Phosphoenolpyruvate regulates the Th17 transcriptional program and inhibits autoimmunity.

Aerobic glycolysis, a metabolic pathway essential for effector T cell survival and proliferation, regulates differentiation of autoimmune T helper (Th) 17 cells, but the mechanism underlying this regulation is largely unknown. Here, we identify a glycolytic intermediate metabolite, phosphoenolpyruvate (PEP), as a negative regulator of Th17 differentiation. PEP supplementation or inhibition of downstream glycolytic enzymes in differentiating Th17 cells increases intracellular PEP levels and inhibits interleukin (IL)-17A expression. PEP supplementation inhibits expression of signature molecules for Th17 and Th2 cells but does not significantly affect glycolysis, cell proliferation, or survival of T helper cells. Mechanistically, PEP binds to JunB and inhibits DNA binding of the JunB/basic leucine zipper transcription factor ATF-like (BATF)/interferon regulatory factor 4 (IRF4) complex, thereby modulating the Th17 transcriptional program. Furthermore, daily administration of PEP to mice inhibits generation of Th17 cells and ameliorates Th17-dependent autoimmune encephalomyelitis. These data demonstrate that PEP links aerobic glycolysis to the Th17 transcriptional program, suggesting the therapeutic potential of PEP for autoimmune diseases.

Human immune and gut microbial parameters associated with inter-individual variations in COVID-19 mRNA vaccine-induced immunity.

COVID-19 mRNA vaccines induce protective adaptive immunity against SARS-CoV-2 in most individuals, but there is wide variation in levels of vaccine-induced antibody and T-cell responses. However, the mechanisms underlying this inter-individual variation remain unclear. Here, using a systems biology approach based on multi-omics analyses of human blood and stool samples, we identified several factors that are associated with COVID-19 vaccine-induced adaptive immune responses. BNT162b2-induced T cell response is positively associated with late monocyte responses and inversely associated with baseline mRNA expression of activation protein 1 (AP-1) transcription factors. Interestingly, the gut microbial fucose/rhamnose degradation pathway is positively correlated with mRNA expression of AP-1, as well as a gene encoding an enzyme producing prostaglandin E2 (PGE2), which promotes AP-1 expression, and inversely correlated with BNT162b2-induced T-cell responses. These results suggest that baseline AP-1 expression, which is affected by commensal microbial activity, is a negative correlate of BNT162b2-induced T-cell responses.

JunB Is Critical for Survival of T Helper Cells.

Clonal expansion and differentiation of various T helper subsets, such as Th1, Th2, and Th17 cells, depend on a complex of transcription factors, IRF4 and a BATF-containing AP-1 heterodimer. A major BATF heterodimeric partner, JunB, regulates Th17 differentiation, but the role of JunB in other T helper subsets is not well understood. Here we demonstrate that JunB is required for clonal expansion of Th1, Th2 and Th17 cells. In mice immunized with lipopolysaccharide (LPS), papain, or complete Freund's adjuvant (CFA), which induce predominantly Th1, Th2 and Th17 cells, respectively, accumulation of antigen-primed, Junb-deficient CD4+ T cells is significantly impaired. TCR-stimulated Junb-deficient CD4+ T cells are more sensitive to apoptosis, although they showed largely normal proliferation and cellular metabolism. JunB directly inhibits expression of genes involved in apoptosis, including Bcl2l11 (encoding Bim), by promoting IRF4 DNA binding at the gene locus. Taken together, JunB serves a critical function in clonal expansion of diverse T helper cells by inhibiting their apoptosis.

Altered pre-existing SARS-CoV-2-specific T cell responses in elderly individuals.

Pre-existing SARS-CoV-2-specific T cells, but not antibodies, have been detected in some unexposed individuals. This may account for some of the diversity in clinical outcomes ranging from asymptomatic infection to severe COVID-19. Although age is a risk factor for COVID-19, how age affects SARS-CoV-2-specific T cell responses remains unknown. We found that pre-existing T cell responses to specific SARS-CoV-2 proteins, Spike (S) and Nucleoprotein (N), were significantly lower in elderly donors (>70 years old) than in young donors. However, substantial pre-existing T cell responses to the viral membrane (M) protein were detected in both young and elderly donors. In contrast, young and elderly donors exhibited comparable T cell responses to S, N, and M proteins after infection with SARS-CoV-2. These data suggest that although SARS-CoV-2 infection can induce T cell responses specific to various viral antigens regardless of age, diversity of target antigen repertoire for long-lived memory T cells specific for SARS-CoV-2 may decline with age; however, memory T cell responses can be maintained by T cells reactive to specific viral proteins such as M. A better understanding of the role of pre-existing SARS-CoV-2-specific T cells that are less susceptible to age-related loss may contribute to development of more effective vaccines for elderly people.

JunB regulates homeostasis and suppressive functions of effector regulatory T cells.

Foxp3-expressing CD4+ regulatory T (Treg) cells need to differentiate into effector Treg (eTreg) cells to maintain immune homeostasis. T-cell receptor (TCR)-dependent induction of the transcription factor IRF4 is essential for eTreg differentiation, but how IRF4 activity is regulated in Treg cells is still unclear. Here we show that the AP-1 transcription factor, JunB, is expressed in eTreg cells and promotes an IRF4-dependent transcription program. Mice lacking JunB in Treg cells develop multi-organ autoimmunity, concomitant with aberrant activation of T helper cells. JunB promotes expression of Treg effector molecules, such as ICOS and CTLA4, in BATF-dependent and BATF-independent manners, and is also required for homeostasis and suppressive functions of eTreg. Mechanistically, JunB facilitates the accumulation of IRF4 at a subset of IRF4 target sites, including those located near Icos and Ctla4. Thus, JunB is a critical regulator of IRF4-dependent Treg effector programs, highlighting important functions for AP-1 in Treg-mediated immune homeostasis.

A molecular epidemiologic study of point mutations for pyrimethamine-sulfadoxine resistance of Plasmodium falciparum isolates from Lao PDR.

To understand the current condition of pyrimethamine-sulfadoxine (PS) resistant falciparum malaria in Lao PDR, the frequency of point mutations in dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genes of Plasmodium falciparum were examined in 50 blood samples collected from the patients with P. falciparum infection in Southern Lao PDR. Point mutations in 5 codons of the DHFR gene, which is known to be related to pyrimethamine resistance, were detected in 15 out of the 50 samples (30%). Among the 15 samples, 10 samples showed a double mutation of codons 59 and 108 (Cys59Arg with Ser108Asn). In the remaining 5 samples, an additional mutation was observed in codon 51 (Asn51 lle), providing a triple mutation of codons 51, 59 and 108. On the other hand, point mutations in the 4 codons of DHPS gene related to sulfadoxine resistance were observed only in 2 samples (4.0%), namely in codon 437 (Ala437Gly). Only one sample showed mutations in both DHFR and DHPS genes. From the results, it should be considered that the frequency of PS resistant malaria is still low in Lao PDR. Continuous monitoring for the PS resistant malaria, however, is necessary because of the increasing use of PS in this country.

Cellulolytic environment in the midgut of the wood-feeding higher termite Nasutitermes takasagoensis.

Unlike lower termites, xylophagous higher termites thrive on wood without the aid of symbiotic protists. In the higher termite Nasutitermes takasagoensis, both endogenous endo-ß-1,4-glucanase and ß-glucosidase genes are expressed in the midgut, which is believed to be the main site of cellulose digestion. To further explore the detailed cellulolytic system in the midgut of N. takasagoensis, we performed immunohistochemistry and digital light microscopy to determine distributions of cellulolytic enzymes in the salivary glands and the midgut as well as the total cellulolytic activity in the midgut. Although cellulolytic enzymes were uniformly produced in the midgut epithelium, the concentration of endo-ß-1,4-glucanase activity and luminal volume in the midgut were comparable to those of the wood-feeding lower termite Coptotermes formosanus, which digests cellulose with the aid of hindgut protists. However, the size of ingested wood particles was considerably larger in N. takasagoensis than that in C. formosanus. Nevertheless, it is possible that the cellulolytic system in the midgut of N. takasagoensis hydrolyzes highly crystalline cellulose to a certain extent. The glucose produced did not accumulate in the midgut lumen. Therefore, the present study suggests that the midgut of the higher termite provides the necessary conditions for cellulolysis.

Digestive beta-glucosidases from the wood-feeding higher termite, Nasutitermes takasagoensis: intestinal distribution, molecular characterization, and alteration in sites of expression.

beta-Glucosidase [EC 3.2.1.21] hydrolyzes cellobiose or cello-oligosaccharides into glucose during cellulose digestion in termites. SDS-PAGE and zymogram analyses of the digestive system in the higher termite Nasutitermes takasagoensis revealed that beta-glucosidase activity is localized in the salivary glands and midgut as dimeric glycoproteins. Degenerate PCR using primers based on the N-terminal amino acid sequences of the salivary beta-glucosidase resulted in cDNA fragments of 1.7 kb, encoding 489 amino acids with a sequence similar to glycosyl hydrolase family 1. Moreover, these primers amplified cDNA fragments from the midgut, and the deduced amino acid sequences are 87-91% identical to those of the salivary beta-glucosidases. Successful expression of the cDNAs in Escherichia coli implies that these sequences also encode functional beta-glucosidases. These results indicate that beta-glucosidases that primarily contribute to the digestive process of N. takasagoensis are produced in the midgut. Reverse transcription-PCR analysis indicated the site-specific expression of beta-glucosidase mRNAs in the salivary glands and midgut. These results suggest that termites have developed the ability to produce beta-glucosidases in the midgut, as is the case for endo-beta-1,4-glucanase, in which the site of expression has shifted from the salivary glands of lower termites to the midgut of higher termites.