MACROPHAGE POLARIZATION: A DUAL ROLE BETWEEN INTERLEUKIN-4 SIGNALING AND SELENOPROTEIN BIOSYNTHESIS

Restricted (Penn State Only)
Author:
Shay, Ashley E
Graduate Program:
Molecular Medicine
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
October 06, 2017
Committee Members:
  • Kumble Sandeep Prabhu, Dissertation Advisor
  • Kumble Sandeep Prabhu, Committee Chair
  • Margherita Teresa-Anna Cantorna, Committee Member
  • Pamela Hankey Giblin, Committee Member
  • Connie Jo Rogers, Outside Member
  • Girish Soorappa Kirimanjeswara, Committee Member
Keywords:
  • macrophage
  • polarization
  • selenium
  • selenoprotein
  • helminth
  • inflammation
  • prostaglandin
  • resolution
  • cyclooxygenase
  • arachidonic acid
  • Nippostrongylus brasiliensis
  • interleukin-4
  • M2 macrophage
  • Peroxisome proliferator-activated receptor gamma
Abstract:
Selenium (Se) is an essential micronutrient that utilizes a unique mechanism to be chemically converted and charged to the 21st amino acid, selenocysteine (Sec), which is incorporated into proteins at a UGA codon. Sequence analysis has revealed 25 known selenoproteins in humans and 24 in mice based on a specific 3’ untranslated region element termed the Sec insertion sequence present in mRNA that forms a distinct stem-loop structure necessary for the recruitment of Sec to the actively translating ribosome. Studies have shown that either dietary Se deficiency or genetic mutations in individual selenoproteins or proteins involved in selenoprotein biosynthesis cause detrimental effects on human health. Selenoproteins have been shown to have a variety of functions, including redox homeostasis, thyroid metabolism, and protein folding. Many selenoproteins remain uncharacterized with new functions being unraveled. The goal of these studies is to identify new, uncharacterized selenoprotein functions involved in IL-4-dependent M2 macrophage polarization. An in vivo model of parasitic infection was used to induce a T-helper type-2 (Th2) response characterized by an IL-4 release in order to mimic IL-4 stimulation in in vitro studies. Mice were kept on three custom Se diets and infected with the murine helminth Nippostrongylus brasiliensis. Wild-type mice kept on Se deficient diet (Se-Def; sodium selenite ≤ 0.01 ppm) had a significantly higher parasite burden compared to mice fed Se adequate (Se-Ade; sodium selenite = 0.08 ppm) or Se supplemented (Se-Supp; sodium selenite = 0.4ppm) diets. Additionally, jejunal macrophages from wild-type mice fed Se-Def diet had higher M1 macrophage gene expression and lower M2 macrophage gene expression compared to mice fed Se-Ade or Se-Supp diets. Macrophage-specific Sec tRNA deficient mice had similar results to wild-type mice fed Se-Def diet despite being fed Se-Supp diet. These Se dependent effects appear to be mediated through arachidonic acid metabolism and peroxisome proliferator-activated receptor gamma signaling since inhibition of either pathway led to increased parasite burden, increased M1 macrophage gene expression, and decreased M2 macrophage gene expression despite being fed Se-Ade or Se-Supp diet and was rescued by exogenous administration of 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2). In order to examine the mechanism by which IL-4 stimulation increases the production of 15d-PGJ2 to drive M2 macrophage polarization, the two isoforms important for the production of 15d-PGJ2, cyclooxygenase (Ptgs; Cox)-1 and Cox-2, were examined. Interestingly, Ptgs-2, which is a highly inducible gene regulated by diverse stimuli via mechanisms involving transcriptional and translational control of expression, was significantly downregulated by IL-4 in bone marrow-derived macrophages (BMDMs), highlighting the importance of Cox-1 in a Th2 environment. This phenomenon not only challenges the dogma that Cox-1 is only developmentally regulated, but also demonstrates a novel mechanism in which IL-4–dependent regulation of Cox-1 involves the activation of the feline sarcoma oncogene kinase-Akt-mechanistic target of rapamycin complex (mTORC) axis. The presence of terminal oligopyrimidine sequences in the vicinity of the transcription start site in the 5’ untranslated region of both murine and human PTGS-1 mRNA further supported the involvement of mTORC. Since Cox-1 does not behave as a bona fide terminal oligopyrimidine sequence with regard to polysome-dependent control, it is likely that mTORC2 may affect Cox-1 protein expression. Downstream targets of mTORC2 are unknown and further work is needed to delineate the functional differences between mTORC1 and mTORC2 in regulating Cox-1 protein expression. That being said mTORC signaling appears to play a role in the accumulation of Cox-1 protein over the course of IL-4 stimulation potentially through either increased abundance of or increased interaction with heat shock protein chaperones. Activation of AMP-activated protein kinase (Ampk) by metformin, inhibition of mTORC by torin 1, or CRISPR/Cas9-mediated genetic knockout of tuberous sclerosis complex-2 blocked the IL-4-dependent expression of Cox-1 and the ability of macrophages to polarize to M2. However, use of 15d-PGJ2 partially rescued the effects of Ampk activation in-vivo, suggesting the importance of Cox-1 in macrophage polarization as well as N.brasiliensis clearance. In summary, these findings suggest a new paradigm where IL-4–dependent upregulation of Cox-1 expression may play a key role in tissue homeostasis and wound healing during Th2-mediated immune responses, as seen during helminth infections. To examine the Se-dependent requirement for prostaglandin J2 production and M2 macrophage polarization during IL-4 stimulation, macrophage specific Cre-Lox mouse models were utilized to knockout various components of the selenoprotein biosynthesis machinery. While loss of Sec-tRNA[Ser]Sec (Trsp) in macrophages significantly reduced the expression of the entire selenoproteome, loss of Sec insertion sequence binding protein-2 (Sbp2) led to only a partial loss of the selenoproteome, reminiscent of a hierarchical control of selenoprotein expression. Complete selenoproteome loss in the TrspLysMCre model lead to decrease in the production of Δ12-PGJ2, M2 macrophage polarization, and the ability to efficiently clear N.brasiliensis despite Se-supplementation. Sbp2LysMCre BMDMs, with a select few selenoproteins upregulated by IL-4 stimulation (Seleno-I, Seleno-K, Txnrd-1, and Gpx-2), were able to produce Δ12-PGJ2 at levels similar to those in Sbp2LysMWT BMDMs. Normal production of Δ12-PGJ2 led to unaltered M2 macrophage polarization and efficient N.brasiliensis clearance in the Sbp2LysMCremodel. These results suggest that these select few selenoproteins may regulate eicosanoid class switching upon IL-4 stimulation to increase M2 macrophages and effectively clear N.brasiliensis infection. Further work needs to be performed to narrow in on which selenoprotein(s) is playing a role in polarization and determining its exact function. This dissertation demonstrates for the first time how IL-4 and Se function in an additive manner to upregulate Cox-1 expression as well as a specific set of selenoproteins, respectively, to enhance eicosanoid class switching to produce the peroxisome proliferator-activated receptor gamma ligands Δ12-PGJ2 and 15d-PGJ2. Enhanced peroxisome proliferator-activated receptor gamma signaling increases the expression of M2 macrophages, which are required to efficiently clear helminth N.brasiliensis.