Diminished secretion and function of IL‐29 is associated with impaired IFN‐α response of neonatal plasmacytoid dendritic cells

Abstract Plasmacytoid dendritic cells (pDCs) are key players in the antiviral immune response and type III IFNs such as IL‐29 appear to play a pivotal role in pDC function. Pronounced susceptibility to viral infections in neonates is partly resulting from diminished antiviral immune mechanisms. Accordingly, the aim of the present study was to investigate the impact of IL‐29 in the altered immune response of neonatal pDCs. PBMCs of adult and term newborns were stimulated with CpG‐ODN2216 in the presence or absence of IL‐29 and assessed for IFN‐α production, downstream‐signaling, and activation marker expression. A significantly lower IL‐29 production after TLR9‐specific stimulation was demonstrated in neonatal pDCs. IL‐29 enhanced the IFN‐α production of pDCs in adults compared to newborns. Newborn pDCs displayed a significantly lower surface expression of IL‐10 and IL‐28Rα receptor resulting in diminished STAT1 and IRF7 activation. Interestingly, concomitant stimulation with CpG‐ODN2216/IL‐29 had no impact on the expression of surface activation and maturation markers of pDCs in neither population. The diminished antiviral immune response of neonatal pDCs is associated with reduced production and cellular responses toward IL‐29. Potential therapeutic agents enhancing the IL‐29 response in neonatal pDCs possibly augment viral protection in newborns.


INTRODUCTION
The susceptibility to infections of newborn infants partly results from a hyporesponsiveness of innate immune responses against bacterial and viral antigens. This "immaturity" of neonatal immune responses is associated with neonatal morbidity and mortality. 1 In recent years, novel insights in immunological impairments of the neonatal immune system have been described, revealing diminished defending responses against infectious triggers. [2][3][4] immunological network of antiviral immune responses. The type III IFN family consists of IL-29 (IFN-1), IL-28A (IFN-2) as well as IL-28B  and was demonstrated to possess potent antiviral activity.
Despite the similarity to type I IFNs, type III IFNs signal through distinct receptors. The type III IFN receptor is composed of the IL-28 receptor (IL-28R )-and the IL-10 receptor 2 (IL-10R2)-chain. 6 Whereas the IL-10R2 chain is ubiquitously expressed, the IL-28R receptor appears to be more selectively expressed on immune cells such as pDCs, B cells, or M s. 7 Both receptor complexes share similar downstream-signaling pathways including JAK/STAT and IRF proteins.
Tyrosine kinase 2 (TYK2) is an important signaling molecule to mount a functional IFN-response. 8 Interestingly, type III IFN signaling and function was not influenced in a patient with TYK2-deficiency. 9 Up to date, few studies investigated the physiological and immunomodulatory effect of type III IFNs, especially of IL-29, on immune cells. IL-29 appears to augment the antiviral immune response in human peripheral blood and exert different immunomodulatory effects on pDCs. [10][11][12] To date, data in respect to type III IFNs in

Donors
Blood samples from healthy adult volunteers (age 18-40 years) were obtained by puncture of an antecubital vein after informed consent.
Umbilical cord blood was obtained after cesarean section from healthy term newborns (38-42 weeks of gestation). Written informed consent from the mother was obtained prior to birth. The study protocol was approved by the ethics committee of the Medical University of Vienna (1989/2015).

Cytokine measurement in cell culture supernatant
PBMCs were stimulated as described above for 24 h at 37 • C.
Cell free supernatant was obtained by 2 centrifugations steps at 10,000 × g for 10 min at 4 • C. Supernatant was aliquoted and frozen at -80 • C until analysis. IFN-(Thermo Fisher Scientific) and IL-29 (R&D Systems) were assessed using an ELISA according to the manufacturer's protocol.

Intracellular IFN-staining
PBMCs were stimulated as described above for 8 h at 37 • C. After

Intracellular IRF7 staining
PBMCs were stimulated as described above for 16 h at 37 • C. for 60 min at room temperature in the dark. Cells were washed twice and immediately analyzed using a Cytoflex LX flow cytometer (Beckman Coulter).

TaqMan quantitative RT-PCR
The ABI PRISM 7500HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) and One-Step RT-qPCR kit (Bio-Rad, Hercules, CA, USA) was used for quantitative RT-PCR analysis.
Primer-probes sets for IFNA FAM and GAPDH VIC were obtained predesigned from Applied Biosystems and tested for primer efficacy (gene expression assays Hs00265051_s1). Multiplex amplification was carried out in a total volume of 20 µl for 40 cycles of 3 s at 95 • C and 30 s at 60 • C. Initial denaturation was performed for 3 min at 95 • C. Target gene expression was normalized to GAPDH housekeeping gene expression. Normalized target gene expression was analyzed by the comparative ΔΔCT method and calculated as x-fold expression.

Statistical analysis
Statistical analysis was performed with R 3.5.1. Shapiro-Wilk test was performed to prove normal distribution and Levene test was applied to verify the homogeneity of variance. Data were then analyzed using 1-way ANOVA in accordance with Tukey. Data that were not normally distributed were analyzed using Kruskal-Wallis test. Flow cytometry data analysis was carried out with FlowJo X (FlowJo LLC, Ashland, Oregon). A P-value ≤ 0.05 was considered as statistically significant.

Concomitant TLR9 and type I or III IFN stimulation differentially activate adult and term PBMCs
Initially, we investigated the potential of different TLR agonists R848 induced a significantly lower production of IFN-after 24-h stimulation compared to CpG2216 (Fig. 1A); poly(I:C) as well as CpG2006 had no effect on the IFN-production (data not shown). Thus, we chose CpG2216 as optimal stimulus and conducted as next investigational step a dose-kinetic response. Increasing concentrations up to 50 µM CpG2216 resulted in comparable IFN-levels. In case of IL-29 secretion, stimulation with 1 µM CpG2216 resulted in a significantly higher production of IL-29 compared to 10 and 50 µM dosages (Fig. 1B), leaving 1 µM CpG2216 as optimal concentration for further experiments.
TLR9 specific stimulation led to a significantly higher secretion of IL-29 in adult PBMCs compared to term newborns (Fig. 1C). Interestingly, CpG2216 had no effect on the secretion of IL-28A and IL28B in our experimental setting (data not shown).
Next, we investigated the immunomodulatory effect of concomitant TLR9 and type I or III IFNs on the secretion of the complementary IFN type. First, we analyzed the dose-response relation of IL-29 showing a significant higher IFN-secretion in adult PBMCs when simultaneously stimulated with 1000 ng/ml IL-29. In contrast, term PBMCs did not respond to concurrent IL-29 stimulation displaying significantly lower IFN-levels ( Fig. 1D). Subsequently, concomitant stimulation with CpG2216 and increasing IFN-dosages resulted in a significant increase of IL-29 secretion in both groups (Fig. 1E). Adult PBMCs demonstrated a significantly higher IL-29 production after stimulation with CpG2216 and 1000 ng/ml IFN-compared to term newborns (Fig. 1F).

Newborn pDCs exhibit diminished response against IL-29
In the next step, we investigated the impact of simultaneous stimu- to CpG alone. Interestingly, a higher fraction as well as higher IFNproduction per pDC was observed in adult probands and with no effect in the newborn group, which is illustrated in the upper panel of Fig. 2C.

Downstream signaling of IL-29 is decreased in neonatal pDCs
To determine the mechanism behind the diminished functional response against IL-29 of newborn pDCs, we investigated the expression of surface receptors and associated signaling molecules of type I and III IFNs pathways. First, we examined the expression of the involved receptors for IFN-(IFNAR1/IFNAR2), IL-29 (IFNLR/IL10R), and CpG2216 (TLR9) recognition. Adult pDCs expressed significantly higher levels of IFNLR and IL10R compared to term newborns ( Fig. 3C and D). IFNAR1, IFNAR2, and TLR9 expression was similar between healthy adults and term newborns (Fig. 3A, B, and E). Next, we inves- Normal distribution was determined with Shapiro-Wilk test. Normal distributed data was analyzed using 1-way ANOVA in accordance with Tukey and not-normal distributed data were analyzed using Kruskal-Wallis test. * P < 0.05; ** P < 0.01; *** P < 0.001; MFI, mean fluorescence intensity levels of STAT1 in both age groups (Fig. 3F). The overall expression of STAT1 in adult and newborn PBMCs showed no difference in both groups (Fig. 3G).
Next, we investigated the expression levels of the transcription factor IRF7 in adult and neonatal pDCs. Adult pDCs showed a significant up-regulation of IRF7 after CpG, type I and III IFNs as well as combined stimulation compared to term infants. In infants, stimulation with the indicated stimuli had no significant effect on IRF7 up-regulation ( Fig. 3H). Unfortunately, we were not able to detect phosphorylated IRF7 via flow cytometry. ICOS-L, and CCR7 in both study groups. (Fig. 4).

DISCUSSION
IL-29 exerts a variety of immunomodulatory effects on various cell types. 10,[13][14][15] In recent years, several reports have shed light onto the type III IFN system and its role during viral infections such as airway-inflammation 16 or hepatitis. 17 Reduced production of IFN-in response to RSV infection is associated with a significant increase in the frequency of upper respiratory infections and pneumonia. 18 Important cellular players of innate immune responses fighting viral pathogens are pDCs. Although low in number in circulating blood, pDCs are the major producers of IFN-after viral pathogen encounter. 19 Recently, several reports described the immunomodulatory effect of IL-29 on the enhancement of IFN-production in human pDCs. 12,15,20 In our previous studies, we had demonstrated that pDCs from term and preterm neonates exhibit diminished IFN-production and decreased expression of activation markers after TLR-specific triggers, which is thought to contribute to the susceptibility toward viral infections in neonates. 3 4 Now, for the first time, we analyzed the production as well as the immunomodulatory effects of IL-29 on adult and neonatal PBMC and pDCs.
First, we determined the role of IL-29 in induction of IFN-upon TLR-specific engagement. In addition to pDCs, BDCA3 + myeloid dendritic cells (mDCs) are described to be a major IL-29 producer in peripheral blood after TLR3-specific stimulation. 21 Therefore, besides TLR7/8 and TLR9-agonists, we included poly(I:C) as TLR3-specific agent. In our experimental setting, we could only detect a robust IL-29 production in adult PBMCs-but not in neonatal PBMCs-upon  Our last investigational step was to investigate the notion that IL-29 may exhibit its effects by influencing the expression profiles of activation and maturation markers of pDCs. Correspondingly, concomitant stimulation with CpG2216 and IL-29 did alter the expression of T cell co-stimulatory molecules and homing receptors of pDCs possibly paving the way for the initiation of an adaptive immune response. Thus, IL-29 mediates its immunomodulatory antiviral effects on pDCs by enhancing the primary cytokine response, but by altering expression profiles of activation and homing markers after TLR9-specific engagement.
The impaired ability of neonatal pDCs to initiate an adequate antiviral immune response upon TLR-specific activation implies clinical and immunological relevance in this infection prone population. Indeed, IL-29 seems to be a promising candidate for the treatment of acute and chronic viral infections such as hepatitis C or E. 25,26 However, based on our data, therapeutic approaches targeting IL-29 might display reduced efficacy in neonates. Megjugorac et al. described that IL-4 lead to higher secretion of IL-1 receptor agonist (IL-1Ra) in monocytes resulting in augmented IL-29 output of pDCs. Thus, targeting pathways such as IL-1Ra might be beneficial to augment the IFN-response of neonatal pDCs. Neonates seem to produce comparable amounts of IL-1Ra during bacterial infections compared to adults. 27 The role of IL-1Ra in neonatal viral infections and the impact on the IFN-response will need to be elucidated in more detail in the future.
We conclude that not only the diminished IL-29 production but moreover lack of efficient IL-29 responsiveness is associated with the impaired capability of neonatal pDCs to secrete IFN-after