For adoptive transfer of effector T cells, 5x106 6 Th17 or Th1 1C6 T cells 7 were injected i.p., in cold PBS (Cellgro) into NOD.Scid recipient mice, which additionally received 200 ng of pertussis toxin (List Biological Labs) i.p. on d0 and d2 post-injection.
Sex differences in the incidence and severity of multiple sclerosis (MS) have long been recognized, but the underlying mechanisms remain poorly defined. Here, we elucidate the cellular and molecular underpinnings of why male sex is associated with a more aggressive and debilitating disease course. Using an adoptive transfer model of EAE to examine disease driven by CD4+ T helper 17 (Th17 cells), we find that male Th17 cells induced disease of increased severity relative to female Th17 cells, irrespective of whether the cells were transferred to male or female recipients. Both female and male peripheral Th17 exhibited phenotypic plasticity as early as disease onset, producing either IL-17 and IFNγ together or IFNγ alone; however, a higher proportion of male Th17 produced IFNγ throughout the disease course, with increased expression of both IFNγ and GM-CSF from CNS-infiltrating Th17 cells correlating with severe disease in males. Further, male cells upregulated signature genes previously associated with phenotypically plastic Th17 responses. Intriguingly, male sex chromosomal complement, rather than androgens, was responsible for increased pathogenicity, and an X-linked immune regulator, Jarid1c, was downregulated in pathogenic male Th17. Additionally, Jarid1c expression is reduced in CD4+ T cells derived from men with MS. Together, our data indicate that male sex is a critical regulator of Th17 cell plasticity and CNS autoimmunity.
Discovery of compounds inhibiting the ADP-ribosyltransferase activity of pertussis toxin
Cells were incubated for 30 minutes 591 at 37°C under normal cell culturing conditions, after which 10 ng/mL of pertussis AB5 holotoxin 592 (List Biological Laboratories Inc., 179A) was added to the cells and incubation was continued for 593 2 h.
Bordetella pertussis causes the highly contagious respiratory disease pertussis, also known as whooping cough. The resurgence of pertussis has been witnessed even in highly vaccinated populations, and macrolide-resistant strains have been isolated. One attractive target for drug development is the pertussis toxin – an important type IV secretion system-dependent virulence factor of B. pertussis. The AB5-topology pertussis toxin is composed of a pentameric PtxS2-S5 (1:1:2:1) complex mediating toxin binding to cell surface receptors, and one ADP-ribosyltransferase subunit PtxS1. Once internalized into the host cell, PtxS1 ADP-ribosylates α-subunits of heterotrimeric Gαi-superfamily members, thereby disrupting G-protein-coupled receptor (GPCR) signaling. Here, we describe protocols to purify mg-levels of truncated but highly active recombinant B. pertussis PtxS1 from E. coli and an in vitro high throughput-compatible assay to quantify NAD+ consumption during PtxS1-catalyzed Gαi ADP-ribosylation. The in vitro NAD+ consumption assay was used to screen compounds inhibiting the PtxS1 activity. Two inhibitory compounds (NSC 29193 and NSC 228155) with low micromolar IC50-values were identified that also were potent in an independent in vitro assay monitoring conjugation of ADP-ribose to Gαi. Docking and molecular dynamics simulations identified plausible binding poses of NSC 228155 and in particular NSC 29193, most likely owing to the rigidity of the latter ligand, at the NAD+-binding pocket of PtxS1. NSC 228155 inhibited the pertussis AB5 holotoxin-catalyzed ADP-ribosylation of Gαi in living human cells in low micromolar concentration. NSC 228155 and NSC 29193 might prove useful as lead compounds in targeted drug development in pertussis.
Macrophages were activated with Salmonella minnesota R595 LPS (100 ng/ml; List Biological Laboratories) 4 hours before nigericin treatment or 16 hours before bacterial infection, unless otherwise noted.
- 7-wk-old female SJL mice were immunized subcutaneously with 75 or 100 μg/mouse of pPLP emulsified in CFA (MT 300 μg/mouse). Mice also received 200 ng of Bordetella pertussis (List Biological Lab) ip on the day of immunization and 2 days later