV infection played a role in the regulation of IL-34 expression. To explore the kinetics of IL-22 and IL-34 induction, both mRNA and protein expression of IL-22 and IL-34 were measured at various time points after infection with IAV at a multiplicity of infection (MOI) of 1. IAV infection of freshly isolated PBMCs from healthy individuals resulted in increased IL-22 and IL-34 mRNA and protein expressions (Fig. 2A). The mRNA levels of IL-22 and IL-34 was increased as early as 24 hours after infection and IL-22 and IL-34 levels continued to increase throughout the course of the experiment. IL-22 and IL-34 levels showed a similar trend as determined by ELISA analyses (Fig. 2C). We next infected freshly isolated PBMCs with various doses of IAV and harvested the cells 48 hours after infection. IL-22 and IL-34 mRNA levels were positively correlated with the doses of IAV (Fig. 2B). Protein levels of IL-22 and IL-34 in ELISA analyses mirrored the transcription data (Fig. 2D). These results suggested that IAV infection could activate the expression of IL-22 and IL-34 in a time- and dose- dependent manner.
IAV stimulates IL-34 through IL-22 pathway during inflammatory response
To define the role of IL-22 in the regulation of IAV induced inflammatory factor IL-34, we constructed mammalian expression plasmids for IL-22 and synthesized multiple RNAi plasmids for IL-22 (Fig. 3A). The result revealed that overexpression of IL-22 potentiated IL-34 mRNA levels in a dose-dependent manner in PBMCs from healthy individuals (Fig. 3B). Elevated levels of secreted IL-34 in culture supernatants were also observed by ELISA (Fig. 3C).
To determine the effects of IL-22 on the activation of IL-34 mRNA level and protein expression, freshly isolated PBMCs transfected with different amounts of RNAi plasmids for IL-22. The result showed that knockdown of IL-22 inhibited IAV-induced activation of the IL-34 (Fig. 3D). Furthermore, IL-34 production in culture supernatants was inhibited by IL-22 RNAi plasmids in a dose-dependent manner (Fig. 3E). Taken together, these data suggested that IL-22 was an upstream regulatory factor of IAV-triggered IL-34 production.
IL-34 feedback inhibits IAV-induced IL-22 expression
To define the role of IL-34 in the regulation of IAV induced inflammatory factor IL-22, we constructed mammalian expression plasmids for IL-34 and synthesized multiple RNAi plasmids for IL-34 (Fig. 4A). To determine the effects of IL-34 on the regulation of IL-22 mRNA expression, freshly isolated PBMCs were transfected with different amounts of IL-34 overexpression plasmids. The result showed that the mRNA level of IL-22 was decreased as the amount of IL-34 increased (Fig. 4B). Furthermore, IL-22 protein production in culture supernatants was suppressed by IL-34 over-expression in a dose-dependent manner (Fig. 4C). To confirm the above results, IL-34-specific RNAi was transfected into freshly isolated PBMCs and infected with 1 MOI IAV for 48 hours. The result showed that the mRNA level of IL-22 was increased by knocking down IL-34 (Fig. 4D). Furthermore, IL-22 protein production in culture supernatants stimulated with IAV was enhanced by knocking down IL-34 (Fig. 4E). Taken together, these results suggested that IL-34 played a very important role in the inflammatory response following an IAV-IL-22-IL-34 dependent positive regulatory order, while a negative feedback to IL-22 biosynthesis was also first observed (Fig.5).
Our results obtained from observations on IAV in patients as well as from experiments on cultures infected with the virus, indicated that IL-34 played an important role in the inï¬‚ammatory reactions occurring during IAV infections. Our study identiï¬ed that IAV infection triggered the production of IL-22, and resulted in subsequent IL-34 expression. However, this signal cascade could control itself through a negative feedback loop that IL-34 suppressed IL-22 production.
PBMCs are good model to investigate inflammation and host immune response (Mihai et al. 2006). Most viruses trigger robust cytokines expression in PBMCs though viral infection is not productive. In this study, using PBMCs model and clinical blood samples analysis, we found that IAV induced IL-22 mRNA expression and protein secretion. IL-22 has redundant, protective, or pathogenic functions during autoimmune, inï¬‚ammatory, and infectious diseases. Currently, a report has shown that IAV infection induced IL-22 expression in iNKT cells, which are important controllers of host pulmonary responses during experimental IAV infection in the mouse system (Christophe et al. 2012). These observations are consistent with our results in another cell model.
Although IL-22 reduces lung inï¬‚ammation during IAV infection, virtually little is currently known regarding the regulation of a newly identified infammatory factor IL-34 in IAV infection or the mechanism whereby IAV upregulates IL-34 expression. The present study provided considerable new information relevant to these issues. We ï¬rst identiï¬ed that IAV could upregulate the expression of IL-34. We then demonstrated a previously unknown loop in which IL-22 upregulated IL-34 expression and IL-34 feedback inhibited IL-22 expression in IAV infected PBMCs. At present, a ‘‘cross-talk’’ between some important pro-inflammatory factors, IL-22, IL-6 and IFN-γ, has been extensively examined (Stoyan et al. 2013), but the relationship between IL-22 and IL-34 has not been evaluated.
In our present study, there was a difference of the IAV infection induced IL-32 level between in vitro and in vivo. There was an over 20-fold increased in cell cultures infected with IAV in vitro, however, IL-34 mRNA levels were approximately 9.9-fold higher in IAV patients than in healthy individuals in vivo. It is likely the inflammatory factor is not able to be increased sharply under physiological conditions due to complex inflammation network in which inflammatory factors regulate each other.
In summary, we proposed a hypothetical model according to which IAV triggered the production of IL-22 and IL-34, resulting in a host inï¬‚ammatory response. In this model, we further demonstrated that IL-22 was located up-stream of IL-34 in the positive regulatory pathway. As shown in Fig. 5, the cross-talk between the two genes was described as follows: IL-22 upregulated IL-34 production, and conversely, IL-34 attenuated IL-22 activity.