A novel mechanism for inducing tolerogenic DCs by inhibition of NPRA signaling in DCs is described. Our demonstration that down-regulation of NPRA levels and reduction in NPRA signaling in DCs increases the population of Tregs may have important applications in treating respiratory disease and inflammatory conditions. Effective allergen immunotherapy involves generation of Treg cells, and targeted NPRA down-regulation may be used as a means to develop tolerogenic DCs that induce Tregs to ameliorate or prevent inflammation.
The discovery of the NPRA inhibitory peptide NP73-102, which reduces expression of NPRA and inhibits NPRA signaling and the activation of several pro-inflammatory transcription factors in epithelial cells [8, 9], has provided the impetus to study the mechanism of how NPRA signaling affects inflammation and immunity. Increased NPRA signaling in DCs leads to a Th2 response, which restricts Treg production, while inhibiting signaling induces more IL-10 and TGF-β production and stimulates Treg formation.
Using an in vitro T cell-DC coculture system, down-regulation of NPRA by NP73-102 resulted in greater DC-mediated generation of Tregs. Analysis of DC surface marker expression and in vitro phagocytosis demonstrated that DC maturation was not significantly affected by NPRA signaling blockade. The mechanism underlying the NP73-102-induced Treg response is not fully understood. It may be due to inactivation of NF-κB activity by NP73-102 inhibition of NPRA signaling, since inhibition of NF-κB in DCs enhances their tolerogenic activity and prevents detrimental autoimmune diseases .
These data demonstrate that pNP73-102 increases the level of IL-10 and TGF-β, but not of IL-12 and IFN-γ, compared to cells given pANP or vector alone. TGF-β, which inhibits Th1 and Th2 development, is critical in Th17 development, in combination with IL-6  and leads to the generation of Foxp3-positive regulatory T cells . However, there was no increase in the number of Th17 lymphocytes, even in the presence of IL-6, in the DC-naïve T cell co-culture system, suggesting that Tregs may inhibit Th17 generation or that different cytokine profiles may produce diverse outcomes . As shown in Figure 3A, the pANP-treated group also induced few Tregs versus the empty vector control, suggesting that TGF-β may be involved in the process (Figure 2E &2F). However, attenuation of NPRA signaling by pNP73-102 induced a greater amount of SOCS3 and generated more Tregs which supports the hypothesis that ANP signaling effects are mediated through production of tolerogenic DCs. Data show that ANP inhibits TGF-β-induced Smad2 and Smad3 nuclear translocation in rat pulmonary arterial smooth muscle cells , which is consistent with our finding that ANP induced less TGF-β production in human DCs than NP73-102.
The SOCS3 protein was strongly induced by both IL-6 and IL-10. SOCS3 selectively inhibits IL-6 signaling via its binding to the IL-6 receptor, but does not inhibit the IL-10 receptor . The suppressive effect of SOCS3 is primarily restricted to STAT3 , and these results show that pNP73-102 inhibits STAT3 activity and enhances SOCS3 expression. This is in marked contrast to pANP and the vector control, which induce STAT3 phosphorylation and decrease SOCS3 expression in hmDCs. These data further support the idea that in this model, NPRA signaling in tolerogenic DCs involves the regulation of SOCS3 expression and STAT3 activity.
Cells exposed to pNP73-102 selectively diminished TLR2 expression compared to cells given pANP or vector. This could be explained by decreased NF-κB-mediated down-regulation of TLR through a binding site for NF-κB on the TLR2 promoter . Reports have indicated that down-regulation of both TLR4 and TLR2 expression in mice decreases the expression of inflammatory cytokines and enhances production of anti-inflammatory cytokines, which induce immune tolerance . Significantly, enhanced MyD88 expression was found in DCs treated with pNP73-102 compared to pANP and vector control. An LPS-inducible MyD88 is defective in its ability to induce IRAK phosphorylation and behaves as a dominant-negative inhibitor of LPS-induced NF-κB activation . Also, MyD88-knockout mice show significantly reduced expression of SOCS3 , which is in part consistent with our data, although it is unclear why pANP induced higher SOCS3 expression in the absence of MyD88 than vector control. Thus, the enhanced expression of both SOCS3 and MyD88 in DCs may be associated with a reduced response to ANP, whereas the specific enhancement of SOCS3 and/or MyD88 expression may explain the generation of tolerogenic DCs.
Indirect inhibition of JAKs due to the binding of SOCS to membrane proximal regions of receptor chains results in steric hindrance of constitutive JAK binding to the receptor . Inhibition of NPRA signaling by pNP73-102 through SOCS3 might occur by this mechanism. The IP data support this hypothesis. NPRA binds to SOCS3, and this interaction might contribute to the effects of NPRA signaling on immunoregulation. NPRA also binds to TLR2 and STAT3. However, TLR2 and SOCS3 involvement in regulation of NPRA expression (unpublished data) might result from these protein interactions rather than STAT3 involvement. Further work is needed to clarify whether the interactions among these proteins are direct or indirect.
In our animal model, we found that NPRA-/- mice had decreased expression of Th2-like cytokines, and that adoptive transfer of DCs from WT to NPRA-/- mice restored levels of these cytokines to those seen in WT. This is an important finding since it complements the in vitro results in an animal model. The lungs from NPRA-/- mice given DCs from WT mice exhibited inflammation similar to that of the WT. OVA-treated NPRA-/- mice given no DCs or given DCs from NPRA-/- mice did not have significant lung inflammation (Figure 6A, B), suggesting that DCs are the key mediators in modulating lung inflammation by NPRA signaling.
Taken together, our results demonstrate a novel mechanism for integration of TLR2, STAT3 and SOCS3 with NPRA signaling to regulate the immunomodulatory activity of DCs. They support the hypothesis that inhibition of NPRA signaling and TLR2 expression in DCs induces more IL-10 and TGF-β secretion and increases SOCS3 expression, thereby promoting the generation of Treg cells.